Testosterone 6beta-hydroxylase Activity Research Articles

Cytochrome P450 3As Gene Expression and Testosterone 6.BETA.-Hydroxylase Activity in Human Fetal Membranes and Placenta at Full Term

Expression levels of cytochrome P450 (CYP) 3A4, CYP3A5 and CYP3A7 mRNAs in placentas and fetal membranes, which were split into amnion and chorion leave attached decidua (chorion/decidua), obtained from pregnant women with normal delivery (5 subjects) and Caesarean section (15 subjects) were determined. These CYP3A mRNAs were also expressed in amnion and chorion/decidua together with placenta, although the expression level of these mRNAs was strikingly different between subjects. The expression level of the CYP3A4 mRNA in the placenta was about 2-fold higher than those in amnion and chorion/decidua. On the other hand, the expression levels of CYP3A5 and CYP3A7 mRNAs were highest in chorion/decidua. The immunologically related protein(s) with CYP3A7 was detected in all tissues examined. Testosterone 6beta-hydroxylase activity in homogenate of human placenta, amnion and chorion/decidua were 26.6, 3.7 and 4.6 pmol/h/mg protein, respectively. These results suggest that CYP3As in fetal membranes have the metabolic function to protect the fetus from exposure to drugs.

Comparison of Cytochrome P450 3A Enzymes in Cynomolgus Monkeys and Humans

Drug metabolizing activities of cytochromes P450 (P450s, or CYPs) 3A4 and 3A5 in liver microsomes from the cynomolgus monkey [Macaca fascicularis (mf)] were investigated and compared with those of human P450 3A enzymes. Low activities for dealkylation of ethoxyresorufin and pentoxyresorufin were seen in recombinant monkey mfCYP3A4 and mfCYP3A5 and in recombinant human CYP3A4 and CYP3A5 expressed in bacterial membranes. Hydroxylation activities of mfCYP3A4 and mfCYP3A5 toward coumarin, paclitaxel, diclofenac, flurbiprofen, and S-mephenytoin were below detectable levels, as was also true for CYP3A4 and CYP3A5. Monkey mfCYP3A5 and mfCYP3A4 were highly active in bufuralol 1'-hydroxylation. mfCYP3A5 was efficient at dextromethorphan O-demethylation, although human CYP3A5 was unable to catalyze this reaction. Apparent bufuralol 1'-hydroxylation and dextromethorphan O-demethylation activities of monkey liver microsomes were higher than those of human liver microsomes, possibly because of contributions of mfCYP3A5 to these P450 2D-dependent drug oxidations. mfCYP3A5 and CYP3A5 catalyzed midazolam 1'-hydroxylation at a low substrate concentration more efficiently than the corresponding CYP3A4. mfCYP3A5 had higher testosterone 6beta-hydroxylase activity than mfCYP3A4, but the reverse relationship was observed in oxidation of nifedipine and hydroxylation of dexamethasone. These results demonstrate that monkey P450 3A enzymes have similar substrate selectivity to that of human P450 3A enzymes, but exhibit wider substrate selectivity toward P450 2D substrates.

The Mibefradil Derivative NNC55-0396, a Specific T-Type Calcium Channel Antagonist, Exhibits Less CYP3A4 Inhibition than Mibefradil

A novel mibefradil derivative, NNC55-0396, designed to be hydrolysis-resistant, was shown to be a selective T-type Ca(2+) channel inhibitor without L-type Ca(2+) channel efficacy. However, its effects on cytochromes P450 (P450s) have not previously been examined. We investigated the inhibitory effects of NNC55-0396 toward seven major recombinant human P450s--CYP3A4, CYP2D6, CYP1A2, CYP2C9, CYP2C8, CYPC19, and CYP2E1--and compared its effects with those of mibefradil and its hydrolyzed metabolite, Ro40-5966. Our results show that CYP3A4 and CYP2D6 are the two P450s most affected by mibefradil, Ro40-5966, and NNC55-0396. Mibefradil (IC(50) = 33 +/- 3 nM, K(i) = 23 +/- 0.5 nM) and Ro40-5966 (IC(50) = 30 +/- 7.8 nM, K(i) = 21 +/- 2.8 nM) have a 9- to 10-fold greater inhibitory activity toward recombinant CYP3A4 benzyloxy-4-trifluoromethylcoumarin-O-debenzylation activity than NNC55-0396 (IC(50) = 300 +/- 30 nM, K(i) = 210 +/- 6 nM). More dramatically, mibefradil (IC(50) = 566 +/- 71 nM, K(i) = 202 +/- 39 nM) shows 19-fold higher inhibition of CYP3A-associated testosterone 6beta-hydroxylase activity in human liver microsomes compared with NNC55-0396 (IC(50) = 11 +/- 1.1 microM, K(i) = 3.9 +/- 0.4 microM). Loss of testosterone 6beta-hydroxylase activity by recombinant CYP3A4 was shown to be time- and concentration-dependent with both compounds. However, NNC55-0396 (K(I) = 3.87 microM, K(inact) = 0.061/min) is a much less potent mechanism-based inhibitor than mibefradil (K(I) = 83 nM, K(inact) = 0.048/min). In contrast, NNC55-0396 (IC(50) = 29 +/- 1.2 nM, K(i) = 2.8 +/- 0.3 nM) and Ro40-5966 (IC(50) = 46 +/- 11 nM, K(i) = 4.5 +/- 0.02 nM) have a 3- to 4-fold greater inhibitory activity toward recombinant CYP2D6 than mibefradil (IC(50) = 129 +/- 21 nM, K(i) = 12.7 +/- 0.9 nM). Our results suggest that NNC55-0396 could be a more favorable T-type Ca(2+) antagonist than its parent compound, mibefradil, which was withdrawn from the market because of strong inhibition of CYP3A4.

Cytochrome P450 Expression and Regulation in CYP3A4/CYP2D6 Double Transgenic Humanized Mice

Analysis of the developmental and sexual expression of cytochrome P450 drug-metabolizing enzymes is impeded by multiple and varied external factors that influence its regulation. In the present study, a CYP2D6/CYP3A4-double transgenic (Tg-CYP2D6/CYP3A4) mouse model was employed to investigate hepatic CYP2D6 and CYP3A4 ontogeny and sexual dimorphism. Both age and sex have considerable effects on hepatic CYP3A4 protein expression in 3- to 8-week-old transgenic mice, whereas neither factor alters CYP2D6 content. Constitutive CYP2D6 expression resulted in 2- to 3-fold higher dextromethorphan O-demethylase activity in Tg-CYP2D6/CYP3A4 mouse liver microsomes compared with wild-type mice. In contrast, expression of CYP3A4 in transgenic mouse livers did not increase dextromethorphan N-demethylase and midazolam 1'-hydroxylase activities. Pretreatment with pregnenolone 16alpha-carbonitrile (PCN) and 1,4-bis-2-(3, 5-dichloropyridyloxy)-benzene (TCPOBOP) elevated CYP3A4 expression in double transgenic mice. Interestingly, induction of hepatic CYP3A4 was greater in females than age- and treatment-matched males. Consequently, the increase in midazolam 1'-hydroxylase activity was markedly higher in 8-week-old female mice than in corresponding males (8-fold versus 6-fold for PCN treatment and 6-fold versus 5-fold for TCPOBOP). Furthermore, increases in testosterone 6beta-hydroxylase activity after CYP3A induction were relatively lower compared with those in midazolam 1'-hydroxylation for age-, sex-, and treatment-matched mice. The difference in CYP3A4 expression and induction between male and female mice suggests that women may be more susceptible to CYP3A4-mediated drug-drug interactions, and the extent of drug-drug interactions could be substrate dependent.

MDR1 genotype is associated with hepatic cytochrome P450 3A4 basal and induction phenotype

Variant cytochrome P450 (CYP) 3A4 alleles cannot explain human variation in CYP3A4 expression. This study investigated whether common single-nucleotide polymorphisms (SNPs) in multidrug resistance 1 (MDR1), encoding P-glycoprotein, or the pregnane X receptor (PXR) were associated with basal or inducible CYP3A4 expression. MDR1 G2677T and C3435T SNPs and a PXR 6-base pair (bp) deletion were genotyped in the deoxyribonucleic acid from 144 human livers in 3 cohorts each phenotyped for basal or rifampin (INN, rifampicin)-inducible hepatic CYP3A4 expression (or both) and in 57 human small bowel biopsy specimens from 3 cohorts each phenotyped for either basal or rifampin-induced CYP3A4 expression (or both). Hepatic CYP3A4 expression/function was significantly higher in persons homozygous for the MDR1 2677T (Ser893) allele compared with persons homozygous for 2677G (Ala893) in all 3 hepatic cohorts. For example, homozygous MDR1 2677 TT livers had higher midazolam hydroxylase activity than homozygous 2677 GG livers (1831 +/- 1336 pmol x min(-1) x mg protein(-1) versus 1060 +/- 552 pmol x min(-1) x mg protein(-1), P = .03). In 2 of the 3 groups the association was observed in men but not in women. For example, homozygous MDR1 2677 TT male hepatocytes had significantly higher testosterone 6beta-hydroxylase activity compared with homozygous 2677 GG livers (0.120 +/- 0.06 pmol x min(-1) x mg protein(-1) versus 0.069 +/- 0.04 pmol x min(-1) x mg protein(-1), P = .0002). Conversely, rifampin induction of testosterone 6beta-hydroxylation in primary human hepatocytes was significantly higher in persons homozygous for 2677G (12.0 +/- 5.7-fold) compared with MDR1 homozygous TT carriers (7.3 +/- 4.6-fold) (P = .01). Suggestive evidence for higher CYP3A4 expression in MDR1 2677T carriers was also observed in human intestines. CYP3A4 expression was also related to a 6-bp deletion in PXR in 2 of the liver cohorts. Two-factor ANOVA analysis revealed a significant interaction between the MDR1 2677 SNP and the PXR 6-bp deletion influencing CYP3A4 expression (P = .007). Individuals homozygous for the MDR1 2677T allele show enhanced constitutive CYP3A4 expression in the liver and intestine, as compared with those homozygous for the MDR1 2677G allele, particularly in men. Conversely, the magnitude of CYP3A4 induction by rifampin is greater in persons with the MDR1 2677G allele and is inversely related to baseline CYP3A4 expression. MDR1 likely influences basal CYP3A4 expression by limiting the intracellular concentration of an endogenous regulator. MDR1 genotype may be a useful predictor of basal CYP3A4 and the extent of some CYP3A4-mediated drug interactions in humans. Moreover, the influence of MDR1 genotype on CYP3A4 expression adds additional complexity to determining the relative contribution of the MDR1 alleles to the disposition of substrates shared by CYP3A4 and MDR1/P-glycoprotein.

CYP1A1 Is a Major Enzyme Responsible for the Metabolism of Granisetron in Human Liver Microsomes

Granisetron, a potent 5-HT3 receptor antagonist, has been reported to be mainly metabolized to 7-hydroxygranisetron and a lesser extent to 9'-desmethylgranisetron in humans. A previous study indicated that cytochrome P450 (CYP)3A4 is a major catalyst of 9'-demethylation, although the major CYP isoform(s) responsible for 7-hydroxylation are unknown. To clarify granisetron 7-hydroxylase, the in vitro metabolism of granisetron using expressed human CYPs and human liver microsomes was investigated. 7-Hydroxygranisetron was produced almost exclusively by CYP1A1, while, apparently, 9'-desmethylgranisetron was preferentially produced by CYP3A4. Marked inter-individual differences in the ratio of the formation of 7-hydroxygranisetron and 9'-desmethylgranisetron in human liver microsomes was observed. Granisetron 7-hydroxylase activity was strongly correlated with benzo[a]pyrene 3-hydroxylase activity (p<0.0001), but not with testosterone 6beta-hydroxylase activity in human liver microsomes. Furthermore, an anti-human CYP1A1 antibody completely inhibited 7-hydroxylation in human liver microsomes, however, the reaction was not inhibited at all by an anti-CYP3A4 antibody. On the other hand, granisetron 9'-demethylase activity correlated significantly not only with testosterone 6beta-hydroxylase activity (p<0.0001) but also with benzo[a]pyrene 3-hydroxylase activity (p<0.01). Consistent with this, both the anti-CYP1A1 and anti-human CYP3A4 antibodies inhibited the 9'-demethylase activity. These data indicate that CYP1A1 is a major enzyme responsible for the metabolism of granisetron via a main 7-hydroxylation pathway and an alternative 9'-demethylation route. This is the first report demonstrating the substantial contribution of CYP1A1 to the metabolism of a drug, although its role in the metabolism of environmental compounds is well established.

Altered Pharmacokinetics of Paclitaxel in Experimental Hepatic or Renal Failure

The aim of this study was to investigate the effect of hepatic or renal insufficiency on the pharmacokinetics of paclitaxel in rats. Rats were treated with carbon tetrachloride (CCl4; 0.5 ml/kg) to induce hepatic failure or were subjected to 5/6 nephrectomy (5/6 Nx) to induce renal failure. Paclitaxel (3 mg/kg) was administered intravenously or intraportally. Testosterone 6beta-hydroxylase activity, which is a marker of CYP3A activity, was measured in rat liver microsomes from CCl4-treated or 5/6 Nx rats. After paclitaxel was administered intravenously, total body clearance was significantly reduced by 73% and 34% relative to each control value in CCl4-treated and 5/6 Nx rats, respectively (control, 1.82+/-0.42 vs. CCl4-treated, 0.49+/-0.11; sham, 1.54+/-0.07 vs. 5/6 Nx, 1.01+/-0.12 L h(-1) kg(-1); mean+/-SE, n = 5 to 6). Testosterone 6beta-hydroxylase activity was reduced by 92% and 59% relative to each control value in rat liver microsomes from CCl4-treated and 5/6 Nx rats, respectively. After the intraportal administration of paclitaxel, apparent clearance was reduced by 85% relative to control value in rats with hepatic failure, while that in rats with renal failure was the same as the reduction in systemic clearance. These results suggested that not only hepatic failure but also renal failure could modify the pharmacokinetics of paclitaxel in vivo.

CYP3A4 is a human microsomal vitamin D 25-hydroxylase.

The human hepatic microsomal vitamin D 25-hydroxylase protein and gene have not been identified with certainty. Sixteen hepatic recombinant microsomal enzymes were screened for 25-hydroxylase activity; 11 had some 25-hydroxylase activity, but CYP3A4 had the highest activity. In characterized liver microsomes, 25-hydroxylase activity correlated significantly with CYP3A4 testosterone 6beta-hydroxylase activity. Activity in pooled liver microsomes was inhibited by known inhibitors of CYP3A4 and by an antibody to CYP3A2. Thus, CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase. Studies were performed to identify human microsomal vitamin D-25 hydroxylase. Sixteen major hepatic microsomal recombinant enzymes derived from cytochrome P450 cDNAs expressed in baculovirus-infected insect cells were screened for 25-hydroxylase activity with 1alpha-hydroxyvitamin D2 [1alpha(OH)D2], 1alpha-hydroxyvitamin D3 [1alpha(OH)D3], vitamin D2, and vitamin D3 as substrates. Activity was correlated with known biological activities of enzymes in a panel of 12 characterized human liver microsomes. The effects of known inhibitors and specific antibodies on activity also were determined. CYP3A4, the most abundant cytochrome P450 enzyme in human liver and intestine, had 7-fold greater activity than that of any of the other enzymes with 1alpha(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1alpha(OH)D2 than with 1alpha(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1alpha(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6beta-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C9*1 diclofenac 4'-hydroxylase activity (r = 0.65, p < 0.05), but not with activity of any of the other enzymes. Activity in recombinant CYP3A4 and pooled liver microsomes was dose-dependently inhibited by ketoconazole, troleandomycin, isoniazid, and alpha-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity. CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1alpha(OH)D2 and 1alpha(OH)D3.

In vitro metabolism of a new neuroprotective agent, KR-31543 in the human liver microsomes: identification of human cytochrome P450.

KR-31543, (2S,3R,4S)-6-amino-4-[N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl)amino]-3,4-dihydro-2-dimethoxymethyl-3-hydroxy-2-methyl-2H-1-benzopyran, is a new neuroprotective agent for preventing ischemia-reperfusion damage. This study was performed to identify the metabolic pathway of KR-31543 in human liver microsomes and to characterize cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-31543. Human liver microsomal incubation of KR-31543 in the presence of NADPH resulted in the formation of two metabolites, M1 and M2. M1 was identified as N-(4-chlorophenyl)-N-(2-methyl-2H-tetrazol-5-ylmethyl)amine on the basis of LC/MS/MS analysis with a synthesized authentic standard, and M2 was suggested to be hydroxy-KR-31543. Correlation analysis between the known CYP enzyme activities and the rates of the formation of M1 and M2 in the 12 human liver microsomes have showed significant correlations with testosterone 6beta-hydroxylase activity (a marker of CYP3A4). Ketoconazole, a selective inhibitor of CYP3A4, and anti-CYP3A4 monoclonal antibodies potently inhibited both N-hydrolysis and hydroxylation of KR-31543 in human liver microsomes. These results provide evidence that CYP3A4 is the major isozyme responsible for the metabolism of KR-31543 to M1 and M2.

Highly selective inhibition of human CYP3Aa in vitro by azamulin and evidence that inhibition is irreversible.

Azamulin [14-O-(5-(2-amino-1,3,4-triazolyl)thioacetyl)-dihydromutilin] is an azole derivative of the pleuromutilin class of antiinfectives. We tested the inhibition potency of azamulin toward 18 cytochromes P450 using human liver microsomes or microsomes from insect cells expressing single isoforms. In a competitive inhibition model, IC(50) values for CYP3A (0.03-0.24 microM) were at least 100-fold lower than all other non-CYP3A enzymes except CYP2J2 ( approximately 50-fold lower). The IC(50) value with heterologously expressed CYP3A4 was 15-fold and 13-fold less than those of CYP3A5 and CYP3A7, respectively. The reference inhibitor ketoconazole was less selective and exhibited potent inhibition (IC(50) values <10 microM) for CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP4F2, and CYP4F12. Inhibition of CYP3A by azamulin appeared sigmoidal and well behaved with the substrates 7-benzyloxy-4-trifluoromethylcoumarin, testosterone, and midazolam. Preincubation of 4.8 microM azamulin in the presence of NADPH for 10 min inhibited approximately 95% of testosterone 6beta-hydroxylase activity compared with preincubation in the absence of NADPH. Catalytic activities of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1 were unaffected by similar experiments. Incubation of azamulin with heterologously expressed CYP3A4 yielded a type I binding spectrum with a spectral dissociation constant of 3.5 microM, whereas no interaction was found with CYP2D6. Azamulin exhibited good chemical stability when stored in acetonitrile for up to 12 days. Aqueous solubility was found to be >300 microM. Azamulin represents an important new chemical tool for use in characterizing the contribution of CYP3A to the metabolism of xenobiotics.

Large-scale Production of Genetically Engineered CYP3A4 in E. coli: Application of a Jarfermenter

To produce a large amount of CYP3A4, we applied a jarfermenter (ABLE, BMJ-PI or BMS-PI) to culture the genetically engineered E. coli cells harboring CYP3A4 along with NADPH-cytochrome P450 reductase (OR). The jarfermenter is a stirred bacterial culture vessel in which the pH, the dissolved oxygen (DO) and the temperature of a culture medium can be controlled. The expression of CYP3A4 in E. coli cells in the 500 mL of culture medium contained in the BMJ-PI (1 L vessel) (JFM-1) was examined by altering the parameters mentioned above. The highest expression of CYP3A4 in E. coli cells was attained when cultured at pH 6.0, at 30 degrees C under the DO of 0.1 ppm. The incubation was performed 18 hr after the addition of 1.5 mM isopropyl beta-D(-)-thiogalactopyranoside. The expression levels of CYP3A4 and the OR in the membrane fraction of E. coli cells were 267 nmol/L culture and 552 units/L culture, respectively. The CYP3A4 level was about three times higher than that obtained by incubation in a 500 mL flask (100 mL of medium) (84 nmol/L culture). The testosterone 6beta-hydroxylase activity of CYP3A4 expressed in the membrane fraction of E. coli obtained with the JFM-1 was examined. The apparent K(m) and V(max) values were 66.4 microM and 57.8 nmol/min/nmol CYP, respectively. Expecting the mass production of the CYP3A4 by a culture of E. coli, the possibility of a scale up of the culture with the BMS-PI (10 L vessel) (JFM-10) was examined. The optimal culture condition to achieve the highest expression of CYP3A4 with JFM-1 was employed. The expression levels of CYP3A4 and the OR obtained with JFM-1 and JFM-10 were almost equal. The total level of CYP3A4 obtained by using JFM-10 (5 L of medium) was calculated to be about 1.4 mumol. Based on these results, we confirm that the jarfermenter is a useful tool to produce large amounts of CYP3A4.

In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.

CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes.

Induction of cytochrome P450 3A4 (CYP3A4) is determined typically by employing primary culture of human hepatocytes and measuring CYP3A4 mRNA, protein and microsomal activity. Recently a pregnane X receptor (PXR) reporter gene assay was established to screen CYP3A4 inducers. To evaluate results from the PXR reporter gene assay with those from the aforementioned conventional assays, 14 drugs were evaluated for their ability to induce CYP3A4 and activate PXR. Sandwiched primary cultures of human hepatocytes from six donors were used and CYP3A4 activity was assessed by measuring microsomal testosterone 6beta-hydroxylase activity. Hepatic CYP3A4 mRNA and protein levels were also analyzed using branched DNA technology/Northern blotting and Western blotting, respectively. In general, PXR activation correlated with the induction potential observed in human hepatocyte cultures. Clotrimazole, phenobarbital, rifampin, and sulfinpyrazone highly activated PXR and increased CYP3A4 activity; carbamazepine, dexamethasone, dexamethasone-t-butylacetate, phenytoin, sulfadimidine, and taxol weakly activated PXR and induced CYP3A4 activity, and methotrexate and probenecid showed no marked activation in either system. Ritonavir and troleandomycin showed marked PXR activation but no increase (in the case of troleandomycin) or a significant decrease (in the case of ritonavir) in microsomal CYP3A4 activity. It is concluded that the PXR reporter gene assay is a reliable and complementary method to assess the CYP3A4 induction potential of drugs and other xenobiotics.

Toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic cytochrome P450 in F344 rats.

The subacute toxicity and effects of 2,6-di-tert-butyl-4-methylphenyl N-methylcarbamate (terbutol) on hepatic microsomal cytochrome P450 (P450) were investigated in male and female F344 rats. Rats were given 0.25, 0.5, and 1.0% terbutol for 28 days. Liver weights of male and female rats increased at all dose levels. The compound did not affect activity or amount of serum biochemical markers related to hepatic damage. The concentrations of terbutol in rat serum were less than 0.1 microM, and its major metabolites in serum were 2,6-di-tert-butyl-4-carboxyphenyl N-methyl-carbamate and 2,6-di-tert-butyl-4-carboxyphenol. In male rats, P450 and cytochrome b5 (b5) contents, and NADPH cytochrome c reductase (fp2) activity in liver microsomes were increased about 2-fold by 1% terbutol administration for 7 to 28 days. Among the P450-dependent monooxygenase activities in liver microsomes, 7-benzyloxyresorufin-O-debenzylase (BROD) activity was greatly increased by 100-fold, and 7-ethoxyresorufin-O-deethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), and aminopyrine-N-demethylase (APND) activities were elevated 2- to 3-fold. 7-Methoxyresorufin-O-deethylase (MROD), erythromycin-N-demethylase (EMND), estradiol 2-hydroxylase (ED2H), chlorzoxazone 6-hydroxylase (CZ6H), and lauric acid omega-hydroxylase (LAOH) activities were unchanged. For the activities of testosterone hydroxylation, testosterone 16beta-hydroxylase (T16BH) activity was markedly increased by 30-fold, and testosterone 6beta-hydroxylase (T6BH) and testosterone 7alpha-hydroxylase (T7AH) activities were slightly elevated. Testosterone 2alpha-hydroxylase (T2AH) activity was not affected. Terbutol 4-methylhydroxylase (T4MH) activity was increased 9-fold by 1% terbutol. In an immunoinhibition study, T4MH activity in liver microsomes from 1% terbutol-treated rats was decreased about 50% by polyclonal anti-rat CYP2B1, whereas polyclonal anti-rat CYP2A1 and CYP2C11 did not affected the activity. These results indicate that terbutol increased CYP2B subfamily in rat liver microsomes, and that the compound did not cause serious hepatic damage.

Amino acid 305 determines catalytic center accessibility in CYP3A4.

Site-directed mutagenesis has been used to replace alanine 305 with phenylalanine (A305F) and serine (A305S) in the active site of cytochrome P450 3A4 (CYP3A4). Enzyme kinetics for diazepam, erythromycin, nifedipine, and testosterone metabolism have been determined for both mutants and wild-type CYP3A4. The A305F mutation abolished diazepam oxidase activity and reduced the S(50) and V(max) for erythromycin N-demethylase activity from 17 to 10 microM and from 3.2 to 1.2 pmol product/min/pmol P450, respectively. The V(max) for testosterone 6beta-hydroxylase activity was also significantly reduced, from 2.3 to 0.6 pmol product/min/pmol P450, whereas the S(50) increased from 33 to 125 microM. The nifedipine oxidase activity was diminished to a lesser extent, down from 6.5 to 4.9 pmol product/min/pmol P450, whereas the S(50) increased from 9 to 42 microM. The K(i) for ketoconazole, a CYP3A4 selective inhibitor, was increased more than 10-fold from 0.050 to 0.55 microM, from 0.052 to 0.73 microM, and from 0.043 to 2.2 microM by the A305F mutation when measured against erythromycin, nifedipine, and testosterone metabolism activities, respectively. Similarly, the inhibition constants of the broader specificity inhibitors; clotrimazole, econazole, and miconazole were increased 3- to 15-fold by the A305F mutation. In contrast, the A305S mutation increased testosterone 6beta-hydroxylase (V(max) = 2.9 pmol product/min/pmol P450) and erythromycin N-demethylase (V(max) = 5.1 pmol product/min/pmol P450) activities, but reduced nifedipine oxidase activity (V(max) = 4.6 pmol product/min/pmol P450). K(i) values for ketoconazole and other azole inhibitors were unchanged by the A305S mutation. It is proposed that in CYP3A4, the mutagenesis of alanine 305 to a phenylalanine increases the steric hindrance of the catalytic center, thereby greatly reducing azole inhibitor binding affinity, but maintaining monoogygenase activity.

Effect of 4-tert-octylphenol on cytochrome P450 enzymes in rat liver.

The effects were studied of 4-tert-octylphenol (OP) on hepatic cytochrome P450 enzymes in rats. Rats were treated intraperitoneally with OP twice, at doses of 5, 10, and 20 mg/kg. Among the cytochrome P450-dependent monooxygenase activities, testosterone 2alpha-hydroxylase activity, which is associated with CYP2C11, was significantly decreased by OP at all doses. The level relative to control activity was 67-22%. CYP3A2-dependent monooxygenase, testosterone 6beta-hydroxylase activity was also decreased by 51% by OP at 20 mg/kg. Furthermore, immunoblotting showed that OP (10 or 20 mg/kg) significantly decreased CYP2C11/6 and CYP3A2/1 protein levels. However, the reduction ratio of CYP2C11/6 and CYP3A2/1 protein levels by OP treatment was lower than that of testosterone 2alpha-hydroxylase and testosterone 6beta-hydroxylase activities. The Cl(int) (Vmax/Km) value for testosterone 2alpha-hydroxylase was significantly decreased by OP at all doses, whereas the Cl(int) value for testosterone 6beta-hydroxylase was only decreased by OP at 20 mg/kg. In addition, 7-ethoxycoumarin O-deethylase activity was significantly decreased by 32% by the highest dose of OP. By contrast, CYP1A1-, CYP1A2-, CYP2A1-, CYP2B1/2-, CYP2D1-, CYP2E1- and CYP4A1/2/3-dependent monooxygenase activities were not affected by OP at any dose. These results suggest that OP changes the male-specific cytochrome P450 isoforms in rat liver, and that these changes closely relate to the toxicity of OP.

Effect of troglitazone on cytochrome P450 enzymes in primary cultures of human and rat hepatocytes

1. Troglitazonewas the first thiazolidinedione approved for clinical use in the treatment of non-insulin-dependent diabetes mellitus. During clinical investigations of drug-drug interactions with therapeutics (terfenadine and cyclosporine) known to be metabolized by CYP3A4, pharmacokinetic interactions were noted upon troglitazone multiple-dose treatments. The nature of the interactions suggested induction of CYP3A enzymes. 2. Primary cultures of human hepatocytes were used to investigate the induction potential of troglitazone with respect to CYP3A4, CYP2B6 and CYP1A1/2. In human hepatocytes, troglitazone induced both immunoreactive CYP3A4 protein and testosterone 6beta-hydroxylase activity in a dose-dependent fashion (EC50 = 5-10 muM), accompanied by an increase in CYP3A4 mRNA. The capacity of troglitazone to induce CYP3A4 was between that of rifampin (EC50 = ~0.8 muM) and dexamethasone (40-50 muM). Troglitazone increased CYP2B6 immunoreactive protein but did not significantly effect CYP1A1/2 activity, immunoreactive protein or mRNA. 3. Troglitazone produced significant increases in CYP3A message, protein and activity in primary rat hepatocytes, a slight increase in CYP2B1/2 activity and no change in CYP1A1/2 message or activity. 4. These results provide evidence that troglitazone can induce CYP3A and CYP2B enzymes while apparently not altering CYP1A. This provides a rationale for the clinically observed interactions of troglitazone with selected CYP3A4 substrates.

Creation of polarized cells coexpressing CYP3A4, NADPH cytochrome P450 reductase and MDR1/P-glycoprotein.

To develop model polarized cell systems expressing cytochrome P4503A4. NADPH P450 reductase, and P-glycoprotein (Pgp). LLC-PK1 and derivative L-MDR1 cells stably expressing Pgp, the product of the multidrug resistance gene (MDR1), were transfected stably using either a mammalian neomycin selectable expression vector (CYP3A4-Neo) or an episomal vector based on Epstein-Barr virus (CYP3A4-Hygro). These CYP3A4 expressing cells were compared with LLC-PK1, L-MDR1, or Caco-2 cells transduced with Adenovirus-3A4 vector (Ad3A4) with or without simultaneous Adenovirus-P450 Reductase (AdRed) transduction. Cells were characterized for expression of CYP3A4 protein and CYP3A4 mediated metabolism towards midazolam and testosterone. Analysis of membrane integrity and drug transport assays were performed to determine whether infection with recombinant Ad3A4 +/- AdRed affected Pgp function. The rank order of optimal CYP3A4 expression and activities in LLC-PKI and L-MDR1 cells from highest to lowest was cells cotransduced with Ad3A4 plus AdRed >> Ad3A4 >>> CYP3A4-Hygro > CYP3A4-Neo. Similarly, coexpression of Ad3A4 plus AdRed led to enhanced CYP3A4 mediated metabolism in Caco-2 cells over cells with Ad3A4 alone. Incubation of transwell cultured cells expressing Ad3A4/AdRed with midazolam led to readily detectable metabolite in the medium. In microsomes from Caco-2 and LLC-PK1 cells, each co-transduced with Ad3A4/AdRed, Vmax values for testosterone 6beta-hydroxylase activity ranged from 414 to 1350 pmoles/min/mg, respectively. For either Caco-2 or LLC-MDR1 cells, TEER values and the rate of apical to basal and basal to apical transport of vinblastine or digoxin were similar in cells with and without Ad3A4/Red transduction. Polarized cellular systems coexpressing Ad3A4, AdRed, and the MDR1/Pgp transporter were developed and characterized. The results document the utility of these polarized model systems for simultaneous drug transport/drug metabolism studies. Since the experimental approach can be adapted to study the interplay of multiple enzyme/ transporting systems, it may find significant application as a screening tool for the pharmaceutical industry and as a more basic research tool to study the kinetics of intestinal drug bioavailability.

Mechanism-based inactivation of cytochrome P450s 1A2 and 3A4 by dihydralazine in human liver microsomes.

Dihydralazine is known to induce immunoallergic hepatitis. Since anti-liver microsome (anti-LM) autoantibodies found in the serum of the patients react with P450 1A2, it is suggested that dihydralazine is biotransformed into a reactive metabolite, which covalently binds to cytochrome P450 1A2 and triggers an immunological response as a neoantigen. We investigated inactivation of P450 enzymes, including P450 1A2, during the metabolism of dihydralazine to evaluate the selectivity of P450 1A2 as a catalyst and a target of dihydralazine. Human liver microsomes or microsomes from lymphoblastoid cells expressing P450 enzymes were preincubated with dihydralazine in the presence of NADPH, followed by an assay of several monooxygenase activities. Preincubation of human liver microsomes with dihydralazine in the presence of NADPH resulted in decreases in phenacetin O-deethylase activity (an indicator of P450 1A2 activity) and testosterone 6beta-hydroxylase activity (P450 3A4), but not in diclofenac 4'-hydroxylase activity (P450 2C9), an indication of inactivation of P450s 1A2 and 3A4 during the dihydralazine metabolism. The inactivation of both of the P450s followed pseudo-first-order kinetics and was saturable with increasing dihydralazine concentrations. Similar time-dependent decreases in the activities were obtained in the case for use in microsomes expressing P450 1A2 and P450 3A4 instead of the human liver microsomes. The data presented here demonstrated that dihydralazine was metabolically activated not only by P450 1A2 but also by P450 3A4, and the chemically reactive metabolite bound to and inactivated the enzyme themselves, suggesting that dihydralazine is a mechanism-based inactivator of P450s 1A2 and 3A4. The data support the postulated covalent binding of a reactive metabolite of dihydralazine to P450 1A2 as a step in the formation of anti-LM antibodies in dihydralazine hepatitis, but it is not the unique factor for determining the specificity of the autoantibodies.

Induction of hepatic xenobiotic metabolizing enzymes in female Fischer-344 rats following repeated inhalation exposure to decamethylcyclopentasiloxane (D5).

Decamethylcyclopentasiloxane (D5) is a cyclic siloxane with a wide range of commercial applications. The present study was designed to investigate the effects of D5 on the expression and activity of selected rat hepatic phase I and phase II metabolizing enzymes. Female Fischer-344 rats were exposed to 160 ppm D5 vapors (6 h/day, 7 days/week, for 28 days) by whole-body inhalation. Changes in the activity and relative abundance of hepatic microsomal cytochromes P450 (CYP1A, CYP2B, CYP3A, and CYP4A), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were measured. Repeated inhalation exposure of rats to D5 increased liver size by 16% relative to controls by day 28. During a 14-day post-exposure period, liver size in D5-exposed animals showed significant recovery. Exposure to D5 did not change total hepatic P450, but increased the activity of hepatic NADPH-cytochrome c reductase by 1.4-fold. An evaluation of cytochrome P450 (CYP) enzymes in hepatic microsomes prepared from D5-exposed rats revealed a slight (1.8-fold) increase in 7-ethoxyresorufin O-deethylase (EROD) activity, but no change in immunoreactive CYP1A1/2 protein. A moderate increase (4.2-fold) in both 7-pentoxyresorufin O-depentylase (PROD) activity and immunoreactive CYP2B1/2 protein (3.3-fold) was observed. Testosterone 6beta-hydroxylase activity was also increased (2.4-fold) as was CYP3A1/2 immunoreactive protein. Although a small increase in 11- and 12-hydroxylation of lauric acid was detected, no change in immunoreactive CYP4A levels was measured. Liver microsomal epoxide hydrolase activity and immunoreactive protein increased 1.7- and 1.4-fold, respectively, in the D5-exposed group. UDPGT activity toward chloramphenicol was induced 1.8-fold, while no change in UDPGT activity toward 4-nitrophenol was seen. These results suggest that the profile for enzyme induction following inhalation exposure of female Fischer-344 rats to D5 vapors is similar to that reported for phenobarbital, and therefore D5 may be described as a weak "phenobarbital-like" inducer.