JP2000512137A - Fragment of leptin (ob protein) - Google Patents

Abstract

  (57) [Summary] Weight-modulating peptides or functional derivatives, analogs or variants thereof that substantially modulate energy utilization, pharmaceutical compositions containing such compounds, methods of preparing such compounds and methods of preparing such compounds Use in medicine.

Description

DETAILED DESCRIPTION OF THE INVENTION                 Fragment of leptin (ob protein)   The present invention relates to novel compounds, in particular novel peptides, pharmaceutical compositions containing such compounds The invention relates to compositions and the use of such compounds in medicine.   Physiological regulation of the energy balance of food intake versus energy output in the body The mechanism has been the subject of discussion for many years. In recent nature In the disclosure, Y. Zhang et al. [Nature, 372, 425-431, 1994]. Is one of the molecules that play an important role in regulating energy balance. Suggests that Zhang and colleagues also added that both mouse and human cloning and sequencing of ob gene protein or leptin You.   Structure of human leptin (or human ob protein) and weight control of the animal Is disclosed in British Patent Application Publication No. GB 229,382. This Also filed a specific fragment of leptin, which is said to be capable of regulating body weight. Is disclosed.   Collins et al. [Nature, 380, 677, 1996] report a reduction in leptin. Quantitatively disclosed that quantity could be explained by improved energy use and reduced food intake. ing.   The present inventors have surprisingly found that by improving energy use, Certain novel fragments of leptin have been found to regulate weight Was. These fragments may therefore cause nutritional and metabolic disorders, especially obesity and It is believed to be particularly useful in the treatment of diabetes and diabetes.   Thus, in a first aspect, the invention substantially modulates energy utilization. Weight controlling peptide or a functional derivative, analog or mutation thereof Provide body.   Preferably, adjusting body weight refers to weight loss.   Preferably, the regulation of energy use is by improving energy use.   Preferably, the peptide is an ob protein, especially a human ob protein, or Or a fragment of a functional derivative, analog or variant thereof.   Hereinafter, the protein fragment (or peptide) is the human ob protein The amino acid sequence is referred to using the following abbreviations: "1 to 6 "Protein fragment consisting of amino acid residues of" ab1-6 "is abbreviated as" ob1-6 " .   Individual peptides were ob21-26 (MVPIQK), ob27-32 (VQ DDTK), ob33-36 (TLIK), ob37-41 (TIVTR), o b42-54 (INDISHTQSVSSK), ob55-56 (QK), ob 57-74 (VTGLDFIPGLHPILTLSK), ob93-105 (N VIQISNDDLENLR), ob106-115 (DLLHVLAFSK), ob116-149 (SCHLPWASGLELDSLGGVLEASGYS TEVALSR) and ob150-167 (LQGSLQDMLWQLDL SPGC), especially ob57-74 (VTGLDFIPGLHPILTLSK) Include.   Suitably, the present invention relates to any one or more of the foregoing individual peptides. And peptides formed therefrom.   Preferably, the present invention relates to two consecutive elements of any of the individual peptides described above. And peptides formed from the same.   As already mentioned, the invention further relates to the peptides described herein. And functional derivatives, analogs and variants of   Functional derivatives include salts and solvates of the peptides described herein. The physical properties of the protein, such as stability or therapeutic properties, For example, chemically by attaching groups or moieties that enhance pharmacokinetic properties Also encompassed are modified peptides of the invention.   Functional analogs are those in which one or more amino acids of the peptides described herein are one or more. Includes functionally similar peptides replacing another amino acid.   Another amino acid is an alternative stereochemical to the amino acid in the ob protein. Includes amino acids with properties.   Functional analogs may further include small molecule agonis of the peptides described herein. G Or an antagonist. Such compounds can be prepared by known procedures such as GB Can be prepared and tested according to the procedures disclosed in US Pat. You.   Salts include pharmaceutically acceptable salts, especially pharmaceutically acceptable acid addition salts.   Acid addition salts of peptides include the parent compound and excess acid such as hydrochloric acid, hydrobromic acid From sulfuric acid, phosphoric acid, acetic acid, maleic acid, succinic acid or methanesulfonic acid It is prepared in standard solvents using standard methods. The acetate form is particularly useful. Conversion Some compounds form acceptable internal salts or zwitterions. Katio The acidic salt is prepared by converting the parent compound into an excess of an alkaline reagent such as a hydroxide containing an appropriate cation. It is prepared by treating with a substance, a carbonate or an alkoxide. Na⁺, K⁺, Ca²⁺ And NH_Four ⁺Are examples of cations present in pharmaceutically acceptable salts It is.   Solvates include pharmaceutically acceptable solvates, such as hydrates.   It is understood that the present invention encompasses both peptide and non-peptide compounds.   In addition, the present invention relates to specific peptides ob21-26, ob27-32, ob33 -36, ob37-4, ob42-54, ob55-56, ob57-74, o b93-105, ob106-115, ob116-149 and ob150- 167, in particular a subfragment of ob57-74; or one of the above peptides Is a peptide formed from more than one, especially any two consecutive components: Encompasses functional derivatives, analogs or variants thereof.   Suitable peptides or subfragments have at least 4 amino acids .   The peptides of the present invention are suitably prepared using conventional digestion techniques, synthetic techniques or standard techniques. Prepared by using expression methods.   Thus, in yet another aspect, the invention relates to a peptide, A process for preparing a derivative, comprising the following steps:   At least two peptides, especially ob proteins, especially human ob proteins Hydrolyzing to a peptide fragment of   Separating the peptide fragment;   Thereafter, if desired, its functional derivative may be prepared. Providing a method comprising:   Hydrolysis of proteins can be performed, for example, by enzymatic digestion with trypsin. Appropriate.   Separation of the required peptide is usually accomplished by appropriate chromatography means, e.g., column chromatography. Performed by using chromatography.   The reaction conditions for the processing of the ob protein will depend on the safety of the products for which they are required. As is qualitative, it is determined by the particular reagents used, and When syn is the reagent, the reaction is usually in a temperature range of 25-40 ° C and a p-value of 7-9. The reaction is performed within the H range, preferably at 37 ° C. and pH 7.4.   As noted, the peptides of the invention may be further synthesized by conventional synthetic methods, such as liquid phase or Prepared by use of solid phase peptide synthesis.   Thus, the present invention provides a means for substantially regulating energy utilization, To provide a synthetic peptide or a functional derivative, analog or variant thereof that regulates You.   Any of the peptides described in this specification may be used as synthetic peptides according to the present invention. Form a part.   The peptide binding units of the proteins relevant to the present invention can be From peptide synthesizers [Atherton, E. and Sheppard, R.C. (eds.) (1989) So lid Phase Peptide Synthesis: A practical approach (LRL Press, Oxford)] And followed by procedures appropriate for direct disulfide or amide bond formation. Can be prepared.   Well-known methods for peptide synthesis are described in Ali et al., J. Med. Chem., 29: 984 (1986). And J. Med. Chem., 30: 2291 (1987). It is expressly incorporated as a part of the present invention. Preferably, the peptide is a Merrifield solid-phase technology [J. Am. Chem. Soc., 85: 2149 (1964)]. But Solid-phase synthesis of di-, tri-, tetra- or penta-peptide fragments Convergent synthesis prepared by and combined or further modified by liquid phase synthesis As in, combinations of solid and liquid phase synthesis are also used.   During synthesis, side chain functional groups (eg, -NH_Two, -COOH, -OH, -SH) Protected during the coupling reaction. Usually, the α-amino group is temporarily fluorenylmeth Kiss Protected as carbonyl (Fmoc) but other acid or base protecting groups, eg For example, t-butoxycarbonyl (Boc) can be used. Amino side of lysine The chain group is t-butoxycarbonyl, benzyloxycarbonyl or p-chlorobenzene. Protected as benzyloxycarbonyl (Z or Cl-Z). Acetamide Tyl, trityl, t-butyl, St-butyl or para-methylbenzyl (p -MBz) Protection is used for cysteine. Hydroxy group is butyl or benzyl Protected as an ether and the carboxyl group is butyl, benzyl (Bz) or cyclo Protected as hexyl ester.   The peptide may be synthesized from either the C-terminus or the N-terminus, preferably from the former. Wear. Prior to coupling, alpha carboxyl (of protected amino acids as appropriate) Activates the sil group. One skilled in the art can activate the protected group in a number of ways. An example For example, N, N'-dicyclohexylcarbodiimide (DCC), 2- (1H-benzene Zotriazol-1-yl) -1,1,3,3-tetramethyluronium hexa Fluorophosphate (HBTU), p-nitrophenyl ester (pNp), Droxybenzotriazole ester (HOBt), N-hydroxysuccinyl A mixed anhydride or a symmetric anhydride of midyl ester (OSu) may be used.   Liquid phase synthesis of the peptide is carried out using a conventional method for forming an amide bond. Typically The protected Boc-amino acid having a free carboxyl group can be converted to a suitable carbodiimide Coupling agents such as N, N'-dicyclohexylcarbodiimide (DCC) And optionally 1-hydroxybenzotriazole (HOBT) and Protected amino groups having free amino groups in the presence of tylaminopyridine (DMAP) Combine with amino acids.   In liquid phase synthesis, the coupling reaction is preferably at a low temperature (eg, -20 ° C). , Dichloromethane (DCM), dimethylformamide (DMF), N-methyl Pyrrolidone (NMP), tetrahydrofuran (THF) acetonitrile (ACN ) Or in a solvent such as dioxane.   When using the solid phase method, starting from the carboxy terminus, the peptide Build continuously towards the mino end. Solid phase synthesis is the C-terminal of protected amino acids To 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) -phenoxy resin [Link amide resin, H. Rink, Tetrahedron Letters, 28, 3787 (198 7)] 4-benzyloxybenzyl alcohol resin [One resin, S.S.Wang, JAC S, 95, 1328 (1973)] or 4-hydroxymethylphenoxyacetic acid Start by covalently bonding to a suitable resin, such as a resin.   In solid-phase synthesis, the first amino acid residue is bound to an insoluble polymer. General. For example, two commonly used polymers are polystyrene (divinyl And 1% cross-linked polyacrylamide. these Polymer binds to the first amino acid (ie, the carboxy terminus) of the target peptide Reactive groups such as -OH, -NH_TwoAnd -CH_TwoFunctional group containing Cl Has been entered. The choice of linkage between the first amino acid and the polymer depends on the peptide Dominated by the end. For example, a peptide having a carboxyl group at the C-terminus Is for a peptide linked by an ester bond and ending with carboxamide Has an amide bond.   Once the first protected amino acid has bound the desired resin, the amino protecting group is The next (protected) amino acid is removed by treatment with a secondary amine such as peridine. Is linked to this amino group. This process is a simple intermediate Continuously, without separation, until the desired peptide is formed. Finished pen The peptide is then subjected to deblocking and / or cleavage from the resin in any order.   Preferred solvents for the coupling reaction include, but are not limited to, dichloromethane Dichloromethane (DCM), dimethylformamide (DMF) and N-methylpyrroli Don (NMP). After the desired sequence is synthesized, the peptide is deprotected, Cleaved from resin using trifluoroacetic acid or trifluoromethanesulfonic acid Let   A preferred method for cleavage of the peptide from the supporting resin is to support the resin. The peptide is scavenged with trifluoroacetic acid by appropriate cation and carbonium ion scavenging. Jars such as phenol, anisole, thioanisole, ethanedithiol , Water or ethyl methyl sulfide.   To obtain the compounds of the present invention, the synthetic peptide can be cyclized using methods well known in the art. Conversion / Coupling.   For example, the disulfide bond of two linear peptides, both containing cysteine residues Coupling by coupling results in free thiol on one strand and appropriate activation on the other strand Selectively achieved by reaction with the cysteine derivative. Substitutable protecting group Particularly useful as is a S- (carbomethoxy-sulfenyl) derivative . This method, for example, removes the cysteine residues of both linear peptides from acetamide Protection with a chill (Acm) group. One chain is treated with mercury (II) acetate and Treated with beta-mercaptoethanol to remove the acetamidomethyl protecting group I do. Treating the second chain with carbomethoxysulfenyl chloride to remove the activated species obtain. When the two peptides are stirred in a dilute aqueous solution having a pH of about 7 to 8, Substitution of the methoxysulfenyl group and formation of interchain disulfides occur.   If an intramolecular disulfide is to be formed, complete the corresponding linear peptide To form a dimercaptan. Dimercaptan Any oxidizing agent known in the art that can be converted to a sulfide may be used. This Examples of such reagents include alkali metal ferricyanides (eg, potassium ferricyanide). Or sodium), oxygen, iodomethane or iodine. Reaction is suitable In an inert solvent such as aqueous methanol or water at a temperature of about 0 to 40 ° C. Performed under high dilution. The pH is usually maintained at about 7-8. Peptides still To the supporting resin or other functional groups are still protected The peptide may be cyclized with Perform on the peptide.   Where two disulfides are to be formed between two linear peptides Use two cysteine thiol protecting groups, such as Acm and trityl be able to. In each peptide, the pair of cysteines to be bound is a trityl group, Contains one of the various types arranged to be protected by Acm. Trityl group Is independently removed from each peptide and the thiol on one peptide is replaced with 2,2'-dipi Activation with lysyl disulfide followed by the addition of other monothiol peptides And a bis (S-acetamido-methyl) disulfide bond Two separate monothiol derivatives are obtained from which the combined peptide can be obtained. second The disulfide of This product was converted to Kamber [B. Kamber, Helv. Chim. Acta, 54, 927 (1971)]. And Kamber et al. [B. Kamber et al., Helv. Chim. Acta, 63, 899, (1980)]. Can be obtained by direct iodination.   The peptide chain further comprises -NH (CH_Two)_nCoupling using a coupling group such as CO- Can be This corresponds to the corresponding amino acid (NH_Two(CH_Two)_nCOOH) N^a-Fmoc induction By incorporating it into a peptide chain that grows during normal solid-phase synthesis. Is most easily achieved. Side chain carboxyl of acidic amino acids such as glutamic acid And lysine and other basic amino acids to couple peptide chains A similar method can be used to perform this. In this case, the following N⁶-G glue Compounds such as tamillysine derivatives are incorporated into peptide chains that grow during normal solid-phase synthesis. May be incorporated.Coupling to the growing peptide chain is via the carboxyl of the glutamic acid residue The removal of the Fmoc group on the amino group of lysine requires the addition of another amino acid. Supply a starting point.   Alternatively, N^a-A trityl protecting group can be used on the glutamic acid residue After coupling, it is removed with 80% acetic acid and N-acetylated with acetic anhydride. . Sa The resulting coupling can proceed as described above.   Acetylation of protein-free amino by removing amino protecting group with acetic anhydride By doing so, an N-terminal N-acetyl group can be introduced. C-terminal cal The boxyamide group can be obtained by using a suitable solid phase synthetic resin such as Rink amide resin. Is obtained.   As noted, the peptides of the present invention can be further purified using recombinant DNA technology. It is prepared by expression of a DNA encoding the peptide sequence.   Thus, the present invention provides a means for substantially regulating energy utilization, And its functional derivatives, analogs or variants Huh.   Any of the peptides described herein can be used as a recombinant peptide of the present invention. Form a part.   In a further aspect, the invention relates to a process for the preparation of a compound according to the invention, comprising Expressing the DNA encoding the compound in the host cell and recovering the product. Provide a way to become.   A DNA polymer comprising a nucleotide sequence encoding the compound is also a part of the present invention. To form   The method of the present invention is described in Maniatis et al., Molecular Cloning-A Laboratory Manual; Col. d Spring Harbor, 1982 and DNA Cloning, Volumes I, II and III (D.M. over edition, IRL Press Ltd). You.   In particular, the method comprises:     i) In a host cell, a DN containing a nucleotide sequence encoding the compound. Preparing a replicable expression vector capable of expressing the A polymer;    ii) transforming a host cell with the vector;   iii) culturing the transformed host cells under conditions that allow expression of the DNA To produce the compound; and    iv) recovering the compound Process.   The invention further relates to the condensation of suitable mono-, di- or oligomeric nucleotide units. To A method for preparing a DNA polymer is also provided.   Preparation may be performed in vitro, chemically, enzymatically or by a combination of the two methods. Or performed in vivo. Thus, the DNA polymer is suitable for DNA fragmentation. Enzymatic treatment, for example, D. M. Roberts et al., Biochemistry, 1985, 2 4, 5090-5098.   A DNA fragment is a DNA fragment containing a necessary nucleotide sequence. Digestion with restriction enzymes, by chemical synthesis, or enzymatic polymerization on DNA or RNA templates Or by a combination of these methods. DNA fragment It may be preferable to use a total synthesis of the   Digestion with restriction enzymes is carried out in a suitable buffer at a temperature of 20-70 ° C., preferably 5 ° C. This is done with 0.1-10 mg of DNA in a volume of 0 ml or less.   Enzymatic polymerization of DNA is performed in vitro, if necessary, using the nucleotide triphosphate dAT. 10-37 in a suitable buffer containing P, dCTP, dGTP and dTTP. DNA polymerase I (Klenow fragment) at a temperature of G) and the like.   Enzymatic ligation of DNA fragments can be performed by using a suitable method such as T4 DNA ligase. Using a suitable DNA ligase in an appropriate buffer at 4 ° C to room temperature, It can be implemented by multiplication.   Chemical synthesis of DNA polymers or fragments involves the use of conventional phosphotriesters. Chemistry and phosphite or phosphoramidite chemistry tic Synthesis of Protein Fragments-A Laboratory Manual "(H.G. Gassen and And A. Lang), Verlag Chemie, Weinheim (1982) or other scientific publications, For example, M.J.Gait, H.W.D.Matthes, M.Singh, B.S.Sproat and R.C.Titmas, Nu cleic Acids Research, 1982, 10, 6243; B.S.Sproat and W.Bannw arth, Tetrahedron Letters, 1983, 24, 5771; M.D. Matteucci and M.H.Caruthers, Tetrahedron Letters, 1980, 21, 719; M.D.Matteucc i and M.H.Caruthers, Journal of the American Chemical Society, 1981 , 103, 3185; S.P. Adams et al., Journal of the American Chemical. Society, 1983, 105, 661; N.D.Sinha, J. Biernat, J. McMannus and And H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H.D. Solids as described in Matthes et al., EMBO Journal, 1984, 3, 801]. This is done using phase technology. Preferably, an automated DNA synthesizer is employed.   The DNA polymer preferably comprises together the DNA sequence encoding the compound. Prepared by ligation of two or more DNA molecules.   A DNA molecule is prepared by appropriate restriction enzymes on a vector having the necessary coding sequences. Obtained by digestion of   The exact structure of the DNA molecule and the method for obtaining it will depend on the structure of the desired product. Exist. Designing an Appropriate Method for Construction of a DNA Molecule Encoding the Compound Is routine for those skilled in the art.   Expression of a DNA polymer encoding the compound in a recombinant host can be effected in a host cell. It is performed by a replicable expression vector capable of expressing a DNA polymer in the cell. Expression The vectors are novel and further form part of the present invention.   A replicable expression vector is a vector that is compatible with the host cell in accordance with the present invention. To obtain a linear DNA segment having an intact replicon, A linear segment together with the linear segment encodes the compound Prepared by ligating further DNA molecules under ligation conditions You.   Ligation of the linear segment and one or more DNA molecules can be performed simultaneously, if desired. Or it is performed continuously.   Thus, the DNA polymer may be pre-formed or base if desired. May be formed during construction of the reactor.   The choice of vector is some prokaryotic cells, such as E. coli Iha mouse C127, mouse myeloma, Chinese hamster ovary, fungus, eg For example, fungal or unicellular yeast or insect cells such as Drosophila It will be determined by the host cell being a eukaryotic cell. The host cell is It may be in a transgenic animal. Suitable vectors are plasmids, Bacteriophages, cosmids and eg baculovirus or vaccinia Recombination of origin Virus.   The preparation of a replicable expression vector involves preparing the DNA using appropriate restriction enzymes, polymerization, and ligation. For the gating, for example, the operation described in Maniatis et al. It can be carried out manually. Polymerization and ligation is the preparation of DNA polymers. The procedure is as described above for production. For digestion with restriction enzymes, use an appropriate buffer Medium, at a temperature of 20-70 ° C., in a volume of 50 ml or less, 0.1-10 mg of DNA This is performed using   Recombinant host cells, according to the present invention, are capable of replicating host cells of the present invention. It is prepared by transforming a vector under transformation conditions. Suitable transformation Conversion conditions are conventional and are described, for example, in Maniatis et al. ng ", Volume II, edited by D.M. Glover, IRL Press Ltd. 1985.   The choice of transformation conditions is determined by the host cell. Therefore, E. coli Bacterial host such as CaCl_Two[Cohen et al., Proc. Nat. Acad. Sci., 1 973, 69, 2110], or RbCl, MnCl_Two, Potassium acetate and glycero And [3-morpholino] -propane- It may be treated with sulfonic acid, RbCl and glycerol. Mammalian cells in culture Is transformed by calcium co-precipitation of vector DNA onto cells.   The invention further extends to a vector comprising a compound of the invention.   The present invention further provides a host cell transformed with the replicable expression vector of the present invention. Extend to   Culture of the transformed host cells under conditions that allow expression of the DNA polymer And generally described, for example, in Maniatis et al. And "DNA Cloning", supra. And so on. Thus, it is preferable to provide nutrients to the cells and to maintain the temperature below 45 ° C. Incubate at a low temperature.   The expression product is recovered by a conventional method depending on the host cell. Thus, if the host cell is a bacterium For example, in the case of E. coli, this is physically, chemically or enzymatically dissolved, The protein product is isolated from the resulting lysate. Where products are secreted from bacterial cells If so, it is recovered from the periplasmic or nutrient medium. When the host cell is a mammal Product Is isolated from the nutrient medium.   DNA polymers can be, for example, bovine papilloma virus vectors or Chinese Stable transgenic mammals expressing the amplified vector in Muster ovary cells Assembled with vectors designed to isolate cell lines [DNA Cloni ng Vol. II, edited by D.M. Glover, IRL Press 1985; Kaufman, R.J. et al., Molecular an d Cellular Biology, 5, 1750-1759, 1985; Pavlakis G.N. and Hamer, D.H., Proceedings of the National Academy of Sciences (USA), 80, 397-401, 1983; Goeddel, D.V. et al., European Patent Application 0. 093619 (1983)].   If necessary, the peptide prepared using the method described above may be purified by various techniques. Can be Preferred embodiments include gel filtration, chromatography, reverse phase HP Including LC and crystallization, especially chromatography is used. Incidentally The prepared product was analyzed for purity by HPLC, amino acid analysis, amino acid sequencing. And fast atom bombardment and / or electron injection mass spectrometry.   Functional derivatives, analogs and variants of the proteins described herein are Prepared using conventional methods similar to those described herein.   As noted, it has been pointed out that the compounds of the present invention have useful pharmaceutical properties. ing. Accordingly, there is also provided a compound for use as an active therapeutic substance.   In particular, the compounds of the present invention have substantial ability to regulate body weight by improving energy utilization. And therefore nutritional and metabolic disorders, especially obesity and diabetes Useful in therapy.   The present invention is further directed to a method of treating nutritional and metabolic disorders, which is effective, There is provided a method comprising administering an acceptable, non-toxic amount of a compound of the present invention.   Therefore, the present invention further provides a compound of the present invention and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising:   In use, the active compound is usually formulated as a human or veterinary pharmaceutical carrier, diluent and And / or used in the form of a pharmaceutical composition with excipients, the exact form of the composition being Depends on the method of administration. The active compound can be administered, for example, for oral administration in tablets, capsules , Rose In the form of a syringe or syrup; for inhalation, olfactory, aerosol or sprayable In the form of a functional solution; in the form of a sterile solution for parenteral administration; About creams, lotions, liniments, gels, ointments or sprays It is. Parenteral routes include intravenous, intramuscular, subcutaneous, transdermal and intraperitoneal administration Is included.   In addition, subcutaneous implant pumps or controlled release devices, use in transdermal patches and Also included are formulations of the above derivatives, suitable for use as a fine powder suitable for intranasal administration. .   Dosage ranges for the administration of the compounds of this invention may vary depending on the condition being treated. Dosage is effective, dosage is age, severity or severity of disease, if contraindicated Will vary. The dose ranges from 0.001 mg / kg / day to 50 mg / kg. kg / day, but preferably between 0.01 and 1.0 mg / kg / day. You.   Solid oral dosage forms include, for example, lactose, microcrystalline cellulose, Cium, mannitol, magnesium carbonate, glycine, dextrose, shoe Such as sucrose, starch, mannitol, sorbitol and calcium carbonate Diluents, for example, liquid glucose, syrup, acacia, gelatin, starch serum Agent, methylcellulose, polyvinylpyrrolidone, alginate, and presella Binders such as tinted starch; for example, starch, alginic acid, microcrystalline cellulose , Pectin, cross-linked polyvinyl pyrrolidone, sodium starch glycolate and Disintegrants such as sodium and sodium carboxymethyl cellulose; talc and silica Lubricating agents such as stearic acid and lubricating agents such as magnesium stearate Agents such as sorbic acid and methyl or propyl parahydroxybenzoate; Any preservatives or pharmaceutically acceptable wetting, for example sodium lauryl sulfate An agent may be contained.   Capsules are usually made of gelatin shells coated with other ingredients, such as glycerol, Tall, surfactants, translucent bulking agents, preservatives, sweeteners, flavoring and coloring agents It is included together with such as. Capsule contents include diluent, lubricant and disintegrant Is included. Tablets consist of compressed powders or granules and may be coated May be dissolved, dispersed or foamed before administration to the patient, or Immediately after incorporation or, for example, for tablets with an acid-insoluble coating Designed to dissolve or disperse in the gastrointestinal tract sometime later Is also good. Tablets Usually contains excipients such as diluents, binders, disintegrants, lubricants, lubricants, etc. And flavoring agents. Effervescent tablets are compatible with carbonates or bicarbonates. Contains acid in the beginning. Tablet coding can be natural or synthetic resin, gum, insoluble Consists of filler, sucrose, plasticizer, polyhydric alcohol and wax, further colored And flavoring agents. Lozenges and pastils dissolve in the mouth Intend to do so. Lozenges are molded or compressed, usually with a flavoring base. May be combined. Pastille is gelatin and glycerol or acacia And molded from sucrose base. These are preservatives, colorings and And flavoring agents.   Film coating resins include cellulose derivatives, zein, vinyl polymers, And acrylic resin, the coating composition is usually a plasticizer, such as castor Includes oil or glycerin triacetate. Enteric coating resin is vinegar Includes copolymers of cellulose acid phthalate and methacrylic acid.   Solid compositions suitable for oral administration can be obtained by standard methods, such as mixing, filling, granulating, and tableting. Can be Using a repetitive mixing operation, these active substances are used in large amounts of fillers. It can be dispersed throughout the composition.   Liquid compositions suitable for oral administration include, for example, elixirs, mixtures, concentrated solutions, suspensions It may be in the form of a liquid, an emulsion or a linktus agent. Make sure they are in water before use. Alternatively, it may be a dry product reconstituted with another suitable vehicle. Such a liquid set The product can be prepared with the usual excipients such as sucrose, sorbitol, gelatin, Chill cellulose, carboxymethyl cellulose, hydroxypropyl methyl cell Loin, sodium alginate, xanthan gum, acacia, carrageenan, Mosquito, precipitation inhibitor such as aluminum stearate gel; lecithin, acacia, Emulsifiers such as rubitan monooleate; edible oils, oily esters such as glycero Or non-aqueous vehicles, including esters of cellulose, ethanol, and glycerol Buffers, such as alkali metal citrates and phosphates; Sodium sorbate, sorbic acid, methyl or propyl parahydroxybenzoate Includes any preservatives and, if desired, conventional flavorings and colorings.   The composition may be implanted subcutaneously, for example, in the form of a compressed tablet or delayed release capsule. May be.   Alternatively, compositions suitable for injection include solutions, suspensions or emulsions, or use It may be in the form of a dry powder, which is first dissolved or suspended in a suitable vehicle.   Fluid unit dosage forms are prepared using a compound and a pyrogen-free sterile vehicle. You. Compounds are either dissolved or suspended in the vehicle, depending on the vehicle and concentration used. It can either be cloudy. Solutions can be administered parenterally in any form , Especially for intravenous infections. In preparing the solution, Dissolve the agent in the vehicle and fill the solution into an appropriate sterile vial or ampoule. Before filling and sealing, if necessary isotonic by addition of sodium chloride and sterile technique Can be sterilized by filtration through a sterilizing filter. As an alternative If the stability of the solution is appropriate, the solution in the sealed container is sterilized by autoclaving. it can. Advantageously, buffers, solubilizers, stabilizers, preservatives or disinfectants, suspending agents Alternatively, additives such as emulsifiers and / or local anesthetics may be dissolved in the vehicle. Good.   Dry powder, dissolved or suspended in a suitable vehicle before use, should be Fill pre-sterilized medicinal substances and other ingredients in sterile containers using bacteria technology It is prepared by Alternatively, the drug substance and other ingredients may be present in an aqueous vehicle. The solution may be sterilized by filtration, and using aseptic techniques in a sterile area, Distribute in the appropriate solution. The product is then lyophilized and the container is aseptically sealed.   Suspend the sterile compound in the sterile vehicle instead of dissolving it and sterilize by filtration. Suitable for intramuscular, subcutaneous or transdermal injections in substantially the same manner except that A parenteral suspension is prepared. Compound is isolated under sterile conditions or otherwise For example, it may be sterilized after isolation by gamma irradiation. Advantageously, a suspending agent For example, polyvinylpyrrolidone may be present in the composition to promote homogeneous distribution of the compound. Be blended.   In yet another embodiment, a method of treating a nutritional and metabolic disorder comprising administering to a patient A method is provided which comprises administering an effective, non-toxic amount of a compound of the present invention.   The invention further relates to a medicament for treating nutritional and metabolic disorders such as obesity and diabetes. There is provided the use of a compound of the invention for the manufacture.   Unexpected toxicity is expected when compounds of the present invention are administered in accordance with the present invention. I can't.   The following examples illustrate the compounds of the present invention.   Example   Pharmacological method: The activity of the compound of the present invention is evaluated according to the method shown below. You. Effect of leptin fragment on food intake in SD rats   Surgery   Rats were pre-treated with Synulox (0.1 ml / 100 g) about 1 hour before anesthesia, Next, Domitor (0.04 ml / 100 g im) and sublimase (0.9 m 1/100 g ip). Each rat is cannulated in the lateral ventricle under sterile conditions. Embedding Then, Antisedan and Nubain (50% v / v: 50% v / v 0.02 ml / 100 g) The anesthesia is restored using IP. After surgery, each rat Is administered 0.05 ml Zenecarp.   Experimental operation:   Experiment 1: After surgery, the weight of each animal was monitored daily throughout the entire journey.   To confirm that the cannula is in the lateral ventricle, angiotensin II (1 (00 ng / 5 μl) was injected icv and water intake was monitored for 5 minutes after injection.   24-hour food intake was measured 5 and 6 days after surgery. Day 6, weigh the animals Divided into three groups (a, b and c, 8 rats per group) and then overnight I fasted. On the day of the experiment (day 7) the rats were injected icv as follows:   group a-vehicle (PBS, phosphate buffer solution, 5 [mu] l / rat);   group b-human leptin (11.5 μg / 5 μl); and   Group c-leptin trypsin digest (30 pg / 5 μl)   Immediately after the completion of the icv injection procedure, rats receive more than the known daily requirement of food. Supplied. Food intake and body weight were measured after 24 hours. Table 1 shows the obtained results. Shown in   Experiment 2: Another group of animals was prepared exactly as above, but after an overnight fast On the day of the experiment, several groups of rats were injected as follows:   group a-vehicle (PBS 5 μl / rat);   group b-human leptin (8.75 μg / 5 μl);   group c-murine leptin (10 μg / 5 μl)   group d-ob57-74, sequence VTGLDIPIPGLHPILTLSK (3.3 3 μg / 5 μl);   Again, an excess of food over the pre-weighed daily requirement is re-administered after injection; Changes and the amount of food consumed were recorded after 24 hours. Table 2 shows the obtained results. You. result:   Table 1 SD rats of human leptin and fragments thereof administered intracerebroventricularly On body weight and food intake in rats * P <0.05                               24-hour food intake Weight change                                     (G) (g / 24: 00) Vehicle 36.68 ± 2.5 31.44 ± 1.2 (5 μl / rat, n = 8) Human-leptin 30.62 ± 1.5 * 22.88 ± 2.05 * (11.5 μg / rat, n = 8) Human-leptin trypsin 34.6 ± 1.99 23.88 ± 1.2 * Digest (30 μg / rat, n = 8)   Table 2 Intracerebroventricularly administered human leptin (h-leptin), murine leptin ( m-leptin) and its leptin fragment 57-74 (VTGLDFIP (GLHPILTLSK) to control body weight change and food intake in SD rats Impact * P <0.05                                 24-hour food intake Weight change                                     (G) (g / 24: 00) Vehicle 32.16 ± 0.8 30.55 ± 0.67 (5 μl / rat, n = 8) h-leptin 33.7 ± 1.6 24.5 ± 3.7 (8.75 μg / rat, n = 8) m-leptin 31.3 ± 1.7 23.7 ± 2.18 * (10 μg / rat, n = 8) Leptin fragment 57-74 33.6 ± 0.9 26.8 ± 1.2 * (3.33 μg / rat, n = 8)

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12P 21/02 A61K 37/02 // (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1 : 19) (31) Priority claim number 9703493.8 (32) Priority date February 20, 1997 (Feb. 20, 1997) (33) Priority claim country United Kingdom (GB) (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), EA (AM, AZ, BY, KG, KZ, MD, RU) , TJ, T M), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, HU, 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, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU (72) Inventor Arch, Jonathan Robert Sanders United Kingdom C.M. 19.5 Able, Essex, Harlow, Third Avenue, New Frontiers Science Park South, SmithKline Beecham Pharmaceuticals (72) Trick UK, CM 19.5 Able, Essex, Harlow, Third Avenue, New Frontiers Science Park South, SmithKline Beecham Pharmaceuticals (72) Inventor Neville, William Arthur UK, CM 19.5 ADB, Essex, Harlow, Third Avenue, New Frontiers Science Park South, SmithKline Beecham Pharmaceuticals

Claims (1)

[Claims]   1. Peptides regulating body weight, a means of substantially regulating energy use Or a functional derivative, analog or variant thereof.   2. Weight loss pepti, a means of substantially regulating energy use Or a functional derivative, analog or variant thereof.   3. The peptide is a fragment of the ob protein, or a functional derivative thereof, or the like. 3. The peptide according to claim 1, which is an analog or a mutant.   4. ob21-26, ob27-32, ob33-36, ob37-41, o b42-54, ob55-56, ob57-74, ob93-105, ob10 6-115, ob116-149 and ob150-167 human ob proteins 4. A pep according to claim 1, wherein the pep is selected from quality fragments. Chid.   5. The peptide has the amino acid sequence VTGLDIPIPGLHPILTLSK The peptide according to any one of claims 1 to 4.   6. A method according to claim 1 wherein the peptide is formed from one or more peptides according to claim 4. The peptide mentioned.   7. A peptide according to claim 4, which is formed from two consecutive elements. The peptide according to claim 1.   8. Synthesis or recombination which is a peptide according to any one of claims 1 to 7 Eh peptide.   9. Nucleotide encoding the peptide according to any one of claims 1 to 7 Pseudo sequence.   10. A nucleic acid encoding the peptide according to any one of claims 1 to 7. A vector containing an otide sequence.   11. A host cell transformed with the replicable expression vector according to claim 9.   12. A method for producing the peptide according to claim 1, or a functional derivative thereof,   Hydrolyzing the peptide to at least two peptide fragments;   Separating the peptide fragment;   Then prepare its functional derivative if desired A method consisting of steps.   13. A method for producing the peptide according to any one of claims 1 to 7, Expressing the DNA encoding the peptide in a recombinant host cell and recovering the product A method consisting of:   14．   i) a DNA polymer containing the nucleotide sequence encoding the required peptide Preparing a replicable expression vector that can be expressed in the main cell;   ii) transforming a host cell with the vector;   iii) transforming the transformed host cells under conditions capable of expressing the DNA polymer. Culturing to produce the peptide;   iv) recover the peptide 14. The method of claim 13, comprising the steps of:   15. A pharmaceutical set comprising the compound according to claim 1 and a pharmaceutically acceptable carrier. Adult.   16. A compound according to claim 1 for use as an active therapeutic substance.   17． 16. The compound according to claim 15, which is used in the treatment of a nutritional disorder and a metabolic disorder. .   18. A method of treating nutritional and metabolic disorders that is effective and pharmaceutically acceptable Administering a non-toxic amount of the compound of claim 1.   19. Claim 1 for the manufacture of a medicament for the treatment of nutritional and metabolic disorders. Use of compounds.