Chronic hepatitis C virus (HCV) infection remains a major global health challenge, affecting more than 180 million individuals worldwide and leading to severe liver-related complications, including cirrhosis and hepatocellular carcinoma (Lavanchy, 2009). The advent of direct-acting antiviral agents (DAAs) targeting specific HCV proteins, such as the NS3/4A protease, has revolutionized treatment strategies, particularly for the difficult-to-treat genotype-1 (GT-1).

Faldaprevir (BI 201335) is a potent, selective HCV NS3/4A protease inhibitor. Preclinical evaluations demonstrated faldaprevir’s robust antiviral activity against HCV genotypes (GTs) 1, 4, 5, and 6 (White et al., 2010), along with a favorable absorption, distribution, metabolism, and excretion (ADME) profile in multiple animal models. These studies also highlighted its high metabolic stability and remarkable liver distribution, predicting promising pharmacokinetics (PK) and target organ exposure in humans (White et al., 2010). Faldaprevir (FDV) is a substrate of CYP 3A, a substrate of OATP 1B1, and a substrate and an inhibitor of P-gp (Chen et al., 2014; Joseph et al., 2014; Huang et al., 2017; 2021). At a therapeutic dose of 120 mg QD, FDV shows inhibitory effects against CYP2C9 and weak inhibitory effects against CYP3A4 but has no effect on other CYPs. Moreover, in combination with pegylated interferon alfa-2a (PegIFN) and ribavirin (RBV), faldaprevir achieved sustained virologic response (SVR) rates of up to 84% in treatment-naïve patients, without exacerbating the adverse event profile of the existing treatment regimen (Ferenci et al., 2015). In addition, it was also shown in Phase II trials that faldaprevir could be used in interferon-free regimens in which faldaprevir was combined with an NS5B polymerase inhibitor, deleobuvir (Zeuzem et al., 2014; 2015). FDV is currently under development for an interferon-free regimen for treatment of HCV infection (NCT02593162, 2024; NCT02716428, 2024) and is also considered for the treatment of COVID-19 (Keretsu et al., 2020; Luan et al., 2020; Gammeltoft et al., 2021).

The objective of this study was to assess the safety, tolerability, and pharmacokinetics (including assessment of dose linearity) of faldaprevir after single ascending oral doses and to preliminarily assess the effect of food on the pharmacokinetics of faldaprevir.

The study was conducted at the PHAROS GmbH, Ulm, Germany, in accordance with the International Conference on Harmonization guidelines for Good Clinical Practice and the principles of the Declaration of Helsinki. Before study initiation, the clinical trial protocol, the subject information, and the informed consent form were reviewed by the responsible local Independent Ethics Committee (Ethikkommittee der Landesärztekammer Baden-Württemberg, Stuttgart, Germany). The clinical trial application was also reviewed by the German Competent Authority (BfArM, Bonn, Germany).

The objectives of this study were 1) to characterize the pharmacokinetics of FDV, an inhibitor of HCV NS3/4A, in healthy male volunteers after single ascending oral-dose administration, 2) to preliminarily evaluate the effect of food on the pharmacokinetics of FDV, and 3) to document its safety and tolerability in these subjects.

For the SRD part of the study, six subjects received active drugs and two received placebos. Due to the lack of prior clinical data, it is usually not possible to accurately determine the sample size for the first time in human studies; however, it is generally believed that the selected sample size (6 + 2) is sufficient for the evaluation of the safety and preliminary pharmacokinetics of the new chemical entity (NCE) (Broom, 1990; Huang et al., 2008b).

This study was a first-in-human trial conducted in healthy male volunteers. However, the pharmacokinetics and safety of FDV in special populations, including HCV-infected patients and those with liver dysfunction, were evaluated in subsequent clinical studies, with findings reported in the previously published literature (Huang et al., 2016; Ferenci et al., 2015).

FDV has been shown to be actively absorbed into the liver. In rats, FDV demonstrated a 42-fold enrichment in the liver compared with plasma (Duan et al., 2012), which was accurately predicted using a rat HepatoPac model (Ramsden et al., 2014a). Using a human Hepato Pac model, 22–32-fold enrichments in the liver was predicted in humans (Ramsden et al., 2014b). Three processes contributed approximately equally to the total absorption of FDV, namely; (i) active uptake inhibited by rifamycin SV (related to OATP); (ii) active uptake not inhibited by rifamycin SV; (iii) and passive permeability (Li et al., 2014). With increased dose and concentration of FDV, the active hepatic uptake of FDV may be saturated and lead to more than a dose-proportional increase in plasma FDV concentrations. This may have contributed to a supra-proportional increase in AUC and C_max observed in the current study. The saturation of uptake of FDV into the liver may result in a concomitant decrease in the volume of distribution and systemic clearance, and it may lead to a minimal impact on the plasma t_1/2. The observed relatively longer t_1/2 at lower dose (≤120 mg) and relatively similar t_1/2 at higher dose [≥240 mg; considering t_1/2 values of 19.6 (CV% = 18%) for 480 mg from the food effect part suggested that, at low doses, FDV may be distributed (uptake) to tissues (compartment) other than the liver or at higher dose, FDV distributed far less to other tissues. A similar pattern of nonlinear pharmacokinetics was observed for grazoprevir (ZEPATIER™, 2024) and simeprevir (OLYSIO^®, 2024); both are NS3/4A HCV protease inhibitor, with hepatic uptake mediated by OATP1B. For simeprevir, a PBPK model suggested that saturation of OATP1B played a major role in the contribution of the observed non-linearity (Snoeys et al., 2016). For grazoprevir, the FDA clinical pharmacology reviewer agreed with the sponsor’s hypotheses that saturation of the OATP1B-mediated uptake process contributed to nonlinear PK observed for grazoprevir (FDA, 2012). The observed saturation of hepatic uptake via OATP1B as a potential cause of nonlinear pharmacokinetics for faldaprevir warrants further investigation. In vitro transporter studies and PBPK modeling could help validate this hypothesis and are considered potential directions for future research.

FDV is metabolized by CYP3A4 and is a substrate of the efflux transporter of P-gp; thus, saturation of these two processes may also contribute to the nonlinear PK observed in the present study. However, since systemic CYP3A4 (in the liver), but not intestinal CYP3A4, contributed to most of the metabolism of FDV (Li et al., 2014), and the half-life of FDV was not prolonged with the increased dose in the current study, saturation of CYP3A4 metabolism was not considered the main cause of the observed non-liner PK of FDV. Given the km values of 11.9 µM, at the dose of 120 mg (assuming intestinal volume of 250 mL), the efflux processes mediated by P-gp administration would be saturated already (Wu et al., 2016); thus, the contribution of the saturation of P-gp to the observed nonlinear PK of FDV was probably limited.

A clinically irrelevant increase in bioavailability by food was observed in the current study. This appeared to be consistent with the fact that FDV is a BCS II compound as it has been shown that food may increase the exposures of BCS II compounds (Lentz, 2008). The observed slight increase in BA under fed conditions in the current pilot study of FDV in solution was in good agreement with the observed increased BA when FDV capsules were given with high-fat and high-calorific breakfast in the later pivotal final food effect study (Wu et al., 2016). The 90% CI exceeding 125% may be attributable to underpowering as this is a pilot food-effect study. The slight increase in FDV exposure under fed conditions is not considered clinically relevant. In the FDV clinical development program, both 120-mg and 240-mg doses were evaluated in Phase II/III trials and demonstrated comparable safety profiles (Ferenci et al., 2015), with 120 mg selected as the therapeutic dose. FDV exposure at 240 mg was approximately 5-fold higher based on trough concentrations (Huang et al., 2016) and up to 7-fold higher based on AUC_0–24,_ss (Huang et al., 2021) compared to the 120-mg dose in HCV-infected patients. Therefore, the modest increase in exposure observed in the fed state falls within the established safety margin, and no dose adjustment is required when FDV is co-administered with high-fat or high-calorie food.

AEs observed in this study were consistent with the known FDV safety profiles (Chen et al., 2014; Huang et al., 2014; 2017; 2021; Joseph et al., 2014; 2015; Wu et al., 2016). Elevation of unconjugated bilirubin was observed in several subjects in the present study. It is likely related to the interaction of FDV with bilirubin. Bilirubin, formed from hemoglobin degradation, binds to albumin in the circulation, dissociates in hepatic blood flow, and enters hepatocytes via OATPs. Once inside hepatocytes, bilirubin binds to cytoplasmic proteins and is conjugated to glucuronic acid by UGTs in the endoplasmic reticulum. This conjugation process helps enhance bilirubin’s water solubility and thus its intrinsic clearance. Glucuronidated bilirubin is actively transported into bile via MRP2 at the canalicular membrane, and then, it is cleared into the feces (Sticova and Jirsa, 2013; Tátrai and Krajcsi, 2020). FDV is an inhibitor of OATP1B1, OATP1B3, UGT1A1, and MRP2, so FDV inhibits all steps of in vivo disposition of bilirubin and thus leads to benign accumulation of unconjugated bilirubin (Joseph et al., 2014; Wu et al., 2016; Huang et al., 2017; 2021); When FDV-mediated UGT inhibition is combined with predisposing genetics causing impaired bilirubin clearance, the incidence of benign hyperbilirubinemia (indirect bilirubin of >3.0 mg/dL) increases. In fact, genotyping in the seven subjects with an elevation of indirect bilirubin of >3.0 mg/dL in the current study showed a polymorphism (UGT1A1*28) associated with benign indirect hyperbilirubinemia in Gilbert’s Syndrome (Rotger et al., 2005; Lankisch et al., 2006) in six of these subjects. For all subjects with an elevation of benign unconjugated bilirubin, no clinically relevant changes in alanine transaminase, hemoglobin, or neutrophil count were observed. Slight elevations in direct bilirubin were observed in 14 subjects, and it is likely due to methodical overestimation of direct bilirubin via the Diazo reaction (Doumas and Wu, 1991; Kazmierczak et al., 2002). After comprehensively analyzed in vitro, preclinical and clinical data on the FDV development program, it was concluded that the FDV-mediated hyperbilirubinemia is not associated with any liver injury or toxicity and is considered to result from decreased bilirubin elimination due to the drug–bilirubin interaction (Sane et al., 2014). Elevated unconjugated bilirubin (hyperbilirubinemia) was also observed in patients treated with atazanavir (an inhibitor of UGTs and OATPs) and simeprevir (inhibitor of OATPs). Due to the benign nature of this observation (resulted from the interaction disposition process and reversible upon discontinuation), the observed hyperbilirubinemia is considered not important as it is not associated with any liver injury (Lankisch et al., 2006; OLYSIO^®, 2024). In clinical studies involving faldaprevir, bilirubin levels were closely monitored, and adverse events related to bilirubin elevation were reported and managed following regulatory guidelines. These findings have been documented in previously published studies (Sulkowski et al., 2013; Ferenci et al., 2015; Jensen et al., 2016; Chen et al., 2014; Huang et al., 2014; 2017; 2021; Joseph et al., 2014; 2015; Sane et al., 2014; Wu et al., 2016).

In summary, both AUC₀–   ∞ and C_max increased supra-proportionally to dose. Urinary excretion (f_e) of FDV was negligible. Food had no clinically relevant effect on FDV exposure (AUC and C_max). The FDV solution given as single doses of 4–1,200 mg was generally well tolerated: AEs were consistent with the known FDV safety profile; tolerability was reasonable, with increases in gastrointestinal symptoms and indirect hyperbilirubinemia at higher doses; increases in indirect bilirubin concentrations were reversible and not associated with decreases in alanine transaminase, hemoglobin, or neutrophil count. In conclusion, at single doses of 4–1,200 mg in healthy male subjects, FDV showed dose-dependent pharmacokinetics and was generally safe and well tolerated.