key: cord-0768515-uu4h5hdh
authors: Humeniuk, Rita; Mathias, Anita; Cao, Huyen; Osinusi, Anu; Shen, Gong; Chng, Estelle; Ling, John; Vu, Amanda; German, Polina
title: Safety, Tolerability, and Pharmacokinetics of Remdesivir, an Antiviral for Treatment of COVID‐19, in Healthy Subjects
date: 2020-06-26
journal: Clin Transl Sci
DOI: 10.1111/cts.12840
sha: e592e1c7cab5f7d430288a5f6b08ba710fc5f170
doc_id: 768515
cord_uid: uu4h5hdh

Remdesivir (RDV), a single diastereomeric monophosphoroamidate prodrug that inhibits viral RNA polymerases, has potent in vitro antiviral activity against SARS‐CoV2. RDV received FDA’s emergency use authorization in United States and approval in Japan for treatment of severe COVID‐19 patients. This report describes two phase 1 studies that evaluated the safety and pharmacokinetics (PK) of single escalating and multiple intravenous (IV) doses of RDV (solution or lyophilized formulation) in healthy subjects. Lyophilized formulation was evaluated for potential future use in clinical trials due to its storage stability in resource‐limited settings. All adverse events were Grade 1 or 2 in severity. Overall, RDV exhibited a linear profile following single‐dose IV administration over 2 hours of RDV solution formulation across the dose range of 3 to 225mg. Both lyophilized and solution formulations provided comparable PK parameters. High intracellular concentrations of the active triphosphate (approximately 220 to 370‐ fold higher than the in vitro EC50 against SARS‐CoV‐2 clinical isolate) were achieved following infusion of 75 mg or 150 mg lyophilized formulation over 30 minutes or 2 hours. Following multiple‐doses of RDV 150mg once daily for 7 or 14 days, RDV exhibited a PK profile similar to single‐dose administration. Metabolite GS‐441524 accumulated approximately 1.9‐fold after daily dosing. Overall, RDV exhibited favorable safety and PK profiles that supported once‐daily dosing.

Coronaviruses (CoVs) are positive-sense, single-stranded, enveloped RNA viruses with high mutation and recombination rates which allow them to cross species barriers and adapt to new hosts. (1) (2) (3) The genetically diverse CoV family, comprised of four groups: alpha-CoV, beta-CoV, gamma-CoV and delta-CoV, infects a variety of avian and mammalian species (such as camels, cats, bats). (1, (4) (5) (6) Human CoVs generally cause mild to moderate upperrespiratory tract illnesses. However, recent years have seen the emergence of more virulent strains, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV), that can cause life-threatening illness. (4, 6, 7) In December 2019, SARS-CoV-2 was identified as the cause of an outbreak of respiratory illness in Wuhan, China. SARS-CoV-2 shared 79% sequence identity with SARS-CoV and both viruses utilized the angiotensin converting enzyme II (ACE-2) receptor for cell entry. (8, 9) This outbreak was declared a Public Health Emergency of International Concern by the World Health Organization (WHO) on 30 January 2020. (10) The clinical disease termed Coronavirus Disease 2019 or COVID-19 is highly transmissible. The incubation period of COVID-19 ranges from 1-14 days. Common clinical symptoms include fever, fatigue, dry cough, shortness of breath, and myalgia. (11, 12) In severe cases, COVID-19 can cause pneumonia, acute respiratory distress syndrome, organ failure, septic shock, and death. (13) Worldwide there are over 7.9 million infections with over 430,000 deaths related to There are currently no approved effective therapeutic agents available for the treatment of Remdesivir (RDV, GS-5734) is a diastereomeric monophosphoramidate prodrug which undergoes metabolic activation to form intracellularly the active triphosphate, GS-443902. Briefly, RDV is extensively metabolized by hydrolase activity and forms an intermediate metabolite, GS-704277 which then is subject to cleavage of the phosphoramidate bond resulting in the formation of the nucleoside analog monophosphate which is further phosphorylated to the pharmacologically active nucleoside

This article is protected by copyright. All rights reserved triphosphate, GS-443902 that selectively inhibits viral RNA polymerases but not host RNA or DNA polymerases ( Figure S1 ). (15) (16) (17) Dephosphorylation of the nucleoside analog monophosphate results in the formation of the nucleoside analog, GS-441524, that is not efficiently re-phosphorylated. RDV and its metabolites (GS-441524 and GS-704277) are detectable in plasma. Since all target cells relevant for SARS-CoV-2 infection are not currently known, peripheral blood mononuclear cells (PBMC) are used as the surrogate to assess the intracellular activation of RDV to In vitro studies in different cell types demonstrated that RDV and GS-441524 are active against SARS-CoV-2, with potency directly related to the intracellular concentrations of GS-443902. In vitro, RDV exhibited antiviral activity against a clinical isolate of SARS-CoV-2 in primary human airway epithelial (HAE) cells with an EC 50 (50% effective concentration) of 9.9 nM after 48 hours of treatment. (18) In Vero cells, the EC 50 values of RDV were 137 nM at 24 hours and 750 nM at 48 hours post-treatment. (19) In SARS-CoV-2-infected rhesus monkeys who received IV bolus injections of 10 mg/kg on the first day (initiated 12 hours post-inoculation), followed by 5 mg/kg daily thereafter, RDV resulted in a significant reduction in clinical scores, signs of respiratory disease, and viral RNA levels compared to vehicle-treated animals. (4, (19) (20) (21) On May 1, 2020, based on available data from two global clinical trials, Federal Drug Administration (FDA) granted emergency use authorization (EUA) to allow RDV to treat adults and children hospitalized with severe COVID-19. (19, 22, 23) Based on these clinical data, remdesivir has been approved for the treatment of adults and pediatric patients in Japan. (24) This article describes the safety and pharmacokinetics of the solution and lyophilized formulations of intravenous (IV) remdesivir administered to healthy participants in the two first-in-human (FIH) Phase 1 studies.

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Protocols and informed consents for both studies were approved by the study center's institutional review board, and subjects provided written consent before study participation.

The single-and multiple-dose studies were randomized, blinded, placebo-controlled, Phase 1 studies to evaluate the PK, safety, and tolerability of single-ascending (Study 1) or multiple (Study 2) IV doses of RDV compared with placebo in healthy subjects conducted at SeaView Research, INC in Miami, FL. Eligible subjects were healthy male and nonpregnant, non-lactating female subjects of non-childbearing potential, 18 to 55 years of age with a body mass index (BMI) between 18 to 30 kg/m 2 . Subjects did not participate in more than one Cohort of the study.

Major inclusion criteria included healthy subjects based on medical history/physical examinations/laboratory evaluations, normal 12-lead electrocardiogram, creatinine clearance >90 mL/min, no evidence of HIV, hepatitis B virus or hepatitis C virus infection, and use of at least 2 forms of contraception, including an effective barrier method. Exclusion criteria included plasma and blood donation within 7 and 56 days of study entry, respectively, active medical illness, use of prescription drugs within 28 days of study drug dosing (except vitamins, acetaminophen, ibuprofen, and/or hormonal contraceptive).

Single-Dose Study: In Study 1, nine dose cohorts were evaluated. In Cohorts 1-6, eligible subjects were randomized 4:1 within each cohort to receive RDV solution formulation at doses ranging from 3 mg to 225 mg (n = 8) or placebo (n = 2) on Day 1, administered as a 2-hour infusion. In Cohorts 7-9, eligible subjects were randomized 5:1 (n=10 active: n=2 placebo) within each cohort to receive RDV lyophilized formulation at single doses of either 75 mg (Cohort 7 and Cohort 9) or 150 mg (Cohort 8) or placebo on Day 1. Subjects in Cohorts 7 and 8 received a single IV dose of study medication administered over a 2-hour period, while subjects in Cohort 9 received a single IV dose of RDV administered over a 30-Accepted Article minute period. Dose escalation in Cohorts 1-6 occurred in sequential order after reviewing the safety data from the previous cohort, and only in the absence of dose-limiting toxicity and/or meeting any prespecified stopping criteria. Cohorts 7 and 8 were conducted in parallel and informed the conduct of Cohort 9. Subjects were discharged on Day 7 and returned 6 days (+/-1 day) later for an in-clinic follow up visit.

Multiple-Dose Study: In Study 2, two cohorts were enrolled and sentinel dosing was utilized within each cohort. Subjects were randomized 2:1 (8 active: 4 placebo) within each cohort to receive RDV 150 mg or matching placebo, respectively. Each cohort was comprised of 2 groups of 6 subjects each (4 who received RDV and 2 who received placebo). Each subject received study drug administered intravenously over a 1-hour period once daily for 7 days in Cohort 1 and 14 days in Cohort 2. Subjects were discharged on Day 9 for Cohort 1 and Day 16 for Cohort 2. All subjects returned 7 days (+/-2 day) after discharge for an in-clinic followup visit.

Dose Selection. The starting dose of 3 mg in Study 1 (single dose) was selected in accordance with the US Food and Drug Administration guidance document for estimating a safe starting dose of a new chemical entity in human test subjects (25) and was expected to provide a 15 and 65-fold safety margin relative to NOAELs from the 14-day toxicology studies in rat and cynomolgus monkey, respectively. (data on file) At the monkey NOAEL, the species that more closely mimics the behavior of RDV in humans in terms of plasma stability, anticipated margins of exposures (AUC) for remdesivir and GS-441524 were at least 70-fold and 75-fold at the 3 mg dose. Single doses up to 225 mg evaluated in this study were guided by emerging safety and pharmacokinetic data from lower dose cohorts.

The evaluated dose range allowed for large dose separation for assessment of safety and pharmacokinetics. The dose of 150 mg in Study 2 (multiple dose) was supported by the 14day toxicology data (data on file) and the favorable safety profile in healthy participants who received single doses up to 225 mg in Study 1. The 150 mg dose was projected to achieve

This article is protected by copyright. All rights reserved systemic exposures similar to exposures expected in the infected rhesus monkeys treated with therapeutic doses of remdesivir 10 mg/kg IV. 20

Subjects in Cohorts 1-6 were administered RDV solution formulation over a 2-hour period, subjects in cohorts 7-8 were administered RDV lyophilized formulation over a 2-hour period and subjects in Cohort 9 were administered RDV lyophilized formulation over a 30-min period. Serial blood samples were collected prior to dosing and post-dose PK sampling was performed over 144 hours. Urine samples were collected prior to dosing and relative to the start of infusion on Day 1 over 48 hours. Blood samples for PBMC isolation was collected at pre-dose and over 144 hours relative to the start of infusion.

For the multiple-dose study, intensive PK sampling occurred on Day 1 and 7 for Cohort 1, and Days 1, 7, and 14 for Cohort 2 prior to dosing, and post-dose PK sampling was 

During and following dosing, safety, and tolerability were assessed through the reporting of treatment-emergent adverse events (AEs), clinical laboratory tests (hematology profile, chemistry profile, and urinalysis) according to the DAIDS adverse events grading table, version 1.0, physical examinations, vital signs, serum pregnancy tests (female subjects) review of concomitant medications, and ECGs at various time points during the study.

PK parameters were estimated using noncompartmental methods using a linear-up/log- 

This article is protected by copyright. All rights reserved under the concentration versus time curve from time zero to the last quantifiable concentration (AUC last ), the area under the concentration versus time curve extrapolated to infinity (AUC inf ), the AUC over a dosing interval (at steady state; AUC tau ), maximum observed plasma concentration (C max ), and time of occurrence of C max (t max ), terminal elimination half-life (t ½ ), clearance (CL), renal clearance (CL r ), and volume of distribution

For both studies, the PK analysis set included all subjects who received at least 1 dose of study drug and had at least 1 non missing post-dose concentration. The safety population included all subjects who received at least 1 dose of RDV or placebo.

No formal power or sample size calculations were used to determine cohort size in these phase 1 studies. Subject demographics and baseline characteristics, plasma, PBMC, and urine PK parameters, and safety data were summarized by descriptive statistics for continuous data and by number and percentage of subjects for categorical data. to the exploratory nature of the study, no dose proportionality boundaries have been prespecified. In the multiple-dose study, accumulation of RDV and metabolites was assessed by comparing Day 7 to Day 1 PK for Cohort 1, and Day 14 to Day 1 PK for Cohort 2. A time to achieve to steady state of RDV and metabolites was examined using the Helmert transformation testing procedure of trough plasma concentrations. (26) 

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In the single-dose study, 96 subjects were randomized and all subjects completed the study.

Of those, 78 subjects received RDV and 18 received placebo. These subjects had a mean age of 44 years (range: 24-55 years). The majority of subjects (active + Placebo) were male (58.3%), white (88.5%), and Hispanic or Latino (96.9%). In the multiple-dose study, 24 subjects were randomized of whom 16 subjects received RDV and 8 subjects received placebo. Twenty-two subjects completed the study. Two subjects, one from Cohort 1 who received RDV and one from Cohort 2 who received PBO, prematurely discontinued study drug and the study due to AE and withdrawal of consent, respectively. Subjects had a mean age of 44 years (range: 19-55 years). The majority of subjects were male (58.3%). Most subjects (83.3%) were white, and all were Hispanic or Latino. For both studies, baseline characteristics were generally balanced across cohorts and are presented in Table 1 .

Study drug was generally well tolerated in both studies. There were no SAEs, Grade 3 or 4

AEs or deaths reported in either study.

All AEs reported during this study were Grade 1 or 2 in severity. There were no AEs leading to study drug or study discontinuation. A total of 17 of 78 subjects (21.8%) who received RDV and 2 of 18 subjects (11.1%) who received placebo had at least 1 AE. Constipation was the only AE reported for > 1 subject occurring in 3 of 78 subjects (3.8%) who received RDV. Two subjects receiving RDV solution formulation experienced AEs considered to be study-drug related (dizziness, Cohort 2; pruritus, Cohort 

This article is protected by copyright. All rights reserved irritation (1 subject in the 75-mg lyophilized formulation/30 minute infusion group). All AEs related to study drug and procedures resolved. Multiple-dose study. All adverse events were Grade 1 in severity. A total of 9 of 16 subjects (56.3%) who received RDV and 4 of 8 subjects (50.0%) who received placebo reported at least 1 AE. Adverse events reported for > 1 subject overall included constipation, dyspepsia, and pain in extremity (each in 3 subjects who received RDV) and headache, dermatitis contact, and pruritus (each in 1 subject who received RDV and 1 subject who received placebo). Nine subjects (6 received RDV and 3 received placebo), had ≥ 1 AE that was considered related to study procedure. These AEs included pain in extremity (3 subjects who received RDV); contact dermatitis and pruritus (each in 1 subject who received RDV and 1 who received placebo); infusion site extravasation, infusion site

This article is protected by copyright. All rights reserved pain, infusion site hemorrhage, and ecchymosis (each in 1 subject who received RDV); and dermatitis and iron deficiency anemia (each in 1 subject who received placebo).

Four subjects (3 received RDV; 1 received placebo) had AEs that were considered by the investigator to be study-drug related. In Cohort 1, one subject who received RDV discontinued the study drug due to nausea and also experienced treatment-related headache, vomiting, and tremor. Additional treatment-related AEs of decreased appetite and constipation occurred after study drug discontinuation. This subject also experienced the concomitant lab abnormalities of Grade 2 elevated ALT and AST. All AEs and graded abnormalities resolved. In Cohort 2, 2 subjects who received RDV had dyspepsia both of which resolved. One of these subjects also had elevated ALT and AST levels which returned to within normal range. One subject (Cohort 2) who received placebo had diarrhea, which also resolved during the conduct of the study. Table 2 and for Cohorts 7-9 are summarized in Table 3 . (Table 4) . Overall, single-dose IV administration of RDV as a solution or lyophilized formulation for up to 2 hours at doses ranging from 3 to 225 mg and multiple-dose IV administration of RDV 150 mg once daily for 7 or 14 days was generally well tolerated. All AEs reported during the

This article is protected by copyright. All rights reserved single-dose study were Grade 1 or 2 in severity. No subject had a graded ALT or AST elevation during the study. In the multiple-dose study, all AEs were low grade in severity and no serious AEs were reported. Elevations in ALT and AST were observed, with mild, reversible prothrombin time prolongation in some subjects but without other evidence of clinical hepatitis. The mechanism of these elevations is currently unknown. and subjects with Ebola viral disease (19) . Preliminary analysis of NIAID data showed a shorter median time to recovery (a metric used in influenza trials) and trends toward lower mortality rate in the RDV group (11 days and 8%) compared to placebo (15 days and 11.6%). The results from Study GS-540-5773 suggested a similar improvement in clinical status in patients receiving a 10-day and a 5-day treatment courses of remdesivir. Based on available data, RDV was granted an emergency use authorization (EUA) for the treatment of COVID-19 in adults and children hospitalized with severe disease. (19) The availability of an effective antiviral agent such as RDV with a favorable benefit/risk profile would address a serious unmet medical need for the treatment of COVID-19 infected adults. Overall, the PK and safety data from the FIH study and preliminary clinical data supports continued investigation of RDV in COVID-19 patients. 

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We thank the investigators and coordinators at the participating centers and the subjects who participated in these studies.The authors would like to thank Sunila Reddy, PharmD for her help in preparation of this manuscript. 

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