JPWO2005065703A1 - Novel uses of ligands for GPR103-like receptor protein - Google Patents

Abstract

An object of the present invention is to clarify the relationship between NPX and a disease state, and to develop a pharmaceutical having a novel mechanism of action by using NPX directly or for screening. According to this invention, the appetite suppressant containing the peptide which consists of an amino acid sequence of sequence number 1 or 3 is provided.

Description

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

In recent years, the appetite regulating mechanism has attracted attention with a marked increase in diseases that exhibit abnormal eating behavior. Examples of such diseases include eating disorders such as anorexia nervosa and bulimia and obesity that causes overeating.
Eating disorders are classified as anorexia nervosa (AN) and bulimia nervosa (BN). AN is a syndrome that causes various types of psychosomatic symptoms such as marked loss of eating and restrictions on eating due to body image disorder, strong desire for thinness and fear of obesity. On the other hand, BN is a syndrome in which body weight gain is prevented by vomiting and abuse of laxatives after repeated episodes of overeating that are compulsorily ingesting large amounts of food and are difficult to control. At that time, vomiting and abuse of laxatives may not be possible, leading to obesity.
In the United States, 59% are clinically obese (Institut of Medicine, 1995).
In this way, anti-obesity drugs and drugs for the treatment of eating disorders are a very large market, and eating disorders are often associated with schizophrenia and neurosis in women adolescents and adolescents, who are prevalent in this disease. It is the third place after.
The etiology of these disorders includes social and psychological factors such as stress and skinny praise. For example, when stress tests are performed on obese patients, it has been reported that there are about twice as many abnormal persons as normal weight persons. In addition, dysfunction of the central control mechanism of eating behavior is considered, such as feeling no hunger despite fasting, or feeling no satiety even after taking a large amount of food. It has been.
The formation of a neural network centering on the hypothalamus is suggested as a control mechanism of eating behavior. The hypothalamus forms a neural network with the visceral function control system of the solitary nucleus and dorsal vagus motor nucleus, cognition of the prefrontal cortex and amygdala-behavior control system and motor control of the basal ganglia and motor area-output system, These information are integrated to play a central role in feeding behavior control. That is, humoral signals such as blood glucose and leptin from the periphery, nerve signals from the digestive tract (vagus nerve), and nerve signals from the upper center (such as serotonin, dopamine, and noradrenaline) are integrated in the hypothalamus. Expressed as eating behavior. Until now, the molecular level of dietary behavior has been focused on the identification of neuronal nuclei, such as glucose-sensitive neurons, and has not been elucidated at the substance level. In recent years, the discovery of leptin has led to rapid elucidation of eating behavior at the substance level, and accordingly, neuropeptides in the hypothalamus have attracted attention as regulators that play a central role.
Known as neuropeptides involved in feeding are agouti-related proteins, NPY, galanin and orexin (increased appetite), leptin, melanocortin, CRF, oxytocin and cholecystokinin (appetite suppression). These neuropeptides are produced and secreted in the hypothalamus and are thought to act by binding to their receptors present in the hypothalamic feeding center. Furthermore, recently, receptor subtypes involved in feeding each neuropeptide have been identified and isolated and have emerged as drug targets. Representative of them are Y1 and Y5 receptors (NPY receptors), GALR1, GALR2 and GALR3 receptors (galanin receptors), OX1 and OX2 receptors (orexin receptors), leptin receptors, MC4 receptors ( Agouti-related protein, melanocortin receptor), CRF2 receptor and CRF binding protein (CRF receptor), and the like, and receptor agonists and antagonists are being developed.
Many receptor proteins derived from human brain 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 physiological actions are involved. As one of such receptor proteins, GPR103-like receptor is known (WO2001 / 16316).
In recent years, it has been revealed that NPX has a high binding affinity with the GPR103-like receptor protein expressed in HEK293 cells, indicating that NPX is a ligand for the GPR103-like receptor protein. NPX is a peptide synthesized from a gene predicted to encode a protein whose function is unknown from the human genome sequence, and belongs to the RF amide family in which the C-terminal is amidated. It is predicted to be amidated from the C-terminal arginine-phenylalanine motif processed from the NPX precursor. It is reported that the peptide is distributed in the brain in a large amount and also in the hypothalamus (The Journal of Biological Chemistry Vol. 278, No. 30, Issue of July 25, pp. 27652-2767, 2003).
However, no relation to NPX's feeding behavior has been reported so far.
Until now, only mazindol (amine pump inhibitor) has been approved as an appetite suppressant in Japan for the treatment of obesity. In the United States, an antidepressant such as SSRI is prescribed (The Journal of Clinical Psychiatry Vol. 59, Suppl 15, pp. 28-34, 1998). Currently, clinical trials in which leptin is administered to obese patients are ongoing. In addition, patients with eating disorders are prescribed antidepressants and anxiolytics.
All of these prophylactic or therapeutic drugs exert their effects within 1 hour after administration, but reports on those that can exert their effects after 3 hours or more after administration and can be maintained for a long time Absent. Furthermore, there are no reports on compounds that specifically target and act on feeding behavior.

An object of the present invention is to clarify the relationship between NPX and a disease state, and to develop a pharmaceutical having a novel mechanism of action by using NPX directly or for screening.
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that NPX exhibits a strong appetite suppressing action after a lapse of a certain time after administration. Furthermore, the present inventors have found that a compound that regulates the binding between NPX and the NPR receptor GPR103-like 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 containing a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3.
(2) A prophylactic or therapeutic agent for obesity or eating disorders containing a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3.
(3) Appetite, 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 having a peptide having anorectic activity Inhibitor.
(4) Obesity 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 having an appetite suppressive activity Preventive or therapeutic agent for sickness or eating disorders.
(5) An appetite suppressant containing a peptide having an appetite suppressive activity that is encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 2 or 4 or a DNA that hybridizes under stringent conditions to its complementary sequence Agent.
(6) Obesity containing a peptide encoded by DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to a complementary sequence thereof and having an appetite suppressive activity Or a preventive or therapeutic agent for eating disorders.
(7) The drug according to any one of (1) to (6), wherein appetite is suppressed after a certain period of time.
(8) The drug according to any one of (1) to (7), which can maintain an appetite suppression effect for a long time by further blending 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 of the following peptides (i) to (iii):
(I) a peptide consisting of the amino acid sequence set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity:
(10) Use of any 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 set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity:
(11) A method for screening for a substance that suppresses or promotes appetite after a certain time by promoting or inhibiting the activity of the peptide, comprising using any of the following peptides (i) to (iii): .
(I) a peptide consisting of the amino acid sequence set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity:
(12) The screening method according to (11), wherein the protein according to any one of (iv) to (vi) below or a partial peptide of the protein is used.
(Iv) A protein comprising the amino acid sequence set forth in SEQ ID NO: 5.
(V) an 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: 5, and (i) to (iii) described in (11) ) A protein having binding activity with any of the peptides;
(Vi) encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 6 or DNA that hybridizes to its complementary sequence under stringent conditions, and (i) to (iii) described in (11) Protein having binding activity with any peptide:
(13) The following (i) to (iii), characterized by using DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence A method of screening for a substance that suppresses or promotes appetite after a certain period of time by promoting or inhibiting the expression of any of the peptides.
(I) a peptide consisting of the amino acid sequence set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity:
(14) An appetite suppressant containing a substance selected by the screening method according to any one of (11) to (13).
(15) A preventive or therapeutic agent for obesity or eating disorders, comprising a substance selected by the screening method according to any one of (11) to (13).
(16) A method for suppressing appetite, and a method for preventing or treating obesity or eating disorders, comprising administering an effective amount of a substance selected by the screening method according to any one of (11) to (13).
(17) Use of a substance selected by the screening method according to any one of (11) to (13) for producing an appetite suppressant, or an agent for preventing or treating obesity or eating disorders.
(18) An appetite suppressant containing a receptor agonist for a peptide comprising the amino acid sequence of SEQ ID NO: 1 or 3.
(19) An appetite promoter containing an antagonist of a receptor for a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3.

  FIG. 1 shows the measurement results of food intake of mice in the examples.

Hereinafter, embodiments of the present invention will be described in detail.
As used herein, “NPX”, “NPX precursor” and “GPR103-like receptor protein” are known peptides or proteins. The amino acid sequences of NPX, NPX precursor and GPR103-like receptor protein are described in SEQ ID NOs: 1, 3 and 5, respectively, and the DNA sequences are described in SEQ ID NOs: 2, 4 and 6 of the sequence listing, respectively.
(A) Drug of the present invention In the appetite suppressant of the present invention, and the preventive or therapeutic agent for obesity or eating disorders,
(I) a peptide consisting of the amino acid sequence set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity:
Any of these is used as an active ingredient.
The number of amino acid mutations and mutation sites 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” according to the present invention are shown in SEQ ID NO: Is not limited as long as the function of the peptide represented by 1 or 3 is maintained, but usually the homology with the peptide represented by SEQ ID NO: 1 or 3 is about 80% or more, preferably about 90% or more, More preferably, the peptide is about 95% or more. The number of amino acid mutations 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 amino acids that constitute protein components differ in hydrophobicity, charge, size, etc., but mean that they do not substantially affect the three-dimensional structure (also referred to as three-dimensional structure) of the entire protein. Some of the relationships that are highly conserved are known empirically and by physicochemical measurements. For example, for substitution of amino acid residues, glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln) ), Aspartic acid (Asp) and asparagine (Asn), cysteine (Cys) and threonine (Thr), Thr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg), and the like. In the present invention, peptides having such highly conserved amino acid substitutions can be used, but are not limited thereto.
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 preferably consists of the amino acid sequence described in SEQ ID NO: 1 or 3 A peptide that is functionally equivalent to a peptide. Here, the functionally equivalent peptide is equivalent in biological properties such as binding activity to the receptor and cell stimulation to the receptor-expressing cell, and the amino acid sequence described in SEQ ID NO: 1 or 3 It has the same appetite suppression effect as the peptide consisting of.
In the present invention, “hybridizes under stringent conditions” means, for example, a salt concentration of about 19 mM to 40 mM, a temperature of 65 to 75 ° C. when no formamide is added, and 35% in the presence of 50% formamide. The degree of hybridization when Southern High Predization is performed under the condition of ˜45 ° C.
Examples of DNA that hybridizes under stringent conditions include DNA having a certain degree of homology with the base sequence of DNA used as a probe, for example, 80% or more, preferably 85% or more, more preferably 90%. More preferably, DNA having homology of 93% or more, particularly preferably 95% or more, and most preferably 98% or more is mentioned.
The term “after a certain period of time” in the present invention varies depending on the administration subject, the administration method, etc., but usually means that 2 hours or more after administration, more preferably 5 hours or more have elapsed, and “rapid effect” means Although it varies depending on the administration subject, administration method and the like, it usually means that the effect is exhibited within 1 hour, more preferably within 30 minutes after administration.
According to the present invention, “maintaining an appetite suppression effect for a long time” means that the appetite suppression effect lasts longer than when the fast-acting appetite suppressant and the appetite suppressant of the present invention are each administered alone. In general, it preferably lasts 2 hours or longer, preferably 5 hours or longer, and more preferably 10 hours or longer.
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 an amidated or esterified peptide may be used. .
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, citric acid, apples) Examples include salts with acids, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) or bases (for example, alkali metals), and physiologically acceptable acid addition salts are particularly preferable.
As the peptide used in the present invention, natural peptides derived from humans and other mammals (eg, rat, mouse, rabbit, chicken, sheep, cow, monkey, 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 that 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, etc., and 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. Known protecting groups such as amino groups and condensing agents for the condensation reaction in peptide synthesis can be used. In the solid phase method, various commercially available peptide synthesizers can be used. If necessary, the synthesis can be efficiently performed by protecting and deprotecting the functional group. Methods for introducing and removing protecting groups are also known to those skilled in the art. Solid phase synthesis and liquid phase synthesis can also be carried out according to the methods described in, for example, Biochemistry Experiment Course 1, Protein Chemistry IV, Haruaki Yajima and Shunpei Sugawara, 205 (1977).
Furthermore, after a recombinant vector containing a DNA sequence encoding a peptide is prepared according to a usual genetic recombination technique, a host (for example, an appropriate host cell such as a mammalian cell or an insect cell) is transformed with the vector to transform the vector. A transformant is prepared, and a desired peptide can be separated and purified as a recombinant peptide from a culture in which the transformant is cultured. Preparation of recombinant peptides is described, for example, in Current Protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. It can be performed according to the method described in John Wiley & Sons Section 16.1-16.9.
Since the peptide used in the present invention is an endogenous ligand present in the living body or a peptide functionally equivalent thereto, it is a safe and low-toxic appetite suppressant, or a preventive or therapeutic agent for obesity or eating disorders. Can be used. In particular, since its efficacy is exhibited after a certain period of time after administration, it can be maintained for a long time by combining with a known fast-acting appetite suppressant (eg, mazindol, MTII (preclinical)). .
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 pharmaceutical preparation. Preferably manufactured and administered. When the peptide is used as a pharmaceutical composition, it can be converted into a tablet, capsule, elixir, microcapsule or the like, or an injection such as a sterile solution or suspension, according to the usual method.
The peptide administration method may be oral administration or parenteral administration, and the form of parenteral administration is not particularly limited, and intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, and the like can be performed.
The dose of the peptide varies depending on the administration subject, administration method, etc. For example, in the case of oral administration, about 0.1 to 100 mg per day, preferably about 1.0 to 50 mg per day for an obese patient (60 kg), More preferably, it is 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 10 mg per day is intravenously injected into an obese patient (60 kg). Can do.
Furthermore, according to the present invention, an appetite suppressant containing an agonist of a receptor for the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3, and a receptor for the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3 An appetite promoter containing an antagonist of As described above in the present specification, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 3 is useful as an appetite suppressant because it has an appetite suppressive effect. Therefore, an agonist of a receptor for the peptide having the same appetite suppressive effect is also useful as an appetite suppressant. Furthermore, the receptor antagonist for the peptide has an appetite promoting effect opposite to the appetite suppressing effect of the peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 3, and is useful as an appetite promoting agent. The kind of the agonist and antagonist of the receptor for the peptide consisting of the amino acid sequence shown in 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 above agonists and antagonists can be selected, for example, by the screening methods described herein. In addition, when using the above-mentioned agonist or antagonist as an appetite suppressant or appetite promoter, the administration form, administration method, dosage, etc. can be selected according to the case described above for peptides.
(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 of the following peptides (i) to (iii): And
(I) a peptide consisting of the amino acid sequence set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity:
In the screening method of the present invention, the protein described in any of (iv) to (vi) below or a partial peptide of the protein can also be used.
(Iv) A protein comprising the amino acid sequence set forth in SEQ ID NO: 5.
(V) consisting of 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: 5, and any one of (i) to (iii) above A protein having binding activity with the peptide of
(Vi) the peptide of any one of (i) to (iii) above, which is encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 6 or DNA which hybridizes under stringent conditions to its complementary sequence Proteins that have binding activity with:
The number of amino acid mutations and mutation sites 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: 5” according to the present invention are as follows: However, the homology with the peptide represented by SEQ ID NO: 5 is usually about 80% or more, preferably about 90% or more, more preferably about 95%. Above, particularly preferably 99% or more of peptides. 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. Specific examples of amino acid substitution include, but are not limited to, the above-described substitution with high preservability in the present specification.
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: 5 is preferably functional with a protein comprising the amino acid sequence described in SEQ ID NO: 5 Is an equivalent protein. Here, functionally equivalent proteins mean that biological properties such as ligand binding activity and signal information transmission action are equivalent.
The “protein having binding activity” as used in the present invention varies depending on the measurement method, measurement conditions, and the like. For example, the EC50 value when the binding activity to the peptide is measured using FDSS6000 (Hamamatsu Photonics) is 1. It means a protein having a concentration of 0 to 15.0 nM.
The term “protein” as used in the present invention means not only a natural type protein but also all functionally equivalent proteins, and further a salt, amidated or esterified product thereof. Also good.
In addition, the “partial peptide” referred to in the present invention may be a partial peptide of any one of the proteins described above, and is particularly an extracellular portion of the protein of the present invention, and receptor binding. Those having activity are preferred.
As the above-mentioned protein used in the present invention or a partial peptide of the protein, natural proteins derived from humans or other mammals (eg, rats, mice, rabbits, chickens, sheep, cows, monkeys, etc.) are used. However, as described above in this specification, it may be synthesized by a chemical method such as a solid phase method and a liquid phase method usually used for peptide synthesis, or may be prepared according to a gene recombination method.
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] the peptide of the present invention or a precursor thereof. And a screening method for compounds that improve the expression level of. A substance that suppresses or promotes appetite can be selected by such a screening method.
Specific examples of the screening method for a substance that improves the binding activity between the peptide of the present invention and the receptor protein include the following method (a) or (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 of the peptide and receptor protein, and (ii) binding in the absence of the test substance The screening method including the process of selecting the substance which improves or reduces the binding activity in (i) compared with activity. If it is a substance which improves binding activity at this time, it can be set as a candidate substance which has an appetite suppression effect, and if it is a substance to reduce, it can be set as a candidate substance which has an appetite promotion effect.
(B) (i) contacting the peptide of the present invention with its receptor protein in the presence of a test substance, and cell stimulating activity (for example, arachidonic acid release, acetylcholine release, intracellular Ca ²⁺ release) via the receptor protein , Intracellular cAMP production, intracellular cGMP production, inositol phosphate production, cell membrane potential fluctuation, intracellular protein phosphorylation, c-fos activation, activity promoting or suppressing pH reduction, etc.) A screening method comprising a step of measuring a binding activity and (ii) selecting a substance that improves or decreases the binding activity in (i) as compared with a cell stimulating activity in the absence of a test substance. At this time, any substance that improves cell stimulating activity can be a candidate substance that has an appetite suppressing effect, and any substance that can be reduced can be a candidate substance that has an appetite promoting effect.
As a screening method for a compound that improves the expression level of the peptide of the present invention or a precursor thereof, specifically, (i) the peptide-expressing cell or tissue of the present invention is cultured in the presence of a test substance, And (ii) selecting a substance that improves or decreases the amount of mRNA in (i) as compared with the amount of mRNA in the absence of the test substance. It is done. If it is a substance which improves the amount of mRNA at this time, it can be set as a candidate substance which has an appetite suppression effect, and if it is a substance which reduces, it can be set as a candidate substance which has an appetite promotion effect.
As described above, by utilizing the knowledge obtained in the present invention, it is possible to screen for a substance that suppresses or promotes appetite, particularly a substance that exerts its efficacy after a lapse of a certain time after administration.
Examples of test substances used in the screening method of the present invention include peptides, proteins, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and the like. The compound may be a novel compound or a known compound. Moreover, a peptide library, a compound library, etc. can also be used.
The effect of suppressing or promoting appetite by the test substance can be confirmed using a known method. For example, it can be confirmed by orally or parenterally administering a substance obtained by screening to an obese or eating disorder model animal (for example, mouse) and measuring the food intake of the model animal. .
Since the substance obtained by the screening method of the present invention has the effect of suppressing or promoting appetite, it can be used as a safe and low-toxic pharmaceutical for treatment / prevention. In particular, since there are those that can exert their efficacy after a certain period of time after administration, obesity and eating disorders can be prevented by combining with known fast-acting therapeutic / prophylactic agents (for example, mazindol, MTII (preclinical)). Or as a therapeutic agent, it is possible to maintain the effect for a long time.
When the obtained substance forms a salt, the salt can also be used as a medicine. 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, citric acid, malic acid, Examples thereof include salts with oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) or bases (for example, alkali metals), and physiologically acceptable acid addition salts are particularly preferable.
The above-mentioned substance having the effect of suppressing appetite may be used as it is, but it is usually a pharmaceutical comprising the substance as an active ingredient using one or more pharmaceutically acceptable additives. Preferably, the composition is manufactured and administered. When the substance is used as a pharmaceutical composition, it can be used as a tablet, capsule, elixir, microcapsule or the like, or an injection such as a sterilized solution or suspension, according to a commonly used means.
The administration method of the substance may be oral administration or parenteral administration, and the form of parenteral administration is not particularly limited, and intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, and the like can be performed.
The dose of the substance varies depending on the administration subject, administration method, etc. For example, in the case of oral administration, it is about 0.1 to 100 mg, preferably about 1.0 to 50 mg per day for an obese patient (60 kg). More preferably, it is 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 10 mg per day is intravenously injected into an obese patient (60 kg). Can do.
EXAMPLES Next, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.

The inhibitory action of NPX on night feeding was examined.
ICR mouse males (this mouse, which is a general strain, was purchased from Nippon Charles River) were bred in 3 animals / cage and divided into 5 cages for each group. A test substance prepared by dissolving MTX of melanocortin-4 receptor agonist as a positive control agent in NPX (synthesized by solid phase synthesis method) in phosphate buffered saline containing 0.1% bovine serum albumin, In addition, phosphate buffered saline containing 0.1% bovine serum albumin was administered into the ventricles of the mice in the test substance non-administration group. At the time of administration, mice are lightly anesthetized with halotane, a 27-gauge needle is attached to the Hamilton syringe, and the needle is pulled from the skull to the intersection of the 2 mm outside from the midline and the line drawn through the front of the ear base. The drug solution was injected vertically into the ventricle. The administration dose was 5 μl. Mice were housed in a 12 hour light / dark cycle (lights on at 7:00 am) and dosing was performed around 7 pm. From 15 minutes after the administration, the mice were allowed to freely take food and water, and the food intake was measured 1, 2, 5, 12, 23, 25 and 26 hours after feeding. In addition, the presence or absence of the inhibitory effect on fasting-induced hyperphagia by administration of the test substance was determined by comparison with the amount of food consumed by the mice in the test substance non-administration control group. The food intake of each group of mice 1, 2, 5, 12, 23, 25 and 26 hours after the start of feeding was shown in FIG.
The symbol * in FIG. 1 indicates P <0.05 when a significant difference test is performed by Dunnett's test (multiple comparison method for simultaneously testing only the paired comparison between the control group and the treatment group with respect to the population mean). It shows that there is a significant difference compared to the physiologic saline group. The symbol ** indicates that there is a significant difference when compared with the phosphate buffered saline group at P <0.01 when a significant difference test is performed by Dunnett test.
As is apparent from the results shown in FIG. 1, when MTII was administered, the amount of food intake was significantly suppressed within 1 hour of administration, and the effect gradually decreased after 5 hours. When 3 nmol or 10 nmol of NPX was administered, the effect of suppressing food intake was not seen until 2 hours after administration, and the food intake was significantly suppressed after 5 hours, and the effect was also observed after 12 hours. It can be seen that is sustained.

  The peptide NPX of the present invention is useful as a prophylactic / therapeutic agent for obesity or bulimia nervosa. In particular, since the effect is exhibited after a lapse of a certain time, it is possible to maintain the effect for a long time by combining with a known fast-acting obesity or bulimia preventive / therapeutic agent. Furthermore, the compound obtained by the screening method of the present invention is useful as a prophylactic / therapeutic agent for obesity or eating disorders.

Claims (19)

An appetite suppressant containing a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3. A prophylactic or therapeutic agent for obesity or eating disorders containing a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3. An appetite suppressant comprising 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 having an appetite suppressive activity. Obesity or catabolism comprising 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 having an appetite suppressive activity. A preventive or therapeutic agent for eating disorders. An appetite suppressant containing a peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or a DNA that hybridizes under stringent conditions to a complementary sequence thereof and having an appetite suppressive activity. Obesity or food intake comprising a peptide consisting of the nucleotide sequence set forth in SEQ ID NO: 2 or 4 or a DNA that hybridizes under stringent conditions to a complementary sequence thereof and containing a peptide having anorectic activity Preventive or therapeutic agent for disorders. The drug according to any one of claims 1 to 6, wherein appetite is suppressed after a certain period of time. Furthermore, the chemical | medical agent in any one of Claim 1 to 7 which can maintain an appetite suppression effect for a long time by mix | blending a rapid-acting appetite suppressant. A method for suppressing appetite, and a method for preventing or treating obesity or eating disorders, comprising administering an effective amount of any of the following peptides (i) to (iii):
(I) a peptide consisting of the amino acid sequence set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity: Use of any 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 set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity: A method of screening for a substance that suppresses or promotes appetite after a certain time by promoting or inhibiting the activity of the peptide, comprising using any of the following peptides (i) to (iii):
(I) a peptide consisting of the amino acid sequence set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity: Furthermore, the screening method of Claim 11 using the protein in any one of following (iv) to (vi), or the partial peptide of this protein.
(Iv) A protein comprising the amino acid sequence set forth in SEQ ID NO: 5.
(V) consisting of 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: 5, and from (i) to (iii) according to claim 11 ) A protein having binding activity with any of the peptides;
(Vi) encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 6 or DNA that hybridizes to its complementary sequence under stringent conditions, and (i) to (iii) according to claim 11 Protein having binding activity with any peptide: Any one of the following (i) to (iii), characterized by using DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence A method of screening for a substance that suppresses or promotes appetite after a lapse of time by promoting or inhibiting the expression of the peptide.
(I) a peptide consisting of the amino acid sequence set forth in 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 having anorectic activity; or (iii) Peptide encoded by DNA consisting of the base sequence set forth in SEQ ID NO: 2 or 4 or DNA that hybridizes under stringent conditions to its complementary sequence and having anorectic activity: An appetite suppressant containing a substance selected by the screening method according to claim 11. A prophylactic or therapeutic agent for obesity or eating disorders, comprising a substance selected by the screening method according to claim 11. An appetite suppression method, and a method for preventing or treating obesity or eating disorders, comprising administering an effective amount of a substance selected by the screening method according to claim 11. 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 eating disorders. An appetite suppressant comprising a receptor agonist for a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 3. An appetite promoter containing an antagonist of a receptor for a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3.

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