WO2022104780A1 - 脂质化合物在预防或治疗糖尿病中的用途 - Google Patents

脂质化合物在预防或治疗糖尿病中的用途 Download PDF

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WO2022104780A1
WO2022104780A1 PCT/CN2020/130843 CN2020130843W WO2022104780A1 WO 2022104780 A1 WO2022104780 A1 WO 2022104780A1 CN 2020130843 W CN2020130843 W CN 2020130843W WO 2022104780 A1 WO2022104780 A1 WO 2022104780A1
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diabetes
pharmaceutically acceptable
acceptable salt
acid
fasting
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PCT/CN2020/130843
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English (en)
French (fr)
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蒋澄宇
马一鸣
杜芯仪
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中国医学科学院基础医学研究所
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Priority to PCT/CN2020/130843 priority Critical patent/WO2022104780A1/zh
Priority to CN202080039371.4A priority patent/CN113993524A/zh
Publication of WO2022104780A1 publication Critical patent/WO2022104780A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A61K31/685—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols one of the hydroxy compounds having nitrogen atoms, e.g. phosphatidylserine, lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • This application relates to biology, medicine, chemistry, and clinical fields.
  • the present application relates to novel uses of lipid compounds for the prevention or treatment of diabetes.
  • IDF International Diabetes Federation 1 in 11 adults aged 20-79 had diabetes in 2015; this number is expected to increase to 642 million by 2040; the fastest growth transition from low-income to middle-income areas. At present, China has become the country with the largest incidence of diabetes in the world, and more than 90% of the diabetic patients are type 2 diabetes.
  • Complications include macrovascular disease (hypertension, hyperlipidemia, heart disease, coronary artery disease, stroke, cerebrovascular disease, and peripheral vascular disease), microvascular disease (retinopathy, nephropathy, and neuropathy), and cancer. Therefore, studying the pathological process of diabetes and finding an efficient drug that can alleviate the complications of diabetes has become a new therapeutic strategy.
  • macrovascular disease hypertension, hyperlipidemia, heart disease, coronary artery disease, stroke, cerebrovascular disease, and peripheral vascular disease
  • microvascular disease retinopathy, nephropathy, and neuropathy
  • the liver is an important organ in the whole body metabolism and plays an important role in the metabolism of the body. Insulin resistance is associated with the development of type 2 diabetes (T2DM). Lipotoxins, mitochondrial function, cytokines, and adipocytokines play important roles in patients with nonalcoholic fatty liver disease and type 2 diabetes. People with nonalcoholic fatty liver disease often have insulin resistance. A large number of patients with type 2 diabetes develop nonalcoholic fatty liver disease with the inflammatory complication nonalcoholic steatohepatitis.
  • Brown fat is the body tissue responsible for breaking down obesity-inducing white fat, converting it into carbon dioxide, water, and heat. Brown fat can speed up the body's metabolism, promote white fat consumption, or can be a potential mechanism against diabetes.
  • Commonly used drugs for diabetes are thiazolidinediones, which are suitable for type 2 diabetic patients with prominent insulin resistance (ie, obese or overweight type 2 diabetic patients).
  • the target of thiazolidinediones is PPAR- ⁇ , which can increase the sensitivity of peripheral tissues to insulin, improve insulin resistance and lower blood sugar, and can improve various cardiovascular risk factors related to insulin resistance.
  • lipid compound which is represented by formula (I):
  • lipid compound that is 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (also known as stearoyl lysolecithin; CAS No: 19420-57 -6; 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine) (hereinafter referred to as S-lysoPC).
  • S-lysoPC 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine
  • the use of the compound represented by the aforementioned formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient in the treatment of diabetes or its complications is also provided.
  • the use of the compound represented by the aforementioned formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament wherein the medicament is used for any one or a combination selected from the following: blood sugar control, weight control , Reduce fasting blood lipid level, reduce fasting blood sugar level, reduce fasting insulin level, reduce fasting alanine aminotransferase level, reduce fasting aspartate aminotransferase level, improve insulin sensitivity, and promote the formation of brown fat.
  • the aforementioned compound of formula (I) or a pharmaceutically acceptable salt thereof is suitable for use in type I diabetes.
  • the aforementioned compound of formula (I) or a pharmaceutically acceptable salt thereof is suitable for use in type II diabetes.
  • the compound represented by the aforementioned formula (I) or a pharmaceutically acceptable salt thereof is suitable for improving diabetes complications, and is selected from any one of the following or a combination thereof: insulin resistance, hyperglycemia, insulin resistance, hyperglycemia Hyperglycemia, Hyperlipidemia, Hypertriglyceridemia, Hypercholesterolemia, HypoHDLemia, HyperLDLemia, Nonalcoholic Fatty Liver, Myocardial Injury, Liver Balloon Lesion, Liver Inflammation, Nonalcoholic Fatty liver, myocardial injury, liver ballooning lesions, liver inflammation.
  • the pharmaceutically acceptable salt of the compound of formula (I) is selected from any one or a combination of the following: acetate, benzenesulfonate, benzoate, propionate, malonic acid Salt, Pyruvate, Carbonate, Bicarbonate, Sulfate, Methylsulfate, Bisulfate, Isothiosulfate, Tartrate, Bistarate, Borate, Citrate, Ethylenediamine Tetraacetate, ethanedisulfonate, propionate dodecyl sulfate, ethanesulfonate, glycolate, fumarate, glucoheptonate, gluconate, glutamate, Caproate, hexylisophthalate, hydrochloride, hydroxynaphthoate, hydrobromide, lactate, lactobionate, malate, maleate, cinnamate, mandelate , Mesylate, Methyl Bromide
  • a medicament or a pharmaceutical composition comprising a prophylactically effective amount or a therapeutically effective amount of the aforementioned compound represented by formula (I) or a pharmaceutically acceptable salt thereof.
  • the medicament or pharmaceutical composition of the present application further comprises a pharmaceutically acceptable carrier or excipient.
  • the compound represented by the aforementioned formula (I) or a pharmaceutically acceptable salt thereof is used as the only active ingredient in the medicament or pharmaceutical composition of the present application.
  • the aforementioned compound of formula (I) or a pharmaceutically acceptable salt thereof may be administered in combination (simultaneous or sequential administration) with drugs known in the art for the treatment of diabetes.
  • the medicament or pharmaceutical composition of the present application can be administered in any of the following ways: oral, inhalation, rectal, nasal, topical, parenteral (such as but not limited to: subcutaneous, intravenous, intramuscular, intraperitoneal).
  • the medicament or pharmaceutical composition of the present application is an oral dosage form.
  • the medicament or pharmaceutical composition of the present application is in a form selected from the group consisting of tablets, lozenges, capsules, granules, powders, syrups, solutions, suspensions, aerosols, sprays agent.
  • Figure 1 shows the SPR results of S-lysoPC and PPAR- ⁇ -LBD.
  • Figure 2A shows glucose tolerance test; ⁇ represents: db/db; ⁇ represents: S-lysoPC (3.3 mg/kg/day); ⁇ represents: S-lysoPC (10 mg/kg/day); ⁇ represents: rosiglitazone (10 mg/kg/day).
  • Figure 2B shows the AUC area.
  • Figure 2C shows the insulin tolerance test; ⁇ represents: db/db; ⁇ represents: S-lysoPC (3.3 mg/kg/day); ⁇ represents: S-lysoPC (10 mg/kg/day); ⁇ represents: rosiglitazone (10 mg/kg/day).
  • Figure 2D shows the AUC area.
  • Figure 3A shows the change of body weight of mice in each group with the time of drug administration.
  • Figures 3B to 3D show the biochemical indicators of fasting blood lipids, insulin, and blood glucose of mice in each group.
  • Figure 4A shows the biochemical indexes of alanine aminotransferase (ALT) in each group of mice.
  • Figure 4B shows the biochemical indexes of aspartate aminotransferase (AST) in each group of mice.
  • Figure 4C shows the oil red test of the livers of mice in each group, and the statistical graph of the positive area.
  • Figure 4D shows the HE experiments of the mouse livers in each group, as well as the NAS pathological scores.
  • Figure 4E shows the detection of the protein expression levels of AMPK and PPAR- ⁇ in the livers of mice in each group; S-lysoPC molecule can significantly increase the protein expression of PPAR- ⁇ , and can activate the AMPK pathway.
  • Figure 5A shows a HE map of white fat and a statistical map of the number of adipocytes.
  • Figure 5B and Figure 5C show the expression levels of genes downstream of PPAR- ⁇ .
  • Figure 6A shows the HE map of brown fat and the statistics of the number of adipocytes.
  • Figure 6B shows the detection of brown adipogenic markers; PGC1a, UCP1 were significantly increased, and PPAR- ⁇ protein expression was increased.
  • Figure 7A shows the immunohistochemical images of oxidative stress-injured iNOS in the myocardial tissues of each group, and the positive statistics.
  • Figure 7B shows the fasting CK index in each group of mice.
  • salts that are pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound (compound of formula I).
  • Such salts include:
  • An acid addition salt formed with an inorganic acid or an organic acid such as but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid; the organic acid such as acetic acid, propionic acid, caproic acid, Cyclopentanoic 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, ethyl acetate Sulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, etc.;
  • the inorganic acid such as but not limited to hydrochloric acid
  • suitable pharmaceutically acceptable salts include, but are not limited to, salts derived from inorganic bases, including, for example, aluminum, ammonium, calcium, copper, ferric, divalent Valence iron, lithium, magnesium, manganese, divalent manganese, potassium, sodium, zinc; particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts;
  • Salts formed with organic bases such as amine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, N-methylglucamine, arginine, betaine, caffeine, choline, Diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, N-ethyl-morpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, isopropylamine, lysine Acid, methylglucamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine.
  • organic bases such as amine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, N-methylglucamine, arginine, betaine, caffeine, choline, Diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,
  • Pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, albumin, buffer substances, oils, sorbic acid, potassium sorbate, water, salts, electrolytes, protamine sulfate Protein, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, polyvinylpyrrolidone, polyethylene glycol, cellulose, sodium carboxymethyl cellulose, polyacrylate, beeswax, lanolin.
  • a pharmaceutically acceptable excipient refers to an addition other than the active ingredient in the pharmaceutical preparation, which is stable in nature and does not produce side effects or affect the curative effect when combined with the active ingredient.
  • binders fillers, disintegrants, lubricants in tablets; ointments in solid preparations; preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers in liquid preparations , osmotic regulators, colorants, etc.
  • administering should be understood to refer to providing a compound of the present application (or a pharmaceutically acceptable salt) or a prodrug thereof to a subject.
  • prodrug refers to any compound that, when administered to an organism, produces a biologically active compound as a result of spontaneous chemical reactions, enzymatic chemical reactions, and/or metabolic chemical reactions. Prodrugs undergo chemical transformations to yield biologically active compounds. In certain instances, prodrug forms of compounds can be used, for example, to improve bioavailability, mask or reduce bitter taste or gastrointestinal irritation, improve acceptability to subjects, alter solubility, provide prolonged or sustained release or delivery , Improve the delivery of formulations.
  • compositions intended for oral use may be prepared according to any method known in the art, and such pharmaceutical compositions may contain, for example, sweetening agents, flavoring agents, coloring agents, preservatives.
  • the active ingredient is mixed with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients can be, for example, inert diluents (such as calcium or sodium carbonate, lactose, calcium or sodium phosphate); disintegrants (such as corn starch); binders (such as starch, gelatin or acacia) ; and lubricants (such as magnesium stearate, stearic acid or talc).
  • Tablets may be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and to provide a sustained action for a longer period of time.
  • Formulations for oral administration can also be presented as hard capsules, in which the active ingredient is mixed with an inert solid diluent, or soft capsules, in which the active ingredient is mixed with water or an oily vehicle (peanut oil, liquid paraffin, or olive oil).
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules or tablets, each containing a predetermined amount of the active ingredient; as granules; as in aqueous liquid or non- solutions or suspensions in aqueous liquids; or as emulsions in oil-in-water or water-in-oil.
  • Formulations suitable for topical administration in the mouth include lozenges comprising a flavored base, usually sucrose and acacia or tragacanth, and the active ingredient.
  • an "effective amount” refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.
  • a prophylactically effective amount refers to an amount sufficient to prevent, arrest or delay the occurrence of a disease (or its symptoms) or complication;
  • a therapeutically effective amount refers to an amount sufficient to cure or at least partially alleviate the disease (or its symptoms) or complication. Determining such effective amounts is within the ability of those skilled in the art.
  • an effective amount for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient, the patient's age, weight, sex, mode of administration of the drug, dosage form, dosing period, time interval, other treatments administered concurrently , drug tolerance, etc.
  • a compound or pharmaceutical composition of the present application is provided chronically to a subject.
  • Long-term treatment includes any form of repeated administration over an extended period of time, such as administration for one month or more, one month to one year, one year or more, or longer.
  • long-term treatment involves repeated administration of a compound or pharmaceutical composition of the present application over the life of the subject.
  • long-term treatment involves regular administration, eg, once or more per day, once or more per week, or once or more per month.
  • a suitable administered dose of a compound or pharmaceutical composition of the present application is that amount of the compound that is minimally effective to produce a therapeutic effect.
  • the dosage of a compound or pharmaceutical composition of the present application for a subject will range from about 0.0001 mg to about 100 mg per kilogram of body weight per day.
  • the daily dose will be between 0.001 mg and 50 mg per kilogram of body weight. Even more preferred is the range of 0.01 mg to 10 mg per kilogram of body weight.
  • the effective daily dose may optionally be divided into two, three, four, five, six or more times in unit dosage form, which are administered separately at appropriate intervals throughout the day.
  • kits comprising a compound or pharmaceutical composition of the application.
  • the compounds or pharmaceutical compositions of the present application can be packaged separately or together.
  • the kit optionally includes instructions for the therapy.
  • kits include multiple doses of a compound or pharmaceutical composition of the present application.
  • a kit can include a complete cycle of therapy.
  • the kit includes multiple cycles of therapy.
  • a subject such as a mammal, including, but not limited to, cattle, sheep, horses, dogs, cats, rodents, primates, can be treated according to the prophylactic or therapeutic methods of the present application.
  • the compound shown in formula I or a pharmaceutically acceptable salt thereof can be used in the methods or purposes for preventing or treating the following diseases, disorders and symptoms: Type II diabetes and related symptoms (or complications), disorders related to Type 2 diabetes (high Glycemia, low glucose tolerance, insulin resistance, dyslipidemia, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL level, high LDL level, nonalcoholic fatty liver disease, myocardial damage , liver ballooning lesions, liver inflammation).
  • Type II diabetes and related symptoms or complications
  • disorders related to Type 2 diabetes high Glycemia, low glucose tolerance, insulin resistance, dyslipidemia, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL level, high LDL level, nonalcoholic fatty liver disease, myocardial damage , liver ballooning lesions, liver inflammation).
  • the compound of formula I or a pharmaceutically acceptable salt thereof can be used for the prevention or treatment of type II diabetes and related symptoms (or complications).
  • Type II diabetes is characterized by impaired insulin secretion, and insulin resistance in tissues.
  • Manifestations of the disease include one or more of the following: impaired glucose tolerance, fasting hyperglycemia, urine sugar, decreased insulin levels, increased glucagon levels, increased hepatic glucose output, decreased hepatic glucose Intake and glycogen storage, reduced systemic glucose uptake and utilization, dyslipidemia, fatty liver, ketoacidosis, microvascular disease (eg, retinopathy), nephropathy.
  • the compound represented by formula I or a pharmaceutically acceptable salt thereof can be used to improve insulin resistance.
  • Insulin resistance is clinically defined as the decreased ability of insulin to promote glucose uptake and utilization.
  • the compound represented by formula I or a pharmaceutically acceptable salt thereof can be used to improve impaired glucose tolerance.
  • IGT is characterized by abnormal blood glucose excursions after meals.
  • Treatment with a compound or pharmaceutical composition of the present application resulted in a significantly higher proportion of subjects with an improvement in NAS score (ie, an improvement of at least 2 points) compared to untreated subjects.
  • Treatment with a compound or pharmaceutical composition of the present application resulted in a significantly higher proportion of subjects with improvement in ballooning lesions compared to untreated subjects.
  • Treatment with a compound or pharmaceutical composition of the present application resulted in a significantly higher proportion of subjects with improved myocardial injury compared to subjects treated with conventional drugs.
  • Example 1 Verification of the binding of S-lysoPC molecule to PPAR- ⁇ by SPR
  • PPAR- ⁇ -LBD protein was immobilized on carboxymethyldextran (CM5) sensor chip by amine coupling chemistry, PPAR- ⁇ -LBD was immobilized in flow cell 4 channel, and flow cell 3 channel was used as activation and deactivation reference channel.
  • PPAR- ⁇ -LBD protein (110 ⁇ g/mL, 10 m sodium acetate, pH 4.5) was immobilized on the surface and the injection time lasted 420 seconds. The remaining activated groups on the surface were blocked by a 420 s injection of 1 M ethanolamine at pH 8.5.
  • the flow rate was kept at 30 ⁇ L/min and 1 ⁇ phosphate buffered saline was used as flow buffer during fixation. Approximately 13,000 reaction units (RU) of protein can be captured to flow cell 4 on the CM5 chip.
  • Example 2 Study on the mechanism of S-lysoPC molecular concentration gradient and rosiglitazone in diabetic mouse model
  • the db/db mice used in the experiment and the control db/m mice in the same cage were purchased from Jiangsu Jicui Yaokang Company, about 4 weeks old, 18-21 g. They were kept in the SPF animal room of the Animal Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences.
  • S-lysoPC molecules and rosiglitazone were each dissolved in 5% DMSO, 10% Solutol HS15, 85% saline.
  • the intragastric doses of -S-lysoPC molecules were: 3.3 mg/kg/day and 10 mg/kg/day.
  • Each mouse in each group was gavaged with 200 ⁇ l of the solution for 14 days.
  • mice were fasted for 16 hours in advance, and the fasting blood glucose of the mice in each group was detected by a blood glucose meter, and then 1 mg/g of glucose solution was injected intraperitoneally for each mouse.
  • the blood glucose values of rats at 15min, 30min, 60min, 90min and 120min.
  • mice were fasted for 4 hours in advance, and the fasting blood glucose of the mice in each group was detected with a blood glucose meter, and then insulin was injected intraperitoneally according to 1 U/kg of each mouse, and then the mice in each group were detected with a blood glucose meter for 15 min, 30min, 60min, 90min, 120min blood glucose values.
  • Example 3 Mechanism study of S-lysoPC molecule and rosiglitazone at the same molar dose in a diabetic mouse model
  • the db/db mice used in the experiment and the control db/m mice in the same cage were purchased from Jiangsu Jicui Yaokang Company, about 4 weeks old, 18-21 g. They were kept in the SPF animal room of the Animal Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences.
  • the S-lysoPC molecule and rosiglitazone were each dissolved in 5% DMSO; 10% Solutol HS15; 85% Saline.
  • the gavage dose of -S-lysoPC molecule was: 10 mg/kg/day.
  • Each mouse in each group was gavaged with 200 microliters of the solution for one month, and the body weight was measured weekly.
  • liver/white fat/brown fat/heart White fat, brown fat and heart were divided into 3 parts, one for RNA extraction and qPCR for gene expression detection; one for protein extraction for Western Blot detection of protein expression; one for paraffin-embedded sections for HE staining. Liver tissue was divided into 4 parts, in addition to the above 3 parts, the other part was frozen section for oil red test.
  • RNA was reverse transcribed into cDNA by reverse transcription kit (High-Capacity cDNA Reverse Transcription Kits, Applied Biosystems, cat. no. 4368813).
  • the reverse transcription system was as follows: template RNA (200ng/ ⁇ L) 10 ⁇ L, 10 ⁇ RT buffer 2.0 ⁇ L, 0.8 ⁇ L of 25 ⁇ dNTP Mix (100 mM), 2.0 ⁇ L of random primers, 1.0 ⁇ L of MultiScribeTM reverse transcriptase, 1.0 ⁇ L of RNase inhibitor, 3.2 ⁇ L of nuclease-free H2O, after transient centrifugation, put it into the PCR machine to react, and the reaction
  • the conditions are as follows: (1) 25°C for 10 min; (2) 37°C for 120 min; (3) 85°C for 5 min; (4) 4°C to terminate the reaction. After the reaction, 20 ⁇ L of RNase-free ddH 2 O was added to make up the final volume to 40 ⁇ L.
  • the total volume of the qPCR reaction system was 10 ⁇ l, including: 5 ⁇ L 2 ⁇ SYBR Green Master Mix, 0.5 ⁇ l forward primer (10 ⁇ M), 0.5 ⁇ l reverse primer (10 ⁇ M), 1 ⁇ l cDNA obtained by reverse transcription, and 3 ⁇ l RNase-free ddH 2 O.
  • the PCR reaction conditions are: 95 °C, 5min pre-denaturation, and start the PCR amplification cycle: (1) 95 °C, 10s; (2) 55 °C, 10s; (3) 72 °C, 20s; a total of 40 cycles; the last cooling at 40°C for 10s.
  • the forward primer and reverse primer of the amplification reaction were designed and synthesized by Beijing Qingke Xinye Biotechnology Co., Ltd.
  • the initial voltage is 80V, when the protein sample is in a straight line, adjust the voltage to 120V, until the desired protein band runs out; prepare the transfer solution and pre-cool at 4°C in advance, and remove the gel after electrophoresis.
  • Dehydration put the dehydration box into the hanging basket and dehydrate with gradient alcohol in sequence in the dehydrator. 75% alcohol 4h-85% alcohol 2h-90% alcohol 2h-95% alcohol 1h- anhydrous ethanol I 30min- anhydrous ethanol II 30min- alcohol benzene 5-10min- xylene I 5-10min- xylene II 5- 10min-wax I 1h-wax II 1h-wax III 1h.
  • Sectioning Place the trimmed paraffin block on a paraffin microtome to section, with a slice thickness of 4 ⁇ m.
  • the slices were floated on warm water at 40°C of a spreader to flatten the tissue, picked up with a glass slide, and placed in a 60°C oven to bake the slices. After the water is dried and the wax is baked, take it out and save it at room temperature for later use.
  • Hematoxylin staining of cell nuclei Sections were stained with Harris hematoxylin for 3-8 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for several seconds, rinsed with tap water, returned to blue with 0.6% ammonia water, and rinsed with running water. The differentiation time can be prolonged if the cytoplasm is blue.
  • Eosin staining of cytoplasm Sections were stained in eosin staining solution for 1-3 min. Do not wash with water,
  • Dehydration and sealing Place the sections in 95% alcohol I for 5min-95% alcohol II for 5min-anhydrous ethanol I5min-absolute ethanol II5min-xylene I 5min-xylene II 5min for dehydration and transparency, and remove the sections from xylene. Take it out to dry a little and seal with neutral gum.
  • Antigen retrieval Place the tissue slices in a repair box filled with citrate antigen retrieval buffer (PH6.0) for antigen retrieval in a microwave oven, high heat for 9 minutes to boiling, cease fire for 7 minutes and then switch to medium heat for 7 minutes. During the process, the buffer should be prevented from over-evaporating, and the slices should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time.
  • PH6.0 citrate antigen retrieval buffer
  • Block endogenous peroxidase Place the slices in 3% hydrogen peroxide solution, incubate them in the dark at room temperature for 25 minutes, place the slices in PBS (pH 7.4), shake and wash 3 times on a decolorizing shaker, each time 5min.
  • DAB color development The slides were placed in PBS (PH7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After the slices were slightly dried, the freshly prepared DAB color developing solution was added dropwise in the circle, and the color developing time was controlled under the microscope.
  • Counterstaining nuclei counterstain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for several seconds, rinse with tap water, return to blue with hematoxylin solution, and rinse with running water.
  • Fixation The frozen sections were rewarmed and dried for 10 minutes; the cells were fixed in 4% paraformaldehyde for 15 minutes, and washed with PBS for 3 times, 5 minutes each time.
  • Counterstaining nuclei Harris hematoxylin was counterstained for about 1-2 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for several seconds, rinsed with tap water, returned to blue with ammonia, and rinsed with running water.
  • lipid droplets were orange-red to bright red, and nuclei were blue.
  • the binding KD (M) of S-lysoPC molecule to PPAR- ⁇ was 2.122 ⁇ 10 -5 , which was specific binding ( FIG. 1 ).
  • Glucose tolerance and insulin tolerance tests were detected, and it was found that rosiglitazone and S-lysoPC molecules could significantly reduce blood sugar and significantly improve insulin sensitivity in diabetic mice (Fig. 2A-Fig. 2D). increased, showing better pharmacological effects (Fig. 2A-Fig. 2D).
  • rosiglitazone and S-lysoPC molecules were detected to transport excess fat compared with the model group mice.
  • NAS is a composite score of three histological features of hepatic steatosis, lobular inflammation, and hepatocyte ballooning lesions to assess the severity of fatty liver damage.
  • Figure 4D is the HE staining of the liver. It can be observed that the rosiglitazone group and the S-lysoPC molecule group can significantly reduce the ballooning lesions and inflammatory lesions compared with the diabetes model group.
  • Figure 4E detects the protein expression of AMPK and PPAR- ⁇ in the liver, and it can be found that the S-lysoPC molecule can significantly increase the protein expression of PPAR- ⁇ and can activate the AMPK pathway.

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Abstract

涉及脂质化合物在预防或治疗糖尿病中的用途。提供的脂质化合物是一种PPAR-γ激动剂，通过口服吸收，可以缓解糖耐量、减轻体重、不对心肌产生损伤。提供的脂质化合物可提取自中药，也可以通过合成的方法获得。与传统药物罗格列酮相比，提供的脂质化合物可以改善传统药物的心肌损伤副作用。

Description

脂质化合物在预防或治疗糖尿病中的用途 技术领域
本申请涉及生物、医学、化学、临床领域。具体地，本申请涉及脂质化合物用于预防或治疗糖尿病的新用途。
背景技术
根据国际糖尿病联合会(IDF)的数据显示，2015年全球20-79岁的成年人中11个人有1人患有糖尿病；预计到2040年，这一人数将增加到6.42亿人；增长最快的地区从低收入地区转换到中等收入地区。目前，中国已成为世界糖尿病发病人数最多的国家，并且超过90％以上的糖尿病患者是2型糖尿病。
2型糖尿病患者更容易受到不同形式的短期和长期并发症的影响。并发症包括大血管疾病(高血压、高脂血症、心脏病、冠状动脉疾病、中风、脑血管疾病和外周血管疾病)、微血管疾病(视网膜病变、肾病和神经病变)和癌症。因此，研究糖尿病的病理过程，寻找一个高效的并能减轻糖尿病并发症的药物成为新的治疗策略。
肝脏是全身代谢中的一个重要器官，对机体的代谢起着重要的作用。胰岛素抵抗与2型糖尿病(T2DM)的发生发展有关。脂毒素、线粒体功能、细胞因子和脂肪细胞因子在非酒精性脂肪肝和2型糖尿病患者中起重要作用。非酒精性脂肪肝患者通常有胰岛素抵抗。大量2型糖尿病患者发生非酒精性脂肪肝，并伴有炎症并发症非酒精性脂肪性肝炎。
棕色脂肪是负责分解引发肥胖的白色脂肪的人体组织，将白色脂肪转化成二氧化碳、水和热量。棕色脂肪可以加快人体新陈代谢，促进白色脂肪消耗，或可以成为对抗糖尿病的潜在机制。糖尿病常用的药物有噻唑烷二酮类药物，适用于胰岛素抵抗突出的2型糖尿病患者(即肥胖或超重的2型糖尿病患者)。噻唑烷二酮的靶点是PPAR-γ，可以增加外周组织对胰岛素的敏感性、改善胰岛素抵抗而降低血糖，并能改善与胰岛素抵抗有关的多种心血管危险因素。
然而，噻唑烷二酮导致的不良反应包括：加重心肌损伤，体重增 加，水肿等。发表于The New England Journal of Medicine的一项临床试验发现，噻唑烷二酮类经典药物(罗格列酮)可以加重心肌损伤和心血管的死亡率，并被欧美等国家率先禁用。至此，PPAR-γ激动剂噻唑烷二酮类药物发展受阻。因此，本领域仍需要开发副作用小的新型糖尿病药物。
发明内容
根据一些实施方案，提供了一种脂质化合物，其是式(I)所示：
Figure PCTCN2020130843-appb-000001
根据一些实施方案，提供了一种脂质化合物，其是1-硬脂酰基-2-羟基-sn-甘油-3-磷酸胆碱(也作硬脂酰溶血卵磷脂；CAS号：19420-57-6；1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine)(以下简称S-lysoPC)。
根据一些实施方案，提供了前述式(I)所示化合物或其可药用盐作为活性成分在预防糖尿病或其并发症中的用途。
根据一些实施方案，还提供了前述式(I)所示化合物或其可药用盐作为活性成分在治疗糖尿病或其并发症中的用途。
根据一些实施方案，还提供了前述式(I)所示化合物或其可药用盐作为活性成分在改善糖尿病或其症状中的用途。
根据一些实施方案，提供了前述式(I)所示化合物或其可药用盐作为活性成分在制备药物中的用途，所述药物用于治疗、预防或改善糖尿病或其并发症。
根据一些实施方案，提供了前述式(I)所示化合物或其可药用盐在制备药物中的用途，其中所述药物用于选自以下的任一项或其组合：血糖控制、体重控制、降低空腹血脂水平、降低空腹血糖水平、降低空腹胰岛素水平、降低空腹谷丙转氨酶水平、降低空腹谷草转氨酶水平、提高胰岛素敏感性、促进棕色脂肪的生成。
在一些实施方案中，前述式(I)所示化合物或其可药用盐适用于 I型糖尿病。
在另一些实施方案中，前述式(I)所示化合物或其可药用盐适用于II型糖尿病。
在一些实施方案中，前述式(I)所示化合物或其可药用盐适用于改善糖尿病并发症，其选自以下的任一项或其组合：胰岛素抵抗、高血糖症、胰岛素抵抗、高血糖症、高脂血症、高甘油三酯血症、高胆固醇血症、低HDL血症、高LDL血症、非酒精性脂肪肝、心肌损伤、肝脏气球样病变、肝脏炎症、非酒精性脂肪肝、心肌损伤、肝脏气球样病变、肝脏炎症。
在一些实施方案中，式(I)所示化合物的可药用盐选自以下的任一项或其组合：醋酸盐、苯磺酸盐、苯甲酸盐、丙酸盐、丙二酸盐、丙酮酸盐、碳酸盐、碳酸氢盐、硫酸盐、甲基硫酸盐、硫酸氢盐、异硫代硫酸盐、酒石酸盐、酒石酸氢盐、硼酸盐、柠檬酸盐、乙二胺四乙酸盐、乙二磺酸盐、丙酸酯十二烷硫酸盐、乙磺酸盐、乙醇酸盐、富马酸盐、葡庚糖酸盐、葡糖酸盐、谷氨酸盐、己酸盐、己基间苯二酸盐、盐酸盐、羟基萘甲酸盐、氢溴酸盐、乳酸盐、乳糖酸盐、苹果酸盐、马来酸盐、肉桂酸盐、扁桃酸盐、甲磺酸盐、甲基溴化物、甲基硝酸盐、粘酸盐、萘磺酸盐、硝酸盐、N-甲基葡萄糖胺铵盐、油酸盐、乙二酸盐、棕榈酸盐、泛酸盐、磷酸盐、二磷酸盐、多聚半乳糖醛酸盐、水杨酸盐、硬脂酸盐、碱式醋酸盐、琥珀酸盐、丹宁酸盐、氯茶碱盐、甲苯磺酸盐、环戊丙酸盐、戊酸盐、无机碱/有机碱衍生的盐(例如、钠盐、钾盐、胺盐等)。
根据一些实施方案，提供了一种药物或药物组合物，其包含预防有效量或治疗有效量的前述式(I)所示化合物或其可药用盐。
在一些实施方案中，本申请的药物或药物组合物还包含药学上可接受的载体或赋形剂。
在一些实施方案中，前述式(I)所示化合物或其可药用盐作为本申请的药物或药物组合物中的唯一活性成分。
在另一些实施方案中，前述式(I)所示化合物或其可药用盐可以和本领域治疗糖尿病的已知药物联合施用(同时或顺序给药)。
在一些实施方案中，本申请的药物或药物组合物可以以下面的任 一方式施用：口服、吸入、直肠用药、鼻腔用药、局部用药、非肠道用药(例如但不限于：皮下、静脉、肌内、腹膜内)。
在一些实施方案中，本申请的药物或药物组合物是口服剂型。在具体的实施方案中，本申请的药物或药物组合物是选自以下的形式：片剂、锭剂、胶囊剂、颗粒剂、散剂、糖浆剂、溶液剂、悬液、气雾剂、喷雾剂。
附图说明
图1显示S-lysoPC与PPAR-γ-LBD的SPR结果。
图2A显示糖耐量实验；■代表：db/db；▲代表：S-lysoPC(3.3mg/kg/天)；▼代表：S-lysoPC(10mg/kg/天)；◆代表：罗格列酮(10mg/kg/天)。
图2B显示AUC面积。
图2C显示胰岛素耐量实验；■代表：db/db；▲代表：S-lysoPC(3.3mg/kg/天)；▼代表：S-lysoPC(10mg/kg/天)；◆代表：罗格列酮(10mg/kg/天)。
图2D显示AUC面积。
图3A显示各组小鼠的体重随着服药时间的变化图。
图3B至图3D显示各组小鼠空腹血脂、胰岛素、血糖的生化指标。
图4A显示各组小鼠谷丙转氨酶(ALT)的生化指标。
图4B显示各组小鼠谷草转氨酶(AST)的生化指标。
图4C显示各组小鼠肝脏的油红实验，以及阳性面积统计图。
图4D显示各组小鼠肝脏的HE实验，以及NAS病理评分。
图4E显示检测各组小鼠肝脏中AMPK和PPAR-γ的蛋白表达水平；S-lysoPC分子可以显著提高PPAR-γ的蛋白表达，并且可以激活AMPK通路。
图5A显示白色脂肪的HE图及脂肪细胞数量的统计图。
图5B和图5C显示PPAR-γ下游基因的表达水平。
图6A显示棕色脂肪的HE图及脂肪细胞数量统计图。
图6B显示检测棕色脂肪形成标志物；PGC1a、UCP1显著升高，同时PPAR-γ蛋白表达升高。
图7A显示检测各组心肌组织中，氧化应激损伤iNOS的免疫组化图，以及阳性统计图。
图7B显示各组小鼠中的空腹CK指标。
具体实施方式
本申请中使用的术语“可药用盐”意指在制药上可接受的、并且具有母体化合物(式I所示化合物)所需药理学活性的盐。这类盐包括：
1)与无机酸或与有机酸形成的酸加成盐；所述无机酸例如但不限于盐酸、氢溴酸、硫酸、硝酸、磷酸；所述的有机酸例如乙酸、丙酸、己酸、环戊丙酸、乙醇酸、丙酮酸、乳酸、丙二酸、琥珀酸、苹果酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、苯磺酸、萘磺酸、樟脑磺酸、葡庚糖酸、葡糖酸、谷氨酸、羟基萘甲酸、水杨酸、硬脂酸等；
2)当本申请化合物带有酸性部分时，合适的药学可接受的盐包括但不限于由无机碱衍生得到的盐，所述无机碱包括例如铝、铵、钙、铜、三价铁、二价铁、锂、镁、三价锰、二价锰、钾、钠、锌；特别优选的是铵、钙、镁、钾和钠盐；
3)与有机碱形成的盐，所述的有机碱例如胺、乙醇胺、二乙醇胺、三乙醇胺、乙二胺、N-甲基葡糖胺、精氨酸、甜菜碱、咖啡因、胆碱、二乙胺、2-二乙基氨基乙醇、2-二甲基氨基乙醇、N-乙基-吗啉、N-乙基哌啶、葡萄糖胺、氨基葡萄糖、组氨酸、异丙胺、赖氨酸、甲基葡萄糖胺、吗啉、哌嗪、哌啶、聚胺树脂、普鲁卡因、嘌呤、可可碱、三乙胺、三甲胺、三丙胺、氨丁三醇。
药学上可接受的载体包括但不限于：离子交换剂、氧化铝、硬脂酸铝、卵磷脂、白蛋白、缓冲物质、油、山梨酸、山梨酸钾、水、盐、电解质、硫酸鱼精蛋白、磷酸氢二钠、磷酸氢钾、氯化钠、锌盐、聚乙烯吡咯烷酮、聚乙二醇、纤维素、羧甲基纤维素钠、聚丙烯酸酯、蜂蜡、羊毛脂。
药学上可接受的赋形剂是指在药物制剂中除活性成分以外的附加物，其性质稳定，与活性成分配伍不产生副作用、不影响疗效。如片剂中的黏合剂、填充剂、崩解剂、润滑剂；固体制剂中的软膏剂；液 体制剂中的防腐剂、抗氧化剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透调节剂、着色剂等。
“给药”和“施用”应当理解为是指向受试者提供本申请的化合物(或可药用盐)或其前药。
本文使用的“前药”是指当被施用于生物时由于自发的化学反应、酶催化的化学反应和/或代谢化学反应而产生生物活性化合物的任何化合物。前药经历化学转化以产生生物活性的化合物。在某些情况下，化合物的前药形式可用于，例如改善生物利用度、掩蔽或减少苦味或肠胃刺激性、改善受治疗者的可接受性、改变溶解度、提供延长的或持续的释放或递送、改善制剂的递送。
预期口服使用的药物组合物可根据本领域公知的任何方法来制备，并且这样的药物组合物可包含例如增甜剂、调味剂、着色剂、防腐剂。活性成分与适于制备片剂的、无毒的、药学上可接受的赋形剂相混合。这些赋形剂可以是，例如，惰性稀释剂(如碳酸钙或碳酸钠、乳糖、磷酸钙或磷酸钠)；崩解剂(如玉米淀粉)；粘合剂(如淀粉、明胶或***胶)；以及润滑剂(如硬脂酸镁、硬脂酸或滑石)。片剂可以是未包衣的、或者可以通过公知技术包衣以延迟在胃肠道中的崩解和吸收并提供较长时期的持续作用。用于口服的制剂还可以呈现为硬胶囊，其中活性成分与惰性固体稀释剂相混合，或者呈现为软胶囊，其中活性成分与水或油性介质(花生油、液体石蜡或橄榄油)相混合。
如上所述，适于口服施用的本发明的药物组合物可呈现为离散单元，例如胶囊剂或片剂，每个均含有预定量的活性成分；呈现为颗粒剂；呈现为在水性液体或非水性液体中的溶液或悬液；或呈现为水包油或油包水的乳剂。
适于在口中局部施用的制剂包括糖锭，所述糖锭包含经调味的基底(flavored base)和活性成分，所述经调味的基底通常为蔗糖和***胶或黄蓍胶。
“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防有效量是指足以预防、阻止或延迟疾病(或其症状)或并发症发生的量；治疗有效量是指足以治愈或至少部分缓解疾病(或其症状) 或并发症的量。确定这样的有效量在本领域技术人员的能力范围之内。例如，对于治疗用途的有效量将取决于待治疗的疾病的严重度、患者的总体状态、患者的年龄、体重、性别、药物施用方式、剂型、给药周期、时间间隔、同时施用的其他治疗、对药物的耐受度等。
在一些实施例中，向受试者长期提供本申请的化合物或药物组合物。长期治疗包括长时间任何形式的重复施用，如施用一个月或一个月以上、一个月到一年、一年或一年以上、或更长时间。
在许多实施例中，长期治疗涉及在受试者一生中重复施用本申请的化合物或药物组合物。优选长期治疗涉及定期施用，例如每天一次或一次以上、每周一次或一次以上或每月一次或一次以上。一般来说，本申请的化合物或药物组合物的合适施用剂量是有效产生治疗作用的最低的化合物的量。本申请的化合物或药物组合物用于受试者的剂量将在每天每公斤体重约0.0001mg到约100mg的范围内。优选地，日剂量将在每公斤体重0.001mg到50mg。甚至更优选地每公斤体重0.01mg到10mg的范围。必要时，有效日剂量可任选地以单位剂型分两次、三次、四次、五次、六次或以上，其在一天内以适当时间间隔分开施用。
本申请提供包含本申请的化合物或药物组合物的试剂盒。本申请的化合物或药物组合物可分开包装或包装在一起。试剂盒任选地包括疗法的说明书。在某些实施例中，试剂盒包括多次剂量的本申请的化合物或药物组合物。试剂盒可包括完整疗法周期。在某些实施例中，试剂盒包括多个疗法周期。
根据本申请的预防或治疗方法可以治疗受试者，如哺乳动物，包括但不限于牛、羊、马、狗、猫、啮齿动物、灵长类动物。
式I所示化合物或其可药用盐可以用于预防或者治疗以下疾病、病症、症状的方法或用途中：II型糖尿病和相关症状(或并发症)、与2型糖尿病相关的病症(高血糖症、低葡萄糖耐量、胰岛素抵抗、脂质异常、血脂异常、高脂血症、高甘油三酯血症、高胆固醇血症、低HDL水平、高LDL水平、非酒精性脂肪肝、心肌损伤、肝脏气球样病变、肝脏炎症)。
式I所示化合物或其可药用盐可以用于预防或者治疗II型糖尿病和相关症状(或并发症)。II型糖尿病的特征包括：受损的胰岛素分 泌、和组织中的胰岛素抵抗。该疾病的表现形式包括以下的一种或多种：葡萄糖耐量降低、空腹高血糖症、尿糖、降低的胰岛素水平、增加的胰高血糖素水平、增加的肝葡萄糖排出量、降低的肝葡萄糖摄入与糖原贮积、降低的全身葡萄糖摄入与利用、血脂障碍(dyslipidemia)、脂肪肝、酮酸中毒、微血管疾病(如视网膜病)、肾病。
式I所示化合物或其可药用盐可以用于改善胰岛素抵抗。胰岛素抵抗被临床定义为胰岛素促进葡萄糖摄取和利用的能力下降。
式I所示化合物或其可药用盐可以用于改善葡萄糖耐量降低。IGT其特征在于在进餐后异常的血糖偏移(blood glucose excursions)。
与未治疗的受试者相比，用本申请的化合物或药物组合物治疗导致NAS评分改善(即改善至少2分)的受试者比例显著更高。
与未治疗的受试者相比，用本申请的化合物或药物组合物治疗导致气球样病变改善的受试者比例显著更高。
与传统药物治疗的受试者相比，用本申请的化合物或药物组合物治疗导致心肌损伤改善的受试者比例显著更高。
实施例1：通过SPR验证S-lysoPC分子与PPAR-γ的结合
SPR实验使用Biacore T200(GE Healthcare)在25℃下进行。PPAR-γ-LBD(PPAR-γ的配体结合域)购自MedChemExpress(美国新泽西州)。
用胺偶联化学方法将PPAR-γ-LBD蛋白固定在羧甲基右旋糖酐(CM5)传感器芯片上，PPAR-γ-LBD固定在流动池(flow cell)4通道，流动池3通道作为激活和关闭的参考通道。将PPAR-γ-LBD蛋白(110μg/mL，10m乙酸钠，pH4.5)固定在表面，注射时间持续420秒。在pH值为8.5的条件下，通过420秒注射1M乙醇胺来阻断表面上剩余的活化基团。对于上述所有步骤，流速保持在30μL/min，并在固定期间使用1×磷酸盐缓冲盐水作为流动缓冲液。大约13000个反应单位(RU)的蛋白质可以被捕捉到CM5芯片上的流动池4。
实施例2：S-lysoPC分子浓度梯度和罗格列酮在糖尿病小鼠模型中的机制研究
1.实验动物：
实验所用db/db和同笼对照db/m雄性小鼠均购自江苏集萃药康公司，约4周龄，18-21g。饲养于中国医科科学院基础医学研究所动物中心SPF级动物房。
2.S-lysoPC分子和罗格列酮的制备：
S-lysoPC分子和罗格列酮各自溶解于5％DMSO、10％Solutol HS15、85％生理盐水中。
3.实验分组：
-罗格列酮(Rosiglitazone)灌胃剂量为：10mg/kg/天。
-S-lysoPC分子的灌胃剂量为：3.3mg/kg/天和10mg/kg/天两组。
-模型组(Vehicle)：灌胃液体溶剂。
各组的每只小鼠用200微升溶液灌胃14d。
4.糖耐量和胰岛素耐量试验：
进行糖耐量试验时，提前将小鼠禁食16h，用血糖仪检测各组小鼠的空腹血糖，之后按照每只小鼠1mg/g腹腔注射葡糖糖溶液，之后用血糖仪检测各组小鼠15min、30min、60min、90min、120min的血糖值。
进行胰岛素耐量试验时，提前将小鼠禁食4h，用血糖仪检测各组小鼠的空腹血糖，之后按照每只小鼠1U/kg腹腔注射胰岛素，之后用血糖仪检测各组小鼠15min、30min、60min、90min、120min的血糖值。
实施例3：相同摩尔剂量的S-lysoPC分子和罗格列酮在糖尿病小鼠模型中的机制研究
1.实验动物：
实验所用db/db和同笼对照db/m雄性小鼠均购自江苏集萃药康公司，约4周龄，18-21g。饲养于中国医科科学院基础医学研究所动物中心SPF级动物房。
2.S-lysoPC分子和罗格列酮的制备：
S-lysoPC分子和罗格列酮各自溶解于5％DMSO；10％Solutol HS15；85％Saline中。
3.实验分组：
-罗格列酮(Rosiglitazone)灌胃剂量为：6.7mg/kg/天。
-S-lysoPC分子的灌胃剂量为：10mg/kg/天。
-模型组(Vehicle)：灌胃液体溶剂。
各组的每只小鼠用200微升溶液灌胃一个月，每周测体重。
4.组织取样：
(1)取血清：灌胃一个月后，提前禁食12h后，眼球取血500μl，离心条件为3000rpm，10min，分离出血清。用全自动生化分析仪检测各项生化指标。
(2)取组织：肝/白色脂肪/棕色脂肪/心。白色脂肪、棕色脂肪和心脏分为3份，一份提取RNA，进行qPCR检测基因表达；一份提取蛋白，进行Western Blot检测蛋白表达；一份进行石蜡包埋切片，进行HE染色。肝脏组织分为4份，除以上3份外，另一份进行冰冻切片，进行油红实验。
5.基因检测：
(1)取1ml组织裂解液，按如下步骤提取RNA：
组织样品加入1ml Trizol Reagent LS，匀浆使其充***解，按200μL氯仿/mL Trizol加入氯仿，充分振荡，混匀后室温放置5min；4℃，12,000rpm，离心15min；吸取上层水相，至另一离心管中，按0.4mL异丙醇/mL Trizol加入异丙醇混匀，低温放置10-20min；4℃，12,000rpm，离心15min，弃上清，RNA沉于管底；加入1mL 75％乙醇，温和振荡离心管，悬浮沉淀；4℃，12,000rpm，离心10min，弃上清，加入1mL 75％乙醇，温和振荡离心管，悬浮沉淀；4℃，12,000rpm，离心10min，弃上清，室温晾干，用50-100μL无RNase的H 2O溶解RNA样品，测OD值定量RNA浓度。
(2)将RNA逆转录为cDNA：
通过逆转录试剂盒(High-Capacity cDNA Reverse Transcription Kits，Applied Biosystems，cat.no.4368813)，将RNA逆转录为cDNA，逆转录体系如下：模板RNA(200ng/μL)10μL，10×RT缓冲液2.0μL，25×dNTP Mix(100mM)0.8μL，随机引物2.0μL，MultiScribeTM逆转录酶1.0μL，RNA酶抑制剂1.0μL，无核酸酶H2O 3.2μL，瞬时离心后，放入PCR仪反应，反应条件如下：(1)25℃，10min；(2)37℃，120min；(3)85℃，5min；(4)4℃，终止反应。反应结束 后加入20μL无RNA酶ddH 2O，补足终体积至40μL。
(3)定量PCR扩增反应：
qPCR反应体系总体积10μl，包括：5μL 2×SYBR Green Master Mix，0.5μl正向引物(10μM)，0.5μl反向引物(10μM)，1μl逆转录得到的cDNA，3μl无RNA酶ddH 2O。使用LightCycler 480荧光定量PCR仪，PCR反应条件是：95℃，持续5min预变性，开始进入PCR扩增循环：(1)95℃，10s；(2)55℃，10s；(3)72℃，20s；总共进行40个循环；最后40℃持续10s降温。扩增反应正向引物和反向引物均由北京擎科新业生物技术有限公司设计和合成。
(4)利用2-ΔCt法计算相对表达量。
6.蛋白检测：
(1)BCA检测蛋白浓度
向96孔酶标板中加入20μL RIPA裂解液(标准品孔内不加)，然后加入5μL待检测的蛋白样品(标准品孔：加入25μL标准品)；每孔加入200ml BCA检测液，震荡混匀后用封口膜完全覆盖上表面，37℃孵育30min；使用Synergy 4多功能酶标仪于562nm处检测其吸光度，并根据标准曲线计算样品的浓度。
(2)蛋白样品的预处理
向蛋白样品中加入上样缓冲液；混匀上述体系后，95℃金属浴变性10min，在转至冰上冷却备用。
(3)蛋白电泳
安装好制胶装置，小心加入下层分离胶，用1mL异丙醇封顶，室温静置20至30min，待下层胶凝固后小心吸去异丙醇，加入上层浓缩胶并***梳子，待上层胶凝固后取下胶块玻璃板，安装至电泳装置上，并完全浸没在1×电泳液之中；将梳子取出后，观察并调整凝胶电泳上样孔壁竖直，依次加入15μL蛋白Marker和样品，链接电泳装置后开始电泳，初始电压80V，待蛋白样品成一条直线时调整电压至120V，直至跑出所需蛋白条带；配制转膜液并提前4℃预冷，电泳结束后取出凝胶保留实验所需部分置于转膜液中备用；4℃电流300mA条件下转膜120min；转膜结束后，将PVDF膜完全浸没于5％牛奶之中置于摇床上，室温封闭1h；按照抗体说明配制一抗，将目的条带置于含有一 抗的PVC袋中，完全排出空气后封口，4℃过夜孵育；弃去一抗，取出PVDF膜，室温在TBST中洗膜10min并重复3次；按照抗体说明配制二抗，将目的条带完全浸没于二抗之中，室温孵育1h；弃去一抗，取出PVDF膜，室温在TBST中洗膜10min并重复3次；配制显色液，将显色液滴加在目的条带之上，吸取多余的显色液，在显影仪中显影并保存图像。
7.HE染色实验
(1)取材：新鲜组织固定于4％多聚甲醛24h以上。将组织从固定液取出在通风橱内用手术刀将目的部位组织修平整，将修切好的组织和对应的标签放于脱水盒内。
(2)脱水：将脱水盒放进吊篮里于脱水机内依次梯度酒精进行脱水。75％酒精4h-85％酒精2h-90％酒精2h-95％酒精1h-无水乙醇I 30min-无水乙醇II 30min-醇苯5-10min-二甲苯I 5-10min-二甲苯II 5-10min-蜡I 1h-蜡II 1h-蜡III 1h。
(3)包埋：将浸好蜡的组织于包埋机内进行包埋。先将融化的蜡放入包埋框，待蜡凝固之前将组织从脱水盒内取出按照包埋面的要求放入包埋框并贴上对应的标签。于-20°冻台冷却，蜡凝固后将蜡块从包埋框中取出并修整蜡块。
(4)切片：将修整好的蜡块置于石蜡切片机上切片，片厚4μm。切片漂浮于摊片机40℃温水上将组织展平，用载玻片将组织捞起，并放进60℃烘箱内烤片。待水烤干蜡烤化后取出常温保存备用。
(5)石蜡切片脱蜡至水：依次将切片放入二甲苯Ⅰ20min-二甲苯Ⅱ20min-无水乙醇Ⅰ10min-无水乙醇Ⅱ10min-95％酒精5min-90％酒精5min-80％酒精5min-70％酒精5min-蒸馏水洗。
(6)苏木素染细胞核：切片入Harris苏木素染3-8min，自来水洗，1％的盐酸酒精分化数秒，自来水冲洗，0.6％氨水返蓝，流水冲洗。如果细胞浆有蓝色可以延长分化时间。
(7)伊红染细胞质：切片入伊红染液中染色1-3min。不要水洗，
(8)脱水封片：将切片依次放入95％酒精I 5min-95％酒精II 5min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-二甲苯Ⅰ5min-二甲苯Ⅱ5min中脱水透明，将切片从二甲苯拿出来稍晾干，中性树胶封片。
(9)显微镜镜检，图像采集分析。细胞核蓝色，细胞质红色。
8.石蜡切片免疫组化实验
(1)石蜡切片脱蜡至水：依次将切片放入二甲苯Ⅰ15min-二甲苯Ⅱ15min-二甲苯III 15min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-85％酒精5min-75％酒精5min-蒸馏水洗。
(2)抗原修复：组织切片置于盛满柠檬酸抗原修复缓冲液(PH6.0)的修复盒中于微波炉内进行抗原修复，高火9min至沸，停火7min保温再转中火7min，此过程中应防止缓冲液过度蒸发，切勿干片。自然冷却后将玻片置于PBS(PH7.4)中在脱色摇床上晃动洗涤3次，每次5min。
(3)阻断内源性过氧化物酶：切片放入3％双氧水溶液，室温避光孵育25min，将玻片置于PBS(PH7.4)中在脱色摇床上晃动洗涤3次，每次5min。
(4)血清封闭：在组化圈内滴加3％BSA均匀覆盖组织，室温封闭30min(一抗是山羊来源的用兔血清封闭，其他来源的用BSA封闭)。
(5)加一抗：轻轻甩掉封闭液，在切片上滴加PBS按一定比例配好的一抗，切片平放于湿盒内4℃孵育过夜。(湿盒内加少量水防止抗体蒸发)
(6)加二抗：玻片置于PBS(PH7.4)中在脱色摇床上晃动洗涤3次，每次5min。切片稍甩干后在圈内滴加与一抗相应种属的二抗(HRP标记)覆盖组织，室温孵育50min。
(7)DAB显色：玻片置于PBS(PH7.4)中在脱色摇床上晃动洗涤3次，每次5min。切片稍甩干后在圈内滴加新鲜配制的DAB显色液，显微镜下控制显色时间，阳性为棕黄色，自来水冲洗切片终止显色。
(8)复染细胞核：苏木素复染3min左右，自来水洗，苏木素分化液分化数秒，自来水冲洗，苏木素返蓝液返蓝，流水冲洗。
(9)脱水封片：将切片依次放入75％酒精5min-85％酒精5min--无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-二甲苯Ⅰ5min中脱水透明，将切片从二甲苯拿出来稍晾干，中性树胶封片。
(10)显微镜镜检，图像采集分析。苏木素染细胞核为蓝色，DAB 显出的阳性表达为棕黄色。
9.油红O染色实验
(1)固定：将冰冻切片复温干燥10min；细胞爬片4％多聚甲醛固定15min，PBS漂洗3次，5min/次。
(2)染色：入油红O工作液孵育10-15min；细胞爬片破膜10-15min，PBS漂洗3次，5min/次，入油红O工作液37°染色1-2h。
(3)分化：75％酒精分化2s，水洗1min。
(4)复染细胞核：Harris苏木素复染1-2min左右，自来水洗，1％的盐酸酒精分化数秒，自来水冲洗，氨水返蓝，流水冲洗。
(5)封片：用纸巾吸去周边水分，甘油明胶封片。
染色结果：脂滴呈橘红色至鲜红色，细胞核蓝色。
10.实验结果和结论
图中所有的数据都按照双尾t检验进行了差异水平计算。对于统计检验有显著性的结果，在图中以星号标记显示，*表示P<0.05，**表示P<0.01，星号越多表示显著性越强。部分数据由于个体差异较大，虽然能看出有明显趋势，但是经过计算没有显著性差异。
S-lysoPC分子与PPAR-γ的结合KD(M)为2.122×10 -5，为特异性结合(图1)。
检测糖耐量和胰岛素耐量试验，发现罗格列酮和S-lysoPC分子可以显著降低糖尿病小鼠的血糖和显著提高胰岛素的敏感性(图2A-图2D)，并且发现随着S-lysoPC浓度的提升，呈现更好的药理作用(图2A-图2D)。
在糖尿病小鼠模型中监测小鼠体重，发现服用药物4周后，S-lysoPC分子可以降低小鼠的体重水平(图3A)，并且空腹检测血脂、血糖和胰岛素，与糖尿病组相比，S-lysoPC分子均可以显著降低各个指标(图3B至图3D)。
在糖尿病小鼠检测空腹血清中谷丙转氨酶和谷草转氨酶水平，发现罗格列酮并没有降低酶的水平，然而S-lysoPC分子可以大幅降低酶的水平(图4A和图4B)。证实其在糖尿病模型中，S-lysoPC分子可以对肝脏起到保护作用。
在图4C中检测罗格列酮和S-lysoPC分子在与模型组小鼠相比， 可以转运多余的脂肪。NAS是对肝脏脂肪变性、小叶炎症和肝细胞气球样病变这三项组织学特征进行综合评分，进而评估脂肪肝受损的严重程度。图4D是肝脏的HE染色，可以观察到罗格列酮组和S-lysoPC分子组与糖尿病模型组相比，可以显著减轻气球样病变和炎性病灶。
图4E检测肝脏中AMPK和PPAR-γ的蛋白表达量，可以发现S-lysoPC分子可以显著提高PPAR-γ的蛋白表达，并且可以激活AMPK通路。
在糖尿病小鼠模型中，检测各组白色脂肪HE染色图，发现罗格列酮组和S-lysoPC组均没有提升白色脂肪细胞的数量(图5A)。
提取白色脂肪的RNA后，检测PPAR-γ下游基因表达量，发现GLUT4、ACOX1显著升高(图5B)，炎性因子IL1b、TNFa显著降低(图5C)，其与提升胰岛素敏感性呈正相关。
在糖尿病模型中，罗格列酮组和S-lysoPC组与糖尿病组相比，发现均能可以显著提升棕色脂肪的生成(图6A)。提取棕色脂肪组织，提取棕色脂肪蛋白，检测棕色脂肪形成MARKER、PGC1a、UCP1显著升高，同时PPAR-γ蛋白表达升高(图6B)。
在糖尿病模型中，检测心肌组织中罗格列酮组的iNOS表达升高，提示其心肌损伤加重，同时S-lysoPC组没有升高(图7A)。之后检测血清中CK水平，显示与糖尿病组相比，罗格列酮组有升高的趋势，而S-lysoPC组显著降低(图7B)。

Claims (10)

  1. 式(I)所示化合物或其可药用盐在制备药物中的用途，其中：
    Figure PCTCN2020130843-appb-100001
    所述药物用于治疗、预防或改善糖尿病或其并发症。
  2. 式(I)所示化合物或其可药用盐在制备药物中的用途，其中：
    Figure PCTCN2020130843-appb-100002
    所述药物用于选自以下的任一项或其组合：血糖控制、体重控制、降低空腹血脂水平、降低空腹血糖水平、降低空腹胰岛素水平、降低空腹谷丙转氨酶水平、降低空腹谷草转氨酶水平、提高胰岛素敏感性、促进棕色脂肪的生成。
  3. 根据权利要求1所述的用途，其中所述糖尿病是I型糖尿病或II型糖尿病；优选II型糖尿病。
  4. 根据权利要求1所述的用途，所述糖尿病并发症选自以下的任一项或其组合：胰岛素抵抗、高血糖症、高脂血症、高甘油三酯血症、高胆固醇血症、低HDL血症、高LDL血症、非酒精性脂肪肝、心肌损伤、肝脏气球样病变、肝脏炎症。
  5. 根据权利要求1或2所述的用途，其中所述可药用盐选自以下的任一项或其组合：醋酸盐、苯磺酸盐、苯甲酸盐、丙酸盐、丙二酸盐、丙酮酸盐、碳酸盐、碳酸氢盐、硫酸盐、甲基硫酸盐、硫酸氢盐、异硫代硫酸盐、酒石酸盐、酒石酸氢盐、硼酸盐、柠檬酸盐、乙二胺 四乙酸盐、乙二磺酸盐、丙酸酯十二烷硫酸盐、乙磺酸盐、乙醇酸盐、富马酸盐、葡庚糖酸盐、葡糖酸盐、谷氨酸盐、己酸盐、己基间苯二酸盐、盐酸盐、羟基萘甲酸盐、氢溴酸盐、乳酸盐、乳糖酸盐、苹果酸盐、马来酸盐、肉桂酸盐、扁桃酸盐、甲磺酸盐、甲基溴化物、甲基硝酸盐、粘酸盐、萘磺酸盐、硝酸盐、N-甲基葡萄糖胺铵盐、油酸盐、乙二酸盐、棕榈酸盐、泛酸盐、磷酸盐、二磷酸盐、多聚半乳糖醛酸盐、水杨酸盐、硬脂酸盐、碱式醋酸盐、琥珀酸盐、丹宁酸盐、氯茶碱盐、甲苯磺酸盐、环戊丙酸盐、戊酸盐、无机碱衍生盐、有机碱衍生盐。
  6. 根据权利要求1或2所述的用途，其中所述药物还包含药学上可接受的载体或赋形剂。
  7. 一种用于治疗、预防或改善糖尿病或其并发症的方法，包括步骤：向受试者施用预防有效量或治疗有效量的式(I)所示化合物或其可药用盐，其中：
    Figure PCTCN2020130843-appb-100003
    所述糖尿病是I型糖尿病或II型糖尿病；优选II型糖尿病。
  8. 根据权利要求7所述的方法，所述糖尿病并发症选自以下的任一项或其组合：胰岛素抵抗、高血糖症、高脂血症、高甘油三酯血症、高胆固醇血症、低HDL血症、高LDL血症、非酒精性脂肪肝、心肌损伤、肝脏气球样病变、肝脏炎症。
  9. 根据权利要求7所述的方法，其中所述可药用盐选自以下的任一项或其组合：醋酸盐、苯磺酸盐、苯甲酸盐、丙酸盐、丙二酸盐、丙酮酸盐、碳酸盐、碳酸氢盐、硫酸盐、甲基硫酸盐、硫酸氢盐、异硫代硫酸盐、酒石酸盐、酒石酸氢盐、硼酸盐、柠檬酸盐、乙二胺四 乙酸盐、乙二磺酸盐、丙酸酯十二烷硫酸盐、乙磺酸盐、乙醇酸盐、富马酸盐、葡庚糖酸盐、葡糖酸盐、谷氨酸盐、己酸盐、己基间苯二酸盐、盐酸盐、羟基萘甲酸盐、氢溴酸盐、乳酸盐、乳糖酸盐、苹果酸盐、马来酸盐、肉桂酸盐、扁桃酸盐、甲磺酸盐、甲基溴化物、甲基硝酸盐、粘酸盐、萘磺酸盐、硝酸盐、N-甲基葡萄糖胺铵盐、油酸盐、乙二酸盐、棕榈酸盐、泛酸盐、磷酸盐、二磷酸盐、多聚半乳糖醛酸盐、水杨酸盐、硬脂酸盐、碱式醋酸盐、琥珀酸盐、丹宁酸盐、氯茶碱盐、甲苯磺酸盐、环戊丙酸盐、戊酸盐、无机碱衍生盐、有机碱衍生盐。
  10. 根据权利要求7所述的方法，所述式(I)所示化合物或其可药用盐制备成口服剂型；优选地，制备成选自以下的形式：片剂、锭剂、胶囊剂、颗粒剂、散剂、糖浆剂、溶液剂、悬液、气雾剂、喷雾剂。
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