CN106860449B - Use of matrine derivatives in the treatment of diabetes - Google Patents
Use of matrine derivatives in the treatment of diabetes Download PDFInfo
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Abstract
The invention relates to an application of a compound shown in a formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof in preparing a medicament for treating diabetes, or in preparing a medicament for preventing or treating diabetic complications, or in preparing a medicament serving as an insulin sensitizer,
Description
Technical Field
The invention relates to a new application of matrine derivatives, in particular to an application of matrine derivatives as insulin sensitizers, and also relates to an application of matrine derivatives in preparing medicines for treating or preventing diabetes or diabetic complications.
Background
Diabetes (diabetes mellitus) is a group of metabolic diseases characterized by hyperglycemia, and is classified into type 1 and type 2 diabetes. The long-term blood sugar is increased, and the great vessels and the capillaries are damaged and endanger the heart, the brain, the kidney, the peripheral nerves, the eyes, the feet and the like, so that a series of complications such as diabetic nephropathy, diabetic eye disease, diabetic cardiovascular complications, diabetic neuropathy and the like can be caused. Once complications arise, drug treatment is difficult to reverse, thus emphasizing early prevention of diabetic complications.
At present, the medicines for treating diabetes mainly comprise insulin and analogues thereof, oral hypoglycemic drugs and the like. The oral hypoglycemic agent mainly comprises sulfonylurea insulin secretagogues, non-sulfonylurea insulin secretagogues, thiazolidinediones, biguanides, glucagon-like peptide 1 analogues, DPP-4 inhibitors, alpha-glucosidase inhibitors and the like. The oral hypoglycemic drugs commonly used in clinic have certain side effects. For example, it has been reported that the use of sulfonylurea hypoglycemic agents may produce side effects such as hypoglycemia, major adverse cardiovascular events, gastrointestinal damage, and the like. Non-sulfonylurea insulinotropic agents may cause gastrointestinal adverse reactions such as nausea, abdominal pain, diarrhea, and the like. Thiazolidinedione drugs have been reported to be associated with the development of atherosclerosis, rosiglitazone may lead to cardiovascular risk, and pioglitazone may lead to the development of bladder cancer. The clinical adverse reactions of biguanide drugs mainly include digestive tract symptoms, such as diarrhea, abdominal distension, nausea, anorexia, epigastric discomfort, and adverse reactions such as lactic acidosis. Biguanide drugs are not suitable for diabetic patients with serious liver, kidney, heart and lung insufficiency. DPP-4 inhibitors may produce a hypoglycemic response. Alpha-glucosidase inhibitors may produce side effects such as flatulence.
Therefore, the research and development of a safer and more effective novel oral hypoglycemic agent are very important.
Disclosure of Invention
The inventor surprisingly finds that the matrine derivative can reduce fasting blood glucose of type 2 diabetes mice, improve oral glucose tolerance, improve insulin tolerance and improve urine related indexes of the mice, does not increase ALT content of serum, and simultaneously reduces liver coefficient, triglyceride content of liver and AGEs content of liver through in vitro screening and animal in vivo pharmaceutical verification. This indicates that the matrine derivative can be used as an insulin sensitizer and/or for the prevention and/or treatment of diabetes or diabetic complications, such as diabetic nephropathy. The present invention has been completed based on the above findings.
The invention relates to an application of a compound shown as a formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof in preparing a medicament for treating diabetes.
The invention also relates to application of the compound shown in the formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof in preparing a medicament for preventing and/or treating diabetic complications.
The invention also relates to application of the compound shown in the formula I, the stereoisomer thereof or the pharmaceutically acceptable salt thereof in preparing a medicament serving as an insulin sensitizer.
The invention also relates to application of the pharmaceutical composition in preparing a medicament for treating diabetes, or in preparing a medicament for preventing or treating diabetic complications, or in preparing a medicament serving as an insulin sensitizer, wherein the pharmaceutical composition contains the compound shown in the formula I, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
The invention also relates to a compound shown in the formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, which is used for treating diabetes.
The invention also relates to a compound shown in the formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, which is used for preventing and/or treating diabetic complications.
The invention also relates to a compound shown in the formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, which is used as an insulin sensitizer.
The present invention is a method of treating diabetes comprising administering to a subject in need thereof an effective amount of a compound of formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
The present invention also relates to a method for preventing and/or treating diabetic complications, which comprises administering an effective amount of a compound represented by formula I, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof to a subject in need thereof.
The structure of the compound shown in the formula I is as follows:
wherein the content of the first and second substances,
R1represents aryl or heterocyclyl, said aryl or heterocyclyl being optionally substituted by R4Mono-or polysubstituted, R4Selected from: c1-4Alkanoyl, cyano, C1-4Alkoxy, halo C1-4Alkoxy, carboxyl, sulfonic acid group, C1-4Alkoxycarbonyl, C1-4Alkanoylamino, nitro, halogen, hydroxy, mercapto, amino, C1-4Alkylsulfonyl radical, C1-4Alkyl and halo C1-4An alkyl group;
R2represents- (CH)2)nR3(ii) a Wherein the content of the first and second substances,
n is 0, 1,2, 3 or 4;
R3selected from hydrogen, amino, mercapto, halogen and C1-6An alkoxy group.
In one embodiment, in the compounds of formula I of the present invention, the heterocyclyl is a 5-6 membered monoheteroaryl.
In any of the above embodiments, in the compound of formula I, the heterocyclic group is selected from imidazolyl, thiazolyl, pyridyl, thienyl.
In any of the above embodiments, the aryl group in the compounds of formula I according to the present invention is phenyl.
In any of the above embodiments, in the compounds of formula I, R4Selected from the group consisting of halomethoxy, haloethoxy, halopropoxy, acetyl, halomethyl, haloethyl, halopropyl, cyano, methoxy, ethoxy, propoxy, carboxyl, methoxycarbonyl, acetylamino, nitro, amino, methanesulfonyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Preferably, R4Selected from the group consisting of trifluoromethoxy, trifluoromethyl, cyano, methoxy, carboxyl, nitro, amino, methylsulfonyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Further preferably, R4Is trifluoromethyl.
In any of the above embodiments, in the compounds of formula I, R2Represents- (CH)2)nR3Wherein n is 1,2 or 3; r3Selected from hydrogen, amino, methoxy, ethoxy.
In any of the above embodiments, in the compounds of formula I, R2Represents methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
In any of the above embodiments, in the compounds of formula I, R2Represents methyl, ethyl, n-propyl or isopropyl.
In any of the above embodiments, the compound of formula I according to the present invention is selected from:
the synthesis method of the compound shown in the formula I can be found in CN 106279167A.
The various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art and, to the extent that the terms and phrases are not inconsistent with their known meaning, the meaning of the present invention as expressed herein applies.
The diabetes mellitus of the invention is type 1 diabetes mellitus or type 2 diabetes mellitus.
The diabetic complication is diabetic nephropathy, diabetic eye disease, diabetic cardiovascular complication or diabetic neuropathy, and is preferably diabetic nephropathy.
The insulin sensitizer of the invention is also called insulin sensitizing factor, and is a substance capable of enhancing insulin sensitivity in human body and promoting full utilization of insulin.
The term "aryl" as used in the present invention refers to a monocyclic or bicyclic aromatic system comprising at least one unsaturated aromatic ring, preferably an aryl group having 6 to 10, i.e. 6, 7, 8, 9 or 10 carbon atoms. Specific examples include, but are not limited to, phenyl, naphthyl, and the like.
The term "heterocyclyl" as used herein refers to a monocyclic or bicyclic saturated, partially saturated or unsaturated aromatic or aliphatic ring system, preferably a mono-or bis-heterocyclyl having 4 to 7 atoms (including 4, 5, 6 or 7 atoms) or 7 to 11 atoms (including 7, 8, 9, 10 or 11 atoms), such as a 5 to 6 membered mono-heteroaryl, 7 to 11 membered bis-heteroaryl, nitrogen heterocycle or a 4 to 6 membered aliphatic nitrogen heterocycle, optionally substituted with at least one and up to four heteroatoms independently selected from N, O or S. Specific examples include, but are not limited to, imidazolyl, thiazolyl, pyridyl, thienyl.
The term "C" as used in the present invention1-4Alkyl "refers to straight or branched chain alkyl groups having 1 to 4 carbon atoms, such as 1,2, 3, or 4 carbon atoms. Specific examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl.
The term "C" as used in the present invention1-6Alkoxy "means C as previously described1-6The radical resulting from the linkage of a carbon atom to an oxygen atom on an alkyl radical, preferably "C1-4Alkoxy ", specific examples include, but are not limited to, methoxy, ethoxy, propoxy, or the like.
The term "C" as used in the present invention1-4Alkanoyl "means a carbonyl group having one end of the carbon atom of the carbonyl group as defined above1-4Specific examples of the group obtained by linking an alkyl group include, but are not limited to, formyl, acetyl, propionyl and the like.
The term "C" as used in the present invention1-4Alkoxycarbonyl "means a carbonyl carbon atom bearing one end of a carbon atom as defined above1-4Alkoxy groups are linked to obtain the group. Specific examples include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, and the like.
The term "C" as used in the present invention1-4Alkanoylamino "means an amino group having one end of the nitrogen atom of the amino group and C as defined above1-4The group obtained after the alkanoyl group is attached. Specific examples include, but are not limited to, formylamino, acetylamino, and the like.
The term C as used in the present invention1-4Alkylsulfonyl "means a sulfonyl sulfur atomOne end of each group is as defined above for C1-4The group obtained after alkyl group attachment. Specific examples include, but are not limited to, methylsulfonyl, ethylsulfonyl, and the like.
The term "halogen" as used in the present invention refers to fluorine, chlorine, bromine, iodine.
The term "subject" as used in the present invention includes mammals and humans, preferably humans.
The term "pharmaceutically acceptable salt" as used herein means a salt of a compound of the invention which is pharmaceutically acceptable and which possesses the desired pharmacological activity of the parent compound. Such salts include: acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with organic acids; such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or salts formed when an acidic proton present on the parent compound is replaced by a metal ion, e.g., an alkali metal ion or an alkaline earth metal ion; or a complex compound with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, or the like.
The pharmaceutical composition contains a compound shown in formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient. Such vectors include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin. The excipient refers to an additive in the medicinal preparation except the main medicament. The composition has stable properties, no incompatibility with main drug, no side effect, no influence on curative effect, no deformation at room temperature, no crack, mildew, moth-eaten feeling, no harm to human body, no physiological effect, no chemical or physical effect with main drug, no influence on content determination of main drug, etc. Such as binders, fillers, disintegrants, lubricants in tablets; wine, vinegar, medicinal juice, etc. in the Chinese medicinal pill; base portion in semisolid formulations ointments, creams; preservatives, antioxidants, flavoring agents, fragrances, solubilizing agents, emulsifiers, solubilizers, tonicity adjusting agents, coloring agents and the like in liquid formulations can all be referred to as excipients, and the like.
Pharmaceutical compositions of the compounds of the present invention may be administered in any of the following ways: oral, aerosol inhalation, rectal, nasal, buccal, topical, parenteral, e.g. subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or via an external reservoir. Among them, oral, intraperitoneal or intravenous administration is preferable.
The term "effective amount" as used herein means an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a prophylactically effective amount is an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.
The amount of the compound of the present invention administered to a subject depends on the type and severity of the disease or condition and the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs, as well as on the type of formulation and mode of administration of the drug, and the period or interval of administration. One skilled in the art will be able to determine the appropriate dosage based on these and other factors. In general, the compounds of the invention may be used in a therapeutic daily dosage of about 1 to 800 mg, which may be administered in one or more divided doses as appropriate. The compounds of the invention may be provided in dosage units, which may be present in an amount of 0.1 to 200 mg, for example 1 to 100 mg.
The invention has the beneficial technical effects
The compound shown in the formula I can reduce fasting blood glucose of type 2 diabetes mice, improve oral glucose tolerance, improve insulin tolerance and improve urine related indexes of the mice, does not increase ALT content of serum, and simultaneously reduces liver coefficient, triglyceride content of liver and AGEs content of liver. The compound shown in the formula I can be used as an insulin sensitizer and/or used for preventing and/or treating diabetes or diabetic complications, such as diabetic nephropathy.
Drawings
FIG. 1 histogram of sugar consumption by HepG2 cells;
FIG. 2L 6 histogram of cellular sugar consumption;
FIG. 33 bar graph of sugar consumption by T3-L2 cells;
FIG. 4 is a bar graph of fasting plasma glucose changes in mice;
FIG. 5 an oral glucose tolerance (OGTT) curve;
FIG. 6 bar graph of the area under the OGTT curve;
FIG. 7 insulin tolerance (ITT) curve;
FIG. 8 is a histogram of the area under the ITT curve;
FIG. 9 bar graph of the change in urine volume in mice at 24 hours before and 49 days after administration;
FIG. 10 is a bar graph of urine glucose concentration changes in mice before and 49 days after dosing;
FIG. 11 is a bar graph showing changes in the amount of microalbumin production in urine in mice before and 49 days after administration;
FIG. 12 is a bar graph of changes in total urine protein production in mice before and 49 days after administration;
FIG. 13 is a bar graph of ALT levels in mouse serum 50 days after dosing;
FIG. 14 is a histogram of AST levels in mouse serum 50 days after dosing;
FIG. 15 is a histogram of CHO content in mouse serum 50 days after administration;
FIG. 16 is a bar graph of LDL-c content in mouse serum after day 50 of dosing;
FIG. 17 is a histogram of TG content in mouse serum 50 days after administration;
FIG. 18 is a histogram of the serum insulin concentration in mice 50 days after administration;
FIG. 19 is a histogram of advanced glycation end-product content in mouse serum 50 days after dosing;
FIG. 20 is a bar graph of mouse liver coefficients 50 days after dosing;
FIG. 21 is a bar graph of triglyceride content in liver tissue 50 days after administration;
FIG. 22 is a bar graph of total cholesterol content in liver tissue 50 days after dosing;
FIG. 23 is a bar graph of AGEs content in liver tissue 50 days after dosing;
FIG. 24 is a histogram of liver tissue SOD activity 50 days after administration;
FIG. 25 is a bar graph of liver tissue MDA content 50 days after dosing.
In FIGS. 1 to 25, the abscissa symbols "DB-1, 10" represent 10. mu.M of compound DB-1, "DB-1, 10+ I" represents 10. mu.M of compound DB-1+0.05nM insulin, "DB-1, 20" represents 20. mu.M of compound DB-1, "DB-1, 20+ I" represents 20. mu.M of compound DB-1+0.05nM insulin, and the other abscissa symbols have similar meanings.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The experimental materials and reagents involved in the following examples are as follows:
HepG2 cells, L6 cells, 3T3-L1 cells were purchased from American Type Culture Collection (ATCC).
DMEM medium, EDTA-pancreatin, fetal bovine serum FBS were purchased from gibco, USA.
Glucose assay kits (glucose oxidase method) were purchased from Landao laboratory diagnostics, Inc. (Randox).
Male C57BL/6J mice and male KK/upj-Ay/J mice, maintenance diet, high-fat high-sugar diet purchased from Beijing Huafukang Biotech GmbH.
Compounds DB-1 and DB-1-3 were synthesized by the institute for Biotechnology and medicine of the national academy of medical sciences chemical laboratory (see example 28 and example 30 of CN 106279167A), berberine (BBR) was provided by Shenyang first pharmaceutical factory of northeast pharmaceutical group, and Rosiglitazone (RGZ) was purchased from sigma.
Glucometers and glucose strips were purchased from roche diagnostic products (shanghai) limited.
D-glucose, SOD kit, MDA kit, hexokinase kit and glycogen detection kit are purchased from sigma company.
0.22 μm filters, mouse insulin ELISA kits were purchased from Millpore.
Recombinant human insulin injection was purchased from li.
EDTA-K2 anticoagulant blood collection tubes were purchased from BD corporation, USA.
AGE content of advanced glycosylation end products was purchased from cell biolabs using ELISA kits.
Protein concentration assay kits were purchased from Thermo corporation.
RNA extraction kits were purchased from Qiagen.
The reverse transcription kit and the fluorescent quantitative PCR kit were purchased from Promega corporation.
The concentration unit "M" used in the following experiments represents mol/L.
Example 1 in vitro HepG2 cell line/L6 cell line/3T 3-L1 cell line sugar consumption and insulin stimulated sugar consumption experiments
1.1 the experimental method is as follows:
① cell culture HepG2 cells were cultured in 10% FBS + DMEM medium at 37 ℃ with 5% CO2And (5) culturing in a cell culture box.
L6 cells were cultured in 10% FBS + DMEM medium at 37 ℃ in 5% CO2Culturing in a cell culture box, inoculating L6 cells into the culture plate, and continuing to induce and differentiate for 5-7 days by using 10% FBS + DMEM medium after the cell density reaches 80% -90%.
3T3-L1 cells were cultured in high-glucose medium containing 10% FBS + DMEM at 37 ℃ and 5% CO2And the cell culture box performs growth culture. Then placed at 37 ℃ with 5% CO2The cell culture box is used for induced differentiation culture, and the induced differentiation culture medium is as follows: 10% FBS + DMEM high-glucose medium supplemented with 1. mu.M dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine (IBMX) and 1. mu.g/ml insulin.
Measurement of sugar consumption:
the test compounds were: compounds DB-1, DB-1-3, berberine (BBR) and Rosiglitazone (RGZ), wherein berberine and rosiglitazone are used as positive control drugs. Each test compound was formulated in DMSO as a solution.
HepG2 cells, L6 cells, 3T3-L1 cells were seeded into 96-well plates, respectively. Wherein:
HepG2 cells were seeded 2 x 10 per well4After 24 hours of culture, adding medicine, and carrying out basal sugar consumption and insulin stimulated sugar consumption experiments;
l6 cells were seeded 5 x 10 cells per well3After culturing and induced differentiation, adding medicine for carrying out basic sugar consumption and insulin stimulated sugar consumption experiments;
identical 3T3-L1 cells were seeded 2 x 10 cells per well3And culturing the growth culture medium for 48 hours, replacing a fresh growth culture medium, continuously culturing for 48 hours, then replacing a differentiation culture medium, inducing differentiation for 48 hours, adding medicine, and performing basic sugar consumption and insulin stimulated sugar consumption experiments.
Basal sugar consumption assay, 0.5% FBS + DMEM medium and test compound were added to the wells, while a control group was set. Control group to wells, the same amount of 0.5% FBS + DMEM medium and DMSO was added. The final concentrations of test compound in each well were set to 10. mu.M, 20. mu.M, 40. mu.M, respectively. And (5) adding medicine, culturing for 24 hours, sucking a certain amount of supernatant to measure the residual amount of glucose in the culture medium, and calculating the consumption amount of glucose. Wherein the glucose consumption is the initial glucose content of the medium-the residual glucose content of the medium.
Insulin-stimulated glucose consumption assay, insulin and test compound were added to wells, while an insulin control group was set. Insulin control group to wells the same amount of insulin and DMSO was added. The final concentrations of test compound in each well were set to 10. mu.M, 20. mu.M, 40. mu.M, respectively, and the final concentration of insulin was set to 0.05 nM. And (5) adding medicine, culturing for 24 hours, sucking a certain amount of supernatant to measure the residual amount of glucose in the culture medium, and calculating the consumption amount of glucose.
1.2 results of the experiment
The experiment was independently repeated three times to obtain the average sugar consumption. The sugar consumption histograms for the different cells were plotted separately, as shown in fig. 1 to 3. The results show that in HepG2 cell line, L6 cell line and 3T3-L1 cell line, under the same determination conditions, the compound DB-1 and the compound DB-3 have equivalent or more significant effects on promoting the sugar consumption of cell basal level and promoting the sugar consumption of insulin-stimulated cells compared with positive control drugs berberine and rosiglitazone, and show that the compound DB-1 and the compound DB-1-3 have better blood sugar reducing effect and insulin sensitivity increasing effect at cell level.
Example 2 pharmacological Effect test for lowering fasting blood glucose, increasing insulin sensitivity and treating diabetic nephropathy in type 2 diabetic mice
2.1 Experimental methods
Animal experiments: 8 male C57BL/6J mice at the age of 10 weeks, 63 male KK/upj-Ay/J mice at the age of 10 weeks, wherein: c57BL/6J mice were given maintenance chow, KK/upj-Ay/J mice were given high-fat high-sugar chow, and fasting blood glucose was measured two weeks after feeding and randomized into groups. C57BL/6J mice as normal control group, KK/upj-Ay/J mice as 5 groups, each group of 9 mice, wherein 1 group is model control group, 4 groups are administration groups, the administration amounts are DB-150 mg/kg, DB-1100 mg/kg, BBR100mg/kg and RZG 5mg/kg respectively.
The formula of the high-fat high-sugar feed comprises: 16.46% of protein, 45.65% of fat and 37.89% of carbohydrate.
Measuring fasting blood glucose: body weight was measured before administration without fasting as an initial value, and every 3 days after administration. After the initial fasting blood glucose was measured, a certain amount of feed was weighed out and administered to each mouse, the remaining amount of feed was weighed out every 5 days, a certain amount of feed was re-administered, and the daily food intake of each mouse was calculated after the administration was completed. Fasting for 12 hours at 8:30 night the previous day, measuring fasting blood glucose at 8:30 morning the next day, cutting the tip of the tail, measuring by the vitality type of a Roche glucometer, measuring fasting blood glucose before administration as an initial value, and measuring fasting blood glucose every 10 days after administration. The bar graph of the change of fasting blood glucose of the mice is drawn, as shown in fig. 4.
③ Oral Glucose Tolerance Test (OGTT): after 30 days of administration, 8:30 fasting is carried out for 12 hours at night, the OGTT experiment is carried out on 8:30 in the morning of the next day, 4g of glucose is weighed and dissolved in 20ml of water, 0.22 mu m of filter membrane is used for filtration sterilization to prepare 20% glucose solution, the weight is weighed, fasting blood glucose is measured to be used as the blood glucose value at 0 point, the blood glucose value is intragastrically administered to a mouse with 20% glucose solution, the blood glucose value is finally administered to be 2g/kg, then the blood glucose value is respectively measured at 30min, 60min, 120min and 180min, the blood glucose value is counted, the OGTT curve is drawn, and the area under the OGTT curve.
(iv) Insulin Tolerance Test (ITT): after 35 days of administration, 8:30 mice were fasted for 6 hours in the morning, the blood sugar value of the mice after 6 hours of fasting was measured as the blood sugar value at the 0 point of the ITT experiment, meanwhile, the weight of the mice after 6 hours of fasting was weighed, the conventional recombinant human insulin injection was administered according to the weight, the administration was performed by intraperitoneal injection, the insulin concentration was finally administered at 0.5U/kg weight, the blood sugar was measured 15min, 30min, 60min, 90min and 120min after the insulin injection, the blood sugar value at the 0 point was 1, the relative blood sugar concentration was calculated at the other time points, the ITT curve was drawn, and the area under the ITT curve was calculated.
Collecting and measuring urine: collecting urine of mice 24 hours before and 49 days after administration by using a metabolism cage respectively, independently placing the mice into the metabolism cage, freely eating and drinking water, collecting urine of each mouse 24 hours, centrifuging at 12000rpm for 10min at 4 ℃, removing impurities, measuring the volume of the urine, measuring the total protein concentration of the urine, the trace albumin concentration of the urine and the glucose concentration of the urine by using a kit, calculating the total protein concentration of the mouse 24 hours urine and the generation amount of the trace albumin according to the urine amount, and purchasing the kit from Beijing Jiuqiangsheng biotechnology limited company.
Sixthly, measuring the blood index: after 48 days of administration to animals, fasting was performed for 12 hours, and whole blood was cut into a EDTA-K2 anticoagulated blood collection tube (purchased from BD), and 10 μ L of anticoagulated whole blood was used to determine glycated hemoglobin concentration and hemoglobin concentration, and the ratio of the two was used as the glycated hemoglobin content (kit purchased from RNADOX).
After 50 days of administration, the mice were bled from the eyeballs and allowed to stand at room temperature for 2 hours, centrifuged at 15 ℃ and 3000rpm for 10min, serum was separated, and alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Triglyceride (TG), total Cholesterol (CHO), low-density lipoprotein cholesterol (LDL-C), and UREA (UREA) were measured using a full-automatic biochemical analyzer (hitachi 7100) and a biochemical detection kit (purchased from north-control of midlife). Creatinine (CRE) is manually determined by using a full-wavelength enzyme-labeling instrument, Total Bile Acid (TBA), total bilirubin (T-Bil) and direct bilirubin (D-Bil) are determined by using the full-wavelength enzyme-labeling instrument, and a kit used for manual determination is purchased from Beijing Jiuqiang organisms. Serum insulin levels were determined using an ELISA kit (purchased from Merck Millipore). Content of AGEs (advanced glycation end products) in serum was measured using ELISA kit (purchased from cell biolabs).
Seventhly, treating animal tissues and organs: animals were sacrificed 50 days after dosing. Animals were fasted for 12 hours before treatment, and after weighing the weight after fasting, after blood was taken, the animals were sacrificed by removing the cervical vertebrae, and the liver, kidney, pancreas, epididymal fat, and perirenal fat were weighed, respectively. Half of the liver large leaves, the left kidney and the pancreas half of the mouse are respectively taken and fixed with 10% formalin overnight, pathological HE staining is carried out, and the left liver large leaves, the right kidney and the left pancreas are immediately frozen in liquid nitrogen for subsequent treatment.
Measuring liver triglyceride and total cholesterol: cutting about 50mg of liver tissue, adding 20 mu L of lysis buffer into each mg of tissue according to a proportion, carrying out subsequent steps according to the instruction, and measuring tissue TG and CHO, wherein the kit is purchased from Beijing prilley gene technology company Limited.
Ninthly liver AGE and SOD activity determination, namely cutting about 30mg of liver tissue, adding 20 mu L T-PER tissue protein extraction reagent (purchased from thermal scientific) into each mg of tissue, then using a tissue homogenizer (purchased from Qiagen) to perform lysis, 12000g, 4 ℃, centrifuging for 15min to obtain supernatant, using the kit to determine the AGE content of the liver advanced glycosylation end product (AGE determination kit purchased from cell biolabs) and SOD activity (SOD activity determination kit purchased from sigma), and calibrating the determination result by using protein concentration (protein concentration determination kit purchased from Thermo).
Content of Malondialdehyde (MDA), hexokinase activity, glycogen assay: a certain amount of liver tissue is cut and added into a lysis solution provided by the kit, the lysis solution is used for lysis, the subsequent steps are carried out according to the kit instructions, and the related results are calibrated by protein concentration (the protein concentration determination kit is purchased from Thermo).
2.2 results of the experiment
Change of fasting blood sugar
The change of fasting plasma glucose in mice is shown in FIG. 4. The results show that in KK/upj-Ay/J2 diabetic mice, after the compound DB-1 and the compound DB-1-3 are orally administered for 20 days, 30 days, 40 days and 48 days according to the administration amount of 50mg/kg and 100mg/kg of body weight, the fasting blood glucose of the animals of the administration group is remarkably reduced, and the blood glucose reducing effect of the compound DB-1 and the compound DB-1-3 is equivalent to that of berberine at the dose of 100mg/kg and rosiglitazone at the dose of 5 mg/kg.
② Oral Glucose Tolerance Test (OGTT) results
The OGTT curve is shown in FIG. 5 and the area under the OGTT curve is shown in FIG. 6. The results showed that the oral glucose tolerance of the group administered with 50mg/kg and 100mg/kg of compound DB-1 and the group administered with 100mg/kg of compound DB-1-3 were improved to different degrees 30 days after oral administration in mice with KK/upj-Ay/J2 diabetes, and that the degree of improvement in oral glucose tolerance of the group administered with 100mg/kg of DB-1 and the group administered with 100mg/kg of DB-1-3 was comparable to that of 100mg/kg of berberine or 5mg/kg of rosiglitazone.
Results of Insulin Tolerance Test (ITT)
The ITT curve is shown in FIG. 7, and the area under the ITT curve is shown in FIG. 8. The results showed that 35 days after oral administration in mice with KK/upj-Ay/J2 diabetes, the insulin resistance of the groups administered with 50mg/kg and 100mg/kg of compound DB-1 and compound DB-1-3 were improved to different degrees. The degree of improvement in insulin tolerance in the DB-1 group and DB-1-3 group administered with an amount of 100mg/kg was comparable to that of berberine administered with an amount of 100mg/kg or rosiglitazone administered with an amount of 5 mg/kg.
(iv) urine index measurement result
The changes in urine volume, urine glucose concentration, urine microalbumin production and urine total protein production in each group of animals over 24 hours before and after administration are shown in FIGS. 9 to 12. As can be seen from the figure, in the animal model of KK/upj-Ay/J2 diabetes, the urine volume, urine glucose concentration, urine microalbumin production and urine total protein production in each group of animals at 24 hours before administration were very significantly increased compared with those of C57BL/6J mice, and there was no statistical difference between each group of KK/upj-Ay/J mice; after administration, the model groups had different degree of elevation compared with the prior administration, and had very significant difference compared with the normal control group, while each administration group had different degree of reduction compared with the model group, and had statistical difference. The results show that the compound DB-1 and the compound DB-1-3 with the dosage of 50mg/kg and 100mg/kg can inhibit the development and the deterioration of diabetic nephropathy of mice with KK/upj-Ay/J2 type diabetes mellitus, and the effect is equivalent to that of berberine with the dosage of 100mg/kg and rosiglitazone with the dosage of 5 mg/kg. The results indicate that the compounds DB-1 and DB-1-3 are expected to be prepared for treating diabetic nephropathy.
Serum biochemical index measuring result
Serum biochemical indicators for each group of animals 50 days after administration are shown in figures 13 to 17. As can be seen from the figure, in the mouse animal model of KK/upj-Ay/J2 diabetes, ALT, AST, CHO, TG and LDL-c in the model group are increased and have very significant difference compared with the normal control group after 50 days of administration. Compared with the model group, the compound DB-1-3 with the dosage of 100mg/kg and the BBR with the dosage of 100mg/kg can reduce serum ALT and have statistical difference in each administration group, while the rosiglitazone with the dosage of 5mg/kg causes the serum ALT to be increased with statistical difference, and is considered to be caused by the side effect of the rosiglitazone; the compound DB-1-3 with the dosage of 100mg/kg can also obviously reduce serum AST; the compound DB-1 with the dosage of 100mg/kg can obviously reduce the CHO and LDL-c in serum; the dosage of 100mg/kg of compound DB-1, 50mg/kg of compound DB-1-3, 100mg/kg of BBR and 5mg/kg of rosiglitazone can obviously reduce serum TG.
Sixthly, the result of measuring the content of the serum insulin
The serum insulin levels in each group of animals 50 days after administration are shown in FIG. 18. As can be seen from the figure, in the mouse animal model of KK/upj-Ay/J2 diabetes, after 50 days of administration, the insulin content of the animal in the model group is obviously increased and has extremely obvious statistical difference compared with the animal in the normal group, and each administration group has different degrees of reduction and has obvious statistical difference compared with the animal in the model group. This shows that the compound DB-1 and the compound DB-1-3 can improve the hyperinsulinemia state in animals and increase the insulin sensitivity in vivo, and are expected to be used for preparing insulin sensitizers.
(vii) measurement of serum AGEs
After 50 days of administration, the serum content of advanced glycation end products in each group of animals is shown in FIG. 19. In mouse animal models of KK/upj-Ay/J2 diabetes, 50 days after administration, blood was taken for determination of advanced glycation end products (AGEs) in serum. AGEs are a generic term for a series of highly active end products formed by non-enzymatic glycosylation reactions between amino groups of proteins, fatty acids or nucleic acids and aldehyde groups of reducing sugars, and accumulation of AGEs in vivo can cause various complications such as diabetic nephropathy. The results show that the content of AGEs in the serum of the animals in the model group is obviously higher than that of the animals in the normal group, the statistics difference is extremely obvious, the content of AGEs in the serum of the animals in each administration group is reduced to different degrees compared with the model group, and the statistics difference is obvious. This shows that both the compound DB-1 and the compound DB-1-3 can significantly reduce the content of AGEs in serum, thereby achieving the purpose of preventing diabetic complications such as diabetic nephropathy.
Measurement of organ coefficients
In mouse animal model of KK/upj-Ay/J2 diabetes, 50 days after administration, animal liver, kidney, pancreas, epididymal fat, perirenal fat were weighed. The ratio of the weight of each organ to the weight of the animal is the coefficient of each organ.
The liver factor profile of each group of animals 50 days after dosing is shown in figure 20. The results show that the liver coefficients of all groups of animals are obviously changed, the liver coefficients of the model group are obviously increased compared with those of the control group, and the model group has extremely obvious statistical difference, and the compound DB-1, the compound DB-1-3 and the BBR can obviously reduce the liver coefficients and improve the liver function. However, when rosiglitazone was administered, the liver coefficients were statistically different and we speculated that these were caused by the side effects of rosiglitazone.
Ninthly determination result of triglyceride and total cholesterol content in liver tissue
The bar graph of triglyceride content and total cholesterol content in liver tissue 50 days after administration is shown in fig. 21 and 22. The results show that the liver triglyceride content and the total cholesterol content of the model animals are obviously increased compared with those of the normal control group and have extremely obvious statistical difference, the BBR control group with the administration amount of 100mg/kg can reduce the liver triglyceride content to different degrees, the rosiglitazone control group with the administration amount of 5mg/kg has statistically different increase instead, and the BBR control group with the administration amount of 100mg/kg can reduce the liver triglyceride content to different degrees. Administration of 50mg/kg and 100mg/kg of compounds DB-1 and DB-1-3, 100mg/kg of BBR and 5mg/kg of rosiglitazone reduced total liver cholesterol levels to varying degrees.
Determination result of AGEs content, SOD activity and MDA content in liver tissue of R
The increase of AGEs content in liver will cause liver damage, and SOD activity and MDA content in liver reflect liver oxidative stress level.
After 50 days of administration, the AGEs content, SOD activity, MDA content of liver tissue are shown in fig. 23-25. The determination result of the AGEs content in the liver tissue shows that the liver AGEs content of the model group animal is obviously increased and has extremely obvious statistical difference compared with the normal control group animal, the compounds DB-1, DB-1-3 and BBR can reduce the liver AGEs content to different degrees, and the rosiglitazone can not reduce the liver AGEs content, which shows that the compounds DB-1 and DB-1-3 have superiority compared with the rosiglitazone. The determination results of liver SOD activity and MDA content show that compared with normal control animals, model animals have significantly reduced SOD activity and significantly increased MDA content, while liver SOD activity of animals in each administration group has different increases and MDA content has different decreases, which shows that the compounds DB-1 and DB-1-3 can improve liver oxidative stress level.
In the results, DB-1 and DB-1-3 related to the invention have good effects on reducing the fasting blood glucose of mice with KK/upj-Ay/J2 diabetes mellitus, improving oral glucose tolerance, improving insulin tolerance and improving relevant indexes of urine of the mice, and 100mg/kg of DB-1 and DB-1-3 have equivalent effects to 100mg/kg of BBR and 5mg/kg of rosiglitazone, thereby indicating that the two compounds can be used for treating the type 2 diabetes mellitus and preventing diabetic nephropathy.
In the result (c), 5mg/kg rosiglitazone in the ninum, the (c) and the (d) sound waves shows certain side effects, such as obviously increasing the ALT content of serum, obviously increasing the liver coefficient, obviously increasing the triglyceride content of liver tissues and being incapable of reducing the AGEs content of liver, while the compounds DB-1 and DB-1-3 related to the invention do not increase the ALT content of serum, reduce the liver coefficient, reduce the triglyceride content of liver and simultaneously reduce the AGEs content of liver, and the results show that the compounds DB-1 and DB-1-3 related to the invention have the advantages in the aspect of preparing drugs for treating type 2 diabetes, preparing insulin sensitizer and preventing diabetic nephropathy.
Claims (9)
1. The use of a compound of formula I or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of diabetes, or
In the preparation of medicaments for the prevention and/or treatment of diabetic complications, or
The application in preparing the medicine used as the insulin sensitizer,
wherein the content of the first and second substances,
R1represents phenyl optionally substituted by R4Mono-or polysubstituted, R4Selected from: c1-4Alkyl and halo C1-4An alkyl group;
R2represents- (CH)2)nR3(ii) a Wherein the content of the first and second substances,
n is 0, 1,2, 3 or 4;
R3is a hydrogen atom, and is,
the diabetes mellitus is type 2 diabetes mellitus,
the diabetic complication is diabetic nephropathy.
2. The use of a pharmaceutical composition in the preparation of a medicament for the treatment of diabetes, or
In the preparation of a medicament for the prevention or treatment of diabetic complications, or
The application in preparing the medicine used as the insulin sensitizer,
wherein the pharmaceutical composition contains the compound shown in the formula I or the pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient,
wherein the content of the first and second substances,
R1represents phenyl optionally substituted by R4Mono-or polysubstituted, R4Selected from: c1-4Alkyl and halo C1-4An alkyl group;
R2represents- (CH)2)nR3(ii) a Wherein the content of the first and second substances,
n is 0, 1,2, 3 or 4;
R3is a hydrogen atom, and is,
the diabetes mellitus is type 2 diabetes mellitus,
the diabetic complication is diabetic nephropathy.
3. The use of claim 1 or 2, wherein R4Selected from the group consisting of halomethyl, haloethyl, halopropyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.
4. The use of claim 3, wherein R4Selected from the group consisting of trifluoromethyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.
5. The use of claim 4, wherein R4Is trifluoromethyl.
6. The use of claim 1 or 2, wherein R2Represents- (CH)2)nR3Wherein n is 1,2 or 3; r3Is hydrogen.
7. The use of claim 1 or 2, wherein R2Represents methyl, ethyl, n-propyl or n-butyl.
8. The use of claim 1 or 2, wherein R2Represents methyl, ethyl or n-propyl.
9. The use according to claim 1 or 2, wherein the compound of formula I is selected from:
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