WO2016131320A1 - Nadph在制备治疗心脏疾病药物中的应用 - Google Patents

Nadph在制备治疗心脏疾病药物中的应用 Download PDF

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WO2016131320A1
WO2016131320A1 PCT/CN2015/095391 CN2015095391W WO2016131320A1 WO 2016131320 A1 WO2016131320 A1 WO 2016131320A1 CN 2015095391 W CN2015095391 W CN 2015095391W WO 2016131320 A1 WO2016131320 A1 WO 2016131320A1
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nadph
myocardial
group
ischemic
injury
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PCT/CN2015/095391
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French (fr)
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秦正红
李梅
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苏州人本药业有限公司
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Priority to US15/551,367 priority Critical patent/US20180036332A1/en
Publication of WO2016131320A1 publication Critical patent/WO2016131320A1/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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • 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
    • 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 present invention relates to novel indications for NADPH, and in particular to the use of NADPH for the preparation of a medicament for the treatment of heart disease.
  • Cardiovascular and cerebrovascular diseases are collectively referred to as diseases of the heart and blood vessels of the heart. Cardiovascular and cerebrovascular diseases have the characteristics of “high incidence, high mortality, high disability rate, high recurrence rate and many complications” (“four highs and one more”), which has become a heavy burden for global health care and health resources. It was the number one enemy of the second health revolution. At present, there are more than 270 million people with cardiovascular and cerebrovascular diseases in China. Especially after China enters an aging society, the incidence of cardiovascular and cerebrovascular diseases is still increasing. Therefore, the pathological mechanism and therapeutic protection of cardiovascular and cerebrovascular diseases have been An important task in the medical profession. The pathogenesis of cardio-cerebral ischemia is very complicated.
  • cardiovascular and cerebrovascular diseases since the heart assumes the role of providing power in the circulatory system, the lesion has a specificity different from that of other organs.
  • NADPH reduced nicotinamide adenine dinucleotide phosphate
  • PPP pentose phosphate pathway
  • GSH glutathione
  • GSH is an important antioxidant in the cell, which protects some thiol-containing proteins, fats and proteases from oxidants, especially in maintaining the integrity of erythrocyte membranes.
  • NADPH is also involved in the hydroxylation of the body and the biotransformation of drugs, poisons and certain hormones.
  • NADPH can use the electron donor of detoxification cells to reduce the oxidized compounds of the organism through metabolism in the body, maintain its redox balance, and play an important role in the oxidative defense system.
  • NADPH can also enter the respiratory chain by means of isocitrate shuttle to produce ATP: due to the low permeability of the mitochondrial inner membrane to the substance, the NADPH produced by the mitochondria in vitro cannot be directly oxidized into the respiratory chain.
  • TIGAR cardiomyocyte energy homeostasis under hypoxic stress
  • the function of TIGAR is to inhibit glycolysis and activate the pentose bypass.
  • the pentose bypass produces two metabolites: -NADPH and 5-pentose pentose. Therefore, knocking out TIGAR increases glycolysis and reduces
  • the activity of pentose metabolism, and inhibition of the pentose pathway means that NADPH is lowered, and it can be inferred that NADPH has an aggravating effect on myocardial injury. Therefore, no research has been conducted on the application of NADPH in the treatment of heart diseases.
  • the technical problem to be solved by the present invention is to provide a new indication for the application of NADPH, that is, the application of NADPH in the preparation of a medicament for treating heart diseases.
  • the invention provides the use of NADPH in the preparation of a medicament for treating heart diseases.
  • heart disease refers to one of myocardial damage, myocardial infarction, and cardiomyopathy.
  • cardiomyopathy is hypertrophic cardiomyopathy.
  • the medicament includes a pharmaceutically effective amount of NADPH and a pharmaceutically acceptable carrier.
  • the invention provides a medicine for treating heart diseases, wherein the medicine uses NADPH as an active ingredient, and a conventional excipient is added to NADPH according to a conventional process to prepare a clinically acceptable mixture, capsule, tablet, film, spray. Agent.
  • the invention provides a medicament for treating any one of myocardial injury, myocardial infarction or cardiomyopathy, wherein the drug uses NADPH as an active ingredient, and a conventional excipient is added to NADPH according to a conventional process to prepare a clinically acceptable mixture, Capsules, tablets, filming agents, sprays.
  • the method of the present invention uses NADPH as an active ingredient for preparing a drug for treating heart diseases, and proves that NADPH has the functions of protecting vascular endothelial cells, maintaining normal blood vessel permeability, and reducing ischemic myocardial injury, and administering exogenous NADPH through research. Is there any treatment for ischemic myocardial injury, and found a new use of NADPH in the treatment of myocardial injury, myocardial infarction. Specifically, NADPH injected into mice can enter the blood-brain barrier and enter the cell.
  • NADPH can maintain normal vascular permeability and reduce blood-brain barrier damage after cerebral ischemia and reperfusion; NADPH can reduce the scope of myocardial infarction and play Protective effects of acute ischemic myocardial injury.
  • NADPH is an endogenous antioxidant, it can also be an energy-supplying substance. It has no toxic side effects during therapeutic applications, so it has the advantages of small dosage and safety.
  • NADPH can be administered orally, injectable, and can be administered through the oral and nasal mucosa and the skin.
  • oxidative stress and energy metabolism disorders are common mechanisms of ischemic damage in other organs, NADPH can also be used in a wide range of other diseases.
  • FIG 1 shows the effect of exogenous NADPH on the survival rate of primary cultured HUVEC cells with low glucose hypoxia
  • Figure 2 shows the therapeutic administration of NADPH in the brain of mice with permanent cerebral ischemic stroke. The effects of immune cells on the blood-brain barrier;
  • Figure 3 is a graph showing the effect of prophylactic administration of NADPH on blood-brain barrier permeability in mice with cerebral ischemic reperfusion;
  • Figure 4 is the effect of therapeutic administration of NADPH on blood-brain barrier injury in mice with permanent ischemic stroke;
  • Figure 4a shows the results of blood-brain barrier damage assay;
  • Figure 4b shows the results of blood-brain barrier permeability test;
  • Figure 5 is the effect of therapeutic administration of NADPH on myocardial injury in myocardial ischemia-reperfusion mice;
  • Figure 5a shows the results of TTC staining;
  • Figure 5b is a comparison of myocardial mass in the ischemic infarct area as a percentage of myocardial mass in the ischemic region.
  • NADPH 20g suspending agent microcrystalline cellulose 60g; preservative tert-butyl 4-hydroxyanisole 0.04g; lubricant magnesium stearate 2g; filler lactose added to 200g.
  • the preparation method comprises the following steps:
  • excipients used as such include, but are not limited to, a mixture of one or more of a filler, a disintegrant, a lubricant, a binder, a flavoring agent, a suspending agent, and a preservative.
  • the filler may also be replaced by a mixture of one or more of pregelatinized starch, mannitol, chitin, microcrystalline cellulose, sucrose;
  • the disintegrant may also be replaced by a mixture of one or more of starch, crospovidone, sodium carboxymethylcellulose, sodium carboxymethyl starch;
  • the lubricant may also be replaced by one of talc, silica, and sodium lauryl sulfate. Or a mixture of multiples;
  • the suspending agent may also be replaced by a mixture of one or more of polyvinylpyrrolidone, sucrose, agar, hydroxypropylmethylcellulose;
  • the preservative may also be replaced by a mixture of one or more of parabens, benzoic acid, sodium benzoate, sorbic acid, sorbate;
  • the binder may also be replaced by a mixture of one or more of polyvinylpyrrolidone and hydroxypropylmethylcellulose;
  • the flavoring agent may also be replaced by a sweetener and/or a fragrance;
  • the sweetener is a mixture of one or more of sodium saccharin, aspartame, sucrose, cyclamate;
  • the pharmaceutical composition of the present invention is preferably an oral dosage form and an injection dosage form, and the oral form is selected from the group consisting of an oral solution, a capsule, an effervescent tablet, an oral drug film or a spray; and the injection dosage form is selected from the group consisting of muscle, subcutaneous injection or intravenous drip. Powder injections and water injections, etc. Further, the medicament of the present invention can be prepared into a corresponding dosage form by a method known in the art.
  • the primary cultured endothelial cells used in the experiments were purchased from the US ATCC cell bank. Cryopreservation conditions: 2 ml cryotubes, 1.6 million cells per tube, containing 70% high glucose DMEM, 20% domestic fetal bovine serum, 10% DMSO.
  • HUVEC culture of endothelial cells culture conditions: 37 ° C (5% CO 2 , 95% air), saturated humidity, high glucose DMEM medium, medium containing 100 U penicillin and 100 U streptomycin per liter; 10% domestic fetal bovine serum; The cells were grown to about 80-90% confluence, digested with trypsin-EDTA solution and passaged.
  • Passage density 5 ⁇ 10 5 / bottle for 2-3 days passage; logarithmic growth of HUVEC cells, add appropriate amount of trypsin - EDTA digestive juice, the adherent cells were detached, the cells were collected, counted, and the cell suspension (5 ⁇ 10 4 /ml) was prepared with the culture medium containing 10% fetal bovine serum, and 100 ⁇ l was added to each well in a 96-well plate. Incubate at 37 ° C, 5% CO 2 for 24 h. Take NADPH dissolved in sterile physiological saline to prepare a mother solution of 10mmol/L, filter and sterilize the cells and add to the cell culture medium to detect the cell viability of MTT assay:
  • Figure 1 shows the effect of exogenous NADPH on HUVEC activity in primary cultured endothelial cells with low glucose hypoxia.
  • the survival rate of 5 ⁇ M NADPH for 48h was about 65.3% (p ⁇ 0.05); the survival rate of 10 ⁇ M NADPH for 48h was about 73.6% (p ⁇ 0.01); the survival rate of 20 ⁇ M NADPH for 48h was about 70.9% (p ⁇ 0.01). ).
  • Tg (Itgax-Venus) 1Mnz mice weighing 22-27 g, male.
  • the Tg (Itgax-Venus) 1Mnz mouse is a product of MGI, which is positive for CD 11c-eYFP and is used to study the immune response of dendritic cells involved in brain injury. Room temperature 22 ° C, humidity 50-60%, good ventilation, artificial day and night (12h / 12h), free feeding water.
  • mice Male mice were acclimated for 2 days in the breeding environment before the experiment.
  • Tg Itgax-Venus 1 Mnz mice, weighing 22-27 g, male.
  • the experimental groups were sham operation group, cerebrovascular injury model group, cerebrovascular injury + NADPH (2.5 mg/kg) treatment group, 6 animals in each group. NADPH was diluted with artificial cerebrospinal fluid, and intracerebroventricular injection was performed. 2 ⁇ l of the lateral ventricle of the mice was administered (intracerebral cerebral vascular injury model group was injected with artificial cerebrospinal fluid).
  • mice were anesthetized with chloral hydrate, fixed, and the median neck incision was used to separate the left common carotid artery, the internal neck, and the external carotid artery.
  • the common carotid artery was ligated near the heart.
  • a nylon wire plug with a uniform diameter was inserted before the bifurcation of the internal and external carotid arteries to block the blood flow for 24 hours.
  • the Dextran-40 solution was injected into the tail vein at 22 h of ischemia.
  • Detection of immune cell response in the blood-brain barrier Dextran-Texas was injected into the tail vein of the mice 22 hours after the injury of the brain microvascular injury. After 2 hours, the experimental animals were decapitated and the brain was removed. Then the formalin was perfused to fix the brain tissue, and the slicer was sliced and sliced. The cells were collected in PBS and immunohistochemically stained by a floating method. CD 11c-eYFP antibody labeled dendritic positive cells, DAPI for nuclear staining, and mounting. Confocal microscopy was used to observe the distribution and expression of dendritic inflammatory cell responses in the striatum brain region.
  • Figure 2 is a graph showing the effect of prophylactic administration of NADPH on the local immune response in mice with permanent ischemic stroke.
  • CD 11c-eYFP cells were scattered in the brain area, the cell body was small, and the dendritic structure was clear.
  • the model group showed significant increase in CD 11c-eYFP reactivity and increased cell body deformation in the striatum of the brain.
  • Intraventricular administration of the NADPH (2.5 mg/kg) group was effective in reducing CD 11c-eYFP reactivity in the striatum region.
  • NADPH has a good protective effect on the blood-brain barrier and is involved in the regulation of local immune responses.
  • mice 1) Establishment of a transient middle cerebral artery occlusion model in mice:
  • ICR normal mice group weighing 23 ⁇ 28g, were randomly divided into two groups, 20 in each group, divided into saline group (vehicle group), NADPH (7.5mg / kg) dose group; NADPH 1 week before ischemia ( Inject twice a day through the tail vein into the body.
  • the rat model of ischemic MCAO was prepared with a slight improvement of the internal carotid artery suture method.
  • the mice were anesthetized with 4% chloral hydrate (400 mg/kg). We used the suture method to prepare the ischemic model.
  • the extra-cervical and internal carotid arteries were ligated to the proximal end of the neck and the proximal end of the neck.
  • the plug (6023, Doccol Corporation, Redlands, USA) was inserted from the outside of the neck to the anterior end of the anterior cerebral artery to block the supply of blood to the middle cerebral artery. After blocking the blood flow for 2 hours, the plug was pulled out to achieve reperfusion.
  • the sham-operated mice were the same as the ischemic group and the treatment group except that the mice were not inserted.
  • the room temperature was maintained at 22-25 °C throughout the operation, and the temperature of the mouse was controlled at 37 ⁇ 0.5 °C using an automatic temperature-controlled heating pad. After the operation, the animals were placed in a feeding box with clean litter, and they were allowed to drink water and eat freely.
  • mice C57BL6 mice, weighing 22-27 g, male.
  • the experimental groups were sham operation group, cerebrovascular injury model group, cerebrovascular injury + NADPH (2.5 mg/kg) treatment group, 6 animals in each group. NADPH was diluted with artificial cerebrospinal fluid, and intracerebroventricular injection was performed. 2 ⁇ l of the lateral ventricle of the mice was administered (intracerebral cerebral vascular injury model group was injected with artificial cerebrospinal fluid).
  • the mice were anesthetized with chloral hydrate, fixed, and the median neck incision was used to separate the left common carotid artery, the internal neck, and the external carotid artery. The common carotid artery was ligated near the heart.
  • a nylon wire plug with a uniform diameter was inserted before the bifurcation of the internal and external carotid arteries to block the blood flow for 24 hours.
  • the Dextran-40 solution was injected into the tail vein at 22 h of ischemia. After 2 hours of internal circulation, anesthetized with 10% chloral hydrate, the thoracic cavity was opened, the heart was exposed, and the left ventricle of the heart was perfused with 10 ml of 10 mmol/L PBS solution, and the residual Dextran-Texas in the brain tissue was washed away.
  • NADPH was administered 2 hours prior to the preparation of a permanent middle cerebral artery occlusion cerebral ischemia model. The blood-brain barrier injury of experimental animals was measured 24 hours after permanent cerebral artery occlusion.
  • Dextran-Texas was injected into the tail vein of mice 22 hours after the injury of brain microvascular injury. After 2 hours, the experimental animals were decapitated, then the brain tissue was fixed by formalin perfusion, sliced by vibrating slicer, and DAPI was used for nucleus. Dyeing, sealing. Dextran-Texas fluorescence intensity in the peri-cerebral parenchyma of the cerebral ischemic brain region was detected by laser confocal microscopy.
  • Figure 3 is a graph showing the effect of prophylactic administration of NADPH on blood-brain barrier permeability in mice with cerebral ischemia-reperfusion stroke. Compared with the vehicle group, NADPH was administered 1 week earlier (2 times per day in the tail vein) to significantly reduce the blood-brain barrier permeability of mice with cerebral ischemic stroke (p ⁇ 0.01). ** indicates p ⁇ 0.01.
  • Figure 4 is a graph showing the effect of prophylactic administration of NADPH on blood-brain barrier permeability in mice with permanent ischemic stroke.
  • Figure 4a shows the results of blood-brain barrier damage assay and
  • Figure 4b shows blood-brain barrier permeability test results.
  • the model group showed significant red fluorescent dye Dextran-Texas leakage in the cerebral cortex and striatum compared to the sham operation group.
  • Intraventricular administration of NADPH (2.5 mg/kg) group can effectively reduce the leakage of Dextran-Texas induced by ischemia in the cerebral cortex and striatum. It is suggested that NADPH shows a good protective effect on the blood-brain barrier.
  • Example 4 therapeutic administration of NADPH reduces myocardial ischemic injury
  • LAD left ventricle
  • LV left ventricle
  • the myocardial tissue was placed in 0.5% TTC, bathed at 37 ° C for 15 min, and 10% formaldehyde was used overnight to fix the tissue.
  • the LV was divided into three parts: normal, ischemic infarction zone (red, risk zone) and ischemic infarction zone (infant white zone, infarct zone), and the myocardial mass of ischemic infarct was weighed.
  • the percentage of mass, myocardial infarction range is expressed as the percentage of myocardial mass in the infarct area as a percentage of myocardial mass in the ischemic area.
  • Figure 5 shows the effect of therapeutic administration of NADPH on myocardial ischemia and infarction.
  • Figure 5a shows the results of TTC staining;
  • Figure 5b is a graph comparing the percentage of myocardial mass in the ischemic infarct area to the myocardial mass in the ischemic area.
  • the results of TTC staining showed that compared with the vehicle group (blank control), NADPH was significantly reduced in the tail vein of 0h after reperfusion, and the myocardial infarct size was significantly reduced (p ⁇ 0.05). It is suggested that NADPH reduces myocardial ischemic injury.
  • the blue portion represents the non-ischemic region
  • the red portion represents the ischemic region
  • the white portion represents the infarcted region.
  • * indicates p ⁇ 0.05.

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Abstract

还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)在制备治疗心脏疾病的药物中的应用以及用于治疗心脏疾病的以NADPH为活性成分的药物，其中所述心脏疾病包括心肌损伤、心肌梗死、心肌病。

Description

NADPH在制备治疗心脏疾病药物中的应用 技术领域
本发明涉及NADPH的新适应症，具体地说是NADPH在制备治疗心脏疾病药物中的应用。
背景技术
心脑血管疾病是指心脏血管和脑血管的疾病统称。心脑血管疾病具有“发病率高、死亡率高、致残率高、复发率高、并发症多”(“四高一多”)的特点，目前已成为全球卫生保健和卫生资源的沉重负担，是第二次卫生革命的头号敌人。目前，我国心脑血管疾病患者已经超过2.7亿人，尤其是我国进入老年化社会后，心脑血管疾病的发病率仍在不断增加，因此，研究心脑血管疾病的病理机制及治疗防护一直是医药界的重要任务。心脑缺血疾病发病机制十分复杂，研究心脑缺血疾病新的致病机制、寻找药物作用新的靶点，对于心脑血管疾病的预防和治疗药物的开发具有重要现实的意义。在心脑血管疾病中，由于心脏承担着提供循环***中的动力的角色，因此，其病变具有不同于其他脏器的特殊性。
还原型烟酰胺腺嘌呤二核苷酸磷酸(triphosphopyridine nucleotide，NADPH)由葡萄糖经过磷酸戊糖途径(PPP)代谢产生，作为细胞内最为重要的电子供体和生物合成的还原剂，可为还原性生物合成提供氢离子。NADPH是谷胱甘肽(GSH)的还原酶的辅酶，可使氧化型谷胱甘肽(GSSG)生成还原型GSH，维持还原型的GSH的正常含量。GSH是细胞内重要的抗氧化剂，可保护一些含巯基的蛋白质、脂肪和蛋白酶类免受氧化剂的破坏，特别在维持红细胞膜的完整性方面起着重要作用。NADPH除了参与胆固醇、脂肪酸、单加氧酶系、类固醇激素等的生物合成，还参与体内羟化反应和药物、毒物及某些激素的生物转化。例如，NADPH可利 用解毒细胞的电子供体，通过体内代谢减少生物体氧化型化合物，维持其氧化还原的平衡，在氧化防御***发挥重要作用。NADPH也可以借助于异柠檬酸穿梭作用进入呼吸链产生ATP：由于线粒体内膜对物质的通透性很低，线粒体外产生的NADPH不能直接进入呼吸链被氧化。NADPH上的H可以在异柠檬酸脱氢酶的作用下被交给NAD+，然后由NAD+进入呼吸链产生能量。细胞能量代谢的维持和减少ROS(活性氧簇)对细胞生存，特别对缺血缺氧的组织至关重要，普遍认为能量代谢障碍和氧化应激是心脑缺血疾病的重要机制，研究表明增加细胞能量代谢能力，降低细胞ROS产生可减轻缺血缺氧引起的细胞损伤。基于NADPH的多重生理功能，目前已有一些研究将NADPH应用于某些疾病的治疗中。
根据国际文献报道《p53与TIGAR在缺氧条件下调节心肌细胞能量平衡的作用》(p53and TIGAR regulate cardiac myocyte energy homeostasis under hypoxic stress)中的研究表明，敲除心肌细胞的TIGAR或TIGAR的上游调节基因有加重缺氧条件下心肌细胞凋亡的作用。TIGAR的功能是抑制糖酵解和激活戊糖旁路，戊糖旁路产生两个代谢产物：-NADPH和5-磷酸戊糖，因此，敲除TIGAR的作用增加了糖酵解，且降低了戊糖代谢的活性，而抑制戊糖通路则意味著降低了NADPH，由此可推断出NADPH对于心肌损伤具有加重的作用，因此，目前尚未有研究将NADPH应用于心脏疾病的治疗中。
发明内容
本发明所要解决的技术问题是提供一种NADPH的新的适应症的应用，即NADPH在制备治疗心脏疾病药物中的应用。
本发明提供了NADPH在制备治疗心脏疾病药物中的应用。
进一步地，所述心脏疾病是指心肌损伤、心肌梗死、心肌病中的一种。
进一步地，所述心肌病为肥厚性心肌病。
所述药物包括药学上有效量的NADPH和药学上可接受的载体。
本发明提供的用于治疗心脏疾病的药物，所述药物以NADPH为活性成分，向NADPH中加入常规辅料按照常规工艺制成临床上可接受的合剂、胶囊剂、片剂、药膜剂、喷雾剂。
本发明提供的用于治疗心肌损伤、心肌梗死或心肌病中任意一种疾病的药物，所述药物以NADPH为活性成分，向NADPH中加入常规辅料按照常规工艺制成临床上可接受的合剂、胶囊剂、片剂、药膜剂、喷雾剂。
本发明所述的方案以NADPH作为制备治疗心脏疾病药物的活性成分，证实了NADPH具有保护血管内皮细胞、维护血管正常通透性、减少缺血性心肌损伤的作用，通过研究给予外源性NADPH是否有治疗缺血性心肌损伤作用，发现了NADPH在治疗心肌损伤、心肌梗死的新用途。具体的，经研究小鼠注射的NADPH可以进入血脑屏障，进入细胞内，NADPH在脑缺血再灌后可以维护血管正常通透性，减少血脑屏障损伤；NADPH可缩小心肌梗死范围，发挥急性缺血性心肌损伤的保护作用。上述结果提示NADPH对缺血性心血管损伤起保护作用，NADPH是一个有效的治疗心脏疾病，尤其是心肌损伤、心肌梗塞和心肌病的药物。
由于NADPH是一个内源性的抗氧化物质，也可以是供应能量的物质，在治疗应用时没有发现有毒副作用，因此，具有用量小、安全的优点。加之，NADPH可通过口服、可注射、可透过口鼻粘膜和皮肤给药，服用方便。并且，由于氧化应激和能量代谢障碍是其它脏器组织缺血损伤的共同机制，因此，NADPH在其它疾病中也可有广泛的用途。
附图说明
为了使本发明的内容更容易被清楚的理解，下面结合附图，对本发明作进一步详细的说明，其中，
图1为外源NADPH对低糖缺氧的原代培养内皮细胞HUVEC细胞存活率的影响；
图2为治疗性给予NADPH对永久脑缺血性中风小鼠脑内参与 血脑屏障的免疫细胞的影响；
图3为预防性给予NADPH对脑缺血性再灌注中风小鼠血脑屏障通透性的影响；
图4为治疗性给予NADPH对永久脑缺血性中风小鼠血脑屏障损伤的影响；图4a为血脑屏障损伤测定结果；图4b为血脑屏障通透性测试结果；
图5为治疗性给予NADPH对心肌缺血再灌注性小鼠心肌损伤的影响；图5a为TTC染色结果；图5b为缺血梗死区心肌质量占缺血区心肌质量的百分比对比图。
具体实施方式
实施例1含有NADPH的胶囊剂
本实施例的胶囊剂包括以下成分：
NADPH 20g；助悬剂微晶纤维素60g；防腐剂叔丁基—4—羟基苯甲醚0.04g；润滑剂硬脂酸镁2g；填充剂乳糖加至200g。
其制备方法包括以下步骤：
称取上述处方量的NADPH和各药用辅料，混合均匀，过60目筛三次，装入胶囊即得。
作为中所用的常规辅料包括但不限于填充剂、崩解剂、润滑剂、粘合剂、矫味剂、助悬剂、防腐剂中的一种或几种的混合物。
具体而言，所述填充剂还可替换为预胶化淀粉、甘露醇、甲壳素、微晶纤维素、蔗糖中的一种或多种的混合物；
所述崩解剂还可替换为淀粉、交联聚维酮、羧甲基纤维素钠、羧甲基淀粉钠中的一种或多种的混合物；
所述润滑剂还可替换为滑石粉、二氧化硅、十二烷基硫酸钠中的一种 或多种的混合物；
所述助悬剂还可替换为聚乙烯吡咯烷酮、蔗糖、琼脂、羟丙基甲基纤维素中的一种或多种的混合物；
所述防腐剂还可替换为尼泊金类、苯甲酸、苯甲酸钠、山梨酸、山梨酸盐中的一种或多种的混合物；
所述粘合剂还可替换为聚乙烯吡咯烷酮、羟丙基甲基纤维素中的一种或多种的混合物；
所述矫味剂还可替换为甜味剂和/或香精；所述甜味剂为糖精钠、阿斯帕坦、蔗糖、甜蜜素中的一种或多种的混合物；
当然，所述常规辅料包括但不限于上述列举的范围，本领域技术人员可根据实际情况做适应性的选择及调整。
本领域技术人员可以使用本领域中已知的任何方式施用本发明的药物，包括但不限于外用、口服、舌下、经鼻、胃肠外、局部、皮下、注射、经皮或直肠的施用途径。本发明的药物组合物优选口腔剂型以及注射剂型，口腔型选自口服液、胶囊剂、泡腾片、口服药膜或喷雾剂；注射剂型选自供肌肉、皮下注射给药或静脉滴注使用的粉针剂和水针剂等。且本发明的药物可以采用本领域中公知的方法制为相应剂型。
实验例
下面，为验证本发明的技术效果进行以下实验：
实验例1外源性NADPH对原代培养内皮细胞HUVEC保护作
(1)实验材料：
试验所使用的原代培养内皮细胞HUVEC细胞均购于美国ATCC细胞库。冻存条件：2ml冻存管，每管160万细胞，含70％高糖DMEM，20％国产胎牛血清，10％DMSO。
(2)实验方案：
内皮细胞HUVEC培养：培养条件：37℃(5％CO2，95％空气)，饱和湿度，高糖DMEM培养基，培养基每升含100U青霉素和100U链霉素；10％国产胎牛血清；待细胞生长至80-90％左右融合，用胰酶-EDTA液消化后进行传代，传代密度：5×105/瓶隔2-3天传代；取对数生长的HUVEC细胞，加入适量胰蛋白酶-EDTA消化液，使贴壁细胞脱落，收集细胞，计数，用含10％胎牛血清的培养液配成细胞悬液(5×104/ml)，于96孔板中每孔加入100μl，在37℃、5％CO2培养24h。取NADPH溶于无菌生理盐水中配制成母液为10mmol/L的溶液，滤膜过滤除菌后加入到细胞培养基中MTT法细胞存活率检测：
细胞活性检测：将细胞接种于96孔板中，待细胞生长至指数阶段时加入终浓度为1，5，10，20,40μM的NADPH。以溶剂为阴性对照，放入培养箱于相同条件下继续培养48h。结束培养前4h加入MTT(5mg/ml，D-Hanks’溶解)10μl，试验结束时吸去上清，加入150μl DMSO，用酶联免疫检测仪于570nm处测定各孔OD值，取6孔OD值求均值，计算抑制率：抑制率(IR％)＝(1-测试孔OD/对照孔OD)×100％。
(3)实验结果
图1为外源NADPH对低糖缺氧的原代培养内皮细胞HUVEC活性的影响。与正常对照组相比，5，10，20μM浓度的NADPH作用48h可以使HUVEC细胞存活率明显降低。5μM的NADPH作用48h细胞存活率约为65.3％(p＜0.05)；10μM NADPH作用48h细胞存活率约为73.6％(p＜0.01)；20μM NADPH作用48h细胞存活率约为70.9％(p＜0.01)。1)对照组；2)缺氧低糖组；3)缺氧低糖组+NADPH 1μM；4)缺氧低糖组+NADPH5μM；5)缺氧低糖组+NADPH 10μM；6)缺氧低糖组+NADPH 20μM；7)缺氧低糖组+NADPH 40μM.**与对照组相比，p＜0.01； #与缺氧低糖组相比，p＜0.05；##与缺氧低糖组相比，p＜0.01。其中**表示p<0.01，#表示p<0.05，##表示p<0.01。
实验例2预防性给予NADPH减轻脑损伤后血脑屏障有关免疫细胞反应
(1)实验材料
清洁级雄性ICR小鼠，质量23～28g，苏州大学实验动物中心提供，实验动物生产许可证号：XCYK(苏)2002-2008，实验动物使用许可证号：SYXK(苏)2002-0037。C57BL6及Tg(Itgax-Venus)1Mnz系小鼠，体重22～27g，雄性。Tg(Itgax-Venus)1Mnz系小鼠为MGI公司产品，该鼠为CD 11c-eYFP阳性，用于研究树突状细胞参与脑损伤后免疫反应情况。室温22℃，湿度50-60％，通风良好，人工昼夜(12h/12h)，自由摄食摄水。实验前，将雄小鼠在饲养环境中适应2d。Dextran-Texas(Red)：Invitrogen公司产品，NADPH试剂购自江苏sigma试剂公司；外源性NADPH药物的来源可以通过人工合成、半合成，生物提取获得。
(2)实验方案：
永久性大脑中动脉栓塞脑缺血模型建立。Tg(Itgax-Venus)1Mnz系小鼠，体重22～27g，雄性。实验分组为假手术组、脑血管损伤模型组，脑血管损伤+NADPH(2.5mg/kg)治疗组，每组动物6只。NADPH用人工脑脊液稀释，采用脑室注射，小鼠侧脑室给药2μl(脑血管损伤模型组脑室注射人工脑脊液)。小鼠采用水合氯醛麻醉，固定，颈正中切口，分离左侧颈总动脉、颈内、颈外动脉。颈总动脉近心端结扎，在距颈内、颈外动脉分叉之前***直径统一的尼龙线栓，阻断血流24h。缺血22h时尾静脉注射Dextran-40溶液。经过2h体内循环， 用10％水合氯醛麻醉，打开胸腔，暴露心脏，用10ml 10mmol/L PBS溶液于心脏左心室灌流，将脑组织中残余的Dextran-Texas冲洗掉。NADPH于永久性大脑中动脉栓塞脑缺血模型制作前2小时给药。永久性大脑中动脉栓塞脑缺血模型24h后测定实验动物血脑屏障有关免疫细胞反应。
(3)实验方法
血脑屏障有关免疫细胞反应的检测:脑微血管损伤损伤后22h小鼠尾静脉注射Dextran-Texas，2h后实验动物断头取脑，随后***灌流固定脑组织，振动切片机切片，切片收集于PBS液内，采用漂浮法对其进行免疫组织化学染色。CD 11c-eYFP抗体标记树突状阳性细胞，DAPI进行细胞核染色，封片。共聚焦显微镜观察血管周围树突状炎症细胞反应在纹状体脑区的分布和表达情况。
(4)实验结果
图2为预防性给予NADPH对永久脑缺血性中风小鼠局部免疫应答反应的影响。假手术对照组小鼠脑区散在分布有CD 11c-eYFP细胞，胞体较小，树突结构清晰。脑缺血损伤后24小时，模型组在大脑纹状体区域均可见明显的CD 11c-eYFP反应性增加，胞体变形增大。NADPH(2.5mg/kg)组脑室给药可以有效减少纹状体区域的CD 11c-eYFP反应性。NADPH具有很好的保护血脑屏障作用，与调节局部免疫反应有关
实施例3预防性给予NADPH减少脑缺血再灌注血脑屏障损伤实验
(1)实验材料同实验例2。
(2)实验过程
1)小鼠短暂性大脑中动脉阻塞模型建立：
取ICR正常小鼠组，体重23～28g，随机分2组，每组20只，分为生理盐水组(vehicle组)，NADPH(7.5mg/kg)剂量组；NADPH在缺血前1周(每天注射2次)通过尾静脉注入体内。采用颈内动脉线栓法，稍加改进制备小鼠然缺血MCAO模型，小鼠以4％水合氯醛(400mg/kg)腹腔注射麻醉，我们采用线栓法制备缺血模型，分离颈总、颈外和颈内动脉，结扎劲外和颈总近心端，线栓(6023，Doccol Corporation,Redlands,USA)从颈外***直到大脑前动脉起始端，阻断大脑中动脉供血。阻断血流2h后，拔出线栓实现再灌注。假手术组小鼠除不插线外，其余步骤均与缺血组和治疗组相同。整个手术过程中室温保持在22～25℃，采用自动控温加热垫将小鼠肛温控制在37±0.5℃。术后将动物置于放有清洁垫料的饲养盒中，自由饮水、进食。
2)永久性大脑中动脉栓塞脑缺血模型建立。
C57BL6系小鼠，体重22～27g，雄性。实验分组为假手术组、脑血管损伤模型组，脑血管损伤+NADPH(2.5mg/kg)治疗组，每组动物6只。NADPH用人工脑脊液稀释，采用脑室注射，小鼠侧脑室给药2μl(脑血管损伤模型组脑室注射人工脑脊液)。小鼠采用水合氯醛麻醉，固定，颈正中切口，分离左侧颈总动脉、颈内、颈外动脉。颈总动脉近心端结扎，在距颈内、颈外动脉分叉之前***直径统一的尼龙线栓，阻断血流24h。缺血22h时尾静脉注射Dextran-40溶液。经过2h体内循环，用10％水合氯醛麻醉，打开胸腔，暴露心脏，用10ml 10mmol/L PBS溶液于心脏左心室灌流，将脑组织中残余的Dextran-Texas冲洗掉。NADPH于永久性大脑中动脉栓塞脑缺血模型制作前2小时给药。永久性大脑中动脉栓塞脑缺血模型24h后测定实验动物血脑屏障损伤。
(3)实验方法
1)血脑屏障通透性测试：脑缺血再灌注23h后经尾静脉注入2％伊文思蓝(EB)生理盐水溶液(4ml/kg)1h后将小鼠断头取脑，称重并放入50％三氯乙酸溶液中，匀浆和离心(10000rpm，20min)，去上清液按照1：3的比例用乙醇稀释。620nm处检测OD值；计算脑组织中EB含量。
2)血脑屏障损伤测定：脑微血管损伤损伤后22h小鼠尾静脉注射Dextran-Texas，2h后实验动物断头取脑，随后***灌流固定脑组织，振动切片机切片，DAPI进行细胞核染色，封片。激光共聚焦显微镜下检测脑缺血脑区的脑血管周围脑实质内的Dextran-Texas荧光强弱。
3)数据统计与分析：数据均以均数±标准误(Mean±SEM)表示，统计分析采用单因素方差分析(one-way ANOVA)，p<0.05为统计学差异有显著性。
(4)实验结果
图3为预防性给予NADPH对脑缺血再灌注性中风小鼠血脑屏障通透性的影响。与vehicle组相比，提前1周(每天尾静脉2次)给予NADPH后显著降低脑缺血性中风小鼠的小鼠血脑屏障通透性(p<0.01)。**表示p<0.01。
图4为预防性给予NADPH对永久脑缺血性中风小鼠血脑屏障通透性的影响。图4a为血脑屏障损伤测定结果，图4b为血脑屏障通透性测试结果。脑缺血损伤后24h，模型组在大脑皮层及纹状体区域均可见明显的红色荧光染料Dextran-Texas的漏出相比假手术组。NADPH(2.5mg/kg)组脑室给药可以有效减少缺血诱导大脑皮层及纹状体区域的Dextran-Texas的漏出。提示NADPH显示了很好的保护血脑屏障作用。
实施例4治疗性给予NADPH减少心肌缺血损伤
(1)实验材料
成年雄性SD大鼠，体重270～350g，清洁级。由苏州大学医学部实验动物中心提供。【许可证号：(苏)SYXK2007-0035】。伊文思蓝(Evans Blue，EB，sigma)取SD正常大鼠组，体重23～28g，随机分2组，每组10只，分为生理盐水组(Model组)，NADPH(7.5mg/kg)剂量组；NADPH在心肌缺血再灌注时立刻通过尾静脉注入体内。
(2)实验过程
大鼠在体心肌缺血再灌注损伤模型建立
雄性成年SD大鼠水合氯醛腹腔内注射麻醉，于右侧颈内静脉及颈内动脉置入充满肝素的导管，用于静脉给药、检测动脉血气分析或监测动脉血压。气管切开并***气管导管，连接ALC-V9动物呼吸机行呼气末正压通气，吸入氧浓度33％，调节呼吸频率或潮气量，维持pH7.35～7.45、PaCO225～40mmHg、PaO290～150mmHg。采用智能恒温控制仪维持大鼠体温在36°C～37℃。于第5肋间行左胸切开术，打开心包，6-0无损伤缝合线在左心耳下缘结扎左冠状动脉前降支(Left Anterior Descending Coronary Artery，LAD)，缝线末端穿入自制圈套管，平衡30min。用止血钳夹紧圈套管以阻断LAD血供，如果心外膜发绀苍白，心电图示一过性心律失常，ST段弓背向上抬高表示缺血模型成功；松开圈套管进行再灌注，可见心外膜重新充血证明再灌注成功。再灌注2h检测心肌梗死范围。
(3)实验方法
心肌梗死范围的测定：大鼠心肌缺血30min再灌注2h时，再次阻断LAD，颈内静脉注射5％EB 1ml使左心室(Left  Ventricle，LV)正常区域蓝染，迅速取出心脏分离LV，使用大鼠心脏切片器横断分割成5～6块2mm厚的组织块(见图1-1-3)。将LV中蓝色染的正常组织与未染色的LV缺血区组织分离。采用TTC染色法，将心肌组织放入0.5％TTC中，37℃水浴15min，10％甲醛过夜以固定组织。解剖显微镜下将LV分成正常、缺血未梗死区(红色，risk zone)和缺血梗死区(染为灰白色，infarct zone)3部分，并分别称重缺血梗死区心肌质量占缺血区心肌质量的百分比，心肌梗死范围以梗死区心肌质量占缺血区心肌质量的百分比表示。
(4)实验结果
图5为治疗性给予NADPH对心肌缺血梗死的影响。图5a为TTC染色结果；图5b为缺血梗死区心肌质量占缺血区心肌质量的百分比对比图。TTC染色结果显示：与vehicle组(空白对照)相比，再灌注0h尾静脉给予NADPH后显著地缩小大鼠心肌梗死范围(p<0.05)。提示NADPH减少心肌缺血损伤。蓝色部分代表未缺血区，红色部分代表缺血区，白色部分代表梗死区。其中*表示p<0.05。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (6)

  1. NADPH在制备治疗心脏疾病药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述心脏疾病是指心肌损伤、心肌梗死、心肌病中的一种。
  3. 根据权利要求2所述的应用，其特征在于，所述心肌病为肥厚性心肌病。
  4. 根据权利要求1-3中任一所述的应用，其特征在于，所述药物包括药学上有效量的NADPH和药学上可接受的载体。
  5. 一种用于治疗心脏疾病的药物，其特征在于，所述药物以NADPH为活性成分，向NADPH中加入常规辅料按照常规工艺制成临床上可接受的合剂、胶囊剂、片剂、药膜剂、喷雾剂。
  6. 一种用于治疗心肌损伤、心肌梗死或心肌病中任意一种疾病的药物，其特征在于，所述药物以NADPH为活性成分，向NADPH中加入常规辅料按照常规工艺制成临床上可接受的合剂、胶囊剂、片剂、药膜剂、喷雾剂。
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CN106902131A (zh) * 2017-02-21 2017-06-30 重庆纳德福实业集团股份有限公司 Nadph在制备治疗心肌肥厚与心力衰竭的药物中的应用

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