WO2019120111A1 - 二甲双胍盐在治疗脑梗死中的用途 - Google Patents

二甲双胍盐在治疗脑梗死中的用途 Download PDF

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WO2019120111A1
WO2019120111A1 PCT/CN2018/120479 CN2018120479W WO2019120111A1 WO 2019120111 A1 WO2019120111 A1 WO 2019120111A1 CN 2018120479 W CN2018120479 W CN 2018120479W WO 2019120111 A1 WO2019120111 A1 WO 2019120111A1
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metformin
monothreonate
shy01
cerebral infarction
salt
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PCT/CN2018/120479
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English (en)
French (fr)
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镇学初
刘宗银
钱林艺
曹慧敏
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镇学初
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Priority claimed from CN201810039233.0A external-priority patent/CN110037998A/zh
Priority claimed from CN201810623095.0A external-priority patent/CN110604730A/zh
Application filed by 镇学初 filed Critical 镇学初
Priority to US16/772,667 priority Critical patent/US11497722B2/en
Publication of WO2019120111A1 publication Critical patent/WO2019120111A1/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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • A61K31/60—Salicylic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the invention belongs to the field of pharmaceutical preparations.
  • the invention relates to the use of metformin monothreonate and various other salts in the manufacture of a medicament for the treatment of cerebral infarction.
  • the invention also relates to a process for the preparation of metformin monothreonate and various other salts.
  • Cerebral infarction is the second leading cause of death worldwide. Cerebral infarction can cause sudden damage to brain tissue. If nerve cells lack sufficient oxygen supply, they will die within a few minutes. Then, the bodily functions controlled by these nerve cells will also lose their effect. Since dead brain cells cannot be replaced, the consequences of stroke are usually permanent.
  • recombinant human tissue plasminogen activator the only drug approved by the FDA for the treatment of cerebral infarction is recombinant human tissue plasminogen activator.
  • the application of recombinant human tissue plasminogen activator in a short time after cerebral infarction plays a role in improving neurological function. Promote, but because of its narrow therapeutic window, less than 5% of patients benefit.
  • the invention relates to the use of a metformin salt for the manufacture of a medicament for the prevention and/or treatment of cerebral infarction in a subject.
  • the invention relates to the use of a metformin salt for the manufacture of a medicament for the treatment of cerebral infarction in a subject, wherein the medicament is used in combination with a medicament for the treatment of cerebral infarction.
  • the metformin salt is selected from one or more of the following: metformin monothreonate, metformin tartrate, metformin citrate, metformin mesylate, metformin maleate, and metformin hydrobromide Acid salt. In one embodiment, the metformin salt is preferably metformin monothreonate.
  • the subject is a non-diabetic patient.
  • the drug is administered orally or by injection.
  • the metformin salt preferably metformin monothreonate, is used to treat cerebral infarction in a subject.
  • the drug for treating cerebral infarction is a human tissue plasminogen activator.
  • the human tissue plasminogen activator is a recombinant human tissue plasminogen activator.
  • the invention relates to metformin monothreonate.
  • metformin monothanoate is represented by the following formula (I):
  • the present invention is directed to a process for preparing metformin monothreonate comprising reacting metformin and threonate under heating.
  • the threonate used in the process for preparing metformin monothreonate is an alkali metal salt of threonic acid or an alkaline earth metal salt of threonic acid, preferably magnesium threonate.
  • the heating conditions are 65-75 ° C, preferably 70 ° C.
  • the method of preparing metformin monothreonate further comprises dissolving the reactant in an organic solvent, and subsequently filtering and drying, the organic solvent comprising an alcohol having 1 to 5 carbon atoms, an ether A solvent for a nitrile, a ketone or an ester, preferably ethanol.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the metformin monothreonate of the foregoing aspect, or the metformin monothreonate prepared by the method of the preceding aspect, and one or more pharmaceutically acceptable Carrier.
  • the pharmaceutical composition is in a form for administration by oral route or by injection. In one embodiment, the pharmaceutical composition is a tablet or injection.
  • the metformin monothreonate of the present invention exhibits excellent safety and efficacy for the prevention and treatment of cerebral infarction, as well as high bioavailability.
  • FIG. 1 TTC staining results of rat brain sections (L and R represent the left and right hemisphere directions, respectively).
  • Figure 2 Results of measurement of cerebral infarct size in rat brain sections.
  • Figure 4 Activation effect of SHY01-001 on p-AMPK protein.
  • Figure 5 Anti-inflammatory effects of a.SHY01-001 and metformin hydrochloride on BV-2 microglia;
  • FIG. 6 TTC staining results of rat brain slices (L and R represent the left and right hemispheres, respectively)
  • Figure 7 Results of measurement of cerebral infarct size in rat brain sections.
  • metformin salts are capable of preventing and/or treating cerebral infarction.
  • the present invention relates to the use of metformin salts for the preparation of a medicament for the prevention and/or treatment of cerebral infarction.
  • the invention also relates to metformin salts for use in the prevention and/or treatment of cerebral infarction.
  • the invention further relates to a method of preventing and/or treating cerebral infarction comprising administering to a subject a prophylactically and/or therapeutically effective amount of a metformin salt.
  • Cerebral infarction refers to a group of diseases that cause brain tissue damage due to blood circulation disorders caused by vascular occlusion of the brain.
  • Cerebral infarction is also known as “ischemic stroke”, “ischemic stroke” or “ischemic stroke”.
  • ischemic stroke ischemic stroke
  • ischemic stroke ischemic stroke
  • ischemic stroke ischemic stroke
  • ischemic stroke ischemic stroke
  • ischemic stroke ischemic stroke
  • treating means that an individual having a disease or condition after administration of a medicament of the invention exhibits partial or complete relief of the condition, or does not continue to increase after treatment. Therefore, treatment includes healing.
  • therapeutic effect refers to an effect caused by a treatment that alters, generally reduces or ameliorates the symptoms of a disease or condition, or cures a disease or condition.
  • prevention refers to the prevention and management of the underlying disease by administering the drug or treating it prior to the occurrence or occurrence of a perceptible symptom and/or sign in the relevant disease or condition.
  • treatment does not include prophylactic administration unless otherwise stated.
  • prophylactically or therapeutically effective amount refers to an amount of a substance, compound, or composition comprising a compound that is at least sufficient to produce a prophylactic or therapeutic effect after administration to a subject. Thus, it is prevented, prevented, cured, ameliorated, retarded, or The amount necessary to partially arrest the symptoms of a disease or condition.
  • the metformin salt is used to treat cerebral infarction in a subject.
  • the metformin salt is selected from one or more of the following: metformin monothreonate, metformin tartrate, metformin citrate, metformin mesylate, metformin maleate, and metformin hydrobromide Acid salt. In one embodiment, the metformin salt is preferably metformin monothreonate.
  • the invention provides the use of metformin monothreate for the manufacture of a medicament for the prevention and/or treatment of cerebral infarction.
  • the invention also provides metformin monothreonate for use in preventing and/or treating cerebral infarction.
  • the invention also relates to a method of preventing and/or treating cerebral infarction comprising administering to a subject a prophylactically and/or therapeutically effective amount of metformin monothreonate.
  • the metformin monothreonate is used to treat cerebral infarction in a subject.
  • Subject as used herein includes human or non-human animals, particularly human subjects, including human individuals (referred to as patients) suffering from a disease, particularly a cerebral infarction as described herein.
  • the invention provides the use of a metformin salt, preferably metformin monothreate, for the manufacture of a medicament for the treatment of cerebral infarction in a subject other than diabetes.
  • the invention provides a metformin salt, preferably metformin monothreonate, for use in treating cerebral infarction in a non-diabetic subject.
  • the invention provides a method of treating cerebral infarction in a non-diabetic subject comprising administering to the subject a therapeutically effective amount of a metformin salt, preferably metformin monothreonate.
  • the invention also relates to a combination of metformin salt and other agents for treating cerebral infarction.
  • the invention provides the use of a metformin salt, preferably metformin monothreate, for the manufacture of a medicament for treating cerebral infarction in a subject, wherein the medicament is used in combination with a medicament for treating cerebral infarction.
  • drug for treating cerebral infarction includes drugs known in the art that can be used to treat cerebral infarction, including but not limited to human tissue plasminogen activator, urokinase plasminogen activation.
  • Agent in one embodiment, the agent for treating cerebral infarction is a human tissue plasminogen activator, preferably a recombinant human tissue plasminogen activator.
  • the metformin salt is metformin monothreonate.
  • the invention further relates to a composition comprising a combination of a metformin salt of the invention, preferably metformin monothreonate, and at least one agent for treating cerebral infarction.
  • the therapeutic cerebral infarction drug is a human tissue plasminogen activator, preferably a recombinant human tissue plasminogen activator.
  • the metformin salt is metformin monothreonate.
  • the invention provides the use of a metformin salt, preferably metformin monothreonate, and at least one composition for treating a cerebral infarction in the manufacture of a medicament for the prevention and/or treatment of cerebral infarction.
  • the invention also provides a metformin salt, preferably metformin monothreonate, and at least one composition for treating a brain infarction for preventing and/or treating cerebral infarction.
  • the invention also relates to a method of preventing and/or treating cerebral infarction comprising administering to a subject a prophylactically and/or therapeutically effective amount of a metformin salt, preferably metformin monothreonate and at least one therapeutic brain A composition of an infarct drug.
  • metformin salt of the present invention particularly metformin monothreonate, has good neuroprotective effects and brain tissue distribution, and is distributed more rapidly in brain tissue, and is therefore suitable for the preparation of a medicament for preventing and/or treating cerebral infarction.
  • the invention also relates to metformin monothreonate.
  • metformin monothreonate is as shown in formula (I):
  • the metformin monothionate of the present invention is suitable for use in the preparation of a pharmaceutical preparation. Accordingly, the present invention is also directed to a pharmaceutical composition comprising the metformin monothreonate of the present invention, together with one or more pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered, and which is suitable for contact within the scope of sound medical judgment. Tissues of humans and/or other animals without excessive toxicity, irritation, allergic reactions, or other problems or complications corresponding to reasonable benefits/risks. Examples of suitable pharmaceutically acceptable carriers are as described in Remington's Pharmaceutical Sciences (1990).
  • the pharmaceutically acceptable carrier includes, but is not limited to, a solvent, a stabilizer, a surfactant, a lubricant, a filler, a sweetener, a disintegrant.
  • metformin monothreonate of the invention or the pharmaceutical composition of the invention may act systemically and/or locally.
  • they may be administered in a suitable route, for example by injection, intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or transdermal administration; or by oral, buccal, nasal, transmucosal, topical
  • a suitable route for example by injection, intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or transdermal administration; or by oral, buccal, nasal, transmucosal, topical
  • the form is administered or by inhalation.
  • the pharmaceutical compositions of the invention may be administered in a suitable dosage form.
  • dosage forms include, but are not limited to, tablets, capsules, lozenges, powders, sprays, ointments, injections and the like.
  • the pharmaceutical composition is an injection, a tablet, a solution, preferably an injection.
  • the metformin monothreonate is prepared as a tablet comprising or consisting of: metformin monothreonate, lactose, dextrin, corn starch, povidone K30, magnesium stearate, and silica. In one embodiment, the metformin monothreonate is prepared as an injectable solution comprising or consisting of: metformin monothreonate, disodium hydrogen phosphate, and HCL or NaOH (appropriate amount) ).
  • the invention provides the use of a pharmaceutical composition comprising a metformin salt, preferably metformin monothreonate, for the manufacture of a medicament for the prevention and/or treatment of cerebral infarction.
  • the invention also provides a pharmaceutical composition comprising a metformin salt, preferably metformin monothreonate, for use in the prevention and/or treatment of cerebral infarction.
  • the invention also relates to a method of preventing and/or treating cerebral infarction comprising administering to a subject a prophylactically and/or therapeutically effective amount of a pharmaceutical composition comprising a metformin salt, preferably metformin monothreonate.
  • the invention further relates to a process for the preparation of metformin monothreonate comprising the reaction of metformin and threonic acid under heating.
  • the "threonate" used in the preparation of metformin monothreonate includes magnesium threonate.
  • the heating conditions are 65-75 ° C, preferably 70 ° C.
  • the method further comprises dissolving the reactant in an organic solvent, and then filtering and drying, the organic solvent comprising an alcohol solvent having 1 to 5 carbon atoms.
  • alcohol solvent preferably means an alcohol having 1 to 5 carbon atoms including, but not limited to, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2 - Butanol and tert-butanol.
  • the alcohol solvent is ethanol.
  • the "drying" in the present invention is preferably carried out under reduced pressure at any temperature (preferably room temperature) until the content of the residual solvent is lowered to the extent generally required in the pharmaceutical field.
  • the drying used in the metformin monothanoate prepared according to the invention is vacuum drying.
  • metformin hydrochloride 55g, 0.33mol
  • sodium hydroxide 14g, 0.35mol
  • the mixture was filtered for 1 hour, and the filter cake was evaporated with ethanol and water to give 7.8 g of a white solid.
  • the yield was 18%.
  • threonic acid paste 61 g was added to a 1 L single-mouth bottle, and after adding 100 ml of water, it was dissolved, and then 51.6 g of metformin and 100 g of water were added. After the addition, the solution was further stirred and dissolved, and the reaction was dropped into 1 L of acetone to precipitate a solid. Filtered to dryness to a white solid (yield: 18g).
  • Example 8 Drug distribution test of SHY01-001 and SHY01-005 in brain tissue
  • the brain was removed, washed with ice physiological saline, and after removing water, all or part of the tissue was selected according to the specific homogenization ratio, accurately weighed, added with physiological saline, and homogenized at high speed in an ice bath (15,000 rpm). .
  • the tissue homogenate was stored in a -80 ° C low temperature freezer for sample analysis.
  • the concentration in the brain tissue of SHY01-001 was greater than that of metformin hydrochloride and SHY01-005 from 0.5 hours, and reached a peak of 763.7 ng/mL in 2 hours, followed by a decrease.
  • Example 9 The role of SHY01-001 in preventing ischemia-reperfusion brain injury
  • a total of 13 male clean-grade SD rats (body weight 200-300 g) were selected to construct the MCAO (brain middle occlusion) model (for model construction methods, see Enrique Zea Longa et al., Stroke, Vol 20, No. 1, 1989; A. Tamura et al. , Journal of Cerebral Blood Flow and Metabolism, 1:53-60, 1981), in which the ischemic time was 90 min, blood flow was restored, and subsequent experiments were performed 24 hours after reperfusion.
  • MCAO brain middle occlusion
  • the 50 mg/kg/day positive control drug metformin hydrochloride and SHY01-001 were administered to the rats immediately, followed by intraperitoneal injection once a day for 3 days.
  • the solvent was selected as physiological saline.
  • the control group was injected with the same amount of normal saline.
  • Thirteen MCAO rats were divided into three groups: group A) saline + MCAO group (4); group B) metformin hydrochloride + MCAO group (3); and group C) SHY01-001 + MCAO group (6) only).
  • the whole brain of the rat was taken and sectioned. From the rat whole brain, a single-sided blade was used to cut 4 pieces of brain slices having a thickness of about 2 mm.
  • the first knife is at the midpoint of the front pole of the brain and the line of intersection; the second knife is at the intersection of the optic chiasm; the third knife is at the shank of the funnel; the fourth knife is between the shank of the funnel and the tail of the posterior leaf; At the end of the posterior leaf.
  • the brain sections were placed in a solution of 2% 2,3,5-triphenyltetrazolium chloride (TTC) which was prepared and dyed at 37 ° C in a dark water bath for about 10 minutes. After the staining, the brain sections were placed in 4% paraformaldehyde for 24 h. Brain sections of the three groups of rats are shown in Figure 1. The area of brain infarction was then measured.
  • TTC 2,3,5-triphenyltetrazolium chloride
  • % cortical infarct volume total cortical infarct volume / total contralateral cortical volume * 100
  • % striatum infarct volume total striatum infarct volume total / contralateral striatum volume * 100
  • % hemisphere infarct volume total hemisphere infarct volume / total contralateral hemisphere volume *100
  • % correction (total side hemisphere total volume - (total hemisphere total volume - total hemispheric infarct volume)) / total volume of contralateral hemisphere * 100
  • the tissue ischemia and cerebral infarct size of the rats receiving SHY01-001 were significantly smaller than those of the rats receiving normal saline and metformin hydrochloride.
  • the cerebral infarct size of the rats receiving SHY01-001 was significantly lower than that of the rats receiving normal saline and metformin hydrochloride. (Fig. 2: **P ⁇ 0.01, ***P ⁇ 0.001).
  • the cerebral infarct size of the rats receiving SHY01-001 decreased more than 1-fold in the striatum and cortex compared to the rats receiving metformin hydrochloride. The above results prove that SHY01-001 can effectively reduce the area of cerebral infarction.
  • the cytotoxicity of various metformin salts was determined using the thiazolyl (MTT) method.
  • HEK293T cells were cultured in 96-well plates, and when the cells were over 60% of the bottom area, metformin hydrochloride, SHY01-001, SHY01-002, SHY01-003 was added at a final concentration of 0.5 mM, 2 mM and 5 mM. Seven compounds of SHY01-004, SHY01-005 and SHY01-006 were cultured for 24 hours at 37 ° C in a 5% CO 2 incubator. Cell viability was measured by MTT assay to observe the effect of the compounds on cell viability.
  • SHY01-003 (labeled "003" in Figure 3) at a concentration of 5 mM significantly affected cell viability, and it also showed some inhibition of cell viability at both 0.5 mM and 2 mM concentrations. It shows that SHY01-003 has high cytotoxicity.
  • Example 11 Activation effect of metformin salt on p-AMPK protein
  • HEK293T cells were cultured in 6-well plates, and when the cells were over 60% of the bottom area, metformin hydrochloride, SHY01-001, SHY01-002, SHY01-003, SHY01 were added at a final concentration of 0.5 mM and 2 mM. -004, SHY01-005 and SHY01-006 a total of 6 compounds, cultured at 37 ° C, 5% CO 2 incubator for 6h, collected cells, lysate treatment, preparation of protein samples, packaging, frozen at -80 ° C to be tested use.
  • the expression level of p-AMPK protein was detected by Western blot to detect the activation of p-AMPK protein by the compound.
  • Example 12 Anti-inflammatory effects of SHY01-001 and SHY01-005 on BV2 microglia
  • microglia cell line BV-2 cells were cultured in vitro in a 12-well plate at a final concentration of 1 mM metformin hydrochloride, SHY01-001, SHY01-005, metformin hydrochloride + LPS, SHY01-001 + LPS, SHY01- 005+LPS (200 ng/mL) was added to the cell culture medium for 24 hours. After the cells were collected, the protein was extracted and the protein expression levels of proinflammatory cytokines iNOS and COX-2 were detected.
  • BV2 microglia After stimulation with LPS, BV2 microglia can rapidly change from a resting state to an activated state, and simultaneously express various inflammatory mediators (iNOS, COX-2, IL-6, etc.) to further activate microglia. Activation of microglia can aggravate the death of ischemia-related nerve cells. A decrease in the expression levels of iNOS and COX-2 protein indicates that the inflammatory response is inhibited, microglia activation is reduced, and cell damage is reduced.
  • iNOS inflammatory mediators
  • SHY01-001 was more effective than metformin hydrochloride in inhibiting the expression of iNOS and COX-2 protein after LPS stimulation, as shown in Figure 5b: after LPS stimulation, SHY01-
  • the inhibition of iNOS protein expression by 005 was comparable to that of metformin hydrochloride, and the inhibition of COX-2 protein expression was slightly worse than that of metformin hydrochloride. (**: p ⁇ 0.01, *: p ⁇ 0.05)
  • Example 13 Solution stability test of SHY01-001 and SHY01-005
  • Example 14 Effect of metformin monothreonate on acute cerebral ischemia-reperfusion injury
  • MCA middle cerebral artery
  • the method of suture was used to induce reversible middle cerebral artery occlusion (MCAO), and a rat model of focal cerebral ischemia was established.
  • SD male rats were used, and blood flow was restored after 1.5 hours of ischemia.
  • 50 mg/kg of positive control drugs Metformin, SHY01-001 and magnesium threonate were injected intraperitoneally within 3 days after operation, and the model control group was given the same dose of normal saline.
  • Cerebral infarction volume after ischemia-reperfusion in rats was measured by TTC (2,3,5-triphenyltetrazolium chloride) staining.
  • TTC is a fat-soluble light-sensitive complex, which is a proton acceptor of the pyridine-nucleoside structural enzyme system in the respiratory chain. It reacts with dehydrogenase in normal tissues and turns red, while dehydrogenase activity in ischemic tissue decreases. The reaction is so pale that it does not change.
  • MCAO rats Twenty-four MCAO rats were divided into four groups: group A) saline + MCAO group (7); group B) magnesium threonate + MCAO group (6); group C) metformin hydrochloride + MCAO group (7) Only); and group D) SHY01-001+MCAO group (9).
  • tissue ischemia and cerebral infarct size of the rats receiving SHY01-001 were significantly smaller than those of the rats receiving the physiological saline and metformin hydrochloride and magnesium threonate groups.
  • the cerebral infarct size of the rats receiving SHY01-001 was significantly lower than that of the rats receiving normal saline and metformin hydrochloride. (Fig. 7: **P ⁇ 0.01, *P ⁇ 0.05).
  • the cerebral infarct size of the rats receiving SHY01-001 decreased more than 1-fold in the cerebral infarct area of the brain hemisphere relative to the rats receiving metformin hydrochloride. The above results prove that SHY01-001 can effectively reduce the area of cerebral infarction.
  • Example 15 Effect of metformin monothreate on neurological deficits caused by long-term ischemia-reperfusion
  • the lactose is crushed, passed through a 80 mesh sieve, and used; the raw material metformin monothreonate, dextrin, and corn starch are passed through an 80 mesh sieve; the prescription amount of povidone K30 is weighed and formulated into a 5% solution; Prescription amount of metformin monothreonate, lactose, dextrin, corn starch is added to a high-efficiency wet granulator, dry mixed for 6-10min, slowly added to the prepared povidone K30 binder, wet granulation, Metformin monothreonate wet granules, the wet granules are dried, the drying temperature is 60-70 ° C, the drying time is 15-30 min, and the dried granules are granulated by a 14 mesh sieve to obtain metformin monothreonate.
  • the granules are dried, and the dry granules, magnesium stearate and silica are respectively added to a multi-directional
  • the raw material metformin monothreonate was taken and pulverized to a fine powder.
  • a sodium dihydrogen phosphate solution was prepared, and the metformin monothreonate fine powder was dissolved in a sodium dihydrogen phosphate solution.
  • the pH was adjusted to 7-8 with 0.1 M sodium hydroxide or hydrochloric acid, then transferred to an injection bottle, and steam-sterilized (121 ° C, sterilized for 15 min) to obtain a metformin monothreonate injection.

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Abstract

本发明提供了二甲双胍盐，特别是二甲双胍单苏糖酸盐在制备用于治疗脑梗死中的药物中的用途。本发明的二甲双胍单苏糖酸盐在治疗脑梗死中具有优异的效果。本发明也提供了二甲双胍单苏糖酸盐的制备方法和组合物。

Description

二甲双胍盐在治疗脑梗死中的用途 技术领域
本发明属于药学制剂领域。具体地，本发明涉及二甲双胍单苏糖酸盐及其他多种盐在制备用于治疗脑梗死中的药物中的用途。本发明也涉及二甲双胍单苏糖酸盐及其他多种盐的制备方法。
背景技术
脑梗死是全世界范围内导致死亡的第二大疾病。脑梗死会对大脑组织造成突发性损坏。如果神经细胞缺乏足够的氧气供给，几分钟内就会死亡。接着，受这些神经细胞控制的身体机能也会随之失去作用。由于死亡的大脑细胞无法替换，因此脑卒中造成的后果通常是永久的。
目前唯一被FDA批准用于脑梗死治疗的药物是重组人组织型纤溶酶原激活物，在脑梗死发生后短时间内应用重组人组织型纤溶酶原激活物对提高神经功能起到一定促进作用，但由于其治疗窗窄，不到5％的患者从中受益。
目前，尚缺少一种能够安全且有效治疗脑梗死的药物。
发明内容
一方面，本发明涉及二甲双胍盐在制备用于预防和/或治疗对象中脑梗死的药物中的用途。
另一方面，本发明涉及二甲双胍盐在制备用于治疗对象中脑梗死的药物中的用途，其中所述药物于治疗脑梗死的药物组合使用。
在一个实施方式中，所述二甲双胍盐选自以下一种或多种：二甲双胍单苏糖酸盐、二甲双胍酒石酸盐、二甲双胍柠檬酸盐、二甲双胍甲磺酸盐、二甲双胍马来酸盐和二甲双胍氢溴酸盐。在一个实施方式中，所述二甲双胍盐优选为二甲双胍单苏糖酸盐。
在一个实施方式中，所述对象为非糖尿病患者。
在一个实施方式中，所述药物为经口服途径给药或注射给药。
在一个实施方式中，所述二甲双胍盐，优选二甲双胍单苏糖酸盐用于治疗对象中脑梗死。
在一个实施方式中，所述治疗脑梗死的药物为人组织型纤溶酶原激活物。在一个实施方式中，所述人组织型纤溶酶原激活物为重组人组织型纤溶酶原激活物。
再一方面，本发明涉及二甲双胍单苏糖酸盐。在一个实施方式中，所述二甲双胍单苏糖酸盐如下式(I)所示：
Figure PCTCN2018120479-appb-000001
又一方面，本发明涉及一种制备二甲双胍单苏糖酸盐的方法，包括将二甲双胍和苏糖酸盐在加热条件下反应制得。
在一个实施方式中，制备二甲双胍单苏糖酸盐的方法中所用的苏糖酸盐为苏糖酸的碱金属盐或苏糖酸的碱土金属盐，优选苏糖酸镁。
在一个实施方式中，所述加热条件为65-75℃，优选70℃。
在一个实施方式中，制备二甲双胍单苏糖酸盐的方法进一步包括将反应物在有机溶剂中溶解，并随后过滤和干燥，所述有机溶剂包括具有1-5个碳原子的醇类、醚类、腈类、酮类或酯类溶剂，优选乙醇。
又一方面，本发明涉及一种药物组合物，其包含前述方面的二甲双胍单苏糖酸盐，或者前述方面的方法制备的二甲双胍单苏糖酸盐，以及一种或多种药学上可接受的载体。
在一个实施方式中，所述药物组合物为经口服途径给药或注射给药的形式。在一个实施方式中，所述药物组合物为片剂或注射液。
本发明的二甲双胍单苏糖酸盐对于脑梗死的预防和治疗表现出优异安全性和有效性，以及高的生物利用度。
附图说明
图1：大鼠脑切片TTC染色结果(L和R分别代表左脑和右脑半球方向)。
图2：大鼠脑切片中脑梗死面积的测定结果。
图3：SHY01-001的MTT实验结果。
图4：SHY01-001对p-AMPK蛋白的激活效应。
图5：a.SHY01-001和二甲双胍盐酸盐对BV-2小胶质细胞的抗炎作用；
b.SHY01-005和二甲双胍盐酸盐对BV-2小胶质细胞的抗炎作用。
图6：大鼠脑切片TTC染色结果(L和R分别代表左脑和右脑半球方向)
图7：大鼠脑切片中脑梗死面积的测定结果。
具体实施方式
除非在下文中另有定义，本文中所用的所有技术术语和科学术语的含义意图与本领域技术人员通常所理解的相同。提及本文中使用的技术意图指在本领域中通常所理解的技术，包括那些对本领域技术人员显而易见的技术的变化或等效技术的替换。虽然相信以下术语对于本领域技术人员很好理解，但仍然阐述以下定义以更好地解释本发明。
如本文中所使用的术语“包括”、“包含”、“具有”、“含有”或“涉及”及其在本文中的其它变体形式为包含性的(inclusive)或开放式的，且不排除其它未列举的元素或方法步骤。
A.二甲双胍盐的药学用途
本发明人意外发现二甲双胍盐，特别是二甲双胍单苏糖酸盐能够预防和/或治疗脑梗死。因此，本发明涉及二甲双胍盐在制备预防和/或治疗脑梗死药物中的用途。本发明还涉及二甲双胍盐，其用于预防和/或治疗脑梗死。本发明还涉及一种预防和/或治疗脑梗死的方法，包括给对象施用预防和/或治疗有效量的二甲双胍盐。
如本文中所使用的术语“脑梗死”是指由于脑部血管阻塞造成血液循环障碍而引起脑组织损伤的一组疾病。“脑梗死”也被称为“缺血性脑卒中”、“缺血性卒中”或“缺血性中风”。除非另有说明，否则“脑梗死”、“缺血性脑卒中”、“缺血性卒中”和“缺血性中风”在本文中可互换使用。
如本文中所使用的术语“治疗”是指施用本发明的药物后患有疾病或疾病状况的个体表现出所述症状部分或全部缓解，或者在治疗后不再持续加重。因此，治疗包括治愈。如本文所用，“疗效”表示由治疗所导致的效果，其改变、通常减轻或改善疾病或疾病状况的症状，或者治愈疾病或疾病状 况。
术语“预防”是指通过在相关疾病或疾病状况发生或出现可感知的症状和/或体征之前施用药物或进行处理以防止潜在疾病的发生和发展。除非另有说明，否则，本发明的术语“治疗”并不包括预防性给药。
如本文所用，“预防或治疗有效量指施用于对象之后至少足以产生预防或治疗效果的物质、化合物或包含化合物的组合物的量。因此，其为预防、防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
在一个实施方式中，所述二甲双胍盐用于治疗对象中脑梗死。
在一个实施方式中，所述二甲双胍盐选自以下一种或多种：二甲双胍单苏糖酸盐、二甲双胍酒石酸盐、二甲双胍柠檬酸盐、二甲双胍甲磺酸盐、二甲双胍马来酸盐和二甲双胍氢溴酸盐。在一个实施方式中，所述二甲双胍盐优选为二甲双胍单苏糖酸盐。
因此，在一个实施方式中，本发明提供了二甲双胍单苏糖酸盐在制备预防和/或治疗脑梗死药物中的用途。在一个实施方式中，本发明还提供了二甲双胍单苏糖酸盐，其用于预防和/或治疗脑梗死。在一个实施方式中，本发明还涉及一种预防和/或治疗脑梗死的方法，包括给对象施用预防和/或治疗有效量的二甲双胍单苏糖酸盐。在一个实施方式中，所述二甲双胍单苏糖酸盐用于治疗对象中脑梗死。
如本文所使用的“对象”包括人或非人动物，特别是人个体，包括患有疾病(特别是本文所述的脑梗死)的人个体(称为患者)。
二甲双胍对于脑梗死的疗效与二甲双胍的降糖作用无关。因此，本文所使用的“对象”也包括非糖尿病患者。在一个实施方式中，本发明提供二甲双胍盐，优选二甲双胍单苏糖酸盐在制备用于治疗非糖尿病的对象中脑梗死的药物中的用途。在一个实施方式中，本发明提供二甲双胍盐，优选二甲双胍单苏糖酸盐，其用于治疗非糖尿病的对象中脑梗死。在一个实施方式中，本发明提供一种治疗非糖尿病的对象中脑梗死的方法，包括给对象施用治疗有效量的二甲双胍盐，优选二甲双胍单苏糖酸盐。
本发明还涉及二甲双胍盐与其它治疗脑梗死药物的联合用药。在一个实施方式中，本发明提供了二甲双胍盐，优选二甲双胍单苏糖酸盐在制备用于治疗对象中脑梗死的药物中的用途，其中所述药物与治疗脑梗死的药 物组合使用。
如本文中所使用的术语“治疗脑梗死的药物”包括本领域已知的可以用于治疗脑梗死的药物，包括但不限于人组织型纤溶酶原激活物、尿激酶纤溶酶原激活剂。在一个实施方式中，所述治疗脑梗死的药物为人组织型纤溶酶原激活物，优选重组人组织型纤溶酶原激活物。在一个实施方式中，所述二甲双胍盐为二甲双胍单苏糖酸盐。
本发明还涉及包括组合本发明的二甲双胍盐，优选二甲双胍单苏糖酸盐和至少一种治疗脑梗死药物的组合物。在一个实施方式中，所述治疗脑梗死药物为人组织型纤溶酶原激活物，优选重组人组织型纤溶酶原激活物。在一个实施方式中，所述二甲双胍盐为二甲双胍单苏糖酸盐。
因此，在一个实施方式中，本发明提供了二甲双胍盐，优选二甲双胍单苏糖酸盐和至少一种治疗脑梗死药物的组合物在制备预防和/或治疗脑梗死药物中的用途。在一个实施方式中，本发明还提供了二甲双胍盐，优选二甲双胍单苏糖酸盐和至少一种治疗脑梗死药物的组合物，其用于预防和/或治疗脑梗死。在一个实施方式中，本发明还涉及一种预防和/或治疗脑梗死的方法，包括给对象施用预防和/或治疗有效量的二甲双胍盐，优选二甲双胍单苏糖酸盐和至少一种治疗脑梗死药物的组合物。
本发明的二甲双胍盐，特别是二甲双胍单苏糖酸盐具有良好的神经保护作用和脑组织分布，更快更多的分布于脑组织，因此适用于制备成预防和/或治疗脑梗死的药物。
B.二甲双胍单苏糖酸盐、其药物组合物和制备方法
本发明还涉及二甲双胍单苏糖酸盐。在一个实施方式中，所述二甲双胍单苏糖酸盐如式(I)所示：
Figure PCTCN2018120479-appb-000002
本发明的二甲双胍单苏糖酸盐适合用于制备药物制剂。因此，本发明还涉及一种药物组合物，其包含本发明的二甲双胍单苏糖酸盐，以及一种或多种药学上可接受的载体。
如本文中所使用的术语“药学上可接受的载体”是指与治疗剂一同给药的稀释剂、辅剂、赋形剂或媒介物，并且其在合理的医学判断的范围内适于接触人类和/或其它动物的组织而没有过度的毒性、刺激、过敏反应或与合理的益处/风险相比相应的其它问题或并发症。适合的药学上可接受的载体的实例如在Remington’s Pharmaceutical Sciences(1990)中所述。在一个实施方式中，所述药学上可接受的载体包括但不限于：溶剂、稳定剂、表面活性剂、润滑剂、填充剂、甜味剂、崩解剂。
本发明的二甲双胍单苏糖酸盐或本发明的药物组合物可以系统地作用和/或局部地作用。为此目的，它们可以适合的途径给药，例如通过注射、静脉内、动脉内、皮下、腹膜内、肌内或经皮给药；或通过口服、含服、经鼻、透粘膜、局部的形式或通过吸入给药。
对于这些给药途径，可以适合的剂型给药本发明的药物组合物。所述剂型包括但不限于片剂、胶囊剂、锭剂、散剂、喷雾剂、膏剂、注射剂等。在一个实施方式中，所述药物组合物为注射剂、片剂、溶液剂，优选注射剂。
在一个实施方式中，所述二甲双胍单苏糖酸盐被制备为片剂，所述片剂包括或由以下组分组成：二甲双胍单苏糖酸盐、乳糖、糊精、玉米淀粉、聚维酮K30、硬脂酸镁、和二氧化硅。在一个实施方式中，所述二甲双胍单苏糖酸盐被制备为注射液，所述注射液包括或由以下组分组成：二甲双胍单苏糖酸盐、磷酸氢二钠、和HCL或NaOH(适量)。
在一个实施方式中，本发明提供了包含二甲双胍盐，优选二甲双胍单苏糖酸盐的药物组合物在制备预防和/或治疗脑梗死药物中的用途。在一个实施方式中，本发明还提供了包含二甲双胍盐，优选二甲双胍单苏糖酸盐的药物组合物，其用于预防和/或治疗脑梗死。在一个实施方式中，本发明还涉及一种预防和/或治疗脑梗死的方法，包括给对象施用预防和/或治疗有效量的包含二甲双胍盐，优选二甲双胍单苏糖酸盐的药物组合物。
本发明还涉及制备二甲双胍单苏糖酸盐的方法，包括将二甲双胍和苏糖酸在加热条件下反应制得。在一个实施方式中，在制备二甲双胍单苏糖酸盐时所使用的“苏糖酸盐”包括苏糖酸镁。
在一个实施方式中，所述加热条件为65-75℃，优选70℃。
在一个实施方式中，所述方法进一步包括将反应物在有机溶剂中溶解，并随后过滤和干燥，所述有机溶剂包括具有1-5个碳原子的醇类溶剂。
如本文中所使用的术语“醇类溶剂”优选意指具有1-5个碳原子的醇，其包括但不限于甲醇、乙醇、1-丙醇、2-丙醇、1-丁醇、2-丁醇和叔丁醇。在一个实施方式中，所述醇类溶剂为乙醇。
本发明中的“干燥”优选是在减压下，于任意温度(优选室温)下进行直到残留溶剂的含量降低至制药领域通常所要求的限度的范围内。在一个实施方式中，本发明制备的二甲双胍单苏糖酸盐时所用的干燥为真空干燥。
实施例
以下将结合实施例更详细地解释本发明，本发明的实施例仅用于说明本发明的技术方案，并非用于限定本发明的范围。
实施例1：二甲双胍的制备
结构式：
Figure PCTCN2018120479-appb-000003
合成路线
Figure PCTCN2018120479-appb-000004
合成过程：
500ml单口瓶中，二甲双胍盐酸盐(55g，0.33mol)溶于200ml水中，室温下加入氢氧化钠(14g，0.35mol)，搅拌溶解后0-5℃析晶(会爆析，爆析后加热溶解再析晶)1h后过滤，滤饼用乙醇带水旋蒸后得白色固体7.8g。收率18％。
实施例2：二甲双胍单苏糖酸盐的制备(SHY01-001)
结构式：
Figure PCTCN2018120479-appb-000005
合成路线
Figure PCTCN2018120479-appb-000006
合成过程
在1L单口瓶中加入苏糖酸膏状物61g，加100ml水后溶清，再加入51.6g二甲双胍和100g水，加毕后继续搅拌溶清，将反应液滴入至1L丙酮中，固体析出，过滤干燥得白色固体18g，收率17％。
1H NMR(400MHz,D 2O)δ2.94(s,6H),3.58(m,4H),3.91(m,2H),3.99(m,2H)
实施例3：二甲双胍酒石酸盐的制备(SHY01-002)
结构式：
Figure PCTCN2018120479-appb-000007
合成路线
Figure PCTCN2018120479-appb-000008
合成过程
100ml单口瓶中，二甲双胍(430mg，3.3mmol)和酒石酸(1g，6.7mmol)混合物于10ml无水乙醇中，加热至70℃溶清，继续搅拌1h后热过滤，将滤液置于-10℃条件下析晶，2h后过滤，干燥后称量得350mg白色固体。收率52％。
1H NMR(400MHz,D 2O)δ2.96(s,12H),4.24(s,2H)
实施例4：二甲双胍柠檬酸盐的制备(SHY01-003)
结构式：
Figure PCTCN2018120479-appb-000009
合成路线
Figure PCTCN2018120479-appb-000010
合成过程
100ml单口瓶中，二甲双胍(430mg，3.3mmol)和一水合柠檬酸(1.4g，6.7mmol)混合物于10ml无水乙醇中，加热至70℃溶清，继续搅拌1h后热过滤，将滤液置于-10℃条件下析晶，2h后过滤，滤饼容易吸潮，收集后再转入单口瓶中，用无水乙醇带走残余水分后真空油泵拉干，氮气交换后称量得310mg白色固体。收率18.3％。
1H NMR(400MHz,D 2O)δ2.67-2.85(m,4H),2.98(s,6H)
实施例5：二甲双胍甲磺酸盐的制备(SHY01-004)
结构式：
Figure PCTCN2018120479-appb-000011
合成路线
Figure PCTCN2018120479-appb-000012
合成过程
100ml单口瓶中，二甲双胍(430mg，3.3mmol)和甲磺酸(640mg，6.7mmol)混合物于10ml无水乙醇中，加热至70℃溶清，继续搅拌1h后热过滤，将滤液置于-10℃条件下析晶，2h后过滤，干燥后称量得450mg白色固体。收率60.6％。
1H NMR(400MHz,D 2O)δ3.08(s,6H),2.71(s,3H)
实施例6：二甲双胍马来酸盐的制备(SHY01-005)
结构式：
Figure PCTCN2018120479-appb-000013
合成路线
Figure PCTCN2018120479-appb-000014
合成过程
100ml单口瓶中，二甲双胍(430mg，3.3mmol)和马来酸(777mg，6.7mmol)混合物于10ml无水乙醇中，加热至70℃溶清，继续搅拌1h后热过滤，将滤液置于-10℃条件下析晶，2h后过滤，干燥后称量得380mg白色固体。收率72.8％。
1H NMR(400MHz,D 2O)δ2.98(s,24H),6.23(s,2H)
实施例7：二甲双胍氢溴酸盐的制备(SHY01-006)
结构式：
Figure PCTCN2018120479-appb-000015
合成路线
Figure PCTCN2018120479-appb-000016
合成过程
100ml单口瓶中，二甲双胍(430mg，3.3mmol)和氢溴酸(40％)(1.33mg，6.7mmol)混合物于10ml无水乙醇中，加热至70℃溶清，继续搅拌1h后热过滤，将滤液置于-10℃条件下析晶，2h后过滤，干燥后称量得480mg 白色固体。收率69.3％。
1H NMR(400MHz,D 2O)δ3.10(s,6H)
实施例8：SHY01-001和SHY01-005在脑组织的药物分布试验
90只SD大鼠(7-8周龄，250±20克)在动物房饲养一周(温度保持在18-26℃，明暗照明各12小时，实验动物可自由进食和饮水)。1周后，选取体征状况良好的81只大鼠进入本实验。实验前一天，3组大鼠禁食不禁水12小时。实验当天，称量体重后，按以下公式计算每只大鼠的实际给药体积。
Figure PCTCN2018120479-appb-000017
详细记录3组SD大鼠的体重和单次口服50mg·kg -1样品的实际给药量。大鼠给药3小时后可恢复进食，实验过程中可自由饮水。
大鼠给药后，分别在30min、1h、2h、4h、6h、8h、12h、16h和24h依次处死三组大鼠。摘取脑，用冰生理盐水清洗干净，吸去水分后，根据特定的匀浆比例，选取全部或部分组织，精密称定，加入生理盐水，在冰浴中高速匀浆(15,000转/分)。组织匀浆液保存在-80℃低温冰箱中，以供样品分析。
采用LC-MS/MS分析方法测定二甲双胍盐酸盐、SHY01-001和SHY01-005在大鼠脑组织中的浓度，试验结果如下表1-3所示：
表1.SHY01-001的在大鼠脑组织浓度测试结果
Figure PCTCN2018120479-appb-000018
表2.二甲双胍盐酸盐的在大鼠脑组织浓度测试结果
Figure PCTCN2018120479-appb-000019
表3.SHY01-005的在大鼠脑组织浓度测试结果
Figure PCTCN2018120479-appb-000020
如表1-3所示，SHY01-001的脑组织中的浓度从0.5小时起就大于二甲双胍盐酸盐和SHY01-005，至2小时即可达到峰值763.7ng/mL，随后降低。相比之下，二甲双胍盐酸盐和SHY01-005则需要6-8小时才可达到峰值浓度，且它们的峰值浓度远远低于SHY01-001(SHY01-001vs.二甲双胍盐酸盐＝763.7vs.288.8ng/mL；SHY01-001vs.SHY01-005＝763.7vs.260.5ng/mL)。这表明SHY01-001能够迅速且大量的分布到脑组织中，具有优异的生物利用度。
实施例9：SHY01-001在预防缺血再灌注脑损伤中的作用
选择共计13只雄性清洁级SD大鼠(体重200-300g)构建MCAO(大 脑中动脉闭塞)模型(模型构建方法参见Enrique Zea Longa等，Stroke,Vol 20,No.1,1989；A.Tamura等，Journal of Cerebral Blood Flow and Metabolism,1:53-60,1981)，其中缺血时间90min，恢复血流，再灌注24h后进行后续实验。
MCAO术后24h立即给予大鼠每只50mg/kg/天剂量的阳性对照药二甲双胍盐酸盐以及SHY01-001，之后每天腹腔注射1次，连续给药3天，溶剂选择生理盐水。对照组注射等量生理盐水。13只MCAO大鼠分为三组：组A)生理盐水+MCAO组(4只)；组B)二甲双胍盐酸盐+MCAO组(3只)；和组C)SHY01-001+MCAO组(6只)。
给药3天后，取大鼠全脑，进行切片。从大鼠全脑用单面刀片切成4片，厚度约为2mm的脑片。第一刀在脑前极与视交叉连线中点处；第二刀在视交叉部位；第三刀在漏斗柄部位；第四刀在漏斗柄与后叶尾极之间；第五刀约在后叶尾极处。
随后，将脑切片放入现配的2％2,3,5-氯化三苯基四氮唑(TTC)溶液中，37℃避光水浴10min左右进行染色。染色结束后，将脑切片放置到4％多聚甲醛中固定24h。三组大鼠的脑切片如图1所示。之后测定脑梗死面积。
测量脑梗死面积的方法：扫描图像，使用图像分析软件(Image J)分别测量大鼠1-4片脑片的梗死侧纹状体、皮层和半球梗死体积以及对侧纹状体、皮层以及半球体积。
％皮层梗死体积＝总皮层梗死体积/总对侧皮层体积*100
％纹状体梗死体积＝总纹状体梗死体积总/对侧纹状体体积*100
％半球梗死体积＝总脑半球梗死体积/总对侧脑半球体积*100
％校正＝(对侧脑半球总体积-(梗侧半球总体积-梗侧半球梗死总体积))/对侧脑半球总体积*100
数据采用GraphPad software Version 6.0(GraphPad Software,Inc.La Jolla,CA,USA)对实验结果进行统计分析。所有结果均以平均值±标准差(Mean±SEM)表示。两组数据间的统计采用t检验，多组数据间的比较采用One-way ANOVA(Bonferroni post hoc test)。P<0.05具有显著性差。
如图1的TTC染色结果所示，接受SHY01-001的大鼠的组织缺血情况及脑梗死面积明显小于接受生理盐水和二甲双胍盐酸盐的大鼠的。
如图2的进一步测量结果可知，在大脑皮层、纹状体和脑半球三个部分，相对于接受生理盐水和二甲双胍盐酸盐的大鼠，接受SHY01-001的大鼠的脑梗死面积显著降低(图2：**P<0.01,***P<0.001)。特别地，相对于接受二甲双胍盐酸盐的大鼠，接受SHY01-001的大鼠的脑梗死面积在纹状体和皮层的脑梗死面积降低幅度均超过1倍。上述结果证明SHY01-001可以有效降低脑梗死面积。
实施例10：二甲双胍盐的细胞毒性测试
采用噻唑兰(MTT)法来测定多种二甲双胍盐的细胞毒性。
在96孔板中培养HEK293T细胞，待细胞长满至孔底面积的60％左右时，加入终浓度为0.5mM，2mM和5mM的二甲双胍盐酸盐，SHY01-001，SHY01-002，SHY01-003，SHY01-004，SHY01-005和SHY01-006共7个化合物，37℃，5％CO 2培养箱中培养24h后，采用MTT法检测细胞活力，以观察化合物对细胞活性的影响。
如图3的MTT检测结果所示，浓度5mM的SHY01-003(图3中标为“003”)显著影响细胞活力，且其在0.5mM和2mM浓度下也均显示出一定的细胞活力抑制作用，表明SHY01-003具有高细胞毒性。
SHY01-001(图3中标为“001”)和其他二甲双胍的盐并未明显影响细胞活力，表明在检测浓度下无细胞毒性。
实施例11：二甲双胍盐对p-AMPK蛋白的激活效应
在6孔板中培养HEK293T细胞，待细胞长满至孔底面积的60％左右时，加入终浓度为0.5mM和2mM的二甲双胍盐酸盐、SHY01-001，SHY01-002，SHY01-003，SHY01-004，SHY01-005和SHY01-006共6个化合物，37℃，5％CO 2培养箱中培养6h后，收集细胞，裂解液处理，制备蛋白样品，分装，冻于-80℃待检测用。
采用Western blot法检测p-AMPK蛋白的表达水平，以检测化合物对p-AMPK蛋白的激活作用。
如图4的p-AMPK蛋白的激活结果显示，SHY01-001、SHY01-002，SHY01-004和SHY01-006在对(图4中分别标为“001”、“002”、“004” 和“006”)p-AMPK激活方面效果相近。化合物SHY01-003和SHY01-005(图4中分别标为“003”和“005”)对p-AMPK的激活作用更加明显。
实施例12：SHY01-001和SHY01-005对BV2小胶质细胞的抗炎作用
选择化合物SHY01-001和SHY01-005来比较其对BV2小胶质细胞的抗炎作用。
体外培养小胶质细胞系BV-2细胞于12孔板中，将终浓度1mM的二甲双胍盐酸盐、SHY01-001、SHY01-005、二甲双胍盐酸盐+LPS、SHY01-001+LPS、SHY01-005+LPS(200ng/mL)加到细胞培养基中培养24h，细胞收集后提取蛋白质，检测促炎症因子iNOS、COX-2的蛋白表达水平。
BV2小胶质细胞在LPS刺激后，其状态会由静息状态迅速转化为激活状态，同时表达多种炎症介质(iNOS、COX-2、IL-6等)，进一步激活小胶质细胞。小胶质细胞的活化会加重缺血相关的神经细胞的死亡。iNOS、COX-2蛋白表达水平的降低表明炎症反应被抑制，小胶质细胞激活减少，细胞损伤减少。
如图5a和5b所示，SHY01-001比二甲双胍盐酸盐抑制LPS刺激后iNOS、COX-2蛋白表达的作用均更明显，有显著性差异；如图5b所示：LPS刺激后，SHY01-005对iNOS蛋白表达的抑制作用与二甲双胍盐酸盐相当，对COX-2蛋白表达的抑制作用较二甲双胍盐酸盐略差。(**：p<0.01，*：p<0.05)
实施例13：SHY01-001和SHY01-005的溶液稳定性试验
取二甲双胍盐酸盐和化合物SHY01-001、SHY01-005的样品适量，置于100ml容量瓶中，加入40ml纯净水，超声使溶解，放至室温，用纯净水稀释至刻度，振摇均匀，40度水浴放置，于0h、4h、8h、12h、16h、20h、24h、36h、48h精密量取供试品溶液10μl，注入液相色谱仪，记录色谱图，按照含量测定项下的方法测定样品含量。结果如表4所示：
表4.SHY01-001和SHY01-005的溶液稳定性试验结果
Figure PCTCN2018120479-appb-000021
Figure PCTCN2018120479-appb-000022
如表4所示，SHY01-001的溶液具有优异的稳定性。
实施例14：二甲双胍单苏糖酸盐对急性期脑缺血再灌注损伤的作用
大脑中动脉(MCA)狭窄或闭塞，是造成供血区卒中常见的原因。选用线栓法造成可逆性大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)，建立大鼠局灶性脑缺血模型。选用SD雄性大鼠，手术缺血1.5小时后，恢复血流。术后3天内连续腹腔注射50mg/kg的阳性对照药Metformin、SHY01-001以及苏糖酸镁组，模型对照组给与相同剂量的生理盐水。通过TTC(2,3,5-氯化三苯基四氮唑)染色法来检测大鼠缺血再灌注后脑梗死体积。
TTC是脂溶性光敏感复合物，是呼吸链中吡啶-核苷结构酶系统的质子受体，与正常组织中的脱氢酶反应而呈红色，而缺血组织内脱氢酶活性下降，不能反应，故不会产生变化呈苍白。
24只MCAO大鼠分为四组：组A)生理盐水+MCAO组(7只)；组B)苏糖酸镁+MCAO组(6只)；组C)二甲双胍盐酸盐+MCAO组(7只)；和组D)SHY01-001+MCAO组(9只)。
如图6的TTC染色结果所示，接受SHY01-001的大鼠的组织缺血情况及脑梗死面积明显小于接受生理盐水和二甲双胍盐酸盐及苏糖酸镁组的大鼠的。
如图7的进一步测量结果可知，在大脑皮层、纹状体和脑半球三个部分，相对于接受生理盐水和二甲双胍盐酸盐的大鼠，接受SHY01-001的大鼠的脑梗死面积显著降低(图7：**P<0.01,*P<0.05)。特别地，相对于接受二甲双胍盐酸盐的大鼠，接受SHY01-001的大鼠的脑梗死面积在脑半球的脑梗死面积降低幅度超过1倍。上述结果证明SHY01-001可以有效降低脑梗死面积。
实施例15：二甲双胍单苏糖酸盐对长期缺血再灌注导致的神经功能缺失的作用
选用SD雄性大鼠，采用线栓法建立大鼠局灶性脑缺血模型，手术缺血1.5小时后恢复血流。术后30天连续腹腔注射50mg/kg的阳性对照药Metformin、SHY01-001以及SHY01-002，模型对照组给与相同剂量的生理盐水。模型前对所有大鼠进行神经功能评分作为基础值，模型后第1，7，14，21，28天进行神经功能行为学测试，测试内容为：mNSS评分、Corner test试验、Rota-rod疲劳转棒仪试验。模型后第30天，进行TTC染色，检测大鼠脑梗死体积。
实施例16：二甲双胍单苏糖酸盐的制剂
A.二甲双胍单苏糖酸盐片剂
配方：
Figure PCTCN2018120479-appb-000023
制备工艺：
将乳糖粉碎，过80目筛，备用；将原料二甲双胍单苏糖酸盐、糊精、玉米淀粉分别过80目筛；称取处方量的聚维酮K30，配制成5％的溶液；称取处方量二甲双胍单苏糖酸盐、乳糖、糊精、玉米淀粉加入到高效湿法制粒机中，干混6-10min，缓慢加入配制好的聚维酮K30粘合剂，进行湿法制粒，得二甲双胍单苏糖酸盐湿颗粒，将湿颗粒进行干燥，干燥温度为60-70℃，干燥时间15-30min，采用14目筛网对干燥后的颗粒进行整粒，得二甲双胍单苏糖酸盐干燥颗粒，将干燥颗粒、硬脂酸镁、二氧化硅分别加入到多向运动混合机中，混合，压片，即得二甲双胍单苏糖酸盐片。
B.二甲双胍单苏糖酸盐注射液
配方：
二甲双胍单苏糖酸盐   250mg
磷酸氢二钠           180mg
0.1M HCL或NaOH       适量
取原料二甲双胍单苏糖酸盐，粉碎至细粉。配制葡磷酸二氢钠溶液，并将二甲双胍单苏糖酸盐细粉溶解在葡磷酸二氢钠溶液中。以0.1M氢氧化钠或盐酸调节pH值至7-8，随后转移至注射剂瓶中，蒸汽灭菌(121℃，灭菌15min)得到二甲双胍单苏糖酸盐注射液。
上述具体实施方式对本发明作进一步的详细描述。但不应将此理解为本发明上述主题的范围仅限于以下的实施例，凡基于本发明内容所实施的技术方案均落入于本发明的范围。

Claims (9)

  1. 二甲双胍盐在制备用于预防和/或治疗对象中脑梗死的药物中的用途。
  2. 权利要求1的用途，其中所述二甲双胍盐选自二甲双胍单苏糖酸盐、二甲双胍酒石酸盐、二甲双胍柠檬酸盐、二甲双胍甲磺酸盐、二甲双胍马来酸盐和二甲双胍氢溴酸盐。
  3. 权利要求1的用途，其中所述二甲双胍盐为二甲双胍单苏糖酸盐。
  4. 权利要求1-3任一项的用途，其中所述药物与人组织型纤溶酶原激活物组合使用。
  5. 权利要求1-3任一项的用途，其中所述对象为非糖尿病患者。
  6. 权利要求1-3的用途，其中所述药物为经口服途径给药或注射给药。
  7. 如式(I)所示的二甲双胍单苏糖酸盐，
    Figure PCTCN2018120479-appb-100001
  8. 制备如式(I)所示的二甲双胍单苏糖酸盐的方法，包括将二甲双胍和苏糖酸在加热条件下反应制得。
  9. 药物组合物，其包含权利要求7的二甲双胍单苏糖酸盐，或者权利要求8的方法制备的二甲双胍单苏糖酸盐，以及一种或多种药学上可接受的载体。
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Publication number Priority date Publication date Assignee Title
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* Cited by examiner, † Cited by third party
Title
YU YONGFEI ET AL.: "Clinical study of thrombolytic therapy with intravenous recombinant tissue-type plasminogen activator for patients with acute cerbral infraction", JOURNAL OF CRITICAL CARE IN INTERNAL MEDICINE, vol. 20, no. 4, 15 August 2014 (2014-08-15), pages 244 - 245 *

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