The present invention provides pharmaceutical compositions of salicylanilide inhibitors of transcription factor NF-xcexaB, and methods for treating diseases in which activation of NF-xcexaB is implicated. More specifically, the present invention provides methods of treatment of a variety of diseases associated with NF-xcexaB activation including inflammatory disorders; particularly rheumatoid arthritis, inflammatory bowel disease, and asthma; dermatosis, including psoriasis and atopic dermatitis; autoimmune diseases; tissue and organ rejection; Alzheimer""s disease; stroke; atherosclerosis; restenosis; cancer, including Hodgkin""s disease; certain viral infections, including AIDS; osteoarthritis; osteoporosis; and Ataxia Telangiestasia by administering to a patient in need thereof a compound of the present invention.

This invention relates in general to salicylanilide inhibitors of transcription factor NF-xcexaB. Such compounds are particularly useful for treating diseases in which activation of NF-xcexaB is implicated. More specifically, these compounds inhibit IxcexaB phosphorylation and subsequent degradation. Such compounds are useful in the treatment of a variety of diseases associated with NF-xcexaB activation including inflammatory disorders; particularly rheumatoid arthritis, inflammatory bowel disease, and asthma; dermatosis, including psoriasis and atopic dermatitis; autoimmune diseases; tissue and organ rejection; Alzheimer""s disease; stroke; atherosclerosis; restenosis; cancer, including Hodgkins disease; and certain viral infections, including AIDS; osteoarthritis; osteoporosis; and Ataxia Telangiestasia.
Recent advances in scientific understanding of the mediators involved in acute and chronic inflammatory diseases and cancer have led to new strategies in the search for effective therapeutics. Traditional approaches include direct target intervention such as the use of specific antibodies, receptor antagonists, or enzyme inhibitors. Recent breakthroughs in the elucidation of regulatory mechanisms involved in the transcription and translation of a variety of mediators have led to increased interest in therapeutic approaches directed at the level of gene transcription.
NF-xcexaB belongs to a family of closely related dimeric transcription factor complexes composed of various combinations of the Rel/NF-xcexaB family of polypeptides. The family consists of five individual gene products in mammals, RelA (p65), NF-xcexaB1 (p50/p105), NF-xcexaB2 (p49/p100), c-Rel, and RelB, all of which can form hetero- or homodimers. These proteins share a highly homologous 300 amino acid xe2x80x9cRel homology domainxe2x80x9d which contains the DNA binding and dimerization domains. At the extreme C-terminus of the Rel homology domain is a nuclear translocation sequence important in the transport of NF-xcexaB from the cytoplasm to the nucleus. In addition, p65 and cRel possess potent transactivation domains at their C-terminal ends.
The activity of NF-xcexaB is regulated by its interaction with a member of the inhibitor IxcexaB family of proteins. This interaction effectively blocks the nuclear localization sequence on the NF-xcexaB proteins, thus preventing-migration-of the dimer to the nucleus. A wide variety of stimuli activate NF-xcexaB through what are likely to be multiple signal transduction pathways. Included are bacterial products (LPS), some viruses (HIV-1, HTLV-1), inflammatory cytokines (TNFxcex1, IL-1), and environmental stress. Apparently common to all stimuli however, is the phosphorylation and subsequent degradation of IxcexaB. IxcexaB is phosphorylated on two N-terminal serines by the recently identified IxcexaB kinases (IKK-xcex1 and IKK-xcex2). Site-directed mutagenesis studies indicate that these phosphorylations are critical for the subsequent activation of NF-xcexaB in that once phosphorylated the protein is flagged for degradation via the ubiquitin-proteasome pathway. Free from IxcexaB, the active NF-xcexaB complexes are able to translocate to the nucleus where they bind in a selective manner to preferred gene-specific enhancer sequences. Included in the genes regulated by NF-xcexaB are a number of cytokines, cell adhesion molecules, and acute phase proteins.
It is well-known that NF-xcexaB plays a key role in the regulated expression of a large number of pro-inflammatory mediators including cytokines such as IL-6 and IL-8. Cell adhesion molecules, such as ICAM and VCAM, and inducible nitric oxide synthase (iNOS). Such mediators are known to play a role in the recruitment of leukocytes at sites of inflammation and in the case of iNOS, may lead to organ destruction in some inflammatory and autoimmune diseases.
The importance of NF-xcexaB in inflammatory disorders is further strengthened by studies of airway inflammation including asthma, in which NF-xcexaB has been shown to be activated. This activation may underlie the increased cytokine production and leukocyte infiltration characteristic of these disorders. In addition, inhaled steroids are known to reduce airway hyperresponsiveness and suppress the inflammatory response in asthmatic airways. In light of the recent findings with regard to glucocorticoid inhibition of NFxcexaB, one may speculate that these effects are mediated through an inhibition of NFxcexaB.
Further evidence for a role of NF-xcexaB in inflammatory disorders comes from studies of rheumatoid synovium. Although NF-xcexaB is normally present as an inactive cytoplasmic complex, recent immunohistochemical studies have indicated that NF-xcexaB is present in the nuclei, and hence active, in the cells comprising rheumatoid synovium. Furthermore, NF-xcexaB has been shown to be activated in human synovial cells in response to stimulation with TNF-xcex1. Such a distribution may be the underlying mechanism for the increased cytokine and eicosanoid production characteristic of this tissue. See Roshak, A. K., et al., J. Biol. Chem., 271, 31496-31501 (1996).
The NF-xcexaB/Rel and IxcexaB proteins are also likely to play a key role in neoplastic transformation. Family members are associated with cell transformation in vitro and in vivo as a result of overexpression, gene amplification, gene rearrangements or translocations. In addition, rearrangement and/or amplification of the genes encoding these proteins are seen in 20-25% of certain human lymphoid tumors. In addition, a role for NF-xcexaB in the regulation of apoptosis has been reported stregthening the role of this transcription factor in the control of cell proliferation.
Several NF-xcexaB inhibitors are described in C. Wahl, et al. J. Clin. Invest. 101(5), 1163-1174 (1998), R. W. Sullivan, et al. J. Med. Chem. 41, 413-419 (1998), J. W. Pierce, et al. J. Biol. Chem. 272, 21096-21103 (1997)
The marine natural product hymenialdisine is known to inhibit NF-xcexaB. Roshak, A., et al., JPET, 283, 955-961 (1997). Breton, J. J and Chabot-Fletcher, M. C., JPET, 282, 459-466 (1997).
Salicylanilides are known compounds. A general solution preparation of salicyanilides is described by M. T. Clark, R. A. Coburn. R. T. Evans, R. J. Genco, J. Med. Chem., 1986, 29, 25-29.
We have now discovered a novel method of inhibiting, the activation transcription factor NF-xcexaB using salicylanilides.
An object of the present invention is to provide a method for treating diseases which may be therapeutically modified by altering the activity of transcription factor NF-xcexaB.
Accordingly, in the first aspect, this invention provides a pharmaceutical composition comprising a compound according to Formula I.
In still another aspect, this invention provides a method of treating diseases in which the disease pathology may be therapeutically modified by inhibiting NFxcexaB.
In a particular aspect, this invention provides methods for treating a variety of diseases associated with NF-xcexaB activation including inflammatory disorders; particularly rheumatoid arthritis, inflammatory bowel disease, and asthma; dermatosis, including psoriasis and atopic dermatitis; autoimmune diseases; tissue and organ rejection; Alzheimer""s disease; stroke; atherosclerosis; restenosis; cancer, including Hodgkins disease; and certain viral infections, including AIDS; osteoarthritis; osteoporosis; and Ataxia Telangiestasia.
The present invention provides a method of treatment of diseases associated with NF-xcexaB activation, comprising administering to an animal, particularly a mammal, most particularly a human in need thereof a compound of Formula I:
wherein:
RA substitutes ring A 0-3 times and is independently selected from the group consisting of: NO2, halogen,C1-6alkyl, trifluoromethyl. O-C1-6alkyl and S-C1-6alkyl; and
RB substitutes ring B 0-3 times and is independently selected from the group consisting of: halogen,C(O)C1-6alkyl, C1-6alkyl, Oxe2x80x94C1-6alkyl. Sxe2x80x94C1-6alkyl, CH2-aryl, and aryl;
and pharmaceutically acceptable salts, hydrates and solvates thereof.
The present invention further provides a preferred method of treatment of diseases associated with NF-xcexaB activation, comprising administering to an animal, particularly a mammal, most particularly a human in need thereof a compound of Formula II:
wherein RA of Formula I occurs once and is R1; and RB of Formula I occurs twice and is independently R2 and R3. More specifically:
R1 is selected from the group consisting of: H, NO2, CF3, F, Cl, Br, and I;
R2 is selected from the group consisting of: H and F; and
R3 is selected from the group consisting of: F, Cl, Br, I, phenyl and C(O)C1-6alkyl, preferably C1-6alkyl is CH3;
and pharmaceutically acceptable salts, hydrates and solvates thereof.
Compounds of Formula II selected from the following group are most preferred for use in the methods of the present invention:
N-(4-phenyl-phenyl)-2-hydroxy-5-trifluoromethylcarboxamide;
N-(2,4-Difluorophenyl)-2-hydroxy-5-nitrocarboxamide;
N-(2,4-Difluorophenyl)-2-hydroxy-5-iodocarboxamide, and
N-(4-acetylphenyl)-2-hydroxy-5-iodocarboxamide.
The present invention includes all hydrates, solvates, complexes and prodrugs of the compounds of this invention. Prodrugs are any covalently bonded compounds which release the active parent drug according to Formulas I and II in vivo. If a chiral center or another form of an isomeric center is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereomers, are intended to be covered herein. Inventive compounds containing a chiral center may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. In cases in which compounds have unsaturated carbon-carbon double bonds, both the cis (Z) and trans (E) isomers are within the scope of this invention. In cases wherein compounds may exist in tautomeric forms, such as keto-enol tautomers, each tautomeric form is contemplated as being included within this invention whether existing in equilibrium or predominantly in one form.
The meaning of any substituent at any one occurrence in Formula I or any subformula thereof is independent of its meaning, or any other substituent""s meaning, at any other occurrence, unless specified otherwise.
xe2x80x9cC1-6alkylxe2x80x9d as applied herein is meant to include substituted and unsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl, pentyl, n-pentyl, isopentyl, neopentyl and hexyl and the simple aliphatic isomers thereof. Any C1-6alkyl group may be optionally substituted independently by one or two halogens, SRxe2x80x2, ORxe2x80x2,N(Rxe2x80x2)2, C(O)N(Rxe2x80x2)2, carbamyl or C1-4alkyl, where Rxe2x80x2 is C1-6alkyl.
xe2x80x9cHalogenxe2x80x9d as applied herein is meant to include F, Cl, Br, and I.
xe2x80x9cArxe2x80x9d or xe2x80x9carylxe2x80x9d as applied herein is meant to include phenyl or naphthyl, optionally substituted by one or more of Ph-C0-6alkyl; Het-C0-6alkyl; C1-6alkoxy; Ph-C0-6alkoxy; Het-C0-6alkoxy; OH, (CH2)1-6NR4R5; O(CH2)1-6NR4R5; C1-6alkyl, ORxe2x80x3, N(Rxe2x80x3)2, SRxe2x80x3, CF3, NO2, CN, CO2Rxe2x80x3, CON(Rxe2x80x3), F, Cl, Br or I; where R4 and R5 are H, C1-6alkyl. Ph-C0-6alkyl, or naphthyl-C0-6alkyl; and Rxe2x80x3 is phenyl, naphthyl, or C1-6alkyl.
The compounds of the present invention may be conveniently prepared by the methods set forth in Schemes 1 and 2 below.
A general solution preparation of salicyanilides is described by M. T. Clark, R. A. Coburn, R. T. Evans, R. J. Genco; J. Med. Chem; 1986; 29; 25-29. These compounds may also be conveniently prepared by solid phase techniques. including in the form of a library.
General Preparation:
The general solution preparation is shown in Scheme 1 and 2. A salicylanilide is prepared by the condensation of a substituted salicylic acid with a substituted aniline in the presence of phosphorous trichloride in dry chlorohenzene (Scheme 1). A salicylanilde can also be prepared by conversion of a substituted salicylic acid to its corresponding acid chloride with thionyl chloride in toluene with catalytic DMF. The resulting salicylic acid chloride and a substituted aniline is then heated in toluene to form a salicyanilide (Scheme 2).
Referring to the methods of preparing the compounds of Formula I set forth in Schemes 1 and 2 above, the skilled artisan will appreciate that the present invention includes all novel intermediates required to make the compounds of Formula I.
The starting materials used herein are commercially available or are prepared by routine methods well known to those of ordinary skill in the art and can be found in standard reference books, such as the COMPENDIUM OF ORGANIC SYNTHETIC METHODS, Vol. I-VI (published by Wiley-Interscience).
Acid addition salts of the compounds of Formula I are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, hydrofluoric, sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic or methanesulfonic. Certain of the compounds form inner salts or zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in pharmaceutically acceptable salts. Halides, sulfate, phosphate, alkanoates (such as acetate and trifluoroacetate), benzoates, and sulfonates (such as mesylate) are examples of anions present in pharmaceutically acceptable salts.
This invention provides a pharmaceutical composition which comprises a compound according to Formula I and a pharmaceutically acceptable carrier, diluent or excipient. Accordingly, the compounds of Formula I may be used in the manufacture of a medicament. Pharmaceutical compositions of the compounds of Formula I prepared as hereinbefore described may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insulation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these compounds may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about {circumflex over (20)} mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
For rectal administration, the compounds of this invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
The methods of the present invention include topical administration of the compounds of Formulas I and II. By topical administration is meant non-systemic administration, including the application of a compound of the invention externally to the epidermis, to the buccal cavity and instillation of such a compound into the ear, eye and nose, wherein the compound does not significantly enter the blood stream. By systemic administration is meant oral, intravenous, intraperitoneal and intramuscular administration. The amount of a compound of the invention (hereinafter referred to as the active ingredient) required for therapeutic or prophylactic effect upon topical administration will, of course, vary with the compound chosen, the nature and severity of the condition being treated and the animal undergoing treatment, and is ultimately at the discretion of the physician.
While it is possible for an active ingredient to be administered alone as the raw chemical, it is preferable to present it as a pharmaceutical formulation. The active ingredient may comprise, for topical administration, from 0.01 to 5.0 wt %. of the formulation.
The topical formulations of the present invention, both for veterinary and for human medical use, comprise an active ingredient together with one or more acceptable carriers therefor, and optionally any other therapeutic ingredients. The carrier must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required such as: liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions and may be prepared by dissolving the active ingredient in a suitable aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container which is then sealed and sterilized by autoclaving or maintaining at 90-100 C for half an hour. Alternatively, the solution may be sterilized by filtration and transferred to the container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturizer such as glycerol or an oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with the aid of suitable machinery, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols. The formulation may incorporate any suitable surface active agent such as an anionic, cationic or non-ionic surface active agent such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or in organic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
The compounds of Formulas I and II are useful as inhibitors of NF-xcexaB. The present invention provides useful compositions and formulations of said compounds, including pharmaceutical compositions and formulations of said compounds.
The present invention also provides methods of treatment of diseases associated with NF-xcexaB activation, which methods comprise administering to an animal, particularly a mammal, most particularly a human in need thereof a compound of Formulas I or II. The present invention particularly provides methods for treating inflammatory disorders; particularly rheumatoid arthritis, inflammatory bowel disease, and asthma; dermatosis, including psoriasis and atopic dennatitis; autoimmune diseases; tissue and organ rejection; Alzheimer""s disease; stroke; atherosclerosis; restenosis; cancer, including Hodgkins disease; and certain viral infections, including AIDS; osteoarthritis; osteoporosis; and Ataxia Telangiestasia.
For acute therapy, parenteral administration of a compound of Formulas I or II is preferred. An intravenous infusion of the compound in 5% dextrose in water or normal saline, or a similar formulation with suitable excipients, is most effective, although an intramuscular bolus injection is also useful. Typically, the parenteral dose will be about 0.01 to about 50 mg/kg; preferably between 0.1 and 20 mg/kg, in a manner to maintain the concentration of drug in the plasma at a concentration effective to inhibit activation of NF-xcexaB. The compounds are administered one to four times daily at a level to achieve a total daily dose of about 0.4 to about 80 mg/kg/day. The precise amount of an inventive compound which is therapeutically effective, and the route by which such compound is best administered, is readily determined by one of ordinary skill in the art by comparing the blood level of the agent to the concentration required to have a therapeutic effect.
The compounds of Formulas I and II may also be administered orally to the patient, in a manner such that the concentration of drug is sufficient to inhibit NF-xcexaB or to achieve any other therapeutic indication as disclosed herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of between about 0.1 to about 50 mg/kg in a manner consistent with the condition of the patient. Preferably the oral dose would be about 0.5 to about 20 mg/kg.
The compounds of Formulas I and II may also be administered topically to the patient, in a manner such that the concentration of drug is sufficient to inhibit NF-xcexaB or to achieve any other therapeutic indication as disclosed herein. Typically, a pharmaceutical composition containing the compound is administered in a topical formulation of between about 0.01% to about 5% w/w.
No unacceptable toxicological effects are expected when compounds of the present invention are administered in accordance with the present invention.
The ability of the compounds described herein to inhibit the activation of NF-xcexaB is clearly evidenced in their ability to inibit NF-xcexaB-driven reporter gene activity (see Table 1). The utility of the present NF-xcexaB inhibitors in the therapy of diseases is premised on the importance of NF-xcexaB activation in a variety of diseases.
NFxcexaB plays a key role in the regulated expression of a large number of pro-inflammatory mediators including cytokines such as IL-6 and IL-8 (Mukaida et al., 1990; Liberman and Baltimore, 1990; Matsusaka et al., 1993), cell adhesion molecules, such as ICAM and VCAM (Marui et al., 1993; Kawai et al., 1995; Ledebur and Parks, 1995), and inducible nitric oxide synthase (iNOS) (Xie et al., 1994; Adcock et al., 1994). (Full reference citations are at the end of this section). Such mediators are known to play a role in the recruitment of leukocytes at sites of inflammation and in the case of iNOS, may lead to organ destruction in some inflammatory and autoimmune diseases (McCartney-Francis et al., 1993; Kleemann et al., 1993). Importantly, the compounds described herein inhibit IL-8 synthesis and the production of nitric oxide, a product of iNOS activity (see Table 2).
Evidence for an important role of NF-xcexaB in inflammatory disorders is obtained in studies of asthmatic patients. Bronchial biopsies taken from mild atopic asthmatics show significant increases in the number of cells in the submucosa staining for activated NFxcexaB, total NF-xcexaB, and NF-xcexaB-regulated cytokines such as GM-CSF and TNFxcex1 compared to biopsies from normal non-atopic controls (Wilson et al., 1998). Furthermore, the percentage of vessels expressing NF-xcexaB immunoreactivity is increased as is IL-8 immunoreactivity in the epithelium of the biopsy specimens (Wilson et al., 1998). As such, inhibition of IL-8 production through the inhibition of NFxcexaB, as has been demonstrated by these compounds would be predicted be beneficial in airway inflammation.
Recent studies suggest that NF-xcexaB may also play a critical role in the pathogenesis of inflammatory bowel disease (IBD). Activated NF-xcexaB is seen in colonic biopsy specimens from Chron""s disease and ulcerative colitis patients (Ardite et al., 1998; Rogler et al., 1998; Schreiber et al., 1998). Activation is evident in the inflamed mucosa but not in uninflamed mucosa (Ardite et al., 1998; Rogler et al., 1998) and is associated with increased IL-8 mRNA expression in the same sites (Ardite et al., 1998). Furthermore, corticosteroid treatment strongly inhibits intestinal NF-xcexaB activation and reduces colonic inflammation (Ardite et al., 1998; Schreiber et al., 1998). Again, inhibition of IL-8 production through the inhibition of NF-xcexaB, as has been demonstrated by these compounds would be predicted be beneficial in inflammatory bowel disease.
Animal models of gastrointestinal inflammation provide further support for NF-xcexaB as a key regulator of colonic inflammation. Increased NF-xcexaB activity is observed in the lamina propria macrophages in 2,4,6,-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice with p65 being, a major component of the activated complexes (Neurath et al., 1996; Neurath and Pettersson, 1997). Local administration of p65 antisense abrogates the signs of established colitis in the treated animals with no signs of toxicity (Neurath et al., 1996; Neurath and Pettersson, 1997). As such, one would predict that small molecule inhibitors of NF-xcexaB would be useful in the treatment of IBD.
Further evidence for a role of NF-xcexaB in inflammatory disorders comes from studies of rheumatoid synovium. Although NF-xcexaB is normally present as an inactive cytoplasmic complex, recent immunohistochemical studies have indicated that NF-xcexaB is present in the nuclei, and hence active, in the cellscomprising human rheumatoid synovium (Handel et al., 1995; Marok et al., 1996; Sioud et al., 1998) and in animal models of the disease (Tsao et al., 1997). The staining is associated with type A synoviocytes and vascular endothelium (Marok et al., 1996). Furthermore, constitutive activation of NF-xcexaB is seen in cultured synoviocytes (Roshak et al., 1996; Miyazawa et al., 1998) and in synovial cell cultures stimulated with IL-1xcex2 or TNFxcex1 (Roshak et al., 1996; Fujisawa et al., 1996; Roshak et al., 1997). Thus, the activation of NF-xcexaB may underlie the increased cytokine production and leukocyte infiltration characteristic of inflamed synovium. The ability of these compounds to inhibit NF-xcexaB and thereby inhibit the production of eicosanoids by these cells would be predicted to yield benefit in rheumatoid arthritis (see Table 2).
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Miyazawa K, Mori A, Yamamoto K, Okudaira H (1998) Constitutive transcription of the human interleukin-6 gene by rheumatoid synoviocytes; spontaneous activation of NF-xcexaB and CBF1. Am J Pathol 152, 793-803
Fujisawa K, Aono H, Hasunuma T, Yamamoto K, Mita S, Nishiola K (1996) Activation of transcription factor NF-xcexaB in human synovial cells in response to tumor necrosis factor xcex1. Arthritis Rheumatism 39: 197-203
Roshak A K, Jackson J R, Chabot-Fletcher M, Marshall L (1997) Inhibition of NFxcexaB-mediated interleukin-1xcex2-stimulated prostaglandin E2 formation by the marine natural product hymenialdisine. J Pharmacol Exp Therapeut 283: 955-961
The compounds of this invention may be tested in one of several biological assays to determine the concentration of compound which is required to have a given pharmacological effect.
Assays of NF-xcexaB activity are conducted using a cell based luciferase reporter assay as described in Breton, J. J and Chabot-Fletcher, M. C. JPET, 282, 459-466 (1997). Briefly, U937 human histiocytic lymphoma cell line permanently transfected with the NF-xcexaB reporter plasmids (see below) are cultured in the above medium with the addition of 250 xcexcg/ml Geneticin (G418 sulfate, Life Technologies, Grand Island, N.Y.). The luciferase reporter assay is conducted in the transfected U937 clones. These are twice centrifuged at 300xc3x97g for 5 min and resuspended in RPMI 1640 with 10% FBS to a density of 1xc3x97106 cells/ml. One ml aliquots are added to the wells of 24-well plates. Compound or dimethyl sulfoxide (DMSO) carrier (1 xcexcl) is added to the appropriate wells and the plates are incubated at 37xc2x0 C., 5% CO2 for 30 min. The stimulus is added (5 ng/ml TNFxcex1, 100 ng/ml LPS, or 0.1 xcexcM PMA) and the samples incubated for 5 hours at 37xc2x0 C., 5% CO2, transferred to 1.9 ml polypropylene tubes, and centrifuged at 200xc3x97g for 5 min. The cell pellets are washed twice in 1 ml PBS without Ca2+ and Mg2+, and centrifuged as indicated above. The resulting cell pellets are lysed in 50 xcexcl 1xc3x97 lysis buffer (Promega Corporation, Madison, Wis.), vortexed and incubated for 15 min at room temperature. A 20 xcexcl aliquot of each lysate is transferred to an opaque white 96-well plate (Wallac Inc., Gaithersburg, Md.) and assayed for luciferase production in a MicroLumat LB 96 P luminometer (EGandG Berthold, Bad Wilbad, Germany). The luminometer dispenses 100 xcexcl luciferase assay reagent (Promega Corporation, Madison, Wis.) into each well and the integrated light output is recorded for 20 sec. Light output is measured in relative light units (RLUs).
NF-xcexaB activity may also be measured in an electrophoretic mobility shift assay (EMSA) to assess the presence of NF-xcexaB protein in the nucleus. The cells of interest are cultured to a density of 1xc3x97106/ml. The cells are harvested by centrifugation, washed in PBS without Ca2+ and Mg2+ and resuspended in PBS with Ca2+ and Mg2+ at 1xc3x97107 cells/ml. To examine the effect of compound on the activation of NF-xcexaB, the cell suspensions are treated with various concentrations of drug or vehicle (DMSO, 0.1%) for 30 min at 37xc2x0 C. prior to stimulation with TNFxcex1 (5.0 ng/ml) for an additional 15 min. Cellular and nuclear extracts are prepared follows Briefly, at the end of the incubation period the cells (1xc3x97107 cells) are washed 2xc3x97 in PBS without Ca2+ and Mg2+. The resulting cell pellets are resuspended in 20 xcexcl of Buffer A (10 mM Hepes (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol (DTT) and 0.1% NP-40) and incubated on ice for 10 min. The nuclei are pelleted by microcentrifugation at 3500 rpm for 10 min at 4xc2x0 C. The resulting supernatant was collected as the cellular extract and the nuclear pellet was resuspended in 15 xcexcl Buffer C (20 mM Hepes (pH 7.9), 0.42M NaCl, 1.5 mM MgCl2, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM phenylmethylsulphonyl fluoride (PMSF)). The suspensions are mixed gently for 20 min at 4xc2x0 C. then microcentrifuged at 14,000 rpm for 10 min at 4xc2x0 C. The supernatant is collected and diluted to 60 xcexcl with Buffer D (20 mM Hepes (pH 7.9), 50 mM KCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF). All samples are stored at xe2x88x9280xc2x0 C. until analyzed. The protein concentration of the extracts is determined according to the method of Bradford (Bradford. 1976) with BioRad reagents.
The effect of compounds on transcription factor activation is assessed in an electrophoretic mobility shift assay (EMSA) using nuclear extracts from treated cells as described above. The double stranded NF-xcexaB consensus oligonucleotides (5xe2x80x2-AGTTGAGGGGACTTTCCCAGGC-3xe2x80x2) are labelled with T4 polynucleotide kinase and [g-32P]ATP. The binding mixture (25 xcexcl) contains 10 mM Hepes-NaOH (pH 7.9), 4 mM Tris-HCl (pH 7.9), 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.3 mg/ml bovine serum albumin, and 1 xcexcg poly(dI-dC)xe2x80xa2poly(dI-dC). The binding mixtures (10 xcexcg nuclear extract protein) are incubated for 20 min at room temperature with 0.5 ng of 32P-labelled oligonucleotide (50,000-100,000 cpm) in the presence or absence of unlabeled competitor after which the mixture is loaded on a 4% polyacrylamide gel prepared in 1xc3x97Tris borate/EDTA and electrophoresed at 200 V for 2 h. Following electrophoresis the gels are dried and exposed to film for detection of the binding reaction.
The effect of compounds on the phosphorylation of IxcexaB may be monitored in a Western blot. Cellular extracts are subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels (BioRad, Hercules, Calif.) and the proteins transferred to nitrocellulose sheets (Hybond(trademark)-ECL, Amersham Corp., Arlington Heights, Ill.). Immunoblot assays are performed using a polyclonal rabbit antibody directed against IxcexaBxcex1 or IxcexaBxcex2 followed with a peroxidase-conjugated donkey anti-rabbit secondary antibody (Amersham Corp., Arlington Heights, Ill.). Immunoreactive bands are detected using the Enchanced Chemiluminescence (ECL) assay system (Amersham Corp., Arlington Heights, Ill.).
Effects on eicosanoid production by human synovial fibroblasts (RSF) are assessed using primary cultures of human RSF. These are obtained by enzymatic digestion of synovium obtained from adult patients with rheumatoid. Cells are cultured in Earl""s Minimal Essential Medium (EMEM) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 xcexcg/ml streptomycin (GIBCO, Grand Island, N.Y.), at 37xc2x0 C. and 5% CO2. Cultures are used at passages 4 through 9 in order to obtain a more uniform type I fibroblast population. For some studies, fibroblasts are plated at 5xc3x97104 cells/ml in 16 mm (diameter) 24 well plates (Costar, Cambridge, Mass.). Cells are exposed to an optimal dose of IL-1xcex2 (1 ng/ml; Roshak et al. 1996a)(Genzyme. Cambridge, Mass.) for the designated time. Drugs in DMSO vehicle (1%) are added to the cell cultures 15 minutes prior to the addition of IL-1. Prostaglandin E2 levels in cell-free medium collected at the termination of the culture period are directly measured using enzyme immunoassay (EIA) kits purchased from Cayman Chemical Co. (Ann Arbor, Mich.). Sample or standard dilutions are made with experimental medium.
Anti-inflammatory activity in vivo is assessed using the phorbol ester-induced ear inflammation model in mice. Phorbol myristate acetate (PMA) (4 xcexcg/20 xcexcl acetone) is applied to the inner and outer surfaces of the left ear of Male Balb/c mice (6/group) (Charles River Breeding Laboratories, Wilmington, Mass.). Four hours later, compound dissolved in 25 xcexcl acetone is applied to the same ear. The thickness of both ears is measured with a dial micrometer (Mitutoyo, Japan) after 20 hours and a second topical dose of compound is applied. Twenty-four hours later, ear thickness measurements are taken and the data expressed as the change in thickness (xc3x9710xe2x88x923 cm) between treated and untreated ears. The inflamed left ears are then removed and stored at xe2x88x9270xc2x0 until assayed for myeloperoxidase (MPO) activity, a measure of inflammatory cell infiltration.
Inflammatory cell infiltration is assessed through the measurement of myeloperoxidase activity present in the inflammed ear tissue. Partially thawed ear tissues are minced and then homogenized (10% w/v) with a Tissumizer homogenizer (Tekmar Co., Cincinnati, Ohio) in 50 mM phosphate buffer (pH 6) containing 0.5% HTAB. The tissue homogenates are taken through three cycles of freeze-thaw, followed by brief sonication (10s). MPO activity in the homogenates is determined as follows. The appearance of colored product from the MPO-dependent reaction of o-dianisidine (0.167 mg/ml, Sigma Chemical, St. Louis, Mo.) and hydrogen peroxide (0.0005%) is measured spectrophotometrically at 460 nm. Supernatant MPO activity is quantified kinetically (change in absorbance measured over 3 min, sampled at 15 s intervals) using a Beckman DU-7 spectrophotometer and a kinetics analysis package (Beckman Instruments, Inc., Sommerset, N.J.). One unit of MPO activity is defined as that degrading one micromole of peroxide per minute at 25xc2x0 C.
Effects on inflammation-mediated cartilage breakdown is measured in an in vitro cartilage explant system. In this model bovine articular cartilage explants are incubated for 4 days/96 hours with or without rHuIL-1 alpha to stimulate cartilage breakdown in the presence or absence of test compound. The supernatants are removed for the nitric oxide assays. Nitric oxide was measured using the Greiss reaction and read spectrophotometrically at 530 nm. This reaction measures nitrite (NO2) which is the stable end product of nitric oxide.
Nuclear magnetic resonance spectra were recorded at either 250, 300 or 400 MHz using, respectively, a Bruker AM 250, Bruker ARX 300 or Bruker AC 400 spectrometer. CDCl3 is deuteriochloroform, DMSO-d6 is hexadeuteriodimethylsulfoxide, and CD3OD is tetradeuteriomethanol. Chemical shifts are reported in parts per million (d) downfield from the internal standard tetramethylsilane. Abbreviations for NMR data are as follows: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, dd =doublet of doublets, dt=doublet of triplets, app=apparent, br=broad. J indicates the NMR coupling constant measured in Hertz. Continuous wave infrared (IR) spectra were recorded on a Perkin-Elmer 683 infrared spectrometer, and Fourier transform infrared (FTIR) spectra were recorded on a Nicolet Impact 400 D infrared spectrometer. IR and FTIR spectra were recorded in transmission mode, and band positions are reported in inverse wavenumbers (cmxe2x88x921). Mass spectra were taken on either VG 70 FE, PE Syx API III, or VG ZAB HF instruments, using fast atom bombardment (FAB) or electrospray (ES) ionization techniques. Elemental analyses were obtained using a Perkin-Elmer 240C elemental analyzer. Melting points were taken on a Thomas-Hoover melting point apparatus and are uncorrected. All temperatures are reported in degrees Celsius.
Analtech Silica Gel GF and E. Merck Silica Gel 60 F-254 thin layer plates were used for thin layer chromatography. Both flash and gravity chromatography were carried out on E. Merck Kieselgel 60 (230-400 mesh) silica gel.
Where indicated, certain of the materials were purchased from the Aldrich Chemical Co., Milwaukee, Wis., TCI America, Portland, Oreg.

Inhibitors of transcription factor-NF-xcexaB


Embodiments of the present invention provide techniques for efficiently coding video data during circumstances of network congestion, or instances when a decoder is unable to handle incoming video at the intended frame rate. A video coder may code frames of a video sequence according to motion estimation prediction in which each frame of the video sequence is constrained to be coded as one of an I-frame or a P-frame. The video coder may determine for each coded P-frame, a distance from the respective P-frame and a most-recently coded reference frame, and if the distance matches a predetermined threshold distance, the respective P-frame may be marked as a reference frame. The coded video data of the I-frame(s), the reference P-frames and the non-reference P-frames may be transmitted in a channel.

The present invention relates to video coding and, in particular, to video coding techniques that conserve bandwidth while at the same time preserving image quality.
Modern video coders typically exploit spatial and/or temporal redundancies in video sequences to reduce the number of bits necessary to represent the video. Coded representations of the video, therefore, are easier to store to transmit over communication networks. The video coders employ a variety of different coding techniques, including intra coding and inter coding. Video decoders receive the encoded video and decode the video so that the video can be displayed on a display device. In some instances, the video decoders may not be as sophisticated as the encoders and may not be able to decode the incoming video at the intended frame rate. In other instances, decoders may not be able to process the incoming video due to network congestion on the decoders' end.
The inventor perceives a need in the art for a video coding system that dynamically adjusts to circumstances of network congestion, or to instances when a decoder is unable to handle incoming video at the intended frame rate by changing decoding schemes.

High quality seamless playback for video decoder clients


A geomatics pole support comprises a body and pole engaging members for connecting the support to a geomatics pole so that the body is retained by the pole engaging members projecting generally laterally outwardly from a longitudinal axis of the pole. The body further includes a terrain engagement surface adapted to engage a ground surface and to hold at least a portion of the pole in a stable position out of contact with the ground surface.

This invention relates generally to devices which support various surveying and geomatics poles, and more particularly to a support suitable for steading poles during measurements, permitting calibration verification of leveling indication vials, and holding a portion of the pole out of contact with the ground.
During a typical survey, multiple and various measurements are often taken requiring varies degrees of accuracies. For example, measurements of property boundaries may require a greater degree of accuracy than measurements locating the position of fire hydrants, yet both measurements may be performed in the same survey using the same geomatics target device. In order to meet the specific accuracy or speed efficiency requirements of a particular measurement, survey crew members may be required to change the support holding a geomatics target device. The target devices include prismatic retroreflectors, global positioning system receivers and optical targets.
Commonly, various types of geomatics poles are used to support geomatic target devices. These geomatics poles typically are fitted with a ground engaging point on a base end, permitting precise indication of a ground point. To provide desired line of sight clearances, the target device is typically located on the apex of the pole, opposite of the base end. A level indication vial is commonly located at a point along the length of the pole to facilitate vertical collimation of the pole over the ground point in a process known as plumbing. Geomatics poles, which may be either fixed length or telescopic, are capable of retaining an established length throughout multiple measurements. Known and constant target heights save measurement calculation time. As a result, measurements can be obtained much faster with geomatics poles than with other supporting devices, such as tripods.
However, geomatics poles have several disadvantages. For one, the level indication vials of these poles often lose their accuracy calibration, which may cause inaccurate measurements. Because of this deficiency, level vial accuracy must be verified periodically. Many prior art level vial checking devices are dedicated only to the function of checking level vials and require being fixed to a wall. Thus, these wall mounted level vial checking devices are not available for use in the field.
For some measurements, geomatics poles may be handheld in vertical collimation over a ground point. Positioning a geomatics pole positioning by hand provides a fast and convenient method of taking measurements. However, the practice of handheld positioning of geomatics poles also has disadvantages. One of these disadvantages is that precise and extended retention of pole position is very difficult. Therefore, measurements taken with handheld poles are less precise than supported ones. Another disadvantage with handheld poles becomes evident when a surveyor must perform a task not involving the pole, such as installing a monument or making a note. In these instances, the pole is often laid directly on the ground, subjecting the pole and target to damage and malfunction as a result of ground borne debris (e.g., sand, mud).
A number of dedicated prior art devices are offered to steady or support geomatics poles, which permit more precise measurement observations to be taken. Many of these prior art pole support devices provide supporting legs in the form of bipods or tripods. One version of a geomatics pole supporting bipod is illustrated in U.S. Pat. No. 3,570,130 to Boehm. A version of a dedicated geomatics pole tripod is illustrated in U.S. Pat. No. 5,749,549 to Ashjaee. These pole support devices have disadvantages, among which is that they only perform one function.
In some instances, tripod stands, which are typically designed to support primary instruments such as transits, theodolites and total stations, are also used to support geomatics targets. In other instances, dedicated target support tripods are used. One version of a dedicated target support tripod is illustrated in U.S. Pat. No. 3,195,234 to Glidden. While the tripods are quite stable, they also have a number of disadvantages. For instance, in the case of instrumentation tripods, adaptors (commonly known as tribrachs) are required to provide a connective interface between the target and the tripod. Additionally, these tripods are slow and difficult to set up with targets because the procedures for establishing precise vertical collimation, and determination of height of targets, are quite time consuming. Dedicated target tripods have an additional disadvantage of performing only one function. The disadvantages of using tripods for geomatic target supports often limit their use to those measurements requiring great accuracy.
Standard (i.e., nondedicated) tripods may be adapted to support geomatics poles so that the target devices can remain on the poles, a practice which saves time. One prior art surveying tripod type pole support device is illustrated in U.S. Pat. No. 4,290,207 to Browning. These types of support devices also have many disadvantages, such as being capable of only one use; providing a pole to tripod interface. Also, the positive coupling connections found in many of these pole supports tend to inhibit free rotation of the poles, which inhibits level vial calibration confirmations. These positive connections also resist sliding the support along the pole thereby resisting gravitational forces on the pole and limiting bearing support of the point on the ground surface. Additionally, these pole supports tend to be complex in design, heavy, and expensive.
One prior art tripod type geomatics pole support, marketed under the name Hold A Pole, permits free rotation, and free sliding of the geomatics pole. However, this pole support also has disadvantages, one of which is that the captivity of the geomatics pole is rather flimsy, as the pole is retained with an elastic band. Additionally, the elastic band must be manually attached and detached when the geomatics pole is installed and removed, which is inconvenient and also requires two hands to perform. Additionally, in order to prevent friction and thus permit free sliding and rotation of the pole, the elastic band must be rather weak in design, which may cause the pole to be unstable and possibly result in inaccurate measurements. Additionally, the force of wind can critically load the elastic band allowing the geomatics pole to fall to the ground thereby subjecting the pole, the level vial and its geomatics target to damage.
In addition to taking measurements, persons performing surveys must also engage in other activities such as installing monuments or making notes. In order to free surveyors hands while engaged in activities other than measuring, geomatics equipment is often placed directly on the surface of the ground. The exposure of survey equipment to ground borne debris such as mud or sand can render the equipment inaccurate or even inoperable. Moreover, field conditions in surveying often include rough and remote terrains, and surveying equipment must often be transported by hand, so features of weight and convenience have particular importance to surveyors.

Multiple function geomatics pole support device


Methods for cylindrical grinding a workpiece are disclosed. The method includes cylindrical grinding, with a bonded abrasive wheel having a permeable structure that includes interconnected porosity, a workpiece at a specific cutting energy of less than about 12 Hp/in3·min (29.7 J/mm3), and a material removal rate of at least about 1 in3/min·in (10.8 mm3/sec/mm)grinding. The bonded abrasive wheel may include at least about 3 volume percent of a filamentary sol-gel alpha-alumina abrasive grain having an average length-to-cross-sectional-width ratio of greater than about 4:1, or agglomerates thereof. In one embodiment, the workpiece is ground in the presence of a water soluble oil.

Typically, cylindrical parts are produced by machining a workpiece and then, if necessary, grinding the workpiece.
Attempts have been made to develop high material removal rate (MRR) grinding processes. For example, high MRR outside diameter (OD) grinding processes have been developed as "high speed grinding" processes. Commonly, these high speed OD grinding processes use wheel speeds on the order of 120 meters/second (m/sec) to 300 m/sec, with the general approach of using the higher wheel speed to improve performance. These high speed OD grinding processes typically involve superabrasive (SA) grinding wheels composed of diamond or boron nitride and often having a metal bond, e.g., an electroplated metal bond.
These conventional grinding processes have employed a 100% oil coolant at high pressure to reduce friction, to induce lubricity in the metal-to-metal contact surfaces, and to prevent thermal damage and residual stress to the workpiece. To prevent occasional sparks that could present a fire hazard, these grinding processes typically use a substantial excess of the oil coolant to immerse the grinding zone with the coolant. Large quantities of chips are produced, are directed into the oil coolant, and are difficult to remove, necessitating complex and expensive waste disposal.
In spite of these limitations, high speed OD grinding is practiced on occasion to replacing traditional machining (e.g., turning, milling, or turn broaching). However, application of high speed OD grinding has been limited to small volume part production. In high volume production, the disadvantages of high speed OD grinding are compounded by another serious constraint of the process—the gradual and constant wear of the superabrasive grains, e.g., cubic boron nitride grains. The gradual and constant wear can lead to constant changes in part quality, especially in the case of long cylindrical parts such as, for example, crank shafts. Furthermore, large volume production is not suited to on-line dressing of superabrasive grinding wheels made of galvanic processes. Such wheels are generally used for high speed cylindrical grinding applications with high MRR. This poses another constraint to the implementation of large volume production using high speed OD grinding.
Therefore, there is a need to develop new grinding methods overcoming or minimizing one or more of the shortcomings associated with conventional processes, such as high speed OD grinding.


A compact, high-yield, bio-reactor for use as small, medium and large-scale production unit for therapeutic proteins, proteins in general and enzymes based on controlled activity of the cells or micro organisms and the production of such as high value antibodies for pharmaceutical and/or bio-diagnostic applications.

In bio-reactor technology large-scale production of bio-active compounds, including bio-polymers, from bacterial micro organisms is currently performed in batch fermenters and requires product purification downstream. One of the major limitations of the batch fermentation technology is the large volume of the fermenter needed to accomplish this task on a mass-scale (in the order of millions of tons/year) and the high cost for installing and operating such fermenters.
As an example, most recombinant antibodies approved for human use are currently produced by large-scale cultivation of mammalian micro organisms (e.g. Chinese Hamster Ovary cells, or CHO cells). The process is expensive because of its limited capacity. The investment needed to establish a culture unit for mammalian cells is substantial (approximately 10 million Euro is required for an antibody production of 1 kg/year).
By 2002 approximately 64 different Monoclonal Antibodies (MAbs) had been approved by FDA (US Food and Drug Administration)—a figure expected to rise to 500 in less than 10 years. Many Mabs are already in clinical trials. The production capacity of MAbs was 4,000 kg in 2005 and the capacity is currently (2009) estimated to 10,000 kg. Most of this capacity is provided by stirred tanks having a capacity of around 1 mio. liters. This capacity may not even be sufficient for the production of the few, so far clinically validated therapeutic antibodies.
A projected demand of an expected capacity of 50,000 kg/year by 2020 will require at least 5 times the present, conventional stainless steel stirred tank capacity. It is clear that a World wide bio-tech industry will need to invest heavily in more production capacity in order to increase the current production capacity.
There is clearly a need for novel and innovative bio-reactor devices as well as for alternative methods for the production of bio-active compounds using such bio-reactor devices.
The market for bio-reactors can be divided into the following groups:                Bench-top equipment market, 1-10 liter size        Pilot-scale equipment market, 10-100 liter size        Small-production equipment market, 100-1000 liter        Large-production equipment market, >1000 liter
Current fermenter technologies exhibit a relatively unattractive surface-to-volume ratios (stirred tanks≈1 m2/40 liters and hollow fibres≈1 m2/10 liters). Scaling up bio-reactors for use in the production of adherent micro organisms today relies exclusively on the possibility of increasing the surface area that is available for the adhesion of the micro organisms. This can be done e.g. by increase the size of the reactors. Classical micro organism culture lab ware ranges from 1 to 500 cm2 per stationary flask. The surface area to volume ratio is the range of from 1,000 to 2,000 cm2/liter for rotating flasks. Rotating wall vessel systems on the market would at best reach a ratio of 7.5 m2 in a 2.5 m3 volume.
Multi-plate systems, like OptiCell from Nunc, require large, thermo-controlled volumes to be efficient when used with a high surface area. To face these limitations, micro-carrier approaches, e.g. Cytodex from GE Healthcare) have been developed and such micro-carriers can bring the surface area up to approx. 9 m2/liter for stirred suspension cultures where micro organisms are sensitive to shear forces due to stirring—thus covering only partially the available surface area with micro organisms.
Besides the limitation of the surface area available for micro organisms anchoring, additional problems exist, including cultivation difficulties, undesirable oxygen gradients, tension control, pH control and a general lack of homogeneity of nutrient delivery throughout the bio-reactor.
Micro organism expression levels depend upon the type of molecule to be produced, which range from approx. 5 mg/liter/day for laboratory clones to up to 1 g/liter/day under ideal industrial condition. Based on such production rates, the expected production achievable with a 20 liter continuous, wall-forced-flow bio-reactor is a daily, steady state production of up to 2 grams of bioactive compound per day. Due to expected benefits related to bio-reactor geometry, surface properties, flow control and distribution of nutrients, these production rates might be further improved. By comparison, a classical batch bio-reactor based on micro carriers and duration of 8-10 days may at best produce 10 mg per liter. Accordingly, a 20 liter bio-reactor would produce at the most 200 mg bioactive compound per batch.
Conventional Methods and System Designs
Bio-reactor design is a complex engineering task. Under optimum conditions, the micro organisms must be able to perform their desired function with a 100 percent rate of success. The bio-reactor's environmental conditions, like gas content (i.e., air, oxygen, nitrogen and carbon dioxide), flow rates, temperature, pH and agitation speed/circulation rate need to be closely monitored and controlled. In a traditional, batch bio-reactor the nutrient volume is constant—it is not exchanged and it is stirred throughout the duration of the fermentation.
In a Continuous Flow Stirred Tank bio-reactor (CSTR or chemostat), the continuous flow of fresh medium is fed into the bio-reactor at a constant rate, and medium is mixed with micro organisms and an undesirable mix of cells and product leaves the bio-reactor at the same rate. A fixed bio-reactor volume is maintained and ideally, the effluent stream should have the same composition as the bio-reactor contents. The culture is fed with fresh medium containing one and sometimes two growth-limiting nutrients, including e.g. glucose. The concentration of the micro organisms in the bio-reactor is controlled by the concentration of the growth limiting nutrient level. A steady state micro organism concentration is reached when the density of the micro organisms and the nutrient concentration are constant.
A bio-reactor is a suitable device for growing both anchorage dependent and suspended micro organism. Ideally, any micro organism culture bio-reactor must maintain a sterile culture of micro organisms under medium conditions which maximize micro organism growth and productivity. In the aerobic process, optimal oxygen transfer and access is perhaps the most difficult task to accomplish. Oxygen is poorly soluble in water at atmospheric pressure, even less in fermentation broths, usually helped by agitation, which is also needed to mix nutrients and to keep the fermentation homogeneous. There are, however, limits to the speed of agitation in stirred tank bio-reactors, due both to high power consumption and damages to the micro organism.
In bio-reactors where the goal is to cultivate micro organisms or tissue cells for experimental or therapeutic purposes, the design is often significantly different from industrial bio-reactors. Many micro organisms and tissues, especially mammalian ones, must have a surface or other structural support in order to grow, and agitated environments are often destructive to these micro organism types and tissues. Higher organisms also need more a complex growth medium composition. The most crucial step in the design of monolithic reactors is the proper distribution of nutrient fluids over the reactor cross section.
Flasks for laboratory use are made from either glass or polycarbonate and very commonly they are used only for small scale productions. The maximum surface available in a single flask is 500 cm2 as offered from Thermo Fishers Danish department NuncBrand in the TripleFlask model. If stacked in a one meter high setup the flasks can offer a surface area of 0.6 m2/20 liters volume into a manually handled process.
The German company Sartorious market CELLine cultivation flasks with a built-in micro organism separation method offering nutrient re-circulation and easier product harvesting. Flask surface areas range from 350 cm2 to 1000 cm2 for anchored micro organisms. Also available is a cassette based, stackable system for small, industrial micro organism cultivation on porous polymeric membranes. Each cassette offers 100 cm2 area and 20 cassettes fit into a ready made cassette support system offering in total adhesion area of 2,000 cm2. Each cassette has its own two resealing access ports which provide a closed, growth environment with a sterile fluid path. It is purely intended for anchorage-dependent cells.
Industrial vessel fermenters from Novaferm in Sweden or NNE (Novo Nordisk Engineering) in Denmark offer cultivation systems based on micro organisms suspended in the nutrient. The contact between nutrients and the micro organism are supported by mechanical stirrers and this is far from optimal.
Packed bed bio-reactors with small polyester fibre bodies, like the FibraCell product from New Brunswick Scientific in the United States, features an axial flow path along the porous bodies typically enclosed in a glass vessel. The FibraCell product is originally developed in the early 1990ties by Mr. Avinoam Kadouri from Yeda and Weizmann Institute of Science, Israel, but already in the 1980ties had exceptionally high yields, typically 108 micro organisms/cm3 of bed volume depending on selected micro organisms, been reported. The small bodies are less than 6 millimeter in diameter and the polyester fibre diameter is approx. 6 μm, bulk gravity 1 g/cm3, and the available growth surface areas >1200 cm2/g. The CelliGen 310 type of bio-reactor, also from New Brunswick Scientific—when packed with 500 gram Fibra Cell mini bodies (and in general for small scale industrial application with 14 liters volume)—offer 500 gram×1200 cm2=25 m2 surface area equivalent to 5 m2/liter. The flow path is exclusively axial in the columns. Greater height is generally the solution when seeking to add more packing volume and this demands a greater volumetric flow in the axial direction and this in turn increases the risk of cell damage along the packed bed. Furthermore, the longer the bed the higher is the risk of experiencing an increased lack of nutrients and CO2 content in the opposite end of the fluid inlet. As the nutrient flow now also becomes the exhaust fluid, the flow in the axial direction has a maximal limit. Cell densities of up to 1×108 and expression levels of approx. 0.5 g/mL have been reported from various sources. Artellis from Belgium has recently started to promote such a technology with small polyester fibre bodies packed in stackable envelopes.
Packed bed bio-reactors with porous micro beads, such as micro spheres provided by GE Healthcare or others, are in general very expensive. The beads are typically filled with macro pores and typical have a diameter of from 0.01 millimeter to up to 5 millimeter. They are manufactured from e.g. cellulose, polyethylene glass, gelatine, silicone rubber, silica, etc. Cell densities of up to 1×108 have been reported with the use of micro beards.
Membrane bio-reactors (MBR) are bio-reactors having a cross flow filtration unit which enables continuous processes with total micro organism retention within the reactor to be realized. Provided that high dilution rates can be applied and that inhibition processes are avoided, high biomass concentrations can be reached, thereby improving the volumetric productivity. These membrane bio-reactors have been successfully applied to various microbial bio conversions, such as alcohol fermentation, solvents, organic acid production, starters, and wastewater treatment. On the basis of the biological reaction characteristics and bibliographic results available, there are several bottlenecks associated with this methodology.
Hollow fibre bio-reactors systems use polymeric round hollow fibres bundles into a circular body with all the fibres passing axially through the core. The hollow fibres system can be used for anchorage dependent and suspension micro organisms. Typically the hollow fibre on the inside of the tube is provided with a membrane giving selective passage of molecules depending on their size. In most cases, an ultra filtration type of membrane is used. The molecular cut-off of the membrane differs according to applications, ranging from a few thousand to a hundred thousand Daltons. The ultra filtration membrane prevents free diffusion of secreted product molecules from passing through the membrane and allows them to accumulate in the extra capillary space to a high concentration. The culture media is pumped usually through the fibre lumen, and micro organism grow in the extra capillary space, or the shell side. Supply of low-molecular weight nutrient to the micro organisms and the removal of waste product occur by diffusive transport across the membrane between the lumen and the shell spaces. Although the use of micro filtration hollow fibre membranes for micro organism culture is infrequent, it does find application in various research uses for studying metabolism and for the cultivation of anchorage-dependent or highly aggregated micro organism for which a convective flow of medium through the extra capillary space to bathe micro organism in medium is desired. Scaling-up of a hollow fibre system eventually is limited by the ability to extend the axial length of the fibre without incoming oxygen transfer limitation, creating gradients. Also scaling up the cartridge diameter eventually runs into flow distribution problems among thousands of fibres. The relatively high surface area to volume ratio that hollow fibre bio-reactors provide (100 cm2/mL or more) and the high flux rates of fibres from FiberCell Systems Inc. can allow micro organism to grow at 100 times the density found in flask culture (10*8 vs. 10*6/mL in flask) and is in fact one of the only cell culture methods that permits micro organism cultures at densities that rival those found in vivo.
Honeycomb ceramic bio-reactor systems are typically in the form of a cylinder made of a porous ceramics with square channels passing through the ceramic cylindrical body in the longitudinal direction. Micro organisms are inoculated into the channels and either adheres to the surface or become entrapped into the pores of the ceramic body walls. Medium is passed through the channels to provide nutrient and to remove the metabolites. In the ceramic system, the side on which the micro organisms is adhered is exposed to a slow stream of permeate. The ceramic bio-reactor, to some extent, can be considered a variant of the hollow fibre system described herein above. As in the hollow fibre system, ceramic reactors are supported by medium perfusion loops. Micro organism culture medium is pumped through the longitudinal channels in the ceramic core from a medium reservoir in a circulating loop configuration. Fresh medium is fed into the system, and harvested bioactive compounds are removed to the medium reservoir. Unlike the hollow fibre system, there is no membrane separating the micro organisms and the bulk medium. Products are secreted directly into the bulk medium. Essentially, the ceramic bio-reactor (like the OptiCell) can be used to conveniently replace a large number of roller bottles. As in the hollow fibre systems, oxygen concentration gradient develops along the axial direction and poses a limitation to the length and/or the physical size of the ceramic reactor.
A laminar flow along a channel is often not sufficient to establish a contact between nutrients and a micro organism. Turbulence is not created along the channels having relatively smooth surfaces and the flow will generally be lower at the edges compared to the flow at the flat wall between the edges. Attempts to increase channel density only serves to increase this problem. The air-lift type of membrane bio-reactors do cause some turbulence, but the bubble sizes prevents the oxygen containing bubbles from coming into contact with the micro organisms in the corners. Also, it has been reported that the bubbles may damage the cells. As there is no force to create an interaction between the many channels the nutrients flow along, the channels are not fed equally. More channels in parallel will not affect the flow, the mass and the nutrient transfer to the cells. The liquid distribution results in the provision of heterogeneous sub-environments and a non-uniform protein production. This in turn seriously limits the applicability and the commercial potential for using ceramic monoliths having square cells as bio-reactor supports for the cultivation of micro organisms in large scale fermentations.
Plastic bags, disposable bags are becoming the biggest seller today below 1,000 liter bio-reactor volume. Bags with media inside only need a mechanism inside the bag in order to constantly mix the nutrient with the suspended micro organism. Many different techniques are on the market based on rotating devices with direct mechanical or magnetic force transfer or waving actions. However, the mixer typically adds shear forces to the media and this may potentially damage the micro organisms. Micro carriers could also be used with the bag operation in a perfusion system without a mixer relying on external, positive displacement pumps.
Wave-bag is a single-use system which rock, wave a volume of nutrient in a flat plastic bag on a tray which is kept in simple motion by an external mechanical force. Typical max cell densities are in the range of from 1-2×107/mL when operating in perfusion mode.
U.S. Pat. No. 4,948,728 (G. Stephanopoules) discloses a porous, ceramic material with a plurality of flow passages and a biofilm in contact with an inner wall in a channel and a gas permeable membrane covering an outer wall. A separate oxygen flow along the outer wall permeates the membrane and ceramic housing to reach the biomaterial. Nutrients flow along the inner wall in direct contact with the biofilm. The basic idea behind the invention is to apply oxygen on one side and nutrient flow on the other side of a porous wall. The monoliths, as supplied in the late 1980ties from Corning, are all flow through monoliths originally intended for automobile catalyst support and described in the patent as having a cell density of either 200 cells/in2 (cpsi) (column 10) or 300 (column 5) cells/in2 (cpsi). The Cordierite ceramic monoliths used are characterised as having a pore size of less than 15 μm. The expression "race track" monoliths which is used in the specification indicates that the catalyst carriers originate from the automobile industry and have very thin walls—typically less than 0.5 millimeter. A maximum cell density—with every second channel blocked and designed for DPF (diesel particulate filter) use—would be 90 cells per square inch (cpsi). The use of a parallel wall DPF is not described in the US patent or any associated literature. In the '728 patent, FIG. 1 illustrates a flow through the monolith. FIG. 4 shows the oxygen appearance on one side of the porous wall and the nutrient appearance on the other side of the porous wall. FIG. 5 illustrates that the penetration of oxygen into the porous ceramic wall is not complete, but only partial. Accordingly, there is no forced, hydraulic nutrient or oxygen flow through the wall through which e.g. glucose and oxygen is supposed to flow. This means that the predominant amount of bio film does not receive oxygen and this in turn results in a reduced production capacity. FIG. 5 also shows that the bio film penetration is limited by the hydrophobic membrane and this results in reducing the total amount of micro organisms inside the matrix. The '728 patent does not disclose e.g. how the channel flow should be adapted and/or controlled, if the channels should be blocked for flow control, or how the separation of inlet/outlet channels should be arranged.
Later documentation from G. Stephanopoules shows that controlling the two different fluid flows is not solved via channel blocking, but can be solved via the introduction of multiple, porous ultra small diameter silicon rubber hoses one of which is placed in each channel in order to supply the oxygen. However, this is very impractical and more or less impossible on a ceramic body with 200 or 300 cells/in2, where each channel width is only 1 millimeter or less.
Also, the '728 patent does not disclose any membrane separating the inlet from the outlet. Such a membrane would be required in order to prevent cells from passing through the bio-reactor and be expelled in the flow after the reactor. Also, a high pressure drop membrane playing an important factor in distribution of oxygen and nutrients is not mentioned at all.
Reference is also made to the article by M. A. Applegate and G. Stephanopoulos, "Development of a Single-Pass Ceramic Matrix Bio-reactor for Large-Scale Mammalian Cell Culture," Biotech methodology and Bio engineering, volume 40, 1992, pp. 1,056-1,068. The use of porous hoses by Applegate and Stephanopoulos in each channel in order to supply oxygen (separate oxygenation system) is also described in the article "Review of Nonconventional Bio-reactor Technology" by C. E. Turick published 1993 by U.S. Department of Energy. On page 13, the cited equation refers to open channel ceramic bodies. FIG. 11 on page 12 illustrates one porous silicon tube in each channel in order to supply air/oxygen. It is obvious that channel closing has not been part of the patented invention. One other drawback of using Cordierite is the well known fact that a considerable amount of the pores as described by the porosity figure are so called closed pores. In other words there is no access to as much as 25% of the pores as they are not interconnected to other pores. The product reached a commercial status and became known as the OptiCell product in the industry.
U.S. Pat. No. 4,514,499 (Corning, Inc) describes a monolithic support for cell growth with the same general problems as describes above for U.S. Pat. No. 4,948,728.
U.S. Pat. No. 4,937,196 (New Brunswick Scientific) introduces the terms "membrane" and "micro porous membrane". A micro porous membrane does NOT allow the cells to pass the membrane. It is disclosed that "the dimension of the system is such that every cell is within 200 μm, preferably within 100 μm, of the oxygen source, i.e., the surface of an oxygen transport membrane". The oxygen supply is separated from the nutrient supply (which is void of oxygen) as the two individual feed spacers are separated by the membrane containing the cells. The laminated set-up disclosed in the '196 patent imposes the physical limit that the membrane containing the cells can have a maximum thickness of about 200 μm—as the oxygen supplying spacer is present only on one side of the cell supporting membrane.
Furthermore, it is described that the membranes are positive and do not allow the cells to pass the membrane. FIG. 9 illustrates the fluid path along the spacers from one end to the other, but no dual path feed spacer system capable of supporting such a teaching is disclosed. Nowhere does the '196 patent disclose how the individual feed spacers are anchored to the central tube in the spiral filter. Also, the legend to FIG. 10 indicate that a significant and undesirable pressure gradient is present, but the legend does not offer any suggestion as to how to avoid this.
U.S. Pat. No. 5,501,971 (New Brunswick Scientific) describes the Celligen system in the form of a packed bed of FibraCell polyester fibre based, flat carriers. The system currently on the market from New Brunswick Scientific demonstrates that cells adhere well to a polyester fibre, but the Celligen system is heavily limited by the thickness of the bed. As an increased bed thickness introduces or increases undesirable gradients in the bio-reactor, the bio-reactor device is not scalable and can be used for fermentations on a bench-top scale only.
U.S. Pat. No. 5,266,476 (Yeda) discloses a matrix of a thickness of from 50 to 500 μm and a surface area of 1 millimeter2. The matrix is based on a polyester fibre and is in the form of a very small carrier know in the industry under the trade name FibraCell (supplied by both NBS and Bibby Sterilin Ltd, UK). FibraCell is a mini body approx. 6 millimeter in diameter and 0.6 millimeter thick. FibraCell has been used for various applications, among others by NBS (New Brunswick Scientific, USA) for manufacturing the Celligen product in which Fibra Cell bodies are randomly and efficiently packed in volumes of up to 1 liter. Larger reactor volumes are not possible due to gradient problems.
U.S. Pat. No. 4,546,083 (Stoll Corp) discloses a cylindrical device with fibres positioned around a tubular inlet—a spindle. The volume for the fibre matrix between the spindle and the cartridge inner wall are filled with fibres only.
U.S. Pat. No. 5,543,047 (Pall, Inc) describes a special pleating method improving among others also the potential area/volume ration and as described for filtration purposes only.
U.S. Pat. No. 5,563,069 (Ohio State University) is concerned with a packed bed structure based on cotton cloth in sheet shape.
WO2007/142664 (AMProtein Corp) discloses a method for increasing dissolved oxygen in a culture medium for a semi suspension culture of mammalian cells in a vessel. The vessel concept exploits constant height and varies the diameter in order to vary the fermenter volume in order to handle the gradient issue.
WO2007/039600 (Artelis) discloses a complicated flow pattern vessel device, which has an integrated mixer operating as circulation pump. The device is not suitable for up-scaling due to an undesirable increase in nutrient and/or oxygen gradients. Media re-circulation is performed by a mixer device internally in the vessel and this feature also seriously limits the prospects for up-scaling. Only perfusion mode operation is described. Gradients occurring in the central inlet zone results in the generation of an un-even cell density in the modules. The invention is based on packed micro porous micro carriers present inside modules, envelopes for cell support and/or empty modules suitable for the suspension of cells. Each radial fluid inlet between two envelopes supply media to only one of two envelopes. As described, most of the cells are not circulated, but those which are will be subjected to strong shear forces generated by the mixer impeller. Such cells are at risk of being seriously damaged. The described mixer physics exploit very limited pressure difference capabilities, both limiting the scale-up of the concept and increase shear forces in the fluid significantly.
U.S. Pat. No. 4,789,634 (KG Biologische Laboratorien) discloses a circular devise used for containing polymer microspheres, such as beads. The beads are located in round, stackable sieve boxes to ensure both a radial and an axial flow path.
The journal "BioProcess International" published in June of 2009 carries an article wherein AMprotein (China) states that they use large, randomly oriented, loosely packed cellulose fibre bodies, or Rasching alike soft elements, as "macro"-carriers for bio-reactor fermentations. It appears that the "Current" bio-reactor packed bed height is constant at approx. 150 millimeter and that the volume is modified by using 3 different diameters according to a reactor size ranging from 5 liters over 50 liters to 150 liters, thereby avoiding the problem of how to solve the problem of gradient formation.
MembroFerm is a product which was marketed by MBR Bio Reactor AG (Switzerland) in the early 1990ties. The product is in the form of a flat membrane bio-reactor separated by a thin film fluorocarbon matrix having a thickness of 0.6 millimeter.
None of the above-cited prior art references have solved the gradient problems associated with "packed bed" bio-reactor fermentations. Also, none of the above-cited prior art references have solved the problem of how to effectively scale a fermentation from a bench-top level to industrial production scale without the concomitant generation of undesirable gradients in a bio-reactor.

Method and device for industrial biolayer cultivation


A method of manufacturing a semiconductor characterized in that, in polycrystallizing an amorphous silicon thin film formed on a substrate through an annealing process, the amorphous silicon thin film has a plane area of 1000 μm2 or less. A thin-film transistor characterized by comprising an active silicon film which is formed of a plurality of island-like regions arranged in parallel to each other, each of the island-like regions being formed of a polycrystal silicon thin film having a plane area of 1000 μm2 or less. A method of manufacturing a thin-film transistor comprising the steps of:                forming an amorphous silicon thin film on a substrate; processing the amorphous silicon thin film into a plurality of island-like regions each having a plane area of 1000 μm2 or less; polycrystallizing an amorphous silicon thin film that forms the island-like regions through an annealing process; and forming a thin-film transistor having at least one of the plurality of island-like regions as an active silicon layer.

1. Field of the Invention
The present invention relates to a semiconductor formed of a polycrystal silicon thin film, which is formed by crystallizing an amorphous silicon thin film on an insulator, and a thin-film transistor using that semiconductor.
2. Description of the Related Art
In recent years, a technique has been popularly researched where an amorphous silicon thin film is formed on an insulator such as a quartz substrate, allowed to grow into a solid-phase crystal (SPC) through a heating treatment or an annealing process due to the irradiation of a laser beam or an intense light to form a polycrystal silicon thin film.
A conventional general technique for obtaining a polycrystal silicon thin-film by allowing an amorphous silicon thin film to grow in a solid phase will be described below.
First, an, amorphous silicon thin film having a thickness of 500 to 5000 Å is formed on a quartz substrate.
Thereafter, the amorphous silicon thin film thus formed is heated to 400 to 1100° C. and subjected to an, annealing process to make the amorphous silicon thin film grow into crystal. In this situation, a heater, infrared rays or the like is used as heating means.
The annealing process may be conducted by the irradiation of a laser beam or an intense light other than heating.
In the above-mentioned manner, a polycrystal silicon thin film is obtained.
The polycrystal silicon thin film thus obtained is used as an active silicon layer of the thin-film transistor (TFT), to thereby provide a thin-film transistor. As a result, a liquid-crystal display unit, image sensor or the like which is high in operation speed and in image quality is obtained using the above thin film transistor.
Up to now, the polycrystal silicon thin film obtained by annealing the amorphous silicon thin film has difficulty in lowering a defect density in crystal.
The thin-film transistor using the polycrystal silicon thin film thus formed as the active silicon film is hindered from realizing an improvement in various characteristics of the thin-film transistor, for example, the lowering of a threshold voltage (Vth), an increase in mobility, a decrease in a leak current (Ioff), etc., because the defect density in the active silicon layer is high.

Method of manufacturing a semiconductor method of manufacturing a thin-film transistor and thin-film transistor

Semiconductor Energy Laboratory Co., Ltd.


An elongated flexible sheet (20) contains numbers (22, 26) and corresponding parallel lines (24) that are aligned with the holes (13) of a harmonica (11). The top portion of the sheet (20) contains rows (30, 37, 40, 47) of respective color-coded blocks (32, 34, 36, 38, 39, 52, 54, 56, 48, 49) superimposed over the parallel lines (24). The color-coded blocks (32, 34, 36, 38, 39, 52, 54, 56, 48, 49) encompass sets of lines (24) that correspond to chords which may be played on the harmonica (11). The colors are chosen to create a mnemonic reference. The sheet (20) attaches to a harmonica (11) and is flexed into a viewing position for ready reference upon grasping the harmonica (11).

The harmonica is a musical instrument that is played by blowing or drawing air through a horizontal row of holes in the instrument. Air may be blown or drawn through the holes either singly or through several adjacent holes simultaneously. One of the difficulties in attempting to play a harmonica is becoming familiar enough with the location of the holes in the harmonica so that music can be played coherently and accurately without removing the harmonica from a playing position upon or near a player's mouth. Individuals attempting to play a harmonica are often frustrated by the difficulty in learning to play a recognizable tune. Thus, it would be advantageous to have an aid that makes playing a harmonica less difficult.

Method and device to aid in learning to play chords on a harmonica