中国中药杂志

2021, (13) 3410-3421

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补骨脂定的肝肠微粒体代谢动力学、代谢酶表型及种属差异研究
Study on metabolic dynamics,metabolic enzyme phenotype and species difference of hepatic and intestinal microsome of psoralidin

秦子飞;张贝贝;邢晗;王培乐;杨晶;张晓坚;姚志红;姚新生;
QIN Zi-fei;ZHANG Bei-bei;XING Han;WANG Pei-le;YANG Jing;ZHANG Xiao-jian;YAO Zhi-hong;YAO Xin-sheng;Department of Pharmacy,the First Affiliated Hospital of Zhengzhou University;International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development Ministry of China,Jinan University;College of Pharmacy,Jinan University;

摘要(Abstract):

研究补骨脂定在人肝微粒体(HLM)和肠微粒体(HIM)的代谢活性,明确参与补骨脂定代谢的细胞色素P450酶(CYPs)和尿苷二磷酸葡萄糖醛酸转移酶(UGTs)及补骨脂定体外代谢的种属差异。将不同浓度的补骨脂定溶液分别与HLM和HIM共同孵育,经HLM孵育可以产生2个氧化产物(M1和M2)和2个葡萄糖醛酸结合物(G1和G2),在HIM中仅产生M1和G1;在HLM和HIM中,补骨脂定代谢生成M1的固有清除率(CLint)分别为104.3、57.6μL·min(-1)·mg(-1)·mg(-1),生成G1的CLint分别为543.3、75.9μL·min(-1),生成G1的CLint分别为543.3、75.9μL·min(-1)·mg(-1)·mg(-1)。利用12种CYPs和12种UGTs酶,分别与不同浓度的补骨脂定溶液共同孵育,结果显示,CYP1A1(39.5μL·min(-1)。利用12种CYPs和12种UGTs酶,分别与不同浓度的补骨脂定溶液共同孵育,结果显示,CYP1A1(39.5μL·min(-1)·mg(-1)·mg(-1))、CYP2C8(88.0μL·min(-1))、CYP2C8(88.0μL·min(-1)·mg(-1)·mg(-1))、CYP2C19(166.7μL·min(-1))、CYP2C19(166.7μL·min(-1)·mg(-1)·mg(-1))、CYP2D6(9.1μL·min(-1))、CYP2D6(9.1μL·min(-1)·mg(-1)·mg(-1))是生成M1的关键CYPs代谢酶,而CYP2C19(42.0μL·min(-1))是生成M1的关键CYPs代谢酶,而CYP2C19(42.0μL·min(-1)·mg(-1)·mg(-1))是生成M2的重要亚型酶; UGT1A1(1 184.4μL·min(-1))是生成M2的重要亚型酶; UGT1A1(1 184.4μL·min(-1)·mg(-1)·mg(-1))、UGT1A7(922.8μL·min(-1))、UGT1A7(922.8μL·min(-1)·mg(-1)·mg(-1))、UGT1A8(133.0μL·min(-1))、UGT1A8(133.0μL·min(-1)·mg(-1)·mg(-1))、UGT1A9(348.6μL·min(-1))、UGT1A9(348.6μL·min(-1)·mg(-1)·mg(-1))、UGT2B7(118.7μL·min(-1))、UGT2B7(118.7μL·min(-1)·mg(-1)·mg(-1))重点参与G1的生成,而UGT1A9(111.3μL·min(-1))重点参与G1的生成,而UGT1A9(111.3μL·min(-1)·mg(-1)·mg(-1))是G2生成的关键亚型酶。采用猴肝微粒体(MkLM)、大鼠肝微粒体(RLM)、小鼠肝微粒体(MLM)、狗肝微粒体(DLM)和猪肝微粒体(MpLM),分别与不同浓度的补骨脂定溶液共同孵育,结果显示,补骨脂定的Ⅰ相代谢和葡萄糖醛酸化代谢均表现出显著的种属差异。总体来说,补骨脂定在肝肠微粒体均可以发生较强的代谢; CYP1A1、CYP2C8、CYP2C19、CYP2D6与UGT1A1、UGT1A7、UGT1A8、UGT1A9、UGT2B7是参与其代谢的关键亚型酶;大鼠和猪分别是研究补骨脂定Ⅰ相代谢和葡萄糖醛酸化代谢合适的模式动物。
This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min(-1))是G2生成的关键亚型酶。采用猴肝微粒体(MkLM)、大鼠肝微粒体(RLM)、小鼠肝微粒体(MLM)、狗肝微粒体(DLM)和猪肝微粒体(MpLM),分别与不同浓度的补骨脂定溶液共同孵育,结果显示,补骨脂定的Ⅰ相代谢和葡萄糖醛酸化代谢均表现出显著的种属差异。总体来说,补骨脂定在肝肠微粒体均可以发生较强的代谢; CYP1A1、CYP2C8、CYP2C19、CYP2D6与UGT1A1、UGT1A7、UGT1A8、UGT1A9、UGT2B7是参与其代谢的关键亚型酶;大鼠和猪分别是研究补骨脂定Ⅰ相代谢和葡萄糖醛酸化代谢合适的模式动物。
This study aims to investigate metabolic activities of psoralidin in human liver microsomes( HLM) and intestinal microsomes( HIM),and to identify cytochrome P450 enzymes( CYPs) and UDP-glucuronosyl transferases( UGTs) involved in psoralidin metabolism as well as species differences in the in vitro metabolism of psoralen. First,after incubation serial of psoralidin solutions with nicotinamide adenine dinucleotide phosphate( NADPH) or uridine 5'-diphosphate-glucuronic acid( UDPGA)-supplemented HLM or HIM,two oxidic products( M1 and M2) and two conjugated glucuronides( G1 and G2) were produced in HLM-mediated incubation system,while only M1 and G1 were detected in HIM-supplemented system. The CLintfor M1 in HLM and HIM were 104. 3,and57. 6 μL·min(-1)·mg(-1)·mg(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min(-1),respectively,while those for G1 were 543. 3,and 75. 9 μL·min(-1)·mg(-1)·mg(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min(-1),respectively. Furthermore,reaction phenotyping was performed to identify the main contributors to psoralidin metabolism after incubation of psoralidin with NADPH-supplemented twelve CYP isozymes( or UDPGA-supplemented twelve UGT enzymes),respectively. The results showed that CYP1 A1( 39. 5 μL·min(-1)·mg(-1)·mg(-1)),CYP2 C8( 88. 0 μL·min(-1)),CYP2 C8( 88. 0 μL·min(-1)·mg(-1)·mg(-1)),CYP2 C19( 166. 7 μL·min(-1)),CYP2 C19( 166. 7 μL·min(-1)·mg(-1)·mg(-1)),and CYP2 D6( 9. 1 μL·min(-1)),and CYP2 D6( 9. 1 μL·min(-1)·mg(-1)·mg(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min(-1)) were identified as the main CYP isoforms for M1,whereas CYP2 C19( 42. 0 μL·min(-1)·mg(-1)·mg(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min(-1)) participated more in producing M2. In addition,UGT1 A1( 1 184. 4 μL·min(-1)·mg(-1)·mg(-1)),UGT1 A7( 922. 8 μL·min(-1)),UGT1 A7( 922. 8 μL·min(-1)·mg(-1)·mg(-1)),UGT1 A8( 133. 0 μL·min(-1)),UGT1 A8( 133. 0 μL·min(-1)·mg(-1)·mg(-1)),UGT1 A9( 348. 6 μL·min(-1)),UGT1 A9( 348. 6 μL·min(-1)·mg(-1)·mg(-1)) and UGT2 B7( 118. 7 μL·min(-1)) and UGT2 B7( 118. 7 μL·min(-1)·mg(-1)·mg(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min(-1)) played important roles in the generation of G1,while UGT1 A9( 111. 3 μL·min(-1)·mg(-1)·mg(-1)) was regarded as the key UGT isozyme for G2. Moreover,different concentrations of psoralidin were incubated with monkey liver microsomes( MkLM),rat liver microsomes( RLM),mice liver microsomes( MLM),dog liver microsomes( DLM) and mini-pig liver microsomes( MpLM),respectively. The obtained CLintwere used to evaluate the species differences.Phase Ⅰ metabolism and glucuronidation of psoralidinby liver microsomes showed significant species differences. In general,psoralidin underwent efficient hepatic and intestinal metabolisms. CYP1 A1,CYP2 C8,CYP2 C19,CYP2 D6 and UGT1 A1,UGT1 A7,UGT1 A8,UGT1 A9,UGT2 B7 were identified as the main contributors responsible for phase Ⅰ metabolism and glucuronidation,respectively. Rat and mini-pig were considered as the appropriate model animals to investigate phase Ⅰ metabolism and glucuronidation,respectively.

关键词(KeyWords): 补骨脂定;肝微粒体;肠微粒体;细胞色素P450酶;尿苷二磷酸葡萄糖醛酸转移酶;种属差异;代谢特征
psoralidin;human liver microsomes;human intestine microsomes;cytochrome P450;UDP-glucuronosyltransferases;species difference;metabolic fates

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金青年基金项目(81903704,81703799);; 广东省基础与应用基础研究基金项目(2019A1515011285);; 新药研究国家重点实验室开放基金项目(SIMM1903KF-07)

作者(Authors): 秦子飞;张贝贝;邢晗;王培乐;杨晶;张晓坚;姚志红;姚新生;
QIN Zi-fei;ZHANG Bei-bei;XING Han;WANG Pei-le;YANG Jing;ZHANG Xiao-jian;YAO Zhi-hong;YAO Xin-sheng;Department of Pharmacy,the First Affiliated Hospital of Zhengzhou University;International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development Ministry of China,Jinan University;College of Pharmacy,Jinan University;

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