CN105709214A - 一种长效促卵泡素的缓释药物制剂 - Google Patents
一种长效促卵泡素的缓释药物制剂 Download PDFInfo
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Abstract
本发明提供一种长效促卵泡素的缓释药物制剂，所述缓释药物制剂的组分如下：促卵泡素、枸橼酸钠?枸橼酸缓冲液、肝素以及鱼精蛋白；所述促卵泡素为垂体促卵泡素，或基因重组促卵泡素，所述缓释药物制剂剂型为注射剂或冻干粉针，所述缓释药物制剂中肝素与鱼精蛋白的比例为10:1至1:10，每1ml生理盐水或注射用水中加入促卵泡素100～1000IU，枸橼酸钠10～50mg，枸橼酸0.1～1.0mg，肝素2～10mg，鱼精蛋白2～10mg。本发明可应用于动物的超数排卵和诱导发情。
Description
【技术领域】
本发明涉及一种以促卵泡素为主要药效成分的缓释药物制剂，属于药物学、分子医学和疾病防治领域。
【背景技术】
促卵泡素(Follicle-stimulating hormone,FSH)是一种由动物脑垂体前叶分泌的糖蛋白类促性腺激素。具有调节机体的生长、性成熟和繁殖等作用。
FSH在雌性动物可促进子宫内膜生长、排卵、刺激多卵泡发育等。靶细胞上存在着FSH的特异性受体,FSH与细胞膜上的卵泡刺激素受体结合后,通过G蛋白偶联机制激活腺苷酸环化酶,使细胞内cAMP增加或引起钙离子内流,从而对细胞的代谢或功能活动产生影响。FSH与受体结合后产生两种作用。一方面活化芳香化酶,另一方面诱导LH受体形成。内膜细胞在LH的作用下提供19碳底物-雄烯二酮或睾酮这些底物通过基底膜进入颗粒细胞,活化的芳香化酶把它们转变为17-β雌二醇(E2),而卵泡内膜本身只能产生很少量的雌激素。雌激素协同FSH使颗粒细胞增生,内膜细胞分化,卵泡液形成卵泡腔扩大,从而使卵泡生长和发育。
繁殖是影响畜牧业整体效益的一个关键因素。如何提高动物繁殖率一直都是畜牧领域攻关的一个重点。在现代农业中，种畜的人工授精已经非常普遍，特别是在奶牛(所有受精类型的75％)及猪(所有受精类型的85％)上的应用。胚胎移植技术是家畜繁殖领域中的一次重大革命，它能够提高优秀种畜的繁殖率，充分利用优质遗传基因，对家畜的育种、保种、引种及经济效益的提高都具有非常重要的意义。然而，通过各种手段进行家畜扩繁的前提条件是动物的适时发情和大量优质卵子来源。给种畜注射适量的动物激素则可以满足这些前提条件：目前国际上通用的同类产品有Ovagen(促卵泡素源于羊垂体提取物，新西兰产)和Folltropin-V(促卵泡素源于猪垂体提取物，加拿大产)，可用于超数排卵(即增加成熟卵泡数目，每次排卵数目可达10个以上)和配合同期发情(人工授精、胚胎移植之前的关键技术)。为了能够达到超数排卵，Ovagen和Folltropin-V均需注射八次。国内近年由浙江宁波第二激素厂生产以及杭州动物药品厂生产的注射用垂体促卵泡素，每疗程注射2-5次，能促进卵巢卵泡生长发育和雌激素的分泌，引起动物正常发情，但尚不推荐用于超数排卵。目前的各种同类产品，均需多次注射，不仅人工成本高，更因多次惊扰动物而影响效果。多年来，行业内纷纷研究长效促卵泡素，包括基因重组、缓释制剂、改变给药途径等不同方法，试图减少注射次数，但至今尚未有兽用长效促卵泡素的产品面市。
【发明内容】
本发明要解决的技术问题，在于提供一种长效促卵泡素的缓释药物制剂，可应用于动物的超数排卵和诱导发情。
本发明是这样实现的：
一种长效促卵泡素的缓释药物制剂，所述缓释药物制剂的组分如下：促卵泡素、枸橼酸钠-枸橼酸缓冲液、肝素以及鱼精蛋白。
进一步地，所述促卵泡素为垂体促卵泡素，或基因重组促卵泡素。
进一步地，所述缓释药物制剂剂型为注射剂或冻干粉针。
进一步地，所述缓释药物制剂中肝素与鱼精蛋白的比例为10:1至1:10。
进一步地，所述缓释药物制剂的注射剂处方中肝素与鱼精蛋白的比例为1:1，每1ml生理盐水或注射用水中加入促卵泡素100～1000IU，枸橼酸钠10～50mg，枸橼酸0.1～1.0mg，肝素2～10mg，鱼精蛋白2～10mg。
进一步地，所述肝素与鱼精蛋白的终浓度为4mg/ml。
进一步地，所述缓释药物制剂的冻干粉针处方中肝素与鱼精蛋白的比例为1:1，每1ml生理盐水或注射用水中加入促卵泡素100～1000IU，枸橼酸钠10～50mg，枸橼酸0.1～1.0mg，甘露醇50～500mg，肝素4～40mg，鱼精蛋白4～40mg。
进一步地，所述肝素为低分子肝素，其分子量为5000。
本发明具有如下优点：
本发明长效促卵泡素缓释药物制剂中含有具有缓控释作用的低分子肝素(分子量5000)和鱼精蛋白，通过调整低分子肝素和鱼精蛋白比例，可以调节药物释放速率。本发明药物可应用于畜牧业的超数排卵和诱导发情。
【附图说明】
下面参照附图结合实施例对本发明作进一步的说明。
图1是本发明实施例垂体促卵泡素长效注射剂A1-3的体外溶出实验结果曲线图。
图2是本发明实施例垂体促卵泡素长效注射剂B1-3的体外溶出实验结果曲线图。
【具体实施方式】
请参阅图1和图2所示，对本发明的实施例进行详细的说明。
本发明所涉及的一种长效促卵泡素的缓释药物制剂，所述缓释药物制剂的组分如下：促卵泡素、枸橼酸钠-枸橼酸缓冲液、肝素以及鱼精蛋白。所述促卵泡素为垂体促卵泡素，或基因重组促卵泡素。所述缓释药物制剂剂型为注射剂或冻干粉针。所述缓释药物制剂中肝素与鱼精蛋白的比例为10:1至1:10。
所述缓释药物制剂的注射剂处方中肝素与鱼精蛋白的比例为1:1，每1ml生理盐水或注射用水中加入促卵泡素100～1000IU，枸橼酸钠10～50mg，枸橼酸0.1～1.0mg，肝素2～10mg，鱼精蛋白2～10mg。所述肝素与鱼精蛋白的终浓度为4mg/ml。
所述缓释药物制剂的冻干粉针处方中肝素与鱼精蛋白的比例为1:1，每1ml生理盐水或注射用水中加入促卵泡素100～1000IU，枸橼酸钠10～50mg，枸橼酸0.1～1.0mg，甘露醇50～500mg，肝素4～40mg，鱼精蛋白4～40mg。
所述肝素为低分子肝素，其分子量为5000。
以下结合具体实施例对本发明作进一步的说明。
实施例1
基因重组促卵泡素的制备：
将稳定高表达FSH的CHO细胞接入摇瓶，逐步扩增，至密度1×106/ml，记为培养第1天。连续14天后，取细胞培养液，用于纯化表达产物。
将CHO细胞培养液离心，转速9000g，4℃低温离心20min，收集培养上清。采用截留分子量为10kDa膜超滤，收集超滤浓缩液。用Protein A亲和层析柱，对浓缩液纯化，得到高纯度FSH蛋白，纯度约98％，比活性为12000IU/mg。2～8℃储存备用。
实施例2
垂体促卵泡素长效注射剂的制备：
取垂体促卵泡素(购自宁波第二激素厂)100000IU，溶于200ml枸橼酸钠-枸橼酸缓冲液(pH6.8-7.2)中。每1毫升枸橼酸钠-枸橼酸缓冲液(pH6.8-7.2)含枸橼酸钠30mg，枸橼酸0.6mg。取浓度为10mg/ml的低分子肝素(分子量5000)溶液(200、或400、或600ml)，与上述垂体促卵泡素溶液混合。再取浓度为10mg/ml的鱼精蛋白溶液(600、或400、或200ml)，与上述垂体促卵泡素与低分子肝素的溶液混合。补充枸橼酸钠-枸橼酸缓冲液至1000ml。所有配制过程需在百级无菌区内进行。所有溶液均经无菌过滤后使用。在搅拌下，将所得到的溶液分装至西林瓶中，每瓶10ml，得到垂体促卵泡素长效注射剂。
根据所加入低分子肝素与鱼精蛋白的体积，将所得到的制剂命名为垂体促卵泡素长效注射剂A1，A2和A3。2～8℃储存备用。制剂中各组分浓度如下表1所示：
表1垂体促卵泡素长效注射剂A1-3的制备
实施例3
体外溶出实验：
取实施例2得到的制剂A1-3，分别置10ml离心管中，置37℃恒温培养箱内放置，于第6，12，24，36，48，72，96小时离心(3000rpm)，取上清200微升，ELISA方法测定FSH浓度。在每次取样后，离心管内补充200微升枸橼酸钠-枸橼酸缓冲液(pH6.8-7.2)。根据测定结果，得到溶出曲线，如图1所示。由图1可知，制剂A2(肝素与鱼精蛋白用量比例为1:1)的溶出速率明显慢于另外两种，且更接近零级释放速率。
实施例4
低分子肝素与鱼精蛋白用量对溶出速率的影响：
按照实施例2方法制备垂体促卵泡素长效注射剂B1-3。B1-3采用低分子肝素与鱼精蛋白用量比例固定为1:1，所不同的是终浓度，具体见表2。
表2垂体促卵泡素长效注射剂B1-3的制备
按照实施例3方法进行体外溶出实验，结果见图2。由图2可知，溶出速率与肝素与鱼精蛋白的用量呈线性关系，可见随着低分子肝素与鱼精蛋白用量的增加，溶出速率逐渐减小。说明可以通过调整低分子肝素与鱼精蛋白用量，调节溶出速率。
实施例5
重组促卵泡素长效注射剂的制备：
取重组促卵泡素1百万IU，溶于200ml枸橼酸钠-枸橼酸缓冲液(pH6.8-7.2)中。每1毫升枸橼酸钠-枸橼酸缓冲液(pH6.8-7.2)含枸橼酸钠30mg，枸橼酸0.6mg。取浓度为10mg/ml的低分子肝素(分子量5000)溶液400ml，与上述垂体促卵泡素溶液混合。再取浓度为10mg/ml的鱼精蛋白溶液400ml，与上述垂体促卵泡素与低分子肝素的溶液混合。补充枸橼酸钠-枸橼酸缓冲液至1000、4000、10000ml。所有配制过程需在百级无菌区内进行。所有溶液均经无菌过滤后使用。在搅拌下，将所得到的溶液分装至西林瓶中，每瓶1ml，得到重组促卵泡素长效注射剂。
根据最终溶液的体积，将所得到的制剂命名为重组促卵泡素长效注射剂C1-3，如下表3所示。2－8℃储存备用。
表3重组促卵泡素长效注射剂的制备
按照实施例3方法进行体外溶出实验，结果与图1-2中肝素与鱼精蛋白用量比例为1:1的A2及B2的结果类似。说明溶出曲线取决于肝素与鱼精蛋白用量与比例，而与FSH种类及终浓度无关。
实施例6
注射用长效重组垂体促卵泡素的制备：
按照第C1－3号制剂处方配制重组促卵泡素注射剂，在处方中另外加入甘露醇，每制剂单位(即每支注射液)中含甘露醇100、250、500mg。按照常规冻干工艺冷冻干燥，得到冻干粉针D1-3。室温条件保存。
取冻干粉针D1-3，溶于10ml枸橼酸钠-枸橼酸缓冲液。按照实施例3方法进行体外溶出实验。结果与图1-2中肝素与鱼精蛋白用量比例为1:1的A2及B2的结果类似。说明溶出曲线取决于肝素与鱼精蛋白用量与比例，而与FSH剂型无关。
实施例7
体内活性实验
参照《中华人民共和国药典》2010版二部，附录“卵泡刺激素生物测定法”，测定所制备FSH制剂的生物学活性。
(1)实验动物：未成熟Sprague Dawley雌性大鼠，40±2g，19-22日龄，每组8只；
(2)稀释溶液：以1mg/ml BSA溶液溶解中人绒毛膜促性腺激素(hCG)，配制成20IU/ml hCG溶液，混匀备用；
(3)标准品溶液：试验当日，取190IU/瓶FSH标准品1支，用上面稀释溶液配制成高中低三个浓度，分别为：10IU/ml、5IU/ml、2.5IU/ml；
(4)供试品溶液：试验当日，用上述溶剂上面稀释溶液按标示效价将供试品配制成高中低三个浓度，分别为：30IU/ml、15IU/ml、7.5IU/ml；
(5)测定法：在每日大致相同时间，分别给每组大鼠皮下注射相应药物，标准品溶液或供试品溶液0.5ml。标准品溶液连续注射三天，每天一次。供试品仅于试验第一天注射一次，后两天注射稀释溶液。于最后一次注入24h后，处死大鼠，摘出卵巢，剥离附着组织，去除输卵管称重。按照药典(附录ⅩⅣ)生物检定统计法中量反应测定法计算供试品长效FSH生物活性。
所测试制剂为A2、B2、C2、D2，结果说明所有测试的四种制剂，注射一次的体内生物学活性，与连续注射3次的相同剂量的标准品一致。说明这四种制剂均可以发挥FSH缓释作用，可作为长效FSH使用。
本发明长效促卵泡素缓释药物制剂中含有具有缓控释作用的低分子肝素(分子量5000)和鱼精蛋白，通过调整低分子肝素和鱼精蛋白比例，可以调节药物释放速率。本发明药物可应用于畜牧业的超数排卵和诱导发情。
虽然以上描述了本发明的具体实施方式，但是熟悉本技术领域的技术人员应当理解，我们所描述的具体的实施例只是说明性的，而不是用于对本发明的范围的限定，熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化，都应当涵盖在本发明的权利要求所保护的范围内。
Claims (8)
1.一种长效促卵泡素的缓释药物制剂，其特征在于：所述缓释药物制剂的组分如下：促卵泡素、枸橼酸钠-枸橼酸缓冲液、肝素以及鱼精蛋白。
2.根据权利要求1所述的一种长效促卵泡素的缓释药物制剂，其特征在于：所述促卵泡素为垂体促卵泡素，或基因重组促卵泡素。
3.根据权利要求1所述的一种长效促卵泡素的缓释药物制剂，其特征在于：所述缓释药物制剂剂型为注射剂或冻干粉针。
4.根据权利要求3所述的一种长效促卵泡素的缓释药物制剂，其特征在于：所述缓释药物制剂中肝素与鱼精蛋白的比例为10:1至1:10。
5.根据权利要求4所述的一种长效促卵泡素的缓释药物制剂，其特征在于：所述缓释药物制剂的注射剂处方中肝素与鱼精蛋白的比例为1:1，每1ml生理盐水或注射用水中加入促卵泡素100～1000IU，枸橼酸钠10～50mg，枸橼酸0.1～1.0mg，肝素2～10mg，鱼精蛋白2～10mg。
6.根据权利要求5所述的一种长效促卵泡素的缓释药物制剂，其特征在于：所述肝素与鱼精蛋白的终浓度为4mg/ml。
7.根据权利要求4所述的一种长效促卵泡素的缓释药物制剂，其特征在于：所述缓释药物制剂的冻干粉针处方中肝素与鱼精蛋白的比例为1:1，每1ml生理盐水或注射用水中加入促卵泡素100～1000IU，枸橼酸钠10～50mg，枸橼酸0.1～1.0mg，甘露醇50～500mg，肝素4～40mg，鱼精蛋白4～40mg。
8.根据权利要求1～7任一项所述的一种长效促卵泡素的缓释药物制剂，其特征在于：所述肝素为低分子肝素，其分子量为5000。
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