Application of MDH2 inhibitor in preparation of medicine for treating abnormal glucose metabolism diseases

Abstract

The invention provides application of an MDH2 inhibitor to treatment of abnormal glucose metabolism diseases and preparation of drugs for treating abnormal glucose metabolism. Through gene knockdown or knockout and drug intervention, it is found that MDH2 plays a key role in central glycometabolism induction, and targeting MDH2 may provide a new treatment method for improving the blood glucose level of diabetic patients. The application also finds that the LW6 can improve glucose metabolism disorder and treat diabetes mellitus by targeting an MDH2 target, verifies the effectiveness and safety of the LW6 under central and peripheral administration conditions, does not cause weight loss, eating reduction or other metabolism-related side effects, and reveals a potential neurological mechanism and clinical application value of the LW6.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and in particular to the use of an MDH2 inhibitor in the treatment of abnormal glucose metabolism diseases and in the preparation of a medicament for treating abnormal glucose metabolism diseases. BACKGROUND

[0002] Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia, including various types such as type 1 diabetes, type 2 diabetes and gestational diabetes. Among them, type 2 diabetes (T2D) accounts for more than 90% of all diabetes cases. The pathogenesis of T2D is complex, mainly caused by insulin resistance and decline of pancreatic beta cell function, often accompanied by metabolic disorders such as obesity and hyperlipidemia. With the changes in social lifestyle and population aging, the incidence of T2D is showing a sustained upward trend, causing a significant burden on public health.

[0003] Currently, the treatment of T2D mainly includes lifestyle intervention, oral hypoglycemic drugs (such as metformin, sulfonylurea drugs, SGLT2 inhibitors, etc.) and insulin injection, etc. However, the existing treatment methods have many limitations, including:

[0004] Poor long-term efficacy: many drugs can only control blood glucose, and it is difficult to stop or reverse pancreatic function decline;

[0005] Side effects: some drugs such as sulfonylureas are prone to cause hypoglycemia, and GLP-1 agonists may cause gastrointestinal adverse reactions;

[0006] Individual differences: due to differences in metabolic status and drug sensitivity in patients, the efficacy fluctuates greatly.

[0007] Abnormal glucose metabolism not only causes diabetes, but also causes various diseases including overweight, obesity, neuropathic pain, etc.

[0008] In recent years, the role of the central nervous system (CNS) in metabolic regulation has received increasing attention. Studies have shown that the hypothalamus is an important central region for regulating energy metabolism and blood glucose homeostasis, and the hypothalamic paraventricular nucleus (PVN) contains glucose-sensitive neurons that can affect circulating glucose levels through autonomic or neuroendocrine pathways. However, current research on diabetes treatment drugs targeting central targets is still in its early stages, and there is an urgent need to develop new drugs and methods.

[0009] MDH2 (Malate dehydrogenase 2) is an important mitochondrial malate dehydrogenase involved in the tricarboxylic acid cycle (TCA) and the malate-aspartate shuttle system. Its main function is to catalyze the conversion of malate to oxaloacetate and vice versa, which is essential for the regeneration of oxaloacetate in the TCA cycle and the malate-aspartate shuttle system. +) generating oxaloacetate and reduced nicotinamide adenine dinucleotide (NADH). As a key metabolic enzyme, MDH2 plays an important role in maintaining mitochondrial metabolic homeostasis, regulating energy production and oxidative stress.

[0010] In 2007, Lee's group first reported a novel inhibitor of hypoxia-inducible factor HIF-1, LW6 (CAS No. 934593-90-5), through structure-activity relationship studies, the structural requirements of the N-(aryloxyacetamido) benzamide skeleton were determined. Subsequently, they synthesized a series of chemical probe molecules derived from LW6, confirming that LW6 directly acts on MDH2 in mitochondria. However, its regulatory effect on glucose metabolism has not been found, and the specific hypoglycemic mechanism and effect under different administration routes have not been determined.

[0011] At present, the function of MDH2 in the central nervous system is less studied, and its specific relationship with glucose metabolism has not been explored. SUMMARY

[0012] The present application provides a new target MDH2 for central glucose homeostasis regulation, and improves glucose metabolism disorder and treats type 2 diabetes by targeting MDH2 target. Through gene knockdown or knockout and drug intervention, the present application finds that MDH2 plays a key role in central glucose metabolism sensing, and targeting MDH2 may provide a new treatment method for improving the blood glucose level of patients with type 2 diabetes. The present application also first finds that LW6 can improve glucose metabolism disorder and treat type 2 diabetes by targeting MDH2 target, verifies the effectiveness and safety of LW6 under the conditions of central and peripheral administration, and does not cause weight loss, food reduction and other metabolic related side effects, reveals the potential neural mechanism and clinical application value of LW6.

[0013] The specific technical solutions of the present application are as follows:

[0014] 1. Use of an MDH2 inhibitor in the preparation of a medicament for the treatment of a glucose metabolism disorder.

[0015] 2. Use of an MDH2 inhibitor in the treatment of a glucose metabolism disorder.

[0016] 3. The use according to item 1 or 2, wherein the glucose metabolism disorder comprises overweight, obesity, diabetes, neuropathic pain, preferably diabetes.

[0017] 4. The use according to any one of items 1 to 3, wherein the MDH2 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule or a gene editor comprising a nucleic acid molecule that hybridizes to an MDH2 nucleic acid molecule.

[0018] 5. The use according to item 4, wherein the MDH2 inhibitor is a small molecule compound, preferably the small molecule compound is LW6.

[0019] 6. A pharmaceutical composition for treating a glucose metabolism disorder, comprising a MDH2 inhibitor.

[0020] 7. The pharmaceutical composition according to item 6, wherein the MDH2 inhibitor comprises a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule or a gene editor comprising a nucleic acid molecule hybridized to a MDH2 nucleic acid molecule.

[0021] 8. The pharmaceutical composition according to item 6, wherein the MDH2 inhibitor is a small molecule compound, preferably the small molecule compound is LW6.

[0022] 9. The pharmaceutical composition according to any one of items 6-8, comprising a pharmaceutically acceptable carrier for delivering the MDH2 inhibitor.

[0023] 10. A method of treatment, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 6-10.

[0024] The present application uses various animal models (normal mice, rats and diabetic model mice) to verify the broad applicability and potential clinical transformation value of LW6. The beneficial effects of the present application are as follows:

[0025] 1. The present application first found that LW6 can effectively reduce blood glucose level by targeting the central nervous system, which has high application potential.

[0026] 2. LW6 can effectively regulate blood glucose by both central administration and peripheral intraperitoneal injection, which provides more extensive feasibility for its clinical application.

[0027] 3. LW6 only has a hypoglycemic effect on hyperglycemia, and does not cause hypoglycemia, significant weight loss, reduced food intake or metabolic side effects such as changes in activity level while lowering blood glucose.

[0028] 4. LW6 as a new type of hypoglycemic drug provides a new idea and direction for the treatment of metabolic diseases including overweight, obesity, diabetes, neuropathic pain, etc. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 a is the Western Blotting experimental result gel chart of MDH2 protein expression level after culturing neurons with 0 mM, 25 mM, 50 mM glucose and 50 mM mannitol for 24 h, respectively.

[0030] Figure 1b is the comparison of the expression level of MDH2 protein after treating neurons with 0 mM, 25 mM, 50 mM glucose and 50 mM mannitol for 24 h, respectively.

[0031] Figure 2 a is the experimental schematic diagram of targeting knock-out of neurons or glial Mh2 in Drosophila by RNAi.

[0032] Figure 2 b is the content of trehalose and triglyceride in Drosophila with neuron-specific knock-out of Mdh2.

[0033] Figure 2 c is the content of trehalose and triglyceride in Drosophila with glial cell-specific knock-out of Mdh2.

[0034] Figure 3 a is the schematic diagram of injecting AAV-U6-shRNA (MDH2)-CMV-EGFP-SV40 virus to knock down Mdh2 in the PVN brain region of WT mice.

[0035] Figure 3 b is the change of blood glucose level over time in mice with PVN brain region-specific knock-down of Mdh2 and control group mice;

[0036] Figure 3 c is the change of blood glucose level over time in mice with PVN brain region-specific knock-down of Mdh2 and control group mice during IPGTT experiment.

[0037] Figure 3 d is the area under the curve of the change of blood glucose level over time in mice with PVN brain region-specific knock-down of Mdh2 and control group mice during IPGTT experiment.

[0038] Figure 3 e is the comparison of the change of body weight in mice with PVN brain region-specific knock-down of Mdh2 and control group mice within 24 hours at the fourth week after virus injection.

[0039] Figure 3 f is the comparison of the food intake in mice with PVN brain region-specific knock-down of Mdh2 and control group mice within 24 hours at the fourth week after virus injection.

[0040] Figure 4 a is the schematic diagram of stereotactic cannulation of the third ventricle (3V) of C57BL / 6J mice.

[0041] Figure 4 b is the brain tissue section of 3V cannulated mice with cannulation traces.

[0042] Figure 4 c is the change of blood glucose level over time in mice with 3V injection of LW6 and control group mice.

[0043] Figure 4 d is the comparison of blood glucose levels in mice 15 minutes after 3V injection of LW6 with control mice.

[0044] Figure 4 e is the change in blood glucose levels over time in mice injected with LW6 in the 3V and control mice during IPGTT experiments.

[0045] Figure 4 f is the area under the curve of the change in blood glucose levels over time in mice injected with LW6 in the 3V and control mice during IPGTT experiments.

[0046] Figure 4 g is the comparison of blood glucose levels in mice 15 minutes after 3V injection of LW6 with control mice.

[0047] Figure 4 h is the comparison of insulin levels in mice 15 minutes after 3V injection of LW6 with control mice.

[0048] Figure 4 i is the comparison of weight change in mice 24 hours after 3V injection of LW6 with control mice.

[0049] Figure 4 j is the comparison of food intake in mice 24 hours after 3V injection of LW6 with control mice.

[0050] Figure 4 a is a schematic of SD rats injected with LW6 (30 mM / 3 μL) via 3V cannulation.

[0051] Figure 5 b is the change in blood glucose levels over time in rats injected with LW6 in the 3V and control rats.

[0052] Figure 5 c is the comparison of blood glucose levels in rats 1 hour after 3V injection of LW6 with control rats.

[0053] Figure 5 d is the change in blood glucose levels over time in rats injected with LW6 in the 3V and control rats during IPGTT experiments.

[0054] Figure 5 e is the area under the curve of the change in blood glucose levels over time in rats injected with LW6 in the 3V and control rats during IPGTT experiments.

[0055] Figure 5 f is the comparison of weight change in rats 24 hours after 3V injection of LW6 with control rats.

[0056] Figure 5 g is the comparison result of food intake of rats within 24 hours after 3V injection of LW6 with that of control rats.

[0057] Figure 5 a is a timeline diagram of high-fat feeding and intraperitoneal injection of streptozotocin (STZ) to construct a type 2 diabetes mouse model, intraperitoneal injection of LW6 treatment, and blood glucose monitoring.

[0058] Figure 6 b is the body weight change of mice during the construction of type 2 diabetes.

[0059] Figure 6 c is the comparison result of blood glucose levels of mice before and after injection of STZ.

[0060] Figure 6 d is the change of blood glucose levels over time after injection of drugs in type 2 diabetes mice treated with intraperitoneal injection of LW6, type 2 diabetes control mice, and normal mice treated with intraperitoneal injection of LW6, respectively.

[0061] Figure 6 e is the comparison result of type 2 diabetes mice treated with intraperitoneal injection of LW6, type 2 diabetes control mice, and normal mice treated with intraperitoneal injection of LW6, respectively, at 2 hours after injection of drugs.

[0062] In the figure, “*” represents P < 0.05, “**” represents P < 0.01, “***” represents P < 0.001, and “ns” indicates no significant difference. DETAILED DESCRIPTION

[0063] Specific embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present application can be more thoroughly understood and the scope of the present application can be accurately conveyed to those skilled in the art.

[0064] In the present application, LW6 refers to a HIF-1α inhibitor, CAS No. 934593-90-5, with a molecular formula of C 26 H 29 NO5, and a molecular weight of 435.51.

[0065] In the present application, the subject refers to a human.

[0066] The technical solution of the present application includes the following contents:

[0067] The inventors found that MDH2 plays a key role in the central glucose metabolism sensing without causing weight loss, food intake reduction and other metabolic related side effects, and based on this, the present application provides use of MDH2 inhibitors in the preparation of a medicament for treating abnormal glucose metabolism.

[0068] The MDH2 inhibitor described in the present application can inhibit the expression, content or activity of the Mdh2 gene or RNA or the protein encoded thereby.

[0069] The MDH2 inhibitor described in the present application can inhibit the expression of the MDH2 gene or MDH2 protein, thereby effectively reducing the blood glucose level. Experimental results show that the MDH2 inhibitor can effectively regulate blood glucose by central administration and peripheral intraperitoneal injection, and it only has a blood glucose-lowering effect on hyperglycemia, and does not cause hypoglycemia, nor does it cause significant weight loss, food intake reduction or metabolic side effects such as changes in activity, and the present application provides a new target for central blood glucose homeostasis regulation.

[0070] The diabetes described in the present application can be one of abnormal glucose metabolism diseases, including hyperglycemia such as overweight, obesity, diabetes, neuropathic pain, etc. The diabetes can be, for example, type 1 diabetes, type 2 diabetes, and some special types of diabetes, such as gestational diabetes.

[0071] The present application provides use of the MDH2 inhibitor in the preparation of a medicament for treating abnormal glucose metabolism diseases, preferably the abnormal glucose metabolism disease is diabetes, preferably the diabetes is type 2 diabetes.

[0072] In some embodiments, the MDH2 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule or a gene editor comprising a nucleic acid molecule that hybridizes to a MDH2 nucleic acid molecule.

[0073] In the present application, examples of the nucleic acid molecule include but are not limited to antisense nucleic acid molecules, small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs). Such inhibitory nucleic acid molecules can be designed to target any region of a MDH2 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within a MDH2 nucleic acid molecule or mRNA molecule and reduces expression of a MDH2 protein in a cell of a subject. In some embodiments, the MDH2 inhibitor comprises an antisense molecule that hybridizes to a MDH2 nucleic acid molecule or mRNA molecule and reduces expression of a MDH2 protein in a cell of a subject. In some embodiments, the MDH2 inhibitor comprises an siRNA that hybridizes to a MDH2 nucleic acid molecule or mRNA molecule and reduces expression of a MDH2 protein in a cell of a subject. In some embodiments, the MDH2 inhibitor comprises an shRNA that hybridizes to a MDH2 nucleic acid molecule or mRNA molecule and reduces expression of a MDH2 protein in a cell of a subject.

[0074] In the present application, the inhibitory nucleic acid molecule can comprise RNA or DNA, or both RNA and DNA. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous nucleic acid sequence, such as a heterologous nucleic acid sequence in a vector, or a heterologous label. For example, the inhibitory nucleic acid molecule can be within a vector comprising the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence, or as an exogenous donor sequence comprising the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous label. The label can be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme. The term "label" can also refer to a "tag" or hapten, which can selectively bind to a conjugate molecule such that the conjugate molecule, when added subsequently with a substrate, is used to generate a detectable signal. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG, or 3XFLAG, 6XHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or Fc portions of immunoglobulins. Many labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent and chemiluminescent substrates, and other labels.

[0075] In the present application, the gene editors comprise DNA gene editors and RAN gene editors. The gene editors comprise a gene editing protein and optionally a gRNA.

[0076] In some embodiments, the gene editing protein is a Cas protein. In the present application, suitable Cas proteins include, for example, wild-type Cas9 protein and wild-type Cpf1 protein (e.g. like FnCpf1). The Cas protein can have full cleavage activity to generate a double-strand break in the Mdh2 genomic nucleic acid molecule, or it can be a nickase that generates a single-strand break in the Mdh2 genomic nucleic acid molecule. Further examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and homologues or modified versions thereof.

[0077] In some embodiments, the MDH2 inhibitor is a small molecule compound.

[0078] In some embodiments, the MDH2 inhibitor is LW6.

[0079] The inventors found that MDH2 plays a key role in the central sugar metabolism response through gene knockdown or knockout and drug intervention, and found that LW6 can improve sugar metabolism disorder and treat type 2 diabetes by targeting the MDH2 target, verify its effectiveness and safety under the conditions of central and peripheral administration, and does not cause weight loss, food reduction or other metabolic related side effects, revealing its potential neural mechanism and clinical application value.

[0080] The present application provides a pharmaceutical composition for treating diabetes, which comprises an MDH2 inhibitor.

[0081] In some embodiments, the MDH2 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule or a gene editor comprising a nucleic acid molecule that hybridizes to an MDH2 nucleic acid molecule.

[0082] In some embodiments, the MDH2 inhibitor is a small molecule compound.

[0083] In some preferred embodiments, the small molecule compound is LW6.

[0084] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier for delivery of the MDH2 inhibitor.

[0085] In this application, a carrier for delivery of the MDH2 inhibitor is a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a compound, material, composition, or dosage form that is suitable for use with human subjects and animal subjects without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, and effective to benefit those in need of such treatment. A "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material, involved in carrying or transporting the MDH2 inhibitor from one organ or portion of the body to another organ or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

[0086] Metabolite assay

[0087] 1. Sample preparation

[0088] (1) Whole fly or thorax tissue homogenate

[0089] Take 5 female flies (de-capitated) or 10 thorax tissues, add 200 μL PBST (PBS, 0.1% Tween 20) for homogenization, and heat at 70°C for 5 min to inactivate endogenous enzymes. Centrifuge the sample at 4000 rpm for 3 min (4°C), and take the supernatant for metabolite assay.

[0090] (2) Hemolymph preparation

[0091] Take 40 flies, puncture the thorax with a sterile tungsten needle, collect onto a filter membrane, and then put into a 1.5 mL EP tube. Centrifuge at 4000 rpm for 3 min (4°C) to obtain the supernatant, which is hemolymph. Repeat the centrifugation step to remove residual debris, and use the hemolymph for metabolite assay.

[0092] 2. Metabolite analysis

[0093] Ten μΐ of supernatant was taken to measure the levels of triacylglycerols (TAGs, StanBio Liquicolor Triglycerides Kit), glucose (Glucose Assay Kit, GAGO-20), trehalose (Megazyme) and protein (Bio-Rad Protein Assay) according to the manufacturer's instructions. TAGs, glucose and trehalose contents were normalized to body weight (whole body) or protein concentration (thoracic body, hemolymph).

[0094] Oil Red O staining

[0095] Sample preparation: Whole insects were dissected (ovaries and intestines removed) and fixed in 4% PFA for 20 min. After two washes in PBS, they were transferred to a freshly prepared Oil Red O solution (6 mL of 0.1% Oil Red O in isopropanol + 4 mL of distilled water, filtered with a 0.45 μιη filter) for 30 min. After extensive washing in distilled water, bright field images were acquired under a Leica M125 stereomicroscope and processed with Adobe Photoshop.

[0096] 3V stereotaxic injection

[0097] 1. 3V cannulation in rats and mice: A 26G stainless steel cannula was fixed to the 3V (mouse: AP-1.82 mm, ML 0.00 mm, DV-5.65 mm; rat: AP-3.10 mm, ML 0.00 mm, DV-7.90 mm), fixed with dental cement and reinforced with a screw.

[0098] 2. Virus injection: AAV virus was injected bilaterally in the PVN area of mice (AP-0.85 mm, ML ± 0.25 mm, DV-4.90 mm) at a rate of 50 nL / min, with a total injection of 120 nL per point. The glass microinjector needle was left for 7 min after injection to ensure diffusion.

[0099] 3. 3V drug injection: Drugs were injected into the 3V through the stereotaxic cannula at a rate of 2 μΐ / ηιίη, leaving the needle for 2 min after injection.

[0100] In vivo experiments

[0101] 1. IPGTT (intraperitoneal glucose tolerance test)

[0102] Mice: After 16 h of fasting, 10% glucose solution (1 g / kg) was injected intraperitoneally and tail blood was collected at 0, 15, 30, 60 and 120 min to measure blood glucose (Roche glucometer).

[0103] Rats: After fasting for 18 h, rats were injected with 30% glucose solution (1 g / kg), and blood samples were taken at 0, 15, 30, 60, 90, 120, 180 min to measure blood glucose.

[0104] Histological analysis

[0105] 1. Immunohistochemistry

[0106] After anesthesia, the brains of mice and rats were perfused with PBS and 4% PFA, and the brain tissues were fixed for 24 h, and then dehydrated in 20% and 30% sucrose solutions. The tissues were embedded in OCT and cryosectioned at low temperature (50 pm).

[0107] The tissue sections were blocked in 5% goat serum + 0.2% Triton X-100 (PBS) for 2 h, and the primary antibody (c-Fos, 1:1000; oxytocin, 1:200) was incubated at 4°C, and the secondary antibody was incubated for 2 h (AF488 / AF594, 1:500). After DAPI staining, the sections were mounted, and Leica THUNDER Imager 3D or VS200 scanning was used.

[0108] 2. Western Blot

[0109] After cell and tissue lysis, ultracentrifugation (13000 x g, 4°C, 15 min) was performed, and the protein concentration was determined by BCA method. The samples (20 pg of cells, 9 pg of CSF) were separated by 10%-15% SDS-PAGE, transferred to PVDF membranes, blocked with 5% skim milk for 1 h, and incubated with primary antibodies (MDH2, b-Actin, b-Tubulin, etc.). ECL was used for development. The band density was analyzed using ImageJ.

[0110] Statistical analysis

[0111] GraphPad Prism 9 was used for data analysis. Independent samples were analyzed by two-sided Student’s t-test, and repeated measurement data were analyzed by paired t-test. Single-factor ANOVA was used for multiple group comparisons, and Tukey’s test was used for post-hoc analysis. Two-way or three-way ANOVA was used for multi-factor experimental data, and Sidak’s post-hoc test was performed.

[0112] Examples

[0113] Example 1: In vitro study

[0114] In vitro cultured neurons were treated with different concentrations of glucose (0 mM, 25 mM, 50 mM) and 50 mM mannitol to detect the relationship between glucose concentration and the expression level of MDH2.

[0115] Figure 6a is the result of Western Blotting experiment of MDH2 protein expression level after culturing neurons with 0 mM, 25 mM, 50 mM glucose and 50 mM mannitol for 24 h respectively, Figure 1 b is the comparison result of MDH2 protein expression level after culturing neurons with 0 mM, 25 mM, 50 mM glucose and 50 mM mannitol for 24 h respectively. As can be seen from the figure, the expression of MDH2 decreases with the increase of glucose gradient. The experimental result of neurons treated with mannitol shows that the decrease of MDH2 is not related to the osmotic pressure of the culture medium.

[0116] Example 2: Knockout of Mdh2 gene in Drosophila glial cells and neurons

[0117] Genetic cross: Drosophila (♀ or ♂) carrying Elav-Gal4 (drives the specific expression of Gal4 transcription factor in neurons) is mated with Drosophila (corresponding ♀ or ♂) carrying UAS-Luciferase RNAi (RNA interference construct for control experiment) or UAS-Mdh2 RNAi (RNA interference construct for Mdh2 gene knockout), and the offspring Drosophila (F1) is obtained to drive the expression of specific RNAi in neurons. Drosophila (♀ or ♂) carrying Repo-Gal4 (a genetic tool for driving the expression of glial cell-specific genes) is mated with Drosophila (corresponding ♀ or ♂) carrying UAS-Luciferase RNAi or UAS-MDH2 RNAi, and the offspring Drosophila (F1) is obtained to express specific RNAi in glial cells.

[0118] Index measurement: F1 offspring Drosophila is grouped according to genotype and fed in standard medium. The effect on the whole body trehalose and triglyceride levels is evaluated by metabolomics analysis.

[0119] Figure 1 b and Figure 2 c are the contents of trehalose and triglyceride in Drosophila with neuron-specific knockout of Mdh2 and glial cell-specific knockout of Mdh2 respectively. According to Figure 2 b and Figure 2 the results of c show that, compared with the control group, neuron knockout of Mdh2 significantly reduces the whole body trehalose (the main sugar component of Drosophila hemolymph) and triglyceride levels of Drosophila, while glial cell knockout of Mdh2 does not change the whole body trehalose content of Drosophila, but increases the triglyceride level.

[0120] Example 3: Specific knockout of Mdh2 gene in PVN brain region of mice

[0121] Experimental animals: 8-10 week old C57BL / 6J mice weighing 20-25 g are selected.

[0122] Model establishment: AAV-U6-shRNA(Mdh2)-CMV-EGFP-SV40 virus was injected into the PVN brain area of mice to specifically knock down the Mdh2 gene in the PVN brain area of mice, PVN-Mdh2 - / - The PVN-scram group was the control group.

[0123] Index measurement: The blood glucose level, glucose tolerance, and metabolic behavior of the mice in the knockdown group and the control group were monitored.

[0124] Figure 2 b is the change of blood glucose level of mice with PVN brain area-specific knockdown of Mdh2 and control mice over time, and it can be seen from the figure that the blood glucose of the control mice has a certain increase over time after injection of the control virus, while the knockdown group significantly inhibits the increase of blood glucose compared with the control group, and has a decreasing trend.

[0125] Figure 3 c is the change of blood glucose level of mice with PVN brain area-specific knockdown of Mdh2 and control mice over time during the IPGTT experiment, Figure 3 d is the area under the curve of the change of blood glucose level of mice with PVN brain area-specific knockdown of Mdh2 and control mice over time during the IPGTT experiment, Figure 3 c and Figure 3 d can reflect that the blood glucose of mice with PVN brain area-specific knockdown of Mdh2 has a slower rising trend compared with the control group after intraperitoneal injection of glucose, and the glucose tolerance is significantly increased.

[0126] Figure 3 e and 3f are the 24-hour weight change and food intake of mice with PVN brain area-specific knockdown of Mdh2 compared with the control mice in the fourth week after virus injection, and it can be seen from the figure that the 24-hour weight and food intake of the knockdown group have no obvious change compared with the control group, indicating that PVN brain area-specific knockdown of Mdh2 does not affect the weight and food intake of mice.

[0127] Example 4: 3V cannulation injection experiment of C57BL / 6J mice

[0128] Experimental animals: 8-10-week-old C57BL / 6J mice weighing 20-25 g were selected.

[0129] Cannulation operation: The third ventricle was cannulated using a stereotaxic instrument. The cannulation position was ensured to be accurate, and the cannulation trace was observed by brain tissue section to verify the position.

[0130] LW6 injection: 2 μL of 3 mM LW6 solution (MCE Company, item number: HY-13671) was injected through the third ventricle (3V), and the control group was injected with an equal amount of dimethyl sulfoxide (DMSO).

[0131] Indicator measurement: The body weight change and food intake of mice in the LW6 injection group and the control group were recorded within 24 hours after injection. The blood glucose levels of mice were monitored at 0, 15, 30, 60, 90 minutes and 2, 4, 24 hours after injection. Blood samples were collected at 15 minutes after injection to determine the insulin levels of mice.

[0132] IPGTT experiment: After injection of LW6, glucose tolerance test was performed, and the blood glucose changes and area under the curve (AUC) of mice after glucose load at 0, 15, 30, 60, 120 minutes were recorded.

[0133] Figure 3 c is the change of blood glucose levels of mice injected with 3V of LW6 and control mice over time, Figure 4 d is the comparison result of blood glucose levels of mice 15 minutes after injection of 3V of LW6 and control mice. Figure 4 c reflects that the blood glucose of mice 15 minutes after injection of 3V of DMSO rises rapidly and then slowly decreases over time until it returns to normal level, but injection of LW6 slows down the rising trend of blood glucose. Figure 4 d reflects that the difference in blood glucose levels of the two groups of mice at 15 minutes after injection is significant. The data results show that injection of 3V of LW6 significantly inhibits the increase of blood glucose in mice.

[0134] Figure 4 e and Figure 4 f is the change of blood glucose levels of mice injected with 3V of LW6 and control mice over time and the area under the curve of the change of blood glucose levels over time in the IPGTT experiment, which can reflect that compared with the control group, the blood glucose of mice injected with 3V of LW6 rises slowly after intraperitoneal injection of glucose, and the glucose tolerance is significantly increased.

[0135] Figure 4 g is the comparison result of blood glucose levels of mice injected with 3V of LW6 and control mice before and 15 minutes after injection. The data results can reflect that the blood glucose of mice 15 minutes after injection of 3V of DMSO rises significantly; after injection of LW6, the blood glucose does not change significantly and does not cause hypoglycemia.

[0136] Figure 4 h is the comparison result of insulin levels of mice 15 minutes after injection of 3V of LW6 and control mice. The results show that the insulin level of mice 15 minutes after injection of 3V of LW6 does not change significantly. Figure 4 i and Figure 4 j is the comparison result of body weight change and food intake of mice 24 hours after injection of 3V of LW6 and control mice. Compared with the control group, the body weight and food intake of the LW6 injection group do not change significantly within 24 hours. This indicates that injection of 3V of LW6 does not affect the body weight and food intake of mice.

[0137] Example 5: 3V cannulation injection experiment of SD rats

[0138] Experimental animals: 8-10 week old SD rats weighing 200-250 g were selected.

[0139] Cannulation operation: Cannulation was performed in the third ventricle through stereotactic technique. The accuracy of the cannula position was ensured and verified by sectioning.

[0140] LW6 injection: 3 μL of 30 mM LW6 solution (MCE, Cat No: HY-13671) was injected through the third ventricle (3V), and the control group was injected with an equal amount of DMSO.

[0141] Index measurement: The body weight change and food intake of the injection group and control group mice were recorded within 24 hours after injection. The blood glucose level of the mice was monitored at 0, 15, 30 minutes and 1, 2, 4, 24 hours after injection.

[0142] IPGTT experiment: Glucose tolerance test was also performed to evaluate the hypoglycemic effect of LW6.

[0143] Figure 4 b is the change of blood glucose level over time of the injection group rats and control group rats injected with LW6 in the 3V, Figure 5 c is the comparison result of the blood glucose level of the rats 1 hour after 3V injection of LW6 with that of the control group rats. Figure 5 b reflects that the blood glucose of the rats rapidly rises within 30 minutes after 3V injection of DMSO, and then slowly decreases over time until it returns to normal level, but the injection of LW6 slows down the rising trend of blood glucose. Figure 5 c reflects that the difference in blood glucose between the two groups of rats 1 hour after injection is significant. The data shows that 3V injection of LW6 significantly inhibits the rise of blood glucose in rats.

[0144] Figure 5 d and Figure 5 e is the change of blood glucose level over time and the area under the curve of the change of blood glucose level over time of the injection group rats and control group rats injected with LW6 in the 3V during the IPGTT experiment. It can be reflected that compared with the control group, the blood glucose of the rats injected with LW6 in the 3V rises slowly after intraperitoneal injection of glucose, and the glucose tolerance is significantly increased.

[0145] Figure 5 f and 5g are the comparison results of the body weight change and food intake of the rats 24 hours after 3V injection of LW6 with those of the control group rats. It can be seen from the figure that there is no significant change in body weight and food intake of the LW6 injection group compared with the control group within 24 hours, indicating that 3V injection of LW6 does not affect the body weight and food intake of the rats.

[0146] Example 6: LW6 treatment experiment on type 2 diabetes mouse model

[0147] Model establishment: C57BL / 6J mice were fed with high-fat diet for 8 weeks, and then streptozotocin (STZ, 40 mg / kg, MCE, Cat: HY-13753) was injected intraperitoneally for 5 consecutive days to establish the T2D model. The body weight changes and blood glucose levels of the mice during model establishment were recorded.

[0148] Drug treatment: After the type 2 diabetes mouse model was successfully established, the mice in each group were treated with drugs.

[0149] Normal mice were injected intraperitoneally with 2 mg / kg LW6. Diabetic mice were randomly divided into 2 groups: control group was injected intraperitoneally with 10% DMSO (solvent of LW6); LW6 treatment group was injected intraperitoneally with 2 mg / kg LW6.

[0150] Blood glucose monitoring: The blood glucose levels of mice in each group were recorded at 0, 15, 30 minutes and 1, 2, 24 hours after injection, and the hypoglycemic effects of different groups were compared.

[0151] Data analysis: The blood glucose changes of each group were statistically analyzed to evaluate the hypoglycemic effect of LW6.

[0152] Figure 5 b is the body weight change of the mice during the establishment of type 2 diabetes. The experimental results show that the body weight of the high-fat diet group (HFD) during modeling is significantly higher than that of the normal diet group (CD). Figure 6 c is the comparison result of blood glucose levels of mice before and after injection of STZ. The experimental results show that the blood glucose of mice significantly increases after intraperitoneal injection of STZ for 5 consecutive days, and is significantly higher than that of the control group, indicating that the modeling is successful. Figure 6 d and Figure 6 e is the change of blood glucose levels over time after intraperitoneal injection of LW6 in type 2 diabetic mice, type 2 diabetic control mice and normal mice injected with LW6, and the comparison results of each group of mice at 2 hours after injection of drugs. Figure 6 d reflects that in type 2 diabetic mice, the blood glucose rapidly rises within 2 hours after intraperitoneal injection of DMSO, and then slowly decreases over time, but the injection of LW6 slows down the rising trend of blood glucose. Figure 6 Figure 6 e can reflect that at 2 hours after injection, the difference in blood glucose between the two groups of type 2 diabetic mice is significant. In type 2 diabetic mice, the blood glucose level of the LW6 treatment group is significantly lower than that of the diabetic control group, and the hypoglycemic effect is significant.

[0153] The present application verifies the wide applicability and potential clinical transformation value of LW6 through gene experiments and the use of various animal models (normal mice, rats and diabetic model mice). The present application verifies that LW6 can effectively reduce blood glucose levels by targeting the central nervous system. LW6 can effectively regulate blood glucose by central administration and peripheral intraperitoneal injection. LW6 only has a hypoglycemic effect on hyperglycemia. While reducing blood glucose, it does not cause hypoglycemia, nor does it cause metabolic side effects such as significant weight loss, reduced food intake or changes in activity. LW6, as a new type of hypoglycemic drug, provides a new idea and direction for the treatment of diabetes and other metabolic diseases.

[0154] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not exist, they should be considered as within the scope of the present disclosure.

[0155] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the present application. It should be pointed out that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. Use of an MDH2 inhibitor for the manufacture of a medicament for the treatment of a glucose metabolism disorder.

2. Use of an MDH2 inhibitor for the treatment of a glucose metabolism disorder.

3. Use according to claim 1 or 2, wherein the glucose metabolism disorder comprises overweight, obesity, diabetes, neuropathic pain, preferably diabetes.

4. Use according to any one of claims 1-3, wherein the MDH2 inhibitor is a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule comprising hybridization to an MDH2 nucleic acid molecule, or a gene editor.

5. Use according to claim 4, wherein the MDH2 inhibitor is a small molecule compound, preferably the small molecule compound is LW6.

6. A pharmaceutical composition for the treatment of a glucose metabolism disorder, comprising an MDH2 inhibitor.

7. The pharmaceutical composition according to claim 6, wherein the MDH2 inhibitor comprises a small molecule compound, a polypeptide, an inhibitory nucleic acid molecule comprising hybridization to an MDH2 nucleic acid molecule, or a gene editor.

8. The pharmaceutical composition according to claim 6, wherein the MDH2 inhibitor is a small molecule compound, preferably the small molecule compound is LW6.

9. The pharmaceutical composition according to any one of claims 6-8, comprising a pharmaceutically acceptable carrier for the delivery of the MDH2 inhibitor.

10. A method of treatment, comprising administering to a subject a therapeutically effective amount of the pharmaceutical composition according to any one of claims 6-10.

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