CN115227639A - 一种温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系、制备方法及应用 - Google Patents
一种温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系、制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系、制备方法及应用。将温敏性的甲壳素衍生物在低温下溶于水,在液体状态下加入局部麻醉镇痛药水溶液或者载有局部麻醉镇痛药的可降解高分子微球并在低温下混合均匀,将其注射到需要局部麻醉镇痛的部位可在体温下快速形成凝胶并缓释药物。该局部麻药缓释镇痛复合水凝胶体系的主要特征是制备简单,不使用有机溶剂,可缓慢释放局部麻醉镇痛药,解决了现有局麻药如盐酸布比卡因、罗哌卡因和利多卡因的释放过快作用时间短的问题;可以完全填充不规则的创面,具有良好的生物相容性和生物降解性,有利于长效局部麻醉镇痛抗炎止痒和促进创面愈合作用。
Description
技术领域
本发明属于药物制剂领域,涉及一种药物缓释体系及其制备方法和应用,更具体涉及一种甲壳素水凝胶载局部麻药缓释镇痛体系、制备方法及应用。
背景技术
疼痛是一种令人不快的感觉和情绪上的感受,伴有实质上的或潜在的组织损伤,它是一种主观感受,被认为是继呼吸、脉搏、体温、血压之后的人体第5大生命体征,是临床上最常见的症状之一。它有急性和慢性之分,急性疼痛发生于创伤或手术后,有自限性,当组织损伤恢复后即减轻,若不减轻即可发展为慢性疼痛。慢性疼痛指持续时间超过急性损伤或疾病的正常痊愈时间,长期复发的疼痛。某些长期的剧烈疼痛,如神经痛、癌痛、关节炎疼痛、腰背痛等,对病人不仅是一种难以忍受的折磨,还会引起生理功能紊乱。因此,镇痛是临床治疗的重要任务和难点之一,也是医学研究的一个重要课题。
目前术后镇痛主要包括全身性镇痛和局部镇痛两种。前者主要指口服或静脉注射镇痛药物,如阿片类药物和非甾体抗炎药,由于药物对多系统都有作用,有较多副作用,如便秘、恶心、呕吐等,更严重的可引起呼吸抑制甚至死亡。另外,长期应用阿片类药物可引起耐受、药物滥用及成瘾问题。局部镇痛主要在局部使用麻醉药(局麻药),局麻药可以通过阻断神经细胞膜上的钠和其他离子通道,阻断痛觉信号向中枢神经系统的传递,导致所有的神经下游的结构的电信号被阻断,从而达到术后镇痛的作用,局麻药作用针对性强,减少了全身的副作用,安全性更佳,同时局部镇痛操作简单,可在门诊完成,费用低廉,减轻患者负担。目前,局麻药已被用于外科手术,如开胸手术,剖腹术,剖宫产术,乳腺癌手术,美容乳房手术,肢体截肢等的术后镇痛,并在各种大型手术后的急慢性疼痛的控制和缓解中起到了重要作用。
然而现有的局麻镇痛药作用时间短,一般不超过数小时,而临床要求的镇痛时间一般为24小时,几天到十几天,这就通过加大用药剂量、重复多次给药、导管体内植入或者自控镇痛泵等技术延长镇痛效果。然而这些麻药的心脑副作用虽然发生率低,但一旦发生对病人生命安全是极大的威胁,导管植入或者自控镇痛泵不仅需要昂贵的设备和连续监护,使用后需要移除,而且易引起导管阻塞、破损、感染并发症等问题。缓释局麻药不仅能提高局麻药镇痛效果,还能减少用药频次和减少大剂量使用的不良反应,因此受到了越来越多的关注。目前有关局麻药长效缓释给药系统主要包括微球、脂质体、植入剂、可注射原位凝胶等。微球(microspheres)是球形或类球形的微小球状实体,微球粒径范围一般为1-500微米,小的可以是几纳米,大的可达800微米。微球比无规则的粉末颗粒具有更好的流动性。1994年法国学者Corre等报道了微球作为局麻药载体的研究,他们使用不同分子量的聚乳酸PLA制备了布比卡因缓释微球,体外释放可长达24小时,并发现增加低分子量PLA的用量会增加初始的药物暴释比例[Preparation and characterization of bupivacaine-loaded polylactide and polylactide-co-glycolide microspheres,InternationalJournal of Pharmaceutics,1994,107N1,41-49,DOI:10.1016/0378-5173(94)90300-X]。传统的微球载药制备方法主要有乳化分散-溶剂挥发法、喷雾干燥法及凝聚法等。乳化分散法常需要使用有机溶剂和表面活性剂,简单易操作,是实验室研究使用最多的方法,但有机溶剂和分散剂的残留问题,并且放大生产影响因素较多,工艺控制要求较高。喷雾干燥法过程简单、成本低廉,且易于规模化、连续化生产,但实验室研究使用有限,微球大小难以控制,微球易聚集。而凝聚法是指材料的混合液中通过外界物理化学因素的影响,如用带相反电荷、脱水、溶剂置换等措施使材料溶解度发生改变,而自溶液中析出。这些常用的成球载药方法制得的微球尺寸分布较宽,不少药物在微球表面沉积结晶,存在明显的高暴释现象。马光辉团队采用快速膜乳化新技术制备得到了粒径均一、包埋率高、低突释的载罗哌卡因聚乳酸-羟基乙酸共聚物(PLGA)微球[CN201810874175.3,载麻醉镇痛药缓释微球、其制备方法及其应用]。然而,微球的原位性较差,注射后可能沿着注射部位肌间隙扩散,另外,载药微球的初始突释问题,可能影响阻滞持续时间,甚至产生局部或全身毒性。
脂质体局麻药研究工作较多,其中脂质体局麻药Exparel已成为第一个用于临床的缓释局麻药,2011年被美国食品药品监督管理局(FDA)首次批准,应用于局部浸润麻醉,其在体内可达到72h的镇痛效果,2018年进一步被批准用于肌间沟录入的臂丛神经阻滞。但Exparel的一个弱点就是脂质体的不稳定性,药物易泄露,储存需要控制温度在2-8℃,运输必须通过冷链,另外所用的载体脂质体本身可能会引起一些肉芽肿炎症[The safety andtolerability evaluation of DepoFoam bupivacaine(bupivacaine extended-releaseliposome injection)administered by incision wound infiltration in rabbits anddogs,Expert Opin.Investig.Drugs 2011,20N10,1327-1341]。目前仅有少数几个国家在临床上使用Exparel,在中国还没有被批准这类脂质体局麻药上市。最近美国和加拿大学者在麻醉学期刊一篇系统性综述和荟萃分析文章,认为布比卡因脂质体外周神经阻滞镇痛效果并不优于布比卡因非脂质体[Perineural Liposomal Bupivacaine Is Not Superiorto Nonliposomal Bupivacaine for Peripheral Nerve Block Analgesia,Anesthesiology 2021,134,147-64]。
作为缓释局麻药的植入剂的研究比较有限,美国哈佛医学院Kohane课题组通过静电纺丝技术,将局部麻醉药布比卡因负载于聚乳酸-羟基乙酸PLGA手术缝合线中,在术后伤口缝合中使用,起到缓慢释放持续镇痛的效果,不过载局麻药手术缝合线的机械强度难以达到商品化缝合线的强度,所以在临床应用中存在着缝合线断裂的风险[Electrospundrug-eluting sutures for local anesthesia,JOURNAL OF CONTROLLED RELEASE161N3,903-909,DOI:10.1016/j.jconrel.2012.05.021]。马超课题组利用电纺含布比卡因的PLGA溶液制得载药无纺布,然后热压制成载药纳米膜,通过植入到大鼠皮下切口显示较长的的镇痛效果和体内安全性[Electrospun PLGA nanomembrane:A novel formulationof extended-release bupivacaine delivery reducing postoperative pain,2020-033AE-MATERIALS&DESIGN 193,108768,DOI:10.1016/j.matdes.2020.108768]。然而这些制备过程中都使用了有机溶剂六氟异丙醇,有机溶剂的完全去除是一个难点,安全性难以得到保障。美国FDA在2020年8月28日已批准无菌外科手术植入物XARACOLL用于开放性腹股沟疝修补术后的疼痛控制。XARACOLL是一种独特的,不可注射的药物-设备组合,其形式为完全生物可吸收的胶原植入物,其中含有盐酸布比卡因。XARACOLL在手术过程中直接放置在手术部位,放置后会立即并随时间推移释放布比卡因,与安慰剂相比,XARACOLL可在24小时内提供统计学上显著的止痛效果[https://www.drugs.com/history/xaracoll.html]。
可注射原位凝胶以流动性含药溶液形态给药,在注射部位由于体内生理环境引发相转变或者发生原位交联反应,由液态转变为非交联的半固体凝胶或者交联凝胶,这里的交联可以是物理交联或者化学交联。物理交联主要通过分子间相互作用力(范德华力、疏水相互作用、电荷作用、氢键等)形成,这类凝胶由于形成过程中不涉及化学反应,因而成胶快,应用更简单、更安全。特别是温敏性可注射水凝胶,主要根据外界温度的变化实现溶液-凝胶转变,不需要化学试剂的参与,具有好的生物相容性,被广泛研究用于医用目的。这类温敏性凝胶在低温时(4℃)保持为可流动的液体,可均匀地包载细胞/药物及无需外科手术植入从而提高患者顺应性,通过注射器注入体内后,聚合物可在体温(37℃)下迅速形成凝胶避免细胞和生物活性药物分子等流失,可作为原位载药系统实现药物的局部缓慢释放。因为温度响应是相对最容易实现和最有效的响应,这类智能水凝胶药物缓释载体具有诱人的发展前景。目前多种温敏型凝胶已被应用于局麻药的长效镇痛研究中。符旭东等公开了一种罗哌卡因长效注射用温敏PLGA-PEG-PLGA共聚物凝胶及其制备方法,其药效学研究表明该罗哌卡因温敏凝胶组能显著延长药效维持时间,可持续发挥48h的镇痛作用[CN201510028754.2,罗哌卡因长效注射用温敏凝胶及其制备方法]。韩国的Lim组用温敏性的泊洛沙姆F-127和透明质酸制成一种含有0.75%罗哌卡因载药可注射凝胶[Preclinicalstudies of ropivacaine extended-release from a temperature responsivehydrogel for prolonged relief of pain at the surgical wound,2019-041HG-INT JPHARM 558,225-230,DOI:10.1016/j.ijpharm.2019.01.011],在大鼠皮下凝胶注射部位的罗哌卡因的峰值浓度和平均浓度都是普通药物溶液注射的2倍,而注射凝胶的血药浓度远低于溶液注射的药物浓度,这些说明温敏性凝胶具有局部缓释效果,虽然凝胶体外释放还有暴释现象。张文婧等制备了一种由原位温敏性PLGA-PEG-PLGA水凝胶和装载布比卡因的PLGA微球组成的水凝胶/微球复合载药系统(Gel-MS),该载药系统可实现布比卡因的原位持续缓慢释放,且无明显初始突释现象,大鼠坐骨神经阻滞模型表明,Gel-MS系统的镇痛效果远远强于单纯的微球和凝胶制剂[Precision guided long-acting analgesia by Gel-immobilized bupivacaine-loaded microsphere,Theranostics 2018;8(12):3331-3347.doi:10.7150/thno.25276]。美国Durect公司开发出了一种缓释盐酸布比卡因原位凝胶注射剂Posimir,它以N-甲基吡咯烷酮及蔗糖醋酸异丁酸酯作为辅料,注射前为具有良好的流动性的溶液,在注射局部用药部位后,与体液接触转变为凝胶状态,高黏度使布比卡因以髙浓度滞留在局部缓慢扩散。美国食品药品监督管理局(FDA)在2021年2月1日已批准将Posimir(布比卡因溶液)用于成年人的浸润渗透,用于治疗关节镜下肩峰下减压后,直接在关节镜下可视化,以达到手术后镇痛效果长达72小时和减少阿片类药物的使用。但该凝胶注射体系受环境影响较大,布比卡因浓度较高,易引起药物泄露,并且使用了有机溶剂N-甲基吡咯烷酮也有部分毒性和可能的致癌风险。[https://www.drugs.com/history/posimir.html]。由于局部麻醉给药方式为神经周围给药,因此对材料的组织相容性、生物可降解性、局部刺激性具有很高的要求。因此研发能延长作用时间、使用方便和可降解相容性好的长效局部麻醉药缓释新剂型具有重要的意义。
因为甲壳素及其衍生物具有非常好的生物相容性、生物可降解性和多种生物活性,适合用于药物缓释载体和生物医用材料。甲壳素与羧基化试剂在氢氧化钠-尿素体系中反应,可制得具有pH敏感性和温敏性的羧基甲壳素[中国发明专利申请公开说明书CN201310641249.6],甲壳素与羟丙基化试剂在氢氧化钠-尿素体系中反应,可制得具有温敏性的羟丙基甲壳素[中国发明专利申请公开说明书CN201410170871.8],侯春林等报道了温敏性羟丁基壳聚糖[中国专利申请公开说明书CN200810033699.6]和温敏性羟戊基壳聚糖[中国专利申请公开说明书CN201210220246.0]的制备。然而利用这些温敏性可降解的甲壳素衍生物制备长效局部麻醉药缓释体系的研究非常有限。因此,本发明将结合温敏性改性甲壳素微球和温敏性改性甲壳素水凝胶,得到可注射可降解局部麻醉镇痛缓释复合体系,期望在长效局部麻醉镇痛方面得到应用。
发明内容
针对现有技术中的不足,结合前人工作的基础,本发明的目的是提供一种温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系、制备方法及应用。
本发明利用温敏性改性甲壳素在低温下是流动的液体,加入局部麻醉镇痛药水溶液或者载有局部麻醉镇痛药的可降解高分子微球并在低温下混合均匀后还具有好的流动性可注射,在体内生理条件下快速物理交联成胶缓释药物,本发明具体采用的技术方案如下:
本发明提供一种温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系,其特征在于:所述局部麻药缓释镇痛体系为由温敏改性甲壳素和局部麻醉镇痛药混合制成可注射混合水凝胶,所述温敏性甲壳素的凝胶转变温度低于体温,按质量百分比计,可注射混合水凝胶中温敏性甲壳素的浓度为0.5-5%,局麻镇痛药浓度为0.1-5%。
优选的,所述温敏性甲壳素为温敏性羟丙基甲壳素、温敏性羟乙基甲壳素或者温敏性羟丁基甲壳素中的任意一种或几种组合。
优选的,所述局部麻药缓释镇痛体系还含有分散在可注射混合水凝胶内作为辅助成分的透明质酸。
优选的,所述局部麻药缓释镇痛体系中透明质酸的质量分数为0.1-1%。
优选的,所述局部麻药缓释镇痛体系中,按质量百分比计,温敏性羟丙基甲壳素1-3%,透明质酸0.2-0.8%,所述局部麻醉镇痛药为盐酸罗哌卡因0.5-3%。
优选的,所述局部麻醉镇痛药选自普鲁卡因、布比卡因、左布比卡因、丁卡因、罗哌卡因、依替卡因、阿替卡因、利多卡因、甲哌卡因、丙胺卡因和羟乙卡因的任意一种或者几种的混合物。
优选的,所述局部麻药缓释镇痛体系还包括负载有局部麻醉镇痛药的可降解高分子微球,所述负载有局部麻醉镇痛药的可降解高分子微球均匀分散在可注射混合水凝胶中。
优选的,所述可降解高分子微球为温敏性羧甲基甲壳素多孔微球,负载的局部麻醉镇痛药为盐酸罗哌卡因。
本发明还提供一种温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系的制备方法,其特征在于,包括以下步骤:
(1)在低温下将温敏性羟丙基甲壳素和透明质酸溶解于生理盐水中或者碱性水中,得到A液;
(2)将局部麻醉镇痛药盐酸盐溶解于生理盐水中或者酸性水中,得到B液;
(3)在凝胶转变温度下,混合A液和B液制成混合水凝胶前体溶液,调节pH到5-6,得到均匀的可注射混合水凝胶,即为局部麻药缓释镇痛体系。
优选的,所述低温是指2-30℃,进一步可优选为4-15℃,温敏性羟丙基甲壳素的凝胶转变温度低于体温,可注射混合水凝胶在体温可以成水凝胶缓慢释放药物。
本发明还提供一种温敏改性甲壳素水凝胶/微球复合载局部麻药缓释镇痛体系的制备方法,其特征在于,包括以下步骤:
(1)在低温下将温敏改性甲壳素溶解于生理盐水中或者碱性水中,得到A液;
(2)制备含有局部麻醉镇痛药的可降解高分子微球;
(3)在低温下,将含有局部麻醉镇痛药的可降解高分子微球均匀混合在A也中制成可注射混合水凝胶,即为局部麻药缓释镇痛体系,所述低温低于温敏性甲壳素的凝胶转变温度。
优选的,步骤(1)制备A液过程中加透明质酸。
优选的,所述低温是指2-30℃,进一步可优选为4-15℃,温敏性甲壳素的凝胶转变温度低于体温,可注射混合水凝胶在体温可以成水凝胶缓慢释放药物。
优选的,步骤(2)中可降解高分子微球制备方法如下:
(2.1)将温敏性羧甲基甲壳素配置成碱性的羧甲基甲壳素水溶液,利用碱液溶解聚乙二醇,得到聚乙二醇溶液,在低温下保存;
(2.2)将羧甲基甲壳素水溶液和聚乙二醇溶液低温搅拌混合均匀,再升温物理交联并通过酸性溶液将反应体系中和,然后洗涤纯化得到羧甲基甲壳素微球,冷冻干燥得到羧甲基甲壳素多孔微球;
(2.3)将局部麻醉镇痛药盐酸盐溶解于生理盐水中或者酸性水中,再加入羧甲基甲壳素多孔微球浸泡,离心干燥制得载药微球,即含有局部麻醉镇痛药的可降解高分子微球。
优选的,所述低温是指2-30℃,进一步可优选为4-15℃,温敏性羧甲基甲壳素的凝胶转变温度低于体温,可注射混合水凝胶在体温可以成水凝胶缓慢释放药物。
优选的,所述聚乙二醇的分子量范围为4-30kDa,其浓度范围为20-50%。
优选的,羧甲基甲壳素的乙酰度范围为0.72-0.92,羧甲基甲壳素的羧甲基取代度为0.07-0.23,羧甲基甲壳素的浓度为0.5-10wt%。
优选的,羧甲基甲壳素水溶液与聚乙二醇溶液体积比为1:1-1:20。
本发明还提供了一种可注射的温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系的应用:在制备包括但不局限于局部麻醉镇痛抗炎止痒作用在内的药物的装载,延长药物的释放时间达到长效的目的和应用。优选地,应用在外周神经阻滞或控制术后伤口疼痛作用的药物的装载和缓释。
与现有技术相比,本技术具有如下的有益效果:
(1)本发明通过直接将温敏性改性甲壳素水溶液,与局部麻醉镇痛药水溶液混合均匀,将其注射到需要局部麻醉镇痛的部位可在体温下快速形成凝胶并缓释药物,明显延长了动物体内局麻药作用镇痛时间,制备过程简单,不使用有机溶剂和化学交联剂。
(2)特别是利用温敏羧甲基甲壳素,通过两种水相聚合物溶液(聚乙二醇和羧甲基甲壳素双水相)方法制备微球和载局部麻醉镇痛药微球,最后制得一种新型温敏性可注射改性甲壳素水凝胶和羧甲基甲壳素微球复合载局部麻醉镇痛药缓释体系,释药速度慢于单独使用温敏性水凝胶或者载药微球,解决了现有局麻药作用时间短的问题,制备过程不使用任何化学交联剂和有机溶剂,载药微球和凝胶没有残留交联剂和有机溶剂的毒性问题,成本低,对环境无污染。
(3)本发明使用的温敏性改性甲壳素材料具有良好的生物相容性和生物降解性,可注射可完全填充不规则的创面,有利于长效局部麻醉镇痛抗炎止痒和促进创面愈合作用。
附图说明
图1为本发明实施例2中载盐酸罗哌卡因药2.0%为代表的2.0%R-HPCH-HA水凝胶缓释体系在PBS缓冲溶液中的药物累积释放曲线,使用同样药物含量的纯药物(R)和只使用HPCH载体(R-HPCH)作为对照组。
图2为本发明实施例2中载不同浓度盐酸罗哌卡因(0.5-5mM)的R-HPCH-HA水凝胶缓释体系和纯药物(R)对照组对人神经母细胞瘤细胞株(SH-SY5Y)的体外细胞毒性。
图3为实施例4中载药量为160mg/g的载盐酸罗哌卡因(简写R或者RPH)羧甲基甲壳素微球CMCH-Ms、载药水凝胶R-loaded HPCH和微球水凝胶复合载药缓释体系CMCH-Ms/HPCH三组在缓冲溶液中的药物累积释放曲线,其中图3(a)在pH5.0缓冲溶液中的药物累积释放曲线,图3(b)为在pH7.4缓冲溶液中的药物累积释放曲线。
图4体内麻醉效果:图4(A)为大鼠坐骨神经周围注射不同载药缓释体系后在不同时间点的最大抑痛效应比例MPE;图4(B)为大鼠坐骨神经周围注射不同载药缓释体系后在不同时间点的运动阻滞评分;图4(C)为大鼠坐骨神经周围注射不同载药缓释体系后在不同时间点的感觉阻滞时间;图4(D)为大鼠坐骨神经周围注射不同载药缓释体系后在不同时间点的运动阻滞时间。数据以平均值±标准方差SD表示(n=8;*P<0.05,**P<0.01)。
具体实施方式
为使本发明更加容易理解,下面将进一步阐述本发明的具体实施例。
下面结合实施例和附图对本发明作进一步的描述,其目的在于帮助更好的理解本发明的内容,但这些具体实施例不以任何方式限制本发明的保护范围。
实施例1:温敏性可注射改性甲壳素的合成
根据我们前期的研究工作[中国发明专利申请公开说明书CN201910768980.2],采用均相法在氢氧化钠-尿素体系中制备低脱乙酰度的温敏性甲壳素衍生物羟丙基甲壳素和羧甲基甲壳素。具体制备方法为:称取2克纯化后的甲壳素搅拌分散于预先冷冻的含有11wt%氢氧化钠和4wt%尿素的100克水溶液中,在-30℃下冷冻24h,取出在室温下机械搅拌将其解冻得到溶解的甲壳素水溶液。向得到的甲壳素溶液(100克,2wt%)中加入11.4克环氧丙烷,体系在2℃下搅拌使反应物混合均匀后,升温至5℃反应24h,随后升温至15℃反应6h。最后将体系冷却至2℃,用3M盐酸调节体系pH值到7,用去离子水透析,冷冻干燥得到白色海绵状羟丙基甲壳素(HPCH),产率为87%。1H NMR谱图计算得出产物的乙酰度为0.89,取代度为0.84。用乌氏粘度计测得其粘均分子量为Mη=410kDa。该均相合成的HPCH溶液具有温度敏感性,流变结果显示出可逆的溶胶-凝胶的转变行为,其中浓度为2wt%的HPCH溶液的凝胶转变温度为18℃。
温敏性羧甲基甲壳素的制备:根据发明人前期的研究工作[中国发明专利申请公开说明书CN201310641249.6],采用均相法在氢氧化钠-尿素体系中制备低脱乙酰度的羧甲基甲壳素。称取2克纯化后的甲壳素搅拌分散于预先冷冻的含有11wt%氢氧化钠和4wt%尿素的100克水溶液中,在-20℃下冷冻6h,取出在室温下机械搅拌解冻,再重复冷冻解冻2次即可得到溶解的甲壳素水溶液。向该甲壳素溶液中缓慢加入5.7克氯乙酸钠,保持机械搅拌,使反应物能均匀反应,在5℃的条件下反应24h。之后将体系冷却至2℃,用3M盐酸调节体系pH值到7,再将得到的中性溶液滴加至丙酮中使其沉淀析出。用80%乙醇(V/V)对沉淀物进行洗涤,除去其中的盐与尿素。洗涤后在60℃下烘干,即得到白色粉末状的羧甲基甲壳素,产率为87%。1H NMR谱图计算得出产物的乙酰度为0.82,取代度为0.13。该均相合成的CMCH溶液具有温度敏感性以及pH敏感性。能在低温、碱性水里的条件下溶解,在温度升高、碱性降低的条件下发生凝胶化转变。通过改变氯乙酸钠的用量,控制氯乙酸钠与甲壳素结构中糖单元的摩尔比,得到一系列不同取代度(0.07-0.23)的CMCH产物。改变配比和反应条件可以制得一系列不同取代度温敏性低脱乙酰度(0.72-0.92)的羧甲基甲壳素,其分子量范围为5kDa-1000kDa。
类似的,采用均相法在氢氧化钠-尿素体系中制备低脱乙酰度的羟乙基甲壳素和羟丁基甲壳素,这些甲壳素衍生物的乙酰度范围在0.7-0.92,其分子量范围为5kDa-1000kDa。
实施例2:温敏性羟丙基甲壳素水凝胶载局麻药罗哌卡因缓释体系
使用实施例1制备的温敏性羟丙基甲壳素,称取100mg羟丙基甲壳素(HPCH)和25mg透明质酸(HA)加入5mL生理盐水中,混合振荡均匀后置于4℃冰箱过夜,待完全溶解后得到无色透明可流动的粘稠液体(HPCH-HA),然后加入37.5mg局麻药盐酸罗哌卡因,混合振荡后溶解,制得0.75%RHCL-HPCH-HA可注射缓释水凝胶前体溶液,注射使用之前在低温下加入少量NaOH溶液调整pH到5.5-6.0之间,得到均匀乳白色可注射悬浮液,在37度或者体内形成载局麻药罗哌卡因水凝胶缓释体系。同样,使用50mg、75mg、100mg或者112.5mg局麻药盐酸罗哌卡因,可以制得1.0%RHCL-HPCH-HA,1.5%RHCL-HPCH-HA,2.0%RHCL-HPCH-HA,2.25%RHCL-HPCH-HA可注射载局麻药罗哌卡因水凝胶缓释体系,分别简写为0.75%R-HPCH-HA,1.0%R-HPCH-HA,1.5%R-HPCH-HA,2.0%R-HPCH-HA,2.25%R-HPCH-HA。
于4℃下使用精密注射器准确抽取1mL上述悬浮液置于透析袋(截留分子量:8000-14,000Da)中,然后将透析袋放入装有30mL PBS溶液置于37℃的恒温水浴摇床的离心管中,摇床以100rpm的速度震荡,分别于不同时间取样,每次取出2mL PBS溶液作为样品,并补充2mL新鲜PBS溶液,取出的样品利用紫光仪进行检测,以PBS溶液为空白对照,检测波长为263nm,依据罗哌卡因的标准曲线,计算罗哌卡因药物浓度和制得累积释放曲线,每组设置三个样本重复。图1是以载盐酸罗哌卡因药2.0%为代表的2.0%R-HPCH-HA水凝胶缓释体系的体外累积释放曲线,使用同样药物含量的纯药物对照组和只使用HPCH载体(没有用透明质酸,R-HPCH组)的对照组作为比较。由图1可见,纯药物罗哌卡因在生理盐水中释放速度较快,6小时内释放率超过80%,而R-HPCH组和R-HPCH-HA组中约有61.2%和38.9%的罗哌卡因在最初的16小时内释放,均表现出了明显的药物缓释效果。但与R-HPCH组相比,R-HPCH-HA的药物暴释更少,缓释更慢。
通过采用人神经母细胞瘤细胞株(SH-SY5Y)细胞进行含罗哌卡因载药缓释体系的体外细胞毒性试验。将盐酸罗哌卡因溶解于PBS缓冲液中,配置10mM罗哌卡因溶液,采用细胞培养基进行稀释制备罗哌卡因组浓度梯度分别为0.5-5mM的细胞培养基;低温配置含HPCH2%和HA0.5%及罗哌卡因浓度10mM的细胞培养基溶液在37℃成胶,再制备含相应凝胶缓释体系的罗哌卡因浓度分别为0.5-5mM的细胞培养基。以104/mL细胞密度将SH-SY5Y细胞接种于96孔板中于恒温培养箱正常培养24h,然后分别加入上述含纯药和含药凝胶的细胞培养基培养24小时,通过CKK-8试剂测试相对活细胞数值(图2),在低浓度下(0.5mM,1mM,2mM),SH-SY5Y细胞在两组之间并没有明显差别,表面HPCH-HA载体系统并未额外增加细胞毒性反应。随着药物浓度增加,虽然R-HPCH-HA组在5mM时产生了细胞毒性作用,但与单独纯药组相比,R-HPCH-HA组的细胞毒性反应显著降低(p<0.05)。
实施例3:无溶剂绿色法制备羧甲基甲壳素多孔微球和载药微球
使用实施例1合成的粉末状温敏性羧甲基甲壳素CMCH,用1mol/L的氢氧化钠溶液配制成2wt%的样品溶液10mL,在2℃下保存。使用PEG-10k(分子量为1万的聚乙二醇),用1mol/L的氢氧化钠溶液配制30wt%的聚乙二醇溶液50mL,置于冰水浴中,开动机械搅拌,将CMCH溶液10mL缓慢滴加入,搅拌半小时。然后用60℃的油浴代替冰水浴,同时用1.0M HCl将体系中和至中性。中和升温物理交联使微球固化20min,不使用任何化学交联剂。然后将这些羧甲基甲壳素微球反复用去离子水洗涤,并冷冻干燥制备得到羧甲基甲壳素多孔微球。改变CMCH水溶液和聚乙二醇溶液体积比(1:10-7:10)都可以制备得到羧甲基甲壳素多孔微球。改变PEG的分子量(从6k到30k),改变PEG的浓度(20-50%),改变CMCH的乙酰度(0.72-0.92)和取代度(0.07-0.23)及其浓度0.5-10wt%,改变CMCH溶液与PEG溶液体积比(1:1-1:20)等都不影响上述的制备。
将盐酸罗哌卡因(R)粉末溶解于0.1M HCl溶液中制得不同浓度的药物溶液(1-80mg/mL),将0.1g冻干的羧甲基甲壳素多孔微球浸泡在0.5-5mL不同浓度的罗哌卡因溶液中一定时间后载入罗哌卡因药,若有流动的液体则以3000rpm的转速离心5min,通过烘箱干燥得到载药微球,若有离心得到的液体则通过测试这些清液的体积和262nm处的紫外吸光度计算出没有载入微球的药物量msup;若没有流动的液体,msup则为0。
其中,madd为初始加入的R的质量,mc为干燥的载药微球的质量。
羧甲基甲壳素多孔微球的盐酸罗哌卡因的载药量随着浸泡时间增加而增加,当浸泡时间达到24h时,载药量基本达到饱和,该载药量随着R浓度的增大而增大。例如当盐酸罗哌卡因R浓度为8mg/mL时,浸泡时间为24h制得的微球载药量为160mg/g的载盐酸罗哌卡因羧甲基甲壳素微球(R-loaded CMCH-Ms)。制备其它载药微球如PLGA载罗哌卡因药需要使用有机溶剂。
实施例4:可注射微球水凝胶复合载药缓释体系制备
在4℃下将10mg干燥的R-loaded CMCH-Ms加到1mL 3%HPCH溶液中并混合均匀得到可注射微球水凝胶复合载药体系前体溶液,该前体溶液在37℃或体内会快速形成凝胶。同时将负载盐酸罗哌卡因的羟丙基甲壳素凝胶(R-loaded HPCH)作为对照组,在4℃将相同质量的R直接加到1mL 3%HPCH溶液中并混合均匀,在37℃下形成凝胶。
在体外药物释放实验中,分别称取10mg R-loaded CMCH-Ms、1mL R-loaded HPCH凝胶、1mL R-loaded CMCH-Ms/HPCH凝胶装于透析袋(截留分子量:8-14kDa)中,然后将透析袋放入装有10mL pH 5.0和pH 7.4的缓冲溶液的离心管中,将离心管置于37℃的水浴摇床中以70rpm的速度震荡,在预定的时间点,从离心管中取2mL释放介质并补充2mL新鲜的缓冲溶液。取出的释放介质中的盐酸罗哌卡因R浓度通过紫外可见光谱仪测定262nm处的吸光度,依据罗哌卡因的标准曲线,计算罗哌卡因药物浓度和制得累积释放曲线,每组实验平行重复三次,测试结果为平均值±标准偏差(SD)值。由图3可知,在pH 5.0时,R-loaded CMCH-Ms/HPCH组在1h时的初始药物释放率为24.7±2.4%,R-loaded CMCH-Ms组和R-loadedHPCH组在1h时的初始药物释放率分别为33.7±1.6%、35.5±1.5%;在pH 7.4时,R-loadedHPCH组和R-loaded CMCH-Ms组在1h时的初始药物释放率分别为28.9±0.14%、25.4±1.5%,而微球水凝胶复合载药体系R-loaded CMCH-Ms/HPCH组仅为18.8±1.7%。所以微球水凝胶复合载药体系表现出了明显的药物缓释效果,比单独使用温敏性水凝胶或微球作为载体药物暴释更少,释放更慢,并表现出长效可持续释放。使用其它载药微球如PLGA载罗哌卡因药代替羧甲基甲壳素载药微球可以得到类似的缓释效果。
实施例5:动物体内长效缓释麻醉镇痛效果和安全性评价
为了评价载药缓释体系的体内镇痛效果,采用超声引导下坐骨神经阻滞模型,将含有罗哌卡因的各种缓释注射剂在超声引导下注射到SD大鼠右后肢坐骨神经周围并观察阻滞效果。将大鼠用2%异氟醚麻醉后俯卧位,在超声引导下采用平面外进针,通过超声定位坐骨神经后,使用23G针头于股骨内侧垂直进针,到达臀中肌的两层筋膜之间,注射0.5mL或以下剂型的药物到坐骨神经周边,之后采用热盘法和运动阻滞4分法对大鼠的右后肢的感觉及运动阻滞情况进行评估。实验分6组,(1)2.25%R-HPCH-HA,(2)1.5%R-HPCH-HA,(3)0.75%R-HPCH-HA,(4)0.75%R,(5)HPCH-HA,(6)NS(生理盐水),每组6只大鼠。
热盘法操作:将大鼠放置在预热过的金属表面(55±0.5℃)上,金属表面周围环绕着一圈柱状玻璃(高20厘米,直径28厘米),以限制大鼠的活动。大鼠放进去即刻计时,当研究人员观察到大鼠右后肢有三种反应则表明疼痛的感觉出现:舔后爪,后爪抖动/颤动或跳跃时,立即停止计时并将大鼠从玻璃柱内取出,这个时间计为大鼠基础热潜伏期(实验开始前2天测试),一般约为3-5s,将基础值过低或过高的大鼠剔除。为防止烫伤,如果20s后大鼠仍没有疼痛表现,则将大鼠取出,时间记为20s。每次测量之间间隔5分钟以防止刺激敏化。评估在注射药物后15min开始,之后每隔2小时测试一次,如果大鼠3次测量均达到基线水平,则每4小时测量一次,直到所有大鼠均完成测试。所有观测任务由三位独立的观测人员完成,测试的数据转化为最大抑痛效应比例(MPE%)表示,MPE%计算公式如下:
MPE%=(Lt-Bt)/(Ct-Bt)×100%
其中,Bt为大鼠的基础热潜伏值;Lt为不同时间点大鼠的热潜伏值;Ct为大鼠的最大允许热潜伏值,即20s,将缓释制剂的有效镇痛效果定义为从注射后MPE值从100%到MPE值恢复为50%所需的时间跨度(感觉阻滞时间)。图4AC所示,所有大鼠在注射含罗哌卡因制剂后15min内均达到了最大热潜伏值(20s),所以说明HPCH-HA缓释系统的应用并未影响药物的起效时间。0.75%R纯药对照组在6小时内MPE值回归到了正常基线,而R-HPCH-HA组的平均MPE%值呈剂量依赖性延长,2.25%R-HPCH-HA组和1.5%R-HPCH-HA组的50%MPE镇痛持续时间分别为16.2h和8.1h,较R纯药对照组的持续时间明显延长(P<0.01),单纯载体本身没有任何神经阻滞作用。大鼠的运动阻滞也与感觉阻滞同时检测,对产生运动障碍的效果进行评分即运动阻滞4分法(1分表示大鼠运动完全正常;2分大鼠的足背屈功能障碍,当抬起大鼠尾巴时大鼠不能完全张开脚趾;3分表示大鼠的跖屈障碍,当抬起大鼠尾巴时大鼠不能完全张开脚趾;4分代表大鼠完全丧失背屈、跖屈功能,出现步态障碍)评价。如图4BD所示,2.25%R-HPCH-HA组和1.5%R-HPCH-HA组运动阻滞时间分别为6.8h和5.6h,与0.75%R纯药对照组相比,运动阻滞时间延长有统计学意义(P<0.01)。12小时后,所有大鼠的运动功能评分均恢复正常。
组织炎性和神经毒性评估:注射到大鼠的HPCH-HA残留凝胶粘附于坐骨神经附近的组织,到第14天几乎观察不到凝胶了,基本完全降解,HE染色组织切片表明2.25%R-HPCH-HA植入部位在第7天出现中度炎症,其他含罗哌卡因制剂组及载体组仅出现轻度炎症,14天后,含罗哌卡因制剂组及载体组的炎症反应均有所减轻,说明HPCH-HA作为罗哌卡因载体所引起的炎症反应具有一定的可逆性。各组炎症均未浸润到神经外膜,说明了HPCH-HA作为罗哌卡因载体并未对坐骨神经造成炎性反应。对注射后第7天和第14天坐骨神经组织进行透射电子显微镜(TEM)、甲苯胺蓝(TB)染色和TUNEL染色测试结果表明,轴突和髓鞘结构在各组中均表现为排列整齐的纤维和均匀的髓鞘结构,各组之间的轴突密度和异常髓鞘百分比无显著差异,TUNEL阳性细胞检测结果无明显差异,说明各组均未发生明显的凋亡变化。综合上述结果表明,含罗哌卡因缓释体系体内可降解,对神经纤维结构影响轻微,无明显结构损伤。在注射含罗哌卡因制剂7天和14天后,各组大鼠的谷草转氨酶(AST),尿素(BUN)和肌酐(Cr)均无明显差异,均在正常范围内,因此说明含罗哌卡因的HPCH-HA缓释体系对大鼠的肝肾功能无明显影响。
温敏性羟丙基甲壳素的浓度从0.5-5%代替上述的2%,局麻药的浓度在0.1-5%范围内,透明质酸的加入浓度在0.1-1%之间,局部麻醉镇痛药选自普鲁卡因、布比卡因、左布比卡因、丁卡因、罗哌卡因、依替卡因、阿替卡因、利多卡因、甲哌卡因、丙胺卡因或者羟乙卡因的一种或者几种的混合物代替上述的实验中的罗哌卡因都可得到类似的长效缓释结果。含有温敏性羟丙基甲壳素1-3%和含有0.2-0.8%的透明质酸辅助成分,所述局部麻醉镇痛药选自盐酸罗哌卡因0.5-3%的长效缓释效果更优。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (11)
1.一种温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系,其特征在于:所述局部麻药缓释镇痛体系为由温敏性改性甲壳素和局部麻醉镇痛药混合制成可注射混合水凝胶,所述温敏性改性甲壳素的凝胶转变温度低于体温,按质量百分比计,可注射混合水凝胶中温敏性改性甲壳素的浓度为0.5-5%,局麻镇痛药浓度为0.1-5%。
2.根据权利要求1所述的温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系,其特征在于:所述温敏改性甲壳素为温敏性羟丙基甲壳素、温敏性羟乙基甲壳素或者温敏性羟丁基甲壳素中的任意一种或几种组合。
3.根据权利要求1所述的温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系,其特征在于:所述局部麻药缓释镇痛体系还含有分散在可注射混合水凝胶内作为辅助成分的透明质酸。
4.根据权利要求1所述的温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系,其特征在于:所述局部麻醉镇痛药选自普鲁卡因、布比卡因、左布比卡因、丁卡因、罗哌卡因、依替卡因、阿替卡因、利多卡因、甲哌卡因、丙胺卡因和羟乙卡因的任意一种或者几种的混合物。
5.根据权利要求1所述的温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系,其特征在于:所述局部麻药缓释镇痛体系还包括负载有局部麻醉镇痛药的可降解高分子微球,所述负载有局部麻醉镇痛药的可降解高分子微球均匀分散在可注射混合水凝胶中。
6.根据权利要求5所述的温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系,其特征在于:所述可降解高分子微球为温敏性羧甲基甲壳素多孔微球。
7.一种温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系的制备方法,其特征在于,包括以下步骤:
(1)在凝胶转变温度下,将温敏性羟丙基甲壳素和透明质酸溶解于生理盐水中或者碱性水中,得到A液;
(2)将局部麻醉镇痛药盐酸盐溶解于生理盐水中或者酸性水中,得到B液;
(3)在低温下混合A液和B液制成混合水凝胶前体溶液,调节pH到5-6,得到均匀的可注射混合水凝胶,即为局部麻药缓释镇痛体系。
8.一种温敏改性甲壳素水凝胶/微球复合载局部麻药缓释镇痛体系的制备方法,其特征在于,包括以下步骤:
(1)在低温下将温敏改性甲壳素溶解于生理盐水中或者碱性水中,得到A液;
(2)制备含有局部麻醉镇痛药的可降解高分子微球;
(3)在低温下,将含有局部麻醉镇痛药的可降解高分子微球均匀混合在A也中制成可注射混合水凝胶,即为局部麻药缓释镇痛体系,所述低温低于温敏改性甲壳素的凝胶转变温度。
9.根据权利要求8所述温敏改性甲壳素水凝胶/微球复合载局部麻药缓释镇痛体系的制备方法,其特征在于,步骤(2)中具体方式如下:
(1)将温敏性羧甲基甲壳素配置成碱性的羧甲基甲壳素水溶液,利用碱液溶解聚乙二醇,得到聚乙二醇溶液,在低温下保存;
(2)将羧甲基甲壳素水溶液和聚乙二醇溶液低温搅拌混合均匀,再升温物理交联并通过酸性溶液将反应体系中和,然后洗涤纯化得到羧甲基甲壳素微球,冷冻干燥得到羧甲基甲壳素多孔微球;
(3)将局部麻醉镇痛药盐酸盐溶解于生理盐水中或者酸性水中,再加入羧甲基甲壳素多孔微球浸泡,干燥制得载药微球,即含有局部麻醉镇痛药的可降解高分子微球。
10.一种权利要求1-6任意一项所述温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系的应用,其特征在于:用于长效局部麻醉镇痛抗炎止痒的药物。
11.一种权利要求1-6任意一项所述温敏改性甲壳素水凝胶载局部麻药缓释镇痛体系的应用,其特征在于:用于外周神经阻滞或控制术后伤口疼痛作用的药物。
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