Research Article Preclinical and randomized phase I studies of plitidepsin in adults hospitalized with COVID-19 Jose F Varona1,2 , Pedro Landete3,4, Jose A Lopez-Martin5 , Vicente Estrada6,7, Roger Paredes8,9, Pablo Guisado-Vasco10,11, Lucia Fernandez de Orueta11,12, Miguel Torralba13,14, Jesus Fortun15 , Roberto Vates12, Jose Barberan1,2, Bonaventura Clotet8,9,16,17, Julio Ancochea3,4,18 , Daniel Carnevali10,11, Noemi Cabello19, Lourdes Porras20, Paloma Gijon21, Alfonso Monereo12, Daniel Abad11,12, Sonia Zuñiga22 , Isabel Sola22, Jordi Rodon23 , Julia Vergara-Alert23, Nuria Izquierdo-Useros24,25 , Salvador Fudio26 , Maria Jose Pontes27, Beatriz de Rivas27, Patricia Giron de Velasco5, Antonio Nieto28, Javier Gomez28, Pablo Aviles29, Rubin Lubomirov26, Alvaro Belgrano28, Belen Sopesen5,30,31, Kris M White32,33 , Romel Rosales32,33, Soner Yildiz32,33, Ann-Kathrin Reuschl34, Lucy G Thorne34 , Clare Jolly34, Greg J Towers34, Lorena Zuliani-Alvarez35,36,37,38, Mehdi Bouhaddou35,36,37,38, Kirsten Obernier35,36,37,38 , Briana L McGovern32,33 , M Luis Rodriguez32,33, Luis Enjuanes22, Jose M Fernandez-Sousa39, Nevan J Krogan32,35,36,37,38 , Jose M Jimeno5,*, Adolfo Garcia-Sastre32,33,40,41,* Plitidepsin, a marine-derived cyclic-peptide, inhibits SARS-CoV-2 more than 5% of patients were nausea (42.2%), vomiting (15.6%), and replication at nanomolar concentrations by targeting the host pro- diarrhea (6.7%). Mean viral load reductions from baseline were 1.35, tein eukaryotic translation elongation factor 1A. Here, we show that 2.35, 3.25, and 3.85 log10 at days 4, 7, 15, and 31. Nonmechanical invasive plitidepsin distributes preferentially to lung over plasma, with similar ventilation was required in 8 of 44 evaluable patients (16.0%); six potency against across several SARS-CoV-2 variants in preclinical patients required intensive care support (13.6%), and three patients studies. Simultaneously, in this randomized, parallel, open-label, (6.7%) died (COVID-19-related). Plitidepsin has a favorable safety proof-of-concept study (NCT04382066) conducted in 10 Spanish profile in patients with COVID-19. hospitals between May and November 2020, 46 adult hospitalized patients with confirmed SARS-CoV-2 infection received either 1.5 mg DOI 10.26508/lsa.202101200 | Received 18 August 2021 | Revised 24 December 2021 | Accepted 28 December 2021 | Published online 10 January 2022 (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; Introduction 45 initiated treatment; one withdrew because of hypersensitivity. Two Grade 3 treatment-related adverse events were observed (hyper- As of December 2021, there have been more than 269 million sensitivity and diarrhea). Treatment-related adverse events affecting confirmed cases of coronavirus disease (COVID-19) reported to the 1 Departamento de Medicina Interna, Hospital Universitario HM Monteprincipe, HM Hospitales, Madrid, Spain 2Facultad de Medicina, Universidad San Pablo-CEU, Madrid, Spain 3Hospital Universitario La Princesa, Madrid, Spain 4Universidad Autónoma de Madrid, Madrid, Spain 5Virology and Inflammation Unit, PharmaMar, SA, Madrid, Spain 6Hospital Clı́nico San Carlos, Madrid, Spain 7Universidad Complutense de Madrid, Madrid, Spain 8Infectious Diseases Department, IrsiCaixa AIDS Research Institute, Barcelona, Spain 9Hospital Germans Trias I Pujol, Barcelona, Spain 10Hospital Universitario Quironsalud Madrid, Madrid, Spain 11Universidad Europea, Madrid, Spain 12Internal Medicine Department, Hospital Universitario de Getafe, Madrid, Spain 13Health Sciences Faculty, University of Alcalá, Madrid, Spain 14 Guadalajara University Hospital, Guadalajara, Spain 15Hospital Universitario Ramón y Cajal, Madrid, Spain 16Universitat Autònoma de Barcelona, Barcelona, Spain 17 Universitat de Vic, Universitat Central de Catalunya, Barcelona, Spain 18Centro de Investigación en Red de Enfermedades Respiratorias (CIBERES), Instituto de Salud Carlos III (ISCIII), Madrid, Spain 19Infectious Diseases Department, Clinico San Carlos University Hospital, Madrid, Spain 20Internal Medicine, Hospital General de Ciudad Real, Ciudad Real, Spain 21Clinical Microbiology and Infectious Diseases Department, Hospital General Universitario Gregorio Marañón, Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain 22Department of Molecular and Cell Biology, Centro Nacional de Biotecnologı́a (CNB-CSIC), Madrid, Spain 23IRTA, Centre de Recerca en Sanitat Animal (CReSA, IRTA-UAB), Campus de la UAB, Bellaterra, Spain 24IrsiCaixa AIDS Research Institute, Barcelona, Spain 25Germans Trias I Pujol Research Institute (IGTP), Badalona, Spain 26Clinical Pharmacology Unit, PharmaMar, Madrid, Spain 27Medical Affairs, PharmaMar, Madrid, Spain 28Bio Statistics Unit, PharmaMar, Madrid, Spain 29Preclinical Unit, Pharmamar, Madrid, Spain 30Sylentis, SAU, Madrid, Spain 31Biocross, SL, Valladolid, Spain 32Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA 33Global Health Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA 34Division of Infection and Immunity, University College London, London, UK 35Quantitative Biosciences Institute (QBI), University of California San Francisco, San Francisco, CA, USA 36J David Gladstone Institutes, San Francisco, CA, USA 37QBI, Coronavirus Research Group (QCRG), San Francisco, CA, USA 38 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA 39Pharmamar, Madrid, Spain 40Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA 41Tish Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA Correspondence: jfvarona@hmhospitales.com *José M Jimeno and Adolfo Garcı́a-Sastre contributed equally to this work. © 2022 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 1 of 22 World Health Organization, including over 5 million deaths (1). More comparison, and consistent with previous results (8, 20), plitidepsin than 1 yr after being officially declared a pandemic, substantial showed nanomolar efficacy against SARS-CoV-2-induced cyto- disease burden remains, as the clinical course from severe lung pathic effects in Vero E6 cells with a half-maximal inhibitory involvement, evolving to respiratory failure continues to be the concentration (IC50) of 0.038 μM, at concentrations where no cy- main cause of death for COVID-19 patients. The lack of effective totoxic effects were observed (CC50 2.9 μM) (Fig 1C). Moreover, antiviral therapies represents a glaring unmet need, not only for the plitidepsin maintained its nanomolar potency against replication of treatment of the current severe acute respiratory syndrome early as well as later SARS-CoV-2 lineages, such as B.1.1.7 (α), B.1.351 coronavirus 2 (SARS-CoV-2) pandemic (2), but also for potential (β), B.1.617.2 (δ), B.1.621 (μ), and B.1.1.529 (o) variants (Fig 2A–F). future pandemics, which may originate from other emergent Noteworthy, in human lung and gastrointestinal cell lines pliti- coronaviruses (3, 4, 5). depsin was more effective against both early and α variants than SARS-CoV-2 is a spherical, enveloped virus, around 80–120 nm in remdesivir (Fig 2G). diameter. Within the lipid bilayer envelope, the viral single-stranded RNA genome is packaged within a protein capsid, which comprised Predicting the effective dose of plitidepsin in COVID-19 the nucleocapsid (N) protein (6). The N protein is produced abun- dantly in infected cells and is a key element involved in packaging of To identify target human plasma concentrations of plitidepsin for the viral RNA genome (7). The SARS-CoV-2 N protein, as well as several SARS-CoV-2 infection we developed an extrapolation from in vitro proteins from other viruses, has been shown to bind directly to results, in line with current recommendations (21). This approach eukaryotic elongation factor 1α (eEF1A), an important host factor for integrated results from nonclinical studies, including in vitro drug the replication of many viral pathogens (7, 8). Down-regulation via sensitivity data for SARS-CoV-2 in Vero cells, human plasma protein small interfering RNA or chemical inhibition of eEF1A has been shown binding data (98%) (Table S2), and in vivo tissue distribution data in to result in a significant reduction in the replication and infectivity of rats (lung-to-plasma partition coefficient ratio of 543-fold; Table S3). several viruses, including SARS-CoV-2 (9, 10, 11). The target plasma and lung concentrations for plitidepsin were Plitidepsin is a cyclic depsipeptide originally isolated from a initially based on in vitro data obtained by Boryung Pharmaceu- Mediterranean marine tunicate (Aplidium albicans) that has been ticals, which established an IC50 of 3.26 nM and an IC90 of 9.38 nM. A shown to interact directly and inhibit the activity of eEF1A (12). Ex- validated pharmacokinetic population model of plitidepsin (22) was periments in cell culture and mice models have shown that pliti- used to simulate plasma exposures at different dose levels and depsin can inhibit SARS-CoV-2 replication, indicating that plitidepsin infusion durations, so that plitidepsin plasma profiles would reach may be a promising candidate for the treatment of COVID-19 (8). 0.33, and 0.96 μg/l, assuring target concentrations in lung above the Plitidepsin has undergone an extensive clinical development aforementioned in vitro IC50 and IC90. A 3-d daily schedule was program for the treatment of cancer. Pharmacokinetic and safety initially selected to achieve sustained active exposures, under the properties of plitidepsin have been gathered from several Phase I hypothesis that an acute reduction of the viral load would prevent and II clinical trials which have also explored different i.v. dosing the onset of the more severe inflammatory phase of COVID-19. The schedules and infusion times (13, 14, 15, 16, 17). Based on the results predicted plasma concentrations of plitidepsin, at a dose of 1.5 mg obtained from a Phase III clinical trial (ADMYRE) (18), the Australian infused i.v. over 90 min, were above the target IC50 for the full Therapeutic Goods Administration approved the combination of treatment period and above the IC90 for half of the treatment plitidepsin with dexamethasone for the treatment of patients with period. The respective predictions after a dose of 2.5 mg were above relapsed/refractory multiple myeloma in 2018 (19). the IC90 during most of the treatment period (Fig 3). Thus, we To date, no clinical trials have evaluated plitidepsin for the anticipated that the proposed range of doses would result in stable treatment of infectious disease. In this proof-of concept clinical active concentrations in critical anatomical compartments, such as trial (APLICOV-PC), we sought to determine the safety and toxico- the lung, for more than 120 h. This model was later supported by logical profile of plitidepsin, as well as to explore any potential White et al, who reported an IC90 of 0.88 nM (8). To reach this target efficacy effects, across three dose levels in patients hospitalized concentration in lung tissue, according to the above reasoning, the with COVID-19. plitidepsin plasma concentration should be above 0.18 μg/l. Patient characteristics Results In total, 46 hospitalized COVID-19 patients were enrolled across 10 Plitidepsin shows potent inhibition of viral replication in vitro sites in Spain (Fig 4). A diagram of the per-protocol treatment can be found in Fig 5, and the complete study protocol can be found in The antiviral activity of plitidepsin was evaluated by three separate Supplemental Data 1. Baseline demographic and clinical charac- teams against different coronavirus species, strains and variants. teristics are summarized in Table 1. The average patient age was 52 Treatment of Huh-7 cells with as little as 0.5 nM of plitidepsin yr (range 31–84 yr). Most patients were male (66.7%) and 80% had inhibited infection of a human coronavirus 229E expressing GFP (Fig co-morbidities (46.7% had two or more). The most commonly re- 1A). A 104-fold decrease in SARS-CoV genomic RNA accumulation ported comorbidities were obesity (22.2%), hypertension (20%), and and a 103-fold decrease in virus SARS-CoV titers were observed in type 2 diabetes mellitus (17.8%). The distribution of comorbidities Vero E6 cells treated with 50 nM plitidepsin (Fig 1B and Table S1). By was similar among the three treatment cohorts. Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 2 of 22 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. Most patients had moderate COVID-19 (51.1%), according to bilateral pneumonia seen in 32 of them (71%); the percentage of the US Food and Drug Administration (FDA) categorization (23), patients with bilateral pneumonia was similar across dose with 13.3% and 35.6% having mild and severe disease, re- cohorts. Viral load was similar across the three cohorts, with spectively. Baseline chest X-rays showed evidence of lower average baseline values for SARS-CoV-2 RNA from nasopha- respiratory infection (infiltrates, unilateral pneumonia, or bi- ryngeal samples of 6.1 log 10 copies/ml as measured by lateral pneumonia) in 41 of 45 treated patients (91%), with quantitative (q)RT-PCR. Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 3 of 22 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) o (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. Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 4 of 22 Plitidepsin treatment was generally well tolerated in hospitalized observation of Grade 3 neutropenia was reported in an asymp- patients with COVID-19 tomatic outpatient during follow-up at Day 31; this patient was also taking metamizole, and the investigator responsible deemed that Study interventions are described in Fig 5, the Materials and the event was neither clinically relevant nor related to plitidepsin. Methods section, and Supplemental Data 1 (protocol). One patient Of the 32 patients who entered with normal platelet counts, only withdrew consent before initiating any study-specific procedure. one had Grade 1 thrombocytopenia, whereas of the 12 patients who Forty-four patients completed the study through day 31. One pa- entered into the study with Grade 1 thrombocytopenia, 5 (41.7%) had tient in the 1.5-mg cohort withdrew from the study before com- counts normalized. pleting the full treatment because of a grade 3 hypersensitivity Regardless of causality, abnormalities in liver function tests were reaction occurring shortly after the initiation of the first infusion of common, transient, mild, and reversible. Elevation of alanine plitidepsin. This happened despite the pre-treatment with oral aminotransferase (ALT) and aspartate aminotransferase (AST) were dexamethasone 8 mg. The study protocol was thereafter amended reported in 29 of 44 (66%) and 13 of 44 (30%) patients, respectively. to require IV premedication with dexamethasone phosphate 8 mg, Two patients had a single and self-limited observation of a Grade 3 instead of oral administration, as well as IV ondansetron 8 mg, increase in ALT, with no associated increase in bilirubin (Fig S1). followed by 4 mg orally every 12 h until 48 h after the last ad- Four patients of the 42 who entered with normal creatinine de- ministration of plitidepsin (previously this was left to physician’s veloped a Grade 1 increase in creatinine on study. A Grade 1 in- discretion) (Fig 5; see Supplemental Data 2 for a summary of all crease in creatinine phosphokinase (CPK) was documented in 3 of study amendments). 37 patients (8.1%) who had normal baseline values, whereas four of All 45 of the treated patients were evaluable for safety. Three five patients (80%) who entered with Grade 1–2 elevation had their patients in this study died (6.7%); all had severe disease at baseline, CPK values decreased on study. and each death was determined to be related to COVID-19. Deaths Hyperglycemia was documented in 8 of 45 patients (18%): three occurred on days 22, 30, and 57 after the start of treatment with patients in the 1.5 mg/day dose cohort, two patients in the 2 mg/ plitidepsin. One patient received plitidepsin 1.5 mg/day and the day, and three patients in the 2.5 mg/day group. All cases of hy- other two received 2.5 mg/day, with no reported tolerability issues. perglycemia were Grade ≤ 2 except one patient, who had Grade 3 Seven additional patients experienced serious adverse events: hyperglycemia lasting for 2 d. For five patients, hyperglycemia was five dosed at 1.5 mg/day, 1 dosed at 2.0 mg/day, and 1 dosed at 2.5 considered related to concomitant medication. None of these mg/d. As previously mentioned, only one serious adverse event events were considered to be related to plitidepsin. (2.2% subjects) was considered related to the study drug: a Grade 3 Finally, protocol-specified analysis of electrocardiograms (ECGs) hypersensitivity reaction occurring ~5 min after the start of the first was conducted by a third-party central laboratory (ERT, Inc.). A total infusion of plitidepsin. of 317 ECGs from 44 patients were machine-readable and eligible for Although nearly all (44 of 45; 97.8%) patients experienced one or this analysis. No single value was above the reference limit values more adverse events (AEs), they were determined to be treatment of concern regarding left ventricular repolarization (namely, no related in only 25 patients (55.5%). Regardless of causality, 14 (31%) corrected QT interval by Fredericia [QTcF] was >480 ms and no δ patients experienced at least one Grade ≥ 3 AE according to Na- QTcF was >60 ms) in any of the evaluable patients. No significant tional Cancer Institute–Common Toxicity Criteria for AEs, version 5.0 effects either on atrioventricular conduction or on depolarization, (NCI-CTCAE v5). The prevalence of grade 3–4 AEs was 40.0% in the 2.5 as measured by mean changes in PR and QRS intervals, were mg cohort, 20.0% in the 2.0 mg cohort, and 33.3% in the 1.5 mg observed. No new clinically relevant morphological changes were cohort. Although almost all Grade ≥ 3 AEs were attributed to COVID- observed, except for a few isolated ST segment, T wave, and 19, two Grade 3 AEs were attributed to plitidepsin: one case each of conduction abnormalities, likely explained by the consequences of anaphylactic reaction (at 1.5 mg/day) and diarrhea (at 2.5 mg/day). acute infection. No Grade 4 AEs were reported. Table 2 presents the frequency of treatment-related AEs in this Results on secondary efficacy end points study. The following treatment-related AEs occurred in more than one patient: nausea (42.2%), vomiting (15.6%), diarrhea (6.7%), ab- After treatment, patients’ viral loads were evaluated by RT-PCR. dominal pain (4.4%), dizziness (4.4%), and dysgeusia (4.4%). These Viral load showed mean declines from baseline of 1.35, 2.35, 3.25, events were all mild to moderate (Grade 1–2) except the one case of and 3.85 log10 copies/ml at days 4, 7, 15, and 31, respectively (Table 3; Grade 3 diarrhea described above. The implementation of the see also Table S4 for individual assessments). The mean time to aforementioned protocol amendment was associated with a re- undetectable viral load was 13 d and was longer in patients with duction in the proportion of patients with nausea (from 55.6% to severe disease at baseline (15 d) than in those with mild or 38.9%) and vomiting (from 22.2% to 13.9%). No new hypersensitivity moderate disease (12 d) (Fig 6A). There were no significant dif- reactions were seen in any of the 36 patients treated after the ferences in change in viral load across dose levels (Table 3 and Fig changes to premedication described above (108 infusions). 6B). Several laboratory abnormalities were reported in these pa- Nonmechanical invasive ventilation was required in one patient tients, most of which were consistent with the acute, inflammatory with moderate disease (4.3%) and in seven patients with severe nature of COVID-19. Of note, plitidepsin did not show signs of disease (46.7%). Six patients required intensive care support clinically relevant hemotoxicity; there were two patients with (13.6%), all of whom had severe disease at baseline (6 of 15, 40%). neutropenia (one Grade 1 and another Grade 2). An isolated Tables 3, S5, and S6 summarize additional outcome measures. Fig S2 Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 5 of 22 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. plots the evolution of a six-category ordinal scale that integrates patients with moderate disease compared with 53.3% (8 of 15) in the need for hospitalization and oxygen therapy (24) for each patients with severe disease, and 100% (6 of 6) in patients with mild participant, at prespecified time points. disease. Similarly, the discharge rate by Day 8 was 73.9% (17 of 23) in While on study, 64.4% (29 of 45) of patients received systemic patients with moderate disease compared with 27% (4 of 15) in corticosteroids for the treatment of COVID-19 manifestations, be- patients with severe disease and 66.7% (4 of 6) in patients with mild yond their use on days 1–3 as the per protocol pre-medication. They disease (Table 3). were similarly distributed across the dosing groups: 9 patients in Baseline viral load was found to be significantly correlated with the 1.5 mg/d and 10 patients each in the 2 mg/d and 2.5 mg/d, for a hospital discharge by Day 15, by logistic and Cox regression models. respective median duration of 16 d (interquartile range [IQR]: 11–32 This occurred despite the limitations for modeling (because of the d), 8.5 d (IQR: 5–31 d), and 17.5 d (IQR: 13–35 d). After completing small number of patients [N = 44] and the high percentage of plitidepsin treatment, 15.5% (7 of 45) of patients received other patients [82%] discharged by Day 15), following a stepwise selection additional treatments for COVID-19, including the anti-viral agent of covariates with a P-value < 0.10 in univariate logistic models. remdesivir (one patient) and/or the anti-IL-6 monoclonal antibody In addition to viral PCR reduction, patients showed analytical tocilizumab (six patients). improvement of biomarkers associated to inflammatory processes, such as lymphocyte count and C-reactive protein (CRP) (Figs S5 and Post hoc analysis of hospital discharge rates S6). Because patients with moderate COVID-19 at baseline repre- All 44 patients who completed the 3-d treatment with plitidepsin sented the largest subgroup in this study and may represent the were evaluated for efficacy analyses. Overall, the discharge rates by potential target population for further development of plitidepsin days 8 and 15 after the start of plitidepsin were 56.8% (25 of 44) and as a COVID-19 therapy, we conducted an additional post hoc ex- 81.8% (36 of 44), respectively. Without adjusting for any covariates ploratory analysis in these patients as part of hypothesis gener- and with the constraints of the small sample size, there was no ation for Phase III study design. This analysis showed that all clear dose effect on the time to hospital discharge (Fig 7A and B) patients with moderate disease at baseline who were allocated to and on discharge rates at Day 15: 78.6% (1.5 mg/day), 93.3% (2 mg/ the highest dose level of 2.5 mg/day (8 of 8) were discharged by day day), and 73.3% (2.5 mg/day). Nevertheless, the proportion of pa- 8, whereas three of seven patients (43%) and three of eight patients tients achieving hospital discharge by Day 8 seemed to increase (38%) treated at 2.0 mg/day and 1.5 mg/day, respectively, were with dose, from 42.9% for those receiving 1.5 mg/day to 60% and discharged beyond that time point. These data are visualized in Fig 66.7% for those receiving 2 and 2.5 mg/day, respectively. 8A, which shows that for patients with moderate disease who re- The median time to discharge was 7 d (IQR: 7–9 d) in patients with ceived plitidepsin 2.5 mg/day, the most probable duration of mild COVID-19, 7 d (IQR: 6–8 d) with moderate COVID-19, and 14 d hospitalization was ~1 wk. Furthermore, the variation in hospital- (IQR: 7–26 d) with severe COVID-19 at baseline (overall log-rank P = ization duration in patients receiving 2.5 mg/day was narrower than 0.001) (Figs S3 and S4 and Table S6). Figs 7B and S4 plot the length of that seen with other doses. the hospitalization and time in the intensive care unit per subject, Although statistical analyses were not performed because of the dose, and severity of the disease. As expected, the length of small sample size, visual exploration of mean trends do not appear hospitalization was greater in patients with severe disease at to capture dose-dependent differences in the kinetics of viral PCR baseline. The discharge rate by Day 15 was 95.7% (22 of 23) in in the subgroup of patients with moderate COVID-19 (data not Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 6 of 22 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. shown). Nevertheless, it suggests that the higher the dose of pli- CoV-2 is yet available. Several strategies have been attempted tidepsin, the faster the increase in lymphocyte count (Fig S7A), the with limited effect (25). One large collaborative effort systematically smaller the peak of CRP at day 7 (Fig S7B), and the faster the re- mapped the interactome between SARS-CoV-2 proteins and human covery of neutrophil-to-lymphocyte ratio (NLR) (Fig S7C), D-Dimer proteins, identifying several dozens of potentially druggable in- (Fig S7D), and in the score of a six-ordinal scale for outcome (Fig 8B, teractions (11). Notably, the authors highlighted the potent antiviral Table S5, and Fig S8) (24) (see footnote on Fig 8B for description of effects after the inhibition of eEF1A, which had been previously the ordinal scale). Corresponding improvement of lung infiltrates described as the target of plitidepsin (12). were also observed in non-protocol chest radiographs, an example In the set of preclinical studies of this and previous reports, of which is shown in Fig S9A–C. plitidepsin showed strong antiviral activity and a positive therapeutic index in in vitro models of SARS-CoV-2 infection, with better per- formance than other drugs, including remdesivir (8, 26 Preprint). In Discussion our study, regardless of the coronavirus species (HCoV 227E, SARS- CoV, and SARS-CoV-2), the host cells, or the quantifying method used, Despite worldwide efforts to identify new treatments, as of this highly consistent results were obtained, with the IC50 of plitidepsin publication, no highly effective antiviral therapy against SARS- always being in the nanomolar range. Notably, a similar in vitro Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 7 of 22 Figure 5. APLICOV-PC: Protocol Treatment and Pre- medication. IV, intravenous; PO, oral; 5-HT3, serotonin (5-hydroxytryptamine) receptor 3. antiviral effect was induced by plitidepsin against the α, β, δ, μ, and o patients was 2.2 mg/day (range 1.9–2.7 mg/day), and the median variants of SARS-CoV-2, which are known to bear several mutations cumulative dose for their first cycle was 11 mg (range 9.6–13.7 mg) affecting the viral spike protein that facilitates viral entry through its over a 5-d period. In the current study, plitidepsin was given daily interaction with the human ACE2 receptor (27 Preprint). for 3 consecutive days, and the maximum daily dose was set at 2.5 Using a drug-resistant mutant host factor, White et al demon- mg (7.5 mg in total). This study therefore explored a dose that was strated that the potent in vitro activity of plitidepsin against SARS- 68% of the median recommended dose for cancer patients. CoV-2 was mediated through eEF1A inhibition (8). In addition, they also Treatment with plitidepsin was well tolerated, with most AEs showed strong antiviral activity in vivo, characterized by a significant being mild and transient in nature. There was no relevant hemotoxicity, reduction in the viral load in lungs, as well as a clear reduction in and the proportion of laboratory abnormalities was consistent with alveolar and peribronchial inflammation. In addition, their data also those expected in COVID-19 patients (28). A central laboratory-based supported a 3-d schedule, as the one used in this clinical study (8). analysis performed on serial ECGs from this study indicated that the A phase I trial explored the daily dosing of plitidepsin in cancer administration of 1.5, 2.0, and 2.5 mg plitidepsin as a 90-min IV infusion patients over 5 consecutive days (13). Dose-limiting toxicities oc- once daily for 3 consecutive days did not induce cardiac dysfunction, curred in four of eight patients receiving total cumulative actual with no alterations seen on left ventricular repolarization, atrioven- doses greater than 13 mg (2.6 mg/day). The recommended dose for tricular conduction or depolarization. subsequent phase 2 studies was defined at 1.2 mg/m2/day (6 mg/ The death rate due to COVID-19 in our study was 6.7%. A recently m2 total dose). The median actual dose received by these seven published meta-analysis on 33 studies on COVID-19 (totaling 13,398 Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 8 of 22 Table 1. Patients’ baseline characteristics. Parameter 1.5 mg/daya (n = 15) 2.0 mg/daya (n = 15) 2.5 mg/daya (n = 15) b Age (yr) 51 (32–75) 49 (34–71) 53 (31–84) Gender—N (%) Male 11 (73.3%) 11 (73.3%) 8 (53.3%) Female 4 (26.7%) 4 (26.7%) 7 (46.7%) Race—N (%) White 13 (86.7%) 9 (60%) 9 (60%) Latino 2 (13.3%) 4 (26.7%) 6 (40%) Asian 0 (0%) 1 (6.7%) 0 (0%) Arab 0 (0%) 1 (6.7%) 0 (0%) Time from symptom onset to first administration (d) b 6 (3–10) 6 (3–10) 6 (2–10) Comorbidities—N (%) One 2 (13.3%) 7 (46.7%) 6 (40%) Two or more 8 (53.3%) 6 (40%) 7 (46.7%) Hypertension 2 (13.3%) 2 (13.3%) 5 (33.3%) Heart disease 1 (6.7%) 0 (0%) 1 (6.7%) COPDc 1 (6.7%) 1 (6.7%) 1 (6.7%) Asthma 2 (13.3%) 0 (0%) 3 (20%) Kidney disease 0 (0%) 1 (6.7%) 0 (0%) Diabetes 1 (6.7%) 5 (33.3%) 2 (13.3%) Obesity 1 (6.7%) 5 (33.3%) 4 (26.7%) Patients assessed at room air—N(%) 9 (60.0) 7 (46.7) 6 40%) b SpO2 at room air (%) 95 (92–99) 95 (91–97) 96.5 (94–97) PaO2/FiO2 ratiob 358 (336–408) 352 (285–396) 343 (253–481) PaO2/FiO2 ratio < 300 – N(%) 0 3 (20.0) 2 (13.3) Disease severity at entry—N (%) (1) Mild COVID-19 2 (13.3%) 3 (20%) 1 (6.7%) Moderate COVID-19 8 (53.3%) 7 (46.7%) 8 (53.3%) Severe COVID-19 5 (33%) 5 (33%) 6 (40%) D-dimer (ng/ml)b 330 (162–1,081) 463.5 (200–1,270) 415 (106–962) b Ferritin (ng/ml) 408 (96.8–1,652.8) 597 (174–1,055.2) 363 (12.2–1,647) b C-reactive protein (mg/l) 17.7 (1.2–120.4) 67.2 (2.1–128) 32.6 (0.3–120) log10 copies/ml viral load median (range)b 6.3 (1.5–9.7) 6.2 (3.8–7) 5.7 (1.5–10.6) d Day 1 six-point ordinal scale—N (%) (2) 2d 6 (40) 6 (40) 4 (26.7) d 3 9 (60) 9 (60) 11 (73.3) a Daily for 3 consecutive days. b Median (range). c Chronic Obstructive Pulmonary Disease. d 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. PaO2, Oxygen partial pressure in arterial blood (imputed from oxygen saturation as described in Supplemental Data 2). FiO2. Oxygen proportion in inspired air. Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 9 of 22 Table 2. Plitidepsin-related adverse events. Pre-amendmenta,b (n = 9) Post-amendmentc (n = 36) Parameter Grade 1 Grade 2 Grade 3 Grade 1 Grade 2 Grade 3 N (%) N (%) N (%) N (%) N (%) N (%) Nausea 3 (33.3%) 2 (22.2%) — 11 (30.6%) 3 (8.3%) — Vomiting 2 (22.2%) — — 3 (8.3%) 2 (5.6%) — Diarrhea — — — 1 (2.8%) 1 (2.8%) 1 (2.8%) Abdominal pain — — — 2 (5.6%) — — Dyspepsia — — — 2 (5.6%) — — Asthenia — — — 1 (2.8%) 1 (2.8%) — Anorexia — — — 1 (2.8%) — — Chest discomfort - - - 1 (2.8%) - - Temperature regulation disorder — — — 1 (2.8%) — — Dysthermia — — — 1 (2.8%) — — Anaphylactic reaction — — 1 (11.1%) — — — Amylase increasedd — — — — 1 (2.8%) — Lipase increasede — — — — 1 (2.8%) — Decreased appetite — — — — 1 (2.8%) — Dizziness — — — 2 (5.6%) — — Dysgeusia — — — 2 (5.6%) — — a Relevant amendment #9 was implemented in Protocol v5.0 dated 13 August 2020 (Supplemental Data 2): It modified prophylactic medication before plitidepsin infusion to add ondansetron 8 mg IV slow infusion and changed the route of administration of dexamethasone, from oral to IV. The dose of dexamethasone was 8 mg (calculated as 8 mg dexamethasone phosphate, which is equivalent to 6.6 mg dexamethasone base). b 25 plitidepsin IV infusions. c 108 plitidepsin IV infusions. d Short lasting, 5 min, retro sternal low intensity pain during first day IV infusion: self-resolved plitidepsin infusion completed days 1, 2, and 3. e Same patient, onset day 2 plitidepsin, self-resolved in 48 h. patients, excluding critical care-only studies), estimated that in studies were not predefined end points in this trial. Thus, this study hospitalized patients the mortality rate was 11.5% (95% CI: 7.7–16.9%) was not designed to evaluate if plitidepsin can improve these pa- (29). Published data from large retrospective studies on patients rameters and ongoing and future controlled clinical studies will admitted into Spanish hospitals report death rates between 20% address these hypotheses. The trial did not generate enough in- and 28% (30, 31, 32, 33, 34). It should be noted, however, that these formation to select the best dose for further clinical development, analyses were performed on data extracted from the first epidemic and further research is currently in progress to address this issue. wave, and APLICOV-PC was run during the second wave, which The antiviral mechanism of plitidepsin may represent significant might account in differences in the availability of health resources, advantages in the treatment of COVID-19. Specifically, the likelihood learning curve, and baseline severity of hospitalized patients. of developing treatment-resistant SARS-CoV-2 strains seems to be Preliminary efficacy data gathered from this clinical trial were in remote given that plitidepsin does not directly target a viral agreement with the preclinical antiviral activity of plitidepsin de- component. Therefore, SARS-CoV-2 variants that carry mutations to scribed above. Patients treated with plitidepsin showed reductions in viral components may be equally sensitive to plitidepsin treatment. viral load with respect to their baseline value, and analytical im- All patients in this study received dexamethasone alongside provement of biomarkers associated to inflammatory processes (Figs plitidepsin (i.e., for at least 3 d), which impedes our ability to fully S5 and S6). There were reports of prompt clearance of pneumonia understand the efficacy of plitidepsin in patients with COVID-19. infiltrates in some participants with available chest imaging per- Dexamethasone is currently recommended for hospitalized pa- formed for medical reasons (i.e., not per protocol) (Fig S9A and B). tients with COVID-19 who have SpO2 < 94%, including patients on Nevertheless, these results should only be considered suggestive supplemental oxygen, or in patients with critical illness (38). On the other given the limitations of this trial. Studies on the natural history of hand, supra-physiologic glucocorticoid treatment leads to lymphocyte COVID-19 have shown that viral load peaks at symptom onset, fol- apoptosis mediated by the glucocorticoid receptor (39). Noteworthy, lowed by a subsequent decline (35, 36, 37). Therefore, in the absence there is evidence that the use of corticosteroids is associated with a of a control group, we are not able to conclude that plitidepsin delayed viral clearance in patients COVID-19 (40). In addition, treatment causally affected viral load in this study. In addition, hospital dis- with glucocorticoids in patients with COVID-19 has been reported charge rates, changes in inflammatory biomarkers, and radiological to induce an early drop in absolute lymphocyte counts, with Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 10 of 22 Table 3. Summary of protocol-specified efficacy end points. Dose cohort End point 1.5 mg (N = 14a) 2.0 mg (N = 15) 2.5 mg (N = 15) Total (N = 44) Mortality from Day 1 to Day 7 — — — — Day 15 — — — — Day 31b 1 (7.1) — 1 (6.7) 2 (4.5) Patients requiring invasive mechanical ventilation and/or intensive care unit admission Day 1 to Day 7 2 (14.3) 1 (6.7) 2 (13.3) 5 (11.4) Day 8 to Day 15 1 (7.1) 1 (6.7) 1 (6.7) 3 (6.8) Day 16 to Day 31 1 (7.1) 1 (6.7) 1 (6.7) 3 (6.8) Day 1 to Day 31 2 (14.3) 1 (6.7) 3 (20.0) 6 (13.6) Patients requiring noninvasive mechanical ventilation Day 1 to Day 7 4 (28.6) 0 1 (6.7) 5 (11.4) Day 8 to Day 15 3 (21.4) 0 2 (13.3) 5 (11.4) Day 16 to Day 31 1 (7.1) 1 (6.7) 1 (6.7) 3 (6.8) Day 1 to Day 31 5 (35.7) 1 (6.7) 2 (13.3) 8 (18.2) Patients requiring oxygen therapy at Day 7 12 (85.7) 12 (80.0) 11 (73.3) 35 (79.5) Day 15 4 (28.6) 1 (6.7) 4 (26.7) 9 (20.5) Day 31 0 2 (13.3) 1 (6.7) 3 (6.8) Day 1 to Day 31 12 (85.7) 12 (80.0) 11 (73.3) 35 (79.5) Mean change in viral load from baseline toc log10 copies/ml Day 4 −1.23 −1.49 −1.32 −1.35 Day 7 −2.55 −2.26 −2.25 −2.35 Day 15 −4.22 −2.70 −2.92 −3.25 Day 31 −4.70 −3.53 −3.49 −3.85 Days Mean time from baseline until undetectable viral loadc 11 14 14 13 a One patient who experienced an anaphylactic reaction during the first plitidepsin infusion had treatment discontinued and was not considered evaluable for efficacy. b One additional patient treated at 2.5 mg/day died on Day 57, because of COVID-19 complications. c Results based on 42 patients at Day 4 (13 at 1.5 mg, 14 at 2.0 mg, 15 at 2.5 mg), 40 patients at Day 7 (13 at 1.5 mg, 14 at 2.0 mg, 13 at 2.5 mg), 38 patients at Day 15 (12 at 1.5 mg, 13 at 2.0 mg, 13 at 2.5 mg), and 39 patients at Day 31 (11 at 1.5 mg, 14 at 2.0 mg, 14 at 2.5 mg). prolonged lymphopenia, along with a significant steady increase in studies are needed to evaluate the full benefit/risk profile of the absolute neutrophil count, altogether driving significant in- the concomitant use of glucocorticoids and plitidepsin and creases in the neutrophil-lymphocyte ratio for at least 2 wk (41). eventually define the appropriate candidate for this therapy. Absolute neutrophil count elevation seen in response to cortico- Elevation of inflammation markers such as CRP is associated steroid administration is similar to trends associated with increased with an increased risk of disease severity and mortality (43, 44). In mortality in several coronavirus studies to include the current SARS- this regard, it is noteworthy that in patients with moderate COVID-19 CoV-2 pandemic (41). Notably, in the present study, despite a large at baseline, intra-patient variations of inflammation markers proportion of patients receiving dexamethasone for more than trended favorably with higher doses of plitidepsin, which may 3 d, patients exhibited a median increase in the absolute number suggest a drug-effect. These observed changes in inflammatory of lymphocytes, along with a drop in NLR and in viral RT-PCR results. biomarkers may partly explain the rapid clearance of lung infil- Again, the lack of a control group prevents conclusive evidence, trates observed in chest imaging (Fig S9A–C), and is in line with the but this could be a very interesting finding given that, in addition preclinical observations reported by White et al that treatment with to glucocorticoid therapy, SARS-CoV-2 infection can also induce plitidepsin prevents severe lung inflammation (8) (Fig S10A–C). an early functional exhaustion of cytotoxic lymphocytes that may Translational research on chronic lymphocytic leukemia has be responsible for delaying immune responses (42). Future identified that plitidepsin can induce cytotoxicity in monocytes at Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 11 of 22 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. nanomolar concentrations that have little effect in normal lymphocytes Materials and Methods (45). Monocytes and macrophages may be infected by SARS-CoV-2, which results in an impairment of the adaptive immune responses Preclinical studies on plitidepsin antiviral activity against the virus, virus spread, and local tissue inflammation, mediated by the production of large amounts of pro-inflammatory cytokines and Enjuanes team studies chemokines (46). We hypothesize that plitidepsin, besides acting as an Cells and viral infection (HCoV-229E and SARS-CoV) Monkey Vero antiviral agent, may also modulate immune response by its effects on E6 cells were kindly provided by E. Snijder (Leiden University Medical monocytes/macrophages. Center). Human liver–derived Huh-7 cells were kindly provided by R Plitidepsin was hypothesized to provide potential benefits Bartenschlager (University of Heidelberg). Cells were cultured in against coronavirus infections, and therefore against SARS- DMEM (Lonza) supplemented with 25 mM Hepes, 10% FBS (HyClone), COV-2. This hypothesis was first successfully tested through 2% glutamine, and 1% nonessential amino acids (Sigma-Aldrich) and various in vitro studies, which have led to in vivo tests that have maintained at 37°C in a humidified atmosphere of 5% CO2. again demonstrated the high potency of plitidepsin in inhibiting HCoV-229E was kindly provided by V Thiel (Institute of Virology SARS-CoV-2. Notably, the putative mechanism of action by and Immunology). SARS-CoV virus was rescued from the corre- which plitidepsin inhibits SARS-CoV-2 (i.e., via a host protein sponding infectious cDNAs (47). All experiments with SARS-CoV target) suggests that the drug could have a substantial impact infectious virus were performed in BSL-3 facilities at CNB-CSIC on SARS-CoV-2 variants, including the current variants of according to institutional guidelines. concern δ and o. The set of in vitro research presented in this work confirms this point. Experimental design Human Huh-7 cells were infected with HCoV- We now report a proof-of-concept clinical study, showing the 229E-GFP virus at an MOI of 0.01. After 8 hours post infection (hpi), safety of administering plitidepsin at the doses and duration de- the medium was replaced by fresh medium containing different scribed here, and suggesting a potential therapeutic benefit in plitidepsin concentrations. The presence of fluorescent foci, indi- patients with COVID-19. Nevertheless, our study has several limi- cating HCoV-229E-GFP infection, was analyzed at 48 hpi. tations, including the small number of patients evaluated, the large SARS-CoV virus stock at 2 × 107 pfu per ml was used. Confluent observed variability, and the lack of a control group. These char- Vero E6 cells were infected with SARS-CoV at a MOI of 0.01. After acteristics prevent us from understanding the efficacy of plitidepsin in patients with COVID-19, limit our ability to observe the presence 1 hpi, virus inoculum was retired and medium was replaced by fresh of significant dose–response effects, and restrict our use of this DMEM-HEPES medium with 2% FBS at different concentrations of data to hypothesis generation for future studies. An international the compounds. Treatments were as follows: (1) mock-infected cells controlled Phase III trial exploring the efficacy and safety of pli- and SARS-CoV–infected cells in the absence of DMSO were used as tidepsin in hospitalized patients with moderate COVID-19 requiring a control to rule out DMSO toxicity effect; (2) DMSO at the same % oxygen supplementation (NEPTUNO; NCT04784559) is currently present in the compound dilutions was used as a negative control; ongoing. Based on the favorable safety profile seen in this study, (3) plitidepsin and didemnin B (data not shown), the most prom- doses of 1.5 and 2.5 mg/day plitidepsin have are being evaluated in ising compounds in the previous HCoV-229E-GFP screening, were the Phase III trial. tested at 0.5, 5, 50, 100, and 500 nM; (3) PM021473 was used as a In summary, we have integrated preclinical and clinical studies control with no effect on CoV infection. It was tested at 0.5, 5, 50, 100, on the use of plitidepsin to treat SARS-CoV-2 and other coro- and 500 (data not shown); (4) remdesivir was used as a positive navirus infections and generated promising patient data sup- control, and tested at 0.5, 5, 100, 500, and 1,000 nM. porting the launch of a Phase III clinical study to demonstrate the Two independent biological replicates were performed for each efficacy of treatment with plitidepsin in moderate COVID-19 pa- condition. At 48 hpi, culture supernatant was collected for virus tients who require oxygen therapy. titration, following standard procedures (48). Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 12 of 22 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. Cells were also collected and total intracellular RNA was purified 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine using RNeasy Mini Kit (QIAGEN). Total cDNA was synthesized using 100 (all Thermo Fisher Scientific). ng of total RNA as a template, random hexamers, and a high-capacity SARS-CoV-2 was isolated from a nasopharyngeal swab and its cDNA transcription kit (Life Technologies). SARS-CoV genomic RNA genomic sequence deposited at GISAID repository (http://gisaid.org) was evaluated using a custom TaqMan assay targeting nsp2 se- with accession ID EPI_ISL_510689 as previously described (26 Preprint). quence. The human hydroxymethylbilane synthase (HMBS) gene (TaqMan code Hs00609297_m1) was used as a reference house- Antiviral activity Plitidepsin was assayed from 5 to 100,000 nM in keeping gene. Data were acquired with a 7500 real-time PCR system duplicates as previously detailed (26 Preprint). (Applied Biosystems) and analyzed with 7500 software v2.0.6. Relative Drug dilutions were added to Vero E6 cells and, immediately quantifications were performed using the 2−ΔΔCt method (49). after, 20 tissue culture infectious dose 50% (TCID50) per well of SARS-CoV-2 were inoculated to 30,000 cells in 200 μl. This viral Rodon team studies concentration achieves a 50% of cytopathic effect 3 d postinfection. Cells and viral isolation Vero E6 cells (ATCC CRL-1586) were cul- Untreated noninfected cells and untreated virus-infected tured in DMEM (Lonza), supplemented with 5% FCS (EuroClone), cells were used as negative and positive controls of infection, Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 13 of 22 respectively. To detect any drug-associated cytotoxic effect, Vero E6 cells were equally cultured in the presence of increasing drug concentrations, but in the absence of virus. Viral-induced cytopathic or drug-induced cytotoxic effects were measured 3 d post infection, using the CellTiter-Glo luminescent cell viability assay (Promega). Luminescence was measured in a Fluoroskan Ascent FL luminometer (Thermo Fisher Scientific). Cells not exposed to the virus were used as negative controls of infection and were set as 100% of viability to normalize data and calculate the percentage of cytopathic effect. Response curves of compounds were adjusted to a nonlinear fit regression model, calculated with a four-parameter logistic curve with variable slope. Krogan/Garcia-Sastre teams’ studies Cell culture and drugs Calu-3 cells (ATCC HTB-55) and Caco-2 cells were a kind gift Dr Dalan Bailey (Pirbright Institute). Cells were cultured in DMEM supplemented with 10% heat-inactivated FBS (Labtech), 100 U/ml penicillin/streptomycin, with the addition of 1% Sodium Pyruvate (Gibco) and 1% Glutamax for Calu-3 and Caco-2 cells. All cells were passaged at 80% confluence. For infections, adherent cells were trypsinized, washed once in fresh medium, and passed through a 70 μm cell strainer before seeding at 0.2 × 106 cells/ml into tissue-culture plates. Calu-3 cells were grown to 60–80% confluence before infection as described previously (50 Preprint). Plitidepsin (PharmaMar) and remdesivir (SelleckChem) were reconstituted in sterile DMSO. Viruses SARS-CoV-2 strain BetaCoV/Australia/VIC01/2020 (NIBSC) and SARS-CoV-2 B.1.1.7 (SARS CoV 2 England/ATACCC 174/2020) strain were propagated by infecting Caco-2 cells at a MOI 0.01 TCID50/cell, in DMEM supplemented with 10% FBS at 37°C. Virus was harvested at 72 hpi and clarified by centrifugation at 2,300g for 15 min at 4°C to remove any cellular debris. Virus stocks were aliquoted and stored at −80°C. Virus titers were determined by quantification of SARS-CoV-2 RNA genomes/ml as previously de- scribed (50 Preprint). Infection and drug assays Calu-3 and Caco-2 cells were pretreated with remdesivir or plitidepsin at the indicated concentrations or DMSO control at an equivalent dilution for 2 h before SARS-CoV-2 infection. Figure 8. Post hoc analysis on hospital discharge by plitidepsin dose in Caco-2 and Calu-3 cells were infected at 1 × 103 copies per cell, patients with moderate COVID-19 at baseline. equivalent to an MOI of 0.01 TCID50 per cell (as titered on Vero.E6). (A) Subgroup of patients with moderate COVID-19 (n = 23 pts): Distribution of the Inhibitors were maintained throughout infection. probability of the duration of the hospitalization, according to the dose of Cells were harvested after 24 h for analysis and viral infection 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 measured by intracellular detection of SARS-CoV-2 nucleoprotein moderate disease at baseline, according to the administered dose of plitidepsin. by flow cytometry. Tetrazolium salt (MTT) assay was performed to The six-point scale was defined as follows (24): 1, discharged or having reached verify cell viability. 10% vol/vol MTT was added to the cell media and discharge criteria (defined as “clinical recovery”: normalization of pyrexia, respiratory rate <24 breaths per minute, saturation of peripheral oxygen >94% on cells were incubated for 24 h at 37°C. Cells were lysed with 10% SDS room air, and relief of cough, all maintained for at least 72 h); 2, hospital and 0.01M HCl and the formation of purple formazan was measured admission but not requiring oxygen supplementation; 3, hospital admission for at 620 nm. 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 Flow cytometry For flow cytometry analysis, adherent cells were ventilation; 6, death. See also Table 3 and Figs S7 and S8. recovered by trypsinization and washed in PBS with 2 mM EDTA Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 14 of 22 (PBS/EDTA). Cells were stained with fixable Zombie NIR Live/Dead (ATCC). SARS-CoV-2 (βCoV/KOR/KCDC03/2020) was provided by dye (BioLegend) for 6 min at room temperature. Excess stain was Korea Centers for Disease Control and Prevention (KCDC) and was quenched with FBS-complemented DMEM. Cells were fixed in 4% propagated in Vero cells. Viral titers were determined by plaque paraformaldehyde before intracellular staining. assays in Vero cells. For intracellular detection of SARS-CoV-2 nucleoprotein, cells were permeabilized for 15 min with Intracellular Staining Perm Wash Buffer Reagents Plitidepsin (batch#: 16 D19) and ampoule (batch#: 60108) (BioLegend). Cells were then incubated with 1 μg/ml CR3009 SARS- were provided by Boryung Pharmaceutical. Anti-SARS-CoV-2 N CoV-2 cross-reactive antibody (a kind gift from Dr. Laura McCoy) in protein antibody was purchased from Sino Biological lnc. Alexa permeabilization buffer for 30 min at room temperature, washed Fluor 488–goat anti-rabbit [IgG (H+L) secondary antibody and once and incubated with secondary Alexa Fluor 488–Donkey-anti- Hoechst 33342 were purchased from Molecular Probes. Human IgG (Jackson Labs). All samples were acquired and analyzed on a NovoCyte 3005 Flow Cytometer System (Agilent). Drug treatment, infection and immunofluorescence staining 2 mg of plitidepsin was dissolved in either 1,801.3 μl of DMSO or in In vitro study of SARS-CoV-2 variants sensitivity to plitidepsin Naso- ampoule at a final concentration of 1 mM and a twofold dilution pharyngeal swab specimens were collected as part of the routine series was made with 20-points. Vero cells were seeded at l.2 × 104 SARS-CoV-2 surveillance conducted by Viviana Simon and the Mount cells per well in DMEM supplemented with 2% FBS and I X Antibiotic- Sinai Pathogen Surveillance program (IRB approved, HS#13-00981). Antimycotic solution (Gibco) in black, 384-well, μClear plates Specimens were selected for viral culture on Vero-E6 cells based on (Greiner Bio-One), 24 h before plitidepsin treatment and virus the complete viral genome sequence information (51). The SARS-CoV- infection. Calu-3 cells were seeded at 2.0 × 104 cells per well in 2 virus USA-WA1/2020 was obtained from BEI resources (NR-52281) Eagle’s Minimel Essential Medium supplemented with 10% FBS and and used as wild-type reference. Viruses were grown in Vero- 1X Antibiotic-Antimycotic solution (Gibco) in black, 384-well, μClear TMPRSS2 cells (BPS Bioscience) for 4–6 d; the supernatant was plates (Greine r Bio-One), 24 h before plitidepsin treatment and clarified by centrifugation at 4,000g for 5 min and aliquots were virus infection. Twenty-point plitidepsin dilution series generated frozen at −80°C for long term use. Expanded viral stocks were above was added to Vero or Calu-3 cells with the highest con- sequence-verified to be the identified SARS-CoV-2 variant and tit- centration at 5 μM. After 1 h, the plates were transferred into the tered on Vero-TMPRSS2 cells before use in antiviral assays. BSL-3 containment facility for virus infection. SARS-CoV-2 was Two thousand (2,000) HeLa-ACE2 cells were seeded into 96-well added at a MO1 of 0.0125 and 0.1 to the plates for Vero cells and plates and incubated for 24 h. 2 h before infection, the medium was Calu-3 cells, respectively. replaced with a new media containing the compound of interest, in- The cells were fixed at 24 hpi with 4% paraformaldehyde. Anti- cluding a DMSO control. Plates were then transferred into the biosafety SARS-CoV-2 N protein antibody and Alexa Fluor 488–conjugated goat level 3 (BSL-3) facility and 1,000 PFU (MOI = 0.25) of SARS-CoV-2 was anti-rabbit IgG antibody were used for immunostaining of viral N added, bringing the final compound concentration to those indicated. protein and Hoechst 33342 were used to stain nuclei of the host cells. SARS-CoV-2/WA1, α (B.1.1.7), β (B.1.351), δ (B.1.617.2), μ (B.1.621), and o (B.1.1.529) variants were used as indicated. Plates were then incubated Image analysis The images acquired with Operetta (Perkin Elmer) for 48 h. Infectivity was measured by the accumulation of viral NP protein were analyzed using our in-house Image-Mining (IM) software to in the nucleus of the HeLa-ACE2 cells (fluorescence accumulation). quantify cell numbers and infection ratio by counting Hoechst- Percent infection was quantified as ((Infected cells/Total cells) − stained nuclei and viral N protein-expressing cells, respectively. Background) × 100, and the DMSO control was then set to 100% infection The infection ratio of each well was normalized to the average of for analysis. Cytotoxicity was also performed at matched concentrations infection percentage of infection group (0.5% DMSO) and mock using the MTT assay (Roche), according to the manufacturer’s instruc- infection group in each plate. The cell ratio was determined tions. Cytotoxicity was performed in uninfected HeLa-ACE2 cells with according to the number of cells of each well versus the average same compound dilutions and concurrent with viral replication assay. number of cells of mock infection in each plate and described as All assays were performed in biologically independent triplicates. “cell number to mock” in the dose–response curve (DRC) graph. The DRCs of plitidepsin (both. dissolved either in DMSO or in ampoule), Animal models of SARS-CoV-2 infection experiments and lung was analyzed using the XLfit equation: Y = Bottom + (Top − Bottom)/ histological analysis These studies were conducted under pro- (1 + (IC50/X)Hillslope). tocols approved by the Institutional Animal Care and Use Com- The IC50 and 50% toxicity concentration (CCso) values were mittee (IACUC) and were performed in animal biosafety level 3 calculated from the fitted DRCs. Selectivity index (SI) was calculated (BSL3) facility at the Icahn school of Medicine in Mount Sinai as CC50/IC50. All IC50 and CC50 values were determined in duplicate Hospital, New York City. They are described in reference 8. experiments. All the experiments were conducted simultaneously. Model-based dose justification Plasma protein binding study The binding of plitidepsin to plasma proteins was determined in Definition of in vitro target dose of plitidepsin (Boryung vitro at three concentrations (100, 250 and 500 ng/ml) in male rat, Pharmaceutical (52)) dog and human plasma (53). Virus and cells Both Vero (ATCC CCL-81) and Calu-3 cells (ATCC HTB- Rat (CD), beagle dog, and human blood was collected at Aptuit Srl 55) were purchased from the American Type Culture Collection from at least three male donors, and plasma prepared by Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 15 of 22 centrifuging blood at 2,000g, 4°C for 10 min. Human plasma was Results from previously described studies show that: (a) in obtained from healthy and fasted male volunteers. Na-heparin was human plasma, plasma-protein binding of plitidepsin was esti- used as the anti-coagulant for all species. All plasma was stored at mated at 98%, independent of drug concentration (Table S2), and approximately −20°C and thawed only once, on the day of the (b) after the administration of a single i.v. bolus dose (0.2 mg/kg) of experiment. plitidepsin (14C-labeled), a significant increased distribution of Plasma protein binding was assessed using Rapid Equilibrium radioactivity was found in lung; this preferential distribution led to Dialysis plates pre-loaded with equilibrium dialysis membrane lung-to-plasma area under the curve ratio (LPR) of ~543-fold, re- inserts (MWCO ~8 kD). Separation of free compound from protein-bound material was spectively (calculated from Table S3). achieved by dialysis of the sample through the membrane under The partition coefficient LPR enables the quantification of the appropriate shaking at 37°C. The suitability of equilibrium dialysis total drug concentration in the tissue, and, by assuming similar lung as a method for protein binding determination of plitidepsin was distribution in humans to that observed in rodents and similar assessed. fraction of unbound drug in plasma and tissue, unbound exposures Before experimentation, the time required to reach equilibrium in human lung can be further estimated. was determined in male rat and human plasma at one concen- Therefore, total plasma concentration (μg/l) of plitidepsin as- tration (250 ng/ml) and six selected time points (2, 3, 4, 5, 6, and 7 h) sociated with lung exposures above the in vitro target concen- at 37°C. tration IC50 and IC90, were estimated at 0.33 (95% CI, 0.30–0.37) μg/l Plitidepsin stability was assessed in plasma (all species) and PBS and 0.96 (95% CI, 0.78–1.18) μg/l, respectively, according to the at two concentrations (100 and 500 ng/ml for plasma; 0.5 and 50 ng/ following equation: ml for PBS) by comparing plitidepsin concentration in spiked and preincubated plasma before and after incubation at 37°C for 5 h as ICFtotal;in vitro ICFtotal;plasma = ; determined previously. fu;human ⋅LPRrat Plitidepsin tissue distribution study where ICFtotal,plasma is the total target plasma concentration (μg/l), An in vivo distribution study was carried out in rats. PM140064 (14C1- ICFtotal,in vitro is the concentration (μg/l) used in the in vitro ex- Plitidepsin) was supplied by PharmaMar at a radiochemical purity periment, fu,human is the unbound fraction in human plasma, and of 95% or greater, with no single impurity 3% or greater. The study LPRrat is the lung-plasma area under the curve (AUC) ratio in the was conducted at Aptuit Srl (54). distribution study in rats. This equation considers plasma protein 24 male and 24 female Sprague–Dawley rats each received a binding and lung-plasma AUC ratio, based on current recom- single IV bolus administration of [14C] Plitidepsin at a target dose of mendations (21). 0.2 mg/kg. After administration, three animals per time-point (0.25, 1, 2, 4, 8, 24, 48, and 72 h post-dose) were exsanguinated from the Simulation of plitidepsin plasma exposures at the selected dose abdominal aorta and blood retained. Actual times of bleeding were regimen recorded. In addition to blood, the following tissues were collected: A validated pharmacokinetic population model of plitidepsin (22) brain, eyeballs, heart, liver, lung, skeletal muscle (quadriceps), fat, updated with data from multiple myeloma patients, was used to kidneys, stomach, skin, small intestine, spleen, thyroid, lymph nodes, and testes or ovaries. At the end of the collection period simulate plitidepsin plasma profiles in typical subjects treated with animals were euthanized by CO2 asphyxiation and the carcasses the selected dose regimen (days 1–3 in 1 h 30 min infusion), to were discarded. Additional animals were bled to obtain control observe whether they would reach the estimated target plasma blood/plasma and tissues. concentrations with antiviral activity. This model was developed based on plasma and blood concentrations from 549 cancer pa- Estimation of target IC50 and IC90 total plasma concentrations tients from four Phase I, nine Phase II and one Phase III study The results from Boryung Pharmaceuticals in infected Vero cells treated with plitidepsin either as monotherapy or in combination demonstrated a very strong antiviral effect induced by plitidepsin, with dexamethasone, at doses ranging from 0.8 to 8.0 mg/m2 as a 1- or 24-h infusion weekly, 3- or 24-h infusion biweekly, or 1-h infusion with half-maximal inhibitory concentration (IC50) as low as 3.26 daily for 5 consecutive days every 3 wk. An open, three- (95%, 2.97–3.59) nM. These were consistent with results from a compartment disposition model with linear elimination and lin- different cell line, Calu-3. Based on the resulting Hill slope (2.08) ear distribution from the central compartment to two peripheral and according to the following equation (55), a 90% maximal in- compartments was used to describe the pharmacokinetics (PK) of hibitory concentration (IC90) of 9.38 (95%CI, 7.65–11.50) nM was plitidepsin in plasma. The model was parameterized in terms estimated. of systemic clearance (Cl), central (V1) and peripheral volume of distribution for the shallow (V2) and deep (V3) compartments, and  1=H F intercompartmental exchange flows for shallow (Q2) and deep (Q3) ICFinvitro = × IC50invitro 100 − F compartments. The concentration of plitidepsin bound to red blood cells was modeled as a nonlinear function and the plitidepsin where F is the percentage of response (i.e., 90%) and H is the Hill blood concentration was estimated according to the following slope. equation (22): Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 16 of 22 Randomization Bmax ⋅ Cplasma Randomization was done through registration in the study’s Cblood = Cplasma ⋅ ð1 − HCT Þ + ⋅ HCT kd + Cplasma electronic case report form. The system assigned a unique ran- domization number for each patient. Patients were enrolled se- where Bmax corresponds to the maximal plitidepsin concentration quentially and allocated by block randomization into the three bound to blood cells, kd is the plitidepsin plasma concentration at dose groups (1.5, 2.0, and 2.5 mg plitidepsin) up to an initial sample which the plitidepsin bound to red blood cells is half-maximal, and size of nine patients per group. To ensure safety, the first three HCT is the baseline hematocrit of each patient. patients in the 2.0 mg group could not be enrolled until the first Based on the deterministic simulations of plasma concentration– three patients in the 1.5 mg cohort had successfully completed Day time profiles, flat doses of 1.5, 2.0, and 2.5 mg will be associated to 15 assessments (12 d after completing plitidepsin dosing); similarly, plasma concentrations above IC50 throughout the whole treatment the first three patients in the 2.5 mg cohort could be not enrolled period and will remain above IC90 during most of the administration until the first three patients in the 2.0 mg cohort had successfully interval, as depicted in Fig 3, whereas accumulation after three re- completed Day 15 assessments (12 d after completing plitidepsin peated administrations is minimal. dosing). Finally, three patients had to be treated in the 2.5 mg cohort to complete the accrual of nine patients per cohort. When multiple dose levels were open for enrollment, patients were randomized 1:1 Proof-of-concept study design or 1:1:1 as appropriate (Fig 4). After an interim safety analysis, a protocol amendment was approved to subsequently expand the Trial design accrual up to 45 patients (six additional patients per dose group). The plitidepsin clinical proof-of-concept study (APLICOV-PC, APL-D- 002-20; EudraCT #2020-001993-31; NCT04382066) was a multicenter, Interventions randomized, parallel, open-label, proof-of-concept clinical trial, Enrolled patients received plitidepsin administered as an i.v. in- exploring three dose levels of plitidepsin (1.5, 2.0, and 2.5 mg/day, fusion over 90 min on days 1, 2, and 3 at dose levels of 1.5, 2.0, and 2.5 flat doses) for 3 consecutive days, as a 90-min i.v. infusion, in adult mg. A single batch (Lot 16D19) was administered to all patients. All patients with COVID-19 who required hospitalization. The study was patients also received, 20–30 min before the infusion of plitidepsin conducted in 10 hospital centers in Spain between 12 May 2020 and (i.e., on days 1–3), a premedication regimen consisting in: di- 26 November 2020. Presented data reflect all analyses completed phenhydramine 25 mg i.v., ranitidine 50 mg i.v., and dexamethasone by the cut-off date of 10 December 2020. The research was per- 8 mg orally. In a protocol amendment dated August 2020, the formed in accordance with good clinical practice guidelines and prophylactic medication was modified, replacing oral dexameth- adhered to the tenets of the Declaration of Helsinki. The study asone with i.v. administration of dexamethasone 8 mg (calculated protocol is available in Supplemental Data 1. Supplemental Data 2 as 8 mg dexamethasone phosphate, which is equivalent to 6.6 mg summarizes the protocol amendments as well as post hoc analyses dexamethasone base), and adding ondansetron 8 mg. After a that were performed. subsequent protocol amendment (September 2020), ondansetron 4 mg oral was administered every 12 h until 48 h after the last Participants administration of plitidepsin (Fig 5 and Supplemental Data 2). Adult (aged ≥18 yr) hospitalized patients with a positive SARS-CoV-2 Concomitant administration of IL-6 receptor inhibitors, immu- infection confirmed by real-time RT-PCR testing of a nasopha- nomodulatory drugs, and chloroquine and derivatives were not ryngeal exudate or the lower respiratory tract were eligible for permitted. Other pharmacological treatments for COVID-19 were inclusion in this study. Despite the eligibility of patients with allowed, at the discretion of the investigator, after 24 h from the samples from the lower respiratory tract, all patients reported here administration of the last dose of plitidepsin. Potent CYP3A4 in- had only nasopharyngeal swab samples. Included patients must hibitors and inducers had to be discontinued before starting have had the onset of SARS-CoV-2 infection symptoms no more treatment with plitidepsin and could be re-administered 24 h after than 10 d before initiating the study. Women of reproductive age its last dose. had a negative pregnancy test and were non-lactating. Women and men with partners of childbearing potential agreed to take effective Outcomes contraception while on study and for 6 mo after the last dose of The primary outcome was safety, which included the proportion of plitidepsin. All included patients provided written informed con- patients who developed AEs as graded in terms of severity (NCI sent before the initiation of the study. CTCAE v.5.0). Changes from baseline in vital signs; complete blood Key exclusion criteria included participation in another COVID- count; coagulation parameters; serum chemistry (alanine amino- 19–related clinical trial; receiving antivirals, chloroquine or de- transferase [ALT], aspartate aminotransferase [AST], γ-glutamyl rivatives, IL-6 receptor inhibitors, or immunomodulatory drugs for transferase [GGT], total bilirubin, alkaline phosphatase, lactate treatment of COVID-19; or requiring mechanical ventilation sup- dehydrogenase [LDH], creatinine phosphokinase [CPK], ferritin, port at enrollment. Patients were also excluded for evidence of troponin, and C-reactive protein [CRP]); and 12-lead ECGs for PR and multi-organ failure, clinically relevant heart disease, clinically QT intervals were also measured. With the exception of vital signs, relevant arrhythmia or history of QTc interval prolongation, or which were measured every 8 h during hospitalization then at neuropathy ≥ grade 2. Full exclusion criteria can be found in protocol-specified visits through Day 31, other safety evaluations Supplemental Data 1. occurred at baseline, days 1–7, 15, and 31. Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 17 of 22 Secondary outcomes included change from baseline of viral In regards with subgroup analyses, safety and efficacy outcomes load, as evaluated by quantitative PCR via central laboratory on also were assessed by age-group (<65 versus ≥65 yr) and efficacy nasopharyngeal exudate or sampled from the lower respiratory outcomes were assessed by disease severity at baseline. Safety tract within 48 h before first administration of plitidepsin and on outcomes were also assessed before and after the implementation days 4, 7, 15, and 31, and the percentage of patients requiring of the protocol amendment. oxygen therapy, noninvasive mechanical ventilation, invasive Continuous and categorical end points for the treatment effect mechanical ventilation and/or intensive care unit admission, and are reported. The differences in proportions are reported with 95% the percentage of patients with a fatal outcome on days 7, 15, and confidence intervals. Formal statistical testing between subgroups 31. were not predefined because of the reduced numbers in each subgroup. Exploratory stepwise logistic regression and Cox re- SARS-CoV-2 quantification gression models have been built to select the independent Centralized assessments of nasopharyngeal samples were per- prognostic factors of higher statistical significance to explain the formed at SYNLAB. Viral RNA extraction from the samples was more relevant efficacy outcomes using a threshold of α 0.10 for performed using the Maxwell HT Viral TNA (AX2340) Promega, on variable selection. All statistical analyses and plots were carried out the Hamilton automated extraction platform. Detection of using SAS software 9.4 (SAS Institute) and R 4.0.3. nCoV2019 was carried out with the kit: TaqPath TM COVID-19 RT-PCR KIT (A48102) Thermo Fisher Scientific. The positivity/negativity criteria used for the interpretation of the results are defined in Data Availability Table S7. Each sample was analyzed in triplicate, and the cycle threshold Plitidepsin is available from PharmaMar for noncommercial use (CT) value obtained was extrapolated to the standard curve, so that under a materials transfer agreement. All relevant data are in- a value of viral load is obtained per reaction for said sample. The cluded within this manuscript and all materials other than pliti- quantification, in triplicate, of the viral load of the patient sample depsin are readily available upon request from the corresponding was carried out at each of the five time points. authors. This work is licensed under a Creative Commons Attri- For quantification, in each sample plate, eight points, in triplicate bution 4.0 International (CC BY 4.0) license, which permits unre- (24 PCR in total), of the plasmid IDT 2019 nCov Kit CDC EUA of known stricted use, distribution, and reproduction in any medium, viral load was used to generate the standard line on which to ex- provided the original work is properly cited. To view a copy of this trapolate the results of the samples of the trial patients. The sen- license, visit https://creativecommons.org/licenses/by/4.0/. This sitivity of the analytical method is 10 mRNA copies/run. Assuming a license does not apply to figures/photos/artwork or other content starting sample volume of 200 μl, from which 100 μl of mRNA can be included in the article that is credited to a third party; obtain obtained, and that a single PCR run requires 5 μl mRNA, it can be authorization from the rights holder before using such material. estimated that the sensitivity for 100% of the replicates is 1,000 mRNA copies/ml (3 log10). For mathematical analysis and graphical visu- alization, an intermediate value of 1.5 log10 has been imputed to samples with undetected SARS-CoV-2 mRNA. Supplementary Information Statistical methods Supplementary Information is available at https://doi.org/10.26508/lsa. 202101200. Analysis of safety data was conducted on all patients who received at least one dose of study drug and the results were reported using descriptive statistics by means of frequency and percentage of patients for categorical variables and medians (range and interquartile Acknowledgements range [IQR])/mean (SD) for continuous variables. AEs were coded by using Medical Dictionary for Regulatory Activities We are indebted to the women and men that gave their consent to par- (version 17.0) and were presented as grouped by system organ ticipate in this study, and to their relatives, for understanding their decision in these exceptional circumstances. We would like to thank Pascal Besman class and preferred term. The associated grades with each of the for his input, Timothy Silverstein for providing editorial support, and Lorena AEs were evaluated using the National Cancer Institute-Common Martin Peña for technical and secretarial assistance. The full clinical re- Terminology Criteria for AEs (version 5.0) toxicity criteria. search teams at each one of the participating sites have played an in- Analysis for preliminary evidence of efficacy was performed on strumental role. We recognize hard work and commitment done by Paz all patients who had efficacy assessments at baseline and at least Cañadas, Daniel Sánchez-Brualla, and Carlota Costa from Synlab Diag- nósticos Globales, SAU as central laboratory in charge of viral RT-PCR as- one subsequent assessment during treatment. sessments. We appreciate the collaboration with Boryung Pharmaceuticals Post hoc analyses were conducted to (1) assess disease severity in the first in vitro study on the activity of plitidepsin in SARS-CoV-2 infection at baseline (mild, moderate, severe COVID-19 by FDA criteria) (23) models. We thank R Albrecht for support with the BSL-3 facility and pro- and (2) assess covariates associated with efficacy end points, in- cedures at the Icahn School of Medicine at Mount Sinai, New York. We thank cluding hospital discharge by Day 15 or Day 8. Ana Tercero, Angelines Barroso, Barbara Garcia, Cora Loste, Daniel Perez- Zsolt, Ester Ballana, Gemma Lladós, Hervé Dhellot, Ivette Casafont, Joaquim The Kaplan–Meier method was used for the description of time Segalés, Jon Cendoya, Jordana Muñoz-Basagoiti, José Ramón Santos, Laura to event end points and the median and the 95% confidence in- Soldevila, Lola Castro, Lourdes Mateu, Lucı́a Gutiérrez-Chamorro, Marı́a Luisa tervals were provided. Ramı́rez, Miriam Ramı́rez, Rafael Fernández Alonso, and Sonia Extremera for Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 18 of 22 their dedication and commitment. Funding: This study has been funded by N Izquierdo-Useros: conceptualization, resources, data curation, Pharmamar, SA (Madrid, Spain). This work was supported by grants from supervision, validation, investigation, project administration, and the Government of Spain (PIE_INTRAMURAL_ LINEA 1 - 202020E079; writing—review and editing. PIE_INTRAMURAL_CSIC-202020E043). The research of CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is supported by Grifols phar- S Fudio: conceptualization, data curation, software, formal analysis, maceutical. We also acknowledge the crowdfunding initiative #Yomecorono visualization, and methodology. (https://www.yomecorono.com). N Izquierdo-Useros has nonrestrictive MJ Pontes: conceptualization, data curation, and project administration. funding from PharmaMar to study the antiviral effect of Plitidepsin. NJ B de Rivas: conceptualization, data curation, and project administration. Krogan was funded by grants from the National Institutes of Health P Giron de Velasco: data curation, formal analysis, investigation, (P50AI150476, U19AI135990, U19AI135972, R01AI143292, R01AI120694, and P01AI063302); by the Excellence in Research Award (ERA) from the Laboratory visualization, and methodology. for Genomics Research (LGR), a collaboration between the University of A Nieto: conceptualization, data curation, and formal analysis. California, San Francisco (UCSF), University of California, Berkley (UCB), and J Gomez: conceptualization, data curation, formal analysis, and GlaxoSmithKline (GSK) (#133122P); by the Roddenberry Foundation, and gifts visualization. from QCRG philanthropic donors. This work was supported by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement P Aviles: investigation. #HR0011-19-2-0020. The views, opinions, and/or findings contained in this R Lubomirov: conceptualization, data curation, software, formal material are those of the authors and should not be interpreted as rep- analysis, visualization, and methodology. resenting the official views or policies of the Department of Defense or the A Belgrano: conceptualization, data curation, and formal analysis. U.S. Government. This research was partly funded by Center for Research for B Sopesen: investigation. Influenza Pathogenesis and Transmission (CRIPT), a National Institute of Allergy and Infectious Diseases (NIAID) supported Center of Excellence for KM White: conceptualization, data curation, formal analysis, vali- Influenza Research and Response (CEIRS, contract # 75N93021C00014), by dation, investigation, project administration, and writing—original DARPA grant HR0011-19-2-0020, by supplements to NIAID grants U19AI142733, draft, review, and editing. U19AI135972, and DoD grant W81XWH-20-1-0270, and by the generous support R Rosales: conceptualization, resources, supervision, validation, of the JPB Foundation, the Open Philanthropy Project (research grant 2020- and investigation. 215611 (5384)), and anonymous donors to A Garcı́a-Sastre. S Yildiz received funding from a Swiss National Foundation Early Postdoc Mobility fellowship S Yildiz: conceptualization, resources, supervision, validation, and (P2GEP3_184202). investigation. A-K Reuschl: formal analysis and investigation. LG Thorne: formal analysis and investigation. Author Contributions C Jolly: formal analysis and investigation. GJ Towers: formal analysis and investigation. JF Varona: conceptualization, investigation, and writing—original L Zuliani-Alvarez: formal analysis and investigation. draft, review, and editing. M Bouhaddou: formal analysis and investigation. P Landete: investigation. K Obernier: formal analysis and investigation. JA Lopez-Martin: conceptualization, data curation, software, formal BL McGovern: conceptualization, resources, supervision, validation, analysis, supervision, validation, investigation, visualization, and investigation. methodology, and writing—original draft, review, and editing. ML Rodriguez: conceptualization, resources, supervision, validation, V Estrada: conceptualization and investigation. and investigation. R Paredes: conceptualization and investigation. L Enjuanes: conceptualization, resources, data curation, formal P Guisado-Vasco: investigation. analysis, supervision, validation, investigation, and project L Fernandez de Orueta: investigation. administration. M Torralba: investigation. JM Fernandez-Sousa: conceptualization, funding acquisition, and J Fortun: conceptualization and investigation. methodology. R Vates: investigation. NJ Krogan: conceptualization, data curation, formal analysis, validation, J Barberan: conceptualization and investigation. investigation, writing—original draft, and project administration. B Clotet: investigation. JM Jimeno: conceptualization, data curation, formal analysis, su- J Ancochea: investigation. pervision, investigation, methodology, project administration, and D Carnevali: investigation. writing—original draft, review, and editing. N Cabello: investigation. A Garcia-Sastre: conceptualization, data curation, formal analysis, L Porras: investigation. validation, investigation, project administration, and writing—re- P Gijon: investigation. view and editing. A Monereo: investigation. D Abad: investigation. Conflict of Interest Statement S Zuniga: conceptualization, resources, data curation, supervision, validation, investigation, and project administration. V Estrada has received personal fees from Janssen, Gilead, and ViiV and I Sola: conceptualization, resources, data curation, supervision, grants from MSD. R Paredes has participated in Advisory Boards from Gilead, validation, investigation, and project administration. MSD, ViiV Healthcare, and Theratechnologies. M Torralba has received consulting fees as a member of Advisory Committee and honoraria and J Rodon: conceptualization, resources, data curation, formal speaking fees from Gilead, Janssen, MSD, and ViiV Companies. J Fortún has analysis, supervision, validation, and investigation. participated in scientific events and received consulting or speaking fees or J Vergara-Alert: conceptualization, data curation, and investigation. oral presentations from Pfizer, Gilead, MSD, Astellas, Novartis, and Roche. Proof-of-concept study of plitidepsin in COVID-19 Varona et al. https://doi.org/10.26508/lsa.202101200 vol 5 | no 4 | e202101200 19 of 22 J Ancochea has received fees for scientific consulting and/or speaking from replication of the paramyxovirus respiratory syncytial virus. PLoS One 9: Actelion, Air Liquide, Almirall, AstraZeneca, Boehringer Ingelheim, Carburos e114447. doi:10.1371/journal.pone.0114447 Médica, Chiesi, Faes Farma, Ferrer, GlaxoSmithKline, InterMune, Linde 10. Zhang X, Shi H, Chen J, Shi D, Li C, Feng L (2014) EF1A interacting with Healthcare, Menarini, MSD, Mundipharma, Novartis, Pfizer, Roche, Rovi, nucleocapsid protein of transmissible gastroenteritis coronavirus and Sandoz, Takeda, and Teva. I Sola, S Zúñiga, and L Enjuanes hold a Technology plays a role in virus replication. Vet Microbiol 172: 443–448. doi:10.1016/ Support contract with Pharmamar. N Izquierdo-Useros is inventor of a patent j.vetmic.2014.05.034 of Plitidepsin (EP20382821.5). The Krogan Laboratory has received research support from Vir Biotechnology and F Hoffmann-La Roche. NJ Krogan has 11. Gordon DE, Jang GM, Bouhaddou M, Xu J, Obernier K, White KM, O’meara consulting agreements with Maze Therapeutics and Interline Therapeutics, MJ, Rezelj VV, Guo JZ, Swaney DL, et al (2020) A SARS-CoV-2 protein and is a shareholder of Tenaya Therapeutics. JM Fernández-Sousa is interaction map reveals targets for drug repurposing. Nature 583: President and Founder of Pharmamar, SA (Madrid, Spain). JM Jimeno holds 459–468. doi:10.1038/s41586-020-2286-9 stocks of Pangaea Oncology, has a non-remunerated role in the Scientific 12. Losada A, Muñoz-Alonso MJ, Garcı́a C, Sánchez-Murcia PA, Martı́nez-Leal Advisory Board and holds stocks of Phosplatin Therapeutics, and is a full- JF, Domı́nguez JM, Lillo MP, Gago F, Galmarini CM (2016) Translation time employee of Pharmamar, SA (Madrid, Spain).The A Garcı́a-Sastre lab- elongation factor eEF1A2 is a novel anticancer target for the marine oratory has received research support from Pfizer, Senhwa Biosciences, natural product plitidepsin. Sci Rep 6: 35100–35100. doi:10.1038/ Kenall Manufacturing, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, srep35100 Pharmamar, ImmunityBio, Accurius, and Nanocomposix. A Garcı́a-Sastre has consulting agreements for the following companies involving cash and/or 13. Maroun JA, Belanger K, Seymour L, Matthews S, Roach J, Dionne J, stock: Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Vaxalto, Pa- Soulieres D, Stewart D, Goel R, Charpentier D, et al (2006) Phase I study of goda, Accurius, Esperovax, Farmak, and Pfizer. 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