1. Inhibitors of the viral RNA synthesis

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SARS-CoV-2 is a single-stranded RNA betacoronavirus. Potential targets are some non-structural proteins such as protease, RNA-dependent RNA polymerase (RdRp) and helicase, as well as accessory proteins. Coronaviruses do not use reverse transcriptase. There is only a total of 82% genetic identity between SARS-CoV and SARS-CoV-2. However, the strikingly high genetic homology for one of the key enzymes, the RdRp which reaches around 96%, suggests that substances effective for SARS may also be effective for COVID19.

RdRp inhibitors

Remdesivir (RDV) is a nucleotide analog and the prodrug of an adenosine C nucleoside which incorporates into nascent viral RNA chains, resulting in premature termination. It received an “Emergency Use Authorization” from the FDA in May and a so-called “conditional marketing” authorization from the EMA in July. In vitro experiments have shown that remdesivir has broad anti-CoV activity by inhibiting RdRp in airway epithelial cell cultures, even at sub-micromolar concentrations. This RdRp inhibition works in rhesus macaques (Williamson 2020). The substance is very similar to tenofovir alafenamide, another nucleotide analogue used in HIV therapy. Remdesivir was originally developed by Gilead Sciences for the treatment of the Ebola virus but was subsequently abandoned, after disappointing results in a large randomized clinical trial (Mulangu 2019). Resistance to remdesivir in SARS was generated in cell culture but was difficult to select and seemingly impaired viral fitness and virulence. However, there is a case report describing the occurrence of a mutation in the RdRp (D484Y) gene following failure of remdesivir (Martinot 2020). Animal models suggest that a once-daily infusion of 10 mg/kg remdesivir may be sufficient for treatment; pharmacokinetic data for humans are still lacking. Safety was shown in the Ebola trial. In the Phase III studies on COVID-19, an initial dose of 200 mg was started on day 1, similar to the Ebola studies, followed by 100 mg for another 4-9 days. The key trials are listed here: • Compassionate Use Program: this was a fragmentary cohort (Grein 2020) on some patients (only 53/61 patients were analyzed) with varying disease severity. Some improved, some didn’t: random noise. We believe, for a number of reasons, that this case series published in the New England

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Journal of Medicine is a cautionary tale for “science in a hurry”, arousing false expectations. It might have been preferable to postpone the publication (Hoffmann 2020). • NCT04257656: This multicentre RCT at ten hospitals in Hubei (Wang 2020) randomized a total of 237 patients with pneumonia, oxygen saturation of 94% or lower on room air and within 12 days of symptom onset to receive 10 days of single infusions or placebo. Clinical improvement was defined as the number of days to the point of a decline of two levels on a six-point clinical scale (from 1 = discharged to 6 = death). Patients were 65 years old (IQR 56–71), and many were co-treated with lopinavir (28%) and corticosteroids. The trial did not attain the predetermined sample size because the outbreak was brought under control in China. However, remdesivir was not associated with a difference in time to clinical improvement. Day 28 mortality was 14% versus 13%. Of note, the viral load decreased similarly in both groups. Some patients with remdesivir had dosing prematurely stopped due to adverse events (12% versus 5%, mainly gastrointestinal symptoms and increases of liver enzymes). The positive message from this trial is that time to recovery was “numerically” shorter in the remdesivir group, particularly in those treated within 10 days of symptom onset. • SIMPLE 1: in this randomized, open-label RCT in 397 hospitalized patients with severe COVID-19 and not requiring IMV, clinical improvement at day 14 was 64% with 5 days and 54% with 10 days of remdesivir (Goldman 2020). After adjustment for (significant) baseline imbalances in disease severity, outcomes were similar. The most common adverse events were nausea (9%), worsening respiratory failure (8%), elevated ALT level (7%), and constipation (7%). Because the trial lacked a placebo control, it was not a test of efficacy for remdesivir. An expansion phase will enroll an additional 5600 (!) patients around the world. • The second open-label SIMPLE trial, NCT04292730 (GS-US-540-5774), evaluated the efficacy of two remdesivir regimens compared to standard of care (SOC) in 584 hospitalized patients with moderate COVID-19, with respect to clinical status assessed by a 7-point ordinal scale on day 11. Clinical status distribution was significantly better for those randomized to a 5-day course of remdesivir compared with those randomized to SOC (Spinner 2020). According to the authors, however, this “difference was of uncertain clinical importance”. The difference for those randomized to a 10-day course (median length of treatment, 6 days) compared with standard of care was not significant. By day 28, 9 patients had died: 2 (1%) and 3 (2%) in the 5-day and 10-day remdesivir groups, and 4 (2%) in the SOC group, respectively. Nausea (10% vs 3%), hypokalemia (6% vs 2%), and

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headache (5% vs 3%) were more frequent among remdesivir-treated patients, compared with SOC. • ACTT (Adaptive COVID-19 Treatment Trial): The conclusion of the final report for this double-blinded RCT that had randomized 1062 patients throughout the world, was remarkably short: remdesivir “was superior to placebo in shortening the time to recovery in adults who were hospitalized with COVID-19 and had evidence of lower respiratory tract infection” (Beigel 2020). Median recovery time was 10 versus 15 days. On an eightcategory ordinal scale, patients who received remdesivir were more likely to improve at day 15. The benefit in recovery persisted when adjustment was made for glucocorticoid use. The Kaplan–Meier estimates of mortality were 6,7% with remdesivir and 11,9% with placebo by day 15. Serious adverse events were reported in 131 of the 532 patients who received remdesivir (24,6%) and in 163 of the 516 patients who received placebo (31,6%). • WHO Solidarity Trial Consortium 2020: In SOLIDARITY, 11.330 adults (405 hospitals in 30 countries) were randomized, with 2750 allocated to remdesivir, 954 HCQ, 1411 lopinavir/r, 651 interferon plus lopinavir/r, 1412 only interferon, and 4088 no study drug. Kaplan-Meier 28-day mortality was 12%. No study drug definitely reduced mortality (in unventilated patients or any other subgroup of entry characteristics), initiation of ventilation or hospitalization duration. 301 of 2743 patients receiving remdesivir died as did 303 of 2708 receiving the control (WHO Solidarity 2020). On 20 November, WHO issued a conditional recommendation against the use of remdesivir in hospitalized patients, regardless of disease severity, as there is currently no evidence that remdesivir improves survival and other outcomes in these patients (WHO Date). What comes next? Several additional trials are ongoing, including combination therapies with other drugs such baricitinib (see below). Let’s wait for the results, before we throw remdesivir out with the bathwater. According to a recent review, remdesivir (5 days) should be prioritized for hospitalized patients requiring low-flow supplemental oxygen as it appears that these patients derive the most benefit (Davis 2020). The data also support some benefit in hospitalized patients breathing ambient air (if there is adequate drug supply). Current data do NOT suggest benefit for those requiring high-flow oxygen or mechanical ventilation (non-invasive or invasive). It has become “clear that treatment with an antiviral drug alone is not likely to be sufficient for all patients” (Beigel 2020).

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Of note, some new ideas on remdesivir as an inhalation therapy have been published (Contini 2020). Local instillation or aerosol in the first phase of infection, both in asymptomatic but nasopharyngeal swab positive patients, together with antiseptic-antiviral oral gargles and povidone-iodine eye drops for conjunctiva would attack the virus directly through the receptors to which it binds, significantly decreasing viral replication and the risk of severe COVID-19. Gilead is working on this (knowing that “early intravenous infusions” are not feasible).

Favipiravir is another broad antiviral RdRp inhibitor that has been approved for influenza in Japan (but was never brought to market) and other countries. Favipiravir is converted into an active form intracellularly and recognized as a substrate by the viral RNA polymerase, acting like a chain terminator and thus inhibiting RNA polymerase activity (Delang 2018). In the absence of scientific data, favipiravir has been granted five-year approval in China under the trade name Favilavir® (in Europe: Avigan®). A loading dose of 2400 mg BID is recommended, followed by a maintenance dose of 1200-1800 mg QD. However, in 7 patients with severe COVID-19, the favipiravir trough concentration was much lower than that of healthy subjects in a previous clinical trial (Irie 2020). Potential drug-drug interactions (DDIs) have to be considered. As the parent drug undergoes metabolism in the liver mainly by aldehyde oxidase (AO), potent AO inhibitors such as cimetidine, amlodipine, or amitriptyline are expected to cause relevant DDIs (review: Du 2020). Some encouraging preliminary results in 340 COVID-19 patients were reported from Wuhan and Shenzhen (Bryner 2020). • A first open-label RCT posted on March 26 (Chen 2020) was conducted in

China, comparing arbidol and favipiravir in 236 patients with pneumonia.

Some improvement in the primary outcome (7-day clinical recovery rate) was found only in a subgroup). In the whole study population, no difference was seen.

• No effect of viral clearance was found in a RCT on 69 patients with asymptomatic to mild COVID-19 who were randomly assigned to early or late favipiravir therapy (same regimen starting day 1 or day 6). Viral clearance occurred within 6 days in 67% and 56%. Neither disease progression nor death occurred in any of the patients (Doi 2020). • In the pilot stage of a Phase II/III clinical trial, 60 patients hospitalized with COVID-19 pneumonia were randomized to two different dosing groups or standard of care (Ivashchenko 2020). The proportion of patients

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who achieved negative PCR on day 5 on both dosing regimens was twice as high as in the control group (p < 0.05). In an RCT on 150 patients from India, the median time to the cessation of viral shedding was somewhat shorter (5 days versus 7 days) with favipiravir, compared to controls (Udwadia 2020).

Molnupiravir (MK-4482/EIDD-2801) is an orally-administered bioavailable prodrug of cytidine nucleoside analogue EIDD-1931. Originally developed for treatment of hepatitis C, some studies indicated potent activity of EIDD-1931 against SARS-CoV-2 in multiple cell types. Molnupiravir is able to mitigate SARS-CoV-2 infection and block transmission when therapeutically administered to ferrets (Cox 2020). The drug, initially developed as an inhibitor of influenza viruses, is currently in Phase II/III clinical trials (NCT04405570 and NCT04405739).

Some other RdRp inhibiting compounds have also been discussed. Sofosbuvir is a polymerase inhibitor which is also used as a direct-acting agent in hepatitis C. It is usually well tolerated. Modelling studies have shown that sofosbuvir could also inhibit RdRp by competing with physiological nucleotides for the RdRp active site (Elfiky 2020). Sofosbuvir could be combined with HCV PIs. The first randomized controlled trial in adult patients hospitalized with COVID-19 in Iran to evaluate the efficacy and safety of the two HCV drugs sofosbuvir and daclatasvir in combination with ribavirin (SDR) compared these drugs with standard of care (Abbaspour Kasgari 2020). Though there were trends in favor of the SDR arm for recovery and lower death rates, the trial was too small to make definite conclusions. In addition, there was an imbalance in the baseline characteristics between the arms. Galidesivir is a nucleoside RNA polymerase inhibitor with broad-spectrum activity in vitro against more than 20 RNA viruses in nine different families, including coronaviruses and other viral families. A NIAID-funded, randomized, double-blind, placebo-controlled clinical trial to assess the safety, clinical impact and antiviral effects of galidesivir in patients with COVID-19 is underway. Of note, the drug also works against Zika: in the study presented here, galidesivir dosing in rhesus macaques was safe and offered postexposure protection against Zika virus infection (Lim 2020).

Protease inhibitors (PIs)

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A promising drug target is the viral main protease Mpro, which plays a key role in viral replication and transcription. Some HIV PIs have been extensively studied in COVID-19 patients.

Lopinavir/r is thought to inhibit the 3-chymotrypsin-like protease of coronaviruses. To achieve appropriate plasma levels, it has to be boosted with another HIV PI called ritonavir (usually indicated by “/r”: lopinavir/r). Due to some uncontrolled trials in SARS and MERS, lopinavir/r was widely used in the first months, despite the lack of any evidence. In an early retrospective study on 280 cases, early initiation of lopinavir/r and/or ribavirin showed some benefits (Wu 2020). • The first open-label RCT in 199 adults hospitalized with severe COVID-19 did not find any clinical benefit beyond standard of care in patients receiving the drug 10 to 17 days after onset of illness (Cao 2020). There was no discernible effect on viral shedding. • A Phase II, multi-center, open-label RCT from Hong Kong randomized 127 patients with mild-to-moderate COVID-19 (median 5 days from symptom onset) to receive lopinavir/r only or a triple combination consisting of lopinavir/r, ribavirin and interferon (Hung 2020). The results indicate that the triple combination can be beneficial when started early (see below, interferon). As there was no lopinavir/r-free control group, this trial does not prove lopinavir/r efficacy. • After preliminary results were made public on June 29, 2020, we are now facing the full paper on the lopinavir/r arm in the RECOVERY trial: In 1616 patients admitted to hospital who were randomly allocated to receive lopinavir/r (3424 patients received usual care), lopinavir/r had no benefit.

Overall, 374 (23%) patients allocated to lopinavir/r and 767 (22%) patients allocated to usual care died within 28 days. Results were consistent across all prespecified subgroups. No significant difference in time until discharge alive from hospital (median 11 days in both groups) or the proportion of patients discharged from hospital alive within 28 days was found.

Although the lopinavir/r, dexamethasone, and hydroxychloroquine groups have now been stopped, the RECOVERY trial continues to study the effects of azithromycin, tocilizumab, convalescent plasma, and monoclonal antibodies.

• There was no effect in the SOLIDARITY trial of lopinavir/r (WHO Solidarity 2020)