Eligible participants included healthy men 18–45 years of age with a body mass index (BMI) of 19–32 kg/m2 who weighed at least 60 kg and had no clinically significant abnormal findings on physical examination, electrocardiogram (ECG), blood pressure, heart rate, medical history, or clinical laboratory assessments. Subjects must have had a corrected QT interval no greater than 450 ms on ECG at screening, morning serum cortisol within normal limits, and normal serum potassium. Participants with partners of childbearing potential must have been using two forms of medically accepted methods of contraception throughout the study and for at least 3 months (90 days) after the last dose of study medication; subjects with pregnant partners were excluded. Subjects with any alkaline phosphatase, total bilirubin, or direct bilirubin outside normal range or an alanine aminotransferase or aspartate aminotransferase value greater than 1.2 times the upper limit were also excluded. Additional exclusion criteria included consumption of alcohol or caffeine-containing products 72 h prior to study baseline or use of concomitant medications or nicotine-containing products.
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964, as revised in 2013. Informed consent was obtained from all patients for being included in the study. The protocol was approved by an independent investigational review board (IntegReview, Austin, Texas). This manuscript was prepared according to the International Society for Medical Publication Professionals’ “Good Publication Practice for Communicating Company-Sponsored Medical Research: the GPP3 Guidelines” and the International Committee of Medical Journal Editors’ “Uniform Requirements for Manuscripts Submitted to Biomedical Journals.”
This was a phase 1, open-label, two-period study conducted at a single US center to evaluate the pharmacokinetic effects in healthy men of multiple doses of ketoconazole 200 mg twice daily orally on concomitantly orally administered multiple doses of mifepristone 600 mg once daily and vice versa. A cross-study comparison using data on file was also conducted to determine whether the effect of ketoconazole on the systemic exposure to coadministered mifepristone exceeded that of the maximum recommended dose of mifepristone (1200 mg) .
The 600-mg mifepristone dose was selected for analysis based on internal data suggesting that the increased mifepristone exposure resulting from concomitant administration with ketoconazole 400 mg total daily would be generally comparable to the exposure following 1200 mg of mifepristone. The 200-mg twice-daily ketoconazole dose was consistent with the highest clinical dosing recommendations in its product labeling . The twice-daily dosing regimen was also used to enhance the inhibitory effect of ketoconazole when given with a substrate with a long half-life, as is the case for mifepristone .
Food can enhance the absorption of mifepristone and ketoconazole [4, 22]; therefore, all medications were administered within 30 min of a typical breakfast consisting of 34% fat. Subjects were admitted to the clinic on day − 2. On day − 1, subjects received a single dose of ketoconazole 200 mg, followed by a 48-h pharmacokinetic sampling period that was completed on day 1 prior to mifepristone administration (Fig. 2). Subjects were discharged from the clinic following the 12-h sample and returned for the 24-, 36-, and 48-h sample. During period 1, mifepristone 600 mg was administered alone on days 1–12. During period 2, which followed on days 13–17, ketoconazole 200 mg twice daily was administered concomitantly with mifepristone 600 mg once daily. The morning dose of ketoconazole was administered approximately 5 min before mifepristone. Subjects were readmitted to the clinic on the evening of day 11 and remained onsite through day 19. Subjects returned to the clinic for pharmacokinetic sampling on days 20, 22, 25, and 28 and a termination visit was conducted on day 32.
Plasma trough concentrations of mifepristone and its three active metabolites were measured from blood samples collected 30 min predose on days 1–11 and days 13–16; on washout days 18, 20, 22, 25, and 28; and on termination visit day 32. Serial blood samples were collected predose and at 0.5, 1, 2, 4, 8, 12, and 24 h on days 12 and 17.
Plasma concentrations of ketoconazole were measured in blood samples collected within 30 min predose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h on day − 1 and within 30 min predose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 h on day 17. Plasma trough concentrations of ketoconazole were also measured 30 min predose from blood samples taken on days 13–16 to ensure steady-state concentrations were reached. In the reference study of mifepristone 1200 mg, plasma concentrations of mifepristone and its metabolites were measured in blood samples collected predose and at 0.5, 1, 2, 4, 8, 12, and 24 h after dosing on days 1 and 7.
Samples were collected in heparinized vacutainer tubes and centrifuged at 2500 rpm for 15 min at 4–6 °C. Harvested plasma samples were stored at approximately − 20 °C or lower and shipped under dry ice for analysis.
Fasting blood samples for the analysis of serum cortisol and plasma ACTH were collected between 07:00 and 09:00 during screening and on days 1 and 6 of period 1, days 13–17 of period 2, and termination day 32. Samples were collected prior to dosing during the study periods.
Plasma concentrations of mifepristone, its metabolites, and ketoconazole were assessed by MicroConstants (San Diego, California) using validated high-performance liquid chromatography (HPLC) methods with mass spectrometric (MS) detection. The assay lower limit of quantification was 10 ng/mL for mifepristone and the three active metabolites, and 5.0 ng/mL for ketoconazole. Data analysis was performed using MassLynx v.4.1 (Waters, Milford, Massachusetts).
Human plasma samples containing C-1073, RU 42633, RU 42698, RU 42848, a solution containing C-1073-d4, RU 42633-d4, RU 42698-d4, and RU 42848-d4 (deCODE Genetics, Reykjavik, Iceland) as the internal standards, and sodium heparin as the anticoagulant were extracted using a mixture of hexane and methyl tert-butyl ether (MTBE). The organic layer was dried down under nitrogen and the residue was reconstituted in water/acetonitrile/formic acid (75:25:0.1, v/v/v). Sample extracts were analyzed by reversed-phase chromatography using a phenyl column maintained at 50 °C. The mobile phase was nebulized using heated nitrogen in a Z-spray source/interface set to electrospray positive ionization mode. The ionized compounds were detected using a tandem quadrupole mass spectrometer (MS/MS).
For determination of ketoconazole concentrations, plasma samples containing ketoconazole-d3 or ketoconazole-d8 (Toronto Research Chemicals Inc., North Yolk, Ontario, Canada) as the internal standard and tripotassium ethylenediaminetetraacetic acid or sodium heparin as the anticoagulant were adjusted to approximately pH 10.0 with ammonium hydroxide and extracted with an MTBE solution. The samples were vortex mixed and centrifuged, and the lower portion was frozen in an ultra-cold freezer. The organic layer was transferred to a clean tube and then evaporated under nitrogen. The residue was reconstituted with acetonitrile. An aliquot was analyzed by reversed-phase HPLC using an Atlantis Hilic Silica column (Waters, Milford, Massachusetts), maintained at 40 °C. The mobile phase was nebulized using heated nitrogen in a Z-spray source/interface and the ionized compounds were detected using MS/MS.
Accuracy and precision (percentage of variation, %CV) were evaluated using replicate analyses of human plasma quality control samples prepared at concentrations of 30.0, 300, and 1600 ng/mL for mifepristone and its metabolites and 15.0, 200, and 4000 ng/mL for ketoconazole. Deviation of the measured concentration from the theoretical values (range ± 2.00% to ± 8.67%) and %CV (range 1.82–6.27%) of quality control samples of mifepristone, its metabolites (RU 42633, RU 42698, and RU 42848), and ketoconazole were all within 15%.
For each individual, pharmacokinetic parameters were computed for mifepristone and its metabolites and ketoconazole using a non-compartmental analysis method using WinNonlin version 6.4 (Certara Inc, St. Louis, Missouri). Parameters included maximum plasma concentration (C
max), area under the curve (AUC) using the linear trapezoidal rule, and time to maximum plasma concentration (t
max). For ketoconazole, AUC was calculated as AUC extrapolated to infinity (AUCinf) for day − 1 and AUC0–12 for day 17.
Safety and tolerability were assessed throughout the study by physical examinations, clinical laboratory tests, vital signs, ECGs, and adverse event (AE) reporting. The causal relationship of each AE to mifepristone and/or ketoconazole was assessed by the investigator as probably related, possibly related, or unrelated.
Statistical analyses for safety were performed using SAS version 9.2 or higher (Cary, North Carolina). Statistical analyses for pharmacokinetic comparisons were performed using WinNonlin version 6.4 (Certara Inc. St. Louis, Missouri). The safety population was defined as all subjects who received at least one dose of study drug. The pharmacokinetic population was defined as all subjects who received study drug and for whom the pharmacokinetic profile could be adequately characterized. Pharmacokinetic steady-state parameters of mifepristone when administered alone (period 1, day 12) were compared to the steady-state parameters when administered with concomitant ketoconazole at steady state (period 2, day 17) to estimate the magnitude of the effect of ketoconazole on mifepristone. A linear mixed-effects model was used to estimate the geometric least squares mean ratios (GMRs) and associated 90% confidence intervals (CIs) from the pharmacokinetic parameters (log-transformed values of C
max and AUC). Comparisons of the steady-state pharmacokinetic parameters were considered not clinically different if the 90% CIs around the GMR were within the standard bioequivalence interval of 80:125 . Median differences in t
max were computed using the Wilcoxon matched-pairs signed rank test for paired comparisons and the Mann–Whitney test for any unpaired comparisons; 90% CIs were computed using the Hodges–Lehmann method. Similar methods were used to compare GMRs and associated 90% CIs for the pharmacokinetic parameters of ketoconazole following multiple doses of ketoconazole (200 mg twice daily) plus mifepristone on day 17 (log-transformed C
max and AUC0–12) to a single dose of ketoconazole (200 mg) on day − 1 (C
max and AUCinf).
Sample size calculations determined that a sample size of 8 would achieve an 82% power to detect a mean of paired differences in AUC0–24 of 18,857 ng h/mL with an estimated standard deviation (SD) of 15,963 ng h/mL and a significance level of 0.05 using a two-sided paired t test. A prespecified sample size of 16 subjects was selected for this study in order to account for an approximate discontinuation rate of up to 50%.
Serum cortisol and plasma ACTH values were summarized with descriptive statistics (mean [SD] or median [range]). A Wilcoxon signed rank test was performed to examine the within-subject changes in cortisol and ACTH on day 17 of mifepristone plus ketoconazole compared with day 13 of mifepristone alone.