Cancer Therapy: Clinical The Effect of Ketoconazole on the Pharmacokinetics and Pharmacodynamics of Ixabepilone: A First in Class Epothilone B Analogue in Late-Phase Clinical Development Sanjay Goel,1Marvin Cohen,4 S. Nilgu« n C¸ o« mezoglu,4 Lionel Perrin,5 Franc¸ois Andre¤ ,5 DavidJayabalan, 1Lisa Iacono,4 Adriana Comprelli,4 Van T. Ly,4 Donglu Zhang,4 Carrie Xu,4 W. Griffith Humphreys,4 Hayley McDaid,1, 2 Gary Goldberg,1, 3 Susan B. Horwitz,1, 2 andSridhar Mani 1 Abstract Purpose:To determine if ixabepilone is a substrate for cytochrome P450 3A4 (CYP3A4) and if its metabolism by this cytochrome is clinically important, we did a clinical drug interaction study in humans using ketoconazole as an inhibitor of CYP3A4. Experimental Design: Human microsomes were usedto determine the cytochrome P450 enzyme(s) involvedin the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone. A follow-up clinical study was done in patients with cancer to determine if 400 mg/d ketoconazole (inhibitor of CYP3A4) altered the pharmacokinet- ics, drug-target interactions, and pharmacodynamics of ixabepilone. Results: Molecular modeling and human microsomal studies predicted ixabepilone to be a good substrate for CYP3A4. In patients, ketoconazole coadministration resulted in a maximum ixabepi- lone dose administration to 25 mg/m2 when comparedwith single-agent therapy of 40 mg/m 2. Coadministration of ketoconazole with ixabepilone resulted in a 79% increase in AUC0-1.The relationship of microtubule bundle formation in peripheral blood mononuclear cells to plasma ixabepilone concentration was well described by the Hill equation. Microtubule bundle formation in peripheral bloodmononuclear cells correlatedwith neutropenia. Conclusions: Ixabepilone is a goodCYP3A4 substrate in vitro ; however, in humans, it is likely to be cleared by multiple mechanisms. Furthermore, our results provide evidence that there is a direct relationship between ixabepilone pharmacokinetics, neutrophil counts, and microtubule bundle formation in PBMCs. Strong inhibitors of CYP3A4 should be used cautiously in the context of ixabepilone dosing. Ixabepilone (BMS-247550; Bristol-Myers Squibb; IXEMPRA) is a Authors’ Affiliations: 1Albert Einstein Cancer Center, 2Department of Molecular semisynthetic analogue of epothilone B that interacts with 3 Pharmacology, and Division of Gynecologic Oncology, Department of Obstetrics microtubules and induces them to form stable bundles in cells andGynecology, Albert Einstein College of Medicine, Bronx, New York; 4Bristol- Myers Squibb Research andDevelopment, Princeton, New Jersey; and 5CEA, (1–3). Epothilones have some similarities to taxanes in targeting iBiTec-URA 2096 du Centre National de la Recherche Scientifique, SB2SM, and stabilizing microtubules but also have important differences; CE-Saclay, Gif sur Yvette, France the class is structurally unrelated to taxanes, and unlike taxanes Received9/10/07; revised1/11/08; accepted1/13/08. and anthracyclines, has low susceptibility to multiple mecha- Grant support: DamonRunyonCancerResearchFoundation(CI:15-02; nisms of drug resistance (reviewed in ref. 1). Promising phase I/II S. Mani), DPI-Centre National de la Recherche Scientifique (contract number ST 00332/2004.PD005; F. Andre¤ ), andBristol-Myers Squibb. studies have led to a completion of a phase III study in breast The costs of publication of this article were defrayed in part by the payment of page cancer and approval by the United States Food and Drug charges. This article must therefore be hereby marked advertisement in accordance Administration for use in the treatment of breast cancer. with 18 U.S.C. Section 1734 solely to indicate this fact. We and others have shown that ixabepilone has moderately Note: Supplementary data for this article are available at Clinical Cancer Research variable interpatient pharmacokinetics due in part to variations Online (http://clincancerres.aacrjournals.org/). M. Cohen, S.N. C¸ o« mezoglu, L. Perrin, andF. Andre¤ contributedequally to this work. in drug metabolism (2, 4, 5). Because related epothilone Current address for D. Jayabalan: Cornell University, Weil Medical College, New analogues serve as cytochrome P450 3A4 (CYP3A4) substrates, Yo r k , N Y 10 0 21. it was hypothesized that ixabepilone would also undergo Requests for reprints: Sridhar Mani, Albert Einstein College of Medicine, Albert metabolism by liver cytochromes (e.g., CYP3A4; ref. 6). Einstein Cancer Center,1300 Morris Park Avenue, Chanin 302D-1,Bronx, NY10461. Phone: 718-430-2871; Fax: 718-904-2830; E-mail: [email protected]. To determine the effect of cytochrome P450s on ixabepilone F 2008 American Association for Cancer Research. biotransformation in vitro, a series of studies with liver doi:10.1158/1078-0432.CCR-07-4151 microsomes were done together with molecular dynamic www.aacrjournals.org2701 Clin Cancer Res 2008;14(9) May 1, 2008 Cancer Therapy: Clinical simulations of CYP3A4 ixabepilone docking within the context human liver microsomes in the presence of NADPH for 15 min in a of data derived from published crystal structures of this enzyme shaking water bath before ixabepilone was added. After substrate (7–9). Finally, a phase Ib study was designed to assess the addition, the samples were incubated for 10 min at 37jC and 0.5 mL effects of ketoconazole (a strong CYP3A4 inhibitor) on of acetonitrile was added to each incubation mixture to stop the reaction. ixabepilone pharmacokinetics and pharmacodynamics (10). Then, the samples were centrifuged for 10 min at 3,000 rpm. The supernatants were then analyzed by LC-MS/MS to determine the concentration of ixabepilone (see Bioanalytical Methods). Immunoinhibition. Monoclonal anti-human CYP antibodies Materials and Methods (anti-1A2, anti-2C8, anti-2C9, anti-2C19, anti-2D6, and anti-3A4) were obtained from Dr. R. Krausz at NIH, Bethesda, MD (12). Amixture In vitro studies containing human liver microsomes, 0.1 mol/L potassium phosphate 14 Enzyme kinetics. Under linear conditions, [ C]ixabepilone buffer [containing 0.5 mmol/L of MgCl2 (pH 7.4)], and monoclonal was incubated in triplicate with pooled human liver microsomes anti-CYP antibody was preincubated for 10 min at 37jC. Then, (BD Biosciences; n = 20; at drug concentrations of 0.1, 0.3, 1, 2.5, 5, 10, NADPH and ixabepilone were added to the incubation mixture. The and 25 Amol/L) and expressed CYP3A4 (at drug concentrations of 0.1, 0.3, final mixtures consisted of 0.5 mg/mL human liver microsomes 1, 2.5, 5, 10, 25, 50, and 100 Amol/L) in the presence of NADPH (preincubated with monoclonal antibody), 0.5 Amol/L ixabepilone, (1 mmol/L). Incubations (0.5 mL) in 0.1 mmol/L phosphate buffer 1 mmol/L NADPH, and 0.1 mol/L phosphate buffer (pH 7.4) that were j containing 0.5 mmol/L of MgCl2 (pH 7.4) were carried out at 37jCfor5 incubated for 10 min at 37 C with shaking. The reaction was stopped or 10 min and stopped with ice-cold acetonitrile (1:1, v/v). The protein by addition of 0.5 mL of acetonitrile. The samples were centrifuged for concentration of human liver microsomes was 0.24 mg/mL 10 min at 3,000 rpm, and aliquots of the supernatant were analyzed by (for 0.1 and 0.3 Amol/L ixabepilone concentrations) or 0.5 mg/mL LC-MS (see Bioanalytical Methods). Positive control incubations were (for 1-25 Amol/L ixabepilone concentrations). The CYP3A4 concentration carried out in the absence of antibody solution as described above. An was 25 pmol/mL (for 0.1 and 0.3 Amol/L ixabepilone concentrations) incubation without NADPH and without antibody served as negative and 50 pmol/mL (for 1-100 Amol/L ixabepilone concentrations). For controls. In addition, another control incubation was carried out in the both human liver microsomes and CYP3A4, the incubation time was same manner described above but the incubation mixture contained 5 min for incubations at 0.1 to 2.5 Amol/L ixabepilone concentrations anti–egg white protein antibody to assess nonspecific reactions. and 10 min for 5 to 100 Amol/L ixabepilone concentrations. After Computational methods for substrate docking. Adetailed description quenching with acetonitrile (0.5 mL), the samples were centrifuged for of the molecular dynamic simulations done and metabolism site 5 min at 3,000 rpm. The supernatants were then analyzed by liquid predictions are presented in Supplementary Information. chromatography tandem mass spectrometry (LC-MS/MS) to determine the concentration of ixabepilone (see Bioanalytical Methods). Clinical ketoconazole interaction study Km and Vmax values for ixabepilone metabolism in human liver Patient eligibility. Advanced cancer patients unresponsive to stan- microsomes and CYP3A4 were determined by fitting the rates of parent dard therapeutic interventions were enrolled in this open label phase I drug disappearance in the presence of ixabepilone to the Michaelis- study (10). Additional criteria included an Eastern Cooperative Menten equation [V =(VmaxS)/(Km+S)] using a nonlinear regression Oncology Group performance score of 0 to 1 and no more than three analysis in Sigmaplot (version 8). prior chemotherapeutic regimens. The remaining criteria were identical Metabolite identification. Metabolites were identified by LC-MS/MS to previously published reports (1, 2, 4, 13–23). after analyzing samples from a [14C]ixabepilone (20 Amol/L) incuba- Study design, treatment administration, and dose escalation. This tion with pooled human liver microsomes. The results from these was a study administering ketoconazole, a nanomolar inhibitor of analyses showed that ixabepilone was mainly metabolized to oxidative CYP3A4, with ixabepilone. In cycle 1 (3 wk), ixabepilone was metabolites. In addition to parent drug (P), the identified metabolites coadministered in escalating doses per treatment cohort with a fixed included P+16, P+14, and P-2 metabolites (11). dose of ketoconazole. In cycle 2, ixabepilone was administered as a cDNA-expressed CYP enzymes. Human cDNA-expressed enzymes fixed dose (40 mg/m2) without administering ketoconazole. (BD Biosciences; CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, During cycle 1, patients received a 3-h i.v.