text
stringlengths 0
1.67M
|
|---|
Soluble rage protein |
The object of this invention is to elucidate the factors involved in the regulation of AGE (advanced glycation endproducts, which are produced in a living body accompanied with diabetes and aging) and RAGE (the receptor for AGE), in order to facilitate investigation on the biological activities, physiological phenomenon and diseases related to AGE and RAGE. Plural molecular species are known to exist for RAGE, and among such molecular species, soluble RAGE exhibits the activity to modulate interaction between AGE and transmembrane-type RAGE. Using this soluble RAGE or a nucleic acid encoding it, the soluble RAGE can be measured, in addition, investigation on various physiological phenomenon, biological activities, and diseases related to interaction between AGE and RAGE can be performed to facilitate development a medicine having further efficacy. |
1. (Canceled) 2. A nucleic acid having a base sequence coding for the polypeptide according to claim 23. 3. The nucleic acid according to claim 2 having a base sequence selected from a group comprising: (i) a base sequence described in the SEQ ID No. 1 of the sequence listing, comprising at least its open reading frame region; (ii) a base sequence which at least hybridizes with the base sequence as described in said (i) under a stringent condition; (iii) a base sequence which hybridizes, under a stringent condition, with a serial base sequence comprising five or more bases of the base sequence as described in the SEQ ID No. 1 of the sequence listing, and encodes for an amino acid sequence substantially equivalent to the soluble RAGE polypeptide; (iv) a base sequence which hybridizes, under a stringent condition, with a serial base sequence comprising ten or more bases of the base sequence as described in the SEQ ID No. 1 of the sequence listing, and encodes for an amino acid sequence substantially equivalent to the soluble RAGE polypeptide; (v) a base sequence which hybridizes, under a stringent condition, with a serial base sequence comprising fifteen or more bases of the base sequence as described in the SEQ ID No. 1 of the sequence listing, and encodes for an amino acid sequence substantially equivalent to the soluble RAGE polypeptide; (vi) a base sequence which hybridizes, under a stringent condition, with a serial base sequence comprising twenty or more bases of the base sequence as described in the SEQ ID No. 1 of the sequence listing, and encodes for an amino acid sequence substantially equivalent to the soluble RAGE polypeptide; and (vii) a base sequence coding for a polypeptide comprising an amino acid sequence having at least 80% homology with the polypeptide described in the SEQ ID No. 2, and/or, a serial amino acid sequence comprising at least 1 to 16 amino acid residues of Glu332 to Met347 out of the amino acid sequence described in SEQ ID No.2, and exhibiting biological activity substantially equivalent to said soluble RAGE polypeptide including AGE binding activity, suppressive or inhibitory activity on interaction between AGE and its receptor, and antigenicity substantially equivalent to said soluble RAGE polypeptide. 4. The nucleic acid according to claim 3 containing the open reading frame region of the base sequence described in the SEQ ID No. 1 of the sequence listing or a base sequence substantially equivalent to the open reading frame region. 5. A vector containing the nucleic acid according to claim 3. 6. A transformant containing the nucleic acid according to claim 3 or the vector according to claim 5. 7. The transformant according to claim 6, wherein the host cell is selected from the group consisting of a prokaryotic cell including E. coli , an yeast and an eukaryotic cell including a plant cell and an animal cell including 293T cell, CHO cell and COS cell. 8. A method for production of the polypeptide or a salt thereof comprising cultivating the transformant according to claim 6. 9. The polypeptide or a salt thereof, obtained by the expression of the transformant according to claim 6. 10. A primer available for PCR amplification of the open reading frame region of the base sequence as described in the SEQ ID No. 1 of the sequence listing, a partial sequence thereof, or a base sequence substantially equivalent to the open reading frame region. 11. A composition containing the nucleic acid according to claim 3, the vector according to claim 5, or the transformant according to claim 6. 12. A pharmaceutical composition containing the nucleic acid according to claim 3, or the vector according to claim 5. 13. The pharmaceutical composition according to claim 12 which is available for treatment of pathological conditions or symptoms selected from a group consisting of initiation and/or development of diabetic complications, aging-related diseases, Alzheimer disease, arteriosclerosis and diseases resulting from glycation of proteins in living bodies; and invasion and diffusion of cancer cells. 14. A diagnostic agent containing the nucleic acid according to claim 3 or the vector according to claim 5, which is adapted for diagnosing diseases resulting from alteration in the interaction between AGE and its receptor; in the amount of expression of the soluble RAGE polypeptide; and/or in the activity to capture AGE. 15. A pharmaceutical composition containing a compound capable of promoting or inhibiting biological activity of the polypeptide according to claim 23 or a salt thereof, or the nucleic acid according to claim 3. 16. A method for screening or a screening kit useful for carrying out the method, for screening a compound capable of promoting or inhibiting the biological activity of the polypeptide according to claim 23 or a salt thereof, or the nucleic acid according to claim 3 using any one selected from the group consisting of the polypeptide according to claim 23 or a salt thereof, the nucleic acid according to claim 3 or the vector according to claim 5. 17. The method for screening or the screening kit according to claim 16, which is a method for screening or a screening kit available for screening a compound effective for preventing initiation and/or development of diabetic complications. 18. A compound that regulates production of the soluble RAGE polypeptide which is obtained by using the method for screening or the screening kit according to claim 16. 19. A chimera molecular compound obtained by conjugating the polypeptide according to claim 23 with an amino acid sequence derived from a organism of different species. 20. A genetic diagnosis agent for diagnosing diseases related to the soluble RAGE polypeptide, which is for detection of presence of a mutated region existing in the soluble RAGE polypeptide, or in the gene or RNA coding for the soluble RAGE, wherein the mutated region is capable of altering expression or activity of the soluble RAGE poltypeptide. 21. The genetic diagnosis agent according to claim 20, using one selected from a group consisting of a restriction enzyme capable of specific recognition of a possible mutated region, if any, existing in one selected from gene, mRNA and hnRNA coding for the soluble RAGE polypeptide, and its isoschizomers; and oligonucleotide primers available for gene amplification of a possible mutated region, if any, existing in one selected from gene, mRNA and hnRNA coding for the soluble RAGE polypeptide. 22. A method for genetic diagnosis for diseases related to the soluble RAGE gene comprising the steps of: (a) obtaining a nucleic acid sample; (b) amplifying the nucleic acid sample obtained by the step (a), thereby obtaining amplified nucleic acid fragments which contain a possible mutated region, if any, existing in the soluble RAGE gene; and (c) detecting the presence of the mutated region in the nucleic acid fragments obtained in the step (b). 23. A polypeptide described in any one of the following (1) to (4) or a salt thereof: (1) (i) a polypeptide having at least 60% homology with the amino acid sequence described in the ID No. 2 of the sequence listing, and; (ii) (a) a polypeptide deleted of the transmembrane domain of the membrane-bound type RAGE protein and having a serial amino acid sequence comprising at least 1 to 16 amino acid residues out of Glu332 to Met347 of the amino acid sequence described in the SEQ ID No. 2 of the sequence listing at its C terminal; (b) a polypeptide having a serial amino acid sequence comprising at least 1 to 117 amino acid residues out of Met1 to Val117 at the N terminal of the amino acid sequence described in the SEQ ID No. 2 of the sequence listing; deleted with the transmembrane domain of the membrane-bound type RAGE protein; and having a serial amino acid sequence comprising at least 1 to 16 amino acid residues out of Glu332 to Met347 of the amino acid sequence described in the SEQ ID No. 2 of the sequence listing at its C terminal; (c) a polypeptide having a serial amino acid sequence comprising at least 1 to 1 17 amino acid residues out of a serial amino acid sequence comprising 1 to 117 amino acid residues at the N terminal of the amino acid sequence of the membrane-bound type RAGE polypeptide, and having a serial amino acid sequence comprising at least 1 to 16 amino acid residues out of Glu332 to Met347 of the amino acid sequence described in the SEQ ID No. 2 of the sequence listing at its C terminal; and (d) a polypeptide having amino acid sequence as described in the SEQ ID No.2 of the sequence listing, or a polypeptide exhibiting substantially equivalent biological activity with the polypeptide described in the SEQ ID No. 2 of the sequence listing; (2) a polypeptide comprising at least 1 to 16 amino acid residues out of Glu332 to Met347 of the amino acid sequence described in the SEQ ID No. 2 of the sequence listing; and comprising the following part of the amino acid sequence described in the SEQ ID No.2 of the sequence listing or a salt thereof: (i) a serial amino acid sequence comprising at least 5 to 115 amino acid residues; (ii) a serial amino acid sequence comprising at least 116 to 230 amino acid residues; (iii) a serial amino acid sequence comprising at least 231 to 347 amino acid residues; (iv) a serial amino acid sequence comprising at least one amino acid residue out of the 1st to 117th amino acid residues; (v) a serial amino acid sequence comprising at least one amino acid residue out of the 332nd to 347th amino acid residues; (vi) an amino acid sequence comprising the 19th to 347th amino acid residues; (vii) a serial amino acid sequence comprising the 1st to 347th amino acid residues; and (viii) a polypeptide having an amino acid sequence substantially equivalent to any one of the above-mentioned polypeptides; (3) a polypeptide selected from the polypeptides previously described in (1) or (2), which has an activity toward diseases caused by alteration in the interaction between AGE and its receptor, the expression of the soluble RAGE and/or the activity to capture AGE, or a salt thereof; and (4) a partial peptide or a salt of a polypeptide selected from the polypeptides as described in (1), (2) and (3). 24. A diagnostic agent containing the nucleic acid according to claim 6 which is adapted for diagnosing diseases resulting from alteration in the interaction between AGE and its receptor; in the amount of expression of the soluble RAGE polypeptide; and/or in the activity to capture AGE. 25. A method for screening or a screening kit useful for carrying out the method, for screening a compound capable of promoting or inhibiting the biological activity of the polypeptide or a salt thereof using a transformant according to claim 6. 26. The method for screening or the screening kit according to claim 25, which is a method for screening or a screening kit available for screening a compound effective for preventing initiation and/or development of diabetic complications. 27. A compound that regulates production of the soluble RAGE polypeptide which is obtained by using the method for screening or the screening kit according to claim 25. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates to a human soluble RAGE (receptor for advanced glycation endproducts) polypeptide, specifically to a native soluble RAGE polypeptide having a characteristic C terminal sequence, a nucleic acid encoding the polypeptide, a recombinant vector containing the nucleic acid, and a transformed cell, as well as use of them including screening, assay, diagnosis and treatment. 2. Description of the Related Art Recently, the number of diabetic patients in Japan is steadily increasing, and according to the statistics published in 1998 by the Ministry of Health and Welfare of Japanese Government, the number of diabetic patients was estimated to be six millions and nine hundreds of thousands, and if sub-clinical patients were included, the number would amount to 14 millions. The factor that directly affects life duration and quality of life of a diabetic patient is systemic derangement in the vascular system secondarily caused by hyperglycemia (i.e. vascular complications), not primary malfunction resulting from deficiency in the insulin supply. In view of this, the mechanism involved in occurrence of such vascular complications should be elucidated and a strategy to conquer such complications based on the knowledge should be constructed, as these are significant problems which need urgent resolution. The present inventors have been studied on the environmental and genetic factors involved in the development and progression of diabetic complications. The inventors performed in-vitro experiments on cultured vascular cells as well as in-vivo experiments on transgenic animals. Through such experiments, the inventors demonstrated that the environmental factors are mainly accounted for by advanced glycation endproducts (AGE), which increasingly accumulate with the development of diabetes. Moreover, the inventors demonstrated that the genetic factors are significantly related to the genes encoding RAGE which specifically recognize and bind to AGE, as well as genes existing downstream thereof encoding signal molecules and effector molecules(J. Biol. Chem., 272, 8723-8730, 1997; 275, 27781-25790, 2000; and J. Clin. Invest., 108, 261-268, 2001). It has been predicted that there may be a genetic factor responsible for the susceptibility/resistance of vascular complications in diabetic patients, but identification of such a factor has not been performed yet. It has been suggested that AGE are involved in the development of complications associated with diabetes and aging. Indeed, it was demonstrated that AGE bind to the receptors present on the surface of monocytes/macrophages, neurons, smooth muscle cells and vascular endothelial cells. It is thought that AGE interacts with these receptors, thereby exerting various physiological and biological actions to the living organisms and cells. For example, AGE act to enhance proliferation of vascular endothelial cells, and in addition, AGE are involved in enhancement of vascular permeability, and of formation of thrombus. As to effect of AGE on monocytes/macrophages, the release of cytokines from those cells would be enhanced, and release of various factors involved in cell proliferation and migration and synthesis of matrix would be also increased. It is further suspected that AGE might be involved in the inflammatory response observed in the wall of vessels. |
<SOH> SUMMARY OF THE INVENTION <EOH>Accumulating evidence indicates that AGE exert various physiological and biological effects on bodies and cells by binding to its receptor, which will lead to development and progression of various diseases. Based on these findings, it has been expected to find substances that could affect interaction between AGE and their receptor, thereby enabling application of such a substance for elucidation, prevention, diagnosis and treatment of various diseases. Quite recently, Yamamoto (one of the inventors) revealed that a RAGE protein expressed on human vascular cell has molecular diversity, which may be ascribed to alternative splicing of RAGE gene transcription products (see FIG. 1 ). According to the finding, one of the major molecular species is the soluble RAGE protein, which is secreted out of the cell because it lacks its transmembrane domain. However, the protein has an extracellular domain identical with the corresponding domain of a matured, membrane-binding type protein, and thus it can also bind to AGE to capture them. Indeed, recombinant human soluble RAGE protein was purified, and the purified protein was revealed to bind to various AGE fractions with high affinity, when binding assay with AGE ligands were performed. If expression of the gene encoding the soluble RAGE protein has diversity among individuals, diabetic patients showing high blood level of the protein would be resistant to vascular complications whereas other patients showing low blood level of the protein would be comparatively susceptible to vascular complications. The present invention provides a novel RAGE polypeptide, a nucleic acid comprising a base sequence encoding the polypeptide, a recombinant vector containing the nucleic acid, and a transformant containing the nucleic acid or the vector, as well as use of them for the purpose of screening, diagnosis and treatment. The present invention provides a technique for genetic diagnosis in relation to the soluble RAGE, for example, genetic diagnosis is performed on expression and polymorphism of the novel soluble RAGE protein, which is assumed to be a factor determining the resistance/susceptibility of a patient to diabetic complications, cancer and Alzheimer disease. Moreover, a technique for gene therapy is provided to reduce the risk of said diseases and related diseases based on the result of the diagnosis. The present invention provides the inventions of following [1] to [22]. [1] A polypeptide described in any one of the following (1) to (4) or a salt thereof: (1) (A) a soluble RAGE polypeptide, or (B) a polypeptide selected from the group consisting of the following or a salt thereof: (i) a polypeptide having at least 60% homology with the amino acid sequence of said soluble RAGE polypeptide, and; (ii) (a) a polypeptide containing a serial amino acid sequence comprising at least 5 to 347 amino acids out of the amino acid sequence of the soluble RAGE polypeptide 2 ; (b) a polypeptide deleted of the transmembrane domain of the membrane-bound type RAGE protein and having a serial amino acid sequence comprising at least 1 to 16 amino acid residues out of Glu 332 to Met 347 of the amino acid sequence described in the SEQ ID No. 2 of the sequence listing at its C terminal; (c) a polypeptide having a serial amino acid sequence comprising at least 1 to 117 amino acid residues out of Met 1 to Val 117 at the N terminal of the amino acid sequence described in the SEQ ID No. 2 of the sequence listing and deleted with the transmembrane domain of the membrane-bound type RAGE protein; (d) a polypeptide having a serial amino acid sequence comprising at least 1 to 117 amino acid residues out of a serial amino acid sequence comprising 1 to 117 amino acid residues at the N terminal of the amino acid sequence of the membrane-bound type RAGE polypeptide, and having a serial amino acid sequence comprising at least 1 to 16 amino acid residues out of Glu 332 to Met 347 of the amino acid sequence described in the SEQ ID No. 2 of the sequence listing at its C terminal,; and (e) a polypeptide having amino acid sequence as described in the SEQ ID No. 2 of the sequence listing, or a polypeptide exhibiting substantially equivalent biological activity with the polypeptide described in the SEQ ID No. 2 of the sequence listing; (2) a polypeptide selected from a group consisting of the following part of the amino acid sequence described in the SEQ ID No.2 of the sequence listing or a salt thereof: (i) a serial amino acid sequence comprising at least 5 to 115 amino acid residues; (ii) a serial amino acid sequence comprising at least 116 to 230 amino acid residues; (iii) a serial amino acid sequence comprising at least 231 to 347 amino acid residues; (iv) a serial amino acid sequence comprising at least one amino acid residue out of the 1st to 117th amino acid residues; (v) a serial amino acid sequence comprising at least one amino acid residue out of the 332nd to 347th amino acid residues; (vi) an amino acid sequence comprising the 19th to 347th amino acid residues; (vii) a serial amino acid sequence comprising the 1st to 347th amino acid residues; and (viii) a polypeptide having an amino acid sequence substantially equivalent to any one of the above-mentioned polypeptides; (3) a polypeptide selected from the polypeptides previously described in (1) and (2), which has a biological activity toward diseases caused by alteration in the interaction between AGE and its receptor, the expression of the soluble RAGE and/or the activity to capture AGE, or a salt thereof; and (4) a partial peptide or a salt of a polypeptide selected from the polypeptides as described in (1), (2) and (3). [2] A nucleic acid having a base sequence coding for the polypeptide according to [1]. [3] The nucleic acid according to [2] having a base sequence selected from a group comprising: (i) a base sequence described in the SEQ ID No.1 of the sequence listing, comprising at least its open reading frame region; (ii) a base sequence which at least hybridizes with the base sequence as described in said (i) under a stringent condition; (iii) a base sequence which hybridizes, under a stringent condition, with a serial base sequence comprising five or more bases of the base sequence as described in the SEQ ID No. 1 of the sequence listing, and encodes for an amino acid sequence substantially equivalent to the soluble RAGE polypeptide; (iv) a base sequence which hybridizes, under a stringent condition, with a serial base sequence comprising ten or more bases of the base sequence as described in the SEQ ID No. 1 of the sequence listing, and encodes for an amino acid sequence substantially equivalent to the soluble RAGE polypeptide; (v) a base sequence which hybridizes, under a stringent condition, with a serial base sequence comprising fifteen or more bases of the base sequence as described in the SEQ ID No. 1 of the sequence listing, and encodes for an amino acid sequence substantially equivalent to the soluble RAGE polypeptide; (vi) a base sequence which hybridizes, under a stringent condition, with a serial base sequence comprising twenty or more bases of the base sequence as described in the SEQ ID No. 1 of the sequence listing, and encodes for an amino acid sequence substantially equivalent to the soluble RAGE polypeptide; and (vii) a base sequence coding for a polypeptide comprising an amino acid sequence having at least 80% homology with the polypeptide described in the SEQ ID No. 2, and/or, a serial amino acid sequence comprising at least 1 to 16 amino acid residues of Glu 332 to Met 347 out of the amino acid sequence described in SEQ ID No.2, and exhibiting biological activity substantially equivalent to said soluble RAGE polypeptide-including AGE binding activity, suppressive or inhibitory activity on interaction between AGE and its receptor, and antigenicity substantially equivalent to said soluble RAGE polypeptide. [4] The nucleic acid according to [2] or [3] containing the open reading frame region of the base sequence described in the SEQ ID No.1 of the sequence listing or a base sequence substantially equivalent to the open reading frame region. [5] A vector containing the nucleic acid according to any one of [2] to [4]. [6] A transformant containing the nucleic acid according to any one of [2] to [4], or the vector according to [5]. [7] The transformant according to [6], wherein the host cell is selected from the group consisting of a prokaryotic cell including E. coli , an yeast and an eukaryotic cell including a plant cell and an animal cell including 293T cell, CHO cell and COS cell. [8] A method for production of the polypeptide according to [1] or a salt thereof, by cultivating the transformant according to [6] or [7]. [9] The polypeptide according to [1] or a salt thereof, obtained by the expression of the transformant according to [6] or [7]. [10] A primer available for PCR amplification of the open reading frame region of the base sequence as described in the SEQ ID No.1 of the sequence listing, a partial sequence thereof, or a base sequence substantially equivalent to the open reading frame region. [11] A composition containing the polypeptide according to [1] or a salt thereof, the nucleic acid according to any one of [2] to [4], the vector according to [5], or the transformant according to [6] or [7]. [12] A pharmaceutical composition containing the polypeptide according to [1] or a salt thereof, the nucleic acid according to any one of [2] to [4], or the vector according to [5]. [13] The pharmaceutical composition according to [12] which is available for treatment of pathological conditions or symptoms selected from a group consisting of initiation and/or development of diabetic complications, aging-related diseases, Alzheimer disease, arteriosclerosis and diseases resulting from glycation of proteins in living bodies; and invasion and diffusion of cancer cells. [14] A diagnostic agent containing a polypeptide according to [1] or a salt thereof, the nucleic acid according to any one of [2] to [4], the vector according to [5], or the transformant according to [6] or [7], which is adapted for diagnosing diseases resulting from alteration in the interaction between AGE and their receptor; in the amount of expression of the soluble RAGE polypeptide; and/or in the activity to capture AGE. [15] A pharmaceutical composition containing a compound capable of promoting or inhibiting biological activity of the polypeptide according to [1] or a salt thereof, or the nucleic acid according to any one of [2] to [4]. [16] A method for screening or a screening kit useful for carrying out the method, for screening a compound capable of promoting or inhibiting the biological activity of the polypeptide according to [1] or a salt thereof, or the nucleic acid according to any one of [2] to [4], using any one selected from the group consisting of the polypeptide according to [1] or a salt thereof, the nucleic acid according to any one of [2] to [4], and the vector according to [5] or a transformant according to [6] or [7]. [17] The method for screening or the screening kit according to [16], which is a method for screening or a screening kit available for screening a compound effective for preventing initiation and/or development of diabetic complications. [18] A compound that regulates production of the soluble RAGE polypeptide which is obtained by using the method for screening or the screening kit according to [16] or [17]. [19] A chimera molecular compound obtained by conjugating the polypeptide according to [1] with an amino acid sequence derived from a organism of different species. [20] A genetic diagnosis agent for diagnosing diseases related to the soluble RAGE polypeptide, which is for detection of presence of a mutated region existing in the soluble RAGE polypeptide, or in the gene or RNA coding for the soluble RAGE, wherein the mutated region is capable of altering expression or activity of the soluble RAGE polypeptide. [21] The genetic diagnosis agent according to [20], using one selected from a group consisting of a restriction enzyme capable of specific recognition of a possible mutated region, if any, existing in one selected from gene, mRNA and hnRNA coding for the soluble RAGE polypeptide, and its isoschizomers; and oligonucleotide primers available for gene amplification of a possible mutated region, if any, existing in one selected from gene, mRNA and hnRNA coding for the soluble RAGE polypeptide. [22] A method for genetic diagnosis for diseases related to the soluble RAGE gene comprising the steps of: (a) obtaining a nucleic acid sample; (b) amplifying the nucleic acid sample obtained by the step (a), thereby obtaining amplified nucleic acid fragments which contain a possible mutated region, if any, existing in the soluble RAGE gene; and (c) detecting the presence of the mutated region in the nucleic acid fragments obtained in the step (b). The other objects, features and advantages of the present invention will be apparent to those skilled in the art upon reading the following description of this Specification. However, it should be understood that the following description of examples herein concerns only with preferred embodiments, and implemented only for illustration purposes. It will be quite obvious to those skilled in the art that one can easily develop various variations and/or modifications by referring to the description hereof without departing the scope and intention of this invention as defined herein. All the patent documents and references cited herein are cited only for illustration purposes, and their contents should be considered as a part hereof. The term “and/or” used herein means existence of both of (1) concomitant conjugation and (2) selective conjugation. For example, “treatment and/or prevention” means both of (1) treatment and prevention, and (2) treatment or prevention. In other status, the term “and/or” is intended to mean both of (1) concomitant conjugation and (2) selective conjugation in the same manner. |
Human secreted proteins |
The present invention relates to human secreted polypeptides, and isolated nucleic acid molecules encoding said polypeptides, useful for diagnosing and treating gastrointestinal diseases, disorders, and/or conditions related thereto. Antibodies that bind these polypeptides and also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antogonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention. |
1-32. (canceled) 33. An isolated nucleic acid molecule comprising a first polynucleotide sequence at least 95% identical to a second polynucleotide sequence selected from the group consisting of: (a) a polynucleotide fragment of SEQ ID NO:X as referenced in Table 1A; (b) a polynucleotide encoding a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (c) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity; (e) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B; (f) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; (g) a polynucleotide encoding a predicted epitope of SEQ ID NO:Y as referenced in Table 1B; and (h) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(g), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues. 34. The isolated nucleic acid molecule of claim 33, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y, as referenced in Table IA. 35. The isolated nucleic acid molecule of claim 33, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:Y or the polypeptide encoded by the cDNA sequence included in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X, as referenced in Table 1A. 36. The isolated nucleic acid molecule of claim 33, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:X or the cDNA sequence included in ATCC Deposit No:Z, which is hybridizable to SEQ ID NO:X, as referenced in Table 1A. 37. The isolated nucleic acid molecule of claim 34, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus. 38. The isolated nucleic acid molecule of claim 35, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus. 39. A recombinant vector comprising the isolated nucleic acid molecule of claim 33. 40. A method of making a recombinant host cell comprising the isolated nucleic acid molecule of claim 33. 41. A recombinant host cell produced by the method of claim 40. 42. The recombinant host cell of claim 41 comprising vector sequences. 43. A polypeptide comprising a first amino acid sequence at least 95% identical to a second amino acid sequence selected from the group consisting of: (a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (b) a secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity; (e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B; (f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; and (g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B. 44. The polypeptide of claim 43, wherein said polypeptide comprises a heterologous amino acid sequence. 45. The isolated polypeptide of claim 43, wherein the secreted form or the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus. 46. An isolated antibody that binds specifically to the isolated polypeptide of claim 43. 47. A recombinant host cell that expresses the isolated polypeptide of claim 43. 48. A method of making an isolated polypeptide comprising: (a) culturing the recombinant host cell of claim 47 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide. 49. The polypeptide produced by claim 48. 50. A method for preventing, treating, or ameliorating a gastrointestinal disorder, comprising administering to a mammalian subject a therapeutically effective amount of the polypeptide of claim 43. 51. A method of diagnosing a gastrointestinal disorder in a subject comprising: (a) determining the presence or absence of a mutation in the polynucleotide of claim 33; and (b) diagnosing the gastrointestinal disorder based on the presence or absence of said mutation. 52. A method of diagnosing a gastrointestinal disorder in a subject comprising: (a) determining the presence or amount of expression of the polypeptide of claim 43 in a biological sample; and (b) diagnosing the gastrointestinal disorder based on the presence or amount of expression of the polypeptide. 53. A method for identifying a binding partner to the polypeptide of claim 43 comprising: (a) contacting the polypeptide of claim 43 with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide. 54. The gene corresponding to the cDNA sequence of SEQ ID NO:X. 55. A method of identifying an activity in a biological assay, wherein the method comprises: (a) expressing SEQ ID NO:X in a cell; (b) isolating the supernatant; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatant having the activity. 56. The product produced by the method of claim 53. |
<SOH> BACKGROUND OF INVENTION <EOH>The human digestive system is a collection of specialized organs and body tissues that prepare food for use by hundreds of millions of body cells. Food when eaten cannot reach ceUs because it cannot pass through the intestinal walls to the bloodstream and, if it could would not be in a useful chemical state. The gastrointestinal system modifies food physically and chemically and disposes of unusable waste. Physical and chemical modification (digestion) depends on exocrine and endocrine secretions and controlled movement of food through the digestive tract. The three fundamental processes of the digestive. system are: secretion (e.g., delivery of enzymes, mucus, ions and the like into the lumen, and hormones into blood), absorption (e.g., transport of water, ions and nutrients from the lumen, across the epithelium and into blood), and motility (e.g., contractions of smooth muscle in the wall of the tube that crush, mix and propel its contents). Control of digestive function is achieved through a combination of electrical and hormonal messages which originate either within the digestive system's own nervous and endocrine systems, as well as from the central nervous system and from endocrine organs such as the adrenal gland. The digestive system is composed of the digestive or alimentary tube and accessory digestive organs, which include the Mouth (e.g., tongue, taste buds, soft palate pharynx, salivary glands, teeth), Esophagus, Stomach, Liver, Gallbladder, Pancreas, Small Intestine (e.g., duodenum, jejunum, and ileum), and Large Intestine (e.g., caecum). Common digestive system disorders including infections, inflammations, ulcers and cancers of the digestive or alimentary tube and above listed accessory digestive organs are described in more detail below. Disorders of the Mouth The mouth comprises an area from the lips to the front of the tonsils (fauces) at the start of the throat. The mouth contains the gums, teeth, and the tongue, together with salivary glands which secrete fluids that lubricate and begin food digestion as it is chewed. The roof of the mouth consists of the hard palate at the front and the soft palate at the back. The floor of the mouth comprises the tongue (controlled by a number of muscles attached to bones in the neck). At the front and sides of the tongue there are a number of taste buds. These respond to different tastes at different places (e.g., sweet, salty, sour, and bitter). At the back of the tongue there are some swellings which consist of lymphoid tissue. Underneath the tongue there is a midline attachment (frenulum) and the opening of several of the salivary ducts. There are other salivary glands (the parotid glands) lying over the angle of the jaw with a duct opening to the inside of the cheek at about the level of the second molar tooth. Diseases and disorders of the mouth are vary greatly in manifested symptoms, frequencies, severities, and causes. Accordingly, diseases and disorders of the mouth may be caused or initiated by viruses, bacteria, genetics (e.g. autoimmune disorders), physical or chemical trauma, etc. For example, diseases and disorders of the mouth include canker sores (aphthous ulcers), herpetic stomatitis leukoplakia, gingivostomatitis, oral cancer, oral lichen lanus, oral thrush, histoplasmosis, salivary gland infections, glossitis, Hand, Foot and Mouth disease, salivary duct stones, mumps, etc. Disorders of the Esophagus Disorders of the Esophagus include dysphagia (e.g., difficulty in swallowing) and odynophagia (e.g., difficulty in swallowing accompanied by pain). Inflammatory disorders of the esophagus result from a variety of causes; for example, ingestion of noxious materials (e.g., corrosive esophagitis), lodgment of foreign bodies, or a complex of events associated with reflux of gastric contents from the stomach into the lower esophagus (e.g., peptic esophagitis). Disorders of the motility of the esophagus tend to be either precipitated or aggravated at times of nervous stress. A disorder commonly due to obesity is gastric reflux. Persisting reflux of gastric contents with acid and digesting enzymes leads to chemical inflammation of the lining of the esophagus and ultimately to (peptic) ulceration. If inadequately treated, the process leads to submucosal fibrosis and stricturing, and, besides the symptoms of heartburn and regurgitation, the patient experiences pain on eating and swallowing. Further disorders of the esophagus include the formation of diverticula. A serious injury to the esophagus is spontaneous rupture. It can occur in patients who have been vomiting or retching and in debilitated elderly persons with chronic lung disease. A rupture of this type confined to the mucosa only at the junction of the linings of the esophagus and stomach is called a Mallory-Weiss lesion. Benign tumors of the esophagus originate in the submucosal tissues and principally are leiomyomas (tumors composed of smooth muscle tissue) or lipomas (tumors composed of adipose, or fat, tissues). Malignant tumors are either epidermal cancers, made up of unorganized aggregates of cells, or adenocarcinomas, in which there are gland-like formations. Cancers arising from squamous tissues are found at all levels of the organ, whereas adenocarcinomas are more common at the lower end where a number of glands of gastric origin are normally present. The prognosis is poor because diagnosis is difficult and the tumor has usually been growing for one or two years before symptoms are apparent. Disorders of the Stomach Any disorder that affects the power of coordination of the stomach muscles is capable of producing symptoms ranging from those that are mildly unpleasant (e.g., anorexia and nausea) to others that are life-threatening. The intrinsic muscles of the stomach are innervated by branches of the vagus nerves, which travel along the esophagus from their point of emergence in the brain stem. Severing these nerves or altering their function by the use of anticholinergic medication may produce temporary or more prolonged change in the ability of the stomach to empty itself. Gastric retention may result from the degeneration of the nerves to the stomach that can result from diabetes merintus. Obstruction due to scarring in the area of the gastric outlet, or to tumors encroaching on the lumen, causes the stomach to fill up with its own secretions as well as with partially digested food. In these circumstances, vomiting leads to dehydration and to electrolyte losses, which threaten life if not corrected. Disorders of the stomach include ulcerative diseases, which involve mucosal breakdown either confined to the superficial layers of the mucosa (e.g., an erosion) or extending through the intrinsic layer of muscle of the mucosa into the tissues below (e.g., an ulcer). The circumstances that contribute to mucosal injury and ulcer formation include physical and chemical trauma that result from hot fluids and food, aspirin and other drugs, irritating spices, and pickling fluids. In addition, genetic factors are involved in the development of ulcers. The complications of peptic ulcers are hemorrhage, perforation, and obstruction of the outlet of the stomach (pyloric stenosis) by scarring of the duodenal bulb or of the pyloric channel. A diffuse inflammation of the stomach lining, gastritis, is usually an acute process caused by contaminated food, alcohol abuse, or by bacterial- or viral-induced inflammation of the gastrointestinal tract (gastroenteritis). The other form of gastritis is gastric atrophy, in which the thickness of the mucosa is diminished. Diffuse gastric atrophy leads to partial loss of the glands and secreting cells throughout the stomach and may be associated with ironeficiency anemia. Malignant tumors of the stomach are common and are probably a result of both genetic and environmental factors. Gastric cancer affects men more often than women and accounts for about 20 percent of all deaths from cancers of the gastrointestinal tract in the United States. Other. malignant tumors that involve the stomach are tumors ordinarily made up of lymphoid and connective tissue. Benign tumors, especially leiomyomas, are common and may, when large, cause massive hemorrhage. Polyps of the stomach are not common except in the presence of gastric atrophy. Disorders of the Duodenum and Small Intestine Primary cancer of the duodenum is an infrequent disease, however, benign tumors of the duodenum, particularly polyps and carcinoids, are more frequent. Cancers of the common bile duct or of the pancreas are important causes of death. A common disorder of the small intestine, distension, is caused by lack of coordination of the inner circular and outer longitudinal muscular layers of the intestinal wall which usually results in an accumulation of excess contents in the lumen. The most common cause of disturbed motility in the small intestine is food that contains an unsuitable additive, organism, or component. One of the most serious problems in small intestine are motor disturbances which arise from an intestinal obstruction that results from an actual encroachment on the bowel by an adhesive band or from an internal block produced by a tumor or gallstone. In addition, as profound an obstruction results when a portion of the intestine undergoes partial necrosis, or death, from failure of its blood supply. The extremely common disorder known as the irritable bowel syndrome is probably due to a disturbance of the motility of the whole intestinal tract. The symptoms vary from watery diarrhea to constipation and the passage of stools with difficulty. When the colon is involved, an excess of mucus is often observed in the stools. Occasionally the irritable bowel syndrome may be due to an allergy to a particular foodstuff. The syndrome may develop following an infection such as bacillary dysentery, after which the small intestine remains irritable for many months. A further disorder, malabsorption occurs when the small intestine is unable to transport properly broken down products of digestive materials from the lumen of the intestine into the lymphatics or mesenteric veins, where they are distributed to the rest of the body. Defects in transport occur either because the absorptive cells of the intestine lack certain enzymes, whether by birth defect or by acquired disease, or because they are hindered in their work by other disease processes that infiltrate the tissues, disturb motility, permit bacteria to overpopulate the bowel, or block the pathways over which transport normally proceeds. A malabsorption disorder of unknown cause, tropical sprue, is associated with partial atrophy of the mucosa of the small intestine. Disorders of the small intestine also include bacterial and parasitic infections. Appendicitis is an inflammation of the vermiforin appendix that may be caused by infection or partial or total obstruction. Chronic inflammations of the small intestine include tuberculosis and regional enteritis (Crohn's disease). Celiac disease causes damage to the mucosa of the small intestine, though it is not clear whether it is caused by an immune reaction, or an inability to break down a toxic protein, gluten, to smaller peptide fractions. Studies of the immune function of those with celiac disease suggest that at least a major part of the process is a delayed hypersensitivity reaction and that the morphological changes are correlated with the presence of circulating antibodies to gluten. The mucosal reaction results in progressive atrophy, with dwarfing, if not complete disappearance, of the microvilli and villi that line the intestinal tract. Disorders of the Large Iytestine A wide variety of diseases and disorders occur in the large intestine. A disease that is analogous to achalasia of the esophagus is an idiopathic condition called aganglionic megacolon, or Hirschsprung's disease. It is characterized by the absence of ganglion cells and normal nerve fibres from the distal (or lower) portion of the large intestine, which results in reduced neuromuscular transmission and ceased peristalsis. The entire colon slowly becomes more and more distended and thick-walled. Abscesses in the perianal area are common complicating features of many diseases and disorders of the large intestine. Fungal and bacterial infections are also common causes of large intestine disorders. The most common form of chronic colitis, ulcerative colitis, is idiopathic. It varies from a mild inflammation of the mucosa of the rectum, giving rise to excessive mucus and some spotting of blood in the stools, to a severe, sudden, intense illness, with destruction of a large part of the colonic mucosa, considerable blood loss, toxemia and, less commonly, perforation. The most common variety affects only the rectum and sigmoid colon and is characterized by diarrhea and the passage of mucus. Apart from the greater tendency for fistulas to form and for the wall of the intestine to thicken until the channel is obstructed, Crohn's disease is distinguishable from ulcerative colitis by microscopic findings. In Crohn's disease, the maximum damage occurs beneath the mucosa, and lymphoid conglomerations, known as granulomata, are formed in the submucosa. Crohn's disease attacks the perianal tissues more often than does ulcerative colitis. Although these two diseases are not common, they are disabling. Tumors of the colon are usually polyps or cancers. A peculiar form of polyp is the villous adenoma, often a slowly growing, fernlike structure that spreads along the surface of the colon for some distance. Cancers compress the colonic lumen to produce obstruction, they attach to neighbouring structures to produce pain, and they perforate to give rise to peritonitis. Cancers also may metastasize to distant organs before local symptoms appear. Anorectal disorders related to defecation are more common in the Western world than elsewhere. These disorders usually take the form of fissures (cuts or cracks in the skin or mucous membrane) at the junction of the anal mucous membrane with the slin between the thighs. Anal fistulas sometimes occur as complications of serious bowel disease, as in tuberculosis or Crohn's disease of the bowel, or in certain parasitic diseases. A more general disorder is the enlargement of veins of the rectum and anus to form external or internal hemorrhoids. Hemorrhoids protrude, are associated with anal itching and pain, and bleed, especially when they come in contact with hard stools. Disorders of the Liver A variety of agents, including viruses, drugs, environmental pollutants, genetic disorders, and systemic diseases, can affect the liver. The resulting disorders usually affect one of the three functional components of the liver: the hepatocyte (liver cell) itself, the bile secretory (cholangiolar) apparatus, or the blood vascular system. Most acute liver diseases are self-limited, and liver functioning returns to normal once the causes are removed or eliminated. In some cases, however, the acute disease process destroys massive areas of liver tissue in a short time, leading to extensive death (necrosis) of hepatic cells and often to death of the patient. Hepatitis may result from viral infections or toxic damage from drugs or poisons. When acute hepatitis lasts for six months or more, a slow but progressive destruction of the surrounding liver cells and bile ducts occurs, a stage called chronic active hepatitis. If hepatocellular damage is severe enough to destroy entire acini (clusters of lobules), they are often replaced with fibrous scar tissue. Bile canaliculi and hepatocytes regenerate in an irregular fashion adjacent to the scar tissue and result in a chronic condition called cirrhosis of the liver. Where inflammatory activity continues after the onset of cirrhosis, the disorderly regeneration of hepatocytes and cholangioles may lead to the development of hepatocellular or cholangiolar cancer. Although a number of viruses affect the liver, including the cytomegalovirus of infancy and childhood and the Epstein-Barr virus of infectious mononucleosis, there are three distinctive transmissible viruses that are specifically known to cause acute damage to liver cells: hepatitis virus A (HAV), hepatitis virus B (HBV), and hepatitis virus non-A, non-B (NANB). The symptoms characteristic of the acute hepatitis caused by the HAV, HBV, and NANB viruses are essentially indistinguishable from one another. Acute hepatitis also may be caused by the overconsumnption of alcohol or other poisons, such as comnmercial solvents (e.g., carbon tetrachloride), acetaminophen, and certain fungi. Such agents are believed to cause hepatitis when the formation of their toxic intermediate metabolites in the liver cell (phase I reactions) is beyond the capacity of the hepatocyte to conjugate, or join them with another substance for detoxification (phase II reactions) and excretion. Acute canalicular (cholestatic) hepatitis is most commonly caused by certain drugs, such as chlorpromazine, that lead to idiosyncratic reactions or, at times, by hepatitis viruses. Acute congestive liver disease usually results from the sudderi engorgement of the liver by fluids after congestive heart failure. A prominent autoimmune liver disease is Wilson's disease, which is caused by abnormal deposits of large amounts of copper in the liver. Granulomatous hepatitis, a condition in which localized areas of inflammation (granulomas) appear in any portion of the liver lobule, is a type of inflammatory disorder associated with many systemic diseases, including tuberculosis, sarcoidosis, schistosomiasis, and certain drug reactions. Granulomatous hepatitis rarely leads to serious interference with hepatic function, although it is often chronic. The end result of many forms of chronic liver injury is cirrhosis, or scarring of liver tissue in reponse to previous acinar necrosis and irregular regeneration of liver nodules and bile ducts. Primary biliary cirrhosis, a widespread, though uncommon, autoimnune inflammatory disease of bile ducts, is a disorder primarily affecting middle-aged and older women. Secondary biliary cirrhosis results from chronic obstruction or recurrent infection in the extrahepatic bile ducts caused by strictures, gallstones, or tumors. Infestation of the biliary tract with a liver fluke, Clonorchis sinensis , is a cause of secondary biliary cirrhosis in Asia. Portal hypertension, the increased pressure in the portal vein and its tributaries that is the result of impediments to venous flow into the liver, is brought about by the scarring characteristic of the cirrhotic process. The increased pressure causes feeders of the portal vein to distend markedly, producing varices, or dilations of the veins. When varices are located in superficial tissues, they may rupture and bleed profusely. Two such locations are the lower esophagus and the perianal region. The accumulation of fluid in the abdominal cavity, or ascites, is related to portal hypertension, significant reduction in serum albumin, and renal retention of sodiun When albumin levels in blood are lower than normal, there is a mnarked reduction in the force that holds plasma water within the blood vessels and normally resists the effects of the intravascular pressure. The resulting increase in intravascular pressure, coupled with the increased internal pressure caused by the portal venous obstruction in the liver, leads to massive losses of plasma water into the abdominal cavity. The associated reduction of blood flow to the kidneys causes increased elaboration of the hormone aldosterone, which, in turn, causes the retention of sodium and water and a reduction in urinary output. In addition, because the movement of intestinal lymph into the liver is blocked by the cirrhotic process in the liver, the backflow of this fluid into the abdominal cavity is greatly increased. A progressive reduction in kidney function that often occurs in persons with advanced acute or chronic liver disease, hepatorenal syndrome, probably results from an inadequate perfusion of blood through the cortical (outer) portions of the kidneys, where most removal of waste products occurs. With advanced hepatocytic dysfunction, a spasm of blood vessels in the renal cortex can occur, often with good blood flow to the rest of the kidney. This spasm results in progressive failure in kidney function and often leads to death. Although not uncommon, cancer originating in the liver, usually in hepatocytes and less frequently in cells of bile duct origin, is rare in the West and is almost always associated with active cirrhosis, particularly the form found in patients with chronic hepatitis. Long exposure to certain environmental poisons, such as vinyl chloride or carbon tetrachloride, has also been shown to lead to hepatic cancer. Cancers arising elsewhere in the body, particularly in abdominal organs, lungs, and lymphoid tissue, commonly lead to metastatic cancer in the liver and are by far the most frequent type of hepatic malignancy. Various benign types of tumors and cysts arise from certain components of the liver, such as the hepatocytes (adenomas) or blood vessels (hemangiomas). While the cause of these lesions is not always clear, hepatic adenomas are associated with the prolonged use of female sex hormones (estrogens). Benign cysts in the liver may occur as congenital defects or as the result of infections from infestation of the dog tapeworm ( Echinococcus granulosus ). Abscesses on the liver result from the spread of infection from the biliary tract or from other parts of the body, especially the appendix and the pelvic organs. Specific liver abscesses also result from infections with the intestinal parasite Entamoeba histolytica. Disorders of the Biliary Tract Cholelithiasis, or the formation of gallstones in the gallbladder, is the most common disease of the biliary tract. There are three types of Gallstones: stones containing primarily calcium bilirubinate (pigment stones); stones containing 25 percent or more of cholesterol; and stones composed of variable mixtures of both bilirubin and cholesterol (mixed gallstones). Pigment stones are the result of an increased amount of bilirubin in the liver (due to hemolytic disease) and the consequent secretion into the biliary tract of increased amounts of the water-soluble conjugate, bilirubin diglucuronide, a pigment that is normally secreted in the urine. Cholesterol and mixed cholesterol-bilirubinate stones occur when the proportion of cholesterol in bile exceeds the capacity of bile acids and lecithin to contain the total amount of cholesterol in micellar colloidal solution. Postcholecystectomy syndrome comprises painful attacks, often resembling preoperative symptoms, that occasionally occur following the surgical removal of gallstones and the gallbladder. These attacks may be related to intermittent muscular spasms of the sphincter of Oddi or of the bile ducts. Cancer of the biliary tract is rare but may occur in almost any area, including the gallbladder, the hepatic ducts, the common bile duct, or the ampulla of Vater. In cancer of the bile duct, congenital cysts and parasitic infections, such as liver flukes, seem to lead to increased risks. Persons with extensive chronic ulcerative colitis also show a greater than normal incidence of bile duct carcinoma. Jaundice, or yellowing of the skin, scleras, and mucous membranes, occurs whenever the level of bilirubin in the blood is significantly above normal. This condition is evident in three different types of disorders including, unconjugated, or hemolytic, jaundice; hepatocellular jaundice; and cholestatic, or obstructive jaundice. Unconjugated jaundice results when the amount of bilirubin produced from hemoglobin by the destruction of red blood cells or muscle tissue (myoglobin) overwhelms the normal capacity of the liver to transport it or when the ability of the liver to conjugate normal amounts of billrubin into billrubin diglucuronide is significantly reduced by inadequate intracellular transport or enzyme systems. Hepatocellular jaundice arises when liver cells are damaged so severely that their ability to transport bilirubin diglucuronide into the biliary system is reduced, allowing some of this yellow pigment to regurgitate into the bloodstream. Cholestatic jaundice, occurs when essentially normal liver cells are unable to transport bilirubin either through the hepatocytic-bile capillary membrane, because of damage in that area, or through the biliary tract, because of anatomical obstructions (e.g., atresias, gallstones, cancer). Disorders of the Pancreas Inflammation of the pancreas, or pancreatitis, is probably the most common disease of this organ. The disorder may be confined to either singular or repeated acute episodes, or it may become a chronic disease. There are many factors associated with the onset of pancreatitis, including direct injury, certain drugs, viral infections, heredity, hyperlipidernia (increased levels of blood fats), and congenital derangements of the ductal system. Localized, severe abdominal and midback pain resulting from enzyme leakage, tissue damage, and nerve irritation is the most common symptom of acute pancreatitis. In severe cases, respiratory failure, shock, and even death may occur. Chronic pancreatitis rarely follows repeated acute attacks. It seems instead to be a separate disorder that results in mucus plugs and precipitation of calcium salts in the snaller pancreatic ducts. Mucous production and plugging of the pancreas in Cystic fibrosis patients almost invariably causes destruction and scarring of the acinar tissue, usually without damaging the islets of Langerhans. A sirnilar process in the hepatic biliary system produces foci of fibrosis and bile duct proliferation, a singular form of cirrhosis. The discovery of new human digestive system associated polynucleotides, the polypeptides encoded by them, and antibodies that immunospecifically bind these polypeptides, satisfies a need in the art by providing new compositions which are useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders of the digestive system, including, but not limited to, dysphagia, odynophagia, congenital disorders of the esophagus, gastric reflux, diverticula, Mallory-Weiss lesions, leiomyomas of the esophagus, lipoma, anorexia, nausea, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, gastric cancer, benign tumors of the duodenum (e.g., polyps and carcinoids), pancreatic cancer, cancer of the bile duct, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine (e.g., Meckel's diverticulum, multiple diverticula), bacterial and parasitic infection (e.g., traveler's diarrhea, typhoid, paratyphoid, cholera, roundworms, tapeworms, amoebae, hookworms, strongyloides, threadworms, and blood flukes), megacolon (e.g., Hirschsprung's disease, aganglionic megacolon, acquired megacolon), colitis (e.g., due to bacterial, fungal, or parasitic infection, ulcerative colitis), tumors of the colon (e.g., polyps or cancers), anorectal disorders (e.g., anal fistulas, hemorrhoids, hepatitis (e.g., acute, chronic, persistent hepatitis, viral (for example, hepatitis caused by hepatitis virus A (HAV), hepatitis virus B (HBV), and hepatitis virus non-A, non-B (NANB) infection), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), cirrhosis, portal hypertension, cholelithiasis, cancer of the biliary tract, jaundice (e.g., unconjugated, hemolytic, hepatocellular, cholestatic, or obstructive jaundice). The discovery of new human gastrointestinal-associated polynucleotides, the polypeptides encoded by them, and antibodies that immunospecifically bind these polypeptides, satisfies a need in the art by providing new compositions which are useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating gastrointestinal-specific diseases and disorders described in more detail below. |
<SOH> SUMMARY OF INVENTION <EOH>The present invention encompasses human secreted proteins/polypeptides, and isolated nucleic acid molecules encoding said proteins/polypeptides, useful for detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating gastrointestinal diseases and disorders. Antibodies that bind these polypeptides are also encompassed by the present invention; as are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention also encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention. detailed-description description="Detailed Description" end="lead"? |
Sex- specific selection of sperm from transgenic animals |
The present invention relates to methods and materials for pre-selecting the sex of mammalian offspring. In particular, the materials and methods described herein permit the enrichment of X- or Y-chromosome-bearing sperm in semen by introducing a transgene into a sex chromosome under control of regulatory sequences that provide for expression of the transgene in a haploid-specific manner. |
1. A mammal comprising a transgene on a sex chromosome, wherein the expression of said transgene is operably linked to a promoter region that confers haploid-specific expression to said transgene. 2. The mammal of claim 1, wherein said promoter also confers tissue-specific expression to said transgene. 3. The mammal of claim 2, wherein said tissue-specific expression is testis-specific expression. 4. The mammal of claim 3, wherein said transgene is expressed in one or more cells selected from the group consisting of primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa. 5. The mammal of claim 1 wherein said promoter region comprises the promoter for the protamine gene. 6. The mammal of claim 1 wherein expression of said transgene selectively kills those cells expressing said transgene. 7. The mammal of claim 1 wherein expression of said transgene selectively disables those cells expressing said transgene. 8. The mammal of claim 1 wherein said transgene encodes a marker protein which can be used to sort those cells expressing said transgene from cells not expressing said transgene. 9. Haploid cells harvested from the mammal of claim 1. 10. Haploid cells according to claim 9 which have been enriched for cells expressing said transgene. 11. The mammal of claim 1 wherein the mammal is an ungulate. 12. The mammal of claim 1 wherein the mammal is selected from the group consisting of porcine, ovine, bovine, and caprine. 13. The mammal of claim 1, wherein expression of said transgene is inducible. 14. The mammal of claim 13, wherein expression of said transgene selectively kills those cells expressing said transgene when exposed to an inducing agent. 15. The mammal of claim 13, wherein expression of said transgene selectively disables those cells expressing said transgene when exposed to an inducing agent. 16. The mammal of claim 13, wherein said transgene encodes a marker protein which can be used to sort those cells expressing said transgene when exposed to an inducing agent from cells not expressing said transgene. 17. Haploid cells harvested from the mammal of claim 13. 18. Haploid cells harvested from the mammal of claim 13 which have been enriched for cells expressing said transgene. 19. A method for producing a population of mammalian haploid cells that is enriched for haploid cells containing a specific sex chromosome, wherein said method comprises: harvesting haploid cells from a mammal comprising a transgene which is capable of killing or disabling cells in cis when expressed, wherein said transgene is selectively expressed in cells comprising said specific sex chromosome, and wherein the expression of said transgene is operably linked to a promoter region that confers haploid-specific expression to said transgene, whereby expression of the transgene kills or disables those haploid cells expressing said transgene. 20. The method of claim 19, wherein said method further comprises removing or discarding said killed or disabled haploid cells. 21. A method for producing a population of mammalian haploid cells that is enriched for haploid cells containing a specific sex chromosome, wherein said method comprises: (a) harvesting haploid cells from a mammal comprising a transgene which is capable of killing or disabling cells in cis when expressed, wherein said transgene is selectively expressed in cells comprising said specific sex chromosome, wherein the expression of said transgene is operably linked to a promoter region that confers haploid-specific expression to said transgene, and wherein expression of said transgene is inducible; and (b) inducing the expression of said transgene to kill or disable the those haploid cells expressing said transgene. 22. The method of claim 19, wherein said method further comprises removing or discarding said killed or disabled haploid cells. 23. A method for producing a population of mammalian haploid cells that is enriched for haploid cells containing a specific sex chromosome, wherein said method comprises (a) harvesting haploid cells from a mammal comprising a transgene which is capable of generating a detectable phenotype in cells in cis when expressed, wherein said transgene is selectively expressed in cells comprising said specific sex chromosome, and wherein the expression of said transgene is operably linked to a promoter region that confers haploid-specific expression to said transgene, whereby expression of the transgene produces said detectable phenotype marker in those haploid cells expressing said transgene; and (b) sorting the haploid cells based on the expression of said detectable phenotype. 24. A method for producing a population of mammalian haploid cells that is enriched for haploid cells containing a specific sex chromosome, wherein said method comprises (a) harvesting haploid cells from an animal comprising a transgene which is capable of generating a detectable phenotype in cells in cis when expressed, wherein said transgene is selectively expressed in cells comprising said specific sex chromosome, wherein the expression of said transgene is operably linked to a promoter region that confers haploid-specific expression to said transgene, and wherein expression of said transgene is inducible, whereby expression of the transgene produces said detectable phenotype in those haploid cells expressing said transgene; and (b) sorting the haploid cells based on the expression of said detectable phenotype. 25. The method of any one of claims 19-24, wherein the mammal is an ungulate. 26. The method of claim 25 wherein the ungulate is selected from the group consisting of porcine, ovine, bovine, and caprine. 27. The method of any one of claims 19-24, wherein the haploid cells harvested are spermatozoa. 28. A method for producing a mammal, comprising contacting an ovum with one or more spermatozoa produced according to the method of claim 27 to fertilize said ovum. 29. A method according to claim 28, wherein said ovum is fertilized by an assisted reproductive technique. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Throughout history, humans have sought the ability to assert control over the sex of offspring; both human and livestock. Homo sapiens' attempts to select sex of offspring prior to conception has been well-documented, as evidenced by historical descriptions of methods. Early techniques, circa 500 B.C., began with monoorchydectomy and progressed through a variety of techniques which have come down to us via folklore (such as placing an egg or scissors under the bed for conception of a girl, and placing a hammer under the bed and tying off the left testicle to conceive a boy) (Fugger, 1999, Theriogenology 52:1435-1440). A more scientific approach began in the last century and included utilizing a reported differential survival between X and Y spermatozoa dependent on the pH of the medium. (Shettles, 1970). Further techniques progressed to exploit differences in motility (Ericsson et al., 1973, Nature 246:241-24, Steeno et al., 75, Botchan et al., 1997) or cell density (e.g., centrifugation in a Percoll gradient, Lin et al., 1998, J. Assist. Reprod. and Genetics 15:565-569) to use in distinguishing X from Y sperm. Other techniques tried include size, head shape, surface properties, surface macromolecules, mass, and swimming velocity (see review by Windsor et al., 1993, Reprod. Fert. Dev. 5:155-71). One group, Fabricant et al., (U.S. Pat. No. 4,722,887), utilized the differential expression of a sperm cell-surface sulfoglycolipid to develop a method for separating X-chromosome-bearing and Y-chromosome-bearing sperm by polymeric phase separation. A recent approach to the problem of sex pre-selection relates to methods that rely on the use of antibodies directed to sex-specific epitopes on sperm, or, alternatively, on fertilized embryos. For example, evidence for a male-specific cell surface antigen was first obtained by Eichwald and Silmser (1955, Transplant Bull 2:148) using the inbred mouse strain C57BL/6, but it remained for Hauscha (Transplant Bull, 1955, 2:154) to later hypothesize the existence an antigen coded for by a Y-linked gene. This surface marker became known as H-Y (bistocompatibility locus on the Y chromosome). Y-sperm-specific surface expression of the H-Y antigen has been suggested to be a target epitope for sex pre-selection, and antibodies raised to the H-Y antigen were expected to allow the routine sorting of sperm using cell sorting or immunological adsorption of H-Y expressing sperm (Peter et al., 1993, Theriogenology 40:1177-1185). Similarly, sex-specific antibodies were disclosed as allowing the selective ablation of sperm or embryos utilizing complement (U.S. Pat. No. 5,840,504). See also, U.S. Pat. No. 4,999,283; U.S. Pat. No. 4,511,661; U.S. Pat. No. 4,191,749; U.S. Pat. No. 4,448,767; U.S. Pat. No. 4,680,258; and U.S. Pat. No. 5,840,504. The locus of at least one of the genes responsible for H-Y expression is on the Y chromosome, and this antigen has been shown to be cross-reactive among numerous speciess ranging from fish to man. It is possible that the H-Y antigen may be the primary sex determinant and may control testicular development in mammals. (Wattle, et al., 1975; Wattle and Ok, 1980); Ok, et al., “Application of Monoclonal Anti-H-Y Antibody for Human H-Y Typing,” Human Genetics, 57: 64-67 (1981). H-Y is a “minor” histocompatibility antigen, which is a separate genetic locus from the major histocompatibility complex (MHC). Minor histocompatibility loci are mainly concerned with cellular immunity; few if any products of these loci are efficient in raising antibodies. Nevertheless, a search for a serological counterpart to the transplantation H-Y antigen appeared to have been successful when a serological “E-Y” method was reported by Goldberg and coworkers (1971, Nature 232: 478). Recent data indicates, however, that the serological detectable “H-Y” antigen may not be the same as the histocompatibility antigen. (Simpson et al., 1990, Arch. Androl. 24:235). The molecule identified by serological methods is now widely referred to as serologically detectable male antigen (SMA). These immunological methods have not always lived up to expectations however (Bradley, 1989). For example, some authors found no evidence that H-Y is preferentially expressed on Y-bearing sperm (e.g. Hendricksen et al. 1993, Mol. Reprod. Devel. 35:189) and, in a review, Windsor et al. (1993, Reprod. Fert. Dev. 5:155) have concluded that no differences between the two classes of sperm can be detected immunologically. Another method recently described as showing utility for sex pre-selection involves the use of Fluorescence Activated Cell Sorting (FACS) for sorting sperm based on the reduced amount of DNA in Y sperm as opposed to X sperm due to the small mass of the Y chromosome. The difference in DNA content between X and Y sperm, ranges from 2.8% in humans and 4.0% in most livestock, to 12.5% in voles (Gillis, 1995). See, e.g. Rath et al., 1999, J. Anim. Sci. 77:3346-3352; Welch and Johnson, 1999, Theriogenology 52:1343-1352; Fugger et al., 1998, Human Reprod. 13: 2367-2370; Cran et al., 1995, Vet. Rec. 135: 495-496; Seidel et al., 1997, Theriogenology 48: 1255-1265. FACS sorting, following by insemination, has been shown to work in bulls, rams (Johnson and Clark, 1988) and humans (Johnson et al., 1993). In spite of these successes, this technique is limited by three factors. First, it requires the sophisticated operation of expensive machines. Second, the reagents used to fluorescently label the DNA and the near UV light used to detect the dyes may lead to chromosomal damage and/or mutations. Third, this technique has a poor yield. Progress in these techniques has recently been summarized in review articles by Reubinoff and Schenker (1996) and Botcham et al (1997). In another example, which combines sorting based on DNA content, followed by immunological selection, Spaulding, (U.S. Pat. Nos. 5,021,244 and 5,346,990, and 5,660,997) first sorted sperm into enriched X- and Y-chromosome bearing preparations via DNA content and cell sorting techniques. Spaulding then used the sorted sperm to screen for sex-specific sperm proteins and then proceeded to predict the use of the sex-specific protein for raising antibodies to allow purification of the sperm population to either X-chromosome bearing or Y-chromosome bearing populations. WO 01/47353 proposes methods by which expression of a transgene inserted into a sex chromosome might alter the sex ratio of offspring. The dairy industry demands a large number of females cows for the production of milk, and currently male calves, except those necessary for breeding, are culled. Similarly, for the production of beef, male cattle are preferred. In spite of recent progress in techniques for sorting male sperm (Y) from female sperm (X), the techniques still lack the robustness needed for routine use for the commercial production of livestock. One reason is that the techniques available are difficult to use to produce the large numbers of viable spermatozoa required for use in the production of livestock. Also, some of the techniques carry with them the threat of creating mutations while sorting sperm. Thus, there remains a need in the art for methods and materials permitting the sex pre-selection of offspring. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention discloses a robust technique for producing semen that is enriched for active sperm containing either the X chromosome or the Y chromosome. Because cows of reproductive age normally will give birth to only a single calf per year, which will randomly either be male or female, the ability to pre-select the sex of an offspring is particularly advantageous for the dairy and meat industries. However, in the agricultural industry generally, methods for sex selection could be used to upgrade the nutritional characteristics and quantities of animals produced. Accurate selection of the sex of the offspring could allow the birth of many genetically superior animals of a single sex as offspring of one genetically desirable parent. Thereby, the desirable genetic characteristics of the parent animals can be propagated with much greater velocity than is possible in nature. The ability to increase the reproductive capacity of genetically prized animals, especially dairy cattle, may be a key to solving the hunger problem which exists in many countries today by allowing a more efficient use of available resources. In a first aspect, this invention relates to animals in which one or more transgenes are incorporated into either the X or Y chromosome, and hence into those sperm cells containing a specific sex chromorome, of the transgenic animal. Preferably, the transgene(s) is (are) under the control of a promoter region and/or an enhancer region which is capable of conferring haploid-specific expression to the coupled trausgene. In these embodiments, the semen produced by the transgenic animal can be enriched for sperm of a given-sex by expression of the transgene. Transgenes useful for this invention include genes that encode a gene product which is toxic for a haploid cell when expressed in cis, e.g., suicide genes such as pertussis toxin or the immunoglobulin heavy chain binding protein (BiP); alternatively, gene products that allow for survival in cis when the sperm cell is exposed to a selective agent may be employed. The term “in cis” is defined hereinafter. In other embodiments, the gene may encode an antisense construct capable of blocking the expression of a gene essential for the continued viability or function of the sperm. The only requirement of the transgene(s) used in the instant invention is that they may be expressed in a haploid-specific manner, and that transgene expression results in enhanced production of offspring having the selected sex. The transgenes of the instant invention need not result in the death of the haploid cells in which it is expressed, however, in order to enrich for sperm of a selected sex. For example, a gene may prevent induction of pregnancy by a haploid cell, for example by preventing fusion of a sperm with an oocyte, or by reducing or preventing motility. Even a minor change in fitness, resulting from the presence of one or more transgenes, may result in enhanced production of offspring having the selected sex. See, e.g., Ellison et al., Mol. Reprod. Dev. 55: 249-55 (2000). The transgenes of the instant invention may also encode gene products that allow the haploid cells expressing the gene to be detected by a detection method, e.g., optically. Genes which can be detected optically include the Green Fluorescent Protein (GFP) (Tsien, 1998, Annu. Rev. Biochem. 67:509-44), drFP83 and the ES mutant (Terskikh, et al., 2000, Science 290:1585-1588). Finally, the transgenes of the instant invention may encode gene products that make a haploid cell apparent to an in vivo immune response. For example, sex chromosome-specific immune infertility may be produced by immunizing an animal against a transgene product expressed in a sex chromosome-specific and haploid-specific manner. Such immunity may be created in either a male or a female, resulting in enhanced production of offspring of the selected sex. See, e.g. Tsuji et al., J. Reprod. Immunol. 46: 31-8 (2000); Mahmoud et al., Andrologica 28: 191-6 (1996). The term “haploid cell” as used herein refers to cells that contain a single set of unpaired chromosomes. In animals, cells that give rise to gametes (i.e., sperm and eggs) undergo meiotic division, whereby a diploid cell divides into four haploid cells. In males, a diploid cell contains both an X and a Y chromosome, referred to herein as “sex chromosomes.” Each haploid cell contains only one sex chromosome. The term “haploid cell” can preferably refer to the following cells produced by a male animal: primary spermatocytes (produced in the first meiotic division); secondary spermatocytes (produced in the second meiotic division); spermatids; differentiating spermatids; and spermatozoa. The term “haploid cell” can also refer to cells produced by a female animal, e.g., oocytes and eggs. The term “transgenic” as used herein refers to a cell or an animal that comprises heterologous deoxyribonucleic acid (DNA). Methods for producing transgenic cells and animals are well known to the ordinarily skilled artisan. See, e.g., Mitani et al., 1993, Trends Biotech, 11: 162-166; U.S. Pat. No. 5,633,067, “Method of Producing a Transgenic Bovine or Transgenic Bovine Embryo,” DeBoer et al., issued May 27, 1997; U.S. Pat. No. 5,612,205, “Homologous Recombination in Mammalian Cells,” Kay et al, issued Mar. 18, 1997; and PCT publication WO 93/22432, “Method for Identifying Transgenic Pre-Implantation Embryos;” Kereso et al., 1996, Chromosome Research 4: 226-239; Holló et al., 1996, Chromosome Research 4: 240-247; U.S. Pat. No. 6,025,155, and U.S. Pat. No. 6,077,697; all of which are incorporated by reference herein in their entirety, including all figures, drawings, and tables. The term “heterologous DNA” refers to DNA having (1) a different nucleic acid sequence than DNA sequences present in cell nuclear DNA; (2) a subset of DNA having a nucleotide sequence present in cell nuclear DNA, where the subset exists in different proportions in the heterologous DNA than in the cell nuclear DNA; (3) a DNA sequence originating from another organism species than the species from which cell nuclear DNA originates; and/or (4) a different nucleic acid sequence than DNA sequences present in cell mitochondrial DNA. An artificial chromosome present in a transgenic cell can comprise heterologous DNA. Heterologous DNA can encode multiple types of recombinant products, as defined hereafter. The term “different nucleic acid sequence” as used herein refers to nucleic acid sequences that are not substantially similar. The term “substantially similar” as used herein in reference to nucleic acid sequences refers to two nucleic acid sequences having preferably 80% or more nucleic acid identity, more preferably 90% or more nucleic acid identity or most preferably 95% or more nucleic acid identity. Nucleic acid identity is a property of nucleic acid sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical bases in the two sequences by the total number of bases and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, while sequences that are less highly conserved and have deletions, additions, or replacements have a lower degree of identity. Those of ordinary skill in the art will recognize that several computer programs are available for performing sequence comparisons and determining sequence identity. A “transgenic animal” is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell. Preferred transgenic animals are mammals, most preferably non-human primates, mice, rats, ungulates (including cows, pigs, horses, goats, and sheep), dogs and cats. Preferably, a transgenic animal expresses one or more gene products in a haploid-specific manner. Additionally, preferred sites of integration of a heterologous DNA in a transgenic animal of the instant invention include the Y chromosome and the X chromosome. Numerous methods are well known in the art for producing transgenic animals. For example, a nucleic acid construct according to the invention can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division (Brinster et al., Proc. Nat. Acad. Sci. USA 82:4438-4442, 1985). Alternatively, embryos can be infected with viruses, especially retroviruses, modified to carry nucleic acid constructs according to the invention, or other gene delivery vehicles. In particularly preferred embodiments, transgenic animals can be produced by nuclear transfer using a transgenic nuclear donor cell. Nuclear transfer methods are well known to the ordinarily skilled artisan, and are described in detail hereinafter. See, e.g., U.S. Pat. No. 6,107,543; U.S. Pat. No. 6,011,197; Proc. Nat'l. Acad. Sci. USA 96: 14984-14989 (1999); Nature Genetics 22: 127-128 (1999); Cell & Dev. Diol 10: 253-258 (1999); Nature Biotechnology 17: 456-461 (1999); Science 289: 1188-1190 (2000); Nature Biotechnol. 18: 1055-1059 (2000); Nature 407: 86-90 (2000). The term “transgene” refers to the heterologous DNA included in a transgenic cell or animal. The transgene may refer to the coding sequence or it may also refer to the coding sequence plus additional 5′ and 3′ DNA sequences necessary for the proper expression of the transgene. A cell may contain multiple transgenes, which may or may not be identical to one another. The term “expression” as used herein refers to the production of the protein encoded by a transgene useful in the invention from a nucleic acid vector containing protease genes within a cell. The nucleic acid vector is transfected into cells using well known techniques in the art as described herein. The nucleic acid vector is preferably integrated into the genome of the host. A nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression. The precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region which directs the initiation of RNA transcription. Such regions will also normally include those 5′-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. The term “promoter” as used herein, refers to nucleic acid sequence needed for gene sequence expression. Promoter regions vary from organism to organism, but are well known to persons skilled in the art for different organisms. For example, in prokaryotes, the promoter region contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5′-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. In preferred embodiments, a promoter is sex-specific, and/or sperm-specific, -and/or inducible. A particularly preferred promoter is the protamine promoter. The term “sex chromosome-specific expression” refers to expression of a gene product in cells with a specific sex chromosome. Particularly preferred is sex chromosome-specific expression in haploid cells, which, by definition, contain only a single sex chromosome. Sex chromosome-specific expression of a gene can be achieved by inserting the gene to be expressed into the specific sex chromosome. In preferred embodiments, a gene is rendered X chromosome-specific by its operable incorporation into the X chromosome. In these embodiments, only haploid cells that contain an X chromosome will exhibit expression of the gene product. In a similar fashion, a gene may be rendered Y chromosome-specific by its operable incorporation into the Y chromosome. The term “haploid-specific expression” refers to expression of a gene product only by haploid cells, such as spermatozoa, spermatids, etc. The gene product may be expressed during assembly, during spermatogenesis, or after at any time prior to fertilization. In particularly preferred embodiments, a gene that is expressed in a haploid-specific fashion is also expressed in a sex chromosome-specific fashion. The transgenes of the instant invention may also be configured and arranged to confer “tissue-specific” expression on the transgene. That is, the expression of the transgene may take place only in specific body tissue(s) of the transgenic animal. Particularly preferred are transgenes that are expressed only in the testis or only in the ovary of the transgenic animal. The term “specific expression” refers to gene expression that is predominantly localized to a desired cell type. Such expression may be “leaky,” i.e., there may be some ectopic expression of the gene in undesired cell types, but the predominant expression may still be in the specific cell type. In preferred embodiments, “specific expression” refers to a gene that is expressed 5-fold higher, 10-fold higher, 20-fold higher, 50-fold higher, and 100-fold higher or more in the desired cell type when compared to expression in undesired cells. Regulatory sequences that may provide for haploid-specific expression and/or tissue-specific expression are well known to the skilled artisan. See, e.g., Yamanaka et al., Biol. Reprod. 62: 1694-1701 (2000); Westbrook et al., Biol. Reprod. 63: 469-81 (2000); Tosaka et al., Genes Cells 5: 265-76 (2000); Reddi et al., Biol. Reprod. 61: 1256-66 (1999); Nayernia et al., Biol. Reprod. 61: 1488-95 (1999); Mohapatra et al., Biochem. Biophys. Res. Comm. 244: 540-5 (1998); Herrada et al., J. Cell Sci. 110: 1543-53 (1997); Rodriguez et al., J. Androl. 21: 414-20 (2000); and Lee et al., Biol. Chem. Hoppe Seyler 368: 807-11 (1987). In preferred embodiments, the gene that is expressed in a haploid-specific manner is under the control of the promoter of the protamine gene. See, e.g., Queralt and Olivia, Gene 133: 197-204 (1993). In certain preferred embodiments, the transgene is capable of killing haploid cells in which it is expressed (“in cis”) and not in cells not expressing the transgene; while in other preferred embodiments, the transgene is capable of functionally disabling haploid cells in cis when expressed. The term “killing haploid cells” refers to the ability of one or more expressed gene products to kill a haploid when expressed. The gene(s) may kill the haploid either directly though the activity of one or more expressed proteins, or indirectly, via metabolizing an exogenously supplied compound to produce a toxic product or by failing to metabolize a toxic chemical supplied exogenously. In preferred embodiments, the gene product(s) are expressed in a haploid-specific manner; in other embodiments, the gene product(s) are expressed in an inducible fashion. Particularly preferred as a gene to kill haploid cells is the immunoglobulin heavy chain binding protein (BiP) gene, mutations of which have been shown to exhibit dominant negative effects in cells. See, e.g., Hendershot et al., Proc. Natl. Acad. Sci. USA 93: 5269-74 (1996). The skilled artisan will recognize that expression of a gene may also render haploid cells in which it is expressed viable in the presence of a molecule that would ordinarily kill or disable the cells. Such a strategy is often used, e.g., by inserting antibiotic resistance genes into cells, then killing those cells that do not express the resistance gene by contacting the cells with an antibiotic. The term “disabling haploid cells” refers to the ability of one or more expressed gene products to prevent the proper functioning of a haploid cell when expressed, without killing the cell. Genes which may disable haploid cells include, but are not limited to, (1) proteins that disturb ionic gradients by forming pores in the membranes of a cell, both extracellular and intracellular, (2) proteins that interfere with the motility of sperm, e.g., by binding to microtubules, by affecting protein tyrosine kinases, etc., (3) enzymes capable of degrading DNA such as those involved in apoptosis, (4) proteins that are directly toxic to the cell, (5) enzymes that produce a compound which is toxic to the cell when supplied with an exogenous metabolite, and (6) proteins that affect energy metabolism. The term “disabling” can also refer to acting upon a haploid cell so as to reduce or destroy its mobility, to disrupt or degrade its DNA so as to block the ability of the DNA to be used in creating a viable offspring, or to prevent it from binding to and combining with another haploid cell (i.e., participating in fertilization). See, e.g., Uma Devi et al., Andrologia 32: 95-106 (2000); Jelks et al., Reprod. Toxicol. 15: 11-20 (2001); Jones & Bavister, J. Androl. 21: 616-24 (2000). In yet another preferred embodiment, the transgene is a marker gene that encodes a product which can be detected and used as a basis for sorting haploid cells. Preferably, the protein encoded allows for optical detection. Such a protein can be a fluorescent protein. The term “marker gene” refers to a gene which can be used to physically separate cells expressing this marker from cells not expressing this marker. One such gene is green fluorescent protein. The term “sort” refers to the process of creating two populations of haploid cells with one population enriched for cells containing a specific sex chromosome. This term can refer to FACS sorting, a technique which is familiar to one skilled in the art. The term may also encompass others means of creating a population of cells enriched for a specific sex chromosome such as affinity purification by a marker found on the surface of cells, or some other means of selection. While the gene(s) described above can be expressed in the final haploid cell types produced by males and females (i.e., spermatozoa and eggs), the skilled artisan will understand that a population of these final cells enriched for cells containing a specific sex chromosome can be obtained by expressing the gene(s) in precursors to those final cells. For example, one or more transgenes can be expressed in primary spermatocytes that kill only those cells containing the transgene(s). As a result, only those cells not expressing the gene can mature into spermatozoa. The term “X sperm” refers to a sperm or spermatozoa which includes only an X sex chromosome. Such cells may also be referred to as X-chromosome sperm or an X-chromosome-bearing sperm. Similarly, the term “Y sperm” refers to a sperm or spermatozoa which includes only a Y sex chromosome. Such cells may also be referred to as Y-chromosome sperm or an Y-chromosome-bearing sperm. The term “enriched” means both purifying in an numerical sense and purifying in a functional sense. “Enriched” does not imply that there are no undesired cells are present, just that the relative amount of the cells of interest have been significantly increased in either a numeric or functional sense. First, by the use of the term “enriched” in referring to haploid cells in a numerical sense is meant that the desired cells constitute a significantly higher fraction (2- to 5-fold) of the total haploid cells present. This would be caused by a person by preferential reduction in the amount of the other haploid cells present. The term “enriched” in reference to haploid cells may also mean that the specific cells desired constitute a significantly higher fraction (2- to 5-fold) of the total, functional haploid cells present. This would be caused by a person by preferential reduction in the amount of functional undesired cells. “Enriched” may also mean that one population of haploid cells is at some competitive disadvantage in comparison to another population. For example, a small decrease in fitness of, say, X chromosome-bearing sperm may dramatically reduce their ability to compete with Y chromosome-bearing sperm to fertilize an ovum. The term “significant” is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to the other of haploid cells of about at least 2-fold, more preferably at least 5- to 10-fold or even more. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired haploid cells. The term “functional sperm” means sperm that are capable of fertilizing ova. In preferred embodiments, a functional sperm is motile, capable of binding to ova, capable of transferring their DNA to the ova, and contain undamaged DNA. The skilled artisan will understand that not all of these characteristics are required for a sperm to function, however. For example, non-motile sperm can be directly injected into eggs to initiate fertilization. In preferred embodiments, a transgenic animal is a mammal, most preferably an ungulate. Particularly preferred transgenic animals are selected from the group consisting of a bovid, ovid, suid, equid, caprid, and cervid. The term “mammalian” as used herein refers to any animal of the class Mammalia. Preferably, a mammal is a placental, a monotreme and a marsupial. Most preferably, a mammalis a canid, felid, murid, leporid, ursid, mustelid, ungulate, ovid, suid, equid, bovid, caprid, cervid, and a human or non-human primate. The term “canid” as used herein refers to any animal of the family Canidae. Preferably, a canid is a wolf, a jackal, a fox, and a domestic dog. The term “felid” as used herein refers to any animal of the family Felidae. Preferably, a felid is a lion, a tiger, a leopard, a cheetah, a cougar, and a domestic cat. The term “murid” as used herein refers to any animal of the family Muridae. Preferably, a murid is a mouse and a rat. The term “leporid” as used herein refers to any animal of the family Leporidae. Preferably, a leporid is a rabbit. The term “ursid” as used herein refers to any animal of the family Ursidae. Preferably, a ursid is a bear. The term “mustelid” as used herein refers to any animal of the family Mustelidae. Preferably, a mustelid is a weasel, a ferret, an otter, a mink, and a skunk. The term “primate” as used herein refers to any animal of the Primate order. Preferably, a prlimate is an ape, a monkey, a chimpanzee, and a lemur. The term “ungulate” as used herein refers to any animal of the polyphyletic group formerly known as the taxon Ungulata. Preferably, an ungulate is a camel, a hippopotamus, a horse, a tapir, and an elephant. Most preferably, an ungulate is a sheep, a cow, a goat, and a pig. Especially preferred in the bovine species are Bos taurus, Bos indicus, and Bos buffaloes cows or bulls. The term “ovid” as used herein refers to any animal of the family Ovidae. Preferably, an ovid is a sheep. The term “suid” as used herein refers to any animal of the family Suidae. Preferably, a suid is a pig or a boar. The term “equid” as used herein refers to any animal of the family Equidae. Preferably, an equid is a zebra or an ass. Most preferably, an equid is a horse. The term “bovid” as used herein refers to any animal of the family Bovidae. Preferably, an bovid is an antelope, an oxen, a cow, and a bison. The term “caprid” as used herein refers to any animal of the family Caprinae. Preferably, a caprid is a goat. The term “cervid” as used herein refers to any animal of the family Cervidae. Preferably, a cervid is a deer. In certain embodiments, this invention relates to animals in which one or more transgenes capable of being expressed in a haploid-specific manner in cells is incorporated into the genome, and hence the haploid cells, of the transgenic animal. This transgene can be under the control of a promoter region and/or an enhancer region which is capable of conferring sex chromosome-specific expression on the coupled transgene; and this transgene can also under the control of a promoter region and/or an enhancer region which only allows expression of its operably linked gene when provided specific inducing agent. The term “inducible” refers to a promoter which is only active in the presence of specific inducing agent. Preferably the inducing agent is supplied exogenously. The inducing factor may require binding to other cellular components in order to achieve the intended result of increasing transcription. Examples of inducible promoters are well known to those skilled in the art. The exogenous inducing agent may be given to the animal producing the sperm, or it may be incubated with isolated sperm. The inducing agent may also be produced endogenously by the animal from which the enriched sperm is to be isolated. For instance, an inducible promoter, such as the IL-8 promoter that is responsive to TNF or another cytokine, can be employed. Other examples of suitable inducible promoter systems include, but are not limited to, the metallothionine inducible promoter system, the bacterial lacZYA expression system, the tetracycline expression system, and the T7 polymerase system. Further, promoters that are selectively activated at different developmental stages (e.g., globin genes are differentially transcribed in embryos and adults) can be employed. Still other possibilities include the use of a glucocorticoid response element or a tetracycline response element. Construction of an exogenous nucleic acid operably linked to a promoter is also well within the skill of the art (See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, (2d ed. 1989) which is hereby incorporated by reference herein in its entirety including any figures, tables, or drawings.). With respect to the transfer and expression of exogenous nucleic acids according to the present invention, one skilled in the art is aware that different genetic signals and processing events control levels of nucleic acids and proteins/peptides in a cell, including transcription, MRNA translation, and post-transcriptional processing. Transcription of DNA into RNA requires a functional promoter. Protein expression is dependent on the level of RNA transcription which is regulated by DNA signals. Similarly, translation of MRNA requires, at the very least, an AUG initiation codon, which is usually located within 10 to 100 nucleotides of the 5′ end of the MRNA. Sequences flanking the AUG initiator codon have been shown to influence its recognition by eukaryotic ribosomes, with conformity to a perfect Kozak consensus sequence resulting in optimal translation (see, e.g., Kozak, J. Molec. Biol., 1987, 196:947-950). Also, successful expression of an exogenous nucleic acid in a cell can require post-translational modification of a resultant protein. Thus, production of a recombinant protein can be affected by the efficiency with which DNA (or RNA) is transcribed into mRNA, the efficiency with which mRNA is translated into protein, and the ability of the cell to carry out post-translational modification. These are all factors of which one skilled in the art is aware and is capable of manipulating using standard means to achieve the desired end result. Along these lines, to optimize protein production, preferably the transgenic nucleic acid sequence further comprises a polyadenylation site following the coding region of the transgenic nucleic acid. Also, preferably all the proper transcription signals (and translation signals, where appropriate) will be correctly arranged such that the transgenic nucleic acid sequence will be properly expressed in the cells into which it is introduced. If desired, the transgenic nucleic acid also can incorporate splice sites (i.e., splice acceptor and splice donor sites) to facilitate mRNA production. Moreover, if the transgenic nucleic acid sequence encodes a protein, which is a processed or secreted protein or functions in intracellular organelles, such as a mitochondria or the endoplasmic reticulum, preferably the transgenic nucleic acid further comprises the appropriate sequences for processing, secretion, intracellular localization, and the like. Such sequences and signals are well known to those skilled in the art. The term “non-functional” in reference to a spermatozoa refers to cells that are no longer capable of fertilizing an ovum. This may be due to deficiencies in chromosome integrity, motility, or composition of the outer membrane. In yet another aspect, the invention relates to methods for producing a population of haploid cells which are enriched for cells containing a specific sex chromosome, either the X or the Y, where the haploid cells are harvested from an animal comprising one or more transgenes that are capable of killing or disabling cells in cis when expressed. The transgene(s) are preferably under the control of a promoter which is only active in sperm containing a specific sex chromosome. In preferred embodiments, this promoter is active only in sperm containing a X chromosome; and this promoter is active only in sperm containing a Y chromosome. The promoter of the invention is also only active in haploid cells. The transgene then is allowed to act to kill or disable haploid cells containing the selected chromosome. Viable and/or functional haploid cells may be optionally purified away from the non-functional sperm by techniques known to those skilled in the art. In still another aspect, the invention relates to methods for producing a population of haploid cells which are enriched for cells containing a specific sex chromosome, either the X or the Y, where the haploid cells are harvested from an animal comprising one or more transgenes which are capable of killing or disabling cells in cis when expressed, where the promoter of the invention is only active in the presence of an inducing agent. In certain preferred embodiments, this promoter is active only in haploid cells containing a X chromosome, and this promoter is active only in haploid cells containing a Y chromosome. The cells are exposed to an inducing agent, and the promoter region of the transgene(s) then acts to express the transgene(s) in cells containing one sex chromsome but not the other. The haploid cells may be exposed in vivo, either in the source animal or in the maternal host, or they may be exposed in vitro. The transgene then acts to kill or disable those haploid cells containing the selected chromosome. In the foregoing aspects, one or more transgenes may optionally be used which do not kill or disable the haploid cells expressing the transgene(s), but rather causes the expression of a marker gene. This expressed marker may then be used to sort X-chromosome-bearing cells from Y-chromosome-bearing cells by techniques well known to those skilled in the art. In another aspect of the invention, the invention relates to methods for producing an animal using a population of spermatozoa that is enriched for cells containing a specific sex chromosome, either the X or the Y. The offspring produced will thus be primarily of the selected sex. In preferred embodiments, if the fertilization of ova using selected sperm has been conducted in vitro, the resultant embryo is transplanted into a maternal host. In yet another aspect, the invention relates to recombinant nucleic acids arranged and configured for performing the aspects described above, whether in vitro or in a cell or an organism. The transgenes of the instant invention are preferably comprised in the transgenic animals of the invention. The recombinant nucleic acids can alternatively contain a transcriptional initiation region functional in a cell, a sequence complementary to an RNA sequence encoding a protease polypeptide and a transcriptional termination region functional in a cell. Specific vectors and host cell combinations are discussed herein. The present invention also relates to cells and/or organisms that contain the foregoing transgenic nucleic acid molecules incorporated into the genome, and thereby which are capable of expressing a polypeptide or other gene of interest. A cell is said to be “altered to express a desired polypeptide or other gene of interest” when the cell, through genetic manipulation, is made to produce a protein or other gene of interest which it normally does not produce or which the cell normally produces at lower levels. One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into eukaryotic cells. A nucleic acid molecule, such as DNA, is said to be “capable of expressing” a polypeptide or other gene of interest if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression. The precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shall in general include a promoter region and other 5′-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. Two DNA sequences (such as a promoter region sequence and a sequence encoding the gene of interest) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of a gene sequence encoding the gene of interest, or (3) interfere with the ability of the gene sequence of the gene of interest to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence. Thus, to express a gene encoding the gene of interest, transcriptional and translational signals recognized by an appropriate host are necessary. The present invention encompasses the expression of a gene encoding the gene of interest (or a functional derivative thereof) in eukaryotic cells. The selection of control sequences, expression vectors, transformation methods, and the like, are dependent on the type of host cell used to express the gene, and their selection is well within the skill of the artisan. As used herein, “cell”, “cell line”, and “cell culture” may be used interchangeably and all such designations include progeny. Thus, the words “transformants” or “transformed cells” include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that of the originally transformed cell. The term “vector” relates to a single or double-stranded circular nucleic acid molecule that can be transfected into cells and replicated within or independently of a cell genome. A circular double-stranded nucleic acid molecule can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of nucleic acid vectors, restriction enzymes, and the knowledge of the nucleotide sequences cut by restriction enzymes are readily available to those skilled in the art. A nucleic acid molecule encoding a protease can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together. Preferred vectors are those designed for performing “gene targeting” procedures. See, e.g., U.S. Pat. Nos. 6,090,554, 6,069,010, 5,792,663, and 5,789,215, each of which is hereby incorporated by reference in its entirety, including all tables, figures, and claims. The term “transfecting” defines a number of methods to insert a nucleic acid vector or other nucleic acid molecules into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, detergent, or DMSO to render the outer membrane or wall of the cells permeable to nucleic acid molecules of interest or use of various viral transduction strategies. A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, cytomegalovirus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, and the like, may be employed. Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabolite) regulation. Expression of the transgenes of the invention in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, for example, the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature (London) 290:304-31, 1981); and the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982; Silver et al., Proc. Natl. Acad. Sci . (USA) 81:5951-5955, 1984). Translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the gene of interest (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in the formation of a fusion protein (if the AUG codon is in the same reading frame as the protease of the invention coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the protease of the invention coding sequence). A nucleic acid molecule encoding the gene of interest and an operably linked promoter may be introduced into a recipient host cell either as a nonreplicating DNA or RNA molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Permanent expression will occur through the integration of the introduced DNA sequence into the host chromosome. A vector may be employed which is capable of integrating the desired gene sequences into the host cell chromosome. Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector. The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of MRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama ( Mol. Cell. Biol. 3:280-289, 1983). The introduced nucleic acid molecule can be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to “shuttle” the vector between host cells of different species. Once the vector or nucleic acid molecule containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and the like. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene(s) results in the production of the gene of interest, or fragments thereof. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like). A variety of incubation conditions can be used to form the peptide of the present invention. The most preferred conditions are those which mimic physiological conditions. |
Method for retrieving documents |
The invention relates to a method for searching a document base in which documents are interlinked by links. A list of documents to be treated is sorted according to priority. The document pertaining to the highest priority is called up and the distance of said document to a document base is determined. All links from the document are entered into the list of documents to be treated, the distance of the document to the document base being used as the priority. |
1. A method of compiling a list of documents maintained as a target queue, comprising: determining a sequence relative to a document base by a weight determined through a predetermined method; assigning references to other documents to the documents to be analyzed, wherein a starting document is initially the current document, comprising: determining, using an evaluator, the weight of the current document and places the document into the target queue on the basis of the weight, removing the references included in the current document, and assigning the previously determined weight of the document, and, together with the weight, are placed into a ranked source queue, and removing the reference having the highest weight from the source queue by an agent, the corresponding document is retrieved and treated as the current document, and the steps are repeated. 2. The method as in claim 1, wherein each of several agents removes from the source queue a reference to the highest weight, retrieves the document, places the document in a buffer queue with the same weight as the reference, and the respective document having the highest weight is taken from the buffer queue and is treated as the current document. 3. The method as in claim 2, wherein a list of the references used is maintained and the references included in the list are not retrieved and analyzed again, such that references are not entered into the source queue or are discarded together with the highest weight during removal of the reference. 4. The method as in claim 1, wherein references having a preset minimum weight are entered into the source queue, and are otherwise discarded. 5. The method as in claim 1, wherein references having a preset minimum weight are entered into the target queue, and are otherwise discarded. 6. The method as in claim 1, wherein the source queue comprises a predetermined maximum number of entries and, when the number is reached, an entry having a low weight is discarded and an entry having a high weight displaces the entry having the lowest weight. 7. The method as in claim 1, wherein the target queue comprises a predetermined maximum number of entries and, when the number is reached, an entry having a low weight is discarded and an entry having a high weight displaces the entry having the lowest weight. 8. The method as in claim 1, wherein the document base comprises several documents. 9. The method as in claim 1, wherein a measure of dissimilarity used to determine dissimilarity between the current document and the document base is formed by a vector space model. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The system known as the World Wide Web (WWW) comprises a large number of documents that contain references to other documents, which in turn may contain references other documents, etc. Documents that conceal such references behind text or image objects are also known as hypertext, and the references themselves are referred to as hyperlinks. The hypertext documents on the WWW are normally coded in the HTML marking language. To find a document in this largest existing pool of identically formatted documents, search engines have been known for some time. These search engines scan the documents at regular intervals and follow the hyperlinks. In this process, the documents are entered into an index consisting of either the index terms specified in the HTML or words extracted from the text. A user of the WWW who is searching for a document triggers a search of such an index using search terms he has specified. Although this method was relatively effective during the early days of the WWW, the outcome set is only small enough to be useable if very specific search terms and key words can be used. Inexperienced users, in particular, often obtain outcome sets that are either too small or too large. Accordingly, based on the search terms and key words, the documents are displayed in their order of relevance, wherein the relevance can contain commercially preferential treatment. The frequencies of words are generally used to establish relevance, as was already proposed in 1958 in the article titled “The Automatic Creation of Literature Abstracts,” by H. P. Luhn, IBM Journal, p. 159-165. Nevertheless, a need continues to exist for an improved method that is also accessible to inexperienced users. In this context, it is proposed, in U.S. Pat. No. 6,167,398, to calculate a dissimilarity between a reference document and each candidate document by means of a dissimilarity metric and then, after having searched through a predetermined or otherwise delimited number of documents, to place the document into a sequence using the established dissimilarities. Several different dissimilarity metrics are to be used in this process. A disadvantage of this solution is that a set of documents is initially made available and then each of the documents is analyzed. Therefore, it is still necessary to determine a subset of the documents using a key word search question. In U.S. Pat. No. 6,144,973, it is proposed, during a search for documents in the WWW, to evaluate the references in a document on the basis of whether a predetermined degree of similarity to the original document exists. The references are either used, if a predetermined threshold is exceeded, or they are discarded, if the threshold is not reached. There are no provisions for parallel work or making adjustments for documents already found. The primary means of limiting the number of documents accessed consists in limiting the depth of search. |
<SOH> SUMMARY OF THE INVENTION <EOH>The present invention is based on the recognition that the established degree of similarity can be advantageously used to control the subsequent search and rank the references to be searched. The use of improved measures of similarity and the vector space model contribute to this. In one embodiment of the invention, there is a method for searching through a document base in which documents are linked by references. A list of the documents to be processed is sorted by priority. The document corresponding to the highest-priority entry is retrieved, and the dissimilarity between this document and a document base is determined. All references from the document are entered into the list of documents to be processed, wherein the dissimilarity of the document to the document base is used as priority. |
Inductively coupled high-density plasma source |
A high-density plasma source (100) is disclosed. The source includes an annular insulating body (300) with an annular cavity (316) formed within. An inductor coil (340) serving as an antenna is arranged within the annular cavity and is operable to generate a first magnetic field within a plasma duct (60) interior region (72) and inductively couple to the plasma when the annular body is arranged to surround a portion of the plasma duct. A grounded conductive housing (400) surrounds the annular insulating body. An electrostatic shield (360) is arranged adjacent the inner surface of the insulating body and is grounded to the conductive housing. Upper and lower magnet rings (422 and 424) are preferably arranged adjacent the upper and lower surfaces of the annular insulating body outside of the conductive housing. A T-match network is in electrical communication with said inductor coil and is adapted to provide for efficient transfer of RF power from an RF power source to the plasma. At least one plasma source can be used to form a high-density plasma suitable for plasma processing of a workpiece residing in a plasma chamber in communication with the at least one source. |
1. A high-density plasma source for forming a plasma within a plasma duct having an interior region, comprising: a) an annular insulating body including a cylindrical inner surface and a first central axis, with an annular cavity formed within said insulating body and having a second central axis that coincides with the first central axis; and b) an inductor coil arranged within said annular cavity operable to generate a first magnetic field within the plasma duct interior region and inductively couple to the plasma. 2. A plasma source as claimed in claim 1, further comprising a plurality of spaced apart magnets arranged in at least one concentric magnet ring adjacent at least one of an upper and a lower surface of the annular insulating body. 3. A plasma source as claimed in claim 1, further comprising a T-match network in electrical communication with said inductor coil. 4. A plasma source as claimed in claim 3, further comprising a plasma source RF power supply electrically connected to said T-match network. 5. A plasma source as claimed in claim 3, wherein said T-match network is electrically connected to first and second ends of said inductor coil. 6. A plasma source as claimed in claim 3, wherein said T-match network comprises first and second variable capacitors arranged in a plane defined by said inductor coil. 7. A plasma source as claimed in claim 3, wherein said T-match network comprises first and second variable capacitors arranged perpendicular to a plane defined by said inductor coil. 8. A plasma source as claimed in claim 1, wherein said inductor coil comprises a copper coil. 9. A plasma source as claimed in claim 1, wherein said annular insulating body comprises a TEFLON body. 10. A plasma source as claimed in claim 1, further comprising a plurality of spaced apart magnets arranged in first and second concentric magnet rings adjacent an upper and a lower surface of the annular insulating body, respectively, wherein said first and second concentric magnet rings comprise an equal number of magnets. 11. A plasma source as claimed in claim 2, wherein said magnets comprise DC field electromagnets. 12. A plasma source as claimed in claim 2, wherein said magnets comprise a ring of magnets arranged in one of a ring-cusp configuration, a mirror field configuration, and a dipole configuration. 13. A plasma source as claimed in claim 4, wherein the plasma density in the plasma duct is equal to or greater than about 5×1012 cm−3 for 500 W RF power provided to said inductor coil by said RF power source. 14. A plasma source as claimed in claim 4, wherein the plasma density in the plasma duct is equal to or greater than about 1-1.3×1013 cm−3 for 1000 W RF power provided to said inductor coil by said RF power source. 15. A plasma source as claimed in claim 1, further comprising a plurality of cooling fluid channels extending from said annular inductor coil cavity within said annular cavity and extending radially inward to an inner surface of the annular insulating body, wherein said inductor coil is capable of carrying a cooling fluid and comprises a plurality of incrementally spaced apertures positioned so as to provide fluid communication between said inductor coil and said coolant channels. 16. A plasma source as claimed in claim 1, further comprising a grounded conductive housing surrounding said annular insulating body. 17. A plasma source as claimed in claim 16, further comprising an electrostatic shield arranged adjacent an inner surface of the annular insulating body and grounded to said conductive housing. 18. A plasma source as claimed in claim 11, further comprising an electrostatic shield arranged adjacent an inner surface of the annular insulating body. 19. A plasma reactor system for processing a workpiece, comprising: a) a plasma reactor chamber having a central axis, an upper wall and sidewalls surrounding a first interior region capable of supporting a plasma; b) at least one plasma duct attached to said upper wall and/or said sidewall of said reactor chamber and having duct sidewalls enclosing a second interior region in communication with said first interior region; c) a chuck arranged opposite said upper wall for supporting the workpiece; and d) a plasma source surrounding a portion of said at least one plasma duct and defining a plasma generation region within said second region, wherein the plasma source comprises: an annular insulating body substantially concentric with the central axis, and an inductor coil arranged within said annular cavity operable to generate a first magnetic field within the plasma duct interior region and inductively couple to the plasma. 20. A plasma reactor system as claimed in claim 19, wherein the at least one plasma duct comprises plural spaced apart plasma ducts attached to said upper wall and corresponding plasma sources, arranged in a disk configuration. 21. A plasma reactor system as claimed in claim 19, wherein the at least one plasma duct comprises plural spaced plasma ducts attached to said sidewall and corresponding plasma sources, arranged in a ring configuration. 22. A plasma reactor system as claimed in claim 19, further comprising at least one of: i) a gas supply system in pneumatic communication with said plasma duct so as to introduce gas into said plasma generation region; ii) a gas distribution system in pneumatic communication with at least one of said plasma duct and said plasma reactor chamber; iii) a T-match network in electrical communication with said inductor coil; and a first RF power supply in electrical communication with said T-match network; iv) a coolant supply system in fluid communication with said inductor coil; v) a vacuum system in pneumatic communication with said first interior region; vi) a second RF power supply system in electrical communication with said chuck; and vii) a load chamber formed in said reactor chamber sidewalls and enclosing a third interior region in communication with said first interior region, with a sealable door attached thereto sized to allow a workpiece to pass through said third interior region to said chuck, and further including a workpiece handling system in operable communication with said load chamber and said chuck. 23. A plasma reactor as claimed in claim 22, further comprising a control system for controlling the operation of said reactor. 24. A method of forming a high-density plasma in an interior region of a plasma chamber, comprising the steps of: a) injecting plasma gas into a plasma generation region of a plasma duct that is in communication with the chamber interior region; b) providing RF power through a T-match network to an annular inductor coil surrounding said plasma generation region, thereby generating a first magnetic field within said plasma generation region; c) measuring an amount of reflected power reflected from said inductor coil passing back through said T-match network; and d) adjusting the capacitance of said T-match network so as to minimize the amount of reflected power measured in said step c). 25. A method as claimed in claim 24, and further comprising the step of passing a cooling fluid through said inductor coil. 26. A method as claimed in claim 24, further comprising the step of injecting a reactive gas into the interior region of the plasma chamber so as to create a reactive plasma. 27. A method as claimed in claim 24, further comprising the step of allowing the plasma to diffuse from the plasma generation region into the interior region of the plasma chamber and interact with a workpiece. 28. A method as claimed in claim 22, further comprising the step of allowing the reactive plasma to interact with a workpiece. 29. A method of forming a high-density plasma within an interior region of a plasma reactor chamber, comprising: a) injecting plasma gas into a plurality of plasma generation regions, one in each of a corresponding plurality of spaced-apart plasma ducts in communication with the chamber interior region and arranged so as to provide a region of uniform, high-density plasma within the interior region of the reactor chamber; b) providing RF power through a plurality of T-match networks each corresponding to an independent annular inductor coil surrounding each said plasma generation region, thereby generating a first magnetic field within each said plasma generation region; c) measuring an amount of reflected power reflected from each said inductor coil passing back through each said T-match network; and d) adjusting the capacitance of each said T-match network so as to minimize the amount of reflected power measured in said step c). 30. A method as claimed in claim 29, further comprising the step of passing a cooling fluid through each said inductor coil. 31. A method as claimed in claim 29, further comprising the step of injecting a reactive gas into the interior region of the plasma chamber to form a reactive plasma. 32. A method as claimed in claim 30, further comprising the step of allowing the plasma to diffuse from the plasma generation regions into the chamber interior region and interact with a workpiece. 33. A method as claimed in claim 31, further comprising the step of allowing the reactive plasma to interact with a workpiece. 34. A method of abating exhaust in a chamber containing a gas and having an exhaust path, comprising the steps of: a) arranging a plasma source, including an inductor coil contained within an annular insulating body, around the exhaust path so as to define a plasma generation region within the exhaust path; and b) activating the plasma source so as to dissociate at least a portion of gas traveling through the exhaust path. 35. A method as claimed in claim 34, further comprising the step of tuning the T-match network of the plasma source to maximize power transfer to gas passing through the plasma generation region. 36. A method as claimed in claim 34, further comprising the step of passing dissociated gas to a scrubbing system downstream of the plasma source. 37. A method as claimed in claim 34, further comprising the step of arranging a plurality of plasma sources along the exhaust path. 38. A match network for regulating a power applied by an RF power supply to first and second ends of an inductor coil, the network comprising: a first variable capacitor having a third end, coupled to the RF power supply, and a fourth end, coupled to a first end of the inductor coil; and a second variable capacitor having a fifth end, connected to a second end of the inductor coil and to ground, and a sixth end, coupled to the third end of the first variable capacitor. 39. The match network as claimed in claim 38, further comprising the RF power supply. 40. The match network as claimed in claim 39, further comprising a power meter coupled between the RF power supply and the first variable capacitor. 41. The match network as claimed in claim 38, wherein the first and second variable capacitors are physically arranged in a plane of the inductor coil. 42. The match network as claimed in claim 38, wherein the first and second variable capacitors are physically perpendicular to a plane of the inductor coil. |
<SOH> BACKGROUND OF THE INVENTION <EOH>1. Field of the Invention The present invention relates generally to plasma processing systems and methods, and in particular relates to inductively coupled high-density plasma sources suitable for use in a plasma processing system. 2. Background of the Invention Ionized gas or “plasma” may be used during processing and fabrication of semiconductor devices, flat panel displays and other products requiring etching or deposition (“plasma processing”) of materials. Plasma may be used to etch or remove material from semiconductor integrated circuit wafers, or sputter or deposit material onto a semiconducting, conducting or insulating surface. Creating a plasma for use in manufacturing or fabrication processes typically is done by introducing a low-pressure process gas into a chamber surrounding a workpiece, such as an integrated circuit (IC) wafer, that resides on a workpiece support member, more commonly referred to as a “chuck.” The molecules of the low-pressure gas in the chamber are ionized into a plasma by a plasma source after the gas molecules enter the chamber. The plasma then flows over and interacts with the workpiece, which may be biased by providing RF power to the chuck supporting the workpiece. To be most effective in plasma processing, the plasma preferably has a high-density (measured as the number of electrons or ions per cubic centimeter) and is uniform. High-density plasma processing can increase throughput and therefore increase production in semiconductor manufacturing. Furthermore, the plasma preferably has a small volume (thin and flat) so that the radicals in the process system have a short residence time. A short radical residence time permits control of the proper dissociation of radicals in the plasma volume for achieving high rate, selective etch in high aspect ratio etch features. One type of plasma source that has been developed and commonly used is a parallel-plate, capacitively coupled plasma (CCP) source. Such a source uses radio-frequency (RF) power sources to generate the plasma through gas discharge. These power sources typically operate at 13.56 MHz, but can operate at other frequencies. Parallel-plate plasma sources usually have small gap spacing and small plasma volume. However, they typically generate low-density plasmas of less than 10 11 ions/cc which limits the etch rate. Another type of plasma source is an electron cyclotron resonance (“ECR”) source, which uses microwave (2.45 GHz) energy sources to generate a plasma having relatively high densities, on the order of 10 11 -10 12 ions/cc and greater. Although an ECR source provides a relatively high plasma density and good control of ion energy, it requires in the plasma source a significant magnetic field, which is normally undesirable in the processing reactor. In addition, difficulties arise in generating uniform plasmas over large wafer areas. A third type of plasma source is an inductively coupled plasma (ICP) source, which uses an inductively coupled radio-frequency power to generate the plasma This type of plasma source provides for a relatively high plasma density (10 12 ions/cc or greater) and operates with a radio-frequency source (typically 13.56 MHz). However, a shortcoming of conventional inductively coupled plasma sources is a non-uniform plasma density in the region above the substrate. The plasma volume is also very large, resulting in very long residence times for the radicals, which limits the etch rate. A fourth type of plasma source is the Helicon plasma source, which uses a relatively constant volume magnetic field. It is capable of generating a very high density (10 13 ions/cc) and operates with a radio-frequency source (typically 2-30 MHz). The Helicon source requires propagation and damping of the low frequency whistler wave in a system with minimum length greater then one half of the propagating wavelength. In short systems, the plasma generating efficiency is usually reduced drastically. For those prior art systems capable of producing a high-density plasma (in excess of 10 12 ions/cc), efficiency is generally sacrificed in producing plasma in a small volume. This inefficiency makes high-density plasma processing a costly proposition for manufacturing purposes. |
<SOH> BRIEF SUMMARY OF THE INVENTION <EOH>The present invention relates generally to plasma processing systems and methods, and in particular relates to an inductively coupled high-density plasma source suitable for use in a plasma processing system. A first aspect of the present invention is a high-density plasma source that includes an annular insulating body having an upper surface, a lower surface, a cylindrical inner surface and a first central axis. An annular cavity is formed within the insulating body having a second central axis that coincides with the first central axis. A single inductor coil that serves as an antenna is arranged within the annular cavity and is operable to generate a first magnetic field within a plasma duct interior region and inductively couple to the plasma formed therein when the annular body is arranged around a portion of the plasma duct. A grounded conductive housing surrounds the annular insulating body, and an electrostatic shield is arranged adjacent the insulating body inner surface and is grounded to the conductive housing. A T-match network is in electrical communication with the inductor coil. The T-match network includes first and second variable capacitors that are tunable to provide for efficient transfer of RF power from the inductor coil antenna to the plasma. In a second aspect of the present invention, the above-described plasma source includes a first plurality of spaced apart magnets arranged in a first concentric magnet ring adjacent the insulating body upper surface, and a second plurality of spaced apart magnets arranged in a second concentric magnet ring adjacent the insulating body lower surface. A third aspect of the invention is a plasma reactor system for processing a workpiece. The system comprises a plasma reactor chamber having a central axis, an upper wall and sidewalls surrounding a first interior region capable of supporting a plasma. At least one plasma duct is attached to the upper wall and/or to the sidewall of the reactor chamber. The plasma ducts each have duct sidewalls that enclose second interior regions that are in communication with the plasma reactor chamber interior region. Plasma sources, as described briefly immediately above and in more detail below, surround a portion of each plasma duct, and define plasma generation regions within each second region. A chuck is arranged opposite the upper wall and supports a workpiece to be processed. A fourth aspect of the invention is a method of forming a high-density plasma in an interior region of a plasma chamber. The method includes the steps of injecting plasma gas into a plasma generation region of a plasma duct that is in communication with the chamber interior region. The next step involves providing RF power through a T-match network to a single annular inductor coil surrounding the plasma generation region, thereby generating a first magnetic field within the plasma generation region. The next step includes measuring an amount of reflected power reflected from the inductor coil passing back through the T-match network. The last step involves adjusting the capacitance of the T-match network so as to minimize the amount of reflected power. As the plasma uniformity requirements are more critical for still larger wafer size, it is desirable to use a plurality of smaller plasma sources, with individual plasma density control to form a high-density plasma uniformly distributed over a large region above the substrate. |
Spark ignition engine control device |
For the purpose of improving the fuel efficiency by lean combustion and enhancing the fuel efficiency improvement effects by performing compression ignition efficiently in some cylinders, a multi-cylinder spark ignition engine is constructed such that exhaust gas, that is exhausted from preceding cylinders 2A, 2D on the exhaust stroke side among pairs of cylinders whose exhaust stroke and intake stroke overlap in a low load, low rotational speed region, is directly introduced through an inter-cylinder gas passage 22 into following cylinders 2B, 2C on the intake stroke side and only gas exhausted from the following cylinders 2B, 2C is fed to an exhaust passage 20, which is provided with a three-way catalyst 24. Combustion controller is provided that controls the combustion of each of the cylinders such that combustion is conducted by forced ignition in a condition in which the air/fuel ratio is a lean air/fuel ratio which is larger by a prescribed amount than the stoichiometric air/fuel ratio in the preceding cylinders 2A, 2D and, in the following cylinders 2B, 2C, fuel is supplied to burnt gas of lean air/fuel ratio introduced from the preceding cylinders 2A, 2D and combustion is conducted by compression ignition. |
1. A control device for a multi-cylinder spark ignition engine having cylinders arranged to perform a cycle consisting of intake, compression, expansion and exhaustion strokes with prescribed phase differences, characterized in that a gas flow path is constituted in a two-cylinder connected condition, at least in a low load, low rotational speed region, such that burnt gas exhausted from a preceding cylinder which is a cylinder on the exhaust stroke side in a pair of cylinders whose exhaustion stroke and intake stroke overlap is directly introduced into a following cylinder which is a cylinder on the intake stroke side through an inter-cylinder gas passage and gas exhausted from this following cylinder is fed to an exhaust passage; and said control device comprising combustion controller that controls combustion in each cylinder such that at least in part of the operating region of the operating region in which said two-cylinder connected condition is produced, combustion is performed by forced ignition in said preceding cylinder in a condition at an air/fuel ratio larger by a prescribed amount than the stoichiometric air/fuel ratio while fuel is supplied in an amount corresponding to the following cylinder to the burnt gas generated by combustion in this preceding cylinder, and combustion is performed by compression self-ignition in the following cylinder. 2. The control device for a spark ignition engine according to claim 1, characterized in that the air/fuel ratio of the following cylinder in said two-cylinder connected condition is made to be at or below the stoichiometric air/fuel ratio and a three-way catalyst or oxidation catalyst is provided in the exhaust passage connected with this following cylinder. 3. The control device for a spark ignition engine according to claim 1, characterized in that a fuel injection valve is provided that injects fuel directly into said preceding cylinder and, when in said two-cylinder connected condition, fuel is injected in the compression stroke from said fuel injection valve and stratified charge combustion is performed by forced ignition while keeping a lean air/fuel ratio in the preceding cylinder. 4. The control device for a spark ignition engine according to claim 3, characterized in that the air/fuel ratio of the preceding cylinder in said two-cylinder connected condition is twice or more the stoichiometric air/fuel ratio. 5. The control device for a spark ignition engine according to claim 3, characterized in that the air/fuel ratio of the following cylinder in said two-cylinder connected condition is an air/fuel ratio larger than the stoichiometric air/fuel ratio. 6. The control device for a spark ignition engine according to any of claims 1 to 5, characterized in that when in said two-cylinder connected condition uniform combustion is performed by injecting fuel in the following cylinder in the intake stroke. 7. The control device for a spark ignition engine according to claim 1, characterized in that it comprises flow path changeover means for changing over the flow paths of new air and gas, in a high load, high rotational speed operating region, such that the intake port and exhaust port of each of the cylinders are made to be independent, so that new air is introduced into the intake port of each cylinder from an intake passage and exhaust gas exhausted from the exhaust port of each cylinder is fed to said exhaust passage; and combustion controller is arranged to set the air/fuel ratio of each of the cylinders to the stoichiometric air/fuel ratio or less than this and to cause combustion to be performed by forced ignition in each of the cylinders in said high load, high rotational speed operating region. 8. The control device for a spark ignition engine according to claim 7, characterized in that, in said preceding cylinder, there are provided an intake port that communicates with said intake passage, a first exhaust port that communicates with said exhaust passage and a second exhaust port that communicates with the inter-cylinder gas passage and, in said following cylinder there are provided a first intake port that communicates with said intake passage, a second intake port that communicates with said inter-cylinder gas passage and an exhaust port that communicates with said exhaust passage; and as said flow path changeover means, there are provided a valve deactivating mechanism that changes over the operating condition and deactivated condition respectively of the first and second exhaust valves that open and close the first and second exhaust ports of said preceding cylinder and of the first and second intake valves that open and close the first and second intake ports of the following cylinder; and valve stop mechanism controller that, in a low load, low rotational speed region, puts said first exhaust valve and said first intake valve in deactivated condition and puts said second exhaust valve and said second intake valve in operating condition and, in a high load, high rotational speed operating condition, puts said first exhaust valve and said first intake valve in operating condition and said second exhaust valve and said second intake valve in deactivated condition. 9. The control device for a spark ignition four-cycle engine according to claim 1, characterized in that said combustion controller which exercise the control mode whereby combustion is performed in said two-cylinder connected condition as the special operating mode, and said combustion controller, in at least part of the operating region of the region corresponding to said special operating mode, controls the fuel supply rate in respect of both the leading and following cylinders such that the fuel supply rate in the preceding cylinder is greater, while the air/fuel ratio during combustion in said following cylinder is substantially the stoichiometric air/fuel ratio, thereby making the air/fuel ratio when combustion is conducted in the preceding cylinder a value of less than twice the stoichiometric air/fuel ratio and conducting combustion in the preceding cylinder by forced ignition and conducting combustion in the following cylinder by compression self-ignition. 10. The control device for a spark ignition four-cycle engine according to claim 9, characterized in that, in said special operating mode, in the intermediate speed region of the operating region in which the following cylinder is made to perform compression self-ignition, the air/fuel ratio when conducting combustion in the preceding cylinder is made to be a value of substantially twice the stoichiometric air/fuel ratio, or more than said stoichiometric air/fuel ratio. 11. The control device for a spark ignition four-cycle engine according to claim 10, characterized in that, in said special operating mode, in the operating region on the lower speed side than the intermediate speed region of the operating region in which the following cylinder is made to perform compression self-ignition, the air/fuel ratio when conducting combustion in the preceding cylinder is made to be a value of less than twice the stoichiometric air/fuel ratio. 12. The control device for a spark ignition four-cycle engine according to claim 10, characterized in that, in said special operating mode, in the operating region on the higher speed side than the intermediate speed region of the operating region in which the following cylinder is made to perform compression self-ignition, the air/fuel ratio when conducting combustion in the preceding cylinder is made to be a value of less than twice the stoichiometric air/fuel ratio. 13. The control device for a spark ignition four-cycle engine according to claim 9, characterized in that, in said special operating mode, in the intermediate load region of the operating region in which the following cylinder is made to perform compression self-ignition, the air/fuel ratio when conducting combustion in the preceding cylinder is made to be a value of substantially twice the stoichiometric air/fuel ratio, or more than the stoichiometric air/fuel ratio. 14. The control device for a spark ignition four-cycle engine according to claim 9, characterized in that, in said special operating mode, in the intermediate speed/intermediate load region of the operating region in which the following cylinder is made to perform compression self-ignition, the air/fuel ratio when conducting combustion in the preceding cylinder is made to be a value of substantially twice the stoichiometric air/fuel ratio, or more than the stoichiometric air/fuel ratio. 15. The control device for a spark ignition four-cycle engine according to claim 9, characterized in that, in said special operating mode, in the operating region in which the following cylinder is made to perform compression self-ignition, the air/fuel ratio when conducting combustion in the preceding cylinder is made smaller as the load becomes lower. 16. The control device for a spark ignition four-cycle engine according to claim 9, characterized in that, when the engine temperature is low, in the entire operating region in which the following cylinder is made to perform compression self-ignition in said special operating mode, the air/fuel ratio when conducting combustion in the preceding cylinder is made to be less than twice the stoichiometric air/fuel ratio. 17. The control device for a spark ignition engine according to claim 1, wherein said combustion controller executes control in which combustion is conducted in said two-cylinder connected condition in a control mode as a special operating mode; and said combustion condition; and said combustion controller including a fuel injection controller that, in an operating region in which the following cylinder is made to perform compression self-ignition in said special operating mode, relatively retards the injection time of the fuel to the following cylinder in an operating condition in which knocking is likely to occur, compared with an operating condition where knocking is unlikely to occur. 18. The control device for a spark ignition engine according to claim 17, characterized in that, in an operating region in which the following cylinder is made to perform compression self-ignition in said special operating mode, in an operating condition in which knocking is likely to occur, the injection time of the fuel to the following cylinder is set more on the retarded side of the compression stroke as the likelihood of knocking increases. 19. The control device for a spark ignition engine according to claim 17, characterized in that, in an operating region in which the following cylinder is made to perform compression self-ignition in said special operating mode, in an operating condition in which knocking is likely to occur, injection of fuel into the following cylinder is performed in divided fashion and the latter injection time of the fuel in said divided injection is set in the latter half of the compression. 20. The control device for a spark ignition engine according to claim 19, characterized in that, in a region in which the following cylinder is made to perform compression self-ignition, the likelihood of occurrence of knocking or the intensity of knocking is ascertained and the latter injection time in said divided fuel injection is retarded so as to approach more closely the compression top dead center as the likelihood of occurrence of said knocking or the intensity of knocking increases. 21. The control device for a spark ignition engine according to claim 3, characterized in that, in a region in which the following cylinder is made to perform compression self-ignition, in an operating condition in which knocking is likely to occur, injection of fuel into the following cylinder is performed in divided fashion and the latter injection rate of the fuel in said divided injection is set to a larger value than the former injection rate. 22. The control device for a spark ignition engine according to claim 21, characterized in that, in a region in which the following cylinder is made to perform compression self-ignition, the likelihood of occurrence of knocking is ascertained and the ratio of the latter injection period rate with respect to the total injection rate of fuel injected in the following cylinder is changed so as to be larger as the likelihood of occurrence of such knocking becomes higher. 23. The control device for a spark ignition engine according to claim 17, characterized in that, in a region in which the following cylinder is made to perform compression self-ignition, when the engine is in an operating region on the high load side, a condition in which knocking is likely to occur is identified. 24. The control device for a spark ignition engine according to claim 17, characterized in that, if fuel of low octane value is employed, the region in which the following cylinder is made to perform compression self-ignition is identified as a condition in which knocking is likely to occur. 25. The control device for a spark ignition engine according to claim 17, characterized in comprising swirl generating means that generates swirl such that a strong intensity of turbulence is maintained in the latter half of the compression stroke in a region in which the following cylinder is made to perform compression self-ignition, in an operating condition in which knocking is likely to occur. 26. The control device for a spark ignition engine according to claim 25, characterized in that swirl is generated in the combustion chamber by directing the tip portion of the inter-cylinder gas passage in the cylinder tangential direction of the following cylinder in plan view and introducing burnt gas into the following cylinder from said inter-cylinder gas passage in the intake stroke of the following cylinder. 27. The control device for a spark ignition engine according to claim 1, characterized in that the combustion controller exercises control whereby combustion is conducted in said two-cylinder connected condition as a special operating mode; and said combustion controller effects a control such that: in at least part of the operating region in which said special operating mode is involved, combustion is conducted by compression self-ignition in the following cylinder, and the air/fuel ratio of the preceding cylinder is made relatively lower in a high load region in the region in which said compression self-ignition is performed compared with the region on the low load side and a new air introduction intake valve that introduces new air into the following cylinder is opened so that new air is introduced into the following cylinder in addition to the burnt gas that is fed from said preceding cylinder. 28. The control device for a spark ignition engine according to claim 27, characterized in that in a region on the low load side in the operating region in which the following cylinder is made to perform compression self-ignition in said special operating mode, the new air introduction intake valve is maintained in closed condition; and, in a region on the high load side in said compression self-ignition region, the new air introduction intake valve is opened in the vicinity of the intake top dead center of the following cylinder and is closed during the course of the intake stroke of the following cylinder. 29. The control device for a spark ignition engine according to claim 27, characterized in that, in a region on the high load side in the operating region in which the following cylinder is made to perform compression self-ignition in said special operating mode, the burnt gas introduction valve of the following cylinder is opened during the course of the intake stroke and the new air introduction intake valve is opened prior to the opening time of said burnt gas introduction valve. 30. The control device for a spark ignition engine according to claim 27, characterized in that, in a region on the high load side in the operating region in which the following cylinder is made to perform compression self-ignition, control is exercised such as to increase the ratio of the new air intake rate with respect to the total gas rate introduced into the following cylinder, in response to enrichment of the air/fuel ratio of the preceding cylinder, compared with a region on the low load side thereof. 31. The control device for a spark ignition engine according to claim 27, characterized in that, at least in a region in which the following cylinder is made to perform compression self-ignition, the air/fuel ratio of the following cylinder is controlled such that the oxygen concentration in the exhaust gas that is exhausted from the following cylinder is a value corresponding to the combustion condition of the stoichiometric air/fuel ratio. 32. The control device for a spark ignition engine according to claim 1, characterized in that control is exercised such as to make the control mode whereby combustion is conducted in said two-cylinder connected condition a special operating mode; and said combustion controller controls such that the total injection quantity of fuel injected into the two cylinders consisting of said preceding cylinder and following cylinder is increased in response to increase in engine load; and control is exercised such that in said following cylinder, combustion is conducted by compression self-ignition in at least part of the operating region in which said special operating mode is involved and, in said preceding cylinder, stratified charge lean combustion is conducted with the injected fuel put in a stratified condition in an intermediate/low load region of the operating region in which compression self-ignition of said following cylinder is performed, and control is exercised such that, on the high load side of the operating region in which said stratified charge lean combustion is conducted, uniform lean combustion is conducted in a condition with the injected fuel uniformly dispersed. 33. The control device for a spark ignition engine according to claim 32, characterized in that, in the operating region on the high load side in which combustion is conducted in a uniform lean condition in said preceding cylinder, the air/fuel ratio of said preceding cylinder is made to be a value of substantially twice the stoichiometric air/fuel ratio, or a value smaller than said stoichiometric air/fuel ratio. 34. The control device for a spark ignition engine according to claim 32, characterized in that, in a low load operating region of the intermediate/low load operating region in which stratified charge lean combustion is conducted in said preceding cylinder, the air/fuel ratio of said preceding cylinder is made to be a value of substantially twice the stoichiometric air/fuel ratio, or a value smaller than said stoichiometric air/fuel ratio. 35. The control device for a spark ignition engine according to claim 32, characterized in that, in a low load operating region of the intermediate/low load operating region in which stratified charge lean combustion is conducted in said preceding cylinder, if compression self-ignition in said following cylinder is difficult, control is exercised such that the air/fuel ratio of said preceding cylinder is made to be substantially twice the stoichiometric air/fuel ratio or a value smaller than said stoichiometric air/fuel ratio and the combustion mode in the preceding cylinder is shifted from the stratified charge lean condition to said uniform lean condition and the ignition mode in said following cylinder is shifted from compression self-ignition to forced ignition. 36. The control device for a spark ignition engine according to claim 1, characterized in that the flow paths of intake and exhaust are arranged to be capable of being changed over, these flow paths being capable of being changed over between an ordinary operating mode in which each of the cylinders are put in an independent condition in which combustion is conducted respectively independently and a special operating mode in which combustion is conducted in said two-cylinder connected condition, and said control device comprising: first fuel injection means that supplies fuel independently to each of the cylinders in said ordinary operating mode; second fuel injection means whereby it is made possible to supply fuel in an amount corresponding to that of the following cylinder to said burnt gas prior to introduction thereof into the following cylinder after completion of combustion in said preceding cylinder, when in said special operating mode; and wherein said combustion controller, when in said ordinary operating mode, conducts combustion in at an air/fuel ratio in each cylinder, made to be equal to the stoichiometric air/fuel ratio by supplying fuel by said first fuel injection means and, when in the special operating mode, said combustion controller conducts combustion in the preceding cylinder by forced ignition in a condition at an air/fuel ratio greater by a prescribed amount than the stoichiometric air/fuel ratio, by supplying fuel by said first fuel injection means, and said combustion controller controls combustion in each cylinder such as to conduct combustion in the following cylinder by compression self-ignition by introducing gas in a condition of the stoichiometric air/fuel ratio by supplying fuel to said burnt gas by said second fuel injection means. 37. The control device for a spark ignition engine according to claim 36, characterized in that said first fuel injection means is arranged such as to inject fuel directly into the combustion chamber in respect of said preceding cylinder; and the first fuel injection means of said preceding cylinder also serves as said second fuel injection means, when in said special operating mode, by constituting said fuel controller such that supply of fuel for the following cylinder to said burnt gas is performed by said first fuel injection means of the preceding cylinder during the exhaustion stroke of said cylinder. 38. The control device for a spark ignition engine according to claim 37, characterized in that said first fuel injection means is arranged such that fuel is injected into an intake passage in respect of said following cylinder. 39. The control device for a spark ignition engine according to claim 36, characterized in that said second fuel injection means is provided at some point along said inter-cylinder gas passage and fuel is supplied thereby to said burnt gas in an amount corresponding to that of the following cylinder after exhaustion from the preceding cylinder prior to introduction thereof into the following cylinder. 40. The control device for a spark ignition engine according to claim 36, characterized in that said fuel controller, when in said special operating mode, is capable of changing over the fuel injection mode between the first injection mode in which combustion is conducted by compression ignition by supplying fuel to said burnt gas in an amount corresponding to the following cylinder by the first fuel injection means of the following cylinder after introduction of burnt gas into the following cylinder from said preceding cylinder; and a second injection mode in which combustion is conducted by compression self-ignition by supplying fuel to said burnt gas in an amount corresponding to the following cylinder by said second fuel injection means prior to introduction thereof into the following cylinder after completion of combustion in said preceding cylinder, and is constituted such as to determine the degree of capability of self-ignition of the following cylinder from information relating to the operating condition and to be capable of changing over said injection mode in accordance with the results of the determination. 41. The control device for a spark ignition engine according to claim 40, characterized in that said combustion controller is constituted such as to put said injection mode into the second injection mode when in an operating condition wherein the degree of capability for self-ignition of the following cylinder is low. 42. The control device for a spark ignition engine according to claim 41, characterized in that said fuel injection means is constituted such as to determine that the operating condition is the condition in which the degree of capability for self-ignition is low if the cylinder temperature is below a specified temperature after warming up operation. 43. The control device for a spark ignition engine according to claim 41, characterized in that said combustion controller is constituted such as to determine that the operating condition is one in which the degree of capability for self-ignition is low when in a very low load region. 44. The control device for a spark ignition engine according to claim 1, characterized in that there are provided a preceding cylinder intake valve for introducing new air into said preceding cylinder and a burnt gas introduction valve for introducing burnt gas into said following cylinder from said inter-cylinder gas passage when in said two-cylinder connected condition; and in at least a prescribed region on the low load side of said operating region that is in a two-cylinder connected condition, the interval between the intake stroke bottom dead center of said following cylinder and the closure time of said burnt gas introduction valve is set to be shorter than the interval between the intake stroke bottom dead center of said preceding cylinder and the closure time of said preceding cylinder intake valve. 45. The control device for a spark ignition engine according to claim 44, characterized in that there is provided a following cylinder exhaust valve that exhausts exhaust gas of said following cylinder; and in at least a prescribed region on the low load side of said operating region that is in a two-cylinder connected condition, the opening time of said burnt gas introduction valve is set to be the intake stroke top dead center of said following cylinder, while said following cylinder exhaust valve is open until the top dead center of the exhaust stroke of said following cylinder. 46. The control device for a spark ignition engine according to claim 44, characterized in that, in a prescribed region on the high load side of said operating region that is in a two-cylinder connected condition, the closure time of said burnt gas introduction valve is set on the delayed side from said time when in the prescribed region on the low load side. 47. The control device for a spark ignition engine according to claim 44, characterized in that, in a prescribed region on the high load, high rotational speed side of said operating region that is in a two-cylinder connected condition, the closure time of said burnt gas introduction valve is set on the delayed side from said time when in the prescribed region on the low load, low rotational speed side. 48. The control device for a spark ignition engine according to claim 44, characterized in that a burnt gas exhaust valve is provided that exhausts burnt gas of said preceding cylinder to said inter-cylinder gas passage when in said two-cylinder connected condition; and in the operating region that is in said two-cylinder connected condition, the closure time of said burnt gas exhaust valve is set on the advancing side of the closure time of said burnt gas introduction valve and while maintaining the open period of said burnt gas exhaust valve and the open period of said burnt gas introduction valve at fixed prescribed values, the opening time of said burnt gas exhaust valve and the opening time of said burnt gas introduction valve are set so as to vary forwards and backwards in accordance with engine load while maintaining the difference of these times fixed. 49. The control device for a spark ignition engine according to claim 1, characterized in that there are provided a preceding cylinder intake valve that introduces new air into said preceding cylinder and a burnt gas introduction valve that introduces burnt gas into said following cylinder from said inter-cylinder gas passage, when in said two-cylinder connected condition; and in at least a prescribed region on the low load side of the operating region that is in said two-cylinder connected condition, the open period of said burnt gas introduction valve is set so as to be shorter than the open period of said preceding cylinder intake valve. 50. The control device for a spark ignition engine according to claim 1, characterized in that combustion is conducted by compression self-ignition in said preceding cylinder while increasing the amount of internal EGR of said preceding cylinder in a prescribed region on the comparatively low load side of the operating region in which combustion is conducted by compression self-ignition in the following cylinder and in said two-cylinder connected condition and wherein the internal EGR ratio is decreased with increase in load. 51. The control device for a spark ignition engine according to claim 50, characterized in that, in part or all of the operating region in which combustion is conducted by compression self-ignition in both said preceding cylinder and said following cylinder, the closure time of the burnt gas exhaust valve that exhausts burnt gas to said inter-cylinder gas passage in the exhaust stroke provided in said preceding cylinder is set earlier than the top dead center of the exhaust stroke of said preceding cylinder. 52. The control device for a spark ignition engine according to claim 51, characterized in that, in part or all of the operating region in which combustion is conducted by compression self-ignition in both said preceding cylinder and said following cylinder, said combustion controller sets the injection time of fuel into said preceding cylinder later than the closure time of said burnt gas exhaust valve and in the vicinity of the top dead center of the exhaust stroke. 53. The control device for a spark ignition engine according to claim 51, characterized in that, in part or all of the operating region in which combustion is conducted by compression self-ignition in both said preceding cylinder and said following cylinder, said combustion controller exercises control such that the air/fuel ratio in said following cylinder is substantially a lean air/fuel ratio. 54. The control device for a spark ignition engine according to claim 53, characterized in that the catalyst provided in said exhaust passage for cleaning exhaust gas consists solely of a three-way catalyst or solely of a three-way catalyst and oxidation catalyst. 55. The control device for a spark ignition engine according to claim 50, characterized in that it comprises a burnt gas introduction valve provided in said following cylinder for introducing burnt gas from said inter-cylinder gas passage in the intake stroke when in said two-cylinder connected condition, and a following cylinder intake valve provided in said following cylinder for introducing new air in the intake stroke when in said two-cylinder connected condition; and in part all of the operating region in which combustion is conducted by compression self-ignition in both said preceding cylinder and said following cylinder, the opening time of said burnt gas introduction valve is set on the delayed side of the top dead center of the intake stroke of the following cylinder, and said following cylinder intake valve is arranged to open earlier than the opening time of said burnt gas introduction valve. 56. The control device for a spark ignition engine according to claim 55, characterized in that said preceding cylinder is of the long stroke type and in that it comprises a preceding cylinder intake valve that introduces new air in the intake stroke when in said two-cylinder connected condition; and in part or all of the operating region in which combustion is conducted by compression self-ignition in both said preceding cylinder and said following cylinder, the closure time of said burnt gas exhaust valve and said burnt gas introduction valve is set on the delayed side of the top dead center of the exhaust stroke of said preceding cylinder, and the opening time of said preceding cylinder intake valve is set earlier than the top dead center of the intake stroke of the preceding cylinder. 57. The control device for a spark ignition engine according to claim 50, characterized in that it comprises a supercharger that supercharges the intake to said preceding cylinder, and in part or all of the operating region in which combustion is conducted by compression self-ignition in at least said preceding cylinder and said following cylinder, supercharging is performed using said supercharger. 58. The control device for a spark ignition engine according to claim 50, characterized in that, in a prescribed region on the comparatively high load side of said operating region in which combustion is conducted by compression self-ignition in said following cylinder, said combustion controller conduct combustion by forced ignition in said preceding cylinder, and set the air/fuel ratio of said preceding cylinder to be substantially larger than that when in an operating region in which combustion is conducted by compression self-ignition in both said preceding cylinder and said following cylinder. 59. A control device for a multi-cylinder spark ignition engine having cylinders arranged to perform a cycle consisting of intake, compression, expansion and exhaustion strokes with prescribed phase differences, characterized in that a gas flow path is constituted in a two-cylinder connected condition, at least in a low load, low rotational speed region, such that burnt gas exhausted from a leading cylinder which is a cylinder on the exhaust stroke side in a pair of cylinders whose exhaustion stroke and intake stroke overlap is directly introduced into a following cylinder which is a cylinder on the intake stroke side through an inter-cylinder gas passage and gas exhausted from the following cylinder is fed to an exhaust passage; and a three-way catalyst is provided in the exhaust passage connected with the following cylinder; and said control device comprising combustion control means that controls combustion in each cylinder such that at least in part of the operating region of the operating region in which said two-cylinder connected condition is produced, combustion is performed in said leading cylinder in a condition at an air/fuel ratio larger by a prescribed amount than the stoichiometric air/fuel ratio while fuel is supplied in an amount corresponding to the following cylinder to the burnt gas generated by combustion in the leading cylinder, and combustion is performed by compression self-ignition at least in the following cylinder while an amount of fuel injection in each of said cylinders is controlled in a manner that a total air/fuel ratio of both of the leading cylinder and the following cylinder is made to be larger. 60. A control device for a four cycled multi-cylinder spark ignition engine having cylinders arranged to perform a cycle consisting of intake, compression, expansion and exhaustion strokes with prescribed phase differences, and each of said cylinders having an ignition plug, characterized in that an inter-cylinder gas passage is provided between a leading cylinder and a following cylinder in a two-cylinder connected condition such that burnt gas exhausted from the leading cylinder which is a cylinder on the exhaust stroke side in a pair of cylinders whose exhaustion stroke and intake stroke overlap is introduced into the following cylinder which is a cylinder on the intake stroke side; characterized in that said leading cylinder is provided with an intake port that communicates with said intake passage, a first exhaust port that communicates with said exhaust passage and a second exhaust port that communicates with the inter-cylinder gas passage, and said following cylinder is provided with a first intake port that communicates with said intake passage, a second intake port that communicates with said inter-cylinder gas passage and an exhaust port that communicates with said exhaust passage; characterized in that a first and a second exhaust valves that open and close the first and second exhaust ports of said leading cylinder and a first and a second intake valves that open and close the first and second intake ports of the following cylinder are provided and said first and second exhaust valves and said first and second intake valves are selectively operated between its activating state and deactivating state, and said control device comprising combustion control means that controls a fuel supplying and an injection in each cylinder in such a manner that: in a low load, low rotational speed region, said first exhaust valve and said first intake valve are set in deactivated condition and said second exhaust valve and said second intake valve in operating condition so that the two-cylinder connected condition in which burnt gas exhausted from the leading cylinder which is a cylinder on the exhaust stroke side is introduced into the following cylinder which is a cylinder on the intake stroke side through an inter-cylinder gas passage is established; characterized in that a three-way catalyst is provided in the exhaust passage to make the exhaust gas exhausted from the exhaust port of the said following cylinder in said two-cylinder connecting condition being passed through the three-way catalyst, and when said two-cylinder connected condition is established, such that combustion is performed in said leading cylinder at an air/fuel ratio larger by a prescribed amount than the stoichiometric air/fuel ratio while fuel is supplied in an amount corresponding to the following cylinder to the burnt gas generated by combustion in the leading cylinder, and combustion is performed in said following cylinder at a stoichiometric air/fuel ratio by compression self-ignition. 61. The control device for a spark ignition four-cycle engine according to claim 60, characterized in that said combustion controller which exercise the control mode whereby combustion is performed in said two-cylinder connected condition as the special operating mode, and said combustion controller, in at least part of the operating region of the region corresponding to said special operating mode, controls the fuel supply rate in respect of both the leading and following cylinders such that the fuel supply rate in the preceding cylinder is greater, while the air/fuel ratio during combustion in said following cylinder is substantially the stoichiometric air/fuel ratio, thereby making the air/fuel ratio when combustion is conducted in the preceding cylinder a value of less than twice the stoichiometric air/fuel ratio and conducting combustion in the preceding cylinder by forced ignition and conducting combustion in the following cylinder by compression self-ignition. 62. The control device for a spark ignition engine according to claim 60, wherein said combustion controller executes control in which combustion is conducted in said two-cylinder connected condition in a control mode as a special operating mode; and said combustion condition; and said combustion controller including a fuel injection controller that, in an operating region in which the following cylinder is made to perform compression self-ignition in said special operating mode, relatively retards the injection time of the fuel to the following cylinder in an operating condition in which knocking is likely to occur, compared with an operating condition where knocking is unlikely to occur. 63. The control device for a spark ignition engine according to claim 60, characterized in that the combustion controller exercises control whereby combustion is conducted in said two-cylinder connected condition as a special operating mode; and said combustion controller effects a control such that: in at least part of the operating region in which said special operating mode is involved, combustion is conducted by compression self-ignition in the following cylinder, and the air/fuel ratio of the preceding cylinder is made relatively lower in a high load region in the region in which the compression self-ignition is performed compared with the region on the low load side and a new air introduction intake valve that introduces new air into the following cylinder is opened so that new air is introduced into the following cylinder in addition to the burnt gas that is fed from said preceding cylinder. 64. The control device for a spark ignition engine according to claim 60, characterized in that control is exercised such as to make the control mode whereby combustion is conducted in said two-cylinder connected condition a special operating mode; and said combustion controller controls such that the total injection quantity of fuel injected into the two cylinders consisting of said preceding cylinder and following cylinder is increased in response to increase in engine load; and control is exercised such that in said following cylinder, combustion is conducted by compression self-ignition in at least part of the operating region in which said special operating mode is involved and, in said preceding cylinder, stratified charge lean combustion is conducted with the injected fuel put in a stratified condition in an intermediate/low load region of the operating region in which compression self-ignition of said following cylinder is performed, and control is exercised such that, on the high load side of the operating region in which the stratified charge lean combustion is conducted, uniform lean combustion is conducted in a condition with the injected fuel uniformly dispersed. 65. The control device for a spark ignition engine according to claim 60, characterized in that the flow paths of intake and exhaust are arranged to be capable of being changed over, these flow paths being capable of being changed over between an ordinary operating mode in which each of the cylinders are put in an independent condition in which combustion is conducted respectively independently and a special operating mode in which combustion is conducted in said two-cylinder connected condition, and said control device comprising: first fuel injection means that supplies fuel independently to each of the cylinders in said ordinary operating mode; second fuel injection means whereby it is made possible to supply fuel in an amount corresponding to that of the following cylinder to said burnt gas prior to introduction thereof into the following cylinder after completion of combustion in said preceding cylinder, when in said special operating mode; and wherein said combustion controller, when in said ordinary operating mode, conducts combustion in at an air/fuel ratio in each cylinder, made to be equal to the stoichiometric air/fuel ratio by supplying fuel by said first fuel injection means and, when in the special operating mode, said combustion controller conducts combustion in the preceding cylinder by forced ignition in a condition at an air/fuel ratio greater by a prescribed amount than the stoichiometric air/fuel ratio, by supplying fuel by said first fuel injection means, and said combustion controller controls combustion in each cylinder such as to conduct combustion in the following cylinder by compression self-ignition by introducing gas in a condition of the stoichiometric air/fuel ratio by supplying fuel to said burnt gas by said second fuel injection means. 66. The control device for a spark ignition engine according to claim 60, characterized in that there are provided a preceding cylinder intake valve for introducing new air into said preceding cylinder and a burnt gas introduction valve for introducing burnt gas into said following cylinder from said inter-cylinder gas passage when in said two-cylinder connected condition; and in at least a prescribed region on the low load side of said operating region that is in a two-cylinder connected condition, the interval between the intake stroke bottom dead center of said following cylinder and the closure time of said burnt gas introduction valve is set to be shorter than the interval between the intake stroke bottom dead center of said preceding cylinder and the closure time of said preceding cylinder intake valve. 67. The control device for a spark ignition engine according to claim 60, characterized in that there are provided a preceding cylinder intake valve that introduces new air into said preceding cylinder and a burnt gas introduction valve that introduces burnt gas into said following cylinder from said inter-cylinder gas passage, when in said two-cylinder connected condition; and in at least a prescribed region on the low load side of the operating region that is in said two-cylinder connected condition, the open period of said burnt gas introduction valve is set so as to be shorter than the open period of said preceding cylinder intake valve. 68. The control device for a spark ignition engine according to claim 60, characterized in that combustion is conducted by compression self-ignition in said preceding cylinder while increasing the amount of internal EGR of said preceding cylinder in a prescribed region on the comparatively low load side of the operating region in which combustion is conducted by compression self-ignition in the following cylinder and in said two-cylinder connected condition and wherein the internal EGR ratio is decreased with increase in load. 69. A control device for a multi-cylinder spark ignition engine having cylinders arranged to perform a cycle consisting of intake, compression, expansion and exhaustion strokes with prescribed phase differences, characterized in that a gas flow path is formed in a two-cylinder connected condition, at least in a low load, low rotational speed region, such that burnt gas exhausted from a preceding cylinder which is a cylinder on the exhaust stroke side in a pair of cylinders whose exhaustion stroke and intake stroke overlap is directly introduced into a following cylinder which is a cylinder on the intake stroke side through an inter-cylinder gas passage and gas exhausted from the following cylinder is fed to an exhaust passage; and said control device comprising a control unit that controls combustion in each cylinder such that at least in part of the operating region of the operating region in which said two-cylinder connected condition is established, combustion is performed by forced ignition in said preceding cylinder in a condition at an air/fuel ratio larger by a prescribed amount than the stoichiometric air/fuel ratio and fuel is supplied to the following cylinder in an amount corresponding to the burnt gas generated by combustion in the preceding cylinder, and combustion is performed by compression self-ignition in the following cylinder. 70. The control device for a spark ignition four-cycle engine according to claim 69, characterized in that said combustion controller which exercise the control mode whereby combustion is performed in said two-cylinder connected condition as the special operating mode, and said combustion controller, in at least part of the operating region of the region corresponding to said special operating mode, controls the fuel supply rate in respect of both the leading and following cylinders such that the fuel supply rate in the preceding cylinder is greater, while the air/fuel ratio during combustion in said following cylinder is substantially the stoichiometric air/fuel ratio, thereby making the air/fuel ratio when combustion is conducted in the preceding cylinder a value of less than twice the stoichiometric air/fuel ratio and conducting combustion in the preceding cylinder by forced ignition and conducting combustion in the following cylinder by compression self-ignition. 71. The control device for a spark ignition engine according to claim 69, wherein said combustion controller executes control in which combustion is conducted in said two-cylinder connected condition in a control mode as a special operating mode; and said combustion condition; and said combustion controller including a fuel injection controller that, in an operating region in which the following cylinder is made to perform compression self-ignition in said special operating mode, relatively retards the injection time of the fuel to the following cylinder in an operating condition in which knocking is likely to occur, compared with an operating condition where knocking is unlikely to occur. 72. The control device for a spark ignition engine according to claim 69, characterized in that the combustion controller exercises control whereby combustion is conducted in said two-cylinder connected condition as a special operating mode; and said combustion controller effects a control such that: in at least part of the operating region in which said special operating mode is involved, combustion is conducted by compression self-ignition in the following cylinder, and the air/fuel ratio of the preceding cylinder is made relatively lower in a high load region in the region in which the compression self-ignition is performed compared with the region on the low load side and a new air introduction intake valve that introduces new air into the following cylinder is opened so that new air is introduced into the following cylinder in addition to the burnt gas that is fed from said preceding cylinder. 73. The control device for a spark ignition engine according to claim 69, characterized in that control is exercised such as to make the control mode whereby combustion is conducted in said two-cylinder connected condition a special operating mode; and said combustion controller controls such that the total injection quantity of fuel injected into the two cylinders consisting of said preceding cylinder and following cylinder is increased in response to increase in engine load; and control is exercised such that in said following cylinder, combustion is conducted by compression self-ignition in at least part of the operating region in which said special operating mode is involved and, in said preceding cylinder, stratified charge lean combustion is conducted with the injected fuel put in a stratified condition in an intermediate/low load region of the operating region in which compression self-ignition of said following cylinder is performed, and control is exercised such that, on the high load side of the operating region in which the stratified charge lean combustion is conducted, uniform lean combustion is conducted in a condition with the injected fuel uniformly dispersed. 74. The control device for a spark ignition engine according to claim 69, characterized in that the flow paths of intake and exhaust are arranged to be capable of being changed over, these flow paths being capable of being changed over between an ordinary operating mode in which each of the cylinders are put in an independent condition in which combustion is conducted respectively independently and a special operating mode in which combustion is conducted in said two-cylinder connected condition, and said control device comprising: first fuel injection means that supplies fuel independently to each of the cylinders in said ordinary operating mode; second fuel injection means whereby it is made possible to supply fuel in an amount corresponding to that of the following cylinder to said burnt gas prior to introduction thereof into the following cylinder after completion of combustion in said preceding cylinder, when in said special operating mode; and wherein said combustion controller, when in said ordinary operating mode, conducts combustion in at an air/fuel ratio in each cylinder, made to be equal to the stoichiometric air/fuel ratio by supplying fuel by said first fuel injection means and, when in the special operating mode, said combustion controller conducts combustion in the preceding cylinder by forced ignition in a condition at an air/fuel ratio greater by a prescribed amount than the stoichiometric air/fuel ratio, by supplying fuel by said first fuel injection means, and said combustion controller controls combustion in each cylinder such as to conduct combustion in the following cylinder by compression self-ignition by introducing gas in a condition of the stoichiometric air/fuel ratio by supplying fuel to said burnt gas by said second fuel injection means. 75. The control device for a spark ignition engine according to claim 69, characterized in that there are provided a preceding cylinder intake valve for introducing new air into said preceding cylinder and a burnt gas introduction valve for introducing burnt gas into said following cylinder from said inter-cylinder gas passage when in said two-cylinder connected condition; and in at least a prescribed region on the low load side of said operating region that is in a two-cylinder connected condition, the interval between the intake stroke bottom dead center of said following cylinder and the closure time of said burnt gas introduction valve is set to be shorter than the interval between the intake stroke bottom dead center of said preceding cylinder and the closure time of said preceding cylinder intake valve. 76. The control device for a spark ignition engine according to claim 69, characterized in that there are provided a preceding cylinder intake valve that introduces new air into said preceding cylinder and a burnt gas introduction valve that introduces burnt gas into said following cylinder from said inter-cylinder gas passage, when in said two-cylinder connected condition; and in at least a prescribed region on the low load side of the operating region that is in said two-cylinder connected condition, the open period of said burnt gas introduction valve is set so as to be shorter than the open period of said preceding cylinder intake valve. 77. The control device for a spark ignition engine according to claim 69, characterized in that combustion is conducted by compression self-ignition in said preceding cylinder while increasing the amount of internal EGR of said preceding cylinder in a prescribed region on the comparatively low load side of the operating region in which combustion is conducted by compression self-ignition in the following cylinder and in said two-cylinder connected condition and wherein the internal EGR ratio is decreased with increase in load. |
<SOH> BACKGROUND ART <EOH>Techniques are previously known for improving fuel consumption in spark ignition engines by performing combustion under lean air/fuel ratio conditions, in which the air/fuel ratio of the mixture in the cylinders is larger than the stoichiometric air/fuel ratio (or theoretical air/fuel ration). For example, as illustrated in Laid-open Japanese Patent Application No. H. 10-274085, a technique is known in which an injection valve that injects fuel directly into the combustion chamber is provided and super-lean combustion is produced by conducting stratified charge combustion in the low rotational speed low load region etc. Specifically, such stratified charge combustion consists in altering the composition ratio of the mixture in the vicinity of the spark plug in the ignition period by injecting fuel in the compression stroke, while controlling the rate of air intake and rate of fuel injection such as to produce a condition in the combustion chamber as a whole that is much leaner than the stoichiometric air/fuel ratio, and performing combustion with forced ignition by the spark plug in this condition. When super-lean combustion is performed by stratified charge combustion as described above, thermal efficiency is improved and the air intake rate becomes large, reducing the intake negative pressure and thereby greatly improving fuel consumption. Also, in such a super-lean stratified charge combustion condition, even if some of the air that is present in excess is replaced by EGR, fully satisfactory combustion is still achieved, so a comparatively large amount of EGR may be employed and this is thereby beneficial in lowering NOx etc. Thus, even though this large amount of EGR is introduced, the benefit of a lowered pumping loss is still obtained and thermal efficiency is also increased compared with ordinary combustion in which the air intake rate and EGR rate are controlled without layering; the benefit of improved fuel consumption is thereby obtained. However, when stratified charge combustion is performed, although, as the air/fuel ratio is made leaner, improved fuel consumption is obtained up to a certain point, if the mixture becomes leaner than a certain degree, the combustion rate becomes too low, with the result that the combustion occurring in the vicinity of the final period does not contribute to work, so, contrariwise, fuel consumption tends to deteriorate. Thus, there were limits to the extent to which fuel consumption improvement could be achieved by increasing leanness in stratified charge combustion. Compression ignition has been studied as another technique for improving fuel consumption. This compression ignition consists in self-ignition of fuel at high temperature and high pressure in a combustion chamber in the latter period of the compression stroke, in the same way as in the case of a diesel engine. If such compression ignition is performed, even under conditions of a super-lean air/fuel ratio or conditions of introduction of a large amount of EGR, combustion occurs at once throughout the entire combustion chamber. Slow combustion, which does not contribute to work, is thereby avoided, which is beneficial in improving fuel consumption. However, in an ordinary spark ignition engine (gasoline engine), forced ignition for combustion is necessary and the temperature and pressure within the combustion chamber in the vicinity of the top dead center in compression are not elevated to a sufficient degree to produce compression ignition; thus special expedients must be adopted if the temperature or pressure in the combustion chamber is to be raised to the considerable degree necessary to achieve compression ignition. However, in a conventional spark ignition engine, it is difficult to raise the temperature or pressure in the combustion chamber to such an extent as to produce compression ignition in the low load region where fuel consumption improvement is required while yet preventing knocking in the high load region, so implementation of such a technique has not been achieved. In view of the aforementioned problems, the present invention provides a control device for a spark ignition engine wherein the benefit of improved fuel consumption is produced by lean combustion and, in addition, the benefit of improved fuel consumption is increased by effectively performing compression ignition in a portion of the cylinders. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 is a diagrammatic plan view of an entire engine comprising a device according to an embodiment of the present invention; FIG. 2 is a diagrammatic cross-sectional view of a main engine unit etc; FIG. 3 is a block diagram of a control system; FIG. 4 is a diagram showing an example of operating region setting for control in accordance with the operating condition; FIG. 5 is a view showing the exhaust stroke, intake stroke, fuel injection period and ignition period etc of each cylinder; FIG. 6 is a diagram showing a flow path for substantially new air and gas during low load, low rotational speed operation; FIG. 7 is a diagram showing a flow path for substantially new air and gas in an operating region on the high load, high/low i rotational speed side; FIG. 8 is a diagrammatic plan view of an entire engine showing a modified example in which the catalyst etc provided in the exhaust passage is changed from that shown in FIG. 1 ; FIG. 9 is a diagram showing an example of operating region setting for exercising control in accordance with the operating condition in accordance with another embodiment; FIG. 10 is a diagram showing a second example in regard to operating region setting for exercising control in accordance with the operating condition; FIG. 11 is a diagram showing a third example in regard to operating region setting for exercising control in accordance with the operating condition; FIG. 12 is a diagram showing a fourth example in regard to operating region setting for exercising control in accordance with the operating condition; FIG. 13 is a diagram showing a fifth example in regard to operating region setting for exercising control in accordance with the operating condition; FIG. 14 is a diagram showing an example of setting of operating region for exercising control in accordance with the operating condition in accordance with yet a further embodiment; FIG. 15 is a diagram showing the combustion cycle of a preceding cylinder and a following cylinder; FIG. 16 is a diagram showing a further example of the combustion cycle of a preceding cylinder and a following cylinder; FIG. 17 is a diagram showing the specific construction of swirl generating means; FIG. 18 is a diagram showing an example of the setting of the operating region for exercising control in accordance with the operating condition in accordance with yet a further embodiment; FIG. 19 is a diagram showing the combustion cycle and valve opening timing of a preceding cylinder and a following cylinder; FIG. 20 is a block diagram of a control system showing yet a further embodiment; FIG. 21 is a diagram showing an example of the setting of the operating region for exercising control in accordance with the operating condition by means of a device according to the embodiment shown in FIG. 20 ; FIG. 22 is a view showing the relationship between burnt gas temperature and air/fuel ratio under the same load in stratified charge lean combustion and uniform lean combustion; FIG. 23 is a view showing the exhaust stroke, intake stroke, fuel injection period and ignition period etc of each cylinder in the case of a special operating mode in which a preceding cylinder is made to perform uniform lean combustion while a following cylinder is made to perform forced ignition; FIG. 24 is a view showing the exhaust stroke, intake stroke, fuel injection period and ignition period etc of each cylinder in the case of a special operating mode in which a preceding cylinder is made to perform uniform lean combustion while a following cylinder is made to perform compression self-ignition; FIG. 25 is a view showing the relationship between load and air/fuel ratio in a preceding cylinder; FIG. 26 is a block diagram of a control system showing yet a further embodiment; FIG. 27 is a view showing the exhaust stroke, intake stroke, fuel injection period and ignition period etc of each cylinder; FIG. 28 is a diagrammatic plan view showing a modified example of an engine; FIG. 29 is a view showing the exhaust stroke, intake stroke, fuel injection period and ignition period etc of each cylinder in the case of the modified example shown in FIG. 28 ; FIG. 30 is a diagrammatic plan view of an entire engine according to yet a further embodiment; FIG. 31 is a block diagram of a control system of the same embodiment; FIG. 32 is a diagram showing an example of the setting of the operating region for exercising control in accordance with the operating condition; FIG. 33 is a diagram showing the opening/closing times of an intake/exhaust valve in a special operating mode, (a) showing the case of comparatively low load, low rotational speed and (b) showing in like manner the case of comparatively high load, high rotational speed; FIG. 34 is a diagram showing the opening/closing times of an intake/exhaust valve in the ordinary operating mode; FIG. 35 is a partial perspective view showing a cam changeover mechanism employed in a yet a further embodiment; FIG. 36 is a plunger action diagram given in explanation of three types of cam changeover mechanism; FIG. 37 is a plunger action diagram given in explanation of two types of cam changeover mechanism; FIG. 38 is a block diagram of a control system in an embodiment employing a cam changeover mechanism; FIG. 39 is a diagram showing the opening/closing times of an intake/exhaust valve in a special operating mode, (a) showing the case of comparatively low load, low rotational speed and (b) showing in like manner the case of comparatively high load, high rotational speed; FIG. 40 is a diagrammatic plan view of an entire engine according to yet a further embodiment; FIG. 41 is a diagrammatic cross-sectional view of the main engine unit etc according to this embodiment; FIG. 42 is a partial perspective view showing a cam changeover mechanism employed in this embodiment; FIG. 43 is a plunger action diagram for a cam changeover mechanism; FIG. 44 is a block diagram of a control system; FIG. 45 is a diagram showing an example of the setting of the operating region for exercising control in accordance with the operating condition; FIG. 46 is a diagram showing the opening/closing times of an intake/exhaust valve in a special operating mode, (a) showing the case of comparatively low load and (b) showing in like manner the case of intermediate load; FIG. 47 is a diagram showing the opening/closing times of an intake/exhaust valve in a special operating mode, showing the case of comparatively high load; FIG. 48 is a diagram showing the opening/closing times of an intake/exhaust valve in the ordinary operating mode; FIG. 49 is a diagram showing the opening/closing times of an intake/exhaust valve in a special operating mode according to a second example of the control of intake/exhaust etc using a device as shown in FIG. 40 to FIG. 44 , (a) showing the case of comparatively low load and (b) showing in like manner the case of a comparatively high load; FIG. 50 is a diagram showing the opening/closing times of an intake/exhaust valve in a special operating mode according to a third example of the control of intake/exhaust etc using a device as shown in FIG. 40 to FIG. 44 , (a) showing the case of comparatively low load and (b) showing in like manner the case of a comparatively high load; FIG. 51 is a diagram showing the opening/closing times of an intake/exhaust valve in a special operating mode according to a fourth example of the control of intake/exhaust etc using a device as shown in FIG. 40 to FIG. 44 , (a) showing the case of comparatively low load and (b) showing in like manner the case of an intermediate load; and FIG. 52 is a diagrammatic plan view of an entire engine showing yet a further embodiment. detailed-description description="Detailed Description" end="lead"? |
Novel peptides for the diagnosis of schizophrenia |
Short peptides are provided, which bind to a body fluid sample obtained from a schizophrenic patient at a substantively higher level than to a body fluid sample obtained from a non-schizophrenic individual. The peptides are no more than 10 amino acids long and comprise a continuous sequence of at least 5 amino acids which consists of at least one positively charged amino acid at one of its ends. The provided peptides, which are the putative binding sites of autoantibodies found in high levels in schizophrenic individuals, are thus useful in diagnosis of schizophrenia. |
1. A peptide which binds to a body fluid sample obtained from a schizophrenic patient at a substantively higher level than its binding to a body fluid sample obtained from a non-schizophrenic individual, said peptide being no more than 10 amino acids (a.a.) long and comprising a continuous sequence of at least 5 amino acids included in any one of the following sequences: i. LVVGLCK (SEQ ID NO. 1) ii. KLVVGLC (SEQ ID NO. 2) iii. LVVGLMK (SEQ ID NO. 3) iv. KLVVGLM; (SEQ ID NO. 4) said continuous sequence consisting of at least one positively charged a.a. at one end of said sequence; and said peptide comprising at least one positively charged a.a at its end being the positively charged a.a of said continuous sequence or at least one additional positively charged a.a.; or analogs of said peptides being no more than 10 a.a long and in which no more than two a.a of said continuous sequence are conservatively substituted, said analogues essentially maintaining the binding characteristics of the peptides. 2. A peptide which binds to a body fluid sample obtained from a schizophrenic patient at a substantively higher level than its binds to a body fluid sample obtained from a non-schizophrenic individual said peptide selected from the group consisting of: i. LVVGLCK (SEQ ID NO. 1) ii. KLVVGLC (SEQ ID NO. 2) iii. LVVGLMK (SEQ ID NO. 3) iv. KLVVGLM (SEQ ID NO. 4) or analogs of said peptides in which no more than two a.a are conservatively substituted, said analogues essentially maintaining the binding characteristics of the peptides. 3. A peptide which binds to body fluid samples obtained from a schizophrenic patient substantively higher than it binds to a body fluid sample obtained from a non-schizophrenic individual having the amino acid sequence LVVGLCK (SEQ ID NO. 1). 4. A peptide which binds to a body sample obtained from a schizophrenic patient substantively higher than it binds to a sample obtained from a non-schizophrenic individual, wherein the peptide binds antibodies that are capable of specific binding to a peptide having the amino acid LVVGLCK (SEQ ID NO. 1). 5. A peptide according to claim 4, having the amino acid sequence KLVVGLC (SEQ ID NO. 2). 6. A peptide according to claim 4, having the amino acid sequence LVVGLMK (SEQ ID NO. 3). 7. A peptide according to claim 4, having the amino acid sequence KLVVGLM (SEQ ID NO. 4). 8. An assay for the diagnosis of schizophrenia in an individual comprising the following steps: (a) obtaining a body fluid sample from said individual being a blood sample, PAA-containing fraction thereof, or a fraction containing platelet-associated antibodies (PAA) shed from the platelets; (b) contacting said sample with a peptide being no more than 10 amino acids (a.a.) long and comprising a continuous sequence of at least 5 amino acids included in any one of the following sequences: i. LVVGLCK (SEQ ID NO. 1) ii. KLVVGLC (SEQ ID NO. 2) iii. LVVGLMK (SEQ ID NO. 3) iv. KLVVGLM; (SEQ ID NO. 4) said continuous sequence consisting of at least one positively charged a.a. at one end of said sequence; and said peptide comprising at least one positively charged a.a at its end being the positively charged a.a of said continuous sequence or at least one additional positively charged a.a.; or analogs of said peptides being no more than 10 a.a long and in which no more than two a.a of said continuous sequence are conservatively substituted, said analogues essentially maintaining the binding characteristics of the peptides. (c) determining the level of binding of said peptide to said sample, a level substantively higher than the binding level of said peptide to a sample obtained from a non-schizophrenic individual indicating that said tested individual has a high likelihood of having schizophrenia. 9. A method according to claim 8, wherein the peptide of step (b) is selected from the group consisting of: i. LVVGLCK (SEQ ID NO. 1) ii. KLVVGLC (SEQ ID NO. 2) iii. LVVGLMK (SEQ ID NO. 3) iv. KLVVGLM (SEQ ID NO. 4) or analogs thereof in which no more than two a.a are conservatively substituted and which essentially maintain said peptides' binding characteristics. 10. A method according to claim 8, wherein the peptide of step (b) has the amino acid sequence LVVGLCK (SEQ ID NO.1). 11. A method according to claim 8, wherein the peptide in step (b) is such which binds antibodies which bind peptides having the amino acid sequence LVVGLCK (SEQ ID NO. 1) or analogs thereof in which no more than two a.a. are conservatively substituted. 12. A kit for use in the diagnosis of schizophrenia comprising a support comprising one or more peptides of claim 1, immobilized onto it, anti-human immunoglobulin (hIg) antibody or fragment thereof, reagents carrying a detection assay whereby said peptides bind to antibodies present in a tested sample, as well as instructions for use. 13. A kit according to claim 12, wherein said anti-hIg antibody is complexed to a detectable marker. 14. A kit according to claim 12, wherein instead of said anti-hIg antibody, the kit comprises one or more non-bound peptides which bind to antibodies present in a tested sample, said peptides complexed to a detectable marker. 15. Use of any of the peptides of claim 1 for the preparation of a diagnostic composition for the diagnosis of schizophrenia in an individual. 16. The assay of claim 8, for use in confirming a high probability of Schizophrenia in an individual determined by at least one other diagnostic assay. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Schizophrenia is a syndrome which encompasses a variety of mental symptoms like auditory hallucinations, paranoia, delusions, catatonia, bizarre behavior and emotional withdrawal. Schizophrenia affects about 1% of the total population and its economical as well as social burden on society are enormous. The onset of the disease occurs in early age and, thus, patients typically need life-long medical and psychiatric supervision. Schizophrenia is, therefore, rated as one of the most costly diseases in the industrial world (Carpenter, et al., 1994). No common parameter associated with schizophrenia has been identified and, therefore, the internationally agreed diagnosis of this disease is still based today solely on psychiatric evaluation. Known risk factors associated with schizophrenia, are genetic predisposition, birth during winter and complications during pregnancy or birth. Viral and/or bacterial infections with a subsequent autoimmune reaction have been proposed as causative factors for the increasing outbreak of schizophrenia (DeLisi, et al, 1987). Schizophrenia has been shown to involve an autoimmune process and lately autoantibodies and cytotoxic T-cells against platelets were demonstrated in schizophrenic patients (Shinitzky, 1991, Deckmann, 1996, Shinitzky, 1999, U.S. Pat. No. 6,008,001). The cytotoxic T-cell reaction in schizophrenic patients was evaluated by a skin test in which most schizophrenic patients reacted positively against their autologous platelets whereas only a very minor number of non-schizophrenic tested individuals reacted positively in this test (Shinitzky, 1999, WO 99/30163). In addition elevated levels of autoantibodies against platelets were observed in schizophrenic patients but not in patients suffering from manic-depressive disorder, depression, personality disorders and schizoaffective disorder (Shinitzky, 1991 and Deckmann, 1996). In the inventors' prior work several proteins which bind autoantibodies that are found in elevated levels in body fluids of schizophrenic patients were identified (Shinitzky et al, 1999, WO 99/51725). These proteins reacted with purified platelet derived autoantibodies (PAA) from schizophrenic patients but could not differentiate between plasma or blood samples of schizophrenic and non-schizophrenic individuals. Enzymatic digestion of one of these proteins, the enzyme Enolase, resulted in a fragment which bound to plasma samples of schizophrenic patients substantially higher than it bound to plasma samples of non-schizophrenic individuals. On the basis of this fragment several additional peptides were synthesized and such having a high binding activity to PAAs of schizophrenic individuals were isolated. The structure of the antigenic epitope of these peptides was found to be a three-dimensional epitope which, by using a computerized program was predicted to be a cyclic structure comprising a hydrophobic core and an extension having about two positive charges. Immunological studies demonstrated that only the oxidized cyclic form of the peptide was reactive with the anti-platelet autoantibodies. These synthesized peptides comprised at least 17 amino acids (a.a.). |
<SOH> SUMMARY OF THE INVENTION <EOH>In accordance with the present invention, it has been realized that the peptide sequences described in the prior art (WO 99/51725) are able to bind to a higher extent to autoantibodies which are found in elevated levels in body fluids of schizophrenic patients and to a lower extent or not at all in body fluids of non-schizophrenic individuals, but these peptides cannot be the natural binding site for such autoantibodies. This is due to the fact that the sequences are not exposed on the surface of the protein from which they were derived (the enzyme enolase). Except for the single amino acid arginine at position 402, the remaining amino acids are buried inside the protein (see FIG. 1 ). In addition, as known, an antibody binding site is usually comprised of about 5-8 a.a. while each of these peptides comprised 17 a.a. which are not all necessary for the binding site. Therefore, in accordance with the present invention, based on the realization that the peptide which is the natural binding site for autoantibodies found in elevated levels in schizophrenic patients will have a more sensitive and specific binding to such autoantibodies and as such may be advantageous in diagnosis of schizophrenia, an attempt was made to identify an equivalent site on the surface of the enolase by using three-dimensional modeling. Using the three-dimensional structure to search the surface of the enolase resulted in the identification of a putative epitope (see FIG. 2 ) consisting of four positively charged amino acids defined as R414, R184, K194 and R402 which surround a cluster of neutral amino acids defined as L412, L 183, L409, L406, P400 and A401. Peptides having such a putative epitope are provided in accordance with the invention. Thus, by its first aspect, the present invention provides a peptide which binds to a body fluid sample obtained from a schizophrenic patient at a substantively higher level than its binding to a body fluid sample obtained from a non-schizophrenic individual, said peptide being no more than 10 amino acids (a.a.) long and comprising a continuous sequence of at least 5 amino acids included in any one of the following sequences: i. LVVGLCK (SEQ ID NO. 1) ii. KLVVGLC (SEQ ID NO. 2) iii. LVVGLMK (SEQ ID NO. 3) iv. KLVVGLM; (SEQ ID NO. 4) said continuous sequence consisting of at least one positively charged a.a. at one end of said sequence; and said peptide comprising at least one positively charged a.a at its end being the positively charged a.a of said continuous sequence or at least one additional positively charged a.a.; or analogues of said peptide being no more than 10 a.a long and in which no more than two a.a of said continuous sequence are conservatively substituted, said analogues essentially maintaining the binding characteristics of the peptide. A “substantively higher level of binding” in accordance with the invention will be determined by using any of the binding assays known in the art such as those described below and wherein the measured level of binding of a peptide to a sample obtained from a schizophrenic patient is significantly higher than the measured level of binding of the same peptide to a sample obtained from a non-schizophrenic patient as determined by a suitable statistic test, e.g. Student's T-test. The term “continuous sequence” concerns an uninterrupted sequence of between 5 and 7 a.a of any of the sequences of SEQ.IDs 1-4 which includes a positively charged a.a at its end. The positively charged a.a is preferably Lysine (indicated as K in the sequences) but may also be Arginine (R) or Histidine (H). The continuous sequence can be part of a longer peptide of up to 10 a.a., wherein the continuous sequence is situated anywhere in the peptide. In case the peptide consists of more than 7 a.a, wherein the continuous sequence is at one of the peptides ends, said positively charged a.a will be at the open end of the sequence (which is not connected to the additional a.a of the longer peptide) so that the large peptide comprises a positively charged a.a at one of its ends. Wherein the continuous sequence is in the middle of the peptide, the peptide comprises at least one additional positively charged a.a. at one of its ends in addition to the positively charged a.a of the continuous sequence. Analogues of the above peptides are also within the scope of the present invention. Such analogues are peptides which comprise no more than 10 a.a including at least 5 a.a which have the same sequence as one of the above mentioned continuous sequences but in which one or two a.a are conservatively replaced, as this term is defined below. The analogues also comprise at least one positively charged a.a at their end and essentially maintain the activity of the peptides as this term is defined below. The term “essentially maintains the binding characteristics” refers to a peptide which level of binding to the tested sample is at least 50%, preferably 70%, most preferably 90% or more than 100% of the level of binding of the peptide to the same tested sample as determined by the same binding assay. By a preferred embodiment, the invention provides a peptide which binds to a body fluid sample obtained from a schizophrenic patient at a substantively higher level than it binds to a body fluid sample obtained from a non-schizophrenic individual said peptide selected from the group consisting of: i. LVVGLCK (SEQ ID NO. 1) ii. KLVVGLC (SEQ ID NO. 2) iii. LVVGLMK (SEQ ID NO. 3) iv. KLVVGLM (SEQ ID NO. 4) or analogs of said peptides in which no more than two a.a are conservatively substituted, said analogues essentially maintaining the binding characteristics of the peptides. By a most preferred embodiment, a peptide is provided which binds to body fluid samples obtained from a schizophrenic patient substantively higher than it binds to a body fluid sample obtained from a non-schizophrenic individual having the amino acid sequence LVVGLCK (SEQ ID NO. 1). By an additional aspect of the invention, a peptide is provided which binds to a body sample obtained from a schizophrenic patient substantively higher than it binds to a sample obtained from a non-schizophrenic individual, wherein the peptide binds antibodies that are capable of specific binding to a peptide having the amino acid LVVGLCK. Several non limiting examples of such peptides are the following: i. KLVVGLC (SEQ ID NO. 2) ii. LVVGLMK (SEQ ID NO. 3) iii. KLVVGLM (SEQ ID NO. 4) or analogs thereof in which no more than two a.a are conservatively replaced and which maintain the binding characteristics of the peptides. The letters used above and throughout the present description to denote specific amino acids (a.a.) are in accordance with the one letter a.a. symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. In view of the fact that the peptides of the invention are the putative natural binding sites for autoantibodies found in elevated levels in body fluids of schizophrenic patients, and due to their high purity and high activity, these peptides are most useful for the diagnosis of schizophrenia. Thus, by an additional aspect, the present invention provides an assay for the diagnosis of schizophrenia in an individual comprising the following steps: (a) obtaining a body fluid sample from said individual being a blood sample, platelet-associated antibodies (PAA) containing fraction thereof, or a fraction containing PAA shed from the platelets; (b) contacting said sample with a peptide being no more than 10 amino acids (a.a.) long and comprising a continuous sequence of at least 5 amino acids included in any one of the following sequences: i. LVVGLCK (SEQ ID NO. 1) ii. KLVVGLC (SEQ ID NO. 2) iii. LVVGLMK (SEQ ID NO. 3) iv. KLVVGLM; (SEQ ID NO. 4) said continuous sequence consisting of at least one positively charged a.a. at one end of said sequence; and said peptide comprising at least one positively charged a.a at its end being the positively charged a.a of said continuous sequence or at least one additional positively charged a.a.; or analogs of said peptide being no more than 10 a.a long and in which no more than two a.a of said continuous sequence are conservatively substituted, said analogues essentially maintaining the binding characteristics of the peptide. (c) determining the level of binding of said peptide to said sample, a level of binding substantively higher than the level of binding of said peptide to a sample obtained from a non-schizophrenic individual indicating that said tested individual has a high likelihood of having schizophrenia. By a preferred embodiment, the peptide of step (b) is selected from the group consisting of: i. LVVGLCK (SEQ ID NO. 1) ii. KLVVGLC (SEQ ID NO. 2) iii. LVVGLMK (SEQ ID NO. 3) iv. KLVVGLM (SEQ ID NO. 4) or analogs thereof in which no more than two a.a are conservatively replaced and which essentially maintain said peptide's binding characteristics. By a preferred embodiment, the peptide of step (b) has the amino acid sequence LVVGLCK. By an additional aspect, the peptides in step (b) are such which bind antibodies which bind peptides having the amino acid sequence LVVGLCK or analogues thereof. Use of the peptides of the invention and analogues thereof as defined above and below for the preparation of a diagnostic composition for diagnosis of schizophrenia in an individual is also within the scope of the present invention. By an additional aspect, the invention provides a kit useful in the above assay, said kit comprising a support comprising one or more peptides of the invention immobilized onto it, an anti-human immunoglobulin (hIg) antibody or fragment thereof, or one or more non-based peptides conjugated to a detectable marker which bind to antibodies present in the tested sample, reagents required for carrying out the detection assay wherein said peptides bind to antibodies present in a tested sample as well as instructions for use. Wherein the detection of the binding of the peptides of the invention to the tested sample is by an anti-hIg antibody the anti-hIg antibody may be conjugated to a detectable marker or alternatively, the kit may also comprise a second type of antibodies directed against said first antibodies, wherein the second antibodies are conjugated to a detectable marker. By one embodiment, the binding of the peptide of the invention to the tested sample is detected using second non-bound peptides complexed with a detectable marker, said second peptides capable of binding to the antibodies present in the tested sample. In accordance with this embodiment, the detection is achieved by a double antigen sandwich text which may be performed as a one step assay or as a two step assay. Wherein the detection is performed by the double antigen sandwich text assay, the kit of the invention will include such peptides conjugated to a detectable marker instead of the anti-human immunoglobulin antibody. The assay of the invention may be used as a single test for detecting a high likelihood of schizophrenia in an individual. However, in accordance with an additional aspect of the Invention, the peptides and assay may be used as a confirmatory diagnostic tool. Thus, for example, wherein a high likelihood of schizophrenia is determined in an individual by methods used to date (mainly psychiatric evaluation, as mentioned above), this could be reaffirmed (or, alternatively, re-evaluated) by using the assay of the Invention. |
Spacer device |
A spacer unit inlet member integrally constructed of rigid or non-flexible material is described. This inlet member is capable of selectively mounting about its opening any one of a plurality of metered dose inhaler (MDI) actuators having different outlet size or shape. The inlet member comprises a channel having a wall substantially parallel to its rotational axis and surrounding or substantially coaxial to the opening of the inlet member. The peripheral edge of this channel is shaped substantially as a truncated oval having a major axis, a pair of curved sides, and a pair of opposite ends directed chordally of the oval and substantially perpendicular to the major axis of the oval. The inlet member further comprises a pair of opposing curved walls surrounding and substantially parallel to the rotational axis of the channel and having an outermost edge comprising a substantially oval shape. The inlet member also comprises a wall substantially perpendicular to the rotational axis of the channel and positioned between the walls of the channel and the opposing curved walls. |
1. A spacer unit inlet member integrally constructed of rigid or non-flexible material, said member capable of selectively mounting about its opening any one of a plurality of metered dose inhaler (MDI) actuators having different an outlet of size or shape, and comprising: (i) a channel having a wall substantially parallel to its rotational axis and surrounding or substantially coaxial to the opening of said inlet member, wherein a peripheral edge of said channel is shaped substantially as a truncated oval having a major axis and having a pair of curved sides and a pair of opposite ends directed chordally of said oval and substantially perpendicular to the major axis of said oval, (ii) a pair of opposing curved walls and substantially parallel to the rotational axis of said channel, and having an outermost edge comprising a substantially oval shape, and (iii) a wall substantially perpendicular to the rotational axis of said channel and positioned between said walls of said channel and said opposing curved walls. 2. The spacer according to claim 1, wherein one or more of external faces of said channel is/are capable of contacting an inner or outer wall of the outlet of the MDI actuator. 3. The spacer unit according to claim 1, wherein one or more of said opposing curved walls is/are capable of contacting an outer wall of the outlet of the MDI actuator. 4. The spacer unit according to claim 1, wherein the wall that is perpendicular to the rotational axis of the channel is also capable of contacting the peripheral edge of the outlet of the MDI actuator. 5. The spacer unit according to claim 1, wherein a sealing engagement is formed between one or more faces of the MDI actuator outlet and one or more of said walls (i) or (ii) or (iii), to prevent a leakage of a medicament. 6. The spacer unit comprising: (i) an inlet member integrally constructed of rigid or non-flexible material, said member capable of selectively mounting about its opening any one of a plurality of metered dose inhaler (MDI) actuators having different outlet size or shape, and comprising: (a) a channel having a wall substantially parallel to its rotational axis and surrounding or substantially coaxial to the opening of said inlet member wherein the peripheral edge of said channel is shaped substantially as a truncated oval having a major axis and having a pair of curved sides and a pair of opposite ends directed chordally of said oval and substantially perpendicular to the major axis of said oval; (b) a pair of opposing curved walls surrounding and substantially parallel to the rotational axis of said channel, and having an outermost edge comprising a substantially oval shape; and (c) a wall substantially perpendicular to the rotational axis of said channel and positioned between said walls of said channel and said opposing curved walls, and (ii) a spacer unit outlet member, and wherein said inlet member and said outlet member are locked together in sealing engagement to form an interior space for holding a medicament during use, and two openings to facilitate a flow of a medicament through the spacer unit. 7. The spacer unit according to claim 6, wherein the inlet member and the outlet member are snap-locked together. 8. The spacer unit according to claim 6, further comprising complimentary threaded portions provided at the edges of the inlet and outlet members for screwing the the inlet and outlet members together. 9. The spacer unit according to claim 6, wherein the spacer outlet member further comprises one or more one way valves, filters of baffles, to confer a unidirectional flow of air and medicament from the spacer unit to a patient. 10. The spacer unit according to claim 6, wherein the spacer outlet member is integrally constructed of rigid or non-flexible material. 11. The spacer unit according claim 6, wherein the spacer unit further comprises a three way conduit or separator element to facilitate an attachment of one or more incentive toy units. |
<SOH> BACKGROUND TO THE INVENTION <EOH>Inhalable medicaments, such as, for example, those for the prophylactic or therapeutic treatment of asthma or bronchitis, are commonly administered to patients using a metered dose inhaler (MDI). An MDI generally comprises a container housing the medicament, an axially extending vent tube from an internal valve, and a hollow actuator unit that houses the container and feeds the medicament to the patient via its outlet portion. The medicament is commonly packed in the container with a suitable propellant, such as, for example, a substance capable of forming a liquid under pressure and entering the gas phase at low pressure. In use, the patient brings the outlet of the actuator to his/her mouth, depresses the container relative to the actuator, thereby activating the internal valve to dispense a measured dose of medicament into the patient's mouth. In this arrangement, it is often necessary for the patient to coordinate his/her inhalation with the depression of the container to ensure that a sufficient dose of the medicament enters the patient's airway. The medicament will generally include a liquid/gas mixture of propellant, wherein small drops of medicament/propellant mixture enter the patient's airway with the medicament and rapidly projected particles of medicament/propellant mixture are deposited in the throat and mouth and are swallowed. Accordingly, the need to coordinate the dispensing of medicament with the patient's inhalation, and the dispensing of medicament/propellant mixture from the MDI, reduce the efficiency of treatment. Moreover, there is a need to allow the deceleration and dispersal of particles to minimize deposition in the throat and mouth. To enhance the efficacy of treatment, a spacer is commonly attached to the outlet of the actuator. Alternatively, the actuator is removed and the spacer is attached directly to the container via the vent tube. A spacer is a simple expansion chamber conveniently in the form of a small cylinder, conical or pear-shaped, into which a medicament that is dispensed from an MDI can be held prior to inhalation by a patient. In use, one end of a spacer is attached to the outlet of the MDI, and the other end of the spacer is received into the patient's mouth. A sealing engagement between the spacer and the MDI is required to minimize drug/medicament leakage, thereby ensuring that an adequate dose of medicament is received by the spacer unit. The patient depresses the container of the MDI relative to the actuator, thereby activating the internal valve to dispense a measured dose of medicament into the spacer. In a separate action to dispensing of the medicament from the MDI, the patient inhales air/medicament/propellant mixture from the spacer into his/her airway. Accordingly, the spacer provides an advantage in so far as there is no need for the patient to coordinate his/her inhalation with the depression of the container of the MDI. Additionally, the spacer facilitates the deceleration and dispersal of particles of the medicament into smaller particles, for efficient inhalation. As will be known to those skilled in the art, a rigid material, such as, for example, polycarbonate, is preferred for construction of a spacer. This is because such a rigid material confers strength and durability on the device. Additionally, polycarbonate is heat-resistant, facilitating steam sterilization and washing of the device in a dishwasher. Standard spacers are constructed with at least two separate pieces: (i) a hollow inlet member having an opening for attaching an MDI actuator to facilitate the flow of medicament from the MDI to the spacer unit, (ii) a hollow outlet member having an opening for attaching a mouthpiece or mask to facilitate delivery of the medicament to the patient, and often (iii) a separate barrel-shaped element between elements (i) and (ii). During assembly, the components are snap-locked; or screwed together in sealing engagement to form an interior space for holding the medicament during use, and two openings to facilitate the flow of a medicament through the assembled unit. Spacer devices are described in detail by Nowacki et al. in U.S. Pat. No. 4,470,412; and by InfaMed Limited in international Application No. PCT/AU99/00290. Notwithstanding the advantages of using a spacer, such devices do increase the costs associated with treatment relative to the cost of the MDI alone. Moreover, as drug companies generally provide their MDI with an actuator having an outlet of a particular shape, not all actuator outlets are capable of being in sealing engagement with all spacer units. Accordingly, it is highly desirable for a spacer unit to be universally adaptable to all MDI devices. One solution to this problem is to provide an “adaptor” or “back piece” that attaches to the end of the spacer and is capable of attaching to a plurality of actuator outlets. For example, the adaptor described in U.S. Pat. No. 5,848,588 (Trudell Medical International) comprises resilient, flexible material such as a rubber or the like, wherein concentric cylinders cover and grip the end of a cylindrical spacer, and a transverse membrane extends inwardly therefrom and is provided with a central opening, and straight and inwardly-directed ribs for receiving and gripping the outlet of the MDI. Pairs of the ribs have cross bracing, to control stretching of the diaphragm so that it provides a proper seal with an inserted MDI, whilst the radial inner ends of the ribs provide support for the MDI. Spacer devices that do not require a separate adaptor have been designed to fit most known MDI units, with varying success. Generally, the provision of a universally-adaptable spacer of rigid construction has been avoided because such a device would have been prone to breaking and/or cracking, during fitting to MDI outlets of different shapes. Occasionally, rigid spacer devices have been designed with prong-shaped protrusions to maximize flexure of the spacer to accommodate an MDI actuator. These protrusions are inherently brittle when made of a rigid plastic and are adaptable to very few types of actuators. Accordingly, known spacers that are usable with a plurality of MDI devices generally require prior removal of the MDI container from the manufacturer's actuator, and subsequent fitting of the container to the spacer unit. Poor sealing between the vent tube of the MDI and the spacer may result during such procedures. Alternatively, it is known to fit the spacer with a flexible inlet to accommodate various shaped MDI actuators. In fact, most conventional spacers that fit a plurality of different MDI actuators provide a flexible adaptor end that stretches or is compressed relative to the actuator. However, such an arrangement cannot be easily produced as an integral unit, because the spacer is generally made from rigid material, such as, for example, polycarbonate. This is a considerable disadvantage in terms of production of the device, because of the additional costs associated with producing separate pieces. Additionally, by providing a spacer in multiple pieces, with an additional rubber-like adaptor end piece, assembly of the device is made more complex, and requires additional effort either by the production team or the end user. |
<SOH> SUMMARY OF THE INVENTION <EOH>In work leading up to the present invention, the inventors sought to produce a cost-effective spacer unit that is capable of fitting a plurality of differently shaped MDI devices without the need for prior removal of the MDI canister from the actuator. The inventors realized that this object of the invention could be achieved by providing a spacer comprising a rigid material, such as that used in the manufacture of a conventional spacer device. To facilitate a reduction in production costs and time, the inventors produced such a spacer unit in as few as two separate pieces, each of said pieces being of an integral construction that could be produced, for example, from a single injection mold or by blow molding. Accordingly, one aspect of the present invention provides a spacer unit inlet member integrally constructed of rigid or non-flexible material, said member capable of selectively mounting about its opening any one of a plurality of metered dose inhaler (MDI) actuators having different outlet size or shape, and comprising: (i) a channel having a wall substantially parallel to its rotational axis and surrounding or substantially coaxial to the opening of said inlet member, wherein the peripheral edge of said channel is shaped substantially as a truncated oval having a major axis and having a pair of curved sides and a pair of opposite ends directed chordally of said oval and substantially perpendicular to the major axis of said oval; (ii) a pair of opposing curved walls surrounding and substantially parallel to the rotational axis of said channel, and having an outermost edge comprising a substantially oval shape; and (iii) a wall substantially perpendicular to the rotational axis of said channel and positioned between said walls of said channel and said opposing curved walls. In use, one or more of the external faces of said channel is/are capable of contacting the inner or outer wall of the outlet of an MDI actuator. Alternatively, or in addition, one or more of said opposing curved walls is/are capable of contacting the outer wall of the outlet of an MDI actuator. Alternatively, or in addition, the wall that is perpendicular to the rotational axis of the channel is also capable of contacting the peripheral edge of the outlet of an MDI actuator. The number and position of the contacts between the spacer inlet member and the MDI actuator outlet will depend upon the size and shape of the MDI actuator outlet, however al sealing engagement is formed between one or more faces of the MDI actuator outlet and one or more of said walls (i) or (ii) or (iii), to prevent the leakage of a medicament. A second aspect of the present invention provides a spacer unit comprising: (i) an inlet member integrally constructed of rigid or non-flexible material, said member capable of selectively mounting about its opening any one of a plurality of metered dose inhaler (MDI) actuators having different outlet size or shape, and including: (a) a channel having a wall substantially parallel to its rotational axis and surrounding or substantially coaxial to the opening of said inlet member, wherein the peripheral edge of said channel is shaped substantially as a truncated oval having a major axis and having a pair of curved sides and a pair of opposite ends directed chordally of said oval and substantially perpendicular to the major axis of said oval; (b) a pair of opposing curved walls surrounding and substantially parallel to the rotational axis of said channel, and having an outermost edge comprising a substantially oval shape; and (c) a wall substantially perpendicular to the rotational axis of said channel and positioned between said walls of said channel and said opposing curved walls, and (ii) a spacer unit outlet member is provided, and wherein said inlet member and said outlet member are locked together in sealing engagement to form an interior space for holding a medicament during use, and two openings to facilitate the flow of a medicament through the assembled unit. The invention will best be understood from the following description of preferred embodiments when taken in connection with the accompanying non-limiting drawings. |
Quantitative diagnostic analysis of hypertonia |
The invention relates to the application of the direct correlation between the overexpression or the functional molecular modification of human homologs of the sgk family and hypertension for quantitative diagnosis of a particular form of genetically determined hypertension. In particular the invention relates to the detection of a direct link between two different polymorphisms of individual nucleotides in the hsgk1 gene and the genetically determined predisposition to hypertension. The invention further relates to the provision of a diagnostic kit containing antibodies or polynucleotides for detecting the diagnostic targets hsgk1, hsgk2 and hsgk3. |
1. The use of the direct correlation between the overexpression or the functional molecular modification of human homologs of the sgk family and hypertension for quantitative diagnosis of a particular form of genetically determined hypertension. 2. Use according to claim 1, characterized in that the human homolog of the sgk family is the hsgk1 gene. 3. Use according to claim 2, characterized in that overexpression or functional modification is caused by the nucleotide polymorphism (SNP) in intron 6 (T→C) in the hsgk1 gene. 4. Use according to claim 2, characterized in that overexpression or functional modification is caused by the nucleotide polymorphism (SNP) in exon 8 (C→T) in the hsgk1 gene. 5. A kit for quantitative diagnosis of a particular form of the genetically determined form of hypertension, containing antibodies that are directed against the human homologs of the sgk protein family, or polynucleotides that can hybridize under stringent conditions with the human homologs of the sgk gene family, or these antibodies and polynucleotides jointly for quantitative determination of the overexpression or the functional molecular modification of these homologs. 6. A kit according to claim 5, characterized in that the human homolog of the sgk family is the hsgk1 gene. 7. A kit according to claim 6, characterized in that the antibodies are directed against a version of the hsgk1 protein mutated by an SNP or that the polynucleotides can hybridize under stringent conditions with a version of the hsgk1 gene mutated by an SNP. 8. A kit according to claim 7, characterized in that the polynucleotides can hybridize under stringent conditions with a version of the hsgk1 gene mutated by the SNP in intron 6 (T→C). 9. A kit according to claim 7, characterized in that the polynucleotides can hybridize under stringent conditions with a version of the hsgk1 gene mutated by the SNP in exon 8 (C→T). 10. A method of quantitative diagnosis of a particular form of the genetically determined form of hypertension, in which the overexpression of a human homolog of the sgk family or the functional molecular modification of these homologs is detected by the quantitative detection of the homologs in the patient's body sample with antibodies that are directed against the proteins of the homologs, or with polynucleotides that can hybridize with DNA or mRNA of the homologs under stringent conditions. 11. A method according to claim 10, characterized in that the human homolog of the sgk family is the hsgk1 gene. 12. A method according to claim 10, characterized in that the polynucleotides can hybridize with DNA or mRNA of a version of the SNP in intron 6 (T→C) in the hsgk1 gene under stringent conditions. 13. A method according to claim 10, characterized in that the polynucleotides can hybridize with DNA or mRNA of a version of the SNP in exon 8 (C→T) in the hsgk1 gene under stringent conditions. |
Shield for high-frequency transmitter/receiver systems of electronic devices, especially of devices for wireless telecommunication |
The invention relates to a shield for high-frequency transmitter/receiver systems of electronic devices, especially of devices for wireless telecommunication. In order to reduce the space requirements of such a high-frequency transmitter/receiver system (2′) on a circuit support element (1′) of an electronic device, a first partial circuit (20′) of the high-frequency transmitter/receiver system (2′) and a second partial circuit (21′) of the high-frequency transmitter/receiver system (2′) are disposed in a single shield chamber (32) in a substantially separate especially locally/partially separate manner. The shield chamber (32) is linked with a grounded area (10′) on the support element (1′) via an electroconducting connection (320) and the connecting element (320) is interposed between the two partial circuits (20′, 21′) on the support element (1′) in such a way that the twp partial circuits (20′, 21′) on the support element (1′) in such a way that the two partial (20′, 21′) do not interfere with each other functionally. |
1. Shielding for radio-frequency transceivers of electronic devices, particularly of wireless telecommunications devices, having the following features: (a) On a circuit substrate (1′) of the electronic device there are disposed under a single shielding chamber (32) a first sub-circuit (20′) of the radio-frequency transceiver (2′) and a second sub-circuit (21′) of the radio-frequency transceiver (2′) essentially separated from one another, in particular locally/spatially separated, (b) the shielding chamber (32) is connected on the substrate (1′) to a ground plane (10′) via an electrically conductive connecting element (320), (c) the connecting element (320) is disposed between the two sub-circuits (20′, 21′) on the substrate (1′) in such a way that the two sub-circuits (20′, 21′) do not interfere with one another functionally. 2. Shielding according to claim 1, wherein the connecting element (320) is an electrical component which can be inserted with the components of the sub-circuits (20′, 21′) on the substrate (1′). 3. Shielding according to claim 1, wherein the connecting element (320) is a spring. 4. Shielding according to claim 1, wherein the connecting element (320) is a dome-like structure. 5. Shielding according to claim 1 or 4, wherein the connecting element (320) and the shielding chamber (32) are made of the same material. 6. Shielding according to claim 5, wherein the material is low-impedance. 7. Shielding according to claim 5 or 6, wherein the material is nickel silver. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Electronic wireless telecommunications devices such as cordless telephones based on the DECT standard (Digital Enhanced Cordless Telecommunication) or mobile telephone handsets based on the GSM standard (Groupe Spéciale Mobile or Global System for Mobile Telecommunication) not only have circuits for signal processing in the low-frequency baseband but also have radio-frequency transceivers. These circuits and RF devices are usually mounted on a substrate, the so-called circuit board, in numerous process engineering and component insertion operations. In the course of electronic device miniaturization, films rather than circuit boards are increasingly being used as substrates. FIG. 1 shows, from known prior art, a substrate 1 with a ground plane 10 on which there is disposed a radio-frequency transceiver 2 connected to a wire antenna 3 , which is likewise disposed on the substrate 1 . The radio-frequency transceiver 2 contains two sub-circuits 20 , 21 ; a first sub-circuit 20 having, for example, an output power amplifier 200 , and a second sub-circuit 21 having, for example, oscillators 210 and synthesizers 211 . The two sub-circuits 20 , 21 , in a certain spatial arrangement, tend to produce mutual interference in respect of the RF fields but must not interfere with one another in terms of their respective functions. Radiated interference and interference from the environment likewise must be prevented. This scenario is schematically indicated in FIG. 1 by the arrows. The double-headed horizontal and vertical arrows indicate the electromagnetic fields emitted by the sub-circuits 20 , 21 which affect the sub-circuits 20 , 21 . The three-dimensionally represented arrow indicates the phenomenon of crosstalk from the first sub-circuit 20 to the second sub-circuit 21 , and vice versa. The remaining arrows indicate the presence of balancing currents in the substrate 1 and shielding chambers. In other words, the device must satisfy the requirements of internal and external electromagnetic compatibility (EMC). To achieve this, the sub-circuits 20 , 21 of the radio-frequency transceiver 2 on the substrate 1 are enclosed by at least two shielding chambers 30 , 31 in such a way that the individual sub-circuits are completely shielded (i.e.,, RF-proof), a first shielding chamber 30 being used as self-contained shielding for the first sub-circuit 20 , while a second shielding chamber 31 is used exclusively as shielding for the second sub-circuit 21 . In accordance with this prior art, there are provided between the sub-circuits 20 , 21 two continuous isolation barriers which prevent interference effects. An object of the present invention is to reduce the space requirement for the shielding of the radio-frequency transceiver on a circuit substrate of an electronic device, particularly of wireless telecommunications equipment. |
<SOH> SUMMARY OF THE INVENTION <EOH>The idea underlying the present invention is that there are disposed on a circuit substrate of an electronic device, under a single shielding chamber, a first sub-circuit of a radio-frequency transceiver and a second sub-circuit of the radio-frequency transceiver which are substantially (in particular, locally/spatially separate from one another. The shielding chamber is connected to a ground plane on the substrate via an electrically conductive connecting element and the conducting element is disposed between the two sub-circuits on the substrate in such a way that the two sub-circuits do not interfere functionally with one another. Because of the space saved on the circuit substrate by not having a second shielding chamber, it is possible, for example, to implement smaller electronic devices with radio-frequency transceivers than ever before. For electronic devices for wireless telecommunications, this results in such devices being able to be incorporated, for example, in a wristwatch. Additional features and advantages of the present invention are described in, and will be apparent from, the following Detailed Description of the Invention and the Figures. |
Processes for producing coated magnetic microparticles and uses thereof |
This invention relates generally to the field of production of coated magnetizable microparticles and uses thereof. In particular, the invention provides a process for producing coated magnetizable microparticles with active functional groups, which process uses, inter alia, conducting polymerization of said coating monomers on the surface of magnetic particle to form coated magnetizable microparticles with active functional groups in the presence of a coupling agent, coating monomers, a functionalization reagent, a cross-linking agent and an initiator in an organic solvent containing a surfactant. The coated magnetizable microparticles produced according to the present processes and uses of the coated magnetizable microparticles, e.g., in isolating and/or manipulating various moieties are also provided. |
1. A process for producing coated magnetizable microparticles with active functional groups, which process comprises: a) dispersing magnetizable microparticles with a diameter ranging from about 5 to about 1,000 nanometers in an organic solvent containing a surfactant; b) adding a coupling agent, coating monomers, a functionalization reagent, a cross-linking agent and an initiator to said organic solvent containing said dispersed magnetizable microparticles, allowing attachment of said coupling agent to the surface of said magnetizable microparticles and dispersing said coating monomers evenly on the surface of said magnetizable microparticles with said attached coupling agent; c) initiating and completing polymerization of said coating monomers to form coated magnetizable microparticles with active functional groups in the absence of oxygen, whereby polymers of said coating monomers are attached to the surface of said coated magnetizable microparticles via said coupling agent, multiple said polymers are crosslinked together via said cross-linking agent and said functionalization reagent is linked to said polymers, cross-linking agent and/or coupling agent so that at least one functional group of said functionalization reagent remains available. 2. The process of claim 1, wherein the magnetizable microparticles comprises a magnetizable substance selected from the group consisting of a paramagnetic substance, a ferromagnetic substance and a ferrimagentic substance. 3. The process of claim 2, wherein the magnetizable substance comprises a metal composition. 4. The process of claim 3, wherein the metal composition is a transition metal composition or an alloy thereof. 5. The process of claim 4, wherein the transition metal is selected from the group consisting of iron, nickel, copper, cobalt, manganese, tantalum, zirconium, nickel-iron alloy and cobalt-tantalum-zirconium (CoTaZr) alloy. 6. The process of claim 3, wherein the metal composition is a metal oxide. 7. The process of claim 1, wherein the organic solvent is selected from the group consisting of toluene, dimethylbenzene, tetratrahydrofuran and ethanol. 8. The process of claim 1, wherein the surfactant is an anionic, a nonionic surfactant or a cationic surfactant. 9. The process of claim 8, wherein the anionic surfactant is dodecyl sulfonic acid sodium salt or sodium dodecyl benzene sulfonate. 10. The process of claim 1, wherein the concentration of the surfactant in the organic solution ranges from about 0.1% (v/v) to about 5% (v/v). 11. The process of claim 1, wherein the magnetizable microparticles are dispersed in an organic solvent containing a surfactant under ultra-sonication and/or stirring. 12. The process of claim 1, wherein the coupling reagent is selected from the group consisting of bis(2-hydroxyethyl methacrylate) phosphate, bis(trimethylopropane diacrylate) phosphate, and bis(pentaerythritol triacrylate) phosphate 13. The process of claim 1, wherein the coating monomers are selected from the group consisting of acrylic acid, methacrylic acid, methyl methacrylate, 2-hydroxyethyl methacrylate, glycoldimethylcrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, diethylene glycol methacrylate, diethylene glycol acrylate, diethylene glycol dimethacrylate, diethylene glycol diacrylate, diethylene glycol methacrylate, diethylene glycol acrylate, triethylene glycol dimethacrylate, diethylene glycol diacrylate, trimethylopropane trimethacrylate, pentaerythritol triacrylate, styrene, dirinylbenzene and a mixture thereof. 14. The process of claim 1, wherein the functionalization reagent is selected from the group consisting of acrylic acid, methacrylic acid, glycidyl acrylate, pentaerythritol diacrylate and methacrolein. 15. The process of claim 1, wherein the cross-linking agent is selected from the group consisting of diethylene glycol dimethacrylate, diethylene glycol diacrylate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylopropane trimethacrylate, pentaerythritol triacrylate and dirinylbenzene. 16. The process of claim 1, wherein the initiator is benzoyl peroxide or 2,2′-azobisisobutyronitrile. 17. The process of claim 1, wherein the ratio between the sum of the coupling agent, the coating monomers, the functionalization reagent and the cross-linking agent versus the magnetizable microparticles ranges from about 1:400 (w/w) to about 1:1 (w/w). 18. The process of claim 1, wherein the percentages of coating monomers, coupling agent, cross-linking agent, functionalization reagent and initiator in the sum of the substances are: from about 0% to about 80% (v/v) coating monomers, from about 1% to about 10% (v/v) coupling agent, from about 10% to about 80% (v/v) cross-linking agent, from about 5% to about 40% (v/v) functionalization reagent and from about 1% to about 5% (w/v) initiator. 19. The process of claim 1, wherein the coating monomers are evenly dispersed on the surface of the magnetic microparticles and the attached coupling agent under stirring. 20. The process of claim 19, wherein the stirring lasts at least 30 minutes. 21. The process of claim 1, wherein the absence of oxygen in step c) is effected via purging air with an inert gas. 22. The process of claim 21, wherein the inert gas is nitrogen, helium or argon. 23. The process of claim 1, wherein the polymerization is initiated by heating the initiator to release free radicals. 24. The process of claim 23, wherein the initiator is heated to a temperature ranging from about 25° C. to about 150° C. 25. The process of claim 24, wherein the initiator is heated to about 80° C. 26. The process of claim 19, wherein the stirring magnitude is lowered prior to the initiation of the polymerization. 27. The process of claim 26, wherein the stirring magnitude is lowered to 30 rpm or less than 30 rpm. 28. The process of claim 1, wherein the polymerization is allowed to proceed for at least 2 hours. 29. The process of claim 1, further comprising removing non-magnetizable microparticles from the reaction mixture. 30. The process of claim 29, wherein the non-magnetizable microparticles are removed from the reaction mixture by depositing magnetizable microparticles under magnetic stirring and removing supernatant. 31. The process of claim 29, further comprising removing non-polymerized substances from the magnetizable microparticles. 32. The process of claim 31, wherein the non-polymerized substances are removed from the magnetizable microparticles via washing and filtration. 33. The process of claim 32, wherein the non-polymerized substances are removed from the magnetizable microparticles via washing the magnetizable microparticles with deionized water and acetone. 34. Coated magnetizable microparticles with active functional groups, which are produced according to the process of claim 1. 35. The coated magnetizable microparticles of claim 34, which have a mean diameter from about 10 nanometers to about 2 micrometers. 36. The coated magnetizable microparticles of claim 34, which comprises a nuclei selected from the group consisting of ferrite oxide, cobalt oxide and nickel oxide. 37. The coated magnetizable microparticles of claim 34, which further comprises a binding partner that is capable of binding to a moiety. 38. The coated magnetizable microparticles of claim 34, wherein the binding partner is capable of specifically binding to a moiety. 39. The coated magnetizable microparticles of claim 34, wherein the binding partner is an antibody or a nucleotide sequence. 40. The coated magnetizable microparticles of claim 34, wherein the binding partner is selected from the group consisting of a cell, cellular organelle, virus, molecule and an aggregate or complex thereof. 41. A method for isolating a moiety, which method comprises: a) providing coated magnetizable microparticles of claim 34 comprising a binding partner that is capable of binding to a moiety to be isolated; b) contacting a sample containing or suspected of containing of said moiety with said coated magnetizable microparticles provided in step a) under conditions allowing binding between said moiety and said binding partner; and c) recovering said coated magnetizable microparticles from said sample with a magnetic force. 42. The method of claim 41, further comprising recovering the moiety from the coated magnetizable microparticles. 43. The method of claim 41, wherein the isolation is conducted on a chip. 44. The method of claim 41, wherein the isolation is conducted in a liquid container selected from the group consisting of a beaker, a flask, a cylinder, a test tube, an eppindorf tube, a centrifugation tube, a culture dish, a multiwell plate and a filter membrane. 45. A method for manipulating a moiety, which method comprises: a) providing coated magnetizable microparticles of claim 34 comprising a binding partner that is capable of binding to a moiety to be manipulated; b) coupling said moiety to said coated magnetizable microparticles provided in step a) via binding between said moiety and said binding partner to form a moiety-coated-magnetizable-microparticles complex; and c) manipulating said moiety-coated-magnetizable-microparticles complex with a magnetic force, thereby said moiety is manipulated. 46. The method of claim 45, further comprising recovering the moiety from the coated magnetizable microparticles. 47. The method of claim 45, wherein the manipulation is conducted on a chip. 48. The method of claim 45, wherein the manipulation is conducted in a liquid container selected from the group consisting of a beaker, a flask, a cylinder, a test tube, an eppindorf tube, a centrifugation tube, a culture dish, a multiwell plate and a filter membrane. 49. The method of claim 45, wherein the manipulation is selected from the group consisting of transportation, focusing, enrichment, concentration, aggregation, trapping, repulsion, levitation, separation, fractionation, isolation and linear or other directed motion of the moiety. 50. A kit for isolating or manipulating a moiety, which kit comprises: a) coated magnetizable microparticles of claim 34 comprising a binding partner that is capable of binding to a moiety to be isolated or manipulated; and b) instruction(s) for using said coated magnetizable microparticles to isolate or manipulate said moiety. |
<SOH> BACKGROUND ART <EOH>It is important and often crucial to separate, purify and test proteins (such as various antigens, antibodies and enzymes), nucleic acids and cells from a sample in biology, clinical medicine and many other fields. The separation and purification of biological materials can sometimes take up to 90% of time and cost of a procedure or task. Present separation and purification methods include precipitation, centrifugation, and chromatography to separate genomic DNA. However, some of them need expensive instruments; some of them have poor separation and purification results in spite of simple operations; and some of them are complicated and time-consuming. The current demands for sample preparation procedures include: automation and miniaturization, not using or using poisonous or toxic reagents as little as possible, rapid and efficient process, resulting prepared products suitable for subsequent manipulation, e.g., biochemical manipulation; and cost effective. In recent decades, magnetic particles have been used in the purification and separation of biological materials. As a relatively new type of functional polymeric materials, magnetic micro-beads have great potential applications in biomedicine (clinical diagnosis, immunization analysis, enzyme label, target drug, etc.), cytology (cell labels, cell separation, etc.), molecular biology (cDNA library, gene sequencing, extraction and hybridization of DNA and mRNA, etc.), biological engineering and burgeoning bio-chip technology. Compared with conventional separation methods, the novel separation technology using magnetic micro-beads does not need large expensive instruments, requires simple and rapid experimental procedure and has high separation efficiency. Up to now the commonly used magnetic beads in biology are micron-sized particles that are mainly used in cell separation. For example, the magnetic micro-beads bound with lectin can be used to separate T cells in medulla. However, the study on the magnetic nano-beads and their directly coating and functionalizing is still relatively rare. The separation technique using magnetic nano-beads has wide application potential. Because of the excellent soft magnetic characteristics, magnetic nano-beads can be used as targeted therapeutic agents, e.g., can be guided to a desired pathological locus by a magnetic force and effecting its therapeutic effect via a therapeutic agent carried by the magnetic nano-beads. The magnetic micro-beads coated with functional groups comprise inner magnetic micro-crystals and surface coating polymer. Certain preparation methods for coat and functionalized magnetic micro-beads are known. One such known method is to prepare magnetic iron oxide particles first and then coat the prepared magnetic iron oxide particles. However, the polarity of the inner magnetic microcrystals is strong and it is difficult to coat the inner magnetic microcrystals with a polymer layer having a weak polarity. During the coating process, the monomer can polymerize in solution instead of on the surface of the magnetic micro-beads. The nano-particles may also aggregate with each other during the coating process. Current coating methods include physical method and chemical method. The embedding method is commonly used in physical coating. In the embedding process, the magnetic micro-beads are dispersed in a polymer solution. Then solvent is removed through pulverization, flocculation, aggradation and evaporation to obtain the polymer-coated magnetic micro-beads. However, the binding between the inner magnetic microcrystals and the outer layer of the coated magnetic micro-beads, mainly via Van der Walls force, is weak and the polymer layer can be broken off easily. The coated micro-beads have wide diameter distribution and irregular shapes. For example, magnetic crystals can be dispersed directly in aminodextron, polymethacrylic acid or the lecithin solution. Then the magnetic crystals are coated with polymer through physical adsorption. This method can not be used to coat the magnetic micro-beads smaller than 100 nanometers because of particle aggregation. The suspension-polymerization is commonly used in the chemical method. In this process, the monomers polymerize on the surface of the magnetic micro-beads. Silanization of the magnetic micro-beads' surface is mainly used to make the polymerization on the surface viable. But the coating process must be performed step by step and is complicated. And in the process, HCl gas is produced and will corrupt the micro-beads. Because the diameter of the nano-crystals is small and surface energy is high, the nano-crystals will be easily corrupted. As a result, the surface can be broken and the diameter can be changed. So the silanization of the surface is not suitable coating and functionalization of the nano-crystals. There exists a need in the art for a new process for producing coated magnetizable microparticles. This invention address this and other related needs in the art. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>FIG. 1 illustrates the magnetization hysteresis loops of coated Fe 3 O 4 magnetic nanoparticles as described in example 1. FIG. 2 illustrates the FT-IR spectra of coated Fe 3 O 4 magnetic nanoparticles as described in example 1. FIG. 3 illustrates, in a magnified view of the enrichment of the coated magnetizable microparticles when the electromagnetic pole is electrified as described in example 6. FIG. 4 illustrates an exemplary agarose gel electrophoresis of genomic DNA isolated using the present coated magnetizable microparticles in different ionic, pH and solvent conditions as described in example 8. Lanes 1: 100 μl NaI+100 μl isopropanol; 2: 50 μl NaI+150 μl isopropanol; 3: 150 μl NaI+50 μl isopropanol; 4: 100 μl NaI+100 μl ethanol; 5: 100 μl NaCl+100 μl ethanol; 6: NaI+200 μl PEG; 7: NaI+200 μl PEG+100 μl ethanol; 8: 100 μl NaCl+100 μl isopropanol; 9: 100 μl NaI+100 μl methanol; 10: NaI+100 μl urea+100 μl isopropanol; 11: 100 μl SDS+100 μl ethanol; 12: 100 μl SDS+100 μl methanol; 13: NaI+100 μl urea+100 μl ethanol; 14: NaI+100 l urea+100 μl methanol; and M: λDNA markers (Hind III single digestion). FIG. 5 illustrates an exemplary agarose gel electrophoresis of genomic DNA isolated from E. coli using different coated magnetizable microparticles as described in example 9. Lanes 1: uncoated magnetizable microparticles; 2: uncoated magnetizable microparticles; 3: magnetizable microparticles coated with polystyrene; 4: magnetizable microparticles coated with methyl methacrylate; 5: magnetizable microparticles coated with glucosamine; 6: magnetizable microparticles coated with methacrylic acid; L: phenol-chloroform isolated DNA; and M: λDNA markers (Hind III single digestion). detailed-description description="Detailed Description" end="lead"? |
Access networks |
An access network is built using Ethernet or IEEE 802.3 technology. The network comprises a plurality of terminals, a hierarchy of concentrator stages and a DHCP server. On startup of the terminals, DHCP discover messages are sent to the server which include the terminals' MAC addresses. These addresses are cached at the concentrators against the ports on which they are received. Thus, unknown MAC addresses are only sent upstream. To avoid the network being flooded with broadcast messages, any time a client PC uses ARP to find the MAC address of any other client, the central server provides a proxy ARP function. |
1-24. (canceled) 25. A method of routing data in an access network including a server, at least one concentrator coupled to the server via an upstream port of the at least one concentrator, and a plurality of terminals coupled to the at least one concentrator via at least one downstream port of the concentrator, the method comprising the steps of: a) sending a unique terminal address for each terminal from the terminal to the server via the at least one concentrator; b) storing the unique terminal address at the at least one concentrator; c) routing future data addressed to a given destination terminal according to the unique terminal address for that terminal stored at the at least one concentrator; and d) if the destination of the data is connected via a downstream port, sending the data to that port, and no other. 26. The method according to claim 25, including the step of, if the data is received via a downstream port and the destination of the data is not connected via a downstream port, sending the data to the upstream port. 27. The method according to claim 25, including the step of, if the data is received via the upstream port and the destination of the data is not connected via a downstream port, discarding the data. 28. The method according to claim 25, wherein broadcast data are only sent upstream and never downstream. 29. The method according to claim 25, wherein the step of sending the unique terminal address comprises sending a media access control (MAC) address of each terminal. 30. The method according to claim 25, wherein the server is a dynamic host configuration protocol (DHCP) server, and the step of sending the unique terminal address to the server comprises sending a DHCP discover message to the server, the DHCP discover message containing the unique terminal address. 31. The method according to claim 25, wherein the step of storing the unique terminal addresses at the at least one concentrator comprises storing the terminal addresses against the port of the at least one concentrator from which they are received. 32. The method according to claim 25, wherein each of the terminals, the server and the at least one concentrator has a timeout period for stored entries, and comprising the step of setting the timeout of the terminals addresses to a timeout shorter than that of a store for the at least one concentrator or the server. 33. The method according to claim 25, including the steps of sending an address resolution protocol (ARP) broadcast message from a terminal to the at least one concentrator, and routing the ARP broadcast message to the server. 34. The method according to claim 33, wherein the server sends out the unique terminal address of a terminal identified in an ARP request to a requesting terminal. 35. The method according to claim 33, wherein the server forwards an ARP request as a unicast message to the unique terminal address of the terminal identified in the ARP request. 36. An access network, comprising: a) a server; b) at least one concentrator coupled to the server via an upstream port of the at least one concentrator; c) a plurality of terminals coupled to the at least one concentrator via at least one downstream port of the at least one concentrator; d) each of the terminals including means for sending a unique terminal address for that terminal to the server via the at least one concentrator; and e) the at least one concentrator including a store for storing the unique terminal addresses, and including means for sending data for which the destination is connected via a downstream port to that port, and no other. 37. The access network according to claim 36, wherein the at least one concentrator includes means for sending data received via a downstream port, and for which the destination is not connected via a downstream port, to the upstream port. 38. The access network according to claim 36, wherein the at least one concentrator includes means for discarding data received via the upstream port and for which the destination is not connected via a downstream port. 39. The access network according to claim 36, wherein the at least one concentrator includes means for sending broadcast data upstream and never downstream. 40. The access network according to claim 36, wherein the unique terminal address sending means at each terminal includes means for sending a media access control (MAC) address of that terminal. 41. The access network according to claim 36, wherein the server is a dynamic host configuration protocol (DHCP) server, and the means for sending the unique terminal address to the DHCP server at each terminal includes means for sending a DHCP discover message to the DHCP server, the DHCP discover message containing the unique terminal address. 42. The access network according to claim 36, wherein the concentrator store stores the unique terminal addresses against the ports on which they were received from the terminals. 43. The access network according to claim 36, wherein each of the terminals, the server and the concentrator store includes a timeout for stored entries, and wherein the timeout of the terminals is set to a time shorter than the timeout of the server or the concentrator store. 44. The access network according to claim 36, wherein the terminals include means for broadcasting an address resolution protocol (ARP) message to the server via the at least one concentrator. 45. The access network according to claim 44, wherein the server comprises means for sending out the unique terminal address of the terminal identified in an ARP request to a requesting terminal. 46. The access network according to claim 44, wherein the server includes means for routing an ARP request to the terminal identified in the ARP request. 47. The access network according to claim 36, wherein the network is an Ethernet or IEEE 802.3 network. 48. The access network according to claim 36, wherein the network comprises a plurality of concentrators arranged between the server and the terminals, a first concentrator being connected between the server and further concentrators, and the further concentrators being connected either to the terminals or indirectly to the terminals via one or more further concentrators. |
Adp-ribosylating bacterial toxins |
ADP-ribosylating toxins from Neisseria meningitidis, Streptomyces coelicolor, Mycoplasma pneumoniae, Salmonella typhimurium Salmonella paratyphi, and Streptococcus pyogenes are disclosed, together with mutant toxins and uses therefor. There is only a low level of sequence identity between these toxins and toxins such as cholera toxin and E. coli heat labile toxin. |
1-26. (canceled) 27. An ADP-ribosylating polypeptide comprising an amino acid sequence having greater than 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:1, 3, 4, 5, 6 or 7 or a functional fragment thereof of at least 7 amino acids in length. 28. The polypeptide of claim 27, further comprising one or more amino acid mutations, wherein the sites for mutations are shown in column 2 of Table 1 and the mutation is shown in the corresponding row of column 3 of Table 1. 29. The polypeptide of claim 28, wherein ADP-ribosylating activity of the polypeptide is reduced or eliminated. 30. The polypeptide of claim 28 comprising the amino acid sequence of SEQ ID NOs:10, 12, 14 or 16. 31. An isolated polynucleotide encoding the polypeptide of claim 27. 32. An isolated polynucleotide encoding the polypeptide of claim 28. 33. An isolated polynucleotide sequence comprising the sequence set forth in SEQ ID NOs: 2, 8, 11, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 or fragments thereof of at least 10 nucleotides in length. 34. A vector comprising the polynucleotide of claim 31. 35. A host cell comprising the polynucleotide of claim 31. 36. An immunogenic composition comprising the polypeptide of claim 27. 37. The immunogenic composition of claim 36, further comprising one or more antigens. 38. An immunogenic composition comprising an adjuvant comprising the polypeptide of claim 28 and an antigen. 39. An antibody which binds to the polypeptide of claim 27. 40. An antibody which binds to the polypeptide of claim 28. 41. A method of generating an immune response in a subject comprising administering a composition according to claim 36. 42. The method of claim 41, wherein the immune response is generated against a bacterial pathogen selected from the group consisting of Neisseria meningitidis, Streptomyces coilicolor, Mycoplasma pneumoniae, Salmonella typhimurium, Salmonella paratyphi, or Streptococcus pyogenes. 43. A method of generating an immune response in a subject comprising administering a composition according to claim 38 to the subject. 44. The method of claim 43, wherein the composition is administered mucosally. |
<SOH> BACKGROUND ART <EOH>ADP-ribosylating bacterial exotoxins are widely known. Examples include diphtheria toxin ( Corynebacterium diphtheriae ), exotoxin A ( Pseudomonas aeruginosa ), cholera toxin (CT; Vibrio cholerae ), heat-labile enterotoxin (LT; E. coli ) and pertussis toxin (PT). The toxins catalyse the transfer of an ADP-ribose unit from NAD + to a target protein. CT, for instance, transfers ADP-ribose to a specific arginine side chain of the α subunit of G S , which blocks the ability of G S to hydrolyse GTP to GDP. This locks the protein in its ‘active’ form, so adenylate cyclase activity is permanently activated. Cellullar cAMP levels rise, leading to the active transport of ions from the cell and the loss of water into the gut [1]. The toxins are typically divided into two functionally distinct domains—A and B. The A subunit is responsible for the toxic enzymatic activity, whereas the B subunit is responsible for cellular binding. The subunits might be domains on the same polypeptide chain, or might be separate polypeptide chains. The subunits may themselves be oligomers e.g. the A subunit of CT consists of A 1 and A 2 which are linked by a disulphide bond, and its B subunit is a homopentamer. Typically, initial contact with a target cell is mediated by the B subunit and then subunit A alone enters the cell. Crystal structures [2] are known for LT [3], CT [4] and PT [5]. The toxins are typically immunogenic, and have been proposed for use in acellular vaccines. One problem, however, is that the proteins retain their toxic activity in the vaccines. To avoid this problem, site-directed mutagenesis of key active site residues has been used to remove toxic enzymatic activity whilst retaining immunogenicity [e.g. refs. 6 (CT and LT), 7 (PT), 8 etc.]. Current acellular whooping cough vaccines include a form of pertussis toxin with two amino acid substitutions (Arg 9 →Lys and Glu 129 →Gly; ‘PT-9K/129G’ [9]). As well as their immunogenic properties, the toxins have been used as adjuvants. Parenteral adjuvanticity was first observed in 1972 [10] and mucosal adjuvanticity in 1984 [11]. It was surprisingly found in 1993 that the detoxified forms of the toxins retain adjuvanticity [12]. It is an object of the invention to provide further ADP-ribosylating bacterial toxins. |
<SOH> BRIEF DESCRIPTION OF DRAWINGS <EOH>FIG. 1 shows an alignment of catalytic domains of various bacterial toxins, including the N. meningitidis toxin of the invention (NMB1343). Residues important for catalytic activity are shown enlarged. FIG. 2 shows a multiple sequence alignment of conserved regions of LT and toxins of the invention. Residues important for catalytic activity are underlined. Residues important for the conservation of structure have a shaded background. Other conserved residues are indicated in bold. FIG. 3 shows the incorporation of radio-labelled NAD into the N. meningitidis toxin. In FIG. 3B , the lanes are: (L1) 95° C.; (L2) Novobiocin, 5 mMol; (L3) GTP, 10 mMol; (L4) ATP, 10 mMol; (L5) ADP-ribose, 10 mmol; (L6) Nicotinamide (NAM), 10 mMol; (L7) control. The arrow shows the position of the toxin. detailed-description description="Detailed Description" end="lead"? |
Electrical power source |
An electric energy source is made by means of a plate capacitor in interposing a set of plasma tubes between the plates. The assembly is subjected to cycles for the charging of the capacitor. These cycles comprise the excitation and the de-excitation of the gas of the plasma tubes. It is shown that the device has an efficiency of more than one. |
1. An electric energy source comprising: a capacitor with at least two metal plates facing each other and connected to two terminals of the source, and means to charge this capacitor at a high voltage, wherein the means to charge this metal plate capacitor at high voltage comprise: a set of plasma plates positioned so as to be facing these metal plates, these plasma plates being connected to a switch circuit or selector switch circuit to periodically form a set of at least two series-connected capacitors each comprising a metal plate and a plasma plate. 2. A source according to claim 1, wherein: the plasma plates comprise a hollow cylindrical ring and a hollow tube forming a hollow mast inside the cylindrical ring. 3. A source according to claim 2, wherein: the metal plates are formed by films placed flat against the ring and the mast. 4. A source according to one of the claims 1 to 3, wherein connections for linking metal plates or plasma plates form a Faraday cage of the source with the plates. 5. An electric energy source comprising: a capacitor with two plates connected to two terminals of the source, a conduction device interposed between the two plates, comprising: a switch circuit or selector switch circuit to make the conduction device conductive or non-conductive. 6- A source according to one of the claims 1 to 4, wherein: the conduction device and/or the plasma plates comprise a gas contained in a chamber, the switch circuit or selector switch circuit to make the device conductive comprises a circuit to excite the gas and convert it into plasma. 7- A source according to one of the claims 1 to 5, wherein: the circuit to excite the gas comprises a set of electrodes, an electric power supply and a circuit to periodically apply an electrical power supply voltage to the electrodes. 8- A source according to one of the claims 1 to 6, wherein the circuit to excite the gases comprises a set of metal plates, an electric power supply and a circuit to periodically apply a voltage to the plasma plates by induction. 9- A source according to one of the claims 6 to 7, wherein the frequency of periodic application is greater than or equal to 1 kHz. 10- A source according to one of the claims 1 to 8, comprising: a circuit for the charging and a circuit for the discharging of the plate capacitor, the charging circuit comprises a direct-current electrical power supply insulated from the discharging circuit by a one-way electric valve in series, preferably a set of diodes placed on either side of the supply. 11- A source according to one of the claims 1 to 9, comprising: a circuit for discharging the plate capacitor, the discharging circuit comprises a spark gap in series with a resistive load. 12- A source according to one of the claims 1 to 10, wherein the switch circuit or selector switch circuit to make the conduction device conductive or non-conductive comprises a switched voltage generator. 13- A source according to claim 11, wherein the switched voltage generator comprises a circuit to be switched over periodically during one or more cycles after the capacitor has been charged. 14- A source according to claim 12, wherein the switched voltage generator comprises a circuit to disconnect a continuous source for the charging of the capacitor after having charged said capacitor. 15- A source according to one of the claims 12 to 13, wherein the switched voltage generator is a variable frequency generator. 16- A source according to claim 14, wherein the variable frequency of the generator is adjusted as a function of the value of a resistive load connected to the terminals of the source. 17- A source according to one of the claims 1 to 15, wherein: the conduction circuit comprises glass or ceramic tubes, preferably doped with barium titanate. 18- A source according to one of the claims 1 to 16, wherein the gas is argon or any other gas or a mixture of rare gases. |
<SOH> BACKGROUND OF THE INVENTION <EOH>An object of the invention is an electric energy source. The aim of the invention is to propose a source of electric energy, i.e. a generator, of exceptional efficiency. The generator is of the type using discharge capacitors, especially repetitive discharge capacitors. The efficiency depends on the discharging frequency of the capacitor and on the number of charging cycles performed. The energy source of the invention is designed to be fitted into fixed or moving apparatuses, as the generator is easily transportable and is also autonomous. To understand the mode of operation of this invention, a few well-known principles of classical physics need to be recalled. If a metal plate capacitor is charged by means of a voltage source, and if the metal plates are moved away from each other after the capacitor has been disconnected from its source through a switch, there is an increase in voltage at the terminals of the capacitor resulting from the law of conservation of charge Q=CV. This operation can be performed symmetrically through the use (as shown in FIG. 1 ) of an assembly of two capacitors CP 1 and CP 2 . The two capacitors CP 1 and CP 2 are plate capacitors. They are series-connected by means of an electric connection. These capacitors CP 1 and CP 2 have external plates facing the outside of the assembly, and internal plates facing the inside of the assembly. The internal plates of the two capacitors are connected to each other electrically by the electric connection. The external plates are fixed and are located at a great distance from each other when compared with the distance between the internal plates and the external plates in each capacitor. A switch S 1 connects the external plates conditionally to a direct-current power supply HT 1 . The internal plates are moveable. When they are removed, after the switch S 1 has been opened, there is an increase in electrostatic energy that is localized in the capacitor formed by the remaining external electrodes. The system is therefore an energy multiplier. This increase in energy is given by the work of the observer who performs the maneuver of removing the internal plates. It is known that the law of conservation of energy is met since the electrostatic forces verify Newton's third principle. Consequently, the efficiency of the operation cannot exceed 100%. The operation of removing the plates can be done rotationally by means of an electric motor as described in the document U.S. Pat. No. 4,127,804, by Breaux, published on 28 Nov., 1978. In this document, it is sought to minimize the mechanical work by taking capacitors in which the position of the internal plate is offset by 90 degrees. A scheme of this kind does not totally eliminate the resistant electrostatic forces and the gain is obtained to the detriment of the multiplication of energy in the capacitors since the capacitance of each capacitor at the initial point in time is no longer equal. In physics, there are two types of capacitors: capacitors of a first type, which are capacitors with total influence like the spherical capacitor, and capacitors of a second type with partial influence like the plate capacitor. The document by Hiddink, U.S. Pat. No. 4,095,162, published on 13 Jun. 1978, describes a capacitor of the first type in which the internal electrode of the spherical capacitor is replaced by a plasma chamber in order to increase the potential of the external electrode. According to the authors of this document, the charge carried to the surface of the external electrode is small or negligible. Tests based on this approach have not given the conclusive results that were proclaimed. In the invention, to increase efficiency, the structure of the Breaux document has been modified by replacing the internal plates by two plasma chambers bonded to the interior of the external faces of a plane capacitor of the second type. As a consequence, the internal metal plates of the two series-mounted capacitors of FIG. 1 are replaced by chambers containing a gas that can be ionized by applying a high voltage. As a variant, a single plasma chamber extends from one internal plate to the other, setting up an electric connection at the same time. A second configuration using four plasma chambers can be envisaged. The second configuration simply increases the output energy from the system by a factor of two. Further below, we shall how this structure enables a reduction in the work to be furnished in order to charge the external plates and hence increase the efficiency exceptionally. |
<SOH> SUMMARY OF THE INVENTION <EOH>An object of the invention therefore is an electric energy source comprising: a capacitor with two plates connected to two terminals of the source, a conduction device interposed between the two plates, wherein the source comprises: a circuit to make the conduction device conductive or non-conductive. For its first charge, the two-plate capacitor may be connected to a DC voltage source. An object of the invention is also an electric energy source comprising: a capacitor with at least two metal plates facing each other and connected to two terminals of the source, and means to charge this capacitor at high voltage, wherein the means to charge this metal plate capacitor at high voltage comprise: a set of plasma plates positioned so as to be facing the metal plates, these plasma plates being connected to a switch or selector switch circuit to periodically form a set of at least two series-connected capacitors each comprising a metal plate and a plasma plate. |
Well device for throttle regulation of inflowing fluids |
A flow arrangement (10, 12) for use in a well through one or more underground reservoirs, and where the arrangement (10, 12) is designed to throttle radially inflowing reservoir fluids produced through an inflow portion of the production tubing in the well, the production tubing in and along this inflow portion being provided with one or more arrangements (10, 12). Such an arrangement (10, 12) is designed to effect a relatively stable and predictable fluid pressure drop at any stable fluid flow rate in the course of the production period of the well, and where said fluid pressure drop will exhibit the smallest possible degree of susceptibility to influence by differences in the viscosity and/or any changes in the viscosity of the inflowing reservoir fluids during the production period. Such a fluid pressure drop is obtained by the arrangement (10, 12) comprising among other things one or more short, removable and replaceable flow restrictions such as nozzle inserts (44, 62), and where the individual flow restriction may be given the desired cross section of flow, through which reservoir fluids may flow and be throttled, or the flow restriction may be a sealing plug. |
1. A flow control device (10, 20) for a well penetrating at least one underground reservoir and being provided with a production tubing having an inflow portion through which fluids from the at least one reservoir are produced, and one or more positions along the inflow portion of the production tubing being provided with a flow control device (10, 20) comprising a flow channel through which said fluids may flow, said flow channel consisting of an annular cavity (38, 52, 64, 72) formed between an external housing (40, 54, 68) and a base pipe (16) and an inlet (26, 36) in one end of the cavity (38, 52, 64, 72), the housing (40, 54, 68) forming an impermeable wall, and the base pipe (16) forming a main constituent of a tubing length (14) of the production tubing, a downstream end of said flow channel comprising at least one through-going wall opening in the base pipe (16), the flow channel thereby connecting the inside of the base pipe (16) with the at least one reservoir, and said flow channel having at least one through-going channel opening (42, 60) provided with a flow restriction, characterised in that each channel opening (42, 60) is provided with a flow restriction selected from the following types of flow restrictions: a nozzle; an orifice in the form of a slit or a hole; or a sealing plug. 2. The flow control device (10, 20) according to claim 1, characterised in that the at least one flow restriction is formed into a removable and replaceable insert (44, 62) that is placed in mating formation in said channel opening (42, 60). 3. The flow control device (10, 20) according to claim 2, characterised in that the device (10, 20), when comprising several removable and replaceable inserts (44, 62), is provided with inserts (44, 62) of identical external size and shape. 4. The flow control device (10, 20) according to claim 2, characterised in that the at least one insert (44, 62) is externally circular, and that the corresponding channel opening (42, 60) is a complementary insert bore. 5. The flow control device (10, 20) according to claim 3, characterised in that the inserts (44, 62) are externally circular, and that the corresponding channel openings (42, 60) are complementary insert bores. 6. The flow control device (10, 20) according to claim 2, characterised in that said flow channel, when comprising more than one channel opening (42, 60), is provided with inserts (44, 62) containing different types of flow restrictions of said types. 7. The flow control device (10, 20) according to claim 5, characterised in that said flow channel is provided with inserts (44, 62) formed from different types of flow restrictions of said types, thereby allowing customised configuration of flow restrictions in the flow channel, thus enabling customised flow rate control of said inflowing fluids. 8. The flow control device (10, 20) according to claim 1, characterised in that said external housing (40) is provided with at least one through-going access bore (48) placed immediately external to a corresponding insert bore (42) in the wall of the base pipe (16). 9. The flow control device (10, 20) according to claim 8, characterised in that the external housing (40) is enclosed by a removable covering sleeve (50) covering the at least one access bore (48), thereby allowing uncovering of the at least one access bore (48) to obtain access to said corresponding insert bore (42). 10. The flow control device (10, 20) according to claim 1, characterised in that said flow restriction is placed in a through-going channel opening (60) in an annular collar section (56) within said external housing (54, 68), the collar section (56) extending into said cavity (52, 64, 72) between said housing (54, 68) and said base pipe (16). 11. The flow control device (10, 20) according to claim 10, characterised in that said external housing (54) comprises an annular housing (68) removably enclosing said collar section (56), thereby allowing removal of the annular housing (68) to obtain access to the at least one insert bore (60) in the collar section (56). 12. The flow control device (10, 20) according to claim 1, characterised in that the total cross sectional area of flow within the device (10, 20), when provided with several flow restrictions, is equally or unequally distributed between the flow restrictions. 13. The flow control device (10, 20) according to claim 1, characterised in that an upstream end of said flow channel is connected to at least one sand screen (20) connecting the flow channel with said at least one reservoir. 14. The flow control device (10, 20) according to claim 3, characterised in that the total cross sectional area of flow within the device (10, 20), when provided with several flow restrictions, is equally or unequally distributed between the flow restrictions. 15. The flow control device (10, 20) according to claim 5, characterised in that the total cross sectional area of flow within the device (10, 20), when provided with several flow restrictions, is equally or unequally distributed between the flow restrictions. 16. The flow control device (10, 20) according to claim 6, characterised in that the total cross sectional area of flow within the device (10, 20), when provided with several flow restrictions, is equally or unequally distributed between the flow restrictions. 17. The flow control device (10, 20) according to claim 7, characterised in that the total cross sectional area of flow within the device (10, 20), when provided with several flow restrictions, is equally or unequally distributed between the flow restrictions. |
<SOH> BACKGROUND OF THE INVENTION <EOH>The invention has been developed to prevent or reduce several problems occurring in a hydrocarbon reservoir and its horizontal well(s) when subjected to production-related changes in the reservoir fluids. Among many things, these production-related changes lead to fluctuating production rates and uneven drainage of the reservoir. More particularly, this invention seeks to remedy problems associated with production-related changes in the viscosity of the reservoir fluids. At the upstream side of a horizontal well the production tubing is placed in the horizontal or near-horizontal section of the well, hereinafter simply termed horizontal section. During production the reservoir fluids flow radially in through orifices or perforations in the production tubing. The production tubing also may be provided with filters or so-called sand screens that prevent formation particles from flowing into the production tubing. When the reservoir fluids flow through the horizontal section of the production tubing, the fluids are subjected to a pressure loss due to flow friction, and the frictional pressure loss normally is non-linear and is increasing strongly in the downstream direction. As a result, the pressure profile in the fluid flow in the production tubing will is non-linear and is decreasing strongly in the downstream direction. At the onset of production, however, the fluid pressure of the surrounding reservoir rock often is relatively homogenous, and it changes insubstantially along the horizontal section of the well. Thus the differential pressure between the fluid pressure of the reservoir rock and the fluid pressure inside the production tubing is non-linear and is increasing strongly in the downstream direction. This causes the radial inflow rate per unit length of horizontal section of the production tubing to be substantially larger at the downstream side (the “heal”) than that at the upstream side (the “toe”) of the horizontal section. Downstream reservoir zones therefore are drained substantially faster than upstream reservoir zones, causing uneven drainage of the reservoir. During the early to intermediate stages of hydrocarbon recovery, and especially in crude oil recovery, this situation may cause water and/or gas to flow into downstream positions of the horizontal section and to mix with the desired fluid. This effect is referred to as so-called water coning or gas coning in the well. This particularly applies to wells having extensive horizontal length, the length of which may be in the order of several thousand meters, and in which the frictional pressure loss of the fluids within the horizontal section is substantial. This situation causes technical disadvantages and problems to the production. Uneven rate of fluid inflow from different zones of the reservoir also cause fluid pressure differences between the reservoir zones. This may result in so-called cross flow or transverse flow of the reservoir fluids, a condition in which the fluids flow within and along an annulus between the outside of the production tubing and the wellbore wall in stead of flowing through the production tubing. Due to said recovery related situations and problems, flow control devices may be used to appropriately choke the partial flows of reservoir fluids flowing radially into the production tubing along its horizontal inflow portion, and in such a way that the reservoir fluids obtain equal, or nearly equal, radial inflow rate per unit length of the well's horizontal section. |
<SOH> BRIEF DESCRIPTION OF THE DRAWINGS <EOH>In the following, two non-limiting embodiments of the flow control device according to the invention are disclosed, referring also to the accompanying drawings thereof. One specific reference numeral refers to the same detail in all drawings in which the detail is shown, in which: FIG. 1 shows a part section through a pipe length of a production tubing, wherein the pipe length is provided with a flow control device according to the invention, and wherein the device comprises, among other things, nozzle inserts placed in radial insert bores in the wall of the pipe length, and FIG. 1 also shows section lines V-V and VI-VI through the pipe length; FIG. 2 is an enlarged section of details of the flow control device shown in FIG. 1 , and FIG. 2 also shows section line V-V through the pipe length; FIG. 3 shows a part section through a pipe length that is provided with another flow control device according to the invention, but wherein this device comprises nozzle inserts placed in axial insert bores in an annular housing surrounding the pipe length, and FIG. 3 also shows section lines V-V and VI-VI through the pipe length; FIG. 4 shows an enlarged circular section of details of the flow control device according to FIG. 1 , and FIG. 4 also shows section line V-V through the pipe length; FIG. 5 shows a radial part section along section line V-V, cf. FIG. 1 and FIG. 3 , wherein the section shows a connecting sleeve mounted between the flow control device and a sand screen, and FIG. 5 also shows section line I-I through the pipe length; and where FIG. 6 shows a part section along section line VI-VI, cf. FIG. 1 and FIG. 3 , wherein the part section shows details of said sand screen, and FIG. 6 also shows section line I-I through the pipe length. detailed-description description="Detailed Description" end="lead"? |
N-(2-arylethyl) benzylamines as antagonists of the 5-ht6 receptor |
The present invention provides compounds of formula (I), which are antagonists of the 5-HT6 receptor. |
1. A compound of the formula wherein X is selected from the group consisting of —O—, —NH—, —S—, —SO2—, —CH2—, —CH(F)—, —CH(OH)—, and —C(O)—; R1 is selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, imidazolyl, optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused; R2 is selected from the group consisting of hydrogen and C1-C3 alkyl; R3 is selected from the group consisting of hydrogen, fluoro, and methyl; R4 is selected from the group consisting of hydrogen, allyl, C2-C4 alkyl, fluorinated C2-C4 alkyl, optionally substituted phenyl, naphthyl, optionally substituted phenylsulfonyl, optionally substituted benzyl, and optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one or two heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, provided that R4 is not optionally substituted phenylsulfonyl when X is —SO2—, —CH2—, —CH(F)—, —CH(OH)—, or —C(O)—; further provided that when R1 is methoxyphenyl, R4—X is not ethoxy; and pharmaceutically acceptable salts thereof. 2. The compound according to claim 1 wherein X is selected from the group consisting of —O— and —NH—. 3. The compound according to claim 2 wherein X is —O—. 4. The compound according to claim 3 wherein R3 is hydrogen. 5. The compound according to claim 4 wherein R4 is selected from the group consisting of C2-C4 fluorinated alkyl and optionally substituted phenyl. 6. The compound according to claim 5 wherein R1 is selected from the group consisting of optionally substituted phenyl and optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused. 7. The compound according to claim 6 wherein R1 is optionally substituted phenyl. 8. The compound according to claim 6 wherein R1 is optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is benzofused. 9. The compound according to claim 8 wherein the benzofused 5 to 6 membered monocyclic aromatic heterocycle is optionally substituted indol-3-yl. 10. The compound according to claim 9 wherein R4 is optionally substituted phenyl. 11. The compound according to claim 9 wherein R4 is C2-C4 fluorinated alkyl. 12. The compound of claim 11 wherein the compound is N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine. 13. The compound of claim 12 wherein the compound is N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine hydrochloride. 14. The compound of claim 2 selected from the group consisting of N-(2-(5-Methoxy-6-fluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethylamino)benzylamine, N-(2-(5-Methoxy-6-fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropylamino)benzylamine, N-(2-(5-Methoxy-6-fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Methoxy-6-fluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Cyano-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Methylsulfonyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Cyano-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(5-Methylsulfonyl-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(4-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(4-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(7-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(7-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(7-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Amido-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(5-Amido-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Methyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Methyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Methyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoro propoxy)benzylamine, N-(2-(6-Ethoxycarbonyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Ethoxycarbonyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Cyano-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Cyano-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Amido-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Amido-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Trifluoromethoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Trifluoromethoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(7-Chloro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(7-Chloro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Trifluorormethyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Trifluorormethyl-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Cyano-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Cyano-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-(4-Flurorophenyl)-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-(4-Flurorophenyl)-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-(4-Flurorophenyl)-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(4-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(7-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(7-Phenyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-N-methyl-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-N-methyl-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(4-Fluoro-1H-indol-3-yl)ethyl)-N-methyl-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Phenyl-1H-indol-3-yl)ethyl)-N-methyl-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Carboxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Carboxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Chloro-7-fluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Chloro-7-fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5,7-Difluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5,7-Difluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6,7-Difluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6,7-Difluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5,6,7-Trifluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5,6,7-Trifluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(4,5,7-Trifluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4,5,7-Trifluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(7-Cyano-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(7-Cyano-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(4-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(4-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(5-Trifluoromethoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Trifluoromethoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Trifluoromethoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Nitro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Nitro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Nitro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Nitro-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Nitro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Amino-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Amino-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Amino-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Amino-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Propoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-n-Propyl amido-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Ethoxycarbonyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Phenoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy) benzylamine, N-(2-(5-n-Butylamido-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Hydroxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Benzyloxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Benzyloxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Benzyloxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Butyloxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Butyloxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Ethoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Phenylsulfonyoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Phenylsulfonyloxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Butyloxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Hydroxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Hydroxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Hydroxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Hydroxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Carboxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy) benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Isopropyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Isopropyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-N-methyl-3-(2,2-difluoroethoxy)benzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-N-methyl-3-(3-fluoropropoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-N-methyl-3-(3-fluoropropoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-N-isopropyl-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-N-propyl-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-N-ethyl-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(6-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(4-Chloro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Methoxy-2-methyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(7-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Methoxy-2-methyl-1H-indol-3-yl)ethyl)-3-(2,2,3,-tetrafluoropropoxy)benzylamine, N-(2-(7-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Methoxy-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(4-Chloro-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(5-Methoxy-2-methyl-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(7-Methoxy-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(6-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(4,6-Dichloro-5-methoxy-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(4-Chloro-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(5-Methoxy-2-methyl-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(7-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(6-Methoxy-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy) benzylamine, N-(2-(4-Chloro-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(5-Methoxy-2-methyl-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(7-Methoxy-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(6-Methoxy1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4-Chloro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Methoxy-2-methyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(7-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Methoxy-1-methyl-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(5-Methoxy-4,6-difluoro-1-methyl-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(5-Methoxy-4,6-difluoro-1-methyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Methoxy-1-methyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Methoxy-1-methyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Trifluoromethyl-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(5-Fluoro-6-chloro-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(5,6-Difluoro-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(5-Fluoro-6-chloro-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(5,6-Difluoro-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(6-Trifluoromethyl-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(6-Trifluoromethyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Fluoro-6-chloro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5,6-Difluoro-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Trifluoromethyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5,6-Difluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5,6-Difluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5,6-Difluoro-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(6-Trifluoromethyl-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(6-Fluoro-1-methyl-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(6-Fluoro-1-methyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Fluoro-1-methyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Methyl-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(5-Methyl-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-(4,4,4-trifluorobutoxy)benzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-(4,4,4-trifluorobutoxy)benzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(5-Methyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(5-Methyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(5-Methyl-1H-indol-3-yl)ethyl)-3-(3-fluoropropoxy)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(4,4,4-trifluorobutoxy)benzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(4,7-Difluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4,5,6,7-Tetrafluoro-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Bromo-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Bromo-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethloxy)benzylamine, N-(2-(6-Methanesulfonyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Metanesulfonyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Benzenesulfonyl-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Benzenesulfonyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(6-Methoxy-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(7-Fluoro-1H-indol-3-yl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(6-Bromo-1H-indol-3-yl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(6-Bromo-1H-indol-3-yl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(6-Methoxycarbonyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-Benzamido-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-Benzamido-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-Methylsulfonylamino-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-Methylsulfonylamino-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-Isopropyl-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-Isopropyl-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-Ethoxy-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-Ethoxy-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-(2,2,2-Trifluoroethoxy)-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-(2,2,2-Trifluoroethoxy)-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-Butoxy-1H-indol-3-yl)-ethyl)-3-(pyrid-2-yloxy)benzylamine, N-2-(5-Benzenesulfonyl-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-Benzenesulfonyl-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-Ethoxycarbonyl-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-(N′-Propylamido)-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-(N′-Propylamido)-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-(N′-Butylamido)-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(1H-Indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-Benzyloxy-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-Benzyloxy-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-Phenoxy-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-Phenoxy-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(5-(Pyrid-3-yloxy)-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(5-(Pyrid-3-yloxy)-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-Methyl-N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-2-(4,7-Difluoro-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(4,5,6,7-Tetrafluoro-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(4,7-Difluoro-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(4,5,6,7-Tetrafluoro-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(7-Trifluoromethyl-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-2-(7-Trifluoromethyl-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-2-(7-Nitro-1H-indol-3-yl)-ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, and N-2-(7-Nitro-1H-indol-3-yl)-ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine. 15. The compound of claim 4 selected from the group consisting of N-(2-Phenylphenylethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(4-Phenylphenylethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(4-Chlorophenyl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(2,2,3,3-tetrafluoropropoxy)benzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(3,5-Dimethoxyphenyl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(3,5-Dimethoxyphenyl)ethyl)-3-(2-fluoroethoxy)benzylamine, N-(2-(3-Trifluoromethyl-4-fluorophenyl)ethyl)-3-(3,3,3-trifluoropropoxy)benzylamine, N-(2-(3-Trifluoromethyl-4-fluorophenyl)ethyl)-3-(2,2-difluoroethoxy)benzylamine, N-(2-(3-Trifluoromethyl-4-fluorophenyl)ethyl)-3-(2,2,3,3,3-pentafluoropropoxy)benzylamine, N-(2-(3-Trifluoromethyl-4-fluorophenyl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, and N-(2-(3-Trifluoromethyl-4-fluorophenyl)ethyl)-3-(3-fluoropropoxy)benzylamine. 16. The compound of claim 6 selected from the group consisting of N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(3-fluorophenoxy)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(3-fluorophenoxy)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(2-fluorophenoxy)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(2-fluorophenoxy)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(4-fluorophenoxy)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(4-fluorophenoxy)benzylamine, N-(2-(5-Hydroxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Phenoxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-p-Tolyloxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-o-Tolyloxy-1H-indol-3-yl)ethyl)-3-phenoxy benzylamine, N-(2-(5-m-Tolyloxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-2-fluoro-3-phenoxy-benzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-6-fluoro-3-phenoxy-benzylamine, N-Methyl-N-(2-(5-m-tolyloxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Nitro-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Amino-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Propoxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Ethoxycarbonyl-1H-indol-3-yl)ethyl)-3-phenoxy benzylamine, N-(2-(5-Phenyl-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-(4-Fluorophenyl)-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(6-Phenyl-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(6-Trifluoromethyl-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine N-(2-(6-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Chloro-5-methoxy-1H-indol-3-yl)ethyl)-3-phenoxy benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(4,6-Dichloro-5-methoxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(6-Methoxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Chloro-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Methoxy-2-methyl-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(7-Methoxy-1H-indol-3-yl)ethyl)-3-phenoxybenzylamin, N-(2-(5-Methoxy-4,6-difluoro-1-methyl-1H-indol-3-yl)ethyl)-3-(2,2,2-trifluoroethoxy)benzylamine, N-(2-(5-Methyl-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-(4-Fluorophenyl)-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Methanesulfonyl-1H-indol-3-yl)-ethyl)-3-phenoxy benzylamine, N-(2-(5-Cyano-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-(2-(5-Methoxycarbonyl-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-(2-(5-Amido-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-Nitro-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-Butoxy-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-Benzamido-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-Methylsulfonylamino-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-Isopropyl-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-Ethoxy-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-(2,2,2-Trifluoroethoxy)-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-Benzenesulfonyl-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-(N′-Propylamido)-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-(N′-Butylamido)-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-2-(5-Phenoxy-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-Methyl-N-2-(5-Phenoxy-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-4-fluoro-3-phenoxybenzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-phenoxybenzylamine, N-(2-(5-Benzyloxy-1H-indol-3-yl)-ethyl)-3-phenoxybenzylamine, and N-(2-(5-Benzyloxy-1H-indol-3-yl)-ethyl)-N-methyl-3-phenoxybenzyl)benzylamine. 17. The compound of claim 4 selected from the group consisting of N-(2-(3-Chlorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3-Trifluorormethylphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Methoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3,4-Dimethoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3-Methoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3,4-Dichlorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(3-trifluoromethylphenoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(4-t-butylphenoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(4-chlorophenoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(4-methoxyphenoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(4-methylphenoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(3,5-dichlorophenoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(3,4-dichlorophenoxy)benzylamine, N-(2-Phenylethyl)-3-phenoxybenzylamine, N-(2-(4-Chlorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(4-fluororphenoxy)benzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(4-fluororphenoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(2-fluororphenoxy)benzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(2-fluororphenoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(3-fluororphenoxy)benzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(3-fluorophenoxy)benzylamine, N-(2-(2-Chlorophenyl)ethyl)-3-phenyloxybenzylamine, N-(2-(3,4-Dimethoxyphenyl)ethyl)-3-phenoxyoxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-N-methyl-3-phenoxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-N-ethyl-3-phenoxybenzylamine, N-(2-(2-Fluorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3-Fluorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Chlorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Hydroxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(2-Methoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3-Bromo-3-methoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Fluorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(2-Chlorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Bromophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Methylphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3-Methoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Methoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(2-Ethoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Ethoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Phenoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(4-Sulfonamidophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3,4-Dichlorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(2,5-Dichlorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(2,6-Dichlorophenyl)ethyl)-3-phenoxybenzylamine, N-(2-(2,5-Dimethoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(2,3-Dimethoxyphenyl)ethyl)-3-phenoxybenzylamine, N-(2-(3,5-Dimethoxyphenyl)ethyl)-3-phenoxybenzylamine, and N-(2-(3-Ethoxy-4-methoxyphenyl)ethyl)-3-phenoxybenzylamine. 18. The compound of claim 8 selected from the group consisting of N-(2-Pyrid-4-ylethyl)-3-phenoxybenzylamine, N-(2-Pyrid-3-ylethyl)-3-phenoxybenzylamine, N-(2-(Pyrid-2-ylethyl)-3-phenoxybenzylamine, N-(2-Imidazol-4-ylethyl)-3-phenoxybenzylamine, N-(2-Naphth-2-ylethyl)-3-phenoxybenzylamine, N-(2-Naphth-1-ylethyl)-3-phenoxybenzylamine, and N-(2-(Thien-2-ylethyl)-3-phenoxybenzylamine. 19. The compound of claim 1 selected from the group consisting of N-(2-(1H-Indol-3-yl)ethyl)-3-thiophenoxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-phenylsulfonylbenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-thiophenoxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-phenylsulfonylbenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(4-methylthiophenoxy)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(4-methylphenylsulfonyl)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(α-fluorobenzyl)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(α-fluorobenzyl)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(benzyl)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(benzyl)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(α-hydroxybenzyl)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(N-phenylamino)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(N-phenylamino)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(N-benzylamino)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(N-benzylamino)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-hydroxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-hydroxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-naphth-1-yloxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-naphth-1-yloxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-naphth-2-yloxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-naphth-2-yloxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-benzyloxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-benzyloxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-pyrimid-5-yloxy benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-pyrimid-5-yloxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-pyrid-4-yloxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-pyrid-4-yloxybenzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-pyrid-4-yloxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-pyrid-3-yloxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-pyrid-3-yloxybenzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-pyrid-3-yloxybenzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-pyrid-3-yloxy benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-pyrid-2-yloxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-pyrid-2-yloxybenzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-pyrid-2-yloxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-thiazol-2-yloxybenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-thiazol-2-yloxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(2,6-difluororsulfonyloxy)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(2,6-difluororsulfonyloxy)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(pyrid-2-ylamino)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(pyrid-2-ylamino)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(pyrid-2-ylamino) benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(pyrid-3-yl amino)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(pyrid-3-ylamino)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(pyrid-3-ylamino)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-(pyrid-4-ylamino)benzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-(pyrid-4-ylamino)benzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-(pyrid-4-ylamino)benzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-benzoylbenzylamine, N-(2-(1H-Indol-3-yl)ethyl)-3-benzoylbenzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(pyrid-4-yloxy)benzylamine, N-(2-(6-Fluoro-1H-indol-3-yl)ethyl)-3-(pyrid-3-yloxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-thiophenoxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-sulfonylphenylbenzylamine, N-(2-Phenylethyl)-3-thiophenoxybenzylamine, N-(2-Phenylethyl)-3-sulfonylphenylbenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(4-methylthiophenoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-sulfonyl-4-methylphenylbenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(N-benzylamino)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(N-phenylamino)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(α-hydroxybenzyl)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-benzylbenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(α-fluorobenzyl)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-naphth-2-yloxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-naphth-2-yloxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-naphth-1-yloxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-naphth-1-yloxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-hydroxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-hydroxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-benzyloxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(2,4-difluoro phenylsulfonyloxy)benzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(2,4-difluoro phenylsulfonyloxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-thiazol-2-yloxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-thiazol-2-yloxy benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-pyrid-3-yloxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-pyrid-3-yloxybenzylamine, N-(2-(3-Methoxyphenyl)ethyl)-3-pyrid-2-yloxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-pyrid-4-yloxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-pyrimid-5-yloxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-pyrimid-5-yloxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-pyrid-2-yloxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-pyrid-3-ylaminobenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-pyrid-3-ylaminobenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-pyrid-4-ylaminobenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-pyrid-4-ylaminobenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-pyrid-2-ylaminobenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-pyrid-2-ylaminobenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-benzyloxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-(phenylamino)benzylamine, N-(2-(7-Fluoro-1H-indol-3-yl)ethyl)-3-(pyrid-4-yloxy)benzylamine, N-(2-(7-Fluoro-1H-indol-3-yl)ethyl)-3-(pyrid-3-yloxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(pyrid-2-ylmethoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(pyrid-3-ylmethoxy)benzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-(pyrid-4-ylmethoxy)benzylamine, N-2-(5-Butoxy-1H-indol-3-yl)-ethyl)-3-(pyrid-2-yloxy)benzylamine, and N-(2-(6,7-Difluoro-1H-indol-3-yl)-ethyl)-3-(pyridin-4-yloxy)benzylamine. 20. The compound of claim 4 selected from the group consisting of N-(2-(3-Chlorophenyl)ethyl)-3-ethoxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Trifluoromethylphenyl)ethyl)-3-ethoxybenzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-ethoxybenzylamine, N-(2-(5-Chloro-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(5-Fluoro-1H-indol-3-yl)ethyl)-3-ethoxybenzylamine, N-(2-(5-Trifluorormethyl-1H-indol-3-yl)ethyl)-3-ethoxybenzylamine, N-(2-(5-Trifluorormethyl-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-ethoxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(4-Chloro-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(4-Methoxy-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(5-Methoxy-2-methyl-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(7-Methoxy-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(6-Chloro-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(6-Fluoro-1-methyl-1H-indol-3-yl)ethyl)-N-methyl-3-propoxybenzylamine, N-(2-(3-Trifluoromethyl-4-fluorophenyl)ethyl)-3-propoxy-4-methylbenzylamine, N-(2-(5-Fluoro-6-chloro-1H-indol-3-yl)ethyl)-3-propoxy-4-methylbenzylamine, N-(2-(6-Fluoro-1-methyl-1H-indol-3-yl)ethyl)-3-propoxy-4-methylbenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-butoxybenzylamine, N-(2-(3-Chlorophenyl)ethyl)-3-hexoxybenzylamine, N-(2-(2-Fluorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Fluorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(4-Fluorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(2-Chlorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(4-Chlorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(4-Bromophenyl)ethyl)-3-propoxybenzylamine, N-(2-(4-Methylphenyl)ethyl)-3-propoxybenzylamine, N-(2-(4-Hydroxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(2-Methoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Methoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(4-Methoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Ethoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(4-Ethoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(4-Phenoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(4-Sulfonamidophenyl)ethyl)-3-propoxybenzylamine, N-(2-(3,4-Dichlorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(2,5-Dichlorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(2,6-Dichlorophenyl)ethyl)-3-propoxybenzylamine, N-(2-(3,4-Dimethoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(2,5-Dimethoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(2,3-Dimethoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(3,5-Dimethoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Bromo-4-methoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Methoxy-4-ethoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Ethoxy-4-methoxyphenyl)ethyl)-3-propoxybenzylamine, N-(2-(Pyrid-2-yl)ethyl)-3-propoxybenzylamine, N-(2-(Pyrid-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(Pyrid-4-yl)ethyl)-3-propoxybenzylamine, N-(2-(7-Methyl-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(6-Methoxy-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(Thien-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(5-Methyl-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-3-propoxybenzylamine, N-(2-(3-Bromophenyl)ethyl)-3-propoxybenzylamine, N-(2-(3-Methoxycarbonylphenyl)ethyl)-3-propoxybenzylamine, and N-(2-(3-(4-Fluorophenyl)phenyl)-ethyl)-3-propoxybenzylamine. 21. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable diluent. 22. A method of treating disorders associated with the 5-HT6 receptor, comprising: administering to a patient in need thereof an effective amount of a compound of claim 1. 23. The method according to claim 22 wherein the disorder is selected from the group consisting of cognitive disorders, schizophrenia, anxiety, and Alzheimer's disease, comprising: administering to a patient in need thereof an effective amount of a compound of claim 1. 24. A method for treating cognitive disorders comprising: administering to a patient in need thereof an effective amount of a compound of claim 1. 25. A method of treating memory disorders, comprising: administering to a patient in need thereof an effective amount of a compound of claim 1. 26. A method of treating psychosis, comprising: administering to a patient in need thereof an effective amount of a compound of claim 1. 27. A method of treating schizophrenia, comprising: administering to a patient in need thereof an effective amount of a compound of claim 1. 28. A method of treating anxiety, comprising: administering to a patient in need thereof an effective amount of a compound of claim 1. 29. The use of a compound of claim 1 as a pharmaceutical. 29. (canceled) 30. (canceled) 31. (canceled) 32. (canceled) 33. (canceled) 34. (canceled) 35. A compound of the formula wherein Y is selected from the group consisting of O, NH, and NR9, wherein R9 is selected from the group consisting of C1-C4 alkyl, and optionally substituted phenyl; R5 and R6 are hydrogen or taken together with the atoms to which they are attached form a benzo ring, provided that R5 and R6 are hydrogen when Y is NR9; R7 is selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused; R8 is selected from the group consisting of hydrogen and C1-C3 alkyl; and pharmaceutically acceptable salts thereof. 36. A compound according to claim 35 wherein the moiety is attached by either the 4-position or the 6-position. 37. A compound according to claim 36 wherein Y is O. 38. A compound according to claim 36 wherein Y is NH. 39. A compound according to claim 36 wherein Y is NR9. 40. A compound according to claim 36 wherein R5 and R6 are taken together with the atoms to which they are attached for a phenyl ring. 41. A compound according to claim 36 wherein R5 and R6 are hydrogen. 42. A compound according to claim 36 wherein R7 is selected from the group consisting of optionally substituted phenyl and optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused. 43. A compound according to claim 42 wherein the optionally benzofused 5 to 6 membered monocyclic aromatic heterocycle is optionally substituted indol-3-ly. 44. A pharmaceutical composition comprising a compound of claim 35 and a pharmaceutically acceptable diluent. 45. A method of treating disorders associated with the 5-HT6 receptor, comprising: administering to a patient in need thereof an effective amount of a compound of claim 35. 46. A method according to claim 45 wherein the disorder is selected from the group consisting of cognitive disorders, schizophrenia, anxiety, and Alzheimer's disease. 47. (canceled) 48. (canceled) 49. (canceled) 50. The compound according to claim 7 wherein R4 is optionally substituted phenyl. 51. The compound according to claim 8 wherein R4 is optionally substituted phenyl. 52. The compound according to claim 7 wherein R4 is C2-C4 fluorinated alkyl. 53. The compound according to claim 8 wherein R4 is C2-C4 fluorinated alkyl. |
<SOH> BACKGROUND REFERENCES <EOH>1. Branchek, T. A., et al. (2000). Annu Rev Pharmacol Toxicol 40: 319-34. 2. Monsma, F. J., Jr., et al. (1993). Mol Pharmacol 43(3): 320-7. 3. Ruat, M., et al. (1993). Biochem Biophys Res Commun 193(1): 268-76. 4. Kohen, R., et al. (1996). J Neurochem 66(1): 47-56. 5. Ward, R. P., et al. (1996). J Comp Neurol 370(3): 405-14. 6. Ward, R. P., et al. (1995). Neuroscience 64(4): 1105-11. 7. Gerard, C., et al. (1997). Brain Res 746(1-2): 207-19. 8. Gerard, C., et al. (1996). Synapse 23(3): 164-73. 9. Glennon, R. A., et al. (2000). J Med Chem 43(5): 1011-8. 10. Roth, B. L., et al. (1994). J Pharmacol Exp Ther 268(3): 1403-10. 11. Sleight, A. J., et al. (1998). Br J Pharmacol 124(3): 556-62. 12. Routledge, C., et al. (2000). Br. J. Pharmacol. 130(7): 1606. 13. Hirst, W. D., et al. (1999). Br. J. Pharmacol. Suppl.((in press)). 14. Hirst, W. D., et al. (2000). Br. J. Pharmacol. 130: 1597. 15. Bourson, A., et al. (1995). J Pharmacol Exp Ther 274(1): 173-80. 16. Bentley, J. C., et al. (1999). Br J Pharmacol 126(7): 1537-42. 17. Bourson, A., et al. (1998). Br J Pharmacol 125(7): 1562-6. 18. Routledge, C., et al. (1999). Br. J. Pharmacol. 127(Suppl.): 21P. 19. Goldberg, T. E., et al. (1993). Br J Psychiatry 162: 43-8. 20. Hagger, C., et al. (1993). Biol Psychiatry 34(10): 702-12. 21. Lee, M. A., et al. (1994). J Clin Psychiatry 55 Suppl B: 82-7. 22. Purdon, S. E., et al. (2000). Arch Gen Psychiatry 57(3): 249-58. 23. Parada, M. A., et al. (1997). J Pharmacol Exp Ther 281(1): 582-8. 24. Rogers, D. C., et al. (1999). Br J Pharamcol 127(suppl.). 22P. 25. Dawson, L. A., et al. (2000). Br J Pharmacol 130(1): 23-6. 26. Dudkin, K. N., et al. (1996). Neurosci Behav Physiol 26(6): 545-51. 27. Koechlin, E., et al. (1999). Nature 399(6732): 148-51. The present invention provides compounds of formula I: wherein X is selected from the group consisting of —O—, —NH—, —S—, —SO 2 —, —CH 2 —, —CH(F)—, —CH(OH)—, and —C(O)—; R 1 is selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, imidazolyl, optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused; R 2 is selected from the group consisting of hydrogen and C 1 -C 3 alkyl; R 3 is selected from the group consisting of hydrogen, fluoro, and methyl; R 4 is selected from the group consisting of hydrogen, allyl, C 2 -C 4 alkyl, fluorinated C 2 -C 4 alkyl, optionally substituted phenyl, naphthyl, optionally substituted phenylsulfonyl, optionally substituted benzyl, and optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one or two heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, provided that R 4 is not optionally substituted phenylsulfonyl when X is —SO 2 —, —CH 2 —, —CH(F)—, —CH(OH)—, or —C(O)—; and pharmaceutically acceptable salts thereof. The present invention also provides compounds of formula II: wherein Y is selected from the group consisting of O, NH, and NR 9 , wherein R 9 is selected from the group consisting of C 1 -C 4 alkyl, and optionally substituted phenyl; R 5 and R 6 are hydrogen or taken together with the atoms to which they are attached form a benzo ring, provided that R 5 and R 6 are hydrogen when Y is NR 9 ; R 7 is selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused; R 8 is selected from the group consisting of hydrogen and C 1 -C 3 alkyl; and pharmaceutically acceptable salts thereof. The present invention also provides for novel pharmaceutical compositions, comprising: a compound of the formula I or II and a pharmaceutically acceptable diluent. Because the compounds of formula I and II are antagonists of 5-HT 6 receptor, the compounds of formula I and II are useful for the treatment of a variety of disorders. Thus, in another embodiment the present invention provides methods of treating disorders associated with 5-HT 6 , comprising: administering to a patient in need thereof an effective amount of a compound of formula I or II. That is, the present invention provides for the use of a compound of formula I or II and pharmaceutical compositions thereof for the treatment disorders associated with 5-HT 6 . More specifically, the present invention provides a method of treating disorders selected from the group consisting of cognitive disorders, age-related cognitive disorder, mild cognitive impairment, mood disorders (including depression, mania, bipolar disorders), psychosis (in particular schizophrenia), anxiety (particularly including generalized anxiety disorder, panic disorder, and obsessive compulsive disorder), idiopathic and drug-induced Parkinson's disease, epilepsy, convulsions, migraine (including migraine headache), substance withdrawal (including, substances such as opiates, nicotine, tobacco products, alcohol, benzodiazepines, cocaine, sedatives, hypnotics, etc.), sleep disorders (including narcolepsy), attention deficit/hyperactivity disorder, conduct disorder, learning disorders, dementia (including Alzheimer's disease and AIDS-induced dementia), Huntington's Chorea, cognitive deficits subsequent to cardiac bypass surgery and grafting, stroke, cerebral ischemia, spinal cord trauma, head trauma, perinatal hypoxia, cardiac arrest, and hypoglycemic neuronal damage, vascular dementia, multi-infarct dementia, amylotrophic lateral sclerosis, and multiple sclerosis, comprising: administering to a patient in need thereof an effective amount of a compound of formula I or an effective amount of a compound of formula II. In addition, the present invention also provides processes for preparing the compounds of formula I and II and intermediate thereof. As used herein, the following terms have the meanings indicated: The term “C 1 -C 3 alkyl” refers to a straight or branched alkyl chain having from one to three carbon atoms, and includes methyl, ethyl, propyl, and iso-propyl. The term “optionally substituted phenyl” refers to a radical of the formula wherein R a is from 1 to 3 groups independently selected from the group consisting of hydrogen, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, benzyloxy, carboxy, C 1 -C 4 alkoxycarbonyl, amido, N—(C 1 -C 4 alkyl)amido, sulfonylamido, cyano, trifluoromethyl, trifluoromethoxy, nitro, and phenyl optionally substituted with C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, cyano, and trifluoromethyl. The term “optionally substituted naphthyl” refers to a radical of the formula wherein R c is from 1 to 2 groups independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, cyano, trifluoromethyl, and nitro. The term “optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused” refers to radicals of the formula wherein Q 1 is selected from the group consisting of —O—, —S—, and —NR g — wherein R g is selected from the group consisting of hydrogen and C 1 -C 4 alkyl; and Q 2 is —N═, R d , each R e , and R f are each independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, cyano, and trifluoromethyl, or R d and R e (or one of R e ) are taken together with the atoms to which they are attached to form an benzo ring which benzo ring is optionally substituted with 1 to 4 substituents independently selected from the group consisting of hydrogen, hydroxy, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, trifluoromethyl, halogen, carboxy, C 1 -C 4 alkoxycarbonyl, amido, N—(C 1 -C 4 alkyl)amido, amino, (C 1 -C 4 alkyl)amino, acylamino wherein the acyl group is selected from the group consisting of C 1 -C 4 alkyl and phenyl; cyano, nitro, sulfonylamido, phenyl optionally substituted with C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, cyano, and trifluoromethyl; phenoxy, benzyloxy, —NHS(O) 2 R h , wherein R h is selected from the group consisting of C 1 -C 4 alkyl and phenyl; and —S(O) p R i , wherein p is 0, 1, or 2 and R i is selected from the group consisting of C 1 -C 4 alkyl and phenyl optionally substituted with C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, cyano, and trifluoromethyl; and R f is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, trifluoromethyl, and halogen. The term specifically includes furyl, thienyl, pyrrolyl, pyridyl, benzofuryl, benzothienyl, indolyl and quinolinyl; each optionally substituted as described above. The term “fluorinated C 2 -C 4 alkyl” refers to a straight or branched alkyl chain having from two to four carbon atoms substituted with one or more fluorine atoms. The term includes 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3-fluoropropyl, 3,3-difluoropropyl, 3,3,3-trifluoropropyl, 2,2,3,3,3-pentafluoropropyl, 2,2,3,3-tetrafluoropropyl, 4,4,4-trifluorobutyl, 3,3,4,4,4-pentafluorobutyl, and the like. The term “optionally substituted phenylsulfonyl” refers to a radical of the formula wherein R j is from 1 to 3 groups independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, cyano, trifluoromethyl, nitro, and phenyl. The term “optionally substituted benzyl” refers to a radical of the formula wherein R k is from 1 to 3 groups independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, cyano, nitro, trifluoromethyl, and halogen. The term “optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one or two heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur” refers to radicals of the formula wherein Q 3 is selected from the group consisting of —O—, —S—, and —NR g′ — wherein R g′ is selected from the group consisting of hydrogen and C 1 -C 4 alkyl; and Q 4 and Q 5 are —CR m , wherein each R m is independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, halogen, and trifluoromethyl or one or both of Q 4 and Q 5 is —N═; and wherein one or two of Q 6 are —N═, while the others are —CR n ; wherein each R n is independently selected from the group consisting of hydrogen, C 1 -C 4 allyl, C 1 -C 4 alkoxy, halogen, cyano, nitro, and trifluoromethyl. The term specifically includes furyl, thienyl, thiazolyl, pyrazolyl, imidazolyl, triazolyl, oxazolyl, thiazolyl, isoxazolyl, thioisoxazolyl, pyridyl, pyrimidyl, pyridazinyl, and pyrazidinyl; each optionally substituted as described above. The term “C 1 -C 4 alkyl” refers to a straight or branched alkyl chain having from one to four carbon atoms, and includes methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl, iso-butyl, and t-butyl. The term “C 2 -C 4 alkyl” refers to a straight or branched alkyl chain having from two to four carbon atoms, and includes ethyl, propyl, iso-propyl, butyl, sec-butyl, iso-butyl, and t-butyl. The term “C 1 -C 4 alkoxy” refers to a straight or branched alkyl chain having from one to four carbon atoms attached to an oxygen atom, and includes methoxy, ethoxy, propoxy, iso-propoxy, butoxy, iso-butoxy, sec-butoxy, and t-butoxy. The term “halogen” refers to a chloro, fluoro, bromo or iodo atom. The term “pharmaceutically-acceptable addition salt” refers to an acid addition salt. The compound of formula I or II and the intermediates described herein form pharmaceutically acceptable acid addition salts with a wide variety of organic and inorganic acids and include the physiologically acceptable salts which are often used in pharmaceutical chemistry. Such salts are also part of this invention. A pharmaceutically-acceptable addition salt is formed from a pharmaceutically-acceptable acid as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in Journal of Pharmaceutical Science, 66, 2-19 (1977) which are known to the skilled artisan. Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydriodic, nitric, sulfuric, phosphoric, hypophosphoric, metaphosphoric, pyrophosphoric, and the like. Salts derived from organic acids, such as aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, may also be used. Such pharmaceutically acceptable salts thus include chloride, bromide, iodide, nitrate, acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, citrate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, oxalate, phthalate, teraphthalate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, benzenesulfonate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1,5-sulfonate, p-toluenesulfonate, xylenesulfonate, tartrate, and the like. As with any group of pharmaceutically active compounds, some groups are preferred in their end use application. Preferred embodiments of the present invention are given for the compounds of formula I below: Compounds in which wherein X is selected from the group consisting of —O—, —NH—, and —S— are preferred, with compounds in which X is —O— being more preferred. Compounds in which R 1 is optionally substituted phenyl or optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused are preferred. When R 1 is optionally substituted phenyl preferred substituents are 1 to 3 groups independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, halogen, benzyloxy, carboxy, C 1 -C 4 alkoxycarbonyl, amido, N—(C 1 -C 4 alkyl)amido, sulfonylamido, cyano, trifluoromethyl, trifluoromethoxy, nitro, and phenyl optionally substituted with C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, cyano, and trifluoromethyl. When R 1 is optionally substituted phenyl more preferred substituents are 1 to 3 groups independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, halogen, cyano, and trifluoromethyl. Compounds in which R 3 is hydrogen or fluorine are preferred. Compound in which R 1 is optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused, the compounds which are benzo fused are preferred, with indolyl being preferred, and indol-3-yl being even more preferred. When R 1 is indol-3-yl, preferred groups are depicted as the radical below: a) R o is selected from the group consisting of hydrogen and C 1 -C 4 alkyl, with hydrogen being more preferred; b) R p is selected from the group consisting of hydrogen and C 1 -C 4 alkyl, with hydrogen being more preferred; c) R q is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, and halogen, with hydrogen being more preferred; d) R q′ is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, trifluoromethoxy, 2,2,2-trifluoroethoxy, trifluoromethyl halogen, and —S(O) p R i wherein p is 2 and R i is phenyl optionally substituted with C 1 -C 4 alkyl, C 1 -C 4 alkoxy, trifluoromethyl, with halogen being more preferred; e) R q″ is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, nitro, cyano, trifluoromethyl, and —S(O) p R i , wherein p 2 and R i is phenyl optionally substituted with C 1 -C 4 alkyl, with halogen being more preferred; and f) R q′″ is selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, trifluoromethyl, cyano, and nitro, with hydrogen and halogen being preferred. Compounds in which R 4 is selected from the group consisting of C 2 -C 4 alkyl, fluorinated C 2 -C 4 alkyl and optionally substituted phenyl are preferred. When R 4 is C 2 -C 4 alkyl, particularly preferred groups include propyl, isopropyl, and butyl. When R 4 is fluorinated C 2 -C 4 alkyl, preferred groups include 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 3-fluoropropyl, 3,3-difluoropropyl, 3,3,3-trifluoropropyl, 2,2,3,3,3-pentafluoropropyl, and 2,2,3,3-tetrafluoropropyl. When R 4 is optionally substituted phenyl preferred groups include 1 to 3 groups independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, cyano, and trifluoromethyl. Preferred embodiments of the present invention are given for the compounds of formula II below: Compounds in which R 7 is optionally substituted phenyl or optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused are preferred. When R 7 is optionally substituted phenyl preferred substituents are 1 to 3 groups independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkoxy, halogen, cyano, trifluoromethoxy, and trifluoromethyl. Compounds in which R 7 is optionally substituted 5 to 6 membered monocyclic aromatic heterocycle having one heteroatom selected from the group consisting of nitrogen, oxygen, and sulfur and which 5 to 6 membered monocyclic aromatic heterocycle is optionally benzofused, the compounds which are benzo fused are preferred, with indolyl being preferred, and indol-3-yl being even more preferred, with the indol-3-yl depicted above for formula I being more preferred. Preferred compounds of formula II having the points of attachment depicted below: While only compounds of formula I are depicted, the compounds of formula I and II are prepared as described in Schemes A and B below. In the Schemes below all substituents, unless otherwise indicated, are as previously defined, and all starting materials and reagents are well known and appreciated in the art and readily available or prepared by methods described herein. In the Schemes below, it is understood that protecting groups can be used, where appropriate to allow for elaboration of a portion of the compounds of formula I or II. The selection, use, and removal of suitable protecting groups is well known and appreciated in the art ( Protecting Groups in Organic Synthesis, Theodora Greene (Wiley-Interscience)). Scheme A depicts alternative methods for the preparation of compounds of formula I by reductive amination. In one alternative of Scheme A, step a, an appropriate compound of formula (1) is contacted with an appropriate compound of formula (2) in a reductive amination reaction to give a compound of formula I. An appropriate compound of formula (1) is one in which R 1 and R 2 are as desired in the final product of formula I or give rise to groups desired in the final product of formula I. An appropriate compound of formula (2) is one in which X, R 3 , and R 4 are as desired in the final product of formula I, or give rise to groups desired in the final product of formula I. In another alternative of Scheme A, step a, an appropriate compound of formula (3) is contacted with an appropriate compound of formula (4) in a reductive amination reaction to give a compound of formula I. An appropriate compound of formula (3) is one in which R 1 and R 2 are as desired in the final product of formula I or give rise to groups desired in the final product of formula I. An appropriate compound of formula (4) is one in which X, R 3 , and R 4 are as desired in the final product of formula I, or give rise to groups desired in the final product of formula I. The reductive amination depicted in Scheme A, step a, can be carried out under a variety of conditions, such as by hydrogenation using a suitable catalyst or using a suitable reducing agent. For example, an appropriate amine of formula (1) is contacted with an appropriate aldehyde of formula (2) (or alternately an appropriate amine of formula (4) and an appropriate aldehyde of formula (3)) and a suitable reducing agent to give a compound of formula I. The reaction is carried out in a suitable solvent, such as methanol, ethanol, tetrahydrofuran, or mixtures of methanol or ethanol and tetrahydrofuran, dichloromethane, and 1,2-dichloroethane. The reaction may be carried out in the presence of a drying agent, such as sodium sulfate, cupric sulfate, or molecular sieves. The reaction is carried out in the presence of from about 1 to 20 molar equivalents of a suitable reducing agent, such as, sodium borohydride, sodium cyanoborohydride, and sodium triacetoxyborohydride. It may be advantageous to allow Schiff base formation to proceed before addition of the suitable reducing agent. When sodium cyanoborohydride is used it may be advantageous to monitor and adjust the pH during the course of the reaction as is known in the art. The reaction is generally carried out at temperatures of from 0° C. to the refluxing temperature of the solvent. Generally, the reactions require 1 to 72 hours. The product can be isolated and purified by techniques well known in the art, such as filtration, extraction, evaporation, trituration, chromatography, and recrystallization. Scheme A, optional step b, not shown, an acid addition salt of a compound of formula I is formed using a pharmaceutically-acceptable acid. The formation of acid addition salts is well known and appreciated in the art. Scheme B depicts alternative methods for the preparation of compounds of formula I by formation and reduction of an amide. In one alternative, Scheme B, step a, depicts contacting an appropriate compound of formula (1) with an appropriate compound of formula (5) in a amide forming reaction to give a compound of formula (6). An appropriate compound of formula (1) is as described in Scheme A. An appropriate compound of formula (5) is one in which A is an activating group, taking the form of an acid halide, activated ester, activated amide, or anhydride, and X, R 3 , and R 4 are as desired in the final product of formula I, or give rise to groups desired in the final product of formula I. In another alternative, Scheme B, step a, depicts contacting an appropriate compound of formula (7) with an appropriate compound of formula (4) in a amide forming reaction to give a compound of formula (8). An appropriate compound of formula (7) is one in which A is an activating group as described above and R 1 is as desired in the final product of formula I. An appropriate compound of formula (4) is as described in Scheme A. Appropriate compounds of formula (4) and (7) are generally available from commercial sources and can also be prepared by methods described herein and by methods described in the art. The amide formation reaction depicted in Scheme B, step a, is readily accomplished by a number of methods readily available to the skilled person, including those which are conventionally conducted for peptide synthesis. Such methods can be carried out on the acid, acid halide, activated esters, activated amides, and anhydrides. For example, well known coupling reagents such as a carbodiimides with or without the use of well known additives such as N-hydroxysuccinimide, 1-hydroxybenzotriazole, etc. can be used to facilitate amide formation. Such coupling reactions are typically use about 1 to 1.5 molar ratios of acid, amine, and coupling reagent and are conventionally conducted in an inert aprotic solvent such as pyridine, dimethylformamide, dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, tetrahydrofuran and the like. It may be advantageous to use a suitable base, such as triethylamine or N,N-diisopropylethylamine, in such coupling reactions. The reaction is preferably conducted at from about 0° C. to about 60° C. until reaction completion which typically occurs within 1 to about 48 hours. Upon reaction completion, the product can be isolated and purified by techniques well known in the art, such as filtration, extraction, evaporation, trituration, chromatography, and recrystallization. Alternatively, for example, an acid halide can be employed in the reaction. It may be advantageous to use a suitable base to scavenge the acid generated during the reaction, suitable bases include, by way of example, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, pyridine, and the like. Typically, about 1 to 1.5 molar ratios of the acid halide and amine are used. The reaction can be carried out in a variety of inert aprotic solvents such as pyridine, dichloromethane, chloroform, 1,2-dichloroethane, tetrahydrofuran, and the like. The reaction is preferably conducted at from about 0° C. to about 60° C. until reaction completion which typically occurs within 1 to about 12 hours. Upon reaction completion, the product can be isolated and purified by techniques well known in the art, such as filtration, extraction, evaporation, trituration, chromatography, and recrystallization. Alternatively, for example, an acid halide can be employed in the reaction under Schotten-Baumann conditions. Typically, under such conditions 1 to 10 molar equivalents of amine are used. Such couplings generally use a suitable base to scavenge the acid generated during the reaction, such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, and the like. The reaction can be carried out in a variety of mixed solvent systems such as dichloromethane, chloroform, ethyl acetate, tetrahydrofuran and the like; and water. The reaction is preferably conducted at from about 0° C. to about 80° C. until reaction completion which typically occurs within 1 to about 6 hours. Upon reaction completion, the product can be isolated and purified by techniques well known in the art, such as filtration, extraction, evaporation, trituration, chromatography, and recrystallization. Alternatively, for example, an anhydride (either symmetrical or mixed) can be employed in the reaction. Such anhydrides are formed by numerous methods known in art. Typically, about 1 to 1.5 molar equivalents of the anhydride and amine are used. It may be advantageous to use a suitable base to scavenge the acid generated during the reaction. Suitable bases include, by way of example, triethylamine, N,N-diisopropylethylamine, N-methylmorpholine, pyridine, sodium carbonate, potassium carbonate, sodium bicarbonate, and the like. The reaction can be carried out in a variety of solvents. The reaction is preferably conducted at from about 0° C. to about 60° C. until reaction completion which typically occurs within 1 to about 12 hours. Upon completion, the product can be isolated and purified by techniques well known in the art, such as filtration, extraction, evaporation, trituration, chromatography, and recrystallization. Scheme B, steps b, depicts reduction of a compound of formula (6) or (8) to give a compound of formula I. For example, a compound of formula (6) or (8) is contacted with a suitable reducing agent to give a compound of formula I. Suitable reducing agents are those which are capable of reducing an amide to an amine and include, borane reagents, such as borane dimethyl sulfide complex, hydride transfer reagents, such as aluminum hydride and lithium aluminum hydride, and the like. The reaction is carried out in a solvent, such as tetrahydrofuran or diethyl ether, typically using 1 to 10 equivalents of reducing agent. The reaction is generally conducted at from about 0° C. the refluxing temperature of the selected solvent and typically occurs within 1 to about 48 hours. The product can be isolated and purified by techniques well known in the art, such as quenching, filtration, extraction, evaporation, trituration, chromatography, and recrystallization. Scheme B, as an optional step, not shown, an acid addition salt of a compound of formula I is formed using a pharmaceutically-acceptable acid. The formation of acid addition salts is well known and appreciated in the art. In Schemes A and B, as an optional step, not shown, as will be appreciated by the skilled person, a compound of formula I in which R 2 is hydrogen can be alkylated to give a compound in which R 2 is not hydrogen. Methods for alkylating such secondary amines are will known in the art and discussed in Scheme C, step c, below. In Schemes A and B, as will be appreciated by the skilled person, compounds of formula II are also prepared by the methods described in Schemes A and B using compounds of the formula (9) and (10), below: An appropriate compound of formula (9) is one in which Y, R 5 and R 6 are as desired in the final product of formula II and an appropriate compound of formula (10) is one in which A is an activating group, as described above, and Y, R 5 and R 6 are as desired in the final product of formula II. Starting material for Schemes A and B are prepared in the Schemes below. In the Schemes below all substituents, unless otherwise indicated, are as previously defined, and all starting material and reagents are well known and appreciated in the art. Scheme C describes methods for preparing compounds of formula (1). Scheme C, step a, depicts the reaction of an appropriate aldehyde of formula (24) and nitromethane to give the compound of formula (25). An appropriate aldehyde of formula (24) is one in which R 1 is as desired in the final product of formula I. The reaction of the anion of nitromethane with aldehydes to give nitro olefins is well known and appreciated in the art. Modern Synthetic Reactions, H. O. House (2nd ed. The Benjamin/Cummings Publishing Company 1972). For example, an appropriate aldehyde of formula (24) is condensed with nitromethane to give the compound of formula (25). Typically the reaction is carried out in the presence of an excess of nitromethane. The reaction is performed in a suitable solvent, such as tetrahydrofuran, nitromethane, and dimethyl sulfoxide. The reaction is performed using from about 1.1 to about 3 molar equivalents of a suitable base, such as sodium bis(trimethylsilyl)amide, potassium t-butoxide, sodium hydride, sodium acetate, triethylamine, N,N-diisopropylethylamine, ammonium salts, such as ammonium acetate. The reaction is carried out at temperatures of from about −20° C. to the reflux temperature of the selected solvent and generally require from 6 hours to 48 hours. The product of the coupling reaction can be isolated and purified using techniques well known in the art, including extraction, evaporation, chromatography and recrystallization. Scheme C, step b, depicts the reduction of a compound of formula (25) to give a compound of formula (1) in which R 2 is hydrogen. For example, an appropriate compound of formula (25) is hydrogenated over a suitable catalyst, such as Raney® nickel or a palladium catalyst. When Raney nickel is used, the reaction is carried out in a suitable solvent, such as ethanol, methanol, water, and mixtures thereof. It may be advantageous to carry out the hydrogenation under acidic conditions, for example, using hydrochloric or sulfuric acid. When a palladium catalyst is used palladium-on-carbon is preferred and the reaction is carried out in a suitable solvent, such as ethanol, methanol, tetrahydrofuran, water, and mixtures thereof. It may be advantageous to carry out the hydrogenation under acidic conditions, for example, using hydrochloric, trifluoroacetic acid, or sulfuric acid. The reaction is generally carried out at temperatures of from ambient temperature to 70° C. The reaction is carried out with hydrogen at pressures of from 15 psi to 120 psi in an apparatus designed for carrying out reactions under pressure, such as a Parr® hydrogenation apparatus. The product can be isolated by carefully removing the catalyst by filtration and evaporation. The product can be purified by extraction, evaporation, trituration, chromatography, and recrystallization. Alternately, for example, an appropriate compound of formula (25) is contacted with a suitable reducing agent. Suitable reducing agents include hydride transfer reagents, such as aluminum hydride and lithium aluminum hydride, and the like. The reaction is carried out in a solvent, such as tetrahydrofuran or diethyl ether, typically using 1 to 10 equivalents of reducing agent. The reaction is generally conducted at from about 0° C. the refluxing temperature of the selected solvent and typically occurs within 1 to about 48 hours. The product can be isolated and purified by techniques well known in the art, such as quenching, filtration, extraction, evaporation, trituration, chromatography, and recrystallization. Additionally, an appropriate compound of formula (25) can be reduced in two steps to a compound of formula (1). For example, the vinyl group of a compound of formula (25) can be reduced using reagents such as sodium borohydride. The reaction is typically carried out using an excess of borohydride in a solvent, such as methanol, ethanol, isopropanol, water, and the like. The intermediate 2-nitroethyl compound can be isolated and purified by techniques well known in the art, such as quenching, filtration, extraction, evaporation, trituration, chromatography, and recrystallization. The intermediate 2-nitroethyl compound can then be reduced using a variety of methods, such as the hydrogenation and hydride transfer reagents as discussed above. Also, the intermediate 2-nitroethyl compound can be reduced using metals such as zinc to give the desired amine of formula (1) in which R 2 is hydrogen. Scheme C, step c, depicts the optional alkylation of a compound of formula (1) in which R 2 is hydrogen to give a compound of formula (1) in which R 2 is not hydrogen. For example, a compound of formula (1) in which R 2 is hydrogen is contacted with a suitable alkylating agent. A suitable alkylating agent is one which transfers a group R 2 as is desired in the final product of formula I. Suitable alkylating agents include C 1 -C 3 alkyl halides. The reaction is carried out in a suitable solvent, such as dioxane, tetrahydrofuran, tetrahydrofuran/water mixtures, or acetonitrile. The reaction is carried out in the presence of from 1.0 to 6.0 molar equivalents of a suitable base, such as sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, triethylamine, or N,N-diisopropylethylamine. The reaction is generally carried out at temperatures of from −78° C. to the refluxing temperature of the solvent. Generally, the reactions require 1 to 72 hours. The product can be isolated and purified by techniques well known in the art, such as extraction, evaporation, trituration, chromatography, and recrystallization. Alternately, for example, a compound of formula (1) in which R 2 is hydrogen undergoes a reductive amination with an aldehyde or ketone which gives a compound of formula (1) in which R 2 is not hydrogen. Suitable aldehydes include formaldehyde, acetaldehyde, propionaldehyde, and acetone. The reaction is carried out as described in Scheme A, step a. In another alternate, for example, a compound of formula (1) in which R 2 is hydrogen undergoes amide or carbamate formation followed by reduction to give a compound of formula (1) in which R 2 is not hydrogen. Suitable aldehydes include formaldehyde, acetaldehyde, propionaldehyde, and acetone. The reaction is carried out as described in Scheme A, step a. Scheme C, steps d and e, depict an alternative approach to preparing the compounds of formula (1) via formation of an amide using an appropriate compound of formula (7) and an appropriate amine of formula (26) to give an amide of formula (27), followed by reduction to give a compound of formula (1). An appropriate compound of formula (7) is as described in Scheme B. An appropriate amine of formula (26) is one which gives R 2 as desired in final compound of formula I. The skilled person will recognize that many of the amides of formula (27) are commercially available and available in the art. The amide formation and reduction in Scheme C are carried out as described in the Scheme B. Scheme D describes methods for preparing compounds of formula (1) in which R 1 is optionally substituted indol-3-yl. Scheme D, step a, depicts the two-step reaction of an appropriate indole of formula (28) with oxalyl chloride followed by an appropriate amine of formula (26), R 2 NH 2 to give a compound of formula (29). An appropriate indole of formula (28) is one in which Z′ represents optional substituents on the indole 2- and 4- to 7-positions as desired in the final product of formula I. An appropriate amine of formula (26) is as described in Scheme C, above. For example, an appropriate indole of formula (28) is contacted with about 1 to 2 molar equivalents of oxalyl chloride to give an intermediate keto-acid chloride. The reaction is carried out in a suitable solvent, such a diethyl ether or tetrahydrofuran. The reaction is generally carried out at temperatures of from 0° C. to 40° C. and generally require from 6 hours to 48 hours. The intermediate keto-acid chloride product can be isolated and purified using techniques well known in the art, including extraction, evaporation, chromatography and recrystallization. Generally, the intermediate keto-acid chloride product is used directly after isolation. The intermediate keto-acid chloride product is contacted with an appropriate amine, R 2 NH 2 , as defined above and using the procedures described above. Scheme D, step b, depicts the reduction of a compound of formula (29) to give a compound of formula (1) in which R 1 is optionally substituted indol-3-yl. For example, a compound of formula (29) is reduced using a suitable reducing reagent such as, lithium aluminum hydride to give a compound of formula (1) which R 1 is optionally substituted indol-3-yl. The reaction is carried out in a solvent, such as tetrahydrofuran or diethyl ether, typically using 1 to 12 molar equivalents of reducing agent. The reaction is generally conducted at from about 0° C. the refluxing temperature of the selected solvent and typically occurs within 12 to about 48 hours. The product can be isolated and purified by techniques well known in the art, such as quenching, filtration, extraction, evaporation, trituration, chromatography, and recrystallization. In Scheme D, step c, an appropriate indole of formula (28) is formylated to give a compound of formula (30). An appropriate indole of formula (28) is as described in step a, above. For example, an appropriate indole of formula (28) is reacted with a suitable formyl transfer reagent, such as the Vilsmeier reagent formed from dimethylformamide. Generally, about 1 molar equivalent of formyl transfer reagent is used. The reaction is performed in a suitable solvent, such as benzene, dimethylformamide, tetrahydrofuran, or diethyl ether. The reaction is carried out at temperature of from about −70° C. to about 20° C. and generally require from 1 hours to 6 hours. The product of the reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. In Scheme D, step d, an appropriate indole of formula (28) is contacted with (CH 3 ) 2 N—CH═CH—NO 2 to give a compound of formula (30). An appropriate indole of formula (28) is as described in step a, above. For example, an appropriate indole of formula (28) is reacted with 1-dimethylamino-2-nitroethylene. Generally, about 1 equimolar amounts of reagents. The reaction is performed in a suitable solvent, such as trifluoroacetic acid or dichloromethane containing about 2 to 15 equivalents of trifluoroacetic acid. The reaction is carried out at temperature of from about −70° C. to about 20° C. and generally require from 1 hours to 24 hours. The product of the reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. Scheme D, steps e and f, depict an the reaction of an aldehyde of formula (30) to give a nitro olefin of formula (31) and the reduction of the nitro olefin to give a compound of formula (1) in which R 1 is optionally substituted indol-3-yl. These steps can be carried out using the methods described in Scheme C. As will be appreciated by the skilled person, in steps not shown, the indole nitrogen of a compound of formula (1) can be substituted, as desired, using suitable amine protecting groups to give compounds in which R 1 is 1-substitued indol-3-yl. Also as will be appreciated by the skilled person, in steps described in Scheme C, R 2 groups which are not hydrogen can be introduced by various methods. Scheme E describes methods for preparing compounds of formula (2) in which X is —O— or —S—. Scheme E, step a, depicts the formation of an acetal of an appropriate compound of formula (11) to give a compound of formula (12). An appropriate compound of formula (11) is one in which X and R 3 are as desired in the final compound of formula I. Such acetal formation reactions are readily accoplished by methods well known in the art. ( Protecting Groups in Organic Synthesis, Theodora Greene (Wiley-Interscience)). For example, a compound formula (11) is contacted under acid catalysis with an appropriate alcohol, HOR. An appropriate alcohol is one which gives an acetal with is stable to the reaction in step b and can be removed in step c to give a compound of formula (2). Appropriate alcohols include methanol, ethanol, propanol, 1,3-propane diol, ethylene glycol, and the like. In Scheme E, step b, an appropriate compound of formula (11), (12), or (14) is reacted with an R 4 group transfer reagent, as desired, to give a compound of formula (2), (13), or (15); respectively. Appropriate compounds of formula (11), (12), and (14) are ones in which X and R 3 are as desired in the final product of formula I. A variety of reagents that transfers an R 4 as desired in the final product are available and suitable for the reaction depicted in Scheme E. Such reagents include halopyridines, halopryidine N-oxides, allyl halides, C 2 -C 4 alkanols, C 2 -C 4 alkyl halides and sulfonates, fluorinated C 2 -C 4 alkanols, fluorinated C 2 -C 4 alkyl halides and sulfonates, optionally substituted phenyl having at least one fluoro or chloro atom, optionally substituted phenylsulfonyl halides or anhydrides, and optionally substituted benzyl halides. For example, where the appropriate R 4 group transfer reagent is a halide, sulfonate, or anhydride, an appropriate compound of formula (11), (12), or (14) is coupled under basic conditions to give a compound of formula (2), (13), or (15); respectively. The reaction is performed in a suitable solvent, such as acetonitrile, dimethylformamide, dimethylacetamide, tetrahydrofuran, pyridine, and dimethyl sulfoxide. The reaction is carried out in the presence of from about 1 to about 3 molar equivalents of a suitable base, such as potassium hydride, sodium hydroxide, sodium hydride, sodium carbonate, potassium carbonate, cesium carbonate, N,N-diisopropylethylamine, triethylamine, and the like. The reaction is carried out at temperature of from about −30° C. to about 100° C. and generally require from 6 hours to 48 hours. The product of the reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. Of course, when a halopyridine N-oxide is used the N-oxide is remove by reduction to give the R 4 as desired in the final product of formula I. Such reductions are readily accomplished by the skilled person, and include catalytic reduction over palladium catalysts using hydrogen or ammonium formate in a suitable solvent such as methanol, ethanol, water, and mixtures thereof. Alternately, for example, where the appropriate R 4 group transfer reagent is an alkanol, the coupling can be carried out under Mitsunobu conditions which are well known in the art. The reaction is carried out in a suitable solvent, such as tetrahydrofuran and diethyl ether using a phosphine, such as triphenylphosphine or a resin bound phosphine and a dialkyl azodicarboxylate, such as diethyl azodicarboxylate, diisopropyl azodicarboxylate or di-t-butyl azodicarboxylate. The reaction is generally carried out at temperatures of from ambient temperatures to 60° C. The reaction generally requires from 1 hour to 12 hours. The product can be isolated by techniques well known in the art, such as extraction and evaporation. The product can then be purified by techniques well known in the art, such as distillation, chromatography, or recrystallization. Scheme E, step c, depicts the deprotection of an acetal of formula (13) to give a compound of formula (2). Such deprotections are readily accoplished by methods well known in the art. ( Protecting Groups in Organic Synthesis, Theodora Greene (Wiley-Interscience)). For example, a compound formula (13) is contacted under acid under aqueous conditions to give a compound of formula (2). In Scheme E, step d, a bromo compound of formula (15) is formylated to give a compound of formula (2). For example, a compound of formula (15) is metalated by treatment with a metalation reagent such as butyl lithium. The reaction is performed in a suitable solvent, such as hexane, benzene, toluene, tetrahydrofuran or diethyl ether. The reaction is typically carried out in the presence of from about 1 to about 1.5 molar equivalents of a metalating reagent. The metalation reaction is carried out at temperature of from about −70° C. to about 20° C. and generally require from 1 hours to 6 hours. The metalated species is then treated with a formyl transfer reagent, such as dimethylformamide or an alkyl chloroformate to give a compound of formula (2) or a alkoxycarbonyl compound which can be elaborated to an aldehyde as described herein. The product of the reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. Scheme F describes methods for preparing compounds of formula (2) from the versatile intermediate, compound (17), which readily prepared by acetal formation as described above. Scheme F, step a, depicts an aromatic displacement reaction of an appropriate compound of formula (17) and an appropriate alcohol (R 4 OH) or an appropriate thiol (R 4 SH) to give a compound of formula (13) in which X is —O— or —S— are defined above in Scheme E. An appropriate compound of formula (17) is one in which R 3 is as desired in the final product of formula I. In an appropriate alcohol (R 4 OH) or an appropriate thiol (R 4 SH), R 4 is as desired in the final product of formula I, and includes C 2 -C 4 alkyl alcohols and thiols, fluorinated C 2 -C 4 alkyl alcohols and thiols, optionally substituted phenols and thiophenols, optionally substituted benzyl alcohols and thiols. For example, an appropriate compound of formula (17) and an appropriate alcohol (R 4 OH) or an appropriate thiol (R 4 SH) are coupled give a compound of formula (13). The reaction is performed in a suitable solvent, such as dimethylformamide, dimethylacetamide, and dimethyl sulfoxide. The reaction is performed using from about 1.1 to about 3 molar equivalents of an appropriate alcohol or thiol. The reaction is carried out in the presence of from about 1 to about 6 molar equivalents of a suitable base, such as potassium hydride, sodium hydroxide, potassium carbonate, sodium carbonate, or sodium hydride. The coupling is performed using a suitable catalyst, such as copper salts. The reaction generally requires from 6 hours to 48 hours. The product of the coupling reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. Scheme F, steps b-e, depict a number of reactions of an appropriate compound of formula (17), after metalation as described in Scheme E, step d, to give compounds of formula (18)-(21), respectively. In these steps an appropriate compound of formula (17) is one in which R 3 is as desired in the final product of formula I and is not adversely affected by the metalation conditions of the reaction. Generally, these reactions are performed in the solvent used and at the temperature used to form the metalated species. The products of these reactions can be isolated and purified using techniques well known in the art, include quenching, extraction, evaporation, trituration, chromatography, and recrystallization. For example, in Scheme F, step b, a metalated compound of formula (17) is contacted with an appropriate disulfide (R 4 S—) 2 , to give a compound of formula (18). An appropriate disulfide is one that gives R 4 as desired in the final product of formula I and gives rise to compounds in which X is —S—. Appropriate disulfides include C 1 -C 4 alkyl disulfides, optionally substituted phenyl disulfides, and optionally substituted benzyl disulfides. The reaction is performed using from about 1 to about 2 molar equivalents of an appropriate disulfide. The reaction is typically carried out in the same solvent used for the metallation and at temperatures of about −78° C. to about 50° C. The reaction generally require from 12 hours to 48 hours. For example, in Scheme F, step c, a metalated compound of formula (17) is contacted with an appropriate sulfonyl fluoride (R 4 SO 2 F) to give a compound of formula (19). An appropriate sulfonyl fluoride is one that transfers R 4 as desired in the final product of formula I and gives rise to compounds in which X is —SO 2 —. Appropriate sulfonyl fluorides include an optionally substituted phenyl sulfonyl fluoride. The reaction is performed using from about 1 to about 3 molar equivalents of an appropriate sulfonyl fluoride. The reaction is typically carried out in the same solvent used for the metallation and at temperatures of about −78° C. to about 0° C. The reaction generally require from 2 hours to 12 hours. For example, in Scheme F, step d, a metalated compound of formula (17) is contacted with an appropriate acid chloride (R 4 C(O)Cl) to give a compound of formula (20). An appropriate acid chloride is one that transfers R 4 as desired in the final product of formula I and gives rise to compounds in which X is —C(O)—. Appropriate acid chlorides include C 2 -C 4 alkyl acid chlorides, fluorinated C 2 -C 4 alkyl acid chlorides, optionally substituted phenyl acid chlorides, optionally substituted benzyl acid chlorides, and optionally substituted 5 to 6 membered monocyclic aromatic heterocycle acid chlorides. The reaction is performed using from about 0.8 to about 1.2 molar equivalents of an appropriate acid chloride. The reaction is typically carried out in the same solvent used for the metallation and at temperatures of about −78° C. to about 50° C. The reaction generally require from 1 hours to 12 hours. For example, in Scheme F, step e, a metalated compound of formula (17) is contacted with an appropriate aldehyde (R 4 C(O)H) to give a compound of formula (21). An appropriate aldehyde is one that transfers R 4 as desired in the final product of formula I and gives rise to compounds in which X is —CH(OH)—. Appropriate aldehydes include C 2 -C 4 alkyl aldehyde, fluorinated C 2 -C 4 alkyl aldehyde, optionally substituted phenyl aldehyde, optionally substituted benzyl aldehyde, and optionally substituted 5 to 6 membered monocyclic aromatic heterocycle aldehyde. The reaction is performed using from about 1 to about 3 molar equivalents of an appropriate aldehyde. The reaction is typically carried out in the same solvent used for the metallation and at temperatures of about −50° C. to about 50° C. The reaction generally requires from 4 hours to 24 hours. As will be appreciate by the skilled person, compounds of formula (18)-(21) can undergo a number of other transformations which are depicted in Scheme F, steps f-i, to give, ultimately, compounds of formula I having various groups at X. These transformations are trivial and well within the ability of the skilled person. These transformations include oxidation of sulfides (step f) which can be accomplished by peroxide, peracids, and other reagents known in the art; reduction of a benzyl alcohol (step g) which can be accomplished by a variety of reagents, such as triethylsilane/trifluoroacetic acid; halogenation of a benzyl alcohol to give fluoro (step h) using reagents such as DAST and fluorinating reagents; reduction of a ketone (step i) using various hydride transfer reagents or oxidation of a benzylic alcohol (step i) which can be accomplished by manganese dioxide or Swern conditions. In Scheme F, step j, compounds of the formula (13) and (18)-(23) are deprotected to give an aldehyde of formula (2) as described in Scheme E, step c. Scheme G describes methods for preparing compounds of formula (5). Scheme G, step a, a bromo compound of formula (15) is carboxylated to give a compound of formula (5) in which A is —OH. For example, a compound of formula (15) is metalated as described in Scheme E, step d, and the metalated species is then treated with carbon dioxide to give a compound of formula (5) in which A is —OH. The product of the reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. Scheme G, step b, a bromo compound of formula (15) is alkoxyformylated using an appropriate chloroformate or carbonate to give a compound of formula (32). An appropriate chloroformate or carbonate is one that transfers an RO(O)C— group in which R is methyl, ethyl, or benzyl. For example, a compound of formula (15) is metalated as described in Scheme E, step d, and the metalated species is then treated with about 1 to 3 molar equivalents of an appropriate chloroformate or carbonate. The reaction is typically carried out in the same solvent used for the metallation and at temperatures of about −78° C. to about 50° C. The reaction typically requires from 1 to 24 hours. The product of the reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. In Scheme G, step c, an appropriate compound of formula (33) is reacted with an R 4 group transfer reagent, as desired, to give a compound of formula (32). An appropriate compound of formula (33) is one in which X and R 3 are as desired in the final product of formula I. Reagents that transfers an R 4 are as described in Scheme E. For example, where the appropriate R 4 group transfer reagent is a halide or anhydride, an appropriate compound of formula (34) is coupled under basic conditions with to give a compound of formula (33). The reaction is performed in a suitable solvent, such as dimethylformamide, tetrahydrofuran, or pyridine. The reaction is typically carried out in the presence of from about 1 to about 3 molar equivalents of a suitable base, such as sodium carbonate, potassium carbonate, cesium carbonate, N,N-diisopropylethylamine, triethylamine, and the like. The reaction is carried out at temperature of from about −30° C. to about 100° C. and generally require from 6 hours to 48 hours. The product of the reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. Alternately, for example, where the appropriate R 4 group transfer reagent is an alkanol, the coupling can be carried out under Mitsunobu conditions which are well known in the art and described in Scheme E. Scheme G, step d, an ester of formula (32) is deprotected to give a compound of formula (5) in which A is —OH. Such deprotections are readily accoplished by methods well known in the art. ( Protecting Groups in Organic Synthesis, Theodora Greene (Wiley-Interscience)). Scheme G, step e, a compound of formula (5) in which A is —OH is converted to a compound of formula (5) in which A is an activating group, such as acid halide, activated ester, activated amide, or anhydride. The formation of such activated intermediates is well known and appreciated in the art. For example, an acid halide can be prepared by a variety of reagent such as oxalyl chloride, oxalyl bromide, thionyl chloride, thionyl bromide, phosphorous oxychloride, phosphorous trichloride, and phosphorous pentachloride;, a mixed anhydride of substituted phosphoric acid, such as dialkylphosphoric acid, diphenylphosphoric acid, halophosphoric acid; of aliphatic carboxylic acid, such as formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, pivalic acid, 2-ethylbutyric acid; an activated ester, such as phenol ester, p-nitrophenol ester, N-hydroxysuccinimide ester, N-hydroxyplhthalimide ester, 1-hydroxybenztriazole ester; or activated amide, such as imidazole, dimethylpyrazole, triazole; are prepared by method which are well known and appreciated in the art. Such activated intermediates may be prepared and used directly or are prepared and isolated before use in the schemes above. Scheme H describes methods for preparing compounds of formula (4). Scheme H, step a, a bromo compound of formula (15) is converted to a nitrile of formula (35). For example, a compound of formula (15) is treated with copper (I) cyanide to give a compound of formula (35). The reaction is performed in a suitable solvent, such as dimethylformamide. The reaction is typically carried out in the presence of from about 1 to about 3 molar equivalents of copper (I) cyanide. The reaction is carried out at temperature of from about ambient temperature to about 100° C. and generally require from 6 hours to 48 hours. The product of the reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. Scheme H, step b, a nitrile compound of formula (35) reduced to give a compound formula (4) in which R 2 is hydrogen. For example, a nitrile compound of formula (35) is contacted with sodium borohydride in the presence of cobalt chloride. The reaction is carried out in a suitable solvent, such as methanol, or ethanol. The reaction is generally carried out at temperatures of from 0° C. to 50° C. Generally, the reactions require 1 to 72 hours. The product can be isolated and purified by techniques well known in the art, such as extraction with aqueous acid, evaporation, trituration, chromatography, and recrystallization. Alternately, for example, a nitrile compound of formula (35) is hydrogenated over a suitable catalyst, such as Raney® nickel. The reaction is carried out in a suitable solvent, when Raney® nickel is used as the catalyst, suitable solvents will generally contain ammonia, such as ethanol/ammonium hydroxide. The reaction is generally carried out at temperatures of from ambient temperature to 50° C. The reaction is carried out at pressures of from 15 psi (103 kPa) to 120 psi (827 kPa) in an apparatus designed for carrying out reactions under pressure, such as a Parr hydrogenation apparatus. The product can be isolated by carefully removing the catalyst by filtration and evaporation. The product can be purified by extraction, evaporation, trituration, chromatography, and recrystallization. Scheme H, step c, a nitrile compound of formula (35) is converted to a amide of formula (36). For example, a compound of formula (35) is treated with acid or base under hydrolysis conditions to give a compound of formula (36). The reaction is performed in a suitable solvent, such as ethanol, isopropanol, dimethylsulfoxide, each containing water. The hydrolysis of an aromatic nitrile to an amide is well known and appreciated in the art. The product of the reaction can be isolated and purified using techniques well known in the art. These techniques include extraction, evaporation, chromatography and recrystallization. Scheme H, step d, depicts formation of an amide of formula (37) by reacting a compound of formula (5) and an appropriate amine of formula H 2 NR 2 in a amide forming reaction. An appropriate amine of formula H 2 NR 2 is one which gives R 2 as desired in the final product of formula I. Suitable methods of forming amides are well known in the art and are described in Scheme B, above. Scheme H, step e, a amide compound of formula (36) or (37) is reduced to a compound of formula (4). Such reductions of amides are readily carried out as described in Scheme B, above, and as known in the art. Scheme H, step f, a compound of formula (2) and an appropriate amine of formula H 2 NR 2 undergo reductive amination to give a compound of formula (4). Such reductive aminations are readily carried out as described in Scheme B, above, and by other methods known in the art. As will be appreciated by the skilled person, the compounds of formula II are readily prepared by methods analogous to those described above. The present invention is further illustrated by the following examples and preparations. These examples and preparations are illustrative only and are not intended to limit the invention in any way. The terms used in the examples and preparations have their normal meanings unless otherwise designated. For example, “° C” refers to degrees Celsius; “N” refers to normal or normality; “M” refers to molar or molarity; “mmol” refers to millimole or millimoles; “g” refers to gram or grams; “mL” refers milliliter or milliliters; “mp” refers to melting point; “brine” refers to a saturated aqueous sodium chloride solution; etc. In the 1 H NMR, all chemical shifts are given in δ, unless otherwise indicated. detailed-description description="Detailed Description" end="lead"? |
Compositions and method for enhancing proteoglycan production |
The present invention provides compositions comprising notochord enriched media and/or one or more factors derived therefrom. Such compositions are useful for enhancing the production of proteoglycan in cells or animals in need thereof, for example for treating degenerative disc disease. The notochord enriched media is preferably obtained from a nonchondrodystrophic animal. |
1. A composition for enhancing the production of proteoglycan comprising notochord enriched media and/or one or more factors derived from notochord enriched media. 2. A composition according to claim 1 for the treatment of degenerative disease of the chondrocyte matrix. 3. A composition according to claim 1, for the treatment of degenerative disc disease. 4. The composition according to claim 1, wherein the notochord enriched media is obtained from nonchondrodystrophic animals. 5. The composition according to claim 4, wherein the animal is canine, feline or is a rabbit. 6. The composition according to claim 1, wherein the factors derived from notochord enriched media are proteins having a molecular weight in the range of 25 to 220 kilodaltons and a pH in the neutral to acidic range. 7. The composition according to claim 1, further comprising a pharmaceutically acceptable carrier. 8. A method of preparing notochord enriched media comprising: (a) providing isolated notochord cells; and (b) culturing the notochord cells in a medium suitable for maintaining the notochord cells. 9. A method of preparing notochord enriched media comprising: (a) separating a nucleus pulposus from an intervertabral disc of a nonchondrodystrophic animal to provide a total nucleus digest; (b) separating notochord cells from the total nucleus digest; and (c) purifying the notochord cells and culturing the notochord cells in media to provide notochord enriched media. 10. The method according to claim 8, wherein the animal is a nonchondrodystrophic canine, feline or is a rabbit. 11. The method according to claim 10, wherein the animal is canine. 12. The method according to claim 9, wherein the notochord cells are separated from the total nucleus digest using a Percoll gradient method. 13. A method of preparing notochord enriched media comprising: (a) separating a nucleus pulposus from an intervertabral disc of a nonchondrodystrophic animal to provide a total nucleus digest; (b) separating notochord cells from the total nucleus digest; (c) mixing notochord cells with alginate; (d) converting the alginate-containing notochord cells to beads; (e) culturing the beads on a medium comprising one or more infection control substances and growth factors; (f) washing the beads to remove the growth factors; and (g) reculturing beads in media to provide notochord enriched media. 14. A composition for enhancing the production of proteoglycan comprising notochord enriched media prepared using a method according to claim 8. 15. A method for enhancing proteoglycan production comprising administering an effective amount of a composition according to claims 1. 16. The method according to claim 15 for treating degenerative disease of the chondrocyte matrix. 17. The method according to claim 15, for treating degenerative disc disease. 18. The method according to claim 15, wherein the composition is administered percutaneously. 19. The method according to claim 15, wherein the animal is a mammal. 20. The method according to claim 19, wherein the animal is human. 21. A use of a composition according to claim 1 to enhance the production of proteoglycan in a cell or animal in need thereof. 22. A use of according to claim 21 to treat degenerative disease of the chondrocyte matrix. 23. A use of a according to claim 21 to treat degenerative disc disease. 24. A use of a composition according to claim 1 to prepare a medicament to enhance the production of proteoglycan in a cell or animal in need thereof. 25. A use according to claim 24 to prepare a medicament to treat degenerative disease of the chondrocyte matrix. 26. A use of according to claim 24 to prepare a medicament to treat degenerative disc disease. 27. The use according to claim 21, wherein the animal is a mammal. 28. The use according to claim 27, wherein the animal is a human. |
<SOH> BACKGROUND OF THE INVENTION <EOH>Degenerative disc disease is one of the most common causes of disability in North American society. The intervertebral disc is an avascular structure made of a sparse amount of cells interspersed in an extracellular matrix composed of mainly collagen, proteoglycan and water. During the aging process the disc experiences certain biochemical, structural and morphological changes. The effects of these changes are most significant in the nucleus pulposus, which is where many believe that disc degeneration begins. Some of the factors implicated in these changes are disc cell nutrition, degradative enzymes, inflammatory mediators, apoptosis and prolonged mechanical loading. A decrease in cell viability and changes in the matrix composition of the intervertebral disc are visible signs of degenerative disc disease that may be detected during the aging process. Not every person develops degenerative disc disease. There is no biological explanation for the disparity in people who do and do not develop degenerative disc disease in the absence of trauma. An important observation in this regard is that some animals do not develop degenerative disc disease and it is these species that maintain a population of disc notochord cells into adult life. The canine species is a case in point with respect to factors that may have a genetic link. Nonchondrodystrophic dogs maintain their notochord cells for many years and are not known to develop degenerative disc disease, whereas other species of purebred dogs such as beagles (the chondrodystrophic breeds) do develop degenerative disc disease. It is considered that the loss of aggregating proteoglycan and the loss of the associated water content of the nucleus leads to a loss of the resiliency of the disc and compromised load-bearing capacity. The result of such matrix degeneration is further internal derangement of the nucleus, which seems to parallel the molecular disorganization of the nucleus extracellular matrix. It has been reported that the non-aggregating proteoglycans, that seem to arise in the process of degenerative disease, lack a binding site at the hyaluronan central protein core. Therefore, the development of substances that can enhance the production of aggregating proteoglycan can lead to effective treatments for degenerative disc disease and other disorders that involve degeneration of the matrix of chondrocytic cells. |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.