WO2005065703A1

WO2005065703A1 - Novel use of ligand to gpr103-like receptor protein

Info

Publication number: WO2005065703A1
Application number: PCT/JP2005/000439
Authority: WO
Inventors: Shigeru Okuyama; Tatsuya Ohnuki; Shigeyuki Chaki
Original assignee: Taisho Pharmaceutical Co., Ltd.
Priority date: 2004-01-09
Filing date: 2005-01-07
Publication date: 2005-07-21
Also published as: JP4760378B2; JPWO2005065703A1

Abstract

It is intended to clarify the relevancy between NPX and pathologic conditions and develop a drug having a novel function mechanism that has never been observed so far by directly using NPX or performing a screening with the use of it. Namely, an appetite suppressant containing a peptide comprising an amino acid sequence represented by SEQ ID NO:1 or 3.

Description

Specification

Novel applications of ligands for GPR 103-1 i ke receptor protein

The present invention relates to a novel use of polypeptide NPX in the brain. More specifically, the present invention relates to an appetite suppressant using the polypeptide NPX in the brain. Furthermore, the present invention relates to a method for screening a preventive / therapeutic agent for obesity and eating disorders using NPX or NPX and its receptor, GPR103-1 ike receptor protein, and a substance obtained by the screening method. Background technology ''

In recent years, the regulation mechanism of appetite has been attracting attention with the remarkable increase in diseases exhibiting abnormal eating behavior. Such diseases include eating disorders such as anorexia nervosa and bulimia and obesity with overeating.

Eating disorders are classified into anorexia nervosa (AN) and bulimia nervosa (BN). AN is a syndrome that causes severe eating and various psychosomatic symptoms as a result of eating disorders and restriction of eating due to impaired body image, strong desire for leanness and fear of obesity. BN, on the other hand, is a syndrome that prevents weight gain by vomiting and laxative abuse by repeatedly compulsively eating large amounts of food and repeatedly eating overeating difficulties. In this case, obesity may occur because vomiting and laxative abuse are not possible.

In terms of ameripower, 59% are clinically obese (Institute of Medicine, 1995).

As described above, antiobesity drugs and drugs for eating disorders are a very large market, and eating disorders are affected by schizophrenia, neurological disorders in adolescents and adolescents, who are the most common age groups of this disease. It is the third largest after the disease.

The social and psychological factors, such as stress and thin praise, are the causes of these disorders. I can get lost. For example, it has been reported that when a stress test is performed on an obese patient, there are approximately twice as many abnormal individuals as those of normal weight. In addition, dysfunction of the central control mechanism of eating behavior, such as losing hunger irrespective of fasting or losing satiation even after ingesting a large amount of food. Is considered.

The formation of a neural network centered on the hypothalamus has been suggested as a control mechanism of eating behavior. The hypothalamus forms a neural network with the visceral function control system of the solitary nucleus ゃ the dorsal vagal motor nucleus, the cognitive-behavioral control system of the prefrontal cortex and the tongue, and the motor control output system of the basal ganglia and motor cortex. It integrates this information and plays a central role in controlling eating behavior. In other words, peripheral blood signals such as blood glucose and leptin, neural signals from the gastrointestinal tract (vagus nerve), and neural signals from the upper center (serotonin, dopamine, noradrenaline, etc.) are located in the hypothalamus. It is integrated and expressed as eating behavior. Until now, studies at the molecular level in eating behavior have mainly focused on the identification of nerve nuclei such as glucose-sensitive dieurons, and have not been clarified at the substance level. In recent years, the discovery of leptin has led to rapid elucidation of the feeding behavior at the substance level, and accordingly, neuropeptides in the hypothalamus have been attracting attention as regulators that play a central role.

The neuropeptides involved in feeding include agouti-related proteins, NPY, garani ^ / and oleoxin (enhancing appetite), leptin, melanocortin,. CRF, oxitocin and cholecystokinin (appetite suppression). ing. These neuropeptides are produced and secreted in the hypothalamus and are thought to act by binding to their receptors in the hypothalamic feeding center. In addition, recently, receptor subtypes involved in the ingestion of each neuropeptide have been identified and isolated, and have emerged as drug discovery targets. Representative of these are the Y1 and Y5 receptors (NPY receptors), GALR1, GALR2 and GALR3 receptors.

(Galanin receptor), 01 and 02 receptor (orexin receptor), leptin receptor, MC4 receptor (agouti-related protein, melanocortin receptor) The CRF 2 receptor CRF binding protein (CRF receptor) and other agonists and receptor agonists are being developed.

Many other human brain-derived receptor proteins have been discovered in addition to the above. However, since many of these receptors have not been identified for their ligands, it is not known at all what their involvement is in their physiological actions. GPR103-1 ike receptor is known as one of such receptor proteins (WO 2001/16316).

In recent years, it has been revealed that NPX has a high binding affinity to GPR103-1 i ke receptor protein expressed in HEK293 cells.

It was shown to be a ligand for the 1 i ke receptor protein. NPX is a peptide synthesized from a gene predicted to encode a protein of unknown function from the human genome sequence, and belongs to the C-terminally amidated RF amide family. The NPX precursor is processed from the C-terminal arginine-phenylalanine motif, and the C-terminal is predicted to be amidated. It has been reported that the peptide is widely distributed in the brain and also in the hypothalamus (The Journal of Biological Chemistry Vol. 278, No. 30, Issue of July 25, pp. 27652-27657) ,

2003).

However, there has been no report on the association of NPX with eating behavior.

To date, only mazindol (an amine pump inhibitor) has been approved as an appetite suppressant in the home country for the treatment of obesity. In the United States, antidepressants such as SSR I are prescribed (The Journal of Clinical Psychiatry Vol. 59, Suppl 15, pp. 28-34, 1998). Currently, clinical trials are ongoing to administer leptin to obese patients. In addition, patients with eating disorders are prescribed antidepressants and anti-anxiety drugs.

All of these prophylactic or therapeutic agents exert their effects within 1 hour after administration, but exert their effects 3 hours or more after administration and maintain them for a long time. There are no reports of what can be done. Furthermore, there are no reports of compounds that target and act specifically on eating behavior. Disclosure of the invention

An object of the present invention is to clarify the relationship between NPX and a disease state and to develop a drug having a novel mechanism of action by using NPX directly or in screening.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that NPX exhibits a strong appetite-suppressing action after a certain period of time after administration. Furthermore, they found that a compound that regulates the binding between NPX and the NPX receptor GPR103-1 ike receptor protein is effective as a prophylactic / therapeutic agent for obesity or eating disorders. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions are provided.

(1) An appetite suppressant comprising a peptide having the amino acid sequence of SEQ ID NO: 1 or 3.

(2) An agent for preventing or treating obesity or eating disorders, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 or 3.

(3) A peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted in the amino acid sequence of SEQ ID NO: 1 or 3, and has an appetite-suppressing activity. Contains an appetite suppressant.

(4) a peptide comprising an amino acid sequence represented by SEQ ID NO: 1 or 3 in which one or more amino acids are substituted, deleted, added, Z- or inserted, and which has an appetite-suppressing activity; An agent for preventing or treating obesity or eating disorders.

(5) an appetite containing a peptide which is encoded by a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or a DNA which hybridizes to a complementary sequence thereof under stringent conditions and has an appetite-suppressing activity; Inhibitors. (6) an obesity, which comprises a peptide having an appetite-suppressing activity, which is encoded by a DNA consisting of the DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or its complementary sequence under stringent conditions, and For preventing or treating illness or eating disorders.

(7) — The drug according to any one of (1) to (6), which suppresses appetite after a lapse of a fixed time.

(8) The drug according to any one of (1) to (7), which can maintain an appetite-suppressing effect for a long time by adding a fast-acting appetite suppressant.

(9) A method for suppressing appetite, and a method for preventing or treating obesity or eating disorders, comprising administering an effective amount of any one of the following peptides (i) to (iii):

(i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3;

(ii) a peptide having an amino acid sequence in which one or more amino acids have been substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 or 3, and which has an appetite-suppressing activity; or

(iii) a peptide which is encoded by a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or a DNA which hybridizes to a complementary sequence thereof under stringent conditions and has an appetite-suppressing activity:

(10) Use of any one of the following peptides (i) to (iii) for producing an appetite suppressant or an agent for preventing or treating obesity or eating disorders.

(i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3;

(ii) a peptide having an amino acid sequence in which one or more amino acids are substituted, deleted, added and Z or inserted in the amino acid sequence of SEQ ID NO: 1 or 3, and which has an appetite-suppressing activity; or

(iii) a peptide which is encoded by a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or a DNA which hybridizes to its complementary sequence under stringent conditions and has an appetite-suppressing activity: (11) A substance that suppresses or promotes appetite after a certain period of time by promoting or inhibiting the activity of the peptide, characterized by using any one of the following peptides (i) to (iii): How to screen.

(i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3;

(iiii) a peptide which is encoded by a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or a DNA which hybridizes to a complementary sequence thereof under stringent conditions and has an anorectic activity:

(12) The screening method according to (11), further using the protein described in any of the following (i V) to (V i) or a partial peptide of the protein.

(iv) a protein consisting of the amino acid sequence of SEQ ID NO: 5;

(V) one or more amino acids in the amino acid sequence of SEQ ID NO: 5 are substituted, deleted, added and Z- or inserted amino acid sequence, and (i) of (11) iii) a protein having an activity of binding to any of the peptides of the above;

(V i) encoded by a DNA consisting of the nucleotide sequence of SEQ ID NO: 6, or a DNA that hybridizes to its complementary sequence under stringent conditions, and

Protein having an activity of binding to any one of the peptides (i) to (iii) according to (11):

(13) A DNA comprising the nucleotide sequence of SEQ ID NO: 2 or 4, or a DNA that hybridizes to a complementary sequence thereof under stringent conditions, using the following (i) to (iii): A method for screening a substance that suppresses or promotes appetite after a certain period of time by promoting or inhibiting the expression of any of the above peptides.

(i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3;

(ii) one or more amino acids in the amino acid sequence of SEQ ID NO: 1 or 3 A peptide having an amino acid sequence in which an acid has been substituted, deleted, added and / or inserted, and having anorectic activity; or

(iii) a peptide which is encoded by a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or a DNA which hybridizes to a complementary sequence thereof under stringent conditions and has an anorectic activity:

(14) An appetite suppressant, comprising a substance selected by the screening method according to any one of (11) to (13).

(15) An agent for preventing or treating obesity or eating disorders, comprising the substance selected by the screening method according to any one of (11) to (13).

(16) A method for suppressing appetite, including administering an effective amount of a substance selected by the screening method according to any of (11) to (13), and preventing or treating obesity or an eating disorder. Method.

(17) Use of a substance selected by the screening method according to any of (11) to (13) for producing an appetite suppressant or an agent for preventing or treating obesity or eating disorders.

(18) An appetite suppressant comprising an agonist of a receptor for a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3.

(19) An appetite enhancer containing an antagonist of a receptor for the peptide having the amino acid sequence of SEQ ID NO: 1 or 3. Brief Description of Drawings

FIG. 1 shows the results of measuring the amount of food consumed by mice in the examples. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail.

“NPX”, “NPX precursor”, and “GPR103-1 ike receptor protein” as used herein are known peptides or proteins. NPX, NPX precursor and The amino acid sequence of GPR103-1 ike receptor protein is described in SEQ ID NOs: 1, 3 and 5 in the Sequence Listing, and the DNA sequence is described in SEQ ID NOs: 2, 4 and 6 in the Sequence Listing, respectively.

(A) Agent of the present invention

The appetite suppressant of the present invention, and the agent for preventing or treating obesity or eating disorders,

(i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3;

(ii) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 or 3, and which has an appetite-suppressing activity; or

Is used as an active ingredient.

In the “amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence described in SEQ ID NO: 1 or 3”, the number of amino acid mutations and the mutation site in the “amino acid sequence described in the present invention” are as follows. Although there is no limitation as long as the function of the peptide represented by SEQ ID NO: 1 or 3 is maintained, usually, the homology with the peptide represented by SEQ ID NO: 1 or 3 is about 80% or more, Preferably it is about 90% or more, more preferably about 95% or more. The number of mutations in the amino acid is generally 1 to 10, preferably 1 to 8, more preferably about 1 to 5, and particularly preferably 1 to 3.

The side chains of the amino acids that constitute the protein are different in hydrophobicity, charge, size, etc., but do not substantially affect the three-dimensional structure (also called three-dimensional structure) of the entire protein In the sense, some highly conservative relationships are known empirically and by physical measurements. For example, for substitution of amino acid residues, glycine (G 1 y) and proline (Pro), G 1 y and alanine (Al a) Or valine (V a 1), leucine (Le u) and isoleucine (lie), daltamic acid (G 1 u) and glutamine (G 1 n), aspartic acid (Asp) and asparagine (A sn), Cysteine (Cys) and threonine (Thr); Thr and serine (Ser) or Ala; lysine (Lys) and arginine (Arg); In the present invention, such a peptide having an amino acid substitution having high storage stability can be used, but is not limited thereto.

The amino acid sequence in which one or more amino acids are substituted, deleted, added and Z- or inserted in the amino acid sequence described in SEQ ID NO: 1 or 3 is preferably the amino acid sequence described in SEQ ID NO: 1 or 3. It is a peptide functionally equivalent to a peptide consisting of an amino acid sequence. Herein, the functionally equivalent peptide refers to the amino acid described in SEQ ID NO: 1 or 3, which has the same biological properties such as receptor-binding activity and cell stimulating activity on receptor-expressing cells. It has the same appetite-suppressing effect as a peptide consisting of an acid sequence.

The term "hybridize under stringent conditions" as used in the present invention means, for example, 65 to 75 when the salt concentration is about 19 mM to 40 mM and the temperature is not added with formamide. C, the degree of hybridization when Southern hybridization is performed at 35-45 ° C in the presence of 50% formamide. DNAs that hybridize under stringent conditions include DNAs having a certain degree of homology with the nucleotide sequence of the DNA used as a probe, for example, 80% or more, preferably 85% or more, and more preferably Is a DNA having a homology of 90% or more, more preferably 93% or more, particularly preferably 95% or more, and most preferably 98% or more.

The term "after a certain period of time" as used in the present invention means that usually 2 hours or more, more preferably 5 hours or more after the administration, although it varies depending on the administration subject, administration method, and the like.

The term “rapid effect” means that the effect is usually exhibited within 1 hour, more preferably within 30 minutes after administration, depending on the administration subject, administration method and the like.

The term “maintaining an appetite suppression effect for a long time” as used in the present invention refers to a fast-acting appetite suppressant or And the appetite suppressant of the present invention, respectively, means that the appetite-suppressing effect lasts longer than when administered alone, and usually lasts 2 hours or more, preferably 5 hours or more, and more preferably 10 hours or more. Is preferred.

The term “peptide” as used in the present invention means not only an unmodified peptide but also all functionally equivalent peptides, for example, a peptide salt, and amidated or esterified peptide. Yeah.

Peptide salts include physiologically acceptable inorganic acids (eg, hydrochloric acid, phosphoric acid), organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citrate, Examples thereof include salts with malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) or bases (eg, alkali metal), and particularly preferred are physiologically acceptable acid addition salts.

As the peptide used in the present invention, natural peptides derived from humans and other mammals (for example, rats, mice, egrets, chicks, sheep, birds, monkeys, etc.) can be used. The peptide used in the present invention can be produced from human or other mammalian cells or tissues by using a purification method which can be usually performed by those skilled in the art. For example, it can be prepared by homogenizing human or other mammalian cells or tissues, extracting with an acid or the like, and then using ion exchange chromatography or the like.

Alternatively, the peptide used in the present invention can be synthesized by a chemical method such as a solid phase method and a liquid phase method which are usually used for peptide synthesis. As the protecting group such as an amino group in peptide synthesis and the condensing agent for the condensation reaction, known ones can be used. In the solid phase method, various commercially available peptide synthesizers can be used. If necessary, the synthesis can be carried out efficiently by protecting and deprotecting the functional groups. Methods for introducing and removing protecting groups are also known to those skilled in the art. Solid-phase synthesis and liquid-phase synthesis are described in, for example, Biochemistry Laboratory Course 1, Protein Chemistry IV, Haruaki Yajima and Shunpei Sakakibara, 205,

(1977) and the like.

Furthermore, according to the usual recombination technique, the DN encoding the peptide After preparing a recombinant vector containing the A sequence, a host (for example, a suitable host cell such as a mammalian cell or an insect cell) is transformed with the vector to prepare a transformant, and the transformant is cultured. The desired peptide can be isolated and purified from the product as a recombinant peptide. The preparation of the recombinant peptide can be performed, for example, according to the method described in Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 16.1-16.9.

Since the peptide used in the present invention is an endogenous ligand existing in a living body or a peptide functionally equivalent thereto, it is a safe and low-toxic appetite suppressant, or a prophylactic or preventive measure for obesity or eating disorders. It can be used as a therapeutic. In particular, since it exerts its efficacy after a certain period of time after administration, its effect can be maintained for a long time by combining it with a known fast-acting appetite suppressant (eg, mazindol, MT II (preclinical)). is there.

In the present invention, the above-mentioned peptide may be used as it is, but usually, a pharmaceutical composition containing the peptide as an active ingredient using one or more pharmaceutically acceptable additives for pharmaceuticals Is preferably produced and administered. When the peptide is used as a pharmaceutical composition, it can be made into tablets, capsules, elixirs, microcapsules and the like, or as injectables such as sterile solutions and suspensions, according to commonly used means. .

The method of administration of the peptide may be oral or parenteral, and the form of parenteral administration is not particularly limited, and may be intravenous, intramuscular, intraperitoneal, or subcutaneous.

The dose of the peptide varies depending on the administration subject, administration method and the like.For example, in the case of oral administration, about 0.1 to 100 mg / day, preferably 0.1 to 100 mg / day for an obese patient (60 kg) About 1.0 to 50 mg, more preferably about 1.0 to 20 mg. In the case of parenteral administration, for example, about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 30 mg / day for an obese patient (60 kg). 1 to; L 0 mg can be injected intravenously. Further, according to the present invention, an appetite suppressant comprising an agonist of a receptor for a peptide comprising the amino acid sequence represented by SEQ ID NO: 1 or 3, and an amino acid sequence comprising the amino acid sequence represented by SEQ ID NO: 1 or 3 An appetite enhancer containing an antagonist of the receptor for the peptide is provided. As described hereinabove, a peptide having an amino acid sequence represented by SEQ ID NO: 1 or 3 is useful as an appetite suppressant because it has an appetite suppressing effect. Therefore, an agonist of a receptor for the peptide having a similar appetite suppressing effect is also useful as an appetite suppressant. Further, an antagonist of the receptor for the peptide has an appetite-promoting effect opposite to the appetite-suppressing effect of the peptide having the amino acid sequence of SEQ ID NO: 1 or 3, and is useful as an appetite-stimulating agent. is there. The type of agonist and antagonist of the receptor for the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3 is not particularly limited, and may be, for example, a peptide, a peptidomimetic or a low-molecular compound. The agonists and antagonists described above can be selected, for example, by the screening method described herein. In addition, when the above-mentioned agonist or antagonist is used as an appetite suppressant or an appetite enhancer, the dosage form, administration method, dosage amount and the like can be selected according to the case described above for the peptide in this specification.

(B) Screening method of the present invention

The screening method of the present invention relates to a method for screening a substance that suppresses or promotes appetite, and is characterized by using any one of the following peptides (i) to (iii).

(i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3;

(ii) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, or Z in the amino acid sequence of SEQ ID NO: 1 or 3, and which has an appetite-suppressing activity Or

(iii) DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4, or a phase thereof Peptide encoded by DNA that hybridizes under stringent conditions to the complement sequence and has anorectic activity:

In the screening method of the present invention, a protein described in any of the following (iv) to (vi) or a partial peptide of the protein can also be used.

(i V) a protein consisting of the amino acid sequence of SEQ ID NO: 5;

(V) one or more amino acids in the amino acid sequence described in SEQ ID NO: 5 are substituted, deleted, added, Z or inserted, and the amino acid sequence described in the above (i) to (iii) A) a protein having an activity of binding to any of the peptides;

(V i) encoded by a DNA comprising the nucleotide sequence of SEQ ID NO: 6 or a DNA hybridizing under stringent conditions to a complementary sequence thereof, and any one of the above (i) to (iii) A protein having a binding activity with the peptide of the present invention: “Amino acid sequence represented by SEQ ID NO: 5 in which one or more amino acids have been substituted, deleted, added and / or inserted” The number of amino acid mutations and mutation sites in are not limited as long as the function of the peptide represented by SEQ ID NO: 5 is maintained, but the homology with the peptide represented by SEQ ID NO: 5 is generally about 8 0% or more, preferably about 90% or more, more preferably about 95% or more, particularly preferably 99% or more. The number of amino acid mutations is generally 1 to 20, preferably 1 to 10, more preferably about 1 to 7, and particularly preferably 1 to 5. In addition, specific examples of amino acid substitution include, but are not limited to, the highly conservative substitution described above in the present specification. The amino acid sequence in which one or more amino acids have been substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 5 is preferably a protein consisting of the amino acid sequence of SEQ ID NO: 5 It is a functionally equivalent protein. Herein, a functionally equivalent protein means that biological properties such as binding activity to a ligand and signal transduction are equivalent.

The term "protein having binding activity" as used in the present invention differs depending on the measurement method, measurement conditions, and the like. For example, the binding activity to the above-mentioned peptide is determined by FDSS 600 (Hamamatsu Photonics). (Tass) means a protein having an EC50 value of 1.0 to 15OnM. -

The term "protein" as used in the present invention means that not only naturally-occurring protein but also all functionally equivalent proteins are included, and furthermore, their salts or amidated or esterified proteins are included. It may be.

The “partial peptide” referred to in the present invention may be a partial peptide of any of the above-mentioned proteins, and is particularly an extracellular portion of the protein of the present invention and a receptor. Those having binding activity are preferred.

As the above-mentioned protein or a partial peptide of the protein used in the present invention, a natural protein derived from human or other mammals (for example, rat, mouse, rabbit, chicken, sheep, chicken, monkey, etc.) is used. However, as described above in the present specification, it may be synthesized by a chemical method such as a solid phase method and a liquid phase method ordinarily used for peptide synthesis, or may be prepared according to a genetic recombination technique. You may.

Specific examples of the screening method of the present invention include: [1] a method for screening a compound that improves the binding activity between the peptide of the present invention and a receptor protein or a partial peptide thereof, and [2] a peptide or a peptide of the present invention. A screening method for a compound that improves the expression level of the precursor can be mentioned. By such a screening method, a substance that suppresses or promotes appetite can be selected.

Specific examples of the method for screening a substance for improving the binding activity between the peptide of the present invention and a receptor protein include the following methods (a) and (b).

(a) (i) contacting the peptide of the present invention with its receptor protein in the presence of a test substance, measuring the binding activity between the peptide and the receptor protein, and (ii) measuring the binding activity in the absence of the test substance. A screening method comprising a step of selecting a substance that improves or decreases the binding activity in (i) as compared with the binding activity. At this time, if the substance enhances the binding activity, it can be used as a catching substance having an appetite suppressing effect, and if the substance decreases it, it can be used as a candidate substance having an appetite promoting effect.

(b) (i) contacting the peptide of the present invention with its receptor protein in the presence of a test substance; Cell stimulating activity through the receptor protein (eg, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release, intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, phosphorylation of the inner proteins, activation of c one fos, measuring the binding activity of the active or suppressing activity, etc.) that promote reduction, etc. _P H, the cell stimulating activity in the absence of (ii) test substance A screening method comprising, in comparison, a step of selecting a substance that enhances or reduces the binding activity in (i). At this time, a substance that improves the cell stimulating activity can be a candidate substance having an appetite suppressing effect, and a substance that decreases the cell stimulating activity can be a candidate substance having an appetite promoting effect.

As a method for screening a compound for improving the expression level of the peptide of the present invention or a precursor thereof, specifically, (i) culturing the peptide-expressing cell or tissue of the present invention in the presence of a test substance, and Measuring the amount of mRNA encoding the peptide of the present invention, and (ii) selecting a substance that improves or reduces the amount of mRNA in (i) compared to the amount of mRNA in the absence of the test substance. And a screening method comprising: At this time, a substance that improves the amount of mRNA can be a candidate substance having an appetite suppressing effect, and a substance that decreases the mRNA amount can be a candidate substance having an appetite promoting effect.

As described above, by utilizing the knowledge obtained in the present invention, it is possible to screen a substance that suppresses or promotes appetite, particularly a substance that exerts its effect after a certain period of time after administration. .

The test substance used in the screening method of the present invention includes, for example, peptide

, Proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc. These compounds may be novel compounds or known compounds. You may. In addition, peptide libraries, compound libraries, and the like can also be used.

The effect of the test substance to suppress or promote appetite can be confirmed using a known method. For example, animal models of obesity or eating disorders (eg, mice) It can be confirmed by administering the substance obtained by cleaning orally or parenterally and measuring the food consumption of the model animal.

Since the substance obtained by the screening method of the present invention has an effect of suppressing or promoting appetite, it can be used as a safe and low-toxicity drug for treatment and prevention. In particular, some drugs can exert their efficacy after a certain period of time after administration, so they can be used in combination with known fast-acting treatments and obesity or eating when combined with prophylactic drugs (eg, mazindol, MTII (preclinical)). As an agent for preventing or treating disorders, it is possible to maintain its effect for a long time.

When the obtained substance forms a salt, the salt can be used as a medicine. Salts include physiologically acceptable inorganic acids (eg, hydrochloric acid, phosphoric acid), and organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid) Oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) or a base (eg, an alkali metal), and a physiologically acceptable acid addition salt is particularly preferable.

The above-mentioned substance having an appetite-suppressing effect may be used as it is, but usually, a drug containing the substance as an active ingredient using one or more pharmaceutically acceptable additives for preparations Preferably, the compositions are prepared and administered. When the substance is used as a pharmaceutical composition, it should be converted into tablets, capsules, elixirs, microcapsules, or the like, or as an injectable solution such as a sterile solution or suspension, according to commonly used means. it can.

The method of administering the substance may be oral or parenteral, and the form of parenteral administration is not particularly limited, and may be intravenous, intramuscular, intraperitoneal, or subcutaneous.

The dose of the substance varies depending on the administration subject, administration method and the like. For example, in the case of oral administration, about 0.1100 mg, preferably about 1 mg / day for an obese patient (60 kg) is administered. 0-5 O mg, more preferably about 1.0-2 O mg. In the case of parenteral administration, for example, about 0.01 to 30 m / day for obese patients (60 kg) g, preferably about 0.1 to 2 Omg, more preferably about 0.1 to 10 mg can be injected intravenously.

Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. Example

The inhibitory effect of NPX on nighttime feeding was examined.

Three ICR mouse males (this mouse, which is a general-purpose strain, purchased from Nippon Chemical Slipper Co., Ltd.) were bred in Z cages and divided into 5 cages in each group. It was prepared by dissolving a predetermined amount of NPX (synthesized by solid-phase synthesis method) or MTII of melanocortin-14 receptor agonist as a positive control drug in phosphate buffered saline containing 0.1% ゥ serum albumin. Mice in the control group were given a test substance and no test substance was administered. A phosphate buffered saline containing 0.1% serum albumin was administered to the intraventricular chamber. At the time of administration, mice were lightly anesthetized with halothane, a 27-gauge needle was attached to a Hamilton syringe, and the injection needle was placed at the intersection of a line drawn 2 mm outside the midline and the front of the base of the ear. The skull was pierced vertically and a drug solution was injected into the ventricle. The administered dose was 5 μl. Mice were housed on a 12-hour light / cycle （(lights on at 7 am) and dosing occurred around 7 pm. Fifteen minutes after the administration, the mice were allowed to freely eat food and water, and the food intake was measured at 1, 2, 5, 12, 23, 25, and 26 hours after feeding. The presence or absence of the effect of the test substance administration on the suppression of fasting-induced hyperphagia was determined by comparison with the amount of mouse fed in the control group not administered with the test substance. Figure 1 shows the amount of food consumed by each group of mice 1, 2, 5, 12, 23, 25, and 26 hours after the start of feeding.

The symbol * in Fig. 1 indicates that when a significant difference test was performed by the Dunnett test (multiple comparison method for simultaneously comparing only the comparison between the control group and the treatment group with respect to the population mean), P <0.05 It shows that there is a significant difference compared to the buffered saline group. The symbol ** indicates that when P <0.01, the phosphoric acid was It shows that there is a significant difference compared to the buffered saline group.

As is clear from the results shown in Fig. 1, when MTII was administered, the amount of food consumed was significantly suppressed within 1 hour after administration, and the effect gradually decreased after 5 hours. When 3 nmol or 10 nmo 1 of NPX was administered, no effect on food intake was observed up to 2 hours after administration, and after 5 hours, food intake was significantly inhibited. It can be seen that the effect persists even after hours. Industrial applicability

The peptide NPX of the present invention is useful as an agent for preventing or treating obesity or bulimia nervosa. In particular, since the effect is exerted after a certain period of time, a long-term effect can be maintained by combining it with a known and fast-acting drug for preventing and treating obesity and bulimia. Further, the compound obtained by the screening method of the present invention is useful as an agent for preventing or treating obesity or eating disorders.

Claims

The scope of the claims

1. An appetite suppressant containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3.

2. An agent for preventing or treating obesity or eating disorders, comprising a peptide comprising the amino acid sequence of SEQ ID NO: 1 or 3.

3. One or more amino acids in the amino acid sequence of SEQ ID NO: 1 or 3 are substituted, deleted, added and / or inserted, and the peptide has an appetite-suppressing activity. Yes, an appetite suppressant.

4. One or more amino acids in the amino acid sequence of SEQ ID NO: 1 or 3 are substituted, deleted, added and / or inserted, and contain a peptide having an appetite-suppressing activity. To prevent or treat obesity or eating disorders.

5. An appetite suppressant which contains a peptide which is encoded by a DNA which hybridizes to a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or its complementary sequence under stringent conditions and has an appetite-suppressing activity Agent.

6. Obesity or obesity, which contains a peptide which is encoded by a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or a complementary sequence thereof under a stringent condition and has an appetite-suppressing activity. Prevention or treatment of eating disorders.

7. The medicament according to any one of claims 1 to 6, wherein appetite is suppressed after a certain period of time.

8. The medicament according to any one of claims 1 to 7, wherein an appetite suppressing effect can be maintained for a long time by adding a fast acting appetite suppressant.

9. A method for suppressing appetite, and a method for preventing or treating obesity or eating disorders, comprising administering an effective amount of any one of the following peptides (i) to (iii). (i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3; (ii) a peptide comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 or 3, and which has an appetite-suppressing activity; or

(iii) SEQ ID NO: Peptide encoded by DNA that hybridizes under stringent conditions to DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or a complementary sequence thereof and has an appetite-suppressing activity:

10. Use of any one of the following peptides (i) to (iii) for producing an appetite suppressant or an agent for preventing or treating obesity or eating disorders.

(i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3;

(ii) a peptide comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted in the amino acid sequence of SEQ ID NO: 1 or 3, and which has an appetite-suppressing activity Or

(iii) SEQ ID NO: Peptide encoded by DNA that hybridizes under stringent conditions to DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or a complementary sequence thereof and has an anorectic activity:

11. A method for screening a substance that suppresses or promotes appetite after a certain period of time by promoting or inhibiting the activity of the peptide, which comprises using the peptide of any one of the following (i) to (iii).

(i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3;

(iii) a peptide which is encoded by a DNA consisting of the nucleotide sequence of SEQ ID NO: 2 or 4 or a DNA which hybridizes to its complementary sequence under stringent conditions and has an appetite-suppressing activity:

12. The screening method according to claim 11, further comprising using the protein according to any one of the following (i V) to (V i) or a partial peptide of the protein. (iv) a protein consisting of the amino acid sequence of SEQ ID NO: 5;

(v) the amino acid sequence of SEQ ID NO: 5 in which one or more amino acids have a substitution, deletion, addition and Z or insertion amino acid sequence; and (i) the amino acid sequence of claim 11 A protein having an activity of binding to any of the peptides of (a) to (iii)

(vi) encoding a DNA consisting of the nucleotide sequence of SEQ ID NO: 6, or a complementary sequence thereof, by a DNA that hybridizes under stringent conditions, and (i) to (iii) according to claim 11, A protein having a binding activity with any of the following peptides:

13. A DNA comprising the nucleotide sequence of SEQ ID NO: 2 or 4, or a DNA that hybridizes to its complementary sequence under stringent conditions, characterized by the following (i) to (iii): A method of screening a substance that suppresses or promotes appetite after a certain period of time by promoting or inhibiting the expression of any peptide.

(i) a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3;

(ii) a peptide comprising an amino acid sequence in which one or more amino acids have been substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 1 or 3, and which has an appetite-suppressing activity; or

14. An appetite suppressant, comprising a substance selected by the screening method according to claim 11.

15. An agent for preventing or treating obesity or eating disorders, comprising a substance selected by the screening method according to any one of claims 11 to 13.

16. An appetite suppression method, comprising administering an effective amount of a substance selected by the screening method according to any one of claims 11 to 13, and obesity or A method for preventing or treating eating disorders.

17. Use of a substance selected by the screening method according to any one of claims 11 to 13 for producing an appetite suppressant or an agent for preventing or treating obesity or an eating disorder.

18. An appetite suppressant containing an agonist of a receptor for a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3.

1 9. An appetite enhancer containing an antagonist of a receptor for a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3.