JP2001501177A - Method of increasing individual sensitivity to OB protein by up-regulating OB protein receptor - Google Patents

Abstract

  (57) [Summary] Disclosed are methods for increasing the sensitivity of functional OB protein receptors and uses thereof.

Description

DETAILED DESCRIPTION OF THE INVENTIONOB Upregulate protein receptors to OB proteins How to increase individual sensitivity Field of the invention   The present invention relates to OB protein sensitive or functional OB protein receptors. The use of substances that increase the affinity or availability of Relates to a method for reducing the dose of an OB protein composition for obtaining a cosmetic effect .background   Little is known about the molecular basis of obesity, but the "OB gene" and Identification of proteins ("OB proteins" or "leptins") The mechanisms used by the body to regulate fat deposition have been elucidated to some extent (PCT Publication WO 96/05309 (12/22/96), Friedman; Zhang et al., Nature372:Four 25-432 (1994); Nature374: 479 (1995)). OB Tan The protein was expressed in ob / ob mutant mice (mouse fertilization due to defective production of the OB gene product). Active in vivo in both full and normal wild-type mice. That raw Physical activity indicates, among other things, weight loss. Generally, Barinaga, "Obese" Protein  Slims Mice, Science269: 475-476 (1995). OB protein, For the induction and control of body weight and steatosis in animals, including mammals and humans Its use as a modulator is described in PCs incorporated herein by reference. It is disclosed in more detail in T publication WO 96/05309 (12/22/96) and the drawings.   Other biological effects of OB proteins have not been well characterized. ob / ob sudden change When OB protein is administered to different mice, for example, serum insulin levels and blood It is known that fresh glucose levels are reduced. Also administer OB protein Then, it is known that body fat decreases. This is due to ob / ob mutant mice and And non-obese normal mice [Pelleymounter et al., Science269: 540- 543 (1995); Halaas et al., Science269: 543-546 (1995); Campfield et al., Scien ce269: 546-549 (1995) (Peripheral and central levels of microgram doses of OB protein Administration reduced food intake and body weight in ob / ob and diet-induced obese mice. But not in db / db obese mice). In these reports Will eventually No toxicity was observed at the highest dose.   Other substances may play a role in regulating insulin metabolism. There is a potential. Thiazolidinedione still has unknown mechanism of action A class of antihyperglycemic agents. Wilson et al. Med Chem.39: 665-668 (1996); Kallen P.N.A.S.,93: 5793-5796 (1996); Sohda et al., Chem. Pharm. Bull.,43: 2168-2172 ( 1995); Swanson et al., Drug Dev. Res.,35: 69-82 (1995); Kletzien et al., Mol. Pharmac ol.,42: 558-62 (1992). This class of drugs is 1) insulin sensation Or 2) transcription factors, peroxisome proliferator response Activates the responsive receptor (PPAR) (gamma) to reduce hyperglycemia It is thought to act on. PPAR (gamma) is derived from primary fibroblasts to fat cells It is thought to play a role in the conversion to. Wilson et al., J . Med Chem.39: 665-668 (1996); Kallen et al., P.N.A.S.,93: 5793-5796 (1996); Swan son et al., Drug Dev. Res.,35: 69-82 (1995). Recently, thiazolidine One of the diones (AD-5057) is found in obese rats and mice to have a low body weight. Elevates but has a substantial effect on food intake No significant decrease in adipocyte leptin mRNA and serum leptin levels (Zhang et al., J. Biol. Chem.,271: 9455-9459 (1996)). Another Reports have shown that another type of thiazolidinedione, BRL496553, has also Have been shown to reduce leptin mRNA levels in fat cells (Kallen et al., P.N. A.S.,93: 5793-5796 (1996)). Therefore, thiazolidinedione is not It is thought to reduce protein.   Thiazolidinedione is thought to increase insulin sensitivity, At present, increases the individual's sensitivity to OB protein (or leptin) There are no known substances. Such an increase in sensitivity is due to a decrease in the required dose or It may be advantageous for leptin users as it will lead to less frequent dosing.Summary of the Invention   The present invention provides a method for increasing the sensitivity of thiazolidinedione compositions to OB proteins. Based on the assumption that Not wishing to be bound by theory, The lysine dione composition “upgrades” the OB protein receptor available for signal transduction. "Regulate", that is, increase the number of OB receptors Or increase the affinity of the OB receptor for its ligand (OB protein) May be Such availability or affinity of a functional OB protein receptor The increase enhances endogenous and / or exogenous OB protein signaling. Surprisingly and importantly, this is not therapeutic and / or cosmetic. Provide a means to reduce the dosage and / or frequency of inducible OB protein Offer.   Thus, in one embodiment, the present invention relates to a method for producing a functional OB protein in an individual. One or more compositions that act to increase the availability or affinity of cytoplasmic receptors (eg, For example, the administration of a thiazolidinedione composition) can control body weight and And / or provide a method for fat deposition.   In another embodiment, the present invention provides a functional OB protein receptor in an individual. One or more compositions that act to increase the availability or affinity of Azolidinedione composition) and the administration of OB protein , Methods of weight control and fat deposition in an individual.   In yet another embodiment, the present invention relates to a method for producing a functional OB protein in an individual. Acts to increase receptor availability Exogenous by administration of one or more compositions (eg, thiazolidinedione compositions) Methods for reducing the dose and / or frequency of administration of OB proteins are provided.   In yet another aspect, the present invention provides a method for treating a secondary morbidity associated with excess fat (eg, co-mobidities), such as diabetes, abnormal or hyperlipidemia, lack of fertility, And potentially also increased insulin sensitivity and / or reduced lean tissue mass Provide an increased treatment method. Also provided are related compositions.BRIEF DESCRIPTION OF THE FIGURES FIG.: Recombinant mouse met OB (double-stranded) DNA (SEQ ID NOS: 1 and 2) No acid sequence (SEQ ID NO: 3).   FIG.: (Double-stranded) DNA of recombinant human met OB analog (SEQ ID NOs: 4 and 5) and Amino acid sequence (SEQ ID NO: 6).Detailed description Composition   Sensitivity to OB protein or functional OB protein receptor in the individual Compositions having the ability to increase utilization are various thiazolidinedione compositions, For example, 2,4-thiazolidinedione, an optionally substituted thiazolidine Nzion, 5- [4- [2- (5-methyl-2-phenyl-4-oxazolyl) -2-hydroxyethoxy] ene Le] -2,4-thiazolidinedione (AD-5070), clofibrate, ciglitazone, Englitazone, pioglitazone, BRL 49653, troglitazone, M16209, oki Sazolidinedione and thiazolidinedione or derivatives of said compounds, similar Isomer, tautomer, enantiomer, diastereomer, epimer, salt, solvate , Esters, prodrugs and metabolites.   The OB protein is a recombinant mouse protein described below (SEQ ID NO: 3 in FIG. 1), (Nature, supra; incorporated herein by reference). The recombinant human protein or glutaminyl residue at position 28 (See Zhang et al., Nature, supra at page 428). Want). Also, 1) arginine is substituted for lysine at position 35, and 2) i Recombinant human of SEQ ID NO: 6 of FIG. 2 containing leucine instead of soleucine OB protein analogs can also be used (abbreviations for this analog are recombinant human R-> K³⁵, L-> I⁷⁴Is). Recombinant human analog and recombinant mouse protein The amino acid sequence has a methionine at position -1. These residues are described below as being the presence of these OB proteins and In both and analogs, the methionyl residue may be absent.   The mouse protein is substantially homologous to the human protein (especially As a protein, and more particularly at the N-terminus). The recombinant human protein A quality analog alters amino acids in the recombinant human sequence that differ from the mouse sequence ( For example, it can be produced by substituting an amino acid residue). The recombination Since human proteins have biological activity in mice, such analogs Probably active in humans. For example, lysine at residue 35 and lysine at residue 74 Has a soleucine (the first amino acid is valine and the amino acid at position 146 is 32, 35, using the human protein (by numbering SEQ ID NO: 6). 50, 64, 68, 71, 74, 77, 89, 97, 100, 105, 106, 107, 108, 111, 118, 136, One or more of the amino acids at positions 138, 142 and 145 can be replaced with another amino acid. You. The amino acid at the corresponding position in the mouse protein (SEQ ID NO: 3) or another amino acid Can be selected.   Furthermore, based on the rat OB protein sequence, Can be produced (Murakami et al., Biochem. Biophys. Res. Comm. .209: 944-952 (1995); incorporated herein by reference. ). The rat OB protein is human at the following positions (using the numbering of SEQ ID NO: 6): Different from OB protein: 4,32, 33,35,50, 68,71,74,77, 78,89,97,1 00 , 101, 102,105,106,107,108,111,118,136,138and145. Differently One or more of the amino acids at these positions can be replaced with another amino acid. Bold characters indicate that mouse OB protein and rat OB protein are human OB proteins. It is a position that differs from the quality and is therefore particularly suitable for modification. Corresponding Amino acids from the rat OB protein or another amino acid It can be substituted at the position.   Both rat and mouse OB proteins differ from mature human OB proteins in the following positions: Position: 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102 , 105, 106, 107, 108, 111, 118, 136, 138, 142 and 145. One of the amino acids The above is deleted or another amino acid (eg, corresponding rat or mouse Sequence number replaced with the amino acid found in the sequence) No. 6 (having lysine at position 35 and isoleucine at position 74) Will be valid.   It is also found in rhesus monkey OB proteins different from mature human OB proteins Amino acids are 8 (S), 35 (R), 48 (V), 53 (Q), 60 (I), 66 (I), 67 (N), 68 (L), 89 (L) , 100 (L), 108 (E), 112 (D) and 118 (L) (the type of amino acid is (Indicated by amino acid abbreviations). Recombinant human OB protein is cynomolgus Active in monkeys, one or more of the different amino acids in rhesus monkeys SEQ ID NO: 6 (with lysine at position 35) substituted with an acid (eg, the amino acid in parentheses) (With isoleucine at position 74) would be effective. Rhesus Certain different amino acids in the monkey are also those found in the mouse species. (35, 68, 89, 100 and 112). Therefore, Position (numbering of SEQ ID NO: 6 with lysine at position 35 and isoleucine at position 74) Mouse / rhesus monkey / with one or more amino acids of Human consensus molecules can be produced: 4, 8, 32, 33,35, 48, 50, 53 , 60, 64, 66, 67,68, 71, 74, 77, 78,89, 97,100, 102, 105, 106, 107, 1 08, 111,112 , 118, 136, 138, 142 and 145.   Other analogs are made by deleting portions of the protein amino acid sequence. Can be For example, the mature protein has a leader sequence (-22 to -1). Lack. The following truncated form of the human OB protein molecule (numbered SEQ ID NO: 6) Can be manufactured):     (A) amino acids 98-146     (B) amino acids 1-32     (C) amino acids 40-116     (D) amino acids 1-99 and (to which it is connected) 112-146     (E) having one or more of amino acids 100-111 located between amino acids 99 and 112 Amino acids 1-99 and 112-146 (to which they are connected).   Furthermore, the truncated form also contains amino acids that differ from the human OB protein ( At least one of the rhesus monkey, rat or mouse human OB protein) You may. In addition, any of the modifications may involve modified amino acids (eg, peptidomimetics or Or D-amino acids).Derivative   The present protein (in the present invention, the term "protein" is used unless otherwise specified. As encompassing “peptides” and OB analogs (eg, those listed below) Use) also involves attaching one or more chemical moieties to the protein moiety. It can be more derivatized. The chemically modified derivative is also Intra, intraperitoneal, intramuscular, subcutaneous, intravenous, oral, nasal, intrapulmonary, topical or other routes Can be formulated for administration. For chemical modification of biologically active proteins Rather, in certain circumstances, additional advantages (eg, the stability and And increased circulation time as well as reduced immunogenicity). 19 See Davis et al., US Pat. No. 4,179,337, issued Dec. 18, 1979. Overview See Abuchowski et al., Enzymes as Drugs (JS Holcerberg and J. Roberts). Ed., Pp. 367-383 (1981)). Protein modification and fusion proteins For a review describing quality, see Francis, Focus on Growth FactorsThree: 4-10 (1 May 992) (Mediscript, Mountview Court, Friern Barnet Lane, London N) 20, OLD, UK).   Chemical moieties suitable for derivatization can be selected from a variety of water-soluble polymers. You. The polymer of choice is one in which the protein to which it binds is in an aqueous environment (eg, physiological It should be water soluble so as not to precipitate in the environment). Use the final product for treatment When used, the polymer is preferably pharmaceutically acceptable. The weight Whether the combination / protein conjugate will be used for treatment and if so, the dose and circulation. Desired based on cycle time, resistance to proteolysis and other considerations The choice of the polymer will be possible to those skilled in the art. With this protein and peptide Indicates that the effectiveness of derivatization depends on the derivative being in the desired form (ie, Or, more preferably, by injection or infusion, or, for example, oral, pulmonary or Or by further formulation for intranasal delivery) and observe the biological effects. Can be confirmed by inspection (as described herein).   The water-soluble polymer is, for example, polyethylene glycol, ethylene glycol / Propylene glycol copolymer, carboxymethyl cellulose, dextran , Polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolan, R-1,3,6- Trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer Or random or non-random copolymers), and dextran or Or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol Homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxy From ethylated polyols, polystyrene maleate and polyvinyl alcohol Can be selected from the group consisting of: Polyethylene glycol propion alde Hyd is stable in water and may be advantageous in manufacturing.   By attaching a polyamino acid to an OB protein (or analog) moiety, Fusion proteins can be produced. For example, the polyamino acid is It can be a carrier protein that acts to increase the circulating half-life of the protein. Departure For therapeutic or cosmetic purposes in Ming, such polyamino acids may be neutralizing antigens It should not cause a sexual response or other adverse response. Such a po The amino acids can be serum albumin (eg, human serum albumin), antibodies or antibodies. (Eg, the antibody constant region, sometimes referred to as “Fc”) or other polypeptides It can be selected from the group consisting of amino acids. As described below, The binding position of the polyamino acid may be N-terminal, C-terminal or other position of the OB protein portion. And can be linked to the OB protein by a chemical "linker" moiety Like.   The polymer may be of any molecular weight and may be branched. It may be unbranched. For polyethylene glycol, the preferred molecular weight is From about 2 kDa to about 100 kDa in terms of handling and ease of manufacture (the term "about" Certain molecules in the polyethylene glycol preparations are larger than the indicated molecular weight. It shows that some molecules are small). The desired treatment profile (eg, Duration of sustained release, existing effects on biological activity, ease of handling, antigenicity Degree or absence, and polyethylene glycol for therapeutic proteins or analogs Other sizes can be used, depending on other known effects of recall).   The number of polymer molecules attached in this manner can vary, and may vary. The merchant will be able to see the effect on the function. Invitation Conductionization may be performed and the same or different chemical moieties (eg, different weights) Polymers such as polyethylene glycol), di-, tri-, tetra- -Derivatization or some combination of derivatizations may be performed. Tampa The ratio of polymer molecules to protein (or peptide) molecules depends on that in the reaction mixture. Like the concentrations, they are not particularly limited. Generally, the optimal ratio (excessive (Reaction efficiency in terms of absence of reactive protein or polymer) Depends on the desired degree of derivatization (e.g., mono-, di-, tri-, etc.) and the selected weight. Determined by factors such as the molecular weight of the union, whether the polymer is branched or unbranched, and reaction conditions. Will be done.   The chemical moiety has an effect on a functional or antigen domain of the protein. Should be coupled to the protein, taking into account the consequences. Numerous coupling methods are known to those skilled in the art. For example, EP 0 401 3 which is incorporated herein by reference. 84 (conjugation of PEG to G-CSF). See also Malik et al., Exp. Hematol .20: 1028-1035 (1992) (Polystyrene of GH-CSF using tresyl chloride) (Reporting ethylene glycolation). For example, polyethylene Glycols are more reactive groups (e.g., free amino or Xyl group). Reactive groups are activated Group to which the ethylene glycol molecule can bind. You. Amino acid residues having a free amino group include lysine residues and N-terminal amino acids. Residues can be included. Those having a free carboxyl group include asparagine Acid residues, glutamic acid residues, and C-terminal amino acid residues . In addition, as a reactive group for binding a polyethylene glycol molecule, Fuhydryl groups can be used. Preferred for therapeutic purposes is the amino group At the N-terminus or lysine group. Receptor binding or hope If so, binding at residues important for receptor binding should be avoided.   Proteins chemically modified at the N-terminus may be particularly desirable. polyethylene When glycol is used as an example of the present composition, various polyethylene glycol components From proteins (by molecular weight, branching, etc.) to proteins (or peptides) in the reaction mixture D) The ratio of polyethylene glycol molecules to molecules, polyethylene glycol The type of coalification reaction and the selected polyethylene glycol-terminated N-terminal You can choose how to get the protein. N-terminal is polyethylene glycol (Eg, if necessary, other monopolyethylene groups) Separation of this part from the recalled part) Polyethylene glycol at the N-terminus from the recalled population of protein molecules The purification can be carried out by purifying the converted substance. Selective N-terminal chemistry Modifications can be made of various types of primary amino acids available for derivatization of a particular protein. Achieved by reductive alkylation using the difference in reactivity of phenyl groups (lysine vs. N-terminus) I can do it. Under appropriate reaction conditions, the reaction with a carbonyl group-containing polymer Substantially selective derivatization at the N-terminus of the protein is achieved. For example, the tongue Differences in pKa between the ε-amino group of lysine residues in proteins and the α-amino group of N-terminal residues By performing the reaction at a pH available for use, the N-terminus of the protein can be selectively ported. It can be converted to ethylene glycol. With such selective derivatization In addition, the binding of the water-soluble polymer to the protein is controlled. That is, with the polymer Binding occurs preferentially at the N-terminus of the protein and may be linked to other reactive groups (eg, lysine No significant modification of the side chain amino group) occurs. When reductive alkylation is used, The water-soluble polymer may be of the type described above and binds to the protein Should have a single reactive aldehyde. Contains a single reactive aldehyde Using polyethylene glycol propionaldehyde It is possible to use   Monopolyethylene glycol at the N-terminus in terms of ease of manufacture of therapeutic agents Derivatives are preferred. In the case of N-terminal polyethylene glycolation, di- and tri- -Or simply characterize the product compared to other multi-polyethylene glycolated products Due to the purification, a homogeneous product is guaranteed. Commercial production of N-terminal products For ease of preparation, it is preferable to use the reductive alkylation method described above.   Complex   The OB protein, analog or derivative is administered as a complex with the binding composition. Can be Such a binding composition may comprise the OB protein, analog or derivative. It may have the effect of extending the circulation time of the body. Such a composition comprises It can be a protein (or, synonymously, a peptide). Binding protein As an example, an OB protein receptor or a portion thereof (eg, a soluble portion thereof) Is mentioned. By examining OB protein in serum or presence of binding Confirm other binding proteins by experimental screening for Can be Such bonds are typically OB The protein or analog or derivative binds to the endogenous OB protein receptor and And / or not impair the ability to cause signal transduction.   Pharmaceutical composition   In yet another aspect of the invention, the use of said protein and derivative pharmaceutical compositions Provide a way. Such pharmaceutical compositions may be for injection administration or orally, pulmonary, nasal It may be for internal, transdermal or other forms of administration. Generally, the proteins of the invention Or an effective amount of the derivative product and a pharmaceutically acceptable diluent, preservative, solubilizer, Pharmaceutical compositions comprising emulsifiers, adjuvants and / or carriers are encompassed by the present invention. It is. Such compositions include various buffer contents (eg, Tris-HCl, acetate, Phosphates), diluents of pH and ionic strength; detergents and solubilizers (eg Tw een 80, Polysorbate 80), antioxidants (eg ascorbic acid, metabisulfite) Sodium), preservatives (eg Thimersol, benzyl alcohol), filler Additives such as (eg, lactose, mannitol); the substance is a polymerizable compound ( For example, in particulate preparations of polylactic acid, polyglycolic acid, etc.) or liposomes Includes those enclosed within. Also, hyaluronic acid (Hylauronic acid), which promotes sustainability in the circulation May have an effect. Such compositions comprise the present proteins and derivatives. Of the physical state, stability, rate of in vivo release, and in vivo clearance of Speed can be affected. For example, Remington's, which is incorporated herein by reference. Pharmaceutical Sciences, 18th edition (1990, Mack Publishing o., Easton, PA 18 042) See pages 1435-1712. The composition may be in liquid form or dry powder ( For example, it can be manufactured as a lyophilized form. Implantable sustained release formulation, Transdermal formulations are also contemplated.   Remington's Pharmaceutical Science, which is incorporated herein by reference. s, 18th Edition, 1990 (Mack Publishing Co., Easton, PA 18042), Chapter 89. The use of the oral solid dosage forms listed is contemplated by the present invention. For solid dosage forms For tablets, capsules, pills, troches or lozenges, cachets or pellets Pills are included. In addition, liposome or proteinoid encapsulation makes this composition Can be formulated (eg, as reported in US Pat. No. 4,925,673). (As L-rotinoid microspheres). Liposome encapsulation may be used , The liposome may be derivatized with various polymers (see, for example, US Pat. 5,013,556). A description of possible solid dosage forms for treatment is included herein by reference. Marshall, K., Modern Pharmaceutics, G.S. Banker and C.T . Rhodes, Chapter 10, Chapter 1979. Generally, the formulation comprises the protein Quality (or analog or derivative) and protection against gastric environment and biological activity And an inactive ingredient that causes intestinal release of the active substance.   Also specifically contemplated are oral dosage forms of the derivatized proteins described above. The Proteins can be chemically modified so that oral delivery of derivatives is effective . The intended chemical modifications generally include (a) inhibition of proteolysis and (b) gastric Or at least one portion that allows uptake from the intestine into the bloodstream, To the quality (or peptide) molecule itself. In addition, the protein It is desirable to increase overall stability and increase circulation time in the body. Such parts include, for example, polyethylene glycol and ethylene glycol. Copolymer with propylene glycol, carboxymethylcellulose, dextra , Polyvinyl alcohol, polyvinylpyro Includes lidone and polyproline. Abuchowski and Davis, Soluble Polym er-Enzyme Adducts, "Enzymes as Drugs", edited by Hocenberg and Roberts, Wiley-I nterscience, New York, NY, (1981), pp 367-383; Newmark et al. Appl. Bioche m.Four: 185-189 (1982). Other polymers that can be used include poly-1,3 -Dioxolane and poly-1,3,6-tioxocane.   In the case of proteins (or derivatives), the site of release is the stomach, small intestine (duodenum, jejunum). Or ileum), or the large intestine. Does not dissolve in the stomach Other intestinal release formulations are available to those skilled in the art. Preferred Or a site that protects the protein (or derivative) or crosses the stomach environment (eg, Release the biologically active substance in the intestine, for example) Is avoided from the adverse effects of the stomach environment.   In order to guarantee full gastric resistance, a cocoon that is impermeable to at least pH 5.0 Is essential. More common used as enteric coating Inactive ingredients include cellulose acetate trimellitate (CAT), hydroxypropyl Lemethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, poly Vinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, phthalic acetate Cellulose (CAP), Eudragit L, Eudragit S and Shellac. this These coatings may be used as mixed films.   The coating or coating mixture can also be used on tablets These are not intended to protect the stomach. This includes sugar coating, or A coating could be included to facilitate swallowing of the tablets. Capsule Is a hard shell (eg, gelatin) for transporting dry therapeutic agents (ie, powders). In the case of a liquid form, a soft gelatin shell is used. You may. The shell material of the cachet can be thick starch or other edible paper. Would be good. For pills, lozenges, molded tablets or tablet powders, istmassing) techniques can be used.   The therapeutic agent is composed of fine multi-particles (mu) in the form of granules or pellets having a particle size of about 1 mm. ltiparticulate) in the formulation. Also administered in capsules If so, as a powder, lightly compressed plugs or even as tablets, The substance could also be formulated. The therapeutic agent is prepared by compression Could be done.   Colorants and flavoring agents may all be included. For example, the protein (or Or derivatives) (eg, by liposome or microsphere encapsulation). ), And then further refrigerated foodstuffs (e.g., containing colourants and flavoring agents) Beverage).   The volume of the therapeutic agent may be diluted or increased with an inert substance. These diluents Include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose Sucrose, modified dextran and starch can be included. Ma Certain inorganic salts, such as calcium triphosphate, magnesium carbonate, sodium chloride Lithium or the like may be used as the filler. Some commercially available rare Excipients include Fast-Flo, Emdex, STA-Rx1500, Emcompress and Avicell Can be   In the case of formulating the therapeutic into a solid dosage form, a disintegrant may be added. Disintegrant and Substances used include Explotab, a commercial disintegrant based on starch. Any starch, including but not limited to. Starch Glico Sodium oleate, Amberlite, carboxymethylcellulose sodium Um, ultramylopectin, sodium alginate, gelatin, orange peel , Acid carboxymethylcellulose, natural sponge and bentonite are all used. Is available. Another form of the disintegrant is an insoluble cation exchange resin. Collapse Powdered gums may be used as disintegrants and binders, including: agar, Karaya , A powdered gum such as tragacanth. Alginic acid and its na Thorium salts are also useful as disintegrants.   Binders can be used to bind therapeutic agents to form hard tablets. Binders include natural gums such as gum arabic, tragacanth, starch, and gelatin. Products from the product are included. Others include methylcellulose (MC), ethylcellulose (EC) and carboxyl Contains cimethylcellulose (CMC). Polyvinylpyrrolidone (PVP) Droxypropyl methylcellulose (HPMC) both granulate the therapeutic Could be used in alcoholic solutions.   In order to prevent sticking during the formulation process, a lubricant was added during the formulation of the therapeutic agent. Is also good. Lubricants may be used as a layer between the therapeutic agent and the die wall, Is stearic acid (the Including nesium and calcium salts), polytetrafluoroethylene (PTFE ), Liquid paraffin, vegetable oils and waxes, It is not limited. Also, sodium lauryl sulfate, magnesium lauryl sulfate Cium, polyethylene glycols of various molecular weights, such as Carbowax 4000 and 6000 Any soluble lubricant may be used.   Can improve the flow properties of drugs during formulation and aid rearrangement during compression A glidant may be added. The slip agent includes starch, talc, exothermic Can contain silica, hydrated silicoaluminate it can.   Surfactant as a wetting agent to assist in dissolving the therapeutic agent in an aqueous environment Agents may be added. Surfactants include sodium lauryl sulfate, sulfosuccinic acid Anionic cleaning of dioctyl sodium, dioctyl sodium sulfonate, etc. Agents can be included. Cationic detergents may be used and include salts Benzalkonium chloride or benzethonium chloride can be included. Surface activity Possible non-ionic detergents that can be added to the formulation as an agent include lauromacrog 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methylcellulose and carboxymethylcell Loin. These surfactants can be used in protein or derivative formulations. Could be present alone or as a mixture in various ratios.   Additives that potentially enhance the uptake of the protein (or derivative) include: Examples include the fatty acids oleic, linoleic and linolenic acids.   Sustained-release preparations may be desirable. The drug can either diffuse or leach Incorporation into an inert matrix (ie gum) that allows release by canism I could do it. In addition, a matrix that gradually denatures May be inserted. Another form of sustained release of this therapeutic is a single Oros encloses the drug in a semi-permeable membrane that allows water to penetrate and extrude the drug through a small opening The method is based on a therapeutic system (Alza Corp.). Also some vesicle soluble The coating has a sustained release effect.   Other coatings may be used for formulation. These include Various sugars that can be applied in the coating pan are included. The therapeutic agent is It can also be obtained as a lum-coated tablet, in which case the substance used is It is divided into two groups. The first are non-enteric substances, including methylcellulose Loin, ethylcellulose, hydroxyethylcellulose, methylhydroxyd Butylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose Lulose, sodium carboxymethylcellulose, providone and polyethylene Contains ren glycol. The second group is the intestine, which is generally an ester of phthalic acid. Consists of soluble substances.   Mixtures of materials may be used to obtain an optimal film coating. Film coating can be performed in a pan coater or fluid bed, or in a compression coater. It may be performed by the ringing.   Also, pulmonary delivery of the present proteins (or derivatives thereof) is contemplated by the present invention. The The protein (or derivative) is delivered to the lungs of a mammal by inhalation and Enters the bloodstream across the lung epithelial lining. (In other reports on this, Adjei et al., Pharmaceutical Research7.: 565-569 (1990); Adjei Et al., International Journal of Pharmaceutics63: 135-144 (1990) (Loupe acetate Loride); Braquet et al., Journal of Cardiovascular Pharmacology13(Extra number 5): s. 143-146 (1989) (endothelin-1); Hubbard et al., Annals of Internal Medicine.Three : 206-212 (1989) (α1-antitrypsin); Smith et al. Clin. Invest.84: 1145-114 6 (1989) (α-1-proteinase); Oswein et al., "Aerosolization of Proteins", Pr oceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colora do, March 1990 (recombinant human growth hormone); Debs et al., The Journal of Immunology.  140: 3482-3488 (1988) (interferon-γ and tumor necrosis factor alpha) and And Platz et al., US Patent No. 5,284,656 (granulocyte colony stimulating factor).   A wide variety of mechanical devices (e.g., nebulizers) designed for pulmonary delivery of the therapeutic products , Metered dose inhalers, powder inhalers, etc., all of which are well known to those skilled in the art. Including, but not limited to) in the practice of the present invention. Intended.   Some examples of commercially available devices suitable for practicing the present invention include: Mallinckrodt, Inc. (St. Louis, Missouri) Ultravent sprayer from Marquest Medical Products (Englewood, Colorado) corn II sprayer, Venxo from Glaxo Inc. (Research Triangle Park, North Carolina) tolin metered dose inhaler and Fisons Corp. (Spinhale from Bedford, Massachusetts) r powder inhalers.   In any such device, the protein (or analog or derivative) It is necessary to use formulations which are suitable for the dosing of body). Typically, each formulation is Diluents, adjuvants and / or therapeutically useful, specific to the type of device used. May involve the use of suitable propellants in addition to the carrier.   The protein (or derivative) is most effective for delivery to the distal lung, Particles having an average particle size of less than 10 mm (or micron), most preferably 0.5-5 mm It should most advantageously be manufactured in form.   Carriers include trehalose, mannitol, xylitol, sucrose, lactos And carbohydrates such as sorbitol. Other ingredients used in the formulation include , DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactant Agents may be used. Polyethylene glycol may be used (this It may be independent of its use in derivatizing the protein or analog. Good). Dextran such as cyclodextran may be used. Bile salts And other related enhancers may be used. Cellulose and cellulose derivatives May be used. Use amino acids (eg, those used in the preparation of buffers) You may.   In addition, liposomes, microcapsules or microspheres, inclusion complexes, Or the use of other types of carriers.   Formulations suitable for use with a nebulizer (either jet or ultrasonic) , Typically about 0.1 to 25 mg of biologically active protein per mL of solution. Will contain the protein (or derivative) dissolved in water. Also, the formulation , Buffers and simple sugars (eg for protein stabilization and regulation of osmotic pressure) ) May be included. In addition, in the formation of an aerosol, the solution is sprayed. The spray formulation to suppress or prevent the interface-induced aggregation of the protein May contain a surfactant.   Formulations for use with metered-dose inhalers are generally converted to propellants with the help of surfactants. Contains suspended proteins (or derivatives) A finely divided powder. The propellant may be any of the customary propellants used for this purpose. Quality (chlorofluorocarbon, hydrochlorofluorocarbon, hydrofur Olocarbon or hydrocarbon, such as trichlorofluoromethane, dichlorodi Fluoromethane, dichlorotetrafluoroethanol, 1,1,1,2-tetrafluoro Ethane, or a combination thereof). To a suitable surfactant Includes sorbitan trioleate and soy lecithin. Oleic acid also It may be useful as a surfactant.   Formulation for dosing from powder inhaler contains protein (or derivative) Micronized dry powder. The formulation also contains the powder from the device. A dispersing-promoting amount (eg, 50-90% by weight of the formulation) of a bulking agent (eg, Source, sorbitol, sucrose, mannitol, trehalose or xylitol May be included.   Also contemplated is the intranasal delivery of the protein (or analog or derivative). . According to intranasal delivery, after administration of the therapeutic product to the nose, the product is deposited in the lungs. The protein can be passed directly into the bloodstream without the need for dressing. Intranasal delivery Preparations include those containing dextran or cyclodextran. You. Delivery via transport across other mucous membranes is also contemplated.   Administration   Persons of ordinary skill in the art can determine effective dosages by administering and observing the desired therapeutic effect. Will be recognized. In the present invention, increased sensitivity to OB receptor [Eg, the affinity of the OB receptor for its ligand (OB protein) Increase sexuality or "up-regulate" the OB protein receptor] When the composition is further administered, the dose of the OB protein alone required for a given effect is obtained. Will decrease.   Preferably, the formulation of the molecule is between about 0.10 μg / kg / day and 100 mg / kg / day with the desired cure. It is performed so that the therapeutic effect can be obtained. Effective doses are diagnostic tools over time. It can be determined by use. For example, first, blood (or plasma or Or serum) using diagnostics to measure the amount of OB protein in May be measured. Such diagnostics include antibody sandwich It may be in the form of an antibody assay such as an assay. First, the endogenous OB protein Quantify and determine baseline You. Endogenous and exogenous OB proteins (ie, self-produced or administered Protein, analog or derivative found in the body, either The therapeutic amount is determined while quantification continues during the course of treatment. Therefore, the dose May be changed during the course of treatment, initially at a rate until therapeutic benefit is realized. Use relatively high doses and lower doses to maintain therapeutic benefit. Can be used.   Theoretically, if you only want to lose fat-free weight, the dose will be Would be insufficient to cause Therefore, during the first course of treatment of obese humans Achieves weight loss and concomitant loss of adipose tissue / increased lean body mass Will be the dosage administered. Once you have achieved sufficient weight loss, you will gain weight again To increase the desired lean body mass (or reduce the fat rib excluded body weight) Doses sufficient to maintain prophylaxis (depletion prevention) may be administered. OB Tan These doses can be determined empirically because the effect of puta is reversible. (Eg, Campfield et al., Science269: 546-549 (1995) p. 547). Therefore If it is observed that weight loss occurs at a certain dose when weight loss is not desired The desired fat-excluded tissue weight Administer lower doses to achieve increased weight while maintaining the desired weight Will do.   To increase the individual's sensitivity to insulin, a similar Consideration will be given. Insulin administered to an individual for the treatment of diabetes (or Potentially to reduce the amount of amylin or other potential diabetes medications) It is possible to achieve a sufficient increase in fat rib excluded weight without weight loss.   Similar considerations will be made regarding dosage, to increase overall strength. all Increasing fat rib excluding weight with increased physical strength is not enough to cause weight loss Smaller doses could be achieved. Increased red blood cells (oxygenation in the blood), bone resorption Or other benefits, such as reduced osteoporosis, without loss of weight. It is possible.   combination   The method is useful for treating drugs useful in the treatment of diabetes (eg, insulin and possibly thiazo Lysedione, amylin or their antagonists), cholesterol and And blood pressure lowering drugs (eg, drugs that reduce blood lipid levels or other cardiovascular drugs) ), Activities It may be used with other drugs such as enhancers (eg, amphetamine). Also food Greed suppressants (eg, also affect serotonin or neuropeptide Y levels May be used. Such administration can be simultaneous or sequential. it can.   In addition, the method may be used in cosmetic surgery intended to alter the overall appearance of the body (eg, For example, liposuction or laser surgery intended to reduce weight, or Transplant surgery intended to increase apparent weight) You may. Caused by inhibition of blood vessel blockage by deposition of fat such as arterial plaques Is heart surgery (bypass surgery or other surgery) intended to alleviate the adverse condition? The resulting health benefits may be enhanced by the combination of the present compositions and methods. Not. Also, a series of books on how to remove gallstones, such as ultrasonic or laser methods, It may be used either before, during or after the method of treatment. Further, the method comprises the steps of: Improved by surgery or therapy of muscle injury, or by increasing the amount of lean tissue It can be used as an adjunct to other therapies.   Thus, the present invention relates to the availability of a functional OB protein receptor in an individual, Compositions that increase compatibility or sensitivity OB protein or analog or derivative thereof comprising administering an effective amount Methods are provided for increasing susceptibility to the body in said individual. Also, if desired The method comprises the simultaneous or sequential administration of OB proteins, analogs or derivatives thereof. Including   The composition for increasing the availability or sensitivity of a functional OB protein receptor comprises: Thiazolidinedione composition, 2,4-thiazolidinedione, optionally substituted Thiazolidinedione, 5- [4- [2- (5-methyl-2-phenyl-4-oxazolyl) 2-Hydroxyethoxy] endyl] -2,4-thiazolidinedione (AD-5070), black Fibrate, ciglitazone, englitazone, pioglitazone, BRL 49653, Troglitazone, M16209, oxazolidinedione and thiazolidinedione Or derivatives, analogs, tautomers, enantiomers, diastereomers of said compounds Selectable from isomers, epimers, salts, solvates, esters, prodrugs and metabolites. You can choose.   The OB protein, analog or derivative thereof may be selected from: it can:   (A) SEQ ID NO: 3 (described below) or SEQ ID NO: 6 (described below) Amino acid sequences 1-146;   (B) SEQ ID NO: 6 having a lysine residue at position 35 and an isoleucine residue at position 74 ( Amino acid sequences 1-146 described below);   (C) 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78 , 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142 and Positions 145 and 145 (using the numbering of SEQ ID NO: 6, a glutaminyl residue at position 28) Even if it is not present, it will remain the same number). The amino acid sequence of paragraph (b) having different amino acids;   (D) The glutaminyl residue at position 28 may be deleted as desired (a), (b) Or the amino acid sequence of section (c);   (E) a), (b), (c) or (d) having a methionyl residue at the N-terminus; Amino acid sequence;   (F) a truncated OB protein analog, comprising the following (i) to (xi) (35) Using the numbering of SEQ ID NO: 6 with lysine at position and isoleucine at position 74 Selected):     (I) amino acids 98-146,     (Ii) amino acids 1-32,     (Iii) amino acids 40-116,     (Iv) amino acids 1-99 and 112-146,     (V) one or more of amino acids 100 to 111 are sequentially located between amino acids 99 and 112; Amino acids 1-99 and 112-146,     (Vi) amino acids 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138 , 142 and 145 are replaced with another amino acid. OB analog,     (Vii) one or more of amino acids 4, 8 and 32 have been replaced with another amino acid (i i) a truncated analogue of the term,     (Viii) amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, One or more of 100, 102, 105, 106, 107, 108, 111 and 112 are replaced with another amino acid. A truncated analog of paragraph (iii),     (Vix) amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71 , 74, 77, 78, 89, 97, 112, 118, 136, 138, 142 and 145 have one or more A truncated analog of paragraph (iv), which is substituted with a noric acid;     (X) amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 7 4, 77, 78, 89, 97, 100, 102, 105, 106, One or more of 107, 108, 111, 112, 118, 136, 138, 142 and 145 is another amino acid A truncated analog of paragraph (v), which is substituted; and     (Xi) The truncation according to any one of (i) to (x) having an N-terminal methionyl residue An analog to be converted;   (G) any of (a) to (f) including a chemical moiety bound to the protein moiety Any OB protein or analog derivative;   (H) the derivative of paragraph (g), wherein the chemical moiety is a water-soluble polymer moiety;   (I) the derivative of item (h), wherein the water-soluble polymer moiety is polyethylene glycol;   (J) the derivative of item (h), wherein the water-soluble polymer moiety is a polyamino acid moiety;   (K) the water-soluble polymer moiety is bound only to the N-terminus of the protein moiety (h )) Derivatives;   (L) the OB protein of any of (a) to (k) in a pharmaceutically acceptable carrier, Analogs or derivatives.   The following examples illustrate the invention more completely and limit the scope of the invention. It is not specified. Example 1 uses thiazoly Zindione composition increases the affinity or availability of OB protein receptor in an individual In addition, the individuals have increased sensitivity to the OB protein Is a prophetic example of use in humans. Then, " Materials and Methods "are described.Example 1   There are obese human patients (BMI> 27) where weight loss is desired. OB protein receptor An effective amount of the thiazolidinedione composition to increase the availability of Administer for a week. The patient is then given a sufficient amount of OB to cause weight loss. Administer the protein or a derivative or analog thereof. Circulating OB protein Or the level of the analog or derivative is determined by the OB protein (or other applicable Can be monitored using a diagnostic kit such as an antibody assay for Wear. The patient loses weight to obtain the desired weight loss and / or fat mass. One To maintain the desired body weight and / or body fat levels. OB protein or analog or derivative thereof in combination with azolidinedione composition The body is administered chronically for the desired period of time.Materials and methods Administration of protein or vehicle protein: SEQ ID NOs: 1, 2, and 3 are mouse recombinant OB DNA and protein SEQ ID NOs: 4, 5 and 6 are analogous recombinant human OBDN A and protein (FIG. 2) are described. Lysine residue at position 35 and isoform at position 74 The recombinant human protein of SEQ ID NO: 6 with a leucine residue was used in Example 1. . As described above, the following mouse and human analog recombinant proteins are It is merely an example of an OB protein that can be used in methods of treatment and in the manufacture of medicaments. other An OB protein or analog or derivative thereof can also be used.   Here, the first amino acid in the amino acid sequence of the recombinant protein is set to +1 and This is Valine. The amino acid at position -1 is methionine. C-terminal amino acid is 146 Th (cysteine).Manufacturing method   Produce biologically active recombinant methionyl mouse or human analog OB protein The following manufacturing method was used for fabrication. Biologically active recombinant methionyl human Produce OB protein For this purpose, a similar method can be used.   Expression vectors and host strains   The plasmid expression vector used is pCFM1656 (ATCC accession No. 69576). . The DNA was ligated into the expression vector pCFM1656 linearized with XbaI and BamHI. Was transformed into E. coli host strain FM5. E. coli FM5 cells Amgen Inc. (Thousand Oaks, CA). chmann et al., Bacteriol. Rev.40: 116-167 (1976)). The cell contains the integrated λ Gi repressor gene cI₈₅₇(Sussman et al., C.R. Acad. Sci.254: 151 7-1579 (1962)). Vector production, cell transformation and colony selection are performed by standard Performed by the standard method. For example, Sambrook et al., Molecular Cloning: A Labora tory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Ha See rbor, NY. Host cells were grown in LB medium.   Fermentation method   A three-phase fermentation protocol known as the fed-batch method was used. Medium composition is as follows It is described below.   Batch: fermentation vessel (Biolafitte, 12 liter capacity) The source of phosphorous and phosphate was sterilized (18 to 20 psi for 35 minutes, by heating to 122 ° C). ). Cool and remove carbon, magnesium, vitamins and trace metal sources Bacterially added. An overnight culture of the recombinant mouse protein producing bacteria (16 Over 500 hours) (grown in LB medium) was added to the fermentor.   Feed I: 4.0 to 6.0 OD₆₀₀Upon reaching Feed I, the culture was fed. Growth speed In order to control the degree (m), glucose was fed at a limited feed rate. Proliferation Automated system (Distributive Control) to control speed to 0.15 generations / hour  System).   Feed II: OD₆₀₀When the temperature reaches 30, slowly increase the culture temperature to 42 ° C. The feed was changed to Feed II below. The fermentation, sampling every two hours For 10 hours. After 10 hours, cool the contents of the fermentor to below 20 ° C and centrifuge. More harvested. Medium composition: Batch: 10g / L yeast extract                     5.25g / L (NH_Four)_TwoSO_Four                     3.5g / L K_TwoHPO_Four                     4.0g / L KH_TwoPO_Four                     5.0g / L glucose                     1.0g / L MgSO_Four・ 7H_TwoO                     2.0mL / L vitamin solution                     2.0mL / L Trace metal solution                     1.0mL / L P2000 defoamer Feed I: 50g / L Bacto-tripton                                         (Bacto-tryptone)                     50g / L yeast extract                     450g / L glucose                     8.75g / L MgSO_Four・ 7H_TwoO                     10mL / L vitamin solution                     10mL / L Trace metal solution Feed II: 200 g / L Bacto-tripton                                         (Bacto-tryptone)                     100g / L yeast extract                     110g / L glucose   Vitamin solutions (batch and feed I):   0.5 g of biotin, 0.4 g of folic acid and 4.2 g of riboflavin in 450 ml of H_TwoO and Dissolve in 3 ml of 10N NaOH and add H_TwoMake up to 500 mL in O. 14 g of pyridoxine-HCl and 6 1g niacin to 150ml H_TwoDissolved in O and 50 ml of 10N NaOH,_Two250 mL in O Was. 54 g of pantothenic acid in 200 mL of H_TwoDissolved in O to make 250 mL. The three solutions Together, the total volume was 10 liters.         Trace metal solutions (batch and feed I):         Ferric chloride (FeCl_Three・ 6H_TwoO): 27g / L         Zinc chloride (ZnCl_Two・ 4H_TwoO): 2g / L         Cobalt chloride (CoCl_Two・ 6H_TwoO): 2g / L         Sodium molybdate (NaMoO_Four・ 2H_TwoO): 2g / L         Calcium chloride (CaCl_Two・ 2H_TwoO): 1g / L         Cupric sulfate (CuSO_Four・ 5H_TwoO): 1.9g / L         Boric acid (H_ThreeBO_Three): 0.5g / L         Manganese chloride (MnCl_Two・ 4H_TwoO): 1.6g / L         Sodium citrate dihydrate: 73.5 g / L   Purification method of mouse OB protein   Purification was performed by the following steps (unless otherwise indicated, the following steps were performed at 4 ° C.) Done).   1. Cell paste   E. coli cell paste was suspended in 5 volumes of 7 mM EDTA (pH 7.0). . The cells in the EDTA are passed twice through a microfluidizer. By doing so, it was further destroyed. The destroyed cells are attached to a JS-4.2 rotor. And centrifuged at 4.2K rpm for 1 hour in a Beckman J6-B centrifuge.   2. Cleaning of inclusion bodies: 1st time   Remove the supernatant from the above and resuspend the pellet in 5 volumes of 7 mM EDTA (pH 7.0). It became cloudy and homogenized. This mixture was centrifuged as in step 1.   3. Cleaning of inclusion bodies: 2nd time   The supernatant from above was removed and the pellet was washed with 10 volumes of 20 mM Tris (pH 8.5), 10 mM Resuspended in MDTT and 1% deoxycholate and homogenized. This mixture And centrifuged as in step 1.   4. Cleaning of inclusion bodies: 3rd time   The supernatant from above was removed and the pellet was resuspended in 10 volumes of distilled water and homogenized. I knew it. This mixture was centrifuged as in step 1.   5. Playback   The pellet was immersed in 15 volumes of 10 mM Hepes (pH 8.5), 1% sarcosine sodium (N- (Lauroyl sarcosine) at room temperature. After 60 minutes, the solution is It was made copper and then stirred overnight.   6. Removal of sarcosine   The regeneration mixture was diluted with 5 volumes of 10 mM Tris buffer, pH 7.5, and And centrifuged as above. Collect the supernatant, MI) (20-50 mesh, chloride form, diluted regeneration mixture See page 89 of WO 89/10932 for more information. Put this mixture on a column And the eluate was collected. Removal of sarcosine was confirmed by reverse phase HPLC.   7. Acid precipitation   Collect the eluate from the previous step, adjust the pH to 5.5, and incubate for 30 minutes at room temperature. I did it. This mixture was centrifuged as in step 1.   8. Cation exchange chromatography   Adjust the pH of the supernatant from the previous step to 4.2 and load onto the CM Sepharose Fast Flow. (At 7% volume). With 20 mM NaOAc (pH 4.2), 0M to 1.0M NaCl, 2 A 0 column volume salt gradient was applied.   9. Hydrophobic interaction chromatography   Pool the CM Sepharose pool of the peak fraction from the above step (confirmed from UV absorbance) , 0.2 M ammonium sulfate. 0.4M to 0M sulfuric acid in 5mM NaOAc (pH4.2) Monium was subjected to a 20 column volume reverse salt gradient. Concentrate this substance in PBS Was diafiltered.   Fermentation of recombinant human OB protein analogs:   Said host cell for producing a recombinant human OB protein analogue (SEQ ID NO: 6) Is fermented using the same conditions and composition as described above for the recombinant mouse material. Can do it.Purification of recombinant human OB protein analog:   The recombinant human protein analog is used in the purification of the recombinant mouse protein described above. It can be purified by the same method as described above. Recombinant human OB protein analog In the production of, the pH of the supernatant from step 7 was adjusted to 5.0 and this was Step 8 should be performed by loading on a moving column. 20 colors The volumetric salt gradient should be run with 20 mM NaOAc (pH 5.5), 0M to 0.5M NaCl . Step 9 involves diluting the CM Sepharose pool 4 times with water and adjusting the pH to 7.5. More to do. This mixture should be 0.7M ammonium sulfate. A 20 column volume reverse salt gradient was run using 5 mM NaOAc (pH 5.5), 0.2 M to 0 M ammonium sulfate. Should be done in the system. Otherwise, it is the same as the above-mentioned process. Ricin 35 and Recombinant human OB protein of SEQ ID NO: 6 with isoleucine 74 was Was formulated in a buffer (pH 6.0) containing 4.3% arginine.   Although the present invention has been described with reference to preferred embodiments, changes and modifications thereof are not It is understood that it will be found in the person skilled in the art. Therefore, the appended claims are particularly remarkable. Scope of the claimed invention It is intended to encompass all such equivalent changes within the box.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/10 A61P 3/10 9/10 9/10 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM ), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, H , IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW

Claims (1)

[Claims] 1. Compositions that increase the affinity or availability of a functional OB protein receptor in an individual OB protein or an analog or derivative thereof, comprising administering a substance A method of increasing the susceptibility to OB in said individual, optionally comprising Those characterized by combination with administration of the protein or its analogs or derivatives Law. 2. Such a composition that increases the affinity or availability of a functional OB protein receptor is 2. The method according to claim 1, which is a thiazolidinedione composition. 3. The OB protein or an analog or derivative thereof,   (A) the amino acid sequence 1-146 of SEQ ID NO: 3 or SEQ ID NO: 6;   (B) SEQ ID NO: 6 having a lysine residue at position 35 and an isoleucine residue at position 74 The described amino acid sequence 1-146;   (C) 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78 , 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142 and And position 145 (numbered SEQ ID NO: 4) (B) having different amino acids substituted at one or more positions Amino acid sequence of item;   (D) The glutaminyl residue at position 28 may be missing if desired (a), (b) Or the amino acid sequence of section (c);   (E) a), (b), (c) or (d) having a methionyl residue at the N-terminus; Amino acid sequence;   (F) a truncated OB protein analog, comprising the following (i) to (xi) (35) Using the numbering of SEQ ID NO: 6 with lysine at position and isoleucine at position 74 Selected):     (I) amino acids 98-146,     (Ii) amino acids 1-32,     (Iii) amino acids 40-116,     (Iv) amino acids 1-99 and 112-146,     (V) one or more of amino acids 100 to 111 are sequentially located between amino acids 99 and 112; Amino acids 1-99 and 112-146,     (Vi) amino acids 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138 , 142 and 145 are substituted with another amino acid. Kated OB analog,     (Vii) one or more of amino acids 4, 8 and 32 have been replaced with another amino acid (f ) (Ii) the truncated analog of paragraph (ii);     (Viii) amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, One or more of 100, 102, 105, 106, 107, 108, 111 and 112 are replaced with another amino acid. (F) (iii) a truncated analog,     (Vix) amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71 , 74, 77, 78, 89, 97, 112, 118, 136, 138, 142 and 145 have one or more A truncated analogue of paragraph (f) (iv), which is substituted with a noric acid;     (X) amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 7 4, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138 , 142 and 145 are substituted with another amino acid. Kated analogs, and     (Xi) any of (f) (i) to (x) having an N-terminal methionyl residue; Uncated analogs;   (G) any of (a) to (f), including a chemical moiety bound to the protein moiety An OB protein or analog derivative of;   (H) the derivative of paragraph (g), wherein the chemical moiety is a water-soluble polymer moiety;   (I) the derivative of item (h), wherein the water-soluble polymer moiety is polyethylene glycol;   (J) the derivative of item (h), wherein the water-soluble polymer moiety is a polyamino acid moiety;   (K) the water-soluble polymer moiety is bound only to the N-terminus of the protein moiety (h )) Derivatives;   (L) the OB protein of any of (a) to (k) in a pharmaceutically acceptable carrier, Analog or derivative The method according to claim 1, wherein the method is selected from: 4. The administration may be due to overweight, diabetes, high blood lipid levels, arteriosclerosis, Reduction or prevention of stone formation, insufficient lean tissue mass, insufficient insulin Claims 1 to treat a condition selected from susceptibility and stroke. 4. The method according to 2 or 3.