JP5054075B2 - Undernutrition Symptom Treatment Agent - Google Patents

Description

  The present invention relates to a novel therapeutic agent for malnutrition symptoms. More specifically, the present invention relates to a therapeutic agent for diseases exhibiting undernutrition including ghrelin or a ghrelin analog as an active ingredient. The present invention also relates to a novel dysphagia or metabolic disorder therapeutic agent using a ghrelin agonist or antagonist.

  In weight control, the balance between food intake and energy consumption is the key, and the balance between the two causes obesity and skinnyness. Since the discovery of leptin, discovered in 1994, as adiposity (body fat mass accumulation) signal, it has been found that many new peptides related to appetite regulation are located downstream of leptin. It was. In particular, the hypothalamus-derived neuropeptide groups that had previously been considered as independent functions function individually downstream of leptin, and information exchange is also carried out between these neuropeptide groups. I know that

  Among these neuropeptides, neuropeptide Y (NPY), orexins (orexins), motilin, melanin-concentrating hormone (MCH) and agouti-related protein (agouti) -related protein (AGRP) is known. In addition, as substances that suppress appetite, α-melanocyte-stimulating hormone (α-MSH), corticotropin-releasing factor (CRF), cocaine- and amphetamine-controlled transcription The thing (cocain-and amphetamine-regulated transcript: CART), the cholecystokinin (cholesystokinin: CCK), etc. are known. These peptides are involved in physiological mechanisms that control gastrointestinal motility and are thought to affect energy homeostasis.

  In particular, NPY is a neurotransmitter consisting of 36 amino acids and is abundantly expressed in the hypothalamus, which is the feeding center. NPY is produced in the hypothalamic arcuate nucleus (ARC) and is secreted mainly through the axon to the paraventricular nucleus (PVN), affecting feeding. If NPY is administered centrally, it exerts a strong food-enhancing effect (Schwartz, MW et al., Am. J. Clin. Nutr., 69: 584, 1999). Conversely, it showed a tendency to suppress. This phenomenon is similarly seen with other PP family peptides. Various physiological actions caused by NPY are performed through NPY receptors. Currently, five subtypes (Y1, Y2, Y4, Y5, y6) of the NPY receptor have been cloned, and the basic structure is a seven-transmembrane G protein-coupled receptor. Y5 receptor has been reported as a receptor closely related to feeding regulation from the analysis of ligand binding specificity and feeding promotion activity, and analysis including antagonist administration experiments (Inui A ., Trends Pharmacol Sic 20: 43-46, 1999).

On the other hand, growth hormone (GH) is a hormone secreted from the anterior pituitary gland, and its secretion is skillfully controlled, stimulated by growth hormone releasing hormone (GHRH) in the hypothalamus and suppressed by somatostatin. . In recent years, a mechanism for regulating GH secretion by a different route from GHRH and somatostatin has been clarified. This alternative pathway for regulating GH secretion has been developed through research on growth hormone secretagogue (GHS), which is a synthetic compound having GH secretion promoting activity. GHS acts by a different pathway than GHRH. That is, GHRH activates the GHRH receptor to increase intracellular cAMP concentration, whereas GHS activates a receptor different from the GHRH receptor to induce intracellular Ca ⁺ via the intracellular IP3 system. ⁺ Increase ion concentration. The structure of GHS-R, which is a receptor on which GHS acts, was elucidated in 1996 by the expression cloning method (Howard AD et al, Science, 273: 974-977, 1996). GHS-R is a typical G protein-coupled receptor that penetrates the cell membrane seven times, and is mainly present in the hypothalamus and pituitary gland.

  Furthermore, since there exists a receptor that binds GHS, which is a synthetic compound that does not exist in the living body, an endogenous ligand that binds to and activates this GHS-R was searched. As a result, ghrelin was purified and identified from the stomach of rats as a specific ligand for GHS-R (Kojima M. et al., Nature, 402: 656-660, 1999).

  Ghrelin is a peptide consisting of 28 amino acid residues, and the third serine residue is n-octanoylated. Human ghrelin differs from rat ghrelin in two amino acid residues. The structural formulas of rat and human ghrelin are shown below.

Chemically synthesized ghrelin has nanomolar order and has an intracellular Ca ⁺⁺ increasing activity of CHO cells in which GHS-R is expressed, and a growth hormone releasing activity in primary cultured pituitary cells. Furthermore, blood growth hormone is also increased in rats in vivo. Ghrelin mRNA is prominently expressed in the stomach, and ghrelin is also present in the blood. Furthermore, GHS-R is also present in the hypothalamus, heart, lung, pancreas, small intestine and adipose tissue (Kojima et al.). In addition, ghrelin has been reported to have an effect of promoting feeding (Wren et al., Endocrinology, 141 (11): 4325-4328, 2000). These findings suggest that ghrelin is produced in the stomach and is transported to the pituitary gland through the blood and then exerts various actions in the brain and periphery, but its physiological role is still sufficient. It has not been elucidated.

  An object of the present invention is to elucidate the action and mechanism of ghrelin in appetite regulation, and to develop a novel therapeutic agent for malnutrition symptoms using the same. It is another object of the present invention to develop a novel dysphagia or metabolic disorder therapeutic agent using a ghrelin agonist or antagonist.

As a result of diligent research to achieve the above object, the present inventors have found that ghrelin exhibits a remarkable appetite promoting action via NPY and Y1 receptors, and have completed the present invention.
That is, this invention provides the therapeutic agent of the disease which shows the malnutrition symptom which contains ghrelin as an active ingredient.

  The present invention further comprises administering a candidate substance to an animal in the presence or absence of ghrelin, and taking food, NPY mRNA expression, NPY binding to NPY Y1 receptor, oxygen consumption, gastric emptying A method of screening for an agonist or antagonist of ghrelin, comprising measuring speed or vagal activity.

The present invention further provides a therapeutic agent for anorexia or weight loss comprising an agonist of ghrelin obtained by the above method as an active ingredient.
The present invention further provides an agent for preventing or treating obesity comprising an ghrelin antagonist obtained by the above method as an active ingredient.

In FIG. 1, A shows the amino acid sequences of human ghrelin and human motilin. B shows the amino acid sequences of human ghrelin receptor and human motilin receptor. The same amino acid is indicated with an asterisk. FIG. 2 shows the effect of ghrelin ICV administration on feeding. FIG. 3 shows the effect of mouse ICV administration of ghrelin compared to NPY, AGRP, orexin A, orexin B and MCH. FIG. 4 shows the expression of the NPY gene in the hypothalamus after ghrelin ICV administration. The upper panel shows a Northern blot of hypothalamic NPY mRNA after administration of ghrelin ICV. The lower graph shows Northern blot data normalized to G3PDH mRNA and expressed as a percentage of the control group. FIG. 5 shows the effect of pretreatment with NPY Y1 receptor antagonist (BIBO3304) and Y5 receptor antagonist (L152804) on ghrelin-induced feeding. FIG. 6 shows the effect of ghrelin ICV administration on oxygen consumption. FIG. 7 shows the effect of ghrelin IP administration on feeding. FIG. 8 shows the effect of ghrelin IP administration on hypothalamic NPY mRNA expression. FIG. 9 shows the effect of ghrelin IP administration on gastric emptying rate. FIG. 10 shows the effect of vagus nerve cutting on the feeding promoting effect of ghrelin. FIG. 11 shows the effect of vagus nerve cutting on the afferent nerve activity of the gastric vagus nerve upon administration of ghrelin. FIG. 12 shows the results of northern blot analysis of ghrelin mRNA expression in lean mice fasted for 48 hours. FIG. 13 shows the results of examination of ghrelin mRNA expression in the stomach by Northern blotting when IL-1β and leptin were administered IP. FIG. 14 shows the results of examination of ghrelin mRNA expression in the stomach of ob / ob obese mice by Northern blot. FIG. 15 shows the results of Northern blotting of ghrelin mRNA expression in the stomach when leptin was repeatedly administered to ob / ob mice. FIG. 16 shows the effect on food intake and body weight when co-administered ghrelin and IL-1β to fasted lean mice. FIG. 17 shows the effects of repeated administration of ghrelin on IL-1β-induced food intake and weight loss. FIG. 18 shows the effect of ghrelin on weight gain in LC-6 transplanted HHM / cahexia model mice. FIG. 19 shows the effect of ghrelin on fat increase in LC-6 transplanted HHM / cahexia model mice.

  Ghrelin used as an active ingredient in the present invention is rat ghrelin or human ghrelin or a ghrelin analog represented by Formula 1.

  Ghrelin analogs include those in which one or more of 28 amino acids are deleted, substituted or added as long as they have an appetite-promoting action, and these various derivatives, for example, peptide-constituting amino acids Substituted derivatives (including those in which a group such as alkylene is inserted between amino acids) and ester derivatives are also included.

  Ghrelin or ghrelin analogs may be produced by any method, including, for example, those isolated and purified from human and rat cells, synthetic products, semi-synthetic products, those obtained by genetic engineering techniques, etc. There is no limit.

  A representative example of one in which one or more of the 28 amino acids are deleted, substituted or added is des-Gln14-ghrelin lacking the 14th Gln residue of ghrelin. Rat des-Gln14-ghrelin is caused by a difference in splicing of the ghrelin gene, and is present in the rat stomach in about one-fourth of ghrelin, and the strength of growth hormone releasing activity is the same as ghrelin.

  Furthermore, J. Med. Chem. 2000, 43, 4370-4376 describes the minimal sequence of ghrelin necessary for the activation of human GHSR1a. Included in ghrelin analogs. For example, among 28 amino acids of ghrelin, the amino acid has 3rd and 4th amino acids from the N-terminus (preferably 4 amino acids at the N-terminus), and the side chain of the 3rd amino acid (Ser) from the N-terminus is substituted. Peptides and derivatives thereof, which have an appetite promoting action.

Examples of the side chain of the third amino acid from the N-terminus include acyl groups other than n-octanoyl, which is the side chain of ghrelin, and substituted alkyl groups (these carbon numbers are preferably 6 to 18). Side chains include the following:
_{-CO- (CH 2) 6 CH 3} , -CO (CH 2) 9 CH 3, -CO (CH 2) 14 CH 3, -CO-CH = CH-CH = CH-CH = CH-CH 3, - _{CO-CH (CH 2 CH 2} CH 3) 2, -CO- (CH 2) 6 CH 2 Br, -CO- (CH 2) 2 CONH (CH 2) 2 CH 3, -CH 2 -NH-CO ( _{CH 2) 8 CH 3, -CH} 2 -O-CO (CH 2) 8 CH 3, -CH 2 -NH-CO (CH 2) 6 CH 3,

As a specific example of a ghrelin analog having the 3rd and 4th amino acids from the N-terminus and the side chain of the 3rd amino acid (Ser) from the N-terminus being substituted, the 37th Peptide Discussion Session ( Compounds reported on October 18-20, 2000):
_{NH 2 - (CH 2) 4} -CO-Ser ( octyl) -Phe-Leu-NH- (CH 2) 2 -NH 2 and the like.

Considering the action mechanism of ghrelin described below, which has been clarified by the present invention, these ghrelin analogs can also be expected to have an appetite promoting activity.
In the therapeutic agent for diseases exhibiting undernutrition symptoms of the present invention, ghrelin or a ghrelin analog may be used in combination of two or more.

  The therapeutic agent for diseases exhibiting undernutrition symptoms of the present invention can be administered centrally (for example, intraventricular administration, spinal cavity injection) or peripheral administration. Preferably, it is used for peripheral administration. As described above, NPY and other PP family peptides are not related to feeding when administered peripherally, but the therapeutic agent of the present invention showed a significant appetite enhancing effect even when administered peripherally. Therefore, the therapeutic agent of the present invention is less painful for patients associated with administration and can be easily taken, and has much greater advantages than conventional appetite regulating peptides.

  Ghrelin or a ghrelin analog can be made into a normal preparation for oral administration and a preparation for parenteral administration by known formulation techniques, alone or together with pharmacologically acceptable carriers, additives and the like. For example, it is formulated into solution preparations (injections such as arterial, intravenous or subcutaneous injections, nasal drops, syrups, etc.), tablets, troches, capsules, powders, granules, ointments, suppositories, etc. be able to. It can also be used in drug delivery systems (such as sustained release agents).

  The dosage of the therapeutic agent for a disease exhibiting an undernutrition symptom of the present invention varies depending on the age, weight, symptom, route of administration, etc. of the patient and is determined by the judgment of a doctor. Usually, for intravenous administration, ghrelin is about 0.1 μg to 1000 mg per kg body weight, preferably about 0.01 mg to 100 mg, more preferably 0.1 mg to 10 mg. However, the dose is not limited to this.

  The therapeutic agent of the present invention can be used for the treatment of diseases exhibiting malnutrition symptoms, and in particular, diseases selected from anorexia, cachexia or malignant diseases, infectious diseases and debilitated states due to concomitant weight loss due to inflammatory diseases It is effective for. In particular, it is useful as a therapeutic agent for anorexia or weight loss associated with cachexia. Cachexia refers to poor general condition mainly caused by gradual weight loss, anemia, skin dryness or edema, loss of appetite, and the end stage of numerous diseases such as infections, parasitic diseases, and malignant tumors. As a symptom. In the present specification, terms such as increased appetite, increased feeding, and accelerated feeding are used interchangeably as terms having the same meaning.

  The present invention further comprises administering a candidate substance to an animal in the presence or absence of ghrelin, and taking food, NPY mRNA expression, NPY binding to NPY Y1 receptor, oxygen consumption, gastric emptying Provided is a screening method for an agonist or antagonist of ghrelin or a ghrelin analog, which comprises measuring speed or vagal activity. As a specific measurement method, for example, the method described in this specification can be used, but is not limited thereto.

An agonist of ghrelin or a ghrelin analog obtained by the above screening method can be used as an active ingredient of the therapeutic agent for anorexia or weight loss of the present invention.
Moreover, the antagonist of ghrelin or a ghrelin analog obtained by the above screening method can be used as an active ingredient of the obesity preventive or therapeutic agent of the present invention. As shown in the examples below, pretreatment with NPY Y1 receptor antagonist significantly inhibited ghrelin-induced feeding promotion. Therefore, administration of an antagonist of ghrelin or a ghrelin analog can not only treat but also prevent obesity.

  In the present invention, motilin or motilin analog or an agonist thereof can be used as an agonist of ghrelin or ghrelin analog, and an antagonist of motilin or motilin analog can be used as an antagonist of ghrelin or ghrelin analog.

  Motilin is a 22 amino acid residue peptide secreted from the endocrine cells of the duodenum and upper jejunum (Itoh, Z., Peptides, 18: 593-608, 1997), the digestive tract interdigestive movement, gallbladder contraction And is involved in the secretion of enzymes from the stomach and pancreas. Motilin has been reported to promote GH secretion and non-peptide motilin agonists have been reported to promote gastric motility (supra, Itoh). As shown in FIG. 1A, human ghrelin and human motilin show 36% amino acid identity with each other (access numbers A59316 and P12872). Furthermore, as shown in FIG. 1B, the human ghrelin receptor exhibits 50% amino acid identity overall with the human motilin receptor (access numbers Q92847, Q92848 and Q43193). Recently, Tomasetto et al. Also isolated a novel peptide from the mouse stomach, which is identical to ghrelin and named a motilin-related peptide (Tomasetto C. et al., Gastroenterology, 119: 395- 405, 2000). In view of the sequence homology between ghrelin and motilin and the sequence homology between ghrelin receptor and motilin receptor, motilin or motilin analogs or agonists thereof can be used as agonists of ghrelin or ghrelin analogs. As an antagonist of a ghrelin analog, motilin or an antagonist of a motilin analog can be used.

  The mechanisms that control feeding and energy balance are complex and not yet fully understood. To date, NPY, AGRP, orexins, MCH, beacon, melanocyte stimulating hormone (MSH), neuromedin U, cocaine- and amphetamine-regulated transcripts (CART), corticotropin releasing factor (CRF) Many peptides, including leptin, have been shown to affect energy homeostasis. As described above, NPY, which is a peptide consisting of 36 amino acids, is one of the key components in the feeding stimulation system and body weight control. In previous studies, centrally administered NPY stimulated feeding in rodents and reduced metabolic rate (Bray G.A. et al., Recent Prog Horm Res, 53: 95-118, 1998). According to the pharmacological characterization of receptors using NPY analogs and specific antagonists, both Y1 and Y5 receptors are considered to be NPY feeding receptors.

  In the present invention, ghrelin administered with ICV, like NPY, significantly stimulated feeding and reduced oxygen consumption, both of which were blocked with Y1 receptor antagonists. The presence of small amounts of ghrelin in the brain has been suggested by amplification by reverse transcription polymerase chain reaction (RT-PCR) and immunohistological analysis. In previous reports, GHS-R is localized in the arcuate nucleus (ARC) and NPY is synthesized in the arcuate nucleus (Tannenbaus GS et al., Endocrinology, 139: 4420-4423, 1998). ). In situ hybridization studies have shown that GHS-R and NPY are colocalized in arcuate nucleus neurons (Guan XM et al., Brain Res MolBrain Res, 48: 23-29, 1997, etc.) ). Furthermore, it is known that a non-peptidic growth hormone release-promoting factor functions in the hypothalamus to change the electrical activity of arcuate nucleus neurons and activate the expression of the transcription factor c-fos (Dickson Sl et al., Neuroendocrinology, 61: 36-43, 1995).

  In the present invention, NPY mRNA expression was significantly increased by central administration of ghrelin. Thus, the mechanism of action of ghrelin that produces a positive energy balance appears to be involved in the hypothalamic NPY and Y1 receptor systems. To date, several peptides have been shown to increase feeding when administered to the brain (Elmquist JK et al., Nat Neurosci, 1: 445-450, 1998, etc.). However, no peptide that shows an appetite-enhancing effect when administered peripherally has been reported so far. In the present invention, it was revealed that peripherally administered ghrelin stimulates feeding through NPY and Y1 receptors. It has been suggested that rapid gastric emptying is closely associated with overeating and obesity, and that delayed gastric emptying is also associated with anorexia and cachexia (Inui A., Cancer Res 59 : 4493-4501, 1999). In the present invention, ghrelin significantly increased gastric emptying rate as did motilin. Previous studies have reported that cholecystokinin (CCK) has a potent antifeedant effect and an inhibitory effect on gastric voiding via afferent activation of the gastric vagus nerve (Schwartz GJ et al , Am J Physiol, 272: R1726-1733, 1997).

  In the present invention, it has been clarified that the anorectic effect of ghrelin is also mediated by the vagus nerve and afferent activity. As is apparent from electrophysiological studies, the effective amount of ghrelin when administered intravenously to rats is lower than the effective amount of CCK. Various anorexigenic molecules, including bombesin, IL-1β, leptin and gastrin releasing peptide (GRP) have been reported to increase the discharge rate of gastric vagal afferents (see above, Schwartz). Therefore, the effect of ghrelin on vagus nerve activity and the effect on feeding are opposite to these feeding-inhibiting molecules, supporting that anorectic activity acts via the vagus nerve.

  Furthermore, in the present invention, it has been shown that the expression of ghrelin mRNA in the stomach is increased by starvation. These results suggest that increased ghrelin mRNA in the starved state is at least partially responsible for activation of the hypothalamic NPY, resulting in feeding. If so, the stomach is not only a source of leptin, a satiety signal from the periphery to the hypothalamus, but also a source of ghrelin, a feeding stimulating signal. In anorexia and cachexia, cytokines such as IL-1, IL-6 and tumor necrosis factor have an important effect on energy balance (Inui A., Cancer Res 59: 4493-4501, 1999, etc.). In addition, cachexia is known to cause a poor general condition with weight loss as the main symptom. Several hormones known to suppress feeding, including leptin, CRF, CCK and insulin are induced by cytokines. In the present invention, ghrelin mRNA expression in the stomach is decreased by both IL-1β and leptin, and is increased in ob / ob mice (mice that have lost leptin and therefore suffered from overeating and thus become obese). Showed that. Repeated administration of leptin decreased not only energy uptake but also ghrelin mRNA expression. Thus, ghrelin gene expression in the stomach is associated with appetite control and nectar, and plays a role in either the adaptive response to starvation or the development of obesity. Furthermore, peripherally administered ghrelin reversed the loss of appetite and weight loss induced by IL-1β and improved cachexia. It is known that ghrelin potently stimulates growth hormone release from the pituitary gland (supra, Kojima et al.). Considering these findings together with the findings obtained in the present invention, it is possible to stimulate weight gain only with ghrelin administered with IP, and this peptide contributes to the control of body growth and the mass of adipose tissue. There is a possibility. While the inventor is not bound by any particular theory, ghrelin controls body weight in the long term rather than short-term diet-related orexigen (the counterpart of CCK and other diet-related satiety factors). It may be a factor that acts as a leptin counterpart. To date, growth hormone has been used as a potential anabolic agent to treat muscle stress associated with surgical stress, sepsis, glucocorticoid administration, HIV infection and cancer. Growth hormone stimulates systemic and muscle protein synthesis, at least under certain conditions. Instead, ghrelin is effective in treating elderly people with reduced growth hormone secretion, reduced muscle mass, and often anorexia.

  The therapeutic agent for diseases exhibiting malnutrition according to the present invention comprising ghrelin or a ghrelin analog as an active ingredient has a positive energy balance state by promoting feeding, reducing energy consumption, and stimulating growth hormone secretion. And induce weight gain.

  The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto. Various changes and modifications can be made by those skilled in the art based on the description of the present invention, and these changes and modifications are also included in the present invention.

Test Material and Method (1) Animal Experiment Seven-week-old ddy male mice (32-35 g) were purchased from JAPAN SLC (Shizuoka, Japan). 10-11 week old obese (ob / ob) C57BL / 6J mice (38-42 g) were purchased from Shionogi Co., Ltd. (Shiga, Japan). These were reared in an individually controlled environment (temperature 22 ± 2 ° C., humidity 55 ± 10%, light cycle started at 7 am with a light / dark cycle every 12 hours). Food and water were given ad libitum unless otherwise noted. Mice were used only once in each experiment. Rat ghrelin, rat NPY, human agouti-related protein 86-132 (AGRP), mouse orexin A, mouse orexin B and mouse melanin-concentrating hormone (MCH) were purchased from Peptide Institute (Osaka, Japan). Recombinant mouse leptin and recombinant mouse IL-1β were purchased from R & D Systems (Minneapolis, USA) and Upstate Biotechnology (New York, USA), respectively. BIBO3304 was a gift from Boeringer-Ingelheim Pharma, Germany) and L152804 and J115814 from Banyu (Banyu Pharmaceutical Co., Ltd., Tokyo, Japan). BIBO3304 and J115814 are NPY Y1 receptor antagonists, and L152804 is a Y5 receptor antagonist. Immediately prior to administration, each drug is diluted with 4 μl of artificial cerebrospinal fluid (ACSF) and administered to the intra-third core broventricular (ICV), or diluted intraperitoneally (IP) with 100 μl of physiological saline. did. Control groups received only ACSF or saline. Each antagonist was administered concurrently with ghrelin. The results are shown as mean ± SE. Analysis of variance (ANOVA) was performed, and differences between groups were determined by Bonferroni t-test. There was a statistically significant difference when P <0.05.
(2) ICV administration In order to administer ICV, mice were anesthetized with sodium pentobarbital (80-85 mg / kg IP), and within the stereotaxic apparatus (SR-6, Narishige, Tokyo, Japan) for 7 days before the experiment. Scented. Each skull was drilled with a needle 0.9mm lateral to the center suture and 0.9mm posterior to the anterior apex. A 24-gauge cannula (Safelet-Cas, Nipro, Osaka, Japan) with one end inclined at a length of 3 mm was implanted in the third ventricle for ICV administration. The cannula was fixed to the skull with dental cement and covered with silicone. A 27 gauge injection insert was attached to the microsyringe with PE-20 tubing. This was inserted into the fixed cannula with tweezers without restraining the mouse or greatly restricting its behavior. At the end of the experiment, dye (Evans blue 0.5% and gelatin 5%) was injected to confirm the position of the cannula end and subjected to histological experiments on frozen brain sections.
(3) Truncal vagotomy
Four days before the experiment, a vagus nerve amputation was performed as follows. Mice were anesthetized with sodium pentobarbital (80-85 mg / kg IP). An incision was made in the midline of the abdominal wall and the stomach was covered with sterile gauze moistened with warm saline. The lower esophagus was exposed and the anterior and posterior branches of the vagus nerve were cut. At the end of the surgery, the abdominal wall was double sutured. Sham (false) surgical mice also exposed the vagus nerve trunk, but did not cut it. Mice from which the vagus nerve had been cut and mice that had undergone siamese surgery were bred in a complete nutrient liquid diet (Oriental Yeast Co., Ltd. Tokyo, Japan).
(4) Feeding test The test started at 10:00. Prior to the feeding test, mice were allowed free access to food and water. However, in a study examining the effect of IP co-administration of ghrelin and IL-1β on feeding, mice were not allowed to feed for 16 hours but were allowed to drink water only freely. The amount of food intake was determined by subtracting the remaining food amount from the food amount measured and given 20 minutes, 1 hour, 2 hours and 4 hours after ICV or IP administration. In lean mice that were not dietary restricted, ghrelin (3 nmol / mouse), IL-1β (5 picomoles / mouse) or saline was administered IP repeatedly for 5 days. Mice were injected daily at 7 and 19:00. The amount of food intake and body weight was measured every day, and the condition of fur was observed.
(5) RNA isolation and Northern blot analysis RNA was isolated from the hypothalamic block and stomach using RNeasy Mini Kit (Qlagen, Tokyo, Japan). Total RNA was denatured with formaldehyde, electrophoresed on a 1% agarose gel, and blotted onto Hybond N ⁺ membrane (Amersham Pharmacia Biotech AB, Uppsala, Sweden) (Ueno N. et al., Gastroenterology, 117: 1427-1432, 1999). The membrane was hybridized with ³² P-labeled cDNA probe to measure NPY mRNA in the hypothalamus, and hybridized with digoxigenin-labeled cDNA probe to measure ghrelin mRNA in the stomach. The total amount of hybridized signal was measured by densitometry (Image Master 1D Elite ver 3.0, Amersham Pharmacia Biotech AB, Uppsala, Sweden). Data were normalized to glyceraldenide 3-phosphate dehydrogenase (G3PDH) mRNA and expressed as a percentage of the control group.
(6) NPY gene expression Mice were fasted for 24 hours. During the fasting period, mice scheduled to receive ICV received ghrelin (1 nmole / mouse) or ACSF every 12 hours, and mice were killed by cervical dislocation 30 minutes after the third final dose. Mice scheduled for IP administration were administered ghrelin (3 nmol / mouse) or saline every 8 hours, and mice were killed by cervical dislocation 30 minutes after the fourth final dose. The brain block was immediately excised, frozen on dry ice, and stored at −80 ° C. until a Northern blot was prepared.
(7) Ghrelin gene expression Lean mice were fasted for 48 hours. During the fasting period, fasted mice were given IP administration of IL-1β (5 picomoles / mouse), leptin (3 nanomoles / mouse) or saline every 12 hours, and the mice were cervical 30 minutes after the fifth final administration. Killed by dislocation. Ob / ob mice that were not fasted were repeatedly administered leptin (3 nmol / mouse) or saline for 7 days. Mice were injected daily at 7 and 19:00 and mice were killed by cervical dislocation 30 minutes after the last dose. The stomach was immediately excised and frozen on dry ice and stored at -80 ° C. until Northern blots were prepared.
(8) Oxygen consumption Oxygen consumption was measured at 22 ° C. using an O ₂ / CO ₂ metabolism measurement system (Model MK-5000, Muromachikikai, Tokyo, Japan) (supra, Ueno N. et al.). The chamber volume was 560 ml and the air flow rate into the chamber was 500 ml / min. Samples were removed every 3 minutes and standard gas samples were removed every 30 minutes. Mice were placed in the light cycle without food, water, and unrestrained in the chamber, and ghrelin (0.3-1 nmol at 10 o'clock) in the presence or absence of BIBO3304 (5 nmol / mouse). / Mouse) was administered ICV, and the oxygen consumption after 2 hours was measured.
(9) Gastric emptying rate Before the gastric emptying rate measurement test, the mice were fasted for 16 hours and allowed to drink water freely. Fasted mice were allowed to eat a pre-weighed diet pellet for 1 hour, after which ghrelin was administered. Mice were fasted again 1 or 2 hours after administration. Eating weight was determined by weighing the remaining pellets. Mice were killed by cervical dislocation 2 or 3 hours after the start of the test. Immediately, the stomach was exposed by laparotomy, the pylorus and cardia were quickly ligated and removed, and the dry content weight was measured. The contents were dried with a vacuum lyophilization system (Model 77400, Labconco, Kansas, USA). The gastric emptying rate was calculated by the following formula:
Gastric emptying rate (%) = {1− (dry weight of food recovered from stomach / feeding weight)} × 100
(10) Electrophysiological study Male Wistar rats (300 g) were anesthetized with urethane (1 g / kg IP) and a tracheal cannula was inserted. Under a dissecting microscope, neurofilaments were cut from the distal cut end of the gastric branch of the vagus nerve, and afferent nerve activity was recorded with a pair of silver wire electrodes. A rate meter (5 second rest period) was used to observe the time course of neural activity (Niijima A., J. Nutr 130: 971S-973S, 2000). Ghrelin (3-300 femtomole / rat) administration was via a small catheter inserted into the inferior vena cava (IV). The effect of ghrelin on vagal activity was measured by comparing the average number of impulses every 5 seconds over 50 seconds before and after injection. Results were expressed as mean ± SE. ANOVA and Scheffe tests were performed to evaluate differences between groups. The case of P <0.05 was determined to be statistically significant.
Example 1 Effect of Ghrelin ICV Administration on Feeding In this example, the effect of ghrelin administration of mouse ventricle (ICV) was tested. Lean mice that were not fasted received ICV administration of ACSF (control) or ghrelin (0.003-1 nmol / mouse). Food intake was tested 20 minutes, 1 hour, 2 hours and 4 hours after drug administration. The obtained results are shown in FIG. Results are expressed as mean ± SE, and n represents the number of animals. ^* P <0.05 and ^** P <0.01 are significant differences compared to the control group by Bonferroni t-test. Ghrelin significantly and significantly increased food intake in a dose-dependent manner. At 24 hours after ICV administration, mice receiving 1 nanomole ghrelin also increased cumulative food intake, which was not statistically significant (6.31 ± 0.10 g vs. 5.68 ± 0. 21 g (control group): P <0.076).

Example 2 Effect of Ghrelin ICV Administration Compared to Other Peptides The effect of ghrelin mouse ICV administration compared to NPY, AGRP, Orexin A, Orexin B and MCH was as in Example 1 at 1 nmole / mouse. Tested. The results are shown in FIG. The ability to increase food intake at 4 hours was in the order of NPY>ghrelin>AGRP> orexin A> orexin B> MCH. Thus, ghrelin was more potent than all appetite peptides except NPY.

Example 3: Effect of ghrelin ICV administration on NPY gene expression In order to examine the possibility that ghrelin acts via the NPY pathway, NPY gene expression in the hypothalamus after administration of ghrelin ICV was described above. It was investigated by. The obtained results are shown in FIG. The upper panel shows a Northern blot of hypothalamic NPY mRNA after administration of ghrelin ICV. The lower graph shows the northern blot data normalized to glyceraldenide 3-phosphate dehydrogenase (G3PDH) mRNA and expressed as a percentage of the control group. Ghrelin increased NPY mRNA expression by 58%.

Example 4 Effect of Pretreatment with NPY Receptor Antagonist on Ghrelin-Induced Increase in Feeding Pretreatment with NPY Y1 receptor antagonists (BIBO3304 and J115814) and Y5 receptor antagonist (L152804) It was tested whether it had an effect on induced feeding. The Y1 receptor antagonists BIBO3304 and J115814 (5 nmol / mouse: ICV administration) significantly inhibited feeding induced by ghrelin (1 nmol / mouse: ICV administration). On the other hand, Y152 receptor antagonist L152804 (5 nmol / mouse: ICV administration) did not show this effect (FIG. 5). Thus, ghrelin was thought to act via the NPY Y1 receptor.

Example 5: Effect of ghrelin on oxygen consumption by ICV administration The effect of ghrelin on oxygen consumption by ICV administration was tested using the method described above. The obtained result is shown in FIG. Ghrelin decreased oxygen consumption at the same dose that promoted feeding (0.3-1 nmol / mouse: ICV administration), but this was a Y1 receptor antagonist (BIBO3304: 5 nmol / mouse: ICV administration). It was inhibited by pretreatment. In addition, ghrelin increases respiratory quotient (RQ) in mice given 1 nanomole ghrelin 1 hour (7.24 ± 6.96%) and 2 hours (5.18 ± 6.01%) after ICV administration However, it did not reach statistical significance.

Example 6: Effect of ghrelin IP administration on feeding and NPY mRNA expression It was tested in lean mice that were not fasted to see if IP-administered ghrelin showed similar feeding enhancement effects. Ghrelin IP administration significantly increased food intake at 3 nmol / mouse (FIG. 7). Accumulated food intake 24 hours after IP administration of 3 nmol / mouse ghrelin was even significantly higher (6.68 ± 0.16 g vs. 6.10 ± 0.17 g (control): P <0.05). This anorectic activity was blocked by ICV administration of the Y1 receptor antagonist BIBO3304, but not with the Y5 receptor antagonist L152804. When the mRNA expression of hypothalamic NPY after IP administration of ghrelin was examined by Northern blot, a 12% increase in expression was observed (FIG. 8: Saline in FIG. 8 was administered with saline as a control group) Group results are shown).

Example 7: Effect of ghrelin IP administration on gastric emptying rate Whether ghrelin increased gastric emptying rate was tested using the method described above. FIG. 9 shows the gastric emptying rate after 1 hour and 2 hours when ghrelin (0.3-1 nmol / mouse) was administered by ICV. With either ICV or IP administration, ghrelin significantly increased gastric emptying rate 1 hour after administration (30.16 ± 3.70% (3 nmoles) vs. 20.34 ± 2.27% (control): P <0.05). Therefore, ghrelin enhances gastric motility as does motilin.

Example 8: Effect of vagus nerve amputation on feeding promotion effect of ghrelin, NPY mRNA expression and afferent nerve activity of gastric vagus nerve Whether or not the feeding promotion effect of ghrelin is related to the route through the vagus nerve is described above. The mice were subjected to vagus nerve amputation by the method described above. The vagus nerve amputation is an invasive operation, but this operation eliminates the feeding promotion effect induced by IP administration of ghrelin (FIG. 10: In FIG. 10, Sham is the sham operation group, and Vagotomy is the vagus. Shows the group that underwent nerve amputation). The surgery also eliminated a significant increase in hypothalamic NPY mRNA expression induced by IP-treated ghrelin (data not shown). In electrophysiological studies conducted using the above method, ghrelin IV administration significantly reduced afferent nerve activity of the gastric vagus nerve (FIG. 11).

Example 9: Ghrelin mRNA expression in the stomach Ghrelin mRNA expression in the stomach was examined by Northern blot analysis. As shown in FIG. 12, 48 hours fasting significantly (16%) increased ghrelin mRNA compared to control fasting mice (note that in FIG. 12, Fed represents a fasting group of mice. Indicates a fasted group of mice).

  To test whether any catabolic substances had an effect on ghrelin mRNA expression in the stomach, IL-1β and leptin were administered IP. As shown in FIG. 13, both IL-1β and leptin significantly reduced ghrelin mRNA expression in the stomach (23 ± 2.8% for IL-1β and 22 ± 3.5% for leptin).

  In addition, Northern blot analysis in the stomach of ob / ob obese mice was performed. Compared to lean mice fed an arbitrary amount of diet, ghrelin mRNA expression in ob / ob mice was significantly (19%) elevated (FIG. 14). Leptin significantly decreased ghrelin mRNA expression in both ob / ob and lean mice (17 ± 3.2%: P <0.01). Repeated administration of leptin to ob / ob mice resulted in a significant (31%) decrease in ghrelin mRNA and a simultaneous decrease in food intake and body weight compared to the control group given saline (FIG. 15). Neither the control group nor the ob / ob mice detected ghrelin mRNA expression on the hypothalamic northern blot.

Example 10: Effects on food intake and body weight when ghrelin and IL-1β were co-administered Effects on food intake and body weight when ghrelin and IL-1β were co-administered were examined. As shown in FIG. 16, decreased feeding induced by IL-1β was blocked by ghrelin. Furthermore, repeated administration of ghrelin reversed the loss of food intake and body weight induced by IL-1β (FIG. 17).

  Moreover, in the group administered with IL-1β alone, the fur was disturbed and the deterioration of the general condition was observed, but in the group administered with ghrelin and IL-1β, the fur was improved and ghrelin was associated with cachexia. It was found to improve the general condition.

Example 11: Effect of ghrelin on LC-6 transplanted HHM / cahexia model mice HHM model mice transplanted with human lung cancer cell line LC-6 were used as model animals. This model mouse is known to exhibit weight loss associated with cachexia (WO 98/13388). 6 mice per group were administered ghrelin 3, 0.3, 0 nmol / individual 10 times (twice a day, every 12 hours, for 5 days). Separately, a normal group (n = 5) in which no tumor was transplanted was prepared.

Body weight was measured on days 0-5, and the fat weight around the testis was measured on day 5.
The obtained results are shown in FIGS. In the 3 nmol administration group, an increase in body weight was observed until the third day after the start of administration as compared with the 0 nmol administration group, and it continued until the fifth day. In the 3 nmol administration group, an increase in fat weight was observed compared to the 0 nmol administration group.

Claims (1)

Of the amino acid sequence described in Ghrelin or SEQ ID NO: 1, the amino acid sequence is 1-4th from the N-terminal, and the side chain of the third amino acid from the N-terminal is any of the following acyl groups and substituted alkyl groups _{: -CO- (CH 2) 6 CH} 3, -CO (CH 2) 9 CH 3, -CO (CH 2) 14 CH 3, -CO-CH = CH-CH = CH-CH = CH-CH 3, _{-CO-CH (CH 2 CH 2} CH 3) 2, -CO- (CH 2) 6 CH 2 Br, -CO- (CH 2) 2 CONH (CH 2) 2 CH 3, -CH 2 -NH-CO _{(CH 2) 8 CH 3,} -CH 2 -O-CO (CH 2) 8 CH 3, -CH 2 -NH-CO (CH 2) 6 CH 3,

In either substituted, or _{NH 2 - (CH 2) 4} -CO-Ser ( octyl) -Phe-Leu-NH- (CH 2) in the presence or absence of a ghrelin analog is ₂ -NH ₂ , The candidate substance is administered to a model animal exhibiting a state of weakness due to cachexia or concomitant weight loss due to malignant disease , food intake, NPY mRNA expression, NPY binding to NPY Y1 receptor, oxygen consumption, A method for screening a therapeutic agent for a disease selected from a debilitating state caused by accompanying weight loss due to cachexia or malignant disease , which comprises measuring gastric emptying rate or vagus nerve activity.