CN113402463A - 一种塞来昔布微晶、负载塞来昔布微晶的温敏性凝胶及其制备方法 - Google Patents
一种塞来昔布微晶、负载塞来昔布微晶的温敏性凝胶及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种塞来昔布微晶、负载塞来昔布微晶的温敏性凝胶及其制备方法,属于医药技术领域。本发明采用反溶剂沉淀法联用超声破碎技术制备得到无定形塞来昔布微晶,粒径为2‑5μm,可有效增加微晶的溶出速率,从而解决塞来昔布难溶、悬浮稳定性差的问题。本发明将泊洛沙姆和聚(氯乙烯‑甲基乙烯基醚/马来酸酐)共聚物热交联制成改性F127温敏性聚合物,并用改性F127温敏性聚合物负载塞来昔布微晶得到温敏性凝胶,该凝胶具有缓释功能,制剂转变成凝胶后,塞来昔布长期缓慢释放,可减少给药次数,延长药物的释放时间,长期保持关节腔内局部治疗浓度,提高塞来昔布治疗效果和患者使用体验。
Description
技术领域
本发明涉及一种塞来昔布微晶、负载塞来昔布微晶的温敏性凝胶及其制备方法,属于医药技术领域。
背景技术
塞来昔布是一种选择性COX-2抑制剂,主要适应症为骨关节炎、风湿性关节炎等。其上市之前的非甾体抗炎药则大多对COX-1和COX-2都有抑制作用,如对乙酰氨基酚、布洛芬、吲哚美辛等。在临床上,塞来昔布给药途径多为口服,口服药物的吸收受首过效应影响,在肝肾药物分布较高,关节部位浓度较低,需大量口服才能达到局部有效浓度。因此,采用关节腔直接注射给药的方式,避免首过效应,可以大幅减少药物用量,继而减轻肝肾负担,对肝肾功能损害者的用药意义重大。但是,塞来昔布为难溶性药物,在注射剂中悬浮稳定性较差。现有研究采用球磨法、高压均质法将塞来昔布转化为粒径较小的微晶,改善药物的悬浮稳定性和溶出性能。但球磨机和高压均质器的价格较为昂贵,维护成本较高,不适合用于在实验室进行难溶性药物的微晶制备。
温度敏感型原位凝胶是一种依赖温度而发生相转变的凝胶。它在储藏条件下是自由流动的液体,注射进入人体后可填充于组织间隙,迅速发生相转变,在注射部位形成半固体状态凝胶,达到局部给药或延缓药物释放的目的。它具有可注射、创伤小、给药方便、控制药物释放等优点,适用于体内局部注射给药。泊洛沙姆407(商品名为F127)是国家药品监督管理局批准的药用辅料,其温敏性和缓释性能在多项研究中已得到广泛共识。其中,浓度为20%-30%的F127水溶液具有受热反向胶凝的性质,即冷藏温度下是自由流动的液体,而室温或体温时形成澄明的凝胶。可是,较高的浓度影响了温敏性F127溶液在临床中使用的安全性,F127为高分子,不能被肾脏代谢,其在肝脏中的聚积可能会影响脂代谢,进而诱发高脂血症。
发明内容
为了解决上述问题,本发明提供了一种塞来昔布微晶的制备方法和一种改性F127温敏性聚合物的制备方法,并提供一种负载塞来昔布微晶的生物相容的可注射温敏性凝胶。
本发明的第一个目的是提供一种塞来昔布微晶的制备方法,所述微晶包括塞来昔布和稳定剂,所述方法包括以下步骤:
(1)将稳定剂溶解于水中形成水相,将塞来昔布溶解于无水乙醇中形成有机相,分别使用水系滤头和有机系滤头过滤;
(2)低温下,将水相置于搅拌下,滴加有机相,获得塞来昔布水分散体;
(3)低温下,将步骤(2)中所得的塞来昔布水分散体置于超声破碎仪探头下破碎10-30min后,得到微晶混悬液;
(4)将步骤(3)得到的微晶混悬液在液氮下冻结,之后冷冻干燥,即得塞来昔布微晶。
优选的,按质量分数计,所述微晶包括塞来昔布50-90份、稳定剂10-50份。
优选的,所述稳定剂包括聚乙烯吡咯烷酮、羟丙甲纤维素、泊洛沙姆、聚乙烯醇、聚乙二醇、吐温、司盘中的一种或多种。
优选的,步骤(1)中,所述水相中稳定剂的质量体积浓度为0.075-0.225%(g/mL),所述有机相中塞来昔布的质量体积浓度为1.5-4.5%(g/mL)。
优选的,所述水系滤头和有机系滤头的尺寸为0.22-0.80μm,优选为0.45μm。
优选的,步骤(2)所述低温是指4-10℃。
优选的,步骤(2)所述搅拌的速率为500-2000rpm。
优选的,步骤(2)所述的滴加有机相的速率为0.5-2.0mL/min。
优选的,步骤(3)所述低温是指4-10℃。
优选的,步骤(3)所述的超声破碎的功率为200-800W,脉冲周期为2-5s,脉冲间隔为2-5s。
优选的,步骤(4)中所述在液氮下冻结5~15min,所述冷冻干燥的时间优选为24~48h。
经研究,本发明采用反溶剂沉淀法联用超声破碎技术,设备需求简单、操作简便,制得的塞来昔布微晶,为无定形晶体,粒径为2-5μm,可有效增加微晶的溶出速率,从而解决塞来昔布难溶、悬浮稳定性差的问题。
本发明的第二个目的是提供上述制备方法制备得到的塞来昔布微晶。
本发明的第三个目的是提供一种改性F127温敏性聚合物的制备方法,所述改性F127温敏性聚合物由泊洛沙姆和聚(氯乙烯-甲基乙烯基醚/马来酸酐)共聚物热交联制成,具体通过以下方法制备得到:无水条件下,加热泊洛沙姆和聚(氯乙烯-甲基乙烯基醚/马来酸酐)共聚物的混合物,优选的,加热温度40℃-60℃之间,加热时间为12-48h。
优选的,所述泊洛沙姆为Pluronic F127,所述聚(氯乙烯-甲基乙烯基醚/马来酸酐)共聚物为GantrezTMS-97BF。
优选的,所述F127和GantrezTMS-97BF的质量比为25:6-100:3。
经研究,本发明获得的改性F127温敏性聚合物的8%-15%的水溶液具有受热反向胶凝的性质,相较于现有技术,可大幅减少F127用量,聚合物生物相容性良好,适合临床应用。
本发明的第四个目是提供一种负载塞来昔布微晶的温敏性凝胶的制备方法,包括以下步骤:将F127温敏性聚合物制成8%-15%的水溶液,15℃-25℃下,将塞来昔布微晶混悬于F127温敏性聚合物水溶液中,搅拌,加热至37℃-45℃,即得负载塞来昔布微晶的温敏性凝胶。
优选的,所述塞来昔布微晶的加入量为5-30g/L。
本发明的第五个目的是提供上述制备方法制备得到的负载塞来昔布微晶的温敏性凝胶。
本发明还提供了一种温敏性凝胶,所述温敏性凝胶包括浓度为10%-15%的上述改性F127温敏性聚合物的水溶液。
本发明还提供了一种用于骨关节炎或风湿性关节炎的药物,所述药物包含上述负载塞来昔布微晶的温敏性凝胶。
本发明还提供了上述负载塞来昔布微晶的温敏性凝胶的制备方法在医药领域的应用。
本发明取得的有益效果:
(1)本发明采用反溶剂沉淀法联用超声破碎技术,设备需求简单、操作简便,制得无定形塞来昔布微晶,粒径2-5μm,与原料塞来昔布粉末相比溶出速率更快,生物利用度更高,解决了塞来昔布难溶、悬浮稳定性差的问题。
(2)本发明制备得到的改性F127温敏性聚合物在37℃-45℃下,8%-15%的水溶液具有受热反向胶凝的性质,可大幅减少F127用量,聚合物生物相容性良好,适合临床应用。
(3)本发明制备得到的负载塞来昔布微晶的温敏性凝胶具有温度敏感性质,当温度低于体温时,制剂以液态形式存在,在注射部位温度升高至体温,制剂由转变成凝胶,该凝胶具有缓释功能,制剂转变成凝胶后,塞来昔布长期缓慢释放,可减少给药次数,延长药物的释放时间,长期保持关节腔内局部治疗浓度,提高塞来昔布治疗效果和患者使用体验。
附图说明
图1为塞来昔布原料粉末、PVP K30、两者混合物和塞来昔布微晶的FT-IR谱图。
图2为塞来昔布微晶溶出曲线。
图3为Pluronic F127、GantrezTMS-97BF和改性F127温敏性聚合物的FT-IR谱图。
图4为10%F127水溶液、10%F127-GantrezTMS-97BF混合水溶液和10%GZF927水溶液的温度扫描情况(实线为储存模量,虚线为损耗模量)。
图5为改性F127温敏性聚合物的细胞活力情况。
图6为负载塞来昔布微晶的温敏性凝胶的37℃药物释放情况。
具体实施方式
下面结合实施例对本发明作进一步的描述,但本发明的实施方式不限于此。
实施例1:
(1)将15mg泊洛沙姆188溶解于20mL水中形成水相,将30mg塞来昔布溶解于2mL无水乙醇中形成有机相,分别使用0.45μm水系滤头和有机系滤头过滤;
(2)4℃下,水相置于磁力搅拌器500rpm下搅拌,0.5mL/min滴加有机相,获得塞来昔布水分散体;
(3)4℃下,将所得的分散体置于超声破碎仪探头下200W(脉冲周期2s,脉冲间隔2s)破碎10min后,得到微晶混悬液;
(4)微晶混悬液在液氮中5min冻结,后冷冻干燥24h,即得塞来昔布微晶,经过动态光散射法测定可知所制得的微晶粒径为4492±660nm。
实施例2:
(1)将45mg PVP K30(聚乙烯吡咯烷酮)溶解于20mL水中形成水相,将90mg塞来昔布溶解于2mL无水乙醇中形成有机相,分别使用0.45μm水系滤头和有机系滤头过滤。
(2)6℃下,水相置于磁力搅拌器2000rpm下搅拌,2.0mL/min滴加有机相,获得塞来昔布水分散体;
(3)6℃下,将所得的分散体置于超声破碎仪探头下800W(脉冲周期5s,脉冲间隔5s)破碎30min后,得到微晶混悬液;
(4)微晶混悬液在液氮中15min冻结,后冷冻干燥48h,即得塞来昔布微晶,经过动态光散射法测定可知所制得的微晶粒径为2508±508nm。
采用FT-IR法验证塞来昔布微晶的制备:塞来昔布原料粉末、PVPK30、塞来昔布原料粉末与PVPK30组成的混合物(二者质量比为2:1)、实施例2制备得到的塞来昔布微晶以4cm-1的分辨率在4000~500cm-1扫描,FT-IR光谱如图1所示:3233cm-1和3341cm-1的塞来昔布特征吸收峰,在塞来昔布微晶谱图中显著减弱,由此可以确定塞来昔布被包封于PVP中。
塞来昔布微晶体外溶出度研究:取塞来昔布原料药、塞来昔布未超声结晶(即省略步骤(3)得到的结晶)和塞来昔布微晶各3份,每份相当于塞来昔布90mg,置于溶出杯中,按照2015版《中国药典》二部附录溶出度测定法第二法,进行体外溶出度试验。其中,溶出介质为含0.2%SDS的pH7.4磷酸盐缓冲溶液,溶出介质用量为900ml,温度37.0℃,转速为50r/min,依法操作,在5、15、25、35、45、60、90min时取样5ml,同时立刻补充同体积、同温度的溶出介质,取出溶液经0.45μm微孔滤膜过滤,取续滤液进HPLC检测。
其中,HPLC色谱条件如下:
色谱柱:Agilent TC-C18柱(250mm×4.6mm,5μm);流动相:甲醇-水(75:25);检测波长:254nm;流速:1.0ml/min;柱温:30℃;进样体积:10μL。
在不同时间点的样品经含量测定并计算累积溶出百分数,结果如图2所示,本发明制备的塞来昔布微晶在含0.2%SDS的pH7.4磷酸盐缓冲溶液中可快速溶出,溶出速率明显高于塞来昔布原料药和塞来昔布未超声的结晶产品。说明,本发明的微晶能够有效增加塞来昔布的溶出速率。
试验组:
与实施例2相同,区别在于塞来昔布投药量不同,设计两组试验,试验组1-1称取30mg的塞来昔布,试验组1-2称取60mg的塞来昔布。
与实施例2相同,区别在于超声破碎功率与时间不同,设计两组试验,试验组2-1将分散体在600W超声下破碎20min,试验组2-2将分散体在300W超声下破碎10min。
将实施例2与对比例1、2中改变塞来昔布投药量以及超声破碎功率和时间不同对所制备所得塞来昔布微晶的粒径进行对比,结果如表1。由此可见,塞来昔布微晶的制备流程中塞来昔布药物量及超声破碎功率与时间在本发明范围内可制备出塞来昔布微晶粒径在2-5μm,且按照上述溶出测试可知本发明方法可有效增加塞来昔布的溶出速率。
表1塞来昔布微晶粒径对比表
对比例编号 | 试验组1-1 | 试验组1-2 | 试验组2-1 | 试验组2-2 |
平均粒径nm | 2736 | 2655 | 2908 | 4179 |
实施例3:
(1)将25mg HPMC E15(羟丙基甲基纤维素)溶解于20mL水中形成水相,将50mg塞来昔布溶解于2mL无水乙醇中形成有机相,分别使用0.45μm水系滤头和有机系滤头过滤;
(2)10℃下,水相置于磁力搅拌器900rpm下搅拌,1.0mL/min滴加有机相,获得塞来昔布水分散体;
(3)10℃下,将所得的分散体置于超声破碎仪探头下400W(脉冲周期3s,脉冲间隔3s)破碎15min后,得到微晶混悬液;
(4)微晶混悬液在液氮中10min冻结,后冷冻干燥36h,即得塞来昔布微晶,经过动态光散射法测定可知所制得的微晶粒径为3891±371nm。
实施例4
室温下,1000mg Pluronic F127和30mgGantrezTMS-97BF混合溶解于去离子水中,冷冻干燥,得到Pluronic F127和GantrezTMS-97BF白色固态混合物。混合物置于敞口玻璃皿中,在40℃烘箱中加热12h,收集皿内的灰白色半透明改性F127温敏性聚合物(GZF927),于干燥器中保存。
实施例5:
室温下,1000mg Pluronic F127和100mg GantrezTMS-97BF混合溶解于去离子水中,冷冻干燥,得到Pluronic F127和GantrezTMS-97BF白色固态混合物。混合物置于敞口玻璃皿中,在50℃烘箱中加热24h,收集皿内的灰白色半透明GZF927,于干燥器中保存。
实施例6:
室温下,1000mg Pluronic F127和240mg GantrezTMS-97BF混合溶解于去离子水中,冷冻干燥,得到Pluronic F127和GantrezTMS-97BF白色固态混合物。混合物置于敞口玻璃皿中,在60℃烘箱中加热48h,收集皿内的灰白色半透明GZF927,于干燥器中保存。
改性F127温敏性聚合物(GZF927)的制备表征
采用FT-IR法验证GZF927的合成,Pluronic F127、GantrezTMS-97BF和实施例5中GZF927以4cm-1的分辨率在4000~500cm-1扫描,FT-IR光谱如图3所示,可见,1700cm-1处为GantrezTMS-97BF的羧酸基团峰;1800~1600cm-1之间Pluronic F127无特征吸收峰;1732cm-1处为GZF927的酯羰基峰,出现酯羰基峰,由此可以确定GZF927合成制备成功。
改性F127聚合物(GZF927)的温敏性表征
采用流变温度扫描法验证GZF927温敏性,10%F127水溶液、10%F127-GantrezTMS-97BF混合水溶液(二者质量比为10:1)和实施例5中10%GZF927水溶液,在15℃-45℃之间测量溶液储存模量和损耗模量变化,频率10rad/s,应变1%。
结果如图4所示,可见,10%F127水溶液和10%F127-GantrezTMS-97BF混合水溶液,在扫描区间内损耗模量大于储存模量,体系呈溶胶状。而10%GZF927在升温过程中,储存模量和损耗模量逐渐上升,体系转变为凝胶态,温敏性明显。可见,本发明制备得到的GZF927溶于水后具有温敏性。
另外,经过实验发现,改性F127温敏性聚合物的8%-15%的水溶液均具有受热反向胶凝的性质,相较于现有技术,可大幅减少F127用量,聚合物生物相容性良好,适合临床应用。
改性F127聚合物(GZF927)的细胞毒性考察
RAW 264.7细胞以每孔1×104个细胞接种于96孔板中,待24h细胞贴壁后,设置正常对照组、实施例5中GZF927组每组6个复孔。正常对照组以无血清DMEM培养基孵育细胞,GZF927组分别以含终浓度为1.0、2.0、3.0、5.0mg/mL GZF927的无血清DMEM孵育细胞。按上述处理方式孵育细胞24h后,收集各孔培养基,在每孔中加入0.5g/L MTT PBS溶液100μL,继续培养4h后弃上清,每孔加入100μL DMSO,震荡10min,在570nm处测量各孔的吸光度。
结果如图5所示,GZF927对RAW 264.7细胞活力均无影响,显示出良好的生物相容性。
实施例7:
15℃下,将实施例2制备得到的15mg的塞来昔布微晶重新混悬于2mL浓度为10%实施例5制备得到的GZF927水溶液中,缓慢搅拌,加热至37℃,即得负载塞来昔布微晶的温敏性凝胶。
实施例8:
25℃下,将实施例2制备得到的60mg的塞来昔布微晶重新混悬于2mL浓度为15%实施例5制备得到的GZF927水溶液中,缓慢搅拌,加热至45℃,即得负载塞来昔布微晶的温敏性凝胶。
体外释放研究
取2mL的实施例7中负载塞来昔布微晶的10%改性F127水溶液3份,每份相当于塞来昔布10mg,置于6孔板中,在37℃保持5min,形成凝胶。释放介质为pH=7.4磷酸盐缓冲液(PBS)。释放介质量为8mL,温度37.0℃,摇床转速100r/min。在不同的时间点(15min、30min、1h、2h、4h、8h、24h、2d、3d、4d和8d),收集1mL上清液并用相同体积的新鲜PBS代替,取出上清经0.45μm微孔滤膜过滤,进HPLC检测。其中,HPLC色谱条件为:
色谱柱:Agilent TC-C18柱(250mm×4.6mm,5μm);流动相:甲醇-水(含0.27%磷酸二氢钾,用磷酸调解pH3.0±0.05)(65:35);检测波长:215nm;流速:1.0ml/min;柱温:30℃;进样体积:10μL。
如图6所示,塞来昔布在第8天累计释放率达80%以上,证明改性F127水凝胶具有缓释功能,塞来昔布微晶可以实现长期储存、缓慢释放的功能。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一种塞来昔布微晶的制备方法,其特征在于,所述微晶包括塞来昔布和稳定剂,所述方法包括以下步骤:
(1)将稳定剂溶解于水中形成水相,将塞来昔布溶解于无水乙醇中形成有机相,分别使用水系滤头和有机系滤头过滤;
(2)低温下,将水相置于搅拌下,滴加有机相,获得塞来昔布水分散体;
(3)低温下,将步骤(2)中所得的塞来昔布水分散体置于超声破碎仪探头下破碎10-30min后,得到微晶混悬液;
(4)将步骤(3)得到的微晶混悬液在液氮下冻结,之后冷冻干燥,即得塞来昔布微晶。
2.根据权利要求1所述的一种塞来昔布微晶的制备方法,其特征在于,按质量分数计,所述微晶包括塞来昔布50-90份、稳定剂10-50份,其中,所述稳定剂包括聚乙烯吡咯烷酮、羟丙甲纤维素、泊洛沙姆、聚乙烯醇、聚乙二醇、吐温、司盘中的一种或多种。
3.根据权利要求1或2所述的一种塞来昔布微晶的制备方法,其特征在于,步骤(3)所述的超声破碎的功率为200-800W,脉冲周期为2-5s,脉冲间隔为2-5s。
4.根据权利要求1~3任一项所述的一种塞来昔布微晶的制备方法制备得到的塞来昔布微晶。
5.一种改性F127温敏性聚合物的制备方法,其特征在于,所述改性F127温敏性聚合物由泊洛沙姆和聚(氯乙烯-甲基乙烯基醚/马来酸酐)共聚物热交联制成,具体通过以下方法制备得到:无水条件下,加热泊洛沙姆和聚(氯乙烯-甲基乙烯基醚/马来酸酐)共聚物的混合物,其中,加热温度为40℃-60℃,加热时间为12-48h。
6.根据权利要求5所述的一种改性F127温敏性聚合物的制备方法制备得到的改性F127温敏性聚合物。
7.一种温敏性凝胶,其特征在于,所述温敏性凝胶为浓度为8%-15%的权利要求7所述的改性F127温敏性聚合物的水溶液。
8.一种负载塞来昔布微晶的温敏性凝胶的制备方法,其特征在于,包括以下步骤:将权利要求7所述的F127温敏性聚合物制成8%-15%的水溶液,15℃-25℃下,将权利要求4所述的塞来昔布微晶混悬于F127温敏性聚合物水溶液中,搅拌,加热至37℃-45℃,即得负载塞来昔布微晶的温敏性凝胶。
9.根据权利要求8所述的一种负载塞来昔布微晶的温敏性凝胶的制备方法制备得到的负载塞来昔布微晶的温敏性凝胶。
10.一种用于骨关节炎或风湿性关节炎的药物,其特征在于,所述药物包含权利要求4所述的塞来昔布微晶或权利要求9所述的负载塞来昔布微晶的温敏性凝胶。
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