Tolbutamide Methylhydroxylation Research Articles

Contribution of the human cytochrome P450 2C subfamily to the metabolism of and the interactions with endogenous compounds including steroid hormones.

The objectives of this study were as follows: 1) to compare the metabolic activities of endogenous compounds and their effects on dopamine formation and hydroxylation of steroid hormones, mediated by human cytochrome P450 (CYP), including CYP2C9.1 and CYP2C19, as well as the variants CYP2C9.2 (Arg144Cys) and CYP2C9.3 (Ile359Leu); and 2) to assess the effects of steroid hormones on the activities of CYP2C9.1, CYP2C9.2, and CYP2C19 to estimate the contribution of the CYP2C subfamily to metabolism and drug-drug interactions of endogenous compounds. Dopamine formation from p -tyramine and 6β- and 21- (for progesterone) hydroxylation of testosterone, cortisol, and progesterone by CYP2C9 variants, CYP2C19, CYP2D6, and CYP3A4 were determined using HPLC. The effects of steroid hormones such as testosterone, cortisol, and progesterone on tolbutamide methyl hydroxylation mediated by CYP2C subfamily members were investigated. Only CYP2D6 catalyzed dopamine formation. The 6β-hydroxylation activities of testosterone, cortisol, and progesterone catalyzed by CYP2C9 variants and CYP2D6 were less than 5% of those by CYP3A4. Although cortisol did not inhibit tolbutamide methyl hydroxylation catalyzed by CYP2C9.1, CYP2C9.2, or CYP2C19 and testosterone did not inhibit CYP2C19 activity, the reactions catalyzed by CY2C9.1 and CYP2C9.2 were inhibited by testosterone. The inhibition of progesterone by CYP2C19 was stronger than that by CYP2C9.1 and CYP2C9.2. CYP2C9.1 and CYP2C19 noncompetitively and competitively inhibited tolbutamide methyl hydroxylation with inhibition constants of 43.2 μM and 1.03 μM, respectively. Clinical interactions among endogenous compounds would vary within the CYP2C subfamily, although the contribution of the CYP2C subfamily may be of minor importance for dopamine formation and the detoxification (6β-hydroxylation) of endogenous steroid hormones.

Substrate Specificity of Human Cytochrome P450 (CYP) 2C Subfamily and Effect of Azole Antifungal Agents on CYP2C8.

The metabolic activities of aminopyrine N-demethylation and tolbutamide methylhydroxylation by the human hepatic cytochrome P450 (P450 or CYP) 2C subfamily were compared and the effects of azole antifungal agent on the drug-metabolizing activity of CYP2C8 were investigated. Aminopyrine N-demethylation and tolbutamide methylhydroxylation by CYP2C8, CYP2C9, and CYP2C19 were determined by the previous reported methods. The effects of five azole antifungal agents, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole, on the aminopyrine N-demethylation activity by CYP2C8 were investigated. With regard to aminopyrine N-demethylation, CYP2C19 had the lowest Michaelis constant (Km) and CYP2C8 had the highest maximal velocity (Vmax) among the CYP2C subfamily members. The Vmax/Km values for CYP2C8 were the highest, followed by CYP2C19. For tolbutamide methylhydroxylation, the Km and Vmax for CYP2C19 were three and six times higher than the corresponding values for CYP2C9, and the Vmax/Km value for CYP2C19 was twice that for CYP2C9, whereas hydroxylated tolbutamide formed by CYP2C8 was not detected. Fluconazole, itraconazole, and voriconazole at a concentration of 2 or 10 µM neither inhibited nor stimulated CYP2C8-mediated aminopyrine N-demethylation activity at substrate concentrations around the Km (5 mM). However, ketoconazole and miconazole noncompetitively inhibited CYP2C8-mediated aminopyrine N-demethylation with the inhibitory constant values of 1.98 and 0.86 µM, respectively. These results suggest that ketoconazole and miconazole might inhibit CYP2C8 clinically.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

In vitro determination of the effect of Andrographis paniculata extracts and andrographolide on human hepatic cytochrome P450 activities

We investigated the effects of Andrographis paniculata (AP) extracts and andrographolide on the catalytic activity of three human cDNA-expressed cytochrome P450 enzymes: CYP2C9, CYP2D6 and CYP3A4. In vitro probe-based high performance liquid chromatography assays were developed to determine CYP2C9-dependent tolbutamide methylhydroxylation, CYP2D6-dependent dextromethorphan O-demethylation and CYP3A4-dependent testosterone 6β-hydroxylation activities in the presence and absence of AP extracts and andrographolide. Our results indicate that AP ethanol and methanol extracts inhibited CYP activities more potently than aqueous and hexane extracts across the three isoforms. Potent inhibitory effects were observed on CYP3A4 and CYP2C9 activities (K (i) values below 20μg/ml). Andrographolide was found to exclusively but weakly inhibit CYP3A4 activity. In conclusion, data presented in this study suggest that AP extracts have the potential to inhibit CYP isoforms in vitro. There was, however, variation in the potency of inhibition depending on the extracts and the isoforms investigated.

Regional Distribution of Drug-metabolizing Enzyme Activities in the Liver and Small Intestine of Cynomolgus Monkeys

The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation, and testosterone 6β-, 16α-, 16β-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1'-hydroxylation and testosterone 6β-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.

고속 스크리닝 기법을 이용한 한약제제의 cytochrome P45O 저해능 탐색

본 연구는 우황청심원을 비롯한 상용되는 20종의 한약제제를 대상으로 9종의 시토크롬 동종효소에 대한 대사능의 저해정도를 고속 스크리닝 기법을 이용하여 탐색함으로써, 한약제제와 약물의 병용으로 인한 약물 상호작용 가능성을 평가하고자 하였다. 인체 간 마이크로좀 시료에 9종의 주요 시토크롬 약물대사효소의 지표약물과NADPH-generating system및 한약제제(500 <TEX>${\mu}g/ml$</TEX>)를 첨가한 후 <TEX>$37^{\circ}C$</TEX>에서 15분간 반응시켜 생성된 각각의 대사물을 LC/MS/MS를 이용하여 정량하여 시토크롬 동종효소 활성의 변화를 평가하였다. 그 결과 우황청심원 현탁액 및 황련해독탕 물 추출물이 각각 CYP2B6 및 CYP2D6 효소 활성을 선택적으로 강력하게 저해하였다. 이러한 결과는 약국에서 쉽게 구입할 수 있는 한약제제들 중 일부는 인체 간 시토크롬 활성 저해능을 가지고 있고, 이들 효소에 의해 대사되는 약물과의 병용 복용시 약물상호작용 발생 가능성이 있음을 의미한다. 향후 한약제제에서 저해능을 나타내는 주된 성분을 규명하여 이 성분의 저해능과 저해 기전을 살피는 노력이 필요할 것이다. We evaluated the potential of 20 herbal medications (HMs), commonly used in Korea, to inhibit the catalytic activities of several cytochrome P450 (CYP) isoforms. The abilities of 500 <TEX>${\mu}g/ml$</TEX> of aqueous extracts of 20 HMs to inhibit phenacetin O-deethylation (CYP1A2), coumarin 6-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), rosiglitazone hydroxylation (CYP2C8), tolbutamide 4-methylhydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1'-hydroxylation (CYP3A) were tested using human liver microsomes. The HMs Woohwangcheongsimwon suspension and Hwanglyeonhaedok-Tang strongly inhibited CYP2B6 and CYP2D6 isoform activity, respectively. These results suggest that some of the HMs used in Korea have potential to inhibit CYP isoforms in vitro. Although the plasma concentrations of the active constituents of the HMs were not determined, some herbs could cause clinically significant interactions because the usual doses of those individual herbs are several grams of freeze-dried extracts.

Inhibition of human cytochrome P450 isoforms and NADPH-CYP reductase in vitro by 15 herbal medicines, including Epimedii herba

We evaluated the potential of 15 herbal medicines (HMs), commonly used in Korea, to inhibit the catalytic activities of several cytochrome P450 (CYP) isoforms and microsomal NADPH-CYP reductase. The abilities of 1-1000 microg/mL of freeze-dried aqueous extracts of 15 HMs to inhibit phenacetin O-deethylation (CYP1A2), tolbutamide 4-methylhydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), midazolam 1-hydroxylation (CYP3A4) and NADPH-CYP reductase were tested using human liver microsomes. The HMs Epimedii herba, Glycyrrhizae radix and Leonuri herba inhibited one or more of the CYP isoforms or NADPH-CYP reductase. Of the three HMs, Epimedii herba extracts were the most potent inhibitors of several CYP isoforms (IC(50) 67.5 microg/mL for CYP2C19, 104.8 microg/mL for CYP2E1, 110.9 microg/mL for CYP2C9, 121.9 microg/mL for CYP3A4, 157.8 microg/mL for CYP2D6 and 168.7 microg/mL for CYP1A2) and NADPH-CYP reductase (IC(50) 185.9 microg/mL ). These results suggest that some of the HMs used in Korea have the potential to inhibit CYP isoforms in vitro. Although the plasma concentrations of the active constituents of the HMs were not determined, some herbs could cause clinically significant interactions because the usual doses of those individual herbs are several grams of freeze-dried extracts. Controlled trials to test the significance of these results are necessary.

Stereoselective inhibition of cytochrome P450 forms by lansoprazole and omeprazole in vitro

The stereoselectivity of the inhibitory interaction potential of lansoprazole and omeprazole isomers on six human cytochrome P450 forms was evaluated using human liver microsomes. Lansoprazole enantiomers showed stereoselective inhibition of CYP2C9-catalysed tolbutamide 4-methylhydroxylation, CYP2C19-catalysed S-mephenytoin 4′-hydroxylation, CYP2D6-catalysed dextromethorphan O-demethylation, CYP2E1-catalysed chlorzoxazone 6-hydroxylation and CYP3A4-catalysed midazolam 1-hydroxylation, whereas omeprazole only inhibited CYP2C19 stereoselectively. Of the P450 forms tested, CYP2C19-catalysed S-mephenytoin 4′-hydroxylation was extensively inhibited by both the lansoprazole and omeprazole enantiomers in a competitive and stereoselective manner; the S-enantiomers of both drugs inhibited the hydroxylation more than the R-enantiomers. The estimated Ki values determined for CYP2C19-catalysed S-mephenytoin 4′-hydroxylation were 0.6, 6.1, 3.4 and 5.7 µM for S-lansoprazole, R-lansoprazole, S-omeprazole and R-omeprazole, respectively. The results indicate that although both lansoprazole and omeprazole are strong inhibitors of CYP2C19, the inhibition of CYP2C19 by lansoprazole is highly stereoselective, whereas the inhibition by omeprazole is less stereoselective. In addition, S-lansoprazole, the most potent CYP2C19 inhibitor, is not a good CYP2C19-selective inhibitor owing to its inhibition of other P450 forms.

LANSOPRAZOLE ENANTIOMER ACTIVATES HUMAN LIVER MICROSOMAL CYP2C9 CATALYTIC ACTIVITY IN A STEREOSPECIFIC AND SUBSTRATE-SPECIFIC MANNER

We recently proposed a possible stereoselective activation by lansoprazole of CYP2C9-catalyzed tolbutamide hydroxylation, as well as stereoselective inhibition of several cytochrome P450 (P450) isoforms. This study evaluated the effects of lansoprazole enantiomers on CYP2C9 activity in vitro, using several probe substrates. For tolbutamide 4-methylhydroxylation and phenytoin 4-hydroxylation, R-lansoprazole was an activator (140 and 550% of control at 100 microM R-lansoprazole, EC50 values of 19.9 and 30.2 microM, respectively). R-Lansoprazole-mediated activation of the formation of 4-hydroxyphenytoin was also seen with recombinant human CYP2C9. R-Lansoprazole increased the Michaelis-Menten-derived V(max) of phenytoin 4-hydroxylation from 0.024 to 0.121 pmol/min/pmol P450, and lowered its K(m) from 20.5 to 15.0 microM, suggesting that R-lansoprazole activates CYP2C9-mediated phenytoin metabolism without displacing phenytoin from the active site. Kinetic parameters were also estimated using the two-site binding equation, with alpha values <1 and beta values >1, indicative of activation. Additionally, phenytoin at 10 to 200 microM had no reciprocal effect on the hydroxylation of R-lansoprazole. Meanwhile, R-lansoprazole had no activation effect on diclofenac and S-warfarin metabolism in the incubation study using both recombinant CYP2C9 and human liver microsomes. These substrate-dependent activation effects suggest that phenytoin has a different binding orientation compared with diclofenac and S-warfarin. Overall, these results suggest that R-lansoprazole activates CYP2C9 in a stereospecific and substrate-specific manner, possibly by binding within the active site and inducing positive cooperativity. This is the first report to describe stereoselective activation of this cytochrome P450 isoform.

The inhibitory effects of herbal components on CYP2C9 and CYP3A4 catalytic activities in human liver microsomes.

Herbal medicines are widely consumed by patients in different clinical settings in the United States and all over the world. In this study, 7 herbal components ginsenosides Rb1, Rb2, Rc, and Rd (from ginseng quercetin) ginkgolides A and B (from ginkgo biloba) were investigated for their inhibitory effects on hepatic CYP2C9 and CYP3A4 catalytic activities in human liver microsomes. Tolbutamide 4-methylhydroxylation and testosterone 6beta-hydroxylation were used as index reactions of CYP2C9 or CYP3A4 catalytic activities, respectively. The metabolites of both reactions were measured by high-performance liquid chromatography and used as indicators of whether enzymes were inhibited or unaffected by these agents. Herbal components were studied at various concentrations (0.1, 1, 10, 100, 200 micromol/L). The herbal compounds investigated were capable of inhibiting CYP2C9 and CYP3A4 catalytic activities, but the potencies differed. Quercetin showed marked inhibitory effects on both tolbutamide 4-methylhydroxylation and testosterone 6beta-hydroxylation with IC(50) values of 35 and 38 micromol/L, respectively. Ginsenoside Rd also had significant inhibitory potency on both CYP2C9- and CYP3A4-mediated index reactions with IC(50) values of 105 and 62 micromol/L, respectively. Ginsenosides Rb1, Rb2, and Rc had limited inhibitory activities on both enzyme reaction systems, whereas the effects of ginkgolides A and B appeared negligible. It is concluded that the components of ginseng and ginkgo biloba screened are capable of inhibiting CYP2C9- and CYP3A4-mediated metabolic reactions. Our findings suggest that quercetin and ginsenoside Rd have the potential to interact with conventional medicines that are metabolized by CYP2C9 and CYP3A4 in vivo.

Potential of pranlukast and zafirlukast in the inhibition of human liver cytochrome P450 enzymes

1. The potential of zafirlukast to inhibit several human cytochrome P450 enzymes is well known. However, pranlukast, a structural analogue of zafirlukast, has not been studied. Accordingly, the inhibitory potential of pranlukast was evaluated and compared with that of zafirlukast, a known CYP2C9 inhibitor, in in vitro microsomal incubation studies.2. Both pranlukast and zafirlukast showed moderate inhibition of CYP2C9-catalysed tolbutamide 4-methylhydroxylation, competitively inhibiting tolbutamide 4-methylhydroxylation with estimated mean Ki values of 3.82 ± 0.50 and 5.86 ± 0.08 μM, respectively.3. Pranlukast had no effect on CYP2C19-catalysed S-mephenytoin 4′-hydroxylation or CYP3A4-catalysed midazolam 1-hydroxylation. However, zafirlukast showed minor inhibition of these reactions. Neither pranlukast nor zafirlukast inhibited CYP1A2-catalysed phenacetin O-deethylation, CYP2D6-catalysed dextromethorphan O-demethylation or CYP2E1-catalysed chlorzoxazone 6-hydroxylation.4. The results suggest that like zafirlukast, pranlukast also has the potential moderately to inhibit CYP2C9-catalysed tolbutamide 4-methylhydroxylation. Therefore, the inhibitory potential of pranlukast should be considered when it is co-administered with CYP2C9 substrates with narrow therapeutic ranges (e.g. S-warfarin, phenytoin).

Effect of cefixime and cefdinir, oral cephalosporins, on cytochrome P450 activities in human hepatic microsomes.

The effects of two kinds of oral cephalosporins, cefixime and cefdinir, on cytochrome P450 (CYP) activities in human hepatic microsomes were investigated. Both cefixime and cefdinir at 2 mM concentration neither inhibited nor stimulated CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, CYP3A4-mediated nifedipine oxidation, or CYP3A4-mediated testosterone 6beta-hydroxylation. The free fractions of cefixime and cefdinir in the incubation mixture, which were measured by ultracentrifugation, were 86.1-93.8% and 94.1-97.8%, respectively. These results suggest that both cefixime and cefdinir would not cause clinically significant interactions with other drugs, which are metabolized by CYPs, via the inhibition of metabolism.

Effect of gamma-oryzanol on cytochrome P450 activities in human liver microsomes.

The effects of gamma-oryzanol, a drug mainly used for the treatment of hyperlipidaemia, on several cytochrome P450 (CYP) specific reactions in human liver microsomes were investigated to predict drug interactions with gamma-oryzanol in vivo from in vitro data. The following eight CYP catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6beta-hydroxylation. gamma-Oryzanol had little inhibitory effects on CYP activities, indicating that this compound would not be expected to cause clinically significant interactions with other CYP-metabolized drugs at expected therapeutic concentrations.

Residues Glutamate 216 and Aspartate 301 Are Key Determinants of Substrate Specificity and Product Regioselectivity in Cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.

Inhibitory effects of H1-antihistamines on CYP2D6- and CYP2C9-mediated drug metabolic reactions in human liver microsomes

To screen the inhibitory effects of H1-antihistamines on hepatic bufuralol 1'-hydroxylation and on tolbutamide 4-methylhydroxylation in human liver microsomes. Bufuralol 1'-hydroxylation and tolbutamide 4-methylhydroxylation were used as index reactions for CYP2D6 and CYP2C9, respectively. The metabolites of both reactions were measured using high-performance liquid chromatography and were used as indicators of whether CYP2D6 or CYP2C9 activities were inhibited or unaffected by the agents. All five H1-antihistamines studied showed a concentration-dependent inhibition of CYP2D6-mediated bufuralol 1'-hydroxylation with 50% inhibitory concentration (IC50) values of 32-109 microM. Cyclizine and promethazine showed inhibitory effects on tolbutamide 4-methylhydroxylation with IC20 values of 85 microM and 88 microM, respectively. Tripelennamine, chlorpheniramine, and diphenhydramine showed no inhibitory effects on CYP2C9. All five H1-antihistamines studied inhibited CYP2D6 markedly, but only cyclizine and promethazine inhibited CYP2C9 at concentrations above that usually seen in plasma. Promethazine and chlorpheniramine inhibited CYP2D6 at concentrations that are very close to their therapeutic plasma concentrations. Further studies in humans, especially in poor metabolizers of CYP2D6, will be required to confirm these findings.

Inhibition of human drug metabolizing cytochrome P450 by buprenorphine.

The effects of buprenorphine, a powerful mixed agonist/antagonist analgesic, on several cytochrome P450 (CYP) isoform specific reactions in human liver microsomes were investigated to predict drug interaction of buprenorphine in vivo from in vitro data. The following eight CYP-catalytic reactions were used in this study: CYPlA1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6beta-hydroxylation. Buprenorphine strongly inhibited the CYP3A4- and CYP2D6-catalyzed reactions with Ki values of 14.7 microM and 21.4 microM, respectively. The analgesic also weakly inhibited specific reactions catalyzed by CYP1A1/2 (Ki=132 microM), CYP2B6 (Ki=133 microM), CYP2C19 (Ki=146 microM), CYP2C8/9 (IC50>300 microM), and CYP2E1 (IC50>300 microM), but not CYP2A6 mediated pathway. In consideration of the Ki values obtained in this study and the therapeutic concentration of buprenorphine in human plasma, buprenorphine would not be predicted to cause clinically significant interactions with other CYP-metabolized drugs.

Studies on the Metabolic Fate of M17055 a Novel Diuretic (4): Species Difference in Metabolic Pathway and Identification of Human CYP Isoform Responsible for the Metabolism of M17055

The metabolic profile of M17055, a novel diuretic, after administration to experimental animals and after incubation with human liver microsomes was investigated. 1. Extensive metabolism was observed in rats and monkeys and the structures of six metabolites (RU1, RU2, and RU3 from rat urine or liver perfusate; MU1, MU2 and MU3 from monkey urine) were assumed or identified. The clear species difference of metabolism was revealed between rats and a monkey with different structures of the isolated metabolites. 2. When these metabolites were quantified using radioactive material, RU3, RU1 and MU3 were considered to be major metabolites in rat urine, rat bile and monkey urine respectively, while in a dog, unchanged drug was observed as the major component indicating only little metabolism occurred in dog, when administered intravenously. 3. RU1 and RU2 were also generated from [(14)C]M17055 after incubation with human liver microsomes, suggesting that the metabolic pathway of M17055 in humans involves that observed in rats. 4. [(14)C]M17055 metabolism in human liver microsomes was inhibited by CYP2C8/9 and CYP3A4/5 inhibitors, and also by the antibodies that recognize CYP2C8/9/19 and CYP3A4. Significant correlations were observed between the rate of [(14)C]M17055 metabolism and the activity of testosterone 6beta-hydroxylation or tolbutamide methyl-hydroxylation. cDNA-expressed CYP3A4 and CYP2C9 could catalyze the metabolism of [(14)C]M17055. These results suggest that the metabolism of M17055 in human liver microsomes is catalyzed mainly by CYP3A4 and CYP2C9.

Effect of probucol on cytochrome P450 activities in human liver microsomes.

The effects of probucol, a cholesterol-lowering agent, on several cytochrome P450 (CYP) isoform-specific reactions in human liver microsomes were investigated to predict drug interactions with probucol in vivo from in vitro data. The following eight CYP catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6beta-hydroxylation. Probucol had neither stimulatory nor inhibitory effects on CYP1Al/2, 2A6, 2B6, 2C8/9, 2C19, 2D6, 2E1, and 3A4 activities at concentrations up to 300 microM, indicating that probucol, at the expected therapeutic concentrations, would not be predicted to cause clinically significant interactions with other CYP-metabolized drugs.

Equally potent inhibitors of cholesterol synthesis in human hepatocytes have distinguishable effects on different cytochrome P450 enzymes.

Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC(50) values: 0.2-8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug-drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6beta-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S-mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O-dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 microM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC(50) values around 200 microM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC(50) value: 4 microM). Using the assay of testosterone 6beta-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC(50) values of about 200 microM and a little more by C and A (IC(50) around 100 microM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug-drug interaction is considered.

CYP2C9 Ile359 and Leu359 variants: enzyme kinetic study with seven substrates.

To assess the effects of Ile359 to Leu359 change on CYP2C9-mediated metabolism, we performed site-directed mutagenesis and cDNA expression in yeast for CYP2C9 and examined in detail the kinetics of seven metabolic reactions by wild-type CYP2C9 (Ile359) and its Leu359 variant. For the metabolism of all the substrates studied, the Leu359 variant exhibited smaller Vmax/Km values than did the wild-type. The differences in the Vmax/Km values between the wild-type and the Leu359 variant varied from 3.4-fold to 26.9-fold. The Leu359 variant had higher Km values than did the wild-type for all the reactions studied. Among the seven reactions studied, the greatest difference in the Vmax values between the wild-type and the Leu359 variant was for piroxicam 5'-hydroxylation (408 versus 19 pmol/min/nmol P450), whereas there were no differences in the Vmax values between the wild-type and the Leu359 variant for diclofenac 4'-hydroxylation and tolbutamide methylhydroxylation. These results indicate that the Ile359 to Leu359 change significantly decreases the catalytic activity of all the CYP2C9-mediated metabolisms studied, whereas the extent of the reduction in activity and changes of the kinetic parameters varies between substrates. Moreover, the amino acid substitution decreased the enantiomeric excess in the formation of 5-(4-hydroxyphenyl)-5-phenylhydantoin from phenytoin.

Characterization of cytochrome P450 enzymes involved in drug oxidations in mouse intestinal microsomes

1. Cytochrome P450 (P450, CYP) enzymes involved in drug oxidations in mouse intestines were characterized for their role in the first-pass metabolism of xenobiotics. 2. Preparation of mouse intestinal microsomes using a buffer containing glycerol and protease inhibitors including (p-amidinophenyl) methanesulphonyl fluoride, EDTA, soybean trypsin inhibitor, aprotinin, bestatin and leupeptine gave the highest testosterone 6β-hydroxylase activity among several preparation buffers tested in this study. Testosterone 6β-hydroxylase activity catalysed by mouse intestinal microsomes subjected to freezing and thawing was lower than that catalysed by unfrozen intestinal microsomes. 3. Low but significant catalytic activities of nifedipine oxidation, midazolam 1′ - and 4-hydroxylation, chlorzoxazone 6-hydroxylation, bufuralol 1′ - and 6-hydroxylations and tolbutamide methylhydroxylation were observed in mouse intestinal microsomes. Testosterone 6β-hydroxylation, chlorzoxazone 6-hydroxylation, and bufuralol 1′ - and 6-hydroxylations were inhibited by ketoconazole, diethyldithiocarbamate and quinine respectively. 4. Immunoblot analysis using anti-rat CYP3A antibodies demonstrated two immunoreactive bands showing similar migration in mouse intestinal and hepatic microsomes, although studies using anti-CYP1A, anti-CYP2C, anti-CYP2D and anti-CYP2E1 antibodies did not detect any band in mouse intestinal microsomes. 5. The results suggest that mouse intestinal microsomes should be prepared with glycerol and several protease inhibitors and that Cyp3a enzymes probably play an important role in drug oxidations catalysed by mouse intestine.