Cornelia M.M. van der Kruijssen

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.

Organic anion transporting polypeptide 1a/1b–knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs

Organic anion transporting polypeptides (OATPs) are uptake transporters for a broad range of endogenous compounds and xenobiotics. To investigate the physiologic and pharmacologic roles of OATPs of the 1A and 1B subfamilies, we generated mice lacking all established and predicted mouse Oatp1a/1b transporters (referred to as Slco1a/1b-/- mice, as SLCO genes encode OATPs). Slco1a/1b-/- mice were viable and fertile but exhibited markedly increased plasma levels of bilirubin conjugated to glucuronide and increased plasma levels of unconjugated bile acids. The unexpected conjugated hyperbilirubinemia indicates that Oatp1a/1b transporters normally mediate extensive hepatic reuptake of glucuronidated bilirubin. We therefore hypothesized that substantial sinusoidal secretion and subsequent Oatp1a/1b-mediated reuptake of glucuronidated compounds can occur in hepatocytes under physiologic conditions. This alters our perspective on normal liver functioning. Slco1a/1b-/- mice also showed drastically decreased hepatic uptake and consequently increased systemic exposure following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine. Importantly, intestinal absorption of oral methotrexate or fexofenadine was not affected in Slco1a/1b-/- mice. Further analysis showed that rifampicin was an effective and specific Oatp1a/1b inhibitor in controlling methotrexate pharmacokinetics. These data indicate that Oatp1a/1b transporters play an essential role in hepatic reuptake of conjugated bilirubin and uptake of unconjugated bile acids and drugs. Slco1a/1b-/- mice will provide excellent tools to study further the role of Oatp1a/1b transporters in physiology and drug disposition.

Methotrexate Pharmacokinetics in Transgenic Mice with Liver-Specific Expression of Human Organic Anion-Transporting Polypeptide 1B1 (SLCO1B1)

Human organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter that can transport a wide variety of drugs. In the present study, we have generated and characterized a transgenic mouse model with specific and functional expression of human OATP1B1 (SLCO1B1) in the liver. Immunohistochemical staining revealed basolateral localization of transgenic OATP1B1 in the liver, whereas no expression of OATP1B1 was found in the kidney and small intestine. Using this transgenic model, the in vivo role of human OATP1B1 in the disposition of the anticancer drug methotrexate (MTX) was studied. In mice on a semisynthetic diet, the area under the plasma concentration-time curve for intravenous methotrexate in SLCO1B1 transgenic mice was 1.5-fold decreased compared with wild-type mice. Furthermore, the amount of MTX in the liver was markedly higher (∼2-fold) in the SLCO1B1 transgenic mice compared with wild-type mice, resulting in 2- to 4-fold higher liver-plasma ratios of MTX. Some murine liver Slco genes were markedly down-regulated on the semisynthetic diet compared with a standard diet, which probably reduced murine Oatp-mediated MTX uptake in the liver and therefore facilitated detection of the function of the transgenic OATP1B1. Taken together, these data demonstrate a marked and possibly rate-limiting role for human OATP1B1 in MTX elimination in vivo. Variation in OATP1B1 activity due to genetic polymorphisms, drug-drug interactions, and possibly dietary conditions may therefore play a role in the severity of MTX-related toxicity. SLCO1B1 transgenic mice could be a useful tool in studying the in vivo role of human OATP1B1 in drug pharmacokinetics.

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.

Functionally Overlapping Roles of Abcg2 (Bcrp1) and Abcc2 (Mrp2) in the Elimination of Methotrexate and Its Main Toxic Metabolite 7-Hydroxymethotrexate In vivo

Purpose: ABCC2 (MRP2) and ABCG2 (BCRP) transport various endogenous and exogenous compounds, including many anticancer drugs, into bile, feces, and urine. We investigated the possibly overlapping roles of Abcg2 and Abcc2 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX). Experimental Design: We generated and characterized Abcc2;Abcg2-/- mice, and used these to determine the overlapping roles of Abcc2 and Abcg2 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX. Results: Compared with wild-type, the plasma areas under the curve (AUC) for MTX were 1.6-fold and 2.0-fold higher in Abcg2-/- and Abcc2-/- mice, respectively, and 3.3-fold increased in Abcc2;Abcg2-/- mice. The biliary excretion of MTX was 23-fold reduced in Abcc2;Abcg2-/- mice, and the MTX levels in the small intestine were dramatically decreased. Plasma levels of 7OH-MTX were not significantly altered in Abcg2-/- mice, but the areas under the curve were 6.2-fold and even 12.4-fold increased in Abcc2-/- and Abcc2;Abcg2-/- mice, respectively. This indicates that Abcc2 compensates for Abcg2 deficiency but that Abcg2 can only partly compensate for Abcc2 absence. Furthermore, 21-fold decreased biliary 7OH-MTX excretion in Abcc2;Abcg2-/- mice and substantial 7OH-MTX accumulation in the liver and kidney were seen. We additionally found that in the absence of Abcc2, Abcg2 mediated substantial urinary excretion of MTX and 7OH-MTX. Conclusions: Abcc2 and Abcg2 together are major determinants of MTX and 7OH-MTX pharmacokinetics. Variations in ABCC2 and/or ABCG2 activity due to polymorphisms or coadministered inhibitors may therefore substantially affect the therapeutic efficacy and toxicity in patients treated with MTX.

Transport of Diclofenac by Breast Cancer Resistance Protein (ABCG2) and Stimulation of Multidrug Resistance Protein 2 (ABCC2)-Mediated Drug Transport by Diclofenac and Benzbromarone

Diclofenac is an important analgesic and anti-inflammatory drug, widely used for treatment of postoperative pain, rheumatoid arthritis, and chronic pain associated with cancer. Consequently, diclofenac is often used in combination regimens and undesirable drug-drug interactions may occur. Because many drug-drug interactions may occur at the level of drug transporting proteins, we studied interactions of diclofenac with apical ATP-binding cassette (ABC) multidrug efflux transporters. Using Madin-Darby canine kidney (MDCK)-II cells transfected with human P-glycoprotein (P-gp; MDR1/ABCB1), multidrug resistance protein 2 (MRP2/ABCC2), and breast cancer resistance protein (BCRP/ABCG2) and murine Bcrp1, we found that diclofenac was efficiently transported by murine Bcrp1 and moderately by human BCRP but not by P-gp or MRP2. Furthermore, in Sf9-BCRP membrane vesicles diclofenac inhibited transport of methotrexate in a concentration-dependent manner. We next used MDCK-II-MRP2 cells to study interactions of diclofenac with MRP2-mediated drug transport. Diclofenac stimulated paclitaxel, docetaxel, and saquinavir transport at only 50 μM. We further found that the uricosuric drug benzbromarone stimulated MRP2 at an even lower concentration, having maximal stimulatory activity at only 2 μM. Diclofenac and benzbromarone stimulated MRP2-mediated transport of amphipathic lipophilic drugs at 10- and 250-fold lower concentrations, respectively, than reported for other MRP2 stimulators. Because these concentrations are readily achieved in patients, adverse drug-drug interactions may occur, for example, during cancer therapy, in which drug concentrations are often critical and stimulation of elimination via MRP2 may result in suboptimal chemotherapeutic drug concentrations. Moreover, stimulation of MRP2 activity in tumors may lead to increased efflux of chemotherapeutic drugs and thereby drug resistance.

High Impact of Oatp1a/1b Transporters on In Vivo Disposition of the Hydrophobic Anticancer Drug Paclitaxel

Purpose: Organic anion-transporting polypeptides (OATP) mediate the cellular uptake of a broad range of drugs. The hydrophobic anticancer drug, paclitaxel (PTX), was recently identified as a substrate for OATP1B3 in vitro. We investigated the role of Oatp1a/1b transporters in the pharmacokinetics of PTX in vivo, as well as their impact at different dose levels of PTX and methotrexate (MTX). Experimental Design: Recently generated Slco1a/1b−/− (lacking all Oatp1a/1b transporters) and wild-type mice were intravenously dosed with 2, 10, or 50 mg/kg of PTX, or with 10, 50, or 500 mg/kg of MTX, and plasma and tissue drug concentrations were measured. Results: In spite of its hydrophobicity, PTX systemic exposure (at 10 mg/kg) was increased by greater than 2-fold in Slco1a/1b−/− mice compared with wild-type, whereas PTX liver uptake was reduced by about 2-fold. Oatp1a/1b transporters displayed a high impact on PTX and MTX pharmacokinetics over a broad dose range. For MTX, even at 500 mg/kg, saturation of Oatp1a/1b was not observed, with a 3.4-fold increase in plasma and 30-fold decrease in liver levels in Slco1a/1b−/− mice compared with wild-type. Although beginning saturation of Oatp1a/1b was observed at the highest dose of PTX, plasma levels in Slco1a/1b−/− mice were still 1.7-fold increased and liver levels 1.5-fold decreased compared with wild-type. Conclusion: Oatp1a/1b transporters play a pronounced role in determining plasma levels and tissue distribution of MTX and PTX, thus affecting even highly hydrophobic drugs. Variation in OATP1A/1B transporter activity, due to genetic variation, inhibition, and/or tumor expression might affect toxicity and therapeutic efficacy of these anticancer drugs. Clin Cancer Res; 17(2); 294–301. ©2010 AACR. Clin Cancer Res; 17(2); 294–301. ©2010 AACR.

Organic anion-transporting polypeptide 1B1 mediates transport of Gimatecan and BNP1350 and can be inhibited by several classic ATP-binding cassette (ABC) B1 and/or ABCG2 inhibitors.

Organic anion-transporting polypeptides (OATPs) are important uptake transporters that can have a profound impact on the systemic pharmacokinetics, tissue distribution, and elimination of several drugs. Previous in vivo studies of the pharmacokinetics of the lipophilic camptothecin (CPT) analog gimatecan suggested that the ATP-binding cassette (ABC) B1 (P-glycoprotein) and/or ABCG2 (breast cancer resistance protein) inhibitors elacridar and pantoprazole could inhibit transporters other than ABCB1 and ABCG2. In this study, we tested the possible role of OATP1B1 in this interaction by screening a number of CPT analogs for their transport affinity by human OATP1B1 in vitro. In addition, the impact of several widely used ABCB1 and/or ABCG2 modulators on this OATP1B1-mediated transport was assessed. We identified two novel CPT anticancer drugs, gimatecan and BNP1350, as OATP1B1 substrates, whereas irinotecan, topotecan, and lurtotecan were not transported by OATP1B1. It is interesting to note that transport of 17β-estradiol 17β-d-glucuronide (control), gimatecan, and BNP1350 by OATP1B1 could be completely inhibited by the classic ABCB1 and/or ABCG2 inhibitors elacridar, valspodar, pantoprazole, and, to a lesser extent, zosuquidar and verapamil. Therefore, the effect of these ABCB1 and ABCG2 modulators on the plasma pharmacokinetics of gimatecan and BNP1350 (and possibly also other OATP1B1 substrates) may be partly because of inhibition of OATP1B1 besides inhibition of ABCB1 and/or ABCG2. The findings of this study suggest that OATP1B1 polymorphisms or coadministration with one of the ABCB1/ABCG2 inhibitors could affect drug uptake, tissue distribution, and elimination of some CPT anticancer drugs, thereby modifying their efficacy and/or safety profile.

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)

ABCC2, ABCC3, and ABCB1, but not CYP3A, Protect against Trabectedin-Mediated Hepatotoxicity

Purpose: Trabectedin (Yondelis, ET-743) is a novel anticancer drug with potent activity against various tumors. However, dose-limiting hepatotoxicity was observed during clinical trials. Because recent reports have suggested that cytochrome P450 3A (CYP3A), as well as the drug transporters ABCB1, ABCC2, and ABCC3 might protect against trabectedin-mediated hepatotoxicity, we investigated the individual and combined roles of these detoxifying systems. Experimental Design: Madin-Darby canine kidney cells expressing ABCC2 and ABCC3 were used to study in vitro trabectedin transport. We investigated the hepatotoxicity of trabectedin, and the plasma and liver levels of this drug and its metabolites in mice deficient for CYP3A, Abcb1a/1b, Abcc2, and/or Abcc3 after i.v. trabectedin administration. Results: Trabectedin was transported by ABCC2 but only modestly by ABCC3. Contrary to our expectation, absence of CYP3A resulted in only a marginal increase in hepatotoxicity. Some hepatotoxicity was observed in Abcc2−/− mice, but very little in Abcb1a/1b−/− and Abcc3−/− mice. Strikingly, severe hepatotoxicity was found in Abcb1a/1b/Abcc2−/− and Abcc2/Abcc3−/− mice. However, hepatotoxicity was drastically decreased in Cyp3a/Abcb1a/1b/Abcc2−/− compared with Abcb1a/1b/Abcc2−/− mice. This suggests that the formation of CYP3A-specific metabolites is an important prerequisite for trabectedin-mediated hepatotoxicity. Further studies revealed that there is increased accumulation of metabolites of trabectedin, but not of trabectedin itself, in the livers of mice that lack Abcc2 but are CYP3A proficient. Conclusions: Our data show that ABCB1, ABCC2, and ABCC3 have a profound and partially redundant function in protection from trabectedin-mediated hepatotoxicity, presumably by clearing the liver from hepatotoxic trabectedin metabolites that are primarily formed by CYP3A. (Clin Cancer Res 2009;15(24):7616–23)