Effect of ketoconazole on the pharmacokinetics of axitinib in healthy volunteers

Adults aged 18–55 years who were assessed as healthy based on a detailed medical examination, which included medical history, blood pressure (BP) and pulse-rate measurements, a 12-lead electrocardiogram (ECG) test, and clinical laboratory evaluations, were eligible to participate in the study. Subjects had a body mass index of 18–30 kg/m², a total body weight >50 kg, and normal ECG and resting BP measurement (<140/90 mm Hg). Volunteers agreed to abstain from strenuous physical activity and consumption of products containing grapefruit, caffeine, xanthine, or alcohol from 2–4 days prior to initiating the study until collection of the final pharmacokinetic samples. Exclusion criteria were conditions that might influence the absorption, distribution, metabolism, or excretion of axitinib or ketoconazole: recent history of drug abuse, high levels of alcohol consumption, or use of products containing tobacco or nicotine; donation of >500 ml of blood within 56 days before beginning study treatment; use of prescription or non-prescription drugs or supplements (other than acetaminophen) within 14 days before initiating treatment (28 days for herbal supplements and hormonal contraception methods or 6 months for depot medroxyprogesterone acetate); or treatment with an investigational agent or use of any known CYP450 enzyme-inducing or -inhibiting drug within 30 days of beginning study medication.

This was a randomized, two-way crossover study conducted in healthy volunteers. The primary objective was to characterize changes in axitinib plasma pharmacokinetic parameters when a single dose of axitinib was co-administered with repeated doses of ketoconazole to healthy subjects. Secondary objectives included assessing the safety and tolerability of axitinib administered with ketoconazole, as well as evaluating the effects of a single axitinib dose on the corrected QT (QTc) interval and BP when administered alone or in combination with ketoconazole.

This study was approved by the institutional review board or ethics committee of each participating center and was carried out in accordance with the ethical principles originating in or derived from the Declaration of Helsinki. Additionally, this trial complied with informed consent regulations and the International Conference on Harmonization Good Clinical Practice guidelines, as well as applicable local laws and regulatory requirements. Written informed consent was obtained prior to subjects entering the study.

Placebo treatment was administered to all subjects on day –1 to characterize QT under the condition of no therapy. Using a computer-generated randomization schedule, subjects were assigned to receive either axitinib alone (treatment A) or combination treatment with ketoconazole plus axitinib (treatment B). Treatments were administered to subjects in one of two different sequences (A→B or B→A) separated by a 14-day wash-out period (Fig. 2). During treatment A, volunteers received a single, oral, 5-mg dose of axitinib on day 1. During treatment B, volunteers received oral doses of ketoconazole 400 mg/day in the morning on days 1–7, and a single 5-mg dose of axitinib administered with ketoconazole on day 4.

Axitinib and placebo treatment were administered in a single-blind manner, and ketoconazole treatment was not blinded. Subjects received axitinib or placebo in the morning after fasting overnight for at least 8 h; food and beverages were permitted 4 h after dosing. The axitinib tablet formulation used in this study was previously shown to produce higher exposures in the overnight fasted state in a phase I study conducted in patients with solid tumors [7] and was the basis for administration of axitinib in the fasted state in the current study. Ketoconazole was administered with breakfast once daily in the morning, except on day 4 when it was administered simultaneously with axitinib after an overnight fast of at least 8 h. During the first 4 h after dosing, subjects were required to refrain from lying down, except while BP, pulse rate, and ECG measurements were obtained.

Blood samples (5 ml) for determination of axitinib pharmacokinetic profiles were collected on days 1–3 at 0 h (pre-dose) and 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 36, and 48 h post-dose during treatment A. During treatment B, samples were collected on days 4–8 at 0 h (pre-dose) and at 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 36, 48, 72, and 96 h post-dose. Blood pressure and pulse rate were recorded in triplicate at screening, and single measurements were obtained on days –2 and –1, days 1–3 during treatment A, on the day before commencing the second treatment, days 4–6 during treatment B, and at the end of the study. Standard 12-lead ECG tests were carried out at screening, days –2 and –1, day 1 during treatment A, days 3 and 4 during treatment B, and at the end of the study. Assessments of ECG, BP, and pulse rate were performed prior to the collection of blood samples at the timepoints indicated above plus an additional assessment 10 h post-dose.

Safety and tolerability were assessed by monitoring adverse events, vital signs, ECGs, and laboratory evaluations, as well as through physical examinations. Adverse events were graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 3.0 (ctep.​cancer.​gov/​protocoldevelopm​ent/​electronic_​applications/​docs/​ctcaev3.​pdf).

Axitinib concentrations in potassium (K₃) EDTA plasma were measured using a validated liquid chromatography/tandem mass spectrometric assay (Charles River Laboratories, Worcester, MA) with a range of 0.1 to 25 ng/ml [7]. Due to axitinib light sensitivity, all sample processing and bioanalytical procedures were performed under red light or with appropriate protection from visible light. Plasma samples were added to a 96-well opaque microtiter plate and fortified with deuterium-labeled (d7) axitinib internal standard, followed by addition of 1.0 M sodium bicarbonate. The samples were extracted with ethyl acetate:hexanes (75:25, v/v), centrifuged, and the supernatants were evaporated to dryness under a gentle nitrogen stream in microtiter plates heated to 35°C. Sample residues were reconstituted in water/methanol/sodium bisulfite (75:25:0.5, v/v/v) and eluted from a C18 column (XTerra® MS C18, 30 × 2.1 mm, 3.5 μm; Waters Corporation, Milford, MA) using a multi-step gradient (mobile phase A = water, mobile phase B = acetonitrile; 10–90% B in 3 min; 0.5 ml/min flow rate) onto an API 3000® mass spectrometer (AB Sciex, Ontario, Canada) with an electrospray ionization source. Detection was in positive ion mode with multiple reaction monitoring (mass transition [m/z] 387→355 and 394→360 for axitinib and axitinib-d7). Duplicate quality control (QC) samples at three concentration levels spanning the assay range (low, medium, and high) were included in each analytical run to measure assay performance. Inter-assay precision and accuracy were determined by QC samples from across all study sample runs. Precision was within 12% coefficient of variation (CV), while overall bias (expressed as percent relative error) was within 5.5%. No plasma metabolites were characterized in the study.

Genotyping of drug-metabolizing enzymes was carried out using polymerase chain reaction (PCR) to amplify DNA samples for all assays performed in this study (Pfizer-Ann Arbor, Worldwide Safety Sciences, Ann Arbor, MI). In particular, the following variants of UGT1A1 were assessed: UGT1A1*60, UGT1A1-3156 (-3156 G → A nucleotide change), UGT1A1 Promoter TA repeat (*28, *36, *37), UGT1A1*6 and UGT1A1*27. Genotyping for the CYP2C19 *2, *3, and *17 alleles was also conducted. The TaqMan® Allelic Discrimination procedure was used for detection of all alleles, with the exception of the CYP3A4*2 (MassEXTEND™; Sequenom, San Diego, CA) and UGT1A1*28 (sequencing) genotyping assays.

TaqMan® allelic discrimination employs the 5′-nuclease activity of AmpliTaq Gold® DNA polymerase to allow direct detection of PCR products by the release of a fluorescent reporter. Two TaqMan® probes (Applied Biosystems, Carlsbad, CA) were used in this assay, one probe for each allele. Each probe consists of an oligonucleotide with a 5′ reporter (TET™ [tetrachloro-6-carboxyfluorescein]/VIC® or FAM® [6-carboxyfluoroscein] dye and a 3′-quencher dye [TAMRA™ (6-carboxytetramethylrhodamine)] or a non-fluorescent quencher). An intact probe results in quenching of the reporter dye fluorescence.

During the annealing phase of PCR, forward and reverse primers hybridize to the flanking region of the polymorphic site. In addition, the TaqMan® probes hybridize to the target polymorphic site within the PCR product. The reporter dye is cleaved by the Taq Gold® enzyme, resulting in an increase in the reporter dye fluorescence. By measuring the intensities of TET™/VIC® (allele 1 or homozygous wild type) and FAM™ (allele 2 or homozygous variant) signal, the specific genotype of an allele is discriminated.

Individual reaction plates with 20 ng of genomic DNA per reaction were used. Master mixes were prepared for each assay performed, and controls for both no template and each allele were included for each plate. After master mix (10X PCR buffer with MgCl₂, forward and reverse primers, TaqMan® probes, deoxynucleotides, and AmpliTaq Gold® DNA polymerase) had been added to the controls and unknown DNA samples, plates were sealed and PCR cycling (GeneAmp® PCR System 9700, Applied Biosystems) performed. The sealed 96-well plates were then transferred to the ABI 7700® (Applied Biosystems) and fluorescence data collected.

A Simcyp®-based simulation (Simcyp® Population-based ADME Simulator, version 6.0 [Simcyp Ltd, Sheffield, UK] on Microsoft Windows® XP [Redmond, WA]) was performed to assess changes in axitinib plasma concentrations with co-administration of ketoconazole (inhibitor) in healthy volunteers. Simcyp® uses the relationship between the inhibitor concentration at the active site in vivo and the inhibition constant (K_i) determined in vitro to predict the effects of drug–drug interactions involving CYP enzymes. Competitive inhibition, induction, and/or mechanism-based inhibition can be investigated using this software [11].

A Simcyp® model was developed for axitinib using in vitro and clinical pharmacokinetic parameters for axitinib (input parameters are shown in Table 1). The required Simcyp® model for ketoconazole was available within Simcyp® as an inhibitor file. Due to the inability to determine glucuronidation clearance from in vitro tissues accurately, the following approach was used. Systemic clearance (CL_sys) for axitinib was determined from a previous study in healthy volunteers who received intravenous axitinib (1 mg). In vivo CL_sys was then converted to intrinsic clearance (CL_int) using amount of microsomal protein per gram of liver and liver weight to yield a CL_int of 500 μl/min/mg microsomal protein. Contribution of CYP3A and glucuronidation to total CL_int was determined through sensitivity analyses to be 50% (CYP3A4 = 250 μl/min/mg microsomal protein) and 30% (non–CYP-mediated clearance = 150 μl/min/mg), respectively, when simulations with ketoconazole were conducted. The remaining 20% of clearance was attributed to non-metabolic pathways (4.2 L/h).

Physiologic variability (healthy Northern European Caucasians: height, weight, age, etc.) and variation in phenotype were calculated using parameters within the Simcyp® software. Dose, dose interval, and duration of administration of axitinib and ketoconazole were set according to the clinical protocol. Simcyp® predicts the mean value as well as the distribution in the population for the effect by applying a Monte Carlo approach. Population simulations were performed using 100 Northern European Caucasians aged 20–50 years (1:1 male/female) in 10 trials, with each trial containing 10 subjects. To prevent bias, the Simcyp® end-user did not have access to the clinical drug–drug interaction results.

Based on an IC₅₀ of 0.4 μM for ketoconazole-mediated inhibition of axitinib metabolism observed in human liver microsomal preparations, ketoconazole 400 mg was predicted to result in a 1.8-fold increase in axitinib geometric mean AUC from time zero to infinity (AUC_0–∞) and a 1.5-fold increase in geometric mean C_max.

The standard deviation (SD) of the difference between (natural log) AUC_0–∞ of axitinib plus ketoconazole and axitinib alone was estimated to be 0.42, based on variability in axitinib pharmacokinetics observed in the FIH phase I study in patients with solid tumors [7]. The sample size was calculated as 24 subjects (12 per sequence) to provide 80% power to ensure that the lower limit of the 90% confidence interval (CI) for the AUC ratio was above 1.6 (a 60% increase), if ketoconazole treatment increased axitinib exposure by 100%.

Data analyses included all subjects who received at least one dose of study medication (placebo, axitinib, or ketoconazole). In addition, evaluable pharmacokinetic data were required for analyses of pharmacokinetic parameters, and at least one baseline and post-baseline BP measurement were needed for BP analyses.

Pharmacokinetic evaluation of axitinib was performed with WinNonlin®, version 4.01 (Pharsight, Mountain View, CA) using a non-compartmental approach. Estimated axitinib pharmacokinetic parameters included AUC, C_max, and half-life (t_1/2). The primary analysis for the study was based on axitinib AUC_0–∞. This value was log-transformed and analyzed using a mixed-effects model with treatment, period, and sequence as fixed effects and subject (nested within sequence) as the random effect. The 90% CI for the difference in the means of the log-transformed AUC_0–∞ was calculated. The antilogs of the confidence limits obtained constituted the 90% CI for the ratio of the geometric mean of ketoconazole plus axitinib compared with axitinib alone. Secondary analyses were performed using axitinib AUC from time zero to the time of last quantifiable concentration (AUC_last) and C_max in a similar fashion.