Pharmacokinetics, safety, and tolerability of the 2‐ and 3‐direct‐acting antiviral combination of AL‐335, odalasvir, and simeprevir in healthy subjects

Abstract This Phase I, open‐label, two‐group, fixed‐sequence study evaluated the pharmacokinetics and safety of AL‐335, odalasvir, and simeprevir in healthy subjects. Group 1 (n = 16) received AL‐335 800 mg once daily (QD) (days 1‐3, 11‐13, and 21‐23), simeprevir 150 mg QD (days 4‐23), and odalasvir 150 mg (day 14) followed by 50 mg QD (days 15‐23). Group 2 (n = 16) received the same AL‐335 regimen as in Group 1 plus odalasvir 150 mg (day 4) followed by 50 mg QD (days 5‐23) and simeprevir 150 mg QD (days 14‐23). Blood samples were collected to determine plasma concentrations of AL‐335 (prodrug) and its metabolites, ALS‐022399 (monophosphate precursor) and ALS‐022227 (parent nucleoside), odalasvir, and simeprevir. Thirty‐two subjects were enrolled. Odalasvir and simeprevir given alone, or in combination, increased AL‐335 area under plasma concentration‐time curve over 24 hours (AUC 0‐24 h) 3‐, 4‐, and 7‐ to 8‐fold, respectively; ALS‐022399 AUC 0‐24 h increased 2‐, 2‐, and 3‐fold, respectively. Simeprevir had no effect on ALS‐022227 AUC 0‐24 h, whereas odalasvir with/without simeprevir increased ALS‐022227 AUC 0‐24 h 1.5‐fold. AL‐335 had no effect on odalasvir or simeprevir pharmacokinetics. Odalasvir and simeprevir AUC 0‐24 h increased 1.5‐ to 2‐fold for both drugs when coadministered irrespective of AL‐335 coadministration. Study medications were well tolerated with no serious adverse events. One subject prematurely discontinued study drugs (unrelated event). This study defined the preliminary pharmacokinetic and safety profiles of the combination of AL‐335, odalasvir, and simeprevir in healthy subjects. These data support the further evaluation of this combination for the treatment of chronic hepatitis C virus infection.

simeprevir in healthy subjects. These data support the further evaluation of this combination for the treatment of chronic hepatitis C virus infection. Current treatment options for HCV infection comprise a combination of two or three oral, direct-acting antiviral (DAA) compounds. 3,4 Evidence suggests that adding a third DAA with a different mechanism of action may increase efficacy and allow for a shorter treatment duration. [5][6][7][8][9][10] In recent studies evaluating 3-DAA regimens that include a nucleotide analog NS5B inhibitor (eg, sofosbuvir or dasabuvir), sustained virological response rates 12 weeks after the end of treatment (SVR12) were 80%-98% in treatment-na€ ıve patients with or without cirrhosis following 6 or 8 weeks of therapy [7][8][9]11 and 96%-98% in patients treated for 12-16 weeks who had previously failed on an NS5A-containing DAA regimen. 6,10 An 8-and 12-week 2-DAA regimen with glecaprevir (NS3/4A protease inhibitor) and pibrentasvir (NS5A inhibitor) has also been evaluated in treatment-na€ ıve or -experienced noncirrhotic genotype 1-infected patients demonstrating 99% and 100% SVR12 rates, respectively. 12 It has been suggested that the high SVR12 rates achieved with nucleotide-based 3-DAA regimens following a shortened treatment duration of 6 or 8 weeks may be partly ascribed to the more rapid first-phase decline in HCV ribonucleic acid (RNA) observed when a third DAA was added. 8 In addition to the high levels of therapeutic efficacy achieved with 2-or 3-DAA regimens, the opportunity to provide patients with an effective shorter course of therapy could also have a favorable impact on affordability, adherence, and patient quality of life due to reduced side effects and decreased pill burden. 8,13,14 Simeprevir is a HCV NS3/4A protease inhibitor licensed for the treatment of chronic HCV infection as a component of combination antiviral therapy. The recommended adult dose of simeprevir is 150 mg (100 mg in Japan) once daily (QD) administered with food.
Simeprevir is a substrate and inhibitor of P-glycoprotein, breast cancer resistance protein (BCRP), and organic anion-transporting polypeptide (OATP) 1B1/3, and is transported into the liver by OATP1B1/3 where it undergoes metabolism by cytochrome P450 (CYP) 3A. It is also a mild inhibitor of intestinal (not hepatic) CYP3A and a mild but not clinically relevant inhibitor of CYP1A2. 15 Coadministration of simeprevir with cyclosporine, an inhibitor of multiple transporters such as OATP and P-glycoprotein, and with CYP3A inhibitors and inducers such as ritonavir and efavirenz, has resulted in clinically significant drug interactions such that coadministration of simeprevir with these drugs is not recommended. 15 Odalasvir is an investigational HCV NS5A inhibitor in develop- The study design is shown in Figure 1. The primary objective of the study was to assess the 2-and 3way interaction between AL-335, odalasvir, and simeprevir in healthy subjects. A secondary objective was to determine the safety and tolerability of the DAAs alone and in combination.

| Study assessments
In both Groups 1 and 2, subjects were admitted to the clinic on day -1. Subjects remained in the clinic for the duration of the study with the exception of days 5-8 and days 15-18, where daily outpatient visits were performed. Subjects were discharged on day 24. The last study assessments were performed at the final follow-up visit 28 days after completion of the study confinement period. Analysis of variance (ANOVA) was applied to the log-transformed parameters and evaluated using a mixed-effects model, including treatment as fixed effect and subject as random effect. Least squares mean ratios of C max , AUC 0-24 h, and minimum observed plasma con- Screened and included in the study (n = 32)

| Bioanalysis and pharmacokinetic analysis
PK population (n = 31) b : all subjects who received at least one dose of study medication without any protocol deviation affecting PK evaluation and with complete profile(s) Safety population (n = 32): all subjects who received at least one dose of study medication, including those who did not complete the study Completed study assessments (n = 14) Completed study assessments (n = 15) Completed dosing (n = 15) F I G U R E 2 Subject disposition. PK pharmacokinetic; QD once daily; TEAE treatment-emergent adverse event. a Risk of interaction between treatment for tooth abscess and study drugs. b The subject that withdrew from the study on day 2, for reasons not related to the study, received two QD administrations of AL-335 800 mg alone (day 1 and day 2) before any PK samples were taken and was therefore excluded from the PK analysis was used to assess T max and a one-way ANOVA was used to assess steady-state on factor 'day'. Pharmacokinetic data were analyzed Unless otherwise stated, statistical tests for analyses were twosided at a 5% level of significance.

| Subjects
A total of 54 subjects were screened; 22 subjects were not included due to screening failures. Thirty-two healthy subjects were screened and included in the study; of these, 29 (91%) completed the study and 3 (9%) discontinued prematurely (Figure 2). Two subjects in Group 1 discontinued due to reasons not related to the study; one withdrew from the study on day 2 and thus received two QD administrations of AL-335 800 mg alone (day 1 and day 2) before any pharmacokinetic samples were taken and was therefore excluded from the pharmacokinetic analysis; the other subject withdrew from the study on day 19 and was included in the pharmacokinetic analysis. The third subject from Group 2 discontinued treatment early on day 11 due to a tooth abscess and was included in the pharmacokinetic analysis.
All 32 subjects were included in the safety analysis population and 31 were included in the pharmacokinetic analysis.
Subject demographic and baseline disease characteristics are summarized in Table 1. All 32 enrolled subjects (100%) were male, the majority were white (94%), mean age (AESD) was 39.0 AE 13.3 years, and mean body mass index (AESD) was 24.8 AE 3.8 kg/m 2 .

| Pharmacokinetics
Odalasvir and simeprevir, individually or in combination, had a marked effect on the pharmacokinetic profile of AL-335 and its metabolite ALS-022399, but less of an effect on ALS-022227 (Tables 2-4).  odalasvir or simeprevir, and a 2.6-to 3.4-fold increase was noted when AL-335 was co administered with both odalasvir and simeprevir (Table 3, Figure 3). No statistically significant difference in ALS-022399 T max was observed between any of the treatment combinations.

| Safety
A total of 20 treatment-emergent AEs (TEAEs) were reported in 12 subjects (37.5%) ( Unless otherwise stated, data for C max , C min, C last , AUC 0-24 h , and t 1/2 are shown as arithmetic mean AE SD and data for T max and T last are median (range).  10 days in Group 1) cannot be ruled out as a possible etiology to the different rates observed. Importantly, no fatigue event was considered to be clinically significant in this study as they were not associated with other abnormalities, premature study-drug discontinuation or other medical interventions.
TEAEs were more common in Group 2 than in Group 1 (5 TEAEs in 3 subjects in Group 1 and 15 TEAEs in 9 subjects in Group 2), with the major difference being the incidence of fatigue (1 vs. 7 events in Groups 1 and 2, respectively). After excluding this term, the incidence of TEAEs across the two groups was comparable (4 vs. 8 TEAEs in Groups 1 and 2, respectively).
Despite the numeric difference in incidence of TEAEs across the two groups, none of the events in either group appear to have been clinically concerning as evidenced by a lack of severe events or requirement of treatment discontinuation (with the exception of the subject that discontinued study treatment due to a tooth abscess).
Only two of the 20 TEAEs reported, oropharyngeal pain and tooth abscess, required concomitant treatment.
One study subject experienced clinically significant laboratory elevations in serum alanine aminotransferase (peak 5.5 9 ULN; grade 3) and aspartate aminotransferase (peak 3.    When the difference in odalasvir treatment duration is accounted for, the effect of simeprevir on odalasvir is reduced with a 10-15% C max increase and 12-19% AUC increase until day 17, compared with a 25% and 50% increase, respectively, at day 20 without accounting for this difference. The data from this study were used to guide the initial dose selection in Phase IIa and Phase IIb studies. The Phase IIa study, AL-335-604 (NCT02569710), is an ongoing study assessing the safety, pharmacokinetics, and efficacy of a 2-and 3-DAA regimen comprising AL-335 in combination with odalasvir with or without simeprevir in HCV-infected patients. 22 The Phase IIb study, OMEGA-1 (ClinicalTrials.gov ID: NCT02765490), is evaluating AL-335, odalasvir, and simeprevir for 6 and 8 weeks' treatment duration in HCV genotype 1-, 2-, 4-, 5-, and 6-infected patients without cirrhosis. 23 In conclusion, in this study, combinations of AL-335, odalasvir, and simeprevir were found to be well tolerated in healthy subjects Tooth abscess after odalasvir alone; ALT increase and fatigue (3 cases) after AL-335 + simeprevir + odalasvir. c Tooth abscess, considered to be unrelated to any of the study medications.