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    • 专利摘要:
    It is an object of the present invention to provide an insulin resistance and / or an obesity prevention agent or an improvement agent and a selection method thereof based on a new concept. We found that GIP causes insulin resistance and obesity as a new mechanism, and the new concept that a compound that inhibits GIP function has an insulin resistance improving action and an anti-obesity action has been accomplished and the present invention has been completed. That is, the present invention provides an insulin resistance and / or an obesity prevention agent or a remedy comprising a compound which inhibits the function of GIP as an active ingredient, as well as a compound which inhibits the function of GIP and / Obesity prevention agent or improvement agent.
    公开号:KR20020093150A
    申请号:KR1020027015204
    申请日:2001-05-15
    公开日:2002-12-13
    发明作者:미야와키카즈마사；야마다유이치로；반노부히로；세이노유타카；츠바모토요시하루；타케다모토히로；하시모토히로유키；야마시타토쿠유키；조모리타카히토
    申请人:가부시키가이샤산와카가쿠켄큐쇼；
    IPC主号:
    专利说明:

    BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to an insulin resistance and /
    [2] GIP is a kind of digestive tract hormone belonging to the glucagon secretin family. GIP is called incretin together with glucagon-like peptide 1 (GLP · 1). It is secreted by K cells present in small intestine when food is ingested. Glucose of pancreatic β cell promotes insulin secretion, In the body. In addition, GIP is known to inhibit gastric motility and stimulate the secretion of gland fluid. However, the inhibitory action of gastric acid secretion at the early stage of discovery is now questionable. The GIP receptor gene has been extensively expressed in pancreatic β cells and adipocytes, and GIP has been known to have action in other tissues, but the details are uncertain. Of course, the association with insulin resistance is unknown.
    [3] Examples of the GIP receptor inhibitor include GIP (6-30) -H 2 (Regulatory Peptide 69, 151-154, 1997) or GIP (7-30) -NH 2 (Am J Physiol 1999, 276 E1049-1054). However, there is no description as an insulin resistance improving agent or an anti-obesity agent at all.
    [4] Insulin resistance is a weak state of absorption promotion of glucose, which is the main action of insulin in skeletal muscle, fat cells and liver. With insulin resistance, even if the amount of insulin in the body is about the same, the hypoglycemic effect is weakened, and more insulin is needed to maintain normal blood sugar. Japanese people with most type 2 diabetes have a high incidence of insulin resistance, so the following course of diabetes develops. Initially, to compensate for insulin resistance, a large amount of insulin is secreted from the islets of Langerhans (a group of cells that secrete insulin in the pancreas) and the blood glucose levels remain normal, but hyperinsulinemia occurs. Soon, the function of the Langerhans islands begins to weaken, making it impossible to maintain a large amount of insulin secretion, insulin resistance can not be supplemented, and blood glucose levels rise. Currently, phyoglitazone (trade name: Actos) is used clinically in Japan as an insulin resistance preventive or remedy. In addition, it is not known that GIP inhibitors lead to the prevention or amelioration of insulin resistance.
    [5] On the other hand, obesity is an increasing lifestyle disease caused by westernization of modern Japanese people, and it causes lifestyle diseases such as fatty liver, diabetes, gout, hypertension and arteriosclerosis. Medically, obesity is recognized as a condition leading to abnormal accumulation of fat as a result of excessive intake of calories due to genetic and environmental factors and is being treated. Treatment of obesity is a combination of diet and exercise therapy, and appetite suppressants are rarely used. Currently, obesity prevention agent or improvement agent that is clinically used in Japan is only Margin stone (sanorex), and studies on β3 adrenergic receptor agonist, central action agonist, digestive absorption inhibitor, lipid synthesis inhibitor, and leptin are being developed.
    [6] Regarding the anti-obesity effect of GIP inhibitors, it is described in WO98 / 24464. The present application only reports that GIP (7-30) -NH 2 is a GIP receptor inhibitor and GIP (7-30) -NH 2 inhibits glucose uptake in the intestinal tract. In this application, the effect of GIP (7-30) -NH 2 as a substantial anti-obesity agent is not suggested, which is an excessive leap to be an anti-obesity agent.
    [7] In addition, there is a report that GIP promotes the uptake of free fatty acids and glucose in adipocytes, but there is no clear relationship with obesity.
    [8] It is an object of the present invention to provide an insulin resistance and / or an obesity prevention agent or an improvement agent based on a new concept, and a selection method thereof.
    [1] The present invention relates to an agent for preventing or ameliorating insulin resistance and / or obesity, and more particularly, to a method for preventing or ameliorating an insulin resistance and / or an obesity prevention agent or an agent for inhibiting the function of GIP (Gastric Inhibitory Polypeptide or Glucose dependent Insulinotropic Polypeptide) On the basis of the technical idea, an insulin resistance and / or an obesity prevention agent or an improvement agent, and a selection method thereof.
    [16] FIG. 1 is a chart showing the result of a high-fat diet loading experiment in which gametophytes were continuously administered GIP or saline and consumed for 4 weeks in a control mode or a high fat diet mode,
    [17] a: a graph showing changes in body weight over 4 weeks,
    [18] b: a graph showing the fasting blood glucose level for 4 weeks,
    [19] c: A graph showing fasting insulin levels after 4 weeks. Also, the symbol * in the figure indicates that the risk rate p is p < 0.05.
    [20] FIG. 2 is a graph showing changes in body weight when mice were fed a control diet or a high fat diet for mice lacking GIP and GIP receptor,
    [21] a: graph showing the change in body weight of wild mice,
    [22] b: graph showing changes in body weight of GIP-receptor-deficient mice. The symbols * and ** in the figure indicate that the risk rate p is p < 0.005 and p < 0.01, respectively.
    [23] FIG. 3 is a graph showing the fasting blood glucose level and fasting insulin level of the wild-type and GIP-receptor-deficient rats fed the control normal diet and high fat diet,
    [24] a: graph showing fasting blood glucose level,
    [25] b: a graph showing fasting insulin levels. Also, symbols * and ** in the figure indicate that the risk rate p is p <0.05 and p <0.01, respectively.
    [26] FIG. 4 is a graph showing the results of oral glucose tolerance test of wild-type mice or GIP-receptor-deficient rats fed with the control normal diet and high fat diet,
    [27] a: a graph showing the blood glucose level transition in the mouse,
    [28] b: graph showing the blood glucose level change of GIP receptor deficient mice,
    [29] c: a graph showing the blood insulin level change in the mouse,
    [30] d: graph showing blood insulin levels in GIP-receptor-deficient mice. Also, symbols * and ** in the figure indicate that the risk rate p is p <0.05 and p <0.01, respectively.
    [31] FIG. 5 is a graph showing the blood lipid markers of the mice fed with the control diet, the high fat diet and the GIP receptor-deficient mice,
    [32] a: a graph showing the total cholesterol value,
    [33] b: graph showing triglyceride value,
    [34] c: graph showing free fatty acid value,
    [35] d: a graph showing the LDL cholesterol value,
    [36] e: a graph showing the HDL cholesterol value.
    [37] FIG. 6 is a photograph showing the results of histological analysis of a rat or a GIP receptor-deficient rat fed with a control diet, a high fat diet,
    [38] a: dissection images showing visceral adipose tissue and subcutaneous adipose tissue,
    [39] b: staining photos of fatty tissue,
    [40] c: Dyeing photograph of liver tissue,
    [41] d: Fatty cell staining of epididymal fat pad.
    [42] 7 is a C57BL / 6j-GIPR + / + / Lep + / +, C57BL / 6j-GIPR + / + / Lep - the three strains rats of the - / - and C57BL / 6j-GIPR - / - / Lep - / As a chart showing the results of an examination of the effect of GIP receptor deficiency in ob / ob mice using,
    [43] a: graph showing weight gain,
    [44] b: graph showing fasting blood glucose level,
    [45] c is a graph showing the blood glucose level transition after glucose load.
    [46] 8 is a graph showing the results of experiments examining the action of 3-bromo-5-methyl-2-phenylpyrazolo [1,5-a] pyrimidin-7-ol (BMPP) ,
    [47] a: A graph showing the inhibitory effect of BMPP on cAMP production when stimulated with 100 pM GIP of CHO cells expressing GIP receptor,
    [48] b: A graph showing the inhibitory effect of BMPP on cAMP production when GLP-1 receptor-expressing CHO cells were stimulated with 100 pM GLP-1,
    [49] c: A graph showing the inhibitory effect of BMPP on the production of cAMP when stimulated with 100 pM glucagon of CHO cells expressing glucagon receptor,
    [50] d: a graph showing the inhibitory effect of BMPP on the production of cAMP when stimulated with 5 mu m phosholin (CHO cells) without receptor gene.
    [9] In the course of studying the function of GI7P, the present inventors found that fasting blood glucose and fasting blood insulin levels were not affected by GIP administration despite the fact that continuous administration of GIP to normal rats and high- The amount of the drug is increased depending on the amount of the drug. In other words, we found that GIP worsened insulin resistance independent of weight gain. In addition, we found that insulin resistance, which occurs in wild type mice, improves fasting glucose, insulin sensitivity, and glucose tolerance in GIP receptor gene deficient rats using GIP receptor gene deficient mice. In addition, ob / ob mice, which are hereditary obesity animals, also showed deficient GIP receptor gene and improved fasting blood glucose level and glucose function and improved insulin resistance.
    [10] From these, it is newly found that GIP is the cause of insulin resistance which has not been known until now, and compounds which inhibit the function of GIP, such as GIP receptor inhibitor or GIP production inhibitor, have the function of preventing or improving insulin resistance Has gained new concept.
    [11] Meanwhile, the present inventors found that obesity occurring in wild rats is inhibited in GIP receptor gene-deficient mice through a high-fat diet experiment using a GIP receptor gene-deficient rat in the process of exploring the function of GIP. In addition, ob / ob mice, which are hereditary obesity animals, could also inhibit obesity by deficient GIP receptor gene. Insulin resistance was also improved in these patients.
    [12] From these results, it is newly found that GIP is a new mechanism that has not been proposed until now and it is known that it is a cause of obesity. Compounds that inhibit the function of GIP such as GIP receptor inhibitor or GIP production inhibitor have anti- The new concept of anti - obesity effect was obtained together with resistance improving action.
    [13] That is, the present invention relates to a pharmaceutical composition for treating insulin resistance and / or obesity, which comprises a compound which inhibits the function of GIP, together with an insulin resistance and / And a method for selecting an improvement agent.
    [14] These pharmacological actions of GIP were not easily predictable, as they were different from the previously known action of GIP on insulin secretion promoting action and gastric acid secretion inhibiting action.
    [15] The present invention provides an entirely novel concept that a compound inhibiting the function of GIP serves as an insulin resistance and / or an obesity prevention or amelioration agent, and it has been found that compounds which inhibit the function of GIP are selected for insulin resistance and / Obesity prevention agent or improvement agent can be obtained.
    [51] The present invention is a novel concept that is completely different from what was previously considered to be a mechanism of action of insulin resistance, obesity prevention or remedy, and is an insulin resistance and / or an obesity prevention agent or remedy which contains a compound that inhibits the function of GIP as an active ingredient.
    [52] In the present invention, it is indicated that a compound inhibiting the function of GIP is a preventive or remedy for insulin resistance, and at the same time, it is a preventive or remedy for obesity. However, it has been experimentally demonstrated that the mechanism of preventing or improving the insulin resistance of a compound that inhibits the function of GIP does not necessarily coincide with the mechanism of preventing or improving the obesity of the compound. That is, it has become clear that the agent for preventing or ameliorating insulin resistance comprising the compound that inhibits the GIP function of the present invention as an active ingredient does not necessarily exhibit the effect of preventing or improving insulin resistance accompanying the prevention or improvement of obesity. Therefore, one function of the agent for preventing or ameliorating insulin resistance comprising an active ingredient of a compound inhibiting the GIP function of the present invention is an agent for preventing or ameliorating insulin resistance against insulin resistance patients without obesity, It is a simultaneous prevention or amelioration of insulin resistance and obesity that improves insulin resistance together with obesity in obese patients.
    [53] Compounds that inhibit the function of GIP are compounds that inhibit the function at the GIP gene or GIP receptor gene level, or at the GIP itself or at the GIP receptor level, for example, GIP receptor inhibitors and GIP production inhibitors. Examples of GIP receptor inhibitors include low molecular weight synthetic compounds and GIP fragmented peptides. Examples of GIP fragmented peptides include GIP (6-30) -NH 2 (Regulatory Peptide, 69, p. 151-154, 1997) and GIP (7-30) -NH 2 E1049 page 54) is known. Other compounds which inhibit the function of GIP include 3-bromo-5-methyl-2-phenylpyrazolo [1,5-a] pyrimidin-7-ol (BMPP) which is assured in the present invention.
    [54] The insulin resistance and / or obesity prevention or ameliorating agent of the present invention may be administered in various dosage and dosage forms depending on the respective compound. If it is necessary to continue administration, orally administrable low molecular weight compounds are preferably orally administered. The dosage can not be quantified uniformly, but it is about 0.1 mg / kg to 10 mg / kg for GIP-fragmented peptides such as GIP (6-30) -NH 2 and GIP (7-30) -NH 2 It is considered to be preferable to administer it by subcutaneous administration as an injection, intramuscular administration, or intravenous administration. In addition, in the case of 3-bromo-5-methyl-2-phenylpyrazolo [1,5-a] pyrimidin-7-ol (BMPP), a dose of about 1 mg / kg to about 100 mg / It is considered necessary to administer it by oral administration as a capsule or by subcutaneous administration, intramuscular administration, or intravenous administration as an injection. In addition, these preparations can use conventional formulation techniques.
    [55] The present invention also provides a method of selecting an insulin resistance and / or an agent for preventing or ameliorating obesity, which comprises selecting a compound that inhibits the function of GIP, and this screening method can also be used for screening insulin resistance and / It is a new concept entirely different from what was thought of as a method.
    [56] Selection of a compound that inhibits the function of GIP (hereinafter, referred to as "GIP function inhibitor") may be performed by, for example, selecting GIP receptor inhibitor or selecting GIP production inhibitor. The compound to be screened is preferably a low molecular weight compound having a molecular weight of 600 or less from the viewpoint of oral administration.
    [57] As a method for selecting a GIP function inhibitor (such as a GIP receptor inhibitor), the following methods can be exemplified.
    [58] 1. A method for screening a drug which has an activity of inhibiting the production of cAMP, which is an intracellular transduction substance of GIP, as an index. According to a conventional method (Diabetes 45: 1701-1705, 1996), human GIP Using receptor expressing cells, cAMP is produced by GIP in the presence of a drug for 30 minutes at 37 DEG C in Crevslingel buffer containing 1 mM isobutylmethylxanthine. Thereafter, cAMP is extracted by 30% trichloroacetic acid, and cAMP is measured by radioimmunoassay. Alternatively, cAMP is measured by SPA (Scintilation Proximity Assay) provided by Amersham Pharmacia Biotech Co.,
    [59] 2. Usdin T.B., a method of screening for agents that inhibit the production inhibitory activity of cAMP, which is a cellular intracellular substance of GIP, as an index. In accordance with the method of the party (Endocrinology 133: 2861-2870, 1993), the GIP cDNA encoding the GIP used in the above 1. was introduced into CHO cells, and the lac Z gene derived from bacteria was added to the cAMP-dependent promoter derived from the VIP gene . The cells were reacted with GIP in Krebslingel buffer containing 1 mM isobutylmethylxanthine at 37 DEG C for 30 minutes in the presence of the drug. Since -Galactosidase accumulates in cells according to cAMP produced by GIP activity, this -Galactosidase activity is used as an index of GIP activity.
    [60] 3. A method for screening a drug using the GIP-binding inhibitory activity of the GIP receptor as an indicator, comprising the steps of culturing the human GIP receptor expressing cells described above in phosphate buffered saline (PBS) containing 5.6 mM glucose, 0.5% bovine serum albumin, (-)). After the drug and 125 I-level GIP were contained in the cells and incubated for 1 hour at 37 ° C, the cells were washed with the above buffer solution, the cells were dissolved with 1M sodium hydroxide, The radioactivity in the genus is measured by gamma-counter.
    [61] 4. It is a method of selecting drugs based on the activity of inhibiting the fatty acid absorption of adipocytes by GIP as an index and is performed according to Beck's method (Cellular and Molecular Biololgy 33 (5), 555-562, 1987). That is, 3T3-L1 cells were differentiated into adipocytes using insulin, dexamethasone, and isobutylmethylxanthine, and then cultured in Krebslingel gelatine buffer solution (pH 7.4) containing 2% serum albumin and 0.1% 0.75 mmol / l of tritium label and palmitic acid and a medicament were added. After incubation at 37 ° C for 1 hour, the mixture was extracted with a heptane potassium hydroxide solution to investigate the inhibition of absorption of palmitic acid by a drug in the radioactivity of palmitic acid present in the heptane layer.
    [62] Next, among the GIP function inhibitors, methods for selecting GIP production inhibitors will be described below.
    [63] 5. GIP is stimulated by nutritional factors from the duodenum and the K-cells of the plant and is secreted into the duodenum by the method of Tseng et al. (Proc. Natl. Acad. Sci, 90: 1992-1996, And the expression of GIP mRNA induced by perfusion is inhibited by a drug. Specifically, SD male rats (250-350 g) were fasted overnight, anesthetized, and then lyophilized. The tube was placed in the pyloric portion of the stomach, and 20% intralipose was perfused through the duodenum for 30 to 60 minutes. At the same time, the drug is administered in the jugular vein by ring gel, and then the duodenum is taken out, RNA is extracted according to a conventional method, and GIP mRNA is detected by RNA blot hybridization or reverse transcription PCR using GIP cDNA as a probe. In addition, as in the later 6, the suppression of GIP mediated by the drug may be examined by quantitating GIP in tissues or blood.
    [64] 6. Small intestine-derived cancer cells that mass-produce GIP obtained according to the method of Kieffer et al. (Am. J. Physiol 269: E316-322, 1995) are stimulated with drugs and then produced in cells or in the culture supernatant By quantifying GIP, we investigate the effects of drugs on GIP secretion. The quantification of GIP was performed by high performance liquid chromatography according to the method of Kieffer et al. (Am. J. Physiol 269: E316-322, 1995) and by radioimmunoassay (GIP RIA kit is available from Peninsula Laboratory, Inc.).
    [65] 7. Using the small intestine-derived cancer cells producing large amounts of GIP as described above, the effect of the drug on GIP mRNA synthesis was examined by quantifying GIP mRNA induced in cells after addition of the drug, do.
    [66] Example
    [67] Test Example 1. Administration of GIP by a normal mouse and high-load method load test
    [68] (1) Method
    [69] 1-1) GIP administration and high-level load
    [70] A 17-week-old C57BL / 6j mouse (male) was used, and 5 mice in each group were implanted with an osmotic pump under the skin of the back of each rat. GIP 20, 60, 200 μg / kg / Were administered continuously or continuously in a high-fat diet or a control diet. The energy composition of the high fat diet was 45% lipid, 38% carbohydrate, 17% protein, energy composition of the control diet was 13% lipid, 60% carbohydrate and 27% protein, high fat diet 4.77 kacl / g, kacl / g of energy. GIP administration and high fat diet loading were performed for 4 weeks.
    [71] 1-2) Comparison of body weight and measurement of fasting blood glucose level and insulin level
    [72] Weights of rats in each arm were measured every week and compared. The blood glucose level and the insulin level were measured by collecting blood at fasting 4 weeks after the start of the experiment.
    [73] (2) Results
    [74] 2-1) Weight gain by GIP (Fig. 1A)
    [75] The weight gain in the group fed normal diet was 5.7% at 4 weeks, while that in the high fat group was 24.1%. Even when GIP was administered to this high-fat diet group, there was no increase in body weight.
    [76] 2-2) Fasting blood glucose level (Fig. 1B)
    [77] Fasting blood glucose levels were elevated in the GIP-fed group compared to the GIP-untreated group, depending on the GIP dose.
    [78] 2-3 fasting plasma insulin levels (Fig. 1c)
    [79] In the GIP - fed group, the fasting plasma insulin level was elevated in a dose - dependent manner compared to the GIP - treated group.
    [80] (3) Consideration
    [81] Continuous administration of GIP did not affect weight gain in high fat diet rats. However, on the other hand, the elevation of fasting plasma glucose and fasting plasma insulin levels by high-fat loading increased dose-dependently with continued administration of GIP. This indicates that the insulin resistance caused by the high fat diet load is exacerbated by GIP without the progress of obesity. In other words, GIP was found to be a factor independent of obesity and to exacerbate insulin resistance. This indicates that a compound that inhibits the function of GIP is independent of prevention or amelioration of obesity, and is a preventive or remedy for insulin resistance.
    [82] Test Example 2. High-load method load test by GIP receptor deficient mice
    [83] (1) Method
    [84] 1-1) Highland method load
    [85] Seven primary GIP receptor deficient mice and wild type rats were used, and five rats in each group were treated with high fat diet and control diet. The energy composition of the high fat diet was 45% lipid, 35% carbohydrate, 20% protein, and the energy content of the control diet was 13% lipid, 60% carbohydrate and protein 27% It has energy. The rats in each group were given diets for 8 weeks to 52 weeks for 45 weeks. Oral glucose tolerance test, blood lipid measurement, and histological comparison were performed in each group. During the first 7 weeks, all groups received the usual diet. GIP receptor-deficient mice were also examined by Miyawaki, K., et al. Proc. Natl. Acsd. Sci. USA 96, 14843-14847, 1999, and male rats were used.
    [86] 1-2) Measurement of fasting blood glucose, insulin and glucose tolerance
    [87] Rats in each experimental group were fasted for 16 hours (6:00 am to 10:00 am), and blood glucose level, insulin level and oral glucose tolerance test were performed. In the oral glucose tolerance test, glucose was orally administered at a dose of 2 g / kg, and blood glucose levels were measured before and 15, 30, 60, 90, and 120 minutes after the administration by taking blood from the subcutaneous vein. The blood glucose level was measured using an enzyme electrode method (Samhwa Chemical Laboratories), and the insulin level in the blood was measured using an enzyme-labeled antibody method (Shibayagi).
    [88] 1-3) Measurement of blood lipid
    [89] The rats in each experimental group were fasted for 16 hours (6:00 am to 10:00 am), and blood plasma was collected and blood lipid markers were measured by the enzyme method (ChionaMedex).
    [90] 1-4) Histological analysis of liver and adipose tissue
    [91] Liver and adipose tissues were wrapped in paraffin, and 3.5 μm thick sections were made. The slice was dyed with hematoxylin eosin method after removing paraffin using xylan and ethanol. The liver cells were prepared separately and frozen sections were stained with Oil O.
    [92] (2) Results
    [93] 2-1) Weight gain due to high fat diet or control normal diet (Fig. 2)
    [94] There was no significant difference in weight gain between the GIP-receptor-deficient and wild-type mice when the control diet was consumed. In the case of high-fat diet, the weight of the wild-type rats was increased by 35% as compared with the control diet (Fig. 2a). In contrast, there was no difference in weight gain between the high-fat diet group and the control fat group in GIP-receptor-deficient rats. (Figure 2b)
    [95] 2-2) Oral sugar load test (Fig. 3)
    [96] In wild-type rats, the fasting blood glucose level and insulin level of the high fat diet group were significantly higher than those of the control group (Fig. 3a, b). In the GIP-receptor-deficient mice group, the fasting hyperglycemia and hyperinsulinemic states, which were observed in the wild-type mouse high-fat diet group, were clearly improved (FIG. In the wild-type mouse high-fat diet group, there was no significant difference in the maximum blood glucose level at the time of glucose loading compared to the control group (Fig. 4A). In the wild-type mouse high-fat diet group, the blood insulin level at the time of glucose loading was significantly higher than that of the control group (Fig. 4C). These results suggest that insulin resistance is caused by high fat loading in wild type rats. In the GIP receptor-deficient rats, there was a slight mild capacity deficit compared with the wild-type mouse control group. There was no difference between fasting and maximum blood glucose levels, basal and stimulated insulin levels, high fat diet and control diet. (Fig. 4b, d).
    [97] 2-3) Blood lipid marker (Figure 5)
    [98] Comparing the blood lipid markers of each group, triglyceride (FIG. 5B) and LDL cholesterol (FIG. 5D) were lowered in GIP receptor-deficient rats as compared with wild-type rats in addition to the normal diet and high fat diet.
    [99] 2-4) Histological analysis (Fig. 6)
    [100] In wild-type rats, there was a significant increase in visceral adipose tissue and subcutaneous adipose tissue due to high fat diet load. In GIP-receptor-deficient rats, the accumulation of visceral adipose tissue and subcutaneous adipose tissue between the high- There was no difference (Fig. 6A, b). Furthermore, in the liver tissues of hematoxycin · eosin staining and oilless O staining, the fatty degeneration of hepatic tissue by wild-type rats was observed in wild type rats, but in the GIP receptor-deficient rats, (Fig. 6C). In the epididymal fat pads, there was a significant hypertrophy of adipocytes by wild type rats in wild type rats. However, in the GIP-receptor-deficient rats, no change was observed in the adipocyte fat pad adipocyte pad even after the high fat loading (Fig. 6d).
    [101] (3) Consideration
    [102] The 45 - week high body weight gain of wild type rats was lost in GIP - receptor homozygous mice. In the autopsy, the visceral fat content of the wild type rats was increased compared with the GIP receptor homo deficient mice. GIP receptor In the case of a homozygous deficient rat, a compound that inhibits the function of GIP (for example, a GIP inhibitor) is inferred to be a compound that inhibits the accumulation of fat in adipose tissue Suppression has been proven. No increase in body weight was observed in any of the rats fed the diet with less fat than the same calorie intake. It is now clear from these results that quantitative changes in fat and energy intake do not determine the amount of fat tissue, but that GIP responds sensitively to food composition and that the amount of fat tissue is actively regulated. As such, GIP is a tripyptic gene that moves according to the saving and accumulation of energy. Therefore, it is considered that the anti-obesity effect due to the inhibition of GIP function is due to inhibition of the absorption and accumulation promotion action of lipids by adipose tissue. Here, it is suggested that lipid not absorbed into adipose tissue due to GIP receptor deficiency does not increase blood lipid in GIP-receptor-deficient rats, but GIP inhibitor may decrease blood lipid. On the other hand, in the oral glucose tolerance test, in the wild type rats, obesity, a significant elevation of insulin level in the blood and fasting glucose and hyperglycemia were observed by the high fat diet method, but insulin resistance was expressed, but this phenomenon was not observed in the GIP receptor homo deficient mice. That is, it has been found that insulin resistance is improved by inhibition of GIP function. They show that compounds that inhibit the function of GIP become a preventive or therapeutic agent for insulin resistance as well as obesity.
    [103] Test Example 3. Effect of GIP Receptor Defect on Insulin Resistance and Obesity in ob / ob Mice
    [104] (1) Method
    [105] GIP receptor gene deletion ob / ob mice were constructed by defecating the obesity-related hormone, leptin, and introducing a GIP receptor-deficient gene into hereditary obese ob / ob mice. GIP receptor gene homozygous deletion mice (C57BL / 6j-GIPR - / - ) and leptin gene heterozygous C57BL / 6j-Lep +/- mice were transfected with the GIP receptor gene and the double heterozygous mouse C57BL / 6j-GIPR +/- / Lep +/- ). By mating the C57BL / 6j-GIPR +/- / Lep +/- sexes of, C57BL / 6j-GIPR + / + / Lep - / -, C57BL / 6j-GIPR - / - / Lep - / -, C57BL / 6j-GIPR + / + / Lep + / + (corresponding to wild-type C57BL / 6j mice). The body weight of these rats was measured and compared every week. Four weeks after the start of the experiment, rats in each experimental group were sacrificed for 16 hours (6:00 am to 10:00 am) and oral glucose tolerance test was performed. Glucose was orally administered at a dose of 2 g / kg in the oral glucose tolerance test, and blood glucose levels were measured by taking blood from the venipuncture at the time points of 15, 30, 60, 90 and 120 minutes before and after the administration. The blood glucose level was measured using an enzyme electrode method (Samhwa Chemical Laboratories). Male rats were also used for the experiment.
    [106] (2) Results
    [107] 2-1) Weight change (Fig. 7A)
    [108] C57BL / 6j-GIPR + / + / Lep - / - body weight C57BL / 6j-GIPR + / + / Lep + / + was significantly increased as compared to the (wild-type), GIP receptor deficient C57BL / 6j-GIPR of - / - / Lep - / - , the weight gain was significantly inhibited. There was no difference in the intake between C57BL / 6j-GIPR + / + / Lep - / - and C57BL / 6j-GIPR - / - / Lep - / - .
    [109] 2-2) Fasting blood glucose level (Fig. 7b)
    [110] C57BL / 6j-GIPR + / + / Lep- / - showed a significantly higher fasting blood glucose level than C57BL / 6j-GIPR + / + / Lep + / + (wild type). GIP receptor deficiency C57BL / 6j-GIPR - / - / Lep - / - clearly showed improvement in fasting hyperglycemia.
    [111] 2-3) Oral sugar load test (Fig. 7c)
    [112] In the oral glucose tolerance test, C57BL / 6j-GIPR + / + / Lep- / - showed an increase in initial blood glucose level compared to C57BL / 6j-GIPR + / + / Lep + / + (wild type) . C57BL / 6j-GIPR - / - / Lep - / - increase in blood sugar is C57BL / 6j-GIPR + / + / Lep - was inhibited as compared to the - /.
    [113] (3) Consideration
    [114] Ob / ob mice are deficient in the leptin, so they have an impairment of feeding central function and become overeated and induce obesity. Abortion of this rat GIP receptor reduced obesity and improved fasting hyperglycemia and cardiovascular dysfunction. They show that a compound that inhibits the function of GIP becomes a preventive or ameliorating agent for obesity, and at the same time, becomes an agent or agent for preventing insulin resistance.
    [115] Test Example 4. Selection of GIP function inhibitor
    [116] (1) Method
    [117] According to Screening Method Example 1, a low molecular weight GIP inhibitor was screened. Specifically, GIP receptor-expressing CHO cells were stimulated with 100 pM GIP in the presence and absence of the test substance, and the amount of cAMP produced was measured to determine GIP function inhibitory activity in the test subject. In order to confirm the receptor specificity of the test subject, GLP-1 belonging to the glucagon · secretin family and CHO cells expressing the receptor of glucagon were stimulated with GLP-1 or glucagon (100 pM) The inhibitory activity of the test substance on cAMP production was confirmed. Furthermore, in order to verify the effects not related to these receptors, the inhibitory effect of the test substance on cAMP production when stimulated with 5 mu m phosholine using CHO cells not introduced with these receptor genes was also confirmed. As a positive control, 500 nM GIP (7-30) -NH 2 (tGIP) was used.
    [118]
    [119] (2) Results
    [120] As a result of selection, 3-bromo-5-methyl-2-phenylpyrazolo [1,5-A] pyrimidin-7-ol (BMPP, as shown in Formula 1) purchased from MAYBRIDGE , And has a specific inhibitory effect on the function of GIP (FIG. 8). BMPP inhibited the cAMP generation-induced action of GIP in GIP receptor-expressing CHO cells in a concentration-dependent manner, and the CE50 thereof was about 30 mu m (Fig. 8A). BMPP barely inhibited the action of glucagon, but its inhibition rate was only 27% (Fig. 8C) even at 80 mu m, and did not substantially inhibit the action of GLP-1 (Fig. 8B). Moreover, BMPP did not exhibit inhibitory action against cAMP production by phosholin stimulation (Fig. 8d).
    [121] (3) Consideration
    [122] As a result of selection, the selected BMPP had high specificity for GIP receptor in its GIP function inhibitory action, and it was a candidate compound to be a lead compound of a low molecular GIP function inhibitor. On the other hand, there is a weak inhibitory effect on glucagon function, but this may be advantageous because it can be expected to lead to the inhibition of blood glucose increase when administered to diabetic patients.
    [123] The detailed mechanism by which GIP causes insulin resistance is unclear at present. As one possibility, the effect of GIP on any molecule that causes insulin resistance can be considered. As a molecule involved in insulin resistance, free fatty acids, TNF-α have been studied. On the other hand, the relationship between GIP and serum free fatty acid concentration has been studied, and there are various theories. However, there was no consistent relationship between the presence of GIP receptors, insulin resistance, and obesity and free fatty acid levels in serum in wild-type and GIP receptor-deficient rats under these experimental conditions. Reports on the association of GIP with other factors thought to be involved in insulin resistance are not particularly recognized.
    [124] There are two mechanisms by which GIP causes obesity. First, the pathway in which GIP increases insulin secretion and promotes the uptake of nutrients into adipose tissue, while insulin hyperactiates the expression of PPARγ. The second is that GIP elevates cAMP through GIP receptors expressed in adipocytes and promotes direct fat cell enlargement. PPARy is essential for the differentiation of adipocytes, but it is also expressed in other organs, and homozygotes are lethal. On the other hand, the homozygous mouse of the GIP receptor has mild functional dysfunction, and does not show abnormalities such as organ formation. At first glance, there is no significant difference from normal individuals, but in the high-fat setting environment, the GIP receptor homozygous rats show no significant increase in body weight as the wild-type mice gain weight. This property of GIP is closely associated with the concept of the tryptic gene form that manifests itself in response to changes in the environment and leads to adult diseases. In the same calorie intake, more meals including fat should be obesity. However, in the absence of the GIP signal, this rule is lost and the calorie intake defines the body weight. Thus, the GIP receptor becomes a new target of antagonism. That is, a compound that inhibits the function of GIP can inhibit fat accumulation to adipose tissue resulting from excessive intake of fat, and can be used for prevention or treatment of obesity.
    [125] Although described in the prior art, it is described in WO98 / 24464 that GIP inhibitors result in an anti-obesity effect. The present application reports that GIP inhibitors can be antioxidants based on the inhibitory action of glucose on absorption of GIP (7-30) -NH 2 , which is an inhibitor of GIP, in the intestinal tract. However, the logical development that GIP inhibitors have an inhibitory effect on glucose absorption by GIP inhibitors and that GIP inhibitors are antioxidants are not in the interest of anyone skilled in the art as a complete invention, and furthermore, GIP inhibitors have insulin resistance It is also not predictable about improvement. In addition, the results of oral glucose tolerance test (Fig. 4 (a) and (b)) on the GIP receptor-deficient mice performed by us showed that the increase in blood glucose level upon glucose administration was the same as that in wild type mice in GIP receptor- The inhibition of glucose uptake is not recognized. Therefore, it is believed that the obesity-suppressing action of the GIP-receptor-deficient mice in the high fat diet load we have found is not due to the inhibition of glucose uptake reported in WO98 / 24464. In addition, in our experimental results, it is not believed that inhibition of glucose uptake inhibiting blood glucose levels occurs even when a GIP antagonist is administered to rats. From these, WO98 / 24464 does not suggest our invention.
    权利要求:
    Claims (7)
    [1" claim-type="Currently amended] A prophylactic or ameliorative agent for insulin resistance comprising a compound which inhibits the function of GIP as an active ingredient.
    [2" claim-type="Currently amended] An agent for simultaneous prevention or amelioration of insulin resistance and obesity comprising a compound that inhibits the function of GIP as an active ingredient.
    [3" claim-type="Currently amended] The insulin resistance preventing or improving agent according to claim 1, wherein the insulin resistance is not accompanied by obesity.
    [4" claim-type="Currently amended] The insulin resistance preventing or ameliorating agent according to any one of claims 1 to 3, wherein the compound that inhibits the function of GIP is a GIP receptor inhibitor.
    [5" claim-type="Currently amended] The agent for preventing or ameliorating insulin resistance according to any one of claims 1 to 3, wherein the compound that inhibits the function of GIP is a production inhibitor of GIP.
    [6" claim-type="Currently amended] A method for selecting an agent for preventing or ameliorating insulin resistance, which comprises selecting a compound that inhibits the function of GIP.
    [7" claim-type="Currently amended] A method for simultaneously preventing or ameliorating insulin resistance and obesity, which comprises selecting a compound that inhibits the function of GIP.
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    同族专利:
    公开号 | 公开日
    US20030157107A1|2003-08-21|
    AU2001256757B2|2006-03-09|
    CA2417590A1|2001-11-22|
    EP1283058A4|2004-06-23|
    EP1283058A1|2003-02-12|
    CA2417590C|2009-08-11|
    WO2001087341A1|2001-11-22|
    AU5675701A|2001-11-26|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    2000-05-16|Priority to JPJP-P-2000-00143749
    2000-05-16|Priority to JP2000143749
    2001-05-15|Application filed by 가부시키가이샤산와카가쿠켄큐쇼
    2001-05-15|Priority to PCT/JP2001/004058
    2002-12-13|Publication of KR20020093150A
    优先权:
    申请号 | 申请日 | 专利标题
    JPJP-P-2000-00143749|2000-05-16|
    JP2000143749|2000-05-16|
    PCT/JP2001/004058|WO2001087341A1|2000-05-16|2001-05-15|Agents for preventing or ameliorating insulin resistance and/or obesity|
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