[二—（4-氯苯甲酰异羟肟酸）二正丁基合锡]（DBDCT）与CYP450相互作用的初步研究
摘要
一、背景
[二-(4-氯苯甲酰异羟肟酸)二正丁基合锡](DBDCT)是一种新型的抗肿瘤化合物,课题组前期对DBDCT体内代谢与药动学研究表明：DBDCT在大鼠体内吸收迅速、良好并广泛代谢,在尿液和粪便均未检测到原形药物,进入机体的DBDCT可能主要经肝微粒体酶代谢转化,大部分经肾或胆汁排出。
二、目的
本论文拟采用大鼠肝微粒体体外孵育实验,系统研究DBDCT在大鼠肝微粒体内的代谢情况,确定细胞色素P450 (CYP450)在DBDCT代谢中的作用,阐明DBDCT对大鼠CYP450的影响,为理解该药代谢的机制、预测药物相互作用和药物代谢多态性以及指导临床合理用药提供依据。
三、方法
采用差速离心法制备了大鼠的肝微粒体,用紫外可见分光光度法测定肝微粒体蛋白浓度及细胞色素P450含量,建立了测定大鼠肝微粒体样品中DBDCT浓度的HPLC法和大鼠肝微粒体体外代谢研究模型。以DBDCT为底物研究了该药在大鼠肝微粒体中的代谢行为；为了鉴定参与DBDCT代谢的CYP450同工酶,考察了CYP450专属性诱导剂和抑制剂对该反应的影响；分别以非那西丁、双氯芬酸钠、奥美拉唑和硝苯地平为底物评价大鼠CYP1A、CYP2C9、CYP2C19和CYP3A4的活性,以考察DBDCT对大鼠CYP450的作用。
四、结果
对大鼠肝微粒体进行诱导,测定肝微粒体蛋白浓度及细胞色素P450含量,各诱导组与空白组比较,含量均有显著性差异。建立了测定大鼠肝微粒体样品中DBDCT浓度的HPLC法,本法线性范围为3.48-174.20μmol·L-1,最低定量浓度为3.48μmol·L-1,检测限为0.73μmol·L-1,方法简便、快速、准确、重现性好,可适用于DBDCT的体外代谢研究。根据Michaelis-Menten方程和Eadie-Hofstee制图法求得最大反应速率(即纵轴的截距)Vmax=2.813μmol·(min·mg)-1,米氏常数(即直线斜率)Km=159.07μmol·L-1和肝清除率Clin=Vmax／Km=0.017L·(minmg)-1。DBDCT在不同酶源中的体外代谢结果提示：PB、DEX诱导组和空白对照组比较有显著性差异,而BNF组和空白对照组无明显差异；将DBDCT分别于各种CYP亚型的专属抑制剂体外共孵育,以确证介导DBDCT代谢的主要酶系,结果显示酮康唑对DBDCT的代谢有较强的抑制作用；此外,磺胺甲(?)唑对DBDCT的代谢也有一定的抑制,而氟伏沙明、甲氧苄氨嘧啶和奥美拉唑对DBDCT的代谢抑制作用较弱；底物试验中,硝苯地平在DBDCT处理过的鼠肝微粒体中代谢明显减弱,与空白组有明显差异(P<0.01,P<0.001),非那西丁、双氯芬酸钠和奥美拉唑在空白和DBDCT处理过的鼠肝微粒体中代谢无明显差异(P>0.05)。
五、结论
DBDCT在大鼠肝微粒体中代谢较快,提示该药可能肝首过效应较强。通过DBDCT在不同酶源中的体外代谢试验和代谢抑制试验证明CYP3A4在催化DBDCT代谢中起了主导作用,CYP2C9可能部分参与,而CYP1A对DBDCT的代谢无明显催化作用；底物试验结果说明DBDCT对CYP3A4有明显的抑制作用而对CYP1A、CYP2C9和CYP2C19均无明显影响。
1. Background
Di-n-butyl-di-(4-chlorobenzohydroxamato) tin (IV) (DBDCT) is a new antitumor compound.Our previous study has shown that DBDCT could be absorbed instantly and extensively metabolized after i.v. administration to rats. There was no parent drug of DBDCT in urine and manure; Presumably DBDCT was gradually biotransformed via stripping off ligand, alkyl and chlorine, eventually eliminated from kidney and bile through hepatic microsomal enzyme.
2. Aims
The primary objectives of the present study were to characterize cytochrome P450 (CYP450) enzyme(s) involved in the metabolism of DBDCT and evaluate the effects of DBDCT on CYP450.It is useful in the further understanding of DBDCT metabolic mechanism, the prediction of pharmacokinetic drug-drug interactions and interpatient variability in drug exposure.
3. Method
Liver microsomes of rats were prepared using ultracentrifuge method, followed by assays to detect changes in the levels of liver microsomel protein and CYP450.The in vitro metabolism of DBDCT was studied by incubation with rat liver microsomes and an HPLC method for the determination of DBDCT in rat liver microsomes was established.The metabolism of DBDCT was studied in rat liver microsomes using itself as substrate;To characterize the CYP450 isozymes involved in the metabolism of DBDCT, the effects of typical CYP450 inducers and of CYP450 inhibitors were investigated.The inhibitory effects of DBDCT on CYP450 were also investigated in the present study.The following CYP450 isoform specific marker reactions were measured; phenacetin(CYP1A), diclofenac sodium (CYP2C9), omeprazole(CYP2C19) and nifedipine(CYP3A4).
4. Results
Liver microsomes of rats were induced by inducer(Dex,BNF,PB),followed by assays to detect changes in the levels of liver microsomel protein and CYP450 content, the induction group compared with the control group were have significant deviation. An HPLC method for the determination of DBDCT in rat liver microsomes was established, the linear range of 3.48-174.20μmol·L-1, the lower detectable concentration was 3.48μmol·L-1,limitation of quantitation was 0.73μmol·L-1 this method proved to be convenient,fast,accurae with good reproducibility,can be applied to in vitro metabolism study of DBDCT. The kinetic parameters, Km, Vmax and liver clearance rate (Vmax/Km) were calculated by Michaelis-Menten equation and Eadie-Hofstee plot.In the metabolic system, the enzyme kinetics parameters of DBDCT were found as follows: Km=157.07μmol·L-1,Vmax=2.189μmol·(min·mg)-1 and liver clearance rate Vmax/Km=0.014L·(min·mg)-1, respectively. The results of in vitro metabolism of DBDCT in different enzyme sources suggest that:the PB, DEX group compared with the control group were have significantly deviation and the BNF group compared with the control group there have no significant deviation. DBDCT was invubated with exclusive inhibitors of various CYP450 isoforms in vitro, respectively, in order to confirm the major enzymes of mediate metabolism of DBDCT. The results showed that the ketoconazole on metabolism of DBDCT have a stronger inhibitory effect, in addition, sulfamethoxazole on DBDCT metabolism also have some inhibitory effect, and inhibition of fluvoxamine, trimethoprim, and omeprazole on DBDCT metabolism was weaker. Substrate test results:the metabolism of nifedipine in the DBDCT treated rat liver microsomes compared with the control group have significantly deviation (P <0.01, P<0.001). The metabolism of phenacetin, diclofenac sodium and omeprazole in the DBDCT treated rat liver microsomes compared with the control group was no significant difference (P> 0.05).
5. Conclusion
The metabolism of DBDCT in rat liver microsomes was faster, suggesting that the liver first-pass effect of the drug may be stronger. By experiments of in vitro metabolism of DBDCT in different enzymes sources and metabolic inhibition test results showed that the leading role of CYP450 isoforms in catalytic DBDCT metabolism was CYP3A4, CYP2C9 may be partly involved, CYP1A on the metabolism of DBDCT have no significant catalytic effect; Substrate test results showed that DBDCT have a stronger inhibitory effect on CYP3A4 and no significant effect by CYP1A, CYP2C9 and CYP2C19.
[二-(4-氯苯甲酰异羟肟酸)二正丁基合锡](DBDCT)是一种新型的抗肿瘤化合物,课题组前期对DBDCT体内代谢与药动学研究表明：DBDCT在大鼠体内吸收迅速、良好并广泛代谢,在尿液和粪便均未检测到原形药物,进入机体的DBDCT可能主要经肝微粒体酶代谢转化,大部分经肾或胆汁排出。
二、目的
本论文拟采用大鼠肝微粒体体外孵育实验,系统研究DBDCT在大鼠肝微粒体内的代谢情况,确定细胞色素P450 (CYP450)在DBDCT代谢中的作用,阐明DBDCT对大鼠CYP450的影响,为理解该药代谢的机制、预测药物相互作用和药物代谢多态性以及指导临床合理用药提供依据。
三、方法
采用差速离心法制备了大鼠的肝微粒体,用紫外可见分光光度法测定肝微粒体蛋白浓度及细胞色素P450含量,建立了测定大鼠肝微粒体样品中DBDCT浓度的HPLC法和大鼠肝微粒体体外代谢研究模型。以DBDCT为底物研究了该药在大鼠肝微粒体中的代谢行为；为了鉴定参与DBDCT代谢的CYP450同工酶,考察了CYP450专属性诱导剂和抑制剂对该反应的影响；分别以非那西丁、双氯芬酸钠、奥美拉唑和硝苯地平为底物评价大鼠CYP1A、CYP2C9、CYP2C19和CYP3A4的活性,以考察DBDCT对大鼠CYP450的作用。
四、结果
对大鼠肝微粒体进行诱导,测定肝微粒体蛋白浓度及细胞色素P450含量,各诱导组与空白组比较,含量均有显著性差异。建立了测定大鼠肝微粒体样品中DBDCT浓度的HPLC法,本法线性范围为3.48-174.20μmol·L-1,最低定量浓度为3.48μmol·L-1,检测限为0.73μmol·L-1,方法简便、快速、准确、重现性好,可适用于DBDCT的体外代谢研究。根据Michaelis-Menten方程和Eadie-Hofstee制图法求得最大反应速率(即纵轴的截距)Vmax=2.813μmol·(min·mg)-1,米氏常数(即直线斜率)Km=159.07μmol·L-1和肝清除率Clin=Vmax／Km=0.017L·(minmg)-1。DBDCT在不同酶源中的体外代谢结果提示：PB、DEX诱导组和空白对照组比较有显著性差异,而BNF组和空白对照组无明显差异；将DBDCT分别于各种CYP亚型的专属抑制剂体外共孵育,以确证介导DBDCT代谢的主要酶系,结果显示酮康唑对DBDCT的代谢有较强的抑制作用；此外,磺胺甲(?)唑对DBDCT的代谢也有一定的抑制,而氟伏沙明、甲氧苄氨嘧啶和奥美拉唑对DBDCT的代谢抑制作用较弱；底物试验中,硝苯地平在DBDCT处理过的鼠肝微粒体中代谢明显减弱,与空白组有明显差异(P<0.01,P<0.001),非那西丁、双氯芬酸钠和奥美拉唑在空白和DBDCT处理过的鼠肝微粒体中代谢无明显差异(P>0.05)。
五、结论
DBDCT在大鼠肝微粒体中代谢较快,提示该药可能肝首过效应较强。通过DBDCT在不同酶源中的体外代谢试验和代谢抑制试验证明CYP3A4在催化DBDCT代谢中起了主导作用,CYP2C9可能部分参与,而CYP1A对DBDCT的代谢无明显催化作用；底物试验结果说明DBDCT对CYP3A4有明显的抑制作用而对CYP1A、CYP2C9和CYP2C19均无明显影响。
1. Background
Di-n-butyl-di-(4-chlorobenzohydroxamato) tin (IV) (DBDCT) is a new antitumor compound.Our previous study has shown that DBDCT could be absorbed instantly and extensively metabolized after i.v. administration to rats. There was no parent drug of DBDCT in urine and manure; Presumably DBDCT was gradually biotransformed via stripping off ligand, alkyl and chlorine, eventually eliminated from kidney and bile through hepatic microsomal enzyme.
2. Aims
The primary objectives of the present study were to characterize cytochrome P450 (CYP450) enzyme(s) involved in the metabolism of DBDCT and evaluate the effects of DBDCT on CYP450.It is useful in the further understanding of DBDCT metabolic mechanism, the prediction of pharmacokinetic drug-drug interactions and interpatient variability in drug exposure.
3. Method
Liver microsomes of rats were prepared using ultracentrifuge method, followed by assays to detect changes in the levels of liver microsomel protein and CYP450.The in vitro metabolism of DBDCT was studied by incubation with rat liver microsomes and an HPLC method for the determination of DBDCT in rat liver microsomes was established.The metabolism of DBDCT was studied in rat liver microsomes using itself as substrate;To characterize the CYP450 isozymes involved in the metabolism of DBDCT, the effects of typical CYP450 inducers and of CYP450 inhibitors were investigated.The inhibitory effects of DBDCT on CYP450 were also investigated in the present study.The following CYP450 isoform specific marker reactions were measured; phenacetin(CYP1A), diclofenac sodium (CYP2C9), omeprazole(CYP2C19) and nifedipine(CYP3A4).
4. Results
Liver microsomes of rats were induced by inducer(Dex,BNF,PB),followed by assays to detect changes in the levels of liver microsomel protein and CYP450 content, the induction group compared with the control group were have significant deviation. An HPLC method for the determination of DBDCT in rat liver microsomes was established, the linear range of 3.48-174.20μmol·L-1, the lower detectable concentration was 3.48μmol·L-1,limitation of quantitation was 0.73μmol·L-1 this method proved to be convenient,fast,accurae with good reproducibility,can be applied to in vitro metabolism study of DBDCT. The kinetic parameters, Km, Vmax and liver clearance rate (Vmax/Km) were calculated by Michaelis-Menten equation and Eadie-Hofstee plot.In the metabolic system, the enzyme kinetics parameters of DBDCT were found as follows: Km=157.07μmol·L-1,Vmax=2.189μmol·(min·mg)-1 and liver clearance rate Vmax/Km=0.014L·(min·mg)-1, respectively. The results of in vitro metabolism of DBDCT in different enzyme sources suggest that:the PB, DEX group compared with the control group were have significantly deviation and the BNF group compared with the control group there have no significant deviation. DBDCT was invubated with exclusive inhibitors of various CYP450 isoforms in vitro, respectively, in order to confirm the major enzymes of mediate metabolism of DBDCT. The results showed that the ketoconazole on metabolism of DBDCT have a stronger inhibitory effect, in addition, sulfamethoxazole on DBDCT metabolism also have some inhibitory effect, and inhibition of fluvoxamine, trimethoprim, and omeprazole on DBDCT metabolism was weaker. Substrate test results:the metabolism of nifedipine in the DBDCT treated rat liver microsomes compared with the control group have significantly deviation (P <0.01, P<0.001). The metabolism of phenacetin, diclofenac sodium and omeprazole in the DBDCT treated rat liver microsomes compared with the control group was no significant difference (P> 0.05).
5. Conclusion
The metabolism of DBDCT in rat liver microsomes was faster, suggesting that the liver first-pass effect of the drug may be stronger. By experiments of in vitro metabolism of DBDCT in different enzymes sources and metabolic inhibition test results showed that the leading role of CYP450 isoforms in catalytic DBDCT metabolism was CYP3A4, CYP2C9 may be partly involved, CYP1A on the metabolism of DBDCT have no significant catalytic effect; Substrate test results showed that DBDCT have a stronger inhibitory effect on CYP3A4 and no significant effect by CYP1A, CYP2C9 and CYP2C19.
引文
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