Directly Acting Antivirals for COVID-19: Where Do We Stand?

The outbreak of a novel coronavirus (SARS-CoV-2) in Wuhan, China in December 2019 has now become a pandemic with no approved therapeutic agent. At the moment, the genomic structure, characteristics, and pathogenic mechanisms of SARS-CoV-2 have been reported. Based upon this information, several drugs including the directly acting antivirals have been proposed to treat people with coronavirus disease 2019 (COVID-19). This rapid review aims to describe the directly acting antivirals that have been examined for use in the management of COVID-19. Searches were conducted in three electronic databases, supplemented with a search on arXiv, bioRxiv, medRxiv, ChinaXiv, ClinicalTrials.gov, and Chinese Clinical Trial Registry for studies examining the use of antivirals in COVID-19 to identify for case reports, case series, observational studies, and randomized controlled studies describing the use of antivirals in COVID-19. Data were extracted independently and presented narratively. A total of 98 studies were included, comprising of 38 published studies and 60 registered clinical trials. These drugs include the broad spectrum antivirals such as umifenovir, protease inhibitors such as lopinavir/ritonavir as well as the RNA-dependent RNA polymerase inhibitors, remdesivir, and favipiravir. Other drugs that have been used include the nucleosidase inhibitors and polymerase acidic endonuclease inhibitors which are currently approved for prevention of influenza infections. While some of the drugs appear promising in small case series and reports, more clinical trials currently in progress are required to provide higher quality evidence.


INTRODUCTION
In December 2019, an outbreak caused by a novel coronavirus was reported in Wuhan city, in Hubei province, China. The outbreak was found to be caused by a novel virus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Cheng and Shan, 2020;Wu and McGoogan, 2020). Since then, the cases of SARS-CoV-2 have been reported in every single continent around the world. With over nine million individuals infected with coronavirus disease 2019  and over 450 thousand death as of mid-June 2020, COVID-19 is now a public health emergency. In many individuals with COVID-19, they often present with a decrease in both CD4 + and CD8 + T-cells count and suffer from acute respiratory syndrome for 7 to 10 days due to the rapid viral replication (Cheng and Shan, 2020;Zhou F. et al., 2020). Clinical features of SARS-CoV-2 infections are similar to SARS-CoV, characterized by fever, dry cough, dyspnoea or shortness of breath, diarrhea, sore throat, muscle ache, and vomiting in some patients (Meo et al., 2020;Wu and McGoogan, 2020).
The SARS-CoV-2 is a member of the family Coronaviridae, a positive-sense, single-stranded RNA virus that enters the mammalian cell through an interaction of viral spike glycoprotein that binds to the angiotensin-converting enzyme 2 (ACE 2 ) receptor (Fehr and Perlman, 2015). Following receptor binding, the virus uses the host cell receptor and endosome to enter the cell and synthesizes viral polyproteins that encode for the replicase-transcriptase complex. The virus then synthesizes RNA using its RNA-dependent RNA polymerase to synthesize structural proteins leading to completion of assembly and release of viral particles (Fehr and Perlman, 2015;Cheng and Shan, 2020). Genomic sequencing of the virus has revealed that SARS-CoV-2 has a high similarity to the bat-derived SARS-CoV, with approximately 79% identity . Studies have shown that SARS-CoV-2 is spread primarily through the respiratory system and droplets, with an incubation period of between 2 and 14 days, and a median period of 4 days (range, 2-7 days) (Livingston et al., 2020). As such, pharmacological agents that target the spike protein or host's ACE 2 proteins used to treat SARS and Middle-East Respiratory Syndrome (MERS) have been suggested as potential agents that could be used to treat patients with COVID-19. Agents proposed to eradicate the coronavirus or at least reduce the effects and hinder the contagion of the SARS-CoV-2 include repurposing currently available drugs such as monoclonal antibodies, antivirals, antimalarial among others (Fehr and Perlman, 2015;Cheng and Shan, 2020).
This intensifying outbreak has led to a surge in registered clinical trials since the infection was first reported . In order to rapidly inform further and better design and conduct of clinical trials, there is an urgent need to provide government agencies on the investigational candidates most suitable for clinical trials. While there are major gaps in knowledge around COVID-19, especially in terms of the effectiveness and safety of various directly acting antiviral agents, a review of the characteristics of published, on-going trials and a synthesis of all available results can help inform current practice and direct future research. This rapid review was performed to provide government bodies on the evidence available in relation to the antiviral drug therapies that have been examined to date.

Search Strategy
We performed a search of PubMed, EMBASE, Cochrane CENTRAL from inception to March 31st, 2020 to search for articles assessing the use of antivirals in patients with SARS-CoV-2 pneumonia without any language restriction. This was supplemented by a search on ClinicalTrials.gov, WHO International Clinical Trials Registry Platform and Chinese Clinical Trial Registry as well as pre-print articles on medRxiv, arXiv, bioRxiv, and ChinaXiv. Keywords used include: novel coronavirus, COVID-19, 2019-nCoV, antivirals, anti-retroviral and humans. Following peer-review, we updated our search to May 31st, 2020 on the database identified previously. We also expanded our keywords to include the following search terms: SARS-Co-v 2, abidol, tenofovir, EIDD-2801, sofosbuvir/ledipasvir, sofosbuvir/daclatasvir.

Study Selection and Data Abstraction
Articles were screened by two authors (SL, NL, and ST) independently for relevant studies. Studies which described the use of direct acting anti-viral therapies, irrespective of study designs conducted in humans were included. These could include case studies, case reports, cohort studies, observational studies or randomized controlled studies since. In vitro, animal studies and reviews were excluded since studies have suggested that these may not directly translate to clinical effects in human. We excluded drugs which does not act directly on virus such as antibiotics and antimalarial since these drugs have limited role in targeting the functions of the virus and preventing it from replicating in the body. All information was extracted independently by authors with discrepancies resolved thorough consensus. Due to the time constraints, the review was not registered in PROSPERO but the corresponding author can be contacted for the full protocol.

Study Quality and Reporting
The quality of all included studies which were registered and currently underway were assessed subjectively by one author, and classified anecdotally to either low, medium or high. This classification was based upon the study population > study design > sample size of trial and finally the presumed importance of results. Using this approach, a study that reports on patients would be given higher priority over those which had involved healthy subjects. In the event that the study recruited similar populations, a randomized controlled trial would be graded higher priority over a quasi-randomized study > observational study > case series > case report. Finally, a study of similar design that had reported clinical outcomes such as mortality, hospitalization days would be graded higher compared to those which had reported laboratory data only such as presence or absence of SARS-CoV-2 in patients. All data were summarized narratively due to the limited available evidence on the topic.
Most of the registered trials were very small in size with sample size of fewer than 100 patients, with a median sample size of 145 (IQR: 60-343). In most trials, participants had to be aged 18 years and above. Most of these trials were in the recruiting stages (n = 39) or the preparation stages (n = 19). There was only limited data available on the efficacy of antivirals on COVID-19 and their clinical impact. Most of the trials will examine a myriad of primary outcomes, including time to clinical improvements,   Most common adverse events were increased hepatic enzymes, diarrhea, rash, renal impairment, and hypotension. 12 patients (23%) had serious adverse events, which all received invasive ventilation at baseline. Most common serioys adverse events were multiple organ dysfunction syndrome, septic shock, acute kidney injury, and hypotension. 4 patients (8%) discontinued remdesivir prematurely, due to worsening of pre-existing renal failure (n = 1), multiple organ failure (n = 1), elevated aminotransferases (n = 1), including one patient with a maculopapular rash.

(Continued)
Frontiers in Microbiology | www.frontiersin.org  The cumulative uninfected rate of health professionals in the Arbidol group was significantly higher than that of individuals in the non-Arbidol group (log-rank test, χ2 = 98.74; P < 0.001). Forty-eight patients (58.5%) in the infection group were hospitalized, with a median age of 39 (31-49) years, of whom 7 (14.6%) were prophylactically administered Arbidol.

Ye et al. (2020) China
Retrospective cohort, n = 47. Lopinavir/Ritonavir along with Arbidol and interferon (n = 42), and "control" (no lopinavir/ritonavir, with Arbidol and interferon only, n = 5). "The per ml of LPV/r oral liquid contained 80 mg lopinavir and 20 mg ritonavir. Usage and dosage: 5 ml/time (400/100 mg) for adults, twice a day or 10 ml/time (800/200 mg) once a day with food" Not classified "Compared with the control group, the patients in the test group returned to normal body temperature in a shorter time (test group: 4.8 ± 1.94 days vs. control group: 7.3 ± 1.53 days, p = 0.0364)." No significant differences between groups otherwise.
The abnormal percentage of ALT and AST in the test group was lower than that in the control group.
All patients were treated with Arbidol (100 mg TDS for 14 days) and moxifloxacin (0.4 g once a day for 7-14 days).
27 severe (ICU) and 57 "ordinary warded" patients After treatment of Arbidol and moxifloxacin for 1 week, the rates of SARS-CoV-2 nucleic acid turning negative were 69.2% in the severe group and 77.8% in the ordinary group. number of individuals requiring mechanical ventilation, number of individuals hospitalized into ICU, length of hospitalization, mortality as well as absence of virological indicators. Three studies also used physical functioning scores based upon an ordinal 7-point scale from the WHO master protocol and the National Early Warning Score 2 (NEWS2).

DIRECT ANTIVIRALS USED IN COVID-19
Protease Inhibitors Successful entry of the SARS-CoV-2 into the cell will depend on the activation of envelope glycoprotein by host cell protease. As such, protease enzyme inhibitors are considered an excellent drug target for patients with COVID-19 ( Table 3). Examples of such drugs include lopinavir, ritonavir, darunavir, danoprevir and the experimental drug ASC-09. Among these agents, the most commonly examined protease inhibitor was lopinavir/ritonavir combination, using a dosing regimen of 400 mg/100 mg lopinavir/ritonavir twice daily for up to 14 days which was reported in 18 published studies Cao et al., 2020;Deng et al., 2020;Giacomelli et al., 2020;Kim et al., 2020;Li et al., 2020;Lim et al., 2020;Liu et al., 2020;Shi et al., 2020;Vizcarra et al., 2020;Yan et al., 2020;Ye et al., 2020;Young et al., 2020;Zhu Z. et al., 2020;Zuo et al., 2020). These studies were conducted in China (n = 13), South Korea (n = 2), Italy, Singapore, and Greece (n = 1 each). Results from the published randomized controlled studies suggest that there is limited clinical efficacy of the combination (Cao et al., 2020;Hung et al., 2020;Li et al., 2020;Zheng F. et al., 2020). In a recently completed RCT in China, the lopinavir/ritonavir combination was reported to have limited efficacy, with no difference in time to clinical improvement (median, 16 days), duration of intensive care unit stay, days of mechanical ventilation, or days of oxygen support (Cao et al., 2020). Authors reported that there appears to be some benefit when patients were given the drug therapy earlier (within 12 days of symptom onset) as they experienced a shorter time to clinical improvement (HR 1.25; 1.77-2.05 versus 1.30; 0.84-1.99). Nevertheless, given the significant drug-drug interaction and potential risk of adverse events including gastrointestinal distress such as nausea and diarrhea and hepatotoxicity, caution should be exercised while using this combination given that nearly 20% to 30% of patients have elevated transaminases at presentation (Wu and McGoogan, 2020;Wu C. et al., 2020). While there are no RCTs on the other protease inhibitors darunavir and danoprevir, real world evidence have been reported from Germany and China Haerter et al., 2020;Shi et al., 2020). Haerter et al. (2020) in Germany reported the outcomes of a case series of patients living with HIV treated with antiretroviral treatment including darunavir. Of the four patients treated, one died while the other three patients recovered. Shi et al. (2020) similarly reported in their case series on the limited efficacy of darunavir in terms of reducing duration from illness onset to admission and clinical symptoms. Only one small study reported the safety of danoprevir in patients with COVID-19. However, taken together these data are difficult to

Broad Spectrum Antiviral
Another drug commonly examined is umifenovir, a broad spectrum antiviral licensed in China and Russia for influenza.
Umifenovir prevents viral host cell entry by inhibiting the membrane fusion of the viral envelope and host cell cytoplasmic membrane (Blaising et al., 2014;Fink et al., 2018;Haviernik et al., 2018). The drug was suggested to have some effects in reducing the risk of COVID-19 transmission and has been examined for post-exposure prophylaxis using a dose of 200 mg orally every 8 h. In an early pilot study from China, treatment with umifenovir was found to reduce SARS-CoV-2 viral loads, with 94% of patients treated with umifenovir reported negative SARS-CoV-2 viral load compared to 53% in the control . Nevertheless, the results from two RCTs suggested limited efficacy in treating COVID-19 Li et al., 2020), as the recovery rates were comparable with control.

RNA−Dependent RNA Polymerase (RdRP) Inhibitor
Favipiravir is another oral antiviral that has been examined recently. Favipiravir is a pyrazinecarboxamide derivative and guanine analog which selectively inhibits the RNA-dependent RNA polymerase (RdRP) of RNA viruses (Furuta et al., 2009). RdRP is required during the replication process of RNA viruses as it determines the replication rates and mutation of the virus to adapt to the new host environment, which ultimately influences its fidelity. As such, targeting of RdRP has become another mainstay in the treatment of SARS-CoV-2. In a pilot prepost study in China, 80 patients with COVID-19 were treated with favipiravir with a loading dose of 1600 mg followed by a maintenance dose of 600 mg three times daily for up to 14 days. After 14 days of treatment, the authors found that patients treated with favipiravir had better treatment outcomes in terms of disease progression and viral clearance compared to those treated with lopinavir/ritonavir . Two recently completed RCTs in China had reported promising clinical results due to the higher 7-day recovery rates, and symptom improvements such as fever and cough Lou et al., 2020). With no significant adverse events were reported, favipiravir is currently being examined in several clinical trials as a potential target drug for SARS-CoV-2. Remdesivir is another nucleotide analog inhibitor of RdRP that have been extensively examined as a potential anti SARS-CoV-2 medication. The earliest report on the use of remdesivir was reported by Holshue et al. (2020), which reported improvement in the patient's condition after treatment. Since then, two RCTs on remdesivir has been conducted using a dose of remdesivir 200 mg on day 1, followed by 100 mg daily for up to 10 days. In the first RCT of 237 patients with COVID-19 by  in China, the authors found that more patients on remdesivir had clinical improvements after 28 days, and they reported faster time to symptoms improvements compared to control. Beigel et al. (2020) meanwhile reported a large multi-center RCT in Europe, Asia, and America on 1,107 patients treated with either remdesivir or placebo for 10 days. In their study, they found that the median time to recovery was much faster with remdesivir treatment, with a significantly higher number of patient who recovered. Nevertheless, there are uncertainties about the adverse effects of the drugs, and more clinical trials are underway to examine the potential of this drug in SARS-CoV-2.

Nucleosidase and Neuroamidase Inhibitors
Another class of drugs that has been used in SARS-CoV-2 is the neuroamidase inhibitors such as oseltamivir. Given that the COVID-19 outbreak in China occurred during the peak influenza season, a large proportion of patients had received oseltamivir therapy prior to the discovery of SARS-CoV-2 as these agents have been used for various influenza subtype and other RNA viruses to inhibit the spread of the influenza virus (Wang et al., 2014;Malosh et al., 2018). Several clinical trials are currently evaluating the effectiveness of oseltamivir either alone or as a combination such as with chloroquine and favipiravir, but given its pharmacological action, there is limited role of these drugs in the management of COVID-19 once influenza has been excluded.
Similarly, the neuroamidase inhibitors ribavirin and azvudine have been recommended in the initial stages for management of COVID-19, given that the symptoms were thought to be due to pneumonia. There is currently no evidence to suggest that ribavirin when used alone offers any benefit in the management of COVID-19. The combination therapy of ribavirin, lopinavir/ritonavir and interferon beta-1b was recently shown to have some positive results and would need to be explored further (Hung et al., 2020). However, as ribavirin causes a dose-dependent hematological toxicity, and is a known teratogen, there is limited value of this drug in the treatment of COVID-19.

Polymerase Acidic Endonuclease Inhibitors
The only drug in its class examined identified in the current review was baloxavir marboxil. This drug targets the viral polymerase acidic protein to block the endonuclease function, resulting in the inhibition of virus mRNA transcription and infection (Koszalka et al., 2019;Locke et al., 2019). Only one small clinical study in China has been identified in the current review, but due to the small sample the implications will be limited (ChiCTR2000029548).

DISCUSSION
With no therapeutic agent is currently known to be effective for COVID-19, multiple different antivirals have been examined based upon the early in vitro evidence against SARS-CoV. While several case series and reports showed improvements with use of lopinavir-ritonavir, the recently published study by Cao et al. (2020) have showed limited benefits highlighting the difficulty in finding an appropriate agents for rapid implementation in such outbreaks. It remains unfortunate that this therapy is ineffective, given that this would have represented an immediate and safe oral therapy for COVID-19. For most of the current trials reported, these are underpowered and unlikely to provide the healthcare community with the necessary high quality evidences needed to combat this pandemic if taken individually. In addition, most of the trials registered will only include patients aged 18 and above, and thus will unlike to provide the necessary information on children, adolescents, pregnant women or even those with respiratory diseases (Lee, 2020).
These trials also included a wide range of primary outcomes including time to clinical improvements, number of individuals requiring mechanical ventilation, number of individuals hospitalized into ICU, length of hospitalization, mortality as well as absence of virological indicators. As most of the outcomes that will be reported varied, and will include subjective outcomes, this may lead to measurement bias. Importantly, few of the current trials have reported on mortality in their study either as a primary or secondary outcome. While the case fatality rates differs between countries, ranging from as low as 0.3% to as high as 11.0%, these reports have not been forthcoming in all the included studies and should be given attention (Rajgor et al., 2020). In addition, most of the current studies are not coordinated, leading to inconsistencies among trials in their definitions of conditions and inclusion criteria, the design and delivery of intervention and comparison, as well as measurement of the outcomes. Cognisant of this, the World Health Organization (WHO) is initiating a clinical trials experts group which will aim to develop a master protocol for a RCT to evaluate efficacy of therapeutics against nCov (World Health Organization, 2020). Other impending initiatives include the strengthening of management and coordination of the promising drugs such as remdesivir and favipiravir, which should be prioritized for clinical studies. This is based upon the potential activity of both agents against RNA polymerase, established use in novel influence and also oral bioavailability. This ideally should involve the pharmacist who can help in the development of treatment protocols, monitoring of drug adverse events as well as assist in the expanded access of these new investigational drugs (Lee et al., 2019;Stevens et al., 2020).
Investigators should also consider using other clinical trial designs including step-wedge design which may reduce the need for large sample sizes (Baio et al., 2015). In addition, a database should also be setup to share all available existing data between sites and countries, which effectively create a real-world evidence study network, which can increase the speed of information dissemination especially in pandemics such as COVID-19. Indeed, there is a need for researchers to report as much details as possible to ensure reproducibility of results especially as these studies currently use very weak outcomes which can limit the efficacy assessment. Nevertheless, the development of clinical trials during an outbreak is an adaptive process, with new evidence being generated at an impressive rate. As such, we believe that these results generated will inform the adaptation of existing and new trials that are being developed. Indeed, with progressive release of trial results, there is a need for a living systematic review to progressively update the pooled results with each additional trial included. This is crucial in view of the small sample size of individual studies.
Nevertheless, we acknowledge that there are some limitations to this review, given that we had only one reviewer who had conducted the search. In addition, this review also included several pre-print articles which have not been peer-reviewed and thus may not provide the academic rigor normally required for published studies. However, in view of the evolving situation of COVID-19 and the need for rapid understanding of this disease, the decision to include these studies were needed in order for us to provide the readers with the most updated information available. Most of the published treatment data to date are derived from observational studies which have relatively small sample size, which may introduce risk regarding the magnitude of effect sizes.
In summary, this updated review of antivirals in COVID-19 showed that there is limited information available to guide clinical practice as well as the need for a more coordinated research network to seek the best therapeutic options especially in pandemics. While several agents reviewed have suggested some potential benefits of therapy, the evidence remains inconclusive.