CN117298113A - 替那帕诺及其脂质体靶向m2巨噬细胞在抗肿瘤药中的应用 - Google Patents
替那帕诺及其脂质体靶向m2巨噬细胞在抗肿瘤药中的应用 Download PDFInfo
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Abstract
本发明公开了替那帕诺脂质体(T7587)靶向M2巨噬细胞在抗肿瘤药中的应用，属于生物医药领域。本发明以替那帕诺脂质体靶向M2巨噬细胞对肿瘤的影响为主要出发点，证实替那帕诺能影响前列腺癌微环境内的M2巨噬细胞，通过YY1、NUSAP1、HIF‑1α复合体影响肿瘤的发生发展，这体现了替那帕诺的老药新用，为抗肿瘤药物的研发提供新的思路。通过IC50获得替那帕诺最佳浓度在100μM，并且替那帕诺明显降低YY1、NUSAP1和HIF‑1α表达，且在缺氧效果下降低YY1/NUSAP1/HIF‑1α共定位。其理想浓度下的替那帕诺脂质体比替那帕诺单药能更有效抑制C57BL/6小鼠的肿瘤生长，但对BALB/c小鼠无明显疗效。
Description
技术领域
本发明涉及生物医药领域，具体涉及替那帕诺及其脂质体靶向M2巨噬细胞在抗肿瘤药中的应用。
背景技术
替那帕诺，英文名为Tenapanor，其结构式为：
作为一种Na+/H+交换体3(NHE3)抑制剂，其通常用于治疗慢性肾脏病患者的高磷血症。通过抑制小肠和结肠中的NHE3通道，减少钠离子的吸收和水分的重吸收。这样可以增加粪便中的水分含量，并促进磷的排泄，从而降低血液中磷的浓度。与此同时，目前发现其可用于慢性腹泻和肠易激综合征的治疗。而在抗肿瘤药物中的应用并无报道。
前列腺癌(prostate cancer)是一种男性生殖系统肿瘤，其中95％以上的前列腺癌为前列腺腺癌(prostate adenocarcinoma)。随着对于肿瘤病因的认识和临床治疗技术的提高，过去几十年中，免疫检查点抑制剂(immune checkpoint inhibitors，ICI)在某些肿瘤类型中具有显著疗效，但其在前列腺癌中的活性仍然有限，主要是由于免疫抑制性肿瘤微环境(tumor microenvironment,TME)。
TME是一个复杂的生态系统，包括肿瘤细胞、免疫细胞、细胞因子和基质成分，并调节肿瘤的建立。抗肿瘤T淋巴细胞的缺乏和巨噬细胞的浸润是前列腺癌免疫抑制性肿瘤微环境的重要特征。在不同的细胞因子和生长因子，巨噬细胞可以被极化为经典的活化M1型和交替活化的M2型。M2亚型被广泛认为是肿瘤促进型TAMs(pTAMs)的主要表型。pTAMs是免疫抑制型肿瘤微环境的驱动力之一，从而加速了恶性肿瘤的增殖和转移。由于免疫抑制性微环境与免疫检查点抑制剂无反应性之间的密切联系，人们注意到了巨噬细胞作为"冷"肿瘤的一个有希望的免疫治疗目标。
虽然上述的肿瘤机理已经较为明了，但在临床治疗中，由于腹腔注射治疗的靶向性限制，药物通过腹腔注射进入体内后并不能局限于肿瘤部位，且长时间滞留于腹腔可能会导致一些副作用的产生，。
发明内容
本发明的目的在于公开替那帕诺及其脂质体靶向M2巨噬细胞抑制肿瘤进展，并在制备抗肿瘤药物中的应用。
本发明的目的可以通过以下技术方案实现：
本发明的第一方面，涉及替那帕诺在制备抗肿瘤药物中的应用。
可选地，所述肿瘤前列腺癌。
可选地，所述替那帕的浓度为100μM。
可选地，所述替那帕诺脂质体能够靶向巨噬细胞。
可选地，所述药物为注射剂、悬浮剂或者乳剂。
可选地，所述替那帕诺为17-[[[3-[(4S)-6,8-二氯-1,2,3,4-四氢-2-甲基-4-异喹啉基]苯基]磺酰基]氨基]-N-[2-[2-[[2-[[[3-[(4S)-6,8-二氯-1,2,3,4-四氢-2-甲基-4-异喹啉基]苯基]磺酰基]氨基]乙氧基]乙氧基]乙基]-8-氧代-12,15-二氧杂-2,7,9-三氮杂十七酰胺二盐酸盐。
可选地，采用脂质体包裹所述替那帕诺，且其上连接有用于靶向M2型巨噬细胞的多肽。
可选地，所述多肽为M2型巨噬细胞靶向肽，氨基酸序列为Tyr-Glu-Gln-Asp-Pro-Trp-Gly-Val-Lys-Trp-Trp-Tyr。
本发明的第二方面，涉及一种用于治疗肿瘤的药物，包括：用于靶向M2型巨噬细胞的多肽、替那帕诺以及包裹所述替那帕诺的脂质体。
在本发明的一些优选的示例中，所述抗肿瘤药物以替那帕诺或其脂质体作为唯一活性成份。所述抗肿瘤药物还可以包含替那帕诺脂质体，及药学上可接受的辅助剂。
本发明揭示了肿瘤免疫微环境中的M2巨噬细胞在肿瘤发生进展中扮演关键作用，巨噬细胞通过YY1、NUSAP1、HIF-1α复合体影响前列腺癌的进展。靶向M2巨噬细胞的替那帕诺脂质体可以有效的抑制肿瘤的发生、发展。
本发明以前列腺癌为例，成功地印证了该假说，即替那帕诺及其脂质体通过靶向M2巨噬细胞可以有效地抑制前列腺癌的发生和发展。由于能有效的抑制肿瘤的发生和发展，替那帕诺脂质体或机制相似的替代物在抗肿瘤药物的制备方面将具有巨大潜力。
本发明的有益效果：
本发明以替那帕诺及其脂质体靶向M2巨噬细胞对肿瘤的影响为主要出发点，证实替那帕能影响前列腺癌微环境内的M2巨噬细胞，通过YY1、NUSAP1、HIF-1α复合体影响肿瘤的发生发展，这体现了替那帕诺的老药新用，为抗肿瘤药物的研发提供新的思路。通过IC50获得替那帕的最佳浓度在100μM，并且替那帕能明显降低YY1、NUSAP1和HIF-1α表达，且在缺氧效果下降低YY1/NUSAP1/HIF-1α共定位。其理想浓度下的替那帕诺脂质体比替那帕诺能更有效抑制C57BL/6小鼠的肿瘤生长，但对BALB/c小鼠无明显疗效。
附图说明
下面结合附图对本发明作进一步的说明。
图1为本申请的实施例1中，替那帕诺处理后的IC50曲线。
图2为本申请的实施例1中，不同浓度替那帕诺处理正常人肾上皮细胞系293T细胞。
图3为本申请的实施例1中，共免疫沉淀(co-IP)实验。
图4为本申请的实施例1中，替那帕诺影响YY1/NUSAP1/HIF-1α共定位。
图5为本申请的实施例2中，腹腔注射替那帕诺脂质体后活体成像。
图6为本申请的实施例3中，通过腹腔注射比较替那帕诺和替那帕诺脂质体的疗效。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。
下述实施例中使用的材料包括：
细胞：正常人肾上皮细胞系293T、人单核细胞白血病细胞系THP-1。
试剂：DMEM培养基，1640培养基，CCK-8试剂，替那帕诺，替那帕诺脂质体，M2型巨噬细胞靶向肽，YY1、NUSAP1和HIF-1α抗体。
其中，所述的M2型巨噬细胞靶向肽(DSPE-PEG-M2pep),其结构式为：
是由12个氨基酸组成的M2型巨噬细胞靶向肽，简称为M2pep,其含有的氨基酸序列为Tyr-Glu-Gln-Asp-Pro-Trp-Gly-Val-Lys-Trp-Trp-Tyr。多项研究证实M2pep的显著靶向M2型TAMs的作用，例如过表达肝癌抗原甲胎蛋微颗粒-M2pep-瑞喹莫德的纳米药物能靶向递送药物到肝癌组织M2型TAMs，使其逆极化成M1型，改善肝癌免疫抑制微环境，促进CD8+T细胞浸润。
脂质体：属于一种人工膜，由卵磷脂和神经酰胺等制得的空心物质。在水中磷脂分子亲水头部插入水中，脂质体疏水尾部伸向空气，搅动后形成双层脂分子的球形脂质体，直径25～1000nm不等。脂质体可用于转基因，或制备的药物，利用脂质体可以和细胞膜融合的特点，将药物送入细胞内部。此外，脂质体可以通过增强滞留效应被动进入肿瘤组织，相较于单纯的放化疗，具有一定的临床治疗优势。
动物：C57BL/6小鼠和BALB/c小鼠。
实施例1
1、细胞培养
正常人肾上皮细胞系293T、单核细胞白血病细胞系THP-1分别培养在含有10％胎牛血清和1％青霉素-链霉素的DMEM培养基和1640培养基中，每2～3天进行一次传代。
2、IC50检测
将正常人肾上皮细胞系293T、单核细胞白血病细胞系THP-1细胞分别种于96孔板，分为空白组、实验组和单纯培养基组。空白组为不加替那帕诺处理的细胞。单纯培养基组为不含替那帕诺且不含细胞的培养基。实验组为用不同浓度替那帕诺的细胞，处理浓度分别为5000μM、500μM、100μM、50μM和5μM。空白组、单纯培养基组和实验组的每个浓度均设置3个复孔，处理48h。每孔加入10uL的CCK-8溶液，在细胞培养箱中孵育1～2h。利用Tecan M200酶标仪，选择450nm波长，检测各孔光吸收值，计算相对细胞数，结果如图1所示，其中(A)为单核细胞白血病细胞系THP-1细胞；(B)为正常人肾上皮细胞系293T细胞。使用浓度分别为1000μM、500μM、100μM、50μM、10μM和5nm/mL的替那帕诺处理293T细胞，显微镜拍照并观察细胞形态和数量，如图2所示。
3、共免疫沉淀(co-IP)实验
将单核细胞白血病细胞系THP-1细胞分别针对YY1进行co-IP实验，而后将所得蛋白复合物通过Western Blot实验检测100μM下THP-1细胞中YY1、NUSAP1、HIF-1α的关系。结果如图3所示。其中，(A)YY1；(B)
NUSAP1；(C)HIF-1α分别进行共免疫沉淀。使用对照组DMSO(NC)和实验组替那帕诺进行实验，NC组分别用YY1、NUSAP1、HIF-1α抗体进co-IP实验。结果表明YY1、NUSAP1、HIF-1α三者结合成复合体；与对照组比较替那帕诺破坏了YY1、NUSAP1、HIF-1α复合体。
4、共聚焦显微镜观察共定位情况
将单核细胞白血病细胞系THP-1细胞用佛波酯(Phorbol 12-myristate 13-acetate,PMA)诱导后分为实验组与对照组进行共免疫沉淀实验。实验组为用100μM的替那帕诺处理的细胞。对照组为不加替那帕诺处理的细胞。而后将实验组与对照组按照YY1和NUSAP1、NUSAP1和HIF-1α、HIF-1α和YY1各自分为三组，在共聚焦显微镜下观察其各自共定位情况。结果如图4所示。其中，(A)HIF-1α和YY1；(B)NUSAP1和YY1；(C)NUSAP1和HIF-1α分别进行免疫荧光染色。使用对照组缺氧处理的THP-1-PMA细胞(NC)和实验组替那帕诺以及缺氧处理的THP-1-PMA细胞进行实验，NC组分别用HIF-1α和YY1、NUSAP1和YY1、NUSAP1和HIF-1α进行免疫荧光染色，并用DAPI作为细胞核的对比染色。结果表明三组均有明显共定位情况出现；与对照组比较替那帕诺使得三组共定位情况均有不同程度的降低。
实施例2
构建C57BL/6小鼠(扬州大学比较医学中心)皮下瘤模型。实验前小鼠在实验室环境中驯化2-3周至6-8周龄，通过小鼠背部皮下注射小鼠前列腺癌细胞(RM-1细胞)，注射后每4天评估一次肿瘤体积。皮下植入RM-1细胞2周后，通过对C57BL/6小鼠皮下瘤模型腹腔注射替那帕诺脂质体，并依次于注射后2h、8h、16h、24h、32h、48h进行活体成像拍照，观察分析其药物富集情况。浓度均为10μg/ml，用量为1.4ml。如图5所示，结果表明替那帕诺脂质体在通过腹腔注射后24-36h明显向C57BL/6小鼠皮下瘤富集。
实施例3
按照上述方法构建BALB/c小鼠以及C57BL/6小鼠(扬州大学比较医学中心)皮下瘤模型，分为四组，(A)C57BL/6小鼠皮下瘤模型腹腔注射替那帕诺脂质体，以及等数量的C57BL/6小鼠皮下瘤模型作为对照组；(B)
C57BL/6小鼠皮下瘤模型腹腔注射替那帕诺以及等数量的C57BL/6小鼠皮下瘤模型作为对照组；(C)BALB/c小鼠皮下瘤模型腹腔注射替那帕诺脂质体，以及等数量的BALB/c小鼠皮下瘤模型作为对照组；(D)BALB/c小鼠皮下瘤模型腹腔注射替那帕诺，以及等数量的BALB/c小鼠皮下瘤模型作为对照组。替那帕诺及其脂质体浓度均为10μg/ml，用量为1.4ml。
结果如图6所示，替那帕诺或替那帕诺脂质体在免疫功能成熟(含有巨噬细胞)的C57BL/6小鼠中作用明显，而在免疫功能缺陷的BALB/c小鼠(不含巨噬细胞)中无疗效。
综合上述实施例所述，首先，通过IC50初步测定100μM为替那帕诺的最佳浓度，随后使用DMSO对照组(NC)和实验组替那帕诺进行实验，在NC分别用YY1、NUSAP1、HIF-1α抗体进行共免疫沉淀(co-IP)实验，结果均表明YY1、NUSAP1、HIF-1α三者共沉淀，形成YY1、NUSAP1、HIF-1α复合体；当我们在实验组分别用YY1、NUSAP1、HIF-1α抗体进行co-IP实验时，结果均表明三者共沉淀状态被破坏，即替那帕诺会导致YY1、NUSAP1、HIF-1α复合体解体。随后，通过YY1/NUSAP1/HIF-1α共定位分析发现，在NC中YY1和NUSAP1、NUSAP1和HIF-1α、HIF-1α和YY1三组均有明显共定位情况出现，而在替那帕诺处理的实验组中，YY1和NUSAP1、NUSAP1和HIF-1α、HIF-1α和YY1三组共定位情况均有不同程度的降低。而后通过腹腔注射替那帕诺脂质体后活体成像拍照发现，替那帕诺脂质体在注射后24-36h会明显向皮下瘤富集，这证实了通过腹腔注射进行替那帕诺脂质体治疗的可靠性；最后，通过动物实验发现，替那帕诺或替那帕诺脂质体在免疫功能成熟(含有巨噬细胞)的C57BL/6小鼠中作用明显，而在BALB/c小鼠(不含巨噬细胞)中无疗效，是因为无论是否靶向巨噬细胞进行治疗，此两种治疗方法都会影响到C57BL/6小鼠中的巨噬细胞，从而抑制肿瘤生长，而BALB/c小鼠对此两种治疗方法无疗效则进一步说明替那帕诺脂质体和替那帕诺都是通过影响巨噬细胞而抑制肿瘤生长。上述结论表明：替那帕诺及其脂质体能明显降低巨噬细胞中YY1，NUSAP1和HIF-1α复合体的功能，且在缺氧效果下降低YY1/NUSAP1/HIF-1α共定位，并以此通过靶向M2巨噬细胞从而明显抑制免疫成熟的C57BL/6小鼠肿瘤生长。
通过上述的实施例可以发现，替那帕诺或替那帕诺脂质体在免疫功能成熟(含有巨噬细胞)的C57BL/6小鼠中作用明显，而在BALB/c小鼠(不含巨噬细胞)中无疗效，这主要是因为无论是否靶向巨噬细胞进行治疗，此两种治疗方法都会影响到C57BL/6小鼠中的巨噬细胞，从而抑制肿瘤生长，而BALB/c小鼠对此两种治疗方法无疗效则进一步说明替那帕诺脂质体和替那帕诺都是通过影响巨噬细胞而抑制肿瘤生长。
综合以上结果表明，替那帕诺及其脂质体能通过靶向M2巨噬细胞，抑制肿瘤细胞的增殖，在肿瘤的临床治疗中有重要应用价值。
在本说明书的描述中，参考术语“一个实施例”、“示例”、“具体示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。
Claims (10)
1.替那帕诺在制备抗肿瘤药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述肿瘤为前列腺癌。
3.根据权利要求1所述的应用，其特征在于，所述药物为注射剂、悬浮剂或者乳剂。
4.根据权利要求1所述的应用，其特征在于，所述替那帕诺为：17-[[[3-[(4S)-6,8-二氯-1,2,3,4-四氢-2-甲基-4-异喹啉基]苯基]磺酰基]氨基]-N-[2-[2-[[2-[[[3-[(4S)-6,8-二氯-1,2,3,4-四氢-2-甲基-4-异喹啉基]苯基]磺酰基]氨基]乙氧基]乙氧基]乙基]-8-氧代-12,15-二氧杂-2,7,9-三氮杂十七酰胺二盐酸盐。
5.根据权利要求1所述的应用，其特征在于，采用脂质体包裹所述替那帕诺，且其上连接有用于靶向M2型巨噬细胞的多肽。
6.根据权利要求5所述的应用，其特征在于，所述多肽为M2型巨噬细胞靶向肽，氨基酸序列为Tyr-Glu-Gln-Asp-Pro-Trp-Gly-Val-Lys-Trp-Trp-Tyr，。
7.一种用于治疗肿瘤的药物，其特征在于，包括：用于靶向M2型巨噬细胞的多肽、替那帕诺以及包裹所述替那帕诺的脂质体。
8.根据权利要求7所述的药物，其特征在于，所述替那帕诺为：17-[[[3-[(4S)-6,8-二氯-1,2,3,4-四氢-2-甲基-4-异喹啉基]苯基]磺酰基]氨基]-N-[2-[2-[[2-[[[3-[(4S)-6,8-二氯-1,2,3,4-四氢-2-甲基-4-异喹啉基]苯基]磺酰基]氨基]乙氧基]乙氧基]乙基]-8-氧代-12,15-二氧杂-2,7,9-三氮杂十七酰胺二盐酸盐。
9.根据权利要求7所述的药物，其特征在于，所述多肽为M2型巨噬细胞靶向肽，氨基酸序列为Tyr-Glu-Gln-Asp-Pro-Trp-Gly-Val-Lys-Trp-Trp-Tyr。
10.根据权利要求7所述的药物，其特征在于，所述药物为注射剂、悬浮剂或者乳剂。
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