【作者】 李智勇；

【导师】 涂瑶生；

【作者基本信息】 广州中医药大学 ， 中药学， 2010， 博士

【摘要】 癌性疼痛(简称癌痛)是由癌症本身以及癌症治疗过程中产生的疼痛。目前癌症已超过了艾滋病、结核、疟疾致死病例的总和,成为人类第一位死因。癌性疼痛的众多临床表现中,疼痛是癌症病人面临的最重要的问题之一,然而由于抗癌痛药物的毒副作用、昂贵的治疗成本等问题,造成临床中癌痛治疗严重不足。癌痛膏为广东省第二中医院临床实践应用于治疗各种恶性肿瘤所致疼痛的经验处方,具有温经通络,消瘤止痛之功,主治各种恶性肿瘤所致的疼痛。在临床使用十余年,病例数达上千例,临床疗效确切,没有出现严重的毒副反应。本研究针对癌痛的临床特点和现有癌痛治疗方面存在的不足,在传统中医“内病外治”的理论指导下,将癌痛的治疗特点和巴布剂的给药特性相结合,采用现代制剂技术将其开发为疗效独特、使用方便、毒副作用小、无创伤给药的中药复方经皮给药制剂----癌痛巴布剂。本论文主要从提取工艺,巴布剂基质筛选、促渗剂的筛选与优化、制备工艺、质量标准、主要药效学和药理毒理安全性方面进行研究。1.提取工艺的研究根据癌痛巴布剂各味药材的功能主治、现代化学成分、药理及临床的研究状况,在中药医药理论的指导下,结合各味药物含有脂溶性或水溶性有效成分不同,采用一定浓度的乙醇或水提取、分离其主要有效部位。1.1肉桂、生姜挥发油的提取：以挥发油的提取量为指标,采用单因素考察法对药材的粉碎度、提取时间等主要影响水蒸汽蒸馏法提取挥发油的影响因素进行考察,结果显示,将肉桂、生姜粉碎成粗粒,采用水蒸汽蒸馏法提取5h,即可达总收油量的97%以上,基本提取完全。1.2赤芍、红花和肉桂与生姜提油后药渣水提工艺研究：以加水量、提取时间、提取次数等为主要考察因素,以芍药苷、羟基红花黄色素A等的提取率为评价指标,采用三因素三水平的正交设计优选的最佳水提工艺,优选的最佳提取工艺为：每次加入8倍量水,煎煮3次,每次1.5小时。1.3草乌、生南星、白芷、乳香中的主要有性成分多为脂溶性成分,因此分别以草乌中的主要有效成分乌头类生物总碱、白芷中的主要有效成分之一欧前胡素的提取率以及出膏率作为评价指标,考察草乌、生南星、白芷、乳香的最佳提取醇浓度、提取时间,并采用正交设计法对醇取提工艺进行优化,优选的最佳醇提工艺为每次加入10倍量60%的乙醇,煎煮2次,每次1小时。2.癌痛巴布剂的基质处方筛选研究中药巴布剂主要存在黏性差,内聚力不足,反复揭扯性能差等问题,以膏体外观性状,气泡多少,凝聚时间,表面粘性为评价指标,采用单因素考察进行癌痛巴布剂基质的初步筛选,确定了以聚丙烯酸钠、甘油、聚乙烯醇、明胶、高岭土、甘羟铝、柠檬酸、蓖麻油为癌痛巴布剂的基质组成。并以初黏力、剥离强度和外观综合评分为指标,采用U17(1716)均匀设计进行拟水平试验,对癌痛巴布剂的基质处方进行优选研究,实验设计结果的二次多项式逐步回归方程显示,癌痛巴布剂基质的最优配比组成为聚丙烯酸钠：明胶：高岭土：甘羟铝：蓖麻油：甘油：聚乙烯醇(6.46：6.0：4.1：0.08：0.1：56：0.05)。验证结果显示,该配比制成的癌痛巴布剂粘度适宜、剥离性好、外观平整。3.癌痛巴布剂的促渗剂筛选研究经皮给药系统经皮给药的最大障碍是皮肤对药物渗透的屏障作用,因此对于经皮给药制剂的研究,选择理想的透皮吸收促进剂以促进足够量的药物进入人体内从而起到治疗作用,具有重要意义。3.1癌痛巴布剂体外经皮渗透试验方法的建立通过考察不同接收介质、不同动物离体皮肤、离体皮肤储存时间等对癌痛巴布剂中芍药苷的体外经皮渗透行为,建立了癌痛巴布剂中芍药苷体外经皮渗透吸收的实验方法：采用脱毛的小鼠腹部皮肤为研究对象,接收介质选用生理盐水,试验温度32℃,磁搅拌子旋转速度为200r／min。3.2不同透皮促渗剂对癌痛巴布剂体外透皮吸收的影响研究以目前最常用的氮酮、油酸、丙二醇、薄荷脑、冰片等透皮促渗剂为研究对象,采用体外透皮吸收法分别考察了0%,1%,2%,3%,5%氮酮；0%,1%,2%,3%,5%,7.5%,10%油酸；0%,5%,6%,7.5%,8%,10%丙二醇；0%,0.5%,0.75%,1%,1.5%,1.75%,2%薄荷脑；0%,0.5%,0.75%,1%,1.5%,1.75%,2%冰片对癌痛巴布剂中芍药苷的透皮吸收的影响,并计算Qn、Q、J、ER和TLAG,并绘制渗透曲线,结果显示除油酸加入(1%)即引起癌痛巴布剂出现漏膏现象,不适宜用作癌痛巴布剂的促渗剂外,氮酮(0%～3%)、丙二醇(5%～8%)、薄荷脑(0.5%～1.5%)、冰片(0.75%～1.5%)在一定浓度范围内对癌痛巴布剂中芍药苷具有不同程度的促渗透作用,并对癌痛巴布剂的外观性状不造成严重影响。3.3癌痛巴布剂多元渗透剂的均匀设计法优选研究透皮吸收促进剂单独使用效果不理想,常联合使用由亲水性分子和亲油性分子构成两元、三元甚至多元复合透皮吸收促渗系统。根据不同浓度氮酮、丙二醇、薄荷脑、冰片的单因素考察结果,以外观综合评分、初黏力、持粘力、芍药苷的透皮吸收速率常数(J)等为评价指标,采用U7(74)均匀设计法优化筛选癌痛巴布剂的多元复合透皮吸收促渗剂。结果最优的癌痛巴布剂多元复合促渗剂的配比组成为：氮酮(1.54%)-丙二醇(7.53%)-薄荷脑(1.26%)-冰片(1.50%),验证试验结果显示,优选的多元复合促渗剂对癌痛巴布剂的促进吸收作用优于各单种促渗剂。4.癌痛巴布剂成型工艺研究在癌痛巴布剂的提取工艺、基质配方以及促渗剂配方确定的情况下,考察浸膏含水量、以及促渗剂同时加入巴布基质后对巴布基质的强度、黏性与成胶时间等性能的影响,并对处方基质适宜载药量、药物基质的添加顺序、搅拌速度和时间、除泡方法、涂布方法、涂布时间、涂布厚度、癌痛巴布剂的干燥方法和温度进行考察,确定癌痛巴布剂的成型工艺为：取处方量的聚乙烯醇,加入150倍(g／ml)蒸馏水,记录重量,置90℃水浴中加热溶解(需要补少量水),放凉,补重,加入明胶,静置溶胀,置50℃水浴中溶解,加入高岭土,50℃水浴中搅拌均匀,得A相；取聚丙烯酸钠,加入少量甘油分散混匀,加入蒸馏水适量,快速搅拌均匀,静置,使充分溶胀,搅拌均匀待用,得B相；取少量蒸馏水溶解柠檬酸,与甘羟铝和剩余甘油混匀,加入肉桂、生姜挥发油、蓖麻油,促渗剂等,混匀,再加入浸膏,搅拌均匀,得C相；在50℃水浴下A相加入B相,搅拌均匀,再加入C相中,搅拌均匀；把调制好的药料置涂布机上,调节好涂布规格,涂布成型、室温晾干,裁成规格为8.5cm×12cm的片材,质检,包装,即得。5.癌痛巴布剂的质量标准研究根据《中国药典》2005年版一部附录贴膏剂项下巴布剂的制剂通则和要求,制定了癌痛巴布剂的质量标准。采用薄层色谱法对该制剂中的肉桂、白芷、赤芍、红花、乳香和生姜等进行了定性鉴别,实验结果表明,鉴别方法简便、可靠、专属性强、阴性无干扰；对含膏量、赋形性、粘附性、重量差异和微生物限度等检查项目进行了研究,规定每100cm2癌痛巴布剂含膏量应不少于9.8g；粘附性照巴布膏剂粘着力试验测定法(附录ⅫE第一法)取7号钢球,测定,应符合规定；照粘着力试验测定法(附录ⅫE第二法)持黏力测试,巴布剂滑落至脱落的时间应大于40s；并采用高效液相色谱法对癌痛巴布剂中的乌头总碱、芍药苷、羟基红花黄色素A、欧前胡素等进行含量测定研究,根据实验研究结果,规定本品每片含草乌以乌头碱(C3H47NO11)、次乌头原碱(C33H45NO10)和新乌头碱(C33H45NO11)的总量计,应为3mg～15mg；含赤芍以芍药苷(C23H28O11)计不得少于18.5mg；含白芷以欧前胡素(C16H14O4)计不得少于1.2mg；含红花以羟基红花黄色素A(C27H30O15)计不得少于7.4mg。从而为控制癌痛巴布剂的内在质量提供可靠依据。6.癌痛巴布剂的稳定性试验研究按照拟定的质量标准草案对三批癌痛巴布剂用纯铝袋包装下,以癌痛巴布剂的外观性状,鉴别、含膏量、黏附性(初黏力以钢球号计、持黏力以时间s计)、微生物检查以及癌痛巴布剂中乌头总碱、芍药苷、欧前胡素、羟基红花黄色素A的含量测定等为考核指标,进行了6个月的加速稳定性试验和长期稳定性试验,初步考察癌痛巴布剂的稳定性。结果表明癌痛巴布剂在样品包装条件下,三批样品在温度40℃±2℃、相对湿度75%±5%的条件下,和在常温条件下,考察6个月,外观无变化,鉴别均呈正反应,检查、含膏量、黏附性、含量测定合格,微生物检查检查符合规定。说明样品6个月内基本稳定。7.癌痛巴布剂和癌痛膏的体外透皮对比实验研究采用体外透皮吸收法对比了癌痛膏和癌痛巴布剂体外离体透皮情况,结果显示,与癌痛膏相比,癌痛巴布剂中芍药苷的体外透皮吸收24h后的累计透过百分率具有显著性提高,达到62.38%±1.84%,相比癌痛膏30.74%±1.50%提高2.03倍,透皮吸收常数达(7.64±0.53)μg·cm-2·h-1,是癌痛膏的透皮吸收常数(2.83±0.17)μg·cm-2·h-1的2.70倍,而平均透皮滞后时间0.75h与癌痛膏的平均透皮滞后时间0.27h相比增加2.78倍。证明癌痛巴布剂无论是在累计透皮吸收百分率、透皮速率常数均优于癌痛膏,其中芍药苷的体外透皮吸收较癌痛膏具有较大的提高,而透皮迟滞时间有所延长。8.癌痛巴布剂的药效学研究和安全性实验研究8.1采用冰醋酸诱发小鼠扭体反应法,以麝香追风膏,吡罗昔康贴片为阳性对照,以巴布剂空白基质为空白对照组,并设立原剂型癌痛膏组,观察癌痛巴布剂冰醋酸诱发小鼠扭体反应的抑制作用,结果显示癌痛巴布剂高、中、低剂量组对冰醋酸诱发小鼠扭体反应均有一定抑制作用。癌痛巴布剂高、中剂量组的镇痛作用明显优于原癌痛膏和麝香追风膏贴,提示癌痛巴布剂的镇痛作用存在一定的量效关系。8.2以新西兰兔为实验对象,观察其对皮肤刺激性反应的作用,结果显示,在实验过程中,未发现实验动物的活动、饮食、粪便等有异常情况,眼、鼻等亦未发现异常分泌物；涂药部位未发现色素沉着,未见出血点等异常情况。未发现癌痛巴布剂对新西兰兔皮肤有明显的刺激性反应。8.3以豚鼠为实验对象,观察癌痛巴布剂的过敏性作用,结果显示在试验过程中,未发现实验动物有哮喘、站立不稳或者休克等全身性过敏性反应。未发现癌痛巴布剂对豚鼠皮肤有明显的致敏性作用。8.4以新西兰兔为实验对象,观察癌痛巴布剂对新西兰兔的急性毒性作用,结果显示在试验过程中,未发现实验动物毛发、眼和粘膜有异常变化,亦未发现实验动物的呼吸、中枢神经系统和四肢活动有异常表现。两周内,实验动物全部成活并未发现癌痛巴布剂对新西兰兔皮肤有明显的急性毒性作用。更多还原

【Abstract】 Cancer pain is caused by the cancer itself or generated during the treatment of cancer. Currently, cancer has become the first cause of death, and the number of fatal cases caused by it has exceeded the sum of the ones caused by AIDS, tuberculosis, malaria. Cancer pain is one of the most important issues the cancer patients are facing in the cancer clinical manifestations. But because of the problems such as the side effects of the anti-cancer pain drug, the expensive treatment costs, etc. they resulted in a serious shortage of clinical treatment of cancer pain. Aitong Paste is an experienced prescription of the treatment of the pain caused by various malignant tumors in the clinical practice, in the second Chinese Medicine Hospital of Guangdong Province. Aitong Paste is of the effect of warming the meridians, eliminating the tumor, releasing the pain, and is mainly used for the cure of the pain caused by a variety of malignancies. Aitong Paste has been in clinical use for many years. The number of clinical curative effect cases is over one thousand. It has good clinical efficacy, and no serious side effects have been found. Aiming at the clinical characteristics of the cancer pain and the existing shortcomings in the therapy of the cancer pain, and under the guidance by the theory of "internal diseases treated with external therapy" in traditional Chinese Medicine, the study compounds the treatment characteristics of the cancer pain and the transdermal drug delivery formulations and exploits it with the the modern technique of drug for Chinese herbal compound transdermal drug delivery formulations—Aitong cataplasm with the efficacy of a unique, easy to use, less toxicity, and non-invasive administration. In this thesis, the extraction process, matrix optimization, penetration enhancers screening and optimization, manufacturing process, quality standards, stability, the main pharmacodynamic and safety pharmacology and toxicology are also studied for Aitong cataplasm.1. Research on the Extraction Process of Aitong cataplasmAccording to modern chemical composition, pharmacological and clinical research of all Chinese herbal medicine in Aitong cataplasm, it decided to extract and separate of the main active site with a certain concentration of ethanol or water1.1 The results of Cinnamon, ginger essential oil extracted optimize:cinnamon and ginger are crushed into coarse powder, and is extracted by steam distillation for 5h. It can reach up to 97% of the total oil 1.2 Study of water extraction process of Paeoniae, Safflower, and Cinnamon and Ginger residue after oil extractionThe amount of water added, extraction time and extraction times were investigated as the main factors. the extraction percentage of Paeoniflorin and HSYA assessment were used as evaluating indicator, three factors and three level orthogonal design were used to optimize the best water extraction process, and the optimum extraction conditions were:8 times per volume of water added, boiled 3 times and 1.5h each time.1.3 Study of ethanol extraction process of Aconitum, Arisaematis, Dahuricae and FrankincenseThe main pharmacological components of Aconitum, Arisaematis, Dahuricae, Frankincense are mostly fat-soluble components, so the extraction percentage of Alkaloids Aconitine and Imperatorin were used as evaluating indicator, and investigated the best extract alcohol concentration and extraction time. Also the orthogonal design method was used to optimize the extraction alcohol process, the best alcohol selection extraction process was to add 10 times 60% ethanol, boiled 2 times and 1h each time.2. Study on screening and optimizing the matrix prescription of Aitong CataplasmAppearance, bubble number, pool time and the surface viscosity as the index for evaluation, single factor was used for screening matrix and determined:sodium polyacrylate, glycerin, polyvinyl alcohol, gelatin, kaolin, Aluminum glycinate, citric acid, castor oil for the matrix composition of Aitong Cataplasm, the initial viscosity, peel strength and appearance of the comprehensive score as the index, Uniform design U17(1716) was used to optimize the matrix formulation of Aitong Cataplasm, and the results of quadratic polynomial stepwise regression equation of the optimization the experimental design shows that the matrix of Aitong cataplasm composed of the optimal ratio of matrix was:Sodium polyacrylate:Gelatin:Kaolin:Aluminium glycinate:Castor oil:Glycerol: Polyvinyl alcohol (6.46:6.0:4.1:0.08:0.1:56:0.05). The results showed that the Aitong Cataplasm prepared in the optimum conditions has a good scalability, smooth appearance, moderate viscosity, good moisture retention and good compatibility with Aitong Cataplasm powder.3. Study on Optimize penetration enhancers of Aitong Cataplasm3.1 Established the in-vitro percutaneous penetration test method of Aitong Cataplasm.By examining the influence of the different receiving medium, the skin of different animals, magnetic stir speed, test temperature on in vitro percutaneous permeation behavior of the paeoniflorin in the Aitong cataplasm, establish experimental methods of Paeoniflorin in vitro percutaneous absorption:the abdominal skin of mice hair removal is for the study object, saline is for receiving medium, test temperature is 32℃, and the magnetic stir bar rotation speed is 200r/min respectively. 3.2 Study on the influence of the different transdermal penetration enhancers on transdermal absorption of Aitong CataplasmThe most commonly used Azone, Oleic acid, Propylene glycol, Menthol, Borneol etc. permeation enhancers were used as the research object, respectively, in vitro percutaneous absorption, study the influence of 0%,1%,2%,3%,5%Azone; 0%,1%,2%,3%,5%, 7.5%,10%oleic acid; 0%,5%,6%,7.5%,8%,10%propylene glycol; 0%,0.5%,0.75%, 1%,1.5%,1.75%,2% menthol; 0%,0.5%,0.75%,1%,1.5%,1.75%,2% of borneol on the Paeoniflorin dose transdermal absorption of Aitong Cataplasm, calculating Qn, Q, J, ER and TLAG, and drew the penetration curve. The results show that adding oleic acid (1%) causes cause Aitong Cataplasm paste leakage phenomenon, and it is not fit to enhance paeoniflorin penetration of Aitong Cataplasm; besides, the Azone (0%～3%), propylene glycol (5%～8%), Menthol (0.5%～1.5%), Borneol (0.75%～1.5%) in a certain range of concentration with different levels enhancement of Paeoniflorin penetration effect, and cannot cause serious change of the appearance characteristics of Aitong Cataplasm3.3 Optimize the multiple penetration enhancers of Aitong Cataplasm with the Uniform Design MethodTransdermal absorption enhancers alone are not effective, often combined elements from the hydrophilic and hydrophobic molecules or two or three or even multiple composite transdermal penetration system. Depending on the single factor results of the concentration of Azone, propylene glycol, menthol, borneol, the appearance of an integrated score, initial adhesion, continued adhesion, Paeoniflorin transdermal absorption rate constants (J) are used as evaluating indicators, and uniform design U7 (74) was used to optimize the prescription of multiple penetration enhancers. The results showed that optimal composite ratio of multiple penetration enhancers of Aitong cataplasm was:Azone (1.54%)-Propylene glycol (7.53%)-Menthol (1.26%)-Borneol (1.50%), and the validation test results showed that the multi-compound penetration enhancers on the promotion of Aitong Cataplasm absorption is better than the single species of penetration enhancers.4. Study on Molding Process of Aitong CataplasmAfter the extraction process, matrix formulation and the penetration enhancers formula had been determined, study on the extract moisture and the influence of the matrix strength, viscosity and gelling time performance caused by adding the penetration enhancers into matrix etc. In the meantime study on appropriate prescription drug matrix can loading, the addition of drug substrates in order, stirring speed and time, the method of reduce bubble number, coating method, coating time, coating thickness, drying temperature and method of Aitong Cataplasm. Cataplasm molding process are:bring the amount of polyvinyl alcohol of prescription, adding 150-fold (g/ml) distilled water, record the weight and solution it in heating 90℃water bath (with a small amount of added water), let cool, and make up weight, adding gelatin, standing swelling, dissolved in 50℃water bath set to include kaolin,50℃water bath and stir well, get A-phase; bring sodium polyacrylate, mix in a small amount of glycerol and mix by adding appropriate amount of distilled water, stir quickly, standing to full swelling, mix well, get B-phase; take a small amount of citric acid dissolved in distilled water, Aluminium Glycinate and residual glycerol and mix, adding cinnamon, ginger essential oil, castor oil, penetration enhancers, then add extract, mix well, get C-phase; add the A-phase into B-phase under 50℃water bath,, mix well, then add C-phase, mix well; the modulation Paste coating with machine and regulate the coating specifications, coating molding, dry and cut specification 8.5cm×12cm of sheet material, quality control, packaging.5. Study on the Quality Standard of Aitong CataplasmThe quality standard of Aitong Cataplasm was developed according to the common requirement of patch described in the appendix of Chinese Pharmacopoeia (Part One) (Edition 2005) and specific character of cataplasm. TLC was used to identify the Aconitum, Dahuricae, Cinnamon, Paeoniae, Safflower, Frankincense, Ginger, etc the qualitative in Aitong cataplasm. Results of experiments show that the identification methods that can be identifiable, reliable, specific, negative control without interference; it should contain the amount of ointment not less than 9.8g per 100cm2, Adhesion test according to adhesion assay (Appendix XII E 1st law) to take on the 7th ball, shall comply with provisions; according to adhesion test assay (Appendix XII E Second Law), cataplasm slipped to the time off should be more than 40 seconds; and study on shaping, weight variation, microbial limit qualification; HPLC were used to determine the content of the Aconitum alkaloids, Paeoniflorin, HSYA A, Imperatorin. According to the experimental results, the provisions of each piece, containing Aconitum calculated by the total amount of aconitine (C34H47NO11), hypaconitine (C33H45NO10) and mesaconitine (C33H45NO11) should be 3mg-15mg; containing Paeoniae calculated by Paeoniflorin (C23H2gO11) should not be less than 18.5mg; containing Dahuricae calculated by imperatorin (C16H14O4) should not be less than 1.2mg; containing Safflower calculated by HSYA A (C27H30O15) should not be less than 7.4mg.6. Investigate Stability of Aitong CataplasmThe three batches of Aitong Cataplasm are packaged with aluminum bag. In accordance with the quality standards draft, regard the appearance of character, identification, ointment content, initial Adhesion, Continued Adhesion, microbiological examination, Aconitum alkaloids, Paeoniflorin, Imperatorin, HSYA A determination of indicators as assessment, carried out accelerated stability test and long-term stability test for 6 months. The results showed that the Aitong Cataplasm is stable within 6 months.7. Study on comparative experiment in vitro transdermal between Aitong Paste and Aitong cataplasm.The results of experiments show that the accumulated percentage of Aitong Cataplasm paeoniflorin after 24h in vitro percutaneous absorption significantly increased, reaching to 62.38%. Compared to the 30.74% of Aitong Paste, it increased by 2.03 times; The transdermal absorption constant is up to 7.64μg·cm-2·h-1 and it is 2.70 times as large as the percutaneous absorption constant 2.83μg·cm-2·h-1; The average lag time was 0.75h, which is 2.78 times as large as 0.27h of Aitong Paste.8. Experimental Study of Pain Cataplasm Pharmacodynamics and safety8.1 In the experiments, Shexiang Zhuifeng Paste and piroxicam patch were used as positive control groups, matrix was used as blank control group, established the Aitong Paste group, and observed Aitong Cataplasm writhing induced by glacial acetic acid-induced inhibition of writhing response in mice. The results showed that the high, medium and low doses of Aitong cataplasm can reduced writhing response in mice writhing reaction to glacial acetic acid-induced; the analgesic effect of high dose group was better than Aitong paste and Shexiang Zhuifeng paste, and analgesic effect of low-dose group was slightly better than Aitong paste; it prompt that there is a certain relationship between pain analgesic effect and dose.8.2 Animal experiments showed that obvious skin irritation was not be found on New Zealand rabbits; obvious acute skin toxicityin was not be found on New Zealand rabbits; and skin significantly allergenic effect was not be found on guinea pig.更多还原