17834-03-6Relevant articles and documents
Inhibitory effects of silibinin on cytochrome P-450 enzymes in human liver microsomes
Beckmann-Knopp, Svane,Rietbrock, Stephan,Weyhenmeyer, Roland,Boecker, Ronald H.,Beckurts, K. Tobias,Lang, Werner,Hunz, Miriam,Fuhr, Uwe
, p. 250 - 256 (2000)
Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7- 300 μM) at 37°in 0.1 M KH2PO4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC50 values were determined for each substrate at respective K(M) concentrations. Silibinin had little effect (IC50 > 200 μM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(+)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC50 = 173 μM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC50 = 29 μM and 46 μM) and S(-)-warfarin 7-hydroxylation (CYP2C9; IC50 = 43 μM and 45 μM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (K(i,c) = 2.3 μM and 2.4 μM). Inhibition was competitive for S(-)-warfarin 7-hydroxylation (K(i,c) = 18 μM and 19 μM) and mainly non-competitive for denitronifedipine oxidation (K(i,n) = 9 μM and 12 μM). With therapeutic silibinin peak plasma concentrations of 0.6 μM and biliary concentrations up to 200 μM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.
Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient
Zhou, Xiao-Yang,Lu, Xiang-Ran,Li, Ying-Hui,Ma, Ya-Qing,Zhao, Shi-Wen,Wang, Fang,Xu, Ren-Ai,Hu, Guo-Xin,Cai, Jian-Ping
, (2021/03/03)
Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0?mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.
Ion mobility spectrometry-mass spectrometry analysis for the site of aromatic hydroxy
Shimizu, Atsushi,Chiba, Masato
, p. 1295 - 1299 (2013/07/26)
Hydroxylated metabolites often retain the pharmacological activity of parent compound, and the position of hydroxylation determines the formation of chemically reactive intermediates, such as quinones and analogs, from para- and/or ortho-hydroxylation of phenols or arylamines. Therefore, the identification of exact position of hydroxylation is often required at the early development stage of new drug candidates. In many cases, liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides identical MS/MS spectra among isomeric hydroxylated metabolites, and therefore, it alone cannot unequivocally identify the exact position(s) of hydroxylation. Ion mobility spectrometry (IMS), integrated with LC-MS/MS, recently showed the capability of separating isomeric species based on differences in their drift times from IMS, which are linearly proportional to the collision cross-section (CCS) reflecting physical size and shape. In the present study, a chemical derivatization of isomeric hydroxylated metabolites with 2-fluoro-N-methyl pyridinium p-toluenesulfonate was found to confer distinct theoretical CCS value on each isomer by forming corresponding N-methyl pyridine (NMP) derivative. The regression lines established by the comparison between theoretical CCS values and observed drift times from IMS for each set of parent compound (labetalol, ezetimibe, atorvastatin, and warfarin) and its MS/MS product ions accurately and selectively projected the actual drift times of NMP derivatives of corresponding aromatic or isomeric hydroxylated metabolites. The established method was used for the accurate assignment of predominant formation of 2-hydroxylated metabolite from imipramine in NADPH- fortified human liver microsomes. The present application expands the versatility of LC-IMS-MS technique to the structure identification of isomeric hydroxylated metabolites at the early stage for drug development.
