Adv Ther (2017) 34:2466–2480 DOI 10.1007/s12325-017-0634-4

ORIGINAL RESEARCH

Pharmacokinetic Evaluation of the Interactions of (ASP2151) with Ketoconazole, , Midazolam, and Warfarin in Healthy Adults

Tomohiro Kusawake . Martin den Adel . Dorien Groenendaal-van de Meent . Alberto Garcia-Hernandez . Akitsugu Takada . Kota Kato . Yoshiaki Ohtsu . Masataka Katashima

Received: September 1, 2017 / Published online: October 26, 2017 Ó The Author(s) 2017. This article is an open access publication

ABSTRACT single dose of midazolam (7.5 mg) and warfarin (25 mg). A drug interaction was considered to Introduction: Amenamevir is a nonnucleoside occur if the 90% confidence interval (CI) of the antiherpes virus compound available for treat- least squares geometric mean ratio (GMR) of ing herpes zoster . Four studies aimed amenamevir to the comparator was outside the to determine any potential interactions prespecified interval of 0.80–1.25. between amenamevir and ketoconazole, rifam- Results: Interactions were observed between picin, midazolam, or warfarin in healthy male amenamevir and ketoconazole, rifampicin, and participants. midazolam, but not between amenamevir and Methods: Two studies were open-label studies warfarin. After a single 400-mg dose of ame- that evaluated the effects of multiple doses of namevir, the GMRs of amenamevir plus keto- ketoconazole (400 mg) and rifampicin (600 mg) conazole or rifampicin versus amenamevir on the of a single oral dose of alone for Cmax and the area under the plasma amenamevir. The other two studies were ran- concentration–time curve from time zero to domized, double-blind, parallel-group studies infinity (AUCinf) were 1.30 (90% CI 1.17–1.45) that evaluated the effects of multiple doses of and 2.58 (90% CI 2.32–2.87), respectively, for amenamevir on the pharmacokinetics of a ketoconazole and 0.42 (90% CI 0.37–0.49) and 0.17 (90% CI 0.15–0.19), respectively, for Enhanced content To view enhanced content for this rifampicin. Following multiple doses of ame- article go to http://www.medengine.com/Redeem/ namevir (400 mg), the GMRs of midazolam plus C8CCF060639C490D. amenamevir versus midazolam alone for AUCinf and C were 0.53 (90% CI 0.47–0.61) and 0.63 T. Kusawake (&) max Astellas Pharma Global Development Inc., (90% CI 0.50–0.80), respectively. After a single Northbrook, IL, USA dose of warfarin, the (S)-warfarin and (R)-war- e-mail: [email protected] farin mean Cmax increased and mean AUCinf decreased in the presence of amenamevir; M. den Adel Á D. Groenendaal-van de Meent Á A. Garcia-Hernandez however, the 90% CIs of the GMRs for these Astellas Pharma Europe B.V., Leiden, The parameters remained within the predefined Netherlands limits. A. Takada Á M. Katashima Conclusion: These findings confirm that ame- Astellas Pharma Inc., Tokyo, Japan namevir (as a cytochrome P450 3A4 substrate) can interact with ketoconazole or rifampicin, K. Kato Á Y. Ohtsu Astellas Pharma Inc., Tsukuba, Japan and (as a cytochrome P450 3A4 inducer) can Adv Ther (2017) 34:2466–2480 2467 interact with midazolam; however, no interac- elimination pathway of amenamevir was hep- tion between amenamevir and (S)-warfarin was atic metabolism in animal studies (unpublished observed, indicating that amenamevir is not an data). In a study in healthy volunteers, urinary inducer of cytochrome P450 2C9. as unchanged drug was approxi- Funding: Astellas Pharma. mately 10% [9]. In vitro data further clarified Trial registration: EudraCT2007-002227-33 the main elimination pathway; the metabolism (study 15L-CL-008), EudraCT2007-002228-14 of amenamevir is significantly correlated with (study 15L-CL-009), EudraCT2007-002761-13 marker-enzyme activities specific for the (study 15L-CL-010), and EudraCT2007-002779- cytochrome P450 (CYP) isozymes CYP2B6, 14 (study 15L-CL-018). CYP2C19, and CYP3A4/5 [10]. The strongest correlation was observed for CYP3A4/5 2 Keywords: Amenamevir; Drug–drug (r = 0.9236, p\0.0001), suggesting CYP3A4/ interactions; Ketoconazole; Midazolam; 5-mediated metabolism is the main pathway for Pharmacokinetics; Rifampicin; Safety; elimination of amenamevir. In addition, Varicella–zoster virus; Warfarin in vitro enzyme induction data indicated that amenamevir potentially induces CYP3A4 and CYP2C9 activities, with a weak potential for INTRODUCTION inducing CYP2C19 (unpublished data). Herpes zoster is a major disease of the elderly Varicella–zoster virus (VZV) is a human her- population, and patients often require con- pesvirus (HHV) that leads to the development of comitant for the treatment of two distinct diseases: varicella (chicken pox) as other comorbidities [11]. Therefore, it is the first episode and herpes zoster () as important to determine any potential interac- the recurrent episode [1]. VZV is neurotrophic tions between medications whose metabolism is and remains latent in dorsal sensory ganglia mediated by CYP3A4 and CYP2C9. after the initial [2]. Herpes zoster is a This article summarizes the results of four manifestation of the reactivation of latent VZV. drug–drug interaction phase 1 studies that It is relatively uncommon in immunocompe- investigated potential interactions between tent individuals younger than 40 years, but the amenamevir and ketoconazole (a strong CYP3A4 incidence of herpes zoster markedly increases in inhibitor), rifampicin (a strong CYP3A4 inducer), those older than 50 years [3]. While the inci- midazolam (a CYP3A4 substrate), and (S)-war- dence rate of developing herpes zoster is 6–8 per farin (a CYP2C9 substrate) in healthy adults. 1000 person-years at 60 years of age, the inci- dence rate in patients aged 80 years or older increases to 8–12 per 1000 person-years [3]. METHODS Nucleoside analogs such as acyclovir [4], valacyclovir [5], and [6] have been Study Design approved for the treatment of virus 1, herpes simplex virus 2, and VZV infec- Each of the four drug–drug interaction studies tions. Although these drugs are used for the was conducted at a single study site in France by treatment of HHV infections, there is a medical the contract research organization SGS-Aster. need for therapies with improved antiviral Study 15L-CL-008 (EudraCT2007-002227-33) activity to provide more rapid and complete was conducted between October 17, 2007, and resolution of signs and symptoms. November 28, 2007, study 15L-CL-009 (Eu- Amenamevir (ASP2151) is a nonnucleoside draCT2007-002228-14) was conducted between antiherpes compound developed for the treat- October 19, 2007, and November 28, 2007, ment of VZV infections. Preclinical studies have study 15L-CL-010 (EudraCT2007-002761-13) indicated that amenamevir targets the viral was conducted between September 13, 2007, helicase–primase complex, which is essential for and November 17, 2007, and study 15L-CL-018 viral DNA replication [7, 8]. The main (EudraCT2007-002779-14) was conducted 2468 Adv Ther (2017) 34:2466–2480 between September 13, 2007, and December 3, respiratory, renal, hepatic, neurological, der- 2007. Two of the studies were open-label studies matological, psychiatric, or metabolic disorders, that evaluated the effects of multiple doses of as judged by the study investigator. Study ketoconazole (study 15L-CL-008) and rifampi- 15L-CL-018 excluded participants with a history cin (study 15L-CL-009) on the pharmacokinet- of and/or signs and symptoms of current ics of a single oral dose of amenamevir. The abnormal hemostasis or blood dyscrasia or other two studies were randomized, dou- abnormal prothrombin time (PT) or activated ble-blind, parallel-group studies that evaluated partial thromboplastin time at screening. the effects of multiple doses of amenamevir (200 and 400 mg) on the pharmacokinetics of a Dosing and Sampling Schedules single dose of midazolam (study 15L-CL-010) and a single dose of warfarin (study In study 15L-CL-008, participants received a 15L-CL-018). single oral dose of amenamevir (400 mg as two The study protocols were approved by the 200-mg tablets) on day 1, after which they institutional review boards at the study site underwent a 48-h washout period. They then ˆ ´ ´ [Hopital Ambroise-Pare, Secretariat du CPP Ile received ketoconazole orally (400 mg as two de France VIII, Laboratoire d’Anatomopatholo- 200-mg tablets) once daily on days 3–13, with a gie (studies 15L-CL-008 and 15L-CL-009) and second single dose of amenamevir (400 mg) ´ CPP Ile de France I, Secretariat du CPP Ile de administered in combination with ketocona- ˆ France I, Hopital Hotel-Dieu (studies zole on day 10. Blood samples for amenamevir 15L-CL-010 and 15L-CL-018)]. All procedures analysis were collected before dosing and followed were in accordance with the ethical 30 min and 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 16, 24, and standards of the responsible committee on 36 h after dosing on day 1, before dosing on day human experimentation (institutional and 3, and before dosing and 30 min and 1, 1.5, 2, 3, national) and with the Helsinki Declaration of 4, 5, 6, 8, 12, 16, 24, 36, 48, 60, 72, 84, and 96 h 1964, as revised in 2000. Informed consent was after dosing on day 10. obtained from all participants for their being In study 15L-CL-009, participants received a included in the study. single oral dose of amenamevir (400 mg) on day 1 and underwent a 48-h washout period, Study Participants after which they received rifampicin orally (600 mg as two 300-mg tablets) once daily on All four studies enrolled healthy male adults (aged days 3–11, with a second single dose of ame- 18–55 years). Studies 15L-CL-008 and 15L-CL-009 namevir (400 mg) administered with rifampi- required participants to have a body weight of at cin on day 10. Blood samples for amenamevir least 60 kg but less than 100 kg and a body mass analysis were collected before dosing and index of at least 18 kg/m2 but less than 30 kg/m2. 30 min and 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 16, 24, Studies 15L-CL-010 and 15L-CL-018 required and 36 h after dosing on day 1, before dosing participants to have a body mass index of at on day 3, and before dosing and 30 min and 1, least 18 kg/m2 but less than 30 kg/m2. The 1.5, 2, 3, 4, 5, 6, 8, 12, 16, 24, 36, and 48 h exclusion criteria included, but were not limited after dosing on day 10. to, the following: a known or suspected hyper- In study 15L-CL-010, participants were ran- sensitivity to any of the study drugs or any of domly assigned to receive amenamevir orally the components of the formulations used; any (200 or 400 mg) once daily on days 3–12, and all clinically significant history of asthma, eczema, participants received a single dose of midazolam or any other allergic or severe hypersensitivity (7.5 mg as one 7.5-mg tablet) on days 1 and 12. to any drug; a history of clinically significant Blood samples for analysis of midazolam were electrocardiogram (ECG) abnormalities or any collected before dosing and 15 and 30 min and clinically relevant history of other diseases or dis- 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 16, 24, and 36 h after orders, including cardiovascular, gastrointestinal, dosing on days 1 and 12. Adv Ther (2017) 34:2466–2480 2469

Study 15L-CL-018 had two treatment peri- Pharmacokinetic Assessments ods: in period 1, participants received a single oral dose of warfarin (25 mg; five 5-mg tablets) Plasma concentrations of amenamevir, mida- on day 1, followed by a 2-week washout period; zolam, (S)-warfarin, and (R)-warfarin were in period 2, participants were randomly determined by validated liquid chromatogra- assigned to receive amenamevir (200 or 400 mg) phy–tandem mass spectrometry methods. The once daily on days 1–17, and all participants amenamevir method included direct plasma received warfarin (25 mg) in combination with injection (MAYI-ODS trapping column), fol- amenamevir on day 10. Blood samples for war- lowed by reverse phase (Xterra MS C18, farin analysis were obtained before dosing and 30 mm 9 4.6 mm) liquid chromatography with 1, 2, 4, 6, 8, 12, 16, 24, 36, 48, 60, 72, 96, 120, tandem mass spectrometry detection (TSQ 144, and 168 h after the first single dose of Quantum with atmospheric pressure chemical warfarin on day 1 of period 1, and on day 10 of ionization interface in negative mode). The period 2, and PT analysis was performed before method is validated over a range of dosing and 1, 4, 8, 12, 24, 48, 72, 120, and 168 h 5–10,000 ng/mL with 0.2 mL plasma. The after the first single dose of warfarin on day 1 of midazolam method involved a liquid–liquid period 1, and on day 10 of period 2. extraction with methyl tert-butyl ether at pH 10, In all four studies, amenamevir was admin- followed by reverse phase (Chromolith Perfor- istered under fed conditions. In studies mance, RP-18e, 100 mm 9 4.6 mm) liquid 15L-CL-008, 15L-CL-010, and 15L-CL-018, chromatography with tandem mass spectrome- amenamevir and the comparator were both try detection (API3000 with atmospheric pres- administered with 240 mL of water within sure chemical ionization interface in positive 5 min after a standardized breakfast. In study mode). The method is validated over a range of 15L-CL-009, rifampicin was administered with 0.1–100 ng/mL with 0.25 mL plasma. The war- 120 mL of water 1 h before the participants ate a farin method involved a solid-phase extraction standardized breakfast, and amenamevir was on Oasis HLB 1 cc 30-mg cartridges, followed by administered with 120 mL of water within chiral (CHIRALCEL ODR-L 250 mm x 4.6 mm) 5 min after breakfast. Participants drank 120 mL liquid chromatography with tandem mass of water 1 h before breakfast on day 1 for con- spectrometry detection (API3000 with electro- sistency with day-10 dosing. Amenamevir was spray ionization interface in negative mode). administered with food on the basis of the The method is validated over a range of results of a phase I study in healthy volunteers 5–2500 ng/mL with 0.1 mL plasma. that showed that the pharmacokinetics of The pharmacokinetic parameters assessed amenamevir were affected by food, with the included plasma maximum concentration area under the plasma concentration–time (Cmax), AUCinf, time to plasma Cmax (tmax), plasma curve (AUC) from time zero to infinity (AUCinf) half-life (t‘), total plasma clearance (CL/F). In all almost doubling when amenamevir was four studies, the least squares (LS) geometric administered with food [12]. mean ratios (GMRs) of Cmax and AUCinf were also In studies 15L-CL-010 and 15L-CL-018, calculated, and if the 90% confidence interval blinding of the amenamevir dose was main- (CI) for both Cmax and AUCinf was within the tained by administration of one active 200-mg prespecified interval of 0.80–1.25, the absence of amenamevir tablet and one matching placebo significant interaction between amenamevir and to participants assigned to receive amenamevir ketoconazole (study 15L-CL-008), amenamevir at a dose of 200 mg, and administration of two and rifampicin (study 15L-CL-009), amenamevir active 200-mg amenamevir tablets to those and midazolam (study 15L-CL-010), or ame- randomized to receive amenamevir at a dose of namevir and warfarin (study 15L-CL-018) was 400 mg. concluded. 2470 Adv Ther (2017) 34:2466–2480

Pharmacodynamic Assessments Cmax) and a power of 85% or 90%, respectively, for each individual test. Assuming a weak In study 15L-CL-018, the pharmacodynamic induction effect, so that the 90% CI for the assessments for warfarin included AUC for PT treatment ratio ranged from 80% to 125%, the from time zero to 168 h (AUCPT;0–168 h), maxi- number of participants required for each ame- mum PT (PTmax), and time to reach PTmax namevir dose group (200 and 400 mg) was 20 (tPT;max). A significant interaction between (study 15L-CL-010) or 16 (study 15L-CL-018). amenamevir and warfarin was also concluded if Assuming a drop-out rate of 8–10%, 22 partici- the 90% CI for the LS GMR of AUCPT;0–168 h and pants per dose group were recruited in study PTmax was outside the prespecified interval of 15L-CL-010, and 18 participants per dose group 0.80–1.25. were recruited in study 15L-CL-018. Calculations were performed with actual Safety Assessments sampling times by noncompartmental methods with use of WinNonlinÒ Professional version 5.0 or higher (Pharsight, Mountain View, CA, In all four studies, safety assessments included Ò USA) or SAS version 8.2 or higher (SAS Insti- the evaluation of the frequency and severity of tute, Cary, NC, USA). Studies 15L-CL-008, treatment-emergent adverse events (TEAEs), 15L-CL-009, and 15L-CL-010 had two analysis 12-lead ECG recordings, clinical laboratory set populations: the safety analysis set (SAF; parameters (biochemistry, hematology, serol- defined as all participants who received at least ogy, and urinalysis), vital signs (blood pressure one dose of the study drug) and the pharma- and pulse), and physical examination findings. cokinetics analysis set (PKAS; defined as all TEAEs were coded according to the Medical participants in the SAF population for whom Dictionary for Regulatory Activities version 9.1 sufficient plasma concentration data were (study 15L-CL-010) or version 10.1 (remaining available to provide at least one pharmacoki- studies), and were summarized by system organ netic parameter). In addition to the SAF and class and preferred term. PKAS populations, study 15L-CL-018 included a pharmacodynamic analysis set, which included Statistical Analysis all participants from the SAF population for whom sufficient plasma concentration data Statistical analysis was conducted by Astellas were available to provide at least one pharma- Pharma. For studies 15L-CL-008 and codynamic parameter. 15L-CL-009, the sample sizes needed to evaluate For each study, absence of a clinically sig- the interaction between amenamevir and keto- nificant pharmacokinetic drug–drug interaction conazole or rifampicin were calculated with an was concluded if the 90% CI for the both the assumed coefficient of variation for the AUC AUCinf ratio and the Cmax ratio fell within the ratio of 18.45% and for the Cmax ratio of prespecified interval of 0.80–1.25 according to 21.40%, so the 90% CI for the treatment ratio the following hypothesis testing: had a multiplicative distance from the ratio to • H0 (null hypothesis): AUCinf ratio less than the outer limits of 1.125. From these calcula- 0.80 or greater than 1.25. tions, the number of participants required was • H1 (alternative hypothesis): AUCinf 20; given an assumed drop-out rate of 8–10%, a ratio 0.80 or greater and 1.25 or less. total of 22 participants were planned and Identical hypothesis testing were performed recruited for each of these studies. For studies for Cmax. AUCinf and Cmax were logarithmically 15L-CL-010 and 15L-CL-018, the sample sizes transformed (natural logarithm) and then sub- needed to demonstrate the absence of a clini- jected to a general linear model including per- cally relevant interaction between amenamevir iod and participant as fixed factors.. and midazolam or warfarin were calculated with In study 15L-CL-018, the same hypothesis an assumed coefficient of variation of 19.4% testing was performed for the pharmacody-

(for midazolam Cmax) or 15.6% (for warfarin namic parameters AUCPT;0–168 h and PTmax. Adv Ther (2017) 34:2466–2480 2471

Table 1 Summary of baseline characteristics Study 15L-CL-008 15L-CL-009 15L-CL-010 15L-CL-018 5 5 (n 22) (n 22) Amenamevir Amenamevir Amenamevir Amenamevir 200 mg 400 mg 200 mg 400 mg (n 5 22) (n 5 22) (n 5 18) (n 5 19) Age (years) 30.0 ± 8.4 31.9 ± 7.9 36.3 ± 9.5 34.1 ± 10.9 30.3 ± 9.4 29.5 ± 8.5 Race White 13 (59.1%) 16 (72.7%) 16 (72.7%) 18 (81.8%) 13 (72.2%) 12 (63.2%) Black or 6 (27.3%) 3 (13.6%) 6 (27.3%) 3 (13.6%) 4 (22.2%) 3 (15.8%) African American Asian 0 0 0 0 0 0 Other 3 (13.6%) 3 (13.6%) 0 1 (4.5%) 1 (5.6%) 4 (21.1%) Weight (kg) 78.2 ± 10.6 77.6 ± 8.9 74.2 ± 11.1 74.6 ± 9.1 74.0 ± 8.8 77.7 ± 9.3 Height (m) 1.77 ± 0.05 1.76 ± 0.06 1.75 ± 0.05 1.75 ± 0.05 1.77 ± 0.05 1.79 ± 0.05 BMI (kg/m2) 25.0 ± 2.5 25.1 ± 2.7 24.1 ± 3.2 24.3 ± 2.8 23.6 ± 2.6 24.3 ± 2.6 All values are presented as the mean ± standard deviation unless stated otherwise BMI body mass index

RESULTS Fig. 1 Mean amenamevir plasma concentration versus c time curve after administration of a single 400-mg dose of amenamevir a alone (open circles) and with ketoconazole Study Population at a dosage of 400 mg once daily (closed circles), and b alone (open circles) and with rifampicin at a dosage of Studies 15L-CL-008 and 15L-CL-009 both 600 mg once daily (closed circles) enrolled a total of 22 healthy male participants, all of whom received the study drug, completed the study, and were included in the SAF and deviations in study drug adherence in study PKAS populations. In study 15L-CL-010, 44 15L-CL-008, 15L-CL-009, or 15L-CL-010. male participants were randomized and treated, The demographics and baseline characteris- and all participants completed the study and tics of the participants in the four studies are were included in the SAF and PKAS populations. summarized in Table 1. In study 15L-CL-018, 37 male participants were randomized and treated, and 35 participants Pharmacokinetics completed the study. In this study, two partic- ipants prematurely discontinued their partici- Effects of Ketoconazole on Amenamevir pation in the study after receiving amenamevir Pharmacokinetics at a dosage of 400 mg once daily for 3 and After a single 400-mg dose of amenamevir in 8 days, respectively, because of withdrawal of study 15L-CL-008, the amenamevir mean Cmax consent; therefore, the SAF population con- and AUCinf were increased in the presence of sisted of 37 participants and the PKAS popula- ketoconazole administered at a dosage of tion consisted of 35 participants. There were no 400 mg once daily compared with amenamevir 2472 Adv Ther (2017) 34:2466–2480 Adv Ther (2017) 34:2466–2480 2473 alone. The plasma concentration versus time amenamevir and ketoconazole was confirmed profile for amenamevir alone and with keto- as the entire 90% CI was above the predefined conazole is shown in Fig. 1a. The amenamevir upper limit. tmax showed no difference between the absence or presence of ketoconazole. However, with Effects of Rifampicin on Amenamevir ketoconazole coadministration, the mean t‘ for Pharmacokinetics amenamevir increased 2.2-fold, while CL/F After a single 400-mg dose of amenamevir in decreased 2.6-fold. A summary of amenamevir study 15L-CL-009, the amenamevir mean Cmax pharmacokinetics in the absence and presence and AUCinf were decreased during administra- of ketoconazole is presented in Table 2. tion of rifampicin at a dosage of 600 mg once On the basis of the LS GMR of amenamevir daily compared with amenamevir alone. The plus ketoconazole versus amenamevir alone for amenamevir mean tmax also tended to decrease Cmax of 1.30 (90% CI 1.17–1.45), the null during rifampicin coadministration. The hypothesis of a relevant interaction between plasma concentration versus time profile for amenamevir and ketoconazole was not rejected amenamevir alone and with rifampicin is as the 90% CI for the Cmax LS GMR was not shown in Fig. 1b. In the presence of rifampicin, within the predefined limits. For the LS GMR for the amenamevir mean t‘ decreased 2.7-fold and AUCinf of 2.58 (90% CI 2.32–2.87), the existence the amenamevir mean CL/F increased sixfold of a significant interaction between compared with amenamevir alone. A summary

Table 2 Summary of amenamevir pharmacokinetic parameters after administration of a single 400-mg dose of amenamevir alone and with ketoconazole at a dosage of 400 mg once daily or rifampicin at a dosage of 600 mg once daily Parameter Study 15L-CL-008 15L-CL-009 Amenamevir alone Amenamevir 1 ketoconazole Amenamevir alone Amenamevir 1 rifampicin (n 5 22) (n 5 22) (n 5 22) (n 5 22)

AUCinf 19,917 ± 5561 51,483 ± 12048 18,658 ± 4935 3115 ± 930 (ng h/mL)a

Cmax 1448 ± 329 1886 ± 448 1325 ± 282 563 ± 218 (ng/mL)a b tmax (h) 4.0 (1.0–5.0) 4.0 (3.0–6.0) 3.0 (0.5–5.0) 1.75 (1.0–5.0) t‘ (h) 8.1 ± 1.9 17.5 ± 2.9 8.0 ± 1.2 2.9 ± 0.6 CL/F (L/h) 20.8 ± 5.9 8.0 ± 2.1 22.1 ± 5.9 133.5 ± 40.0 LS GMRc Amenamevir ? ketoconazole vs amenamevir alone Amenamevir ? rifampicin vs amenamevir alone

AUCinf 2.58 (2.32–2.87) 0.17 (0.15–0.19)

Cmax 1.30 (1.17–1.45) 0.42 (0.37–0.49) All values are presented as the arithmetic mean ± standard deviation unless stated otherwise AUCinf area under the plasma concentration versus time curve from time zero to infinity, CL/F clearance, Cmax maximum plasma concentration, LS GMR least squares geometric mean ratio, t‘ half-life, tmax time to maximum plasma concentration a Values are presented as the geometric mean ± standard deviation b Values are presented as the median, with the range in parentheses c The 90% confidence interval is given in parentheses 2474 Adv Ther (2017) 34:2466–2480

Fig. 2 Mean midazolam plasma concentration versus time alone (open triangles) and with amenamevir at a dosage of curve after administration of a single 7.5-mg dose of 400 mg once daily (closed triangles) midazolam either alone (open circles) and with ame- namevir at a dosage of 200 mg once daily (closed circles) or of the amenamevir pharmacokinetics in the administration of amenamevir at a dosage of absence and presence of rifampicin is presented 400 mg once daily compared with midazolam in Table 2. alone, although during administration of ame- On the basis of the LS GMR of amenamevir namevir at a dosage of 200 mg once daily, only plus rifampicin versus amenamevir alone for the midazolam AUCinf decreased. The plasma both Cmax and AUCinf of 0.42 (90% CI concentration versus time profile for midazo- 0.37–0.49) and 0.17 (90% CI 0.15–0.19), lam after administration of midazolam alone respectively, a significant interaction between and with amenamevir at dosages of 200 and amenamevir and rifampicin was confirmed as 400 mg once daily is shown in Fig. 2. The the 90% CIs for both parameters were entirely changes in midazolam exposure were associated outside the predefined limits. with a reduction in midazolam mean t‘ and an increase in mean CL/F. A summary of the Effects of Amenamevir on Midazolam midazolam pharmacokinetics in the absence Pharmacokinetics and presence of amenamevir at a dosage of 200 In study 15L-CL-010, multiple doses of ame- or 400 mg once daily is presented in Table 3. namevir (200 and 400 mg) once daily both On the basis of the LS GMR for AUCinf (0.72), affected the pharmacokinetics of a single the amenamevir 200-mg dose shows a signifi- 7.5-mg dose of midazolam, with the 400-mg cant interaction with midazolam, as the 90% CI dose showing the most pronounced effects. for this ratio (0.65–0.79) was entirely outside After a single dose of midazolam, the midazo- the predefined limits. On the basis of the LS C lam mean AUCinf and Cmax decreased during GMRs for both max and AUCinf of 0.63 (90% CI Adv Ther (2017) 34:2466–2480 2475

Table 3 Summary of midazolam pharmacokinetic parameters after administration of a single 7.5-mg dose of midazolam alone and with amenamevir at a dosage of 200 or 400 mg once daily in study 15L-CL-010 Parameter Amenamevir 200 mg Amenamevir 400 mg Midazolam alone Midazolam 1 amenamevir Midazolam alone Midazolam 1 amenamevir (n 5 22) (n 5 22) (n 5 22) (n 5 22)

AUCinf (ng h/ 104.5 ± 64.7 74.8 ± 37.5 107.5 ± 55.8 57.3 ± 25.7 mL)a

Cmax (ng/ 26.3 ± 13.7 25.9 ± 19.1 30.2 ± 22.7 19.0 ± 12.9 mL)a b tmax (h) 1.5 (0.5–3.0) 1.0 (0.3–4.0) 1.5 (0.3–3.0) 1.0 (0.5–4.0) t‘ (h) 5.7 ± 2.5 4.8 ± 1.9 5.3 ± 1.8 4.3 ± 1.4 CL/F (L/h) 80.1 ± 37.0 109.0 ± 43.4 78.2 ± 39.2 143.8 ± 68.0 LS GMRc Midazolam ? amenamevir 200 mg vs midazolam Midazolam ? amenamevir 400 mg vs midazolam alone alone

AUCinf 0.72 (0.65–0.79) 0.53 (0.47–0.61)

Cmax 0.98 (0.82–1.18) 0.63 (0.50–0.80) All values are presented as the arithmetic mean ± standard deviation unless stated otherwise AUCinf area under the plasma concentration versus time curve from time zero to infinity, CL/F clearance, Cmax maximum plasma concentration, LS GMR least squares geometric mean ratio, t‘ half-life, tmax time to maximum plasma concentration a Values are presented as the geometric mean ± standard deviation b Values are presented as the median, with the range in parentheses c The 90% confidence interval is given in parentheses

0.50–0.80) and 0.53 (90% CI 0.47–0.61), pharmacokinetic interaction between ame- respectively, which both have 90% CIs that namevir and (S)-warfarin or (R)-warfarin. were entirely outside the predefined limits, a The mean (S)-warfarin t‘ and CL/F values significant interaction occurred between ame- were similar during administration of both namevir at a dosage of 400 mg once daily and doses of amenamevir compared with warfarin midazolam. The LS GMR of an amenamevir alone. The mean (S)-warfarin tmax was similar dose of 400 mg versus an amenamevir dose of to that of warfarin plus amenamevir at dose of

200 mg was 0.64 (90% CI 0.48–0.86) for Cmax 200 mg and warfarin alone, but tended to be and 0.75 (90% CI 0.63–0.88) for AUCinf. lower with warfarin plus amenamevir at a dose of 400 mg versus warfarin alone. The mean Effects of Amenamevir on Warfarin (R)-warfarin t‘ values in the presence of ame- Pharmacokinetics namevir at a dosage of 200 or 400 mg once After a single 25-mg dose of warfarin in study daily were slightly lower than those observed 15L-CL-018, the (S)-warfarin and (R)-warfarin with warfarin alone, while the mean (R)-war- t mean Cmax values were slightly higher and the farin CL/F and max values remained similar AUCinf values were slightly lower during both in the presence and in absence of ame- administration of amenamevir at a dosage of namevir. A summary of the (S)-warfarin and 200 or 400 mg once daily compared with war- (R)-warfarin pharmacokinetic parameters in the farin alone. However, as the 90% CIs of the LS absence and presence of amenamevir at a GMRs for these parameters remained within the dosage of 200 or 400 mg once daily is pre- predefined limits, there was no significant sented in Table 4. 2476

Table 4 Summary of (S)-warfarin and (R)-warfarin pharmacokinetic parameters after administration of a single 25-mg dose of warfarin alone and with amenamevir at a dosage of 200 or 400 mg once daily in study 15L-CL-018 Parameter (S)-Warfarin (R)-Warfarin Warfarin alone Warfarin 1 Warfarin Warfarin Warfarin Warfarin Warfarin Warfarin (n 5 18) amenamevir alone (n 1 amenamevir alone 1 amenamevir alone 1 amenamevir 200 mg 5 17) 400 mg (n 5 18) 200 mg (n 5 17) 400 mg (n 5 18) (n 5 15) (n 5 18) (n 5 15)

AUCinf 48,072 ± 19,712 46,725 ± 18,437 50,329 ± 24,609 45,032 ± 23,503 69,581 ± 18,385 67,092 ± 18,315 75,652 ± 18,160 68,723 ± 12,736 (ng h/ mL)a

Cmax (ng/ 1194 ± 132 1217 ± 134 1168 ± 162 1251 ± 237 1197 ± 133 1240 ± 134 1187 ± 149 1262 ± 232 mL)a b tmax (h) 4.0 (2.0–4.0) 4.0 (1.0–4.0) 4.0 (1.0–4.0) 2.0 (1.0–4.0) 4.0 (2.0–4.0) 4.0 (1.0–12.0) 4.0 (2.0–12.0) 4.0 (2.0–12.0) t‘ (h) 34.6 ± 8.6 35.0 ± 7.1 36.4 ± 12.2 36.4 ± 9.7 42.0 ± 7.0 39.7 ± 6.3 45.7 ± 8.5 40.8 ± 6.0 CL/F (L/ 0.25 ± 0.07 0.26 ± 0.07 0.25 ± 0.08 0.27 ± 0.08 0.17 ± 0.04 0.18 ± 0.04 0.16 ± 0.04 0.17 ± 0.03 h) LS GMRc (S)-Warfarin ? amenamevir (S)-Warfarin ? amenamevir (R)-Warfarin ? amenamevir (R)-Warfarin ? amenamevir 200 mg vs (S)-warfarin alone 400 mg vs (S)-warfarin alone 200 mg vs (R)-warfarin alone 400 mg vs (R)-warfarin alone

AUCinf 0.97 (0.94–1.01) 0.92 (0.89–0.96) 0.96 (0.94–0.99) 0.91 (0.88–0.95)

Cmax 1.02 (0.98–1.06) 1.08 (1.02–1.15) 1.04 (1.01–1.07) 1.07 (1.01–1.13)

All values are presented as the arithmetic mean ± standard deviation unless stated otherwise 34:2466–2480 (2017) Ther Adv AUCinf area under the plasma concentration versus time curve from time zero to infinity, CL/F clearance, Cmax maximum plasma concentration, LS GMR least squares geometric mean ratio, t‘, half-life, tmax time to maximum plasma concentration a Values are presented as the geometric mean ± standard deviation b Values are presented as the median, with the range in parentheses c The 90% confidence interval is given in parentheses Adv Ther (2017) 34:2466–2480 2477

Table 5 Summary of warfarin pharmacodynamic (prothrombin time, PT) parameters after administration of a single 25-mg dose of warfarin alone and with amenamevir at a dosage of 200 or 400 mg once daily in study 15L-CL-018 Parameter Amenamevir 200 mg Amenamevir 400 mg Warfarin alone Warfarin 1 amenamevir Warfarin alone Warfarin 1 amenamevir (n 5 18) 200 mg (n 5 18) (n 5 17) 400 mg (n 5 15)

AUCPT;0–168 h 2480 ± 246 2411 ± 199 2556 ± 254 2425 ± 163 (s h)a

PTmax (s) 17.2 ± 2.3 17.3 ± 2.5 17.9 ± 2.4 16.7 ± 1.9 tPTmax (h) 36.0 ± 12.4 35.9 ± 12.3 39.5 ± 14.6 36.0 ± 13.6 LS mean ratiob Warfarin ? amenamevir 200 mg vs warfarin alone Warfarin ? amenamevir 400 mg vs warfarin alone

AUCPT;0–168 h 0.97 (0.94–1.00) 0.96 (0.95–0.98)

PTmax 1.00 (0.96–1.04) 0.95 (0.93–0.97) All values are presented as the arithmetic mean ± standard deviation unless stated otherwise AUCPT;0–168 h area under the prothrombin time versus time curve from time zero to 168 h, LS least squares, PTmax maximum prothrombin time, tPTmax time to maximum prothrombin time a Values are presented as the geometric mean ± standard deviation b The 90% confidence interval is given in parentheses

Pharmacodynamics Safety

Effects of Amenamevir on Warfarin Across all four studies, there were no deaths, seri- Pharmacodynamics ous TEAEs, or TEAEs that resulted in discontinu-

After a single dose of warfarin, mean PTmax ation of participation in the study. In addition, no values were slightly lower during administra- clinically significant changes from the baseline tion of amenamevir at a dosage of 400 mg were observed in laboratory parameters, vital once daily and were reached slightly earlier signs, or 12-lead ECG recordings, and physical with both amenamevir doses than with war- examination findings remained normal. farin alone. Compared with warfarin alone, In study 15L-CL-008, 13 TEAEs were reported mean AUCPT;0–168 h values showed no differ- by eight participants (36.4%). Headache was the ence with amenamevir at a dosage of 200 mg only TEAE reported by more than one partici- once daily and were slightly lower with ame- pant (five participants in total; 22.7%), and was namevir at a dosage of 400 mg once daily. On reported during administration of ketoconazole the basis of the LS mean ratios for PTmax and alone (four participants; 18.2%) and ame- AUCPT;0–168 h, there was no significant phar- namevir plus ketoconazole (one participant; macodynamic interaction between ame- 4.5%). All cases of headache were considered to namevir and warfarin, as the 90% CIs for both be TEAEs related to the study drug. ratios remained within the predefined limits In study 15L-CL-009, four participants for both doses of amenamevir. A summary of (18.2%) reported four TEAEs overall. The only the warfarin pharmacodynamic parameters in TEAE to be reported by more than one participant the absence and presence of amenamevir at a (two participants in total; 9,1%) was flatulence. dosage of 200 or 400 mg once daily is pre- All TEAEs in this study were considered by the sented in Table 5. investigator to be related to the study drug. 2478 Adv Ther (2017) 34:2466–2480

In study 15L-CL-010, TEAEs were reported by rifampicin on the pharmacokinetic profile of two participants (9.1%) in the group that amenamevir were to be expected. received the 200-mg dose of amenamevir and In these clinical studies, a significant inter- by two participants (9.1%) in group that action between amenamevir and midazolam (a received the 400-mg dose of amenamevir. None CYP3A4 substrate) was also confirmed, with of the TEAEs were reported by more than one significant decreases in midazolam exposure participant. Palpitations and asthenia (midazo- when a single 7.5-mg dose of midazolam was lam plus amenamevir at a dose of 400 mg) were coadministered with amenamevir at a dosage of TEAEs that were considered to be related to the 400 mg once daily compared with midazolam study drug. alone. This pharmacokinetic interaction with In study 15L-CL-018, six TEAEs were repor- amenamevir appears to be related to the dose, ted by five participants (27.8%) in the group with numerically greater decreases in midazo- that received amenamevir at a dose of 200 mg, lam exposure with 400 mg versus 200 mg once and six TEAEs were reported by three partici- daily administration of amenamevir. The pants (15.8%) in the group that received ame- interaction between amenamevir and midazo- namevir at a dose of 400 mg. None of the TEAEs lam confirms that amenamevir is a CYP3A4 were reported by more than one participant. In enzyme inducer. this study, eight TEAEs were considered to be In these studies, no significant pharmacoki- related to the study drug: two TEAEs with war- netic or pharmacodynamic interactions were farin alone (nausea and dyspepsia), four TEAEs observed between amenamevir and (S)-warfarin with amenamevir alone (myalgia with the (a CYP2C9 substrate). In contrast to in vitro 200-mg dose, and back pain, abdominal pain, data, which suggested that amenamevir is a and dyspepsia with the 400-mg dose), and two potential CYP2C9 inducer (unpublished data), TEAEs with warfarin plus amenamevir (head- data from these human studies indicate that ache with 200-mg amenamevir dose and oral amenamevir is not an inducer of CYP2C9. herpes with the 400-mg amenamevir dose). Amenamevir was safe and generally well tolerated in these studies, with no deaths or serious adverse events reported and no clini- DISCUSSION cally significant changes in vital signs, clinical laboratory parameters, or ECG recordings. The viral helicase inhibitor amenamevir is a These observations are consistent with those of nonnucleoside anti-HHV drug available for the previous clinical studies of amenamevir in treatment of VZV infections. On the basis of patients with recurrent genital herpes infection in vitro data, amenamevir is thought to be [13]. predominantly metabolized by CYP3A4/5, and These studies have some limitations that is a potential inducer of CYP3A4 and CYP2C9 may affect the interpretation of the findings. As (unpublished data). However, no clinical with most drug–drug interaction studies, the drug–drug interaction studies have previously study populations were relatively small, and been conducted. randomized controlled studies with larger Data from the four clinical studies in healthy patient populations are needed to confirm the humans described here confirm the existence of safety and tolerability of amenamevir in clinical interactions between amenamevir and keto- practice. conazole (a strong CYP3A4 inhibitor) and between amenamevir and rifampicin (a strong CYP3A4 inducer). Amenamevir exposure was CONCLUSIONS significantly increased during coadministration with ketoconazole and significantly decreased These studies confirm that amenamevir (as a during coadministration with rifampicin. Given CYP3A4 substrate) can interact with ketocona- that amenamevir is known to be a CYP3A4 zole or rifampicin, and (as a CYP3A4 inducer) substrate, the effects of ketoconazole and can interact with midazolam in a Adv Ther (2017) 34:2466–2480 2479 dose-dependent fashion; however, no signifi- integrity of the work as a whole, and have given cant interaction was observed between ame- final approval for the version to be published. namevir and (S)-warfarin, indicating that amenamevir may not be an inducer of CYP2C9. Disclosures. Tomohiro Kusawake is an Information regarding potential interactions employee of Astellas Pharma. Martin den Adel is between amenamevir and other drugs is partic- an employee of Astellas Pharma. Dorien Groe- ularly important in elderly or immunocompro- nendaal-van de Meent is an employee of Astel- mised patient populations (who may be las Pharma. Alberto Garcia-Hernandez is an receiving treatment with other drugs), and employee of Astellas Pharma. Akitsugu Takada appropriate caution will be needed in patients is an employee of Astellas Pharma. Kota Kato is receiving concomitant medications. The rec- an employee of Astellas Pharma. Yoshiaki Ohtsu ommended dose of amenamevir for VZV infec- is an employee of Astellas Pharma. Masataka tion is 400 mg/day by oral administration for Katashima is an employee of Astellas Pharma. 7 days. Because of this, the drug interactions The authors have no other relevant affiliations seen in these studies may not be a significant or financial involvement with any organization issue to manage in the clinical setting, as the or entity with a financial interest in, or a amenamevir administration period is relatively financial conflict with, the subject matter or short. However, appropriate caution will be materials discussed in the article other than needed in the following situations: those disclosed. 1. CYP3A4 substrates: CYP3A4 substrates should be used with caution because of a Compliance with Ethics Guidelines. The potential decrease in their exposure. study protocols were approved by the institu- 2. CYP3A4 inducers: Amenamevir and strong tional review boards at the study site [Hoˆpital CYP3A4 inducers should not be coadminis- Ambroise-Pare´, Secre´tariat du CPP Ile de France tered. Amenamevir exposure was demon- VIII, Laboratoire d’Anatomopathologie (studies strated to decrease, which could possibly 15L-CL-008 and 15L-CL-009) and CPP Ile de result in reduction or loss of antiviral France I, Secre´tariat du CPP Ile de France I, efficacy. Hoˆpital Hotel-Dieu (studies 15L-CL-010 and 3. CYP3A4 inhibitors: Strong CYP3A4 inhibi- 15L-CL-018)]. All procedures followed were in tors should be used with caution because of accordance with the ethical standards of the a potential increase in amenamevir responsible committee on human experimen- exposure. tation (institutional and national) and with the Helsinki Declaration of 1964, as revised in 2000. Informed consent was obtained from all par- ACKNOWLEDGEMENTS ticipants for their being included in the study. Data Availability. The datasets generated Sponsorship for the studies, article processing during and/or analyzed during the current charges, and open access fee were funded by study are not publicly available because these Astellas Pharma. All authors had full access to studies were small single-center studies and all of the data in these studies and take com- anonymization of these data is difficult to plete responsibility for the integrity of the data achieve. and accuracy of the data analysis. We thank Sarah Grieg of Springer Healthcare Communi- Open Access. This article is distributed cations who wrote the first draft of the manu- under the terms of the Creative Commons script. This medical writing assistance was Attribution-NonCommercial 4.0 International funded by Astellas Pharma. All named authors License (http://creativecommons.org/licenses/ meet the International Committee of Medical by-nc/4.0/), which permits any noncommer- Journal Editors (ICMJE) criteria for authorship cial use, distribution, and reproduction in any for the manuscript, take responsibility for the medium, provided you give appropriate credit 2480 Adv Ther (2017) 34:2466–2480 to the original author(s) and the source, provide 7. Chono K, Katsumata K, Kontani T, et al. ASP2151, a a link to the Creative Commons license, and novel helicase-primase inhibitor, possesses antiviral activity against varicella-zoster virus and herpes indicate if changes were made. simplex virus types 1 and 2. J Antimicrob Che- mother. 2010;65(8):1733–41.

8. Himaki T, Masui Y, Chono K, et al. Efficacy of REFERENCES ASP2151, a helicase-primase inhibitor, against thy- midine kinase-deficient herpes simplex virus type 2 infection in vitro and in vivo. Antiviral Res. 1. Arvin AM. Varicella-zoster virus. Clin Microbiol 2012;93(2):301–4. Rev. 1996;9(3):361–81. 9. Ohtsu Y, van Trigt R, Takama K, et al. Quantifica- 2. Gilden D, Mahalingam R, Nagel MA, Pugazhenthi S, tion of ASP2151 in human plasma and urine: a Cohrs RJ. Review: the neurobiology of varicella pitfall associated with supersaturation of analyte in zoster virus infection. Neuropathol Appl Neurobiol. urine. Chromatographia. 2017;80(2):217–27. 2011;37(5):441–63. 10. Katsumata K, Chono K, Kato K, et al. Pharmacoki- 3. Kawai K, Gebremeskel BG, Acosta CJ. Systematic netics and pharmacodynamics of ASP2151, a heli- review of incidence and complications of herpes case-primase inhibitor, in a murine model of herpes zoster: towards a global perspective. BMJ Open. simplex virus infection. Antimicrob Agents Che- 2014;4(6):e004833. mother. 2013;57(3):1339–46.

4. GlaxoSmithKline. ZOVIRAXÒ (acyclovir) capsules, 11. Johnson RW, Alvarez-Pasquin MJ, Bijl M, et al. tablets, suspension: prescribing information. 2005. Herpes zoster epidemiology, management, and https://www.accessdata.fda.gov/drugsatfda_docs/ disease and economic burden in Europe: a multi- label/2005/018828s030,020089s019,019909s020lbl. disciplinary perspective. Ther Adv Vaccines. pdf. Accessed 21 Jul 2017. 2015;3(4):109–20.

5. GlaxoSmithKline. VALTREXÒ (valacyclovir 12. Kusawake T, Keirns J, Kowalski D, et al. Pharma- hydrochloride) caplets: prescribing information. cokinetics and safety of amenamevir in healthy 2008. https://www.accessdata.fda.gov/drugsatfda_ subjects: analysis of four randomized phase 1 docs/label/2008/020487s014lbl.pdf. Accessed 21 studies. Advances in Therapy (under review). Jul 2017. 13. Tyring S, Wald A, Zadeikis N, Dhadda S, Take- 6. . FamvirÒ (famciclovir) tablets: prescribing nouchi K, Rorig R. ASP2151 for the treatment of information. 2006. https://www.accessdata.fda. genital herpes: a randomized, double-blind, pla- gov/drugsatfda_docs/label/2006/020363s026lbl.pdf. cebo- and valacyclovir-controlled, dose-finding Accessed 21 July 2017. study. J Infect Dis. 2012;205(7):1100–10.