CN111346223A - 用于治疗乙型肝炎的药物制剂及其制备方法和用途 - Google Patents
用于治疗乙型肝炎的药物制剂及其制备方法和用途 Download PDFInfo
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Abstract
本发明涉及一种用于治疗乙型肝炎的药物制剂及其制备方法和用途。该药物制剂包含乙肝表面抗原溶液，进一步包含乙肝核心抗原溶液，更进一步包含寡聚脱氧核苷酸溶液，所述药物制剂不含糖，组成成分简单，无铝佐剂导致的不良反应，抗原稳定性好，在长期稳定性实验中表现出良好的保存效果，可满足疫苗的生产、存储和使用需要，适合治疗性乙肝疫苗的大规模生产使用。
Description
本申请要求2018年12月24日提交的名称为“用于治疗乙型肝炎的药物制剂及其制备方法和用途”，申请号为2018115804704的中国发明专利申请的优先权。
技术领域
本发明属于生物医药领域，涉及一种用于治疗乙型肝炎的药物制剂及其制备方法和用途。
背景技术
乙型肝炎病毒(HBV)感染是世界范围的严重公共卫生问题之一。HBV感染是导致慢性乙型肝炎、肝硬化和肝细胞癌的重要原因(Fattovich G.J.Hepatol.2008；48：335-352)。临床治疗慢性HBV感染的常用药物主要有核苷类似物和干扰素。核苷类似物无法完全清除肝细胞内的cccDNA，且长期使用易导致耐药突变株的出现以及停药后反弹(Kwon H，LokAS.Nat Rev Gastroenterol Hepatol.2011；8：275-284)。干扰素不适合用于无症状的HBV携带者，在慢性HBV患者中，使用半年后HBeAg血清学转换发生率仅33％，而且干扰素副作用较大也限制其应用(Tang SX，Yu GL.Lancet 1990；335(8684)：302)。
目前广泛应用的乙肝蛋白疫苗通过诱导体液免疫，产生保护性中和抗体，达到预防的目的。大量的研究发现中和抗体仅能够消除胞外病毒颗粒，而清除胞内感染的病毒则主要依赖特异性的细胞免疫反应，辅助性T细胞、CD4+T细胞产生的IFN-γ等Th1型细胞因子，尤其是病毒特异性的杀伤性T淋巴细胞(cytotoxic T lymphocyte，CTL)来清除(ChinR，Lacamini S.Rev Med Viorl.2003：13(4)：255-72)。细胞免疫反应的强弱直接决定着乙肝的预后。因此，理想的治疗性乙肝疫苗需要同时诱导特异性的体液免疫和细胞免疫，突破乙肝的免疫耐受。
CN104043120B提供了一种治疗性乙肝疫苗，其包括乙肝表面抗原(HBsAg)、乙肝核心抗原(HBcAg)、寡聚脱氧核苷酸(CpG)，可以突破乙肝的免疫耐受，用于病毒性乙型肝炎，尤其是慢性乙型肝炎的治疗。
传统的乙肝疫苗通常含有氢氧化铝作为佐剂，铝佐剂还能使抗原吸附在其上以减少抗原分子间的相互作用，从而增加抗原的稳定性。但是铝佐剂也有一些不足之处。例如，接种含铝佐剂的疫苗会导致一些不良反应，如红斑、皮下结节、接触性超敏、肉芽肿、肌筋膜炎等。使用CpG佐剂避免了氢氧化铝佐剂的上述不良反应。同时实验出人意料地发现，添加糖类，如蔗糖，并未起到对抗原的保护作用，反而降低了抗原的稳定性，因此如何进一步提高疫苗的抗原稳定性是需要解决的问题。
发明内容
本发明的目的是在现有技术基础上，进一步提供一种抗原稳定性更好的用于治疗乙型肝炎的药物制剂，其在不含氢氧化铝等铝佐剂，且不含糖类的情况下，抗原稳定性好，适合治疗性乙肝疫苗的规模化生产和应用。
为实现上述目的，本发明提供了一种用于治疗乙型肝炎的药物制剂，所述药物制剂含有乙肝表面抗原溶液，其中，乙肝表面抗原溶液含有乙肝表面抗原或其片段和聚山梨酯80。
优选地，所述乙肝表面抗原溶液还含有磷酸盐缓冲剂。
优选地，所述药物制剂还含有乙肝核心抗原溶液，其中，乙肝核心抗原溶液中含有乙肝核心抗原或其片段、磷酸盐缓冲剂和聚山梨酯80。
更优选地，所述药物制剂还含有寡聚脱氧核苷酸溶液，其中，寡聚脱氧核苷酸溶液中含有寡聚脱氧核苷酸、磷酸盐缓冲剂和聚山梨酯80。
优选地，所述磷酸盐缓冲剂包含Na2HPO4和NaH2PO4；更优选地，所述磷酸盐缓冲剂包含Na2HPO4、NaH2PO4和NaCl；进一步优选地，Na2HPO4、NaH2PO4和NaCl的重量比为2:2.4:6～18，优选为2:2.4:6～9，更优选为2:2.4:9。
优选地，所述聚山梨酯80的浓度为0.03％(0.03g/100ml)至0.1％(0.1g/100ml)，优选为0.05％(0.05g/100ml)。
进一步优选地，所述药物制剂不含糖，例如蔗糖。
优选地，所述乙肝表面抗原的氨基酸序列为SEQ ID NO.1；优选地，所述乙肝核心抗原的氨基酸序列为SEQ ID NO.2；优选地，所述寡聚脱氧核苷酸的核苷酸序列选自SEQ IDNO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8或SEQ ID NO.9中的一个或多个，优选为SEQ ID NO.3。
优选地，所述乙肝核心抗原片段由SEQ ID NO.2中的149～183个连续氨基酸，优选为152～183个连续氨基酸组成。
上述药物制剂为注射剂，所述注射剂选自注射液、冻干粉针或无菌分装制剂。
本发明还提供了所述用于治疗乙型肝炎的药物制剂的制备方法，其包括以下步骤：
1)将磷酸盐缓冲剂加水溶解制成磷酸盐缓冲溶液并调节pH值至7.0～7.4，优选为7.2；
2)将聚山梨酯80用步骤1)制得的磷酸盐缓冲溶液稀释，制得聚山梨酯80溶液；
3)将乙肝表面抗原原液加入步骤2)制得的聚山梨酯80溶液，制得乙肝表面抗原溶液；
4)将乙肝核心抗原原液加入步骤2)制得的聚山梨酯80溶液，制得乙肝核心抗原溶液；
5)将寡聚脱氧核苷酸用步骤1)制得的磷酸盐缓冲溶液溶解并稀释为原液，加入步骤2)制得的聚山梨酯80溶液，制得寡聚脱氧核苷酸溶液；
6)将步骤3)制得的乙肝表面抗原溶液、步骤4)制得的乙肝核心抗原溶液和步骤5)制得的寡聚脱氧核苷酸溶液混合后即得所述药物制剂。
本发明还提供了所述治疗乙型肝炎的药物制剂或上述制备方法制备的药物制剂在制备用于治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的药物中的用途。
其中，所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
本发明还提供了上述***的药物制剂或上述制备方法制备的药物制剂在制备用于在对象中产生针对乙型肝炎病毒的体液免疫和细胞免疫应答的药物中的用途。
本发明还提供了上述***的药物制剂或上述制备方法制备的药物制剂在制备用于使对象中抗乙肝核心抗体发生亚型转变的药物中的用途。
优选地，上述药物为治疗性疫苗。
本发明还提供了一种治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的方法，所述方法包括将治疗有效量的上述治疗乙型肝炎的药物制剂或上述制备方法制备的药物制剂给予有此需要的对象；优选地，所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
本发明还提供了一种在对象中产生针对乙型肝炎病毒的体液免疫和细胞免疫应答的方法，所述方法包括将治疗有效量的上述治疗乙型肝炎的药物制剂或上述制备方法制备的药物制剂给予有此需要的对象。
本发明还提供了一种使对象中抗乙肝核心抗体发生亚型转变的方法，所述方法包括将治疗有效量的上述治疗乙型肝炎的药物制剂或上述制备方法制备的药物制剂给予有此需要的对象。
本发明人经过大量筛选和研究，确定了磷酸盐缓冲液和聚山梨酯80作为组成成分能够有效保持用于乙肝治疗性疫苗的药物制剂中的抗原稳定性，且出人意料的是，实验证明在抗原溶液中添加糖类，如蔗糖，不仅没有起到糖类对蛋白质的保护作用，反而会降低抗原的稳定性。因此，本发明提供的药物制剂组成成分简单，不含糖，无需使用铝佐剂且抗原稳定性好，在长期稳定性实验中表现出良好的保存效果，可满足疫苗的生产、存储和使用的需要，适合治疗性乙肝疫苗的大规模生产和使用。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了在不同浓度NaCl的PBS缓冲溶液内，HBsAg制剂在2～8℃、25℃和40℃下放置1个月的稳定性研究结果；
图2显示了HBsAg制剂在40℃下放置的稳定性研究结果；
图3显示了HBsAg制剂在2～8℃下放置的稳定性研究结果；
图4显示了HBsAg对照制剂和含0.05％的Tween 80的制剂在40℃下放置后的HPLC图谱；
图5显示了HBcAg制剂在40℃下放置的稳定性研究结果；
图6显示了HBcAg制剂在2～8℃下放置的稳定性研究结果；
图7显示了各组分独立存储的药物制剂在25℃下放置后HBsAg抗原特异性IFN-γ的分泌水平；
图8显示了各组分独立存储的药物制剂在25℃下放置后HBcAg抗原特异性IFN-γ的分泌水平；
图9显示了各组分独立存储的药物制剂在2～8℃下放置后HBsAg抗原特异性IFN-γ的分泌水平；
图10显示了各组分独立存储的药物制剂在2～8℃下放置后HBcAg抗原特异性IFN-γ的分泌水平；
图11显示了各组分混合存储的药物制剂在25℃下放置后HBsAg抗原特异性IFN-γ的分泌水平；
图12显示了各组分混合存储的药物制剂在25℃下放置后HBcAg抗原特异性IFN-γ的分泌水平；
图13显示了各组分混合存储的药物制剂在2～8℃下放置后HBsAg抗原特异性IFN-γ的分泌水平；
图14显示了各组分混合存储的药物制剂在2～8℃下放置后HBcAg抗原特异性IFN-γ的分泌水平。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
实施例1治疗性乙肝疫苗制剂各组分的制备
1.HBsAg原液的制备：
HBsAg蛋白的氨基酸序列如SEQ ID NO.1所示。
HBsAg蛋白由HBsAg基因重组酵母细胞制备，酵母细胞种类包括汉逊酵母、酿酒酵母和毕赤酵母，优选为汉逊酵母。
具体制备步骤参考中国专利申请CN108330145A，HBsAg基因重组汉逊酵母细胞经发酵培养，收获菌体。经破菌处理和硅胶吸附、柱层析和TFF等步骤纯化，制备HBsAg原液。制备的HBsAg原液要求其纯度大于95％，蛋白含量不低于200μg/ml。宿主DNA残留量、宿主蛋白残留量和其它化学物质残留量均符合药典规定。
2.HBcAg原液的制备：
HBcAg蛋白的氨基酸序列如SEQ ID NO.2所示。
HBcAg蛋白由HBcAg基因重组酵母细胞制备，酵母细胞种类包括汉逊酵母、酿酒酵母和毕赤酵母，优选汉逊酵母。
具体制备步骤参考中国专利申请CN108047316A，HBcAg基因重组汉逊酵母细胞经发酵培养，收获菌体。经破菌处理和硫酸铵、柱层析和TFF等步骤纯化，制备HBcAg原液。制备的HBcAg原液要求其纯度大于95％，蛋白含量不低于200μg/ml。宿主DNA残留量、宿主蛋白残留量和其它化学物质残留量均符合药典规定。
3.寡聚脱氧核苷酸(CpG)的制备：
寡聚脱氧核苷酸是合成制备的寡聚脱氧核苷酸序列片段，其含有一个或多个CpG基序，优选含有18～25个核苷酸，为全硫代修饰的序列，包括：
TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO.3)、GTG CCA GCG TGC GCC ATG(SEQID NO.4)、TCG TCG TTT TGT CGT TTT GTC GTT(SEQ ID NO.5)、TGA CTG TGA ACG TTC GAGATG A(SEQ ID NO.6)、TCG TTC GTT CGT TCG TTC GTT CGT T(SEQ ID NO.7)、TCG TCG TCGTCG TCG TCG TCG(SEQ ID NO.8)或TCC ATG ACG TTC CTG ACG TT(SEQ ID NO.9)等，优选TCG TTC GTT CGT TCG TTC GTT(SEQ ID NO.3)。
具体制备步骤参考中国专利CN104043120B的实施例1，经合成的CpG(SEQ IDNO.3)经冻干制成粉末状CpG原料药，要求其纯度大于90％，含水量小于15％，含量不小于70％。重金属杂质、微生物限度检查符合药典规定。
实施例2治疗性乙肝疫苗制剂的制备
1.PBS缓冲溶液：Na2HPO4·12H2O 2.0g，NaH2PO4·2H2O 2.4g，NaCl 9g，加水溶解并稀释至1000ml，用盐酸或氢氧化钠调节pH值至7.20；
2.0.5％聚山梨酯80(Tween 80)溶液：称取Tween 80 5.0g，用PBS缓冲溶液稀释至1000ml；
3.HBsAg溶液：取HBsAg原液(实施例1制得)，加入0.5％Tween 80溶液，用PBS缓冲溶液稀释Tween 80的浓度至0.05％；
4.HBcAg溶液：取HBcAg原液(实施例1制得)，加入0.5％Tween 80溶液，用PBS缓冲溶液稀释Tween 80的浓度至0.05％；
5.CpG溶液：取CpG(实施例1制得)用PBS缓冲溶液溶解稀释后得CpG原液，加入0.5％Tween 80溶液，用PBS缓冲溶液稀释Tween 80的浓度至0.05％；
6.将步骤3制得的HBsAg溶液、步骤4制得的HBcAg溶液和步骤5制得的CpG溶液混合后即得所述药物制剂。
实施例3治疗性乙肝疫苗制剂处方的筛选试验
在HBsAg、HBcAg和CpG三个组分中，HBsAg受各种因素影响最大，稳定性最差。因此，以HBsAg为指标筛选制剂处方。
HBsAg主要以病毒样颗粒(以下简称VLP)形式存在，HPLC检测主峰面积代表着制剂中VLP的数量。因此，主峰面积的减少代表着制剂中VLP形式的HBsAg量的减少。
1)缓冲体系的筛选
溶液配制：
1.0.5％聚山梨酯80(Tween 80)溶液由实施例2制得；
2.组氨酸缓冲溶液：组氨酸1.55g，盐酸组氨酸2.095g，加水溶解并稀释至1000ml。
3.HBsAg溶液(PBS缓冲溶液)：取HBsAg原液，加入0.5％Tween 80溶液，用PBS缓冲溶液稀释Tween 80的浓度至0.05％。
4.HBsAg溶液(组氨酸缓冲溶液)：取HBsAg原液，经半透膜过夜透析，换液至组氨酸缓冲溶液中，加入0.5％Tween 80溶液，用PBS缓冲溶液稀释Tween 80的浓度至0.05％。
上述两种HBsAg溶液在40℃下放置1个月后进行HPLC检测，结果见表1。
表1制剂缓冲体系筛选试验结果
HPLC检测结果显示，以组氨酸缓冲溶液制备的HBsAg溶液在40℃下放置1个月后，主峰面积下降了36.4％，而PBS缓冲溶液制备的HBsAg溶液在40℃下放置1个月后，主峰面积仅下降了6.1％。因此，选择PBS缓冲溶液作为制剂的缓冲体系。
2)PBS缓冲溶液的筛选
制剂配方使用PBS缓冲溶液，使用盐酸或氢氧化钠调节pH值至7.0～7.4，优选为7.2。
在PBS缓冲溶液中，NaCl用于调节缓冲液的渗透压，对HBsAg蛋白也有保护作用，因此进行试验筛选合适的NaCl浓度，其中，PB为磷酸盐缓冲对，即Na2HPO4·12H2O和NaH2PO4·2H2O。
溶液配制：将下列各组磷酸盐缓冲剂(表2)分别加水溶解并稀释至1000ml，配制为PBS缓冲溶液。取HBsAg原液，经半透膜过夜透析，换液至下列各组PBS缓冲溶液中。
表2 PBS缓冲溶液的条件筛选
上述五种HBsAg溶液在2～8℃、25℃和40℃下放置1个月后进行HPLC检测，结果见图1。
HPLC检测结果显示，当缓冲溶液中NaCl浓度在0～18g/L之间时，随着NaCl浓度的增加，放置1个月后HBsAg溶液主峰面积的下降幅度逐渐减小。2～8℃放置1个月，五种HBsAg溶液主峰面积都没有明显下降；40℃放置1个月，五种HBsAg溶液主峰面积均有明显下降，下降幅度在13.9％～25.4％之间，随着NaCl浓度的增加，主峰面积的下降幅度逐渐减小；25℃放置1个月，NaCl浓度为0g/L和3g/L时主峰面积有明显下降，下降幅度分别为14.8％和18.2％，NaCl浓度在6～18g/L之间时，主峰面积下降幅度不超过3.7％。因此选择PB+6～18g/L NaCl作为制剂的缓冲液，优选6～9g/L，更进一步优选9g/L。
3)辅料筛选
溶液配制：
1.50％蔗糖溶液：称取蔗糖50g，用PBS缓冲溶液稀释至100ml；
2.HBsAg溶液(Tween 80)：取HBsAg原液，加入0.5％Tween 80溶液，用PBS缓冲溶液稀释Tween 80的浓度至0.05％。
3.HBsAg溶液(蔗糖+Tween 80)：取HBsAg原液，加入50％蔗糖溶液和0.5％Tween80溶液，用PBS缓冲溶液稀释蔗糖溶液的浓度至5％、Tween 80的浓度至0.05％。
上述两种HBsAg溶液在40℃下放置1个月后进行HPLC检测，结果见表3。
表3制剂辅料筛选试验结果
HPLC检测结果显示，使用蔗糖+Tween 80制备的HBsAg制剂在40℃下放置1个月后，主峰面积下降了31.6％，而单独使用Tween 80辅料制备的HBsAg制剂，在40℃下放置1个月后，主峰面积仅下降了6.1％。即蔗糖并未起到对抗原的保护作用，反而降低了抗原的稳定性。因此选择Tween80作为制剂的辅料，不仅具有良好的抗原稳定性，还可降低复杂成分潜在的不良反应风险。
实施例4治疗性乙肝疫苗制剂的稳定性实验
1.实验组设置如表4、表5、表6，制备方法同实施例2。
表4 HBsAg溶液中Tween 80浓度的筛选
表5 HBcAg溶液中Tween 80浓度的筛选
表6 CpG溶液中Tween 80浓度的筛选
2.稳定性实验方案
表7制剂稳定性研究设计
对各稳定性研究样品，在规定的取样时间点取样，进行HPLC检测。
3.实验结果
1)HBsAg制剂稳定性结果分析：
将不同处方的HBsAg溶液和HBsAg对照制剂在40℃下共同放置，分别于第7、15和30天取样进行检测。结果显示(见图2和图3)：不添加Tween 80的HBsAg对照制剂的HPLC主峰面积下降最快，在40℃下放置30天，主峰面积下降了23.5％；其次是0.01％Tween 80处方的溶液，下降了15.9％；0.03％～0.1％Tween 80处方的溶液，三者之间下降幅度基本一致，下降了6％左右。
将不同处方的HBsAg溶液和HBsAg对照制剂于2～8℃下共同放置6个月的稳定性数据与在40℃下放置的结果一致。6个月时间点样品相对于零点样品，对照制剂(处方中不含Tween 80)和处方中含0.01％、0.03％、0.05％和0.1％Tween 80处方的溶液，其主峰面积分别下降了15.4％、15.8％、12.2％、6.5％和4.8％。说明Tween 80对HBsAg VLP有保护作用。
图4显示，HBsAg对照制剂在40℃下放置7天就有明显的聚集峰出现，随着放置时间的增加，其聚集峰面积逐渐增大。而0.05％Tween 80处方的制剂在40℃下放置30天都没有明显的聚集峰出现。说明Tween 80保护HBsAg VLP，使其一致地保持单一VLP状态，免于聚集。同时，0.05％Tween 80处方与0.1％Tween 80处方效果相当，减少辅料添加量也能降低潜在的不良反应风险。
2)HBcAg制剂稳定性结果分析：
HBcAg同样主要以VLP形式存在，HPLC检测主峰面积代表着制剂中VLP的数量。因此，主峰面积的减少代表着制剂中VLP形式的HBcAg量的减少。
HPLC结果(见图5和图6)显示HBcAg VLP比较稳定，在2～8℃下放置6个月和40℃放置30天，HPLC主峰面积没有明显变化。
3)CpG制剂稳定性结果分析：
对各稳定性研究样品，在规定的取样时间点取样，进行纯度和相对活性检测。
其中纯度使用RP-UPLC方法检测，相对活性使用细胞法检测，表征CpG溶液与人TLR9受体的结合活性，结果以与参考品(由发明人制备的CpG原料，经过充分结构表征和含量标定，作为参考品，其相对活性值定义为100％)的活性的百分比值表示。
纯度结果和相对活性结果显示(见表8和表9)：CpG溶液比较稳定，在2～8℃下放置6个月和40℃放置30天，纯度和相对活性没有明显变化。
表8 CpG溶液在40℃下放置稳定性的研究结果
表9 CpG溶液在2～8℃下放置稳定性的研究结果
实施例5治疗性乙肝疫苗制剂各组分独立存储的药效验证
1、实验动物：C57BL/6小鼠，雌性，5周龄，160只，8只/组，上海灵畅生物科技有限公司。
2、实验组设置：按照实施例2中步骤1-5所述制备方法分别制得HBsAg溶液、HBcAg溶液和CpG溶液，三种组分独立存储放置，在规定的取样时间点取样(见表10)，取样后按照实施例2中步骤6将HBsAg溶液、HBcAg溶液和CpG溶液三种组分混合，即得所述药物制剂，其分别含有50μg/ml的HBsAg、25μg/ml的HBcAg和25μg/ml的CpG，Tween 80的浓度为0.05％。对该药物制剂进行药效验证。
表10治疗性乙肝疫苗各组分独立存储的药效验证
3、动物免疫：第0天小鼠采用腿部肌肉注射方法进行免疫接种，共免疫1次，接种体积为200μl/只，在免疫后第7天评价细胞免疫水平，在免疫后第28天评价体液免疫水平。
4、细胞免疫：
1)检测步骤：免疫后第7天取脾，按常规方法制备脾淋巴细胞，具体如下：无菌操作取脾脏：用无菌镊子及剪刀剪取脾脏，放于70μm细胞滤网中，置于含有2ml预冷处理的2％FBS(购自GIBCO公司)-PBS的平皿中；用研磨棒研磨脾脏，脾脏细胞通过筛目进入平皿中，得到细胞悬液，用巴氏吸管将悬液放入经40μm细胞滤网过滤(购自BD公司)的50ml无菌离心管；500×g，4℃离心5分钟；弃去上清，加入2ml 1×破红剂(购自BD公司)重悬细胞，4℃避光静置5分钟，以破碎红细胞；加入10ml 2％FBS-PBS终止破红反应；500×g，4℃离心5分钟；弃去上清，加入5ml 2％FBS-PBS重悬细胞备用。分别使用刺激物HBsAg特异性肽库PS4和HBcAg特异性肽库PCP刺激脾细胞；按照试剂盒说明书，使用ELISPOT试剂盒(BD公司)检测HBsAg和HBcAg抗原特异性IFN-γ分泌水平；使用ImmunoSPOT Series 3酶联斑点分析仪读取ELISPOT试剂盒测出的斑点数(具体操作步骤参考中国专利申请CN104043120B的实施例7)。
其中，HBsAg特异性肽库PS4参考中国专利申请CN104043120B的实施例7；HBcAg特异性肽库PCP序列见SEQ ID NO.10～24。
2)评价指标：若无血清培养基孔斑点数≤5SFC，样品孔斑点数≥10SFC，判定为阳性；若5SFC＜无血清培养基孔斑点数≤10SFC，样品孔斑点数/无血清培养基孔斑点数≥2，判定为阳性；若无血清培养基孔斑点数＞10SFC，样品孔斑点数/无血清培养基孔斑点数≥3，判定为阳性。
5、体液免疫：
1)检测步骤：免疫后第28天进行采血，分离血清(全血置于37℃恒温培养箱放置40min,12000rpm，4℃离心10min；吸取上清，冻存于-20℃备用)，按照试剂盒说明书，使用ELISA试剂盒(上海科华)检测产生的HBsAb和HBcAb抗体阳转率。检测设空白对照、阴性对照和待测样品，每种两个平行孔，其中阴性对照为阴性小鼠血清；除空白对照外，各孔分别加入阴性对照或待测样品，再加入酶结合物，混匀封板后37℃孵育30分钟；使用洗涤液洗涤各孔，每孔加入显色剂A液和显色剂B液，混匀封板后37℃孵育15分钟；每孔加入终止液混匀；使用酶标仪读取450nm波长处的各孔OD值。
2)评价指标：HBsAb的参考值cut-off value(COV)＝阴性对照平均OD值×2.1；样本的OD值小于COV值，检测结果为阴性；样本的OD值大于或等于COV值，检测结果为阳性。
HBcAb的参考值cut-off value(COV)＝阴性对照平均OD值×0.3；样本的OD值小于COV值，检测结果为阳性；样本的OD值大于或等于COV值，检测结果为阴性。
6、实验结果：
1)25℃稳定性试验条件：
表11 25℃稳定性试验条件药物制剂的药效结果
结果分析：
细胞免疫水平检测结果：ELISPOT斑点结果见图7和图8，分析结果显示，0-6个月的HBsAg特异性IFN-γ阳转率均为100％，HBcAg特异性IFN-γ阳转率均为100％，均高于70％，细胞免疫检测合格。体液免疫水平检测结果显示：HBsAb阳转率和HBcAb阳转率均在62.5％以上，均高于50％，体液免疫检测结果合格。
2)2～8℃稳定性试验条件：
表12 2～8℃稳定性试验条件药物制剂的药效结果
结果分析：
细胞免疫水平检测结果：ELISPOT斑点结果见图9和图10，分析结果显示，0-12个月的HBsAg特异性IFN-γ阳转率均为100％，HBcAg特异性IFN-γ阳转率高于87.5％，均高于70％，细胞免疫检测合格。体液免疫水平检测结果显示：HBsAb阳转率和HBcAb阳转率均在62.5％以上，均高于50％，体液免疫检测结果合格。
实施例6治疗性乙肝疫苗制剂各组分混合存储的药效验证
1、实验动物：C57BL/6小鼠，雌性，5周龄，128只，8只/组，上海灵畅生物科技有限公司。
2、实验组设置：按照实施例2中的制备方法制得所述药物制剂，其分别含有50μg/ml的HBsAg、25μg/ml的HBcAg溶液和200μg/ml的CpG溶液，Tween 80的浓度为0.05％。存储放置后在规定的取样时间点取样(见表13)，进行药效验证。
表13治疗性乙肝疫苗各组分混合存储的药效验证
3、动物免疫：第0天小鼠采用腿部肌肉注射方法进行免疫接种，共免疫1次，接种体积为200μl/只，在免疫后第7天评价细胞免疫水平，在免疫后第28天评价体液免疫水平。
4、细胞免疫：检测步骤及评价指标同实施例5。
5、体液免疫：检测步骤及评价指标同实施例5。
6、实验结果：
1)25℃稳定性试验条件：
表14 25℃稳定性试验条件下药物制剂的药效结果
结果分析：
细胞免疫水平检测结果：ELISPOT斑点结果见图11和图12，分析结果显示，0-3个月的HBsAg特异性IFN-γ阳转率均为100％，HBcAg特异性IFN-γ阳转率高于75％，均高于70％，细胞免疫检测合格。体液免疫水平检测结果显示：HBsAb阳转率和HBcAb阳转率均在62.5％以上，均高于50％，体液免疫检测结果合格。
2)2～8℃稳定性试验条件：
表15 2～8℃稳定性试验条件下药物制剂的药效结果
结果分析：
细胞免疫水平检测结果：ELISPOT斑点结果见图13和图14，分析结果显示，0-6个月的HBsAg特异性IFN-γ阳转率均为100％，HBcAg特异性IFN-γ阳转率均为100％，均高于70％，细胞免疫检测合格。体液免疫水平检测结果显示：HBsAb阳转率和HBcAb阳转率均在62.5％以上，均高于50％，体液免疫检测结果合格。
综上所述，本发明提供的用于治疗乙型肝炎的药物制剂不含糖，在使用CpG佐剂避免氢氧化铝佐剂造成的不良反应的同时，通过加入适量的聚山梨酯80和磷酸盐缓冲剂，使得整个处方成分简单，抗原稳定性好，在长期稳定性实验中表现出良好的保存效果，满足疫苗的生产、存储和使用的需要，适合治疗性乙肝疫苗的大规模生产使用，具有广阔的市场前景。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，所述修改和改变应归属于权利要求书的范围。
序列表
<110> 南京远大赛威信生物医药有限公司
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Claims (18)
1.一种治疗乙型肝炎的药物制剂，所述药物制剂含有乙肝表面抗原溶液，其中，乙肝表面抗原溶液含有乙肝表面抗原或其片段和聚山梨酯80。
2.如权利要求1所述的药物制剂，所述乙肝表面抗原溶液还含有磷酸盐缓冲剂。
3.如权利要求1-2任一项所述的药物制剂，所述药物制剂还含有乙肝核心抗原溶液，其中，乙肝核心抗原溶液中含有乙肝核心抗原或其片段，磷酸盐缓冲剂和聚山梨酯80。
4.如权利要求1-3任一项所述的药物制剂，所述药物制剂还含有寡聚脱氧核苷酸溶液，其中，寡聚脱氧核苷酸溶液中含有寡聚脱氧核苷酸，磷酸盐缓冲剂和聚山梨酯80。
5.如权利要求1-4任一项所述的药物制剂，其中，所述磷酸盐缓冲剂包含Na2HPO4和NaH2PO4；更优选地，所述磷酸盐缓冲剂包含Na2HPO4、NaH2PO4和NaCl；进一步优选地，Na2HPO4、NaH2PO4和NaCl的重量比为2:2.4:6～18，优选为2:2.4:6～9，更优选为2:2.4:9。
6.如权利要求1-4任一项所述的药物制剂，其中，所述聚山梨酯80的浓度为0.03g/100ml至0.1g/100ml，优选为0.05g/100ml。
7.如权利要求1-4任一项所述的药物制剂，其中，所述药物制剂不含糖，例如蔗糖。
8.如权利要求1-7任一项所述的药物制剂，其中所述乙肝表面抗原的氨基酸序列为SEQID NO.1；优选地，所述乙肝核心抗原的氨基酸序列为SEQ ID NO.2；优选地，所述寡聚脱氧核苷酸的核苷酸序列选自SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ IDNO.7、SEQ ID NO.8或SEQ ID NO.9中的一个或多个，优选为SEQ ID NO.3。
9.如权利要求8所述的药物制剂，其中，所述乙肝核心抗原片段由SEQ ID NO.2中的149～183个连续氨基酸，优选为152～183个连续氨基酸组成。
10.如权利要求1-9任一项所述的药物制剂，其中，所述药物制剂为注射剂，所述注射剂选自注射液、冻干粉针或无菌分装制剂。
11.如权利要求1-10任一项所述的药物制剂的制备方法，其包括以下步骤：
1)将磷酸盐缓冲剂加水溶解制成磷酸盐缓冲溶液并调节pH值至7.0～7.4，优选为7.2；
2)将聚山梨酯80用步骤1)制得的磷酸盐缓冲溶液稀释，制得聚山梨酯80溶液；
3)将乙肝表面抗原原液加入步骤2)制得的聚山梨酯80溶液，制得乙肝表面抗原溶液；
4)将乙肝核心抗原原液加入步骤2)制得的聚山梨酯80溶液，制得乙肝核心抗原溶液；
5)将寡聚脱氧核苷酸用步骤1)制得的磷酸盐缓冲溶液溶解并稀释为原液，加入步骤2)制得的聚山梨酯80溶液，制得寡聚脱氧核苷酸溶液；
6)将步骤3)制得的乙肝表面抗原溶液、步骤4)制得的乙肝核心抗原溶液和步骤5)制得的寡聚脱氧核苷酸溶液混合后即得所述药物制剂。
12.如权利要求1至10中任一项所述的治疗乙型肝炎的药物制剂或权利要求11所述的制备方法制备的药物制剂在制备用于治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的药物中的用途；优选地，所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
13.如权利要求1至10中任一项所述的治疗乙型肝炎的药物制剂或权利要求11所述的制备方法制备的药物制剂在制备用于在对象中产生针对乙型肝炎病毒的体液免疫和细胞免疫应答的药物中的用途。
14.如权利要求1至10中任一项所述的治疗乙型肝炎的药物制剂或权利要求11所述的制备方法制备的药物制剂在制备用于使对象中乙肝核心抗体发生亚型转变的药物中的用途。
15.如权利要求12至14中任一项所述的用途，其中所述药物为治疗性疫苗。
16.一种治疗乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病的方法，所述方法包括将治疗有效量的如权利要求1-10中任一项所述的治疗乙型肝炎的药物制剂或权利要求11所述的制备方法制备的药物制剂给予有此需要的对象；优选地，所述乙型肝炎病毒感染和/或乙型肝炎病毒介导的疾病选自乙型肝炎、肝硬化和肝癌。
17.一种在对象中产生针对乙型肝炎病毒的体液免疫和细胞免疫应答的方法，所述方法包括将治疗有效量的如权利要求1-10中任一项所述的治疗乙型肝炎的药物制剂或权利要求11所述的制备方法制备的药物制剂给予有此需要的对象。
18.一种使对象中抗乙肝核心抗体发生亚型转变的方法，所述方法包括将治疗有效量的如权利要求1-10中任一项所述的治疗乙型肝炎的药物制剂或权利要求11所述的制备方法制备的药物制剂给予有此需要的对象。
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