The study protocol was reviewed and approved by an institutional review board (IRB; Schulman IRB; 4445 Lake Forest Drive, Suite 300; Cincinnati, OH 45242). The study was carried out in accordance with the Declaration of Helsinki and the International Council for Harmonisation Good Clinical Practice guidelines. All participants provided written informed consent prior to study participation.

Eligible participants included males and nonpregnant, nonlactating females. All participants were nonsmokers, aged between 18–45 years, had a body mass index (BMI) 19–30 kg/m2, and were required to have a normal or clinically insignificant 12-lead electrocardiogram (ECG), normal renal function, no significant medical history, and general good health at the time of screening (≤ 28 days prior to the first dose), as determined by the investigators. Exclusion criteria included pregnancy or lactation; any serious or active medical or psychiatric illness; liver disease including Gilbert’s disease; receipt of any investigational drug or device (30 days prior to first dose); use of any prescription or over-the-counter medications (except vitamins, acetaminophen, ibuprofen, and/or hormonal contraceptive medications) or herbal products within 28 days of commencing study drug dosing; a positive test result for human immunodeficiency virus 1 antibody, hepatitis B surface antigen, or hepatitis C antibody; treatment with systemic steroids, immunosuppressant therapies, or chemotherapeutic agents within 3 months prior to screening or expected to receive these agents during the study; and no current alcohol or substance abuse.

This was a Phase 1, open-label, multicenter, multiple-cohort study in healthy participants. Following completion of screening and Day 1 assessments, eligible participants were enrolled in 1 of 6 cohorts, including 5 prespecified cohorts (Cohorts 1, 2, 3, 5, and 6) and 1 adaptive cohort (adaptive Cohort 4 [initiated based on preliminary data from Cohort 2]).

Cohort 1 assessed the effect of the administration of a single dose of cyclosporine (OATP/P-gp/CYP3A inhibitor) or rifampin (OATP1B1/1B3 inhibitor) on the exposure of a single dose of cilofexor. Participants in Cohort 1 (n = 24) were randomized and received a single dose of cilofexor 100 mg, a single dose of cyclosporine 600 mg with a single dose of cilofexor 100 mg, and a single dose of rifampin 600 mg with cilofexor 100 mg in 1 of 6 treatment sequences. Between each treatment there was a washout period on Days 2–8 and Days 10–16.

Cohort 2 assessed the effect of the administration of multiple doses of voriconazole (CYP3A4 inhibitor) or gemfibrozil (CYP2C8/OATP1B1 inhibitor) on the exposure of a single dose of cilofexor. Participants in Cohort 2 (n = 18) received a single dose of cilofexor on Day 1 followed by voriconazole 200 mg twice daily (BID) for 4 days starting on Day 9 with a single dose of cilofexor 100 mg administered on Day 9. The last dose of voriconazole was administered on Day 12 in the evening. Gemfibrozil 600 mg BID was administered for 4 days starting on Day 20 with a single dose of cilofexor administered on Day 20. The last dose of gemfibrozil was administered in the morning on Day 23. There was a 7-day washout period between each treatment.

Cohort 3 assessed the effect of the administration of multiple doses of rifampin (OATP/CYP/P-gp inducer) on the exposure of a single dose of cilofexor. Participants in Cohort 3 (n = 18) received a single 100-mg dose of cilofexor on Day 1 followed by a 7-day washout period. Rifampin 600 mg once daily was administered in the evening 1 hour before a meal for 7 days starting on Day 9. Finally, a single 100-mg dose of cilofexor was administered on Day 16 in the morning.

Cohort 4 assessed the effect of the administration of grapefruit juice (Everfresh Juice company), an intestinal OATP (and CYP3A) inhibitor, on the exposure of a single dose of cilofexor. Participants in Cohort 4 (n = 24) were randomized to 1 of 2 treatment sequences and received a single dose of cilofexor 100 mg and 16 ounces of grapefruit juice with cilofexor 100 mg. Treatments were administered on either Day 1 or Day 9 with a 7-day washout period between each treatment.

Cohort 5 assessed the effect of multiple-dose administration of cilofexor on the exposure of single doses of midazolam (CYP3A4 substrate) and atorvastatin (OATP/CYP3A4 substrate). Participants in Cohort 5 (n = 24) received a single 2-mg dose of midazolam on Day 1, followed by a single 10-mg dose of atorvastatin on Day 2, followed by a washout period on Days 3–6. Participants received cilofexor 100 mg once daily on Days 7–11 with a single dose of midazolam 2 mg on Day 7 and a single dose of atorvastatin 10 mg on Day 8.

Cohort 6 assessed the effect of multiple-dose administration of cilofexor on the exposure of single doses of dabigatran etexilate (intestinal P-gp substrate), pravastatin (OATP substrate), and rosuvastatin (OATP/BCRP substrate). Participants in Cohort 6 (n = 24) received a single dose of a cocktail of dabigatran etexilate 75 mg + pravastatin 40 mg + rosuvastatin 10 mg on Day 1 followed by a 5-day washout period. Participants received cilofexor 100 mg once daily on Days 7–10 with a single dose of the cocktail on Day 7.

A summary of cohorts and a treatment schematic are presented in Fig. 1. Participants were confined to the clinic from Day − 1 until completion of assessments on Day 21 (Cohort 1), Day 24 (Cohort 2), Day 20 (Cohort 3), Day 13 (Cohort 4), Day 12 (Cohort 5), or Day 11 (Cohort 6).

Study schematic for each cohort. ATV atorvastatin, BCRP breast cancer resistance protein, BID twice daily, CIL cilofexor, CsA cyclosporine, CYP cytochrome P450 enzyme, DE dabigatran etexilate, GFJ grapefruit juice, GFZ gemfibrozil, MDZ midazolam, OATP organic anion transporting polypeptide, P-gp P-glycoprotein, PRA pravastatin, RIF rifampin, ROS rosuvastatin, VORI voriconazole, WO washout

In all cohorts, study drugs were administered within 5 min of participants completing a standardized, moderate-fat meal. The meal was initiated 30 min prior to administration of the study drug. Participants then fasted until after collection of the PK sample at 4 hours post-dose.

Intensive PK sampling occurred pre-dose (< 5 min) and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 48, 72, and 96 hours post-dose on Days 1, 9, and 17 (Cohort 1); Days 1, 9, and 20 (Cohort 2); Days 1 and 16 (Cohort 3); Days 1 and 9 (Cohort 4); Days 1, 2, 7, and 8 (Cohort 5); and Days 1 and 7 (Cohort 6).

Concentrations of cilofexor, midazolam, atorvastatin, o-hydroxyatorvastatin (o-OH-ATV), pravastatin, rosuvastatin, and dabigatran (total and free) in human plasma samples were quantified using fully validated high-performance liquid chromatography-tandem mass spectroscopy (LC-MS/MS) methods. All samples were analyzed within the time frame supported by frozen stability storage data. Lab vendor(s) analyzed plasma samples for cilofexor, midazolam, atorvastatin, o-OH-ATV, dabigatran (total and free), pravastatin, and rosuvastatin concentrations. Once the pharmacokinetic concentration data were deemed final (quality assurance performed by Covance Madison, QPS, and PPD labs), the pharmacokinetic parameters were calculated.

Pharmacokinetic parameters were estimated with Phoenix WinNonlin 8.2 software (Certara, LP, Princeton, NJ, USA) using standard noncompartmental methods. Samples with concentrations below the limit of quantitation of the bioanalytical assays occurring prior to the achievement of the first quantifiable concentration were assigned a concentration value of zero and at all other time points were treated as missing data in the noncompartmental analyses. Pharmacokinetic parameters included area under the plasma concentration-time curve (AUC) from time 0 to the last quantifiable concentration (AUClast), AUC extrapolated to infinity (AUCinf), maximum observed plasma concentration (Cmax), time to maximal concentration (Tmax), and terminal-phase elimination half-life (t½).

For each cohort, the selected sample size was projected to achieve at least 80% power such that the 90% CI for the geometric least-squares mean (GLSM) ratio of AUCinf, AUClast, and Cmax in test (victim drug administered with perpetrator drug) versus reference (victim drug administered alone) treatments would be within 0.70 to 1.43, if the true GLSM ratio was 1.0. For each cohort, analyte, and PK parameter, a parametric (normal theory) mixed-effects ANOVA model was fitted to the natural log-transformed values of the single-dose PK parameter under evaluation using SAS PROC MIXED. For Cohorts 1 and 4, the statistical model included treatment, sequence, and period as fixed effects and participant within sequence as a random effect. For Cohorts 2, 3, 5, and 6, the statistical model included treatment as a fixed effect and participant as a random effect. The test versus reference ratio and associated 90% CI were calculated by taking the exponential of the point estimate and the corresponding lower and upper limits, which was consistent with the two 1-sided tests approach. A lack of DDI was concluded if the GLSM ratios and corresponding 90% CIs for selected PK parameters fell within the prespecified lack of PK alteration boundaries of 70% to 143%.

Safety was monitored throughout the study. Safety was evaluated by assessment of clinical laboratory tests, ECGs, periodic physical examinations (including vital sign measurements), and documentation of adverse events (AEs). Clinical and laboratory AEs were coded using the Medical Dictionary for Regulatory Activities, Version 19.1.