Use of p2RY12 inhibitor in preparation of drug for treating obesity, overweight, and metabolic syndrome diseases

Abstract

Use of a P2RY12 inhibitor in the preparation of a drug for treating obesity, overweight, and metabolic syndrome diseases. A low-dose nasal formulation of the P2RY12 inhibitor can significantly reverse obesity, overweight, and insulin resistance, thereby safely and efficiently achieving the treatment of obesity, overweight, and metabolic syndrome diseases.

Description

Use of P2RY12 inhibitors in the preparation of drugs for treating obesity, overweight and metabolic syndrome Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the use of a P2RY12 inhibitor in preparing a medicament for treating obesity, overweight and metabolic syndrome. Background Art

[0002] In recent years, the prevalence of overweight, obesity, and related metabolic diseases has steadily increased, evolving into a global healthcare crisis. A complex interplay of genetic, environmental, and behavioral factors fuels this epidemic. Lifestyle modifications, including diet, physical activity, and behavioral therapy, are the cornerstones of management, with evidence demonstrating their efficacy in reducing weight and improving metabolism. Current treatment paradigms encompass a broad range of interventions, from surgical to pharmacological. Bariatric surgery, including gastric bypass and sleeve gastrectomy, offers a cure for patients with severe obesity, while pharmacological therapies such as orlistat, liraglutide, GLP-1 receptor agonists such as semaglutide and tizepatide, and dual glucose-dependent insulinogenic polypeptide (GIP) / GLP-1RA offer a less invasive approach to treating these conditions. Existing treatment modalities are fraught with limitations, such as the inherent risks of surgical intervention and the potential side effects associated with pharmacological therapy. Furthermore, long-term weight maintenance remains a major challenge, highlighting the urgent need for new and more effective therapeutic strategies.

[0003] At present, the main drugs for long-term weight maintenance are mainly injectable and oral drugs. These dosage forms of drugs are delivered through the intestines and blood. In order to ensure effective absorption and utilization in the body, the dosage is usually large, and long-term use is prone to produce strong side effects.

[0004] The development of centrally acting drugs targeting the brain is a major endeavor in the field of neuropharmacology. These drugs, carefully designed to modulate neural activity, show great promise in treating a wide range of neurological and psychiatric disorders. However, the efficacy of these drugs is often hampered by the formidable blood-brain barrier (BBB), which restricts the delivery of systemic therapeutics to the central nervous system (CNS). To circumvent this challenge and increase drug concentrations in the brain, intranasal delivery is an emerging strategy. This innovative route not only has the potential to bypass the BBB but also mitigates the peripheral side effects associated with conventional systemic delivery.

[0005] Intranasal drug delivery exploits the unique anatomical and physiological connection between the nasal cavity and the brain, facilitating drug transport directly to the central nervous system. This is particularly advantageous because it bypasses systemic circulation, reducing the risk of adverse reactions while ensuring more targeted delivery. Over the past decade, the scientific and pharmaceutical fields have witnessed a boom in interest in this non-invasive and patient-compliant drug delivery method. The appeal of intranasal drug delivery stems from its ability to maintain effective drug concentrations in the brain, which is crucial for the therapeutic efficacy of centrally acting drugs.

[0006] Therefore, there is an urgent need in the art to develop drugs and dosage forms thereof that can safely and effectively treat obesity, overweight and metabolic syndrome in the long term.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a medicine and its application for treating obesity, overweight and metabolic syndrome safely and effectively.

[0009] In a first aspect, the present invention provides a use of a P2RY12 inhibitor for preparing a medicament for treating obesity, overweight and metabolic syndrome in a subject, wherein the medicament is a brain-targeted preparation.

[0010] In another preferred embodiment, the brain-targeted agent is an agent targeting hypothalamic cells.

[0011] In another preferred embodiment, the brain-targeted preparation is a preparation targeting oxytocin neurons.

[0012] In another preferred embodiment, the brain-targeted preparation is a nasal preparation.

[0013] In another preferred embodiment, the dosage form of the nasal preparation is selected from the following group: aerosol, nasal drops, powder, gel, microsphere preparation, liposome preparation, and emulsion.

[0014] In another preferred embodiment, the P2RY12 inhibitor is selected from the following group: clopidogrel, prasugrel, ticlopidine, ticagrelor, congrelor, antibodies, phages, nanobodies and other small molecules or macromolecules that selectively or semi-selectively target P2RY12.

[0015] In another preferred embodiment, the subject has one or more characteristics selected from the following group:

[0016] (a) the expression level of P2RY12 in the hypothalamic neuronal cells of the subject is relatively high;

[0017] (b) low cyclic adenosine monophosphate (cAMP) levels in hypothalamic neurons of the subject;

[0018] (c) the level of ERK1 / 2 phosphorylation in the hypothalamic neuronal cells of the subject is low;

[0019] (d) The expression level of c-Fos in the hypothalamic neuronal cells of the subject is relatively low.

[0020] In another preferred embodiment, the low level refers to an expression amount, content or level that is significantly lower than a reference value; the high level refers to an expression amount, content or level that is significantly higher than a reference value.

[0021] In another preferred embodiment, the reference value is the expression amount, content or level of the corresponding gene in normal subjects (subjects not suffering from obesity, overweight and metabolic syndrome).

[0022] In another preferred embodiment, the significantly lower value refers to an expression level ≤ 2 / 3 of the benchmark value, preferably ≤ 1 / 2 of the benchmark value, and more preferably ≤ 1 / 3 of the benchmark value.

[0023] In another preferred embodiment, the significantly higher refers to an expression level ≥ 4 / 3 of the benchmark value, preferably ≥ 3 / 2 of the benchmark value, and more preferably ≥ 2 times of the benchmark value.

[0024] In another preferred embodiment, the P2RY12 inhibitor is a reversible P2RY12 inhibitor or an irreversible P2RY12 inhibitor.

[0025] In another preferred embodiment, the obesity, overweight and metabolic syndrome diseases are selected from the following group: simple obesity, secondary obesity and insulin resistance.

[0026] In another preferred embodiment, the simple obesity is selected from the group consisting of constitutional obesity and overeating obesity.

[0027] In another preferred embodiment, the secondary obesity is selected from the group consisting of hypothalamic obesity, pituitary obesity, hypercortisolism, insulopathy obesity, hypothyroidism obesity, hypogonadism obesity, and hypogonadism obesity.

[0028] In another preferred embodiment, the insulin resistance is selected from the following group: primary insulin resistance and hereditary insulin resistance.

[0029] In another preferred embodiment, the unit dose of the nasal preparation is 0.1 to 10 mg / kg, preferably, 0.1 to 8 mg / kg, more preferably, 0.1 to 5 mg / kg, based on the weight of the subject.

[0030] The second aspect of the present invention provides a pharmaceutical composition, which is a brain-targeting preparation;

[0031] And the pharmaceutical composition contains a P2RY12 inhibitor selected from the group consisting of clopidogrel, prasugrel, ticlopidine, ticagrelor, congrelor, antibodies, phages, nanobodies and other small molecules or macromolecules that selectively or semi-selectively target P2RY12, or a combination thereof;

[0032] and a pharmaceutically acceptable carrier.

[0033] In another preferred embodiment, in the pharmaceutical composition, the content of the active ingredient is 0.01-99 wt %, preferably 0.1-90 wt %, based on the total weight of the pharmaceutical composition.

[0034] The third aspect of the present invention provides use of the pharmaceutical composition according to the second aspect of the present invention, wherein the pharmaceutical composition is used to prepare a medicament for treating obesity, overweight and metabolic syndrome in a subject.

[0035] In another preferred embodiment, the pharmaceutical composition is used as a separate brain-targeted preparation in combination with other effective therapeutic agents for obesity, diabetes, overweight and metabolic syndrome in the comprehensive treatment of obesity, overweight and metabolic syndrome.

[0036] In another preferred embodiment, the other effective therapeutic agents for obesity, overweight diseases and metabolic syndrome include oral preparations and injection preparations.

[0037] In another preferred embodiment, the other effective therapeutic agents for obesity, diabetes, overweight and metabolic syndrome include: orlistat, semaglutide, liraglutide, semaglutide, tezetide, metformin, bupropion, lorcaserin, phentermine, and dual glucose-dependent insulinogenic polypeptide.

[0038] In another preferred embodiment, the pharmaceutical composition is used as a separate nasal preparation in combination with other effective treatment methods for obesity, overweight and metabolic syndrome (surgical treatment, etc.) in the comprehensive treatment of obesity or overweight and metabolic syndrome.

[0039] In a fourth aspect, the present invention provides a method for treating obesity, overweight and metabolic syndrome, comprising: administering a therapeutically effective amount of a P2RY12 inhibitor to the nasal cavity of a subject in need thereof.

[0040] In another preferred embodiment, the P2RY12 inhibitor contains a P2RY12 inhibitor selected from the following group: clopidogrel, prasugrel, ticlopidine, ticagrelor, congrelor, antibodies, phages, nanobodies and other small molecules or macromolecules or their combinations that selectively or semi-selectively target P2RY12.

[0041] In another preferred embodiment, the subject is a primate mammal, such as a monkey or a human.

[0042] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 shows abnormal expression of P2RY12 in oxytocin-secreting (OXT) neurons in the paraventricular nucleus of the hypothalamus in patients with diabetes. Iba-1, a microglial marker, and NeuN, a neuronal marker, are used. Scale bars are 200 μm and 20 μm, respectively.

[0044] Figure 2 shows the expression of P2RY12 in oxytocin neurons in the paraventricular nucleus of the hypothalamus (OXT) in mice induced by a lipid diet. Immunofluorescence analysis of P2RY12 expression in OXT neurons in mice fed a lipid diet and mice fed a normal diet was performed. Scale bar, 100 μm.

[0045] Figure 3 shows the expression of P2RY12 in oxytocin (OXT) neurons in the paraventricular nucleus of the hypothalamus (PVH) of mice induced by a high-fat diet. Adult mice were stereotaxically injected with an OXT probe (OXT-Venus) into the paraventricular nucleus (PVH) of the hypothalamus. Following surgery, mice were fed a high-fat diet or a standard diet for four weeks. Body weights were measured, and PVH tissue was obtained. After digestion, filtration, and flow cytometry sorting, Venus-positive OXT cells were isolated. RNA was extracted, and cDNA was reverse-transcribed. QPCR was then performed to examine changes in P2RY12 expression. (n = 3) Error bars represent SEM; **: p < 0.01, analyzed by Student's t-test.

[0046] Figure 4 shows a schematic diagram of the potential molecular mechanism of P2RY12 inhibiting the MC4R signaling pathway. Under normal circumstances, satiety signals are input through the melanocortin signaling system. α-MSH in the OXT neurons of the PVH activates the MC4R / AC / cAMP / ERK / cFos signaling pathway, which then leads to the transcription and expression of important neural signaling-related genes, maintaining energy homeostasis by reducing appetite and increasing energy expenditure. Metabolic stress stimulates NFkB to induce the overexpression of P2RY12, which in turn activates the MC4R / AC / cAMP / ERK / cFos signaling pathway. i The protein inhibits adenylate cyclase AC and its downstream cAMP / ERK / cFos signaling pathway, leading to the inhibition of gene transcription and expression related to neural signals, increased appetite, reduced energy expenditure, imbalance of energy homeostasis and obesity.

[0047] Figure 5 shows a flowchart of the in vitro experimental operation of overexpressing P2RY12 in GT1-7 cells; molecular cloning was used to construct a lentiviral vector for overexpressing P2RY12, which was then infected into hypothalamic GT1-7 cells after viral packaging and titer detection. Target cells with strong GFP positivity were then screened by flow cytometry, and stably transfected P2RY12-overexpressing cell lines were obtained after amplification. After cell starvation and α-MSH drug induction together with control cells, relevant phenotypes were detected in vitro using fluorescence quantitative PCR, enzyme-linked immunosorbent assay (ELISA), and Western blot.

[0048] Figure 6 shows Western Blot showing changes in the phosphorylation level of ERK1 / 2 at Thr202 / Tyr204 and c-Fos expression in GT1-7 cells overexpressing P2RY12.

[0049] FIG7 shows the results of Western Blot quantitative analysis (n=3), error bars represent SEM; *: p<0.05; **: p<0.01; analyzed by one-way ANOVA (Tukey).

[0050] Figure 8 shows the mRNA level of P2RY12 detected by fluorescent quantitative QPCR after α-MSH treatment of GT1-7 cells overexpressing P2RY12, (n=3) error bars represent SEM; ***: p<0.001; ****: p<0.0001; analyzed by one-way ANOVA (Tukey).

[0051] FIG9 shows the cyclic AMP levels in P2RY12-overexpressing GT1-7 cells before and after α-MSH treatment detected by ELISA (n=3). Error bars represent SEM; **: p<0.01; ***: p<0.001; results were analyzed by one-way ANOVA (Tukey).

[0052] FIG10 shows the mRNA level of c-Fos detected by fluorescent quantitative QPCR after α-MSH treatment of GT1-7 cells overexpressing P2RY12, (n=3) error bars represent SEM; ****: p<0.0001; analyzed by one-way ANOVA (Tukey).

[0053] FIG11 shows the body weight changes of adult mice first fed with a normal diet and then fed with a high-fat diet and administered with ticlopidine (90 mg / kg / day) via drinking water (n=4 for high-fat diet + normal saline, n=4 for high-fat diet + ticlopidine); **: p<0.01, by one-way ANOVA RM, Dunnet).

[0054] Figure 12 shows the changes in body weight of obese adult mice fed a high-fat diet (HFD) after administration of three different gradient concentrations of the P2RY12 inhibitors ticlopidine, clopidogrel, and prasugrel in drinking water (high-fat diet + control n=6, high-fat diet + ticlopidine n=6, high-fat diet + clopidogrel n=6, and high-fat diet + prasugrel n=6), analyzed by two-way ANOVA, Tukey).

[0055] Figure 13 shows the experimental design flow chart for obese adult mice fed a high-fat diet (HFD) to which three P2RY12 inhibitors, ticlopidine, clopidogrel, and prasugrel, were intranasally administered, respectively, while mice of the same age were fed a normal diet as a control. The experimental design flow chart then included testing of the mice's cumulative food intake, metabolic cage analysis, analysis of the mice's lean / fat body composition, and glucose / sugar insulin tolerance tests.

[0056] Figure 14 shows the body weight changes after intranasal injection of the P2RY12 inhibitors ticlopidine, clopidogrel, and prasugrel (normal diet + control n=5, high-fat diet + control n=5, high-fat diet + ticlopidine n=7, high-fat diet + clopidogrel n=5, and high-fat diet + prasugrel n=5), error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Dunnett).

[0057] Figure 15 shows the changes in body weight of mice 8 days after intranasal injection of the P2RY12 inhibitor clopidogrel (normal diet + control n=5, high-fat diet + control n=5, high-fat diet + clopidogrel n=5) Error bars represent SEM; *: p<0.05; analyzed by Student's t test.

[0058] FIG16 shows the morphological characteristics of mice 4 weeks after intranasal injection of P2RY12 inhibitors ticlopidine and prasugrel.

[0059] Figure 17 shows low-field nuclear magnetic resonance analysis of changes in fat / lean content in mice after treatment with the P2RY12 inhibitors ticlopidine and prasugrel (normal diet + control n=5, high-fat diet + control n=5, high-fat diet + ticlopidine n=5, and high-fat diet + prasugrel n=5), error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0060] FIG18 shows images of white adipose tissue (iWAT) of mice stained with hematoxylin-eosin after treatment with the P2RY12 inhibitors ticlopidine and prasugrel.

[0061] Figure 19 shows the quantitative analysis of fat area based on the results of Figure 16 using ImageJ image analysis software; (normal diet + control n=3, high-fat diet + control n=3, high-fat diet + ticlopidine n=3 and high-fat diet + prasugrel n=3) error bars represent SEM; **: p<0.01; analyzed by one-way ANOVA (Tukey).

[0062] Figure 20 shows the glucose tolerance test analysis of blood glucose levels in mice after treatment with P2RY12 inhibitors ticlopidine and prasugrel (normal diet + control n=5, high-fat diet + control n=5, high-fat diet + ticlopidine n=5, and high-fat diet + prasugrel n=5), error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0063] Figure 21 shows the insulin tolerance test analysis of blood glucose levels in mice after treatment with P2RY12 inhibitors ticlopidine and prasugrel (normal diet + control n=5, high-fat diet + control n=5, high-fat diet + ticlopidine n=5, and high-fat diet + prasugrel n=5), error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0064] Figure 22 shows changes in cumulative food intake in mice after treatment with the P2RY12 inhibitors ticlopidine and prasugrel. (N=5 for normal diet + control, n=5 for high-fat diet + control, n=7 for high-fat diet + ticlopidine, and n=5 for high-fat diet + prasugrel) Error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; two-way ANOVA, Tukey).

[0065] Figure 23 shows body weight changes in mice induced by intranasal administration of the P2RY12 inhibitor ticlopidine at different doses (human daily dose or half of it) and high-fat diet. (Normal diet + control, n=5; high-fat diet + control, n=5; high-fat diet + ticlopidine, n=7) Error bars represent SEM; *: p<0.05; **: p<0.01; two-way ANOVA, Tukey).

[0066] Figure 24 shows changes in oxygen consumption in mice after treatment with the P2RY12 inhibitor prasugrel in metabolic cage analysis (normal diet + control, n=4; high-fat diet + control, n=4; high-fat diet + prasugrel, n=4). Error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0067] Figure 25 shows changes in carbon dioxide emissions in mice after treatment with the P2RY12 inhibitor prasugrel in metabolic cage analysis (normal diet + control, n=4; high-fat diet + control, n=4; high-fat diet + prasugrel, n=4). Error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0068] Figure 26 shows changes in thermogenesis in mice following treatment with the P2RY12 inhibitor prasugrel as analyzed in metabolic cages (normal diet + control, n=4; high-fat diet + control, n=4; high-fat diet + prasugrel, n=4). Error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0069] Figure 27 shows changes in respiratory rate in mice after treatment with the P2RY12 inhibitor prasugrel by metabolic cage analysis. (N=4 for control group on a normal diet, n=4 for control group on a high-fat diet, and n=4 for control group on a high-fat diet plus prasugrel) Error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0070] Figure 28 shows the changes in body weight of obese adult mice fed a high-fat diet (HFD) after intranasal administration of the P2RY12 inhibitor ticagrelor. (N=5 for control group, n=5 for HFD group, and n=6 for HFD group + ticagrelor group) Error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0071] Figure 29 shows that after 24 days of intranasal injection of the P2RY12 inhibitor ticagrelor into obese adult mice fed a high-fat diet (HFD), the hypothalamic tissue was extracted and homogenized in deuterated dimethyl sulfoxide (DMSO). At the same time, ticagrelor was added to the deuterated dimethyl sulfoxide as a standard reference, and the tissue samples were compared and analyzed using a 700 MHz nuclear magnetic resonance spectrometer.

[0072] Figure 30 shows a flow chart of the experimental design for intranasal administration of different doses (human daily dose or half of it) of the P2RY12 inhibitors ticagrelor, congrelor, and prasugrel to obese adult mice fed a high-fat diet (HFD). The mice were then tested for cumulative food intake, analyzed for lean / fat body composition, and subjected to glucose / sugar insulin tolerance tests.

[0073] Figure 31 shows the body weight changes after intranasal injection of the P2RY12 inhibitors ticagrelor, congrelor and prasugrel (normal diet + control n=5, high-fat diet + control n=5, high-fat diet + ticagrelor n=6, high-fat diet + congrelor n=6 and high-fat diet + prasugrel n=5), error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Dunnett).

[0074] Figure 32 shows low-field nuclear magnetic resonance analysis of changes in fat / lean content in mice after treatment with the P2RY12 inhibitors ticagrelor and congrelor (normal diet + control n=5, high-fat diet + control n=5, high-fat diet + ticagrelor n=6, high-fat diet + congrelor n=6), error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0075] Figure 33 shows the glucose tolerance test analysis of blood glucose levels in mice after treatment with the P2RY12 inhibitors ticagrelor and congrelor (normal diet + control n=5, high-fat diet + control n=5, high-fat diet + ticagrelor n=6, high-fat diet + congrelor n=6), error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0076] Figure 34 shows the insulin tolerance test analysis of blood glucose levels in mice after treatment with the P2RY12 inhibitors ticagrelor and congrelor (normal diet + control n=5, high-fat diet + control n=5, high-fat diet + ticagrelor n=6, high-fat diet + congrelor n=6), error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0077] Figure 35 shows changes in cumulative food intake in mice after treatment with the P2RY12 inhibitors ticagrelor and congrelor (normal diet + control, n=5; high-fat diet + control, n=5; high-fat diet + ticagrelor, n=6; high-fat diet + congrelor, n=6). Error bars represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

[0078] FIG36 shows a flow chart of an experiment in which caloric restriction was stopped in elderly cynomolgus monkeys (Macaca fascicularis) and a P2RY12 inhibitor nasal spray was injected once daily at a dose equivalent to the daily congrelor dose in humans (0.18 mg / kg / d).

[0079] Figure 37 shows the changes in body weight after five weeks of intranasal administration of a P2RY12 inhibitor nasal spray once daily (Congrelor n=3, saline n=3), error bars represent SEM; *: p<0.05; **: p<0.01; two-way ANOVA, ).

[0080] Figure 38 shows the results of once-daily intranasal administration of a P2RY12 inhibitor nasal spray, equivalent to the daily dose of congrelox in humans (0.18 mg / kg / d). Body weight change was calculated as the area under the normalized weight curve (congrelox n=3, saline n=3). Error bars represent SEM; *: p<0.05; analyzed by Student's t-test.

[0081] Figure 39 shows a flowchart of the P2RY12 overexpression experiment in OXT neurons using the FLEX (flip-excision) overexpression strategy, followed by treatment with the P2RY12 inhibitor prasugrel, and testing of the mice's cumulative food intake, metabolic cage analysis, and blood glucose analysis.

[0082] Figure 40 shows the changes in body weight of male mice after overexpression of P2RY12 in OXT neurons and intranasal administration of the P2RY12 inhibitor prasugrel. (Control group + saline n = 9, control group + prasugrel n = 9, P2RY12 OXT Overexpression + saline n = 10, P2RY12 OXT Overexpression + prasugrel (n = 9), error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; two-way ANOVA, Dunnett).

[0083] Figure 41 shows immunofluorescence analysis of c-Fos expression levels after overexpression of P2RY12 in OXT neurons and intranasal administration of the P2RY12 inhibitor prasugrel. Scale bar: 50 μm.

[0084] FIG42 shows the glucose tolerance test analysis of blood glucose levels after overexpression of P2RY12 in OXT neurons and after intranasal administration of the P2RY12 inhibitor prasugrel (control group + saline n = 9, control group + prasugrel n = 9, P2RY12 OXT Overexpression + saline n = 10, P2RY12 OXT Overexpression + prasugrel (n = 9), error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; analyzed by two-way ANOVA, Tukey).

[0085] Figure 43 shows the changes in cumulative food intake in mice after overexpression of P2RY12 in OXT neurons and intranasal administration of the P2RY12 inhibitor prasugrel. (Control group + saline n = 9, P2RY12 OXT Overexpression + saline n = 10, P2RY12 OXT Overexpression + prasugrel (n = 9), error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; analyzed by two-way ANOVA, Tukey).

[0086] Figure 44 shows the changes in body weight of female mice after overexpression of P2RY12 in OXT neurons. (Control group n=7, P2RY12 OXTOverexpression (n = 7), error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; two-way ANOVA, Dunnett).

[0087] Figure 45 shows the changes in cumulative food intake in female mice after overexpression of P2RY12 in OXT neurons (control group n=7, P2RY12 OXT Overexpression (n = 7), error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; analyzed by two-way ANOVA, Tukey).

[0088] Figure 46 shows the metabolic cage analysis of the changes in oxygen consumption in mice after overexpression of P2RY12 in OXT neurons and treatment with the inhibitor prasugrel. (Control group + saline n = 4, P2RY12 OXT Overexpression + saline n = 4, P2RY12 OXT Overexpression + prasugrel (n = 4) (Error bars indicate SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; analyzed by two-way ANOVA, Tukey).

[0089] Figure 47 shows the metabolic cage analysis of the changes in carbon dioxide emissions in mice after treatment with the P2RY12 inhibitor prasugrel. (Control group + saline n = 4, P2RY12 OXT Overexpression + saline n = 4, P2RY12 OXT Overexpression + prasugrel (n = 4) (Error bars indicate SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; analyzed by two-way ANOVA, Tukey).

[0090] Figure 48 shows the changes in thermogenesis of mice after treatment with the P2RY12 inhibitor prasugrel by metabolic cage analysis. (Control group + saline n = 4, P2RY12 OXT Overexpression + saline n = 4, P2RY12 OXT Overexpression + prasugrel (n = 4) (Error bars indicate SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; analyzed by two-way ANOVA, Tukey).

[0091] Figure 49 shows the metabolic cage analysis of the respiratory rate changes of mice after treatment with the P2RY12 inhibitor prasugrel. (Control group + saline n = 4, P2RY12 OXT Overexpression + saline n = 4, P2RY12 OXT Overexpression + prasugrel (n = 4) (Error bars indicate SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; analyzed by two-way ANOVA, Tukey). DETAILED DESCRIPTION

[0092] After extensive and in-depth research, the inventors unexpectedly discovered for the first time that the P2RY12 receptor, which was originally highly expressed only in microglia, was unexpectedly abnormally highly expressed in oxytocin neurons.

[0093] Based on these surprising findings, the inventors attempted to target the brain with a P2RY12 inhibitor. Nasal administration at a dose approximately one-twenty-seventh the dose of a conventional oral P2RY12 inhibitor (e.g., ticlopidine) significantly improved obesity, overweight, and insulin resistance in HFD mice. This invention provides a new avenue for the clinical therapeutic application of P2RY12 inhibitors and offers a safe and effective new drug for the treatment of obesity, overweight, and metabolic syndrome. Based on this foundation, the inventors completed the present invention.

[0094] P2RY12

[0095] P2RY12 (i.e., purinergic receptor P2Y12) is considered a specific marker for microglia, and its expression levels vary in different central nervous system (CNS) diseases. Microglia are the primary immune cells of the central nervous system and are involved in the activation and migratory behavior of microglia. In addition, the P2RY12 receptor is a key element in the purinergic signaling pathway and plays a key role in platelet aggregation and its impact in various clinical diseases, especially cardiovascular disease. As a G protein-coupled receptor (GPCR), P2RY12 is mainly activated by adenosine diphosphate (ADP) and plays a vital role in the regulation of platelet activation and aggregation, which are key processes in thrombosis. Therapeutic targeting of P2RY12 has become a cornerstone in the treatment of thrombotic diseases, especially in acute coronary syndromes and percutaneous coronary intervention.

[0096] P2RY12 inhibitors

[0097] P2RY12 inhibitors primarily fall into two categories: thienopyridines and non-thienopyridines. The former, including first-generation ticlopidine, second-generation clopidogrel, and third-generation prasugrel, are prodrugs that require conversion to active metabolites via hepatocyte P450 enzymes before irreversibly binding to P2RY12 receptors to produce their effects. The latter, including first-generation ticagrelor and second-generation cangrelor, are novel P2RY12 receptor inhibitors characterized by direct, reversible interaction with P2RY12, rapid onset of action, and short duration of action.

[0098] First-generation ticlopidine is an inactive prodrug and has been gradually withdrawn from clinical use due to the high incidence of hematologic side effects, such as thrombotic thrombocytopenic purpura (TTP), neutropenia, and aplastic anemia. Second-generation clopidogrel, the most widely used P2RY12 inhibitor, is an inactive prodrug. Following oral administration, its active metabolite, produced in vivo, irreversibly binds to the P2RY12 receptor, rendering it unable to respond to ADP. This blockade inhibits platelet function and effectively reduces ADP-mediated platelet activation and aggregation. Disadvantages include a slow onset of action and significant variability in efficacy, resulting in varying inhibitory effects in different populations. Third-generation prasugrel, like clopidogrel, is a prodrug that must be converted to an active metabolite to inhibit platelet function. After oral administration, prasugrel has a faster onset of action and less individual variability in its effects than clopidogrel.

[0099] Ticagrelor is a first-generation thienopyridine P2RY12 receptor inhibitor. Its molecular structure is similar to ATP, making it also known as an ATP analogue. It directly targets the P2RY12 receptor. Its action is direct, reversible, and rapid. Its long-lasting effect and slow clearance make it more effective against thrombosis than irreversible inhibitors. However, a side effect of ticagrelor is an increased incidence of dyspnea. Cangrelor, a second-generation thienopyridine P2RY12 receptor inhibitor, is similar to ticagrelor in that it directly inhibits the P2RY12 receptor. However, cangrelor cannot be taken orally and must be administered intravenously. Its short half-life allows for targeted antiplatelet therapy within a prescribed timeframe, tailored to clinical needs.

[0100] Oxytocin neurons

[0101] Oxytocin (OXT) neurons in the paraventricular hypothalamus are secondary neurons of the melanocortin system that regulates energy intake and expenditure. They are mainly responsible for the regulation of neuroendocrine and autonomic nervous functions, and are also involved in regulating various behavioral activities such as social interaction, emotion, eating and rhythm.

[0102] cAMP

[0103] Cyclic adenosine monophosphate (cAMP) is an important second messenger in cell signaling. When ligands bind to receptors on the cell membrane, they activate G proteins, which in turn activate adenylate cyclase, catalyzing the conversion of ATP to cyclic AMP. It has a wide range of physiological functions.

[0104] ERK1 / 2

[0105] Extracellular regulated protein kinases, collectively known as ERK1 / 2, have two ERK isoforms: ERK1 and ERK2, which share 84% of their amino acid residues. ERK1 / 2 possess a typical protein kinase structure and regulate cellular life processes by phosphorylating substrates. ERK1 / 2 is a key component of the RAF-MEK-ERK signaling pathway. Activated by phosphorylation at Thr202 and Tyr204, ERK1 / 2 activates a variety of substrates (over 160) involved in cell proliferation, differentiation, migration, and angiogenesis. Therefore, phosphorylation of ERK1 / 2 at (Thr202, Tyr204) / (Thr185, Tyr187) is a key step in ERK activation and an essential indicator for related research.

[0106] c-Fos

[0107] c-Fos is a nuclear phosphoprotein that forms a heterodimer with the c-Jun protein, which then forms the AP-1 (activator protein-1) complex. This complex binds to DNA at specific AP-1 sites in the promoter and enhancer regions of target genes, translating extracellular signals into changes in gene expression. c-Fos not only functions as a transcription factor but also influences neuronal gene expression through epigenetic mechanisms.

[0108] Obesity or overweight

[0109] Obesity or overweight disease is determined by body mass index (BMI) for adults who are overweight or obese, 24.0 kg / m 2 ≤BMI<28.0 kg / m 2 Overweight, BMI ≥ 28.0 kg / m 2 Central obesity can be determined by waist circumference. A waist circumference of ≥90cm for men and ≥85cm for women is considered central obesity in adults. Visceral obesity is defined as a body composition test result indicating that the visceral fat area is >100cm. 2 .

[0110] Simple obesity is the most common type of obesity, accounting for approximately 95% of obese individuals. Simply put, it's obesity not caused by disease. Patients with this condition have relatively even body fat distribution, no endocrine disorders, and no metabolic disorders. They often have a family history of obesity. Simple obesity can be categorized into constitutional obesity and overeating-related obesity.

[0111] Constitutional obesity, also known as parental obesity, is caused by genetics and an increased number of fat cells in the body, and is also related to overnutrition before the age of 25. This type of person has a slower and lower metabolic rate, with anabolism exceeding catabolism.

[0112] Overeating obesity, also known as acquired obesity, is caused by people consciously or unconsciously overeating in adulthood, which causes the calorie intake to far exceed the needs of body growth and activity. The excess calories are converted into fat, which promotes hypertrophy of fat cells and an increase in cell number, resulting in a large accumulation of fat and leading to obesity.

[0113] Secondary obesity differs from simple obesity in that it is caused by a disease. This condition, which accounts for approximately 2%-5% of obese individuals, is caused by endocrine or metabolic disorders. While similarly characterized by excessive fat deposition, secondary obesity primarily manifests as the clinical symptoms of the primary disease, with obesity being only one of the key symptoms. These patients may also experience a variety of other clinical manifestations, including those associated with increased cortisol, hypothyroidism, and hypogonadism.

[0114] Medication-induced obesity accounts for approximately 2% of the obese population. While some medications are effective in treating certain conditions, they can also cause obesity. For example, the use of adrenocortical hormones (such as dexamethasone) to treat allergic diseases, rheumatism, rheumatoid arthritis, and asthma can also lead to secondary obesity. Estrogen and contraceptives containing estrogen can sometimes cause women to gain weight, or at least predispose them to weight gain.

[0115] Secondary obesity is divided into the following seven categories:

[0116] (1) Hypothalamic obesity;

[0117] (2) pituitary obesity;

[0118] (3) hypercortisolism (also known as Cushing's syndrome);

[0119] (4) pancreatic obesity;

[0120] (5) hypothyroidism-related obesity;

[0121] (6) hypogonadal obesity;

[0122] (7) Drug-induced obesity.

[0123] metabolic syndrome

[0124] The core of metabolic syndrome is insulin resistance. The causes of insulin resistance are both hereditary (genetic defects) and acquired (environmental factors). Insulin resistance refers to a decrease in insulin's ability to promote glucose utilization. This reduced glucose utilization leads to elevated blood sugar levels, which in turn leads to a compensatory increase in insulin production, manifesting as hyperinsulinemia.

[0125] Pharmaceutical compositions and methods of administration

[0126] Since the P2RY12 inhibitors of the present invention have excellent activity in inhibiting obesity or overweight diseases, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate obesity or overweight diseases.

[0127] The pharmaceutical composition provided by the present invention preferably contains an active ingredient in a weight ratio of 0.001-99wt%, preferably a ratio in which the active compound of the present invention as the active ingredient accounts for 0.1wt% to 90wt% or 1wt% to 50wt% of the total weight, with the remainder being a pharmaceutically acceptable carrier, diluent or solution or saline solution.

[0128] The pharmaceutical composition of the present invention comprises a safe and effective amount of a compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 10-1000 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0129] "Pharmaceutically acceptable carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0130] The pharmaceutical composition is an aerosol, nasal drops, powder, gel, microsphere preparation, liposome preparation, and emulsion.

[0131] In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof.

[0132] Liquid dosage forms for nasal administration include pharmaceutically acceptable emulsions, solutions, suspensions or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, or mixtures thereof.

[0133] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0134] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0135] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0136] The treatment method of the present invention can be used alone or in combination with other treatment methods or therapeutic drugs.

[0137] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 50 to 1000 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0138] Compared with the prior art, the present invention has the following main advantages:

[0139] (1) The P2RY12 inhibitor of the present invention has unexpectedly excellent effects in alleviating and treating obesity, overweight and metabolic syndrome as a brain-targeting agent.

[0140] (2) The P2RY12 inhibitor of the present invention, as a brain-targeted preparation, requires a low unit dose and has good safety when treating obesity, overweight, and metabolic syndrome, avoiding serious side effects caused by large-dose administration (for example, as described in Marina Paul et al., Int. J. Mol. Sci. 2023, 24, 11706).

[0141] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0142] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0143] Materials and Methods

[0144] 1. Animal Model

[0145] Experimental procedures for all sexes of mice were performed on a C57BL / 6J genetic background (at least 9 backcrosses) according to international standards. Animals were maintained on a 12 / 12 h light / dark cycle with free access to water and food (11.1% from fat, feed catalog number: 1010088) or HFD diet (60% from fat, feed catalog number: XTHF60), both purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd. The diet-induced obesity (FLEX) mouse model was fed an HFD for 12 weeks starting at 5 weeks of age, unless otherwise stated in the results section. Oxt-Ires-Cre (JAX: 024234, further referred to as OXT Cre ) mice, as described previously (Wu Z et al., PLoS One 2012;7:e45167). Cynomolgus macaques serve as an important model for investigating the pathogenesis of human obesity, overweight, and metabolic diseases, as well as for evaluating new drugs and the efficacy of existing drug interventions. Six obese, elderly male cynomolgus macaques (Macaca fascicularis), aged 8 to 16 years and weighing 8.7 to 10.85 kg, were used. They were intranasally administered the P2RY12 antagonist congrelor daily for five weeks at a dose equivalent to the human daily dose of congrelor (0.18 mg / kg / day). Body weight changes before and after administration were analyzed and compared.

[0146] 2. Medication

[0147] Animals were administered a ticlopidine aqueous solution (31.5 mg / kg / day or 90 mg / kg / day; APExBIO, USA) or normal drinking water daily. Animals were then administered intranasally with ticlopidine (3.57 mg / kg; B2164, APExBIO, USA), clopidogrel (1 mg / kg; A5183, APExBIO, USA), prasugrel hydrochloride (0.14 mg / kg; B1283, APExBIO, USA), ticagrelor (2.4 mg / kg; B2166, APExBIO, USA), cangrelor (0.09 mg / kg; 163706-36-3, MedChemExpress, China), and normal drinking water daily.

[0148] 3. Metabolic Cage

[0149] For metabolic analysis, animals were acclimated in metabolic cages for 3 days before metabolic data were collected over the next three days. Parameters such as oxygen consumption, carbon dioxide and heat production, and respiratory exchange rate were measured in a comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments).

[0150] 4. Glucose tolerance test

[0151] After fasting for 16 hours, the mice were first tested for baseline blood glucose levels. Subsequently, 2 g / kg body weight of D-glucose was intraperitoneally injected into the mice. Blood glucose levels were measured at 15, 30, 60, 90, and 120 minutes using an ACCU-CHEK blood glucose meter (Roche).

[0152] 5. Insulin tolerance test

[0153] For the insulin tolerance test, mice were fasted for 4 hours, and their basal blood glucose levels were measured. Then, 0.5 U / kg body weight of insulin was injected intraperitoneally. Blood glucose levels were measured 15, 30, 60, 90, and 120 minutes after injection.

[0154] 6. Magnetic resonance imaging

[0155] Fat / lean body composition analysis was performed on a MesoMR23-060H-I (Newmai Technology) magnetic resonance imaging system according to the instrument manufacturer's experimental operating instructions.

[0156] 7. Nuclear Magnetic Resonance

[0157] After the experiment, the mice were decapitated and their brains were removed for hypothalamic dissection. The tissues were then immediately homogenized in deuterated dimethyl sulfoxide (DMSO). Ticagrelor was added to the deuterated dimethyl sulfoxide as a standard reference, and the tissue samples were analyzed using a 700 MHz nuclear magnetic resonance spectrometer (AVANCE NEO, Bruker, Germany).

[0158] 8. Histological Analysis

[0159] Adipose tissue was fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. Paraffin sections were cut into 5-μm sections and stained with hematoxylin and eosin (Beyotime, C0105S). Images were acquired using an optical microscope (Olympus). Adipocyte area was analyzed using ImageJ.

[0160] 9. Statistical Analysis

[0161] Statistics were performed using GraphPad Prism software (version 9.0). Data normality was tested using the Shapiro-Wilk normality test. Statistical comparisons between groups were performed using the unpaired Student's t-test. Multivariate comparisons were performed using two-way analysis of variance with Bonferroni multiple comparisons. Results are presented as mean ± SEM. P < 0.05 was considered statistically significant; *P < 0.05, **P < 0.01, ***: p < 0.001; ****: p < 0.0001.

[0162] Example 1. Detection of P2RY12 in the brains of diabetic patients revealed abnormal upregulation of oxytocin (OXT) neurons in the paraventricular nucleus of the hypothalamus

[0163] By using multiple immunofluorescence staining to detect brain slice specimens collected from a human brain bank, we found that compared with non-diabetic subjects, the expression of P2RY12 in OXT neurons in the paraventricular nucleus of the hypothalamus of diabetic patients was significantly increased (Figure 1).

[0164] Example 2. Abnormal upregulation of P2RY12 expression in oxytocin (OXT) neurons in the paraventricular nucleus of the hypothalamus of mice induced by high-fat diet

[0165] Subsequently, immunofluorescence was used to detect the expression level of P2RY12 in OXT neurons of mice fed a high-fat diet and mice fed a normal diet. It was found that the P2RY12 signal in OXT neurons of mice induced by a high-fat diet was significantly enhanced (Figure 2). At the same time, adult mice were injected with OXT-venus oxytocin neuron probes into the PVH region of the hypothalamus by brain stereotaxic injection. After 4 weeks of high-fat diet feeding, under the condition that there was no difference in body weight, it was found that the expression of microglial marker P2RY12 in OXT neurons of the hypothalamus PVH of mice fed a high-fat diet was significantly increased (Figure 3). Based on the above results, it is speculated that the abnormal expression of microglial marker P2RY12 in OXT neurons of the PVH is upregulated under metabolic stress.

[0166] Example 3. Overexpression of P2RY12 reduces the activity of hypothalamic cells

[0167] Overexpression of P2RY12 in hypothalamic cells leads to inactivation of the AC / cAMP / ERK / cFos signaling pathway in the G protein-coupled system (Figure 4). Molecular cloning was used to construct a P2RY12-overexpressing lentiviral vector. After viral packaging and titer testing, the vector was infected with hypothalamic GT1-7 cells. Flow cytometry was then used to screen for strongly GFP-positive cells. After cell expansion, a stably transfected P2RY12-overexpressing cell line was obtained. Finally, α-MSH was used to induce in vitro phenotypes. The flow chart is shown in Figure 5. Western blot analysis revealed a significant increase in P2RY12 expression in P2RY12 stably transfected cells, decreased ERK1 / 2 phosphorylation, and decreased c-Fos expression in overexpressing cells (Figures 6 and 7). Quantitative PCR revealed a significant increase in P2RY12 mRNA expression in P2RY12 overexpressing cells (Figure 8). ELISA revealed that P2RY12 overexpression decreased cyclic adenosine monophosphate (cAMP) levels (Figure 9). Furthermore, the induction of c-Fos by α-MSH was also counteracted by P2RY12 overexpression (Figure 10). Based on these results, we speculate that overexpression of P2RY12 in PVH OXT neurons may lead to a decrease in the reactivity of these melanocortin cells, thereby contributing to obesity.

[0168] Example 4. Obesity in HFD-fed mice can be reversed by inhibiting P2RY12

[0169] To further verify the above inference, rodent mice were used as animal models to conduct in vivo research. As an inhibitor of P2RY12, ticlopidine is a prodrug that can be converted into an irreversible P2RY12 inhibitor and is widely used in clinical practice to block platelet aggregation. IRES-CreGFP fl / wt Obese mice were administered the P2RY12 inhibitor ticlopidine at a dose of 90 mg / kg / day via drinking water (Figure 11). The results showed that the high dose of ticlopidine (equivalent to 27 times the human daily dose) led to rapid weight loss (Figure 11). Furthermore, the ticlopidine inhibitor reversed obesity in the control mice fed a high-flow diet (HFD), suggesting that P2RY12 may also be abnormally overexpressed in the control obese mice, suggesting that P2RY12 may play a pathogenic role in the pathogenesis of obesity in these mice. It is also worth noting that other P2RY12 inhibitors, such as clopidogrel and prasugrel (27 times the human daily dose) or ticlopidine at a lower dose (approximately 9 times the human daily dose), were administered to obese mice via drinking water. The HFD did not significantly reverse the obese phenotype of the mice (Figure 12).

[0170] Example 5. Nasal injection of a P2RY12 inhibitor can reverse obesity and insulin resistance

[0171] To reduce the dose of P2RY12 inhibitors to levels comparable to those used in human clinical practice while also ensuring high brain concentrations for their efficacy, we employed intranasal administration. We tested the therapeutic effects of five P2RY12 inhibitors in obese mice. The experimental procedures are shown in Figures 13 and 30. Obese mice fed a high-fat diet were intranasally administered with irreversible inhibitor prodrugs (ticlopidine, prasugrel, and clopidogrel) and clinically equivalent doses of reversible direct inhibitors (congrelor and ticagrelor). With the exception of clopidogrel, the other four inhibitors significantly suppressed appetite (Figures 22 and 35) and rapidly reversed the obese phenotype (Figures 14-19, 31-32). Furthermore, in a glucose tolerance test, congrelor and prasugrel significantly improved glucose clearance compared to ticlopidine and ticlopidine (Figures 20 and 33). In addition, insulin tolerance tests revealed that prasugrel, ticlopidine, ticagrelor, and congrelor significantly improved fasting blood glucose levels and insulin resistance in mice fed a high-fat diet (Figures 21, 34). Unlike the other four P2RY12 inhibitors, we found that intranasal administration of the irreversible prodrug clopidogrel at normal doses resulted in a transient, significant decrease in body weight in obese mice only during the first week of treatment; there was a non-significant trend toward weight loss at other times (Figures 14, 15). For the other inhibitors, intranasal administration of congrelor at half the human daily intravenous dose was still effective in treating obesity. Intranasal administration of ticlopidine, ticagrelor, or prasugrel at human oral doses also demonstrated effective treatment of obesity, with the effective intranasal dose of ticlopidine being 27-fold lower than the oral dose (Figures 14, 31).

[0172] Example 6. Effect of Nasal Administration of P2RY12 Inhibitors on Weight Loss in Obese Cynomolgus Monkeys

[0173] We systematically investigated and evaluated the efficacy of various P2RY12 antagonists in obese mice using in vivo mouse models. Further testing and validation in larger mammalian models, such as monkeys, dogs, and pigs, is warranted. The metabolism and distribution of adipose tissue in cynomolgus monkeys closely resemble those in humans, and the obesity symptoms induced by a high-fat diet in cynomolgus monkeys mirror those in humans, making them an excellent model for studying obesity and metabolic diseases. After removing elderly obese cynomolgus monkeys from dietary restriction, we administered the P2RY12 antagonist congrelor daily intranasally for five weeks at a dose equivalent to that in humans (0.18 mg / kg / day) (Figure 36). We found that compared to cynomolgus monkeys receiving a placebo (normal saline), those receiving congrelor significantly inhibited weight gain, demonstrating a significant therapeutic effect (Figures 37, 38).

[0174] Example 7. Overexpression of P2RY12 in PVH OXT neurons leads to obesity in mice

[0175] To further verify the obesogenic effect of P2RY12, FLEX adeno-associated virus vector was injected into OXT via stereotaxic brain localization. Cre In adult mice, P2RY12 was specifically overexpressed in OXT neurons in the PVH hypothalamus (PVH) (Figure 39). Consistent with our expectations, both male and female mice overexpressing P2RY12 in OXT neurons experienced significant weight gain. Intranasal administration of the P2RY12 inhibitor prasugrel at the experimental concentrations described above reversed the obese phenotype (Figures 40-49). Mechanistically, the P2RY12 inhibitor restored c-Fos expression in OXT neurons (Figure 41). Thus, we have demonstrated in vivo that overexpression of P2RY12 in PVH OXT neurons leads to obesity, and treatment with a specific inhibitor of the ADP / ATP receptor reverses this obese phenotype, confirming that ectopic expression of P2RY12 in OXT neurons contributes to obesity. Furthermore, male mice appear to be more sensitive to P2RY12 than female mice. Following P2RY12 overexpression, female mice exhibited a later onset of the obesity phenotype, which was less severe than in males (Figure 44). The reason for this difference may be related to gender.

[0176] discuss

[0177] As shown in Table 1 below, when the P2RY12 inhibitor of the present invention is used as a brain-targeted formulation for the treatment of obesity and overweight, the required dosage is much lower than the conventional dosage required for oral administration. Moreover, when administered at a low dose as a brain-targeted formulation, the weight loss effect is significant, with the maximum weight loss percentage being much higher than that of the corresponding oral formulation. Furthermore, the inhibitor also exhibits significant metabolic activity in tests of food intake, glucose tolerance, and insulin tolerance.

[0178] Table 1 Metabolic activity of P2RY12 inhibitors measured

[0179] Note: N / A means not measured;

[0180] Comparison of body weight before and after administration of HFD mice; Maximum weight loss percentage: the highest weight loss ratio measured after administration, p value calculated by two-tailed paired t test;

[0181] The percentage decrease in food intake, glucose or insulin area under the curve (AUC) after drug administration compared with that of the HFD control group was statistically analyzed by one-way ANOVA (Tukey) followed by Dunnet's post hoc test, using 0 g and 4 mmol / L as the baselines for food intake and tolerance testing, respectively.

[0182] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A use of a P2RY12 inhibitor, characterized in that: Used for preparing a medicament for treating obesity, overweight and / or metabolic syndrome in a subject, wherein the medicament is a brain-targeted preparation.

2. The use according to claim 1, characterized in that The brain-targeted agent is an agent that targets hypothalamic cells.

3. The use according to claim 1, characterized in that The brain-targeted formulation is a nasal formulation.

4. The use according to claim 1, wherein The P2RY12 inhibitor is selected from the group consisting of clopidogrel, prasugrel, ticlopidine, ticagrelor, congrelor, antibodies, phages, nanobodies and other small molecules or macromolecules that selectively or semi-selectively target P2RY12.

5. The use according to claim 1, characterized in that The subject has one or more characteristics selected from the group consisting of: (a) the expression level of P2RY12 in the hypothalamic neuronal cells of the subject is relatively high; (b) low cyclic adenosine monophosphate (cAMP) levels in hypothalamic neurons of the subject; (c) the level of ERK1 / 2 phosphorylation in the hypothalamic neuronal cells of the subject is low; (d) The expression level of c-Fos in the hypothalamic neuronal cells of the subject is relatively low.

6. The use according to claim 1, wherein The P2RY12 inhibitor is a reversible P2RY12 inhibitor or an irreversible P2RY12 inhibitor.

7. The use according to claim 1, characterized in that The obesity, overweight and metabolic syndrome diseases are selected from the group consisting of simple obesity, secondary obesity and insulin resistance.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition is a brain-targeting formulation; The pharmaceutical composition contains a P2RY12 inhibitor selected from the group consisting of clopidogrel, prasugrel, ticlopidine, ticagrelor, congrelor, antibodies, phages, nanobodies and other small molecules or macromolecules that selectively or semi-selectively target P2RY12, or a combination thereof; and a pharmaceutically acceptable carrier.

9. The use of the pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is used for preparing medicine for treating obesity, overweight and metabolic syndrome diseases of a subject.

10. The use of the pharmaceutical composition according to claim 8, characterized in that: The pharmaceutical composition is used as a separate nasal preparation in combination with other effective obesity, overweight and metabolic syndrome treatment methods (surgical treatment, etc.) in the comprehensive treatment of obesity or overweight and metabolic syndrome.

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