Part II: Treatment Strategies for COVID-19

Different Antiviral Drugs against COVID-19 and Future Perspective

Iyad Ibrahim Shaqura^{1, 2}, Ahmed Abdelmajed Alkhodary^{2, 3}, Abdullah⁴, Muhammad Ibrar⁵, Fazlullah Khan^{6, *}

¹ Department of Health Management and Economics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

² Ministry of Health, Gaza Governorates, Palestine

³ International Centre for Casemix and Clinical Coding, Faculty of Medicine, National University of Malaysia, Kuala Lumpur, Malaysia

⁴ Department of Pharmacy, University of Malakand, Chakdara Dir Lower, KPK, Pakistan

⁵ Department of Allied Health Sciences, Iqra University Peshawar, Swat Campus, KPK, Pakistan

⁶ Department of Allied Health Sciences, Bashir Institute of Health Sciences, Bharakahu, Islamabad, Pakistan

Abstract

The outbreak of coronavirus disease-19 (COVID-19) across the world has caused serious health issues in terms of physical and psychological damage to human health. The spread of this virus was rapid and shortly spread to almost every country in the world. Due to the high infection rate and occurrence of complications in the infected individuals, the research and development of anti- COVID-19 drugs became the utmost necessity of time as no specific drug is available to relieve the clinical symptoms of COVID-19. We reviewed reliable information on targeted severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) candidate drugs in the present chapter. Also, we summarized novel insight into the future development of safe, effective, and less-toxic antiviral drugs available and employed for the management of COVID-19. Similarly, we focused on antiviral medications under investigation for this purpose; several medications with different mechanisms of action were noticed for the treatment of COVID-19.

Keywords: COVID-19, Drug repurposing, Hydroxychloroquine, Remdesivir, Umifenovir.

^* Corresponding author Fazlullah Khan: Department of Allied Health Sciences, Bashir Institute of Health Sciences, Bharakahu, Islamabad, Pakistan; Tel: +92-3469433155; E-mail: fazlullahdr@gmail.com

INTRODUCTION

According to the International Committee on Taxonomy of Viruses, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the virus causing COVID-19 disease. This virus is belonging to the Coronaviridae family of viruses [1]. Acute respiratory distress syndrome is considered the most widespread critical feature of this disease. It is well known that this category among viruses is responsible for severe acute respiratory syndrome with up to 10% mortality rate [2]. In focus, COVID-19 disease is significantly linked with notifiable morbidity and mortality [3]. As a severe and infectious respiratory disease, several suggested clinical guidelines are currently available for treating COVID-19 [4^-6]. Within these treatment guides, various types of medications are indicated as a part of the treatment strategy. Among the literature, the most commonly investigated treatment choices for COVID-19 disease were antiviral agents, immunomodulatory agents, antibiotics, analgesics, antipyretics, herbal medications, and some other supportive medications [7–9]. Currently, over 38 medications are under investigation for being considered as a treatment for COVID-19 disease [7^-8].

Several medications with different mechanisms of action were noticed for the treatment of COVID-19 disease. These mechanisms encompass acting as protease inhibitors; RNA polymerase inhibitors, cell membrane fusion inhibitors, ACE2 receptor connection inhibitors, serine protease receptor inhibitors for the TMPRSS2, host cell immunostimulatory agents against virus invasion, mesenchymal stromal cells therapies, monoclonal antibodies, and passive immunization plasma [7^-10]. One of the most pivotal antiviral medication groups includes protease inhibitors, which affect the 3-chymotrypsin like proteinase and Papain-like protease enzymes that act directly on the processing of the viral polyprotein. The second group of antivirals encompasses RNA polymerase inhibitors which inhibit the viral genome replication through competing natural nucleosides of the virus RNA-dependent RNA polymerase active site and then block the virus RNA synthesis process. A third group works mainly on the inhibition of intracellular viral replication through alkalization of the intracellular medium to inhibit the uncoating process of the virus envelope. Fourthly, the group of medications acting particularly on the avoidance of virus entry and membrane fusion into host cells by several mechanisms, most explicitly, preventing the binding of virus spike protein to host cell wall receptors such as ACE2 and TMPRSS2 receptors. The fifth group including those medications which work mostly extracellularly exerts its effect through binding to interleukin-6 receptor and inhibition of cytokine release to protect from amplified immune response [10].

Many clinical experiments are under investigation to reveal the safety and efficacy of these medications in use for treating COVID-19 infection [7, 11^-13]. The perspective beyond these trials focuses mainly on repurposing those approved medications that are already existing and utilized for other indications before COVD-19. It is noteworthy that any of the investigated medications have been approved for such indication so far [8, 12, 13]. In this chapter, our main focus will be on the antiviral drugs under study for COVID-19 treatment.

Mechanisms of Different Antivirals Actions in COVID-19 Treatments

Nonstructural proteins (NSPs) are conserved proteins that function in gene expression during virus replication. It is thought that NSPs are fundamentally associated with the replication and function of coronavirus. Structural and accessory proteins are the other two types that are less conserved than the NSPs and only embraced in the virion synthesis. Hence, the antivirals might be pivotal in controlling the proteins during viral transcription and replication as well, then they could be a likely choice to recuperate the emanating COVID-19 infections and clinical consequences [14, 15]. This information sounds crucial especially when talking about the mechanisms of antiviral agents' actions. A brief about these mechanisms of antiviral agents is illustrated in Fig. (1).

G-441524 is the active metabolite of remdesivir that veils viral RNA polymerase and shirks proofreading by viral exonuclease, lowering the production of viral RNA. Remdesivir works by slowing up chain discontinuation of nascent viral RNA and positively affects SARS-CoV, MERS-CoV, and bat-CoV strains [16]. Drawing on the repurposing concept, Umifenovir is one of the portentous antiviral drugs with an unrivaled mechanism acting on the S protein/ACE2 interaction and dampening membrane fusion in the viral envelope [17]. It mainly blocks both cell membranes and the fusion of viral endosomes through integration into cell membranes and intercession with the hydrogen bonding network of phospholipids [18]. Favipiravir (FPV) is a guanine analog with a pyrazine carboxamide structure, and competitively decreases purine nucleosides [19]. Through h phosphoribosylation and phosphorylation, FPV as a prodrug is transformed into active FPV ribofuranosyl phosphates as soon as it gets in the infected cells by endocytosis [19].

Oseltamivir (GS4104) is an ester prodrug, which is hydrolyzed by hepatic carboxylesterases to produce the active metabolite (GS4071), a potent, selective inhibitor of influenza virus neuraminidase. Viral neuraminidase disconnects the binding between the cell receptor and the virus during virus spreading from the cellular space to its periphery (budding). Thus, neuraminidase inhibitors (NAIs) inhibit neuraminidase. Renal filtration and tubular secretion are responsible for metabolite excretion [20]. Ribavirin acts by intracellular guanosine diminution inhibiting the inosine monophosphate dehydrogenase enzyme as an antiviral drug, interrupting guanosine synthesis. It can also affect the virus replication indirectly by accelerating IFN gene expression and modulating immune responses. Ribavirin is an inhibitor of both RNA synthesis and mRNA capping [21, 22]. Lopinavir (LPV) is a peptide-mimetic molecule augmenting the non-self-cleavage peptide linkage which is the main target of the HIV-1 protease enzyme, thus blocking the activity of that enzyme [23], while Ritonavir acts through an important step in the HIV life cycle through which protease enzyme cleaves polyproteins, afterward, immature proteins inside the virus aggregate into particles which bloom from the cell as infectious virions. Thereby, the main role of protease inhibitors is dedicated to compete for the active cleavage site on the protease enzyme to prevent the cleavage of these polyproteins and thus the formation of mature virions [24]. Given Baloxavir marboxil, it is an RNA polymerase inhibitor that hampers mRNA synthesis by hindering the cap-dependent endonuclease of PA, repressing virus propagation. PA, PB1, and PB2 are the components of the viral polymerase. Therefore, this antiviral agent limits the viral infection which takes place when hemagglutinin on the surface of the viral envelope ties up to receptors located on the cell surface of the airway in the human body. It is noteworthy that the virus ribonucleoprotein (RNP) complex is liberated in the intracellular space (shelling), and moves into the nucleus. Viral RNA transcription and viral genome RNA replication, which accounts for synthesizing mRNA, are independently spurred [25].

Regarding SARSCoV-2, Chloroquine can suppress the entrance of the virus and prohibit its cell-binding by impeding ACE2 receptor glycosylation and its fusion with spike protein, indicating earlier probable effectiveness of Chloroquine against COVID-19 infection, before reducing ACE2 expression and action [26^-28].

Different Antiviral Drugs used in COVID-19 Treatment

Despite lacking definitive and specific treatment regimens, treating cases with SARS-CoV-2 infection is principally depending on those already well-known drugs and drawing on symptoms experienced by the infected patients. Antiviral medications are mainly used against COVID-19, especially in secondary infections triggered by ARDS [29]. However, the efficacy of these medications needs to be investigated by well-designed clinical experiments. Briefly, drugs targeting RNA respiratory viruses have guided some efficacious therapies, indicating obstacles for antivirals in COVID-19 infections treatment. Multiple antivirals are under study for symptomatic COVID-19 but no clear information boosting the usage of those agents. Remdesivir sounds promising in vitro against SARS-CoV2 highlighting desirable results for inpatients in a compassionate use series with a short-term restoration and a mild decline in deaths. Presently, Remdesivir is under investigation in phase III clinical trials, the compassionate use series, and finally to approve its use in emergency cases. A randomized-controlled trial of Lopinavir/Ritonavir (LPV/r) showed no obvious clinical or virologic advantage, in addition, drug-drug interactions and adverse reactions restrict its usage. Antivirals such as Oseltamivir, which is commonly used in treating influenza, have restricted action against SARS-CoV-2, while those agents like FPV and Umifenovir target the virus through specific sites, and demand further examination. These antiviral drugs which have shown activity against the virus should be investigated to approve their usability as treatment and prophylaxis for those patients severely infected with COVID-19 [30].

Fig. (1))

Mechanism of action for medications used in the treatment of COVID-19 infection [10]. ACE2R: Angiotensin-converting Enzyme 2 receptor; TMPRSS2: Type II transmembrane serine protease; RdRp: RNA-dependent RNA polymerase.

Broad-spectrum Antiviral Agents (BSAA)

Remdesivir

Remdesivir is a phosphoramidite prodrug of an adenosine C nucleoside. In 2017, it was first synthesized, developed, and introduced by Gilead Sciences as a wide-spectrum antiviral drug against the Ebola virus, and used these days as a promising medication for COVID-19 treatment [31]. In mice infected with the virus MERS-CoV, it was found that Remdesivir can significantly improve lung function and alleviate pulmonary pathological damage via reducing the viral load in the lungs [32]. In another study, it was also revealed that low micromolar concentrations of Remdesivir can potently block SARS-CoV-2 infection with a high selectivity index (half-maximal effective concentration (EC50), 0.77 μM; half-cytotoxic concentration (CC50) > 100 μM; SI > 129.87) [26]. Holshue et al. revealed interesting findings of IV Remdesivir when has been utilized in a trial on a patient who had been cured of pneumonia but was infected with COVID-19 in the United States [33]. For evaluating its efficacy and safety COVID-19 infected cases, China has launched a randomized, placebo-controlled, double-blind, multicenter, phase III clinical trial on 308 and 452 cases, respectively, in February 2020. A 200 mg as an initial dose of Remdesivir has given to the patients in the case group followed by a subsequent IV infusion dose of 100 mg for 9 consecutive days accompanied by routine treatment, meanwhile, the same dose in placebo was given to patients in the control group. It was estimated that the trial results are going to be provided in mid-April but it has been suspended as no eligible patients can be recruited [34]. Currently, a 10-days regimen is recommended for Remdesivir including 200 mg loading dose on day 1, then 100 mg once-daily maintenance doses for 9 days. It is worthwhile that this experiment has been terminated as the epidemic has been controlled well in China [35]. Recently, case series in the USA investigating the compassionate use of Remdesivir on 61 inpatient adults infected with COVID-19 have shown an amelioration amongst 68% of those patients regarding oxygen therapy during an average of 18-day prosecution period, while the clinical status had worsened among 15% of the patients. Clinical amelioration was detected among 84% of ages less than 50 years old, but this was less noticed in patients aged 70 years old or more, and in patients who were on mechanical ventilation compared with patients on extubated ventilation. The mortality rate was 13% among old patients≥70 years old, and patients with higher baseline serum creatinine indicating a more elevated threat. The most common side effects observed in 60% of cases were; elevated liver enzymes (transaminases) (23%), diarrhea (9%), rash (8%), kidney dysfunction (8%), and hypotension (8%) [36]. Remdesivir exhibited changes in its characteristics depending on the abovementioned dose (linear pharmacokinetics), and an intracellular half-life when exceeds 35 hours. Reversible aspartate aminotransferase and alanine transaminase elevations took place following multiple doses. Liver or kidney adjustments are not currently recommended, but at the start-up, the point might be necessary when the glomerular filtration rate is approximately lower than 30 mL/min [37, 38]. Accordingly, some other studies have supported the trials on Remdesivir against new COVID-19 based on its safety profile in vivo [16, 39]. It should be noted that the US Food and Drug Administration (FDA) authorized the use of Remdesivir for treating inpatients infected with COVID-19 in the emergency department, and this was in May 2020 [40]. It is noteworthy that Remdesivir has drawn a conditional marketing authorization from the European Commission for one year, due to the urgent need against the Covid-19 pandemic. The intravenous drug, manufactured by US pharma company Gilead Sciences, will be used for adults and children over the age of 12 who require supplemental oxygen. Subsequently, the authorization can be extended or became unconditional when further data is submitted. That encompasses data from presently ongoing phase III clinical trials [41].

Umifenovir (Arbidol)

Umifenovir (brand name, Arbidol), an indole carboxylic acids derivative, was firstly approved in Russia, then China as a prophylactic treatment in patients infected with influenza A and B, and some other arbovirus [42]. Subsequently, Umifenovir (brand name is Arbidol) has shown in vitro antiviral efficacy against multiple virus strains such as the Ebola, human herpesvirus 8 (HHV-8), hepatitis C virus (HCV), and Tacaribe arenavirus [43]. In the influenza virus, it acts on reducing the probability to reach the acidic media which is essential for the conversion of hemagglutinin (HA) into the fusogenic form [44]. Some studies have revealed the effectiveness of Umifenovir against both SARS-CoV-1 and SARS-CoV-2 in vitro [45, 46]. In China, an observational study has shown a decline in mortality rates in 67 COVID-19-infected patients, in addition to reduced length of hospital stay among 36 patients who used Umifenovir [47]. In contrast, in another Chinese retrospective study, Umifenovir failed to show negative tests for SARS-CoV-2 among 81 inpatients (not in the intensive care unit), nor in the length of stay when compared with the standard treatment [48]. Currently, there are two Chinese randomized and open-label trials aim at examining the efficacy and safety of Umifenovir while used in COVID-19 cases. One of these trials (NCT04252885) is for comparing the efficacy of the combination (Umifenovir and standard treatment) versus (LPV/r and standard treatment), whereas the other trial (NCT04260594) is for comparing (Umifenovir plus standard treatment) with standard treatment alone.

Favipiravir (T-705 or Avigan)

Favipiravir (FPV) (brand name, Avigan) was developed in 2014 by Fujifilm Toyama Chemical's Japanese company to treat avian influenza or other types of influenza resistant to neuraminidase inhibitors. FPV shows its broad-spectrum antiviral action via selective working on conservative catalytic RNA domain-dependent RNA polymerase (RdRp), interfering with the incorporation process of the nucleotide during RNA replication in the virus [19]. Those frequent transition mutations can be attributed to dysregulation occurring during RNA replication encompassing. For example, permutation of guanine (G) and cytosine (C) occurs due to adenine (A) and thymine (T), respectively. It leads to disables mutagenesis in RNA viruses [19]. Accordingly, FPV can play an important role in infections caused by RNA viruses such as influenza, Ebola, and norovirus [49]. One trial has concluded that FPV is needed in large doses for safe and efficacious clinical plans drawing on its activity in Vero E6 cells infected with SARS-CoV-2 with half-maximal effective concentration (EC50) of 61.88 μM and half-cytotoxic concentration (CC50) at over 400 μM [26]. In China, a randomized-controlled trial (ChiCTR200030254) has demonstrated that FPV was more effective than Umifenovir when both have been used to treat COVID-19 patients. This supremacy in terms of recovery rate was (71.43%) with FPV but (55.86%) with Umifenovir, moreover, and the time needed for relieving fever and cough also was profoundly shorter in the FPV group [50]. In a Chinese hospital, Jianjun Gao has carried out a study on 80 inpatients infected with COVID-19. It was found that FPV had a more potent antiviral activity than the LPV/r with no adverse effects [19]. The effective dose of FPV against COVID-19 is 600 mg Tid with 1,600 mg as a loading dose firstly for no more than 14 days, the half-life is approximately 5 hours [51]. However, FPV has exhibited teratogenic and embryotoxic effects in animals, therefore, it is contraindicated in pregnancy [51], [52]. FPV is used these days in Japan, but not in the United States, for clinical use. Up to Apr 15, 2020, eight clinical trials have been currently conducted in China and two in Japan to investigate the antiSARS-CoV-2 potential of FPV. These trials embrace non-randomized- and randomized-controlled trials to evaluate efficacy and safety of FPV as a single therapy (ChiCTR2000030113, JPRN-jRCTs031190226, JPRNjRCTs041190120), or when combined with IFN-α (ChiCTR2000029600), also with Baloxavir marboxil (ChiCTR2000029544, ChiCTR2000029548), with Tocilizumab (ChiCTR2000030894, NCT04310228), or with Chloroquine phosphate (ChiCTR2000030987, NCT04319900).

Oseltamivir (Tamiflu)

Oseltamivir (brand name is Tamiflu) is an antiviral medication approved to treat influenza A and B. Oseltamivir carboxylate is the active form of Oseltamivir, distinguished in a longer half-life, and formed by the hydrolysis of the prodrug, which is an ethyl ester in nature [53, 54]. The potential of Oseltamivir in decreasing death in cases infected with the H5N1 influenza virus was before the respiratory failure takes place and when the dose was 75 mg bid [55, 56]. Generally, patients require to take this drug for 5 days. Still, in influenza cases accompanied by acute respiratory distress syndrome (ARDS), pneumonia, or in immune-deficient patients, Oseltamivir should be used for 10 days [57]. Oseltamivir and Zanamivir are deemed as treatment and prophylactic first-line therapy in influenza, as shown in Table 1. In Wuhan, China, a study has revealed that no positive results were detected after receiving Oseltamivir when treating COVID-19 [58]. Oseltamivir is in a clinical trial for Phase 3 to examine its efficacy against 2019-nCoV in COVID-19 therapy with an HIV protease inhibitor (ASC09F) as a 3CLpro inhibitor is in use individually in Phase 4 2019-nCoV [59].

Furthermore, Oseltamivir has been utilized per oral for treating 2019-nCoV and hospitalized cases in China, yet, its effectiveness in treating COVID-19 patients [59]. Oseltamivir has shown a wide safety margin while used in animals. In the clinical trials program, severe adverse reactions were documented at the same frequency as with placebo (1.3% with 75 mg bid, 0.7% with 150 mg bid, and 1.2% placebo) [20]. In addition, teratogenicity or fertility-related reactions were not evident. Oseltamivir has also been investigated as a prophylactic agent or even as a medication against influenza A and B. Transient gastrointestinal disturbances are the most prominent adverse effects that might appear with Oseltamivir. These disturbances can diminish when the drug is taken with a light meal [60].

Ribavirin

Ribavirin is a broad-spectrum antiviral drug, guanosine nucleoside analog in structure. It is used to treat several viral infections, such as respiratory syncytial virus (RSV), hepatitis C virus, bunyavirus, herpesvirus, adenovirus, poxvirus, and some viral hemorrhagic fevers. In studies on coronaviruses in animals, Ribavirin has shown a weak inhibitory effect. Nevertheless, it can minimize the release of pro-inflammatory cytokines from the macrophages through its immune-modulatory effect in mice and convert the Th-2 response to Th-1 response [61]. However, one of the adverse reactions of Ribavirin is the reduction in hemoglobin concentrations when given to ill-persons. It has limited use as an antiviral agent against SARS-CoV-2 [62, 63].

Lopinavir-Ritonavir

Lopinavir (LPV) is a highly specific inhibitor for HIV-1 protease; however, it is used exclusively in combination with Ritonavir. Kaletra was the brand name for this combination which was firstly produced by Abbott company in 2000 [64]. Ritonavir is responsible for enzyme inhibition and enhancing Lopinavir exposure by boosting oral bioavailability and reducing biotransformation [64]. In addition, Ritonavir results in increasing the area under curve related to LPV plasma concentration more than 100 folds in healthy volunteers [65]. In SARS, the protocol including lopinavir-ritonavir (LPV/r) (usually combined with corticosteroids) is effective in reducing deaths, lowering mechanical ventilation needs, decreasing rescue corticosteroid treatment, and minimizing the post-therapy load of the virus [66, 67]. According to these results, this combination was evaluated in patients with COVID-19. Early reports of LPV/r for COVID-19 treatment were mostly case reports of small-scale retrospective and non-randomized cohort studies. Hence, endorsing the role of this medication is not an easy task in this regard [67, 68]. Recently, in an open-label, individually randomized, controlled trial, LPV/r was investigated among patients with COVID-19 using a dose of 400mg/100 mg, orally twice daily plus standard of care, against others who were on the standard of care alone. No benefit was observed for LPV//r beyond standard care. The most frequent adverse effects related to LPV//r use were; diarrhea, nausea, and asthenia [69]. In a Korean report, LPV//r administration has significantly improved the clinical symptoms of coronavirus such as fever and cough.

Interestingly, also coronavirus titers were few or even undetected in the follow-up study. Nevertheless, the analysis encompassed only one infected person in the initial phase of the epidemic in Korea [70]. Another retrospective cohort study revealed a better effect of the combination of Umifenovir with LPV/r when compared with LPV/r alone when both groups have been applied containing 16 and 17 patients, respectively [71]. Undesirable reactions of LPV/r were abdominal disturbances such as nausea and diarrhea (up to 28%) and liver toxicity (2%-10%). Commonly, side effects of LPV/r might be escalated by using combination therapy. A percentage of patients may experience elevated transaminases [72]. Consequently, LPV/r-induced hepatotoxicity could limit the use of this combination [73].

Baloxavir Marboxil

Baloxavir marboxil is a new antiviral agent that can inhibit the process of RNA replication in influenza by acting on various parts of the proteins in the influenza polymerase complex. In short, the Baloxavir marboxil prohibits cap-dependent endonuclease [74]. As both are RNA viruses, Baloxavir marboxil seems to have a significant effect against SARS-CoV-2 by preventing its RNA synthesis. At the same time, it was revealed that the Baloxavir marboxil possesses antiviral activity against SARS-CoV-2 in vitro. Based on non-linear regression fitting, EC50 against SARS-CoV-2 was estimated to be 3.32, 5.48, and 10.4 μM for Umifenovir, Baloxavir acid, and LPV, respectively. In 24 patients (82.8%), the findings showed undetected viral RNA in two successive tests within 14 days post to the trial establishment. After 14 days, 70%, 77%, and 100% who have been treated with Baloxavir, FPV, and those in the control group, respectively, showed negative results according to the virus test. In this trial, the time needed for those patients for improvement was 14, 14, and 15 days in the cases treated with Baloxavir, FPV, and control group, respectively. In total, 7 days were sufficient for 15 patients (51.7%) to be free of the virus (60%, 44%, and 50% in Baloxavir, FPV, and control group, respectively. One and two patients in the Baloxavir marboxil, and FPV groups were admitted for intensive care within 7 days post to trial establishment based on the case aggravation, but no deaths have been reported. That patient in Baloxavir marboxil group needed to be treated with extracorporeal membrane oxygenation ten days after trial initiation. In short, it was concluded that adding Baloxavir or FPV in their usual dose to the existing standard treatment had not any contribution to clinical improvement in this study [75].

Chloroquine and Hydroxychloroquine

Chloroquine and Hydroxychloroquine are long-standing aminoquinolines, with similar structures. They are often clinically used in the treatment of various diseases such as malaria, amebiasis, rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) [76]. The hydroxyl group in Hydroxychloroquine, when compared with chloroquine, leads to a decrease in its toxicity while giving similar action. In humans, graft-versus -host disease can be controlled by targeting lysosomes which is one of these mechanisms of action of Chloroquine and Hydroxychloroquine [77]. These drugs accumulate in lysosomes and significantly change the entire pH. They are directly responsible for affecting lysosomal proteases' activity by influencing the disintegration of proteins and glycosaminoglycan [77, 78]. The effectiveness of Chloroquine is evident in mice models on viruses such as human coronavirus OC43, enterovirus EV-A71, Zika virus, and influenza A H5N1 virus, as well as ex vivo conditions on Ebola virus [47, 79, 80]. In both healthy and ill persons experiencing SLE and RA, Hydroxychloroquine has an anti-inflammatory effect on Th17-related cytokines (IL-6, IL-17, and IL-22) [81]. Based on one analysis, cytokine storm triggered by COVID-19 has a significant role in ARD, leading to death in infected patients. Interestingly, Chloroquine and Hydroxychloroquine can suppress cytokine storms in accordance with some evidence [82]. In vitro, it has been reported that Hydroxychloroquine has shown its effectiveness in repressing SARS-CoV-2 infection [28, 83, 84]. Wang et al. (2020) revealed that the EC50 of chloroquine against COVID-19 tested on VERO E6 was 1.13 μM and the EC90 was 6.90 μM. It is noteworthy that after 24 hours of growth, Hydroxychloroquine demonstrated in vitro activity with a lower EC50 for SARS-CoV-2 when compared with Chloroquine (Hydroxychloroquine: EC50 = 6.14 μM and Chloroquine: EC50 = 23.90 μM) [85]. Nevertheless, the evidence on the safety and efficacy of these medications in the treatment of COVID-19, therefore, the risk/benefit relationship should be considered [86]. Accordingly, more clinical experiments to investigate the efficacy and safety of Hydroxychloroquine as a prophylactic and treatment agent against COVID-19 are underway. In the USA, Chloroquine and Hydroxychloroquine have been authorized for the emergency treatment of COVID-19 by the FDA. In one study, Chloroquine was not recommended for COVID-19 treatment (Table 1) because of the high dosage utilized during the 10 days of needed for treatment plan (12 g) may result in serious cardiac effects [87]. Meanwhile, this was contrasted by another study in which Chloroquine was safely used in the treatment and resulted in radiologic findings improvement, viral clearance enhancement, and disease progression reduction [88]. Nevertheless, the design and outcomes data of this clinical trial have not yet been published for peer review which makes the validity of the results questionable. In France, a recent open-label non-randomized study recruited 36 patients have been divided into two groups, 20 in the Hydroxychloroquine group and 16 in the control group. The dose of Hydroxychloroquine was 200 mg bid per oral versus standard supportive care in the control group. By the sixth day, 70% of patients (14/20) in the Hydroxychloroquine group demonstrated virologic clearance using nasopharyngeal swabs, while this had happened in only 12% of patients (2/16) in the control group, the difference was statistically significant (p=0.001). In another Chinese prospective study in which 30 patients had been selected randomly. All were treated with 400 mg Hydroxychloroquine daily for 5 days combined with standard of care (i.e. supportive care, IFN, and other antivirals) versus standard care alone. The virologic outcomes were not different. On the seventh day, the difference in the virologic clearance between the two groups was not statistically significant (p> 0.05), this was 86.7% in the hydroxychloroquine plus standard of care group versus 93.3% in the standard care group, respectively [89]. Presently, there are numerous RCTs on both Chloroquine and Hydroxychloroquine evaluating their efficacy in COVID-19 treatment. Chloroquine prophylaxis was studied among health care employees (NCT04303507) and hydroxychloroquine for post-exposure prevention following risky exposures (NCT04308668) are planned or enrolled [90]. Chloroquine dose in COVID-19 is 500 mg per oral od or bid with scarceness of data about the maximum dose to assess its activity and safety profile [84]. Not withstanding the general indicated dose for hydroxychloroquine in most related diseases is 400 mg once a day per oral, but one study had probed the pharmacokinetics of the drug based on its physiological characteristics, and indicated that the optimal dose for hydroxychloroquine in COVID-19 is a loading dose of 400 mg bid once for 1 day followed by 200 mg bid daily [85]. Conversely, another regimen for Whipple disease suggests the total daily dose of hydroxychloroquine 600 mg based on safety and clinical experience [84], hence, some more research is required to set out the optimal dose of hydroxychloroquine for COVID-19. Adverse reactions associated with chloroquine and hydroxychloroquine are rarely occurred as they relatively well-tolerated in SLE and malaria patients. However, serious side effects might appear in <10% of patients embracing QTc prolongation, hypoglycemia, neuropsychiatric effects, and retinopathy [91]. This necessitates baseline electrocardiography to check out heart status pre and post the use of these drugs in order to prevent arrhythmias, particularly in patients with a previous history, as well as to avoid drug-drug interaction with medications which prolong QT-interval such as azithromycin and fluoroquinolones [92]. Generally considered, chloroquine and hydroxychloroquine can be used safely in pregnancy [93], nevertheless, ocular toxicity has been reported as a side effect for these agents in one study had been conducted on 588 patients as a part of a review including 12 papers [94].

Other Antiviral Agents

Darunavir is one of the several antiretrovirals claimed to show in vitro activity when used against SARS-CoV-2. Clinically, still, there is no evidence on humans regarding the use of this medication in the treatment of COVID-19 [10]. Darunavir/Cobicistat (Prezcobix) and Darunavir /Cobicistat /Emtricitabine/ Tenofovir/ Alafenamide (Symtuza) are two Darunavir-based combinations produced by Johnson & Johnson, which has recently declared the lack of evidence to assure their usage as a therapy for SARS-CoV-2 [95]. Moreover, out of the three, one randomized, open-label clinical trial investigating Darunavir/Cobicistat for COVID-19 latterly indicated that this treatment was not efficient at clearing the virus on the seventh day after randomization when compared with the standard care [96].

BSAA Combination Therapy

Efficacious antiviral therapy is fundamental for combating the pandemic related to the novel coronavirus disease (COVID-19). One of these promising combinations in this regard is LPV/r, Ribavirin, and IFN beta-1b for treating COVID-19 infected patients. This combination was examined through a multicenter, prospective, open-label, randomized, phase 2 trial in adult patients infected with COVID-19. Those patients were admitted to six hospitals in Hong Kong, randomly selected (2:1), and divided into groups; combination and control. The patients in the combination group have received a combination of LPV 400 mg and Ritonavir 100 mg bid, Ribavirin 400 mg bid, and three doses of 8 million international units of IFN beta-1b on reciprocal days for 14 days. In the control group, the patients were given LPV 400 mg and Ritonavir 100 mg every bid for 14 days. Nasopharyngeal swabs were obtained in the incipient final stage, and they were negative for SARS-CoV-2 RT-PCR and were carried out to treat the population deliberately. This trial was registered with (ClinicalTrials.gov: NCT04276688). In this trial, of the 127 recruited patients, 86 and 41 were randomly selected to the combination and control groups, respectively. Symptoms appearance and then starting the treatment was in the median duration, 5 days (IQR 3–7). Based on the negative nasopharyngeal swab, the time needed for getting this negative result in the combination group (7 days [IQR 5–11]) was significantly shorter than that required for the control group (12 days [8^-15]; hazard ratio 4·37 [95% CI 1·86–10·24], p=0·0010). It should be noted that adverse reactions that accompanied the use of this combination were self-limited nausea and diarrhea in both groups. In the control group, only one patient paused LPV/r due to biochemical hepatitis. No deaths among patients have been reported during the study. Among those mild to moderate COVID-19 patients, this combination, triple antiviral therapy, was safe and more effective than LPV/r alone in mitigating symptoms and diminishing the duration of viral shedding, and reducing the length of hospital stay [97].

Umifenovir with LPV/r is another combination under investigation. However, this combination has demonstrated efficacy in one study [71]. Another prospective study (ChiCTR200030254) revealed the superiority of Favipiravir on Umifenovir concerning the clinical recuperation rate and alleviation of fever and cough [50]. Moreover, Darunavir with Umifenovir was a suggested combination to combat the SARS-CoV-2 outbreak reported in different recovering cases from the disease in China [98].

Many clinical experiments are currently investigating the effectiveness of Oseltamivir in the treatment of SARS-CoV-2 infection. In addition, Oseltamivir is also examined in clinical trials in numerous combinations, such as chloroquine and FPV [98].

Although the results of ribavirin and IFNα-2b in a MERS-CoV rhesus macaque model were promising, its results with IFN (either α2a or β1) on MERS-CoV infected patients were different. Mostly, Ribavirin exists in combinations with IFN but the findings vary from one type to another. It is noteworthy that the combination of IFNα-2b and Ribavirin for treating MERS-CoV demonstrated that their single-dose regimen had been decreased by 8 folds and 16 folds, respectively. They further activate cytokines, enhance immune system responses, reduce viral replication, host response modification, and clinical outcomes enhancement [63].

Table 1 Characteristics of the current treatments against COVID-19.

Some other researches exhibited reduced ARDS and mortality among SARS patients when they are given a combination of Ribavirin plus LPV/r. Moreover, the reduction was significant by adding corticosteroids to treat patients with SARS-CoV [66, 99]. An in vitro study has been conducted recently and revealed that Ribavirin should be used in a high effective concentration (EC50= 109.50 μM) against SARS-CoV-2 [67]. There is no evidence on the benefit of inhaled Ribavirin over enteral or intravenous administration for nCoV or RSV treatment [100]. Four studies demonstrated possible undesirable reactions, including hematologic and hepatotoxicity [101]. In MERS therapy, the combination of Ribavirin with IFNs is generally showed no noticeable clinical outcomes or viral clearance [102, 103]. Ribavirin leads to severe dose-dependent hematologic toxicity. The large doses used in the SARS trials resulted in hemolytic anemia in more than 60% of cases. In addition, 75% of patients have experienced transaminase elevation [101]. Similarly, in the broadest MERS investigational experiment, around 40% of patients required blood transfusion after receiving a combination of Ribavirin plus IFN [103]. Ribavirin also has a known teratogenic effect, so it is contraindicated in pregnancy [104]. The combination therapy could be a good suggestion to overcome the indecisive efficacy data with Ribavirin for other nCoVs and its significant toxicity to treat COVID-19. In the present time, there is an experiment to evaluate the efficacy and safety of IFN-α. IFN-α combined with Ribavirin, LPV/r, or Ribavirin with LPV/r for SARS-CoV-2 cases in China (ChiCTR2000029387) work effectively.

Zinc inhibits SARS-CoV and retrovirus RNA polymerase in vitro models. It works as ionophores to prevent viral replication in cell culture. Zinc can act in combination withChloroquine or Hydroxychloroquine. This combination is currently under investigation [105]. Moreover, Zinc enhances Chloroquine intracellular uptake [106].

The combination of Azithromycin with Hydroxychloroquine has been reported in 6 patients led to viral clearance in all patients (100%) better than that of hydroxychloroquine monotherapy (57%) [107]. Notwithstanding these auspicious results, several major limitations had been reported, such as the small sample size and the exclusion of 6 patients in the hydroxychloroquine group from analysis due to early termination of treatment. These limitations might lead to critical illness or drug intolerance, variable baseline viral loads between Hydroxychloroquine alone and combination therapy groups, and no clinical or safety outcomes reported. Many clinical experiments areunder trial to assess the combined effect of Azithromycin plus Hydroxychloroquine in people infected with SARS-CoV-2. Three clinical studies are currently conducted in France using azithromycin (500 mg on the first day, then 250 mg od for four days) in combination with hydroxychloroquine (600 mg od for 10 days). One of these trials is in an open-label non-randomized study (6 patients) [107], the second is in an open-label uncontrolled study (11 patients) [108], and the third is an uncontrolled observational study (80 patients) [108]. Gautret et al. revealed a 100% viral clearance among their 6 patients by testing nasopharyngeal swabs [107]. Nonetheless, Molina et al. [106] have contrasted the results of another scientist after repeating the trials, thought the prompt and complete viral clearance was quite unpredictable as they revealed that 8 of 11 patients had serious comorbidities [108]. Depending on these findings, data exhibited so far are inadequate to weigh up possible clinical advantages of azithromycin in COVID-19 patients [108]. It is also worthwhile to take into account the cardiac toxicity resulting from the additive effect of hydroxychloroquine and azithromycin combination. Protraction of the QT interval is a common side effect for both agents and may potentiate the risk in people with cardiac-related comorbidities.

Clinical Trials on Treatments for COVID-19

COVID-19 has turned out to be one of the most severe challenges in medical history. Hence, the quick development and approval of effective and safe treatments are necessary for reducing the deaths resulting from this pandemic. Consequently, many companies seek to repurpose those current therapies of known efficacy and safety profiles that can act on COVID-19. Up to the date of writing, a search of ClinicalTrials.gov revealed almost 1,000 results for COVID-19 [114]. Here, some of the most promising clinical trials will be shown.

The first clinical trials on traditional antiviral drugs reported non-promising early results [69, 115]. To address the risk and weak evidence of multiple small trials, the WHO has established the international Solidarity study, which embraces more than 100 countries to compare the findings related to four treatments: Remdesivir, hydroxychloroquine, and Chloroquine, LPV/r, and LPV/r in combination with IFN β-1a [116]. Interestingly, the Solidarity Trial's International Steering Committee has recommended stalling the trials on both Hydroxychloroquine and LPV/r arms, and the WHO accepted this. These recommendations have been built in light of the temporal results and by reviewing the evidence of all trials presented in the WHO summit on COVID-19 research and innovation on 1-2 July 2020. These interim trial results indicated that Hydroxychloroquine and LPV/r as a combination have limited or with no effect in reducing mortality among inpatients infected with COVID-19 compared to standard care. For each of these medications, the tentative findings do not introduce robust evidence on the deadrise. However, the clinical lab results indicated the safety of this combination to some extent. This decision was only made concerning the conduct of the Solidarity trial in hospitalized patients, but will not affect the investigatory efforts to examine the use of Hydroxychloroquine or LPV/r in non-hospitalized patients or as pre- or post-exposure prophylaxis for COVID-19. The transitory solidarity findings are currently available for peer-reviewed publication [117].

Ribavirin (Virazole^®, Bausch Health Companies Inc., Laval, Canada) is under investigation in an open-label, interventional trial to assess its safety and efficacy in inpatients adults who were COVID-19 positive and experienced severe respiratory distress [116]. A similar antiviral drug, Favipiravir (Avigan^®, FUJIFILM Toyama Chemical Co., Ltd., Tokyo, Japan), is currently in phase II development (NCT04358549) [116]. Moreover, preclinical and clinical data imply the antiviral properties of Azithromycin (Zithromax^®, Pfizer Inc., New York, NY, USA), and this is expected to be examined in COVID-19 patients soon [116].

Antibodies such as SLB (Kevzara^®, Sanofi, New York, NY, USA, and Regeneron Pharmaceuticals, Inc., Tarrytown, NY, USA) and TZB (Actemra/RoActemra^®, F. Hochmann-La Roche AG, Basel, Switzerland), which can effectively block IL-6 signal transduction. They are prioritized as the first agents to be examined in patients suffering complications from COVID-19 infection. Preliminary analysis of the phase II portion of Sanofi's trial investigating SLB (NCT04327388) manifested that it promptly decreased C-reactive protein, a key marker of inflammation. Nevertheless, SLB did not exhibit positive trends among severe cases that required oxygen supplementation but not mechanical, the results in critical cases that required mechanical ventilation were positive. Accordingly, the trial has been modified so that only critical patients are going to be enrolled to receive treatment. Evidence about TZB appears more auspicious, but the findings of the Roche global, randomized, double-blind, placebo-controlled phase II COVACTA trial (NCT04320615) will provide the pivotal answer about its profile [116]. Regarding Canakinumab, an IL-1β blocker, Novartis has also proclaimed a venture to launch a phase III clinical trial to investigate this treatment in patients with COVID-19 pneumonia, based on the reports of elevated levels of IL-1β in COVID-19-infected patients [116].

Evidence drawn from animal models on viral pneumonia indicates that complement inhibition may relieve lung injury, referred to as COVID-19 infection [118]. Complement is one of the innate immune response components to viruses that initiate pro-inflammatory responses. For the compassionate-use cases, Alexion is about initiating a global phase III trial to examine the use of Ravulizumab-cwvz (Ultomiris^®, Alexion Pharmaceuticals, Boston, MA, USA) on mechanical ventilation term, hospitalization length of stay, and survival of patients with COVID-19 compared to best supportive care [119]. Eculizumab (Soliris^®, Alexion Pharmaceuticals, Boston, MA, USA) is under investigation by Alexion in COVID-19 cases.

A preliminary study on 10 Chinese patients shows that immunotherapy using convalescent plasma-containing neutralizing antibodies improved clinical symptoms, raise blood oxygen levels and lymphocytes, decrease C-reactive protein levels and undetectable viral loads. Only two patients were detached from ventilators. It should be noted here that treatment with plasma was particularly successful when given within 14 days of symptoms appearance and with no detected adverse effects [120]. This result has to be assessed on a large scale.

Kiniksa Pharmaceuticals (Bermuda) recently has also declared evidence regarding Mavrilimumab treatment. This fully-human monoclonal antibody acts on granulocyte-macrophage colony-stimulating factor receptor alpha (GM-CSFRα) in 6 COVID-19-infected patients experiencing serious pneumonia and hyper-inflammation. All patients demonstrated an improvement in fever alleviation and oxygen levels within 1–3 days. There was no need for mechanical ventilation among those patients. However, further studies are still required [121].

Eli Lilly and Company has also established a phase II study (A study of LY3127804 in hospitalized COVID-19 participants with likely progressive ARDS or mechanical ventilation; NCT04342897) LY3127804, which is a selective monoclonal antibody against angiopoietin 2. Scientists hope that inhibition of angiopoetin 2 will reduce the advancement to ARDS or the demand for mechanical ventilation in COVID-19 patients [121]. With new trials being announced daily, many potential therapeutics will be in clinical development. Despite the accelerated rate of progress, wide gaps subsist in grasping the COVID-19 immunopathology. Collaborative efforts between pharmaceutical companies may embark and be reinforced to come up with effective therapies; however, this might need time for further investigation to be utilized in humans. Hopefully, these efforts will succeed sooner in possessing suitable and effective treatment and bringing people's life back to normal.

Only a few clinical trials have been undertaken these days to investigate Baloxavir for the treatment of COVID-19. In an exploratory, open-label, randomized controlled study at a single center in China (CHiCTR2000029544) was carried out. 29 COVID-19-infected inpatients on LPN/r, Darunavir/cobicistat, or Umifenovir, in combination with inhaled IFN-α, were randomized to treatment with Baloxavir marboxil (80 mg per oral on day 1 and day 4, and 80 mg per day on day 7 as required) (n=10), FPV (1600 or 2200 mg orally on day 1, followed by 600 mg Tid for up to 14 days) (n=9), or control (standard antiviral treatment) (n=10). After 14 days of treatment, 70, 77, and 100% of patients in the Baloxavir, FPV, and control groups, respectively, have shown negative viral RNA results according to 2 consecutive tests [75]. Another randomized controlled trial registered in China: (CHiCTR2000029548) [111].

FUTURE PERSPECTIVE

The COVID-19 pandemic is evident as a surpassing disastrous outbreak, particularly in the broadly influenced countries such as China, Italy, Iran, and the USA in enormous features, particularly health, social, and economic. However, it is too early to predict any de facto scenario, but definitely, it will significantly affect all countries around the world [122]. Special attention is paid to the future premises, which comprise identifying accurate and prompt diagnostic tools, proper control of disease transmission chain, manufacturing effective, safe, approved medications for the treatment of the infection. Meanwhile, efficacious prevention (vaccines) and treatment, especially for COVID-19 pneumonia, will be the time required for the real global threat to health attributed to SARS-CoV-2. Nevertheless, no official approval for any of COVID-19 drugs as they still represent a big problem for humans. Given the epidemiological aspects of SARS-CoV-2, it is substantial to interrupt the virus proliferation via some common methods such as quarantining the confirmed positive cases and controlling the spread of infection. Additionally, it is fundamental to benefit from the little evidence about medications these days to establish strategies to avoid and manage the SARS-CoV-2 transference. Based on the confirmation which stated that the genome sequence of SARS-CoV-2 is highly similar to both bat and human SARS-CoVs, some antiviral drugs for SARS-CoV infection have also been investigated for treating and preventing COVID-19 pneumonia. It seems that the most common concentration of ongoing clinical trials is on the repurposing of medications that has approval for other diseases into a COVID-19 disease treatment. Finally, among the literature, one novel perspective was recognized as an effective tool in the process of identifying new promising medications for the treatment of diseases generally. Furthermore, the need for designing and developing vaccines for SARS-CoV-2 cannot be ignored, moreover, developing new drugs and repurpose the previously used ones is of special concern. The establishment of animal models is one of the lessons extracted during the epidemics of SARS-CoV and MERS-CoV that should emphasize to retreat the various aspects of human disease as well as the safety and efficacy of the vaccine [123].

Interestingly, artificial intelligence is one of the fundamental suggestions that should be considered an effective method for early investigation of medications activity and safety in general and for COVID-19-related infections particularly. This technique can be used for both new medications design as well as for medication repurposing. Such applications are expected to introduce new medications with the adjusted estimation of safety and efficacy before any clinical trial. Through using different artificial intelligence techniques, several studies indicated mediations as potential treatments for COVID19 disease [124^-126]. Within these studies, suggested medications include remdesivir, Atazanavir, Efavirenz, Grazoprevir, Ganciclovir, Daclatasvir, Simeprevir, Asunaprevir, Dolutegravir [126], while, other agents such as Carfilzomib, Valrubicin, LPV, Eravacycline, and Elbasvir as available medications for COVID19 disease based on computational drug repurposing study [125]. Some of the mentioned medications are involved in current clinical trials while others are still in the theoretical phase. In the literature, the most commonly promising COVID19 treatment based on clinical findings embraces Remdesivir [127], FPV [127], Chloroquine/Hydroxychloroquine, and Azithromycin combination [127]. Melatonin can also be considered as a promising treatment as adjuvant therapy [128]. Additionally, plasma from patients recovered from COVID-19 can be deemed as a potent medication as well [129].

CONCLUSION

Many studies have been published recently to address the possibilities of different antiviral drug repurposing and to develop new therapeutic agents to treat COVID-19. Every idea presented in these published studies revealed that two concepts are ideal, one is the repurposing of existing antiviral agents and the second is the design and development of multi-targeted drugs. This will provide a valid approach to the scientific community and hence will narrow the knowledge gap. The practical applications of such concepts will provide an opportunity to avail of new chemical entities that are most likely to be used in the fight against COVID-19.

CONSENT FOR PUBLICATION

Not Applicable.

CONFLICT OF INTEREST

The authors confirm that this chapter contents have no conflict of interest.

ACKNOWLEDGEMENT

Declared none.

REFERENCES