WO2025189623A1 - Use of p2ry12 inhibitor in preparation of drug for treating obesity, overweight, and metabolic syndrome diseases - Google Patents

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

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

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.

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 strong side effects.

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.

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.

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.

Summary of the Invention

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

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.

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

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

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

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.

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.

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

(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.

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.

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).

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.

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.

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

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

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

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.

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

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.

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

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;

and a pharmaceutically acceptable carrier.

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.

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.

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.

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

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.

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.

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.

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.

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

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

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.

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.

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.

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, and α-MSH in OXT neurons of the PVH is activated. The MC4R/AC/cAMP/ERK/cFos signaling pathway, followed by important neural signaling-related gene transcription and expression, maintains the balance of energy homeostasis by reducing appetite and increasing energy consumption; metabolic stress stimulates NFkB to induce the overexpression of P2RY12, and P2RY12 inhibits adenylate cyclase AC and its downstream cAMP/ERK/cFos signaling pathway through Ga _i protein, resulting in the obstruction of neural signaling-related gene transcription and expression, increased appetite, reduced energy consumption, energy homeostasis imbalance and obesity.

Figure 5 shows the flowchart of P2RY12 overexpression in GT1-7 cells and its in vitro experimental operation; the P2RY12 overexpression lentiviral vector was constructed by molecular cloning, and after viral packaging and titer detection, the hypothalamic GT1-7 cells were infected, and then the GFP-positive target cells were screened by flow cytometry. After amplification, a stably transfected P2RY12 overexpression cell line was obtained. After cell starvation and α-MSH drug induction together with the control group cells, the relevant phenotypes were detected in vitro by fluorescence quantitative PCR, enzyme-linked immunosorbent assay (ELISA) and Western Blot.

Figure 6 shows Western Blot analysis of changes in ERK1/2 phosphorylation levels at Thr202/Tyr204 and c-Fos expression in GT1-7 cells overexpressing P2RY12.

Figure 7 shows the quantitative analysis of Western Blot results (n=3) error bars represent SEM; *: p<0.05; **: p<0.01; analyzed by one-way ANOVA (Tukey).

Figure 8 shows the mRNA level of P2RY12 detected by fluorescence 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).

Figure 9 shows the cyclic AMP levels in GT1-7 cells overexpressing P2RY12 before and after α-MSH treatment detected by ELISA (n=3). Error bars represent SEM; **: p<0.01; ***: p<0.001; analyzed by one-way ANOVA (Tukey).

Figure 10 shows the mRNA level of c-Fos detected by fluorescence 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).

Figure 11 shows the changes in body weight of adult mice that were first fed with a normal diet and then fed with a high-fat diet and administered with ticlopidine (90 mg/kg/day) through drinking water (high-fat diet + normal saline n = 4, high-fat diet + ticlopidine n = 4); **: p < 0.01, one-way ANOVA RM, Dunnet).

Figure 12 shows the changes in body weight of obese adult mice fed with a high-fat diet (HFD) after administration of three different gradient concentrations of the P2RY12 inhibitors ticlopidine, clopidogrel, and prasugrel through drinking water (high 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) were analyzed by two-way ANOVA (Tukey).

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.

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).

Figure 15 shows the changes in mouse body weight 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 t test.

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

Figure 17 shows the 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) analyzed by low-field nuclear magnetic resonance. Error bars represent SEM; *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; analyzed by two-way ANOVA, Tukey).

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

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).

FIG20 shows 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).

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; analyzed by two-way ANOVA, Tukey).

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

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) of a high-fat diet. (Number of mice fed a normal diet + control: n = 5; number of mice fed a high-fat diet + control: n = 5; number of mice fed a high-fat diet + ticlopidine: n = 7). Error bars represent SEM; *: p < 0.05; **: p < 0.01; two-way ANOVA, Tukey).

Figure 24 shows changes in oxygen consumption in mice after treatment with the P2RY12 inhibitor prasugrel in metabolic cage analysis. (N = 4 for control group on a regular 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; analyzed by two-way ANOVA (Tukey).

Figure 25 shows changes in carbon dioxide emissions in mice after treatment with the P2RY12 inhibitor prasugrel in metabolic cage analysis. (N = 4 for control group on a regular 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; analyzed by two-way ANOVA (Tukey).

Figure 26 shows changes in thermogenesis in mice following treatment with the P2RY12 inhibitor prasugrel as analyzed in metabolic cages. (N = 4 for control group on a regular 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).

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).

Figure 28 shows body weight changes in 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; analyzed by two-way ANOVA (Tukey).

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.

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.

Figure 31 shows the body weight changes of intranasally injected 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).

Figure 32 shows the 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; analyzed by two-way ANOVA, Tukey).

Figure 33 shows the glucose tolerance test analysis of the blood glucose levels of 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; analyzed by two-way ANOVA, Tukey).

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).

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

Figure 36 shows a flow chart of an experiment in which caloric restriction in elderly crab-eating macaques (Macaca fascicularis) was stopped and a P2RY12 inhibitor nasal spray was injected once daily at a dose equivalent to the daily dose of congrelor in humans (0.18 mg/kg/d).

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, ).

Figure 38 shows that the P2RY12 inhibitor nasal spray was injected once daily into the nasal cavity, which is equivalent to the daily dose of canagrelor in humans (0.18 mg/kg/d). The area under the normalized weight curve was calculated to represent the body weight change (canagrelor n=3, saline n=3). The error bars represent SEM; *: p<0.05; analyzed by Student's t test.

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.

Figure 40 shows the changes in body weight in 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's method).

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.

FIG42 shows 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; two-way ANOVA, Tukey).

Figure 43 shows 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; two-way ANOVA, Tukey).

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

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

Figure 46 shows metabolic cage analysis of oxygen consumption changes 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 represent SEM; *: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; two-way ANOVA, Tukey).

Figure 47 shows changes in carbon dioxide emissions in 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 represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

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

Figure 49 shows changes in respiratory rate in 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 represent SEM; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; two-way ANOVA, Tukey).

DETAILED DESCRIPTION

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.

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.

P2RY12

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.

P2RY12 inhibitors

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.

The first-generation ticlopidine is an inactive prodrug and has been gradually withdrawn from clinical use due to the high incidence of hematological side effects, such as thrombotic thrombocytopenic purpura (TTP), neutropenia, and aplastic anemia. Clopidogrel is currently the most widely used P2RY12 inhibitor. It is an inactive prodrug. After oral administration, the active metabolite produced in the body irreversibly binds to the P2RY12 receptor, rendering the receptor unable to respond to ADP, thereby blocking platelet function and effectively reducing ADP-mediated platelet activation and aggregation. Disadvantages include a slow onset of action and significant individual 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. Oral prasugrel has a more rapid onset of action and less individual variability in efficacy than clopidogrel.

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 can act directly on the P2RY12 receptor. Its action is direct, reversible, and rapid. Its long-lasting effect and slow clearance make it more effective in preventing 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 can only be administered intravenously. Its short half-life allows for targeted antiplatelet therapy within a prescribed timeframe, based on clinical needs.

Oxytocin neurons

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.

cAMP

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. This molecule has a wide range of physiological functions.

ERK1/2

Extracellular regulated protein kinases, collectively known as ERK1/2. There are two ERK isoforms: ERK1 and ERK2, which share 84% of their amino acid residues. ERK1/2 has a typical protein kinase structure and regulates cellular life activities by phosphorylating substrates. ERK1/2 is a key component of the RAF-MEK-ERK signaling pathway. Phosphorylation at Thr202 and Tyr204 leads to activation, which in turn activates a variety of substrates (over 160) related to 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 indispensable detection indicator in related research.

c-Fos

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.

Obesity or overweight

Obesity or overweight is determined based on the Body Mass Index (BMI) in adults. A BMI of 24.0 kg/ ^m² ≤ < 28.0 kg/ ^m² is considered overweight, and a BMI ≥ 28.0 kg/ ^m² is considered obese. Central obesity can be determined based on waist circumference: A waist circumference of 90 cm or greater for men and 85 cm or greater for women is considered central obesity in adults. Visceral obesity is defined as a visceral fat area > 100 ^cm² as indicated by body composition testing.

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.

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.

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.

The difference between secondary obesity and simple obesity is that secondary obesity is obesity caused by disease. Secondary obesity is a type of disease caused by endocrine disorders or metabolic disorders, accounting for about 2%-5% of obese people. Although it also has the characteristics of excessive fat deposition in the body, it still has the clinical symptoms of primary diseases as the main manifestation. Obesity is only one of the important symptoms of this type of patients. This type of patients also have various other The clinical manifestations of thyroid disease are mostly manifested in various diseases such as increased cortical enzymes, hypothyroidism, and hypogonadism.

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 can be more susceptible to weight gain.

Secondary obesity is divided into the following seven categories:

(1) Hypothalamic obesity;

(2) pituitary obesity;

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

(4) pancreatic obesity;

(5) hypothyroidism-related obesity;

(6) hypogonadal obesity;

(7) Drug-induced obesity.

metabolic syndrome

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.

Pharmaceutical compositions and methods of administration

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.

The pharmaceutical composition provided by the present invention preferably contains an active ingredient in a weight ratio of 0.001-99wt%, preferably a ratio of the active compound of the present invention as an active ingredient accounting for 0.1wt% to 90wt% of the total weight or 1wt% to 50wt%, and the rest is a pharmaceutically acceptable carrier, diluent or solution or saline solution.

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.

"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.

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

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.

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, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, Castor oil and sesame oil or a mixture of these substances, etc.

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

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.

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

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

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.

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

(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.

(2) The P2RY12 inhibitor of the present invention is used as a brain-targeted preparation to treat obesity, overweight and metabolic syndrome diseases. The required unit dose is low and the safety is good, avoiding the serious side effects caused by large-dose administration (for example, as described in Marina Paul et al., Int. J. Mol. Sci. 2023, 24, 11706).

The present invention will be further described below with reference to 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. Experimental methods in the following examples where specific conditions are not specified are 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

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.

Materials and Methods

1. Animal Model

Experimental procedures were performed in mice of all sexes on a C57BL/6J genetic background (at least nine backcrosses) according to international standards. Animals were maintained on a 12/12-hour light/dark cycle with free access to water and food (11.1% from fat, chow product number: 1010088) or a HFD diet (60% from fat, chow product 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 noted in the Results section. Oxt-Ires-Cre (JAX: 024234, further referred to as OXT- ^Cre ) mice were used 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 crab-eating macaques (Macaca fascicularis), aged between 8 and 16 years old and weighing between 8.7 and 10.85 kg, were used. They were given daily nasal injections of the P2RY12 antagonist congrelor, at a dose equivalent to the daily dose of congrelor in humans (0.18 mg/kg/d), for 5 consecutive weeks. The weight changes of the crab-eating macaques before and after administration were analyzed and compared.

2. Medication

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.

3. Metabolic Cage

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).

4. Glucose tolerance test

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).

5. Insulin tolerance test

Insulin tolerance test, mice were fasted for 4 hours, and the basal blood glucose level was measured first, followed by 0.5 U/kg Insulin was injected intraperitoneally based on body weight, and blood glucose levels were measured 15, 30, 60, 90, and 120 minutes after injection.

6. Magnetic resonance imaging

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.

7. Nuclear Magnetic Resonance

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).

8. Histological Analysis

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.

9. Statistical Analysis

Statistics were performed using GraphPad Prism software (version 9.0). The Shapiro-Wilk normality test was used to test the normality of the data. Statistical comparisons between the two groups were performed using the unpaired Student’s t-test. Multivariate comparisons were performed using two-way analysis of variance with Bonferroni multiple comparisons. The results are shown as the mean ± SEM. Statistical significance was considered to be statistic- ally significant at P < 0.05; *P < 0.05, **P < 0.01, ***: p < 0.001; ****: p < 0.0001.

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

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).

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

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). After injecting a neuronal probe into the PVH region of the hypothalamus and feeding mice a high-fat diet for four weeks, the researchers found that the expression of the microglial marker P2RY12 was significantly increased in OXT neurons in the PVH of the hypothalamus of mice fed a high-fat diet, even though their body weight remained unchanged (Figure 3). These results suggest that metabolic stress may be responsible for the abnormal upregulation of microglial marker P2RY12 in OXT neurons of the PVH.

Example 3. Overexpression of P2RY12 reduces the activity of hypothalamic cells

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 led to decreased 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 reduce the reactivity of these melanocortin cells, leading to obesity.

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

To further verify the above inferences, rodent mice were used as an animal model for in vivo research. Ticlopidine, as a P2RY12 inhibitor, is a prodrug that can be converted into an irreversible P2RY12 inhibitor and is widely used clinically to block platelet aggregation. Transgenic OXT ^IRES-Cre GFP ^fl/wt obese mice fed a high-fat diet (HFD) 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) caused rapid weight loss (Figure 11). At the same time, the ticlopidine inhibitor also reversed the obesity of the control mice fed an HFD, indicating that P2RY12 may also be abnormally overexpressed in the control obese mice, suggesting that P2RY12 may play a pathogenic role in the onset of obesity in these mice. It is also worth noting that for other P2RY12 inhibitors, the same high dose (27 times the human daily dose) of clopidogrel and prazidor also reversed the obesity of the control mice fed an HFD. After administration of ticlopidine, or a lower dose (about 9 times the human daily dose) of ticlopidine to the drinking water of obese mice, it was found that the obese phenotype of the mice was not significantly reversed by HFD ( Figure 12 ).

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

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).

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

We systematically explored and evaluated the efficacy of various P2RY12 antagonists on obese mice using an in vivo mouse model. On this basis, it is necessary to test and verify in animal models such as large mammals, monkeys, dogs, and pigs. The metabolism and distribution of adipose tissue in crab-eating macaques are very similar to those in humans. The obesity symptoms in crab-eating macaques caused by a high-fat diet are also the same as those in humans. It is the best model for studying obesity and metabolic diseases. Elderly obese crab-eating macaques stopped restricting their diet, and we injected the P2RY12 antagonist congrelo daily through the nose at a dose equivalent to the daily dose of congrelo in humans (0.18 mg/kg/d) for 5 consecutive weeks (Figure 36). The results showed that compared with the crab-eating macaques injected with a placebo (normal saline), the experimental group injected with congrelo through the nose had a significantly lower incidence of obese mice. The weight gain of cynomolgus monkeys was significantly inhibited, showing a significant therapeutic effect (Figures 37, 38).

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

To further validate the obesogenic effect of P2RY12, we overexpressed P2RY12 specifically in OXT neurons of adult mice by stereotaxic injection of a FLEX adeno-associated viral vector into the PVH region of the hypothalamus of OXT- ^Cre mice (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 OXT neurons of the PVH leads to obesity, and that treatment with a specific inhibitor of the ADP/ATP receptor can reverse this obese phenotype, confirming that ectopic expression of P2RY12 in OXT neurons has a pathogenic obesogenic effect. Furthermore, male mice appear to be more sensitive to P2RY12 than female mice. After overexpression of P2RY12, female mice developed an obesity phenotype later and with a milder phenotype than male mice ( Figure 44 ). This difference may be due to sex.

discuss

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.

Table 1 Metabolic activity of P2RY12 inhibitors measured

Note: N/A means not measured;

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;

Compared with the food intake, glucose or insulin area under the curve (AUC) of the HFD control group, the The percentage of decline after drug administration 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.

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 (10)

A use of a P2RY12 inhibitor, characterized in that it is used to prepare a medicament for treating obesity, overweight and/or metabolic syndrome in a subject, wherein the medicament is a brain-targeted preparation. The use according to claim 1, wherein the brain-targeting preparation is a preparation targeting hypothalamic cells. The use according to claim 1, wherein the brain targeting preparation is a nasal preparation. The use according to claim 1, characterized in that 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. The use according to claim 1, wherein 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. The use according to claim 1, characterized in that the P2RY12 inhibitor is a reversible P2RY12 inhibitor or an irreversible P2RY12 inhibitor. 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. A pharmaceutical composition, characterized in that the pharmaceutical composition is a brain-targeting preparation; 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. The use of the pharmaceutical composition according to claim 8, characterized in that the pharmaceutical composition is used to prepare a medicament for treating obesity, overweight and metabolic syndrome in a subject. 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 disease treatment methods (surgical treatment, etc.) in the comprehensive treatment of obesity or overweight and metabolic syndrome diseases.