INTRODUCTION:

Viruses represents the most teeming biological organisms on earth.¹ They are responsible for killing approximately twenty percent of the microbial biomass in oceans per day.²

In the recent decade, there have been alarming outbreaks of several viruses like Zika virus, Ebola virus, Severe Acute Respiratory Syndrome Corona virus (SARS-CoV), Middle East Respiratory Syndrome Corona virus (MERS –CoV) and now a novel corona virus.³ This article provides a basic understanding of SARS- COV-2 virus and overview of plausible mechanism of Chloroquine / Hydroxychloroquine in management of COVID-19.

In year 2019, in Wuhan (Province of China), a novel corona virus was first detected.⁴ In February 2020, it was assigned the name COVID-19 by World Health Organization and on 11th March,2020it was declared a pandemic.⁵ The corona virus study group of the International Committee on Taxonomy of Viruses (ICTV) has named it Severe Acute Respiratory Syndrome Corona virus 2 (SARS-COV-2).⁶ Three pandemics have been caused by corona viruses-SARS- CoV, MERS- CoV and SARS-CoV-2.³ In spite of all the necessary measures taken to halt this emerging virus, it has spread worldwide.⁷ Due to pandemic, astronomical efforts are going on to search molecules effective against SARS-CoV-2.

Structure and characteristics of virus:

SARS-CoV-2 belongs to the family of Coronaviridae, which has subfamily Coronavirinae. Coronavirinae subfamily is subdivided into four genus, one of which is betacoronavirus containing different species like human coronavirus 229E, OC43, SARS or SARS-COV-2.^8,9Main characteristic of this family of viruses include enveloped particles with long club shaped surface projections and single stranded positive genomic RNA.^[10]For long, coronaviruses have been found in bats (BtCoV) which act as largest CoV reservoirs. In 2002 there was an outbreak of severe acute respiratory syndrome which was found to be caused by new Coronavirus (SARS -CoV) with 10% mortality. In 2014 another corona virus named Middle East Respiratory syndrome corona virus (MERS-CoV) was detected which caused 35% mortality.^{11, 12, 13}

Corona virus virions are enveloped spherical particles with flexible nucleocapsid consisting of genomic RNA associated with nucleoprotein (N),spike (S) protein, membrane(M) and envelope(E) proteins.¹⁴ Major glycoprotein that projects from the envelope is spike(S) protein (peplomeres) which in electron microscope gives the appearance of the solar corona and hence its name.^[15] The spike protein is responsible for attachment of the virus with host cells and consequent release of their genome via fusion.¹²S₁forms the large receptor binding domain of the spike protein while S₂ forms the stalk of the spike.¹³The S protein receptor interaction is the main factor for corona viruses to infect specific tissues in specific host species. Some like MERS-CoV binds to Dipeptidyl-peptidase- 4(DPP-4) to enter host cells while SARS-CoV binds regions of Angiotensin Converting Enzyme-2 (ACE-2) which is a zinc binding carboxypeptidase enzyme responsible for regulation of cardiac function and blood pressure.¹⁵ ACE-2 is expressed in epithelial cells of the lungs and small intestine as well as many other organs. SARS-COV-2 has also been shown to bind to ACE-2. The overall structure of SARS-COV-2 resembles that of SARS-COV with some conformational differences in the position of their receptor binding domains on S protein.^{14, 16} During infection, the S protein is cleaved into subunits, S₁ and S₂by cathepsin or other proteases. S₁contains the receptor binding domain (RBD) at C-terminus which allows coronaviruses to directly bind to the peptidase domain (PD) of ACE2. S₂ then likely plays a role in membrane fusion.¹⁷

Vankadri et al studied detailed structure of SARS-COV-2 spike glycoprotein. They predicted the unique glycosylation sites of S protein that distinguishes it from SARS-CoV.¹⁸ Corona viruses enter the host cell by pH dependent endosomal transport.^19,20,21,22 In other words, fusion of virus with host cells generally occurs within acidified endosomes. In the cytoplasm, virus uses an enzyme replicase (RNA dependent RNA polymerase) for its replication and virions (new virus particles) are formed. ¹⁴

The S glycoprotein is the major inducer of antibodies. Corona viruses infect a wide range of host organisms producing illnesses ranging from mild flu to severe systemic disease. No vaccines or other antiviral agents are available till now for its treatment. ^{23, 24}The remedies recommended for managing SARS- CoV-2 are based on previous data available, in treating SARS or MERS-CoV. The drugs that might be successful in treating SARS-CoV-2 patients include Chloroquine, Remedesivir, lopinavir/ritonavir in combination with interferon-β, monoclonal antibodies (mAbs) or convalescent plasma.^24,25 The RNA dependent RNA polymerase (RdRp) sequence in SARS-CoV-2 is similar to RdRp in SARS-CoV (96%). ²⁶Hence a lucid option is to repurpose agents showing efficacy against SARS-CoV towards SARS-CoV- 2. Drug repurposing (DR) involves assigning new indications for the already existing drugs by studying new molecular pathways and targets for intervention or in simple words it is exploring new therapeutic indications for existing drugs. ^{27, 28,29}

Advantages with repurposed drugs are their well-known pharmacokinetic, pharmcodynamic properties and dosage regimens as well as their safety profile.^[30]Another advantage of drug repurposing is that such drugs can quickly enter clinical trials and manufacturing chains are already in place for their large-scale production.¹⁴Three studies have demonstrated DR for anti-CoV strategy.^{31, 32, 33}

History of Chloroquine/Hydroxychloroquine:

Quinine, a parent compound of chloroquine was isolated from cinchona bark by two French chemists in 1820. It was primarily used in the treatment of malaria. During World War II, Japanese occupied cinchona growing regions in South East Asia which led to discovery of synthetic compounds for malaria.³⁴ In 1934 German scientists synthesized Resochin(chloroquine) and Sontochin (3-methylchloroquine). During World War II French soldiers found Sontochin and gave it to Americans who in order to enhance the efficacy of Sontochin did chemical alterations in its structure. The product was chloroquine. Later on it was realized that Resochin and chloroquine were identical.^[35]Addition of β-hydroxy to the chloroquine molecule led to the development of new molecule hydroxychloroquine, which demonstrated far less toxicity than original molecule of chloroquine.³⁶

The non-malarial indications of these drugs were observed serendipitously. These drugs have been of proven value in rheumatoid arthritis, cardiovascular diseases and various dermatological conditions and even in some viral infections and malignancies. Chloroquine possesses pleiotropic pharmacological actions including immunomodulatory, anti-inflammatory actions and also induction of apoptosis.³⁷

Pharmacological characteristics:

Chloroquine belongs to the class of 4-aminoquinolines. They have flat aromatic core structure with basic side chain which contributes to the cumulation of the drug intracellularly based on Handerson-Hasselbach law. Hence, chloroquine and hydroxychloroquine are concentrated within endosomes, golgi vesicles and lysosomes where pH is acidic. Both chloroquine and hydroxychloroquine occur as R and S enantiomers. ³⁸Chloroquine is water soluble weak base with good absorption from gastrointestinal tract with about75% bioavailability.³⁹ The elimination half-life ranges between 40 and 50 days because of its widespread tissue uptake.⁴⁰ The serum concentrations however show inter-patient variability.³⁸ These drugs have extensive volume of distribution and long half-life. Both drugs are metabolized by CYPP450 enzymes and are liable to many drug- drug interactions.^{41, 42} Chloroquine is administered as its phosphate salt while hydroxychloroquine is administered as its sulphate salt.³⁸In 2018, Collins et al came out with a pharmacokinetic model to study Absorption, Distribution, Metabolism, Excretion (ADME) of Hydroxychloroquine.⁴³ Hydroxychloroquine has affinity for melanin containing tissues especially skin and eyes. However, these drugs are generally well tolerated and toxicity depends upon dosing and duration of use.^44,45 These drugs have strong affinity for blood cells particularly thrombocytes and granulocytes. In plasma chloroquine and its analogues are bound to albumin and α-acid glycoprotein. It also has great propensity to avidly bind to various tissues in the body leading to slower excretion. Main elimination pathway is via kidneys though small amount is excreted in bile, sweat and saliva also.^{46, 47}

Chloroquine exists in both protonated and unprotonated forms. Unprotonated chloroquine can locate itself readily into the acidic cytoplasmic vesicles like lysosomes and late endosomes. Once inside the cells, chloroquine gets protonated and is trapped. ^[48,49] There is excess accumulation of chloroquine in lysosomes which interferes with lysosomal acidification which is required for lysosomal enzymes to act optimally.^[38] Hence phagocytosis, chemotaxis, and antigen presentation is halted and so is phagolysosomal fusion.^[50]Chloroquine has anti-inflammatory and immunomodulatory properties based on its inhibition of antigen processing and presentation, inhibition of stimulation of Toll-like receptor (TLR9) cells which participate in immune responses or inhibition of cytokine production and release by T cells.⁵¹Several in vitro studies have demonstrated that chloroquine and hydroxychloroquine decrease the production of anti-inflammatory cytokines like interleukins IL-1, IL-6, Tumor necrosis Factor (TNF) and Interferon ( IFNγ).Immune pathways targeted by these drugs include TLR7 and TLR9 signalling T cell receptor and B cell receptor activation.³⁸

Mechanism of action of chloroquine differs according to pathogens. In viruses, chloroquine inhibits uncoating and glycosylation of newly synthesized protein. This has been demonstrated in vitro studies against HIV. As antibacterial, its role has been studied in coxiella burnetti (Q fever) where it causes change in pH of acidic vacuoles. ⁴⁹

Mechanism of action as antiviral agent:

Chloroquine and hydroxychloroquine disrupts the replication of viruses by affecting the endocytic pathway utilized by viruses to infect their target cells or by interfering with late stages of replication of enveloped viruses.⁴⁹It has been established that many viruses including corona viruses use endocytic pathway and need pH dependent conformational changes that enable fusion, penetration or uncoating for entry into their host cells.^52,53 Chloroquine has been found to prevent uncoating of influenza B and hepatitis A virus by interfering with the lysosomal acidification which is required for fusion between virus envelope and lysosomal membranes. Secondly, Chloroquine and hydroxychloroquine are involved in posttranslational modifications of the virus envelope glycoproteins in golgi network and endoplasmic vesicles by various enzymes like proteases and glycosyltransferases. These enzymes need acidic pH for their optimal activity and chloroquine by causing alkalization of acidic vesicles disrupts functioning of these enzymes resulting in decreased viral infectivity.⁴⁹ In Mayaro virus-infected monkey kidney cells, it was found that chloroquine caused a significant reduction in virus yield. It was observed that chloroquine inhibited budding of virus particles through plasma membrane of host cells.⁵⁴

Harley et al demonstrated that chloroquine by causing neutralization of Trans Golgi Network (TGN) ph, resulted in accumulation of non-infectious herpes simplex 1 in TGN. Chloroquine also resulted in inhibition of infectious virus production. ^[55]With chloroquine treatment in Human Immunodeficiency virus (HIV-1) there was significant reduction in surface glycoproteins gp120 and infectivity of virion particles. It was proposed that the most likely reason was due to interference in the terminal glycosylation pathway in TGN.⁵⁶

Chloroquine also leads to inhibition of enzymes involved in sialic acid biosynthesis.⁵⁷ Sialic acid is a component of receptor of SARS-CoV and orthomyxoviruses.⁵⁶ Chloroquine has been found to interfere with terminal glycosylation of ACE-2 receptor in corona virus cell cultures.⁵⁸

SARS corona virus induces immunopathological damage which can lead to worsening of symptoms in second week of infection. Involvement of proinflammatory cytokines like TNFα and IL-6 leading to immune activation is demonstrated in porcine respiratory corona virus which shows similar symptomology and lung damage as SARS-CoV. Hydroxychloroquine and chloroquine by decreasing production of proinflammatory cytokines TNFα and IFNα may also help in decreasing the disease severity. ⁴⁹

Adverse effects:

Main adverse effects include nausea, vomiting diarrhea and abdominal discomfort. Chloroquine and Hydroxychloroquine can cause QT interval prolongation leading to dysrhythmia. These drugs cause inhibition of degradation of photo receptors in retinal pigment epithelium by lysosomes which causes retinal damage resulting in bulls eye maculopathy.³⁸

In vitro Studies demonstrating Chloroquine / Hydroxychloroquine efficacy:

Way back in 2004, Keyaert et al described growth inhibition by chloroquine of SARS-CoV in Vero E6 cell cultures. They observed that the EC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concentration of chloroquine reached during treatment of acute malaria⁵⁹ In 2005 Vincent et al demonstrated that preinfection chloroquine treatment renders Vero E₆ cells refractory to SARS- CoV infection and when chloroquine was added post viral infection, there was considerable decrease in the number of virus antigen positive cells.⁶⁰Dewilde et al in 2014 demonstrated chloroquine effectiveness against MERS-CoV, SARS coronavirus and human corona virus 229E in vero E6 lines.⁶¹Dyall et al in 2014 found chloroquine diphosphate to have activity against MERS CoV and SARS- CoV in vero E6 lines.⁶²

In March 2020, the study by Yao et al demonstrated Hydroxychloroquine (Half maximal effective concentration EC50=0.72μm) to be more potent than chloroquine (EC50=5.4μm) in inhibiting SARS-CoV-2 infected Vero cells. Physiologically based pharmacokinetic model was used, which indicated a loading dose of 400mg twice daily orally of hydroxychloroquine sulfate followed by maintenance dose of 200mg twice daily for four days for possible use in SARS-CoV-2.⁶³Another study by Wang et al evaluated the efficacy of many antiviral drugs in SARS-CoV-2 infected Vero cells. Two drugs Remdesivir and Chloroquine were found to be highly effective.⁶⁴

As hydroxychloroquine is a less toxic option than chloroquine, Liu et al recently completed in-vitro study to see the antiviral effect of hydroxychloroquine as against chloroquine. They concluded that Hydroxychloroquine can effectively inhibit SARS-CoV-2 though careful design of clinical trial is needed to achieve efficient control of SARS-CoV-2 as selectivity index (SI= CC50 cytotoxic concentration/ EC50 maximal effective concentration) of hydroxychloroquine is low as compared to chloroquine. To explore the detailed mechanism of action of chloroquine and hydroxychloroquine, they also used immunoflorescence analysis and confocal microscopy which suggested that chloroquine and hydroxychloroquine inhibited the transport of SARS-CoV-2 from early endosomes to endolysosomes, which may be needed to release the viral genome.⁶⁵

Clinical Studies:

Gao et al, in a letter to editor which was published in February 2020, highlighted various clinical trials which were carried out in China in various hospitals to test the efficacy and safety of Chloroquine and Hydroxychloroquine. Results from more than hundred patients demonstrated that Chloroquine phosphate is effective in inhibiting the exacerbation of pneumonia, improving lung imaging findings, promoting a virus negative conversion and shortening the disease course. Severe adverse effects were also not reported from these patients.⁶⁶

A study by Gautret et al, 2020, in their open label non-randomized clinical trial reported that hydroxychloroquine when combined with azithromycin resulted in optimum level of clearance of virus in respiratory secretions.⁶⁷Similarly, Purvati et al in 123 mild to moderate COVID-19 patients demonstrated that 200mg of hydroxychloroquine and 500mg of azithromycin produced a significantly rapid PCR conversion to negative in 3day period.⁶⁸

Systematic review by Chaudhary et al, which included seven clinical trials evaluating efficacy of hydroxychloroquine or Chloroquine observed that five trials demonstrated effectiveness of HCQS/CQ when compared to supportive care or to lopinavir/ritonavir in treatment of SARS-CoV-2. But it was admitted that all trials may involve bias. ⁶⁹

However, in a study conducted on 150 patients with persistent mild to moderate COVID-19, addition of Hydroxychloroquine did not show significant results in virus clearance as compared to standard of care.^[70] In some trials done in United States, China, Brazil and France no benefit was reported with use of chloroquine, instead more complications including increased mortality were seen.^71,72,73,74

Similarly, Maganoli et al in retrospective analysis in U.S found no evidence that use of Hydroxychloroquine with or without azithromycin reduced the risk of mechanical ventilation in hospitalized patients with COVID 19.⁷⁵

In 2020, Recovery (Randomized evaluation of COVID-19 therapy) trial in UK was conducted at 176 hospitals in COVID 19 patients to assess the efficacy of hydroxychloroquine. 1567 patients were administrated hydroxychloroquine and 3155 patients received usual care. The primary outcome was mortality within 28 days. Death was reported in 421 out of 1561 patients (27%) in HCQS group. It was concluded that among patients hospitalized with COVID-19, HCQS did not achieve lower incidence of death at 28 days than those who received usual care.⁷⁶ However, some researchers still question the use of higher dose of hydroxychloroquine (4 g cumulated over four days) in RECOVERY trial. It was argued that high doses may be useless as hydroxychloroquine concentrates around 1500 times more in lysosomes as compared to plasma. Hence low doses of hydroxychloroquine can suffice. ⁷⁷

The treatment with hydroxychloroquine or chloroquine faced a major blow when WHO in Solidarity trial stopped the hydroxychloroquine arm because of no mortality benefit in COVID-19 hospitalized patients.⁷⁸

Axforas et al, in their meta-analysis of 28 RCTs on hydroxychloroquine or chloroquine concluded that treatment with HCQS is associated with increased mortality in SARS-CoV-2 infected hospitalized patients and no mortality gain was seen with chloroquine. However, this meta-analysis does not address prophylactic use or other outcomes.⁷⁹

Some studies are underway to examine its preventive role. A randomized controlled trial in Iran is initiated to study effect of hydroxychloroquine on COVID-19 prevention in cancer patients undergoing treatment. Prophylactic single dose 200mg tab of hydroxychloroquine will be given on alternate day.^[80]

A randomized open label phase 2 clinical trial is commenced in Uganda on adult patients to study the efficacy and safety of hydroxychloroquine for the treatment of SARS-CoV-2 infection in non-severe patients. Its objective is to find if hydroxychloroquine can lead to early viral clearance.⁸¹

A study in France is being conducted to determine the efficacy of pre-exposure treatment with hydroxychloroquine on risk and severity of COVID-19 infection. A patient population with SLE or Gougerot’s disease who are treated for a long time with HCQS are assessed if HCQS which was initiated before the pandemic COVID-19 has an independent protective effect on the risk or severity of infection with COVID-19.⁸²

Conclusion and Future Perspective:

There is always a conflict between clinical bedside therapy and academic plausibility of how a drug works. With pandemic lurking overhead it was but expected to pin high hopes on these drugs. With course of time, it is clear that it does not provide any mortality benefit in hospitalized patients. However, few studies are underway to learn more about these drugs and its role in COVID-19. History has shown us the comeback of older drugs like polymyxins in clinical use despite initial hiccups with toxic effects. With each passing day, as our understanding about these viruses is increasing, only time will tell whether these drugs stand any role in fight against SARS-CoV-2.

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Received on 20.10.2021 Modified on 14.12.2021

Res. J. Pharmacology and Pharmacodynamics.2022;14(2):110-116.

DOI: 10.52711/2321-5836.2022.00019