Emerging pharmacotherapies for COVID-19

Graphical abstract


Background
In December 2019, Wuhan city, the capital of Hubei province in China, became the centre of a virulent disease of pneumonia of unknown cause [1]. By Jan 7, 2020, Chinese scientists had isolated a completely unique coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; previously referred to as 2019-nCoV), from these patients with virus-infected pneumonia [2]. The outbreak was declared a Public Health Emergency of International Concern on 30 January 2020 by WHO [3]. WHO later designated this SARS-Cov-2 as coronavirus disease 2019  in February 2020. By March 11, 2020 around 114 countries were affected globally by coronavirus disease then WHO declared COVID-19 as a deadly disease [3]. Although the outbreak is probably going to possess started from a zoonotic transmission event related to an oversized seafood market (Huanan seafood market, Wuhan) that also traded in live wild animals, it soon became clear that efficient person-to-person transmission was also occurring [4]. From phylogenetics analyses undertaken with available full genome sequences, bats and possibly pangolins appear to be the reservoir of COVID-19 virus, but the intermediate host (s) has not yet been identified [5,6].

Epidemiology
As of 6 April 2020, over 1,280,000 cases of COVID-19 are reported in over 200 countries and territories, leading to approximately 70,500 deaths. Over 270,000 people have recovered globally [6]. The primary case of the 2019-20 coronavirus pandemic in India was reported on 30 January 2020, originating from China. As of 6 April 2020, the Ministry of Health and Family Welfare have confirmed a complete of 4067 cases, 292 recoveries (including 1 migration) and 109 deaths within the country. Experts suggest the amount of infections may be much higher as India's testing rates are among the bottom within the world. The infection rate of COVID-19 in India is reported to be 1.7, significantly less than within the worst affected countries [7].

Management guidelines
At present clinical management includes infection prevention and control measures and supportive care, including supplementary oxygen and mechanical ventilatory support when indicated. WHO and CDC also recommend safety measures like avoiding direct contact with the patients stricken by acute respiratory infections, frequent washing of hands and people with respiratory infections to hide their nose and mouth while sneezing or coughing. Currently there are many International furthermore as country-specific government management guidelines for COVID- 19

Potential pharmacotherapies
There aren't any US-FDA, EMA or the other regulatory approved drugs specifically for the treatment of patients with COVID-19. However, on March 22, 2020 the Indian Council of Medical Research (ICMR) recommended the employment of hydroxychloroquine as prophylaxis for asymptomatic health care workers and asymptomatic household contacts of laboratory confirmed cases of COVID-19 [8]. Further, on March 28, 2020 the FDA granted Emergency Use Authorization (EUA) to be used of Chloroquine Phosphate or Hydroxychloroquine Sulphate for Treatment of COVID-19 [9]. WHO has identified a listing of "promising candidates" for COVID-19 treatment which include remdesivir (an investigational agent); lopinavir-ritonavir (approved to be used in HIV) with or without interferon; investigational immunotherapies like monoclonal and polyclonal antibodies; and convalescent sera. In its January 27 statement, WHO failed to support the antimalarial chloroquine or hydroxychloroquine, ribavirin (used for hepatitis), or corticosteroids/steroids for COVID-19 clinical studies [10]. WHO later launched SOLIDARITY trial on March 20, 2020 involving 45 countries and counting which tests four therapy groups: Remdesivir, lopinavir-ritonavir with or without interferon, and hydroxychloroquine. The rationale for selecting these drugs was stated as there was some evidence of effectiveness against the SARS-CoV 2 virus, which caused Covid-19, either in vitro and/or animal studies [11].
As shown in in the figure, the envelope is like HIV virus consisting of spike glycoprotein S, E protein and membrane protein M which are essential for entry of virus in the cell. This is a RNA virus and thus RNA genome is present in the core.  Fig. 3, the viral cycle is like other viruses consisting of attachment, integration, uncoating, use of cell machinery for replication, assembly and finally release of virions. Steps in coronavirus replication are potential targets for antiviral drugs and vaccines [2]. The spike glycoprotein S of coronavirus is a good candidate for vaccines because neutralizing antibodies are directed against glycoprotein S. Blockade of the specific virus  Protease inhibitors may block replication. The polymerase functions in a unique membrane-bound complex in the cytoplasm, and the assembly and functions of this complex are potential drug targets. Viral mRNAs made by discontinuous transcription are shown in the cytoplasm with the protein that each encodes indicated at the right. The common 70 base long leader sequence on the 5′ end of each mRNA is shown in red. Budding and exocytosis are processes essential to virus replication that may be targets for development of antiviral drugs. Apart from the pathophysiology of virus itself, it is now believed that the primary cause of mortality in susceptible patients is either due to pro-inflammatory cytokine storm or due to secondary bacterial infection. Secondary bacterial infections can be treated by using various antibiotics. Research is currently focussed for innovation of new therapies to treat this potentially fatal cytokine storm. Besides, most microbes including COVID-19 are known to bind to Toll like Receptor (TLR) which in turn induces a highly proinflammatory cytokine, Interleukin 1 (IL-1). IL-1 is the mediator of fever and fibrosis. This can lead to further deterioration in susceptible COVID positive patients. Thus, drugs supressing IL-1 or IL-1 receptor can be taken into consideration for treatment of COVID -19 [3].

Pathophysiology and potential therapeutic targets
A study has found out that women are much less susceptible to being affected by COVID 19 compared to men. Initially, the reason was hypothesised to be only environmental stating that as smoking is more prevalent in men and hence their respiratory system is compromised leading to greater risk of COVID infection. However, new research suggests that there are some innate differences between both sexes leading to the difference in the susceptibility. The presence of an extra X chromosome in females lead to lower viral load levels, and less inflammation than in men, while CD4 + T cells are higher with better immune response. Besides, women produce higher titre of antibodies which tends to remain in circulation for longer periods. The levels of activation of the immune cells are higher in women than in men, and it is correlated with the trigger of TLR7 and the production of interferon (IFN). TLR7 is higher in women than in men and it's biallelic expression leads to higher immune responses and increases the resistance to viral infections. TLR7 is expressed in innate immune cells which recognizes single strand RNA virus by promoting the production of antibodies against the virus and the generation of pro-inflammatory cytokines including IL-6 and IL-1 family members. Moreover, in women the production of inflammatory IL-6 after viral infection is lower than in males and is often correlated with a better longevity. In addition, on the X chromosome there are loci that code for the genes involved in the regulation of immune cells such as FOXP3 and transcription factor for T regulatory cells (T reg ) involved in virus pathogenesis. The X chromosome influences the immune system by acting on many other proteins, including TLR8, CD40 L and CXCR3 which can be over-expressed in women, and influence the response to viral infections and vaccinations. However, greater production of pro-inflammatory cytokines enhances risk of cytokine storm. Thus, drugs acting against pro-inflammatory cytokines can be theoretically more effective in women compared to men and in future, there may be gender wise differential therapy for treatment of COVID 19 [4].  interferes with the glycosylation of cellular receptor of SARS-CoV and thereby it has the potential to block viral infection. It also inhibits cathepsins, that leads to the formation of the autophagosome which cleaves SARS-CoV-2 spike protein. Furthermore, chloroquine through the inhibition of MAP-kinase interferes with SARS-CoV-2 molecular crosstalk, besides altering the virion assembly, budding and interfering with the proteolytic processing of the M protein.
• In-vitro: Chinese researchers who studied the effect of chloroquine in vitro (using Vero E6 cell line infected by SARS-CoV-2) found chloroquine to be highly effective in reducing viral replication that can be easily achievable with standard dosing due to its favourable penetration in tissues including the lung [5].
• Animal model: Antiviral activity of chloroquine against human coronavirus OC43 infection in new born C57BL/6 mice was highly effective [6].
• Key Clinical Trial: A Chinese study involving more than 100 patients of COVID-19 found chloroquine superior to the control group in reducing symptom duration, exacerbation of pneumonia including radiological improvement and promoting virus-negative seroconversion without any severe side effects [7].

B. Hydroxychloroquine (HCQ)
• Mechanism of Action: Same as that of chloroquine since same structure except for an additional hydroxy moiety in one terminal in HCQ.
• Safety Profile: Addition of hydroxyl molecule makes HCQ less permeable to blood-retinal barrier and allows faster clearance from retinal pigment cell, thereby suggesting a lesser risk of retinal toxicity with HCQ, as compared to chloroquine. Furthermore, the narrow therapeutic and safety index margin with chloroquine makes HCQ a safer option than chloroquine. Long-term clinical safety profile of HCQ is better than that of chloroquine since it allows higher daily dose of HCQ with less drug-drug interactions [5]. • Key Clinical Trial: A single case report by Holshue et al. described clinical improvement after RDV used to treat the first U.S. case of COVID-19. There are several randomized control trials are currently being conducted to evaluate the efficacy and safety of RDV in patients with COVID-19. Two phase III trials initiated in China in February 2020, aimed to evaluate RDV in hospitalized adult patients with mild/moderate (NCT04252664) or severe (NCT04257656) COVID-19 (RDV 200 mg on day 1 and 100 mg once daily for 9 days vs. placebo). Preliminary results of these trials are expected to be announced at the end of April 2020 [12].

D. Lopinavir-ritonavir
• Mechanism of Action: The polyprotein of the replicase protein is cleaved into functional units by virus-encoded proteinases which is inhibited by these protease inhibitor combinations. Thus, inhibiting proteolysis.
• In-vitro: The lopinavir/ritonavir combination was first considered a potentially useful treatment after in-vitro studies showed it had antiviral activity against SARS coronavirus [13].
• Key Clinical Trial: Chan and colleagues compared outcomes in people who received lopinavir/ritonavir as initial treatment, and as rescue therapy, with matched controls; all patients were given ribavirin and steroids according to a standardised protocol. The addition of lopinavir/ritonavir as initial treatment was associated with a statistically significant reduction in the overall death rate and intubation rate compared with matched controls (p < 0·05) [15].
[Note: Camostat Mesylate is another drug of same class (Protease inhibitors) having same mechanism of action, has shown evidence of effectively blocking SARS-CoV-2 in lung cells in-vitro.] [5].
E. Convalescent plasma • The US-FDA has accepted emergency investigational new drug applications for use of convalescent plasma for patients with severe or life-threatening COVID-19 [16].
• A case series described administration of plasma from donors who had completely recovered from COVID-19 to five patients with severe COVID-19 on mechanical ventilation and persistently high viral titres despite investigational antiviral treatment. The patients had decreased nasopharyngeal viral load, decreased disease severity score, and improved oxygenation by 12 days after transfusion, but these findings do not establish a causal effect. Finding appropriate donors and establishing testing to confirm neutralizing activity of plasma may be logistical challenges [17,18].

a) Interleukin 1
Interleukin 1 (IL-1) consists of two molecules namely IL-1a and IL-1b, associated with innate immunity. IL-1b is associated with most of the biological pleiotropic properties and hence it is directly referred as IL-1. IL-1 is proinflammatory cytokine and it binds to IL-1 receptor to modulate it's action. Association with IL-1 receptor leads to recruitment of other pro-inflammatory cytokines. Anakinra is one of the anti-interleukin 1 antagonist which is used to treat rheumatoid arthritis and currently being repurposed for the treatment of COVID-19 [19].
• Mechanism of Action: Inhibits actions of IL-1 leading to inhibition of cytokine storm which is common reason for fatality in COVID-19 • Current Evidence: No current in-vitro and in vivo evidence specifically for COVID 19. Currently, there is no published clinical trial. However, as per the data on clinicaltrials.gov, there are 8 trials using Anakinra against COVID-19 either in recruitment or pre-recruitment stage [20]. One of the major phase 3 trials using this drug is ongoing in Italy and is initiated by the manufacturer (Swedish Orphan Biovitrum) to evaluate efficacy and safety of anakinra or emapalumab with standard of care in reducing hyperinflammation and respiratory distress in patients with COVID-19. b) Interleukin 6 IL-6 is one of the key pro-inflammatory cytokines. IL-6 activates its downstream Janus kinase (JAK) signal by binding the transmembrane (cis-signalling) or soluble form (trans-signalling) of the IL-6 receptor (IL-6R) and interacting with membrane-bound gp130. Excessive IL-6 signalling leads to a myriad of biological effects that contribute to organ damage, such as maturing naïve T cells into effector T cells, inducing vascular endothelial growth factor (VEGF) expression in epithelial cells, increasing vessel permeability and reduces myocardial contractility [21].
• Mechanism of Action: Tocilizumab is a recombinant humanized monoclonal anti-IL-6R antibody. It binds both soluble and membrane-bound IL-6R to inhibit IL-6-mediated cis-signalling and transsignalling.
• Current Evidence: Case study/series describing use of tocilizumab in patients with COVID-19 have been reported from various areas of the world. In preliminary data from a non-peer reviewed, single-arm Chinese trial involving 21 patients with severe or critical COVID-19 infection, showed rapid fever reduction and a reduced need for supplemental oxygen within few days after receiving tocilizumab (initially given as a single 400-mg dose by IV infusion; this dose was repeated within 12 h in 3 patients because of continued fever). In China: Randomized, multicentre, controlled clinical trial evaluating efficacy & safety in 188 patients with COVID-19 is under way. Results are not yet available. In US/Global scenario, randomized, placebo-controlled trial is in phase 3 (NCT04320615) is in collaboration with the US Health and Human Services Biomedical Advanced Research and Development Authority (BARDA); the study will evaluate safety and efficacy of tocilizumab in combination with standard of care compared with placebo. In this study, there is expectation of enrolment of about 330 patients globally, including in the U.S., beginning in April 2020 [22].

G. Anti-inflammatory cytokines as drugs a) Interleukin 37 (IL-37)
Though it is a member of IL-1 family and is structurally like IL-1, it has anti-inflammatory activity. IL-37 has several mechanisms for immunosuppression but it ultimately leads to suppression of IL-1. It inhibits histocompatibility complex (MHC) molecules and thus inflammation by suppressing IL-1, IL-6, TNF & CCL2 [23]. Currently, there is no evidence regarding safety and efficacy of IL-37.

b) Interleukin 38 (IL-38)
Like IL-37, this is one of the most recently discovered anti-inflammatory cytokine belonging to the family of IL-1. It binds to the receptor of Interleukin 1 receptor type 6 and leads to suppression of inflammation. In-vitro cultures of activated peripheral blood mononuclear cells (PBMCs) are inhibited by IL-38 in the production of several cytokines including IL-1, IL-17 and IL-22. IL-38 gene knock out mice are more susceptible to inflammatory conditions. Currently there are no clinical trials using either IL 38 or its analogues. However, IL-38 can also be a potential new therapy [24].

Conclusion
As this is still an evolving topic, only the key medications mentioned in WHO and CDC guidelines as of 9th April 2020 have been incorporated in this review. Besides, we have added potential immunosuppressive therapies in the review as cytokine storm is one of the most common reasons for mortality in susceptible individuals. However, there are many more drugs currently under investigation including drugs like ivermectin, Vitamin C, Baloxavir, colchicine and Tacrolimus. The efficacy and safety of these drugs is still unknown and will be clearer in the coming months.

Funding
None. The research was self funded.

Declaration of Competing Interest
None.