Preclinical and randomized phase I studies of plitidepsin in adults hospitalized with COVID-19

Abstract

Plitidepsin, a marine-derived cyclic-peptide, inhibits SARS-CoV-2 replication at nanomolar concentrations by targeting the host protein eukaryotic translation elongation factor 1A. Here, we show that plitidepsin distributes preferentially to lung over plasma, with similar potency against across several SARS-CoV-2 variants in preclinical studies. Simultaneously, in this randomized, parallel, open-label, proof-of-concept study (NCT04382066) conducted in 10 Spanish hospitals between May and November 2020, 46 adult hospitalized patients with confirmed SARS-CoV-2 infection received either 1.5 mg (n = 15), 2.0 mg (n = 16), or 2.5 mg (n = 15) plitidepsin once daily for 3 d. The primary objective was safety; viral load kinetics, mortality, need for increased respiratory support, and dose selection were secondary end points. One patient withdrew consent before starting procedures; 45 initiated treatment; one withdrew because of hypersensitivity. Two Grade 3 treatment-related adverse events were observed (hypersensitivity and diarrhea). Treatment-related adverse events affecting more than 5% of patients were nausea (42.2%), vomiting (15.6%), and diarrhea (6.7%). Mean viral load reductions from baseline were 1.35, 2.35, 3.25, and 3.85 log10 at days 4, 7, 15, and 31. Nonmechanical invasive ventilation was required in 8 of 44 evaluable patients (16.0%); six patients required intensive care support (13.6%), and three patients (6.7%) died (COVID-19-related). Plitidepsin has a favorable safety profile in patients with COVID-19.

Conflict of interest statement

V Estrada has received personal fees from Janssen, Gilead, and ViiV and grants from MSD. R Paredes has participated in Advisory Boards from Gilead, MSD, ViiV Healthcare, and Theratechnologies. M Torralba has received consulting fees as a member of Advisory Committee and honoraria and speaking fees from Gilead, Janssen, MSD, and ViiV Companies. J Fortún has participated in scientific events and received consulting or speaking fees or oral presentations from Pfizer, Gilead, MSD, Astellas, Novartis, and Roche. J Ancochea has received fees for scientific consulting and/or speaking from Actelion, Air Liquide, Almirall, AstraZeneca, Boehringer Ingelheim, Carburos Médica, Chiesi, Faes Farma, Ferrer, GlaxoSmithKline, InterMune, Linde Healthcare, Menarini, MSD, Mundipharma, Novartis, Pfizer, Roche, Rovi, Sandoz, Takeda, and Teva. I Sola, S Zúñiga, and L Enjuanes hold a Technology Support contract with Pharmamar. N Izquierdo-Useros is inventor of a patent of Plitidepsin (EP20382821.5). The Krogan Laboratory has received research support from Vir Biotechnology and F Hoffmann-La Roche. NJ Krogan has consulting agreements with Maze Therapeutics and Interline Therapeutics, and is a shareholder of Tenaya Therapeutics. JM Fernández-Sousa is President and Founder of Pharmamar, SA (Madrid, Spain). JM Jimeno holds stocks of Pangaea Oncology, has a non-remunerated role in the Scientific Advisory Board and holds stocks of Phosplatin Therapeutics, and is a full-time employee of Pharmamar, SA (Madrid, Spain).The A García-Sastre laboratory has received research support from Pfizer, Senhwa Biosciences, Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, and Nanocomposix. A García-Sastre has consulting agreements for the following companies involving cash and/or stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pagoda, Accurius, Esperovax, Farmak, and Pfizer. A García-Sastre is inventor on patents and patent application on the use of antivirals for the treatment of virus infections, owned by the Icahn School of Medicine at Mount Sinai, New York. A patent application based on this work has been filed (EP20382821.5). JA Lopez-Martin, S Fudio, MJ Pontes, B de Rivas, A Nieto, J Gómez, P Girón de Velasco, P Avilés, R Lubomirov, A Belgrano, and B Sopesén are employees and shareholders of Pharmamar, SA (Madrid, Spain). JA Lopez-Martin is a co-inventor of a patent for plitidepsin (WO2008135793A1). JM Jimeno is a co-inventor on a patent for didmenin (WO99/42125) and on patents for aplidine (WO03/033013 and WO 2004/080421).

© 2022 Varona et al.

Figures

Figure 1.. Plitidepsin shows strong antiviral activity in vitro against different coronavirus species.

(A) Treatment of Huh-7 cells with 0.5–50 nM of plitidepsin inhibited infection of a human coronavirus 229E expressing green fluorescent protein. All cells were treated 8 h after infection and fluorescent foci were analyzed at 48 h. (B) Accumulation of SARS-CoV genomic RNA is inhibited with increasing doses of plitidepsin. Confluent Vero E6 cells were infected with SARS-CoV and subsequently treated with plitidepsin at varying concentrations 1 hour post infection. Viral genomic RNA was measured 48 hours post infection. (C) Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of plitidepsin. Plitidepsin was used at a concentration ranging from 5 nM to 100 μM. Nonlinear fit to a variable response curve from one representative experiment with two replicates is shown (blue), excluding data from drug concentrations with associated toxicity; cytotoxicity in the absence of virus is also shown (green). Error bars represent SD; points without error bars have a SD that is too small to visualize. DMSO, dimethyl sulfoxide; RLU, relative light unit.

Figure 2.. Plitidepsin shows strong antiviral activity in vitro against SARS-CoV-2 variants.

(A, B, C, D, E, F) Plitidepsin inhibits SARS-CoV-2 variants. HeLa-ACE2 cells were pretreated with plitidepsin or DMSO control 2 h after infection with (A) SARS-CoV-2/WA1, (B) α (B.1.1.7), (C) β (B.1.351), (D) δ (B.1.617.2), (E) μ (B.1.621), or (F) ο (B.1.1.529). Virus infectivity was measured 48 h postinfection. Cytotoxicity was performed in uninfected HeLa-ACE2 cells with same compound dilutions and concurrent with viral replication assay. Error bars represent SD across biologically independent triplicates. (G) Plitidepsin efficacy against early and α (B.1.1.7) variants compared to remdesivir. Calu-3 and Caco-2 cells were pre-treated with plitidepsin, remdesivir, or DMSO control at the indicated concentrations at an equivalent dilution for 2 h before SARS-CoV-2 infection. Cells were harvested after 24 h for analysis, and viral infection measured by intracellular detection of SARS-CoV-2 nucleoprotein by flow cytometry. Tetrazolium salt (MTT) assay was performed to verify cell viability. Error bars represent standard error of the mean. IC50: half maximal inhibitory concentration.

Figure 3.. Pharmacological estimation of active plasma concentrations of plitidepsin.

Predicted plasma concentrations achieved by a 90 min i.v. infusion of plitidepsin (1.5, 2, and 2.5 mg) and plasma IC50 and IC90 thresholds to assure concentrations in lung above IC50 and IC90 established in vitro, respectively (8, 52). Results were used to support the study doses and schedule. IC50: half maximal inhibitory concentration; IC90: 90% of maximal inhibitory concentration.

Figure 4.. Study Flow (CONSORT).

* For safety reasons, the first three patients of the study were sequentially allocated at the lowest dose level. Inclusion in the highest dose group was opened when three patients had been randomized to the intermediate dose. For that reason, the last three patients treated at the highest dose were also sequentially allocated. ** One patient withdrew consent before starting any study procedure and was replaced. *** All treated patients were evaluated for safety. All patients who completed treatment were assessed for efficacy. One patient experienced a grade 3 hypersensitivity reaction, shortly after the start of day 1 infusion of plitidepsin. This patient did not complete therapy, discontinued the study for safety reasons and was not evaluable for efficacy. This patient was not replaced. d, days; pts, patients.

Figure 5.. APLICOV-PC: Protocol Treatment and Pre-medication.

IV, intravenous; PO, oral; 5-HT3, serotonin (5-hydroxytryptamine) receptor 3.

Figure S1.. Intra-patient time-course variation in ALT, per dose-cohort.

Shadowed area represents treatment with plitidepsin on days 1 to 3. Each color line represents one patient. Bold line represents median values. ALT, alanine aminotransferase; ULN, upper limit of normal.

Figure 6.. APLICOV-PC Study: Preliminary Efficacy Outcomes.

(A) Viral load kinetics (qRT-PCR from nasopharyngeal exudates), by baseline severity of the disease (23). (B) Viral load kinetics (qRT-PCR from nasopharyngeal exudates), by dose of plitidepsin. LoQ: limit of quantification. See Table S4 for individual data results.

Figure S2.. Individual assessment of a six-category ordinal scale over time according to the severity of the disease at baseline.

The six-point scale was defined as follows (24): 1, discharged or having reached discharge criteria (defined as “clinical recovery”: normalization of pyrexia, respiratory rate 94% on room air, and relief of cough, all maintained for at least 72 h); 2, hospital admission but not requiring oxygen supplementation; 3, hospital admission for oxygen therapy (but not requiring high-flow or ventilation support); 4, hospital admission for noninvasive ventilation or high-flow oxygen therapy; 5, hospital admission for extracorporeal membrane oxygenation or invasive mechanical ventilation; 6, death.

Figure 7.. Post hoc analysis on hospital discharge by plitidepsin dose.

(A) Reverse Kaplan Meier plot showing the cumulative incidence of hospital discharge by plitidepsin dose. (B) Length of hospitalization, by plitidepsin dose and disease severity at baseline (23). Orange bars represent admission in intensive care units. Dashed lines labeled D8 and D15 are days 8 and 15, respectively, considering the start of therapy with plitidepsin as Day 1; this is equivalent to stays of 7 or 14 d from the start of therapy. See Figs S2S–SFigs S2–S4 for post hoc analysis on hospital discharge and respiratory support according to the severity of the disease at baseline.

Figure S3.. Post hoc analysis on hospital discharge, by baseline disease severity.

Reverse Kaplan Meier plot showing the cumulative incidence of hospital discharge by baseline severity (mild, moderate or severe COVID-19, according to FDA definition) (23).

Figure S4.. Post hoc analysis on hospital discharge, based on baseline disease severity and plitidepsin dose.

Length of hospitalization, by disease severity at baseline (23) and dose of plitidepsin. Orange bars represent admission in intensive care units. Dashed lines labeled D8 and D15 are days 8 and 15, respectively, considering the start of therapy with plitidepsin as Day 1; this is equivalent to stays of 7 or 14 d from the start of therapy.

Figure S5.. Intra-patient time-course variation in lymphocytes, per dose-cohort.

Shadowed area represents treatment with plitidepsin on days 1 to 3. Each color line represents one patient. Bold line represents median values.

Figure S6.. Intra-patient time-course variation in C-Reactive Protein, per dose-cohort.

Shadowed area represents treatment with plitidepsin on days 1 to 3. Each color line represents one patient. Bold line represents median values.

Figure 8.. Post hoc analysis on hospital discharge by plitidepsin dose in patients with moderate COVID-19 at baseline.

(A) Subgroup of patients with moderate COVID-19 (n = 23 pts): Distribution of the probability of the duration of the hospitalization, according to the dose of plitidepsin administered. (B) Subgroup of patients with moderate COVID-19 (n = 23 pts): Mean score over time of a six-category ordinal scale in patients with moderate disease at baseline, according to the administered dose of plitidepsin. The six-point scale was defined as follows (24): 1, discharged or having reached discharge criteria (defined as “clinical recovery”: normalization of pyrexia, respiratory rate <24 breaths per minute, saturation of peripheral oxygen >94% on room air, and relief of cough, all maintained for at least 72 h); 2, hospital admission but not requiring oxygen supplementation; 3, hospital admission for oxygen therapy (but not requiring high-flow or ventilation support); 4, hospital admission for noninvasive ventilation or high-flow oxygen therapy; 5, hospital admission for extracorporeal membrane oxygenation or invasive mechanical ventilation; 6, death. See also Table 3 and Figs S7 and S8.

Figure S7.. Inflammatory biomarkers in patients with moderate COVID-19 throughout follow-up.

(A, B, C, D) Infection and inflammation parameters in patients with moderate COVID-19 receiving plitidepsin, including (A) lymphocyte counts, (B) C-reactive protein, (C) neutrophil to lymphocyte ratio, and (D) D-dimers. Shadowed area represents treatment with plitidepsin on days 1 to 3. Mean and standard error of the mean are represented in the plots.

Figure S8.. Individual assessment of a six-category ordinal scale over time in patients with moderate COVID-19 at baseline, according to the administered dose of plitidepsin.

In patients with moderate COVID-19 at baseline (n = 23 pts), more patients treated at 2.5 mg/d were taken off supplementary oxygen (category 2) and were discharged from the hospital (category 1) by the end of the first week, with respect to other doses (non-statistically significant post hoc finding). The six-point scale was defined as follows (24): 1, discharged or having reached discharge criteria (defined as “clinical recovery”: normalization of pyrexia, respiratory rate 94% on room air, and relief of cough, all maintained for at least 72 h); 2, hospital admission but not requiring oxygen supplementation; 3, hospital admission for oxygen therapy (but not requiring high-flow or ventilation support); 4, hospital admission for noninvasive ventilation or high-flow oxygen therapy; 5, hospital admission for extracorporeal membrane oxygenation or invasive mechanical ventilation; 6, death. Categories 5 and 6 are not represented in the legend as there were no patients fulfilling the criteria. See also Fig 8B.

Figure S9.. Comparisons of radiological and inflammatory biomarkers in a 41-yr-old patient treated with 2.5 mg/day plitidepsin.

(A, B) Baseline CT scan of a participant subject, a 41 yr old male with moderate COVID-19 at baseline, (A) shows multiple bilateral lung infiltrates before plitidepsin treatment and (B) their status 7 d after treatment with plitidepsin 2.5 mg/day. (C) Changes to C-reactive protein and absolute lymphocyte count in the same patient.

Figure S10.. Lung alveoli from murine models.

Hematoxylin Eosin stained sections of lung from K18 hACE2 mice. (A, B, C) Lungs were harvested on day 3 postinfection (8): (A) lung tissue from a noninfected mouse, (B) alveolar inflammation in a SARS-CoV-2-infected mouse receiving vehicle, and (C) alveolar inflammation in a SARS-CoV-2 infected mouse receiving plitidepsin. Scale bar represents 100 μm.