The inhibition activity of 36 flavonoids against CYP1A2 was determined by our previously developed in vitro method. The Comparative Molecular Similarity Indexes Analysis (CoMSJA) approach was used to probe the quantitative relationships between the flavonoids' molecular structural descriptors and their inhibitory activities. A reliable CoMSIA model with the combined electrostatic and hydrophobic fields was derived with the regression coefficient R2 of 0.948 and the cross-validation regression coefficient q2 of 0.630, separately, which is capable of elucidating the quantitative relationship between the 3D structural descriptors of the flavones and their bioactivities. Comparing with flavone, the larger pi-pi conjugated system of alpha-naphthoflavone significantly improved the biologically inhibitory ability. Based on the core structure of the latter, either electropositive substituents or hydrophobic groups at the 6, 3', and 4' ring positions or electronegative counterparts at the 5 ring position, can enhance the inhibitory potency against CYP1 A2 according to the CoMSIA contour maps.
Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP1A2 Inhibitors ; Flavonoids ; chemistry ; pharmacology ; Humans ; Microsomes, Liver ; metabolism ; Models, Molecular ; Molecular Structure ; Quantitative Structure-Activity Relationship

<p>AIMTo identify the cytochrome P450 (CYP) isoform (s) involved in daidzein mono-hydroxylated metabolites using human liver microsomes.p><p>METHODSKinetic analysis of the rates of formation of mono-hydroxylated metabolites of daidzein, including 7,8,4'-trihydroxyisoflavone (7,8,4'-THI), 7,3',4'-trihydroxyisoflavone (7,3',4'-THI) and 6,7,4'-trihydroxyisoflavone (6,7,4'-THI), was performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 micromol x L(-1). Nine selective inhibitors or substrate probes specific for different CYP isoforms were applied for screening the isoform(s) responsible for mono-hydroxylated metabolism of daidzein.p><p>RESULTSMichaelis-Menten kinetic parameters were best fitted to one-component enzyme kinetic model. The mean Km (micromol x L(-1) ) and V(max) (micromol x g(-1) x min(-1)) values were 27 +/- 10 and 4. 8 +/- 2.1, 54 +/- 22 and 2.3 +/- 1.0, 51 +/- 29 and 2.2 +/- 0.8, for the formation rates of 7,8,4'-THI, 7,3',4'-THI, and 6,7,4'-THI, respectively. Furafylline, the CYP1A2 specific inhibitor, estrogen, and monoclonal antibody raised against human CYP1A2 (MAB-1A2) apparently inhibited the formation of mono-hydroxylated metabolites, The IC50 of Fur for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 1.0, 0.9 and 0. 8 mol x L(-1), respectively. The IC50 of estrogen for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI were 51, 60 and 64 mol x L(-1) respectively. The IC50 of MAB-1A2 for the formation of the mono-hydroxylated products was 1 mol x L(-1), but neither other selective inhibitor nor substrate probes, including coumarin (CYP2D6), sulphaphenzole ( CYP2C9/10), omeprazole ( CYP2C19), quinidine (CYP2D6), diethyldithiocarbamate (CYP2E1), troleandomycin (CYP3A4) and keteconazole (CYP3A4), did so with human liver microsomes.p><p>CONCLUSIONThe in vitro studies indicated that daidzein mono-hydroxylated products were principally metabolized by CYP1A2 in human.p>
Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP1A2 Inhibitors ; Estrogens ; pharmacology ; Humans ; Hydroxylation ; Isoflavones ; metabolism ; Microsomes, Liver ; metabolism ; Theophylline ; analogs & derivatives ; pharmacology

The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.
Animals ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 CYP2C9 ; metabolism ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; metabolism ; Drug Interactions ; Glucuronosyltransferase ; metabolism ; Humans ; Ligusticum ; chemistry ; Microsomes, Liver ; enzymology ; NADP ; metabolism ; pharmacology ; Pyrazines ; metabolism ; pharmacokinetics ; Rats ; Uridine Diphosphate Glucuronic Acid ; metabolism ; pharmacology

To study the enzyme kinetics of ligustilide metabolism and the effects of selective CYP450 inhibitors on the metabolism of ligustilide in liver microsomes of rat, a LC-MS method was established for quantitative analysis of ligustilide in liver microsomes incubation system with nitrendipine as internal standard. The determination m/z for ligustilide was 173, and for nitrendipine, 315. An optimum incubation system was found and various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of ligustilide. The results showed that enzyme kinetics of ligustilide could be significantly inhibited by ketoconazole, trimethoprim and a-naphthoflavon but scarcely inhibited by omeprazole, 4-methylpyrazole and quinidine. Therefore, CYP3A4, CYP2C9 and CYP1A2 are the major isoenzyme participated in in vitro metabolism of ligustilide.
4-Butyrolactone ; analogs & derivatives ; metabolism ; Animals ; Benzoflavones ; pharmacology ; Cytochrome P-450 CYP1A2 ; Cytochrome P-450 CYP3A ; Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; Cytochromes ; antagonists & inhibitors ; Ketoconazole ; pharmacology ; Kinetics ; Male ; Microsomes, Liver ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trimethoprim ; pharmacology

To illustrate the compability rule of Jinlingizi powder, by investigating the effects of Jinlingzi Powder with different compatibility on the enzymatic activity of cytochrome P1 A2 (CYP1A2) from rat liver microsome. The different compability of Jinlingizi powder is designed, based on the orthogonal array L9 (3(4)). In vitro test, rat liver microsomes incubation system is applied to detect the 50% inhibitory concentraton of Jinlingzi powder with different compatibility to cytochrome P1A2 (CYP1A2) enzyme. In vivo experiments, rats is treated orally with the different compability of Jinlingizi powder for 5 days, then be injected with probe drug phenacetin. The biosample from liver tissue is obtained by microdialysis probe, then analysisd by HPLC. The concentration-time data are modulated by software WinNonlin. IC50 data show no significant inhibitory activty to cytochrome P1 A2. Acetaminophen and phenacetin PK parameters indicate that the different compability of Jinlingizi powder can modulate the CYP 1A2 mediated metabolism, which is associate with the compatibility of Jinlingzi powder.
Animals ; Cytochrome P-450 CYP1A2 Inhibitors ; Drugs, Chinese Herbal ; pharmacology ; Male ; Medicine, Chinese Traditional ; Microsomes, Liver ; drug effects ; enzymology ; Powders ; Rats ; Rats, Wistar

Rats were continuously given different doses of water extract of Polygonum multiflorum (1, 10 g x kg(-1)) for 7 days to prepare liver microsomes. Cocktail in vitro incubation approach and Real-time quantitative PCR technology were used to observe the effect of water extract of P. multiflorum on CYP450 enzymatic activities and mRNA expressions in rat liver. Compared with the blank control group, both 1, 10 g x kg(-1) water extract of P. multiflorum treated groups showed significant inhibitions in CYP2E1 enzymatic activities and mRNA expressions (enzymatic activities of CYP2E1, P < 0.01; mRNA expression of CYP2E1, P < 0.05 in 1 g x kg(-1) group, P < 0.01 in 10 g x kg(-1) group). They revealed a significant increase in the enzymatic activity of CYP3A1 (P < 0.01), but without significant change in mRNA expressions. The 10 g x kg(-1) group showed a significant inhibition in CYP1A2 enzymatic activities and mRNA expressions in rat livers (P < 0.01).
Animals ; Cytochrome P-450 CYP1A2 ; genetics ; metabolism ; Cytochrome P-450 CYP2E1 ; genetics ; metabolism ; Cytochrome P-450 Enzyme Inhibitors ; administration & dosage ; isolation & purification ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; Liver ; drug effects ; enzymology ; Male ; Microsomes, Liver ; drug effects ; enzymology ; Polygonum ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley