Phase I study of PF‐04895162, a Kv7 channel opener, reveals unexpected hepatotoxicity in healthy subjects, but not rats or monkeys: clinical evidence of disrupted bile acid homeostasis

Abstract During a randomized Phase 1 clinical trial the drug candidate, PF‐04895162 (ICA‐105665), caused transaminase elevations (≥grade 1) in six of eight healthy subjects treated at 300 mg twice daily for 2‐weeks (NCT01691274). This was unexpected since studies in rats (<6 months) and cynomolgus monkeys (<9 months) treated up to 100 mg/kg/day did not identify the liver as a target organ. Mechanistic studies showed PF‐04895162 had low cytotoxic potential in human hepatocytes, but inhibited liver mitochondrial function and bile salt export protein (BSEP) transport. Clinical relevance of these postulated mechanisms of liver injury was explored in three treated subjects that consented to analysis of residual pharmacokinetic plasma samples. Compared to a nonresponder, two subjects with transaminase elevations displayed higher levels of miRNA122 and total/conjugated bile acid species, whereas one demonstrated impaired postprandial clearance of systemic bile acids. Elevated taurine and glycine conjugated to unconjugated bile acid ratios were observed in two subjects, one before the onset of elevated transaminases. Based on the affinity of conjugated bile acid species for transport by BSEP, the profile of plasma conjugated/unconjugated bile acid species was consistent with inhibition of BSEP. These data collectively suggest that the human liver injury by PF‐04895162 was due to alterations in bile acid handling driven by dual BSEP/mitochondrial inhibition, two important risk factors associated with drug‐induced liver injury in humans. Alterations in systemic bile acid composition were more important than total bile acids in the manifestation of clinical liver injury and may be a very early biomarker of BSEP inhibition.


| INTRODUCTION
Severe drug-induced liver injury (DILI) is a challenging issue for health care providers, regulators, and pharmaceutical companies. In severe cases patients may require a liver transplant or experience death. Hepatotoxic agents can be broadly classified into two categories: intrinsic hepatotoxicants (those that cause liver injury predictably in humans and animals when given in sufficiently high doses) and idiosyncratic hepatotoxicants (those that cause liver injury in "susceptible" individual humans, are more varied in their clinical presentation, and generally do not cause hepatotoxicity in animals). 1 Hepatotoxic agents in the former category are detected in standardized nonclinical safety assessment studies and generally removed from further drug development. However, due to the low incidence of idiosyncratic hepatotoxicity, potential pharmaceutical agents in the latter category are more difficult to detect in standardized nonclinical/clinical studies and may not become evident until well after marketing approval is granted. There is a third category in practice where drug candidates cause a relatively high incidence of transaminase elevations in early clinical trials that were not detected in nonclinical safety assessment studies. Trying to mitigate these risks are the subject of many initiatives within the pharmaceutical industry 2-4 that can vary in their approach. [5][6][7][8] Integrating these approaches into decisions regarding medicinal design and compound selection is important since standard animal models only predict about 55% 9,10 of human transaminase elevations.PF-04895162 (ICA-105665, discovered by Icagen, Inc., Durham, NC), is a novel small molecule that showed signs of efficacy for the treatment of epilepsy 11 by opening neuronal Kv7.2/7.3 and Kv7.3/7.5 potassium channels. 12 In nonclinical studies, only a single 7-day exploratory toxicity study in rats showed a dose-dependent alanine aminotransferase (ALT) elevation that was not accompanied by any histological correlate. This finding was not confirmed in a repeat 7 day study at a higher dose in rats or in longer term safety assessment studies in rats and cynomolgus monkeys of 6 and 9 months in duration, respectively. Therefore, this drug candidate advanced into clinical studies in healthy subjects and patients with epilepsy. No evidence of liver injury in healthy subjects was observed in single dose studies up to 600 mg 11 or multiple doses up to 200 mg twice daily (BID) for 7 days. 13 Mild/moderate transaminase elevations were noted in one of 12 subjects each at 250 mg BID and 300 mg BID PF-04895162 for 7-days (Pfizer data on file). However, in a 14-day multiple dose study in healthy subjects, transaminase elevations as high as 5x the ULN were noted in six of eight subjects who received 300 mg BID PF-04895162.
This high incidence rate in a small subset of subjects led to the discontinuation of this drug candidate from further clinical development.
To investigate mechanistic factors possibly contributing to the observed liver injury, a broad suite of assays were conducted where inhibition of both mitochondrial function and BSEP transporter activity were highlighted as possible mechanisms. To establish a clinical correlation to these postulated mechanistic liabilities, that are known independent or combined drivers of liver injury, a few treated subjects from the 14-day clinical study consented to additional biomarker examination using residual pharmacokinetic samples. Elevations in miRNA122 levels coincided with ALT elevations, confirming liver origin of ALT. Total and individual tauro-and glyco-conjugated bile acids, which are purportedly preferential substrates for BSEP transport, were elevated in plasma from affected, but not unaffected subjects, suggesting some specific effect of PF-04895162 on hepatobiliary elimination of bile acids concurrently or in advance of liver injury. Although

| Bioethical statements for conduct of nonclinical studies
These studies were conducted in accordance with the United States

| Clinical study design and clinical trial objectives
A randomized, double-blind, third-party open (i.e., subject and Investigator blind, and Sponsor open), placebo-controlled study was conducted in healthy subjects to investigate the safety, tolerability, and pharmacokinetics of single and multiple oral doses of PF-04895162 and to characterize the effect of food on the pharmacokinetics of a single oral dose of PF-04895162. The study was conducted in accordance with the International Conference on Harmonization guideline for Good Clinical Practice and the principles of the Declaration of Helsinki and was approved by the local independent ethics committee. All subjects gave written informed consent prior to participation in the study. The study was planned as a parallel group, dose escalation study in four cohorts of 10 subjects each, however, the study was terminated prematurely and only Cohort 1 was conducted. After an initial screening visit to confirm eligibility, subjects in Cohort 1 ( Figure 1

| Subjects
Subjects were healthy males and/or females of nonchildbearing potential, aged 18 to 55 years inclusive with body mass index of 17.5 to 30.5 kg/m 2 and total body weight of >50 kg. Healthy was defined as no clinically relevant abnormalities identified by a detailed medical history, full physical examination, including blood pressure and pulse rate measurement, 12 lead electrocardiograms (ECGs) or clinical laboratory tests. Subjects with evidence or history of clinically significant disease (including drug allergies, but excluding

| Clinical assessments
Safety was assessed throughout the study by monitoring adverse events, vital signs (blood pressure, pulse rate, and body temperature),

| Statistical analysis used in the clinical study
The sample size of 10 subjects per cohort was chosen based on No formal hypothesis testing was conducted.

| Summary methods for in vitro investigative work
Methodology for the following in vitro assays and biomarker determinations were as follows: THLE/HepG 2 72 hour cytotoxicity assays based on ATP depletion; 14 mitochondrial functional assessment based on state 3/4 respiration determination in the presence of glutamate/ malate 15,16 or fatty acid oxidation inhibition using palmitate; 17 BSEP inhibition in vesicles using tritium labeled taurocholic acid (TCA); 18 human hepatocyte imaging assay determination of cell loss, mitochondrial membrane permeability, glutathione content, and production of reactive oxygen species; 19 miRNA-122 determination using Quanti-Gene 2.0 miRNA; 20 human multidrug resistance protein 3 inhibition/ activation (MDR3, encoded by ABCB4) was determined at Biotranex based on biotransformation of d 9 -choline to d 9 -phosphatidylcholine in human hepatocytes; 21 and fractionated bile acid determinations were conducted by LC-MS/MS methodology. 22 These various assays have been published in detail and used by us in previous investigative works. 2,3,21 Examination of human sodium/taurocholate cotransporting polypeptide (NTCP, encoded by SLC10A1) was determined based on uptake of tritium labeled TCA in CHO cells and human multidrug resistance-associated protein 3/4 (MRP3/4, encoded by ABCC3/4) transporter inhibition in vesicles obtained from HEK293 cells using tritium labeled estradiol 17 β-D-glucuronide and dehydroepiandrosterone sulfate as substrates, respectively. Both assays were conducted at Solvo Biotechnology (Hungary) according to their protocols. 23,24 Calculations for the Heuman index for hydrophobicity of bile acids in serum were based on the sums of bile acid hydrophobicity for each measured bile acid (AUC 0-12 h) where larger values are considered a more hydrophobic environment than smaller values. 25

| Nonclinical safety assessment summary
A summary of liver effects along with a detailed synopsis of each nonclinical study that was used to support the Investigational New Drug Application filing along with longer term studies can be found in the supplemental information associated with this article. In brief, this compound advanced into clinical studies without any histopathological evidence of liver injury or reproducible transaminase elevations in rats and monkeys. The addition of total bile acid analysis in nonclinical safety assessment studies, although germane to this investigation retrospectively, is not routine. 26 Absorption, distribution, metabolism, and excretion of parent and metabolites were examined in albino and pigmented male rats after administration of a single oral dose (target 15 mg/kg) of 14 C-labeled parent. Elimination of radioactivity occurred mostly within the first 24 hours after dosing with approximately 71 and 22% of the administered dose excreted in feces and urine, respectively. An equivalent human study was not performed.

| Healthy subject clinical study summary
Ten subjects were enrolled and received treatment in the clinical study. All subjects were male, aged 19 to 44 years, eight subjects were Black and two subjects were White. One subject was withdrawn due to an adverse event following 300 mg BID PF-04895162.
All other subjects completed the study.
Safety: All subjects reported at least 1 adverse event during the study ( Table 1). None of the adverse events were severe or serious.
One subject (Subject 7) discontinued after 3 days of 300 mg BID PF-04895162 due to clinically significant laboratory abnormalities

| Pharmacokinetics of PF-04895162
Peak concentrations of PF04895162 were achieved on Days 7 and 14 at a median of 2 hours postdose, which was earlier than the median observed following a single dose (5 hours under fasting conditions) (

| Evaluation of potential hepatotoxicity mechanisms
Since hepatotoxicity was not identified as a hazard in nonclinical studies, we performed a series of in vitro mechanistic studies. PF-04895162 did not display potent cytotoxic properties in THLE and

| Examination of residual plasma pharmacokinetic samples for biomarkers of liver injury
Three of eight treated healthy subjects consented for additional analyses of residual plasma PK samples for biomarkers of liver injury  AUC tau , area under the plasma concentration-time profile from over the dosing interval (12 h); C max , maximum plasma concentration; CV, Coefficient of variation; N, Number of subjects; NA, Not Applicable (t ½ , R ac , R ac ,C max , and R ss were determined only for Day 14); SD, Standard deviation; T max , time to maximum plasma concentration in hours (h); t ½ , terminal half-live, R ac , accumulation ratio (AUC tau,day14 /AUC tau,day1 ); R ac , C max , accumulation ratio (C max,day14 / C max,day 1 ); R ss , accumulation at steady state (AUC tau,day14 /AUC inf,day 1 ). a Values are presented as geometric mean along with their %Geometric CV in parenthesis (%Geometric CV) for all except: median (range) for T max , and arithmetic mean ± SD for t ½ .   initially and approach 0.22-0.25x at steady state (see Table 3). Morgan et al. 40  Potent BSEP inhibition by various drugs has been associated as a mechanism of liver injury in humans. 40 In cases like bosentan, elevations in total bile acids preceded transaminase elevations at high administered doses. 46 Subsequent analysis showed bosentan was a potent BSEP inhibitor (~12 μmol/L) and could raise total bile acids in rats. 46 While the association between high total serum bile acids preceding transaminase elevations is strong, it is interesting to note that genetic analysis in another clinical study showed a polymorphism in CYP2C9*2 (*2/rs1799853), and not BSEP (ABCB11, rs2287622), was highly associated with bosentan-induced transaminase elevations 47 despite known associations of BSEP polymorphisms with liver injury. 48 Regardless, potent BSEP inhibition, along with inhibition of other hepatic transporters, has been implicated in two recent clinical DILI examples. For example, BSEP inhibition was later implicated as causing liver injury in humans by AMG 009, an adverse finding that was not detected in preclinical safety studies. 40  examination of this small clinical study only made possible by access to clinical samples. This incident prompted the addition of standardized language in early clinical protocols to collect, retain and examine serum samples, leftover from the conduct of safety labs, for exploratory safety biomarker work in with subject consent in advance of the clinical trial start. Only in this way can we truly establish whether drug-induced liver injury is causally or only associated with alterations in bile acid homeostasis and the potential use of bile acids as surrogate markers. 51 In summary, the incidence of transaminase elevations in human healthy subjects treated with PF-04895162 led to its termination from clinical development before its therapeutic potential could be fully examined. Although preclinical safety assessment studies did not highlight the liver as a target organ, retrospective investigation using in vitro assays designed to detect potential mechanisms of liver injury demonstrated combined BSEP and mitochondrial inhibition as potential mechanisms that are recognized as dual risk factors for human DILI. 2 In our historical experience, for drug candidates that entered into clinical development without evidence of adverse liver injury in preclinical safety studies, 74% had BSEP inhibition properties where 55% also had mitochondrial inhibition/uncoupling activity (unpublished data). It remains to be determined why these risk factors may become apparent in early clinical trials vs postmarketing events. Weak inhibition of BSEP and MRP4 also correlated with a genetic analysis that showed polymorphisms in BSEP and MRP4 were also associated with both absolute and increased ALT levels in these responders. 52