Regulation of CYP3A4 by the bile acid receptor FXR: evidence for functional binding sites in the CYP3A4 gene

CYP3A4, the most abundant cytochrome P450 in human liver, is responsible for the metabolism of numerous xenobiotics and endobiotics. CYP3A4 expression is highly variable and is induced by numerous compounds of exogenous and endogenous origin, including elevated concentrations of secondary bile acids via the pregnane X receptor (PXR). We show that physiological concentrations of the primary bile acid chenodeoxycholic acid regulate the expression of CYP3A4 via the bile acid receptor FXR. Experiments performed in vitro in different cell culture systems, gel-mobility shift assays and experiments performed in vivo in transgenic mice lacking FXR or PXR and treated with the synthetic FXR agonist GW4064 were undertaken to study the implication of FXR in the regulation of CYP3A. Our data provide evidence for the presence of two functional FXR recognition sites located in a 345-bp element within the 5'-flanking region of CYP3A4. Mutational analysis of these sites and experiments in transgenic mice lacking FXR or PXR support the relevance of FXR activation for CYP3A regulation. Thus, whereas elevated concentrations of precursors of bile acids and secondary bile acids induce CYP3A via PXR, primary bile acids can modulate the expression of CYP3A via FXR. These findings may explain elevated CYP3A expression in cholestasis and part of the variability of drug responsiveness and toxicity between individuals.     

Effect of ursodeoxycholic acid administration on bile acid composition in hamster bile

The modification in the composition of bile acids in hamster by the administration of high dose of ursodeoxycholic acid (UDCA) was investigated. Male Golden Syrian hamsters were divided into five groups: a control group, two groups that received 0.5 g of UDCA per 100 g of standard diet during 30 and 60 days and another two groups that received 1 g of UDCA per 100 g of standard diet during 30 and 60 days. After ether anaesthesia the gallbladder was removed and bile was immediately aspirated. Bile acids were determined by high performance liquid chromatography (HPLC). Taurolithocholic (TLCA) and glycolithocholic acids (GLCA) increased significantly in all treated groups. The glyco/tauro ratio of 0.69 in controls became more than 1 in treated animals except in the case of lithocholic acid (LCA) conjugates which remained less than 1. UDCA derivatives increased proportionally to the administered dose and the cholic/cheno ratio diminished significantly. A moderate increase of 3- and 7-keto derivatives of chenodeoxycholic acid (CDCA) was observed in all treated groups but the above mentioned increment was especially evident in 3-keto derivatives. A high percentage of UDCA administered in the hamster was likely transformed to CDCA and the glyco conjugates of the bile acids were the predominant species except for the LCA derivatives.     

Pioglitazone is metabolised by CYP2C8 and CYP3A4 in vitro: potential for interactions with CYP2C8 inhibitors

Our objective was to identify the cytochrome P450 (CYP) enzymes that metabolise pioglitazone and to examine the effects of the CYP2C8 inhibitors montelukast, zafirlukast, trimethoprim and gemfibrozil on pioglitazone metabolism in vitro. The effect of different CYP isoform inhibitors on the elimination of a clinically relevant concentration of pioglitazone (1 microM) and the formation of the main primary metabolite M-IV were studied using pooled human liver microsomes. The metabolism of pioglitazone by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5 was investigated using human recombinant CYP isoforms. In particular, the inhibitors of CYP2C8, but also those of CYP3A4, markedly inhibited the elimination of pioglitazone and the formation of M-IV by HLM. Inhibitors selective to other CYP isoforms had a minor effect only. Of the recombinant isoforms, CYP2C8 (20 pmol/ml) metabolised pioglitazone markedly (56% in 60 min.), and also CYP3A4 had a significant effect (37% in 60 min.). Montelukast, zafirlukast, trimethoprim and gemfibrozil inhibited pioglitazone elimination in HLM with IC50 values of 0.51 microM, 1.0 microM, 99 microM and 98 microM, respectively, and the formation of the metabolite M-IV with IC50 values of 0.18 microM, 0.78 microM, 71 microM and 59 microM, respectively. In conclusion, pioglitazone is metabolised mainly by CYP2C8 and to a lesser extent by CYP3A4 in vitro. CYP2C9 is not significantly involved in the elimination of pioglitazone. The effect of different CYP2C8 inhibitors on pioglitazone pharmacokinetics needs to be evaluated also in vivo because, irrespective of their in vitro CYP2C8 inhibitory potency, their pharmacokinetic properties may affect the extent of interaction.     

Amino acid residues critical for differential inhibition of CYP2B4, CYP2B5, and CYP2B1 by phenylimidazoles

The molecular basis for reversible inhibition of rabbit CYP2B4 and CYP2B5 and rat CYP2B1 by phenylimidazoles was assessed with active-site mutants and new three-dimensional models based on the crystal structure of CYP2C5. 4-Phenylimidazole was 17- to 32-fold more potent toward CYP2B4 and CYP2B1 than CYP2B5. The 3D models, along with site-directed mutagenesis data, revealed the importance of residue 114 for sensitivity to inhibition of all three CYP2B enzymes. Besides Ile 114, Val 367 was also found to be critical for inhibition of CYP2B4 and CYP2B1. The most interesting new insights were obtained from analysis of the CYP2B5 model and the CYP2B5 active-site mutants. Simultaneous substitution of residues 114, 294, 363, and 367 with the corresponding residues of CYP2B4 decreased the IC(50) value for inhibition by 4-phenylimidazole 12-fold. Docking 4-phenylimidazole into the models of CYP2B5 mutants demonstrated that the inhibitor-binding site is strongly influenced by residue-residue interactions, especially between residues 114 and 294. A chlorine substitution at position 4 of the phenyl moiety of 4- and 1-phenylimidazole resulted in IC(50) values 95- and 130-fold lower for CYP2B4 than for CYP2B5, respectively, suggesting that these compounds are selective inhibitors of CYP2B4. Overall, the study revealed that differences in the determinants of inhibition between CYP2B4 and CYP2B5 are caused not only by single residue inhibitor contacts but also by residue-residue interactions. This new generation of CYP2B models may provide valuable information for the design of selective inhibitors of human CYP2B6 and for the development of drugs that avoid drug interactions due to P450 inhibition.     

Contribution of CYP4A8 to the formation of 20-hydroxyeicosatetraenoic acid from arachidonic acid in rat kidney

20-Hydroxyeicosatetraenoic acid (20-HETE) has been shown to be an arachidonic acid metabolite of the cytochrome P450 (CYP) enzymes belonging to the CYP4A subfamily and is a predominant regulator of renal vascular tone and tubular ion reabsorption in rat kidney. CYP4A8 is one of the CYP4A enzymes expressed in rat kidney, but its contribution to 20-HETE formation has not been assessed. In order to clarify that the role of CYP4A8, we have developed bacterial expression systems for the expression of recombinant CYP4A8 (rCYP4A8). We also produced an antibody against rCYP4A8 which was used for immunoinhibition and immunohistochemical studies. In a reconstituted system, rCYP4A8 sufficiently catalyzed 20-HETE formation as well as prostaglandin A(1) omega-hydroxylation, a marker activity for CYP4A8. In addition, anti-rCYP4A8 sera significantly inhibited prostaglandin A(1) omega-hydroxylation and strongly inhibited arachidonic acid omega-hydroxylation in rat kidney microsomes. These observations suggested for the first time that CYP4A8 also contributed to 20-HETE formation in rat kidney. Furthermore, immunohistochemstry suggested that CYP4A8 is present in preglomerular arteries, where 20-HETE has been established to be a vasoconstrictor.     

Stereoselectivity in metabolism of ifosfamide by CYP3A4 and CYP2B6

The aim was to identify the hepatic cytochromes P450 (CYPs) responsible for the enantioselective metabolism of ifosfamide (IFA). The 4-hydroxylation, N2- and N3-dechloroethylation of IFA enantiomers were monitored simultaneously in the same metabolic systems using GC/MS and pseudoracemate techniques. In human and rat liver microsomes, (R)-IFA was preferentially metabolized via 4-hydroxylation, whereas its antipode was biotransformed in favour of N-dechloroethylation. CYP3A4 was the major enzyme responsible for metabolism of IFA enantiomers in human liver. The study also revealed that CYP3A (human CYP3A4/5 and rat CYP3A1/2) and CYP2B (human CYP2B6 and rat CYP2B1/2) enantioselectively mediated the 4-hydroxylation, N2- and N3-dechloroethylation of IFA. CYP3A preferentially supported the formation of (R)-4-hydroxyIFA (HOIF), (R)-N2-dechloroethylIFA (N2D) and (R)-N3-dechloroethylIFA (N3D), whereas CYP2B preferentially mediated the generation of (S)-HOIF, (S)-N2D and (S)-N3D. The enantioselective metabolism of IFA by CYP3A4 and CYP2B1 was confirmed in cDNA transfected V79 cells.     

Stereoselectivity in metabolism of ifosfamide by CYP3A4 and CYP2B6

The aim was to identify the hepatic cytochromes P450 (CYPs) responsible for the enantioselective metabolism of ifosfamide (IFA). The 4-hydroxylation, N²- and N³-dechloroethylation of IFA enantiomers were monitored simultaneously in the same metabolic systems using GC/MS and pseudoracemate techniques. In human and rat liver microsomes, (R)-IFA was preferentially metabolized via 4-hydroxylation, whereas its antipode was biotransformed in favour of N-dechloroethylation. CYP3A4 was the major enzyme responsible for metabolism of IFA enantiomers in human liver. The study also revealed that CYP3A (human CYP3A4/5 and rat CYP3A1/2) and CYP2B (human CYP2B6 and rat CYP2B1/2) enantioselectively mediated the 4-hydroxylation, N²- and N³-dechloroethylation of IFA. CYP3A preferentially supported the formation of (R)-4-hydroxyIFA (HOIF), (R)-N²-dechloroethylIFA (N2D) and (R)-N³-dechloroethylIFA (N3D), whereas CYP2B preferentially mediated the generation of (S)-HOIF, (S)-N2D and (S)-N3D. The enantioselective metabolism of IFA by CYP3A4 and CYP2B1 was confirmed in cDNA transfected V79 cells.     

Pioglitazone, an in vitro inhibitor of CYP2C8 and CYP3A4, does not increase the plasma concentrations of the CYP2C8 and CYP3A4 substrate repaglinide

Objective Pioglitazone, a thiazolidinedione antidiabetic, inhibits cytochrome P450 (CYP) 2C8 and CYP3A4 enzymes in vitro. Repaglinide, a meglitinide analogue antidiabetic, is metabolised by CYP2C8 and CYP3A4. In patients with type 2 diabetes, the pioglitazone-repaglinide combination has acted synergistically on glycaemic parameters. Our aim was to determine whether pioglitazone increases the plasma concentrations of repaglinide. Methods In a randomized, 2-phase cross-over study, 12 healthy volunteers received 30 mg pioglitazone or placebo once daily for 5 days. On day 5, they ingested a single 0.25 mg dose of repaglinide 1 h after the last pretreatment dose. Plasma repaglinide and pioglitazone, and blood glucose concentrations were measured for 12 h. Results During the pioglitazone phase, the mean peak plasma repaglinide concentration (C_max) and the total area under the concentration-time curve [AUC(0-∞)] of repaglinide were 100% (range 53–157%, P=0.99) and 90% (range 63–120%, P=0.22), respectively, of those during the placebo phase. Also the half-life of repaglinide was unaffected, but the median peak time of repaglinide was shortened from 40 min to 20 min by pioglitazone (P=0.014). The short-term pioglitazone administration did not modify the blood glucose-lowering effect of a single dose of repaglinide. Conclusions Pioglitazone does not increase the plasma concentrations of repaglinide, indicating that the inhibitory effect of pioglitazone on CYP2C8 and CYP3A4 is very weak in vivo, probably due to its extensive plasma protein binding. The synergistic effect of repaglinide and pioglitazone on the glycaemic parameters, seen in patients with type 2 diabetes during their long-term use, is unlikely to be caused by inhibition of repaglinide metabolism by pioglitazone.     

Species differences in bile acids I. Plasma and urine bile acid composition

Maintenance of bile acid (BA) homeostasis is essential to achieve their physiologic functions and avoid their toxic effects. The marked differences in BA composition between preclinical safety models and humans may play a major role in the poor prediction of drug‐induced liver injury using preclinical models. We compared the composition of plasma and urinary BAs and their metabolites between humans and several animal species. Total BA pools and their composition varied widely among different species. Highest sulfation of BAs was observed in human and chimpanzee. Glycine amidation was predominant in human, minipig, hamster and rabbit, while taurine amidation was predominant in mice, rat and dogs. BA profiles consisted primarily of tri‐OH BAs in hamster, rat, dog and mice, di‐OH BAs in human, rabbit and minipig, and mono‐OH BA in chimpanzee. BA profiles comprised primarily hydrophilic and less toxic BAs in mice, rat, pig and hamster, while it primarily comprised hydrophobic and more toxic BAs in human, rabbit and chimpanzee. Therefore, the hydrophobicity index was lowest in minipig and mice, while it was highest in rabbit, monkey and human. Glucuronidation and glutathione conjugation were low in all species across all BAs. Total concentration of BAs in urine was up to 10× higher and more hydrophilic than plasma in most species. This was due to the presence of more tri‐OH, amidated, sulfated and primary BAs, in urine compared to plasma. In general, BA profiles of chimpanzee and monkeys were most similar to human, while minipig, rat and mice were most dissimilar to human.     

Mesophase formation during cholesterol gallstone dissolution in human bile: effect of bile acid composition

Duodenal bile obtained from patients with gallstones who were acutely infused with chenodeoxycholic acid, ursodeoxycholic acid, or cholic acid were examined for the propensity toward the formation of a liquid crystalline mesomorphic phase when cholesterol gallstones were incubated in these bile acids. Bile taken from patients infused with ursodeoxycholic acid was found to be enriched in ursodeoxycholic acid; mesophase formation was detected in these samples but not in bile from patients infused with chenodeoxycholic acid or cholic acid.     

A novel constitutive androstane receptor-mediated and CYP3A-independent pathway of bile acid detoxification

Cytosolic sulfotransferase (SULT)-mediated sulfation plays an essential role in the detoxification of bile acids and is necessary to avoid pathological conditions, such as cholestasis, liver damage, and colon cancer. In this study, using transgenic mice bearing conditional expression of the activated constitutive androstane receptor (CAR), we demonstrate that activation of CAR is both necessary and sufficient to confer resistance to the hepatotoxicity of lithocholic acid (LCA). Surprisingly, the CAR-mediated protection is not attributable to the expected and previously characterized CYP3A pathway; rather, it is associated with a robust induction of SULT gene expression and increased LCA sulfation. We have also provided direct evidence that CAR regulates SULT expression by binding to the CAR response elements found within the SULT gene promoters. Interestingly, activation of CAR was also associated with an increased expression of the 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2), an enzyme responsible for generating the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate. Analysis of gene knockout mice revealed that CAR is also indispensable for ligand-dependent activation of SULT and PAPSS2 in vivo. Therefore, we establish an essential and unique role of CAR in controlling the mammalian sulfation system and its implication in the detoxification of bile acids.     

Territrems B and C metabolism in human liver microsomes: major role of CYP3A4 and CYP3A5

Human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and control and CYP3A4 cDNA-transfected V79 Chinese hamster cells were tested for their ability to metabolize territrem B (TRB) and territrem C (TRC). Two TRB metabolites, designated MB(2) and MB(4), and one TRC metabolite, designated MC, were formed by all of these preparations. Of the nine supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5), only those expressing CYP3A4 or CYP3A5 metabolized TRB and TRC. MB(2), MB(4), and MC were formed by CYP3A4 cDNA-transfected V79MZ Chinese hamster cells, but not by non-transfected cells. In order to investigate which CYP450 isoforms were responsible for MB(2), MB(4) and MC formation in human liver microsomal preparations, six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketoconazole, and diethyldithiocarbamate) and antibodies against CYP3A4 were used. MB(2), MB(4), and MC formation was markedly inhibited by ketoconazole, but less affected by quinidine and sulfaphenazole. Anti-CYP3A4 antibody markedly inhibited MB(2), MB(4), and MC formation and also 6 beta-hydroxytestosterone formation from testosterone. The CYP3A-dependent reaction of testosterone 6 beta-hydroxylation showed a high correlation with 4 beta-C hydroxylation of TRB (r(2)=0.97, P<0.0001), O-demethylation of TRB (r(2)=0.95, P<0.0001), and 4 beta-C hydroxylation of TRC (r(2)=0.99, P<0.0001). Immunoblotting and RT-PCR showed that CYP3A4 and CYP3A5 were expressed in all four human liver microsomal preparations tested (HLM1-HLM4). The amount of MB(2), MB(4), and MC formed using different HLM preparations was related to the 6 beta-testosterone hydroxylase activity of the preparations. However, the extent of MB(2), MB(4), and MC formation was not related to the age or gender of the person from whom the microsomal sample was prepared. It was therefore suggest that CYP3A4 and CYP3A5 are the major enzymes responsible for TRB and TRC metabolism by human liver microsomes.     

Metabolism of repaglinide by CYP2C8 and CYP3A4 in vitro: effect of fibrates and rifampicin

Repaglinide is an antidiabetic drug metabolised by cytochrome P450 (CYP) 2C8 and CYP3A4 enzymes. To clarify the mechanisms of observed repaglinide drug interactions, we determined the contribution of the two enzymes to repaglinide metabolism at different substrate concentrations, and examined the effect of fibrates and rifampicin on CYP2C8, CYP3A4 and repaglinide metabolism in vitro. We studied repaglinide metabolism using pooled human liver microsomes, recombinant CYP2C8 and recombinant CYP3A4 enzymes. The effect of quercetin and itraconazole on repaglinide metabolism, and of gemfibrozil, bezafibrate, fenofibrate and rifampicin on CYP2C8 (paclitaxel 6alpha-hydroxylation) and CYP3A4 (midazolam 1-hydroxylation) activities and repaglinide metabolism were studied using human liver microsomes. At therapeutic repaglinide concentrations (<0.4 microM), CYP2C8 and CYP3A4 metabolised repaglinide at similar rates. Quercetin (25 microM) and itraconazole (3 microM) inhibited the metabolism of 0.2 microM repaglinide by 58% and 71%, and that of 2 microM repaglinide by 56% and 59%, respectively. The three fibrates inhibited CYP2C8 (Ki: bezafibrate 9.7 microM, gemfibrozil 30.4 microM and fenofibrate 92.6 microM) and repaglinide metabolism (IC50: bezafibrate 37.7 microM, gemfibrozil 111 microM and fenofibrate 164 microM), but had no effect on CYP3A4. Rifampicin inhibited CYP2C8 (Ki 30.2 microM), CYP3A4 (Ki 18.5 microM) and repaglinide metabolism (IC50 13.7 microM). In conclusion, both CYP2C8 and CYP3A4 are important in the metabolism of therapeutic concentrations of repaglinide in vitro, but their predicted contributions in vivo are highly dependent on the scaling factor used. Gemfibrozil is only a moderate inhibitor of CYP2C8 and does not inhibit CYP3A4; inhibition of CYP-enzymes by parent gemfibrozil alone does not explain its interaction with repaglinide in vivo. Rifampicin competitively inhibits both CYP2C8 and CYP3A4, which can counteract its inducing effect in humans.     

Derivation of CYP3A4 and CYP2B6 degradation rate constants in primary human hepatocytes: A siRNA-silencing-based approach

The first-order degradation rate constant (k_deg) of cytochrome P450 (CYP) enzymes is a known source of uncertainty in the prediction of time-dependent drug–drug interactions (DDIs) in physiologically-based pharmacokinetic (PBPK) modelling. This study aimed to measure CYP k_deg using siRNA to suppress CYP expression in primary human hepatocytes followed by incubation over a time-course and tracking of protein expression and activity to observe degradation. The magnitude of gene knockdown was determined by qPCR and activity was measured by probe substrate metabolite formation and CYP2B6-Glo™ assay. Protein disappearance was determined by Western blotting. During a time-course of 96 and 60 h of incubation, over 60% and 76% mRNA knockdown was observed for CYP3A4 and CYP2B6, respectively. The k_deg of CYP3A4 and CYP2B6 protein was 0.0138 h⁻¹ (±0.0023) and 0.0375 h⁻¹ (±0.025), respectively. The k_deg derived from probe substrate metabolism activity was 0.0171 h⁻¹ (±0.0025) for CYP3A4 and 0.0258 h⁻¹ (±0.0093) for CYP2B6. The CYP3A4 k_deg values derived from protein disappearance and metabolic activity were in relatively good agreement with each other and similar to published values. This novel approach can now be used for other less well-characterised CYPs.     

Effects of vercirnon on the activity of CYP3A4, CYP2C19 and CYP2C8 enzymes and BCRP and OATP1B1 transporters using probe substrates

Purpose

Vercirnon is a CCR9 chemokine receptor antagonist being developed for the treatment of Crohn’s disease. As a variety of concomitant medications are often required for the treatment of Crohn’s disease, it is important to characterise the drug interaction profile of vercirnon. To confirm the results of previous in vitro inhibition studies, this study assessed the in vivo effect of vercirnon on the activity of cytochrome P450 enzymes (CYP3A4, CYP2C19 and CYP2C8) and drug transport proteins (BCRP and OATP1B1) using probe substrates.

Methods

This was an open-label, single-sequence, repeat-dose study conducted in 24 healthy adult subjects. On days 1–4, subjects received probe substrates (midazolam, pioglitazone, omeprazole and rosuvastatin; in that order), followed by administration of vercirnon 500 mg twice daily (BID) on days 5–14. On days 11–14, in addition to vercirnon 500 mg BID, subjects also received probe substrates as on days 1–4. Blood samples were collected for pharmacokinetic analysis of probe substrates, vercirnon and two of its metabolites.

Results

Geometric least-squares mean ratios (90 % confidence interval) of area under the concentration-time curve from time zero to infinity for probe administered with vercirnon (test) compared with probe alone (reference) for midazolam, pioglitazone, omeprazole and rosuvastatin were 0.92 (0.85, 0.99), 1.01 (0.95, 1.07), 0.99 (0.76,1.31) and 0.98 (0.88, 1.09), respectively.

Conclusions

Co-administration of probe substrates midazolam, pioglitazone, omeprazole, and rosuvastatin following repeat dosing of vercirnon 500 mg BID demonstrated vercirnon had no clinically significant effect on CYP3A4, CYP2C8, CYP2C19 enzyme activity or BCRP or OATP1B1 transporter activity.     

CYP2C8 but not CYP3A4 is important in the pharmacokinetics of montelukast

AIM

According to product information, montelukast is extensively metabolized by CYP3A4 and CYP2C9. However, CYP2C8 was also recently found to be involved. Our aim was to study the effects of selective CYP2C8 and CYP3A4 inhibitors on the pharmacokinetics of montelukast.

METHODS

In a randomized crossover study, 11 healthy subjects ingested gemfibrozil 600 mg, itraconazole 100 mg (first dose 200 mg) or both, or placebo twice daily for 5 days, and on day 3, 10 mg montelukast. Plasma concentrations of montelukast, gemfibrozil, itraconazole and their metabolites were measured up to 72 h.

RESULTS

The CYP2C8 inhibitor gemfibrozil increased the AUC(0,∞) of montelukast 4.3-fold and its t_1/2 2.1-fold (P < 0.001). Gemfibrozil impaired the formation of the montelukast primary metabolite M6, reduced the AUC and C_max of the secondary (major) metabolite M4 by more than 90% (P < 0.05) and increased those of M5a and M5b (P < 0.05). The CYP3A4 inhibitor itraconazole had no significant effect on the pharmacokinetic variables of montelukast or its M6 and M4 metabolites, but markedly reduced the AUC and C_max of M5a and M5b (P < 0.05). The effects of the gemfibrozil-itraconazole combination on the pharmacokinetics of montelukast did not differ from those of gemfibrozil alone.

CONCLUSIONS

CYP2C8 is the dominant enzyme in the biotransformation of montelukast in humans, accounting for about 80% of its metabolism. CYP3A4 only mediates the formation of the minor metabolite M5a/b, and is not important in the elimination of montelukast. Montelukast may serve as a safe and useful CYP2C8 probe drug.     

Participation of CYP2C8 in retinoic acid 4-hydroxylation in human hepatic microsomes.

Cytochromes P450 (CYPs) catalyze the 4-hydroxylation of all-trans-retinoic acid (ATRA), an agent used in the treatment of certain malignancies. Literature studies have implicated several CYPs in this reaction, but the relative importance of individual CYPs is unclear. Human microsomal CYPs that contribute to the activity were evaluated by correlation with activities of hepatic drug-metabolizing CYPs, the capacity of cDNA-derived CYPs to catalyze the reaction, and inhibition of the microsomal activity by chemicals. 4-HydroxyATRA formation in microsomes varied 7-fold (8.7 to 61 pmol/mg protein/min) and correlated partially with activities mediated by CYPs 3A, 2C, and 1A (p = 0.53 to 0.66). cDNA-derived CYPs 2C8, 2C9, and 3A4, but not 1A1 or 1A2, catalyzed ATRA 4-hydroxylation (2.53, 4.68, and 1.29 pmol/pmol CYP/hr). The Km for the reaction was 9 +/- 3 microM in hepatic microsomes (N = 3) and 6 microM in microsomes containing cDNA-derived CYP2C8; by comparison, Km values for the activity mediated by CYPs 2C9 and 3A4 were 100 and 74 microM, respectively. Inhibition of microsomal ATRA 4-hydroxylation was elicited by chemicals that interact with CYP2C8 (paclitaxel and diclofenac), but not those that interact with CYP2C9 (sulfaphenazole, tolbutamide, and torasemide). The CYP3A inhibitor troleandomycin and an anti-CYP3A IgG inhibited the activity slightly. Greater inhibition was produced by the less selective CYP3A inhibitors parathion, quinidine, and ketoconazole; CYP1A inhibitors were ineffective. These findings suggest that CYP2C8 is a major contributor to ATRA 4-hydroxylation in human liver and that 3A subfamily CYPs may be minor participants. Individual variation in CYP2C8 and 3A4 expression may influence ATRA pharmacokinetics and drug interactions during therapy.     

Evidence supporting the interaction of CYP2B4 and CYP1A2 in microsomal preparations.

Recent studies have demonstrated that the catalytic behavior of one cytochrome P450 (P450) enzyme can be influenced by the presence of a second P450. This effect has been observed using reconstituted systems containing reductase, CYP2B4, and CYP1A2, primarily at subsaturating reductase. Addition of 1A2 caused a 75% inhibition of CYP2B4-dependent 7-pentoxyresorufin-O-dealkylation (PROD). Conversely, CYP2B4-dependent benzphetamine (bzp) demethylation did not exhibit this response after CYP1A2 addition. Addition of CYP2B4 to a reconstituted system containing reductase and CYP1A2 caused synergism of CYP1A2-dependent 7-ethoxyresorufin-O-dealkylation (EROD). This behavior was consistent with the formation of heteromeric CYP1A2-CYP2B4 complexes with altered catalytic properties. Although such responses have been documented in reconstituted systems, they have not been demonstrated in microsomal preparations. The goal of the present study was to determine whether such interactions were observed in rabbit liver microsomes. In an effort to detect such changes, we took advantage of the differential effect of CYP1A2 on CYP2B4-selective PROD and bzp metabolism. Rabbits were treated with phenobarbital (PB), beta-naphthoflavone (betaNF), and both PB + betaNF-conditions that enrich microsomes with CYP2B4, CYP1A2, or both enzymes, respectively. Benzphetamine demethylation activity was equivalently elevated in both the PB and the PB + betaNF groups, consistent with the induction of CYP2B4 in both groups. In contrast, PROD activity in the PB + betaNF group was less than 25% of that found in the PB-treated rabbits. These results demonstrate that the interactions observed in reconstituted systems are not an artifact of reconstitution but are observed under the more natural conditions of the microsomal membrane.     

Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver

The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.