CN113350509B - 一种药物组合物及其制备方法与应用 - Google Patents

一种药物组合物及其制备方法与应用 Download PDF

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CN113350509B
CN113350509B CN202110728891.2A CN202110728891A CN113350509B CN 113350509 B CN113350509 B CN 113350509B CN 202110728891 A CN202110728891 A CN 202110728891A CN 113350509 B CN113350509 B CN 113350509B
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田华雨
陈杰
林琳
胡莹莹
郭兆培
陈学思
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Changchun Institute of Applied Chemistry of CAS
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Abstract

本发明涉及医药技术领域，尤其涉及药物组合物及其制备方法与应用。本发明提供的药物组合物中，包括免疫检查点抑制剂、降解肿瘤组织细胞外基质的药物、小分子抑制剂中的至少两种，其中，免疫检查点抑制剂、降解肿瘤区域细胞外基质药物通过载体担载相关基因，能够特异性在肿瘤微环境中表达和释放，与小分子抑制剂联合实现治疗癌症的目的。研究表明，三者联用可以提高对肿瘤的抑制效果，其效果优于三种组分分别单独使用或两两联用。

Description

一种药物组合物及其制备方法与应用
技术领域
本发明涉及医药技术领域，尤其涉及一种药物组合物及其制备方法与应用。
背景技术
恶性肿瘤是威胁人类健康和生命的主要杀手之一，其发病率、死亡率和年轻化呈现逐年走高的趋势。[参见W.Q.Chen,R.S.Zheng,P.D.Baade,S.W.Zhang,H.M.Zeng,F.Bray,A.Jemal,X.Q.Yu,J.He,Cancer Statistics in China,2015,Ca-CancerJ Clin 2016,66,115-132.]目前，癌症的临床治疗手段主要是手术、放射治疗和化学药物治疗。但是，这些传统的治疗方式均无法彻底清除肿瘤细胞并有效控制肿瘤细胞的转移和复发。近年来，肿瘤的免疫治疗被评为【Science】2013年度十大科学突破之首，开启了肿瘤免疫治疗的新篇章。
免疫检查点封锁(ICB)是癌症免疫疗法的一种典型疗法。ICB可以干扰免疫检查点及其受体之间的相互作用，在许多癌症类型中都取得了令人鼓舞的治疗效果。但是，ICB的低响应率在很大程度上限制了其进一步的发展。可能的原因就是肿瘤部位的免疫抑制微环境促使瘤内T细胞耗竭，丧失了增值和转变为记忆细胞的能力。另一方面，肿瘤组织高渗透压，外周T细胞难以进入肿瘤组织。
清除肿瘤细胞外基质(ECM)具有促进外周T细胞浸润的潜力。研究已经证明透明质酸(HA)是肿瘤ECM中的关键成分，并具有通过多种机制促进肿瘤进展的功能。HA降解依赖于透明质酸酶(HAase)家族。HAase联合化疗或肿瘤疫苗可以取得较好的抗肿瘤治疗效果。[参见X.W.Guan,J.Chen,Y.Y.Hu,L.Lin,P.J.Sun,H.Y.Tian,X.S.Chen,Biomaterials,2020,171,198-206.]。然而，静脉注射免疫检查点抗体和HAase会引起不良反应，如心脏毒性或严重的免疫相关不良反应(irAEs)。近年来，已经开发了多种针对肿瘤微环境的纳米递送系统，以实现更好的肿瘤靶向释放。因此，亟需开发肿瘤微环境响应性的纳米递送策略来提高抗肿瘤作用，同时降低不良反应。
另外，肿瘤组织是一个免疫抑制微环境，不仅肿瘤特异性T细胞数量减少且免疫抑制性细胞显著上调，如Treg细胞、M2巨噬细胞以及MDSCs细胞等。这也导致了肿瘤细胞能够逃脱机体的免疫监视，阻碍树突状细胞呈递肿瘤抗原，抑制肿瘤特异性T细胞的杀伤活性，最终导致肿瘤细胞的免疫耐受和免疫逃逸。[参见J.Yang,Y.Yang,N.Kawazoe,G.Chen.Encapsulation of individual living cells with enzyme responsivepolymer nanoshell,Biomaterials,2019,197,317-326.]。依鲁替尼(Ibrutinib)小分子BTK(布鲁顿氏酪氨酸激酶)抑制剂抑制单核细胞(MDSCs，巨噬细胞等)细胞因子受体信号通路，从而抑制其增值作用。[参见J.Conniot,A.Scomparin,C.Peres,E.Yeini,S.Pozzi,A.Matos,R.Kleiner,L.Moura,E.Zupancic,A.Viana,H.Doron,P.Gois,N.Erez,S.Jung,R.Fainaro,H.Florindo.Immunization with mannosylated nanovaccines andinhibition ofthe immune-suppressing microenvironment sensitizes melanoma toimmune checkpointmodulators,NatNanotechnol,2019,14,891-901.]。
综上，肿瘤及其微环境形成的机制较为复杂，单一疗法很难对恶性肿瘤进行根治，这就需要针对肿瘤细胞及其微环境进行综合调控，利用多种药物进行组合治疗，可有效控制肿瘤的发生和发展。
发明内容
有鉴于此，本发明提供了一种药物组合物及其制备方法与应用。该药物组合物将免疫检查点阻断、细胞外基质清除和小分子抑制剂组合，明显提高了对肿瘤的抑制效果，其效果优于三种组分分别单独使用或者两两联用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供一种药物组合物，包括以下组分中的至少两种：
I)、免疫检查点抑制剂；
II)、降解肿瘤细胞外基质的物质；
III)、小分子抑制剂；
所述免疫检查点为PD-L1/PD-1、CTLA-4、IDO-1、TIM3、LAG-3中的至少一种；
所述降解肿瘤区域细胞外基质的物质包括：纤维细胞酶、肝素酶、透明质酸酶和胶原蛋白酶中的至少一种；
所述小分子抑制剂为依鲁替尼和/或索拉菲尼。
本发明中，所述免疫检查点抑制剂为表达PD-L1/PD-1、CTLA-4、IDO-1、TIM3、LAG-3、KIR、4-1BB、OX40、B7-H3、GITRVISTA和CD27中的至少一种的shRNA。所述免疫检查点抑制剂还可以是表达以上至少一种免疫检查点的siRNA、质粒DNA或mRNA。
本发明中，所述药物组合物包括：表达shPD-L1基因的质粒、表达透明质酸酶基因的质粒和依鲁替尼中至少两种。
一些实施方案中，所述药物组合物包括表达shPD-L1基因的质粒、表达透明质酸酶基因的质粒和依鲁替尼。
本发明研究表明，shPD-L1、pSpam-1和Ibrutinib三者连用可以提高对肿瘤的抑制效果，其效果优于三种组分分别单独使用，或者两者联用。
为了使基因药物具有良好的可降解性和靶向性，本发明将免疫检查点抑制剂和/或降解肿瘤区域细胞外基质的物质担载于靶向纳米载体中。
一些实施方案中，所述靶向纳米载体由透明质酸和阳离子聚合物制成；所述阳离子聚合物为胍基对甲基苯磺酸修饰的聚赖氨酸。
本发明中，胍基对甲基苯磺酸修饰的聚赖氨酸简称为PLL-RT。本发明所述靶向纳米载体为HA/PLL-RT纳米粒子。
本发明中，免疫检查点抑制剂和/或降解肿瘤区域细胞外基质的物质担载于HA/PLL-RT纳米粒子中。所述HA/PLL-RT纳米粒子由线性聚α-赖氨酸、对甲苯磺酰基保护的精氨酸和透明质酸靶向分子制成。一些实施方案中，所述聚α-赖氨酸与对甲苯磺酰基保护的精氨酸的摩尔比为1:(10～100)；所述聚α-赖氨酸与甘露糖的摩尔比为1:(1～30)。一些实施方案中，所述的聚α-赖氨酸的数均分子量为3000～30000。
首先将聚-α-赖氨酸接枝对甲苯磺酰基保护的精氨酸(Arg(Tos))获得PLL-RT纳米粒子，通过静电作用将透明质酸吸附到纳米载体表面，获得HA/PLL-RT纳米粒子。一些实施方案中，HA与PLL-RT纳米粒子的质量比为(0.2～1)：(2～10)。
其中，PLL-RT纳米粒子的制备方法，包括以下步骤：
A)对甲苯磺酰基和叔丁氧羰基双保护的精氨酸进行活化，再加入线性聚-α-赖氨酸的水溶液进行反应；
所述线性聚-α-赖氨酸与对甲苯磺酰基和叔丁氧羰基双保护的精氨酸的摩尔比为1：(10～100)；
B)将A)中反应后的溶液进行透析、冻干；
C)将B)中冻干产物与三氟乙酸进行反应，加入无水乙醚沉降，真空抽干和透析后，得到阳离子聚合物PLL-RT。
其中，优选的，所述步骤A)甲苯磺酰基和叔丁氧羰基双保护的精氨酸的N,N-二甲基甲酰胺溶液的浓度为0.02～0.5mg/mL，线性聚-α-赖氨酸水溶液的浓度为0.05～0.5mg/mL，反应温度为20～37℃，反应的时间为24～96小时。优选的，所述步骤B)透析时间为2～5天，冻干温度为-30～-80℃。优选的，所述步骤C)反应时间为0.5～24小时。优选的，所述步骤C)透析时间为2～5天，冻干温度为-30～-80℃。
一些实施例方案中，本发明药物组合物包括HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒和依鲁替尼。
一些实施方案中，所述HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒与依鲁替尼以表达shPD-L1的质粒、表达Spam1的质粒和依鲁替尼的质量比计为(0.5～2)：(0.5～2)：(5～20)。一些具体实施例中，所述HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒与依鲁替尼以shPD-L1、Spam1和依鲁替尼的质量比计为1:1:10。
所述HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒中，HA/PLL-RT、表达shPD-L1的质粒和表达Spam1的质粒的质量比为：(1～10)：(0.5～2)：(0.5～2)。
本发明中HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒的粒度为50～500nm。
本发明还提供了所述药物组合物的制备方法，包括以下步骤：
将透明质酸(HA)溶于蒸馏水至0.1～1mg/mL，制成HA溶液；
将PLL-RT溶于蒸馏水中，制成0.5～5mg/mL的PLL-RT溶液；
将Spam1质粒和shPD-L1质粒的混合溶液与PLL-RT溶液混合，反应，涡旋振荡20min，然后加入HA溶液，涡旋振荡20min，制成HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒；
将蓖麻油、乙醇和蒸馏水按照体积比为1:1:18混合，加入依鲁替尼溶解，获得依鲁替尼溶液。
一些实施方案中，所述PLL-RT纳米粒子的浓度为0.8～4mg/mL；
一些实施方案中，所述透明质酸HA的浓度为0.2～1mg/mL；
一些实施方案中，所述反应的温度为15～30℃，时间为10～30分钟；
一些实施方案中，所述Spam1质粒和shPD-L1质粒在水溶液中的浓度均为0.1～1mg/mL。
本发明还提供了所述的药物组合物在制备抗肿瘤药物中的应用。
本发明还提供一种抗肿瘤药物，包括本发明所述的药物组合物。
所述抗肿瘤药物还包括药学上可接受的辅料。本领域技术人员可选择制成不同剂型所需的辅料，采用本领域熟知的方法将所述药物组合物制成不同的剂型，如注射剂、粉针剂等。
本发明还提供一种治疗癌症的方法，给予本发明所述的药物组合物。
本发明中，所述药物组合物中，小分子抑制剂的存在形式为注射剂，其浓度为0.8～1.2mg/mL。可通过静脉或腹腔注射。
本发明提供的药物组合物包括：包括以下组分中的至少两种：I)、免疫检查点抑制剂；II)、降解肿瘤细胞外基质的物质；III)、小分子抑制剂；所述免疫检查点为PD-L1/PD-1、CTLA-4、IDO-1、TIM3、LAG-3中的至少一种；所述降解肿瘤区域细胞外基质的物质包括：纤维细胞酶、肝素酶、透明质酸酶和胶原蛋白酶中的至少一种；所述小分子抑制剂为依鲁替尼和/或索拉菲尼。该组合物能够实现肿瘤组织的免疫检查点阻断、提高肿瘤部位免疫细胞的浸润以及改善肿瘤部位的免疫抑制微环境，最终实现了高效抗肿瘤作用。实现表明，三者联用可以明显提高对肿瘤的抑制作用，效果优于三组分单独使用或两两组合的效果。
附图说明
图1本发明药物组合物的抗肿瘤治疗效果。
具体实施方式
本发明提供了一种药物组合物及其制备方法与应用。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中，所述靶向纳米载体由阳离子聚合物担载免疫检查点抑制剂和降解肿瘤组织细胞外基质的基因通过静电复合作用形成，再通过静电作用复合透明质酸靶向分子，最终形成具有肿瘤细胞靶向能力的基因递送系统。其中，所述阳离子聚合物优选为聚乙烯亚胺、聚乙烯亚胺衍生物、聚赖氨酸和聚赖氨酸衍生物中的一种或几种；采用上述阳离子聚合物能够与带负电的基因形成纳米复合物，通过静电作用可在表面吸附透明质酸靶向分子，最终形成基因递送系统。该靶向纳米载体可有效提高基因物质的表达效率，从而调控肿瘤组织免疫检查点抑制剂和降解肿瘤组织细胞外基质药物的表达，联合小分子抑制剂药物后，实现了高效抗肿瘤治疗效果。本发明对所述靶向纳米载体的种类没有特殊限制，为本领域技术人员熟知的即可。
本发明中引发肿瘤免疫检查点封锁的药物采用担载shPD-L1沉默基因的HA/PLL-RT纳米粒子。在本发明中，所述能够引发免疫检查点封锁的基因包括其余检查点沉默基因，例如CTLA-4、IDO-1、TIM-3和LAG-3等。
本发明中引发肿瘤细胞外基质清楚的药物采用担载Spam-1基因的HA/PLL-RT纳米粒子。在本发明中，所述能够引发肿瘤细胞外基质清除的基因包括其余对细胞外基质有破坏作用的基因，例如胶原酶基因、肝素酶基因和杀伤成纤维细胞基因等。
本发明中调控肿瘤免疫抑制微环境的小分子抑制剂选用依鲁替尼，溶解在蓖麻油、乙醇和蒸馏水按照体积比为1:1:18的混合溶剂中，制备成药剂。本发明中，所述能够调控肿瘤免疫抑制微环境的小分子抑制剂还可以使其他对肿瘤组织中各种细胞组分具有调控作用的小分子，例如索拉菲尼等。
本发明提供的药物组合物能够实现肿瘤组织的免疫检查点阻断、提高肿瘤部位免疫细胞的浸润以及改善肿瘤部位的免疫抑制微环境，最终实现了高效抗肿瘤作用。
1.基因递送系统的制备和表征：阳离子聚合物PLL-RT，靶向分子HA以及带负电的基因物质可通过静电作用组装成纳米颗粒，通过粒度仪和扫描电镜可表征基因递送系统的表面形貌。
2.抗肿瘤实验：HA/PLL-RT/pSpam-1/shPD-L1纳米粒子与小分子抑制剂依鲁替尼协同给药可以发挥最好的抗肿瘤效果。
3.降解肿瘤细胞免疫检查点实验：HA/PLL-RT/shPD-L1纳米粒子可以在体内高效降低肿瘤细胞表达PD-L1蛋白的水平。PD-L1蛋白的水平通过ELISA检测。
4.降解肿瘤细胞外基质实验：HA/PLL-RT/pSpam-1纳米粒子可以在体内提升透明质酸酶的表达量，有利于肿瘤区域透明质酸的降解，从而破坏细胞外基质。透明质酸酶的表达量通过ELISA检测。
5.增加瘤内CD8+T细胞浸润数量的实验：HA/PLL-RT/pSpam-1/shPD-L1纳米粒子与小分子抑制剂依鲁替尼协同给药可以增加肿瘤内部CD8+T细胞的数量。CD8+T细胞的数量通过流式细胞术检测。
6.逆转肿瘤免疫抑制微环境实验：HA/PLL-RT/pSpam-1/shPD-L1纳米粒子与小分子抑制剂依鲁替尼协同给药可以降低肿瘤内部M2和MDSCs细胞数量。M2和MDSCs的数量通过流式细胞术检测。
本发明采用的试材皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1实施例1PLL-RT基因载体的制备
将线性聚-α-赖氨酸(分子量15000Da)溶解于去离子水中，对甲苯磺酰基和叔丁氧羰基双保护的精氨酸溶解于DMF中。然后，加入EDC·HCl和HOBT，室温下活化反应1小时，再缓慢加入PLL的水溶液，室温反应72小时。透析和冻干后，将产物在三氟乙酸的条件下反应4小时，加无水乙醚沉降，真空抽干，透析，冻干得到白色固体产物PLL-RT。
聚-α-赖氨酸接枝对甲苯磺酰基保护的精氨酸(Arg(Tos))的接枝摩尔比为1:90。
实施例2HA/PLL-RT/pSpam-1/shPD-L1基因递送系统的制备
将靶向分子HA、阳离子载体PLL-RT、pSpam-1和shPD-L1分别溶于超纯水中，形成水溶液，浓度分别为0.1mg/mL、1mg/mL、0.2mg/mL和0.2mg/mL。再将PLL-RT、pSpam-1和shPD-L1等体积复合，复合比例为5:1:1。涡旋30s混匀后，室温下孵育20分钟，然后再加入等体积的HA溶液，涡旋30s混匀后，室温下再孵育20分钟，最后得到HA/PLL-RT/pSpam-1/shPD-L1。
对比例1制备PLL-RT/pSpam-1/shPD-L1纳米粒子
制备PLL-RT/pSpam-1/shPD-L1纳米粒子，制备步骤和参数参见实施例2。
实施例3靶向纳米载体的表征
对上述靶向纳米载体进行粒径和电位测试，结果参见表1。
表1实施例3的电位粒径测试结果
平均直径(nm) 电位(mV)
PLL-RT/pSpam-1/shPD-L1 138.5 16.3
HA/PLL-RT/pSpam-1/shPD-L1 142.3 13.5
由以上测试结果可知，本发明靶向纳米载体HA/PLL-RT能够有效地包裹和压缩质粒DNA，形成带正电的纳米颗粒，有利于肿瘤细胞的内吞作用。
实施例4靶向纳米载体的内吞
培养小鼠黑色素瘤B16F10细胞，按照2.5×105细胞/孔的密度接种于24孔培养板中，继续培养24小时。加入荧光标记材料(样品分别为：PBS、FAM-DNA，PLL-RT/FAM-DNA和HA/PLL-RT/FAM-DNA)，每孔FAM-DNA上样量为0.5微克，继续培养4小时后，用流式细胞仪检测基因递送系统的内吞。
表2实施例4的细胞内吞结果
DNA内吞效率(％)
PBS 0.95
FAM-DNA 1.25
PLL-RT/FAM-DNA 38.3
HA/PLL-RT/FAM-DNA 50.6
结果表明，与PLL-RT/FAM-DNA和单纯FAM-DNA相比，本发明的靶向纳米载体HA/PLL-RT/FAM-DNA内吞量均显著增加，说明本发明靶向纳米载体HA/PLL-RT有利于促进基因物质的内吞。
实施例5HA/PLL-RT/pSpam-1/shPD-L1联合Ibrutinib抗肿瘤治疗
4-6周龄的C57BL6小鼠，随机分为8组，每组5只。在皮下注射1×106细胞，构建小鼠皮下肿瘤模型，治疗方案见照图1。各组小鼠分别接受不同的疗法，即：PBS、HA/PLL-RT/pHAase、HA/PLL-RT/shPD-L1、Ibrutinib、HA/PLL-RT/pHAase+Ibrutinib、HA/PLL-RT/pHAase/shPD-L1、HA/PLL-RT/shPD-L1+Ibrutinib、HA/PLL-RT/pHAase/shPD-L1+Ibrutinib。
PBS组小鼠作为对照，仅给予等量PBS缓冲液；
HA/PLL-RT/pHAase/shPD-L1的质量比为0.5:5:1:1；
每只小鼠pHAase的给药剂量为0.6mg/kg；
每只小鼠shPD-L1的给药剂量为0.6mg/kg；
Ibrutinib的给药量为6mg/kg；
肿瘤的抑制效果显示(表3)，三者联合用药的效果相对于其他单独疗法或二者联合存在显著性的优势。
表3肿瘤抑制率
肿瘤抑制率(％)
PBS 0
HA/PLL-RT/pHAase 45.2
HA/PLL-RT/shPD-L1 31.5
Ibrutinib 5.4
HA/PLL-RT/pHAase+Ibrutinib 54.7
HA/PLL-RT/pHAase/shPD-L1 65.3
HA/PLL-RT/shPD-L1+Ibrutinib 38.5
HA/PLL-RT/pHAase/shPD-L1+Ibrutinib 86.3
实施例6肿瘤细胞免疫检查点阻断研究
治疗结束后，将小鼠肿瘤取出，对肿瘤组织进行匀浆，离心后取上清，利用ELISA试剂盒检测肿瘤组织内免疫检查点PD-L1的含量，检测结果如表4所示。
表4联合治疗对PD-L1的阻断效果
Figure BDA0003138594660000101
实施例7肿瘤组织免疫细胞浸润研究
治疗结束后，将小鼠肿瘤取出，对肿瘤组织进行匀浆，离心后取上清，利用ELISA试剂盒检测肿瘤组织内透明质酸酶的含量，检测结果如表5所示。
表5联合治疗产生透明质酸酶的含量
Figure BDA0003138594660000102
实施例8依鲁替尼逆转肿瘤组织免疫抑制微环境
治疗结束后，将小鼠肿瘤取出，利用流式细胞术检测肿瘤内部MDSCs和M2细胞的数量。检测结果如表6所示。
表6依鲁替尼降低肿瘤内部MDSCs和M2细胞的数量
Figure BDA0003138594660000103
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (5)

1.一种药物组合物，其特征在于，包括：HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒和依鲁替尼，其中，PLL-RT为胍基对甲基苯磺酸修饰的聚赖氨酸。
2.根据权利要求1所述的药物组合物，其特征在于，所述HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒与依鲁替尼以表达shPD-L1的质粒、表达Spam1的质粒和依鲁替尼的质量比计为1:1:10；
所述HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒中，HA/PLL-RT、表达shPD-L1的质粒和表达Spam1的质粒的质量比为：(1～10)：(0.5～2)：(0.5～2)。
3.根据权利要求1或2所述的药物组合物的制备方法，其特征在于，包括：
将透明质酸(HA)溶于蒸馏水至0.1～1mg/mL，制成HA溶液；
将PLL-RT溶于蒸馏水中，制成0.5～5mg/mL的PLL-RT溶液；
将Spam1质粒和shPD-L1质粒的混合溶液与PLL-RT溶液混合，反应，涡旋振荡20min，然后加入HA溶液，涡旋振荡20min，制成HA/PLL-RT/(Spam1+shPD-L1)纳米颗粒；
将蓖麻油、乙醇和蒸馏水按照体积比为1:1:18混合，加入依鲁替尼溶解，获得依鲁替尼溶液。
4.权利要求1～2任一项所述的药物组合物在制备抗肿瘤药物中的应用。
5.一种抗肿瘤药物，其特征在于，包括权利要求1～2任一项所述的药物组合物。
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