CN111803475A - 喷他脒在制备抑制pd1和pd-l1蛋白之间相互作用药物中的应用 - Google Patents
喷他脒在制备抑制pd1和pd-l1蛋白之间相互作用药物中的应用 Download PDFInfo
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Abstract
喷他脒在制备抑制PD1和PD‑L1蛋白之间相互作用药物中的应用，属于医药领域。本发明经研究发现：Pentamidine可以作为PD1/PD‑L1蛋白相互作用的抑制剂，其中，PD1浓度为10μg/ml，PD‑L1浓度为10μg/ml，喷他脒的浓度为0.5‑4μM。PD1/PD‑L1信号通路与免疫应答、肿瘤免疫逃逸密切相关，抑制PD1/PD‑L1信号通路的异常激活能够起到预防及治疗肿瘤的作用。本发明的Pentamidine能够抑制PD1/PD‑L1蛋白质之间相互作用。
Description
技术领域
本发明属于医药领域，具体涉及喷他脒在制备抑制PD1和PD-L1蛋白之间相互作用药物中的应用。
背景技术
目前治疗免疫治疗的方法主要包括两大部分，免疫增强化和免疫正常化，免疫增强化用来增强那些被认为对肿瘤免疫过程中起到关键作用的过程，主要包括主动性免疫疗法和被动性免疫疗法。主动性免疫疗法包括抗体靶向治疗，肿瘤疫苗，细胞因子治疗，过继性免疫细胞治疗以及CAR-T细胞治疗。这种疗法通过使用免疫系统的效应细胞和相应的效应分子，直接对肿瘤细胞进行杀伤或者增强其对肿瘤细胞的杀伤作用。将免疫应答提高到更高更强的水平，以达到杀伤肿瘤细胞的目的。
其次免疫增强化还可以通过调节内源性的细胞信号或者免疫激活和抑制的相关机制来实现，增强相对应的免疫反应的具体步骤，这一类疗法被称为是被动性免疫疗法，比如增强对抗原的摄取加工过程，以及通过抗原呈递细胞向T细胞呈递的过程，这类药物包括针对肿瘤抗原的肿瘤疫苗以及增强免疫的佐剂，此外，通过某些免疫增强通路的额外激活，同样可以达到相应的目的，这类药物包括Ⅰ型干扰素、Toll样受体的激动剂和STING通路激动剂，用以增强抗原呈递细胞的活性。而另一类药物，包括树突状细胞疫苗和抗细胞效应T淋巴细胞抗原-4（CTLA-4）的单抗，甚至体外扩增和激活肿瘤侵润性T细胞并回输的过继性免疫疗法，属于增强初始T细胞的活化与扩增过程和增强效应T细胞的杀伤能力进行免疫增强化治疗。
发明内容
本发明目的在于克服现有技术缺陷，提供一种Pentamidine（喷他脒）在抑制PD1和PD-L1蛋白之间相互作用药物中的应用。
Pentamidine是一种小分子化合物，分 子 式： C19H24N4O2，分 子 量：340.427 ，Pentamidine; 100-33-4; 4,4'-(Pentane-1,5-diylbis(oxy))dibenzimidamide。FDA批准用于内脏利什曼病(黑热病)和卡氏肺囊虫病的一线治疗，可以直接购买市售产品。
本发明提供了一种天然化合物Pentamidine作为PD1和PD-L1蛋白之间作用的抑制剂。
具体的，喷他脒在制备免疫治疗药剂中的应用，PD1浓度为10μg/ml，PD-L1浓度为10μg/ml，喷他脒的浓度为0.5-4 μM。
进一步的，通过增强T细胞对癌细胞的杀伤能力来发挥增强免疫作用。所述癌细胞为肺癌细胞。癌细胞数量1*105个每毫升，喷他脒的浓度为0.5-4 μM。
本发明发现：Pentamidine在制备抑制PD1/PD-L1信号通路药物中的应用，也就是发现了Pentamidine对PD1/PD-L1信号通路的抑制作用，Pentamidine对其产生抑制作用的适宜浓度为：0.5-4 μM。
本发明经研究发现：Pentamidine可以作为PD1/PD-L1蛋白相互作用的抑制剂。PD1/PD-L1信号通路与免疫应答、肿瘤免疫逃逸密切相关，抑制PD1/PD-L1信号通路的异常激活能够起到预防及治疗肿瘤的作用。本发明的Pentamidine能够抑制PD1/PD-L1蛋白质之间相互作用。
附图说明
图1和表1为Pentamidine在体外结合人源PD1和PD-L1的能力，其中，Pentamidine在能够结合人源PD-L1，KD值为3.498*10-4，能够结合鼠源PD-L1，KD值为1.588*10-4，而对人源和鼠源的PD-1没有明显结合：图中为以商品化PD1/PD-L1抑制剂为对照组，测试了不同浓度梯度下Pentamidine体外结合人源PD1和PD-L1的能力；
图2和图3为Pentamidine增强原代T细胞杀伤肿瘤细胞的能力，其中，图2中Pentamidine在浓度范围为:1μM时能够促进原代T细胞对非小细胞肺癌细胞H1975进行杀伤；图中为加药不同浓度在不同时间点的肿瘤细胞死亡比例；
图4为不同浓度Pentamidine处理不同时间后H1975细胞的细胞活力。
具体实施方式
以下结合实施例对本发明的技术方案作进一步地详细介绍，但本发明的保护范围并不局限于此。
实施例1
材料与方法
1.材料
1.1试剂
Pentamidine购自santa cruz公司，纯度为98%，PI购自索莱宝公司。
1.2仪器与器材：
海尔医用低温保存箱（青岛海尔特种电器有限公司）；酶联免疫检测仪（thermo公司）：Biacore T200（GE Healthcare Life Sciences）；电子天平（梅特勒-托利多仪器上海有限公司）； 细胞培养箱（thermo公司）；流式细胞仪（BD FACS Calibur）；Thermo 超净工作台：多肽氨基偶联试剂盒（Series S sensor chip cm5，GE Healthcare Life Sciences）：PD1/PD-L1 inhibitor 1Catalog No：DC9280 (CAS:1675201-83-8，购自DC Chemicals）。
在体外结合人源PD1和PD-L1的能力
具体实验流程如下，人重组PD1蛋白（组蛋白标签），人重组PD-L1蛋白（组蛋白标签），小鼠重组PD1蛋白（组蛋白标签），小鼠重组PD-L1蛋白（组蛋白标签）购买自北京义翘神州科技有限公司，使用终浓度10μg/ml的重组蛋白通过多肽氨基偶联试剂盒（Series S sensorchip cm5）交联在Series S sensor chip cm5芯片（根据GE公司提供的操作手册，在SeriesS sensor chip cm5芯片PD1蛋白和PD-L1蛋白的偶联量为200RU）上，并在Biacore T200中运行，检测蛋白交联水平，重组蛋白完成交联之后，对化合物pentamidine与四种重组蛋白人重组PD1蛋白（组蛋白标签），人重组PD-L1蛋白（组蛋白标签），小鼠重组PD1蛋白（组蛋白标签），小鼠重组PD-L1蛋白（组蛋白标签）的体外物理结合能力进行检测。将化合物Pentamidine 25mg用DMSO稀释为5mM作为储备液后，稀释为终浓度5μM,10μM,20μM,40μM,80μM,160μM，320μM的溶液各160μl，并在Biacore T200中检测与四种重组蛋白之间的体外物理结合能力。在实验中，以PD1/PD-L1 inhibitor 1作为阳性对照，以DMSO为溶剂，将PD1/PD-L1 inhibitor 1分别稀释至终浓度为5μM,10μM, 20μM, 40μM, 80μM,160μM,320μM的溶液各160μl，阳性对照用来证明购买的PD1和PD-L1重组蛋白能够与PD1/PD-L1的inhibitor互作，该实验通过表面等离子体共振实验分析分子之间相互作用，得出pentamidine与PD-L1重组蛋白相结合的KD 值（动力学参数），具体结果详见图1，并通过图1计算出Kd值，具体见表1，通过表1可以得出Pentamidine和PD1/PD-L1 inhibitor 1对重组蛋白的体外物理结合能力的Kd值，Pentamidine能够与人源和小鼠原PD-L1重组蛋白在体外存在明显的物理结合能力，并随着浓度的增加而增加。
表1
表1中：mPD-1代表人重组PD1蛋白，mPD-L1代表人重组PD-L1蛋白，hPD-1代表小鼠重组PD1蛋白，hPD-L1代表小鼠重组PD-L1蛋白，NA表示未检出。
增强原代T细胞杀伤肿瘤细胞的能力
具体实验流程如下，培养中的人原代PBMCs生长于GT-T551培养基（TAKARA公司）含10%胎牛血清（BI 公司）和200U/ml的IL-2进行维持培养。H1975肺癌细胞系使用RPMI-1640培养基（BI 公司），含10%胎牛血清（BI 公司）。在实验进行第一日，对H1975细胞进行CFSE活细胞染料标记，标记按照说明书进行操作，标记后细胞于24孔板中以每孔2万个细胞，铺上H1975细胞，并进行过夜培养，等待其贴壁，贴壁后加入人原代T细胞进行共培养，人原代PBMCs和H1975细胞以细胞数量20:1的比例在24孔板中进行共培养，并加入PD1/PD-L1 inhibitor 1终浓度4μM，Pentamidine 0.5μM持续共培养约16-18h，同时用不加人原代PBMCs的同样数量H1975细胞加入相同浓度的PD1/PD-L1 inhibitor 1或Pentamidine作为阴性对照实验，最终可以看到人原代PBMCs和H1975共培养组出现大量H1975细胞漂浮于培养基中，消化细胞并收集培养基上清后，加入1ml的PBS清洗细胞，离心除去多余的PBS后，加入100μl的PBS和5μl的PI进行染色，染色20分钟后进行流式细胞分析，分析结果如图2，首先根据FSC/SSC圈出H1975主要细胞群，然后用FL-1H通道标记处CFSE细胞染料阳性的H1975细胞，然后对CFSE阳性的细胞的FL-3H通道分析死亡的H1975细胞比例，如图2所示。
根据不同处理的实验组中，死亡的H1975细胞的比例进行非配对T检验统计学分析，可以得出在人原代T细胞与H1975肺癌细胞的共培养体系中加入化合物Pentamidine后，H1975细胞的死亡比例明显增加，并相对于非加药组有显著地统计学差异，并与阳性对照组化合物Inhibitor (PD1/PD-L1 inhibitor 1)的效应相似，如图3所示。并通过不同浓度的化合物Pentamidine与H1975细胞系的在不同时间进行孵育来探索，Pentamidine作为PD1/PD-L1抑制化合物的最大使用剂量，使用MTT实验测定Pentamidine处理后H1975细胞的细胞活力，首先选择在96孔板中每个孔加入1mL浓度为1*105个每毫升H1975细胞，5%CO2，37℃孵育分别培养24/48/72小时，每孔加入20μl MTT溶液（5mg/ml），继续培养2h，终止培养，除去培养基和MTT溶液后，每孔加入100 μl DMSO溶液，震荡混匀后在酶联免疫检测仪OD 560nm处测量各孔的吸光值。如图4所示，当Pentamidine使用剂量为4μM时，在24h,48h和72h均不影响H1975细胞系的细胞活力，当浓度增加后，H1975细胞活力出现明显的减少，故Pentamidine的最大使用剂量为4μM。
实验中最低浓度为0.5μM， 通过MTT得知更大浓度可以达到44μM，而不影响细胞增殖。
综上可以看出：本发明经研究发现Pentamidine能够抑制PD1/PD-L1蛋白之间的体外相互作用，并且增强原代T细胞对肺癌细胞系的体外杀伤作用。
Claims (5)
1.喷他脒在制备免疫治疗药剂中的应用，其特征在于，作为PD1和PD-L1蛋白之间作用的抑制剂。
2.如权利要求1所述喷他脒在制备免疫治疗药剂中的应用，其特征在于，PD1浓度为10μg/ml，PD-L1浓度为10μg/ml，喷他脒的浓度为0.5-4 μM。
3.如权利要求1所述喷他脒在制备免疫治疗药剂中的应用，其特征在于，通过增强T细胞对癌细胞的杀伤能力来发挥增强免疫作用。
4.如权利要求3所述喷他脒在制备免疫治疗药剂中的应用，其特征在于，所述癌细胞为肺癌细胞。
5.如权利要求4所述喷他脒在制备免疫治疗药剂中的应用，其特征在于，癌细胞数量1*105个每毫升，喷他脒的浓度为0.5-4 μM。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050215629A1 (en) * | 2001-09-07 | 2005-09-29 | Taolin Yi | PTPase inhibitors and methods of using the same |
| CN102573826A (zh) * | 2009-05-01 | 2012-07-11 | 奥克兹美制药公司 | 用于治疗癌症的喷他脒组合 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102573826A (zh) * | 2009-05-01 | 2012-07-11 | 奥克兹美制药公司 | 用于治疗癌症的喷他脒组合 |
Non-Patent Citations (1)
| Title |
|---|
| JULIETTE MÉRIAN ET AL.: "Development of a liposome formulation for improved biodistribution and tumor accumulation of pentamidine for oncology applications", 《INTERNATIONAL JOURNAL OF PHARMACEUTICS》 * |
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|---|---|---|---|---|
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