川阿格雷的毒代动力学及药物暴露对代谢的影响
|
摘要
川阿格雷是基于传统中药机理，运用现代技术合成的新一代血小板凝集抑制剂。本文的工作旨在对其毒代动力学和体内相互作用作一整体评价，以支持人体药代动力学研究并为临床用药提供参考依据。
一、生物样品中川阿格雷分析方法的建立
本文采用液液萃取法处理血浆样品，建立了以桂皮醛为内标的反相高效液相色谱—紫外光谱检测川阿格雷的体内分析方法。色谱分离条件为：色谱柱Diamonsil C18（5μm，100×4.6mm）；流动相组成为乙腈、水及用来调节pH的乙酸铵（pH4.4），流速为1mL/min；紫外检测波长为276nm，柱温30℃。采用本文建立的生物样品预处理方法和反相高效液相色谱法分离测定生物样品中川阿格雷的含量，生物样品预处理方法回收率高，色谱分离选择性好。本法的准确度、精密度、灵敏度、专属性和定量线性范围均达到生物样品分析的要求，该方法成功用于川阿格雷在Beagle犬与SD大鼠体内的毒代动力学研究。
二、肝微粒体中CYP450酶亚型特异性底物及其代谢产物定量分析方法的建立
本文采用液液萃取法处理肝微粒体温孵样品，建立了以原儿茶酸、利眠宁和非那西丁为内标的反相高效液相色谱—紫外光谱检测CYP450酶亚型特异性底物及其代谢产物的分析方法。色谱分离条件为：色谱柱迪马Diamonsil C18（5μm，150×4.6mm）；非那西丁（CYP1A2底物）：流动相甲醇（0.1%甲酸）（A）—水（0.1%甲酸）（B），梯度洗脱0~5min18%A、5~10min18%~60%A、10~15min60%A，流速1.0mL/min，紫外检测波长为247nm，柱温30℃；咪达唑仑（CYP3A4底物）：流动相甲醇（A）—水（0.02%甲酸）（B），梯度洗脱0~11min40%~60%A，流速1.0mL/min，紫外检测波长为223nm，柱温30℃；氯唑沙宗（CYP2E1底物）：流动相甲醇（A）—水（B），梯度洗脱0~10min37%~75%A，流速1.0mL/min，紫外检测波长为287nm，柱温25℃。采用本文建立的样品预处理方法和反相高效液相色谱法分离测定温孵样品中特征底物及代谢产物的含量，方法回收率高，色谱分离选择性好，准确度、精密度、专属性和定量线性范围均达到生物样品分析的要求，该方法成功用于川阿格雷对SD大鼠肝微粒体酶活性影响的研究。
三、川阿格雷在动物体内的毒代动力学研究
本试验设计成年、健康Beagle犬多剂量静脉注射给药川阿格雷生理盐水注射液40、80和160mg/Kg三个剂量组进行毒代动力学研究。选择18条Beagle犬，雌雄各半，按体重随机分成低、中、高剂量三个剂量组，每一剂量组6条Beagle犬。每组Beagle犬一周给药6次（每天给药1次），第七天停药。采用缓慢静脉推注方式给药，给药周期为90天。于给药前及给药后第1，43，88天取血，每次取血点分别为给药后0、0.5、1.0、2.0、4.0、8.0、12.0、24.0h。用上述建立的HPLC-UV法测定血浆中川阿格雷的浓度，根据所得的血浆药物浓度—时间曲线，采用非房室模型推算药物动力学参数。结果显示药物在低剂量、中剂量长期静脉注射给药时对机体没有明显损伤，在高剂量长期静脉注射给药时对机体存在一定损伤，引起代谢或排泄功能减弱而导致最大血药浓度（C_0）增大。
本试验设计健康SD大鼠多剂量腹腔注射给药川阿格雷生理盐水注射液80、160和320mg/Kg三个剂量组进行毒代动力学研究。选择18只SD大鼠，雌雄各半，按体重随机分成低、中、高剂量三个剂量组，每一剂量组6只SD大鼠。每组SD大鼠一周给药6次（每天给药1次），第七天停药，采用缓慢腹腔注射方式给药，给药周期为90天。于给药前及给药后第1，44，91天取血，每次取血点分别为给药后0.833、0.5、1.0、2.0、4.0、8.0、12.0、24.0h。用上述建立的HPLC-UV法测定血浆中川阿格雷的浓度，根据所得的血浆药物浓度—时间曲线，采用非房室模型推算药物动力学参数。结果显示药物在低剂量、中剂量长期腹腔注射给药时对机体没有明显损伤，在高剂量长期腹腔注射给药时对机体存在一定损伤，引起代谢或排泄功能减弱而导致最大血药浓度（Cmax）增大。
四、川阿格雷暴露水平影响大鼠肝微粒体代谢酶活性的研究
本试验选择健康SD大鼠多剂量注射川阿格雷注射液15mg/Kg、80mg/Kg、160mg/Kg、320mg/Kg，进行川阿格雷影响大鼠肝微粒体代谢酶活性的研究。选择36只SD大鼠，雌雄各半，分成对照组和给药组，每组6只，给药组尾静脉注射给予川阿格雷15mg/Kg，腹腔注射给予川阿格雷80mg/Kg、160mg/Kg、320mg/Kg，每天2次，连续7天；对照组给予相同剂量注射用生理盐水，7天后制备肝微粒体蛋白温孵探针底物，采用BCA蛋白浓度测定方法测定蛋白含量，采用高效液相色谱法测定CYP1A2，CYP3A4和CYP2E1探针底物的代谢产物含量，评价CYP1A2，CYP3A4和CYP2E1的活性。结果显示给药15mg/Kg后，CYP1A2，CYP2E1的活性未受影响，CYP3A4的活性受到抑制；给药80mg/Kg后，CYP1A2，CYP2E1的活性未受影响，CYP3A4的活性受到抑制；给药160mg/Kg后，CYP1A2，CYP2E1、CYP3A4的活性稍受抑制；给药320mg/Kg后，CYP1A2，CYP2E1、CYP3A4的活性受到抑制。
Chuan’agelei, as a new platelet aggregation inhibitor, has been composed by moderntechnology which was based on mechanism of traditional Chinese medicine. The work ofthis paper was designed to evaluate the toxicokinetics and interaction features ofChuan’agelei in animals, so as to support the pharmacokinetics study in human and offerthe reference for clinical dosage and dosage form changing.
1. The establishment of analytical method of Chuan’agelei in biological matrix
A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has beendeveloped for the determination of Chuan’agelei in beagle dog plasma and rat plasma,using cinnamic aldehyde as an internal standard. The sample pretreatment wasliquid-liquid extraction. Separation was obtained on a Diamonsil C18column (5μm,100×4.6mm) with UV detection, using a wavelength of276nm. The isocratic mobile phaseconsisted of acetonitrile-10mM ammonium acetate buffer (pH4.4)(30:70, v/v) was run at aflow rate of1ml/min for different biological matrix. The chromatographic system usedprovided good separation of the compound without interfering peaks from endogenoussubstances. The calibration curves were linear in each sample range with correlationcoefficients above0.99. The precision of intra-day and inter-day were evaluated byanalysis of variance with the qualified results. The method has been successfully used tosupport the toxicokinetics study of Chuan’agelei in beagle dogs and SD rats.
2. The establishment of analytical method of of substrates and metabolites of CYP450in liver microsomes
A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has beendeveloped for the determination of substrates and metabolites in liver microsomes, usingprotocaechuic, chlordiazepoxide and phenacetin as internal standards. The samplepretreatment was liquid-liquid extraction.Separation was obtained on a Diamonsil C18column (5μm,150×4.6mm) with UV detection, using wavelengths of247nm(CYP1A2),223nm(CYP3A4),287nm(CYP2E1). The gradient mobile phase consisted of methanol(0.1%formic acid)-water (0.1%formic acid)(CYP1A2), methanol-water (0.02%formicacid)(CYP3A4), methanol-water (CYP2E1), was run at a flow rate of1ml/min fordifferent biological matrix.The chromatographic system used provided good separation ofthe compound without interfering peaks from endogenous substances. The calibration curves were linear in each sample range with correlation coefficients above0.99. Themethod has been successfully used to support the effect of chuan’agelei on enzyme acitivity ofrat liver microsomes.
3. The toxicokinetics study of Chuan’agelei in animals
The beagle dogs were allocated to three groups: low dose (group1, n=6), mid dose(group2, n=6), and high dose (group3, n=6). Group1,2and3doses corresponded to40,80and160mg/kg/day, respectively. Following a single intravenous administration withdifferent doses of Chuan’agelei to beagle dogs (40,80and160mg/kg); the blood sampleswere collected at different time after dosing. Blood samples for determination of plasmaChuan’agelei concentrations were obtained just prior to drug administration (0h) on1stdayand at0,0.5,1.0,2.0,4.0,8.0,12.0and24.0hours after administration on days1,43and88. All collected blood samples were centrifuged to obtain plasma and the concentrationsof Chuan’agelei in plasma were determined by HPLC method described as above.Toxicokinetics parameter calculations were carried out using non-compartmental analysismethod. The results showed that there was no accumulation in dogs after low doseadministration, but there was little accumulation in dogs after mid and high doseadministration of Chuan’agelei.
The SD rats were allocated to three groups: low dose (group1, n=6), mid dose (group2, n=6), and high dose (group3, n=6). Group1,2and3doses corresponded to80,160and320mg/kg/day, respectively. Following a single intraperitoneal administration withdifferent doses of Chuan’agelei to SD rats (80,160and320mg/kg); the blood sampleswere collected at different time after dosing. Blood samples for determination of plasmaChuan’agelei concentrations were obtained just prior to drug administration (0h) on1stdayand at0.833,0.5,1.0,2.0,4.0,8.0,12.0and24.0hours after administration on days1,44and91. All collected blood samples were centrifuged to obtain plasma and theconcentrations of Chuan’agelei in plasma were determined by HPLC method described asabove. Toxicokinetics parameter calculations were carried out using non-compartmentalanalysis method. The results showed that there was no accumulation in dogs after low doseadministration, but there was little accumulation in dogs after mid and high doseadministration of Chuan’agelei.
4. The research on the effect of drug expourse on the rat liver microsomal metabolism
Thirty-six rats were equally distributed into control group and drug administrationgroup and were evenly composed of male and female. Among which, drug administration group was given a vein dose of15mg/kg, an abdominal dose of80mg/kg,160mg/kg,320mg/kg chuan’agelei in seven days, twice a day. Control group was given the same dose ofsaline. After this, liver microsomes protein was prepared to incubate the probe drug.Concentrations of protein were determined by BCA method. Concentrations of metaboliteswere determined by HPLC method and used to evaluate the effect of CYP1A2, CYP3A4and CYP2E1.The result showed that after a vein dose of15mg/kg, Chuan’agelei have noeffect on CYP1A2and CYP2E1, but can restrain CYP3A4; after a abdominal dose of80mg/kg, Chuan’agelei have no effect on CYP1A2and CYP2E1, but can restrain CYP3A4;after a abdominal dose of160mg/kg, Chuan’agelei have effect on CYP1A2, CYP2E1,CYP3A4; after a abdominal dose of320mg/kg, Chuan’agelei have effect on CYP1A2,CYP2E1, CYP3A4.
一、生物样品中川阿格雷分析方法的建立
本文采用液液萃取法处理血浆样品，建立了以桂皮醛为内标的反相高效液相色谱—紫外光谱检测川阿格雷的体内分析方法。色谱分离条件为：色谱柱Diamonsil C18（5μm，100×4.6mm）；流动相组成为乙腈、水及用来调节pH的乙酸铵（pH4.4），流速为1mL/min；紫外检测波长为276nm，柱温30℃。采用本文建立的生物样品预处理方法和反相高效液相色谱法分离测定生物样品中川阿格雷的含量，生物样品预处理方法回收率高，色谱分离选择性好。本法的准确度、精密度、灵敏度、专属性和定量线性范围均达到生物样品分析的要求，该方法成功用于川阿格雷在Beagle犬与SD大鼠体内的毒代动力学研究。
二、肝微粒体中CYP450酶亚型特异性底物及其代谢产物定量分析方法的建立
本文采用液液萃取法处理肝微粒体温孵样品，建立了以原儿茶酸、利眠宁和非那西丁为内标的反相高效液相色谱—紫外光谱检测CYP450酶亚型特异性底物及其代谢产物的分析方法。色谱分离条件为：色谱柱迪马Diamonsil C18（5μm，150×4.6mm）；非那西丁（CYP1A2底物）：流动相甲醇（0.1%甲酸）（A）—水（0.1%甲酸）（B），梯度洗脱0~5min18%A、5~10min18%~60%A、10~15min60%A，流速1.0mL/min，紫外检测波长为247nm，柱温30℃；咪达唑仑（CYP3A4底物）：流动相甲醇（A）—水（0.02%甲酸）（B），梯度洗脱0~11min40%~60%A，流速1.0mL/min，紫外检测波长为223nm，柱温30℃；氯唑沙宗（CYP2E1底物）：流动相甲醇（A）—水（B），梯度洗脱0~10min37%~75%A，流速1.0mL/min，紫外检测波长为287nm，柱温25℃。采用本文建立的样品预处理方法和反相高效液相色谱法分离测定温孵样品中特征底物及代谢产物的含量，方法回收率高，色谱分离选择性好，准确度、精密度、专属性和定量线性范围均达到生物样品分析的要求，该方法成功用于川阿格雷对SD大鼠肝微粒体酶活性影响的研究。
三、川阿格雷在动物体内的毒代动力学研究
本试验设计成年、健康Beagle犬多剂量静脉注射给药川阿格雷生理盐水注射液40、80和160mg/Kg三个剂量组进行毒代动力学研究。选择18条Beagle犬，雌雄各半，按体重随机分成低、中、高剂量三个剂量组，每一剂量组6条Beagle犬。每组Beagle犬一周给药6次（每天给药1次），第七天停药。采用缓慢静脉推注方式给药，给药周期为90天。于给药前及给药后第1，43，88天取血，每次取血点分别为给药后0、0.5、1.0、2.0、4.0、8.0、12.0、24.0h。用上述建立的HPLC-UV法测定血浆中川阿格雷的浓度，根据所得的血浆药物浓度—时间曲线，采用非房室模型推算药物动力学参数。结果显示药物在低剂量、中剂量长期静脉注射给药时对机体没有明显损伤，在高剂量长期静脉注射给药时对机体存在一定损伤，引起代谢或排泄功能减弱而导致最大血药浓度（C_0）增大。
本试验设计健康SD大鼠多剂量腹腔注射给药川阿格雷生理盐水注射液80、160和320mg/Kg三个剂量组进行毒代动力学研究。选择18只SD大鼠，雌雄各半，按体重随机分成低、中、高剂量三个剂量组，每一剂量组6只SD大鼠。每组SD大鼠一周给药6次（每天给药1次），第七天停药，采用缓慢腹腔注射方式给药，给药周期为90天。于给药前及给药后第1，44，91天取血，每次取血点分别为给药后0.833、0.5、1.0、2.0、4.0、8.0、12.0、24.0h。用上述建立的HPLC-UV法测定血浆中川阿格雷的浓度，根据所得的血浆药物浓度—时间曲线，采用非房室模型推算药物动力学参数。结果显示药物在低剂量、中剂量长期腹腔注射给药时对机体没有明显损伤，在高剂量长期腹腔注射给药时对机体存在一定损伤，引起代谢或排泄功能减弱而导致最大血药浓度（Cmax）增大。
四、川阿格雷暴露水平影响大鼠肝微粒体代谢酶活性的研究
本试验选择健康SD大鼠多剂量注射川阿格雷注射液15mg/Kg、80mg/Kg、160mg/Kg、320mg/Kg，进行川阿格雷影响大鼠肝微粒体代谢酶活性的研究。选择36只SD大鼠，雌雄各半，分成对照组和给药组，每组6只，给药组尾静脉注射给予川阿格雷15mg/Kg，腹腔注射给予川阿格雷80mg/Kg、160mg/Kg、320mg/Kg，每天2次，连续7天；对照组给予相同剂量注射用生理盐水，7天后制备肝微粒体蛋白温孵探针底物，采用BCA蛋白浓度测定方法测定蛋白含量，采用高效液相色谱法测定CYP1A2，CYP3A4和CYP2E1探针底物的代谢产物含量，评价CYP1A2，CYP3A4和CYP2E1的活性。结果显示给药15mg/Kg后，CYP1A2，CYP2E1的活性未受影响，CYP3A4的活性受到抑制；给药80mg/Kg后，CYP1A2，CYP2E1的活性未受影响，CYP3A4的活性受到抑制；给药160mg/Kg后，CYP1A2，CYP2E1、CYP3A4的活性稍受抑制；给药320mg/Kg后，CYP1A2，CYP2E1、CYP3A4的活性受到抑制。
Chuan’agelei, as a new platelet aggregation inhibitor, has been composed by moderntechnology which was based on mechanism of traditional Chinese medicine. The work ofthis paper was designed to evaluate the toxicokinetics and interaction features ofChuan’agelei in animals, so as to support the pharmacokinetics study in human and offerthe reference for clinical dosage and dosage form changing.
1. The establishment of analytical method of Chuan’agelei in biological matrix
A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has beendeveloped for the determination of Chuan’agelei in beagle dog plasma and rat plasma,using cinnamic aldehyde as an internal standard. The sample pretreatment wasliquid-liquid extraction. Separation was obtained on a Diamonsil C18column (5μm,100×4.6mm) with UV detection, using a wavelength of276nm. The isocratic mobile phaseconsisted of acetonitrile-10mM ammonium acetate buffer (pH4.4)(30:70, v/v) was run at aflow rate of1ml/min for different biological matrix. The chromatographic system usedprovided good separation of the compound without interfering peaks from endogenoussubstances. The calibration curves were linear in each sample range with correlationcoefficients above0.99. The precision of intra-day and inter-day were evaluated byanalysis of variance with the qualified results. The method has been successfully used tosupport the toxicokinetics study of Chuan’agelei in beagle dogs and SD rats.
2. The establishment of analytical method of of substrates and metabolites of CYP450in liver microsomes
A reversed-phase high-performance liquid chromatographic (RP-HPLC) method has beendeveloped for the determination of substrates and metabolites in liver microsomes, usingprotocaechuic, chlordiazepoxide and phenacetin as internal standards. The samplepretreatment was liquid-liquid extraction.Separation was obtained on a Diamonsil C18column (5μm,150×4.6mm) with UV detection, using wavelengths of247nm(CYP1A2),223nm(CYP3A4),287nm(CYP2E1). The gradient mobile phase consisted of methanol(0.1%formic acid)-water (0.1%formic acid)(CYP1A2), methanol-water (0.02%formicacid)(CYP3A4), methanol-water (CYP2E1), was run at a flow rate of1ml/min fordifferent biological matrix.The chromatographic system used provided good separation ofthe compound without interfering peaks from endogenous substances. The calibration curves were linear in each sample range with correlation coefficients above0.99. Themethod has been successfully used to support the effect of chuan’agelei on enzyme acitivity ofrat liver microsomes.
3. The toxicokinetics study of Chuan’agelei in animals
The beagle dogs were allocated to three groups: low dose (group1, n=6), mid dose(group2, n=6), and high dose (group3, n=6). Group1,2and3doses corresponded to40,80and160mg/kg/day, respectively. Following a single intravenous administration withdifferent doses of Chuan’agelei to beagle dogs (40,80and160mg/kg); the blood sampleswere collected at different time after dosing. Blood samples for determination of plasmaChuan’agelei concentrations were obtained just prior to drug administration (0h) on1stdayand at0,0.5,1.0,2.0,4.0,8.0,12.0and24.0hours after administration on days1,43and88. All collected blood samples were centrifuged to obtain plasma and the concentrationsof Chuan’agelei in plasma were determined by HPLC method described as above.Toxicokinetics parameter calculations were carried out using non-compartmental analysismethod. The results showed that there was no accumulation in dogs after low doseadministration, but there was little accumulation in dogs after mid and high doseadministration of Chuan’agelei.
The SD rats were allocated to three groups: low dose (group1, n=6), mid dose (group2, n=6), and high dose (group3, n=6). Group1,2and3doses corresponded to80,160and320mg/kg/day, respectively. Following a single intraperitoneal administration withdifferent doses of Chuan’agelei to SD rats (80,160and320mg/kg); the blood sampleswere collected at different time after dosing. Blood samples for determination of plasmaChuan’agelei concentrations were obtained just prior to drug administration (0h) on1stdayand at0.833,0.5,1.0,2.0,4.0,8.0,12.0and24.0hours after administration on days1,44and91. All collected blood samples were centrifuged to obtain plasma and theconcentrations of Chuan’agelei in plasma were determined by HPLC method described asabove. Toxicokinetics parameter calculations were carried out using non-compartmentalanalysis method. The results showed that there was no accumulation in dogs after low doseadministration, but there was little accumulation in dogs after mid and high doseadministration of Chuan’agelei.
4. The research on the effect of drug expourse on the rat liver microsomal metabolism
Thirty-six rats were equally distributed into control group and drug administrationgroup and were evenly composed of male and female. Among which, drug administration group was given a vein dose of15mg/kg, an abdominal dose of80mg/kg,160mg/kg,320mg/kg chuan’agelei in seven days, twice a day. Control group was given the same dose ofsaline. After this, liver microsomes protein was prepared to incubate the probe drug.Concentrations of protein were determined by BCA method. Concentrations of metaboliteswere determined by HPLC method and used to evaluate the effect of CYP1A2, CYP3A4and CYP2E1.The result showed that after a vein dose of15mg/kg, Chuan’agelei have noeffect on CYP1A2and CYP2E1, but can restrain CYP3A4; after a abdominal dose of80mg/kg, Chuan’agelei have no effect on CYP1A2and CYP2E1, but can restrain CYP3A4;after a abdominal dose of160mg/kg, Chuan’agelei have effect on CYP1A2, CYP2E1,CYP3A4; after a abdominal dose of320mg/kg, Chuan’agelei have effect on CYP1A2,CYP2E1, CYP3A4.
引文
1.杨秀伟，杨晓华，蒲小平等.创新药物研究中的吸收、分布、代谢、排泄/毒性（ADME/Tox.）平台建设[J].北京大学学报（医学版）.2004，36（1）：5
2.郝琨，柳晓泉，王广基.临床前毒代动力学研究进展.中国药科大学学报.2004，35（3）：195.
3.史国兵，马艳，于洋.液相色谱技术在体内药物分析中的应用[J].中国药师.2003，6（2）：105
4.吴小曼，蔡梅，陈玉英.毛细管电泳应用于体内药物分析的新技术[J].药学进展.1999，23（6）：325
5.童珊珊，余江南，刘文英等.色谱技术在体内药物分析中的进展[J].国外医药学分册.2000，27（6）：360
6.钟大放.液相色谱-质谱联用法在药物研究中的应用[J].世界科学技术——中医药现代化.2003，5（4）：44
7.金科涛，王宇光，徐彭等. HPLC测定大鼠肝微粒体P450活性方法学研究进展[J].药物分析杂志.2006，26（3）：415
8.李丹，韩永龙，余涛等.体外肝代谢模型的优缺点及其应用[J].中国临床药理学与治疗学.2011，16（6）：688-690
9.盛日正，李家明，张飞龙等.新型川芎嗪阿魏酸衍生物的合成及其抗血小板聚集活性[J].合成化学，2011，19（2）：157-158
10.邬皓，李家明，盛日正等.川芎嗪芳酸醚甘氨酸衍生物合成及抗血小板聚集活性[J].安徽医药，2011，15（8）：937-938
11.吴民淑，王广基.国外相伴毒代动力学研究进展.药学进展1999，23（6）：321
12.刘昌孝.临床前药物安全性评价研究中的药物毒代动力学问题.中国临床药理学与治疗学杂志.1999，4（2）：157
13.袁守军.当前新药毒代动力学（TK）的研究概况与不足[J].毒理学杂志.2007，21（4）：297-298
14.林飞.毒代动力学在新药安全性评价中的意义及应用[J].毒理学杂志.2005，19（3）：245-246
15.李江，王爱平，王晓良等.前药dl-PHPB在Beagle犬中反复给药毒性及伴随毒代动力学研究[J].2011，13（8）：1-3
16.刘昌孝.新药安全性评价中的毒代动力学研究.毒理学杂志.2007，21（4）：275~276
17.王阳，刘茂.基于生理毒代动力学木星和剂量——反应模型的苯暴露健康风险评价方法.中国工业医学杂志，2009，22（1）：34-37
18.孙严彤，高峰，张梦亮等.川丁特罗对大鼠细胞色素P450酶的影响.[J]吉林大学学报.2010，48（6）：1061-1064
19.郭海方，邹晓丽，许卉等.丹参酚酸A对大鼠肝微粒体细胞色素P450酶系的影响.[J]中国中药杂志.2010，35（3）：348-350
20.陈荣，谢陶吟，邹素兰等.探针药物法测定大黄对大鼠干细胞色素CYP2E1酶的影响.[J]中国临床药学杂志.2009，18（2）：110-112
21.唐坤，付翔. RP-HPLC法测定人血浆中奥扎格雷的浓度.[J]中国药房.2009，20（32）：2519
22.石杰，张新惠，庄安士.候选药物对大鼠肝细胞色素P4503A4影响的研究[J].中国现代应用药学杂志.2007，24（2）：108-110
23.和凡，钟国平，赵立子等. LC-MS/MS法研究黄酮类化合物对人肝微粒体细胞色素P450酶6种亚型的体外抑制作用[J].中国新药杂志.2009，18（24）：2340-2342
24.段俊敏，王晓莹，郭宾等.底物消除法测定微粒体酶动力学参数的实用性分析[J].中国卫生检验杂志.2011，21（7）：1691-1694
25.陆国才，袁伯俊.新药毒性机制研究进展.[J]2006，15（4）：244-246
26. Buchanan JR, Burka LT, Melnick RL. Purpose and guidelines for toxicokinetic studieswithin the National Toxicology program [J]. Environ Health Perspect,1997,105(5):468
27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with theFolin Phenol reagent.J Biol Chem,1951,193(1):265-275
28.简礅昱，徐帆，廖春龙等.3种方法测定大鼠肝微粒体蛋白含量的比较[J].中国药师.2011，14（3）：342-344
29.柳晓泉，赵阳，周丹等.人肝微粒体中西尼地平及其代谢物的HPLC测定法及代谢动力学[J].中国药科大学学报.2002，33（4）：304
2.郝琨，柳晓泉，王广基.临床前毒代动力学研究进展.中国药科大学学报.2004，35（3）：195.
3.史国兵，马艳，于洋.液相色谱技术在体内药物分析中的应用[J].中国药师.2003，6（2）：105
4.吴小曼，蔡梅，陈玉英.毛细管电泳应用于体内药物分析的新技术[J].药学进展.1999，23（6）：325
5.童珊珊，余江南，刘文英等.色谱技术在体内药物分析中的进展[J].国外医药学分册.2000，27（6）：360
6.钟大放.液相色谱-质谱联用法在药物研究中的应用[J].世界科学技术——中医药现代化.2003，5（4）：44
7.金科涛，王宇光，徐彭等. HPLC测定大鼠肝微粒体P450活性方法学研究进展[J].药物分析杂志.2006，26（3）：415
8.李丹，韩永龙，余涛等.体外肝代谢模型的优缺点及其应用[J].中国临床药理学与治疗学.2011，16（6）：688-690
9.盛日正，李家明，张飞龙等.新型川芎嗪阿魏酸衍生物的合成及其抗血小板聚集活性[J].合成化学，2011，19（2）：157-158
10.邬皓，李家明，盛日正等.川芎嗪芳酸醚甘氨酸衍生物合成及抗血小板聚集活性[J].安徽医药，2011，15（8）：937-938
11.吴民淑，王广基.国外相伴毒代动力学研究进展.药学进展1999，23（6）：321
12.刘昌孝.临床前药物安全性评价研究中的药物毒代动力学问题.中国临床药理学与治疗学杂志.1999，4（2）：157
13.袁守军.当前新药毒代动力学（TK）的研究概况与不足[J].毒理学杂志.2007，21（4）：297-298
14.林飞.毒代动力学在新药安全性评价中的意义及应用[J].毒理学杂志.2005，19（3）：245-246
15.李江，王爱平，王晓良等.前药dl-PHPB在Beagle犬中反复给药毒性及伴随毒代动力学研究[J].2011，13（8）：1-3
16.刘昌孝.新药安全性评价中的毒代动力学研究.毒理学杂志.2007，21（4）：275~276
17.王阳，刘茂.基于生理毒代动力学木星和剂量——反应模型的苯暴露健康风险评价方法.中国工业医学杂志，2009，22（1）：34-37
18.孙严彤，高峰，张梦亮等.川丁特罗对大鼠细胞色素P450酶的影响.[J]吉林大学学报.2010，48（6）：1061-1064
19.郭海方，邹晓丽，许卉等.丹参酚酸A对大鼠肝微粒体细胞色素P450酶系的影响.[J]中国中药杂志.2010，35（3）：348-350
20.陈荣，谢陶吟，邹素兰等.探针药物法测定大黄对大鼠干细胞色素CYP2E1酶的影响.[J]中国临床药学杂志.2009，18（2）：110-112
21.唐坤，付翔. RP-HPLC法测定人血浆中奥扎格雷的浓度.[J]中国药房.2009，20（32）：2519
22.石杰，张新惠，庄安士.候选药物对大鼠肝细胞色素P4503A4影响的研究[J].中国现代应用药学杂志.2007，24（2）：108-110
23.和凡，钟国平，赵立子等. LC-MS/MS法研究黄酮类化合物对人肝微粒体细胞色素P450酶6种亚型的体外抑制作用[J].中国新药杂志.2009，18（24）：2340-2342
24.段俊敏，王晓莹，郭宾等.底物消除法测定微粒体酶动力学参数的实用性分析[J].中国卫生检验杂志.2011，21（7）：1691-1694
25.陆国才，袁伯俊.新药毒性机制研究进展.[J]2006，15（4）：244-246
26. Buchanan JR, Burka LT, Melnick RL. Purpose and guidelines for toxicokinetic studieswithin the National Toxicology program [J]. Environ Health Perspect,1997,105(5):468
27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with theFolin Phenol reagent.J Biol Chem,1951,193(1):265-275
28.简礅昱，徐帆，廖春龙等.3种方法测定大鼠肝微粒体蛋白含量的比较[J].中国药师.2011，14（3）：342-344
29.柳晓泉，赵阳，周丹等.人肝微粒体中西尼地平及其代谢物的HPLC测定法及代谢动力学[J].中国药科大学学报.2002，33（4）：304