title: Characterization of phase I metabolism of resibufogenin and evaluation of the metabolic effects on its antitumor activity and toxicity.
abstract: Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has displayed great potential as a chemotherapeutic agent in oncology. However, it is a digoxin-like compound that also exhibits extremely cardiotoxic effects. The present study aimed to characterize the metabolic behaviors of RB in humans as well as to evaluate the metabolic effects on its bioactivity and toxicity. The phase I metabolic profile in human liver microsomes was characterized systemically, and the major metabolite was identified as marinobufagenin (5β-hydroxylresibufogenin, 5-HRB) by liquid chromatography-mass spectrometry and nuclear magnetic imaging techniques. Both cytochrome P450 (P450) reaction phenotyping and inhibition assays using P450-selective chemical inhibitors demonstrated that CYP3A4 was mainly involved in RB 5β-hydroxylation with much higher selectivity than CYP3A5. Kinetic characterization demonstrated that RB 5β-hydroxylation in both human liver microsomes and human recombinant CYP3A4 obeyed biphasic kinetics and displayed similar apparent kinetic parameters. Furthermore, 5-HRB could significantly induce cell growth inhibition and apoptosis in A549 and H1299 by facilitating apoptosome assembly and caspase activation. Meanwhile, 5-HRB displayed very weak cytotoxicity of human embryonic lung fibroblasts, and in mice there was a greater tolerance to acute toxicity. In summary, CYP3A4 dominantly mediated 5β-hydroxylation and was found to be a major metabolic pathway of RB in the human liver, whereas its major metabolite (5-HRB) displayed better druglikeness than its parent compound RB. Our findings lay a solid foundation for RB metabolism studies in humans and encourage further research on the bioactive metabolite of RB.
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Jing
authors.initials: J
authors.lastname: Ning
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Zhen-Long
authors.initials: ZL
authors.lastname: Yu
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Liang-Hai
authors.initials: LH
authors.lastname: Hu
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Chao
authors.initials: C
authors.lastname: Wang
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Xiao-Kui
authors.initials: XK
authors.lastname: Huo
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Sa
authors.initials: S
authors.lastname: Deng
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Jie
authors.initials: J
authors.lastname: Hou
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Wu
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Guang-Bo
authors.initials: GB
authors.lastname: Ge
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.) maxc1978@163.com.
authors.forename: Xiao-Chi
authors.initials: XC
authors.lastname: Ma
authors.affiliation: Research Institute of Integrated Traditional and Western Medicine, College of Pharmacy, Dalian Medical University, Dalian, China (J.N., Z.-L.Y., C.W., X.-K H., S.D., J.H., X.-C.M.); Key Laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Jilin University, Changchun, China (L.-H., H); Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China (J.N., J.-J.W., G.-B.G., L.Y.); State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China (C.W.).
authors.forename: Ling
authors.initials: L
authors.lastname: Yang
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000009:adverse effects / Q000378:metabolism* / Q000494:pharmacology*; D002018:Bufanolides / Q000009:adverse effects / Q000378:metabolism* / Q000494:pharmacology*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004285:Dogs; D006168:Guinea Pigs; D006801:Humans; D006900:Hydroxylation / Q000502:physiology; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008252:Macaca fascicularis; D008297:Male; D050216:Metabolic Detoxication, Phase I / Q000502:physiology*; D051379:Mice; D008813:Mice, Inbred ICR; D008862:Microsomes, Liver / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000970:Antineoplastic Agents; D002018:Bufanolides; C095591:chan su; C005734:bufogenin; C093896:marinobufagenin; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A