Effect of ketoconazole-mediated CYP3A4 inhibition on clinical pharmacokinetics of panobinostat (LBH589), an orally active histone deacetylase inhibitor

Patient selection was performed at the phase I outpatient department. Patients with histologically or cytologically confirmed advanced or metastatic solid tumors for whom no standard therapy was available and with an Eastern Cooperative Oncology Group (ECOG) performance status ≤2 were eligible. Since clinical responses were observed in phase I/II trials with panobinostat, the selection of this phase I patients population with no other therapeutic options was considered appropriate for this study where a prolonged treatment with a potentially active drug was offered to these patients, although, based on tumor characteristics no potential benefit was anticipated beforehand. The serum potassium, total corrected calcium, magnesium and phosphorus had to be above the lower limit of normal. QTc had to be ≤450 ms. Concomitant treatment with CYP3A inhibitors and medications which increase the risk of Torsades de Point was not allowed. Significant cardiac disease or other concurrent uncontrolled medical conditions were exclusionary criteria. The study was designed and conducted under the appropriate institutional review boards’ approvals and in accordance with the principles embodied in the Declaration of Helsinki. Written informed consent was obtained from each participant. This study is registered as NCT00503451 (http://​www.​ClinicalTrials.​gov).

Patients received single-agent panobinostat at 20 mg orally after at least 2 h fasting on day 1 and single-agent ketoconazole once daily at 400 mg on days 5–9 (Fig. 1). On day 8, panobinostat was administered 1 h after ketoconazole. If at interim analysis after evaluation of 12 patients any of the four parameters (AUC_0–24, AUC_0–tlast, AUC_0–∞ and C _max) had the paired t statistic (calculated based on log-transformed data) falling outside of (−1.98405, 1.98405), then the recruitment was to be stopped. If not, the trial was to proceed until 16 patients completed the study. The study design also stipulated that, while completer status for 12 patients was being confirmed, enrollment was not halted as long as there were no safety concerns.

All patients enrolled before this interim analysis were to proceed to the extension phase regardless of the interim result. From day 15 onward, the extension phase of the study started in which patients received single-agent 20 mg oral panobinostat each Monday, Wednesday and Friday (MWF) until disease progression, patient withdrawal of consent or unacceptable toxicity. Toxicity was evaluated using the Common Terminology Criteria for Adverse Events (CTCAE) v 3.0.

Close monitoring by evaluating signs and symptoms, physical examination, laboratory assessments and ECGs was performed during the trial. Patients had scheduled visits on days 1, 2, 3, 5, 8, 9, 10 and 15. Laboratory assessments were scheduled at days 1, 5, 8, 9, 10 and 15. Twelve-lead ECGs were scheduled as follows: predose day 1 and at 1.5, 3, 6, 24 and 48 h post-dose; preketoconazole dose on day 5 and at 1.5 and 3 h post-dose; preketoconazole on day 8 and 1.5, 3, 6, 24 and 48 h post-dose panobinostat; and predose on day 15. All ECGs were taken at least in triplicate separated by 2–5 min and were reviewed independently at a central laboratory (eRT, Bridgewater, NJ). During the extension phase, single ECGs were performed weekly as were laboratory assessments. Bazett’s formula was used to correct QT intervals for heart rate.

During cycle 1 at days 1 and 8, patients had serial blood sampling collections for pharmacokinetic evaluation of panobinostat. Blood samples (3 ml/sample) were collected in tubes containing sodium heparin prior to panobinostat dose and 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 24 and 48 h post-dose. Samples were centrifuged at 800 g for 15 min at 4°C to yield plasma within 60 min after blood collection and stored below −60°C until analyses.

Blood samples (3 ml) for ketoconazole concentration determinations were collected predose and 2 h post-dose of ketoconazole on days 5, 8 and 9 (Fig. 1).

Panobinostat is analyzed in human plasma samples using a specific LC–MS/MS method with the lower limit of quantification (LLOQ) of 0.5 ng/ml and a dynamic range of 0.5–500 ng/ml of the analyte using 0.1 ml sample volume. A semi-automated protein precipitation extraction of human plasma samples was used to separate panobinostat from human plasma protein. The obtained sample extracts were evaporated to dryness, reconstituted with 10% aqueous acetonitrile (containing 0.2% formic acid) and analyzed by LC–MS/MS on a Sciex API3000 or a Sciex API4000 tandem mass spectrometer. A Waters Xbridge C8 column (2.5 μm particle size, 50 × 2.1 mm) was employed in reversed-phase chromatography with gradient elution using 10% acetonitrile in water (containing 0.1% formic acid and 0.1% acetic acid, mobile phase A) and 90% acetonitrile in water (containing 0.25% formic acid and 0.25% acetic acid, mobile phase B) at a flow rate of 0.3 ml:/min. The assay was linear from 0.5 to 500 ng/ml with the bias (%) and CV(%) values of the QC sample results ranging from -0.7 to 0.7% and 2.3 to 11.6%, respectively, based on the within-study validation during the sample analysis.

For determination of ketoconazole in human plasma, a 100 μl aliquot of each sample (Standards, QC samples, study samples, blanks, etc.,) was pipetted into the appropriate well in a 96-deep-well plate. A 100 μl aliquot of the internal standard working solution (250 ng/ml in 50% acetonitrile, v/v) was added to all wells except for the blanks, to which a 100 μl aliquot of 50% acetonitrile was added. A 400 μl aliquot of acetonitrile was added to each well, the plate covered, vortexed for approximately 10 min and centrifuged at approximately 4,000 rpm for approximately 10 min at room temperature. Using a TomTec device, a 50 μl aliquot of sample extract was transferred to a clean 96-deep-well plate, followed by an addition of 450 μl of 30% acetonitrile in water (v/v). After a brief vortex mixing, a 10 μl aliquot was injected onto the HPLC system connected to a Sciex API5000 mass spectrometer. The assay was linear from 50.0 to 15,000 ng/ml with the bias (%) and CV(%) values of the QC sample results ranging from -4.2 to 3.3% and 6.0 to 8.6%, respectively, based on the within-study validation during the sample analysis.

Panobinostat plasma concentration–time curve following each dose from individual patient was used to calculate pharmacokinetic parameters that included C _max, T _max and AUC_0–∞. PK parameters were obtained using non-compartmental methods implemented in WinNonlin Pro software (Version 5.01, Pharsight Corporation, Mountain View, CA, USA). C _max and T _max were obtained by visual inspection of the concentration–time curve. AUC_0–∞ was calculated using the linear up/log down method up to the last measured concentrations and the additional area estimated from that concentration and the value of apparent terminal half-life estimated for the targeted administration. The apparent terminal half-life was estimated using the best-fit variables of a single exponential to the log-linear portion of the plasma concentration–time curve using non-weighted linear regression.

Ratio was calculated using the following formula: \(([\hbox{PK-parameter}]_{{\rm panobinostat}+{\rm ketoconazole}}/[\hbox{PK-parameter}]_{\rm panobinostat})\).

Blood samples (1.5 ml in EDTA-containing tubes) were obtained at baseline for genotyping analysis of CYP3A4*1B, CYP3A5*2, *3, *6 and *7 and performed using AmpliChip CYP450 at Epidauros, Bernried, Germany. DNA preparation and sequencing followed the method described by Sanger et al. (16), using commercial kits provided by Applied Biosystems. Wild-type allele CYP3A4*1A is defined as the exclusion of allele CYP3A4*1B. Wild-type allele CYP3A5*1 is defined as the exclusion of alleles *2, *3, *6 and *7.

Pharmacokinetic evaluations to determine the influence of the potent CYP3A inhibitor ketoconazole on the pharmacokinetics of panobinostat were conducted during cycle 1 at days 1–14. A sample size of 16 would have provided enough power (82.4%) to detect a minimum increase of 30% (ketoconazole plus panobinostat vs. panobinostat alone) with an alpha level of 0.1. Since strong interaction between ketoconazole and panobinostat is possible, a ratio higher than 1.3 (ketoconazole plus panobinostat vs panobinostat alone) might be observed. Thus, a drug–drug interaction (DDI) could become apparent with fewer patients, and stopping if this occurs avoids unnecessary exposure of patients to study medications. An interim analysis of PK data was carried out when 75% of anticipated patients (12 completed patients out of 16 planned completers) were enrolled and had completed the core phase.

The number of 12 patients was chosen with two considerations. A sample size of 12 is generally accepted to evaluate safety [9], and secondly, it provided reasonable power for stopping at interim. The O’Brien–Fleming method was used to calculate the boundaries at interim and final analyses [10].

A linear mixed effects model was fit to the log-transformed PK parameters (C _max and AUC_0–24, AUC_0–t and AUC_0–∞). Included in the model was treatment (ketoconazole + panobinostat or panobinostat alone) as fixed effects and patient as a random effect. For the DDI analysis, the combination treatment (ketoconazole + panobinostat) was the test and panobinostat alone the reference. Proper contrasts were used to estimate the treatment difference (i.e., ketoconazole + panobinostat vs. panobinostat alone). The point estimate of the treatment difference and the corresponding 90% confidence intervals were calculated and back-transformed to obtain the point estimate and CI on the linear scale for the ratio of geometric means of the test as compared with the reference. The median, minimum and maximum of T _max difference between test and reference were also presented.