CN112316153B - 一种抗肿瘤的药物组合物及其应用 - Google Patents
一种抗肿瘤的药物组合物及其应用 Download PDFInfo
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Abstract
本发明涉及及生物制药技术领域,具体公开了一种抗肿瘤的药物组合物及其应用,本发明所述药物组合物包括阿伦磷酸钙和外源基因,本发明制备的药物组合物安全性高,可以降低人卵巢癌细胞SKOV3细胞的存活率,降低了肿瘤细胞的生长,而对人肾小管上皮细胞HK2的毒性较弱。
Description
技术领域
本发明涉及生物制药技术领域,尤其是涉及一种抗肿瘤的药物组合物及其应用。
背景技术
随着基因治疗的不断发展,在基因导入载体方面出现了两大主流:一是非病毒载体系统;二是病毒载体系统。在基因治疗领域中,病毒载体是最为常见的一类基因载体,大约70%的治疗方案采用了病毒载体,病毒载体主要包括各种逆转录病毒、腺病毒,腺相关病毒(AAV),疱疹病毒、痘病毒等。此类病毒载体是利用病毒对宿主细胞的感染将外源基因导入宿主细胞内。病毒载体具有较高的转染效率,但是病毒载体装载基因容量有限,并且具有较强的免疫原性,存在潜在的生物安全性隐患。
目前常用的非病毒基因载体主要包括阳离子脂质体以及各种阳离子多聚物材料,比如聚乙烯亚胺、多聚赖氨酸、鱼精蛋白和壳聚糖等。此类投递系统结合DNA分子的基本原理是:依靠本身结构中的大量阳性基团(氨基)产生的强大正电性,通过与DNA上带负电的磷酸根进行电性结合,从而形成纳米级的正电性颗粒。这种递送系统具有本身的优势:(1)电性结合,浓缩DNA分子,所以基因装载容量大;(2)通过调节材料与DNA分子的比例,实现基因递送颗粒粒径和电性的可控性;(3)形成的基因递送颗粒带正电,利于通过电性作用与靶细胞膜结合,提高了导入效率和靶向性。
膀胱是暂时储存尿液的肌性囊,膀胱壁由粘膜、粘膜下膜、肌层和外膜组成,外膜在膀胱上面为浆膜,其余部分为纤维膜,肌层由平滑肌纤维构成,在尿道内口处环形成膀胱括约肌,粘膜主要由尿路上皮细胞组成。膀胱癌是指发生在膀胱黏膜上的恶性肿瘤,是泌尿系统常见的恶性肿瘤。膀胱灌注药物或者全身应用抗体治疗是膀胱癌常见的治疗方式,但是膀胱内灌注的药物会随着尿液排出体外,不能在体内长时间滞留,起到持久的抗肿瘤疗效。因此,还有改善的空间。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种抗肿瘤的药物组合物及其应用,本发明的药物组合物安全性高,可以抑制肿瘤细胞的生长,并且Luciferase基因可以有效检测外源基因分泌蛋白质表达量的情况,使得外源基因可以达到治疗或预防膀胱疾病。
为实现上述目的,本发明采取的技术方案为:
本发明的第一目的是提供一种抗肿瘤的药物组合物,所述药物组合物包括阿伦磷酸钙和外源基因。
在本发明的技术方案中,本发明人首次将阿伦磷酸钙和外源基因复配形成药物组合物,意外发现阿伦磷酸钙可作为外源基因的载体,由于阿伦磷酸钙上有氨基,可通过化学反应在阿伦磷酸钙表面修饰靶向肿瘤或者其他组织的活性基团,使得药物组合物具有发挥抗肿瘤的效果,在试验例一中将药物组合物分别与SKOV3及HK2细胞培养48h后细胞存活率实验中,可以明确得知药物组合物可以降低人卵巢癌细胞SKOV3及人肾小管上皮细胞HK2细胞的存活率,显著降低了肿瘤细胞的生长,而对人肾小管上皮细胞HK2的毒性较弱。
作为本发明所述抗肿瘤的药物组合物的优选实施方式,所述外源基因包括微环DNA、质粒DNA、siRNA中的至少一种。
在一些实施例中,外源基因不仅仅局限于微环DNA、质粒DNA和siRNA中的一种,还可以是各种形式的外源核酸物质。
作为本发明所述抗肿瘤的药物组合物的优选实施方式,所述外源基因为微环DNA。
作为本发明所述抗肿瘤的药物组合物的优选实施方式,所述微环DNA编码抗EpCAM/CD3双特异性抗体基因。
EpCAM为多数上皮来源肿瘤细胞表面高表达的基因,CD3为T细胞表面特异性表达抗原。抗EpCAM/CD3双特异性抗体可以特异性结合T细胞表面CD3抗原和肿瘤细胞表面EpCAM抗原,特异性激活T细胞,释放干扰素、白介素、颗粒酶和肿瘤坏死因子等杀伤肿瘤细胞。
本发明的第二目的是提供一种上述药物组合物在制备抗肿瘤药物中的应用。
作为本发明所述应用的优选实施方式,所述抗肿瘤药物制成片剂、丸剂、粉剂、袋装剂、胶囊、混悬剂、乳剂、溶液剂、糖浆剂、气溶胶、注射用无菌溶液或无菌粉剂。
作为本发明所述抗肿瘤的药物组合物的优选实施方式,所述阿伦磷酸钙的制备包括以下步骤:
将可溶性钙盐溶解于蒸馏水中形成可溶性钙盐的水溶液,将阿伦磷酸盐溶解于蒸馏水中形成阿伦磷酸盐的水溶液,将可溶性钙盐的水溶液加入至阿伦磷酸盐的水溶液中充分混合形成混合物,所述可溶性钙盐中的钙离子与阿伦磷酸盐的摩尔比为(2~4):(4~7),加热得阿伦磷酸钙粗产物,洗涤并干燥,制得阿伦磷酸钙。
在一些实施例中,阿伦磷酸钠可以替换为其他双磷酸盐,例如依替膦酸钠、氯膦酸钠、伊班膦酸钠、唑来膦酸等。在本发明的技术方案中,CaCl2·2H2O与阿伦磷酸钠的质量比为特定比例,使得制备的阿伦磷酸钙质量较佳,外观形状较佳。
本发明的第三目的是提供一种抗肿瘤制剂,包括上述的药物组合物。
在一些实施例中,本发明提供的药物组合物可根据常规方法制成抗肿瘤制剂,抗肿瘤制剂可以是片剂、丸剂、粉剂、袋装剂、胶囊、混悬剂、乳剂、溶液剂、糖浆剂、气溶胶、软和硬明胶胶囊、注射用无菌溶液、无菌粉剂等形式,抗肿瘤制剂还可以包括阳离子脂质体、阳离子多聚物、磷酸钙、二氧化硅和金纳米棒等。
作为本发明所述抗肿瘤制剂的优选实施方式,所述抗肿瘤制剂中所述外源基因的质量浓度为0.1~0.3μg/μl。
作为本发明所述抗肿瘤制剂的优选实施方式,所述抗肿瘤制剂中所述阿伦磷酸钙的质量浓度为10~100mg/ml。
在本发明的技术方案中,通过在抗肿瘤制剂中设置阿伦磷酸钙的浓度为10~100mg/ml,外源基因的质量浓度为0.1~0.3μg/μl,可以较好的抑制人卵巢癌细胞SKOV3及人肾小管上皮细胞HK2的活性,使药物组合物可以有效抑制肿瘤细胞的生长。
通过动物实验发现,将药物组合物制成制剂通过肌肉注射、皮下注射、皮内注射、腹腔注射等方式导入至小鼠体内,经过24h和48h后,发现Luciferase基因检测外源基因分泌的蛋白质表达量较多,其中经过48h的Luciferase基因检测外源基因分泌的蛋白质表达量多于经过24h的Luciferase基因检测外源基因分泌的蛋白质表达量,本发明外源基因选择为中国专利CN106810610A的抗EpCAM和CD3特异性靶向抗体基因。
与现有技术相比,本发明具有以下有益效果:
本发明提供了一种抗肿瘤的药物组合物,本发明的药物组合物包括阿伦磷酸钙和外源基因,有效降低人卵巢癌细胞SKOV3细胞的存活率,降低了肿瘤细胞的生长,并且对人肾小管上皮细胞HK2的毒性较弱。
附图说明
图1为本发明制备的阿仑磷酸钙透射电子显微镜示意图;
图2为本发明制备的阿仑磷酸钙元素分布图;
图3为本发明制备的药物组合物与人卵巢癌细胞SKOV3及人肾小管上皮细胞HK2共培养48h后的细胞存活率示意图;
图4为本发明制备的药物组合物在小鼠膀胱内的转染图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合附图和具体实施例对本发明作进一步说明。
在本发明的实施例和试验例中,小鼠来源于北京维通利华实验动物技术有限公司,其小鼠的年龄为6-8周龄。
在本发明的实施例和试验例中,抗EpCAM/CD3特异性靶向抗体为中国专利CN106810610A的特异性双靶向抗体。
实施例1
一种抗肿瘤的药物组合物,包括0.1mg阿伦磷酸钙和50μl抗EpCAM/CD3双特异性抗体基因。
其中阿伦磷酸钙的制备包括以下步骤:将0.1gCaCl2·2H2O溶解在40mL蒸馏水中形成可溶性钙盐的水溶液,将0.33g阿伦磷酸钠溶解于40mL蒸馏水中形成阿伦磷酸钠的水溶液,将可溶性钙盐的水溶液加入至阿伦磷酸钠的水溶液中充分混合形成混合物,使用NaOH溶液将混合物的pH值调到5.5,在温度为100℃条件下微波加热10min,制得白色的阿伦磷酸钙粗产物;将阿伦磷酸钙粗产物用蒸馏水洗涤1次,无水乙醇洗涤2次,在60℃烘箱中烘干24小时,制得阿伦磷酸钙,干燥备用。
在本实施例中,将制备的抗肿瘤的药物组合物与药学上常用的辅料或/载体制备得抗肿瘤制剂,其中辅料可以包括蒸馏水、生理盐水、葡萄糖等,在抗肿瘤制剂中抗EpCAM/CD3双特异性抗体基因的质量浓度为0.2μg/μl,阿伦磷酸钙的质量浓度为100mg/ml。
实施例2
一种抗肿瘤的药物组合物,包括0.2mg阿伦磷酸钙和50μl抗EpCAM/CD3双特异性抗体基因。
其中阿伦磷酸钙的制备包括以下步骤:将0.1gCaCl2·2H2O溶解在40mL蒸馏水中形成可溶性钙盐的水溶液,将0.4g阿伦磷酸钠溶解于40mL蒸馏水中形成阿伦磷酸钠的水溶液,将可溶性钙盐的水溶液加入至阿伦磷酸钠的水溶液中充分混合形成混合物,使用用NaOH溶液将混合物的pH值调到4.5,在温度为80℃条件下微波加热10min,制得白色的阿伦磷酸钙粗产物;将阿伦磷酸钙粗产物用蒸馏水洗涤1次,无水乙醇洗涤2次,在60℃烘箱中烘干24小时,制得阿伦磷酸钙,干燥备用。
在本实施例中,将制备的抗肿瘤的药物组合物与药学上常用的辅料或/载体制备得抗肿瘤制剂,其中辅料可以包括蒸馏水、生理盐水、葡萄糖等,在抗肿瘤制剂中抗EpCAM/CD3双特异性抗体基因的质量浓度为0.1μg/μl,阿伦磷酸钙的质量浓度为10mg/ml。
实施例3
一种抗肿瘤的药物组合物,包括0.1mg阿伦磷酸钙和60μl抗EpCAM/CD3双特异性抗体基因。
其中阿伦磷酸钙的制备包括以下步骤:将0.2gCaCl2·2H2O溶解在40mL蒸馏水中形成可溶性钙盐的水溶液,将0.45g阿伦磷酸钠溶解于40mL蒸馏水中形成阿伦磷酸钠的水溶液,将可溶性钙盐的水溶液加入至阿伦磷酸钠的水溶液中充分混合形成混合物,使用用NaOH溶液将混合物的pH值调到6.0,在温度为95℃条件下微波加热10min,制得白色的阿伦磷酸钙粗产物;将阿伦磷酸钙粗产物用蒸馏水洗涤1次,无水乙醇洗涤2次,在60℃烘箱中烘干24小时,制得阿伦磷酸钙,干燥备用。
在本实施例中,将制备的抗肿瘤的药物组合物与药学上常用的辅料或/载体制备得抗肿瘤制剂,其中辅料可以包括蒸馏水、生理盐水、葡萄糖等,在抗肿瘤制剂中抗EpCAM/CD3双特异性抗体基因的质量浓度为0.2μg/μl,阿伦磷酸钙的质量浓度为50mg/ml。
实施例4
一种抗肿瘤的药物组合物,包括0.15mg阿伦磷酸钙和50μl抗EpCAM/CD3双特异性抗体基因。
其中阿伦磷酸钙的制备包括以下步骤:将0.2gCaCl2·2H2O溶解在40mL蒸馏水中形成可溶性钙盐的水溶液,将1.0g阿伦磷酸钠溶解于40mL蒸馏水中形成阿伦磷酸钠的水溶液,将可溶性钙盐的水溶液加入至阿伦磷酸钠的水溶液中充分混合形成混合物,使用用NaOH溶液将混合物的pH值调到6.5,在温度为110℃条件下微波加热10min,制得白色的阿伦磷酸钙粗产物;将阿伦磷酸钙粗产物用蒸馏水洗涤1次,无水乙醇洗涤2次,在60℃烘箱中烘干24小时,制得阿伦磷酸钙,干燥备用;
在本实施例中,将制备的抗肿瘤的药物组合物与药学上常用的辅料或/载体制备得抗肿瘤制剂,其中辅料可以包括蒸馏水、生理盐水、葡萄糖等,在抗肿瘤制剂中抗EpCAM/CD3双特异性抗体基因的质量浓度为0.3g/μl,阿伦磷酸钙的质量浓度为20mg/ml。
其中,实施例1制备的阿伦磷酸钙的透射电子显微镜示意图如图1所示,从图1可知,阿伦磷酸钙为一维棒状结构,棒的直径约为0.1微米,长度为1微米至20微米不等,有些棒聚集在一起形成纳米簇结构。
实施例2-4制备的阿伦磷酸钙在透射电子显微镜下其性状与实施例1制备的阿伦磷酸钙性状相似。
此外,实施例1制备的阿伦磷酸钙元素分布图参考图2,实施例2-4制备的阿伦磷酸钙元素分布图与实施例1类似。
对比例1
与实施例1相同,抗肿瘤的药物组合物包括0.1mg阿伦磷酸钠和50μl抗EpCAM和CD3双特异性抗体基因,其他参数与实施例1相同。
试验例一、本发明的药物组合物对人卵巢癌细胞SKOV3及肾小管上皮细胞HK2的影响
将实施例1-4及对比例1中制备的药物组合物分别与人卵巢癌细胞SKOV3及人肾小管上皮细胞HK2共培养,48小时后由CCK8测定细胞存活率,结果见图3所示。
根据图3,当药物组合物中的阿伦磷酸钙的浓度为10~20mg/ml时,人卵巢癌细胞SKOV3及人肾小管上皮细胞HK2存活率迅速下降,且人卵巢癌细胞SKOV3的存活率低于肾小管上皮细胞HK2存活率;当药物组合物中的阿伦磷酸钙的浓度为20~100mg/ml时,人卵巢癌细胞SKOV3与肾小管上皮细胞HK2的存活率缓慢下降,当药物组合物中的阿伦磷酸钙的浓度为100mg/ml时,人卵巢癌细胞SKOV3的存活率低于肾小管上皮细胞HK2的存活率,综上说明,药物组合物可以抑制人卵巢癌细胞SKOV3与肾小管上皮细胞HK2的存活,有效控制肿瘤细胞的数量。
对比例1中将阿伦磷酸钙替换成阿伦磷酸钠时,则不能抑制人卵巢癌细胞SKOV3与肾小管上皮细胞HK2的存活,效果不及实施例1-4抗肿瘤的效果。
试验例二、本发明制备的药物组合物在小鼠膀胱内转染实验
取实施例1药物组合物中的阿伦磷酸钙0.1mg溶解于50ul PBS缓冲液中,向PBS缓冲液中添加50μl Luciferase基因,将溶解的阿伦磷酸钙与Luciferase基因充分混合,然后通过膀胱注射递送至小鼠体内,在6小时和24小时后,将小鼠麻醉,通过小动物成像仪观察Luciferase基因检测外源基因分泌的蛋白质在小鼠体内的表达情况,本试验例中使用的外源基因为中国专利CN106810610A的抗EpCAM/CD3双特异性抗体基因,结果见图4。
根据图4可知,采用实施例1的药物组合物在小鼠膀胱内Luciferase基因检测外源基因分泌的蛋白质表达量较多,在48h后Luciferase基因检测外源基因分泌的蛋白质表达量高于经过24h的Luciferase基因检测外源基因分泌的蛋白质表达量,本发明实施例2-4中Luciferase基因检测外源基因分泌的蛋白质表达量的结果与实施例1中Luciferase基因检测外源基因分泌的蛋白质表达量的结果类似。
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 (4)
1.一种抗肿瘤的药物组合物在制备抑制人卵巢癌细胞SKOV3及人肾小管上皮细胞HK2药物中的应用,其特征在于,所述药物组合物包括阿仑膦酸钙和外源基因;所述外源基因为微环DNA;所述微环DNA编码抗EpCAM/CD3双特异性抗体基因;
所述阿仑膦酸钙的制备方法包括以下步骤:
将可溶性钙盐溶解于蒸馏水中形成可溶性钙盐的水溶液,将阿伦磷酸盐溶解于蒸馏水中形成阿伦磷酸盐的水溶液,将可溶性钙盐的水溶液加入至阿伦磷酸盐的水溶液中充分混合形成混合物,所述可溶性钙盐中的钙离子与阿伦磷酸盐的摩尔比为(2~4):(4~7),加热得阿仑膦酸钙粗产物,洗涤并干燥,制得阿仑膦酸钙。
2.如权利要求1所述的应用,其特征在于,所述抗肿瘤药物制成片剂、丸剂、粉剂、胶囊、乳剂、溶液剂或糖浆剂。
3.如权利要求1所述的应用,其特征在于,所述药物组合物制成抗肿瘤制剂,所述抗肿瘤制剂中所述外源基因的质量浓度为0.1~0.3μg/μl。
4.如权利要求3所述的应用,其特征在于,所述抗肿瘤制剂中所述阿仑膦酸钙的质量浓度为10~100mg/ml。
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