CN108459094A - 一种大鼠肝cyp450酶诱导的评价方法 - Google Patents
一种大鼠肝cyp450酶诱导的评价方法 Download PDFInfo
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- G—PHYSICS
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Abstract
本发明属于医药检测的技术领域,涉及一种大鼠肝CYP450酶诱导的评价方法,其包括如下步骤:(1)大鼠给药诱导;(2)大鼠肝组织采集;(3)肝S9的制备;(4)孵育体系的处理;(5)样品分析。本发明所述的方法简单便捷,且成本低廉,可以有效的定性检测并评价药物对大鼠肝CYP450酶的诱导作用。
Description
技术领域
本发明属于医药检测的技术领域,涉及一种大鼠肝CYP450酶诱导的评价方法。
背景技术
CYP450酶是介导大部分外源性和内源性物质的代谢酶,包括CYP1A2、CYP2B6、CYP2C8、CYP2C9、CYP2C19、CYP2D6和CYP3A4等与药物代谢密切相关的同工酶。药物研发阶段候选化合物的淘汰率很高,原因之一是候选化合物对代谢酶的诱导或抑制活性有可能导致临床的代谢性药物相互作用(DDI)。其中CYP450酶诱导所致的DDI能显著改变联合用药的药动学行为、药效及毒性。新药候选化合物的酶诱导能力评价已成为国际制药企业新药研发中的必要环节(Lin,J.H.,CYP induction-mediated drug interactions:in vitroassessment and clinical implications,Pharmaceutical Research,2006,23(6),第1089-1116页)。
目前对于CYP450酶诱导的能力可通过体内外技术评价。体内技术中人源化的转基因鼠则是预测人体酶诱导能力较好的整体模型。体外技术包括应用新鲜或冻存的原代人肝细胞的酶诱导方法、PXR受体激活法(艾常虹等,基于细胞色素P450酶诱导的药物相互作用及评价,《国际药学研究杂志》,2011年,第1期,第52-57页)。利用体外方法进行药物代谢研究,可有效缩短新药研发周期,直接观察酶对底物的选择性代谢和底物对酶的诱导和抑制情况,确定药物代谢途径,预测体内潜在的药物相互作用,是新药发现早期和临床前评价最常用的评价技术。目前,体外方法均是通过探针底物孵育,根据底物的代谢物生成量变化来判断是否有诱导,该方法通过建立肝S9温孵体系,从而建立了大鼠CYP450酶诱导的评价模型。
发明内容
本发明目的在于开发一种快速方便且成本较低的大鼠肝CYP450酶诱导评价方法,其对给药后所采集的大鼠肝组织简单制备为肝S9后,即可进行肝药酶活性考察,整个制备过程时间大大缩短,且对于仪器设备要求相对较低,更有利于肝药酶活性的保持。另外本发明开发出CYP1A2、CYP2B6、CYP3A4以及2C家族亚酶(CYP2C9)的合适孵育体系,能准确评价药物的肝CYP450酶诱导情况。
本发明涉及一种大鼠肝CYP450酶诱导检测评价方法,其包括如下步骤:
(1)大鼠给药诱导;
(2)大鼠肝组织采集;
(3)肝S9的制备;
(4)孵育体系的处理;
(5)样品分析;
其中,所述的肝S9的制备包括如下步骤:称取500mg左右大鼠肝组织于5mL离心管中,于冰上剪碎肝组织,按1:3(w:v)比例加入匀浆液,用手持匀浆机于冰上以6000rpm匀浆1min,将匀浆后的混合液转入2mL离心管中,9000rpm,4℃离心30min,每管取200μL上清液冻存于-70~-80℃冰箱中;
其中,所述匀浆液为PH为7.4的0.25M蔗糖、0.01M Tris-HCl和1mM EDTA的混合。
在一些实施例中,所述孵育体系的处理包括:往96孔板中加入15μL的底物溶液和30μL S9混合溶液,平行测试两孔,37℃预孵10min;然后加入15μL 8mM的NADPH溶液,孵育45min,加入150μL含有浓度为100ng/mL的普萘洛尔作为内标的ACN溶液终止反应;最后,将孵育后的混合液以4000rpm离心5min,取出100μL上清液,再加入100μL水稀释,得到待分析的样品。
在一些实施例中,所述底物溶液为CYP1A2探针底物溶液、CYP2B6探针底物溶液、CYP2C9探针底物溶液或CYP3A4探针底物溶液。
在一些实施例中,所述CYP1A2探针底物为非那西汀,CYP2B6探针底物为安非他酮,CYP2C9探针底物为双氯芬酸钠,CYP3A4探针底物为睾酮。
在一些实施例中,所述的CYP1A2探针底物溶液为将非那西汀溶于0.1M磷酸钾缓冲液中形成的浓度为120μM的溶液;所述CYP2B6探针底物溶液为将安非他酮溶于0.1M磷酸钾缓冲液中形成的浓度为40μM的溶液;所述CYP2C9探针底物溶液为将双氯芬酸钠溶于0.1M磷酸钾缓冲液中形成的浓度为40μM的溶液;所述CYP3A4探针底物溶液为将睾酮溶于0.1M磷酸钾缓冲液中形成的浓度为320μM的溶液。
在一些实施例中,所述S9混合溶液是将本发明所述方法制备得到的肝S9用0.1M磷酸钾缓冲液稀释100倍得到的混合溶液。
在一些实施例中,所述8mM的NADPH溶液为将NADPH溶于0.1M磷酸钾缓冲液中形成的浓度为8mM的溶液。
在一些实施例中,所述大鼠给药诱导包括:对大鼠进行灌胃给予地塞米松,给药剂量为50mg/kg,连续给药7天。
在一些实施例中,所述大鼠肝组织采集包括:末次给药24h后,采用二氧化碳安乐处死大鼠,取其肝组织上叶用生理盐水冲洗干净,吸干表面生理盐水,冻存于-70~-80℃冰箱中。
在一些实施例中,所述样品分析的体检为:
分析仪器:LC/MS/MS;
色谱分析条件:
色谱柱:Waters XBridgeTMC18,2.1*50mm 3.5μm;
柱温:40℃;
进样体积:10-20μL;
进样器温度:10℃;
运行时间:3.7min;
流速:0.3-0.4mL/min;
流动相:
A相:H2O+2mM乙酸铵+0.1%甲酸;
B相:MeOH+2mM乙酸铵+0.1%甲酸;
梯度:
0.20min,B相2%;
0.22min,B相20%;
1.80min,B相80%;
2.60min,B相90%;
2.70min,B相2%;
3.70min,停止;
洗针液:ACN:MeOH:H2O=1:1:1(V:V:V),另加0.1%甲酸;
洗针时间:10s;
质谱条件为:
具体实施方式
本发明采用以下方案,针对地塞米松给药后大鼠肝中CYP1A2、CYP2B6、CYP2C9、CYP3A4酶诱导进行定性检测,其包括以下步骤:
1)大鼠给药诱导
实验设置空白溶媒组和阳性药对照组:空白溶媒组采用生理盐水给药;阳性药对照组采用已报道的具肝CYP450酶诱导的化合物地塞米松灌胃给药,给药剂量为50mg/kg,连续口服给药7天。
2)大鼠肝组织采集
末次给药24小时后采用二氧化碳安乐处死大鼠,取肝组织上叶用生理盐水冲洗干净,吸干表面生理盐水,冻存于-70~-80℃冰箱中以备匀浆。
3)肝S9制备
每只大鼠称取约500mg左右肝组织于5mL离心管中,于冰上剪碎肝组织,按1:3(w:v)比例加入匀浆液(0.25M蔗糖、0.01M Tris-HCl、1mM EDTA,PH 7.4),用手持匀浆机于冰上以6000rpm/min匀浆约1min,将匀浆后的混合液转入2mL离心管中,4℃离心30min,上层液即为肝S9,每管取200μL上清液冻存于-70~-80℃冰箱中备用。
4)孵育体系设计
为了确保线性反应,本发明对缓冲体系、NADPH浓度、各亚酶探针底物的浓度、S9浓度以及孵育时间进行了探索,最终确定各亚酶的孵育体系如下:
缓冲体系:0.1M磷酸钾缓冲液;
NADPH溶液:将NADPH溶解于缓冲体系中,溶液浓度为8mM;
非那西汀(CYP1A2探针底物):将非那西汀溶解于缓冲体系中,溶液浓度为120μM;
双氯芬酸钠(CYP2C9探针底物):将双氯芬酸钠溶解于缓冲体系中,溶液浓度为40μM;
盐酸安非他酮(CYP2B6探针底物):将盐酸安非他酮溶解于缓冲体系中,溶液浓度为40μM;
睾酮(CYP3A4探针底物):将睾酮溶解于缓冲体系中,溶液浓度为320μM;
S9浓度:对已制备的肝S9用0.1M磷酸钾缓冲液稀释100倍(例如:10μL肝S9加到1mL的0.1M磷酸钾缓冲液中);
孵育时间:45min。
5)孵育体系处理:
在各CYP酶探针底物(CYP1A2探针底物为非那西汀、CYP2B6探针底物为安非他酮、CYP2C9探针底物为双氯芬酸钠、CYP3A4探针底物为睾酮)对应的孔中加入15μL相对应的底物溶液。
在各探针底物的位置中加入30μL S9混合溶液,每个药物每个时间点平行做两孔,37℃预孵10min。
预孵后,先将15μL NADPH溶液加入到各孔中,启动反应开始计时,孵育45min;孵育结束后,将150μL ACN(含内标普萘洛尔,浓度为100ng/mL)加入各孔中终止反应。以4000rpm离心5min,取出100μL上清液,加100μL水稀释,用于样品分析。
6)样品分析方法:
分析仪器:LC/MS/MS
色谱条件:
流动相:
A相:H2O+2mM乙酸铵+0.1%甲酸;
B相:MeOH+2mM乙酸铵+0.1%甲酸;
洗脱梯度:
质谱条件:
内标以及各亚酶底物的代谢物以及其检测条件:
注:普萘洛尔为分析用内标;对乙酰氨基酚为CYP1A2探针底物代谢物;羟基安非他酮为CYP2B6探针底物代谢物;4'-羟基双氯酚酸为CYP2C9探针底物代谢物;羟睾酮为CYP3A4探针底物代谢物。
实施例及测试数据
实施例1地塞米松给药后大鼠肝中CYP3A4酶诱导检测
按以上技术方案对地塞米松连续给药后的大鼠肝中CYP3A4亚酶的诱导情况进行了检测,结果如下表:
结论:
从上表数据可以看出地塞米松给药组肝S9加CYP3A4探针底物后其代谢物生成量是空白溶媒组肝S9代谢物生成量的4.2倍,根据2009版《药物相互作用研究指导原则》中指出酶活性达到2倍以上即认为有酶诱导,本发明能准确的判断药物对大鼠肝中CYP3A4的酶诱导情况。
实施例2地塞米松给药后大鼠肝中CYP2C9酶诱导检测
按以上技术方案对地塞米松连续给药后的大鼠肝中CYP2C9亚酶的诱导情况进行了检测,结果如下表:
结论:
从上表数据可以看出地塞米松给药组肝S9加CYP2C9探针底物后其代谢物生成量是空白溶媒组肝S9代谢物生成量的3.5倍,诱导倍数高于2倍。根据2012版《药物相互作用研究指导原则》指出CYP2C、CYP2B与CYP3A具有协同诱导作用,2C家族与CYP3A4的基因表达均是由PXR核受体调控,所以对于CYP3A4有诱导的化合物对CYP2C9也可能会有诱导,本发明的试验结果与机制一致,准确的判断了药物对大鼠肝中CYP2C9的酶诱导情况。
实施例3地塞米松给药后大鼠肝中CYP2B6酶诱导检测
按以上技术方案对地塞米松连续给药后的大鼠肝中CYP2B6亚酶的诱导情况进行了检测,结果如下表:
结论:
从数据可以看出地塞米松给药组肝S9加CYP2B6探针底物后其代谢物生成量是空白溶媒组肝S9代谢物生成量的2.3倍,诱导倍数高于2倍。根据2012版《药物相互作用研究指导原则》指出CYP2C、CYP2B与CYP3A具有协同诱导作用,所以对于CYP3A4有诱导的化合物对CYP2B6也可能会有诱导,本发明的试验结果与机制一致,准确的判断了药物对大鼠肝中CYP2B6的酶诱导情况。
实施例4地塞米松给药后大鼠肝中CYP1A2酶诱导检测
按以上技术方案对地塞米松连续给药后的大鼠肝中CYP2B6亚酶的诱导情况进行了检测,结果如下表:
结论:
从数据可以看出地塞米松给药组肝S9加CYP1A2探针底物后其代谢物生成量是空白溶媒组肝S9代谢物生成量的1.2倍,低于2倍表明无诱导,这与文献报道(Stejskalova L等,Dexamethasone accelerates degradation of aryl hydrocarbon receptor(AHR)andsuppresses CYP1A1induction in placental JEG-3cell line,Toxicology Letters,2013,223(2),第183-191页)一致。表明本发明所述的检测方法不会带来假阳性结果,实验结果较可靠。
综上所述,本发明所述技术方案可以有效的定性检测药物对于大鼠肝CYP450酶的诱导作用,可以简单快捷的定性评价药物对大鼠肝CYP450酶的诱导效果。
Claims (8)
1.一种大鼠肝CYP450酶诱导检测评价方法,其包括如下步骤:
(1)大鼠给药诱导;
(2)大鼠肝组织采集;
(3)肝S9的制备;
(4)孵育体系的处理;
(5)样品分析;
其中,所述的肝S9的制备包括如下步骤:称取大鼠肝组织于离心管中,于冰上剪碎肝组织,按1:3(w:v)比例加入匀浆液,用手持匀浆机于冰上以6000rpm匀浆1min,将匀浆后的混合液转入2mL离心管中,9000rpm,4℃离心30min,每管取200μL上清液冻存于-70~-80℃冰箱中;
其中,所述匀浆液为PH为7.4的0.25M蔗糖、0.01M Tris-HCl和1mM EDTA的混合。
2.根据权利要求1所述的方法,其中,所述孵育体系的处理包括:往96孔板中加入15μL的底物溶液和30μL S9混合溶液,平行测试两孔,37℃预孵10min;然后加入15μL 8mM的NADPH溶液,孵育45min,加入150μL含有浓度为100ng/mL的普萘洛尔作为内标的ACN溶液终止反应;最后,将孵育后的混合液以4000rpm离心5min,取出100μL上清液,再加入100μL水稀释,得到待分析的样品;
其中,所述底物溶液为CYP1A2探针底物溶液、CYP2B6探针底物溶液、CYP2C9探针底物溶液或CYP3A4探针底物溶液。
3.根据权利要求2所述的方法,其中,所述CYP1A2探针底物为非那西汀,CYP2B6探针底物为安非他酮,CYP2C9探针底物为双氯芬酸钠,CYP3A4探针底物为睾酮。
4.根据权利要求2所述的方法,其中,所述的CYP1A2探针底物溶液为将非那西汀溶于0.1M磷酸钾缓冲液中形成的浓度为120μM的溶液;所述CYP2B6探针底物溶液为将安非他酮溶于0.1M磷酸钾缓冲液中形成的浓度为40μM的溶液;所述CYP2C9探针底物溶液为将双氯芬酸钠溶于0.1M磷酸钾缓冲液中形成的浓度为40μM的溶液;所述CYP3A4探针底物溶液为将睾酮溶于0.1M磷酸钾缓冲液中形成的浓度为320μM的溶液。
5.根据权利要求2所述的方法,其中,所述S9混合溶液是将根据权利要求1制备得到的肝S9用0.1M磷酸钾缓冲液稀释100倍得到的混合溶液。
6.根据权利要求1所述的方法,其中,所述大鼠给药诱导包括:对大鼠进行灌胃给药,连续给药7天。
7.根据权利要求1所述的方法,其中,所述大鼠肝组织采集包括:末次给药24h后,采用二氧化碳安乐处死大鼠,取其肝组织上叶用生理盐水冲洗干净,吸干表面生理盐水,冻存于-70~-80℃冰箱中。
8.根据权利要求1所述的方法,其中,所述样品分析的体系为:
分析仪器:LC/MS/MS;
色谱分析条件:
色谱柱:Waters XBridgeTMC18,2.1*50mm 3.5μm;
柱温:40℃;
进样体积:10-20μL;
进样器温度:10℃;
运行时间:3.7min;
流速:0.3-0.4mL/min;
流动相:
A相:H2O+2mM乙酸铵+0.1%甲酸;
B相:MeOH+2mM乙酸铵+0.1%甲酸;
洗脱梯度:
0.20min,B相2%;
0.22min,B相20%;
1.80min,B相80%;
2.60min,B相90%;
2.70min,B相2%;
3.70min,停止;
洗针液:ACN:MeOH:H2O=1:1:1(V:V:V),另加0.1%甲酸;
洗针时间:10s;
质谱条件为:
扫描类型:MRM;
离子源:ESI+;
干燥器温度:350℃;
干燥气流速:9L/min;
雾化器压力:40psi;
毛细管电压:4000V。
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