Why Remdesivir Failed: Preclinical Assumptions Overestimate the Clinical Efficacy of Remdesivir for COVID-19 and Ebola

Abstract

Remdesivir is a nucleoside monophosphoramidate prodrug that has been FDA approved for coronavirus disease 2019 (COVID-19). However, the clinical efficacy of remdesivir for COVID-19 remains contentious, as several trials have not found statistically significant differences in either time to clinical improvement or mortality between remdesivir-treated and control groups. Similarly, the inability of remdesivir to provide a clinically significant benefit above other investigational agents in patients with Ebola contrasts with strong, curative preclinical data generated in rhesus macaque models. For both COVID-19 and Ebola, significant discordance between the robust preclinical data and remdesivir's lackluster clinical performance have left many puzzled. Here, we critically evaluate the assumptions of the models underlying remdesivir's promising preclinical data and show that such assumptions overpredict efficacy and minimize toxicity of remdesivir in humans. Had the limitations of in vitro drug efficacy testing and species differences in drug metabolism been considered, the underwhelming clinical performance of remdesivir for both COVID-19 and Ebola would have been fully anticipated.

Keywords: GS-441524; GS-5734; in vitro models; in vivo models; nonhuman primates; pharmacokinetics; prodrug; remdesivir.

FIG 1

Remdesivir is preferentially bioactivated in hepatocytes. (A) RDV NTP (GS-443902) formation in PHH and HEp-2 cells is about 4-fold higher than in HAE cells and about 12-fold higher in PC-3 cells than in HAE cells. Data are means of data replotted from references (HAE) and (PHH, HEp-2, and PC-3). Cells were seeded at approximately 1 × 10⁶ cells/well and were incubated with 1 μM RDV for 24 h. Levels of NTP in HAE cells represent the average of quadruplicate technical replicates from 2 donors. (B) Comparison of selectivity indices (SIs) for CC₅₀s of RDV in HEp-2, HepG2, PC-3, and PHH and RDV EC₅₀s obtained in A549, Calu3, HAE, and Vero E6 cells infected with SARS-CoV-2. EC₅₀s used were obtained from references (A549) and (Calu3, HAE, and Vero E6).

FIG 2

Duration of drug exposure impacts the antiviral efficacy of remdesivir. (A) Continuous exposure to RDV leads to durable in vitro inhibition of SARS-CoV-2, but pulsed treatment with RDV results in diminished antiviral activity in Vero E6 cells. Data are means of triplicate data replotted from reference . Across the three experiments, RDV was tested at 3.7 μM and CQ was tested at 10 μM. Due to its long in vivo t_1/2, CQ serves as a positive control. (B) Corresponding treatment procedures for prophylactic, entry, and postentry experiments described for panel A. Pulsed experiments correspond to the entry trial. (C) Transcriptome sequencing (RNA-seq) data showing expression of relevant prodrug bioactivating enzymes for RDV in Vero E6 cells, presented as means and standard deviations (SD) from 3 experiments (see the supplemental material). ADK, adenosine kinase; AK2, adenylate kinase 2. AK2 and SLC29A3 were found to mediate RDV potency and toxicity in a genome-wide CRISPR screen (55). (D) Formation of active triphosphate (GS-443902) in normal human bronchial epithelial (NHBE) cells following pulsed (open circles) and continuous (filled circles) incubation. Data are adapted from reference and are presented as means for at least 2 independent replicates. The dotted line at 10.6 pmol/million cells corresponds to the mean C₂₄ of triphosphate formed by RDV in SARS-CoV-2-infected HAE cells when the EC₅₀ was determined to be 10 nM, as reported in reference .

FIG 3

Humans exhibit higher hepatic extraction of McGuigan prodrugs than nonhuman primates. (A) Doses of RDV trialed in the single ascending dose (SAD) and multiple ascending dose (MAD) arms of the phase 1 trial for a 50- to 70-kg human compared to the dose administered in a 3.6- to 5.7-kg NHP. The human and NHP doses have been converted to milligrams per kilogram and milligrams, respectively. Values were obtained from references , , and . (B) ALT and AST levels in NHP (rhesus macaques; 6 per group) challenged with EBOV and treated with RDV for a total of 12 days (adapted from reference 8). Regions shaded in gray correspond to normal ALT/AST ranges in NHP (ALT,5 to 61 U/liter; AST, 12 to 63 U/liter) (56). NHPs treated at 10 mg/kg for 12 days (light blue) did not experience significant elevations in ALT/AST. *, No ALT/AST values were obtained at 14 days postinfection (dpi) in vehicle control animals because 100% of animals had died. (C) Human equivalent dose (HED) of NHP RDV doses tested in reference via direct conversion and allometric scaling as described in reference . Allometric scaling (1) uses an exponent of 0.75, while allometric scaling (2) uses an exponent of 0.80. HEDs were calculated for rhesus macaques weighing 3 kg (light gray) and 5 kg (dark gray) using the Clymer allometric scaling calculator. (D) Sofosbuvir (SOF) is another McGuigan prodrug that is more readily metabolized in PHH than primary monkey hepatocytes in vitro (left) and in vivo (right). (Left) Levels of SOF NTP in primary hepatocytes following a 2-h pulse of 10 μM SOF. (Right) Levels of SOF NTP in human explanted livers and in preclinical model species at the allometrically scaled human dose (400 mg). Open bars, C_max; shaded bars, C₂₄. C₂₄ in primary monkey livers was below the detection threshold. Values were replotted from references and . Formation of SOF NTP is higher in PHH than primary monkey hepatocytes in vitro and in vivo, suggesting more efficient metabolism of McGuigan prodrugs in human than monkey livers.

FIG 4

Human equivalent dose of RDV in rhesus macaque models of COVID-19 or EBOV does not yield robust reductions in viral titers. (A and B) SARS-CoV-2-infected rhesus macaques (2.6 × 10⁶ 50% tissue culture infective doses [TCID₅₀] of nCoV-WA1-2020) treated with the allometrically scaled dose of 10 mg/kg 1 dpi followed by 5 mg/kg 2 to 6 dpi yields a modest global reduction in SARS-CoV-2 viral titers. Data are adapted from the supplemental information in reference . (A) Heat map of organs profiled for the presence of SARS-CoV-2 RNA in RDV-treated (n = 6) and control (CT) (n = 6) animals. LN, lymph node; UL, upper lobe; ML, middle lobe; LL, lower lobe. (B) Viral loads in nose swabs and throat swabs between RDV-treated and CT animals. (C) No significant decrease in viral loads in the upper and lower respiratory tracts in patients treated with RDV (200 mg on day 1, 100 mg on days 2 to 10) was seen compared to placebo. Data are adapted from reference and are presented as means (n = 107 [RDV] and 52 [placebo]). (D) Rhesus macaques were infected with EBOV (1,000 PFU; EBOV H. sapiens-tc/COD/1995/Kikwit) and treated i.v. with either 10 mg/kg RDV at 2 dpi for 12 days (light blue), a 10-mg/kg loading dose at 3 dpi and then 3 mg/kg for 11 days (dark purple), a 10-mg/kg loading dose at 3 dpi and then 3 mg/kg for 11 days (light purple), or vehicle (black). Animals (6 per group) were monitored for a total of 28 days. The most distinct reductions in viral titers occurred for NHPs in the 10-mg/kg treatment group (light blue). (E) Survival curves for NHPs at 28 dpi for treatment groups indicated. Though viral titers dropped to the limit of detection (LOD) for the 10/3-mg/kg D2 group (D, dark purple D) by day 12, only 2/6 NHPs in this group survived (E, dark purple), skewing viral titer data. At the same time, this reinforces the close relationship between stark reductions in viral titers and improved disease outcomes, as the remaining 2/6 animals in the 10/3-mg/kg D2 (2 days postinfection) group survived at 28 dpi. Data are adapted from reference .