Pharmacokinetics, pharmacodynamics, safety and tolerability of cilofexor, a novel nonsteroidal Farnesoid X receptor agonist, in healthy volunteers

Abstract

Cilofexor is a nonsteroidal farnesoid X receptor (FXR) agonist being evaluated for treatment of nonalcoholic steatohepatitis (NASH) and primary sclerosing cholangitis (PSC). This work characterized the pharmacokinetics, pharmacodynamic, safety, and tolerability of cilofexor in healthy participants. Cilofexor single and multiple once‐daily doses (10 to 300 mg fasting or fed and twice‐daily doses [15 and 50 mg; fed]; tablet formulation) were evaluated. In each cohort, participants were randomized to active drug or placebo in a 4:1 ratio (planned n = 15/cohort). Multiple dosing was for 14 days. Pharmacokinetic and pharmacodynamic samples were collected and safety and tolerability were assessed. Overall, 120 participants were enrolled in the study and 118 participants received at least one dose of study drug. Cilofexor pharmacokinetics followed bi‐exponential disposition and its exposure increased in a less‐than‐dose‐proportional manner over the 10 to 300 mg dose range, with no significant accumulation with repeated dosing. Moderate‐fat meal reduced cilofexor area under the plasma concentration versus time curve (AUC) by 21% to 45%. Cilofexor increased plasma levels of fibroblast growth factor19 (FGF19) and reduced the serum bile acid intermediate 7α‐hydroxy‐4‐cholesten‐3‐one (C4) and bile acids in an exposure‐dependent manner. Cilofexor doses >30 mg appeared to achieve the plateau of intestinal FXR activation. Cilofexor was generally well tolerated; all treatment‐emergent adverse events (TEAEs) were mild or moderate in severity, with headache being the most frequently observed TEAE. The pharmacokinetics pharmacodynamic safety, and tolerability results from this study supported further evaluations, and informed dose selection, of cilofexor in phase II studies in patients with NASH and PSC.

Study Highlights.

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

Nonalcoholic steatohepatitis (NASH) and primary sclerosing cholangitis (PSC) are liver disease with increased morbidity and mortality and no approved treatments. Therefore, there is need for new drugs that can treat NASH and PSC and reduce their burden.

• WHAT QUESTION DID THIS STUDY ADDRESS?

This study characterized the pharmacokinetics, pharmacodynamics, safety, and tolerability of cilofexor, a nonsteroidal farnesoid X receptor agonist, following single and multiple dose administration to healthy participants.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

The pharmacokinetic and pharmacodynamic data indicate that cilofexor doses of 30 to 100 mg once daily are pharmacologically active and support evaluation of this dose range in patients with NASH and PSC. Cilofexor was safe and well‐tolerated by healthy participants.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

The study results paved the way for efficacy trials and informed dose selection of cilofexor in phase II studies in patients with NASH and PSC.

INTRODUCTION

Nonalcoholic steatohepatitis (NASH) represents a spectrum of liver pathology that resembles alcohol‐induced fatty liver damage but occurs in subjects with no history of excessive alcohol consumption. It is characterized by the excess accumulation of lipid droplets within the liver that can result in progressive fibrosis, ultimately leading to cirrhosis in 10%–15% of affected patients and is associated with increased morbidity and mortality. ¹ , ² , ³ , ⁴ In the United States, it has been estimated that 1.5%–6.45% of the population have NASH ⁴ which is similar to the estimated NASH global prevalence of 2%–6%. ⁵ Primary sclerosing cholangitis (PSC) is a chronic and progressive cholestatic liver disease, characterized by fibro‐obliterative destruction of intrahepatic and/or extrahepatic bile ducts, which results in progressive biliary fibrosis and cirrhosis. ⁶ The overall incidence of PSC is 0.77 per 100,000 person‐years. ⁷ Currently, there are no approved treatments for either NASH or PSC.

Cilofexor is a nonsteroidal farnesoid X receptor (FXR) agonist being evaluated for the treatment of NASH (in combination with other investigational agents) and PSC (as monotherapy). ⁶ , ⁸ , ⁹ FXR is a critical regulator for bile acid homeostasis, glucose and lipid metabolism, intestinal bacterial growth, and hepatic regeneration. ¹⁰ In the intestine, FXR agonists results in the release of fibroblast growth factor 19 (FGF19) from intestinal epithelial cells. FGF19 is an endocrine peptide which drives a signaling cascade to decrease lipogenesis, gluconeogenesis, hepatic triglyceride accumulation, and bile acid synthesis. The reduction in bile acid synthesis can be measured by the concentration of bile acid precursor, 7α‐hydroxy‐4‐cholesten‐3‐one (C4). Administration of cilofexor reduced hepatic steatosis, improved liver function tests, and reduced serum bile acids accumulation in patients with NASH, ⁹ and improved liver function tests and markers of cholestasis in patients with PSC. ⁶ FXR agonists currently in preclinical and clinical development were recently reviewed by Fiorucci et al. ¹¹

Cilofexor shows high protein binding in human plasma (>99%). In vitro data demonstrated that cilofexor is metabolized mainly by CYP2C8 and CYP3A (data on file, Gilead Sciences, Inc.). Cilofexor is a substrate for the transporters P‐gp, BCRP, and OATPs (data on file, Gilead Sciences, Inc.).

Here, we report the results from the phase I first‐in‐human (FIH) study which characterized the pharmacokinetics, pharmacodynamics, safety, and tolerability of escalating single‐ and multiple‐ oral doses of cilofexor in healthy participants. Results from this study supported advancing the compound to phase II clinical evaluation and guided dose selection for phase II.

METHODS

Ethics statement

The study was conducted in accordance with recognized international scientific and ethical standards, including but not limited to the International Council for Harmonization guideline for Good Clinical Practice and the original principles embodied in the Declaration of Helsinki. The study was conducted at a single site in the United States (SeaView Research, Inc). The study protocol (ClinicalTrials.gov NCT02654002) was reviewed and approved by the institutional review board (Schulman Associates IRB, 4445 Lake Forest Drive, Suite 300, Cincinnati, OH 45242, USA). Informed consent was obtained from all individual participants included in the study.

Study participants

Eligible participants included men and nonpregnant, nonlactating women, nonsmoker between 18 and 45 years of age. Eligibility criteria also mandated a body mass index (BMI) between 19 and 30 kg/m², normal or clinically insignificant 12‐lead electrocardiogram (ECG) findings, normal renal function, no significant medical history, and general good health at the time of screening as determined by the investigators. Major exclusion criteria included pregnancy or lactation, any serious or active medical or psychiatric illness, receipt of any investigational drug or device (starting 30 days prior to first dose), the presence of alcohol or substance abuse, a positive test result for human immunodeficiency virus 1 antibody, hepatitis B surface antigen, or hepatitis C antibody, and any liver disease including Gilbert's disease. In addition, the use of any prescription or over‐the‐counter medications (except vitamins, acetaminophen, ibuprofen, and/or hormonal contraceptive medications) was prohibited, as was treatment with systemic steroids, immunosuppressant therapies, or chemotherapeutic agents within 3 months prior to screening or expected to receive these agents during the study.

Study design

This was a phase I, randomized, double‐blind, placebo‐controlled, single‐ and multiple‐dose study to evaluate the pharmacokinetics, pharmacodynamics, safety, and tolerability of cilofexor in healthy participants. The study design is depicted in Figure 1.

FIGURE 1.

Study design. Lower panel shows the study procedures for parts (a) and (b)

The study consisted of two parts (A and B). Within each cohort in both parts of the study, all participants received placebo on day −1 so that each participant can serve as their own control for evaluation of changes in pharmacodynamic biomarkers. Participants were then randomized to receive a single dose of either cilofexor (N = 12) or matching placebo (N = 3) on day 1, followed by a washout period (days 1 to 7), followed by multiple doses of cilofexor or placebo (same treatment assignment as the single dose portion) for 14 days (study days 7 to 20).

Part A: Single‐ and multiple‐ascending doses (cohorts 1 to 4)

Part A consisted of four randomized, staggered, cohorts of single‐ and multiple ascending doses of cilofexor. The administered doses of cilofexor were 10 mg (cohort 1), 30 mg (cohort 2), 100 mg (cohort 3), and 300 mg (cohort 4). Study drug was administered in the fasted state once daily.

Part B: Adaptive cohorts (cohorts 5 to 8)

Part B consisted of four adaptive cohorts. For each cohort, total daily doses, frequency of dosing (once or twice daily), and the meal condition for dosing (fasted vs. fed) were chosen based on available safety, pharmacokinetics, and/or pharmacodynamic data from part A. Once determined, the dose level, frequency of dosing, and meal condition remained consistent within a cohort. The administered doses of cilofexor were 100 mg single dose followed by multiple once daily doses (cohort 5), 50 mg single twice daily dose followed by multiple twice daily doses (cohort 6), 15 mg single twice daily dose followed by multiple twice daily doses (cohort 7), and 10 mg single dose followed by multiple once daily doses (cohort 8). All doses of study drug were administered following a moderate‐fat meal containing ~600 kcal, ~27% of which from fat.

Pharmacokinetic and pharmacodynamic sampling

Pharmacokinetic sampling occurred on study day 1 and day 20 (last day of multiple dosing) and pharmacodynamic sampling (FGF19, C4, and serum total and individual bile acids) occurred on day ‐1, day 1, and day 20. Sampling occurred at predose (≤5 min prior to dosing) and at 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 16, 24, 48, 72, and 96 h postdose. Trough samples for cilofexor plasma concentration analysis were collected predose daily on study days 8 to 19. Blood samples for pharmacodynamic biomarkers were also collected on study days 7, 9, 11, 13, 15, 17, and 19 at predose and 2, 4, and 12 h postdose. Dosing was at approximately the same time every day so the pharmacodynamic sampling on the different days was approximately time matched.

Bioanalytical procedures

Pharmacokinetics

A bioanalytical method for the determination of cilofexor in human plasma was developed and validated at Covance Laboratories, Inc. This method involved liquid extraction of cilofexor and its internal standards (CILO‐d₄) from human plasma followed by liquid chromatography tandem mass spectrometry (LC–MS/MS). Fifty μl aliquots of plasma samples were spiked with 25 μl of the internal standard followed by the addition of 300 μl of acetonitrile to precipitate plasma proteins. The chromatographic analysis was performed on a binary Shimadzu LC‐20 AD (Shimadzu Corporation) using a Luna C18 column (2.0 × 50 mm, 3 μm, Phenomenex) and optimized mobile phases consisting of water: formic acid (100:0.1; mobile phase A) and acetonitrile: formic acid (100:0.1; mobile phase B). Gradient elution was used with flow rate of 0.6 ml/min. Ionization and detection of cilofexor and internal standard were carried out on an API‐5000 triple quadrupole mass spectrometer (AB Sciex), equipped with Turbo Ion Spray MS/MS detection. Positive ions were monitored for both cilofexor and internal standard in multiple reaction monitoring mode. Quantitation was performed using parent → product ion (m/z) transitions of 586.2 → 212.1 for cilofexor and 592.1 → 218.1 for internal standard. Analyst software version 1.4.1 was used for LC–MS/MS parameter control and data collection. Cilofexor: internal standard peak area ratios obtained from a set of eight calibration standards were subjected to weighted (1/x ², where x is nominal concentration) linear least‐squares regression to generate a calibration curve equation, which was then used to calculate concentrations of cilofexor in a given sample from the cilofexor: internal standard peak area ratio obtained for that sample.

The calibration curve range was one to 1000 ng/ml. The percent coefficient of variation for interassay precision was <5.3%. The percent relative error for interassay accuracy range was 1.3% to 8.8%. Validation met the expectations presented in the US Food and Drug Administration (FDA) guidance for bioanalytical method validation. ¹² All the clinical samples were analyzed using the validated method within established sample stabilities.

Pharmacodynamics

Plasma concentrations of FGF19 were measured using an R&D Systems Quantikine enzyme‐linked immunosorbent assay kit (Pacific Biomarkers). The validated concetration range was 39 to 5000 pg/ml. Serum C4 concentrations were measured by LC–MS/MS (Metabolon). The validated concetration range was 2 to 500 ng/ml.

Primary and secondary bile acids were measured by LC–MS/MS (Metabolon) and summed to derive total serum bile acid concentrations.

Pharmacokinetic analyses

Pharmacokinetic parameters were estimated with Phoenix WinNonlin 6.4 software (Certara, LP) using standard noncompartmental methods. Samples quantified below the lower limit of quantification (LLOQ) that occurred prior to the achievement of the first quantifiable concentration were assigned a concentration value of zero. Samples that were below the LLOQ at all other timepoints were treated as missing data.

Pharmacokinetic parameters included area under the plasma concentration versus time curve (AUC) from time zero to the last quantifiable concentration (AUC_last), AUC extrapolated to infinite time (AUC_inf; single dose), AUC over the dosing interval (AUC_tau; multiple dose), maximum observed plasma concentration (C _max), time of C _max, concentration at 24 h postdose (C _tau; multiple dose), the elimination half‐life (t _½), and apparent oral clearance.

Pharmacodynamic analyses

Pharmacodynamic parameter calculated for FGF19 and C4 included the relative magnitude of change from day −1 to day 1 (day 1/day −1) and to day 20 (day 20/day −1) of partial AUC from 2 h postdose to 12 h postdose (AUC_2‐12). The area under the curve was calculated by the trapezoidal rule. Values below the LLOQ were imputed as one half of the LLOQ. Values above the upper limit of quantification (ULOQ) were imputed as the ULOQ plus 1 unit.

Pharmacokinetic/pharmacodynamic analyses

The relationship between cilofexor dose/exposure and changes in FGF19 and C4 AUC_2‐12 ratio were explored graphically. A Spearman correlation analysis was conducted between cilofexor plasma concentrations AUC_tau and day 20/day −1 FGF19 and C4 AUC_2‐8 and AUC_2‐12 ratios across all active cilofexor treatments.

Statistical analyses

No formal sample size calculation was performed. Empirically, a sample size of 15 subjects per cohort, including 12 on active drug and three on placebo was selected to characterize cilofexor pharmacokinetic, pharmacodynamic, safety, and tolerability in this phase I setting.

Dose proportionality

Dose proportionality was evaluated based on AUC_last, AUC_inf, and C _max on day 1 and AUC_tau, C _max, and C _tau on day 20 using an analysis of variance (ANOVA) and power model methods.

An ANOVA model was used to estimate the ratio of the dose‐normalized pharmacokinetic parameter at each dose level to the dose‐normalized pharmacokinetic parameter at the reference dose (30 mg) for each regimen (fasted once daily, fed once daily, and fed twice daily). The geometric least‐squares means ratio and the corresponding 90% confidence intervals (CIs) were estimated for each pharmacokinetic parameter of interest.

The power model had the general equation y = β0 × dose^β1, where y represented the dependent variables (e.g., AUC_last, AUC_inf, and C_max) and the exponent β1 in the power model was estimated by regressing the natural log‐transformed pharmacokinetic parameter on natural log dose for each regimen (fasted once daily, fed once daily, and fed twice daily). The population mean slope β1 and corresponding 90% CI for log (dose) was estimated.

Food effect

The effect of food on the pharmacokinetics of cilofexor was evaluated by comparing cilofexor pharmacokinetic parameters obtained following single‐ and multiple‐dose administration under fed (cohorts 5 and 8) and fasted conditions (cohorts 1 and 3) using a mixed‐effects model with fixed effects of dose (10 or 100 mg), food (fasted or fed), and food by dose interaction and a random subject effect.

Safety assessments

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 (MedDRA), version 19.1.

RESULTS

Participants’ disposition and demographics

Overall, 120 participants were enrolled in the study. Of the 120 randomized participants, 118 participants received at least one dose of study drug and were included in the Safety Analysis Set. Two participants (1 in cohort 6 and 1 in cohort 8) received the day −1 placebo dose but withdrew consent before receiving the first dose of study drug. Of the 118 participants who received study drug, 117 participants completed the study. One participant in cohort 7 was withdrawn from the study on day 7 due to an AE of grade 3 elevated alanine transaminase (ALT). The mean participants age was 35 years (range 18–45 years). Most participants were men (n = 49, 52.1%), White (n = 80, 85.1%), and Hispanic or Latino (n = 92, 97.9%). The mean (SD) BMI at baseline was 26.2 (2.6) kg/m². Demographics and baseline characteristics for each cohort are presented in Table S1.

Pharmacokinetics

Cilofexor plasma concentrations reached peak levels at ~1–4 h after oral dosing. Cilofexor plasma concentrations declined bi‐exponentially afterward with median terminal t _½ ranging from 2 to 13 h across the dose levels (Figure 2 and Table 1).

FIGURE 2.

Cilofexor mean plasma concentration versus time profiles following administration of single and multiple once daily (part (a)) and twice‐daily (part (b)) oral doses to healthy participants

TABLE 1.

Pharmacokinetic parameters for cilofexor following single‐dose and multiple‐dose administration

PK parameters	Cohort 1 10 mg q.d. fasted N = 12	Cohort 2 30 mg q.d. fasted N = 12	Cohort 3100 mg q.d. fasted N = 12	Cohort 4300 mg q.d. fasted N = 12	Cohort 5100 mg q.d. fed N = 12	Cohort 6 50 mg b.i.d. fed N = 12	Cohort 7 15 mg b.i.d. fed N = 12	Cohort 8 10 mg q.d. fed N = 11
Single dose
AUCinf (h•ng/ml)	1260 (30.4)	2470 (37.2)	7740 (93.9)	12,500 (33.9)	5020 (40.3)	3560 (30.7)	1410 (30.4)	924 (23.4)
C max (ng/ml)	304 (41.6)	580 (48.9)	2590 (118)	3060 (66.2)	928 (58.3)	666 (22.9)	340 (36.6)	210 (30.1)
T max (h)	3.0 (2.3, 4.0)	3.0 (2.3, 3.8)	1.3 (1.3, 2.8)	1.3 (1.0, 3.3)	3.8 (2.8, 4.5)	2.5 (2.0, 3.0)	3.0 (2.0, 3.8)	3.0 (2.0, 3.5)
t ½ (h)	5.0 (4.8, 6.2)	8.1 (5.0, 8.7)	10.7 (7.7, 11.9)	12.6 (11.3, 14.0)	10.4 (8.4, 11.7)	2.3 (2.0, 2.93)	1.8 (1.6, 2.1)	4.1 (3.9, 5.6)
CL/F (ml/h)	8660 (29.7)	13,500 (30.6)	20,400 (63.5)	28,800 (62.3)	24,600 (56.9)	15,300.1 (30.0)	11,300 (23.2)	11,400 (23.4)
Multiple dose
AUCtau (h•ng/ml)	1280 (35.8)	2890 (23.5)	6720 (59.2)	8490 (48.8)	4180 (46.8)	36,903 (22.7)	1290 (19.4)	848 (25.2)
C max (ng/ml)	322 (42.4)	718 (32.8)	2230 (75.9)	2330 (81.5)	819 (53.4)	698 (20.9)	290 (27.1)	187 (30.5)
C tau (ng/ml)	2.7 (55.5)	7.9 (50.3)	36.3 (42.5)	70.1 (56.3)	22.6 (37.4)	108 (78.6)	28 (49.7)	3.0 (50.6)
T max (h)	2.5 (1.5, 4.0)	3.0 (1.8, 3.3)	1.5 (1.0, 2.8)	3.3 (1.0, 5.0)	3.5 (3.0, 4.5)	3.0 (2.5, 3.5)	2.5 (2.50, 3.00)	2.5 (2.0, 4.0)
t ½ (h)	5.6 (5.1, 7.1)	10.4 (9.6, 13.3)	15.4 (11.4, 17.6)	15.9 (13.8, 16.9)	11.9 (10.3, 14.8)	2.50 (2.3, 2.7)	2.11 (2.1, 2.2)	4.7 (4.2, 5.2)
CLss/F (ml/h)	8640 (31.3)	11,000 (25.1)	19,700 (50.9)	47,500 (61.7)	29,400 (48.6)	14,200 (24.5)	12,000 (20.5)	12,500 (25.0)

Note: All PK parameters are reported to three significant digits as mean (%CV), except for T _max, and t _½, which are reported as median (Q1, Q3).

Abbreviations: AUC, area under the plasma concentration versus time curve; AUC_inf, AUC extrapolated to infinite time; AUC_last, AUC from time zero to the last quantifiable concentration; AUC_tau, AUC across the dosing interval; C₂₄, concentration at 24 h postdose; CL/F, apparent oral clearance; CL_ss/F, apparent oral steady‐state clearance; C _max, maximum observed plasma concentration; CV, coefficient of variation; PK, pharmacokinetic; T _max, time of maximum observed plasma concentration; t _½ = plasma half‐life.

In general, minimal to no accumulation of cilofexor was observed after multiple‐dose administration of cilofexor once daily or twice daily under fed or fasted conditions (Figure 2).

Cilofexor exposure parameters after single‐dose or multiple‐dose administration of cilofexor under fasted once daily, fed once, or twice daily administration increased in a less‐than‐proportional manner with dose over the evaluated single and once‐daily multiple dose range under fasting condition (Figure S1). Similarly, less‐than‐proportional increase in cilofexor exposure with dose was observed in the evaluated once‐daily or twice‐daily dose range under fed conditions (Table 1).

Cilofexor C _max was ~31% to 64% lower and cilofexor AUC was 27% to 38% lower when cilofexor (10 mg or 100 mg q.d. single and multiple doses) was administered with moderate‐fat meal compared to the administration of the same dose of cilofexor under fasting condition (Figure S2).

Pharmacodynamics

Plasma FGF19 and serum C4 steady‐state concentrations versus time profile are depicted in Figure 3.

FIGURE 3.

Steady‐state (day 20) mean plasma FGF19 (a) and serum C4 (b) concentration time profile following the administration of cilofexor once daily under fasted conditions to healthy participants. FGF19, fibroblast growth factor 19; C4, 7α‐hydroxy‐4‐cholesten‐3‐one

Plasma FGF19

FGF19 pharmacodynamic parameter ratios were unchanged in the placebo group on day 1 or day 20 (Table 2). FGF19 PD parameter ratios were higher following the administration of cilofexor on day 1 or day 20 compared with the relevant placebo group across all cilofexor doses levels (Table 2). Administration of cilofexor 100 mg dose with food resulted in a prolonged and elevated plasma concentration profile of FGF19 compared with administration of cilofexor under fasted conditions, particularly between 5 and 10 h postdose (Figure S2D).

TABLE 2.

Plasma FGF19 and serum C4 pharmacodynamic (AUC_2‐12) parameter ratios following single‐ (day 1) and multiple‐ (day 20) dose cilofexor once‐daily administration, normalized to baseline (day −1)

PD parameters	Cohort 1 10 mg q.d. fasted (N = 12)	Cohort 2 30 mg q.d. fasted (N = 12)	Cohort 3100 mg q.d. fasted (N = 12)	Cohort 4300 mg q.d. fasted (N = 12)	Cohort 5100 mg q.d. fed (N = 12)	Cohort 6 50 mg b.i.d. fed (N = 11)	Cohort 7 15 mg b.i.d. fed (N = 12)	Cohort 8 10 mg q.d. fed (N = 11)	All cohorts placebo fasted (N = 12)	All cohorts placebo fed (N = 12)
FGF19 day 1/day ‐1	2.04 (58.7)	1.96 (61.9)	2.04 (28.7)	2.31 (25.0)	2.41 (13.9)	2.63 (15.9)	1.74 (28.2)	1.37 (34.1)	0.98 (41.0)	0.92 (23.9)
FGF19 day 20/day ‐1	2.27 (82.9)	2.26 (41.9)	2.05 (27.1)	2.18 (52.5)	1.80 (25.1)	2.12 (33.0)	1.65 a (38.2)	1.51 (19.7)	1.25 (51.2)	0.96 (39.3)
C4 day 1/day ‐1	0.681 (113)	0.640 (29.1)	0.427 (33.0)	0.347 (36.7)	0.837 (50.5)	0.65 (18.6)	0.86 (23.1)	1.11 (39.7)	1.15 (37.2)	1.06 (25.8)
C4 day 20/day ‐1	0.66 (76.7)	0.38 (52.2)	0.50 (65.8)	0.33 (73.2)	0.62 (67.8)	0.17 (66.0)	0.62 a (38.4)	0.97 (47.2)	1.21 (91.2)	0.84 (36.7)

Note: All PD parameter ratios are reported to three significant digits as geometric mean (%CV).

Abbreviations: AUC, area under the plasma concentration versus time curve; AUC_2–8, partial AUC from time 2 h postdose to time 8 h postdose; AUC_2–12, partial AUC from time 2 h postdose to time 12 h postdose; C _max, maximum observed concentration; C _min, minimum suppression; CV, coefficient of variation; PD, pharmacodynamic.

^a For cohort 7, N = 11 for multiple dose administration.

Serum C4

C4 pharmacodynamic parameter ratios were unchanged in the placebo group on day 1 or day 20 (Table 2). C4 pharmacodynamic parameter ratios were lower following the administration of cilofexor on day 1 or day 20 compared with the relevant placebo group across all cilofexor doses levels (Table 2). Similar maximum suppression of C4 was observed after administration of cilofexor 100 mg with food or under fasted conditions (data not shown).

Bile acids

Modest dose‐dependent reductions in serum total bile acids from baseline were noted after multiple doses, q.d. administration of cilofexor (Figure S3).

Pharmacokinetic/pharmacodynamic

Cilofexor dose–response relationships (Figure S4) showed that all doses of cilofexor resulted in increased FGF19 ratios and doses >30 mg resulted in reductions of C4 ratios compared with placebo. Spearman correlation analyses for all active cilofexor treatment showed a modest positive relationship (r = 0.35, p = 0.003) between cilofexor AUC_tau and the day 20 FGF19 AUC_2‐12 ratio (Figure 4a) with the plateau in response observed at AUC ≥1000 ng * h/ml (cilofexor dose 30 mg and above). A stronger, inverse relationship (r = −0.61, p < 0.0001) was observed between cilofexor AUC_tau and the day 20/day −1 C4 AUC_2‐12 ratio (Figure 4b). The reduction in C4 continued to increase with the increase in cilofexor dose and the plateau of response was not observed. An inverse relationship (r = −0.40, p = 0.0017) was observed between FGF19 and C4 day 20/day −1 AUC_2‐12 ratios (Figure 4c).

FIGURE 4.

Cilofexor pharmacokinetic/pharmacodynamic analyses. Cilofexor AUC – FGF19 AUC_2‐12 relationship (a). Cilofexor AUC – C4 AUC_2‐12 relationship (b). FGF19 AUC_2‐12 – C4 AUC_2‐12 relationship (c). AUC, area under the plasma concentration–time curve; C4, 7α‐hydroxy‐4‐cholesten‐3‐one; FGF19, fibroblast growth factor 19

Safety

Cilofexor was generally well‐tolerated. No grade 3 or 4 AEs, serious AEs, or deaths were reported in any group. A total of 33 (35%) and seven (29%) participants who received cilofexor and placebo, respectively, experienced AEs (Table 3). The most frequently reported AEs were headache (12 participants), back pain (5 participants), contact dermatitis (4 participants), iron deficiency anemia (4 participants), diarrhea (3 participants), and viral infection (3 participants). No dose‐dependent trends in AEs were noted and all AEs were mild or moderate (grade 1 or 2) in severity. A total of four participants reported grade 2 AEs (back pain and presyncope, toothache, generalized pruritus, and sciatica). Only the AE of generalized pruritis was considered related to cilofexor (one participant who received cilofexor 100 mg under fed conditions).

TABLE 3.

Adverse events following once daily administration of cilofexor or placebo

Number of subjects (%) Experiencing	Cohort 1 10 mg q.d. fasted (N = 12)	Cohort 2 30 mg q.d. fasted (N = 12)	Cohort3100 mg q.d. fasted (N = 12)	Cohort 4300 mg q.d. fasted (N = 12)	Cohort 5100 mg q.d. fed (N = 12)	Cohort 6 50 mg b.i.d. fed (N = 11)	Cohort 7 15 mg b.i.d. fed (N = 12)	Cohort 8 10 mg q.d. fed (N = 11)	Pooled placebo N = 24 a
AEs	4 (33.3)	3 (25.0)	4 (33.3)	3 (25.0)	5 (41.7)	3 (27.3)	9 (75.0)	2 (18.2)	7 (29.2)
Treatment‐related AEs	0	0	0	0	1 (8.3)	0	4 (33.3)	0	1 (4.2)
Grade 3 or 4 AEs	0	0	0	0	0	0	0	0	0
Grade 2, 3, or 4 AEs	0	1 (8.3)	1 (8.3)	0	1 (8.3)	0	0	1 (9.1)	0
SAEs	0	0	0	0	0	0	0	0	0
Treatment‐related SAEs	0	0	0	0	0	0	0	0	0
AEs leading to permanent study drug discontinuation	0	0	0	0	0	0	1 (8.3)	0	0
AEs leading to permanent study discontinuation	0	0	0	0	0	0	1 (8.3)	0	0
Deaths	0	0	0	0	0	0	0	0	0

Abbreviations: AEs, adverse events; SAEs, serious adverse events.

^a Composite across cohorts.

Most laboratory abnormalities were grade 1 or 2 in severity. At total of four different grade 3 laboratory abnormalities were observed; of which only one was considered related to cilofexor. The participant received cilofexor 15 mg b.i.d. under fed conditions with a subsequent grade 3 elevation of ALT observed on day 7, resulting in the discontinuation of cilofexor. The ALT returned to normal range by day 19. No clinically relevant changes in 12‐lead ECGs, lipids (total cholesterol, high‐density lipoprotein [HDL], low‐density lipoprotein [LDL], and triglycerides) or vital signs (systolic blood pressure, diastolic blood pressure, pulse, temperature, and respiration rate) were observed during the study.

DISCUSSION

This study is the first clinical experiences with cilofexor, a novel, potent and selective FXR receptor agonist, in healthy participants. Cilofexor was well‐tolerated following the administration of single and multiple doses up to 300 mg daily. All AEs were mild to moderate in nature, with comparable frequency between subjects who received cilofexor or placebo. Most laboratory abnormalities were grade 1 or 2 in severity. One participant had a grade 3 elevated ALT that returned to normal range after discontinuation of cilofexor. Although this participant was withdrawn from the study, drug‐induced liver injury (DILI) was not suspected because there were no AEs related to DILI and the participant did not meet criteria for Hy's law. No clinically significant changes in vital signs, lipids, hematology, hepatobiliary, or renal laboratory metrics were observed.

Cilofexor displayed bi‐exponential plasma disposition with median t _½ ranging between 8 to 13 h at doses ≥30 mg. At doses <30 mg, shorter cilofexor t _1/2 was observed likely a result of plasma concentrations falling below the LLOQ during the distribution phase. Minimal to no accumulation of cilofexor was observed, consistent with a short effective half‐life of cilofexor. Administration of cilofexor 100 mg dose with moderate‐fat meal decreased steady‐state exposure of cilofexor. Cilofexor exposure increased in a less than dose proportional manner in the dose range 10 mg to 300 mg likely due to the low and pH‐dependent solubility of cilofexor.

The pharmacodynamic biomarkers (FGF19, C4, and bile acids) assessed in this study provide evidence of biological activity of cilofexor. FGF19 is a direct target gene of FXR in the ileum and C4 is an intermediate molecule in the bile acid synthesis pathway. FGF19 and C4 have been evaluated as biomarkers of disease severity in patients with NASH. ¹³ Placebo was administered to all participants on day −1 so as each participant acted as their own control for pharmacodynamic assessments. Administration of cilofexor increased plasma FGF19 levels and reduced serum C4 and bile acids levels which is consistent with cilofexor biological activity of FXR agonism. Cilofexor doses ≥30 mg appeared to achieve the plateau of intestinal FXR activation, as assessed by increased FGF19 exposure. The pharmacodynamic parameter ratios of FGF19 and C4 were unchanged in the placebo group confirming the biologic activity of cilofexor across all doses evaluated. Short‐term (14 days) administration of cilofexor also resulted in a modest dose‐dependent reduction in serum bile acids. This is consistent with observation in phase II studies where a reduction in bile acid pool was observed without significant changes in the proportion of bile acids. ⁶ , ⁹ Administration of the same total daily dose of cilofexor resulted in a comparable effect on pharmacodynamic biomarkers irrespective of the frequency of cilofexor administration (15 mg b.i.d. vs. 30 mg q.d. or 50 mg b.i.d. vs. 100 mg q.d.; Table 1). Both the 30 and 100 mg cilofexor doses maximized intestinal FXR agonism, whereas the 100 mg dose resulted in additional systemic FXR agonism as evidenced by the further reduction of serum C4 without further increase in plasma FGF19. These findings supported further evaluation of cilofexor at doses of 30 mg (in combination with other agents) and 100 mg (monotherapy) once daily in longer‐duration phase II studies patients with NASH or PSC, respectively.

This study used a design where multiple daily dosing was initiated after single dose administration (after a washout period) which is different from the typical sequential FIH study design where multiple dosing cohorts are initiated after completing few single ascending dose cohorts. This was possible because the FXR agonism mechanism of action had been explored in humans previously, the available nonclinical data suggested no accumulation of cilofexor upon with multiple dosing in human, and large nonclinical safety margin relative to the projected exposures.

CONCLUSION

Cilofexor displayed favorable safety and tolerability profiles following single‐ and multiple‐oral doses administration in the dose range 10 to 300 mg. Administration of cilofexor results in decreased FGF19 exposure and C4 suppression, confirming its biological activity. Cilofexor doses ≥30 mg appeared to achieve the plateau of intestinal FXR activation. Results from this study supported further evaluations, and informed dose selection, of cilofexor in phase II studies in patients with NASH and PSC.

AUTHOR CONTRIBUTIONS

I.R.Y., B.J.K., A.N.B., D.X., Q.S., T.W., and A.A.O. wrote the manuscript. B.J.K., A.N.B., D.X., and Q.S. designed the research. B.J.K., A.N.B., D.X., and Q.S. performed the research. I.R.Y., B.J.K., A.N.B., D.X., Q.S., T.W., and A.A.O. analyzed the data.

FUNDING INFORMATION

This research was funded by Gilead Sciences, Inc.

CONFLICT OF INTEREST

I.R.Y., A.N.B., Q.S., D.X., T.W., and A.A.O. are employees of, and may own stock in, Gilead Sciences, Inc. B.J.K. is a previous employee of Gilead Sciences, Inc., and may own stocks in Gilead Sciences, Inc.

Supporting information

Figure S1

Figure S2

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Table S1

Younis IR, Kirby BJ, Billin AN, et al. Pharmacokinetics, pharmacodynamics, safety and tolerability of cilofexor, a novel nonsteroidal Farnesoid X receptor agonist, in healthy volunteers. Clin Transl Sci. 2023;16:536‐547. doi: 10.1111/cts.13469

DATA AVAILABILITY STATEMENT

Anonymized individual patient data will be shared upon re‐quest for research purposes dependent on the nature of the request, the merit of the proposed research, the availability of the data, and its intended use. The full data‐sharing policy for Gilead Sciences, Inc., can be found at https://www.gileadclinicaltrials.com/transparency‐policy/.