CN116687954A - 一种用于治疗皮肤恶性黑色素瘤的组合物 - Google Patents
一种用于治疗皮肤恶性黑色素瘤的组合物 Download PDFInfo
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Abstract
本发明提供一种用于治疗皮肤恶性黑色素瘤的组合物，其中包含有达卡巴嗪和壳寡糖；本发明所提供的组合物用于制备用于治疗皮肤恶性黑色素瘤的制品。本发明的组合物通过联合用药，与对照组相比显著降低达卡巴嗪的用药量，提高了达卡巴嗪的药物敏感性。
Description
技术领域
本发明属于肿瘤治疗组分筛选制备技术领域，具体涉及一种用于治疗皮肤恶性黑色素瘤的组合物。
背景技术
黑色素瘤是一种由异常黑素细胞过度增殖引起的皮肤黏膜肿瘤。皮肤恶性黑色素瘤是一种高度恶性、高度侵袭性的皮肤肿瘤。目前，治疗黑色素瘤的方法主要有手术切除、化疗、靶向治疗等。虽然黑色素瘤占所有皮肤癌的5％，但它占皮肤癌死亡的75％。主要是因为皮肤恶性黑色素瘤在早期易发生转移，手术切除预后效果较差。转移性黑色素瘤患者的5年生存率仅为23％。
迄今为止，近十几种黑色素瘤的新疗法/治疗方案已获FDA批准，包括4种全身免疫治疗/组合(易普利姆玛、纳武单抗、派姆单抗、伊匹单抗-纳武单抗联合用药)等。虽然靶向性和免疫疗法延长了生存期，但大多数患者都出现了治疗耐药性。由于毒性、内在耐药性和其他耐药性不完全清楚的原因，60-70％的黑色素瘤患者对检查点抑制剂治疗没有反应。对于没有BRAF基因突变的患者，无法使用BRAF抑制剂单药、BRAF-MEK抑制剂联合治疗进行治疗。目前，针对晚期黑色素瘤，FDA批准使用药物只有达卡巴嗪(DTIC)。
达卡巴嗪是烷化剂，通过将烷基引入鸟嘌呤碱基来破坏DNA，最终通过细胞凋亡和其他细胞死亡机制导致细胞死亡。给药后，达卡巴嗪在肝脏中去甲基化为3-甲基-(三氮杂-1-基)咪唑-4-甲酰胺(MTIC)，随后转化为重氮甲烷。达卡巴嗪已成为治疗晚期黑色素瘤的“护理标准”基准。但是临床试验表明达卡巴嗪部分反应(PR)率低，仅为15-28％，完全反应(CR)率约为3-5％，并且很少有小于2％的持久响应。达卡巴嗪治疗转移性黑色素瘤患者的随机III期试验表明，达卡巴嗪治疗的患者中位总生存期为6.4个月，达卡巴嗪治疗的患者总体反应率为12.1％。据文献报道，在达卡巴嗪治疗中加入化疗药物卡莫司汀并无临床优势，并未提高患者的总体生存期。
黑色素瘤的发展可引起全身炎症反应、自身免疫反应和肝脏中毒性损伤。因此，生物化疗被广泛用于治疗黑色素瘤，即化疗与免疫治疗相结合，增强体内的免疫反应。联合用药提高化疗敏感性将会是黑色素瘤治疗的有效手段。
发明内容
本发明的目的是提供一种用于治疗皮肤恶性黑色素瘤的组合物，从而克服达卡巴嗪(DTIC)在目前临床治疗皮肤恶性黑色素瘤中存在的缺陷。
本发明首先提供一种用于治疗皮肤恶性黑色素瘤的组合物，其中包含有达卡巴嗪(DTIC)和壳寡糖；
所述的壳寡糖，其分子量为<1000Da；
所述的组合物中，达卡巴嗪(DTIC)(分子量：182.18)和壳寡糖的质量比为1:1.5-8，优选质量比为1：5。
本发明所提供的组合物用于制备用于治疗皮肤恶性黑色素瘤的制品；
所述的制品，为药品或功能性食品。
本发明的组合物通过联合用药，与对照组相比显著降低达卡巴嗪的用药量，提高了达卡巴嗪的药物敏感性。
附图说明
图1：黑色素瘤小鼠造模过程图，其中将B16细胞植入C57BL/6J小鼠皮下，然后在指定时间点用COS-3和/或DTIC治疗。
图2：COS-3和/或DTIC治疗14天的肿瘤图像；
图3：COS-3和/或DTIC治疗14天的肿瘤体积和大小统计图；
图4：在14天治疗期间各组小鼠的体重图；
图5：MTT法检测COS-3对B16细胞的细胞毒性图，其中(±s，n＝3)。与对照组相比，*P<0.05，**P<0.01。
图6：MTT法检测COS-3与DTIC联合用药对B16细胞的细胞毒性图；
图7：死活细胞染色检测COS-3与DTIC联合用药对B16细胞的衰老情况的影响图；
图8：Ki67染色检测COS-3与DTIC联合用药对B16细胞的增殖情况的影响图，其中(±s，n＝3)，与对照组相比，*P<0.05，**P<0.01。
图9：划痕实验检测COS-3与DTIC联合用药对B16细胞的侵袭情况的影响图，(±s，n＝3)。与对照组相比，*P<0.05，**P<0.01；与COS-3组相比，其中#P<0.05，##P<0.01，与DTIC组相比，&P<0.05，&&P<0.01。
具体实施方式
本发明使用海洋天然产物壳寡糖，发现其能有效提高黑色素瘤细胞对达卡巴嗪的化疗敏感性。当壳寡糖与达卡巴嗪联合使用时，能有效抑制黑色素瘤细胞增殖。
本发明建造黑色素瘤小鼠模型，体内实验观察COS-3(本实验采用的样品主要是有聚合度为2或3壳寡糖组成，分子量为<1000Da，其脱乙酰度>90％)与达卡巴嗪(DTIC)联合用药对黑色素瘤小鼠肿瘤的影响。同时，以体外培养小鼠恶性黑色素瘤细胞株B16为研究对象，从细胞增殖、侵袭、迁移等方面，探究COS-3以及与达卡巴嗪联合用药对B16细胞生物学行为的影响，进一步从分子机制上探讨其抗肿瘤机理。
实施例1：COS-3、DTIC单独用药以及COS-3与DTIC联合用药对黑色素瘤小鼠肿瘤体积的影响
实验动物分组：对照组小鼠腹腔注射100μL 0.9％生理盐水2周；达卡巴嗪高剂量组小鼠腹腔注射达卡巴嗪(给药体积为100μL，药物终剂量为80mg/kg)；达卡巴嗪低剂量小鼠腹腔注射达卡巴嗪(给药体积为100μL，药物终剂量为40mg/kg)；COS-3组小鼠灌胃COS-3溶液(给药体积为200μL，药物终剂量为200mg/kg)；联合给药组小鼠腹腔注射达卡巴嗪(给药体积为100μL，药物终剂量为40mg/kg)并灌胃COS-3溶液(给药体积为200μL，药物终剂量为200mg/kg)。所有小鼠每日给药一次，共计持续14天。
通过评估COS-3和DTIC联合治疗在体内的抗肿瘤作用，证明了COS-3和/或DTIC在黑色素瘤异种移植模型中的疗效。
当平均肿瘤直径达到5mm，将老鼠分成五组：对照组、DTIC(80mg/kg)、DTIC(40mg/kg)、COS-3(200mg/kg)，以及DTIC(40mg/kg)和COS-3(200mg/kg)联合给药治疗组。
在整个研究过程中，每2天使用卡尺测量肿瘤的长度和宽度，并给小鼠称重，实验共计给药14天。
黑色素瘤小鼠造模过程如图1所示。与对照组相比，COS-3组、DTIC(80mg/kg)组和联合治疗组的肿瘤生长和体积显著降低，如图2与图3所示；并且，联合用药(DITC(40mg/kg)+COS-3(200mg/kg))组与达卡巴嗪组(DITC 80mg/kg)组肿瘤体积没有显著差异，这说明联合给药可以减少DTIC的使用用量，提高DTIC的疗效。其中联合用药(DITC(40mg/kg)+COS-3(200mg/kg))组肿瘤生长抑制率为82.28％，DTIC(80mg/kg)组肿瘤生长抑制率为74.34％，COS-3组肿瘤生长抑制率为60.67％，DTIC(40mg/kg)组抑制率仅为49.04％。根据实验结果图4所示，未发现各组小鼠重量有显著变化，这说明小鼠在给药期间未出现明显毒性作用。
实施例2：MTT法检测COS-3、DTIC以及COS-3与DTIC联合用药对B16细胞活性的影响
采用MTT法测定COS-3以及COS-3与DTIC联合用药的细胞毒性。
B16接种于96孔培养板中，每孔密度为1.2×105个细胞/mL，标准条件下孵育24h。将细胞吸附在96孔平板上，分别用不同浓度的COS-3以及COS-3与DTIC联合用药进行给药处理。处理48h后，每孔加入20μL MTT试剂，37℃孵育4h。然后，除去上清液，将形成的MTT紫色结晶甲瓒溶于150μL的二甲基亚砜(DMSO)中。在490nm波长下，使用酶标仪测量吸光度。记录各孔吸光度值，并按照下方公式计算给药组各孔中B16细胞的抑制率：
抑制率＝(给药组OD值-空白组OD值)/(对照组OD值-空白组OD值)×100％
如图5所示，通过MTT检测COS-3对B16细胞活力的影响。COS-3以剂量依赖性抑制黑色素瘤B16细胞活力。选取COS-3浓度为0.5mg/ml进行后续实验，细胞存活率为80.09％。
采取DTIC给药浓度为40、50、60、70、80、90μM与COS-3(0.5mg/ml)联合给药进行，重复三次。如表1所示，发现DTIC给药浓度为50、60、70、80、90μM与COS-3(0.5mg/mL)联合给药时，计算所得CI值均小于1，证明有协同作用。并且通过对IC50进行计算，如图6所示，发现联合给药后IC50明显降低(联合用药时IC50为51.64μM，DTIC单独使用时的IC50浓度为64.57μM)。后续实验采取的DTIC浓度为70μM，细胞存活率为65.79％。DTIC(70μM)与COS-3(0.5mg/ml)联合给药的细胞存活率为42.57％。
表1：COS-3与DTIC联合用药CI值统计表
实施例3：死活细胞染色检测B16细胞衰老情况
对B16细胞进行了活死细胞毒性试验。将B16细胞(2×105细胞/mL)接种于24孔板中。培养过夜后，肿瘤细胞暴露于COS-3、DTIC以及COS-3+DTIC，在37℃、5％ CO2环境下孵育48h。然后在黑暗中同时加入Calcein-AM(钙黄绿素)(2.0μmol/L)和PI(碘化丙啶)(4.0μmol/L)孵育30min。用磷酸盐缓冲盐水(PBS)清洗培养板两次，以去除多余的荧光染料。最后，将细胞放置在荧光倒置显微镜下观察荧光分布情况，其中细胞质呈绿色，死亡细胞中细胞核呈红色。
根据图7所示实验结果，发现经过COS-3与DTIC联合给药处理后，死细胞数量增多，说明COS-3与DTIC联合给药具有诱导肿瘤细胞发生坏死，发挥抗肿瘤的作用。
实施例4：Ki67染色检测B16细胞增殖情况
增殖细胞核抗原Ki67是一种与细胞周期相关的蛋白质，可以反映细胞的增殖情况。Ki67抗原是目前多种恶性肿瘤研究中的热门检测指标，可以通过Ki67抗原的检测，了解恶性肿瘤的细胞增殖活性。其中红色荧光代表Ki67阳性细胞，蓝色为DAPI染细胞核。经过检测，如图8所示，COS-3组平均荧光强度为72.68％，DTIC组平均荧光强度为44.03％，COS-3与DTIC联合给药组平均荧光强度仅为21.02％。COS-3与DTIC联合给药组Ki67阳性细胞少，联合给药可以显著抑制B16细胞增殖。
实施例5：划痕实验检测B16细胞侵袭情况
细胞在24孔板中100％融合培养。用微移液管尖在细胞单层上划一条线。除去培养基，用PBS冲洗单层膜三次。然后在每个孔中加入COS-3、DTIC以及COS-3+DTIC处理。孵育48小时后，观察细胞迁移并在光学显微镜下拍照。实验结果如图9所示，与对照组相比，经过给药处理后，B16细胞的迁移能力明显降低，说明COS-3以及COS-3与DTIC联合给药可以有效抑制黑色素瘤B16细胞侵袭。并且从第二天开始，COS-3+DTIC联合给药组与COS-3组具有显著性差异。从第三天开始，COS-3+DTIC联合给药组与DTIC组具有显著性差异。
Claims (8)
1.一种用于治疗皮肤恶性黑色素瘤的组合物，其特征在于，所述的组合物中包含有达卡巴嗪和壳寡糖。
2.如权利要求1所述的组合物，其特征在于，所述的壳寡糖的分子量为<1000Da。
3.如权利要求1所述的组合物，其特征在于，所述的壳寡糖的聚合度为2或3壳寡糖组成，其脱乙酰度>90％。
4.如权利要求1所述的组合物，其特征在于，所述的组合物中，达卡巴嗪和壳寡糖的质量比为1：4-1：6。
5.如权利要求1所述的组合物，其特征在于，所述的组合物中，达卡巴嗪和壳寡糖的质量比为1：5。
6.权利要求1-5任一项所述的组合物在制备用于治疗皮肤恶性黑色素瘤的制品中的应用。
7.如权利要求6所述的应用，其特征在于，所述的制品为药品或功能性食品。
8.一种功能性食品或药品，其特征在于，所述的功能性食品或药品中包含有药理有效浓度的权利要求1-5任一项所述的组合物。
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