WO2021227572A1 - 微纳米MgH2化合物颗粒在抑制利什曼原虫感染及治疗利什曼病中的应用 - Google Patents

微纳米MgH2化合物颗粒在抑制利什曼原虫感染及治疗利什曼病中的应用 Download PDF

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WO2021227572A1
WO2021227572A1 PCT/CN2021/075141 CN2021075141W WO2021227572A1 WO 2021227572 A1 WO2021227572 A1 WO 2021227572A1 CN 2021075141 W CN2021075141 W CN 2021075141W WO 2021227572 A1 WO2021227572 A1 WO 2021227572A1
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mgh
nano
micro
compound particles
leishmania
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French (fr)
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袁广银
莫妮卡•埃切弗里•伦登
金亮
莎拉•玛丽亚•罗布列多•雷斯特雷波
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上海交通大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the technical field of medicine, particularly, to a micro-nano-particles in inhibiting compound MgH2 Leishmania infection and leishmaniasis in therapeutic applications; particularly to a micro-nano-particles MgH 2 inhibiting compound mucocutaneous type Leish Mania and its application in the treatment of skin and/or mucosal ulcers caused by Leishmania infection and the function of internal organs (spleen, liver, bone marrow, etc.) caused by visceral leishmaniasis.
  • Leishmaniasis is a disease endemic in tropical regions. This disease is mainly an intracellular parasite (leishmania) disease caused by the spread of sand flies or sand flies. Leishmaniasis is widely distributed all over the world, mainly in tropical and subtropical regions. 98 countries are affected by it. More than 15 million people in Asia, Africa, Southern Europe, and Central and South America are infected with the disease. Leishmaniasis cases in my country Mainly distributed in Xinjiang, Inner Mongolia, Gansu, Sichuan, Shaanxi, and Shanxi provinces. The global death toll is more than 70,000 annually, and the disease is a conditioned parasite for AIDS patients. (Reference: https://www.who.int/leishmaniasis/en/ ).
  • Leishmaniasis is mainly manifested as visceral type (Leishmaniadonovani) and skin mucosal type (Leishmania braziliensis), of which the skin mucosal type has the highest incidence.
  • Visceral leishmaniasis is caused by Leishmania invading internal organs (spleen, liver, bone marrow, etc.). If not treated in time, it can often cause death.
  • Cutaneous mucosal leishmaniasis is mainly caused by Leishmania brasiliensis parasitism in the skin, and it can also invade the nasopharynx or ear mucosa via lymph or blood, causing mucosal lesions.
  • the clinical manifestations are local lesions of the skin all over the body after infection.
  • the nodules are granulomas of varying sizes or dark papules. They do not ulcerate and rarely heal themselves. The nodules can be connected into pieces similar to tumor-like leprosy. Leishmania without amastigotes can be found in the nodules. . Skin lesions are common on the face and neck, and the course of the disease can be as long as decades or even life, severely reducing the patient's quality of life.
  • the pathological mechanism of the disease is that Leishmania infects the human body with the participation of an intermediate host, and then swims in the tissues and finally is specifically engulfed by macrophages and continues to survive and reproduce in the cytoplasmic phagolysosome, becoming a kind of macrophage Specific infection of intracellular parasites.
  • Skin-type pathology is mainly manifested as a large number of infected macrophages infiltrating the skin lesions and leading to local chronic inflammatory reactions, and skin tissues are prolonged and unhealed to form skin lesions.
  • Leishmaniasis is currently treated clinically with antimony agents: 5-valent antimony agent sodium antimony gluconate, which has a strong insecticidal effect on Leishmania but its side effects are also obvious, such as the treatment of white blood cells, especially neutrophils Continue to decrease.
  • antimony agents include 5-valent antimony agent sodium antimony gluconate, which has a strong insecticidal effect on Leishmania but its side effects are also obvious, such as the treatment of white blood cells, especially neutrophils Continue to decrease.
  • patients with heart disease and liver and kidney disease are poorly tolerated.
  • the use of systemic treatment for skin-type Leishmaniasis has little effect, large side effects, and is not cost-effective.
  • the patent document CN109453167A discloses the application of a maleimide compound in the preparation of Leishmania insecticide, and records that the maleimide compound has higher Anti-Leishmaniasis activity, and some compounds are higher than existing drugs.
  • the maleimide compound has a single type, is not easy to separate, and some of them are highly toxic, and their antibacterial activity is highly dependent on the pH value. It is a passive antibacterial material that depends on an acidic environment (reference: Shen Zhenzhong, new type Preparation of antibacterial agent containing maleimide structure and its antifungal activity, Master's thesis of Zhejiang University of Technology, May 2012, P.16).
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide a micro-nano MgH 2 compound particle for inhibiting Leishmania infection and treating leishmaniasis, specifically to provide a micro-nano MgH 2 compound particle Application in inhibiting Leishmania and treating skin and mucous membrane ulcers caused by its infection, as well as the treatment of damage to internal organs caused by its infection.
  • the present invention provides an application of MgH 2 in the preparation of a pharmaceutical composition for inhibiting Leishmania.
  • the present invention provides an application of MgH 2 in the preparation of a pharmaceutical composition for treating skin or mucosal ulcers caused by Leishmania infection.
  • the present invention provides an application of MgH 2 in the preparation of a pharmaceutical composition for treating visceral damage caused by Leishmania infection.
  • the particle diameter of the micro-nano MgH 2 compound particles is 1 nm-10 ⁇ m.
  • the particle diameter of the micro-nano MgH 2 compound particles is 100 nm-1000 nm.
  • the effective concentration of MgH 2 is 1-15 mg/100 ⁇ l.
  • the present invention provides a pharmaceutical composition for inhibiting Leishmania, which is characterized by comprising micro-nano MgH 2 compound particles, and pharmaceutically acceptable carriers and/or excipients.
  • the present invention provides a pharmaceutical composition for treating skin or mucosal ulcers caused by Leishmania infection, which is characterized by comprising micro-nano MgH 2 compound particles, and pharmaceutically acceptable carriers and/or excipients .
  • the present invention provides a pharmaceutical composition for treating visceral damage caused by Leishmania infection, comprising micro-nano MgH 2 compound particles, and pharmaceutically acceptable carriers and/or excipients.
  • the particle diameter of the micro-nano MgH 2 compound particles is 1 nm-10 ⁇ m.
  • the particle diameter of the micro-nano MgH 2 compound particles is 100 nm-1000 nm.
  • micro-nano MgH 2 compound particles of the present invention produce a large amount of active oxygen species (such as hydroxyl radicals, superoxide anions, etc.) through hydrolysis reaction, and the active oxygen species can effectively kill Leishmania.
  • active oxygen species such as hydroxyl radicals, superoxide anions, etc.
  • micro-nano MgH 2 compound particles of the present invention can be hydrolyzed into hydrogen and magnesium hydroxide in the cytoplasm, thereby changing the intracellular microenvironment.
  • the magnesium hydroxide in the product can quickly alkalize the cytoplasmic environment, change the acidic environment in the phagolysosome that is conducive to the growth of Leishmania, thereby inhibiting the growth of Leishmania.
  • Magnesium ions and hydrogen molecules in the degradation products of micro-nano MgH 2 compound particles can inhibit the expression of inflammatory factors in macrophages through anti-inflammatory effects, and promote inflammation and resolution of skin lesions; in addition, magnesium ions can also promote endothelial cell growth and enrichment
  • the capillaries in the skin lesions promote skin tissue repair. Thus, the effect of treating skin and/or mucosal ulcers caused by Leishmania can be achieved.
  • the medication mode of the present invention can adopt firstly dispersing MgH 2 particles or a combination product in a glycerol or PBS solution to prepare a solution with a concentration of MgH 2 particles of 1-15 mg/100ul. Then apply or spray 1-3 times a day on the ulcer of the skin (mucosa), and the wound will heal within 1-2 weeks. It can also be taken orally in the form of capsules containing micro-nano MgH 2 particles to act on damaged internal organs (mainly liver, spleen, etc.).
  • the present invention has the following beneficial effects:
  • micro-nano MgH 2 particles can significantly reduce the number of Leishmania in macrophages, inhibit their proliferation, and have a very good insecticidal inhibitory effect.
  • micro-nano MgH 2 particles of the present invention can quickly and effectively cure skin and/or mucosal ulcers caused by Leishmania and damaged internal organs (mainly liver and spleen organs) caused by Leishmania. Condition.
  • the experimental results of the present invention show that the curing time of sodium antimony gluconate with a 5-valent antimony preparation takes 1-3 months, while the curing time of the present invention is shortened to within 2-4 weeks.
  • micro-nano MgH 2 compound particles used in the present invention have been proved to have good biological safety through experiments, and can be applied to treat skin and mucous membrane ulcers directly or capsules can be taken orally to act on damaged organs of internal organs.
  • the 5-valent antimony preparation antimony gluconate has obvious side effects and requires injection treatment, which leads to lower patient compliance. Therefore, the micro-nano MgH 2 compound particles have great clinical application value.
  • Figure 1 shows the effects of three micro-nano MgH 2 compound particles on the hydrogen content, magnesium ion concentration and pH in the cell culture medium (RPMI1640);
  • Figure 1A shows the hydrogen content;
  • Figure 1B shows the magnesium ion concentration;
  • Figure 1C shows the pH value;
  • Figure 2 shows the IC50 effect of micro/nano MgH 2 compound particles on macrophages
  • Figure 3 is a picture of Leishmania (visceral Leishmaniadonovani) infected with macrophages;
  • Figure 4 is a diagram showing the relationship between the concentration of micro-nano MgH 2 compound particles and cells infected with Leishmania (Leishmaniadonovani);
  • Figure 5 is the in vivo biological safety assessment of micro-nano MgH 2 compound particles;
  • Figure 5A is the weight change;
  • Figure 5B is the spleen weight;
  • Figure 5C is the liver and kidney function indicators.
  • the MgH 2 compound particles of different particle sizes are continuously monitored for hydrogen release, magnesium ion concentration and pH value.
  • the specific method is as follows:
  • MgH 2 particles with particle diameters of 1nm-100nm, 100nm-1000nm, 1um-10um were dropped into the cell culture dish, the final concentration was 1mg/ml, cultured for 48h, and the hydrogen release, magnesium ion concentration and pH were continuously monitored. value.
  • Figure 1 It can be seen from the results that the three kinds of MgH 2 particles with different diameters can generate high concentration of hydrogen in the initial stage and continue to decrease over time; while the magnesium ion concentration continues to rise, the pH value continues to rise and gradually stabilizes at about 8.5.
  • micro-nano MgH 2 compound particles are co-cultured with macrophages infected with Leishmania (Leishmaniadonovani), the specific method is as follows:
  • micro/nano MgH 2 compound particles 100nm-1000nm
  • infected macrophages were co-cultured (medium RPMI1640) for 48h, and Giemsa staining was used to observe the number of infected cells and the number of protozoa.
  • concentration of magnesium hydride increases, the number of infected cells and protozoa decreases in a concentration-dependent manner.
  • control group is a pure cell culture medium without adding micro/nano MgH 2 particles.
  • the unit of the number of infected cells and the number of parasites is "piece".
  • Table 3 The effect of different doses of micro/nano MgH 2 compound particles on skin ulceration of hamsters infected by Leishmania
  • the MgH 2 compound particle treatment group can improve the clinical symptoms of the skin lesion in a concentration-dependent manner, inhibit the number of Leishmania in macrophages, and reduce the inflammatory cells in the skin lesion.
  • tissue repair is significantly enhanced, the area of the skin lesion is significantly reduced, and the hair follicle grows vigorously, which can speed up the healing of the wound in the skin lesion area.

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Abstract

一种微纳米MgH 2化合物颗粒在抑制利什曼原虫感染及治疗利什曼病中的应用，所述微纳米MgH 2化合物颗粒可用于制备抑制利什曼原虫的药物组合物，以及制备治疗利什曼原虫感染引起的皮肤、黏膜溃疡或内脏受损的药物组合物。本研究首次发现了微纳米MgH 2化合物颗粒可以显著减少巨噬细胞内利什曼原虫的数量，抑制其增殖，具有非常好的杀虫作用；其可以快速、有效的治愈利什曼原虫感染引起的皮肤和/或粘膜溃疡以及肝脏、脾脏等内脏器官功能受损，且生物安全性好。

Description

微纳米MgH 2化合物颗粒在抑制利什曼原虫感染及治疗利什曼病中的应用 技术领域
本发明属于医药技术领域，具体涉及一种微纳米MgH2化合物颗粒在抑制利什曼原虫感染及治疗利什曼病中的应用；尤其涉及一种微纳米MgH 2化合物颗粒在抑制粘膜皮肤型利什曼原虫及治疗利什曼原虫感染引起的皮肤和/或粘膜溃疡中的应用以及内脏型利什曼病引起的内脏器官(脾、肝、骨髓等)功能受损。
背景技术
利什曼病是一种热带地区流行的疾病。这种病主要是由沙蝇或白蛉的传播导致的细胞内寄生虫(利什曼原虫)病。利什曼病广泛分布于世界各地，主要分布在热带和亚热带地区，有98个国家受其影响，在亚洲、非洲、南欧和中南美洲超过1500万人感染此病,我国的利什曼病病例主要分布与新疆、内蒙古、甘肃、四川、陕西、和山西六省。全球每年死亡人数在7万以上，且该病是艾滋病患者的条件致病性寄生虫。(参考： https://www.who.int/leishmaniasis/en/)。
利什曼病主要表现为内脏型(Leishmaniadonovani)和皮肤粘膜型(Leishmaniabraziliensis)两大类型，其中皮肤粘膜型的发病率最高。内脏型利什曼病，由利什曼原虫侵害内脏器官(脾、肝、骨髓等)而致病，如未得到及时治疗，常可致患者死亡。皮肤粘膜型利什曼病主要由巴西利什曼原虫寄生于皮肤内引起皮肤病变，也可经淋巴或血液侵入鼻咽部或耳粘膜内，导致粘膜病变。临床表现为感染后全身皮肤发生局部病变。皮肤损伤除少数为褪色型外，多数为结节型。结节呈大小不等的肉芽肿，或呈暗色丘疹状，不破溃很少自愈，且结节可连成片类似瘤型麻风，在结节内可查到无鞭毛体的利什曼原虫。皮损常见于面部及颈部，病程可长达数十年甚至终身，严重降低患者生活质量。
该病的病理机制为利什曼原虫在中间宿主参与下感染人体，随后在组织内游弋最终被巨噬细胞特异性吞噬并在胞质的吞噬溶酶体内继续生存繁殖，成为一种巨噬细胞特异性感染的胞内寄生虫。皮肤型病理主要变现为皮损处存在大量受感染的巨噬细胞浸润并导致局部的慢性炎症反应，皮肤组织迁延不愈形成皮损。
利士曼原虫病治疗目前临床采用锑剂：5价锑制剂葡萄糖酸锑钠，对利什曼原虫有很强的杀虫作用但是其副作用同样明显，如治疗中血白细胞尤其中性粒细胞继续减少。此外有心脏病，肝肾疾病患者耐受性差。还有部分锑剂治疗三个疗程仍未愈者，称之为“抗锑剂”患者。此外对于皮肤型利士曼原虫病使用全身治疗，效果小，副作用大，性价比不高。
尽管在寄生虫相关的生物化学和生物学领域，人们的认识已经有了很大进步，但目前由于治疗费用昂贵、持续时间长、副作用严重，急需开发治疗疾病的新方法。不幸的是，利什曼病多发生在贫困地区，制药行业无意研究，是一类容易被忽视的疾病。相关国家的医疗卫生机构正在集中寻找替代方案，来降低目前主要使用含锑药物的毒性、成本和治疗时间。
据检索，专利文献CN109453167A中，公开了一种顺丁烯二酰亚胺类化合物在制备利士曼原虫杀虫剂中的应用，并记载了顺丁烯二酰亚胺类化物具有较高的抗利士曼原虫病活性，且部分化合物高于现有药物。然而顺丁烯二酰亚胺类化合物种类单一，不易分离，部分毒性较高，而且其抗菌活性对PH值依赖性大，是一种依赖酸性环境的被动性的抗菌材料(参考：沈振忠，新型含顺丁烯二酰亚胺结构抗菌剂的制备及其抗真菌活性研究，浙江工业大学硕士论文，2012年5月，P.16)。同时该专利文献也只是给出了体外细胞实验和前景效果预测，并没有给出动物体内的实际治疗效果的例证。由此可见，如何有效治疗或者缓解皮肤型利什曼病成为紧迫的课题。目前需要开发一种持续稳定且无副作用的用于治疗利士曼原虫病导致的皮肤组织迁延不愈形成皮损以及内脏器官受损的有效治疗药物。
发明内容
本发明的目的是为了克服现有技术的不足，提供一种微纳米MgH 2化合物颗粒在抑制利什曼原虫感染及治疗利什曼病中的应用，具体是提供一种微纳米MgH 2化合物颗粒在抑制利什曼原虫及治疗其感染引起的皮肤及粘膜溃疡中的应用以及其感染引起的内脏器官受损的治疗用途。
本发明的目的是通过以下技术方案实现的：
第一方面，本发明提供了一种MgH 2在制备抑制利什曼原虫的药物组合物中的应用。
第二方面，本发明提供了一种MgH 2在制备治疗利什曼原虫感染引起的皮肤或黏膜溃疡的药物组合物中的应用。
第三方面，本发明提供了一种MgH 2在制备治疗利什曼原虫感染引起的内脏受损的药物组合物中的应用。
优选地，所述微纳米MgH 2化合物颗粒的颗粒直径为1nm-10μm。
优选地，所述微纳米MgH 2化合物颗粒的颗粒直径为100nm-1000nm。
优选地，所述药物组合物中，MgH 2的有效浓度为1-15mg/100μl。
第四方面，本发明提供了一种抑制利什曼原虫的药物组合物，其特征在于，包括微纳米MgH 2化合物颗粒，以及药学上可接受的载体和/或辅料。
第五方面，本发明提供了一种治疗利什曼原虫感染引起的皮肤或黏膜溃疡的药物组合物，其特征在于，包括微纳米MgH 2化合物颗粒，以及药学上可接受的载体和/或辅料。
第六方面，本发明提供了一种治疗利什曼原虫感染引起的内脏受损的药物组合物，包括微纳米MgH 2化合物颗粒，以及药学上可接受的载体和/或辅料。
优选地，所述微纳米MgH 2化合物颗粒的颗粒直径为1nm-10μm。
优选地，所述微纳米MgH 2化合物颗粒的颗粒直径为100nm-1000nm。
本发明的药理作用如下：
1、本发明所述的微纳米MgH 2化合物颗粒通过水解反应产生大量活性氧物质(如羟基自由基，超氧阴离子等)，该活性氧物质可以有效杀死利什曼原虫。
2、本发明所述的微纳米MgH 2化合物颗粒可以在胞质内水解成氢气和氢氧化镁，从而改变细胞内微环境。其产物中的氢氧化镁可以迅速碱化胞质环境，改变吞噬溶酶体内有利于利什曼原虫生长的酸性坏境，从而起到抑制利什曼原虫生长的目的。
3、微纳米MgH 2化合物颗粒的降解产物中镁离子和氢气分子可以通过抗炎效应抑制巨噬细胞的炎症因子表达，促进皮损部位的炎症消解；此外镁离子还可以促进内皮细胞生长，丰富皮损部位的毛细血管，促进皮肤组织修复。由此可达到治疗由利什曼原虫引起的皮肤和/或粘膜溃疡的作用。
本发明的用药方式可采用将MgH 2颗粒或其组合产品首先弥散分布在甘油或PBS溶液中，配制成MgH 2颗粒浓度为1-15mg/100ul的溶液。然后每天在皮肤(粘膜)溃烂处涂抹或喷涂1-3次，1-2周时间内伤口即可痊愈。也可采用含有微纳米MgH 2颗粒的胶囊形式口服，作用于受损内脏器官(主要是肝脏、脾脏等)。
与现有技术相比，本发明具有如下的有益效果：
1、本发明首次发现了微纳米MgH 2颗粒可以显著减少巨噬细胞内利什曼原虫的数量，抑制其增殖,具有非常好的杀虫抑制作用。
2、本发明的微纳米MgH 2颗粒可以快速、有效的治愈利什曼原虫引起的皮肤和/或粘膜溃疡以及由于利什曼原虫引起的内脏器官(主要是肝脏、脾脏器官)功能受损的情况。本发明的实验结果显示，5价锑制剂葡萄糖酸锑钠的治愈时间需1-3个月，而本发明的治愈时间缩短到了2-4周内。
3、本发明采用的微纳米MgH 2化合物颗粒通过实验证实生物安全性好，可以涂抹治疗直接作用于皮肤粘膜溃疡处或者胶囊口服作用于内脏受损器官。而5价锑制剂葡萄糖酸锑毒副作用明显且需要注射治疗，导致患者依从性较低。因此，微纳米MgH 2化合物颗粒具有巨大的临床应用价值。
附图说明
图1为三种微纳米MgH 2化合物颗粒对细胞培养基(RPMI1640)内的氢气含量、镁离子浓度和pH值的影响；其中图1A为氢气含量；图1B为镁离子浓度；图1C为pH值；
图2为微纳米MgH 2化合物颗粒对巨噬细胞的IC50作用；
图3为利什曼原虫(内脏型利什曼原虫Leishmaniadonovani)感染巨噬细胞的图片；
图4为是微纳米MgH 2化合物颗粒浓度和受利什曼原虫(内脏型利什曼原虫Leishmaniadonovani)感染细胞关系图；
图5为微纳米MgH 2化合物颗粒体内生物安全性评估；其中，图5A为体重变化；图5B为脾脏重量；图5C为肝肾功能指标。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
实施例1
将不同粒径的MgH 2化合物颗粒进行氢气释放、镁离子浓度和pH值的连续监测。具体方法如下：
将颗粒直径分别为1nm-100nm，100nm-1000nm,1um-10um的三种MgH 2颗粒滴入细胞培养皿中，终浓度为1mg/ml，培养48h，并连续监测氢气释放，镁离子浓度和pH值。其结果如图1所示。从该结果可见，三种不同直径的MgH 2颗粒均可在初期产生高浓度的 氢气，并随时间持续降低；而镁离子浓度持续升高，pH值持续升高并逐渐稳定在8.5左右。
实施例2
将微纳米MgH 2化合物颗粒与受利什曼原虫(内脏型利什曼原虫Leishmaniadonovani)感染的巨噬细胞共培养，具体方法如下：
1)首先确定MgH 2化合物颗粒对巨噬细胞的IC50(半数抑制浓度，即细胞活性被抑制一半的浓度)：我们把氢化镁颗粒(100-1000nm)和THP-1来源的巨噬细胞共培养72小时，之后用CCK-8检测细胞的毒性(图2)，发现不同培养时间点的IC50是不同的：
24H:0.261mg/mL
48H:0.290mg/mL
72H:0.306mg/mL
2)检测微纳米MgH 2化合物颗粒对受利什曼原虫(内脏型利什曼原虫Leishmaniadonovani)感染的THP-1来源巨噬细胞的作用：
首先，确定最佳感染数目和感染时间，通过设置不同的增殖期原虫(5天或6天)，感染比例(5-30条虫/细胞)，感染时间(3h或24h)，采用Giemsa染色观察感染细胞数和原虫数获取最佳感染效率组和最低感染效率组。结果发现5天增殖期原虫、5条虫/细胞感染3h的实验组的感染效率最低，而5天增殖期原虫、15条虫/细胞、感染24h的实验组的感染效率最高(表1和图3)。如此，我们选择上述两实验组进行后续研究。
表1不同比例的内脏型利什曼原虫和巨噬细胞共培养后的感染结果
Figure PCTCN2021075141-appb-000001
注：感染细胞数和寄生虫数的单位均为“个”。
随后，采用微纳米MgH 2化合物颗粒(100nm-1000nm)和受感染的巨噬细胞共培养(培养基为RPMI1640)48h，用Giemsa染色观察受感染细胞数目和原虫数目。如图4和表2所示，随着氢化镁浓度的升高，受感染细胞和原虫数目呈浓度依赖性下降，我们计算发现其EC50＝0.043mg/ml(EC50是指：半数效应浓度，引起受试对象50％个体产生特定效应的剂量)，显著低于IC50＝0.290mg/ml。这表明微纳米MgH 2化合物颗粒可以在不影响生物安全性的前提下，显著抑制巨噬细胞的感染。
表2不同浓度微纳米MgH 2化合物颗粒对受内脏型利什曼原虫感染巨噬细胞的治疗效果
Figure PCTCN2021075141-appb-000002
注：对照组为不加微纳米MgH 2颗粒化合物组，为纯细胞培养基。感染细胞数和寄生虫数的单位均为“个”。
实施例3动物实验验证
1)生物安全性验证：选C57BL/6小鼠，每天灌胃不同浓度(0,60,120,250和500mg/kg)的微纳米MgH 2化合物颗粒(100nm-1000nm)，至第八天观察各项生理指标。如图5所示，小鼠存活率100％，体重，脾脏重量和肝肾指标均正常。
2)微纳米MgH 2化合物颗粒治疗研究：
2.1首先把颗粒直径为100nm-1000nm的MgH 2化合物在磷酸缓冲液中溶解后，制备成5个浓度组(1mg/100ul，3mg/100ul，5mg/100ul，10mg/100ul和15mg/100ul)。
2.2然后建立粘膜皮肤型利什曼原虫(Leishmaniabraziliensis)感染的小鼠模型，具体方法为：
取仓鼠(Mesocrvcetus Guratus)若干只，背部下缘剃毛，在尾部上源2cm处皮下注射利士曼原虫(100万条/100ul/只)，每天观察注射部位皮肤变化，培养4-8周后， 观察到注射部位皮肤组织呈鲜红色，有出血倾向伴新鲜肉芽组织增生，毛发脱落，迁延不愈，表明皮肤溃疡已经形成，模型建立成功。
2.3最后把受利什曼原虫感染的仓鼠模型35只，分为7组，每组5只。将不同浓度组的MgH 2化合物(颗粒尺寸100-1000nm)分别涂抹(100ul)在感染仓鼠的皮损部位，每24h涂抹一次，持续14天。同时设置未处理组(即空白对照组，不对感染小鼠做任何处理)和锑剂皮下注射组(采用5价锑制剂葡萄糖酸锑钠，锑剂(200μg/ml)溃疡处皮下注射100μl/只/天)。各实验组处理后，皮肤溃烂伤口愈合率结果如下表3所示。
表3不同剂量的微纳米MgH 2化合物颗粒对受利什曼原虫感染的仓鼠皮肤溃烂的疗效
Figure PCTCN2021075141-appb-000003
通过前述的动物研究发现，相比于未处理组，MgH 2化合物颗粒治疗组可以浓度依赖性地改善皮损部位临床症状，抑制巨噬细胞内的利什曼原虫数目，减少皮损处炎症细胞和炎症因子的浸润，组织修复明显增强，皮损面积显著缩小，毛囊生长旺盛，可加快皮损区伤口的愈合。此外，研究表明口服氢化镁颗粒对小鼠的肝肾功能无明显损害(图5C)，体重正常(图5A)，脾脏正常(图5B)，心肺脑组织均正常，血常规正常，未见到任何器官组织受损，而已有研究表明锑剂可以引起较严重的肝肾功能损害，甚至致死等副作用。
本发明具体应用途径很多，以上所述仅是本发明的优选实施方式。应当指出，以上实施例仅用于说明本发明，而并不用于限制本发明的保护范围。对于本技术领域的普通 技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进，这些改进也应视为本发明的保护范围。

Claims (10)

  1. 一种微纳米MgH 2化合物颗粒制备抑制利什曼原虫的药物组合物中的应用。
  2. 一种微纳米MgH 2化合物颗粒在制备治疗利什曼原虫感染引起的皮肤或黏膜溃疡的药物组合物中的应用。
  3. 一种微纳米MgH 2化合物颗粒在制备治疗利什曼原虫感染引起的内脏受损的药物组合物中的应用。
  4. 根据权利要求1或2或3所述的应用，其特征在于，所述微纳米MgH 2化合物颗粒的颗粒直径为1nm-10μm。
  5. 根据权利要求4所述的应用，其特征在于，所述微纳米MgH 2化合物颗粒的颗粒直径为100nm-1000nm。
  6. 根据权利要求1或2或3所述的应用，其特征在于，所述药物组合物中，MgH 2的有效浓度为1-15mg/100μl。
  7. 一种抑制利什曼原虫的药物组合物，其特征在于，包括微纳米MgH 2化合物颗粒，以及药学上可接受的载体和/或辅料。
  8. 一种治疗利什曼原虫感染引起的皮肤或黏膜溃疡的药物组合物，其特征在于，包括微纳米MgH 2化合物颗粒，以及药学上可接受的载体和/或辅料。
  9. 一种治疗利什曼原虫感染引起的内脏受损的药物组合物，其特征在于，包括微纳米MgH 2化合物颗粒，以及药学上可接受的载体和/或辅料。
  10. 根据权利要求7或8或9所述的药物组合物，其特征在于，所述微纳米MgH 2化合物颗粒的粒直径为1nm-10μm。
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