CN114984094B - 白头翁皂苷组合物在制备治疗car-t疗法引起crs的药物中的应用 - Google Patents
白头翁皂苷组合物在制备治疗car-t疗法引起crs的药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种白头翁皂苷组合物在制备治疗CAR‑T疗法引起CRS的药物中的应用,白头翁皂苷组合物包括重量比为3‑9:1的白头翁皂苷B4和白头翁皂苷B5。本发明对CD19‑CAR‑T细胞诱导肿瘤细胞死亡无影响,但能够显著抑制细胞释放炎症因子水平,对CAR‑T治疗产生的炎症因子风暴具有保护作用。
Description
技术领域
本发明涉及药物技术领域。更具体地说,本发明涉及一种白头翁皂苷组合物在制备治疗CAR-T疗法引起CRS的药物中的应用。
背景技术
嵌合抗原受体T细胞(CAR-T)疗法作为通过改造病人自身免疫系统而实现特异性杀死癌症细胞的一种免疫疗法,为人类战胜癌症带来胜利曙光。2017年,美国食品药品监督管理局(FDA)已经批准了用于治疗白血病和淋巴瘤的CAR-T疗法,从而拯救了那些传统疗法无法治愈的癌症患者。然而,研究人员发现在CAR-T的临床的应用中,会伴有明显副作用——炎症因子风暴(Cytokine release syndrome,CRS)。而且CAR-T杀伤肿瘤细胞的效果越明显,炎症因子风暴(CRS)也越强烈,过强的炎症反应反而会引起病人的死亡。2019年,有37%-93%的淋巴瘤患者和77%-93%的白血病患者患有CRS。CRS也是CAR-T治疗患者死亡的重要原因。因此,抑制CRS对CAR-T治疗患者死亡具有一定的保护作用。白头翁皂苷是从毛茛科多年生草本植物白头翁中提取具有药理活性的五环三萜类皂苷,其中白头翁皂苷B4(Anemoside B4,B4)和白头翁皂苷B5(Hederasaponin C,HSC)是其主要有效成分,但关于白头翁皂苷对CAR-T导致CRS的作用及机制研究尚不明确。
发明内容
本发明提供一种白头翁皂苷组合物在制备治疗CAR-T疗法引起CRS的药物中的应用,其对CD19-CAR-T细胞诱导肿瘤细胞死亡无影响,但能够显著抑制细胞释放炎症因子水平,对CAR-T治疗产生的炎症因子风暴具有保护作用。
为了实现根据本发明的这些目的和其它优点,提供了一种白头翁皂苷组合物在制备治疗CAR-T疗法引起CRS的药物中的应用,白头翁皂苷组合物包括重量比为3-9:1的白头翁皂苷B4和白头翁皂苷B5。
优选的是,所述药物含有所述白头翁皂苷组合物和药学上可接受的载体,白头翁皂苷B4、白头翁皂苷B5纯度不低于60%。
优选的是,所述药物含有治疗有效量的白头翁皂苷的盐酸盐、高氯酸盐、甲磺酸盐、磷酸盐、柠檬酸盐或硫酸盐和药学上可接受的载体。
优选的是,药学上可接受的载体包括稀释剂、增溶剂、潜溶剂、崩解剂、分散剂、润滑剂、矫味剂、抗氧剂、粘合剂、吸收剂、湿润剂、缓冲剂、交联剂。
优选的是,所述药物被制成药学上允许的剂型。
优选的是,所述剂型包括丸剂、片剂、粉剂、胶囊剂、颗粒剂、散剂、滴丸、滴剂、喷雾剂、注射剂、混悬液、凝胶剂、栓剂。
优选的是,白头翁皂苷组合物的给药剂量为不低于5mg/kg·d。
本发明至少包括以下有益效果:
白头翁皂苷组合物可以提高CAR-T治疗小鼠生存率,降低CAR-T治疗后小鼠发热,改善小鼠体重降低,对CAR-T治疗产生的炎症因子风暴如ATP、IL-6、IL-1的和SAA水平具有显著的抑制作用。体外细胞实验证明,白头翁皂苷组合物对CD19-CAR T细胞诱导肿瘤细胞死亡无影响,但能够显著抑制细胞释放炎症因子水平。说明白头翁皂苷组合物对CAR-T治疗产生的炎症因子风暴具有保护作用。
本发明的其它优点、目标和特征将部分通过下面的说明体现,部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
附图说明
图1为本发明的B4+HSC对小鼠生存率的影响;
图2为本发明的B4+HSC对小鼠体温的影响;
图3为本发明的B4+HSC对小鼠体重的影响;
图4为本发明的B4+HSC对小鼠血清中ATP、IL-6、IL-1β和SAA水平的影响;
图5为本发明的B4+HSC对CD19-CAR T诱导Raji细胞死亡的影响;
图6为本发明的B4+HSC对CD19-CAR T诱导细胞释放炎症因子水平的影响。
具体实施方式
下面结合附图对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
应当理解,本文所使用的诸如“具有”、“包含”以及“包括”术语并不排除一个或多个其它元件或其组合的存在或添加。
需要说明的是,下述实施方案中所述实验方法,如无特殊说明,均为常规方法,所述试剂和材料,如无特殊说明,均可从商业途径获得。
1材料
1.1实验动物及细胞
雌性SCID-beige小鼠(购自北京维通利华生物有限公司),8周龄,SPF级,体重(20±2)g,饲养于可控环境中,室温20-24℃、湿度40%-50%,实验期间动物自由进食、饮水,昼夜节律正常。Raji细胞购自ATCC,10%FBE DMEM培养基培养,37℃、5%CO2孵箱中。
1.2主要药品和试剂
白头翁皂苷提取物,其中白头翁皂苷B4+白头翁皂苷B5(以下简称B4+HSC)的含量在60%以上,经测试重量比为4:1;白头翁皂苷提取物由实验室提取分离制备;IL-6、IL-1β和SAA Elisa Kit(欣博盛);穿孔素试剂盒、LDH试剂盒、颗粒酶B试剂盒(碧云天)。
1.3实验仪器
1/1000精密天平(梅特勒-托利多仪器上海有限公司,型号:ME204E);低温高速离心机(Eppendorf公司,型号:5425R);酶联免疫检测仪(美国BioTek公司,型号:SYNERGYH1)。
2实验方法
2.1动物模型分组与给药
30只小鼠随机分为空白对照组(附图1-5中简述为空白组),模型对照组(附图1-5中简述为模型组)、B4+HSC 5mg/kg灌胃组(附图1-5中简述为白头翁皂苷提取物5mg/kg)、B4+HSC 10mg/kg灌胃组(附图1-5中简述为白头翁皂苷提取物10mg/kg)、B4+HSC 20mg/kg灌胃组(附图1-5中简述为白头翁皂苷提取物20mg/kg),每组6只。
除空白对照组外,其余各组每只小鼠腹腔注射Raji细胞悬液(3×106细胞/200μL),接种21天后内眦采血,并记录肛温和体重。采血前10天采正常人外周血,分离培养CD8+T细胞(起始1×107naive T细胞)并感染CD19-CAR慢病毒制备CAR-T细胞,大量扩增。在采血后4-6小时,除空白组外其它小鼠按照3×107细胞回输CD19-CAR T细胞,回输细胞前一周,B4+HSC 5mg/kg灌胃组、B4+HSC 10mg/kg灌胃组、B4+HSC 20mg/kg灌胃组静脉注射白头翁皂苷组合物,每天一次,持续9天。空白对照组和模型对照组注射等体积生理盐水,每天一次,持续9天。回输CD19-CAR T细胞前12h及后48h内,每12h检测记录小鼠肛温和体重,同时记录死亡数。
2.1.1检测指标
(1)记录小鼠体重和体温;
(2)记录小鼠回输CD19-CAR T细胞后小鼠死亡情况;
(3)回输48小时后内眦采血检测ATP、IL-6、IL-1β和SAA。
2.2细胞实验
2.2.1 ATP和LDH检测细胞活力
CD19-CAR T细胞按照效靶比1:1与Raji细胞共孵育6h,共孵育前4h,Raji细胞加入白头翁皂苷提取物30μg/mL,再与肿瘤细胞共培养。ATP和LDH试剂盒检测细胞活力。
2.2.2 ELISA试剂盒检测炎症因子IL-6和IL-1β释放
CD19-CAR T细胞按照效靶比1:1与Raji细胞共孵育6h,共孵育前4h,Raji细胞加入白头翁皂苷提取物30μg/mL,再与肿瘤细胞共培养。ELISA剂盒检测炎症因子IL-6和IL-1β释放。
3实验结果
3.1白头翁皂苷提取物对小鼠生存率的影响
CD19-CAR T细胞回输后观察小鼠48h内死亡情况,实验结果如图1所示,模型对照组小鼠存活率约10%,而B4+HSC 5mg/kg灌胃组、B4+HSC 10mg/kg灌胃组、B4+HSC 20mg/kg灌胃组能够显著改善CD19-CAR T治疗导致的小鼠死亡,且B4+HSC 20mg/kg灌胃组存活率达到80%。以上结果说明白头翁皂苷提取物能够显著改善CAR-T治疗导致的小鼠死亡。
3.2白头翁皂苷提取物对小鼠体温的影响
CD19-CAR T细胞回输后观察小鼠48h内体温情况,实验结果如图2所示,模型对照组小鼠体温显著升高,而B4+HSC 5mg/kg灌胃组、B4+HSC 10mg/kg灌胃组、B4+HSC 20mg/kg灌胃组能够显著降低CD19-CAR T治疗导致小鼠发热,且B4+HSC 20mg/kg灌胃组效果最显著。以上结果说明,白头翁皂苷提取物能够显著降低CAR-T治疗导致的小鼠发热。
3.3白头翁皂苷提取物对小鼠体重的影响
CD19-CAR T细胞回输后观察小鼠48h内体温情况,实验结果如图3所示,模型对照组小鼠体重显著降低,而B4+HSC 5mg/kg灌胃组、B4+HSC 10mg/kg灌胃组、B4+HSC 20mg/kg灌胃组能够显著改善CD19-CAR T治疗导致小鼠体重降低,且B4+HSC 20mg/kg灌胃组效果最显著。以上结果说明白头翁皂苷提取物能够显著改善CAR-T治疗导致的小鼠体重降低。
3.4白头翁皂苷提取物对小鼠血清中ATP、IL-6、IL-1β和SAA水平的影响
CD19-CAR T细胞回输后48h,取小鼠血清检测ATP、IL-6、IL-1β和SAA水平。实验结果如图4所示,###P<0.001vs空白对照组,***P<0.001vs模型对照组,模型对照组小鼠血清中ATP、IL-6、IL-1β和SAA水平显著升高,而B4+HSC 5mg/kg灌胃组、B4+HSC 10mg/kg灌胃组、B4+HSC 20mg/kg灌胃组能够显著抑制CD19-CAR T治疗后小鼠血清中ATP、IL-6、IL-1β和SAA水平,且B4+HSC 20mg/kg灌胃组效果最显著。以上结果说明,白头翁皂苷提取物能够抑制CAR-T治疗后小鼠血清中ATP、IL-6、IL-1β和SAA水平,而IL-6和IL-1β是CAR-T治疗导致CRS的重要因素。
3.5白头翁皂苷提取物对CD19-CAR T诱导Raji细胞死亡的影响
如图5所示,图5A为ATP检测细胞活力,图5B为LDH试剂盒检测细胞释放LDH水平,***P<0.001vs PBS组,CD19-CAR T细胞与Raji细胞共培养后,细胞活力显著降低,LDH释放增加,而白头翁皂苷提取物对CD19-CAR T诱导细胞死亡无影响。
3.6白头翁皂苷提取物对CD19-CAR T诱导细胞释放炎症因子水平的影响
如图6所示,图6A为白头翁皂苷提取物对IL-6水平的影响;图6B为白头翁皂苷提取物对IL-1β水平的影响,###P<0.001vs PBS,***P<0.001vs CD19组,CD19-CAR T细胞与Raji细胞共培养后,诱导细胞死亡并释放大量炎症因子,IL-6和IL-1β水平显著升高。而白头翁皂苷提取物对CD19-CAR T细胞于Raji细胞共培养诱导炎症因子水平升高具有显著的抑制作用。以上结果说明,白头翁皂苷提取物对CD19-CAR T细胞诱导Raji细胞死亡无影响,但能够抑制CD19-CAR T细胞诱导炎症因子的释放,从而发挥抑制CAR T治疗导致的CRS。
4结论
白头翁皂苷提取物可以提高CAR-T治疗小鼠生存率,降低CAR-T治疗后小鼠发热,改善小鼠体重降低,对CAR-T治疗产生的炎症因子风暴如ATP、IL-6、IL-1β和SAA水平具有显著的抑制作用。体外细胞实验证明,白头翁皂苷提取物对CD19-CAR T细胞诱导肿瘤细胞死亡无影响,但能够显著抑制细胞释放炎症因子水平。说明白头翁皂苷提取物对CAR-T治疗产生的炎症因子风暴具有保护作用。
这里说明的设备数量和处理规模是来简化本发明的说明的。对本发明的应用、修改和变化对本领域的技术人员来说是显而易见的。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用,它完全可以被适用于各种适合本发明的领域,对于熟悉本领域的人员而言,可容易地实现另外的修改,因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的图例。
Claims (5)
1.白头翁皂苷组合物在制备治疗CAR-T疗法引起炎症因子风暴的药物中的应用,其特征在于,白头翁皂苷组合物由重量比为4:1的白头翁皂苷B4和白头翁皂苷B5组成,白头翁皂苷B4、白头翁皂苷B5纯度不低于60%。
2.如权利要求1所述的应用,其特征在于,所述药物含有治疗有效量的白头翁皂苷的盐酸盐、高氯酸盐、甲磺酸盐、磷酸盐、柠檬酸盐或硫酸盐和药学上可接受的载体。
3.如权利要求2所述的应用,其特征在于,药学上可接受的载体选自稀释剂、增溶剂、潜溶剂、崩解剂、分散剂、润滑剂、矫味剂、抗氧剂、粘合剂、吸收剂、湿润剂、缓冲剂、交联剂。
4.如权利要求1所述的应用,其特征在于,所述药物被制成药学上允许的剂型。
5.如权利要求1所述的应用,其特征在于,所述剂型选自丸剂、片剂、粉剂、胶囊剂、颗粒剂、散剂、滴丸、滴剂、喷雾剂、注射剂、混悬液、凝胶剂、栓剂。
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