WO2022061962A1 - 一种l型氨基酸转运蛋白抑制剂或拮抗剂有效干预糖尿病的方法 - Google Patents

一种l型氨基酸转运蛋白抑制剂或拮抗剂有效干预糖尿病的方法 Download PDF

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WO2022061962A1
WO2022061962A1 PCT/CN2020/119572 CN2020119572W WO2022061962A1 WO 2022061962 A1 WO2022061962 A1 WO 2022061962A1 CN 2020119572 W CN2020119572 W CN 2020119572W WO 2022061962 A1 WO2022061962 A1 WO 2022061962A1
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amino acid
acid transporter
diabetes
type amino
antagonist
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PCT/CN2020/119572
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English (en)
French (fr)
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陈雁
韦思颖
王滔
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中国科学院上海营养与健康研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to the field of biomedicine. Specifically, the present invention relates to a method for effectively intervening diabetes by an L-type amino acid transporter inhibitor or antagonist, more particularly, the present invention relates to a method for effectively intervening diabetes by blocking the L-type amino acid transporter by an inhibitor or antagonist.
  • Diabetes mellitus is a complex metabolic disease characterized by hyperglycemia, which is a chronic syndrome caused by the body's inability to secrete or utilize insulin. Dietary patterns and diet composition are considered to be one of the most important factors influencing the development of chronic diseases. Meanwhile, dietary restriction is an extremely effective dietary pattern for the treatment of numerous metabolic diseases such as diabetes, obesity, tumors, neovascular diseases, and the like.
  • diabetes treatment including metformin, alpha-glucosidase inhibitors, insulin secretagogues, dipeptidyl peptidase IV (DPP4) inhibitors, thiazolidinediones (TZDs), sodium-glucose cotransporters 2 (SGLT2) inhibitor, insulin, glucagon-like peptide 1 (GLP1) receptor agonist, or a combination thereof.
  • DPP4 dipeptidyl peptidase IV
  • ZTDs thiazolidinediones
  • SGLT2 sodium-glucose cotransporters 2
  • insulin glucagon-like peptide 1 (GLP1) receptor agonist, or a combination thereof.
  • the biggest disadvantage of all current diabetes treatment programs is that they can only alleviate disease progression and reduce complications, but cannot effectively restore the function and number of pancreatic ⁇ cells to achieve the purpose of curing the disease.
  • Exercise and nutritional interventions can also lead to unfavorable factors such as motor function strain and poor patient compliance.
  • There are two main factors in the pathogenesis of diabetes one is insulin resistance in peripheral tissues, and the other is the impairment of islet ⁇ -cell function.
  • Most of the current diabetes treatment drugs reduce blood sugar (ie, symptomatic therapy) by inhibiting glucose absorption, increasing muscle uptake and metabolism of glucose, inhibiting hepatic glucose production and output, and improving the insulin sensitivity of peripheral tissues to relieve symptoms.
  • blood sugar ie, symptomatic therapy
  • the purpose of the present invention is to provide a new method for controlling diabetes by inhibiting L-type amino acid transporter, and improving the number and function of pancreatic ⁇ cells, and finding that L-type amino acid transporter is a new target for controlling diabetes.
  • an L-type amino acid transporter inhibitor or antagonist for preparing a composition or preparation for preventing and/or treating diabetes.
  • the L-type amino acid transporter inhibitor or antagonist is selected from the group consisting of small molecule compounds, antibodies, polypeptides, nucleic acids, or combinations thereof.
  • the L-type amino acid transporter inhibitor or antagonist includes 2-amino-2-norbornanecarboxylic acid (BCH) or an analog thereof.
  • BCH 2-amino-2-norbornanecarboxylic acid
  • the diabetes is selected from the group consisting of type 1 diabetes mellitus, type 2 diabetes mellitus, other types of diabetes mellitus (such as diabetes caused by single gene mutation), prediabetes mellitus with impaired glucose tolerance, or a combination thereof.
  • the L-type amino acid transporter inhibitor or antagonist reduces the intracellular content of methionine and/or leucine.
  • the cells are selected from the group consisting of pancreatic beta cells, hepatocytes, skeletal muscle cells, or a combination thereof.
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the mammal includes a mammal suffering from diabetes.
  • the mammal includes a human or a non-human mammal.
  • non-human mammals include rodents (eg, mice, rats, or rabbits) and primates (eg, monkeys).
  • the composition includes a pharmaceutical composition.
  • the pharmaceutical composition contains (a) an L-type amino acid transporter inhibitor or antagonist; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is liquid, solid, or semi-solid.
  • the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral liquids, or injections.
  • the component (a) accounts for 1-99 wt % of the total weight of the pharmaceutical composition, preferably 10-90 wt %, more preferably 30-70 wt % %.
  • the composition further includes other drugs for preventing and/or treating diabetes.
  • the other drugs for preventing and/or treating diabetes are selected from the group consisting of metformin, ⁇ -glucosidase inhibitor, insulin secretagogue, dipeptidyl peptidase IV (DPP4) inhibitor, thiazole Alkanediones (TZDs), sodium-glucose cotransporter 2 (SGLT2) inhibitors, insulin, glucagon-like peptide 1 (GLP1) receptor agonists, or combinations thereof.
  • composition or preparation can be used alone or in combination in the prevention and/or treatment of diabetes.
  • the combined use includes: combined use with other drugs for preventing and/or treating diabetes.
  • a second aspect of the present invention provides a pharmaceutical composition, comprising:
  • a first active ingredient for preventing and/or treating diabetes comprising: an L-type amino acid transporter inhibitor or antagonist;
  • a second active ingredient for preventing and/or treating diabetes includes: other drugs for preventing and/or treating diabetes; and
  • the component (a1) accounts for 1-99wt% of the total weight of the pharmaceutical composition, preferably 10-90wt%, more preferably 30-70wt% %.
  • the component (a2) accounts for 1-99 wt % of the total weight of the pharmaceutical composition, preferably 10-90 wt %, more preferably 30-70 wt % %.
  • the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.
  • the other drugs for preventing and/or treating diabetes are selected from the group consisting of metformin, ⁇ -glucosidase inhibitor, insulin secretagogue, dipeptidyl peptidase IV (DPP4) inhibitor, thiazole Alkanediones (TZDs), sodium-glucose cotransporter 2 (SGLT2) inhibitors, insulin, glucagon-like peptide 1 (GLP1) receptor agonists, or combinations thereof.
  • the pharmaceutical composition may be a single compound or a mixture of multiple compounds.
  • the pharmaceutical composition is used to prepare a medicine or preparation for treating or preventing diabetes.
  • the pharmaceutical dosage form is an oral administration or a non-oral administration dosage form.
  • the oral dosage form is tablet, powder, granule or capsule, or emulsion or syrup.
  • parenteral dosage form is injection or injection.
  • the total content of the active ingredient (a1) and the active ingredient (a2) is 1-99 wt % of the total weight of the composition, more preferably 5-90 wt %.
  • a third aspect of the present invention provides a medicine box, comprising:
  • an optional second container and the active ingredient (a2) other medicaments for the prevention and/or treatment of diabetes, or a medicament containing the active ingredient (a2), located in the second container.
  • first container and the second container are the same or different containers.
  • the medicine in the first container is a single preparation containing L-type amino acid transporter inhibitor or antagonist.
  • the medicine in the second container is a unilateral preparation containing other medicines for preventing and/or treating diabetes.
  • the dosage form of the drug is an oral dosage form or an injection dosage form.
  • the kit further contains instructions, which describe the instructions for co-administering the active ingredient (a1) and the active ingredient (a2) to prevent and/or treat diabetes.
  • the dosage forms of the preparation containing the active ingredient (a1) L-type amino acid transporter inhibitor or antagonist or the preparation containing other drugs for preventing and/or treating diabetes include capsules, tablets, respectively. pills, suppositories, or intravenous injections.
  • the fourth aspect of the present invention provides the use of the pharmaceutical composition of the second aspect of the present invention or the kit of the third aspect of the present invention for preparing a medicament for preventing and/or treating diabetes.
  • a fifth aspect of the present invention provides a method for preventing and/or treating diabetes, comprising the steps of:
  • the L-type amino acid transporter inhibitor or antagonist, the pharmaceutical composition of the second aspect of the present invention, or the kit of the third aspect of the present invention is administered to a subject in need.
  • the administration comprises oral administration.
  • the subject includes a human or a non-human mammal.
  • the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
  • the administration frequency of the L-type amino acid transporter inhibitor or antagonist is 1-7 consecutive days per week, preferably, 2-5 consecutive days per week, more preferably, weekly 2-3 days in a row.
  • the administration time of the L-type amino acid transporter inhibitor is 1-20 weeks, preferably, 2-12 weeks, more preferably, 4-8 weeks.
  • a sixth aspect of the present invention provides a method for reducing fasting blood sugar in mammals, comprising:
  • the L-type amino acid transporter inhibitor or antagonist, the pharmaceutical composition of the second aspect of the present invention, or the kit of the third aspect of the present invention is administered to a subject in need.
  • the administration comprises oral administration.
  • the subject includes a human or a non-human mammal.
  • the subject includes a diabetic human or a non-human mammal.
  • the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
  • a seventh aspect of the present invention provides a method for screening candidate drugs for the treatment of diabetes, comprising the steps of:
  • test substance is a candidate drug for treating diabetes.
  • the cells are mammalian cells.
  • the cells are selected from the group consisting of pancreatic beta cells, hepatocytes, skeletal muscle cells, or a combination thereof.
  • the cells are cells cultured in vitro.
  • the "significantly lower than” refers to E1/E2 ⁇ 1/2, preferably, ⁇ 1/3, more preferably ⁇ 1/4.
  • the "significantly lower” means that A1/A2 ⁇ 1/2, preferably, ⁇ 1/3, more preferably ⁇ 1/4.
  • the method is non-diagnostic and non-therapeutic.
  • the method includes step (c): administering the drug candidate determined in step (a) to a non-human mammal, so as to determine its effect on diabetes in the non-human mammal.
  • test substance is selected from the group consisting of small molecule compounds, antibodies, polypeptides, nucleic acids, or combinations thereof.
  • the eighth aspect of the present invention provides a method for screening candidate compounds for the treatment of diabetes, comprising the steps of:
  • the compound in the test compound library binds to the L-type amino acid transporter, it indicates that the compound that binds to the L-type amino acid transporter is a candidate compound.
  • the method includes step (ii): administering the drug candidate determined in step (a) to a non-human mammal, thereby determining its effect on diabetes in the non-human mammal.
  • the method is non-diagnostic and non-therapeutic.
  • a ninth aspect of the present invention provides a method for screening candidate drugs for the treatment of diabetes, comprising the steps of:
  • the cells are mammalian cells.
  • the cells are selected from the group consisting of pancreatic beta cells, hepatocytes, skeletal muscle cells, or a combination thereof.
  • the cells are cells cultured in vitro.
  • the labeling substance includes dye, fluorescein, biotin, and radioactive element.
  • the "significantly lower” refers to C1/C2 ⁇ 1/2, preferably, ⁇ 1/3, more preferably ⁇ 1/4.
  • the method is non-diagnostic and non-therapeutic.
  • the method includes step (c): administering the drug candidate determined in step (a) to a non-human mammal, so as to determine its effect on diabetes in the non-human mammal.
  • test substance is selected from the group consisting of small molecule compounds, antibodies, polypeptides, nucleic acids, or combinations thereof.
  • Figure 1 shows the effect of supplementation of different essential amino acids on fasting blood glucose in low-protein diets
  • Figure 1A shows the animal experimental protocol, during the use of a low-protein diet, supplemented with different essential amino acids to determine which essential amino acid can reduce low-protein diets. The effect of diet to lower blood sugar.
  • Figure 1B shows that the hypoglycemic effect of the low-protein diet was attenuated after the addition of methionine or leucine, suggesting that these two amino acids were involved in the hypoglycemic effect of the low-protein diet.
  • Figure 2 shows that depletion of methionine and leucine can effectively reduce fasting blood glucose in diabetic mice
  • Figure 2A shows the scheme of another animal experiment, in which methionine or leucine was specifically depleted in the mouse diet for three days a week, and then the study Whether amino acid deletion can lower blood sugar
  • Figure 2B shows that the lack of methionine or leucine in the rat diet can effectively reduce blood sugar, which further suggests that these two amino acids are involved in the function of low-protein diet to lower blood sugar.
  • Figure 3 shows that the L-type amino acid transporter inhibitor BCH can effectively improve fasting blood glucose
  • Figure 3A shows another new animal test protocol.
  • the mice were fed a low-protein diet, or a normal diet but added the L-type amino acid transporter inhibitor BCH. , to study whether BCH has the effect of lowering blood sugar.
  • Figure 3B shows that intermittent use of a low-protein diet can effectively reduce blood sugar, and intermittent use of BCH can also significantly reduce blood sugar, revealing that intermittent use of L-type amino acid transporter inhibitors is a new way to improve diabetes.
  • Figure 4 shows that inhibition of L-type amino acid transporter can significantly increase the number of pancreatic ⁇ cells.
  • the mouse used is a genetically traced mouse, and the islet sections of this mouse are used to observe under a fluorescence immunomicroscope, the green cells are islet beta cells, and the red cells are non-beta cells (the normal control is group 1).
  • the islet structure was disorganized and the number of beta cells was reduced.
  • Islet beta cells were significantly increased after application of a low-protein diet (group 3).
  • the number of islet ⁇ cells was also significantly increased after the use of L-type amino acid transporter inhibitor BCH (group 4), revealing that inhibition of L-type amino acid transporter can effectively improve the number of islet ⁇ cells.
  • L-type amino acid transporter inhibitors or antagonists can effectively reduce fasting blood glucose in mammals and can effectively treat diabetes.
  • the inventors also unexpectedly found that methionine and leucine played the most critical role in the effect of low-protein food on fasting blood sugar. Intermittent use of a diet low in methionine and leucine was effective in improving fasting blood glucose in diabetic mice. On this basis, the present inventors have completed the present invention.
  • Diabetes mellitus is a complex metabolic disease characterized by hyperglycemia, which is a chronic syndrome caused by the body's inability to secrete or utilize insulin.
  • diabetes mellitus is a group of systemic metabolic diseases characterized by chronic hyperglycemia caused by defects in insulin secretion or action caused by multiple etiologies.
  • Long-term sugar, fat and protein metabolism disorders can cause multiple organ damage and homeostasis imbalance, which can lead to chronic diseases, functional impairment, and even failure of the heart, kidneys, eyes, nerves, blood vessels and other tissues and organs.
  • Type 1 diabetes diabetes (diabetes mellitus type 1, T1DM)
  • type 2 diabetes diabetes mellitus type 2, T2DM) are two important types of diabetes, and their pathogenesis is different.
  • Continuously elevated blood sugar is a key factor in multi-organ damage caused by diabetes. Therefore, how to reduce the organ damage caused by blood sugar while using insulin to lower blood sugar for treatment is an important scientific issue.
  • the present invention starts with dietary restriction, and studies a brand-new nutritional intervention method, and more importantly, it is found that intermittently inhibiting the biological activity of L-type amino acid transporter is a new method for treating diabetes, and can improve pancreatic islet beta cell number and whether it effectively alleviates type 1 and type 2 diabetes in mice.
  • the present invention found in the research that the mechanism and control of dietary restriction intervening in diabetes are closely related to the levels of two essential amino acids, methionine and leucine, in the body.
  • the present invention finds for the first time that the content of methionine and leucine in the body is reduced by inhibiting the transport of specific amino acids, so that the effect of intervening diabetes is similar to that of nutrient restriction without controlling the body's nutrient intake.
  • the L-type amino acid transporter family consists of four Na ion neutral amino acid transporters, which are divided into LAT1-LAT4, which are mainly responsible for the transport of neutral amino acids such as methionine and leucine on the cell membrane.
  • an "L-type amino acid transporter inhibitor or antagonist” is a small molecule that is specific for an L-type amino acid transporter and inhibits or antagonizes its transport function (primarily the transport of methionine, and/or leucine). molecules or macromolecular compounds.
  • the L-type amino acid transporter inhibitor or antagonist of the present invention reduces the intracellular content of specific amino acids by blocking the transport of specific amino acids, so as to play a role in intervening diabetes similar to nutritional restriction without controlling the body's nutritional intake. Effect.
  • blocking the transport of specific amino acids in the present invention can also be used in combination with other substances selected from the following group (such as other drugs for preventing and/or treating diabetes).
  • Representative examples include (but are not limited to): selected from the group consisting of: metformin, alpha glycosidase inhibitors, insulin secretagogues, dipeptidyl peptidase IV (DPP4) inhibitors, thiazolidinediones (TZDs) , a sodium-glucose cotransporter 2 (SGLT2) inhibitor, insulin, a glucagon-like peptide 1 (GLP1) receptor agonist, or a combination thereof.
  • DPP4 dipeptidyl peptidase IV
  • ZDs thiazolidinediones
  • SGLT2 sodium-glucose cotransporter 2
  • insulin a glucagon-like peptide 1 (GLP1) receptor agonist, or a combination thereof.
  • L-type amino acid transporter inhibitors or antagonists include, but are not limited to: - amino-2-norbornylcarboxylic acid (BCH), based on L-type amino acid transporter to establish in vitro screening drug model screening Obtained inhibitors (including macromolecular drugs, antibodies or nucleic acid drugs).
  • BCH - amino-2-norbornylcarboxylic acid
  • compositions and methods of administration are provided.
  • the present invention also provides a pharmaceutical composition, which contains (a) a safe and effective amount of the L-type amino acid transporter inhibitor or antagonist of the present invention; and (b) a pharmaceutically acceptable carrier or excipient Form.
  • the dosage of the L-type amino acid transporter inhibitor or antagonist of the present invention is usually 10 micrograms-100 mg/dose, preferably 100-1000 micrograms/dose.
  • an effective dose is about 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg body weight of an L-amino acid transporter inhibitor or antagonist of the present invention administered to an individual.
  • the L-type amino acid transporter inhibitors or antagonists of the present invention can be used alone or in combination with other therapeutic agents (eg, formulated in the same pharmaceutical composition).
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
  • the term refers to pharmaceutical carriers that do not themselves induce the production of antibodies detrimental to the individual receiving the composition, and are not undue toxicity upon administration. These vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • Such carriers include, but are not limited to: saline, buffers, dextrose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • Pharmaceutically acceptable carriers in therapeutic compositions can contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • therapeutic compositions can be prepared as injectables, eg, liquid solutions or suspensions; solid forms suitable for solution or suspension, liquid carriers, prior to injection can also be prepared.
  • compositions of the present invention can be administered by conventional routes including, but not limited to, intratumoral, intramuscular, intravenous, subcutaneous, intradermal, or topical administration.
  • the subject to be prevented or treated can be an animal; especially a human.
  • the pharmaceutical composition of the present invention When used for actual treatment, various pharmaceutical compositions in different dosage forms can be adopted according to the usage. Preferably, it is an intravenous drug preparation or an intratumoral drug injection.
  • compositions can be formulated according to conventional methods by mixing, diluting or dissolving, with occasional addition of suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.
  • ophthalmic eye drops can be prepared by dissolving the L-type amino acid transporter inhibitor or antagonist of the present invention in sterile water (with a surfactant dissolved in sterile water) together with a base substance, adjusting Osmotic pressure and pH can be adjusted to physiological state, and suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and viscosity-increasing agents can be optionally added, and then completely dissolved.
  • suitable pharmaceutical additives such as preservatives, stabilizers, buffers, isotonic agents, antioxidants and viscosity-increasing agents can be optionally added, and then completely dissolved.
  • compositions of the present invention can also be administered in the form of sustained release formulations.
  • the L-type amino acid transporter inhibitors or antagonists of the present invention can be incorporated into pellets or microcapsules supported by a slow release polymer, which are then surgically implanted into the tissue to be treated.
  • sustained-release polymers ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, Lactic acid-glycolic acid copolymers and the like are preferably exemplified by biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.
  • the dose of the L-type amino acid transporter inhibitor or antagonist of the present invention as an active ingredient may be determined according to the body weight, age, sex, symptoms of each patient to be treated extent and reasonably determined.
  • the present invention finds for the first time that by inhibiting the expression and/or activity of the L-type amino acid transporter, the fasting blood sugar of mammals can be effectively reduced, and the blood sugar level of mammals can be better controlled.
  • the present invention finds for the first time that by inhibiting the transport of specific amino acids (such as methionine and/or leucine) to reduce their content in the body, it is similar to nutritional restriction without controlling the body's nutritional intake. The effect of intervention in diabetes.
  • specific amino acids such as methionine and/or leucine
  • the present invention can increase the number of pancreatic islet beta cells and restore the insulin secretion function, which is superior to the currently used diabetes drugs.
  • mice 8-week-old male C57BL/6 mice were purchased from Slack Laboratory Animal Co., Ltd.; STZ and LAT inhibitors were purchased from Sigma Company; blood glucose meter blood glucose test strips were purchased from Abbott Laboratories, USA.
  • STZ builds a type 1 diabetes model: C57 mice fasted for 5 hours were injected with STZ (40 mg/kg body weight) for 5 consecutive days, and the blood glucose of the mice was observed to rise significantly after one week, and the model was successfully established;
  • Experiment 1 Low-protein diet plus a single amino acid experiment. A total of 50 8-week-old male C57BL/6 mice, 5 in each group, were divided into ten groups. The first group of mice ate ordinary food ad libitum every day; the second group of mice ate low-protein food for the first three days of the week and normal food for the last 4 days; the third to tenth groups ate the first 3 days of each week with a A low-protein food with essential amino acids, followed by regular food for the next 4 days; repeated weekly for a total of 6 weeks.
  • Experiment 2 Lack of a single amino acid experiment. A total of 25 8-week-old male C57BL/6 mice, 5 in each group, were divided into five groups. The first group of mice ate ordinary food ad libitum every day; the second group of mice ate low-protein food for the first three days of the week, and normal food for the last 4 days; the third group of mice ate methionine-deficient food for the first 3 days of the week, and then The mice in the fourth group ate leucine-deficient food for the first 3 days and normal food for the last 4 days. The mice in the fifth group ate methionine- and leucine-deficient food for the first 3 days of the week. , after 4 days of eating ordinary food. Repeat weekly for a total of 5 weeks.
  • mice in each group were divided into four groups.
  • the mice in the first, second, and fourth groups ate ordinary food ad libitum every day; the mice in the third group ate low-protein food for the first three days of the week, and normal food for the last 4 days; the mice in the fourth group were intraperitoneally injected with 200 (mg/kg) L-type amino acid transporter inhibitor BCH. Repeat weekly for a total of 4 weeks.
  • mice in experiment 3 were of the INS Cre mTmG strain, and the islets were sectioned for fluorescence microscopy analysis. According to the characteristics of the genetically traced mice, islet beta cells are shown in green and non-beta cells are shown in red.
  • Low-protein rat food was purchased from Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd., containing 5% protein, 12.3% fat, and 26.7% total sugar.
  • the main ingredients were sweet potato powder, vegetable fat powder, vitamin mixture, mineral mixture, etc.
  • Normal rat diet formula normal rat diet containing 20.5% protein was purchased from Shanghai Proton Biotechnology Co., Ltd. The main nutrients are fish meal, wheat, corn, bran, vitamins, minerals, amino acids, etc.
  • mice diets deficient in methionine, leucine, and pairs of methionine and leucine were designed for experiments. Changes in fasting blood glucose of mice after 6 consecutive weeks of dietary intervention were detected at the end of each nutritional intervention session. It was found that the blood glucose of the mice was significantly higher than that of the wild-type mice (the first group) after the STZ model was successfully established. After 6 weeks of continuous intervention, intermittent feeding of either methionine-deficient, leucine-deficient, or both methionine and leucine-deficient diets could effectively reduce fasting blood glucose in mice after STZ modeling ( Figure 2, A, B ). Therefore, reducing the content of two essential amino acids, methionine or leucine, can indeed regulate fasting blood sugar.
  • Example 3 L-type amino acid transporter inhibitor can effectively improve fasting blood sugar
  • the L-type amino acid transporter protein is mainly responsible for the transport of methionine, leucine and other amino acids, so we tried an L-type amino acid The transporter inhibitor BCH to see if it can also effectively reduce fasting blood sugar.
  • BCH an amino acid transporter transporter inhibitor
  • Example 4 L-type amino acid transporter inhibitor can effectively increase the number of pancreatic ⁇ cells
  • islet slices were observed under a fluorescence immunomicroscope, green cells were islet ⁇ cells, and red cells were non- ⁇ cells.
  • the islet structure is disorganized and the number of beta cells is reduced.
  • islet ⁇ cells increased significantly. It is especially important that the number of islet ⁇ cells also increased significantly after the use of L-type amino acid transporter inhibitor BCH, suggesting that inhibition of L-type amino acid transporter can effectively improve the number of islet ⁇ cells ( Figure 4).
  • Group 1 STZ-induced diabetes was group 2
  • low protein intervention group was group 3
  • L-type amino acid transporter inhibitor was group 4.

Abstract

一种L型氨基酸转运蛋白抑制剂或拮抗剂有效干预糖尿病的方法，具体地，提供了一种L型氨基酸转运蛋白抑制剂或拮抗剂的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗糖尿病，并且能够改善胰岛β细胞数量。首次发现，L型氨基酸转运蛋白抑制剂或拮抗剂可有效降低哺乳动物的空腹血糖，并可有效治疗糖尿病。

Description

一种L型氨基酸转运蛋白抑制剂或拮抗剂有效干预糖尿病的方法 技术领域
本发明涉及生物医药领域。具体地，本发明涉及一种L型氨基酸转运蛋白抑制剂或拮抗剂有效干预糖尿病的方法，更具体地，本发明涉及通过抑制剂或拮抗剂阻断L型氨基酸转运蛋白有效干预糖尿病的方法。
背景技术
糖尿病是一种以高血糖为特征的复杂的代谢性疾病，它是由于机体无法分泌或者利用胰岛素而导致的慢性综合征。饮食模式和饮食的组成被认为是影响慢性疾病发生发展最为重要的因素之一。同时，饮食限制是治疗众多代谢性疾病例如糖尿病、肥胖、肿瘤、新血管疾病等的一个极为有效的饮食模式。
目前的糖尿病治疗有多种方法，包括二甲双胍，α糖苷酶抑制剂，胰岛素促泌素，二肽基肽酶IV(DPP4)抑制剂，噻唑烷二酮类(TZD)，钠-葡萄糖协同转运蛋白2(SGLT2)抑制剂，胰岛素，胰高血糖素样肽1(GLP1)受体激动剂，或其组合。
但是目前所有糖尿病治疗方案的最大缺点是只能缓解疾病进展、减少并发症，但不能有效修复胰岛β细胞功能和数量，以达到治愈疾病的目的。运动与营养干预等手段也会导致运动机能劳损、患者依从性差等不利因素。糖尿病发病有两大主要因素，一是外周组织胰岛素抵抗，二是胰岛β细胞功能受损。目前糖尿病治疗药物大多数为通过抑制葡萄糖吸收、增加肌肉对葡萄糖的摄取和代谢、抑制肝糖产生和输出等手段来降低血糖(即对症疗法)，以及提高外周组织的胰岛素敏感性来缓解症状，尚没有针对提升胰岛β细胞的数量和恢复胰岛素分泌功能的治疗手段。
因此，本领域迫切需要开发一种在不控制机体营养摄入、生活习惯的情况下起到和营养限制类似的干预糖尿病的效果，尤其是能够提升胰岛β细胞的数量和恢复胰岛素分泌功能的方法和新型药物。
发明内容
本发明的目的在于提供一种通过抑制L型氨基酸转运蛋白进而控制糖尿病，并有提升胰岛β细胞数量与功能的新方法，发现L型氨基酸转运蛋白是控制糖尿病的一个新靶点。
在本发明第一方面，提供了一种L型氨基酸转运蛋白抑制剂或拮抗剂的用途，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗糖尿病。
在另一优选例中，所述L型氨基酸转运蛋白抑制剂或拮抗剂选自下组：小分子化合物、抗体、多肽、核酸、或其组合。
在另一优选例中，所述L型氨基酸转运蛋白抑制剂或拮抗剂包括2-氨基-2-去甲冰片羧酸(BCH)或其类似物。
在另一优选例中，所述糖尿病选自下组：1型糖尿病、2型糖尿病、其它类型的糖尿病(比如单基因突变导致的糖尿病)、糖耐量受损的前糖尿病、或其组合。
在另一优选例中，所述L型氨基酸转运蛋白抑制剂或拮抗剂降低蛋氨酸和/或亮氨酸在细胞内的含量。
在另一优选例中，所述细胞选自下组：胰岛β细胞、肝细胞、骨骼肌细胞、或其组合。
在另一优选例中，所述组合物或制剂还用于选自下组的一种或多种用途：
(a)降低哺乳动物的空腹血糖；
(b)改善哺乳动物的胰岛β细胞的数量和功能；
(c)改善哺乳动物的胰岛素敏感性。
在另一优选例中，所述哺乳动物包括患有糖尿病的哺乳动物。
在另一优选例中，所述哺乳动物包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠、或兔)、灵长类动物(如猴)。
在另一优选例中，所述组合物包括药物组合物。
在另一优选例中，所述药物组合物含有(a)L型氨基酸转运蛋白抑制剂或拮抗剂；和(b)药学上可接受的载体。
在另一优选例中，所述药物组合物为液态、固体、或半固体。
在另一优选例中，所述药物组合物的剂型包括片剂、颗粒剂、胶囊、口服液、或注射剂。
在另一优选例中，所述的药物组合物中，所述组分(a)占所述药物组合物总重量的1-99wt％，较佳地10-90wt％，更佳地30-70wt％。
在另一优选例中，所述组合物还包括其他的预防和/或治疗糖尿病的药物。
在另一优选例中，所述其他的预防和/或治疗糖尿病的药物选自下组：二甲双胍、α糖苷酶抑制剂、胰岛素促泌素、二肽基肽酶IV(DPP4)抑制剂、噻唑烷二酮类(TZD)、钠-葡萄糖协同转运蛋白2(SGLT2)抑制剂、胰岛素、胰高血糖素样肽1(GLP1)受体激动剂、或其组合。
在另一优选例中，所述组合物或制剂在预防和/或治疗糖尿病的应用中，可单独使用，或联合使用。
在另一优选例中，所述的联合使用包括：与其它预防和/或治疗糖尿病的药物联合使用。
本发明第二方面提供了一种药物组合物，包括：
(a1)用于预防和/或治疗糖尿病的第一活性成分，所述第一活性成分包括：L型氨基酸转运蛋白抑制剂或拮抗剂；
(a2)任选的，预防和/或治疗糖尿病的第二活性成分，所述第二活性成分包括：其他的用于预防和/或治疗糖尿病的药物；和
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物中，所述组分(a1)占所述药物组合物总重量的1-99wt％，较佳地10-90wt％，更佳地30-70wt％。
在另一优选例中，所述的药物组合物中，所述组分(a2)占所述药物组合物总重量的1-99wt％，较佳地10-90wt％，更佳地30-70wt％。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为1:100至100：1，较佳地为1:10至10：1。
在另一优选例中，所述其他的预防和/或治疗糖尿病的药物选自下组：二甲双胍、α糖苷酶抑制剂、胰岛素促泌素、二肽基肽酶IV(DPP4)抑制剂、噻唑烷二酮类(TZD)、钠-葡萄糖协同转运蛋白2(SGLT2)抑制剂、胰岛素、胰高血糖素样肽1(GLP1)受体激动剂、或其组合。
在另一优选例中，所述药物组合物中可以是单一化合物，也可以是多个化合物的混合物。
在另一优选例中，所述的药物组合物用于制备治疗或预防糖尿病的药物或制剂。
在另一优选例中，所述的药物剂型为口服给药或非口服给药剂型。
在另一优选例中，所述的口服给药剂型是片剂、散剂、颗粒剂或胶囊剂，或乳剂或糖浆剂。
在另一优选例中，所述的非口服给药剂型是注射剂或针剂。
在另一优选例中，所述的活性成分(a1)和活性成分(a2)的总含量为组合物总重的1～99wt％，更佳地为5～90wt％。
本发明第三方面提供了一种药盒，包括：
(i)第一容器，以及位于该第一容器中的活性成分(a1)L型氨基酸转运蛋白抑制剂或拮抗剂，或含有活性成分(a)的药物；和
(ii)任选的第二容器，以及位于该第二容器中的活性成分(a2)其他的用于预防和/或治疗糖尿病的药物,或含有活性成分(a2)的药物。
在另一优选例中，所述的第一容器和第二容器是相同或不同的容器。
在另一优选例中，所述的第一容器的药物是含L型氨基酸转运蛋白抑制剂或拮抗剂的单方制剂。
在另一优选例中，所述的第二容器的药物是含其他的用于预防和/或治疗糖尿病的药物的单方制剂。
在另一优选例中，所述药物的剂型为口服剂型或注射剂型。
在另一优选例中，所述试剂盒还含有说明书，所述说明书中记载了联合给予活性成分(a1)和活性成分(a2)从而预防和/或治疗糖尿病的说明。
在另一优选例中，所述含有活性成分(a1)L型氨基酸转运蛋白抑制剂或拮抗剂的制剂或含有其他的用于预防和/或治疗糖尿病的药物的制剂的剂型分别包括胶囊、片剂、栓剂、或静脉注射剂。
本发明第四方面提供了一种本发明第二方面所述的药物组合物或本发明第三方面所述药盒的用途，用于制备用于预防和/或治疗糖尿病的药物。
本发明第五方面提供了一种预防和/或治疗糖尿病的方法，包括步骤：
给需要的对象，施用L型氨基酸转运蛋白抑制剂或拮抗剂、本发明第二方面所述的药物组合物、或本发明第三方面所述的药盒。
在另一优选例中，所述的施用包括口服。
在另一优选例中，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物和灵长目动物，优选小鼠、大鼠、兔、猴。
在另一优选例中，所述L型氨基酸转运蛋白抑制剂或拮抗剂的施用频率为每周连续1-7天次，较佳地，每周连续2-5天，更佳地，每周连续2-3天。
在另一优选例中，所述L型氨基酸转运蛋白抑制剂的施用时间为1-20周， 较佳地，2-12周，更佳地，4-8周。
本发明第六方面提供了一种降低哺乳动物空腹血糖的方法，包括：
给需要的对象，施用L型氨基酸转运蛋白抑制剂或拮抗剂、本发明第二方面所述的药物组合物、或本发明第三方面所述的药盒。
在另一优选例中，所述的施用包括口服。
在另一优选例中，所述的对象包括人或非人哺乳动物。
在另一优选例中，所述对象包括患有糖尿病的人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物和灵长目动物，优选小鼠、大鼠、兔、猴。
本发明第七方面提供了一种筛选治疗糖尿病的候选药物的方法，包括步骤：
(a)在测试组中，在培养体系中，在测试物质的存在下，培养表达L型氨基酸转运蛋白的细胞一段时间T1，检测测试组所述培养体系中的L型氨基酸转运蛋白的表达量E1和/或活性A1；
并且在不存在所述测试物质且其他条件相同的对照组中，检测对照组所述培养体系中L型氨基酸转运蛋白的表达量E2和/或活性A2；
(b1)对E1和E2进行比较，如果E1显著低于E2，则表示所述测试物质是治疗糖尿病的候选药物；和/或
(b2)对A1和A2进行比较，如果A1显著低于A2，则表示所述测试物质是治疗糖尿病的候选药物。
在另一优选例中，所述细胞为哺乳动物细胞。
在另一优选例中，所述细胞选自下组：胰岛β细胞、肝细胞、骨骼肌细胞、或其组合。
在另一优选例中，所述细胞为体外培养的细胞。
在另一优选例中，所述“显著低于”指E1/E2≤1/2,较佳地，≤1/3，更佳地≤1/4。
在另一优选例中，所述所述“显著低于”指A1/A2≤1/2，较佳地，≤1/3，更佳地≤1/4。
在另一优选例中，所述的方法是非诊断和非治疗性的。
在另一优选例中，所述的方法包括步骤(c)：将步骤(a)中所确定的候选药物施用于非人哺乳动物，从而测定其对非人哺乳动物的糖尿病的影响。
在另一优选例中，所述测试物质选自下组：小分子化合物、抗体、多肽、核酸、 或其组合。
本发明第八方面提供了一种筛选治疗糖尿病的候选化合物的方法，包括步骤：
(i)将L型氨基酸转运蛋白与化合物库混合，测定化合物库中的化合物与所述L型氨基酸转运蛋白的结合情况；
其中，如果所述测试化合物库中的化合物与所述L型氨基酸转运蛋白有结合，则表明所述与L型氨基酸转运蛋白结合的化合物为候选化合物。
在另一优选例中，所述方法包括步骤(ii)：将步骤(a)中所确定的候选药物施用于非人哺乳动物，从而测定其对非人哺乳动物的糖尿病的影响。
在另一优选例中，所述的方法是非诊断和非治疗性的。
本发明第九方面提供了一种筛选治疗糖尿病的候选药物的方法，包括步骤：
(a)在测试组中，在培养体系中，在测试物质的存在下，培养表达L型氨基酸转运蛋白的细胞一段时间T1，检测测试组培养细胞中的亮氨酸、蛋氨酸或其标记物质的含量C1；
并且在不存在所述测试物质且其他条件相同的对照组中，检测对照组所述培养体系中亮氨酸、蛋氨酸或其标记含量C2；
(b)对C1和C2进行比较，如果C1显著低于C2，则表示所述测试物质是治疗糖尿病的候选药物。
在另一优选例中，所述细胞为哺乳动物细胞。
在另一优选例中，所述细胞选自下组：胰岛β细胞、肝细胞、骨骼肌细胞、或其组合。
在另一优选例中，所述细胞为体外培养的细胞。
在另一优选例中，所述标记物质包括染料、荧光素、生物素、放射元素。
在另一优选例中，所述“显著低于”指C1/C2≤1/2,较佳地，≤1/3，更佳地≤1/4。
在另一优选例中，所述的方法是非诊断和非治疗性的。
在另一优选例中，所述的方法包括步骤(c)：将步骤(a)中所确定的候选药物施用于非人哺乳动物，从而测定其对非人哺乳动物的糖尿病的影响。
在另一优选例中，所述测试物质选自下组：小分子化合物、抗体、多肽、核酸、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施 例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了添加不同种必需氨基酸对低蛋白粮食干预空腹血糖的影响，图1A显示了动物试验方案，在使用低蛋白饮食期间，添加不同的必需氨基酸，用于判断哪个必需氨基酸可以降低低蛋白饮食降低血糖的作用。图1B显示了添加了蛋氨酸或亮氨酸之后，低蛋白饮食降低血糖的作用有所减弱，从而提示这两种氨基酸参与了低蛋白饮食的降糖作用。
图2显示了缺蛋氨酸和亮氨酸可以有效降低糖尿病小鼠空腹血糖，图2A显示了另一个动物试验的方案，在鼠粮中特异缺失蛋氨酸或亮氨酸，每周缺失三天，然后研究缺失氨基酸之后是否能降低血糖。图2B显示了在鼠粮中缺失蛋氨酸或亮氨酸之后，能有效的降低血糖，从而进一步提示这两种氨基酸参与了低蛋白饮食降低血糖的功能。
图3显示了L型氨基酸转运蛋白抑制剂BCH能有效改善空腹血糖，图3A显示了另一个新的动物试验方案，小鼠采用低蛋白饮食，或正常饮食但添加L型氨基酸转运蛋白抑制剂BCH，研究BCH是否有降低血糖的功效。图3B显示了间歇性使用低蛋白饮食可以有效降低血糖，而间歇性使用BCH也能显著的降低血糖，从而揭示间歇性使用L型氨基酸转运蛋白抑制剂是一种新的改善糖尿病的方法。
图4显示抑制L型氨基酸转运蛋白能够显著增加胰岛β细胞数量。使用的小鼠是一种遗传示踪小鼠，用这种小鼠的胰岛切片在荧光免疫显微镜下观察，绿色细胞为胰岛β细胞，红色细胞为非β细胞(正常对照为第1组)。在STZ诱导的糖尿病小鼠模型中(第2组)，胰岛结构混乱，β细胞数量降低。应用低蛋白饮食后，胰岛β细胞明显增加(第3组)。尤其重要的是，在使用L型氨基酸转运蛋白抑制剂BCH后，胰岛β细胞数量也明显增加(第4组)，揭示抑制L型氨基酸转运蛋白能够有效改善胰岛β细胞的数量。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现，L型氨基酸转运蛋白抑制剂或拮抗剂可有效降低哺乳动物的空腹血糖，并可有效治疗糖尿病。
此外，本发明人还意外发现，蛋氨酸和亮氨酸对低蛋白粮食干预空腹血糖的影响中起了最为关键的作用。间歇性的使用低蛋氨酸和亮氨酸饮食可以有效改善糖尿病小鼠的空腹血糖。在此基础上，本发明人完成了本发明。
糖尿病
糖尿病是一种以高血糖为特征的复杂的代谢性疾病，它是由于机体无法分泌或者利用胰岛素而导致的慢性综合征。
具体地，糖尿病(diabetes mellitus，DM)是一组由多病因引起的胰岛素分泌或者作用缺陷，并以慢性高血糖为特征的全身代谢性疾病。长期的糖、脂肪及蛋白质代谢紊乱可引起多器官损害和稳态失衡，会导致心、肾、眼、神经、血管等组织器官慢性病变，功能减退，甚至衰竭等并发症。1型糖尿病(diabetes mellitus type 1，T1DM)和2型糖尿病(diabetes mellitus type 2，T2DM)是糖尿病的两个重要分型，两者的发病机制有所不同。血糖持续升高是糖尿病所致多器官损伤的关键因素，因此利用胰岛素降低血糖进行治疗的同时如何降低血糖导致的器官损伤作用是一个重要意义的科学问题。
本发明以饮食限制入手，研究了一种全新的营养干预手段，尤其重要的是，发现了间歇性抑制L型氨基酸转运蛋白的生物活性是一种新的治疗糖尿病的方法，并且能够提高胰岛β细胞数量，从而否有效的缓解小鼠的1型和2型糖尿病。
并且本发明在研究中发现，饮食限制干预糖尿病的机制和控制与蛋氨酸和亮氨酸这两种必需氨基酸的在机体的水平是密切相关。
本发明首次发现，通过抑制特定氨基酸的转运从而降低蛋氨酸和亮氨酸在体内的含量，从而在不控制机体营养摄入的情况下起到和营养限制类似的干预糖尿病的效果。
L型氨基酸转运蛋白
L型氨基酸转运蛋白家族由4种Na离子中性氨基酸转运蛋白组成，分为LAT1-LAT4，主要负责了细胞膜上对于蛋氨酸、亮氨酸等中性氨基酸的转运。
L型氨基酸转运蛋白抑制剂或拮抗剂
如本文所用，“L型氨基酸转运蛋白抑制剂或拮抗剂”为特异针对L型氨 基酸转运蛋白，并抑制或拮抗其的转运功能(主要是对于蛋氨酸、和/或亮氨酸的转运)的小分子或大分子化合物。
本发明的L型氨基酸转运蛋白抑制剂或拮抗剂通过阻断特定氨基酸的转运从而降低它们在细胞内的含量，从而在不控制机体营养摄入的情况下起到和营养限制类似的干预糖尿病的效果。
此外，本发明中阻断对特定氨基酸的转运，还可同选自下组其他物质(如其他的预防和/或治疗糖尿病的药物)联用。代表性的例子包括(但并不限于)：选自下组：二甲双胍、α糖苷酶抑制剂、胰岛素促泌素、二肽基肽酶IV(DPP4)抑制剂、噻唑烷二酮类(TZD)、钠-葡萄糖协同转运蛋白2(SGLT2)抑制剂、胰岛素、胰高血糖素样肽1(GLP1)受体激动剂、或其组合。
在一优选实施方式中，L型氨基酸转运蛋白抑制剂或拮抗剂包括，但并不限于：-氨基-2-去甲冰片羧酸(BCH)、基于L型氨基酸转运蛋白建立体外筛药模型筛选获得的抑制剂(包括大分子药物、抗体或核酸药物)。
药物组合物和施用方法
另一方面，本发明还提供了一种药物组合物，它含有(a)安全有效量的本发明的L型氨基酸转运蛋白抑制剂或拮抗剂；以及(b)药学上可接受的载体或赋形剂。本发明L型氨基酸转运蛋白抑制剂或拮抗剂的剂量通常为10微克-100毫克/剂，较佳地为100-1000微克/剂。为了本发明的目的，有效的剂量为给予个体约0.01毫克/千克至50毫克/千克，较佳地0.05毫克/千克至10毫克/千克体重的本发明的L型氨基酸转运蛋白抑制剂或拮抗剂。此外，本发明的L型氨基酸转运蛋白抑制剂或拮抗剂可以单用，也可与其他治疗剂一起使用(如配制在同一药物组合物中)。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在Remington’s Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的赋形剂的充分讨论。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂及其组合。
治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙 醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。
通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：瘤内、肌内、静脉内、皮下、皮内、或局部给药。待预防或治疗的对象可以是动物；尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地为静脉用药制剂或瘤内用药注射剂。
这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂(isotonicities)、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
例如，眼部滴眼液的配制可这样进行：将本发明的L型氨基酸转运蛋白抑制剂或拮抗剂与基本物质一起溶解于无菌水(在无菌水中溶解有表面活性剂)中，调节渗透压和酸碱度至生理状态，并可任意地加入合适的药物添加剂如防腐剂、稳定剂、缓冲剂、等渗剂、抗氧化剂和增粘剂，然后使其完全溶解。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明的L型氨基酸转运蛋白抑制剂或拮抗剂可被掺入以缓释聚合物为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。作为缓释聚合物的例子，可例举的有乙烯-乙烯基乙酸酯共聚物、聚羟基甲基丙烯酸酯(polyhydrometaacrylate)、聚丙烯酰胺、聚乙烯吡咯烷酮、甲基纤维素、乳酸聚合物、乳酸-乙醇酸共聚物等，较佳地可例举的是可生物降解的聚合物如乳酸聚合物和乳酸-乙醇酸共聚物。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明的L型氨基酸转运蛋白抑制剂或拮抗剂的剂量，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定。
本发明的主要优点包括：
(1)本发明首次发现，通过抑制L型氨基酸转运蛋白的表达和/或活性可有效降低哺乳动物空腹血糖，可以更好的控制哺乳动物的血糖水平。
(2)本发明首次发现，通过抑制特定氨基酸(如蛋氨酸和/或亮氨酸)的转运从而降低它们在体内的含量，从而在不控制机体营养摄入的情况下起到和营养限制类似的干预糖尿病的效果。
(3)本发明能够增加胰岛β细胞的数量和恢复胰岛素分泌功能，优于目前使用的糖尿病药物。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非特别说明，否则本发明实施例中所用材料和试剂均为市售产品。
材料与方法：
1.1材料：8周龄雄性C57BL/6小鼠购自斯莱克实验动物有限公司；STZ和LAT抑制剂购自Sigma公司；血糖仪血糖试纸购自美国雅培公司。
1.2方法
1.2.1 STZ构建一型糖尿病模型：连续5天给空腹5h的C57小鼠注射(40mg/kg体重)的STZ，一周后观察到小鼠血糖显著上升，造模成功；
1.2.2空腹血糖监测：小鼠早晨空腹6h后鼠尾采血用血糖仪检测血糖含量。
1.2.3实验方案：
实验一：低蛋白饮食加单一氨基酸实验。8周龄雄性C57BL/6小鼠共50只，每组5只，共分为十组。第一组小鼠每天自由进食普通粮食；第二组小鼠每周前三天进食低蛋白粮食，后4天进食普通粮食；第三到第十组小鼠每周前3天进食含某一种必需氨基酸的低蛋白粮食，后4天进食普通粮食；每周重复，共持续6周。
实验二：缺单一氨基酸实验。8周龄雄性C57BL/6小鼠共25只，每组5只，共分为五组。第一组小鼠每天自由进食普通粮食；第二组小鼠每周前三天进食低蛋白粮食，后4天进食普通粮食；第三组小鼠每周前3天进食缺蛋氨酸的粮食，后4天进食普通粮食；第四组小鼠每周前3天进食缺亮氨酸的粮食，后4天进食普通粮食；第五组小鼠每周前3天进食缺蛋氨酸和亮氨酸的粮食，后4天进食普通粮食。每周重复，共持续5周。
实验三：L型氨基酸转运蛋白抑制剂实验。8周龄雄性遗传示踪小鼠(INS CremTmG品系)小鼠共20只，每组5只，共分为四组。第一、二、四组小鼠每天自由进食普通粮食；第三组小鼠每周前三天进食低蛋白粮食，后4天进食普通粮食；第四组小鼠每周前3天腹腔注射200(mg/kg)L型氨基酸转运蛋白抑制剂BCH。每周重复，共持续4周。
实验四：小鼠胰岛切片分析。实验三的小鼠为INS CremTmG品系，胰岛切片后进行荧光显微镜分析。根据该遗传示踪小鼠的特征，绿色为胰岛β细胞，红色为非β细胞。
1.3鼠粮配方
1.3.1低蛋白鼠粮购自江苏协同医药生物工程有限责任公司，含蛋白5％，脂肪12.3％，总糖26.7％，主要成份有红薯粉，植物脂肪粉、维生素混合物、矿物质混合物等。
1.3.2正常鼠粮配方：含20.5％蛋白的正常鼠粮购自上海普路腾生物科技有限公司。主要营养成份有鱼粉，小麦，玉米，麸皮，维生素，矿物质，氨基酸等。
1.3.3缺氨基酸鼠粮配方：缺蛋氨酸，亮氨酸以及同时缺蛋氨酸和亮氨酸的饲料购自戴茨生物科技(无锡)有限公司。主要营养成分有氨基酸，麦芽糊精，玉米淀粉，玉米油，纤维素，复合矿物质，碳酸氢钠，复合维生素，酒石酸胆碱，黄色色素等。
实施例1 添加不同种必需氨基酸对低蛋白粮食干预空腹血糖的影响
在每个营养干预疗程末检测了连续5周的饮食干预后小鼠空腹血糖的变化，发现在打完STZ造模成功后小鼠的血糖明显升高，在通过低蛋白粮食干预五周后，第二组小鼠血糖和对照组相比显著下降，空腹血糖接近于正常血糖。当将8种不同的必需氨基酸添加到低蛋白粮食中后，发现除了添加了蛋氨酸或者亮氨酸的两组小鼠血糖逐渐回升到和对照组小鼠一样的高空腹血糖，其他氨基酸的添加并不会影响低蛋白营养干预对糖尿病小鼠空腹血糖降低的效果(图1，A、B)。因此，缺蛋氨酸和亮氨酸可能是介导低蛋白饮食降低空腹血糖关键因素。
实施例2 缺蛋氨酸和亮氨酸可以有效降低糖尿病小鼠空腹血糖
接下来，设计了缺蛋氨酸、亮氨酸以及蛋氨酸和亮氨酸对的小鼠饲料用作实验。在每个营养干预疗程末检测了连续6周的饮食干预后小鼠空腹血糖的变化。发现在 打完STZ造模成功后，小鼠的血糖和野生型小鼠(第一组)相比明显升高。在连续干预6周后，无论是缺蛋氨酸、缺亮氨酸还是同时却蛋氨酸和亮氨酸的饲料的间歇性喂养都可以有效降低STZ造模后小鼠的空腹血糖(图2，A、B)。因此，降低蛋氨酸或亮氨酸这两大必需氨基酸的含量确实可以调控空腹血糖。
实施例3 L型氨基酸转运蛋白抑制剂能有效改善空腹血糖
因为蛋氨酸和亮氨酸在体内的含量与糖尿病小鼠的空腹血糖有很大的关联，L型氨基酸转运蛋白蛋白主要负责转运蛋氨酸、亮氨酸等氨基酸，所以，我们尝试了一种L型氨基酸转运蛋白抑制剂BCH来看它是否也能有效降低空腹血糖。我们每周的前三天用腹腔注射BCH这一氨基酸转运蛋白转运蛋白抑制剂的方法来代替食物中缺少特定氨基酸的效果，连续四周实验后发现，这种间歇性的注射氨基酸转运蛋白转运蛋白抑制剂的方法可以有效降低空腹血糖，这种效果甚至比之前的间歇性低蛋白饮食的效果更为明显(图3，A、B)。因此，这一实验结果提示L型氨基酸转运蛋白抑制剂是一个治疗糖尿病的有效方法。
实施例4 L型氨基酸转运蛋白抑制剂能有效提高胰岛β细胞数量
利用遗传示踪小鼠的特征，用胰岛切片在荧光免疫显微镜下观察，绿色细胞为胰岛β细胞，红色细胞为非β细胞。在STZ诱导的糖尿病小鼠模型中，胰岛结构混乱，β细胞数量降低。应用低蛋白饮食后，胰岛β细胞明显增加。尤其重要的是，在使用L型氨基酸转运蛋白抑制剂BCH后，胰岛β细胞数量也明显增加，提示抑制L型氨基酸转运蛋白能够有效改善胰岛β细胞的数量(图4)，其中正常对照为第1组，STZ诱导的糖尿病为第2组，低蛋白干预组为第3组，L型氨基酸转运蛋白抑制剂为第4组。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种L型氨基酸转运蛋白抑制剂或拮抗剂的用途，其特征在于，用于制备组合物或制剂，所述组合物或制剂用于预防和/或治疗糖尿病。
  2. 如权利要求1所述的用途，其特征在于，所述L型氨基酸转运蛋白抑制剂或拮抗剂选自下组：小分子化合物、抗体、多肽、核酸、或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述L型氨基酸转运蛋白抑制剂或拮抗剂降低蛋氨酸和/或亮氨酸在细胞内的含量。
  4. 如权利要求1所述的用途，其特征在于，所述组合物或制剂还用于选自下组的一种或多种用途：
    (d)降低哺乳动物的空腹血糖；
    (e)改善哺乳动物的胰岛β细胞的数量和功能；
    (f)改善哺乳动物的胰岛素敏感性。
  5. 一种药物组合物，其特征在于，包括：
    (a1)用于预防和/或治疗糖尿病的第一活性成分，所述第一活性成分包括：L型氨基酸转运蛋白抑制剂或拮抗剂；
    (a2)任选的，预防和/或治疗糖尿病的第二活性成分，所述第二活性成分包括：其他的用于预防和/或治疗糖尿病的药物；和
    (b)药学上可接受的载体。
  6. 一种药盒，其特征在于，包括：
    (i)第一容器，以及位于该第一容器中的活性成分(a1)L型氨基酸转运蛋白抑制剂或拮抗剂，或含有活性成分(a)的药物；和
    (ii)任选的第二容器，以及位于该第二容器中的活性成分(a2)其他的用于预防和/或治疗糖尿病的药物,或含有活性成分(a2)的药物。
  7. 一种权利要求5所述的药物组合物或权利要求6所述药盒的用途，其特征在于，用于制备用于预防和/或治疗糖尿病的药物。
  8. 一种筛选治疗糖尿病的候选药物的方法，其特征在于，包括步骤：
    (a)在测试组中，在培养体系中，在测试物质的存在下，培养表达L型氨基酸转运蛋白的细胞一段时间T1，检测测试组所述培养体系中的L型氨基酸转运蛋白的表达量E1和/或活性A1；
    并且在不存在所述测试物质且其他条件相同的对照组中，检测对照组所述培养 体系中L型氨基酸转运蛋白的表达量E2和/或活性A2；
    (b1)对E1和E2进行比较，如果E1显著低于E2，则表示所述测试物质是治疗糖尿病的候选药物；和/或
    (b2)对A1和A2进行比较，如果A1显著低于A2，则表示所述测试物质是治疗糖尿病的候选药物。
  9. 一种筛选治疗糖尿病的候选化合物的方法，其特征在于，包括步骤：
    (i)将L型氨基酸转运蛋白与化合物库混合，测定化合物库中的化合物与所述L型氨基酸转运蛋白的结合情况；
    其中，如果所述测试化合物库中的化合物与所述L型氨基酸转运蛋白有结合，则表明所述与L型氨基酸转运蛋白结合的化合物为候选化合物。
  10. 一种筛选治疗糖尿病的候选药物的方法，其特征在于，包括步骤：
    (a)在测试组中，在培养体系中，在测试物质的存在下，培养表达L型氨基酸转运蛋白的细胞一段时间T1，检测测试组培养细胞中的亮氨酸、蛋氨酸或其标记物质的含量C1；
    并且在不存在所述测试物质且其他条件相同的对照组中，检测对照组所述培养体系中亮氨酸、蛋氨酸或其标记含量C2；
    (b)对C1和C2进行比较，如果C1显著低于C2，则表示所述测试物质是治疗糖尿病的候选药物。
PCT/CN2020/119572 2020-09-28 2020-09-30 一种l型氨基酸转运蛋白抑制剂或拮抗剂有效干预糖尿病的方法 WO2022061962A1 (zh)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170348398A1 (en) * 2016-06-03 2017-12-07 Vanderbilt University Compositions and methods for decreasing blood glucagon levels
US20190059433A1 (en) * 2017-08-24 2019-02-28 Wisconsin Alumni Research Foundation Compositions and methods for restoring metabolic health
CN110215448A (zh) * 2018-03-02 2019-09-10 中国医学科学院基础医学研究所 一种含有转运蛋白抑制剂的药物、药物组合物及用途

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE602004019062D1 (de) * 2003-03-07 2009-03-05 Human Cell Systems Inc Methode zur identifizierung von substraten oder inhibitoren von transporterproteinen für verzweigte neutrale aminosäuren
JP2019533676A (ja) * 2016-10-13 2019-11-21 ジュノー セラピューティクス インコーポレイテッド トリプトファン代謝経路調節剤を含む免疫療法の方法および組成物
EP3529278A1 (en) * 2016-10-20 2019-08-28 Regeneron Pharmaceuticals, Inc. Methods of lowering blood glucose levels

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170348398A1 (en) * 2016-06-03 2017-12-07 Vanderbilt University Compositions and methods for decreasing blood glucagon levels
US20190059433A1 (en) * 2017-08-24 2019-02-28 Wisconsin Alumni Research Foundation Compositions and methods for restoring metabolic health
CN110215448A (zh) * 2018-03-02 2019-09-10 中国医学科学院基础医学研究所 一种含有转运蛋白抑制剂的药物、药物组合物及用途

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CASTAÑO-MARTINEZ TERESA, SCHUMACHER FABIAN, SCHUMACHER SILKE, KOCHLIK BASTIAN, WEBER DANIELA, GRUNE TILMAN, BIEMANN RONALD, MCCANN: "Methionine restriction prevents onset of type 2 diabetes in NZO mice", THE FASEB JOURNAL, vol. 33, no. 6, 1 June 2019 (2019-06-01), US, pages 7092 - 7102, XP055915488, ISSN: 0892-6638, DOI: 10.1096/fj.201900150R *
WU, XIN ET AL.: "Progress on L-Type Amino Acid Transporter-1", LIFE SCIENCES, vol. 20, no. 2, 30 April 2008 (2008-04-30), pages 253 - 257, XP055915498 *
YAMAMOTO YU, SAWA RAN, WAKE IKUMI, MORIMOTO AYAKA, OKIMURA YASUHIKO: "Glucose-mediated inactivation of AMP-activated protein kinase reduces the levels of L-type amino acid transporter 1 mRNA in C2C12 cells", NUTRITION RESEARCH, vol. 47, 1 November 2017 (2017-11-01), AMSTERDAM, NL, pages 13 - 20, XP055915501, ISSN: 0271-5317, DOI: 10.1016/j.nutres.2017.08.003 *

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