JP5914906B2 - Insulin secretagogue - Google Patents

Description

  The present invention relates to a medicament comprising a peptide having an activity of promoting insulin secretion, and a substance that inhibits or enhances the insulin secretion promoting activity of the peptide, or an agonist or antagonist for the receptor of the peptide. The present invention relates to a screening method and the like.

  Diabetes mellitus is characterized by hyperglycemia caused by metabolic abnormalities such as glucose metabolism based on insufficient secretion of insulin or decreased insulin sensitivity on the target cell side. When hyperglycemia persists, serious complications occur in various organs and nerves, such as retinopathy, nephropathy, and neuropathy, mainly due to vascular disorders. Therefore, in the treatment of diabetes, it is extremely important to control the blood glucose level and maintain it at a normal level, and means for that purpose have been studied for a long time.

  Among the types of diabetes, in which the onset is slow and does not necessarily require insulin treatment for life support (type 2 diabetes), treatment for controlling blood glucose levels by exercise therapy, diet therapy and a combination of drugs is performed. As a drug, a sulfonylurea insulin secretagogue, which is a kind of oral hypoglycemic agent, is widely used clinically. Recently, insulin secretagogues that are non-sulfonylurea K + ATP channel blockers such as nateglinide and lipaglinide are also commercially available. However, all of these insulin secretagogues promote insulin secretion independent of blood glucose levels, so there is a problem that if the dose is wrong, serious hypoglycemia occurs or blood sugar cannot be sufficiently controlled, It is not always satisfactory.

On the other hand, several hormones that control blood sugar are secreted in vivo. Among them, incretin is known as a hormone that lowers blood sugar levels. Incretin is a general term for gastrointestinal hormones secreted from the gastrointestinal tract with meal intake, and has an action of promoting insulin secretion from the pancreas. Incretin includes glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). GLP-1 having particularly strong activity is a product from proglucagon, which is a precursor consisting of 180 amino acids (Non-patent Document 1). GLP-1 not only has the effect of promoting insulin secretion in normal humans, but also reduces glucagon levels, delays gastric excretion, stimulates (pro) insulin biosynthesis, and is insulin sensitive It also has the effect | action which improves (nonpatent literature 2). However, natural GLP-1 has a short blood half-life. In humans and rats, GLP-1 is rapidly degraded by dipeptidyl peptidase-IV (DPP-IV) to GLP-1 (9-36) amide, which acts as an endogenous GLP-1 receptor antagonist (Non-patent document 3). Due to these problems, natural GLP-1 has not been sufficiently effective as a therapeutic agent for diabetes.
Therefore, exendin-4, which is a GLP-1 analog containing an amino acid substitution at the cleavage site of DLP-IV of GLP-1, has been marketed in the United States. However, side effects such as vomiting and acute pancreatitis are serious problems with this drug (Non-patent Document 4).

  Cachexia is a systemic debilitating disease associated with chronic respiratory disease, heart disease, advanced cancer, and other diseases caused by weight loss, muscle mass loss, loss of appetite, etc. (Non-Patent Document 5). Although the detailed expression mechanism of cachexia is not known, for example, in the case of cancer cachexia, it is known that inflammatory cytokines released from tumor tissue may induce muscle atrophy. At present, there is no established cure for cachexia, and symptomatic treatment such as increasing appetite by administering steroids is currently being used. Insulin is also administered as one of the symptomatic treatments. Effects such as an increase in carbohydrate intake and an increase in fat content are recognized by insulin administration, and an improvement effect on cachexia has been reported (Non-patent Document 6).

  The VGF gene was identified as a gene whose expression is increased by nerve growth factor stimulation of rat PC12 cells (Non-Patent Document 7), and then the human VGF gene was isolated (Non-Patent Document 8). In the VGF gene-disrupted mice, food intake was unchanged, weight loss, body fat loss, increased oxygen consumption, increased exercise capacity, and abnormal reproductive function. In particular, increased energy metabolism has been observed (Non-patent Document 9).

VGF gene is expressed in central, peripheral nervous system, endocrine, neuroendocrine cells, and its expression and distribution are neuropeptide Y, peptide YY, ghrelin, and cholecystokinin that control feeding behavior and gastrointestinal motility It exists in the part related to energy metabolism (Non-patent Document 10).
The protein encoded by the VGF gene (hereinafter referred to as VGF) is 615 amino acids in humans and 617 amino acids in rats / mouses. The 1st to 22nd amino acids of VGF are signal peptides, and there are sequences that undergo processing such as cleavage and amidation by prohormone convertase. The processing of VGF and the functions of derived peptides have been investigated mainly in rats, and several peptide fragments have been identified from rat brain and the like (Non-patent Document 11). For example, the peptide of rat VGF (556-576) (the number in parenthesis represents the position of the peptide sequence in the amino acid sequence of VGF. The same applies to the following peptides) has been confirmed in the rat brain extract. Has been reported to have an activity to suppress weight gain during high-calorie dieting (Non-patent Document 12). Further, for example, rat VGF (577-617), rat VGF (588-617), and rat VGF (599-617), which are partial peptides of rat VGF (556-617) found in the brain, are the rat thalamus. By direct administration to the lower paraventricular nucleus (PVN), the activity causing erection was shown, but this activity was not confirmed in rat VGF (556-576) (Non-patent Document 13). Moreover, when VGF (588-596) was intraperitoneally administered to a VGF gene-disrupted mouse, it was reported that the body weight increased by about 10% to 15% (Patent Document 1).

A plurality of peptides derived from human VGF have been identified so far (Non-Patent Document 14, Patent Document 2). In addition, a peptide having the same sequence as that of the 586-615th sequence of human VGF, which is equivalent to rat VGF (588-617), has also been isolated from the posterior lobe of bovine pituitary gland (Non-patent Document 15). ). VGF (281-306) and VGF (310-347) identified from human thyroid cancer cell lines have been reported to have physiological activity that suppresses secretion of vasopressin from the pituitary gland (Non-patent Document 16). ). Some peptides including human VGF (485-503) have been reported to have an activity to increase intracellular calcium concentration in various tissues (Patent Document 3). However, the specific physiological activity associated with the increase in intracellular calcium concentration is not known.
As an antibody specifically recognizing VGF or a peptide derived from VGF, a polyclonal antibody having a peptide consisting of the 573-617th amino acid sequence corresponding to the C-terminus of rat VGF as an antigen (see Non-patent Document 17), human Polyclonal antibody using a peptide consisting of amino acids 556 to 565 of VGF as an antigen (see Non-patent Document 10), polyclonal antibody using a peptide consisting of amino acids 443 to 588 of rat VGF as an antigen (Non-patent Document 18) Reference) etc. have been reported.

International Publication No. 01/07477 International Publication No. 02/82075 International Publication No. 08/90678

Drucker, Diabetes (1998) 47: 159-169 Nauck, Horm. Metab. Res. (1997) 47: 1253-1258 Deacon, Diabetologia (1998) 41: 271-278 Robles, Drug Des. Devel. Ther. (2009) 3: 219-240 Ashitani, Peptides (2009) 30: 1951-1956 Lundholm, Clin. Cancer Res. (2007) 13: 2699-2706 Lvi, Science (1985) 229: 393-395 Canu, Genomics (1997) 45: 443-446 Hahm, Neuron (1999) 23: 537-548 Levi, Cell. Mol. Neurobiol. (2004) 24: 517-533 Trani, J. Neurochem. (2002) 81: 565-574 Bartolomucci, Proc. Natl. Acad. Sci. USA (2006) 39: 14584-14589 Succu, Eur. J. Neurosci. (2004) 20: 3035-3040 Stark, J. Chromatogr. B Biomed. Sci. Appl. (2001) 754: 357-367 Liu, Endocrinology (1994) 135: 2742-2748 Yamaguchi, J. Biol. Chem. (2007) 282: 26354-26360 Possenti, Endocrinology (1994) 140: 3727-3735 Possenti, EMBO J. (1989) 8: 2217-2223

  An object of the present invention is to provide a compound useful for the prevention or treatment of diabetes or cachexia. It is another object of the present invention to provide a screening method for a substance that inhibits or enhances the insulin secretion promoting activity of the compound, or an agonist or antagonist for the receptor of the compound, using the compound.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a peptide derived from VGF identified from within a biological sample has insulin secretion enhancing activity in a glucose concentration-dependent manner, thereby completing the present invention. It came to.
That is, the present invention relates to the following.
[1] A medicament comprising the peptide of any one of (a) to (d) or a pharmacologically acceptable salt thereof:
(A) a peptide comprising the amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, and comprising an amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 1;
(B) 1 to 5 amino acids are substituted, deleted or added in the amino acid sequence consisting of the amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof and including the amino acid sequence represented by SEQ ID NO: 1 A peptide comprising a defined amino acid sequence;
(C) a peptide comprising an amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, and comprising an amino acid sequence having 80% or more identity with the amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 1; And (d) Formula (I)

Wherein R ¹ is a hydrogen atom, substituted or unsubstituted alkanoyl, substituted or unsubstituted aroyl, substituted or unsubstituted heteroarylcarbonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, Or substituted or unsubstituted heteroaryloxycarbonyl, R ² represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino, and A represents any of the peptides (a) to (c) above Peptide represented by the above).
[2] The medicament according to [1], wherein the peptide is a peptide having an activity of secreting insulin.
[3] The medicament according to [1] or [2], which is a preventive or therapeutic agent for diabetes.
[4] The medicament according to [1] or [2], which is an agent for preventing or treating cachexia.
[5] A response when the peptide according to [1] or a pharmacologically acceptable salt thereof and a test substance are contacted with a mammalian pancreas, islet, pancreatic cell or pancreatic β cell line is measured. Comparing the response of the peptide or a pharmacologically acceptable salt thereof to the pancreas, pancreatic islet, pancreatic cell or pancreatic β cell line of the mammal in the absence of the test substance, As a result, a test substance for which response is suppressed is selected as a candidate substance that inhibits the insulin secretagogue activity of the peptide, and a screening method for a substance that inhibits the insulin secretagogue activity of the peptide.
[6] The response when the peptide according to [1] or a pharmacologically acceptable salt thereof and a test substance are brought into contact with a mammalian pancreas, islet, pancreatic cell or pancreatic β cell line is measured. Comparing the response of the peptide or a pharmacologically acceptable salt thereof to the pancreas, pancreatic islet, pancreatic cell or pancreatic β cell line of the mammal in the absence of the test substance, A screening method for a substance that enhances the insulin secretagogue activity of the peptide, comprising selecting a test substance when the response is enhanced as a candidate substance that enhances the insulin secretagogue activity of the peptide.
[7] When the peptide according to [1] or a pharmacologically acceptable salt thereof and a test substance are contacted with a mammalian pancreatic cell or pancreatic β cell line, or a cell membrane fraction of these cells The amount of the peptide or pharmacologically acceptable salt thereof bound to the cell or cell membrane fraction of the cell is measured, and the peptide or pharmacologically acceptable salt thereof is measured in the absence of the test substance. Compared with the binding amount of the peptide or a pharmacologically acceptable salt thereof when contacted with the cell or a cell membrane fraction of the cell, the test substance when the binding amount decreases as a result of the comparison is the peptide A method for screening an agonist or antagonist for a receptor of the peptide, comprising selecting as an agonist or antagonist candidate substance for the receptor of the peptide.
[8] The peptide according to [1] or a pharmacologically acceptable salt thereof for use in the prevention or treatment of diabetes.
[9] The peptide according to [1] or a pharmacologically acceptable salt thereof for use in the prevention or treatment of cachexia.
[10] A method for preventing or treating diabetes in a mammal, comprising administering to the mammal a prophylactically or therapeutically effective amount of the peptide of [1] or a pharmacologically acceptable salt thereof. .
[11] Prevention or prevention of cachexia in a mammal, comprising administering to the mammal a prophylactically or therapeutically effective amount of the peptide according to [1] or a pharmaceutically acceptable salt thereof. Method of treatment.

  According to the present invention, a prophylactic or therapeutic agent for diabetes or cachexia is provided. Also provided is a method for screening for a substance that inhibits or enhances the insulin secretion promoting activity of the peptide, or an agonist or antagonist for the receptor of the peptide, using the peptide.

FIG. 1 shows promotion of insulin secretion from mouse islets by peptide A (SEQ ID NO: 1). FIG. 2 shows the effect of peptide A on blood insulin concentration in the rat glucose tolerance test. FIG. 3 shows the promotion of insulin secretion from mouse pancreatic islets by peptide A and its derivatives [peptide 1 (SEQ ID NO: 6), peptide 2 (SEQ ID NO: 7), peptide 3 (SEQ ID NO: 9)]. FIG. 4 shows the promotion of insulin secretion from mouse islets by the peptide derivative [Peptide 4 (SEQ ID NO: 10)]. FIG. 5 shows the suppression of blood glucose during the intravenous glucose tolerance test in normal rats by the peptide A derivative (peptides 3 and 4).

<1. Peptide having insulin secretion promoting activity and pharmacologically acceptable salt thereof>
Examples of the peptide used in the present invention (also referred to as the peptide of the present invention) include any of the following peptides (a) to (d).
(A) a peptide comprising the amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, and comprising an amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 1;
(B) 1 to 5 amino acids are substituted, deleted or added in the amino acid sequence consisting of the amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof and including the amino acid sequence represented by SEQ ID NO: 1 A peptide comprising a defined amino acid sequence;
(C) a peptide comprising an amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, and comprising an amino acid sequence having 80% or more identity with the amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 1; And (d) Formula (I)

Wherein R ¹ is a hydrogen atom, substituted or unsubstituted alkanoyl, substituted or unsubstituted aroyl, substituted or unsubstituted heteroarylcarbonyl, substituted or unsubstituted alkoxycarbonyl, substituted or unsubstituted aryloxycarbonyl, Or substituted or unsubstituted heteroaryloxycarbonyl, R ² represents hydroxy, substituted or unsubstituted alkoxy, or substituted or unsubstituted amino, and A represents any of the peptides (a) to (c) above Peptide represented by the above).

  The amino acid sequence consisting of the amino acid sequence represented by SEQ ID NO: 8 or a part thereof and including the amino acid sequence represented by SEQ ID NO: 1 is the amino acid sequence represented by SEQ ID NO: 7 or a partial sequence thereof And the amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 1 is preferred, comprising the amino acid sequence represented by SEQ ID NO: 6 or a partial sequence thereof, and comprising the amino acid sequence represented by SEQ ID NO: 1 An amino acid sequence is more preferable, and an amino acid sequence including the amino acid sequence represented by SEQ ID NO: 1 is particularly preferable.

  The amino acid sequence represented by SEQ ID NO: 1 includes amino acids 485-503 in the amino acid sequence of human VGF (NCBI accession number: NP_003369 (SEQ ID NO: 3)), and amino acid sequences of rat and mouse VGF (rat: NCBI accession). This is an amino acid sequence corresponding to the 489th to 507th amino acids in the mouse sequence number: NP_112259 (SEQ ID NO: 4) and mouse: NCBI accession number: NP_001034474 (SEQ ID NO: 5).

  In the amino acid sequence consisting of the amino acid sequence represented by SEQ ID NO: 8 or a partial sequence thereof, and including the amino acid sequence represented by SEQ ID NO: 1, 1 to 5 amino acid residues are substituted, deleted or added Means that there is a substitution, deletion or addition of 1 to 5 amino acid residues at any one or a plurality of positions in the same sequence, and the substitution, deletion or addition may occur simultaneously. . Examples of the amino acid sequence in which 1 to 5 amino acid residues in the amino acid sequence are substituted, deleted or added include, for example, (1) 1 to 5 (preferably 1 to 3) in the amino acid sequence. Preferably, 1 or 2 amino acid sequences deleted, (2) 1-5 (preferably 1-3, more preferably 1 or 2) amino acids added to the amino acid sequence A sequence, (3) an amino acid sequence in which 1 to 5 (preferably 1 to 3, more preferably 1 or 2) amino acids in the amino acid sequence are substituted with other amino acids, or (4) the above (1 ) To (3) combined amino acid sequences (in this case, the sum of the mutated amino acids is 1 to 5 (preferably 1 to 3, more preferably 1 or 2)).

  As amino acids to be substituted or added, for example, 20 kinds of L-amino acids known as amino acids constituting a protein, that is, L-alanine, L-asparagine, L-aspartic acid, L-arginine, L-glutamine, L- Glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L- Examples include, but are not limited to, valine and L-cysteine, such as tert-leucine, norleucine, norvaline, 2-aminobutanoic acid, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine. , Isoaspartic acid, isoglutamine Other amino acids such as 2-aminoadipic acid, 2-aminosuberic acid, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid, 3-hydroxyproline, 4-hydroxyproline, homoserine, Further, it may be a D-amino acid or a β-amino acid.

  The amino acid sequence represented by SEQ ID NO: 1 has the possibility of being degraded by DPP-IV because the second amino acid residue from the N-terminal side is alanine. Therefore, the second amino acid residue from the N-terminal side remains as long as it has an activity to increase intracellular calcium ion concentration, activity to enhance insulin secretion and / or activity to decrease serum glucose concentration in cells contained in the pancreas of mammals. Substitution of the group with an amino acid other than alanine and proline is preferred for DPP-IV resistance.

Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine, tert-leucine B group: aspartic acid, glutamic acid, Isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid group C: asparagine, glutamine group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid group E : Proline, 3-hydroxyproline, 4-hydroxyproline F group: serine, threonine, homoserine G group: phenylalanine, tyrosine

  An amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 8 or a part of the amino acid sequence and having 80% or more identity with the amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 1 is a homology analysis program BLAST 2 Sequences [FEMS Microbiol Lett. 174, 247 (1999)] using default conditions (program: blastp, matrix: BLOSUM62, open gap: 11 penalties, extension gap: 1 penalty, gap x_dropoff: 50, expect: 10.0 , Word size: 3), and when alignment is performed, the identity (the number of identical amino acids between the subject sequence and the sequence subjected to homology analysis of the peptide sequence / of the peptide) The total number of amino acids of the sequence subjected to the homology analysis of the above sequence + the number of gaps inserted at the time of alignment) is 80% or more, preferably 90% or more. Although higher amino acid sequence identity is preferred, the peptide of the present invention or a pharmacologically acceptable salt thereof increases the intracellular calcium ion concentration of cells contained in the pancreas of mammals and enhances insulin secretion As long as it has activity to reduce and / or reduce serum glucose concentration.

  The length of the peptide used in the peptide of the present invention or a pharmacologically acceptable salt thereof is the activity of increasing intracellular calcium ion concentration in cells contained in the pancreas of mammals, the activity of enhancing insulin secretion, and / or Or it will not restrict | limit especially as long as it has the activity which reduces a serum glucose level, According to the intended purpose, the peptide of desired length can be selected. The length of the peptide of the present invention is preferably 68 amino acids or less, more preferably 48 amino acids or less, and even more preferably 33 amino acids or less.

  Preferable peptides include a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 6 to 8, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, and the like.

In the above formula (I), R ¹ is the N-terminal amino group of any of the peptides (a) to (c), and R ² is the C-terminal of any of the peptides (a) to (c). In the carboxyl group of each. Specifically, R ¹ is substituted with one or both hydrogen atoms in the N-terminal amino group, and R ² is substituted with a hydroxy group in the C-terminal carboxyl group.

  In the definition of each group of the above formula (I), alkanoyl includes, for example, linear or branched alkanoyl having 1 to 20 carbon atoms such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl , Heptanoyl, lauroyl, eicosanoyl and the like.

  Examples of the aryl moiety of aroyl and aryloxycarbonyl include aryl having 6 to 15 carbon atoms such as phenyl and naphthyl.

  Heteroarylcarbonyl and heteroaryloxycarbonyl heteroaryl moieties include, for example, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, indazolyl, benzimidazolyl, quinolyl, isoquinolyl, cinnolinyl Quinazolinyl, quinoxalinyl, naphthyridinyl and the like.

  Examples of alkoxycarbonyl and the alkyl part of alkoxy include linear or branched alkyl having 1 to 20 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, decyl, dodecyl, eicosyl and the like. can give.

  Substituents in substituted alkanoyl, substituted alkoxycarbonyl and substituted alkoxy are the same or different and have 1 to 3 substituents such as hydroxy, carboxy, 3 to 8 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl And cycloaliphatic alkyl such as cyclooctyl, substituted or unsubstituted phenyl, fluorenyl and the like. Substituents of substituted phenyl are the same or different and have 1 to 3 substituents, for example, alkyl, alkoxy, hydroxy, nitro, sulfo, cyano, halogen, etc. Examples of halogen include fluorine, chlorine, bromine and iodine. Each atom is listed. The alkyl part of the substituted phenyl and the alkyl part of alkoxy have the same meaning as the alkyl part of alkoxycarbonyl and alkoxy.

  Substituents in substituted aroyl, substituted aryloxycarbonyl, substituted heteroarylcarbonyl and substituted heteroaryloxycarbonyl are the same or different and have 1 to 3 substituents, and have the same meaning as the substituent of the above substituted phenyl.

  Examples of the substituent in the substituted amino include the same or different substituents having 1 to 2 substituents, such as substituted or unsubstituted alkyl, substituted or unsubstituted aryl, and the like. Alkyl has the same meaning as the alkyl moiety such as alkoxy, and the substituent of the substituted alkyl has the same meaning as the substituent such as substituted alkoxy. Aryl is synonymous with the aryl moiety of the aroyl or aryloxycarbonyl, and the substituted aryl substituent is synonymous with the aroyl or aryloxycarbonyl substituent.

  Moreover, the functional group of the side chain of the amino acid residue which comprises the peptide residue of A in Formula (I) may be chemically modified or protected. Examples of amino acid residues whose side chain functional groups are chemically modified or protected include aspartic acid residues or glutamic acid residues in which the side chain carboxy group is protected with a benzyl ester, and the side chain thiol group is a carboxy group. Examples include methylated cysteine residues.

  The peptide of the present invention may be modified. Such modifications include addition of lipid chains (aliphatic acylation (palmitoylation, myristoylation, etc.)), prenylation (farnesylation, geranylgeranylation, etc.), phosphorylation (serine residue, threonine residue, tyrosine residue, etc.) Phosphorylation), acetylation, amidation, sugar chain addition (N-glycosylation, O-glycosylation) and the like.

  For example, the amino acid sequence represented by SEQ ID NO: 1 has the possibility of being degraded by DPP-IV because the second amino acid residue from the N-terminal side is alanine as described above. Therefore, as long as it has activity to increase intracellular calcium ion concentration, activity to enhance insulin secretion and / or activity to decrease serum glucose concentration in cells contained in the pancreas of mammals, the N-terminal side of the peptide is other than hydrogen Modification with a functional group of is particularly preferred in order to make it DPP-IV resistant. By making it DPP-IV resistant, it can be expected to extend the half-life in the blood of mammals.

  In addition, by amidating the C-terminal of the peptide, an activity that increases intracellular calcium ion concentration in cells contained in the pancreas, an activity that enhances insulin secretion, and / or an activity that decreases serum glucose concentration can be expected. Is preferably amidated at the C-terminus.

  Preferable examples of such a peptide include a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 or 10.

The peptide of the present invention contains a suitable labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase). , Malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.), affinity tags (eg, biotin, etc.), etc. It may be labeled with.

  Examples of the pharmacologically acceptable salt of the peptide of the present invention include acid addition salts, metal salts, and organic base addition salts. Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate and phosphate, and organic acid salts such as acetate, maleate, fumarate, tartrate and citrate. Examples of the metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, zinc salt and the like. Examples of organic base addition salts include primary amines such as methylamine, ethylamine and aniline, secondary amines such as dimethylamine, diethylamine, pyrrolidine, piperidine, morpholine and piperazine, trimethylamine, triethylamine, N, N-dimethylaniline and pyridine. And salts formed with tertiary amines such as ammonium salts and the like.

  The peptide of the present invention or a pharmacologically acceptable salt thereof is preferably isolated or purified. “Isolation or purification” means that an operation for removing components other than the target component has been performed. The purity of the peptide of the present invention or a pharmacologically acceptable salt thereof isolated or purified (the peptide of the present invention or its pharmacologically acceptable relative to the total weight of the peptide or its pharmacologically acceptable salt) The ratio of the weight of the salt formed) is usually 50% or more, preferably 70% or more, more preferably 90% or more, and most preferably 95% or more (for example, 100%).

  The peptide of the present invention or a pharmacologically acceptable salt thereof preferably has insulin secretion promoting activity. Insulin secretion-promoting activity means the activity of promoting insulin secretion from the pancreas of a mammal.

  Mammals include laboratory animals such as rodents and rabbits such as mice, rats, hamsters and guinea pigs, domestic animals such as pigs, cows, goats, horses, sheep and minks, pets such as dogs and cats, humans, monkeys, Primates such as cynomolgus monkey, rhesus monkey, marmoset, orangutan, chimpanzee and the like are included, but not limited thereto. The mammal is preferably a primate (such as a human) or a rodent (such as a rat).

  It can be confirmed by the following method that the peptide of the present invention or a pharmacologically acceptable salt thereof has insulin secretion promoting activity.

The islets isolated from the mice were measured at 37 ° C., 5% CO ₂ in Hepes Krebs-Ringer bicarbonate (HKRB) buffer containing the peptide to be measured or a pharmacologically acceptable salt thereof and 16.7 mmol / L glucose. For 1 hour (measuring group). The concentration of the peptide to be measured or a pharmacologically acceptable salt thereof is in the range of 1 pmol / L to 10 μmol / L. As a control, the same operation as in the measurement target group is performed except that the peptide to be measured or a pharmacologically acceptable salt thereof is not added to HKRB. The insulin concentration in the culture solution after 1 hour of culture is measured by an immunological measurement method. If the concentration of insulin in the culture medium in the measurement target group is significantly higher than the control at at least one point in the above range, the peptide or pharmacologically acceptable salt thereof has insulin secretion promoting activity. Then, it is determined.

  The insulin secretion promoting activity of the peptide of the present invention or a pharmacologically acceptable salt thereof may be dependent on glucose concentration. Glucose concentration-dependent means that there is a positive correlation between glucose concentration and insulin secretagogue activity. That is, in the physiologically possible glucose concentration range (0 to 30 mmol / L), the higher the glucose concentration to which the mammal's pancreas is exposed, the more effective it is due to the peptide of the present invention or a pharmacologically acceptable salt thereof. Increased insulin secretion promoting activity.

  The fact that the peptide of the present invention or a pharmacologically acceptable salt thereof has a glucose concentration-dependent insulin secretagogue activity indicates that the glucose concentration added to HKRB in the above-mentioned confirmation test of insulin secretagogue activity is determined. Except for 5.5 mmol / L, it can be performed by providing a group (low glucose concentration group) that performs the same operation as the measurement target group. In this case, as a low glucose concentration control, the same operation as in the low glucose concentration group is performed except that the peptide to be measured or a pharmacologically acceptable salt thereof is not added to HKRB. Insulin concentration in the culture solution after 1 hour culture is measured by immunological measurement method, insulin secretion promoting activity of the measurement target group (insulin concentration of measurement target group / control insulin concentration), and insulin of low glucose concentration group Secretion promoting activity (insulin concentration in low glucose concentration group / insulin concentration in low glucose concentration control) is calculated. When the insulin secretion promoting activity of the measurement target group exceeds that of the low glucose concentration group, the peptide or a pharmacologically acceptable salt thereof is determined to have a glucose concentration-dependent insulin secretion promoting activity.

  The peptide of the present invention or a pharmacologically acceptable salt thereof preferably has an activity of reducing serum glucose concentration.

  Whether the peptide of the present invention or a pharmacologically acceptable salt thereof has an activity of lowering serum glucose concentration, preferably mammalian serum glucose concentration, can be confirmed by, for example, intravenous glucose tolerance test in normal rats. Can do.

  The peptide to be measured or its pharmacologically acceptable salt dissolved in PBS (Phosphate-buffered saline) is administered to the tail vein of normal rats that have been fasted for about 16 hours, and after 5 minutes a glucose solution (1 g / kg) Is administered intravenously. Serum glucose concentration is measured 5 minutes and 10 minutes after administration of the glucose solution (measuring group). The concentration of the peptide to be measured or a pharmacologically acceptable salt thereof is in the range of 1 pmol / L to 10 μmol / L. As a control, the same procedure as in the measurement target group is performed except that the peptide to be measured or a pharmacologically acceptable salt thereof is not added to PBS.

  When the serum glucose concentration in the measurement target group is significantly lower than the control, it is determined that the peptide or a pharmacologically acceptable salt thereof has an activity of decreasing the serum glucose concentration.

<2. Method for producing peptide of the present invention having insulin secretion promoting activity>
(1) Production Method by Chemical Synthesis The peptides of the present invention are prepared, for example, by Nobuo Izumiya, Tetsuo Kato et al., “Basics and Experiments of Peptide Synthesis”, Maruzen, (1985), Saburo Aimoto et al. 4th Edition, Volume 22, “Organic Synthesis IV Acids / Amino Acids / Peptides”, Maruzen, (1999), Int. J. Pept. Protein Res. 35, 161-214 (1990), Fields, GB, Solid-Phase Known in Peptide Synthesis, Methods in Enzymology, vol. 289, Academic Press, (1997), Pennington, MW and Dunn, BM, Peptide Synthesis Protocols, Methods in Molecular Biology, vol. 35, Humana Press, (1994), etc. It can obtain by refine | purifying after the synthesis | combination by the peptide synthesis method. Specific examples of the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a dichloromethane method, an active ester method, a carboimidazole method, and a redox method. Moreover, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. That is, the amino acid constituting the peptide of the present invention and the remaining part are condensed, and when the product has a protecting group, the desired peptide can be synthesized by removing the protecting group.

  The peptide of the present invention can also be synthesized using an automatic peptide synthesizer. Peptide synthesis using a peptide synthesizer was carried out using a commercially available peptide synthesizer such as a peptide synthesizer manufactured by Shimadzu Corporation or a peptide synthesizer manufactured by Advanced ChemTech, Inc. Fmoc (9-fluorenylmethyloxycarbonyl) -amino acid or Nα-Boc (t-butyloxycarbonyl) -amino acid or the like can be used according to each synthesis program. Protected amino acids and carrier resins used as raw materials are Applied Biosystems, Inc., Shimadzu, Kokusan Chemicals, EMD Biosienses, Inc., Watanabe Chemical, Advanced It can be obtained from Chemtech, AnaSpec, Inc., Peptide Institute, Inc.

  Furthermore, in the peptide of the present invention, the side chain of the amino acid residue constituting the peptide and / or the amino terminus (also referred to as N-terminus) and / or the carboxy terminus (also referred to as C-terminus) of the peptide are chemically modified or protected. Peptide synthesis chemistry such as a method of chemical modification after peptide synthesis, a method of peptide synthesis using chemically modified amino acids, or a method of appropriately selecting reaction conditions for final deprotection of peptide synthesis, etc. [Nobuo Izumiya et al., “Basics and Experiments of Peptide Synthesis”, Maruzen, (1985), Supervised by Yajima Haruaki, “Development of Continuing Drugs”, Volume 14, “Peptide Synthesis”, Hirokawa Bookstore, (1991), "Biochemistry Experiment Course" edited by Japanese Biochemical Society, Volume 1, "Chemistry of Protein IV-Chemical Modification and Peptide Synthesis", Tokyo Chemical Doujin, Ohno Motonori et al., "Biochemical Experiment Method", 12, Vol. 13, "protein of chemical modification (top) (bottom)", Gakkai Shuppan Center, can be produced by (1981)].

  The peptide of the present invention can be purified by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization and the like.

(2) Production method by genetic engineering method When the peptide of the present invention consists of 20 types of L-amino acids constituting the protein and has not been modified at the side chain, N-terminal or C-terminal, Molecular Cloning: A Using the method described in Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press (2001), etc., for example, by the following method, DNA encoding the peptide can be expressed in host cells and produced. .

  The DNA encoding the peptide of the present invention can be synthesized by designing a base sequence encoding the amino acid sequence of the peptide of the present invention and using a DNA synthesizer. It can also be isolated by PCR using cDNA of human, rat or mouse brain or pancreatic cells as a template. When the DNA encoding the peptide of the present invention is used for the production of the peptide of the present invention, the end of the region encoding the peptide of the present invention is made to be a stop codon.

The production method for expressing the N-terminus of the peptide of the present invention as methionine in advance is described below.
A recombinant vector is prepared by inserting the DNA encoding the peptide of the present invention obtained above downstream of the promoter of an appropriate expression vector, and the recombinant vector is transferred to a host cell suitable for the expression vector. Introduce.
Any host cell can be used as long as it can express the target gene, such as bacteria, yeast, animal cells, insect cells, and plant cells.

  As the expression vector, one that can autonomously replicate in the host cell or can be integrated into the chromosome and that contains a promoter at a position where the DNA encoding the peptide of the present invention can be transcribed is used.

  When a prokaryote such as a bacterium is used as a host cell, the recombinant vector containing the DNA encoding the peptide of the present invention can autonomously replicate in the prokaryote, and at the same time, a promoter, a ribosome binding sequence, and a transcription A vector comprising a termination sequence is preferred. A gene that controls the promoter may also be included.

  Examples of the expression vector include pSE420 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by QIAGEN), pKYP10 (Japanese Patent Laid-Open No. 58-110600), pKYP200 [Agric.Biol. Chem., 48, 669 (1984)], pLSA1 (Agric. Biol. Chem., 53, 277 (1989)), pGEL1 (Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)), pBluescript II SK (-) (Stratagene), pTrs30 [prepared from transformed E. coli strain (FERM BP-5407)], pTrs32 [prepared from transformed E. coli strain (FERM BP-5408)], pGHA2 [transformed E. coli strain ( FERM BP-400)], pGKA2 [prepared from transformed E. coli strain (FERM P-6798)], pTerm2 (Japanese Patent Laid-Open No. 3-22979), pGEX-2T (manufactured by GE Healthcare), pET (Novagen) PKK223-2 (manufactured by GE Healthcare), pMAL-c2X (manufactured by New England Biolabs), and the like.

  Any promoter can be used as long as it functions in the host cell. For example, promoters derived from Escherichia coli, phage, etc., such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter, T7 promoter and the like can be mentioned. Further, artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series (Ptrp × 2), a tac promoter, a lacT7 promoter, and a let I promoter can be used.

  It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases).

  In the recombinant vector of the present invention, a transcription termination sequence is not necessarily required for the expression of the DNA of the present invention, but it is preferable to place the transcription termination sequence immediately below the structural gene.

As the host cell, Escherichia (Escherichia) genus Serratia (Serratia) genus Bacillus (Bacillus) genus Brevibacterium (Brevibacterium) genus Corynebacterium (Corynebacterium) genus-mycobacterial (Microbacterium) genus Pseudomonas (Pseudomonas ) microorganisms belonging to the genus or the like, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49 , Escherichia coli W3110, Escherichia coli TB1 , Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum ATCC14068, Brevibacteriumsaccharolyticum ATCC14066, Brevibacterium flavum ATCC14067, Brevibacterium lactoferm entum ATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonas putida , Pseudomonas sp. D-0110 and the like.

  As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into the above host cell. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 ( 1972)], protoplast method (Japanese Patent Laid-Open No. 63-2483942), Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979).

  When yeast is used as a host cell, examples of expression vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like.

  Any promoter can be used as long as it can be expressed in yeast strains. For example, a glycolytic gene promoter such as hexose kinase, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MFα1 promoter, CUP 1 promoter and the like.

As the host cell, Saccharomyces (Saccharomyces) genus Schizosaccharomyces (Schizosaccharomyces) genus Kluyveromyces (Kluyveromyces) genus Trichosporon (Trichosporon) genus Schwanniomyces (Schwanniomyces) genus Pichia (Pichia) sp., Candida (Candida ) Microorganisms belonging to the genus, such as Saccharomyces cerevisiae , Schizosaccharomyces pombe , Kluyveromyces lactis , Trichosporon pullulans , Schwanniomyces alluvius , Candida utilis and the like.

  As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into yeast. For example, electroporation method [Methods Enzymol., 194, 182 (1990)], spheroplast method [ Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)], lithium acetate method (J. Bacteriology, 153, 163 (1983)), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) Examples of the method can be given.

  When animal cells are used as a host, examples of expression vectors include pcDNA3.1 (+) (manufactured by Invitrogen), pAGE107 (Japanese Patent Laid-Open No. 3-22979, Cytotechnology, 3, 133 (1990)), pAS3- 3 (Japanese Patent Laid-Open No. 2-227075), pCDM8 [Nature, 329, 840 (1987)], pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987)], etc. .

  Any promoter can be used as long as it functions in animal cells. For example, cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein Examples include promoters, heat shock promoters, SRα promoters, and the like. In addition, an enhancer of human CMV IE gene may be used together with a promoter.

  Examples of host cells include human cells, such as Namalwa cells, monkey cells, COS cells, Chinese hamster cells, CHO cells, and HBT5637 (Japanese Patent Laid-Open No. 63-299). it can.

  As a method for introducing a recombinant vector into animal cells, any method can be used as long as it is a method for introducing DNA into animal cells. For example, electroporation method [Cytotechnology, 3, 133 (1990)], calcium phosphate method (JP-A-2-27075), lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], Virology, 52, 456 (1973) and the like.

When using insect cells as a host, for example, Current Protocols in Molecular Biology, Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Bio / Technology, 6, 47 ( 1988) and the like, the peptide can be expressed.
In other words, the recombinant gene transfer vector and the defective baculovirus genome are co-introduced into insect cells to obtain recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the peptide. Can be made.

  Examples of the gene transfer vector used in the method include pVL1392, pVL1393 (Becton Dickinson), pBlueBac4.5 (Invitrogen), and the like.

  As the baculovirus, for example, an autographa californica nuclear polyhedrosis virus, which is a virus that infects a serpentine insect, can be used.

  Examples of insect cells include Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)), which is an ovary cell of Spodoptera frugiperda, and High 5 (Invitrogen), an ovary cell of Trichoplusia ni. Etc.) can be used.

  Examples of a method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofection method [Proc. Natl Acad. Sci. USA, 84, 7413 (1987)].

  When plant cells are used as host cells, examples of expression vectors include Ti plasmids and tobacco mosaic virus vectors.

  Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include cauliflower mosaic virus 35S promoter and rice actin 1 promoter.

  Examples of host cells include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.

As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, Agrobacterium (JP 59-140885, JP 60-60) No. 70080, WO94 / 00977 pamphlet), electroporation method (Japanese Patent Laid-Open No. 60-251887), method using particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No. 2517813) ) Etc.

The peptide according to the present invention can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the peptide according to the present invention in the culture, and collecting from the culture. .
The method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.

  When the transformant is a transformant obtained by using a prokaryote such as E. coli or a eukaryote such as yeast as a host, as a medium for culturing the transformant, a carbon source that can be assimilated by the transformant, Either a natural medium or a synthetic medium may be used as long as it contains a nitrogen source, inorganic salts, and the like and can efficiently culture the transformant.

  Any carbon source may be used as long as the transformant can assimilate, for example, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, etc. Alcohols such as ethanol and propanol can be used.

  Examples of the nitrogen source include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.

  Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

  The culture is preferably performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually preferably 16 hours to 7 days. The pH during the culture is preferably maintained at 3.0 to 9.0. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

  Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.

  When culturing a microorganism transformed with a recombinant vector using an inducible promoter as a promoter, it is preferable to add an inducer to the medium as necessary. For example, when cultivating a microorganism transformed with a recombinant vector using the lac promoter, when culturing a microorganism transformed with isopropyl-β-D-thiogalactopyranoside or the like with a recombinant vector using the trp promoter. Indoleacrylic acid or the like may be added to the medium.

  As a medium for culturing a transformant obtained using animal cells as a host, for example, RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's minimal essential medium (MEM) [Science, 122, 501 (1952)], Dulbecco's modified Eagle medium (Virology, 8, 396 (1959)), 199 medium (Proceeding of the Society for the Biological Medicine, 73, 1 (1950)) or fetal bovine serum etc. Examples include added media.

In general, the culture is preferably performed in the presence of 5% CO ₂ , the pH is preferably 6 to 8, the temperature is preferably 30 to 40 ° C., and preferably 1 to 7 days.

  Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.

  Further, according to the method described in JP-A-2-27075, the production amount can also be increased using a gene amplification system using a dihydrofolate reductase gene or the like.

As a medium for culturing a transformant obtained using insect cells as a host, commonly used TNM-FH medium (Becton Dickinson), Sf-900 II SFM medium (Invitrogen), ExCell400, ExCell405 (Both manufactured by JRH Biosciences), Grace's insect medium [Nature, 195, 788 (1962)], and the like can be used.
In general, the culture is preferably carried out at a pH of 6 to 7 and a temperature of 25 to 30 ° C., preferably 1 to 5 days.

  Moreover, you may add antibiotics, such as a gentamicin, to a culture medium as needed during culture | cultivation.

  A transformant obtained using a plant cell as a host can be cultured as a cell or differentiated into a plant cell or organ. As a medium for cultivating the transformant, a commonly used Murashige and Skog medium, a white medium, or a medium in which a plant hormone such as auxin or cytokinin is added to these mediums can be used. .

  In general, the culture is preferably performed at a pH of 5 to 9 and a temperature of 20 to 40 ° C., and preferably 3 to 60 days. Moreover, you may add antibiotics, such as kanamycin and a hygromycin, to a culture medium as needed during culture | cultivation. As described above, a microorganism, animal cell, or plant cell-derived transformant having a recombinant vector incorporating the DNA encoding the peptide of the present invention is cultured according to a normal culture method to produce the peptide. The peptide can be produced by accumulating and collecting the peptide from the culture.

  In order to avoid degradation in the host cell, the peptide of the present invention is produced by producing a fusion protein of an arbitrary polypeptide (hereinafter referred to as polypeptide X) and the peptide of the present invention, and then using the peptide of the present invention as a fusion protein. You may isolate and manufacture from. An expression vector for expressing the fusion protein is prepared by adding a DNA encoding a specific protease recognition sequence or methionine to the 5 ′ end of the DNA encoding the peptide of the present invention described above. The peptide X expression vector can be prepared by ligating DNA encoding polypeptide X together in frame. However, the DNA encoding methionine is added only when the peptide of the present invention does not contain methionine. As the polypeptide X, any polypeptide can be used, and examples thereof include glutathione S-transferase, maltose binding protein, DsbA, DsbC, protein A and the like. Examples of specific protease recognition sequences include factor Xa recognition sequence (Ile-Glu-Gly-Arg) and enterokinase recognition sequence (Asp-Asp-Asp-Asp-Lys). The expression vector for polypeptide X can be prepared by inserting a DNA encoding polypeptide X in place of the DNA encoding the peptide of the present invention in accordance with the expression vector for the peptide of the present invention described above. In addition, commercially available fusion protein expression vectors such as fusion protein expression vector pGEX-3 (manufactured by GE Healthcare) with glutathione S-transferase, fusion protein expression vectors pMAL-c2X, pMAL-p2E with maltose binding protein (New England Biolabs), DsbA fusion protein expression vector pET-39b (+) (EMD Biosciences) or the like may be used. When the peptide of the present invention is fused to the polypeptide X through a recognition sequence of a specific protease, the peptide of the present invention can be cleaved from the fusion protein by protease treatment corresponding to the recognition sequence. When the peptide of the present invention is fused to the C-terminus of polypeptide X via methionine, the peptide is fused by treating with cyanogen bromide according to the method described in JP-A-1-02096. Can be cleaved from protein. After treatment with protease or cyanogen bromide, the peptide can be isolated and purified by combining gel filtration, reverse phase HPLC, affinity chromatography and the like.

  Literature [J. Biol. Chem., 264, 17619 (1989); Proc. Natl. Acad. Sci. USA, 86, 8227 (1989); Genes Develop., 4, 1288 (1990); [Patent Publication No. WO94 / 23021 pamphlet], when a DNA encoding the above peptide is prepared, a DNA encoding a secretory protein signal peptide is added to its 5 ′ end. Then, using the DNA, a recombinant vector is prepared in the same manner as described above, and introduced into a host cell, whereby the peptide can be secreted into the medium for production.

In order to make the N-terminus of the peptide of the present invention asparagine, it can be produced by a method of isolating and then secreting it into the medium after producing the above fusion protein. For example, a DNA consisting of the base sequence represented by SEQ ID NO: 2 and a DNA consisting of a base sequence complementary to SEQ ID NO: 2 are chemically synthesized with a DNA synthesizer and then annealed to prepare double-stranded DNA. The double-stranded DNA and pMAL-c2X cleaved with Xmn I are ligated to produce a plasmid in which the double-stranded DNA is inserted into the Xmn I site of pMAL-c2X. The obtained plasmid encodes a fusion protein in which the peptide of the present invention consisting of the amino acid sequence represented by Factor Xa recognition sequence (Ile-Glu-Gly-Arg) and SEQ ID NO: 1 is fused to the C-terminus of the maltose binding protein. To do. Escherichia coli is transformed with the obtained plasmid. The obtained transformant is cultured in a medium, and the fusion protein is expressed in the cells of the transformant. After the cultured cells are isolated by centrifugation, the cells are crushed to obtain a solution containing the fusion protein. The fusion protein is isolated from the resulting solution by affinity chromatography using a column on which maltose is immobilized, and then the fusion protein is treated with factor Xa, and the peptide comprising the amino acid sequence represented by SEQ ID NO: 1 is fused to the fusion protein. Disconnect from. The peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 can be isolated and purified by gel filtration or reverse phase HPLC.

  In order to isolate and purify the peptide produced by the transformant, a normal protein isolation and purification method can be used.

  For example, when the peptide of the present invention is expressed in a cell in a dissolved state, the cells are collected by centrifugation after culturing, suspended in an aqueous buffer, and then subjected to an ultrasonic crusher, a French press, a Manton Gaurin homogenizer. Then, the cells are disrupted with dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylamino Anion exchange chromatography using resin such as ethyl (DEAE) -Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Kasei), cation exchange chromatography using resin such as S-Sepharose FF (manufactured by Pharmacia) Methods such as electrophoresis, hydrophobic chromatography using resin such as butyl sepharose, phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing etc. A purified preparation can be obtained by using alone or in combination.

  In addition, when the peptide of the present invention is expressed by forming an insoluble substance in cells, the cells are similarly collected, crushed, and centrifuged to recover the peptide insoluble matter as a precipitate fraction. The recovered peptide insolubles are solubilized with a protein denaturant. The peptide is returned to a normal three-dimensional structure by diluting or dialyzing the solubilized solution and reducing the concentration of the protein denaturant in the solubilized solution. After the operation, a purified preparation of the peptide can be obtained by the same isolation and purification method as described above.

  When the peptide of the present invention is secreted extracellularly, the peptide can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using the same isolation and purification method as described above. Can be obtained.

<3. Pharmaceutical containing the peptide of the present invention or a pharmacologically acceptable salt thereof>
The peptide of the present invention and a pharmacologically acceptable salt thereof have an activity of promoting insulin secretion from the pancreas of a mammal. Therefore, the peptide of the present invention and a pharmacologically acceptable salt thereof can be used for diabetes (eg, type 1 diabetes, type 2 diabetes, gestational diabetes, etc.), glucose intolerance or diabetic complications (eg, neuropathy, nephropathy). , Retinopathy, cataract, macrovascular disorder, diabetic gangrene), or an active ingredient of an insulin secretagogue. Furthermore, the peptides of the present invention and pharmacologically acceptable salts thereof can also be used as active ingredients of preventive or therapeutic agents for metabolic disorders such as cachexia.

  In particular, the peptide of the present invention and a pharmacologically acceptable salt thereof are useful as a glucose concentration-dependent insulin secretagogue that exhibits an excellent insulin secretion promoting action in the presence of high concentration glucose. Therefore, the peptide of the present invention and a pharmacologically acceptable salt thereof are particularly useful as a safe prophylactic / therapeutic agent for diabetes with a low risk of vascular complications and hypoglycemia induction, which are harmful effects of insulin. is there.

  The medicament containing the peptide of the present invention or a pharmacologically acceptable salt thereof may be used as an active ingredient alone or as an active ingredient for any other treatment. It can contain as a mixture of these. In addition, these pharmaceutical preparations are produced by any method well known in the technical field of pharmaceutics by mixing the active ingredient with one or more pharmacologically acceptable carriers.

  The content of the peptide of the present invention or a pharmacologically acceptable salt thereof in the pharmaceutical preparation varies depending on the dosage form, the dose of the peptide of the present invention or the pharmacologically acceptable salt thereof, etc. 0.01 to 100% by weight. In addition, the content of the pharmacologically acceptable carrier in the pharmaceutical preparation varies depending on the dosage form, the dose of the peptide of the present invention or a pharmacologically acceptable salt thereof, and is, for example, 0 to 99.9. % By weight.

It is desirable to use the most effective route for treatment, and it is administered orally or parenterally, for example, intracerebroventricularly or intravenously.
Administration forms include tablets, powders, granules, syrups, injections and the like.
Liquid preparations such as syrups suitable for oral administration include saccharides such as water, sucrose, sorbit, fructose, glycols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, p -It can be produced using preservatives such as hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint. Tablets, powders and granules include excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxy It can be produced using a binder such as propylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin.

Formulations suitable for parenteral administration preferably comprise a sterile aqueous solution containing the active compound that is isotonic with the blood of the recipient. For example, in the case of an injection, a solution for injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of salt water and a glucose solution.
Also in these parenteral agents, one kind selected from diluents, preservatives, flavors, excipients, disintegrants, lubricants, binders, surfactants, plasticizers and the like exemplified for oral agents. Or more auxiliary components can be added.

  A medicament containing the peptide of the present invention or a pharmaceutically acceptable salt thereof is used for mammals (eg, human, mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, etc.) It can be safely administered. A sufficient amount of the peptide of the present invention and a pharmacologically acceptable salt thereof to promote insulin secretion from the pancreas of a mammal is used to develop the above-mentioned disease or a potential to develop it. By administering to a mammal, the disease can be prevented or treated.

  The dose and frequency of administration of the peptide of the present invention or a pharmacologically acceptable salt thereof vary depending on the dosage form, patient age, body weight, nature or severity of the condition to be treated. 0.01 mg to 1 g, preferably 0.05 to 50 mg per adult is administered once to several times a day. In the case of parenteral administration such as intravenous administration, 0.001 to 100 mg, preferably 0.01 to 10 mg per adult is administered once to several times a day. However, the dose and the number of doses vary depending on the various conditions described above.

  In addition, the peptide can be expressed in the body by transplanting a recombinant vector that expresses the peptide of the present invention or a predetermined cell into which the recombinant vector has been introduced. The peptide of the present invention expressed in the body can be expected to have the same effect as the case where itself or a pharmacologically acceptable salt thereof is administered from outside the body. The recombinant vector that expresses the peptide and the predetermined cell into which the recombinant vector has been introduced are prepared as described in “2. Method for producing peptide of the present invention having insulin secretion promoting activity, (2) Method for producing by genetic engineering technique”. Can be produced by the method described in the above.

<4. Antibody specifically recognizing the peptide of the present invention>
The antibody that specifically recognizes the peptide of the present invention is an antibody that binds to an epitope present in the amino acid sequence of the peptide of the present invention, and can specifically recognize the peptide of the present invention. Here, “specifically recognize” means that the affinity of a certain antibody for a specific antigen is higher than the affinity for another antigen. This antibody may be a polyclonal antibody or a monoclonal antibody. The antibody also includes antibody fragments such as Fab, Fab ′, and F (ab ′) ₂ prepared from polyclonal antibodies or monoclonal antibodies. Monoclonal antibodies include human chimeric antibodies composed of variable regions of monoclonal antibodies prepared in non-human animals and constant regions of human antibodies, and complementarity determining regions (CDRs) of monoclonal antibodies prepared in non-human animals. Also included are human CDR-grafted antibodies comprising a variable region inserted in the framework region and a constant region of a human antibody.

(1) Preparation of polyclonal antibody Administration of a peptide comprising an amino acid sequence having all or part of the amino acid sequence of the peptide of the present invention as an antigen, intradermally, intravenously, intraperitoneally, intramuscularly, etc. in a non-human animal. Thus, a polyclonal antibody that binds to an epitope present in the amino acid sequence represented by SEQ ID NO: 1 can be produced. In the case where it is difficult to produce an antibody due to problems such as solubility of the peptide, it is also possible to use an epitope obtained by modifying the peptide with a hydrophilic modifying group such as polyethylene glycol. This polyclonal antibody can specifically bind to the peptide of the present invention. In this case, it is desirable to administer an antigen peptide, which is covalently bound to a carrier protein such as keyhole, limpet, hemocyanin, bovine thyroglobulin, or ovalbumin, together with an adjuvant. The covalent bond of the peptide serving as the antigen to the carrier protein can be carried out by a reaction using a cross-linking agent such as maleimide, carbodiimide, or glutaraldehyde. When carrying out the reaction of maleimide, a peptide having a cysteine residue added to the N-terminal or C-terminal of the amino acid sequence of the peptide to be used as an antigen is prepared by the method described in 2 above and covalently bonded via cysteine. Examples of adjuvants include Freund's complete adjuvant, aluminum hydroxide gel, pertussis vaccine, and the like. Rabbits, goats, rats, mice, hamsters and the like can be used as non-human animals to administer the antigen, and the dosage is preferably an amount containing 50 to 200 μg of the peptide serving as the antigen per animal.

  The antigen is preferably administered 3 to 10 times, for example, every 1 to 3 weeks after the first administration, until the antibody titer of the serum is sufficiently increased. The antibody titer of serum can be measured by collecting blood on 3 to 7 days after each administration, preparing serum, and using enzyme immunoassay, radioimmunoassay or the like. The enzyme immunoassay is based on the literature [Enzyme Immunoassay, Medical School (1976); Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. Is immobilized on a suitable plate with (II) blocking and washed, (iii) reacted with serum prepared from the immunized animal, washed (iv ) Reaction with an enzyme-labeled antibody against IgG of animal used for immunization and washing, (v) Reaction using a substrate that develops or emits light with the labeled enzyme, and measures the amount of color or the amount of luminescence to measure the antibody titer. It can be performed by the procedure of using as an index.

  Serum is prepared by collecting blood from a non-human animal whose serum showed a sufficient antibody titer against the peptide as an antigen. This serum, that is, antiserum can be used as a polyclonal antibody, or a polyclonal antibody can be purified from the antiserum.

  Methods for purifying polyclonal antibodies from antisera include, for example, centrifugation, salting out with 40-50% saturated ammonium sulfate, caprylic acid precipitation (Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, (1988)), or DEAE-Sepharose. Examples include a method of treating a column, an anion exchange column, a chromatography using a protein A or G column, a gel filtration column, or the like alone or in combination.

(2) Production of monoclonal antibody A monoclonal antibody that binds to an epitope present in the amino acid sequence of the peptide of the present invention can be produced. In the case where it is difficult to produce an antibody due to problems such as solubility of the peptide, it is also possible to use an epitope obtained by modifying the peptide with a hydrophilic modifying group such as polyethylene glycol. This monoclonal antibody can specifically bind to the peptide of the present invention.

(A) Preparation of antibody-producing cells A mouse or rat is used as an animal to which an antigen is administered, and the same antigen is administered as in the production of the polyclonal antibody in (1). A mouse or rat that shows a titer can be used as a source of antibody-producing cells. Spleen cells can be used as antibody-producing cells. Antibody-producing cells can be prepared from mice or rats that have shown a sufficient antibody titer, for example, as follows.

  Three to seven days after the final administration of the antigen to the mouse or rat showing the antibody titer, the spleen is removed. The spleen is shredded in MEM, loosened with tweezers, centrifuged, the supernatant is discarded, and the precipitated splenocytes are collected. The obtained spleen cells are treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes, and then washed three times with MEM can be used as antibody-producing cells.

(B) Preparation of myeloma cells As myeloma cells, cell lines established from mouse or rat myeloma cells can be used. Examples of established myeloma cells include 8-azaguanine-resistant mouse (BALB / c-derived) myeloma cell line P3-X63Ag8-U1 [Curr. Topics. Microbiol. Immunol., 81, 1 (1978); Europ. J Immunol., 6, 511 (1976)], SP2 / 0-Ag14 [Nature, 276, 269 (1978)], P3-X63-Ag8653 [J. Immunol., 123, 1548 (1979)], P3-X63. -Ag8 [Nature, 256, 495 (1975)] and the like. These cell lines consisted of 8-azaguanine medium [RPMI-1640 medium with glutamine (1.5 mmol / L), 2-mercaptoethanol (5 × 10 ⁻⁵ mol / L), gentamicin (10 μg / mL) and fetal calf. It is preferable to subculture with medium supplemented with serum (10%) (hereinafter referred to as normal medium) and medium further added with 8-azaguanine (15 μg / mL), and 3 to 4 days before cell fusion. It is preferable to culture in a normal medium. It is preferable to use 2 × 10 ⁷ or more of the cells for fusion.

(C) Production of Hybridoma A hybridoma can be produced by fusing the antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) using, for example, polyethylene glycol as follows. The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) are prepared by MEM or PBS (1.83 g disodium phosphate, 0.21 g monopotassium phosphate, 7.65 g sodium chloride, 1 liter distilled water). Wash well with pH 7.2), mix so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1, centrifuge at 1,200 rpm for 5 minutes, and discard the supernatant. The cell group of the obtained precipitate fraction was thoroughly loosened, and the cell group was mixed with polyethylene glycol-1000 2 g, MEM 2 mL, and dimethyl sulfoxide 0.7 mL per 10 ⁸ antibody-producing cells at 37 ° C. while stirring. Add 0.2-1 mL of the solution and add 1-2 mL of MEM several times every 1-2 minutes. After the addition, MEM is added to prepare a total volume of 50 mL. The preparation is centrifuged at 900 rpm for 5 minutes, and the supernatant is discarded.

The cells after cell fusion can be cultured, for example, as follows, and hybridomas with high antibody production can be selected. After the precipitated fraction of the cells obtained was gently loosened, discharging from a graduated pipette, hypoxanthine ⁽¹⁰ -4 mol / L) to gently HAT medium [normal medium in blowing, thymidine (1.5 × 10 ^{- 5} mol / L) and aminopterin (4 × 10 ⁻⁷ mol / L) added medium] are suspended in 100 mL. The suspension is preferably dispensed at 100 μL / well into a 96-well culture plate and cultured at 37 ° C. for 7 to 14 days in a 5% CO ₂ incubator. After culturing, a part of the culture supernatant is collected, and using the culture supernatant instead of serum, the antibody titer is measured as described in (1) above, and a hybridoma corresponding to the culture supernatant having a high antibody titer is obtained. It can be selected as a hybridoma with high antibody production.

  The hybridoma is cloned, and a hybridoma cell that stably produces a high amount of antibody can be obtained by selecting those that produce a high amount of antibody from the obtained clones of each hybridoma. Cloning can be performed, for example, by a limiting dilution method or the like, and is preferably repeated twice (the first time is HT medium (a medium obtained by removing aminopterin from HAT medium), and the second time is a normal medium). . Using the culture supernatant of each clone obtained by cloning, the above-mentioned antibody titer is measured, and a clone of a hybridoma corresponding to the culture supernatant in which a strong antibody titer is recognized is stably produced with high antibody. It can be selected as a hybridoma cell.

(D) Preparation of monoclonal antibody The monoclonal antibody can be prepared from ascites after the hybridoma selected in (c) is grown as ascites cancer in nude mice as follows. The monoclonal antibody of the present invention obtained in (c) was administered to 8-10 week-old mice or nude mice that were intraperitoneally administered with 2,6,10,14-tetramethylpentadecane (pristane) 0.5 mL and bred for 2 weeks. Preferably, 5-20 × 10 ⁶ cells / animal producing hybridoma are injected intraperitoneally. After 10 to 21 days, ascites is collected from the mouse in which the hybridoma has developed ascites tumor, and the solid content is removed by centrifugation at 3,000 rpm for 5 minutes. A monoclonal antibody can be purified and obtained from the obtained ascites supernatant by the same method as that used for the polyclonal antibody.

  The subclass of the antibody can be determined using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. The amount of peptide can be calculated from the Raleigh method or the absorbance at 280 nm.

(3) Method for Measuring the Peptide of the Present Invention The above antibody can be used to immunologically detect or quantify the peptide of the present invention. Immunological detection or quantification methods include competitive methods, sandwich methods, immunohistochemistry, Western blot, agglutination (monoclonal antibody experiment manual, Kodansha Scientific, 1987; secondary biochemistry experiment course 5, immunobiochemistry Research method, Tokyo Chemical Doujin, 1986).

  In the competitive method, the antibody of the present invention is reacted with a sample solution and a certain amount of a competitive substance (the peptide of the present invention to be measured labeled with an enzyme, biotin, a radioisotope, a fluorescent substance, etc.), and the sample solution In this method, the peptide of the present invention and the competitive substance are competitively bound to the antibody, the amount of the competitive substance bound to the antibody is measured using a label, and the peptide of the present invention is quantified from the bound amount. is there. For example, the antibody is immobilized on a solid phase such as a plate or a bead, the peptide of the present invention and the competitive substance are competitively bound to the antibody, the solid phase is washed, and the binding amount of the competitive substance to the antibody on the solid phase The peptide of the present invention and a competitive substance are competitively bound to an antibody, and then an immune complex is precipitated with γ-globulin and polyethylene glycol to separate it from a competitor that is not bound to the antibody. And a method for measuring the amount of the competitive substance bound to. For example, a solution of the peptide of the present invention having a concentration of 5 to 10 points is prepared, and when the solution is used as a sample solution, the binding amount of the competitor to the antibody is measured, and the peptide concentration and the competitor A calibration curve in which the amount of binding is plotted is prepared, and the binding amount of the competitor for the sample solution for quantifying the peptide of the present invention is applied to the calibration curve, whereby the peptide of the present invention in the sample solution can be quantified.

  The sandwich method is a method using two types of antibodies that specifically bind to the peptide of the present invention. For example, a sample solution is reacted with one antibody immobilized on a solid phase such as a plate or a bead, and a sample is obtained. After binding the peptide of the present invention to the antibody on the solid phase, the other antibody labeled with enzyme, biotin, radioisotope, fluorescent substance, etc. is reacted to bind to the antibody on the solid phase. Examples include a method in which the labeled antibody is further bound to the peptide of the invention, the binding amount of the labeled antibody is measured using a labeling substance, and the peptide of the invention is quantified from the binding amount. For example, a peptide solution of the present invention having a concentration of 5 to 10 points is prepared, and when this solution is used as a sample solution, the binding amount to the labeled antibody is measured, and the peptide concentration and the binding amount of the label are measured. Is prepared, and the binding amount of the labeled antibody for the sample solution for quantifying the peptide of the present invention is applied to the calibration curve, whereby the peptide of the present invention in the sample solution can be quantified.

  The enzyme immunoassay method is the above competitive method or sandwich method, wherein the competitor or antibody is labeled with an enzyme such as alkaline phosphatase or peroxidase, reacted with a reagent that develops or emits light with the labeled enzyme, and the color or amount of emitted light is determined. This is a quantitative method for measuring the binding amount of a competitor or labeled antibody. Radioimmunoassay is a quantitative method in which the competitive substance or antibody is labeled with a radioisotope in the competition method or the sandwich method, and the binding amount of the competitive substance or labeled antibody is measured from the radioactivity.

  Immunohistochemistry is the reaction of the antibodies of the present invention labeled with enzymes, biotin, radioisotopes, fluorescent substances, gold colloids, etc., on sections prepared by freezing tissues or cells embedded in paraffin, This is a method for detecting the peptide of the present invention in tissues or cells by detecting the antibody of the present invention using a labeling substance.

  In Western blotting, proteins and peptides contained in a sample are separated by SDS-polyacrylamide gel, and then the proteins and peptides are blotted from the gel onto a polyvinylidene fluoride (PVDF) membrane, nitrocellulose membrane, etc. In this method, the antibody of the present invention labeled with an element or the like is reacted and then the antibody of the present invention is detected using a labeling substance to detect the peptide of the present invention on the membrane.

  In the aggregation method, particles such as latex to which the antibody of the present invention is immobilized are reacted with a sample solution, and the aggregation of particles caused by binding of the antibody on the particle to the peptide of the present invention in the sample is measured by absorbance. It is a method of detecting or quantifying by measuring the above.

<5. Screening method for substances that inhibit or enhance the activity of the peptide of the present invention>
Substances that inhibit the insulin secretagogue activity of the peptide of the present invention include (i) the pancreatic, pancreatic islet, pancreatic cell or pancreatic β cell line of the mammal, the peptide of the present invention or a pharmacologically acceptable salt thereof, and a test. And (ii) the peptide of the present invention or a pharmacologically acceptable peptide thereof in the absence of the test substance in the pancreas, pancreatic islet, pancreatic cell or pancreatic β cell line of the mammal. And (iii) a candidate that inhibits the insulin secretory activity of the peptide of the present invention when the response is suppressed in the presence of the test substance as a result of the comparison. Screening can be performed by selecting as a substance.

  Similarly, (i) a response when a peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance are contacted with a mammalian pancreas, islet, pancreatic cell or pancreatic β cell line is measured. (Ii) the response when the peptide of the present invention or a pharmacologically acceptable salt thereof is contacted with the mammal's pancreas, pancreatic islet, pancreatic cell or pancreatic β cell line in the absence of the test substance. And (iii) when the response is promoted in the presence of the test substance as a result of the comparison, the test substance is selected as a candidate substance that enhances the insulin secretion promoting activity of the peptide of the present invention. A substance that enhances the insulin secretion promoting activity of the peptide can be screened.

  As the mammal, the above-mentioned 1. The ones listed in the section are mentioned. The mammal is preferably a primate (such as a human) or a rodent (such as a rat).

  Contact of the peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance to the pancreas, pancreatic islet, pancreatic cell or pancreatic β cell line is performed in vivo or in vitro.

  An in vivo test can be performed by administering to a non-human mammal the peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance so as to reach the pancreas, islets or pancreatic cells. It is. When administering the peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance, it is also preferable to induce glucose secretion and induce basal insulin secretion (WO 00/01388 pamphlet).

  An in vitro test is performed by using a pancreas, pancreatic islet or pancreatic cell isolated from a mammal, or an established pancreatic β cell line, an appropriate medium containing the peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance. It can be carried out by culturing in. As culture conditions, normal culture conditions for mammalian tissues and cells can be used, and those skilled in the art can easily set them.

  Isolation of islets from the pancreas can be performed, for example, according to the method described in Transplantation, 43, 725 (1987).

  Pancreatic cells include all cells that make up islets. Examples of pancreatic cells include α cells, β cells, δ cells, PP cells, and the like. The pancreatic cell is preferably a pancreatic β cell. In the in vitro test, primary cultured pancreatic β cells having insulin secretion ability are preferably used. In addition to the method shown in the Examples, pancreatic β cells may be prepared by inducing differentiation of pancreatic β cells from, for example, embryonic stem cells (ES cells) [Proc. Natl. Acad. Sci. , 99, 16105 (2002)].

  Examples of pancreatic β cell lines include BRIN-BD11 cells (Diabetes, 45, 1132 (1996)), INS-1 cells (Endocrinology, 130, 167 (1992)), βTC cells (Proc. Natl. Acad. Sci., 85, 9037 (1988)], or MIN6 cells [Endocrinology, 127, 126 (1990)], RINm5F cells [Diabetes, 31, 521 (1982)], HIT-T15 (ATCC CRL-1777) cells, etc. it can.

  The peptide of the present invention or a pharmacologically acceptable salt thereof can be used in mammals so that a concentration sufficient to induce an insulin secretagogue effect is achieved in the pancreas, islet, pancreatic cell or pancreatic β cell line. Or added to the medium. Such concentration is usually in the range of 1 pmol / L to 10 μmol / L, preferably in the range of 0.001 to 1 μmol / L.

  The insulin secretion-promoting effect of the peptide of the present invention or a pharmacologically acceptable salt thereof is dependent on glucose concentration, so that it can be applied to mammalian pancreas, islets, pancreatic cells or pancreatic β cell lines in the presence of glucose. The peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance are preferably contacted. The glucose concentration is, for example, 10 mmol / L or more, preferably 16.7 mmol / L or more. Further, if the glucose concentration is too high, cytotoxicity may develop, and therefore the glucose concentration is usually 50 mmol / L or less, preferably 30 mmol / L or less. Therefore, in an in vivo test, the glucose load is measured before or after administration of the peptide of the present invention or a pharmaceutically acceptable salt thereof and a test substance so that the glucose concentration in the pancreas, pancreatic islet or pancreatic cell falls within the above range. Is preferred. In the in vitro test, it is preferable to add glucose to the medium so that the concentration is within the above range.

  The response may be any measurable biological response that occurs when the peptide of the present invention or a pharmacologically acceptable salt thereof is contacted with a pancreas, islet, pancreatic cell or pancreatic β cell line. For example, a response may be a change in intracellular calcium ion concentration, a change in intracellular cAMP concentration, or a change in insulin secretion. Preferably, it is a change in intracellular calcium ion concentration or a change in insulin secretion, and more preferably a change in insulin secretion.

The intracellular calcium ion concentration can be measured, for example, by the method described in (i) or (ii) below.
(I) Use of apoaequorin-expressing transgenic mice From the transgenic mouse that expresses apoaequorin expressed throughout the body (International Publication No. 2002/010371 pamphlet) produced by introducing an apoaequorin gene expression vector into a fertilized egg, the pancreas Collect islets or pancreatic cells. The obtained pancreas, pancreatic islet or pancreatic cell is suspended and cultured in a medium containing coelenterazine, and coelenterazine is taken into the cell to form aequorin (complex of apoaequorin and coelenterazine). Aequorin emits light by binding to intracellular calcium ions, so the relative luminescence of the cells per second before and after the addition of the medium containing the peptide or its pharmacologically acceptable salt is measured with a luminometer over time. And used as an index of intracellular calcium ion concentration. It can be confirmed that the test substance has an activity to increase the intracellular calcium ion concentration when the amount of relative luminescence increases by the addition of the test substance. In the case of an in vitro test, the peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance are administered to apoaequorin-expressing transgenic mice, and fluorescence in the pancreas, islets or pancreatic cells is measured over time.

(Ii) Use of a calcium-binding fluorescent reagent Fura-2, Indo-1, such that the excitation wavelength, fluorescence wavelength, or fluorescence intensity of pancreas, pancreatic islets, pancreatic cells, or pancreatic β cell line changes with or without calcium ions, It is suspended in a buffer solution containing a calcium ion-binding fluorescent reagent such as Fluo-3, cultured, and taken up into cells. Since Fura-2 binds to calcium ions and the peak of the fluorescence excitation wavelength shifts from 380 nm to 340 nm, the wavelength of 340 nm with respect to the fluorescence intensity when excited at a wavelength of 380 nm before and after the addition of the test substance. The ratio of the fluorescence intensity when excited with is measured with a fluorescence measuring device and used as an indicator of intracellular calcium ion concentration. When the ratio of the fluorescence intensity is increased by the addition of the test substance, it can be confirmed that the test substance has an activity to increase the intracellular calcium ion concentration. Since Indo-1 binds to calcium ions and the fluorescence wavelength shifts from 480 nm to 400 nm, the ratio of the fluorescence intensity at the wavelength of 400 nm to the fluorescence intensity at the wavelength of 480 nm is added before and after the addition of the test substance. Measured with a measuring device and used as an index of intracellular calcium ion concentration. When the ratio of the fluorescence intensity is increased by the addition of the test substance, it can be confirmed that the test substance has an activity to increase the intracellular calcium ion concentration. Fluo-3 significantly increases the fluorescence intensity at a wavelength of 520 nm by binding to calcium ions, so the ratio of the fluorescence intensity at a wavelength of 520 nm is measured with a fluorescence measuring device before and after the addition of the test substance, It is used as an index of intracellular calcium ion concentration. When the ratio of the fluorescence intensity is increased by the addition of the test substance, it can be confirmed that the test substance has an activity to increase the intracellular calcium ion concentration.

  Intracellular cAMP and insulin secretion can be measured using well-known immunological techniques such as ELISA.

  When responses other than changes in insulin secretion are used for evaluation, the candidate substance obtained as a result of screening actually inhibits the insulin secretion promoting effect of the peptide of the present invention or a pharmacologically acceptable salt thereof. It is preferable to confirm whether (or increase). In this case, the obtained candidate substance is again subjected to the above screening method, and by adopting a change in insulin secretion as a response, the candidate substance is actually the peptide of the present invention or a pharmacologically acceptable salt thereof. It can be determined whether or not the insulin secretion promoting effect is inhibited (or enhanced).

  A candidate substance that enhances the insulin secretion promoting activity of the peptide of the present invention obtained by the above screening method or a pharmacologically acceptable salt thereof is a pharmaceutical substance as a candidate substance for a prophylactic or therapeutic agent for diabetes or cachexia. Useful for development. Therefore, this screening method is also useful as a screening method for candidate substances for preventive or therapeutic agents such as diabetes or cachexia. The present invention also provides a screening method for candidate substances for such preventive or therapeutic agents for diabetes or cachexia. In this case, a candidate substance that enhances the insulin secretion promoting activity of the peptide of the present invention or a pharmacologically acceptable salt thereof is selected as a candidate substance for a prophylactic or therapeutic agent for diabetes or cachexia.

  In this case, it is preferable to confirm whether the obtained candidate substance actually prevents or treats diabetes or cachexia. For example, the presence or absence of activity for preventing or treating diabetes is determined by, for example, administering a candidate substance to a diabetes model non-human mammal, and after a certain period of time has elapsed since administration, diabetes such as blood glucose level in the non-human mammal. Can be determined by evaluating the symptom and comparing it with the case where no candidate substance is administered. The same applies to cachexia.

Non-human mammals with diabetes model include KK mice (for example, KK / Ta mice, KK / Snk mice), KK-A ^y mice (for example, KK-A ^y / Ta mice), C57BL / KsJ db / db mice, C57BL / 6J db / db mice, ob / ob mice, type 2 diabetes model mice such as high-fat diet loaded mice, and type 2 diabetes model rats such as GK rats, ZDF rats, and high-fat diet loaded rats it can. Examples of diabetes model non-human mammals include type 1 diabetes model animals such as streptozotocin (STZ) -induced diabetes model animals and multiple low doses of STZ models.

  And the candidate substance confirmed to have the activity to prevent or treat diabetes can be selected as the substance (or candidate substance) for preventing or treating diabetes.

  The substance thus obtained is diabetic (eg, type 1 diabetes, type 2 diabetes, gestational diabetes, etc.), glucose intolerance, diabetes, as well as the peptide of the present invention and pharmacologically acceptable salts thereof. It may be used as an active ingredient of a prophylactic or therapeutic agent for sexual complications (eg, neuropathy, nephropathy, retinopathy, cataract, macrovascular disorder, diabetic gangrene) and an insulin secretagogue. Furthermore, it may be used as an active ingredient of preventive or therapeutic drugs for metabolic disorders such as cachexia.

  In particular, the peptide of the present invention and a pharmacologically acceptable salt thereof are useful as a glucose concentration-dependent insulin secretagogue that exhibits an excellent insulin secretion promoting action in the presence of high concentration glucose. Therefore, the substance obtained by the above screening method is low in risk, such as vascular complications and hypoglycemia induction, which are harmful to insulin, as well as the peptide of the present invention and pharmacologically acceptable salts thereof. It may be particularly useful as a prophylactic or therapeutic agent for diabetes.

  On the other hand, the substance that inhibits the insulin secretion-promoting activity of the peptide of the present invention or a pharmacologically acceptable salt thereof is useful for the development of a medicament as a candidate substance for the prevention or treatment of hypoglycemia due to excessive insulin, for example . Therefore, this screening method is also useful as a screening method for candidate substances for preventive or therapeutic agents such as hypoglycemia due to excessive insulin. The present invention also provides a method for screening a candidate substance for a prophylactic or therapeutic agent for such hypoglycemia due to excessive insulin. In this case, a candidate substance that inhibits the insulin secretion promoting activity of the peptide of the present invention or a pharmacologically acceptable salt thereof is selected as a candidate substance for a prophylactic or therapeutic agent for hypoglycemia due to excessive insulin.

  In this case, it is preferable to confirm whether or not the obtained candidate substance actually prevents or treats hypoglycemia caused by excessive insulin. For example, the presence or absence of activity to prevent or treat hypoglycemia due to excessive insulin is determined by, for example, administering a candidate substance to a model non-human mammal with hypoglycemia due to excessive insulin, and after a certain time has elapsed since the administration, It can be determined by evaluating symptoms of hypoglycemia such as blood glucose level in human mammals and comparing it with the case where no candidate substance is administered.

  A candidate substance that has been confirmed to have an activity of preventing or treating hypoglycemia due to excessive insulin can be selected as a substance (or candidate substance) for preventing or treating hypoglycemia due to excessive insulin.

<6. Method for screening agonist or antagonist for receptor of peptide of the present invention>
The agonist or antagonist for the receptor of the peptide of the present invention is (i) a mammalian pancreatic cell or pancreatic β cell line, or a cell membrane fraction of these cells, and the peptide of the present invention or a pharmacologically acceptable product thereof. Measuring the binding amount of the peptide or a pharmacologically acceptable salt thereof to the cell or a cell membrane fraction of the cell when the salt and the test substance are contacted, and (ii) the cell membrane fraction of the cell or the cell When the peptide of the present invention or a pharmacologically acceptable salt thereof is contacted in the absence of a test substance, the peptide or its pharmacologically acceptable for the cell or a cell membrane fraction of the cell. (Iii) If the result of the comparison shows that the binding amount of the peptide or a pharmacologically acceptable salt thereof decreases in the presence of the test substance, It can be screened by selecting as a candidate substance of agonist or antagonist for the receptor of the peptide.

  Examples of mammalian pancreatic cells and pancreatic β cell lines are those described in 5. above. Those described in the section can be used.

  The cell membrane fraction can be obtained by homogenizing pancreatic cells or pancreatic β cell line, removing cell debris by low speed centrifugation, and then centrifuging the supernatant at high speed to precipitate the cell membrane containing fraction. If necessary, the cell membrane fraction may be purified by density gradient centrifugation or the like.

  Pancreatic cells or pancreatic β cell lines, or cell membrane fractions of the cells are suspended in a suitable buffer. The buffer may be any buffer as long as it does not inhibit the binding of the peptide of the present invention or a pharmacologically acceptable salt thereof to these cells or the cell membrane fraction of these cells. Preferably, a phosphate buffer solution having a pH of 6 to 8) or a Tris-HCl buffer solution is used. For the purpose of reducing non-specific binding, surfactants such as CHAPS, Tween-80, digitonin and deoxycholic acid, and various proteins such as bovine serum albumin and gelatin can be added to the buffer. Furthermore, protease inhibitors such as PMSF, leupeptin, E-64, and pepstatin can be added for the purpose of suppressing degradation of the peptide or ligand of the present invention by protease.

For example, a binding experiment in which 10 μL to 10 mL of the cells or a cell membrane fraction of the cells coexist with the peptide of the present invention having a certain amount of radioactivity labeled with a radioisotope such as ¹²⁵ I and ³ H. I do. The reaction is carried out at 0 to 50 ° C., preferably 4 to 37 ° C., for 20 minutes to 24 hours, preferably 30 minutes to 3 hours. After the reaction, the mixture is filtered with a glass fiber filter or the like, washed with an appropriate amount of the same buffer, and then the radioactivity remaining on the glass fiber filter is measured with a γ-counter or a liquid scintillation counter. The binding amount at this time is defined as the total binding amount (A). The same reaction is carried out under the condition where a large excess of the same unlabeled compound is added, and the binding amount at that time is defined as the nonspecific binding amount (B). The same reaction is performed under the condition where the test substance is added, and the binding amount at that time is C. The binding inhibition rate of the test substance can be determined by the following formula.

  When the binding amount of the peptide of the present invention or a pharmacologically acceptable salt thereof decreases in the presence of the test substance, the test substance is used as an agonist or antagonist candidate substance for the receptor of the peptide of the present invention. You can choose.

  Furthermore, it may be confirmed that the obtained candidate substance is an agonist or antagonist for the receptor of the peptide of the present invention.

  Confirmation that it is an agonist can be carried out by testing whether the obtained candidate substance has the same insulin secretion promoting activity as the peptide of the present invention. For example, (i) the amount of insulin secretion when a candidate substance is contacted with a pancreatic, pancreatic islet, pancreatic cell or pancreatic β cell line of a mammal is measured, and (ii) the pancreas, pancreatic islet, pancreatic cell or Compared with the amount of insulin secreted when the candidate substance is not contacted with the pancreatic β cell line, and (iii) as a result of comparison, when insulin secretion is promoted in the presence of the candidate substance, the candidate substance is added to the peptide of the present invention. It can be selected as an agonist (or candidate substance) for the receptor.

  Mammalian pancreas, pancreatic islets, pancreatic cells, pancreatic β cell lines that can be used in this test are described in 5. above. It is the same as that described in the section.

  In addition, contact of a candidate substance with a mammalian pancreas, pancreatic islet, pancreatic cell or pancreatic β cell line is also described in 5. In the screening method, the peptide of the present invention or a pharmacologically acceptable salt thereof and a test substance are contacted with a mammalian pancreas, islet, pancreatic cell or pancreatic β cell line in vivo or in vitro. Can be implemented.

  Since the insulin secretion promoting effect of the peptide of the present invention or a pharmacologically acceptable salt thereof is dependent on the glucose concentration, the insulin secretion promoting effect of the agonist for the receptor of the peptide of the present invention is also the glucose concentration. May be dependent. Therefore, in this confirmation test, it is preferable to contact a candidate substance for an agonist with a mammalian pancreas, islet, pancreatic cell or pancreatic β cell line in the presence of glucose. The glucose concentration is, for example, 10 mmol / L or more, preferably 16.7 mmol / L or more. Further, if the glucose concentration is too high, cytotoxicity may develop, and therefore the glucose concentration is usually 50 mmol / L or less, preferably 30 mmol / L or less. Therefore, in an in vivo test, the glucose load is measured before or after administration of the peptide of the present invention or a pharmaceutically acceptable salt thereof and a test substance so that the glucose concentration in the pancreas, pancreatic islet or pancreatic cell falls within the above range. Is preferred. In the in vitro test, it is preferable to add glucose to the medium so that the concentration is within the above range.

  Confirmation of the antagonist is whether or not the obtained candidate substance inhibits the insulin secretion promoting activity of the peptide of the present invention. It can carry out by testing by the screening method of the substance which inhibits the activity of the peptide of this invention. When the candidate substance inhibits the activity of the peptide of the present invention, the candidate substance can be selected as an antagonist (or candidate substance) for the receptor of the peptide of the present invention.

  The agonist (or candidate substance thereof) for the receptor of the peptide of the present invention obtained by the screening method of the present invention has an insulin secretion enhancing activity, like the peptide of the present invention or a pharmacologically acceptable salt thereof. Therefore, it is useful for the development of a medicament as a candidate substance for a preventive or therapeutic agent for diabetes, cachexia and the like. The agonist for the receptor of the peptide of the present invention includes diabetes (eg, type 1 diabetes, type 2 diabetes, gestational diabetes, etc.), glucose intolerance, There is a possibility that it can be used as a prophylactic or therapeutic agent for diabetic complications (eg, neuropathy, nephropathy, retinopathy, cataract, macrovascular disorder, diabetic gangrene) and an active ingredient of an insulin secretagogue. Furthermore, it may be used as an active ingredient of preventive or therapeutic drugs for metabolic disorders such as cachexia.

  In addition, since the antagonist of the peptide receptor of the present invention inhibits the insulin secretion enhancing activity of the peptide of the present invention, it is useful for the development of a medicament as a candidate substance for a prophylactic or therapeutic agent for hypoglycemia and the like.

  Examples of the present invention are shown below. However, the present invention is not limited by these examples.

Example 1: Insulin secretion experiment in mouse islets Collagenase (Nitta Gelatin Co., Ltd.) was dissolved in Hanks' Balanced Salt Solution (HBSS) to a concentration of 1 mg / ml. Anesthetized C57BL6 mice (8 weeks old) were laparotomized, and a plastic tube with an adjusted diameter was inserted through the duodenal opening into the pancreatic duct ligated on the liver side. After injecting about 1.5 mL of collagenase solution, the mice were exsanguinated. The pancreas filled with collagenase solution was excised with scissors, transferred to a 50 mL tube containing about 1.5 mL of collagenase solution, and minced and incubated at 37 ° C. for 15 minutes. After the incubation, about 40 mL of HBSS was added, and after vigorous stirring for 1 minute, the mixture was allowed to stand for 15 minutes under ice cooling, and the supernatant was discarded. About 40 mL of HBSS was added, stirred gently, allowed to stand for 10 minutes, then the supernatant was discarded, and this was repeated once more. The precipitate was observed under a stereomicroscope, and islets were separated and collected with a pipetman.
The isolated islets were cultured in RPMI 1640 medium containing 10% fetal calf serum at 37 ° C. in a CO ₂ incubator for 2-3 hours. After culturing, the isolated islets were transferred into Hepes Krebs-Ringer bicarbonate (HKRB) buffer containing 0.5 mg / mL glucose and further cultured for 1 hour, and then transferred to HKRB buffer.
After adding 200 μL of HKRB buffer per well to a 24-well plate, 5 isolated islets were collected with a micropipette together with 100 μL of HKRB buffer and added to each well. Thereafter, 100 μL each of HKRB buffer or a solution of a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 at a 5-fold concentration (peptide A) was added to a final concentration of 0.1 μmol / L. Further, 100 μL of a 5.0 or 15 mg / mL glucose solution was added to adjust the final glucose concentration to 5.5 or 22 mmol / L. After the addition, the mixture was reacted for 1 hour in a CO ₂ incubator at 37 ° C. After completion of the reaction, the supernatant was collected, and the amount of insulin was quantified with a Mouse Insulin ELISA kit (Mercodia).

  As a result, peptide A did not promote insulin secretion under low glucose conditions (5.5 mmol / L), but significantly enhanced insulin secretion under high glucose conditions (22 mmol / L) (FIG. 1). Peptide A was found to promote insulin secretion only under high glucose conditions in a glucose concentration-dependent manner.

Example 2: Glucose tolerance test in rats under anesthesia Wistar male rats (11-13 weeks old) after fasting for 18 hours were anesthetized by intraperitoneal administration of pentobarbital (25 mg / rat). The skin inside the thigh of an anesthetized rat was incised to expose the hip vein. 200 mg glucose and 50 μg peptide A were dissolved in 400 μL of physiological saline and administered through the hip vein. As a comparative control, a solution in which only 200 mg glucose was dissolved in 400 μL of physiological saline was used. Blood was collected at each time point 20 minutes before, 15 minutes before, 10 minutes before, 5 minutes before, and 1, 3, 5, 10, 20, 30, 40, 50, 60 minutes after each sugar load. Medium insulin concentration was measured. Insulin concentration was measured according to the manufacturer's instructions using a rat ultrasensitive insulin measurement kit (Molinaga) after separating the collected blood from serum.

  As a result, it was clarified that in the group administered with peptide A and glucose simultaneously, the increase in blood insulin concentration after 1, 3, 5 and 10 minutes after the load was significantly increased as compared with the group administered with glucose alone. .

Example 3 Preparation of Derivative Peptide of Peptide A Peptide 1 (SEQ ID NO: 6), Peptide 2 (SEQ ID NO: 7) and Peptide 5 (SEQ ID NO: 8) were prepared using an automatic peptide synthesizer PSSM-8 (manufactured by Shimadzu Corporation). And synthesized. Specifically, Fmoc amino acids (Watanabe Chemical Industries) with side chains protected as necessary were introduced into the resin (Watanabe Chemical Industries) from the C-terminal side, and peptide chains were synthesized on the resin. For peptide chain synthesis, after amino acid introduction, resin washing is performed to remove unreacted amino acids, and then piperidine is removed by treatment with piperidine to remove Fmoc groups from Fmoc amino acids whose side chains are protected. In order to achieve this, the operation of washing the resin was repeated.

  Coupling reagents include 0.5 mol / L HBTU [2- (1H-Benzotriazole-1-yl) -1,1,3,3-Tetramethyluronium] / HOBT (1-hydroxybenzotriazole) solution, 1 mol / L DIEA (N, N-diisopropylethylamine) solution was used. The Fmoc amino acid was used in an amount 10 times the amount of resin introduced (resin substitution rate × resin amount). The reagent amount was calculated according to the usage standard of the peptide synthesizer PSSM-8. 1 mL of DMF (N, N-dimethyl formamide) was added to a reaction vessel containing a resin with a peptide whose side chain was protected after synthesis, and nitrogen was blown to allow DMF to pass through. The same washing operation was performed 5 times with 1 mL of DMF, 3 times with 1 mL of methanol, and 2 times with 1 mL of butyl ether, and then dried overnight in a vacuum. To the dried product, 1 mL of crevice cocktail (90% TFA, 5% Thioanisol, 5% 1,2-ethanedithiol) was added and reacted at room temperature for 2 hours to cleave the peptide and remove the side chain protecting group. After nitrogen was blown, the solvent was transferred to a 15 mL tube, 10 mL of diethyl ether was added and stirred, and then centrifuged (5000 rpm, room temperature, 5 min) to obtain a precipitate. Again, diethyl ether was added to the precipitate, and the above ether precipitation operation was repeated three times. The precipitate was dried overnight with a vacuum decompressor to obtain a crude peptide. The crude peptide was dissolved in 1 ml of 90% acetic acid, 4 mL of 2 mol / L acetic acid was added and mixed to obtain a crude peptide solution. The crude peptide solution was analyzed by reverse phase HPLC to confirm the synthesis purity. Peptides 1, 2 and 5 were preparatively purified by identifying chromatographic peaks using MALDI-TOF-MS positive ion measurement using reverse phase HPLC.

  Peptide 3 (SEQ ID NO: 9) in which the N-terminal of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 is acetylated was washed 5 times with 1 mL of DMF, and the acetic anhydride ( 20 equivalents) / DMF was added and the reaction was allowed to stir at room temperature for 1 hour. Peptide 4 (SEQ ID NO: 10) obtained by amidating the C-terminus of the peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 was synthesized as described above using an amidated resin Rink amide MBHA Resin (Nova Biochem). Washing was performed 5 times with 1 mL of DMF, 3 times with 1 mL of methanol, and 2 times with 1 mL of butyl ether, and then dried overnight in a vacuum. The dried product was treated in the same manner as in the above peptides 1, 2 and 5, and preparative purification of peptides 3 and 4 was performed.

Example 4: Insulin secretion experiment in mouse islets using peptide A derivative peptide Collagenase (Wako Pure Chemical Industries, Ltd.) and Dispase (Invitrogen Corporation) were each 1 mg / mL Hanks' Balanced Salt Solution ( An enzyme solution was prepared by dissolving with HBSS. An 8-10 week old male mouse subjected to cervical dislocation was opened, and about 1 mL of enzyme solution was injected from the common bile duct toward the pancreas. The swollen pancreas was exfoliated from the intestine, etc., and cut into small pieces of about 1 mm ³ using a detailed scissors. 5 mL of the enzyme solution was newly added and incubated at 37 ° C. for 15 minutes. After incubation, 10 mL of HBSS was added and stirred vigorously for about 1 minute to separate the tissue, and then dispensed into two 15 mL tubes. The supernatant was collected by centrifugation at 1,000 rpm for 5 minutes. Each sediment was suspended in 4 mL of HBSS, centrifuged at 1,000 rpm for 5 minutes, and the supernatant was collected. The same operation was repeated once to collect the supernatant. A fibrous mass was collected from each supernatant, transferred to 2 ml of enzyme solution, and incubated at 37 ° C. for 50 minutes. After incubation, the mixture was centrifuged at 1,600 rpm for 5 minutes. The supernatant was removed, and the sediment containing the islets was suspended in RPMI1640 medium containing 10% (v / v) fetal calf serum and incubated at 37 ° C. for 1 hour in a CO ₂ incubator.

  The culture solution containing the islets cultured at 37 ° C. was collected and centrifuged at 1,600 rpm for 5 minutes. The supernatant was removed and washed once with HBSS containing 0.01% (w / v) bovine serum albumin (Wako Pure Chemical Industries). After centrifuging at 1,600 rpm for 5 minutes and removing the supernatant, it was washed once with phosphate buffered saline containing 0.01% (w / v) bovine serum albumin. The supernatant was removed by centrifugation at 1,600 rpm for 5 minutes. The suspension was suspended in phosphate buffered saline containing 3.7 mmol / L glucose and 0.01% (w / v) bovine serum albumin, and dispensed to a 24-well plate at 200 μL / well.

After pre-incubation for 30 minutes in a CO ₂ incubator, phosphate buffered saline is added at 200 μL / well, and final concentrations of 16.7 mmol / L glucose, peptide A, and 1 to 4 each have a final concentration of 1 μmol / L. It was prepared as follows. After incubation for 60 minutes in a CO ₂ incubator, the supernatant was recovered and centrifuged at 5,000 rpm for 5 minutes, and the supernatant was recovered.

  The amount of insulin in the supernatant was quantified using a Mouse Insulin ELISA (Mercodia) according to the instructions. As a result, as shown in FIG. 3, Peptide A, Peptide 1 and Peptide 2 significantly increased insulin secretion, and Peptide 3 also increased insulin secretion. In addition, as shown in FIG. 4, peptide 4 also increased insulin secretion.

Example 5: Intravenous glucose tolerance test in normal rats After male Sprague Dawley rats (Nippon Charles River) were fasted for about 16 hours and weighed, blood glucose was collected from the tail vein. did. Peptide 3 and peptide 4 were dissolved in PBS (Phosphate-buffered saline) to a concentration of 50 nmol / mL. The prepared PBS solutions of peptide 3 and peptide 4 were each administered to the tail vein at a dose of 2 mL / kg, and after 5 minutes, a 50 w / v% glucose solution was administered to the tail vein at a dose of 2 mL / kg ( Dose of glucose: 1 g / kg). Blood collected from the tail vein 5 minutes and 10 minutes after administration of glucose solution was collected in a serum separation tube and centrifuged at 1,850 xg (cooled centrifuge Hitachi 05PR-22, manufactured by Hitachi Koki Co., Ltd.) at 4 ° C for 20 minutes. Serum was collected. Glucose CII test Wako (Wako Pure Chemical Industries) was used for measurement of serum glucose concentration.

  As a result, as shown in FIG. 5, the PBS solution of peptide 3 and peptide 4 decreased both the serum glucose concentration at 5 minutes and 10 minutes after intravenous administration of glucose compared to the control to which the solvent (PBS) was administered. I let you.

According to the present invention, there is provided a preventive or therapeutic agent for diabetes or cachexia containing a peptide having an activity of promoting insulin secretion as an active ingredient. Furthermore, a screening method for a substance that inhibits or enhances the insulin secretion promoting activity of the peptide, or an agonist or antagonist for the receptor of the peptide is provided. The substance that enhances the activity of the peptide obtained by the screening method provided by the present invention and the agonist for the receptor of the peptide are useful for the treatment of diabetes or cachexia, and the substance that inhibits the activity of the peptide And antagonists to the receptor of the peptide are useful in the treatment of hypoglycemia excessively caused by other diabetes therapeutics.
This application is based on Japanese Patent Application Nos. 2010-293612 (filing date: December 28, 2010) and 2011-141237 (filing date: June 24, 2011) filed in Japan. All are included in the specification.

Claims (4)

  A prophylactic or therapeutic agent for diabetes comprising a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, 6, 7, 9 or 10 or a pharmacologically acceptable salt thereof.   A response when the peptide according to claim 1 or a pharmacologically acceptable salt thereof and a test substance are brought into contact with a mammalian pancreas, islet, pancreatic cell or pancreatic β cell line, The pharmacologically acceptable salt is compared with the response when contacted with the mammal's pancreas, pancreatic islet, pancreatic cell or pancreatic β-cell line in the absence of the test substance. A screening method for a substance that inhibits the insulin secretagogue activity of the peptide, wherein the test substance to be suppressed is selected as a candidate substance that inhibits the insulin secretagogue activity of the peptide.   A response when the peptide according to claim 1 or a pharmacologically acceptable salt thereof and a test substance are brought into contact with a mammalian pancreas, islet, pancreatic cell or pancreatic β cell line, The pharmacologically acceptable salt is compared with the response when contacted with the mammal's pancreas, pancreatic islet, pancreatic cell or pancreatic β-cell line in the absence of the test substance. A screening method for a substance that enhances the insulin secretion promoting activity of the peptide, wherein the test substance to be enhanced is selected as a candidate substance that enhances the insulin secretion promoting activity of the peptide.   A method for preventing or treating diabetes in a non-human mammal, comprising administering a prophylactically or therapeutically effective amount of the peptide according to claim 1 or a pharmaceutically acceptable salt thereof to the non-human mammal.