WO2014187225A1

WO2014187225A1 - Use of mefloquine in preparation of medication for preventing neuropathic pain

Info

Publication number: WO2014187225A1
Application number: PCT/CN2014/076335
Authority: WO
Inventors: 王智如; 胡文浩; 王英伟; 赵政
Original assignee: 华东师范大学; 上海交通大学医学院附属新华医院
Priority date: 2013-05-21
Filing date: 2014-04-28
Publication date: 2014-11-27
Also published as: CN104173347B; CN104173347A

Abstract

Disclosed are a use of mefloquine in the preparation of a medication for preventing neuropathic pain and a pharmaceutical composition comprising a prophylactically effective amount of mefloquine and a pharmaceutical acceptable carrier.

Description

甲氟喹在制备预防神经病理性疼痛药物中的应用技术领域 Application of mefloquine in preparing medicine for preventing neuropathic pain

本发明涉及医药领域，具体涉及 Ψ氟喹在刺备预防神经病理性疼痛的药物中的应用。背景技术 The invention relates to the field of medicine, in particular to the application of fluoroquine in the medicine for preventing neuropathic pain. Background technique

神经病理性疼痛（ Neuropathic Pain, N P P) 是由于神经机械受损（例如运动、车祸、外科手术）而引发的一类疾病。它属于一种慢性疼痛，疼痛表现为自发性疼痛、痛觉过敏、异常疼痛和感觉异常等临床特征（Neuron, 2012, 73, 638-652 ) 。据 2009年美国国家科学院医学研究所（I0M) 报告，美国最少有 L 16亿成年人遭受慢性疼痛疾病，其中神经源性疼痛病人比例高达 17. 9%，约占美国总人口的 3. 5%；欧洲成年人中大约 5个人中有 1个有慢性疼痛，大约 25%的糖尿病患者有神经病理疼痛。神经病理性疼痛的发病机制比较复杂，包括外周神经***、脊髓及大脑均有参与。最近研究发现 NPP与大脑的记忆机制具有相似性（Science, 2011， 331, 87-91 ) ：该病的一个关键机制在于大脑的前扣带回皮层（anterior c ngu late cortex; ACC )内部的神经连接发生增强，及长时程突触增强（ long- term potentiation; LTP)。己有基础研究表明（Lancet, 2010, 9, 807-819 ) : 在神经受损初期的几天内，一些 NMDA受体阻断剂、 cAMP抑制剂等可以阻断 ACC的 LTP发生，可有效预防神经源性疼痛的发生。 Neuropathic Pain (NPP) is a type of disease caused by neuromechanical damage (such as sports, car accidents, and surgical procedures). It is a chronic pain that manifests as a clinical feature of spontaneous pain, hyperalgesia, abnormal pain, and paresthesia (Neuron, 2012, 73, 638-652). According to the 2009 National Institute of Medicine (I0M) report, at least L 1.6 billion adults in the United States suffer from chronic pain, of which the proportion of patients with neuropathic pain is as high as 17.9%, accounting for 3.5% of the total US population. One in five adults in Europe has chronic pain, and about 25% of people with diabetes have neuropathic pain. The pathogenesis of neuropathic pain is complex, including the involvement of the peripheral nervous system, the spinal cord, and the brain. Recent studies have found that NPP has similarities to the memory mechanisms of the brain (Science, 2011, 331, 87-91): A key mechanism of the disease is the nerves inside the anterior c ngu late cortex (ACC) of the brain. Increased connectivity and long-term potentiation (LTP). Basic research has shown (Lancet, 2010, 9, 807-819): In the early days of neurological damage, some NMDA receptor blockers, cAMP inhibitors, etc. can block the occurrence of ATP LTP, which can effectively prevent The occurrence of neuropathic pain.

甲氟喹，化学名：（+/ ) - α - 2-哌啶基 - 2, 8-双三氟甲基- 4-喹琳甲醇，结构式为 Mefloquine, chemical name: (+/ ) - α - 2-piperidinyl - 2, 8-bistrifluoromethyl-4-quinoline methanol, the structural formula is

(-)-(1 1 12S)— 氟喹（÷)- (1 1 S, 12R)-甲氟喹 (-)-(1 1 12S)-fluoroquinoline (÷)- (1 1 S, 12R)-methylfluoroquine

其商品名 Lariam, 临床应用的是其盆酸盐，其分子式： C_]7H_;7C1F 0. HC1 , 分子量为： 414. 77, 为白色结晶，易溶于水，熔点！叩为 259 260Ό (分解）。临床应用的甲氟喹（盐酸盐）主要用于预防和治疗脑型疟疾（恶性疟）和间日疟或控制耐氯喹的脑型疟疾（恶性疟），由 Roche., 美国军事医学研究机构和 WHO合作开发，于 1984年在泰国最先上市，其后分别在瑞士、法国、澳大利亚、西德、英国等多国上市， 1989年 5月被 FDA批准。 ψ氟喹常 ^作抗疟药，用于治疗恶性疟和间日疟，对耐氯喹及耐乙胺嘧啶虫铢的原虫红细胞内期无性体具有较强的杀灭作用，其作用机制尚不清楚，可能与奎宁相似与铁原卟琳 IX中的铁作用，铁原 Π卜琳 IX是疟色素的主要成分，因而引起疟色素凝集，但发展缓慢，很少形成团块。然而与奎宁不同之处是它不嵌入疟原虫的去氧核糖核酸。血药浓度 18h达高峰，其血浆半衰期较长，但个体差异较大，平均 14日，有效水平可维持 30日以上。在体内代谢为羧酸代谢物，代谢物对疟原虫无作用，主要由粪便及胆汁排出体夕卜。目前， ¥氟喹广泛用于疟疾的预防和治疗，尚未见任何报道公开甲氟喹具有预防神经病性疼痛的作用。 Its trade name Lariam, its clinical application is its pot acid, its molecular formula: C _]7 H _{; 7} C1F 0. HC1, molecular weight: 414. 77, is white crystal, soluble in water, melting point!叩 is 259 260 Ό (decomposed). Clinical application of mefloquine (hydrochloride) is mainly used for the prevention and treatment of cerebral malaria (falciparum malaria) and vivax malaria or cerebral malaria (palmophilia) that controls chloroquine-resistant, by Roche., American Military Medical Research Institute Developed in cooperation with WHO, it was first listed in Thailand in 1984, and subsequently listed in Switzerland, France, Australia, West Germany, the United Kingdom and other countries, and was approved by the FDA in May 1989. It is often used as an antimalarial drug for the treatment of falciparum malaria and vivax malaria. It has a strong killing effect on chloroquine and pyrimethamine-resistant protozoal erythrocyte endoplasms, and its mechanism of action is not yet Clearly, it may be similar to quinine and iron in iron 卟卟 Lin IX. Iron Π Π 琳 IX is the main component of malaria pigment, which causes malaria pigmentation, but it develops slowly and rarely forms a mass. However, the difference with quinine is It does not intercalate the DNA of Plasmodium. The plasma concentration reached a peak at 18h, and its plasma half-life was longer, but the individual differences were larger, with an average of 14 days, and the effective level could be maintained for more than 30 days. Metabolized in the body to a carboxylic acid metabolite, the metabolite has no effect on the malaria parasite, mainly excreted by feces and bile. At present, fluoroquine is widely used in the prevention and treatment of malaria, and no report has been published that the disclosure of mefloquine has the effect of preventing neuropathic pain.

发明内容 Summary of the invention

本发明首次提出了甲氟喹在制备预防神经病理性疼痛的药物中的应用。本发明提出了甲氟喹作为有效活性成分的新的医药用途。 The present invention is the first to propose the use of mefloquine in the preparation of a medicament for preventing neuropathic pain. The present invention proposes a new medical use of mefloquine as an effective active ingredient.

本发明中，所述神经病理性疼痛是指疾病、运动、车祸、外科手术等原因，由机械、化学刺激造成神经受损而引发的一类疼痛，它属于一种慢性疼痛，疼痛表现为自发性疼痛、痛觉过敏、异常疼痛和感觉异常等临床特征。 In the present invention, the neuropathic pain refers to a kind of pain caused by mechanical and chemical stimulation caused by nerve damage caused by diseases, sports, car accidents, surgery, etc. It belongs to a kind of chronic pain, and the pain is spontaneous. Clinical features such as pain, hyperalgesia, abnormal pain, and paresthesia.

本发明中，甲氟喹的化学名为（+/ ) - α 2-哌啶基 2, 8 -双三氟甲基 4 喹啉醇，结构式包括： In the present invention, the chemical name of mefloquine is (+/)-α2-piperidinyl 2,8-bistrifluoromethyl 4 quinolol, and the structural formula includes:

(-)-(1 1 12S)- 氟喹（+)-(1 1 S, 12R)-甲氟喹分子式： C₁₇H₁₇Cl i ), 分子量为： 378,3—1。 (-)-(1 1 12S)-Fluoroquine (+)-(1 1 S, 12R)-Methylfluoroquinol Molecular Formula: C ₁₇ H ₁₇ Cl i ), Molecular Weight: 378, 3-1.

本发明中，在预防神经病理性疼痛的作用中，甲氟喹在神经受损早期，通过抑制前扣带皮层 Cx36 蛋白的活动，预防神经病理性疼痛的发生。本发明中， Ψ氟喹作为活性成分，可制成溶液或固体状的剂型，如注射剂、末剂、散¾、片剂、糖衣剂、胶囊剂、颗粒剂、悬浮剂、溶液剂、糖浆剂、滴剂等进行给药，或采用口服或注射给药。 In the present invention, in the action of preventing neuropathic pain, mefloquine prevents the occurrence of neuropathic pain by inhibiting the activity of the anterior cingulate cortex Cx36 protein in the early stage of nerve damage. In the present invention, fluoroquine as an active ingredient can be formulated into a solution or a solid form, such as an injection, a terminal, a dispersion, a tablet, a sugar coating, a capsule, a granule, a suspension, a solution, a syrup. , drops, etc. are administered or administered orally or by injection.

本发明还提出一种药物组合物，应用于预防神经病理性疼痛的药物中，其包括预防有效量的 φ氟喹和药学上可接受的载体或药学上允许的辅料或添加剂。 The present invention also provides a pharmaceutical composition for use in a medicament for preventing neuropathic pain comprising a prophylactically effective amount of φ fluoroquine and a pharmaceutically acceptable carrier or a pharmaceutically acceptable adjuvant or additive.

本发明药物组合物中可含有常规药学上可接受的辅助物质、稳定剂、湿润别或其它常用的添加剂，例如，乳糖、柠檬酸、酒石酸、硬脂酸、硬脂酸镁、石膏粉、蔗糖、玉米淀粉、滑石粉、明胶、琼脂、果胶、花生油、橄榄油、可可脂、乙二醇、抗坏血酸、 —甘-露醇等。施用本发明药物组合物的载体可以是一种及以上的固体、半固体或液体状的稀释剂、填充剂和其他处方用的辅料。本发明可以经口服、注射、局部组织给药等方式，可以采用适合各给药方式的剂型及用量进行施药。本发明药物组合物可以是药学上的任意溶液或固体状的剂型，包括滴剂、软膏剂、凝胶剂、片剂、水剂、颗粒剂、溶液剂等。药物组合物可以是固体形式如片剂、胶囊剂、散剂、丸齐颗粒剂、软膏剂、凝胶剂、脂质体、栓剂，或是液体形式如水剂、注射剂、滴眼剂、乳剂等。例如， ¥氟喹与有机或无机的固体或液体赋形剂混合。 The pharmaceutical composition of the present invention may contain conventional pharmaceutically acceptable auxiliary substances, stabilizers, wetting or other commonly used additives, for example, lactose, citric acid, tartaric acid, stearic acid, magnesium stearate, gypsum powder, sucrose. , corn starch, talc, gelatin, agar, pectin, peanut oil, olive oil, cocoa butter, ethylene glycol, ascorbic acid, mannitol, etc. The carrier to which the pharmaceutical composition of the present invention is applied may be one or more solid, semi-solid or liquid diluents, fillers and other prescription excipients. The invention can be administered orally, by injection, by local tissue administration, or the like, and can be administered by using a dosage form and an amount suitable for each administration mode. The pharmaceutical composition of the present invention may be in any pharmaceutically acceptable solution or solid dosage form, Including drops, ointments, gels, tablets, liquids, granules, solutions, and the like. The pharmaceutical composition may be in a solid form such as a tablet, a capsule, a powder, a granule, an ointment, a gel, a liposome, a suppository, or a liquid form such as a liquid, an injection, an eye drop, an emulsion or the like. For example, fluoroquine is mixed with an organic or inorganic solid or liquid excipient.

本发明研究发现，甲氟喹能通过血脑屏障，并且一个重要的靶点是神经元之间的缝隙连接 (也称电突触）构成的关键蛋白。已有研究提示，缝隙连接对记忆的前期巩固过程具有重要影响。基于这类疾病与大脑记忆巩固的类似机制，本发明首次提出了一种新的预防神经性病理疼痛的机制，将甲氟喹用于神经源性疼痛疾病的预防。 The present inventors have found that mefloquine can pass the blood-brain barrier, and an important target is a key protein composed of gap junctions (also called electrical synapses) between neurons. Studies have suggested that gap junctions have an important impact on the early consolidation process of memory. Based on a similar mechanism of such diseases and brain memory consolidation, the present invention proposes for the first time a new mechanism for preventing neuropathic pain, using mefloquine for the prevention of neuropathic pain diseases.

本发明研究表明，通过注射 Ψ氟喹有效预防 CCI模型大鼠病理性疼痛样症状的发生；甲氟喹对 CCI模型大鼠病理性疼痛样症状产生影响作用的有效给药时间窗口；前扣带皮层微量导入甲氟喹，通过阻断前扣带皮层 Cx36表达，能有效防治神经病理性疼痛样症状的发生；注射甲氟喹能有效防治 SNI模型大鼠病理性疼痛样症状的发生；表明活性成分 Ψ氟喹能广泛有效地应用亍制备预防神经病理性疼痛的药物中。同时，本发明研究表明，甲氟喹对大鼠运动能力、平衡能力及焦虑状态无明显影响，无毒副作用，具有良好的应用前景。 The present invention shows that the injection of fluoroquine is effective in preventing the pathological pain-like symptoms in the CCI model rats; the effective administration time window of the effect of mefloquine on the pathological pain-like symptoms of the CCI model rats; Microinjection of mefloquine into the cortex can effectively prevent the occurrence of neuropathic pain-like symptoms by blocking the expression of Cx36 in the anterior cingulate cortex; injection of mefloquine can effectively prevent the occurrence of pathological pain-like symptoms in SNI model rats; The fluoroquine can be widely and effectively applied to the preparation of a medicament for preventing neuropathic pain. At the same time, the research of the present invention shows that mefloquine has no significant effect on the exercise capacity, balance ability and anxiety state of rats, has no toxic and side effects, and has a good application prospect.

酎图说明 Description

图 1是实施例 1的腹腔注射不同给药剂量甲氟喹对 CCI模型大鼠的机械痛阈的效果。图 2是实施例 1的腹腔注射不同给药剂量甲氟喹对 CCI模型大鼠的热敏痛阈的效果。图 3是实施例 2的不同时间腹腔注射甲氟喹对 CCI模型大鼠的机械痛的影响。 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the effect of intraperitoneal injection of different doses of mefloquine on the mechanical pain threshold of CCI model rats. Figure 2 is a graph showing the effect of intraperitoneal injection of different doses of mefloquine on the thermal pain threshold of CCI model rats by Example 1. Figure 3 is a graph showing the effect of intraperitoneal injection of mefloquine on mechanical pain in CCI model rats at different times in Example 2.

图 4是实施例 2的不同时间腹腔注射甲氟喹对 CCI模型大鼠的热痛的影响。 Figure 4 is a graph showing the effect of intraperitoneal injection of mefloquine on the thermal pain of CCI model rats at different times in Example 2.

图 5是实施例 3的前扣带回皮层埋管微量注射甲氟喹对 (XI手术后大鼠的机械痛的效果。图 6是实施例 3的前扣带回皮层埋管微量注射甲氟喹对 CCI手术后大鼠的热痛的效果。图 7是实施例 4的慢病毒干扰起效的基因序列 Figure 5 is a graph showing the effect of microinjection of mefloquine on the anterior cingulate cortex tube of Example 3 (the mechanical pain of rats after XI surgery. Fig. 6 is a microinjection of methyl fluoride into the anterior cingulate cortex of Example 3; Effect of quinone on heat pain in rats after CCI surgery. Figure 7 is a gene sequence of lentiviral interference effect of Example 4.

图 8是实施例 4的前扣带回皮层注射慢病毒使该脑区 Cx36蛋白表达下调的免疫印迹图图 9是实施例 4的前扣带回皮层注射慢病毒使该脑区 Cx36蛋白表达下调的免疫组化图图 10是实施例 4的前扣带回皮层显微注射 Cx36表达下调病毒后对 CCI手术后大鼠机械疼痛的效果 Figure 8 is an immunoblot of the anterior cingulate cortex injection of lentivirus in Example 4 to downregulate Cx36 protein expression in the brain region. Figure 9 is a anterior cingulate cortex injection of lentivirus in Example 4 to downregulate Cx36 protein expression in the brain region. Immunohistochemistry map Figure 10 is the effect of the anterior cingulate cortex microinjection of Cx36 expression on the mechanical pain in rats after CCI surgery.

图 11-是实施例 4的前扣带回皮层显微注射 Cx36表达下调病毒后对（XI手术后大鼠热痛的效果 Figure 11 - is the effect of microinjection of the anterior cingulate cortex of Example 4 on the expression of Cx36 down-regulated virus (the effect of heat pain in rats after XI operation)

图 12是实施例 5的腹腔注射甲氟喹对于 SNI模型大鼠机械痛的效果 Figure 12 is a graph showing the effect of intraperitoneal injection of mefloquine on mechanical pain in SNI model rats in Example 5.

图 1-3是实施例 6的腹腔注射甲氟喹幵场实验的表现 Figure 1-3 shows the performance of the intraperitoneal injection of the mefloquine field in Example 6.

图 14是实施例 6的腹腔注射甲氟喹转棒实验的表现具体实施方式结合以下具体实施例和附图，对本发明作进一歩的详细说明，本发明的保护内容不局限于以下实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本实^新型中，并以所附的权利要求书为保护范围。实施本发明的过程、条件、试剂、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。 Figure 14 is a representation of the performance of the intraperitoneal injection of the mefloquinol rotating rod experiment of Example 6. The present invention will be further described in detail with reference to the following specific embodiments and drawings. Variations and advantages that may be made by those skilled in the art are intended to be included within the scope of the invention and the scope of the appended claims. The processes, conditions, reagents, experimental methods, and the like of the present invention are generally known in the art and common knowledge, except for the contents specifically mentioned below, and the present invention is not particularly limited.

本发明各实施例中，所有动物实验均在华东师范大学脑功能基因组学研究所 SPF (高清洁级）动物房内完成，所有动物操作均符合研究所动物管理委员会的规章制度。 CCi手术在 300— 350g 体重的成年 SD大鼠上开展。手术参照按照 Bennett和 Xie于 1988年建立、目前广泛被采用的实验流程：大鼠腹腔注射戊巴比妥钠施行全身麻醉，剪除手术侧后肢皮肤、消毒；手术暴露坐骨神经并将其与其它组织分离，用铬制肠线（规格， 5-0 ) 环绕神经千做 3个轻度结 ¾ (间距约 1mm) ；缝合肌肉和皮趺。待动物清醒后，将其放回饲养笼内，自由进食。 In the various embodiments of the present invention, all animal experiments were performed in the SPF (High Definition Clean) animal room of the Institute of Brain Functional Genomics of East China Normal University, and all animal operations were in accordance with the rules and regulations of the Institute of Animal Management. CCi surgery was performed on adult SD rats weighing 300-350 g. The surgical procedure was established according to Bennett and Xie in 1988. The experimental procedure is widely used: intraperitoneal injection of sodium pentobarbital for general anesthesia, removal of the skin of the hind limb of the surgical side, disinfection; surgical exposure of the sciatic nerve and separation from other tissues Use a chrome gut (size, 5-0) to make three light knots around the nerve (3 mm apart); suture the muscles and skin. After the animals are awake, put them back in the cage and eat freely.

CCI术后的当天幵始（含当天），连续 5天（每天一次）腹腔注射不同剂量（ 10 mg/kg_; 20 mg/kg; 30 mg/kg) 甲氟喹（用药组）、以及同等体积溶齐 ij (空白对照组， vehkle) 。分别在术后 7、 14天，鉴定大鼠的疼痛阈值。甲氟喹需要溶解于吐温 80, 后用生理盐水稀释，因此采用的空白对照溶剂为吐温一 -80和生理盐水的混合液，比例和配置甲氟喹溶液一致。具体地， 100毫克¥氟喹先后配置溶解于 0.05毫升 DMSO , L5毫升吐温、 3毫升 PBS或生理盐水。 The day after CCI (including the day), 5 days (once a day), intraperitoneal injection of different doses (10 mg / kg _; 20 mg / kg; 30 mg / kg) mefloquine (medication group), and the same volume Dissolve ij (blank control, vehkle). The pain threshold of the rats was identified at 7 and 14 days after surgery, respectively. Mefloquine needs to be dissolved in Tween 80 and then diluted with physiological saline. Therefore, the blank control solvent used is a mixture of Tween-80 and physiological saline, and the ratio is the same as that of the mefloquine solution. Specifically, 100 mg of fluquine was dissolved in 0.05 ml of DMSO, L5 ml of Tween, 3 ml of PBS or physiological saline.

实施例 1 腹腔注射甲氟喹对 CCI模型大鼠病理性疼痛样症状发生的防治 Example 1 Prevention and treatment of pathological pain-like symptoms in rats with CCI by intraperitoneal injection of mefloquine

一、机械痛域指标测定。 First, the determination of mechanical pain domain indicators.

参照目前普遍采用的 Dixon报道的 Up-down法 ( Pain 85 (2000) 493-502) 。检测前 3天，将动物放入底部为金属网的有机玻璃箱的检测笼适应环境，每天一次、每次 30分钟。检测当天，待动物进入检测笼后半小时，使用不同弯曲强度（1 , 1.4, 2, 4, 6, 8, 10, 15g) 的 von Fray纤维丝刺激接受手术侧的后肢足底，测定大鼠对该机械刺激的缩足反应。当大鼠对某一弯曲强度的 voti Fray f维丝剌激（直至其弯曲）出现缩足反应，则认定为大鼠出现疼痛样行为, 将该 voii Fray弯曲强度定义为机械痛的阈值。 Refer to the currently widely used Up-down method reported by Dixon (Pain 85 (2000) 493-502). Three days before the test, the animals were placed in a test cage of a plexiglass box with a metal mesh at the bottom to adapt to the environment once a day for 30 minutes. On the day of detection, half an hour after the animals entered the test cage, the von Fray filaments with different flexural strengths (1, 1.4, 2, 4, 6, 8, 10, 15 g) were used to stimulate the hind paws of the surgical side, and the rats were measured. The contraction response to this mechanical stimulus. When the rat showed a contraction response to a flexural intensity of the voti Fray f stimuli (until it was bent), it was determined that the rat had a pain-like behavior, and the voii Fray bending strength was defined as the threshold of mechanical pain.

自 CCI手术当天起连续 5天内，大鼠腹腔注射 10、 20、 30 mg kg甲氟喹，每天注射一次，在实验观察的长期时间范围（21天）内，分别在术后 7、 14天鉴定大鼠的疼痛阈值，检测结果如图 1所示， ¥氟喹均能使模型鼠的机械痛域值保持较高水平，即机械痛阈值回升。从图 1可见，腹腔注射 30 mg/kg甲氟喹，机械痛阈值回升效果最佳。实验表明， CCI手术后腹腔注射甲氟喹，机械痛阈值回升，能有效预防神经病理性疼痛样症状的发生。 Rats were injected intraperitoneally with 10, 20, 30 mg kg of mefloquine once a day for 5 days from the day of CCI surgery. They were injected once a day for 7 days and 14 days after the long-term time range (21 days). The pain threshold of the rats, the test results are shown in Figure 1. The fluoroquine can maintain the high level of mechanical pain in the model rats, that is, the mechanical pain threshold rises. It can be seen from Figure 1 that intraperitoneal injection of 30 mg/kg mefloquine has the best effect on mechanical pain threshold recovery. Experiments have shown that intra-abdominal injection of mefloquine after CCI surgery, the threshold of mechanical pain rises, can effectively prevent the occurrence of neuropathic pain-like symptoms.

二、热痛阈指标鉴定将大鼠置于一有机玻璃箱内的玻璃板上。 30分钟后，用热辐射剌激仪照射大鼠手术侧后肢足部表面，记录大鼠对该热刺激表现缩足反应的时间，该时间定义为大鼠对该热刺激的耐受 '间，用于衡量对热刺激的痛敏指标。 Second, the identification of thermal pain threshold indicators The rats were placed on a glass plate in a plexiglass box. After 30 minutes, the surface of the hind limb of the rat's surgical side was irradiated with a thermal radiation stimulator, and the time of the rat's response to the thermal stimulation was recorded. This time was defined as the tolerance of the rat to the thermal stimulus. Used to measure pain sensitivity indicators for thermal stimulation.

自 CCI手术当天起连续 5天内，大鼠内腹腔注射 0、 20、 30 mg/kg甲氟喹，在所观察的长期时间范围（21天）内，分别在术后 7、 4天鉴定大鼠的疼痛阈值，检测结果如图 2所示，甲氟喹均能够使模型鼠的热痛域值保持较高水平，即热痛阈值回升。从图 2可见，腹腔注射 30 mg/kg甲氟喹，热痛阈值回升效果最佳。实验表明： CC】手术后腹腔注射，热痛敏阈值回升，能有效预防神经病理性疼痛样症状的发生。 Rats were intraperitoneally injected with 0, 20, 30 mg/kg of mefloquine for 5 consecutive days from the day of CCI surgery. Rats were identified at 7 and 4 days after surgery in the long-term time range (21 days) observed. The pain threshold, the test results shown in Figure 2, mefloquine can maintain a high level of thermal pain in the model rats, that is, the thermal pain threshold rises. It can be seen from Figure 2 that the intraperitoneal injection of 30 mg/kg of mefloquine has the best effect on the rebound of thermal pain. Experiments show that: CC] intraperitoneal injection after surgery, the threshold of heat sensitivity is increased, which can effectively prevent the occurrence of neuropathic pain-like symptoms.

实施例 2: 甲氟喹对 CCI模型大鼠病理性疼痛样症状防治作用的有效给药时间窗口 Example 2: Effective administration time window of mefloquine against the prevention and treatment of pathological pain-like symptoms in CCI model rats

将初始给药时间分别放在 CCI手术的第≡天或第四天开始，其余步骤与实施例 1相似，即每天一次、连续五天给药；采^较佳的给药剂量 30mg/kg甲氟喹；在 CCI手术后当天、第 14天和第 2 天分别检测大鼠的机械痛域值、热痛阈值。 The initial administration time is respectively started on the third or fourth day of the CCI operation, and the remaining steps are similar to those in the first embodiment, that is, once a day for five consecutive days; the preferred administration dose is 30 mg/kg A. Fluoroquine; The mechanical pain field value and thermal pain threshold of the rats were measured on the day after CCI surgery, on the 14th day and the 2nd day.

实验结果如图 3、图 4所示， CCI手术第三天开始给药的，大鼠的机械痛域值、热痛阈值有显著回升。而术后第四天起给药，大鼠的机械痛域值、热痛阈值无显著回。实验结果表明， CCI手术后，给药时间越晚，药效越弱；术后两天之内给药的效果较佳。 The results of the experiment are shown in Fig. 3 and Fig. 4. When the CCI operation was started on the third day, the mechanical pain field value and thermal pain threshold of the rats significantly increased. On the fourth day after surgery, there was no significant change in the mechanical pain field and thermal pain threshold of the rats. The experimental results show that after CCI surgery, the later the administration time, the weaker the drug effect; the effect of administration within two days after surgery is better.

实施例 3: 前扣带皮层微量导入 ¥氟喹对 CCI模型大鼠病理性疼痛样症状发生的防治 Example 3: Microintroduction of anterior cingulate cortex: The prevention and treatment of pathological pain-like symptoms in rats with CCI model

为鉴定腹腔注射甲氟喹的疾病防治作用是否发生在大脑前扣带皮层，本实验采用局部药物导入技术，将甲氟喹定点注入前扣带皮层，并测定机械痛及热痛阈值。首先将大鼠用戊巴比妥钠麻醉、头部固定于立体定位仪，暴露颅骨并在前扣带皮层上方实施开颅手术。根据大鼠脑图谱的前扣带皮层坐标定位，将一有活动内芯的不锈钢套管埋入前扣带皮层区域，牙科水泥固定。术后当天及其后 4天，每天导入微量 ¥氟喹（给药组）或同等体积溶剂（对照组）。术后 3、 7、 21天，测定机械痛及热痛阈值。导入微量 ^氟喹的用量为 100mM。 In order to identify whether the prevention and treatment of intraperitoneal injection of mefloquine occurs in the anterior cingulate cortex of the brain, this experiment uses a local drug introduction technique to inject a mefloquine into the anterior cingulate cortex and determine the mechanical pain and thermal pain threshold. The rats were first anesthetized with sodium pentobarbital, the head was fixed to a stereotaxic instrument, the skull was exposed and craniotomy was performed above the anterior cingulate cortex. According to the coordinates of the anterior cingulate cortex of the rat brain map, a stainless steel cannula with a movable inner core is embedded in the cortex of the anterior cingulate and fixed by dental cement. On the day after surgery and 4 days after the operation, a small amount of fluoroquine (administered group) or an equivalent volume of solvent (control group) was introduced every day. Mechanical pain and thermal pain threshold were measured 3, 7, and 21 days after surgery. The amount of trace fluoroquine introduced was 100 mM.

实验结果如图 5、图 6所示：前扣带皮层徵量导入甲氟喹，在术后 3、 7、 21天所检测到的机械痛阈值显著回升、热痛阈值显著回升。此外，本实施例中的阈值回升效果与实施例 1 中腹腔注射产生的阈值回升效果相当，见图 1、图 2。实验表明：腹腔注射 Ψ氟喹产生的药效是通过阻断前扣带皮层 Cx36 蛋白起作用的，前扣带皮层徵量甲氟喹能有效防治神经病理性疼痛样症状的发生。 The results of the experiment are shown in Fig. 5 and Fig. 6. The anterior cingulate cortex was introduced into the form of mefloquine. The mechanical pain threshold detected on the 3, 7 and 21 days after surgery was significantly increased, and the thermal pain threshold was significantly increased. Further, the threshold recovery effect in the present embodiment is equivalent to the threshold recovery effect produced by the intraperitoneal injection in the embodiment 1, as shown in Figs. 1 and 2. Experiments have shown that the effect of intraperitoneal injection of fluvoquine is through blocking the anterior cingulate cortex Cx36 protein. The anterior cingulate cortex cerebral granule can effectively prevent neuropathic pain-like symptoms.

实施例 4: 前扣带皮层局部千扰 Cx36表达对药物脱靶可能性的鉴定 Example 4: Local scrambling of the anterior cingulate cortex Cx36 expression identifies the possibility of drug off-target

为进一歩确认甲氟喹的药效是通过阻断前 ¾带皮层 Cx36 蛋白的活动、而非药物脱靶引起。本实验采用 RNA干扰技术，下调 Cx36蛋白，根据 Cx36的基因序列设计千扰序列（序列见图 7)，均包装到质粒中，并构建慢病毒转载体系 ShRNA- Cx36- GFP。参考文献: Pios One, 201 3 , 8( 1) , e55198 > To further confirm the efficacy of mefloquine is caused by blocking the activity of the former 3⁄4 cortical Cx36 protein, rather than drug off-target. In this experiment, RNA interference technology was used to down-regulate Cx36 protein, and the scrambling sequence was designed based on the gene sequence of Cx36. Columns are shown in Figure 7), packaged into plasmids, and the lentiviral vector shRNA-Cx36-GFP was constructed. References: Pios One, 201 3 , 8( 1) , e55198 >

将携带千扰序列的病毒注入前扣带皮层。一周后，处死大鼠并提取相关脑区分别进行蛋白免疫印迹鉴定和荧光免疫组化鉴定，结果分别见图 8、图 9所示，均检测到 Cx36表达的显著下调，实验表明前扣带皮层 Cx36蛋白表达量被有效下调。 The virus carrying the interference sequence is injected into the anterior cingulate cortex. One week later, the rats were sacrificed and the relevant brain regions were extracted for Western blotting and fluorescent immunohistochemistry. The results are shown in Figure 8 and Figure 9, respectively. Significant down-regulation of Cx36 expression was detected. The experiment showed that the anterior cingulate cortex The amount of Cx36 protein expression was effectively down-regulated.

在术后 3、 7、 2 天分别测定接受了病毒转入的大鼠的机械痛及热痛阈值，实验结果分别见图〗 0、图 1 1 , 接受病毒转入的大鼠在术后 3、 7、 21天所表现的机械痛阈值显著回升、热痛阈值显著回升。并且，本实施例中的阈值回升效果与实施例 1中腹腔注射产生的阈值回升效果相当。实验表明，甲氟喹的药效通过作用于前扣带皮层的 Cx36 而发生作用。前扣带皮层内的 Cx36表达下调，可有效防治神经病理性疼痛样症状的发生。 The mechanical pain and thermal pain threshold of the rats that received the virus transfer were measured on the 3rd, 7th, and 2nd day after surgery. The experimental results are shown in Fig. 0, Fig. 1 1 , and the rats receiving the virus transfer are in the postoperative 3 The mechanical pain thresholds exhibited in 7, 7 and 21 days significantly increased, and the thermal pain threshold significantly increased. Further, the threshold recovery effect in the present embodiment is equivalent to the threshold recovery effect produced by the intraperitoneal injection in the first embodiment. Experiments have shown that the efficacy of mefloquine works by acting on Cx36 in the anterior cingulate cortex. The expression of Cx36 in the anterior cingulate cortex is down-regulated, which can effectively prevent the occurrence of neuropathic pain-like symptoms.

实施例 5: 腹腔注射甲氟喹对 SNI模型大鼠病理性疼痛样症状发生的防治 Example 5: Prevention and treatment of pathological pain-like symptoms in rats with SNI model by intraperitoneal injection of mefloquine

实验针对另一类常用的神经病理性疼痛模型鼠一 SNI 模型，进一歩检验甲氟喹对神经病理性疼痛发生的防治作用。参考 Decosterd和 wooif 报道的方法 ( Pain, 2000, 87, 149- 158. ) ：切开一侧后肢皮肤并分离肌肉，暴露坐骨神经主干及其下的分支即胫神经、腓总神经和勝肠神经；结扎并剪断胫神经和勝总神经，保留细小的離肠神经；将肌肉、皮肤缝合。采用 30mg/k¾ 剂量，在 SNI手术的当天及其后 4天、每天一次腹腔注射甲氟喹；同等体积溶剂注射作为对照组。在术后 3、 7、 21天，测定机械痛阈值。 The experiment aimed at another commonly used neuropathic pain model mouse-SNI model, and further examined the preventive and therapeutic effects of mefloquine on neuropathic pain. Refer to the method reported by Decosterd and wooif (Pain, 2000, 87, 149- 158.): Cut one side of the hind limb skin and separate the muscle, expose the main sciatic nerve and its branches, namely the phrenic nerve, the common peroneal nerve and the venous nerve; Ligation and cutting of the phrenic nerve and the total nerve, retaining the small off-intestinal nerve; suturing the muscles and skin. At the dose of 30 mg/k3⁄4, intraperitoneal injection of mefloquine was given once a day on the day of SNI surgery and 4 days after; the same volume of solvent injection was used as the control group. The mechanical pain threshold was measured at 3, 7, and 21 days after surgery.

实验结果如图 12所示： SNI手术后短期（4天）内腹腔注射 3() _mg/kg ^氟喹，在所观察的长期间范围（21天）内，均能够使模型鼠的机械痛域值保持较高水平，即机械痛阈值回升。实验结果表明：甲氟喹能有效防治 SNI模型鼠神经病理性疼痛样症状的发生。实施例 6: 相关药物毒理检测 The results of the experiment are shown in Figure 12: short-term (4 days) SNI intraperitoneal injection of 3 () _mg / kg ^ fluoroquine, in the long-term range (21 days) observed, can cause mechanical pain in the model rats The domain value remains at a high level, ie the mechanical pain threshold rises. The experimental results show that: mefloquine can effectively prevent the occurrence of neuropathic pain-like symptoms in SNI model rats. Example 6: Related drug toxicology test

给予正常鼠腹腔注射甲氟喹（30mg/kg) , 在当天对药物毒性作用作鉴定。 Normal mice were given intraperitoneal injection of mefloquine (30 mg/kg), and the toxic effects of the drug were identified on the same day.

采用开场实验测定动物基础运动量、焦虑水平等是否发生变化，实验结果见图 13 : 将单只大鼠放入四周壁透明、底部灰色的方形敞口观察箱中心，采用追踪分析***通过红外装置记录并分析大鼠的水平穿格次数。记录动物行为共计 15 mm; 分析运动距离。 The opening experiment was used to determine whether the animal's basic exercise volume, anxiety level, etc. changed. The experimental results are shown in Figure 13. A single rat was placed in the center of the transparent open, bottom gray square open observation box, and recorded by an infrared device using a tracking analysis system. The number of horizontal crossings of the rats was analyzed. Record animal behavior totaling 15 mm; analyze exercise distance.

采 ^转棒实验测定动物运动及平衡能力，实验结果见图 14 : 对大鼠进行三.天运动训练：第-一天, 将大鼠放置在转棒上，转棒以每 5转 /分钟转速开始匀速转动，训练 5分钟 /次、共 2 次；第二天，转速在 5分钟内从 5转 /分至】 5转 /分逐歩增加，训练 5分钟 /次、共 2次；第三天，转速在 5分钟内从 8转 /分一 15转 /分逐步增加，每只大鼠训练 2次训练 5分钟 /次、共 2 次。第四天（测试、该天给药）：转速在 3分钟内由 8转 Z分增加至 25转 /分，记录大鼠留在转棒上而不掉落^间；反复 3次测量。 The experiment was carried out by measuring the movement and balance of the animals. The experimental results are shown in Figure 14. Three-day exercise training was performed on the rats: On the first day, the rats were placed on a rotating rod, and the rotating rod was rotated at 5 rpm. Start to rotate at a constant speed, training for 5 minutes / time, a total of 2 times; the next day, the speed is increased from 5 rpm to 5 rpm in 5 minutes, training 5 minutes / time, a total of 2 times; In days, the speed is gradually increased from 8 rpm to 15 rpm in 5 minutes. Each rat is trained 2 times for 5 minutes/time, total 2 Times. Day 4 (test, dosing on the day): The rotational speed was increased from 8 to Z in 3 minutes to 25 rpm, and the rats were recorded on the rotating rod without dropping; the measurement was repeated 3 times.

实验结果表明：与未接受甲氟喹注射大鼠相比较，接受甲氟喹（30mg/kg)注射的大鼠在开场实验及转棒实验的行为指标未见异常，表明 Ψ氟喹对大鼠运动能力、平衡能力及焦虑状态无明显影响，表明无毒副作用。 The experimental results showed that: compared with the rats that did not receive mefloquine injection, the behavioral indicators of the rats in the start-up experiment and the rotating rod test were not abnormal in the rats receiving mefloquine (30 mg/kg), indicating that the fluoroquine was administered to rats. There was no significant effect on exercise capacity, balance ability and anxiety status, indicating no toxic side effects.

上述实施例只是举例说明本发明技术构思及特点，便于本领域技术人员能够了解并实施本发明，但并不能以此限制本发明的保护范围。凡是根据本发明实质所作出的等效的变化或修饰，都应涵盖在本发明保护范围内。 The above embodiments are merely illustrative of the technical concept and the features of the present invention, and the present invention can be understood and implemented by those skilled in the art, but the scope of the present invention is not limited thereto. Equivalent variations or modifications made in accordance with the spirit of the invention are intended to be included within the scope of the invention.

Claims

fx. 利要豕 Fx.

1. ¥氟喹在制备预防神经病理性疼痛药物中的应用。 1. The application of fluoroquine in the preparation of drugs for preventing neuropathic pain.

2. ¥氟喹作为活性成分在制备预防神经病理性疼痛的药物中的应用 2. The application of fluoroquine as an active ingredient in the preparation of drugs for preventing neuropathic pain

3. 一种药物组合物，应用于预防神经病理性疼痛的药物中，其特征在于，所述组合物包括预防有效量的甲氟喹和药学上接受载体。 A pharmaceutical composition for use in a medicament for preventing neuropathic pain, characterized in that the composition comprises a prophylactically effective amount of mefloquine and a pharmaceutically acceptable carrier.