Metabolism and transporter-mediated drug–drug interactions of the endothelin-A receptor antagonist CI-1034

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Abstract

CI-1034, an endothelin-A receptor antagonist was being developed for pulmonary hypertension. Drug–drug interaction studies using human hepatic microsomes were conducted to assess CYP1A2, CYP2C9, CYP2C19, CYP3A4 and CYP2D6 inhibition potential; CYP3A4 induction potential was evaluated using primary human hepatocytes. CI-1034 moderately inhibited CYP2C9 (IC50 39.6 μM) and CYP3A4 activity (IC50 21.6 μM); CYP3A4 inhibition was metabolism-dependent. In human hepatocytes, no increase in CYP3A4 activity was observed in vitro, while mRNA was induced 15-fold, similar to rifampin, indicating that CI-1034 is both an inhibitor and inducer of CYP3A4. A 2-week clinical study was conducted to assess pharmacokinetics, pharmacodynamics and safety. No significant changes were observed in Cmax0AUC between days 1 and 14. However, reversible elevations of serum liver enzymes were observed with a 50 mg BID dose and the program was terminated. To further understand the interactions of CI-1034 in the liver and possible mechanisms of the observed hepatotoxicity, we evaluated the effect of CI-1034 on bile acid transport and previously reported that CI-1034 inhibited biliary efflux of taurocholate by 60%, in vitro. This indicated that inhibition of major hepatic transporters could be involved in the observed hepatotoxicity. We next evaluated the in vitro inhibition potential of CI-1034 with the major hepatic transporters OATP1B1, OATP1B3, OATP2B1, MDR1, MRP2 and OCT. CI-1034 inhibited OATP1B1 (Ki 2 μM), OATP1B3 (Ki 1.8 μM) and OATP2B1 activity (Ki 3.3 μM) but not OCT, MDR1 or MRP2 mediated transport. Our data indicates that CI-1034 is an inhibitor of major hepatic transporters and inhibition of bile efflux may have contributed to the observed clinical hepatotoxicity. We recommend that in vitro drug–drug interaction panels include inhibition and induction studies with transporters and drug metabolizing enzymes, to more completely assess potential in vivo interactions or toxicity.

Introduction

Adverse clinical drug interactions have been implicated in up to 10.6% of emergency room visits in the elderly [1], 6.2% of all hospital admissions and nearly 10,000 deaths in the United States each year [2]. Drug interactions may result in clinical toxicity when there is an inhibition of activity of one or more relevant drug metabolizing enzyme(s) and/or transporters leading to decreased metabolism or drug efflux, thereby elevating drug levels. Conversely, if drug levels decrease over time as in the case of induction or auto-induction of drug metabolizing enzymes or transporters, the net result could be a reduction in, or even loss of efficacy [3], [4], [5].

Interactions are typically assessed early in drug development by using in vitro models to evaluate the effect of a new chemical entity on drug metabolizing enzymes, particularly the cytochrome P450 enzymes (P450) and on the multi-drug resistant efflux transporter, MDR1. Predicting drug interactions is complicated, particularly when chronic administration of potent P450 inhibitors results in up-regulation of inhibited enzymes. In the case of CYP3A4, this up-regulation has been documented for the protease inhibitors [6], macrolide antibiotics [7] and imidazole antimycotic drugs [8]. Since inhibitors can also be inducers, both inhibition and induction studies need to be conducted to obtain meaningful predictions. Another confounding factor in predicting drug interactions is that often both drug metabolizing enzymes and transporters are involved, and it is difficult to dissect out the contribution of each, in vivo. For example, CYP3A4 and MDR1 are present in high levels in the intestine and liver, are typically induced by the same compounds, have similar substrate and inhibitor specificities and may act synergistically [9]. Historically, predictions of clinical drug interactions based solely on in vitro P450 inhibition, have been less than successful, resulting in unexpected or larger than expected clinical drug interactions. For example, the hypertension drug mibefradil was predicted to be safe based on preclinical studies [10], but severe clinical drug interactions with CYP3A4, CYP2D6 and MDR1 substrates resulted in withdrawal from the market. Retrospective in vitro studies established mibefradil to be a MDR1 substrate as well as a potent inhibitor of both MDR1 and CYP3A4 [11]. Inhibition (or induction) of other transport proteins has also been shown to have clinical relevance, e.g. grapefruit juice inhibits CYP3A4, MDR1 and OATP activity in vitro and decreases the oral absorption of fexofenadine, a MDR1 and OATP substrate, in vivo [12]. Chronic treatments of glyburide, bosentan and some HIV protease inhibitors have resulted in clinical hepatotoxicity and these are all potent inhibitors of the bile salt transporter BSEP [13].

CI-1034, an endothelin-A receptor antagonist was being developed for the treatment of pulmonary hypertension (Fig. 1). Our preliminary in vitro studies focused on evaluating the reversible and metabolism-dependent inhibition potential of CI-1034 with the major drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 using human hepatic microsomes. We also assessed the induction potential of CI-1034 using microarray technology and activity assays for CYP3A4. Phase 2 clinical trials were conducted with four patients: three CI-1034-treated and one placebo-treated. There was a moderate elevation of serum hepatic enzymes in two of the three patients administered active drug. This contributed to the discontinuation of development of this compound. Subsequently, preclinical studies were conducted to more fully understand the drug interaction spectrum of this compound utilizing transporter-based assays. These studies included the effect of CI-1034 on the major hepatic transporters OATP1B1, OATP1B3, OATP2B1, MDR1, MRP2, OAT1 and OCT.

Section snippets

Materials and methods

CI-1034 (4-(7-ethyl-benzo[1,3]dioxol-5-yl)-1,1-dioxo-2-(2-trifluoromethyl-phenyl)-1,2-dihydro-11,6-benzo[e][1,2]thiazine-3-carboxylic acid) was obtained from Parke Davis Pharmaceutical Research and Pfizer Global Research and Development, Ann Arbor, MI. Human liver microsomes (pool of at least 15 donors) were obtained from Xenotech LLC (Kansas City, KS) and hepatocytes were from CellzDirect (Pittsboro, NC). ITS+ (insulin, transferrin, selenium, linoleic acid and BSA supplement), hepatostim

Cytochrome P450 inhibition by CI-1034

CI-1034 inhibited CYP2C9 and CYP3A4 activities with IC50 values of 39.6 ± 5.9 and 21.6 ± 3.4 μM, respectively, and did not inhibit CYP1A2, CYP2C19 and CYP2D6 at the concentrations tested (0–90 μM). The inhibition for CYP3A4 was competitive with an estimated Ki value of 14.0 ± 1.7 μM (Table 1). Fig. 2A illustrates the time-dependent loss of human liver microsomal CYP3A4 activity by CI-1034. Fig. 2B shows the effect of CI-1034 on the enzymatic activity of CYP2C9 in human liver microsomes. Overall, the

Discussion

The drug interactions most commonly evaluated during a compound's development are those associated with inhibition of Phase I drug metabolizing enzymes, particularly P450. The safety of a new chemical entity is based in part, on in vitro safety assessments, drug–drug interaction studies for metabolizing enzymes and in vivo rodent and dog or monkey, multiple dose safety studies. Despite this, 50–70% of drug development programs are terminated after preliminary human testing due to unexpected

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