Modeling human cytochrome P450 2D6 metabolism and drug-drug interaction by a novel panel of knockout and humanized mouse lines

The highly polymorphic human cytochrome P450 2D6 enzyme is involved in the metabolism of up to 25% of all marketed drugs and accounts for significant individual differences in response to CYP2D6 substrates. Because of the differences in the multiplicity and substrate specificity of CYP2D family members among species, it is difficult to predict pathways of human CYP2D6-dependent drug metabolism on the basis of animal studies. To create animal models that reflect the human situation more closely and that allow an in vivo assessment of the consequences of differential CYP2D6 drug metabolism, we have developed a novel straightforward approach to delete the entire murine Cyp2d gene cluster and replace it with allelic variants of human CYP2D6. By using this approach, we have generated mouse lines expressing the two frequent human protein isoforms CYP2D6.1 and CYP2D6.2 and an as yet undescribed variant of this enzyme, as well as a Cyp2d cluster knockout mouse. We demonstrate that the various transgenic mouse lines cover a wide spectrum of different human CYP2D6 metabolizer phenotypes. The novel humanization strategy described here provides a robust approach for the expression of different CYP2D6 allelic variants in transgenic mice and thus can help to evaluate potential CYP2D6-dependent interindividual differences in drug response in the context of personalized medicine.     

Induction of human cytochrome P450 3A4 by the irreversible myeloperoxidase inactivator PF-06282999 is mediated by the pregnane X receptor

1. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl) acetamide (PF-06282999) is a member of the thiouracil class of irreversible inactivators of human myeloperoxidase enzyme and a candidate for the treatment of cardiovascular disease. PF-06282999 is an inducer of CYP3A4 mRNA and midazolam-1′-hydroxylase activity in human hepatocytes, which is consistent with PF-06282999-dose dependent decreases in mean maximal plasma concentrations (C_max) and area under the plasma concentration time curve (AUC) of midazolam in humans following 14-day treatment with PF-06282999.

2. In the present study, the biochemical mechanism(s) of CYP3A4 induction by PF-06282999 was studied. Incubations in reporter cells indicated that PF-06282999 selectively activated human pregnane X receptor (PXR). Treatment of human HepaRG cells with PF-06282999 led to ～14-fold induction in CYP3A4 mRNA and 5-fold increase in midazolam-1′-hydroxylase activity, which was nullified in PXR-knock out HepaRG cells. TaqMan® gene expression analysis of human hepatocytes treated with PF-06282999 and the prototypical PXR agonist rifampin demonstrated increases in mRNA for CYP3A4 and related CYPs that are regulated by PXR.

3. Docking studies using a published human PXR crystal structure provided insights into the molecular basis for PXR activation by PF-06282999. Implementation of PXR transactivation assays in a follow-on discovery campaign should aid in the identification of back-up compounds devoid of PXR activation and CYP3A4 induction liability.     

Induction of cytochrome P450 3A4 in primary human hepatocytes and activation of the human pregnane X receptor by tamoxifen and 4-hydroxytamoxifen.

Tamoxifen is a widely utilized antiestrogen in the treatment and chemoprevention of breast cancer. Clinical studies document that tamoxifen administration markedly enhances the systemic elimination of other drugs. Additionally, tamoxifen enhances its own clearance following repeated dosing. The mechanisms that underlie these clinically important events remain unresolved. Here, we report that tamoxifen and its metabolite 4-hydroxytamoxifen markedly induce cytochrome P450 3A4, a drug-metabolizing enzyme of central importance, in primary cultures of human hepatocytes. Tamoxifen and 4-hydroxytamoxifen (1-10 microM) significantly increased the CYP3A4 expression and activity (measured as the rate of testosterone 6beta-hydroxylation). Maximal induction was achieved at the 5 microM level. At this level, tamoxifen and 4-hydroxytamoxifen caused a 1.5- to 3.3-fold (mean, 2.1-fold) and 3.4- to 17-fold (mean, 7.5-fold) increase in the CYP3A4 activity, respectively. In comparison, rifampicin treatment resulted in a 6- to 16-fold (mean, 10.5-fold) increase. We also observed corresponding increase in the CYP3A4 immunoreactive protein and mRNA levels. Furthermore, tamoxifen and 4-hydroxytamoxifen efficaciously activated the human pregnane X receptor (hPXR; also known as the steroid xenobiotic receptor), a key regulator of CYP3A4 expression. The efficacy of tamoxifen and 4-hydroxytamoxifen relative to rifampicin for hPXR activation was approximately 30 and 60%, respectively. Our results indicate that the mechanism of tamoxifen-mediated alteration in drug clearance pathways in humans may involve CYP3A4 induction by the parent drug and/or its metabolite. Furthermore, the CYP3A4 induction may be a result of hPXR activation. These findings have important implications for optimizing the use of tamoxifen and in the development of newer antiestrogens.     

Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies.     

The PREgnane X receptor gene-humanized mouse: a model for investigating drug-drug interactions mediated by cytochromes P450 3A.

The most common clinical implication for the activation of the human pregnane X receptor (PXR) is the occurrence of drug-drug interactions mediated by up-regulated cytochromes P450 3A (CYP3A) isozymes. Typical rodent models do not predict drug-drug interactions mediated by human PXR because of species differences in response to PXR ligands. In the current study, a PXR-humanized mouse model was generated by bacterial artificial chromosome (BAC) transgenesis in Pxr-null mice using a BAC clone containing the complete human PXR gene and 5'- and 3'-flanking sequences. In this PXR-humanized mouse model, PXR is selectively expressed in the liver and intestine, the same tissue expression pattern as CYP3A. Treatment of PXR-humanized mice with the PXR ligands mimicked the human response, since both hepatic and intestinal CYP3As were strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16alpha-carbonitrile, a rodent-specific PXR ligand. In rifampicin-pretreated PXR-humanized mice, an approximately 60% decrease was observed for both the maximal midazolam serum concentration (C(max)) and the area under the concentration-time curve, as a result of a 3-fold increase in midazolam 1'-hydroxylation. These results illustrate the potential utility of the PXR-humanized mice in the investigation of drug-drug interactions mediated by CYP3A and suggest that the PXR-humanized mouse model would be an appropriate in vivo tool for evaluation of the overall pharmacokinetic consequences of human PXR activation by drugs.     

Regulation of human pregnane X receptor and its target gene cytochrome P450 3A4 by Chinese herbal compounds and a molecular docking study

1. The pregnane X receptor (PXR) plays a critical role in the regulation of human cytochrome P450 3A4 (CYP3A4) gene. In this study, we investigated the effect of an array of compounds isolated from Chinese herbal medicines on the activity of PXR using a luciferase reporter gene assay in transiently transfected HepG2 and Huh7 cells and on the expression of PXR and CYP3A4 in LS174T cells. Furthermore, molecular docking was performed to investigate the binding modes of herbal compounds with PXR.

2. Praeruptorin A and C, salvianolic acid B, sodium danshensu, protocatechuic aldehyde, cryptotanshinone, emodin, morin, and tanshinone IIA significantly transactivated the CYP3A4 reporter gene construct in either HepG2 or Huh7 cells. The PXR mRNA expression in LS174T cells was significantly induced by physcion, protocatechuic aldehyde, salvianolic acid B, and sodium danshensu. However, epifriedelanol, morin, praeruptorin D, mulberroside A, tanshinone I, and tanshinone IIA significantly down-regulated the expression of PXR mRNA in LS174T cells.

3. All the herbal compounds tested can be readily docked into the ligand-binding cavity of PXR mainly through hydrogen bond and aromatic interactions with Ser247, Gln285, His407, and Arg401.

4. These findings suggest that herbal medicines can significantly regulate PXR and CYP3A4 and this has important implication in herb–drug interactions.

     

Regulation of human pregnane X receptor and its target gene cytochrome P450 3A4 by Chinese herbal compounds and a molecular docking study

The pregnane X receptor (PXR) plays a critical role in the regulation of human cytochrome P450 3A4 (CYP3A4) gene. In this study, we investigated the effect of an array of compounds isolated from Chinese herbal medicines on the activity of PXR using a luciferase reporter gene assay in transiently transfected HepG2 and Huh7 cells and on the expression of PXR and CYP3A4 in LS174T cells. Furthermore, molecular docking was performed to investigate the binding modes of herbal compounds with PXR. Praeruptorin A and C, salvianolic acid B, sodium danshensu, protocatechuic aldehyde, cryptotanshinone, emodin, morin, and tanshinone IIA significantly transactivated the CYP3A4 reporter gene construct in either HepG2 or Huh7 cells. The PXR mRNA expression in LS174T cells was significantly induced by physcion, protocatechuic aldehyde, salvianolic acid B, and sodium danshensu. However, epifriedelanol, morin, praeruptorin D, mulberroside A, tanshinone I, and tanshinone IIA significantly down-regulated the expression of PXR mRNA in LS174T cells. All the herbal compounds tested can be readily docked into the ligand-binding cavity of PXR mainly through hydrogen bond and aromatic interactions with Ser247, Gln285, His407, and Arg401. These findings suggest that herbal medicines can significantly regulate PXR and CYP3A4 and this has important implication in herb-drug interactions.     

新型抗抑郁药的代谢与细胞色素P450介导的药物相互作用

新型抗抑郁药(选择性5-羟色胺再摄取抑制剂,SSRI)疗效肯定,耐受性好,已得到广泛应用。SSRI剂既是P 450酶的底物又是P 450酶的抑制剂,其重要特征是对P 450酶的抑制。当SSRI剂与P 450酶的其他底物合用时,可能发生明显的具有临床意义的药物相互作用。     

Heterotropic cooperativity of cytochrome P450 3A4 and potential drug-drug interactions

Cytochromes P450 (CYP) 3A4 is the most abundant human hepatic CYP isoform catalyzing the metabolism of approximately 50% of therapeutic agents. In addition to inhibition or induction, CYP3A4 is subject to stimulation, termed homotropic (substrate stimulation) and heterotropic (stimulation by effectors) cooperativity. The heterotropic cooperativity of CYP3A4 may result from an increase in Vmax, a decrease in Km or a combination of the two and sometimes exhibits regio-selectivity when the enzyme is involved in two or more metabolic pathways for a single substrate. An effector of CYP3A4 can also be a substrate; its metabolism may or may not be inhibited by another substrate. These characteristics of heterotropic cooperativity of CYP3A4 have been interpreted in the context of two binding domains in the active site of the enzyme, two substrate binding plus a distinct allosteric binding site, multiple enzyme conformations or multiple binding sites accompanied by conformational changes. Examples of in vivo CYP cooperativity are rare; representative cases include flavone-dependent stimulation of zoxazolamine metabolism in rats and enhancement of CYP3A-mediated hepatic clearance of diclofenac by quinidine in monkeys. Effector-induced increases in CYP3A4 activity were observed during the 1'-hydroxylation of midazolam and 4'- and 10-hydroxylation of warfarin in human hepatocyte systems. These data imply that CYP cooperativity has the potential to cause in vivo drug-drug interactions. Because cooperative and inhibitory responses from CYP3A4 are known to be substrate-dependent, projection of the pharmacokinetics of an investigational drug and CYP-associated risks of drug-drug interactions in humans can be very complex. Further investigation of CYP cooperativity is warranted.     

Quantitative prediction of macrolide drug-drug interaction potential from in vitro studies using testosterone as the human cytochrome P4503A substrate

Objective Macrolide antibiotics are mechanism-based inactivators of CYP3A enzymes that exhibit varying degrees of inhibitory potency. Our aim was to predict quantitatively the drug-drug interaction (DDI) potential of five macrolides from in vitro studies using testosterone as the CYP3A substrate, and to compare the predictions generated from human liver microsomal and recombinant CYP3A4 data. Methods The in vitro kinetic constants of CYP3A inactivation (K _I and k _inact) were estimated by varying the time of pre-incubation and the concentration of troleandomycin, erythromycin, clarithromycin, roxithromycin or azithromycin. CYP3A activity was determined from the measurement of testosterone 6β-hydroxylation with human liver microsomes (HLM) and recombinant CYP3A4 as the enzyme sources. The mechanism-based pharmacokinetic model was fitted with inactivation data to predict the increase in oral area under the plasma concentration-time curve (AUC) for midazolam. Results All five macrolides inactivated testosterone 6β-hydroxylation by HLM and recombinant CYP3A4 with k _inact values in the range of 0.023 to 0.058 min⁻¹. The potency of inactivation (K _I) was higher using recombinant CYP3A4 as the enzyme source. The oral AUCs for midazolam were predicted from HLM data to increase 16.6, 5.3, 4.6, 1.6 and 1.2-fold due to the inhibition of metabolic clearance by troleandomycin, erythromycin, clarithromycin, roxithromycin and azithromycin, respectively. These results are within the range of the AUC ratios reported for clinical DDI studies. The predicted AUC increases generated using recombinant CYP3A4 overestimated the magnitude of the DDIs. Conclusions The DDI potential of five macrolide antibiotics was quantitatively predicted from in vitro studies using testosterone as the CYP3A substrate with HLM as the enzyme source.     

Human pregnane X receptor: genetic polymorphisms, alternative mRNA splice variants, and cytochrome P450 3A metabolic activity

Human pregnane X receptor (hPXR) gene polymorphisms (spanning exon 2 to exon 5) and alternative mRNA splicing were investigated as possible contributors to individual variability in CYP3A metabolic activity measured both in vivo and in vitro. None of the 9 variants evaluated, including the 2 most common nonsynonymous variants (Pro27Ser and Gly36Arg), was found to be associated with midazolam 1'-hydroxylation rate measured in a bank of human livers (48 European Americans, 4 African Americans, 2 Hispanics). In contrast, 3 linked hPXR variants (g.252A > G, g.275A > G, and g.4760G > A) were significantly (P < .05) associated with oral midazolam clearance in a mixed race/ethnicity population (n = 26) and the African American subpopulation (n = 14) but not in European Americans (n = 9). Although the amount of hPXR mRNA normally spliced at the exon 4-5 junction correlated well with midazolam 1'-hydroxylation activities (P < .05), none of the 6 hPXR mRNA splice variants identified was associated with midazolam 1'-hydroxylation. In conclusion, several hPXR polymorphisms have been identified that may have predictive value for oral midazolam clearance, particularly in African Americans.     

Induction of cytochrome P450 3A by paclitaxel in mice: pivotal role of the nuclear xenobiotic receptor, pregnane X receptor.

Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.     

Pregnane X receptor is required for interleukin-6-mediated down-regulation of cytochrome P450 3A4 in human hepatocytes

Cytochrome P450 3A4 (CYP3A4) is the most abundant cytochrome P450 enzyme in human liver and metabolizes more than 60% of prescribed drugs in human body. Patients with liver conditions such as cirrhosis show increased secretion of cytokines (e.g., interleukin-6) and decreased capacity of oxidation of many drugs. In this study, we provided molecular evidence that cytokine secretion directly contributed to the decreased capacity of oxidative biotransformation in human liver. After human hepatocytes were treated with IL-6, the expression of CYP3A4 decreased at both mRNA and protein levels, so did the CYP3A4 enzymatic activity. Meanwhile, the repression of CYP3A4 by IL-6 occurred after the decrease of pregnane X receptor (PXR) in human hepatocytes. The PXR-overexpressed cells (transfected with human PXR) increased the CYP3A4 mRNA level, and the repression of CYP3A4 by IL-6 was greater in the PXR-overexpressed cells than in the control cells. Further, PXR knockdown (transfected with siPXR construct) decreased the CYP3A4 mRNA level with less repression by IL-6 than in the control cells transfected with corresponding vector. Collectively, our study suggests that PXR is necessary for IL-6-mediated repression of the CYP3A4 expression in human hepatocytes.     

Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent.

Testosterone, terfenadine, midazolam, and nifedipine, four commonly used substrates for human cytochrome P-450 3A4 (CYP3A4), were studied in pairs in human liver microsomes and in microsomes from cells containing recombinant human CYP3A4 and P-450 reductase, to investigate in vitro substrate-substrate interaction with CYP3A4. The interaction patterns between compounds with CYP3A4 were found to be substrate-dependent. Mutual inhibition, partial inhibition, and activation were observed in the testosterone-terfenadine, testosterone-midazolam, or terfenadine-midazolam interactions. However, the most unusual result was the interaction between testosterone and nifedipine. Although nifedipine inhibited testosterone 6beta-hydroxylation in a concentration-dependent manner, testosterone did not inhibit nifedipine oxidation. Furthermore, the effect of testosterone and 7,8-benzoflavone on midazolam 1'-hydroxylation and 4-hydroxylation demonstrated different regiospecificities. These results may be explained by a model in which multiple substrates or ligands can bind concurrently to the active site of a single CYP3A4 molecule. However, the contribution of separate allosteric sites and conformational heterogeneity to the atypical kinetics of CYP3A4 can not be ruled out in this model.     

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.     

Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes

Freshly prepared human hepatocytes are considered as the 'gold standard' for in vitro testing of drug candidates. However, several disadvantages are associated with the use of this model system. The availability of hepatocytes is often low and consequently the planning of the experiments rendered difficult. In addition, the quality of the available cells is in some cases poor. As an alternative, cryopreserved human hepatocytes were validated as a model to study cytochrome P450 1A2 (CYP1A2) and cytochrome P450 3A4 (CYP3A4) induction. In a single blinded experiment, hepatocytes from three separate lots were incubated with three concentrations of different compounds, and compared to non-treated cells and cells incubated with omeprazole or rifampicin. CYP1A2 and CYP3A4 induction was determined by measuring 7-ethoxyresorufin-O-deethylation activity and 6beta-hydroxytestosterone formation, respectively. CYP1A2 and CYP3A4 mRNA and protein expression were analyzed by TaqMan QRT-PCR and immunodetection. Cells responded well to the prototypical inducers with a mean 38.8- and 6.2-fold induction of CYP1A2 and CYP3A4 activity, respectively. Similar as with fresh human hepatocytes, high batch-to-batch variation of CYP1A2 and CYP3A4 induction was observed. Except for 1 and 10 microM rosiglitazone, the glitazones did not significantly affect CYP1A2. A similar result was observed for CYP3A4 activity although CYP3A4 mRNA and protein expression were dose-dependently upregulated. In conclusion, cryopreserved human hepatocytes may be a good alternative to fresh hepatocytes to study CYP1A and 3A induction.