Antagonists of glucagon-like peptide 2, have been identified. Their effects on the growth of gastrointestinal tissue are described. Its formulation as a pharmaceutical, and its therapeutic and related uses in treating bowel tissue, are described. Also described are methods of identifying antagonists of glucagon-like peptide 2.

This invention relates to glucagon-related peptides which are functional antagonists of glucagon-like peptides-2, and to their use therapeutically to counter hyperplasia or induce hypoplasia particularly in intestinal tissue.
Expression of the glucagon gene yields a tissue-determined variety of peptide products that are processed from the 160 residue proglucagon product. The organization of these peptides within the proglucagon precursor was elucidated by the molecular cloning of preproglucagon cDNAs from the anglerfish, rat, hamster and bovine pancreas. These analyses revealed that preproglucagon contains not only the sequence of glucagon and glicentin, but also two additional glucagon-like peptides (GLP-1 and GLP-2) separated from glucagon and each other by two spacer or intervening peptides (IP-I and IP-II). These peptides are flanked by pairs of basic amino acids, characteristic of classic prohormone cleavage sites, suggesting they might be liberated after posttranslational processing of proglucagon (Drucker, Pancreas, 1990, 5(4):484). Analysis of the peptides liberated from proglucagon in the pancreatic islets of Langerhans, for instance, suggests the primary pancreatic peptide liberated is the 29-mer glucagon, whereas glicentin, oxyntomodulin, IP-II and the glucagon-like peptides are more prevalent in the small and large intestines. This demonstration that the glucagon-like peptides are found in the intestine has prompted research into the precise structure and putative function(s) of these newly discovered gut peptides. Most studies have focussed on GLP-1, because several lines of evidence suggested that GLP-1 may be an important new regulatory peptide. Indeed, it has been determined that GLP-1 is the most potent known peptidergic stimulus for insulin release, an action mediated in a glucose-dependent manner through interaction with receptors on pancreatic xcex2cells. GLP-1 and its derivatives are in development for use in the treatment of diabetics.
With respect to the biological role of GLP-2, co-pending U.S. application Ser. No. 08/422,540 (PCT Publ. No. WO 96/32414), incorporated in its entirety herein by reference, discloses that mammalian GLP-2 acts as a trophic agent, to promote growth of intestinal tissue. The effect of GLP-2 is marked particularly by increased growth of the small intestine. Furthermore, co-pending U.S. application Ser. No. 08/631,273 and PCT Application No. PCT/CA 97/00252, both of which are incorporated in its entirety herein by reference, disclose that analogs of vertebrate GLP-2 can have enhanced intestinotrophic activity.
It has now been discovered that alteration of GLP-2 peptide structure can yield peptides capable of inhibiting the intestinotrophic activity of GLP-2. More particularly, and according to one aspect of the invention, there are provided antagonists comprising an amino acid sequence corresponding to that of a first reference mammalian GLP-2 which has been mutated so that from one to four of any of the first four N-terminal residues are deleted. In another aspect of the invention, the antagonists correspond to a reference mammalian GLP-2 that has been mutated so that at least one amino acid selected from the amino acid positions corresponding to the amino acid positions of human GLP-2 at Asp15, Phe22, Thr29, Thr32 and Asp33 is substituted with an amino acid which does not naturally occur at that position in the reference GLP-2. In another aspect of the invention, position Ala2 is substituted with an amino acid selected from the group consisting of Leu, Cys, Glu, Arg, Trp, and PO3-Tyr2. In yet another aspect of the invention, the antagonist corresponds to a polypeptide with any combination of the above substitutions and deletions mutated relative to the reference mammalian GLP-2.
Also provided as an aspect of the invention are methods of producing and identifying GLP-2 antagonists.
For use in medical or veterinary treatment, there is further provided by the present invention a pharmaceutical or veterinary composition comprising an amount of a GLP-2 antagonist effective to antagonize GLP-2 activity in vivo, and a pharmaceutically or veterinarily acceptable carrier.
The GLP-2 antagonist activity of the present GLP-2 antagonists is manifest in vivo as a reduction in the mass of small bowel tissue or as an ability to inhibit the intestinotrophic activity of GLP-2 or intestinotrophic analogs thereof. Accordingly, there is provided, in another aspect of the invention, a method for reducing the mass or suppressing the proliferation of small bowel tissue in a subject, including an animal or a human, which comprises the step of delivering to that subject an amount of a GLP-2 antagonist of the invention effective to cause a reduction in the mass of small bowel tissue.
Subjects for whom such treatment would be useful include those suffering from hyperplastic conditions of the small intestine, for example, as a result of GLP-2 overdose or of GLP-2 overproducing tumors, and conditions wherein prophylactic inducement of small bowel hypoplasia would be useful, for example, in the treatment of clinical obesity as a non-surgical alternative to resection of the small intestine.
The present invention relates to therapeutic and related uses of a novel class of GLP-2 antagonists, particularly for decreasing the growth rate of gastrointestinal tissue, most particularly small bowel. The biological effect of the present GLP-2 antagonists manifests as a decrease in small bowel weight, relative to a mock treated control or as an ability to inhibit the intestinotrophic activity of GLP-2 or an intestinotrophic analog of GLP-2, relative to a control animal given either GLP-2 or an intestinotrophic analog of GLP-2 alone.
The present GLP-2 antagonists are structural analogs of the intestinotrophic GLP-2 peptides. GLP-2 peptides refers collectively to the various vertebrate forms of GLP-2 and to modified forms (characterized by at least one addition, deletion, substitution, and/or incorporation of an amino acid residue with a blocking group) of the GLP-2 analogs which still retain intestinotrophic activity. However, as described herein, certain site-specific alterations of these intestinotrophic GLP-2 peptides can confer antagonist activity to the site-specifically altered analog.
Without being limited by following explanation, it is believed that the site specific alterations which confer antagonist activity interfere with one of the functional activities of the GLP-2 hormone peptide, but not all functional activities. For example, an alteration conferring antagonist activity to a GLP-2 analog may be one which does not inhibit hormone binding to its cognate receptor, but does prevent the subsequent signal transduction through the bound receptor. For example, the site specific alteration of the hormone may prevent dimerization of the hormone receptor which is necessary to transmit a signal to the interior of the cell. Such a mechanism for antagonistic activity has been observed with other hormones such as, for example, human growth hormone (see Fuh et al., Science, 1992, 256:1677-1680).
Generally, sites which are highly conserved among mammalian GLP-2""s are candidates for modification in order to obtain an antagonist. Among mammals, at least residues 1-5, 7, 15, and 22, 29 and 32-33 are highly conserved. Therefore, deletion or substitution of the residues at these sites can result in a GLP-2 antagonist. Additionally, certain modifications of sites near these conserved sites may also cause antagonist activity by disrupting local tertiary amino acid structure or placement of the adjacent conserved residues.
The GLP-2 antagonists of the invention include peptide derivatives with a sequence derived from a vertebrate GLP-2 in that one or more of any of the first four N-terminal amino acids (relative to the sequence of human GLP-2) are deleted. These analogs are referred to herein as the deletion class of GLP-2 antagonists. From the deletion class of GLP-2 antagonists, it will be appreciated that GLP-2 antagonism can result from disruption of the N-terminal structure of GLP-2 within the first four amino acids. Thus, the deletion class of GLP-2 antagonists comprises: GLP-2(2-33), GLP-2(3-33), GLP-2(4-33) and GLP-2(5-33), [desAla2]GLP-2, [desAsp3]GLP-2 and [desGly4] GLP-2.
Additionally, the GLP-2 antagonists of the invention include substitution derivatives of vertebrate GLP-2""s. The substitution class of GLP-2 antagonists includes those antagonists which replace one of the following amino acids at the following positions (relative to sequence of the human GLP-2) with another amino acid: residues 15, 22, 29, 32, and 33. Also included in the substitution class are those incorporating certain Ala2 substitutions.
It is to be understood that, in embodiments of the invention, the GLP-2 antagonists may incorporate any combination of a deletion and a substitution, or may incorporate two or more substitutions at the sites noted.
The GLP-2 antagonists may be analogs of human GLP-2, which has the following sequence:
Unless otherwise specified, the term xe2x80x9cGLP-2xe2x80x9d refers to the sequence of human GLP-2.
The antagonists of the invention are polypeptides which comprise amino acid sequences corresponding to that of a first reference mammalian GLP-2 which has been mutated so that:
(i) from one to four of any of the first four N-terminal residues are deleted; or
(ii) at least one amino acid selected from the amino acid positions corresponding to the amino acid positions of human GLP-2 at Asp15, Phe22, Thr29Thr32 and Asp33 is substituted with an amino acid which does not naturally occur at that position in the reference GLP-2; or
(iii) position Ala2 is substituted with an amino acid selected from the group consisting of Leu, Cys, Glu, Arg, Trp, and PO3-Tyr2; or
(iv) a combination of (i) and (ii), or (ii) and (iii) is mutated.
In specific embodiments of the invention, for example, the GLP-2 antagonists of the invention which are altered at residue positions 1, 2, 3, 4, 22, 29, 32, and/or 33 may be derivatives of rat GLP-2 which is an Ala19 variant of human GLP-2; degu GLP-2, ox GLP-2, porcine GLP-2, guinea pig GLP-2 and hamster GLP-2, the sequences of which have been reported by many authors including Buhl et al in J. Biol. Chem., 1988, 263(18):8621. GLP-2 residues which occur at a specific position are determined by aligning the sequences of GLP-2""s isolated from different vertebrate species and comparing the sequence to the human sequence, reproduced above.
Further, the GLP-2 antagonists of the invention which are altered at residue positions 1, 2, 3, 4, 22, 29, 32, and/or 33 may be derivatives of GLP-2 agonists such as are described in co-pending U.S. application Ser. Nos. 08/632,533 and 08/631,273, and PCT Publication No. Wo 96/32414 and PCT Application No. PCT/CA 97/00252.
Amino acids substitutions appropriate at these sites to yield an antagonist can readily be determined using the murine model of GLP-2 antagonism herein described. That is, a GLP-2 compound incorporating a structural alteration is obtained and then screened in the murine model exemplified herein for GLP-2 antagonism activity. Those GLP-2 compounds which elicit a decrease in bowel growth and/or inhibit the intestinotrophic activity of GLP-2 or a GLP-2 agonist, are identified in this screen as GLP-2 antagonists.
GLP-2 antagonists of the present invention are considered to be functional antagonists of GLP-2 if, when assessed in the murine model exemplified herein, the antagonist: (1) consistently mediates a measurable decrease in small bowel weight relative to a control animal receiving vehicle alone; and/or (2) when assessed by co-administration in said murine model with GLP-2 or a GLP-2 agonist (in a molar excess ratio of preferably 10:1, and more preferably 4:1 over agonist) results consistently in a measurable inhibition of the intestinotrophic effect of GLP-2 or the GLP-2 agonist, as revealed by a reduction in the increase in small bowel weight induced by GLP-2 administered alone.
Particularly suitable for therapeutic use are those functional antagonists of GLP-2 which mediate a bowel weight decrease of at least about 10% relative to a control animal receiving vehicle alone; preferred for therapeutic use are those which mediate a decrease in small bowel weight of at least 15% or more.
The small intestine-mass reducing activity of the present GLP-2 antagonists is noted most significantly in relation to the jejunum, and particularly the proximal jejunum, and is also noted in the distal ileum. Additionally, the activity of GLP-2 antagonists may also be noted as a reduction in the crypt/villus height of the small intestine.
Alternatively, GLP-2 antagonists can be assessed using the co-administration model detailed above. In this case, antagonists are considered to be useful antagonists of GLP-2 if, when co-administered with GLP-2, or an intestinotrophic analog thereof, at a molar ratio of about 10:1, or more preferably at a molar ratio of about 4:1, they diminish the activity of GLP-2 or an intestinotrophic analog thereof by at least 10%, as manifest by a reduction in the increase in small bowel weight relative to a control animal treated with either GLP-2 or the GLP-2 agonist alone.
In another aspect of the invention, there is provided a method useful to identify antagonists of GLP-2, such as those described above, comprising the steps of:
1) obtaining a GLP-2 analog that incorporates an alteration within the peptide sequence:
2) treating a mammal with said analog using a regimen capable of eliciting a measurable loss of the mass of the intestine; and
3) determining the effect of said analog on small bowel weight and/or on the crypt/villus height of the crypt cells of the small intestine relative to a mock treated control animal, whereby a functional GLP-2 antagonist is identified as an analog of GLP-2 which elicits a decrease in said weight and/or said height.
In a related aspect of the invention, there is provided another method useful to identify functional GLP-2 antagonists comprising the steps of:
1) obtaining a GLP-2 analog which incorporates an alteration within the peptide sequence;
2) treating a mammal with said analog in a regimen capable of inhibiting the intestinotrophic activity of GLP-2 or a GLP-2 agonist; and
3) determining the effect of said analog on small bowel weight and/or on the crypt/villus height of the crypt cells of the small intestine relative to a control animal given GLP-2 or a GLP-2 agonist, whereby said functional GLP-2 antagonist is identified as an analog of GLP-2 which inhibits the intestinotrophic activity of GLP-2 and/or the intestinotrophic activity of a GLP-2 agonist.
In a preferred version of the methods described above useful to identify functional GLP-2 antagonists, the GLP-2 analog is chosen from the GLP-2 antagonists described herein.
The substituting amino acids can be chosen from the wide variety of amino acids available to peptide chemists, and include the D-amino acids as well as the L-amino acids and their numerous derivatives. Most practically, chosen amino acids will be amenable to incorporation by solid phase or solution phase synthesis, or by recombinant DNA production means.
In a first screen, analogs which are candidates for antagonistic activity are identified by alanine scanning imutagenesis or other systematic mutagenesis method. These alanine substitutions are tested for antagonistic activity using the methods described in detail herein.
In another aspect of the invention, more effective GLP-2 antagonists may then be made by drastically changing the character of the naturally occuring amino acid residue that is important in forming structural interactions (hydrogen bonding, salt bridging, hydrophobic interactions, positioning of residues) of the GLP-2 hormone with its target molecule (e.g. receptor). With this goal in mind, it is not normally necessary to screen each site with replacements by all 18 of the other naturally occurring residues. Instead, representative members of residue groups are selected. Generally, these groups are:
a. positively charged residues: His, Arg and Lys
b. negatively charged residues: Asp and Glu
c. amides: Asn and Gln
d. aromatic residues: Phe, Tyr, Trp.
e. hydrophobic residues: Ala, Pro, Gly, Val, Leu, Ile, and Met
f. uncharged hydrophilic residues: Ser and Thr.
When preparing these antagonist candidates, one would choose a residue from a group other than the type of residue which is naturally occuring at that position. Extreme substitutions are generated by selecting a residue from a group with opposed combinations of features. For example, a negatively charged residue may be substituted by a positively charged residue.
In the case of the Ala2 substitutions, the substituting amino acids are selected carefully so that antagonists of GLP-2 activity result. It should be noted that substitutions at position 2 can have the effect of enhancing the intestinobtrophic activity of GLP-2. For instance, when Ala2 is replaced by Gly, the result is dramatically enhanced intestinotrophic activity as well as resistance to digestion of the GLP-2 peptide by DPP-IV enzyme. The GLP-2 antagonists of the present invention can surprisingly also be generated by substituting Ala2. In embodiments of the invention, substituting amino acids at position 2 that are useful to generate GLP-2 antagonists are selected from Leu, Cys, Glu, Arg, Trp and PO3-Tyr. GLP-2 antagonists incorporating these substitutions have the added advantage that they render the peptide resistant to digestion by DPP-IV enzyme. Preferably, the Ala2 substituting amino acid is selected from Cys, Glu, Leu, and Arg.
Amino acids substituting for Asp15, Phe22, Thr29, Thr32, and Asp33 are desirably selected from those incorporating a small hydrophobic side chain, such as Ala, Gly and Val.
In embodiments of the invention, the substitution class of GLP-2 antagonists includes: [Gly2, Ala15]GLP-2, [Ala15]GLP-2, [Ala15]GLP-2(2-33), [Ala15]GLP-2(3-33), [Ala15]GLP-2(4-33)[Ala15]GLP-2[(5-33) , [Gly2, Ala22]GLP-2, [Gly2, Ala29]GLP-2, [Gly2, Ala32]GLP-2, [Gly2, Ala33]GLP-2, [Leu2]GLP-2, [GLu2]GLP-2, [Trp2]GLP-2, [PO3-Tyr2]GLP-2, [Cys2]GLP-2, [Ala15]GLP-2, [Ala29]GLP-2, [Ala32]GLP-2 and [Ala33]GLP-2.
The present GLP-2 antagonists, while incorporating a structural alteration of the type noted, may have various amino acid sequences consistent with the sequences of GLP-2 per se or of GLP-2 agonists. The GLP-2 antagonists may also be analogs of vertebrate GLP-2 agonists, in which collateral modifications have been made to enhance other biochemical, biological or physiological properties of the peptide. Such modifications include, for example (in those peptides for which antagonism is conferred by substitution other than at position 2), the substitution of native Ala2 by an amino acid that renders the GLP-2 antagonist resistant to digestion by the enzyme DPP-IV. An amino acid suitable for this purpose includes particularly Gly. Also, the Met10 residue can be replaced by an oxidatively more stable amino acid, such as Leu, Nle, Ile or Ala. Such Met10-substituted analogs are accordingly more stable during synthesis, work-up, and storage. Another modification in this context is replacement of the amino acid at position 20 by an amino acid other than Arg. In certain applications, particularly for the synthetic generation of pharmaceutically or veterinarily acceptable peptides, this modification is desirable to avoid the retention by the Arg residue of counterions from solvents such as TFA.
Within the scope of the present invention are also molecules in which the N- or C-terminus has been modified to incorporate a blocking group of the type used conventionally in the art of peptide chemistry to protect peptide termini from undesired biochemical attack and degradation in vivo.
Suitable N-terminal protecting groups include, for example, C1-5alkanoyl groups such as acetyl. Also suitable as N-terminal protecting groups are amino acid analogs lacking the amino function. Suitable C-terminal protecting groups include groups which form ketones or amides at the carbon atom of the C-terminal carboxyl, or groups which form esters at the oxygen atom of the carboxyl. Ketone and ester-forming groups include alkyl groups, particularly branched or unbranched C1-5alkyl groups, e.g., methyl, ethyl and propyl groups, while amide-forming groups include amino functions such as primary amine, or alkylamino functions, e.g., mono-C1-5alkylamino and di-C1-5alkylamino groups such as methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like. Amino acid analogs are also suitable for protecting the C-terminal end of the present compounds, for example, decarboxylated amino acid analogs such as agmatine.
Embodiments of the invention specifically include such analogs in which the N-terminal blocking group is acetyl; and analogs in which the C-terminal blocking group is an amine, e.g., xe2x80x94NH2.
The present GLP-2 antagonists can be synthesized using standard techniques of peptide chemistry and can be assessed for GLP-2 antagonist activity, all according to the guidance provided herein. Those GLP-2 antagonists that incorporate only L-amino acids can be produced in commercial quantities by application of recombinant DNA technology. For this purpose, DNA coding for the desired GLP-2 antagonist is incorporated into an expression vector and transformed into a microbial, e.g., yeast, or other cellular host, which is then cultured under conditions appropriate for GLP-2 antagonist expression. A variety of gene expression systems have been adapted for this purpose, and typically drive expression of the desired gene from expression regulatory elements used naturally by the chosen host. Because GLP-2 does not require post translational glycosylation for its activity, its production may conveniently be achieved in bacterial hosts such as E. coli. For such production, DNA coding for the selected GLP-2 antagonist may usefully be placed under expression controls of the lac, trp or PL genes of E. coli. As an alternative to expression of DNA coding for the GLP-2 antagonist-per se, the host can be adapted to express GLP-2 antagonist as a fusion protein in which the GLP-2 antagonist is linked releasably to a carrier protein that facilitates isolation and stability of the expression product.
In an approach universally applicable to the production of selected GLP-2 antagonists, and one used necessarily to produce GLP-2 antagonist forms that incorporate non-genetically encoded amino acids and N- and C-terminally derivatized forms, the well established techniques of automated peptide synthesis are employed, general descriptions of which appear, for example, in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Edition, 1984, Pierce Chemical Company, Rockford, Ill.; and in M. Bodanszky and A. Bodanszky, The Practice of Peptide Synthesis, 1984, Springer-Verlag, New York; Applied Biosystems 430A Users Manual, 1987, ABI Inc., Foster City, Calif. In these techniques, the GLP-2 antagonist is grown from its C-terminal, resin-conjugated residue by the sequential addition of appropriately protected amino acids, using either the Fmoc or tBoc protocols, as described for instance by Orskov et al, 1989, supra.
For the incorporation of N- and/or C-protecting groups protocols conventional to solid phase peptide synthesis methods can also be applied. For incorporation of C-terminal protecting groups, for example, synthesis of the desired peptide is typically performed using, as solid phase, a supporting resin that has been chemically modified so that cleavage from the resin results in a peptide having the desired C-terminal protecting group. To provide peptides in which the C-terminus bears a primary amino protecting group, for instance, synthesis is performed using a p-methylbenzhydrylamine (MBHA) resin so that, when peptide synthesis is completed, treatment with hydrofluoric acid releases the desired C-terminally aminated peptide. Similarly, incorporation of an N-methylamine protecting group at the C-terminus is achieved using N-methylaminoethyl-derivatized DVB resin, which upon HF treatment releases peptide bearing an N-methylamidated C-terminus. Protection of the C-terminus by esterification can also be achieved using conventional procedures. This entails use of resin/blocking group combination that permits release of side-chain protected peptide from the resin, to allow for subsequent reaction with the desired alcohol, to form the ester function. FMOC protecting groups, in combination with DVB resin derivatized with methoxyalkoxybenzyl alcohol or equivalent linker, can be used for this purpose, with cleavage from the support being effected by TFA in dichloromethane. Esterification of the suitably activated carboxyl function e.g. with DCC, can then proceed by addition of the desired alcohol, followed by deprotection and isolation of the esterified peptide product.
Incorporation of N-terminal protecting groups can be achieved while the synthesized peptide is still attached to the resin, for instance by treatment with suitable anhydride and nitrile. To incorporate an acetyl protecting group at the N-terminus, for instance, the resin-coupled peptide can be treated with 20% acetic anhydride in acetonitrile. The N-protected peptide product can then be cleaved from the resin, deprotected and subsequently isolated.
Once the desired peptide sequence has been synthesized, cleaved from the resin and fully deprotected, the peptide is then purified to ensure the recovery of a single oligopeptide having the selected amino acid sequence. Purification can be achieved using any of the standard approaches, which include reversed-phase high-pressure liquid chromatography (RP-HPLC) on alkylated silica columns, e.g. C4-, C8-, or C18-silica. Such column fractionation is generally accomplished by running linear gradients, e.g. 10-90%, of increasing % organic solvent, e.g. acetonitrile, in aqueous buffer, usually containing a small amount (e.g. 0.1%) of pairing agent such as TFA or TEA. Alternatively, ion-exchange HPLC can be employed to separate peptide species on the basis of their charge characteristics. Column fractions are collected, and those containing peptide of the desired/required purity are optionally pooled. In one embodiment of the invention, the peptide is then treated in the established manner to exchange the cleavage acid (e.g. TFA) with a pharmaceutically or veterinarily acceptable acid, such as acetic, hydrochloric, phosphoric, maleic, tartaric, succinic and the like, to provide a water soluble salt of the peptide.
According to the present invention, the GLP-2 antagonist is administered to treat subjects, including animals and humans, that would benefit from decreased gastrointestinal tissue growth rate. In one aspect, subject candidates are those who would benefit from decreased mass of small intestine tissue. The effects of GLP-2 antagonists on this tissue, as evidenced by the results exemplified herein, is dramatic and would clearly benefit those subjects suffering from diseases or conditions marked by hyperplasia in the small intestinal tract mucosa, which include GLP-2 producing tumors. Another group of subjects who would clearly benefit from the effects of GLP-2 antagonists are those in whom it would be useful to induce hypoplasia of small intestine tissue, for example, subjects who will in the near future be receiving radiotherapy or chemotherapy or subjects who are receiving radiotherapy or chemotherapy. Small intestine epithelial cells are characterized by rapid cell division and are thus particularly susceptible to damage by radiotherapy or chemotherapy. Indeed, cell damage to the small intestinal epithelial cells is the cause of significant mortality and morbidity in cancer subjects undergoing therapy. Thus, it would be desirable to slow the growth rate of these cells immediately prior to initiation of these therapies and during the course of the treatment. The ability to decrease the growth rate of small intestine cells in these subjects, and thus achieve bowel rest prior to treatment with chemotherapy or radiotherapy, would have the additional benefit of allowing higher doses of radiotherapeutic and chemotherapeutic agents.
Another clinical situation wherein a functional antagonist of GLP-2 would be clinically useful is the treatment of a subject, including an animal or a human, who has been chronically or acutely overdosed with GLP-2 or a GLP-2 agonist. Yet another potential application of functional antagonists of GLP-2 is to block transport of toxins or other drugs across the mucosal layer. Pathogenic effects in some diseases arise as a result of absorption of toxins or drugs via the intestinal epithelium. Elimination of the absorptive capacity of the small bowel by reducing the intestinal epithelium may be beneficial. For example, some diseases such as cholera are lethal because cholera toxin binds to receptors in the intestinal epithelium itself, leading to dehydration and death. GLP-2 antagonists may produce bowel rest, eliminating the target tissue for the toxin (intestinal epithelium) and hence the pathological response to cholera.
Yet another group of subjects who would benefit from a decrease in the mass of the small intestine are those suffering from obesity, as a alternative to surgical intervention such as resection of the small intestine.
Thus, in one aspect the invention provides a method for causing a decrease in the proliferation of small bowel tissue in a subject in need thereof, comprising the step of delivering to the subject an amount of a GLP-2 antagonist of the present invention effective to antagonize GLP-2. The therapeutic efficacy of the GLP-2 antagonist treatment may be monitored by enteric biopsy to examine the villus morphology or by biochemical assessment of nutrient absorption. Additionally, efficacy may be assessed using a clinical endpoint relevant to the particular condition treated, for example, weight loss. In a related aspect the invention provides a method of treating a subject suffering from a gastrointestinal disease, by administering a therapeutically effective amount of a functional antagonist of the present invention, together with a pharmaceutically or veterinarily acceptable carrier, in order to reduce a pathological symptom of the gastrointestinal disease. For example, subjects with small bowel cancer may be administered GLP-2 antagonist to decrease the size of the tumor. Alternatively, subjects with bowel motility disorders, irritable bowel, and chronic diarrhea may benefit from GLP-2 antagonist to increase motility and/or reduce diarrhea.
In another of its aspects, the invention provides a method for treatment of subjects to reduce gastrointestinal tissue mass as part of regimen involving radiotherapy or chemotherapy, in which there is administered to a subject a small bowel mass reducing amount of the GLP-2 antagonist. The invention embraces both co-administration of the GLP-2 antagonist with the radiotherapeutic agent or the chemotherapeutic agent or alternatively administration of the GLP-2 antagonist so as to reduce the growth of small bowel tissue prior to initiation of the radiotherapy or chemotherapy. Appropriate dosing regimens for GLP-2 antagonists may determined by monitoring the subsequent reduction in intestinal damage and/or recovery time after radiotherapy or chemotherapy.
In a further aspect, the invention provides a method for treatment of obesity, which comprises administering to an obese subject an effective amount of a GLP-2 antagonist capable of antagonizing the effects of GLP-2, as evidenced by weight loss under controlled dietary intake conditions.
In another aspect, the invention provides a method of treatment of a subject suffering from a GLP-2 producing tumor which comprises administering to a subject in need thereof an effective amount of a GLP-2 antagonist capable of antagonizing the effects of GLP-2, as evidenced by decrease in size of the intestinal epithelium and small bowel mass over the treatment period.
Another use for antagonists of GLP-2 is as a therapy for correcting a fluid imbalance due to a malabsorption problem across the small intestine. The efficacy of the GLP-2 antagonist treatment is monitored by assessing stool volume, ICF and ECF volume, urine volume and osmolarity, blood pressure, and plasma electrolytes.
As used herein, the term xe2x80x9csubjectxe2x80x9d includes a human or other mammal, including livestock and pets.
For administration to subjects, including humans and animals, the GLP-2 antagonists are provided, in one aspect of the invention, in pharmaceutically or veterinarily acceptable form (e.g., as a preparation that is sterile-filtered e.g. through a 0.22 xcexc filter) and substantially pyrogen-free. Desirably, the GLP-2 antagonist to be formulated migrates as a single or individualized peak on HPLC, exhibits uniform and authentic amino acid composition and sequence upon analysis thereof, and otherwise meets standards set by the various national bodies which regulate quality of pharmaceutical or veterinary products.
For therapeutic use, the chosen GLP-2 antagonist is formulated with a carrier that is pharmaceutically or veterinarily acceptable and is appropriate for delivering the peptide by the chosen route of administration. Suitable pharmaceutically or veterinarily acceptable carriers are those used conventionally with peptide-based drugs, such as diluents, excipients and the like. Reference may be made to xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d, 17th Ed., Mack Publishing Company, Easton, Penn., 1985, for guidance on drug formulations generally. In one embodiment of the invention, the compounds are formulated for administration by infusion or by injection, either sub-cutaneously or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered to a slightly acidic or physiological pH. Thus, the compounds may be administered in distilled water or, more desirably, in saline, buffered saline or 5% dextrose solution. Water solubility may be enhanced, if desired, by incorporating a solubility enhancer, such as acetic acid.
The aqueous carrier or vehicle can be supplemented for use as injectables with an amount of gelatin that serves to depot the GLP-2 antagonist at or near the site of injection, for its slow release to the desired site of action. Concentrations of gelatin effective to achieve the depot effect are expected to lie in the range from 10-20%. Alternative gelling agents, such as hyaluronic acid, may also be useful as depoting agents.
The GLP-2 antagonists of the invention may also be formulated as a slow release implantation device for extended and sustained administration of GLP-2 antagonist. Examples of such sustained release formulations include composites of biocompatible polymers, such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including, A. Domb et al., Polymers for Advanced Technologies3:279-292 (1992). Additional guidance in selecting and using polymers in pharmaceutical or veterinary formulations can be found in the text by M. Chasin and R. Langer (eds.), xe2x80x9cBiodegradable Polymers as Drug Delivery Systems, xe2x80x9d Vol. 45 of xe2x80x9cDrugs and the Pharmaceutical Sciences,xe2x80x9d M. Dekker, New York, 1990. Liposomes may also be used to provide for the sustained release of a GLP-2 antagonist. Details concerning how to use and make liposomal formulations of drugs of interest can be found in, among other places, U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,311,712; U.S. Pat. No. 4,370,349; U.S. Pat. No. 4,372,949; U.S. Pat. No. 4,529,561; U.S. Pat. No. 5,009,956; U.S. Pat. No. 4,725,442; U.S. Pat. No. 4,737,323; U.S. Pat. No. 4,920,016. Sustained release formulations are of particular interest when it is desirable to provide a high local concentration of a GLP-2 antagonist or prolonged circulating levels.
The GLP-2 antagonist can be utilized in the form of a sterile-filled vial or ampoule, that contains an amount of the peptide effective to antagonize endogenous GLP-2 activity, in either unit dose or multi-dose amounts. The vial or ampoule may contain the GLP-2 antagonist and the desired carrier, as an administration-ready formulation. Alternatively, the vial or ampoule may contain the GLP-2 peptide in a form, such as a lyophilized form, suitable for reconstitution in a suitable carrier, such as phosphate-buffered saline.
As an alternative to injectable formulations, the GLP-2 antagonist may be formulated for administration by other routes. Oral dosage forms, such as tablets, capsules and the like, can be formulated in accordance with standard pharmaceutical or veterinary practice.
Finally, chronic delivery of the GLP-2 antagonist for weight loss or other therapeutic indications may be achieved through gene therapy techniques. For example, cells may be engineered ex vivo to express high levels of the GLP-2 antagonist, and then such cells may be implanted into the subject animal or human for therapeutic efficacy.
The therapeutic dosing and regimen most appropriate for treatment will of course vary with the disease or condition to be treated, and according to the subject""s weight and other parameters. The results presented herein below demonstrate that a dose of GLP-2 antagonist equivalent to about 1 mg/kg to 100 xcexcg/kg (or less) administered twice daily over days can generate very significant decrease in small bowel mass. It is conceivable that much smaller doses, e.g., in the xcexcg/kg range, and shorter or longer duration or frequency of treatment, will also produce therapeutically useful results, i.e., a statistically significant decrease particularly in small bowel mass. The dosage sizes and dosing regimen most appropriate for human use are guided by the results herein presented, and can be confirmed in properly designed clinical trials.
An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Primary among these is the amount of GLP-2 normally circulating in the plasma, which is on the order of 151 pmol/ml in the resting state, rising to 225 pmol/ml after nutrient ingestion for healthy adult humans (Orskov, C. and Holst, J. J., 1987, Scand. J. Clin. Lav. Invest. 47:165). Additional factors include the size of the subject, the age of the subject, the general condition of the subject, the particular disease being treated, the severity of the disease, the presence of other drugs in the subject, the in vivo activity of the GLP-2 antagonist and the like. The trial dosages would be chosen after consideration of the results of animal studies and the clinical literature. It will be appreciated by the person of ordinary skill in the art that information such as binding constants and Ki derived from in vitro GLP-2 binding competition assays may also be used in calculating dosages, as well as the calculated half-life of the GLP-2 antagonist in vivo.
A typical human dose of a GLP-2 antagonist would be from about 10 xcexcg/kg body weight/day to about 10 mg/kg/day, preferably from about 50 xcexcg/kg/day to about 5 mg/kg/day, and most preferably about 100 xcexcg/kg/day to 1 mg/kg/day. As it is conceivable that the GLP-2 antagonists of the invention could be up to 10 to even 100 times more potent than GLP-2, a typical dose of such a GLP-2 antagonist may be lower, for example, from about 100 ng/kg body weight/day to 1 mg/kg/day, preferably 1 xcexcg/kg/day to 500 xcexcg/kg/day, and even more preferably 1 xcexcg/kg/day to 100 xcexcg/kg/day.

Antagonists of intestinotrophic GLP-2 peptides


An apparatus for manufacturing a complete wire harness from a plurality of wire harnesses have a first transferring unit, an inserting unit and a removing unit. The first transferring unit transfers a wire clamping bar, which holds a partial harness having a plurality of electric wires, to a wire-harness fabricating station. The inserting unit inserts terminals disposed at ends of the plurality of electric wires into predetermined terminal accommodating chambers of predetermined connectors set on a connector receiving jig. The removing unit removes the emptied wire clamping bar.

1. Field of the Invention
This invention relates to a wire harness and a method and an apparatus for manufacturing the same, and more particularly to the manufacture of one wire harness (or one sub-harness) by combining a plurality of partial harnesses.
The present application is based on Japanese Patent Application No. Hei. 9-230711, which is incorporated herein by reference.
2. Description of the Related Art
As is known, there are a plurality of wire harnesses used in the electrical wiring of an automobile, including such as an engine harness, an instrument panel harness, a cowl side harness, a door harness, and a roof harness.
As a method and an apparatus for manufacturing such wire harnesses, as shown in FIG. 13, a method and an apparatus are known which use a wire clamping bar 101 in which a multiplicity of wire clamps 100 are juxtaposed at equal pitches. Incidentally, each wire clamp 100 comprises a pair of mutually opposing clamp members 100a, and electric wires are clamped therebetween.
After a plurality of electric wires 102 for forming one wire harness are cut to a predetermined length, opposite end portions thereof are retained by-mutual adjacent wire clamps 100 and are held in U-shapes on the wire clamping bar 101. In this state, end-processing such as stripping (removal of an insulating sheath) of a wire end and crimping of a terminal is performed.
This wire clamping bar 101 is set in or transferred to a case fitting station 103, and terminals 104 at wire ends are respectively inserted and fitted into terminal accommodating chambers (not shown) of a connector housing (hereafter, simply referred to as the connector) 105 by using an automatic terminal inserting device 107.
A plurality of connectors 105 are arranged in advance on a setting table 106 as shown at reference numerals 1051 to 1054. The terminals 104 are inserted into and retained in predetermined terminal accommodating chambers of connectors selected from the plurality of connectors 1051 to 1054 in a predetermined order.
As shown in FIG. 14, the automatic terminal inserting device 107 has a mounting head 108, a pair of terminal gripping claws 109 for a wire connecting portion 104a of the terminal 104 and a pair of wire gripping claws 110 are provided at a lower end of the mounting head 108 in such a manner as to be capable of being raised or lowered and of being opened or closed. Further, a pair of wire clamp plates 111 which can be opened or closed is provided at the lower end of the mounting head 108, and a terminal holding plate 112 is provided between the pair of terminal gripping claws 109 in such a manner as to be capable of being raised or lowered.
Further, the mounting head 108 is provided in such a manner as to be capable of moving close to or away from the wire clamp 100 (or the wire clamping bar 101) and of being raised or lowered with respect to the same. Further, a moving plate 113 for supporting the mounting head 108 is mounted on a supporting frame 114 of the case fitting station 103, and is provided in such a manner as to be horizontally movable with respect to the setting table 106 on which the plurality of connectors 105 are arranged.
Accordingly, according to the automatic terminal inserting device 107, the terminal 104 can be automatically inserted into the connector 105 by repeating the following steps a to d.
a. The mounting head is moved to immediately above desired wire clamp 100 of the wire clap bar 101 by means of the moving plate 113.
b. At the same time as the lowering and raising operation of the mounting head 108, an end portion of the wire 102 including the terminal 104 is gripped by the opening/closing operation of the terminal gripping claws 109, the wire gripping claws 110, and the wire clamp plates 111, and is lifted up from the wire clamp 100 which retained the wire.
c. Then, the mounting head 108 is moved in parallel to the wire clamping bar 101, and is stopped at a position opposing a desired terminal accommodating chamber of the desired connector 105.
d. The mounting head 108 is advanced forward toward the connector 105, and while the terminal 104 is being inserted into the terminal accommodating chamber, the terminal gripping claws 109, the wire gripping claws 110, and the wire clamp plates 111 are opened consecutively beginning at the front side, thereby releasing the wire end portion. Namely, the terminal 104 is automatically inserted and fitted into the predetermined terminal accommodating chamber of the predetermined connector.
Thus, there is an advantage, among others, in that, by using the wire clamping bar 101 in which the multiplicity of wire clamps 100 are juxtaposed, all the end processing of wires, including the cutting, retention, stripping, terminal crimping, and case fitting (insertion and fitting of a terminal into a connector) of a multiplicity of wires which make up one wire harness, can be handled on one side of the wire clamping bar. A method and an apparatus similar to the above-described method and apparatus have been disclosed in Japanese Patent Publication No. Hei. 5-55994.
However, since the engine harness, for example, is made up by numerous wires which generally comprise as many as 200 circuits (the number of wires: 200, the number of both end portions that are retained in U-shapes: 400), the number of connectors that are fitted to the wire ends is also numerous. As such, if end processing is performed with respect to these wires by using a single wire clamping bar, troubles are liable to occur, such as erroneous wiring of wires, erroneous crimping of a terminal, erroneous-insertion into a connector, and entanglement of wires or terminals.
On the other hand, to handle numerous wires by a single wire clamping bar, an extremely long bar is required, and not only is a wide working space required, but also a pitch time from the cutting of the wire until completion of final case fitting becomes very long. Hence, the apparatus becomes large and automatic control becomes complex and expensive. Furthermore, if there occurs erroneous wiring or erroneous insertion or the like, since it takes time and trouble in correcting them in an ensuing process, wastage can occur such as discarding the entire wires on the wire clamping bar as defective products.

Apparatus for manufacturing a complete wire harness from a plurality of partial harnesses


Provided is a method of manufacturing a thin film magnetic head. In particular, a method of manufacturing a thin film magnetic head is provided in which a flow process of a photoresist is applied to separate a hard magnetic layer and a metal multi-layer and a photoresist is used to insulate an upper electrode from a lower electrode in manufacturing a magnetic reproduction device, thereby simplifying and optimizing a manufacturing process and significantly reducing a manufacturing time.

1. Field of the Invention
The present invention relates to a method of manufacturing a thin film magnetic head and, more particularly, to a method of manufacturing a thin film magnetic head in which a flow process of a photoresist is applied to separate a hard magnetic layer and a metal multi-layer and a photoresist is used to insulate an upper electrode from a lower electrode in manufacturing a magnetic reproduction device, thereby simplifying and optimizing a manufacturing process and significantly reducing a manufacturing time.
2. Discussion of Related Art
In general, a method of manufacturing a thin film magnetic head which records and replays magnetic information is performed such that a magnetic metal layer is surrounded by an insulator such as an oxide film and a ferromagnet layer, i.e., a hard magnet layer is deposited and then etched or lifted off.
Meanwhile, an insulating layer such as an oxide layer is used for insulation between upper and lower electrodes or between a hard magnet layer and a metal multi-layer.
In the method of manufacturing the thin film magnetic head according to a conventional art, when the hard magnet layer is formed, a basic condition such as fineness or insulation of a device is satisfied, but for the sake of insulation between the hard magnet layer and the metal multi-layer, a mask process should be additionally performed, and a process of removing an oxide layer should be simultaneously performed.
Further, in the method of manufacturing the thin film magnetic head according to the conventional art, an oxide layer deposition process and an etching process should be additionally performed to form an insulating layer between the upper and lower electrodes.

Method of manufacturing thin film magnetic head

Electronics and Telecommunications Research Institute


Disclosed herein is a display including: a pixel array part configured to include pixels that are arranged in a matrix, each having an electro-optical element, a write transistor for writing a video signal, a drive transistor for driving the electro-optical element based on the video signal written by the write transistor, and a holding capacitor connected between the gate and source of the drive transistor, wherein the holding capacitor includes a first electrode, a second electrode disposed to face one surface of the first electrode for forming a first capacitor, and a third electrode disposed to face the other surface of the first electrode for forming a second capacitor, and the first capacitor and the second capacitor are connected in parallel to each other electrically.

1. Field of the Invention
The present invention relates to a display, a method for driving a display, and electronic apparatus, and particularly to a flat-type (flat-panel) display in which pixels, each including an electro-optical element, are arranged on rows and columns (in a matrix), a method for driving the display, and electronic apparatus including the display.
2. Description of the Related Art
In recent years, in the field of displays for image displaying, flat-type display obtained by arranging pixels (pixel circuits) each including a light-emitting element in a matrix is prevailing rapidly. As the flat-type display, an organic electroluminescence (EL) display is being developed and commercialized. The organic EL display includes organic EL elements as the light-emitting elements of the respective pixels. The organic EL element is a so-called current-driven electro-optical element whose light-emission luminance varies depending on the value of the current flowing through the element, and is based on a phenomenon that light emission occurs in response to electric field application to an organic thin film.
The organic EL display has the following features. Specifically, the organic EL display has low power consumption because the organic EL element can be driven by an application voltage lower than 10 V. Furthermore, because the organic EL element is a self-luminous element, the organic EL display provides higher image visibility compared with a liquid crystal display, which displays an image by controlling, for each pixel including a liquid crystal cell, the intensity of light from a light source (backlight) by the liquid crystal cell. In addition, the organic EL display does not need to have an illuminating unit such as a backlight, which is necessary for the liquid crystal display, and therefore reduction in the weight and thickness of the organic EL display can be easily achieved. Moreover, the response speed of the organic EL element is as high as several microseconds, which causes no image lag in displaying of a moving image by the organic EL display.
As the drive system for the organic EL display, a simple-(passive-)matrix system or an active-matrix system can be employed, similar to the liquid crystal display. However, a display of the simple-matrix system involves a problem that it is difficult to realize a large-size and high-definition display since the light-emitting period of the light-emitting element is decreased due to the increase of the scanning line (i.e. number of pixels) although the configuration thereof is simple.
For this reason, in recent years, a display of the active-matrix system is being actively developed in which the current flowing through an electro-optical element is controlled by an active element, such as an insulated gate field effect transistor (typically, thin film transistor (TFT)), provided in the same pixel circuit as that including this electro-optical element. In the display of the active-matrix system, the electro-optical element continues light emission over the one-frame period. This easily realizes a display having a large size and high definition.
It is generally known that the I-V characteristic (current-voltage characteristic) of an organic EL element deteriorates as the time elapses (so-called age deterioration). In a pixel circuit including an N-channel TFT as a transistor for driving an organic EL element by current (hereinafter, referred to as a "drive transistor"), the organic EL element is connected to the source side of the drive transistor. Therefore, the age deterioration of the I-V characteristic of the organic EL element leads to change in the gate-source voltage Vgs of the drive transistor, which results in change in the light-emission luminance of the organic EL element.
A more specific description will be made below about this point. The source potential of the drive transistor is dependent on the operating point of the drive transistor and the organic EL element. The deterioration of the I-V characteristic of the organic EL element varies the operating point of the drive transistor and the organic EL element. Therefore, even when the same voltage is applied to the gate of the drive transistor, the source potential of the drive transistor varies. This varies the source-gate voltage Vgs of the drive transistor, which changes the value of the current flowing through the drive transistor. As a result, the value of the current flowing through the organic EL element also changes, which varies the light-emission luminance of the organic EL element.
Furthermore, a pixel circuit employing a poly-silicon TFT involves, in addition to the age deterioration of the I-V characteristic of the organic EL element, changes over time in the threshold voltage Vth of the drive transistor and the mobility μ in the semiconductor thin film serving as the channel of the drive transistor (hereinafter, referred to as "the mobility of the drive transistor"), and differences in the threshold voltage Vth and the mobility μ from pixel to pixel due to variation in the manufacturing process (variation in transistor characteristics among the respective drive transistors).
If the threshold voltage Vth and mobility μ of the drive transistor are different from pixel to pixel, variation in the value of the current flowing through the drive transistor arises on a pixel-by-pixel basis. Therefore, even when the same voltage is applied to the gate of the drive transistor among the pixels, variation in the light-emission luminance of the organic EL element among the pixels arises, which results in lowered uniformity of the screen.
To address this problem, there has been proposed a configuration aiming to allow the light-emission luminance of the organic EL element to be kept constant without being affected by the age deterioration of the I-V characteristic of the organic EL element and changes over time in the threshold voltage Vth and mobility μ of the drive transistor. Specifically, in this configuration, each of pixel circuits is provided with a compensation function against change in the characteristic of the organic EL element, and correction functions for correction against variation in the threshold voltage Vth of the drive transistor (hereinafter, referred to as "threshold correction") and correction against variation in the mobility μ of the drive transistor (hereinafter, referred to as "mobility correction") (refer to e.g. Japanese Patent Laid-Open No. 2006-133542 (hereinafter referred to as Patent Document 1)).
By thus providing each pixel circuit with the compensation function against change in the characteristic of the organic EL element and the correction functions against variation in the threshold voltage Vth and mobility μ of the drive transistor, the light-emission luminance of the organic EL element can be kept constant without being affected by the age deterioration of the I-V characteristic of the organic EL element and changes over time in the threshold voltage Vth and mobility μ of the drive transistor.
The compensation function against change in the characteristic of the organic EL element is implemented through the following series of circuit operation. Initially, at the timing when a video signal supplied via a signal line is written by a write transistor and is held in a holding capacitor connected between the gate and source of the drive transistor, the write transistor is turned to the non-conductive state to thereby electrically separate the gate electrode of the drive transistor from the signal line for turning the gate electrode to the floating state.
If the gate electrode of the drive transistor enters the floating state, in response to change in the source potential Vs of the drive transistor, the gate potential Vg of the drive transistor also changes in linkage with (in such a manner as to follow) the change in the source potential Vs, due to the connection of the holding capacitor between the gate and source of the drive transistor. This is a bootstrap operation. Due to the bootstrap operation, the gate-source voltage Vgs of the drive transistor can be kept constant. Thus, even when the I-V characteristic of the organic EL element changes over time, the light-emission luminance of the organic EL element can be kept constant.
In this bootstrap operation, the ratio of the rise amount ΔVg of the gate potential Vg of the drive transistor to the rise amount ΔVs of the source potential Vs thereof (hereinafter, referred to as a bootstrap ratio Gbst) is an important factor. Specifically, if this bootstrap ratio Gbst is low, the gate-source voltage Vgs of the drive transistor becomes lower than the voltage obtained at the timing when the video signal is held in the holding capacitor.
The low bootstrap ratio Gbst is equivalent to the fact that the rise amount ΔVg of the gate potential Vg is small with respect to the rise amount ΔVs of the source potential Vs. Therefore, the gate-source voltage Vgs becomes lower. This leads to failure in ensuring of the current necessary as the drive current to be applied to the organic EL element, i.e., the current corresponding to the video signal written by the write transistor, which results in luminance lowering. Thus, luminance unevenness occurs, which causes the deterioration of the image quality.
The bootstrap ratio Gbst depends on the capacitance of the holding capacitor and the capacitances of parasitic capacitors attaching to the gate of the drive transistor. The higher the capacitances of these capacitors are, the higher the bootstrap ratio Gbst is (details of this point will be described later). The capacitance of the parasitic capacitances depends on circuit elements, such as a transistor, connected to the gate electrode of the drive transistor. If the number of elements included in the pixel circuit is reduced and consequently the number of transistors connected to the gate electrode of the drive transistor is decreased, the capacitance of the parasitic capacitors becomes lower correspondingly.
Therefore, increasing the capacitance of the holding capacitor is effective to increase the bootstrap ratio Gbst. The capacitance of the holding capacitor is in proportion to the area of two metal electrodes that form the holding capacitor and are disposed to face each other, and is in inverse proportion to the distance between these two metal electrodes. Therefore, the capacitance of the holding capacitor can be increased by increasing the area of two metal electrodes or decreasing the distance between two metal electrodes. Because there is a limit to the decreasing of the distance between two metal electrodes, the increasing of the area of two metal electrodes, i.e., increasing of the size of the holding capacitor, is preferentially attempted.
However, because the holding capacitor is formed under the condition of the limited pixel size, there is also a limit to the increasing of the size of the holding capacitor. On the contrary, the recent trend toward miniaturization of the pixel size accompanying definition enhancement makes it difficult to increase the bootstrap ratio Gbst through the increasing of the size of the holding capacitor.
As another scheme, it would be available to ensure the drive current corresponding to the video signal by, instead of increasing the bootstrap ratio Gbst, originally designing a large current as the drive current to be applied to the organic EL element via the drive transistor in anticipation of the voltage decrease corresponding to the bootstrap ratio Gbst. However, this scheme involves a problem of an increase in the power consumption.

Display, method for driving display, and electronic apparatus having parallel holding capacitors


The present invention relates to modulators of muscarinic receptors. The present invention also provides compositions comprising such modulators, and methods therewith for treating muscarinic receptor mediated diseases.

The neurotransmitter acetylcholine binds to two types of cholinergic receptors: the ionotropic family of nicotinic receptors and the metabotropic family of muscarinic receptors. Muscarinic receptors belong to the large superfamily of plasma membrane-bound G protein coupled receptors (GPCRs). To date, five subtypes of muscarinic receptors (M1-M5) have been cloned and sequenced from a variety of species, and show a remarkably high degree of homology across species and receptor subtype. These M1-M5 muscarinic receptors are predominantly expressed within the parasympathetic nervous system which exerts excitatory and inhibitory control over the central and peripheral tissues and participate in a number of physiologic functions, including heart rate, arousal, cognition, sensory processing, and motor control.
Muscarinic agonists such as muscarine and pilocarpine, and antagonists, such as atropine have been known for over a century, but little progress has been made in the discovery of receptor subtype-selective compounds, thereby making it difficult to assign specific functions to the individual receptors. See, e.g., DeLapp, N. et al., "Therapeutic Opportunities for Muscarinic Receptors in the Central Nervous System," J. Med. Chem., 43(23), pp. 4333-4353 (2000); Hulme, E. C. et al., "Muscarinic Receptor Subtypes," Ann. Rev. Pharmacol. Toxicol., 30, pp. 633-673 (1990); Caulfield, M. P. et al., "Muscarinic Receptors-Characterization, Coupling, and Function," Pharmacol. Ther., 58, pp. 319-379 (1993); Caulfield, M. P. et al., International Union of Pharmacology. XVII. "Classification of Muscarinic Acetylcholine Receptors," Pharmacol. Rev., 50, pp. 279-290 (1998), the disclosures of which are incorporated herein by reference.
The Muscarinic family of receptors is the target of a large number of pharmacological agents used for various diseases, including leading drugs for COPD, asthma, urinary incontinence, glaucoma, Alzheimer's (AchE inhibitors). Despite the large therapeutic value of this family, cholinergic drugs are limited by the lack of selectivity of these agents, with significant activation of the parasympathetic autonomous system and elevated incidence of adverse effects. The molecular cloning of the muscarinic receptors and the identification of the physiological role of specific isoforms using knock-out mice, has recently delineated novel opportunities for selective muscarinic ligands, and has helped to define the selectivity profile that is required for enhanced efficacy and reduced side effects.
There is a need for modulators of muscarinic receptors M1-M5. There is also a need for methods for treating muscarinic receptor-mediated diseases.
There is also a need for modulators of muscarinic receptors that are selective as to subtypes M1-M5.


A catheter assembly includes a catheter 1 having a catheter hub 11 provided at proximal end thereof, a wing member 2 supporting the catheter 1, a tubular member 3 which is fixed to the wing member 2 and into which the catheter is slidably inserted, a sheath 4 slidably covering around the tubular member 3 and the catheter 1 and fixed to the catheter hub 11 coaxially with the catheter 1, and a piercing member 5 comprising a piercing needle 51 provided at distal end thereof, a wire 52 and a wire hub 53, where the piercing member 5 is slidably inserted into the catheter 1 such that the distal end of the piercing needle 5 is disposed to extend beyond the distal end of the catheter 1. A long accommodating tube 6 for accommodating a piercing member therein may be provided between the catheter hub 11 and the wire hub 53.

1. Field of the Invention
The present invention relates to a catheter assembly capable of sanitarily and safely inserting a catheter into a desired position in a human body.
2. Description of Related Art
A catheter assembly is used for introducing a cannula made of a synthetic resin into veins, arteries or a coelom of a patient and injecting a liquid medicine or removing a fluid. When a catheter is inserted into, for example, a vein, there has been conventionally used a method where a piercing needle having an inner diameter larger than an outer diameter of a catheter is first pierced into a vein. A catheter is then guided to a desired position of the vein through a bore (lumen) of the piercing needle and, thereafter, only the catheter is left in the vein by removing the piercing needle from the vein. With this method there is a fear that blood will leak from the skin surface when the piercing needle is removed from the vein or that bacteria will invade the vein or the like when the piercing needle is removed from the vein. Accordingly, the insertion operation of the catheter must be carried out under an aseptic condition.
Therefore, there has been proposed a catheter assembly capable of aseptically inserting a catheter into a human body by guiding the catheter using a sheath without touching the catheter (Japanese Unexamined Patent Publication No. JP-A-2-13471, Japanese Unexamined Patent Publication No. JP-A-2-111376).
The catheter assembly according to Japanese Unexamined Patent Publication No. JP-A-2-13471, is comprised of a cannula, an inserter for slidably holding the cannula and a hub structure attached to a proximal end of the cannula, and is characterized in that a sheath is attached to the hub structure and surrounds the cannula, and the inserter is provided with a sheath stripping means and sheath locking members. After a distal end of the catheter is inserted into a human body along with a needle and the needle is removed from the catheter, the cannula can be inserted further into the body by pulling a tab of the sheath projecting at a distal end thereof. The cannula integrated with the sheath by the hub structure is prevented from retracting by sheath locking members.
Meanwhile, the catheter assembly according to Japanese Unexamined Patent Publication No. JP-A-2-111376, is comprised of a cannula, an inserter for slidably holding the cannula and a hub structure attached to a proximal end of the cannula, and characterized in that a long accommodating tube for accommodating a needle (long piercing member) is provided therein. After a distal end of the catheter is inserted into a human body along with the needle and the needle is removed from the catheter, the needle contaminated with blood can be accommodated in the long accommodating tube.
However, with the a catheter assembly disclosed in Japanese Unexamined Patent Publication No. JP-A-2-13471, there is a danger of exposing the catheter to the outer atmosphere immediately in front of the sheath stripping means when stripping the sheath from the catheter, and insertion failure or breakage of catheter may be caused because the stripping resistance of the sheath from the catheter is large. Further, erroneous insertion may be caused since the resistance in inserting the catheter into a blood vessel is difficult to be perceived by an operator since the stripping resistance of the sheath from the catheter is large. Further, a manipulatively familiar conventional method in which a catheter is inserted into a blood vessel by pushing it is preferably used if safety in inserting the catheter is regarded as important. However, according to the above-described catheter assembly, although the catheter can be inserted into a blood vessel by pushing it if the rigidity of the sheath is increased, the stripping resistance of the sheath is also increased. On the other hand when the rigidity of the sheath is weakened, the sheath cannot be inserted by pushing it since kinking or bending is caused in the sheath.
Meanwhile, according to the catheter assembly of Japanese Unexamined Patent Publication No. JP-A-2-111376, although the needle can be accommodated in the long accommodating tube, if blood is adhered to a wire, the operator can touch the wire with his hand and, accordingly, the danger of blood borne infections still remains.
The present invention has been developed in view of the above-described situation and it is an object of the present invention to provide a catheter assembly capable of sanitarily and safely inserting a catheter into a desired position in a human body by pushing it. Further, it is an object of the present invention to provide a catheter assembly capable of safely disposing of a piercing needle which has been removed after inserting a catheter.


A method of determining whether or not a nucleic acid having an expected sequence or one or more variants of the expected sequence are present in a sample containing nucleic acids after replication, transcription or editing (or other transformation) of a substrate nucleic acid. The method involves deciding an expected sequence likely to be formed in the sample upon the replication, transcription or editing of the substrate nucleic acid, and possible variants of the expected sequence, providing primer pairs for a polymerase chain reaction, reverse transcriptase polymerase chain reaction or ligase chain reaction, carrying out the polymerase chain reaction or reverse transcriptase polymerase chain reaction in one or more steps to form amplicons, and analyzing the amplicons to determining whether or not a nucleic acid having the expected sequence and/or variants are present in the sample. The primers of the primer pairs are designed to anneal to regions of the nucleic acid of the expected sequence and the variants, the regions being selected to reveal unambiguously the presence or absence in the sample of the nucleic acid of the expected sequence or the variants thereof according to the presence or absence of specific amplicons amplified by the primers.

1. Field of the Invention
This invention relates to means for determining variants of an expected nucleic acid sequence in the population formed when a substrate nucleic acid sequence is replicated, transcribed, edited, or transformed in similar ways. More particularly, the invention relates to the determination of the formation of expected sequences or variants upon such transformations to establish a better understanding and utilization of the role of such variants in genetic processes.
2. Background Art
All life forms have specific genomes that are based either on DNA or RNA (referred to collectively herein as nucleic acids). During cell processing, nucleic acids derived from the genome or other sources are frequently and routinely copied (replicated), transcribed, edited (in the case of eukaryotic organisms), and eventually translated into proteins. During such processing, there is normally an "expected" nucleic acid sequence that results from "normal" replication, transcription or editing, but frequently one or more variants of the expected sequence are formed either in addition to the expected sequence or in place of it. The formation of such variants can result from "errors" in the replication, transcription or editing processes, or may possibly result from normal cell processes, e.g. when genes overlap.
Knowledge of the formation of one or more variants of an expected sequence when nucleic acid transformations take place can provide useful information for scientists in various fields. For example, the formation of sequence variants may be indicative of disease in a particular individual or may help to explain a particular cellular process.
For example, in higher life forms (based on eukaryotic cells), including humans, the primary RNA transcript formed directly from a genetic DNA sequence undergoes editing by the cell before it is used as a template for protein formation (translation). During this process, non-coding regions of the gene (introns) are removed and coding regions (exons) are spliced together to form a functional mRNA template used for protein synthesis. Often, in nature, this editing process leads to the formation of different versions of mRNA (often referred to as "splice variants"). These different versions or variants may differ in the number and/or order of the exons incorporated into the mRNA, as well as the variation of nucleotides across the junction or splice points of adjacent exons.
It is estimated that more than 70% of gene expression events encounter splice variations, thus resulting in variations in expressed proteins, which undermines the established concept that one gene leads to only one version of the protein. The presence or level of specific splice variants may be the cause or an indicator of a disease, disorder, pathological condition or normal condition. Understanding the distribution of splice variants in various tissues is extremely important for understanding the physiological function of genes and for targeting pharmaceuticals in drug discovery, drug evaluation, as well as for diagnostic purposes.
The formation of variants in this way is not limited to the translation of DNA to mRNA in eukaryotic cells. Even in prokaryotic cells (where there are no introns and thus no editing of RNA transcripts), there may be minor variations (mutations) in some of the transcripts. Moreover, during cell division in both prokaryotic and eukaryotic cells, DNA to DNA copies are made, involving a number of enzymes (e.g RNA primase, DNA polymerase, exonuclease, ligase, etc.). Although these steps have built-in proof-reading mechanisms, such replication may result in variations in the copies of DNA formed leading to various abnormalities or diseases (Ref. 1, see References at the end of this disclosure).
A common way of determining the transcription products of cells of a tissue is to employ a DNA micro-array (often called a "gene chip"). In this method, short probes (each representing a gene) are printed on and secured to a slide or chip. The cDNA of the tested tissue, fluorescently labeled, is hybridized to the micro-array. The level of the resulting signal corresponds to the relative expression level of the associated nucleic segment in the population. However, such micro-arrays do not account for the variants among the population formed that carry such nucleic acid segments. Hence it is not the presence or absence of a nucleic acid segment that is important, but to determine and characterize the nucleic sequence species that carry those segments, e.g. if there is a nucleic segment (for example, an exon) that determines a drug binding site on the resultant protein, it is essential to know whether that exon is carried by a specific mRNA so that there will be proper folding of the protein resulting in specific protein structure conducive for binding of the drug.
In the recent past, computational modeling has been adopted as an alternative method for predicting protein structure, which is based on the amino acid sequences (primary structures) of proteins. Technical limitations of determining an amino acid sequence directly from the protein have made it necessary to predict the amino acid sequence from the nucleotide sequence of the mRNA template. The existence of splice variants formed during gene expression interferes with such predictions and has created two main problems for accurate prediction of protein structures; (a) exon composition in various mRNA transcripts formed (b) errors in splicing itself at the exon/exon junction of mRNA templates.
Presently, there is no method available to accurately determine all of the different transcripts that may be simultaneously expressed from a gene.
A method of determining all the sequence variants resulting from nucleic acid transformations would therefore be of practical significance.

Determination of variants produced upon replication or transcription of nucleic acid sequences


A method and system for regulating tasks of background processes so as to reduce interference with foreground processes. The progress rate of a background task (e.g., amount of work performed per unit time) is measured and evaluated against a target amount. If the progress rate appears degraded, the background task is suspended for a computed time interval so as to back off from its interference with a foreground process. Each time the progress rate appears degraded, the time interval is exponentially increased from its previous value up to a maximum, however if the performance appears normal, the time interval is reset to a minimum. Evaluation of the work is statistically based so as to eliminate variations in measurements, and automatic calibration of the target amount is provided, as is a mechanism for prioritizing multiple background tasks.

Many computer systems execute background processes. The tasks of these processes are often directed to performing maintenance (housekeeping) functions, such as file compression, disk defragmentation, file-system content indexing and file archiving. While these tasks are relatively important to complete at some time, there is ordinarily no need to do so at any particular time. Thus, they are often run in the background, operating when no higher-priority foreground process, such as a process of an application program interacting with a user, is operating.
The traditional approach to operating a background process is to assign the process a priority level that is just above the system idle process, whereby CPU (central processing unit) cycles are allotted to the background process only when no normal priority process is ready to use the CPU. For example, in the Unix programming environment, the command "nice" initiates a process with reduced scheduling priority, which may be set as low as the system idle priority level.
Governing the execution of a process by priority scheduling of CPU cycles assumes that the CPU is the limiting resource. However, in many instances, the CPU is not the limiting resource, but instead, process performance may be limited by I/O (input/output) rate and/or contention for another system resource, such as the disk, processor cache, memory capacity and/or memory bandwidth. Since background processes contend for these resources, background processes interfere with higher priority foreground processes, and scheduling priority is insufficient as a mechanism for limiting this interference. The interference increases with background processes related to file-system maintenance activities, since such activities are resource intensive, but often use few CPU cycles.
By way of example, consider a background process that wants to access the same I/O resource (e.g., a disk) as a foreground process, wherein both processes are regularly performing I/O operations to the disk resource that take longer than the amount of time each process needs the CPU. When the foreground process is performing disk I/O operations, the background process is given some CPU cycles, during which time the background process desires access to the disk resource. As can be appreciated, the background process has to wait for completion of the I/O by the foreground process.
When the foreground process completes its I/O operation, the background process is given access to the disk and the foreground process is given CPU cycles, during which time the foreground process again wants access to the disk resource. However, because the foreground process has to wait for access to the disk resource, the foreground process has to idle, and at that moment, the CPU is essentially not being used by either the foreground or background process. Accordingly, the CPU is not the limiting resource, but rather the disk is, i.e., contention for the disk resource by the background process causes the interference with the foreground process. Unless the background process is entirely halted, however, the background process will receive CPU cycles when the foreground process is performing I/O, and thus will continue to interfere with the foreground process because the background process also will request access to the disk resource during its allotted cycles.
A foreground application program's process that is of immediate consequence to a user is thus negatively impacted by (non-CPU) resource contention, unless the background process is canceled. However, simply canceling the background process defeats the many advantages of having a background process run, e.g., to perform useful work during the many times when the foreground process is not contending with the background process for the CPU or other resource.

This patent is subject to a terminal disclaimer.

Method and system for regulating background tasks using performance measurements