Robert A.B. van Waterschoot

Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism

Cytochrome P450 3A (CYP3A) enzymes constitute an important detoxification system that contributes to primary metabolism of more than half of all prescribed medications. To investigate the physiological and pharmacological roles of CYP3A, we generated Cyp3a-knockout (Cyp3a-/-) mice lacking all functional Cyp3a genes. Cyp3a-/- mice were viable, fertile, and without marked physiological abnormalities. However, these mice exhibited severely impaired detoxification capacity when exposed to the chemotherapeutic agent docetaxel, displaying higher exposure levels in response to both oral and intravenous administration. These mice also demonstrated increased sensitivity to docetaxel toxicity, suggesting a primary role for Cyp3a in xenobiotic detoxification. To determine the relative importance of intestinal versus hepatic Cyp3a in first-pass metabolism, we generated transgenic Cyp3a-/- mice expressing human CYP3A4 in either the intestine or the liver. Expression of CYP3A4 in the intestine dramatically decreased absorption of docetaxel into the bloodstream, while hepatic expression aided systemic docetaxel clearance. These results suggest that CYP3A expression determines impairment of drug absorption and efficient systemic clearance in a tissue-specific manner. The genetic models used in this study provide powerful tools to further study CYP3A-mediated xenobiotic metabolism, as well as interactions between CYP3A and other detoxification systems.

Brain accumulation of dasatinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by elacridar treatment.

Purpose: Imatinib, a BCR-ABL tyrosine kinase inhibitor, is a substrate of the efflux transporters P-glycoprotein (P-gp; ABCB1) and ABCG2 (breast cancer resistance protein), and its brain accumulation is restricted by both transporters. For dasatinib, an inhibitor of SCR/BCR-ABL kinases, in vivo interactions with P-gp and ABCG2 are not fully established yet. Experimental Design: We used Abcb1a/1b−/−, Abcg2−/−, and Abcb1a/1b;Abcg2−/− mice to establish the roles of P-gp and ABCG2 in the pharmacokinetics and brain accumulation of dasatinib. Results: We found that oral uptake of dasatinib is limited by P-gp. Furthermore, relative brain accumulation, 6 hours after administration, was not affected by Abcg2 deficiency, but absence of P-gp resulted in a 3.6-fold increase after oral and 4.8-fold higher accumulation after i.p. administration. Abcb1a/1b;Abcg2−/− mice had the most pronounced increase in relative brain accumulation, which was 13.2-fold higher after oral and 22.7-fold increased after i.p. administration. Moreover, coadministration to wild-type mice of dasatinib with the dual P-gp and ABCG2 inhibitor elacridar resulted in a similar dasatinib brain accumulation as observed for Abcb1a/1b;Abcg2−/− mice. Conclusions: Brain accumulation of dasatinib is primarily restricted by P-gp, but Abcg2 can partly take over this protective function at the blood-brain barrier. Consequently, when both transporters are absent or inhibited, brain uptake of dasatinib is highly increased. These findings might be clinically relevant for patients with central nervous system Philadelphia chromosome–positive leukemia, as coadministration of an inhibitor of P-gp and ABCG2 with dasatinib might result in better therapeutic responses in these patients.

Breast Cancer Resistance Protein and P-glycoprotein Limit Sorafenib Brain Accumulation

Sorafenib is a second-generation, orally active multikinase inhibitor that is approved for the treatment of patients with advanced renal cell carcinoma and patients with unresectable hepatocellular carcinoma. We studied active transport of sorafenib in MDCK-II cells expressing human P-glycoprotein (P-gp/ABCB1) or ABCG2 (breast cancer resistance protein) or murine Abcg2. Sorafenib was moderately transported by P-gp and more efficiently by ABCG2 and Abcg2. Because sorafenib is taken orally, we orally administered sorafenib to wild-type, Abcb1a/1b−/−, Abcg2−/−, and Abcb1a/1b;Abcg2−/− mice, completely lacking functional Abcb1a/1b, Abcg2, or both, respectively, and we studied plasma pharmacokinetics and brain accumulation. The systemic exposure on oral administration was not different among all strains. However, brain accumulation was 4.3-fold increased in Abcg2−/− mice and 9.3-fold increased in Abcb1a/1b;Abcg2−/− mice. Moreover, when wild-type mice were treated with sorafenib in combination with the dual P-gp and ABCG2 inhibitor elacridar, brain accumulation was similar to that observed for Abcb1a/1b;Abcg2−/− mice. These results show that the brain accumulation of sorafenib is primarily restricted by ABCG2. This contrasts with previous studies using shared ABCG2 and P-gp substrates, which all suggested that P-gp dominates at the blood-brain barrier, and that an effect of ABCG2 is only evident when both transporters are absent. Interestingly, for sorafenib, it is the other way around, that is, ABCG2, and not P-gp, plays the dominant role in restricting its brain accumulation. Clinically, our findings may be relevant for the treatment of renal cell carcinoma patients with central nervous system relapses, as a dual ABCG2 and P-gp inhibitor might improve the central nervous system entry and thereby the therapeutic efficacy of sorafenib. Mol Cancer Ther; 9(2); 319–26

Midazolam metabolism in cytochrome P450 3A knockout mice can be attributed to up-regulated CYP2C enzymes.

The cytochrome P450 3A (CYP3A) enzymes represent one of the most important drug-metabolizing systems in humans. Recently, our group has generated cytochrome P450 3A knockout mice to study this drug-handling system in vivo. In the present study, we have characterized the Cyp3a knockout mice by studying the metabolism of midazolam, one of the most widely used probes to assess CYP3A activity. We expected that the midazolam metabolism would be severely reduced in the absence of CYP3A enzymes. We used hepatic and intestinal microsomal preparations from Cyp3a knockout and wild-type mice to assess the midazolam metabolism in vitro. In addition, in vivo metabolite formation was determined after intravenous administration of midazolam. We were surprised to find that our results demonstrated that there is still marked midazolam metabolism in hepatic (but not intestinal) microsomes from Cyp3a knockout mice. Accordingly, we found comparable amounts of midazolam as well as its major metabolites in plasma after intravenous administration in Cyp3a knockout mice compared with wild-type mice. These data suggested that other hepatic cytochrome P450 enzymes could take over the midazolam metabolism in Cyp3a knockout mice. We provide evidence that CYP2C enzymes, which were found to be up-regulated in Cyp3a knockout mice, are primarily responsible for this metabolism and that several but not all murine CYP2C enzymes are capable of metabolizing midazolam to its 1′-OH and/or 4-OH derivatives. These data illustrate interesting compensatory changes that may occur in Cyp3a knockout mice. Such flexible compensatory interplay between functionally related detoxifying systems is probably essential to their biological role in xenobiotic protection.

A Critical Analysis of the Interplay between Cytochrome P450 3A and P-Glycoprotein: Recent Insights from Knockout and Transgenic Mice

CYP3A is one of the most important drug-metabolizing enzymes, determining the first-pass metabolism, oral bioavailability, and elimination of many drugs. It is also an important determinant of variable drug exposure and is involved in many drug-drug interactions. Recent studies with CYP3A knockout and transgenic mice have yielded a number of key insights that are important to consider during drug discovery and development. For instance, studies with tissue-specific CYP3A-transgenic mice have highlighted the importance of intestinal CYP3A-dependent metabolism. They also revealed that intestinal CYP3A plays an important role in the regulation of various drug-handling systems in the liver. Intestinal CYP3A activity can thus have far-reaching pharmacological effects. Besides CYP3A, the active drug efflux transporter P-glycoprotein also has a strong effect on the pharmacokinetics of numerous drugs. CYP3A and P-glycoprotein have an extensive overlap in their substrate spectrum. It has been hypothesized that for many drugs, the combined activity of CYP3A and P-glycoprotein makes for efficient intestinal first-pass metabolism of orally administered drugs as a result of a potentially synergistic collaboration. However, there is only limited in vitro and in vivo evidence for this hypothesis. There has also been some confusion in the field about what synergy actually means in this case. Our recent studies with Cyp3a/P-glycoprotein combination knockout mice have provided further insights into the CYP3A-P-glycoprotein interplay. We here present our view of the status of the synergy hypothesis and an attempt to clarify the existing confusion about synergy. We hope that this will facilitate further critical testing of the hypothesis and improve communication among researchers. Above all, the recent findings and insights into the interplay between CYP3A and P-glycoprotein may have implications for improving oral drug bioavailability and reducing adverse side effects.

P-Glycoprotein Limits Oral Availability, Brain Penetration, and Toxicity of an Anionic Drug, the Antibiotic Salinomycin

ABSTRACT Salinomycin is a polyether organic anion that is extensively used as a coccidiostatic antibiotic in poultry and commonly fed to ruminant animals to improve feed efficiency. However, salinomycin also causes severe toxicity when accidentally fed to animals in high doses. In addition, humans are highly sensitive to salinomycin and severe toxicity has been reported. Multidrug efflux transporters like P-glycoprotein (P-gp), BCRP, and MRP2 are highly expressed in the intestine and can restrict the oral uptake and tissue penetration of xenobiotics. The purpose of this study was to investigate whether the anionic drug salinomycin is a substrate for one or more of these efflux pumps. Salinomycin was actively transported by human MDR1 P-gp expressed in polarized MDCK-II monolayers but not by the known organic anion transporters human MRP2 and murine Bcrp1. Using P-gp-deficient mice, we found a marked increase in plasma salinomycin concentrations after oral administration and decreased plasma clearance after intravenous administration. Furthermore, absence of P-gp resulted in significantly increased brain penetration. P-gp-deficient mice also displayed clearly increased susceptibility to salinomycin toxicity. Thus far, P-gp was thought to affect mainly hydrophobic, positively charged or neutral drugs in vivo. Our data show that P-gp can also be a major determinant of the pharmacokinetic behavior and toxicity of an organic anionic drug. Variation in P-gp activity might thus directly affect the effective exposure to salinomycin and possibly to other anionic drugs and toxin substrates. Individuals with reduced or absent P-gp activity could therefore be more susceptible to salinomycin toxicity.

Individual and combined roles of CYP3A, P‐glycoprotein (MDR1/ABCB1) and MRP2 (ABCC2) in the pharmacokinetics of docetaxel

Docetaxel is one of the most widely used anticancer drugs. A major problem with docetaxel treatment, however, is the considerable interpatient variability in docetaxel exposure. Another disadvantage of the drug is that it has a very low oral bioavailability and can, therefore, only be administered intravenously. The drug‐metabolizing enzyme CYP3A and the drug transporter MDR1 (P‐glycoprotein) are major determinants of docetaxel pharmacokinetics. In vitro studies have indicated that docetaxel is also a substrate for the drug transporter MRP2, but the in vivo importance of MRP2 for docetaxel is currently unknown. We, therefore, investigated the role of MRP2 in the pharmacokinetics of docetaxel by utilizing Mrp2−/− mice. We also generated and characterized Cyp3a/Mdr1a/b/Mrp2−/− combination knockout mice to get more insight into how these drug‐handling systems work together in determining docetaxel pharmacokinetics. The systemic exposure in Mrp2−/− mice was not significantly different from wild‐type, after either oral or intravenous administration. Strikingly, however, in Cyp3a/Mdr1a/b/Mrp2−/− mice, systemic docetaxel exposure was increased 166‐fold after oral administration when compared with wild‐type mice, and 2.3‐fold when compared with Cyp3a/Mdr1a/b−/− mice. Interestingly, this 166‐fold increase was disproportionate compared with that for the separate Cyp3a (12‐fold) or Mdr1a/b/Mrp2 (4‐fold) knockouts. The oral bioavailability was increased to 73% in the Cyp3a/Mdr1a/b/Mrp2−/− strain, versus only 10% in wild‐type mice. Our data thus indicate that in the absence of CYP3A and Mdr1a/b activity, Mrp2 has a marked impact on docetaxel pharmacokinetics. These findings could have important implications for improving the oral bioavailability and reducing the variability in docetaxel exposure.

Intestinal cytochrome P450 3A plays an important role in the regulation of detoxifying systems in the liver

CYP3A4 is an important xenobiotic metabolizing enzyme. We previously found that CYP2C55 is highly up‐regulated in Cyp3a‐/‐ mice. Here, we have further investigated the mechanism of regulation of CYP2C55 and other detoxifying systems in Cyp3a‐/‐mice. Induction studies with prototypical inducers dem‐onstrated an important role for the nuclear receptors PXR and CAR in the up‐regulation of CYP2C55. Sub‐sequent diet‐switch experiments revealed that food‐derived xenobiotics are primarily responsible for the increased induction of CYP2C55, as well as of several other primary detoxifying systems in Cyp3a‐/‐ mice. Our data suggest that CYP3A normally metabolizes food‐derived activators of PXR and/or CAR, explaining the increased levels of such activators in Cyp3a‐/‐mice and subsequent up‐regulation of a range of detox‐ifying systems. Interestingly, our studies with tissue‐specific CYP3A4 transgenic Cyp3a‐/‐ mice revealed that not only hepatic but also intestinal expression of CYP3A4 could reduce the hepatic expression of detox‐ifying systems to near wild‐type levels. Apparently, intestinal CYP3A4 can limit the hepatic exposure to food‐derived activators of nuclear receptors, thereby regulating the expression of a range of detoxifying systems in the liver. This broad biological effect further emphasizes the importance of intestinal CYP3Aactivity and could have profound implications for the prediction of drug exposure.—Van Waterschoot, R. A. B., Rooswinkel, R. W., Wagenaar, E., van der Kruijssen, C. M. M., van Herwaarden, A. E., Schinkel, A. H. Intestinal cytochrome P450 3A plays an important role in the regulation of detoxifying systems in the liver. FASEB J. 23, 224‐231 (2009)

PXR-mediated induction of human CYP3A4 and mouse Cyp3a11 by the glucocorticoid budesonide

Budesonide, a glucocorticoid with a high first-pass metabolism, is used for the oral treatment of inflammatory bowel disease. Cytochrome P450 3A4 (CYP3A4) is an enzyme involved in the metabolism of numerous drugs, including budesonide. Since inhibition or induction of CYP3A4 is often the cause of drug-drug interactions we analyzed how budesonide affects the activity and expression of this enzyme. CYP3A4 activity was assessed by the metabolism of a luminogenic substrate (luciferin-benzylether) using recombinant human CYP3A4 protein. We observed no inhibition of the metabolism in the presence of budesonide at concentrations up to 25 microM. Induction experiments in human LS180 colon carcinoma cells showed an increased expression of CYP3A4 mRNA after budesonide treatment. Transactivation assays revealed that budesonide activates the CYP3A4 promoter via the pregnane X receptor (PXR). In mice, oral budesonide administration (25mg/kg) for 4 days induced the murine homolog Cyp3a11 in the intestine 3-fold, whereas liver expression was notably less influenced. In knockout mice devoid of PXR, budesonide-mediated inductions were reduced compared to wild-type mice. In conclusion, we could demonstrate that budesonide is not an efficient inhibitor but rather an inducer of CYP3A via a PXR-mediated mechanism. In vivo, however, oral budesonide administration to mice showed only modest gene induction, which occurred mainly in the intestine. Therefore, the risk for budesonide-mediated drug interactions seems to be low but cannot be ruled out entirely.

P-Glycoprotein, Multidrug-Resistance Associated Protein 2, Cyp3a, and Carboxylesterase Affect the Oral Availability and Metabolism of Vinorelbine

We investigated the interactions of the anticancer drug vinorelbine with drug efflux transporters and cytochrome P450 3A drug-metabolizing enzymes. Vinorelbine was transported by human multidrug-resistance associated protein (MRP) 2, and Mrp2 knockout mice displayed increased vinorelbine plasma exposure after oral administration, suggesting that Mrp2 limits the intestinal uptake of vinorelbine. Using P-glycoprotein (P-gp), Cyp3a-, and P-gp/Cyp3a knockout mice, we found that the absence of P-gp or Cyp3a resulted in increased vinorelbine plasma exposure, both after oral and intravenous administration. Surprisingly, P-gp/Cyp3a knockout mice displayed markedly lower vinorelbine plasma concentrations than wild-type mice upon intravenous administration but higher concentrations upon oral administration. This could be explained by highly increased formation of 4′-O-deacetylvinorelbine, an active vinorelbine metabolite, especially in P-gp/Cyp3a knockout plasma. Using wild-type and Cyp3a knockout liver microsomes, we found that 4′-O-deacetylvinorelbine formation was 4-fold increased in Cyp3a knockout liver and was not mediated by Cyp3a or other cytochrome P450 enzymes. In vitro incubation of vinorelbine with plasma revealed that vinorelbine deacetylation in Cyp3a and especially in P-gp/Cyp3a knockout mice but not in P-gp-deficient mice was strongly up-regulated. Metabolite formation in microsomes and plasma could be completely inhibited with the nonspecific carboxylesterase (CES) inhibitor bis(4-nitrophenyl) phosphate and partly with the CES2-specific inhibitor loperamide, indicating that carboxylesterase Ces2a, which was appropriately up-regulated in Cyp3a and especially in P-gp/Cyp3a knockout liver was responsible for the 4′O-deacetylvinorelbine formation. Such compensatory up-regulation can complicate the interpretation of knockout mouse data. Nonetheless, P-gp, Mrp2, Cyp3a, and Ces2a clearly restricted vinorelbine availability in mice. Variation in activity of their human homologs may also affect vinorelbine pharmacokinetics in patients.