Non-Systematic Review

An overview of antiviral strategies for coronavirus 2 (SARS-CoV-2) infection with special reference to antimalarial drugs chloroquine and hydroxychloroquine

Viktorija Dragojevic Simica, Milijana Miljkovica, Dusica Stamenkovicb, Berislav Vekicc, Nenad Ratkovicd, Radoje Simice, Nemanja Rancica*

aCenter for clinical pharmacology, Military Medical Academy; Faculty of Medicine of Military Medical Academy, University of Defence, Belgrade, Serbia
bDepartment of Anesthesiology and Intensive Care, Military Medical Academy; Faculty of Medicine of Military Medical Academy, University of Defence, Belgrade, Serbia
cDepartment of Surgery, Clinical Center Dr. Dragisa Misovic; Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia; Ministry of Health of the Republic of Serbia dSector for Treatment, Military Medical Academy; Faculty of Medicine of Military Medical Academy, University of Defence, Belgrade, Serbia
eDepartment for Plastic Surgery, Institute for Mother and Child Health Care of Serbia Dr. Vukan Cupic, Belgrade, Serbia; Faculty of Medicine, University of Belgrade, Belgrade, Serbia

*Corresponding author: Nemanja Rancic, MD, PhD, Military Medical Academy, Crnotravska 17, Belgrade, Serbia. Phone: +381 63 852 44 43 E-mail address: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/ijcp.13825

Accepted Article

At present, neither specific antiviral drugs, nor vaccine is recommended for coronavirus disease 2019 (COVID-19) treatment. In this review we discuss the drugs suggested as therapy for COVID-19 infection, with a focus on chloroquine and hydroxychloroquine. The list of drugs used for COVID-19 treatment includes a combination of lopinavir and ritonavir, remdesivir, favipiravir, alpha-interferon, ribavirin, atazanavir, umifenovir, and tocilizumab. As their efficacy and safety are under investigation, none of the regulatory agencies approved them for the treatment of COVID-19 infection. Although chloroquine and hydroxychloroquine possess antiviral and immunomodulatory effects, in practice benefit of their use for COVID-19 treatment is controversial. Several studies investigating hydroxychloroquine were stopped and the French national medicines regulator suspended its use in clinical trials due to safety concerns. The results from the double-blind, randomized clinical trials, including large number of participants, will add better insight into the role of these two drugs as already available and affordable, antimalarial therapy. The ethical issue on emergency use of chloroquine and hydroxychloroquine in the settings of COVID-19 should be carefully managed, with adherence to the “monitored emergency use of unregistered and experimental interventions” (MEURI) framework or be ethically approved as a trial, as stated by the WHO. Potential shortage of chloroquine/hydroxychloroquine on the market can be overbridged with regular prescriptions by medical doctors and national drug agency should ensure sufficient quantities of these drugs for standard indications.

Keywords: SARS-CoV-2; Chloroquine; Hydroxychloroquine; Lopinavir/ritonavir; Remdesivir; Favipiravir; Ribavirin; Atazanavir; Umifenovir; Interferon-alpha 2b; Tocilizumab

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COVID-19- coronavirus disease 2019
SARS-CoV-2- severe acute respiratory syndrome corona virus 2 ARDS- acute respiratory distress syndrome
ACE-2- Angiotensin-Converting Enzyme 2 CRS- cytokine release syndrome
IL- interleukin
LDH- lactate dehydrogenase enzyme
SIRS- systemic inflammatory response syndrome WHO- World Health Organization
ECG- Electrocardiogram IU- international unit
s.c. – subcutaneously
p.o. – per os
DRESS syndrome- Drug rash with eosinophilia and systemic symptoms syndrome lapp- referring to Sámi people, indigenous Finno-Ugric people
FDA- Food and Drug Administration
IC50- half-maximal inhibitory concentrations CQ- chloroquine
HCQ- hydroxychloroquine q.d.- once a day
b.i.d.- twice a day t.i.d.- three times a day
CDC- Center for Disease control and Prevention SOC- standard of care
EMA- European Medicines Agency
CHM- The United Kingdom Commission on Human Medicines NIH- National Institutes of Health
n/a- not available

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1. Accepted Article

A coronavirus is a group of highly diverse, enveloped, positive-sense, and single-stranded RNA viruses that cause respiratory tract infection 1,2. Coronaviruses are common in animals; some of them can be transmitted into humans, such as a novel coronavirus 1. The first cases of coronavirus disease 2019 (COVID-19), caused by a severe acute respiratory syndrome corona virus 2 (SARS- CoV-2), were recognized in Wuhan, Hubei Province, China, in December 2019 3, 4. Common signs of infection include respiratory symptoms, fever, and cough 1,5 6. The illness is ranging from the common cold to more severe disease, such as pneumonia, acute respiratory distress syndrome (ARDS), and complications caused by systemic hyperinflammation.
Genetic sequencing of the virus suggests that SARS-CoV-2 is a beta coronavirus closely linked to the SARS virus4. The COVID-19 infection has higher levels of transmissibility and pandemic risk than that of SARS CoV 1. The entry receptor utilized by SARS-CoV-2 and SARS-CoV is Angiotensin-Converting Enzyme 2 (ACE-2) receptor 2.
Recent research reported that SARS-CoV-2 likely originated in bats, based on the similarity of its genetic sequence to that of other CoVs. The intermediate animal species of SARS-CoV-2 between a bat and humans is still unknown 7. Human-to-human transmission happens mainly by the respiratory route or through contact with infected secretions 2. Asymptomatic individuals transmit the virus 8. Based on data from cases in Wuhan, the incubation time ranges from 3 to 7 days, up to two weeks 8.

2. Coronavirus disease 2019

2.1 Pathogenesis

COVID-19 is capable of producing an excessive immune reaction in humans resulting in extensive tissue damage, known as a “cytokine release syndrome” (CRS) 8. The SARS-CoV-2 binds to alveolar epithelial cells, activates innate and adaptive immune response, leading to the release of various cytokines, including interleukin (IL)-6 9. Proinflammatory factors increase vascular permeability, and this leads to the appearance of blood cells and fluid in alveoli, resulting in dyspnea and respiratory failure.

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Recent findings indicate that SARS-CoV-2 bounds to heme groups on hemoglobin molecule, separate iron with consequent dissociation of heme into porphyrin, which is “captured” by virus proteins 10. Attack of oxidized hemoglobin by viral proteins will decrease the quantity of hemoglobin that can carry oxygen and carbon dioxide. This study found that viral non-structural proteins (ORF1ab, ORF10, and ORF3a) can simultaneously attack the heme on the 1-beta chain of hemoglobin to dissociate the iron leading to a formation of the porphyrin 10. Additionally, E2 glycoprotein, envelope protein, nucleocapsid phosphoprotein, ORF1ab, ORF7a, and ORF8 of the virus could bind to porphyrin 10. In summary, the virus is in a massive demand for porphyrin to survive.

2.2 Clinical course

The treatment depends on the clinical stage of the disease 6, 11-15. The report from the Chinese Center for Disease Control and Prevention analyzed 44,672 confirmed cases of COVID-19 and described three clinical stages 6, 16. Stage I covers mild disease that usually refers to early infection and is present in 81% of patients [6]. The clinical symptoms of stage I are fever, fatigue, cough, anorexia, malaise, muscle pain, sore throat, dyspnea, nasal congestion, headache, diarrhea, nausea, and vomiting. Prominent clinical signs include lymphopenia, increased prothrombin time, elevated values of D-dimer, and lactate dehydrogenase enzyme (LDH) 12.
Stage II is a severe (pulmonary) phase found in 14% of patients 6. Based on clinical presentation, there are two types of this stage IIa and IIb. Stage IIa is characterized by pneumonia without hypoxia and no need for supplemental oxygen. While in stage IIb patients have severe pneumonia with hypoxia, fever, or suspected respiratory infection. Clinical presentation includes respiratory rate > 30 breaths/min; severe respiratory distress; or SpO2 ≤ 93% on room air, hypoxia PaO2/FiO2a ≤ 300 mmHg or >50% of lung involvement on imaging within 24 to 48 hours [6]; transaminitis, low-normal procalcitonin level 12.
The critical stage III, known as systemic hyperinflammation, is found in 5% of patients 6. In this stage, patients are critically ill with ARDS, systemic inflammatory response syndrome (SIRS) or shock, cardiac failure, PaO2/FiO2 < 150 mmHg, elevated inflammatory markers (CRP, LDH, IL- 2, IL-6, IL-7, D-dimer, ferritin), troponin and NT-proBNP elevation 12. The case-fatality rates vary between countries, ranging from 0.1 % to 27.5% 15.

3. Accepted Article
Pharmacotherapeutic approach

3.1 Antiviral medications used for COVID-19 treatment

At present, no pharmacological agent, except remdesivir, has been approved by regulatory agencies for the treatment of COVID-19. Medical practitioners need a critical analysis of drugs proposed to be effective against COVID-19. So far, the recommended treatment for patients with severe COVID-19 is symptomatic, as supportive treatment interventions with oxygen therapy and different modes of mechanical ventilation. The World Health Organization (WHO) released a document summarizing guidelines and scientific evidence based on the treatment of previous epidemics caused by a human coronavirus 13, 17. The numerous antiviral drugs with different mechanisms of action are explored in clinical trials for COVID-19 treatment (Table 1) (Graph 1). Among the listed drugs, chloroquine and hydroxychloroquine are the most examined and used drugs in the current outbreak of SARS-CoV-2 (Table 1). The majority of pharmacotherapeutic guidelines for COVID-19 treatment suggest the use of chloroquine and hydroxychloroquine 38-41.

3.2 Chloroquine and hydroxychloroquine

3.2.1 Antiviral activity of chloroquine and hydroxychloroquine in vitro

An old drug for malaria treatment, chloroquine, has been in clinical use since 1944 42. Chloroquine is a weak base, which concentrates on the highly acidic digestive vacuoles of susceptible Plasmodium, where it binds to heme and disrupts heme sequestration. Hydroxychloroquine (β- hydroxylated N-ethyl substituents of chloroquine) was synthesized in 1946 and introduced in clinical use as equivalent to chloroquine in chemoprophylaxis or treatment of acute attacks of malaria 42. Chloroquine and hydroxychloroquine belong to disease-modifying antirheumatic drugs used in dermatology and rheumatology 43. Additionally, hydroxychloroquine is approved by the Food and Drug Administration (FDA) for treating lupus erythematosus 43.
Both drugs, chloroquine, and hydroxychloroquine, possess the immunomodulatory effects. They increase pH within intracellular vacuoles, therefore change the ability of acid hydrolases and molecular assembly needed for antigen peptide processing 43. Finally, this will lead to decreased stimulation of autoimmune CD4+ T cells and downregulation of autoimmune responses.

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Moreover, both drugs reduce the secretion of proinflammatory cytokines, particularly TNFα in many cells, including human peripheral blood mononuclear cells and human blood 44.

Researchers suggest that chloroquine inhibits viral replication, TNFα, and IL 6 production and the subsequent cascade of events leading to ARDS 45, 46. In vitro, chloroquine is an effective inhibitor of the SARS-CoV replication 47, 48. Chloroquine half-maximal inhibitory concentrations (IC50) for SARS-CoV in vitro inhibition in Vero E6 cells are 1000-fold below the human plasma concentrations of chloroquine following acute malaria treatment with this drug in a dose of 25mg/kg, over three days 48. Since the chloroquine dosage used for the treatment of rheumatoid arthritis (3.6 mg/kg) produced the same plasma as the IC50 for inhibition of SARS-CoV, one study claimed that chloroquine should be considered for immediate use in the prevention and treatment of SARS-CoV infections 48.

Results from the in vitro studies investigating chloroquine antiviral activities showed compromised virus/cell fusion with increased endosomal pH and interference with the terminal glycosylation of cellular ACE-2 receptors 49. These changes result in strong chloroquine antiviral effects on SARS-CoV infected primate cells. Decreased glycosylation of ACE-2 receptors lowers their affinity for SARS-CoV spike (S) protein, potentially preventing infection 49. On the other hand, reduced virus-endosome fusion generated by rapid elevation of endosomal pH may be an antiviral mechanism under post-treatment conditions 47. Based on in vitro studies, chloroquine has prophylactic and therapeutic antiviral activity.
Although SARS-CoV-2 S-protein possesses a weaker binding affinity for ACE-2 receptors than the corresponding protein of SARS-CoV, it still maintains a strong binding affinity to human ACE-2 receptors to result in significant risk for human transmission 9, 50. One of the studies tested the pharmacological activity of chloroquine using SARS-CoV-2 infected Vero E6 cells 34. The use of time-of-addition assay demonstrated that chloroquine influenced both entry and post-entry stages of the SARS-CoV-2 infection in Vero E6 cells 51. Chloroquine EC90 value was 6.90 μM, which equals plasma levels measured in patients with rheumatoid arthritis treated with 500 mg daily dose 51. The chemical components in chloroquine phosphate compete with the porphyrin and bind to the viral proteins, preventing the attack of viral proteins on heme or binding to the porphyrin 10. The chloroquine can inhibit E2 glycoprotein and non-structural protein ORF8 from binding to the porphyrin 10. Additionally, chloroquine can prevent viral ORF1ab, ORF3a, and ORF10 attack on the heme to form the porphyrin 10.

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In vitro, hydroxychloroquine exhibited lower EC50 values than chloroquine, indicating the former has a more potent antiviral activity 52. Dosing recommendations for hydroxychloroquine rely on a physiologically-based pharmacokinetic model 52. Oral application of hydroxychloroquine sulfate (a loading dose of 400 mg given twice daily, followed by 200 mg twice daily, for four days) reached three times the potency of chloroquine phosphate when given 500 mg twice daily for five days (Table 2) 52. Based on the superior antiviral and prophylactic activity of hydroxychloroquine in vitro study, the authors suggested that hydroxychloroquine in combination with anti- inflammatory drugs might be used in the treatment of severe SARS-CoV-2 infection 52.

3.2.2 Animal toxicity, pharmacokinetics in animals and humans and drug safety

Animal toxicity studies compared both drugs in short-term and longer-term assays in five species and by four routes of administration 53. The overall weighted margin of safety was 2.5/1.0 in favor of hydroxychloroquine, meaning that hydroxychloroquine has 40% toxicity of chloroquine 53. The tissue levels of chloroquine averaged about 2.5 times those of hydroxychloroquine when an identical dosage regimen of two drugs were given to albino rats 54. It was apparent that a more rapid and/or extensive transformation of the parent drug took place in rats administered with hydroxychloroquine, and it accounts for virtually all of its lower extent of accumulation in tissues. In human volunteers, in equal doses, two drugs were nearly interchangeable regarding plasma levels 53. The half-life of both drugs was about 50 hours 53. Hydroxychloroquine achieved peak plasma concentration after four hours, and chloroquine after five hours 53. The absorption of the two drugs, after oral intake, is nearly complete 53. The difference between drugs exists in the pattern of excretion since hydroxychloroquine excretion in the urine is three times lower and in the feces three times higher than chloroquine 53, 55.

Better hydroxychloroquine long-term clinical safety profile allows a higher daily dosage and fewer drug-drug interactions compared with chloroquine 42, 56. The addition of the hydroxyl molecule makes hydroxychloroquine less permeable to the blood-retinal barrier and allows faster clearance from retinal pigment cells 57. Therefore, hydroxychloroquine exhibits lower retinal toxicity in comparison to chloroquine 42, 57. Since the cumulative effect on cardiac conduction was seen in a combination of hydroxychloroquine and azithromycin, electrocardiography monitoring is indicated, and surveillance for QT interval prolongation 39. Chloroquine and hydroxychloroquine are metabolized by cytochrome P450 (CYP) isoenzymes CYP2C8, CYP2D6, and CYP3A4;

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therefore, inhibitors and inducers of these enzymes may result in altered pharmacokinetics of these agents 58.

3.2.3 Antiviral activity of chloroquine and hydroxychloroquine in clinical trials and current treatment guidelines

Patients with severe COVID-19 are critically ill on mechanical ventilation with multiple organ failure and possible comorbidities. Pharmacokinetic studies are needed to define the optimal dosing regimen in critically ill since pathophysiological changes may modify hydroxychloroquine pharmacokinetic profile 59. So far, the dosage in critically ill patients is based on population study in patients with rheumatoid arthritis 59. The recommended hydroxychloroquine dosage is 800 mg once daily, on the first day, that can rapidly reach therapeutic levels in critically ill patients, followed by 200 mg, twice daily for seven days. These encouraging findings were, to some extent, supported by the results of the exploratory clinical observations suggesting the superiority of chloroquine versus control to inhibit exacerbation of COVID-19 pneumonia 60. The use of chloroquine treatment in more than 100 patients with COVID-19 resulted in improved radiologic findings, enhanced viral clearance, and shortening disease course 60. However, clinical trial design and outcomes were missing; therefore, the value of the result is questionable.

Another study enrolled 36 COVID-19 positive patients to receive oral hydroxychloroquine sulfate, 200 mg, three times per day, during ten days, in a hospital setting 61 (Table 2). The primary endpoint was virological clearance at day six following inclusion in the study. The secondary endpoints were clinical signs (body temperature, respiratory rate, length of stay at hospital and mortality rate), and the occurrence of side effects 61. At post-inclusion day six, 70% of hydroxychloroquine-treated patients were virologically cured, compared with 12.5% in the control group (p=0.001) 61. Moreover, 100% of patients treated with hydroxychloroquine and azithromycin combination were virologically cured compared with 57.1% in patients treated with hydroxychloroquine 61. Significant limitations and concerns of drugs cardiotoxicity questioned the safety of this regimen.
In another prospective clinical trial conducted in China, 15 patients were randomized to receive hydroxychloroquine 400 mg per day and standard of care (SOC), for five days, or SOC in another 15 patients 68. On day seven, the authors detected similar virologic clearance between groups, such as 86.7% clearance for hydroxychloroquine plus SOC vs. 93.3% in the SOC group 68.

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Multicentric, parallel, open-labeled randomized clinical trial included 150 hospitalized adult COVID-19 patients (Table 2) 66. The addition of hydroxychloroquine to the SOC did not increase anti-viral response but accelerated the alleviation of clinical symptoms and recovery of lymphopenia 66. The efficacy of hydroxychloroquine on the reduction of symptoms (Hazard ratio, 8.83, 95%CI, 1.09 to 71.3) became more evident after posthoc subgroup analysis, which removed the confounding effects of anti-viral agents 66. However, this study demonstrated no difference in viral clearance between hydroxychloroquine and SOC group against the SOC group, which was the primary outcome 66.
A randomized, double-blind, phase 2b study aimed to evaluate the safety and efficacy of two chloroquine dosage regimens in 81 patients with severe COVID-19 infection (Table 2) 67. All patients received a combination of ceftriaxone and azithromycin, while 89.6% of the patients received oseltamivir 67. The primary outcome was mortality 13 days after starting treatment. Mortality was higher in the high-dose arm than in the low-dose arm [death in 16 of 41 patients (39%) vs. in 6 of 40 patients (15%) exacerbation; p = 0.03] [67]. Moreover, QTcF >500 ms occurred more frequently among patients in the high-dose arm (18.9%) compared with the low- dose (11.1%) 67. This study raised concerns at increased mortality rate caused with a combination of high-dose chloroquine (600 mg twice daily), azithromycin and oseltamivir 67.

In a randomized controlled trial, 62 hospitalized patients with mild COVID-19 pneumonia were randomized to receive hydroxychloroquine 200 mg twice daily for five days, plus SOC or SOC 68. The hydroxychloroquine-treated patients had a one day shorter mean duration of fever, and the cough remission time was significantly reduced in that group 68. None of the hydroxychloroquine treated patients experienced the progression of illness 68. According to the analyzed chest computerized tomography, a more significant proportion of patients had improved pneumonia in the hydroxychloroquine treated group (80.6%, 25 of 31) compared with the control group (54.8%, 17 of 31). Moreover, 61.3% of patients in the former group had a significant pneumonia resolution. While adverse events occurred among two (6.4%) of the hydroxychloroquine treated patients, none of the patients in the control group experienced them 68. However, the methodological limitations of this study preclude firm evidence of hydroxychloroquine efficacy and safety.
Differences exist in chloroquine and hydroxychloroquine dosage regimen between national guidelines. National Health Commission of the People’s Republic of China issued “Guidelines for the Prevention, Diagnosis, and Treatment of Pneumonia Caused by COVID-19” and

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recommended dosage and duration of chloroquine treatment (Table 2) 52. The suggested course of chloroquine treatment ranges from a minimum of five days to a maximum of ten days (Table 2) 52,
63. Dutch Centre for Disease Control proposed chloroquine regimen in adults for up to five days, while guidelines by the Italian Society of Infectious and Tropical disease (Lombardy section) recommended ten days (Table 2) 62. Kapoor et al. suggested a dosage regimen of hydroxychloroquine for up to seven days (Table 2) 63.

Chloroquine and hydroxychloroquine therapy were suggested for the treatment of a broad spectrum of COVID-19 severity ranging from mild respiratory symptoms in patients with comorbidities to severe respiratory failure 62. However, according to Infectious Diseases Society of America Guidelines on the Treatment and Management of Patients with COVID-19, hydroxychloroquine/chloroquine should only be used in the context of a clinical trial due to the knowledge gap 39. The same applies to the combination of hydroxychloroquine/chloroquine and azithromycin 39. This is supported by the statement of the US National Institute of Health “The COVID-19 Treatment Guidelines” that insufficient clinical data prevents for or against the recommendation of chloroquine or hydroxychloroquine for the treatment of COVID-19 (Rating of Recommendation grade/Levels of evidence –AIII) 69. The same body recommends against using high-dose chloroquine (600 mg twice daily for ten days) for the treatment of COVID-19 (AI) 69.

Since studies have documented serious dysrhythmias in patients with COVID-19 treated with chloroquine or hydroxychloroquine, especially in combination with azitromycine, authorities of the FDA strongly recommend use of chloroquine or hydroxychloroquine in hospital settings and for the propose of clinical trials 70. When chloroquine or hydroxychloroquine is used, patients should be monitored for adverse effects, especially prolonged QTc interval (A, III) 71. European Medicines Agency (EMA) also considers the use of chloroquine and hydroxychloroquine only in clinical trials or emergency programs 71. Outside clinical trials, it should be prescribed per nationally established protocols. The United Kingdom Commission on Human Medicines (CHM) advises ministers on the safety, efficacy, and quality of medicinal products released the same recommendation as EMA 69. The latest issue of Interim clinical guidelines for adults with suspected or confirmed COVID-19 in Belgium states that it was decided not to recommend hydroxychloroquine off-label use for COVID-19 in this country anymore, except within ongoing clinical registered trials after careful reassessment of the study-related risk/benefit 40.

Significant concerns were raised after publication of the analysis of extensive multinational registry 96,000 admitted COVID-19 patients, the vast majority with mild disease, including about

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15,000 exposed to chloroquine or hydroxychloroquine, alone or in combination with macrolide 72. This study did not find any benefit in the groups treated with these drugs, after adjustment, and even found increased mortality and higher frequency of ventricular arrhythmia. These data attracted so much attention that the Executive group of Solidarity Trial, an international clinical trial dedicated to an effective treatment search for COVID-19, launched by the WHO and partners, temporarily paused the hydroxychloroquine use, during the safety data review by the Data Safety Monitoring Board 73. Then, suddenly due to the unavailability to provide sufficient data to the third party peer review, which raised a question about data accuracy and validity, the authors retracted the article 72. This added even more confusion in the medical community in an already stressful situation as doctors worldwide are struggling to save the lives of millions of patients affected with COVID-19.

The Data Monitoring Committee of the Randomised Evaluation of COVid-19 thERapY (RECOVERY) Trial on hydroxychloroquine, stated that the trial would continue recruitment since mortality reported in the analysis by Mehra were not consistent with those observed in the RECOVERY trial 74. RECOVERY Trial is a large, randomized trial of treatments for patients admitted to hospitals with COVID-19 75. Over 10,000 patients have been randomized to the following arms: Lopinavir-Ritonavir, Low-dose Dexamethasone, Hydroxychloroquine, Azithromycin, and Tocilizumab, or no additional treatment 74. On the other hand, on Tuesday 27th May, French public health agency advised against using hydroxychloroquine outside of clinical trials 76. Shortly after that, the national medicines regulator suspended its use in clinical trials due to the safety concerns 76, 77. Recently, the National Institutes of Health (NIH) stopped study known as ORCHID (The Outcomes Related to COVID-19 treated with hydroxychloroquine among In- patients with symptomatic Disease study) 78. NIH based the decision on results that showed no beneficial effects of hydroxychloroquine for the treatment of COVID-19 in hospitalized patients

Chloroquine and hydroxychloroquine use for COVID-19 treatment are controversial. The results from the double-blind, randomized clinical trials, including a large number of participants, will add better insight into the role of these two drugs as already available and affordable antimalarial therapy.The risk of serious adverse events associated with chloroquine and hydroxychloroquine was recently reanalyzed within the pharmacovigilance data from the EudraVigilance Database of the EMA 40. Across Europe, 182 cases of QTc prolongation have been reported with

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hydroxychloroquine since the beginning of the epidemic, mostly if used in high dosages and/or in combination with the antibiotic azithromycin or other drugs known to prolong the QTc interval 40. Up to date, the QTc-prolonging potential of chloroquine was estimated after a single drug dose since its different posology in malaria treatment 42. The first study involving a large number of patients 79, which examined the QTc-prolonging potential of chloroquine in the context of COVID-19 treatment, indicated that this drug could significantly prolong the QTc interval in a clinically relevant manner 79. In this study, the QTc interval was measured after multiple doses of chloroquine during 48 hours 79. Since chloroquine has a substantial volume of distribution, and long half-life, the QTc interval prolongation risk persists for days after discontinuation of therapy for a prolonged period.

4. The ethical issue on emergency use of chloroquine and hydroxychloroquine

Based on the above-discussed data, the ethical question is whether administration of chloroquine and hydroxychloroquine in the settings of COVID-19 is experimental and requires ethical committee approval or an off-label position ethically justifiable as the best available treatment despite its registration status. The current epidemiological situation justifies the prioritization of ethical review of study proposals for fast track institutional ethical review. Namely, according to WHO guidance, the use of experimental interventions under the emergency use in infectious disease outbreak is referred to as “monitored emergency use of unregistered and experimental interventions” (MEURI) 82. Experimental interventions on an emergency basis outside clinical trials should be provided in several situations like nonexistent proven effective treatment; inability to start clinical studies immediately; data support the efficacy and safety of the drug, at least from laboratory or animal studies 82. Additionally, an appropriately qualified scientific committee can suggest drugs use outside clinical trials approved by relevant country authorities or suitably qualified ethical committees 82. In the countries where clinical trials are conducted, it is a moral obligation to refer patients to the hospital with an active clinical trial on chloroquine/hydroxychloroquine for COVID-19 treatment 83.

Potentially, the increased use of chloroquine/hydroxychloroquine can cause a shortage on the market and inaccessibility for standard indications as treatment of autoimmune disease 71. To prevent unnecessary strain on supply chains, patients should only receive their regular supply of drugs prescribed by their medical doctor. Also, each national drug agency should ensure sufficientAccepted Article
quantities of these drugs, primarily if no clinical trials are conducted in a country during an outbreak.

5. Conclusions

There is rationale from pre-clinical evidence of chloroquine and hydroxychloroquine effectiveness and their safety from long-time clinical use in other indications to justify clinical research of these drugs in patients with COVID-19. However, these drugs have been used in small randomized trials, case series and clinical trials with conflicting study reports. Therefore, data from high- quality, coordinated, multicentric clinical trials are urgently needed to establish the position of chloroquine and hydroxychloroquine in COVID-19 infection treatment. The ethical issue on emergency use of chloroquine and hydroxychloroquine should be carefully managed, with adherence to the MEURI framework or be ethically approved as a trial, as stated by the WHO.


This work was supported in part by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Grant No 175014 and Grant No 175093).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Data availability statement
The authors confirm that the data supporting the findings of this study are available within the article.
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16. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395:497-506. https://doi.org/10.1016/S0140-6736(20)30183-5.
17. 17 World Health Organization. Novel Coronavirus (2019-nCoV) Situation Report – 8. January 28, 2020. Available from: https://www.who.int/docs/default- source/coronaviruse/situation-reports/20200128-sitrep-8-ncov- cleared.pdf?sfvrsn=8b671ce5_2. Accessed on June 20, 2020.
18. Liverpul drug interactions group. Interactions with experimental COVID-19 Therapies. March 16, 2020. Available from: https://www.sefq.es/_pdfs/CV19_19.pdf. Accessed on June 20, 2020.

19. Accepted Article
1. Medicines and Medical Devices Agency of Serbia. Summary of product characteristics: Aluvia®: lopinavir/ritonavir. May, 2019. Available from: 1. https://www.alims.gov.rs/ciril/files/lekovi/smpc/515-01-02931-18-001.pdf. Accessed on June 20, 2020.
20. Medicines and Medical Devices Agency of Serbia. Summary of product characteristics: Actemra®: tocilizumab. September, 2019. Available from: https://www.alims.gov.rs/ciril/files/lekovi/smpc/515-01-02510-19-001.pdf. Accessed on June 20, 2020.
21. Medicines and Medical Devices Agency of Serbia. Summary of product characteristics: Copegus®: ribavirin. May, 2019. Available from: https://www.alims.gov.rs/ciril/files/lekovi/smpc/515-01-01427-18-001.pdf. Accessed on June 20, 2020.
22. ClinicalTrials.gov. Tocilizumab in COVID-19 Pneumonia (TOCIVID-19). Available from: https://clinicaltrials.gov/ct2/show/NCT04317092. Accessed on June 29, 2020.
23. Drugs.com. Atazanavir Side Effects. Last updated on May 24, 2019. Available from: https://www.drugs.com/sfx/atazanavir-side-effects.html. Accessed on June 29, 2020.
24. Drugs.com. Hydroxychloroquine. Last updated on April 23, 2020. Available from: https://www.drugs.com/ppa/hydroxychloroquine.html. Accessed on June 29, 2020.
25. Drugs.com Chloroquine Side Effects. Last updated on July 30, 2019. Available from: https://www.drugs.com/sfx/chloroquine-side-effects.html. Accessed on June 29, 2020.
26. European Medicines Agency. Summary of product characteristics: IntronA: recombinant interferon alfa-2b. Available from: https://www.ema.europa.eu/en/documents/product- information/introna-epar-product-information_en.pdf. Accessed on June 29, 2020.
27. Bergin C, Philbin M, Gilvarry P, O’Connor M, King F. Protocol: Specific Antiviral Therapy in the Clinical Management of Acute Respiratory Infection with SARSCoV-2 (COVID-19). Last updated on April 30, 2020. Available from: https://www.hse.ie/eng/about/who/acute-hospitals-division/drugs-management- programme/guidelines/specific-antiviral-therapy-in-the-clinical-management-of-acute- respiratory-infection-with-sars-cov-2-covid-19.pdf. Accessed on June 29, 2020.
28. European Medicines Agency. Summary of product characteristics: Ribavirin Teva Pharma B.V: ribavirin. Available from: https://www.ema.europa.eu/en/documents/product-
Accepted Article
information/ribavirin-teva-pharma-bv-epar-product-information_en.pdf. Accessed on June 27, 2020.
29. European Medicines Agency. Summary of product characteristics: Reyataz: atazanavir. Available from: https://www.ema.europa.eu/en/documents/product-information/reyataz- epar-product-information_en.pdf. Accessed on June 27, 2020.
30. Van Ierssel S, Dauby N, Bottieau E. Interim Clinical Guidance for adults with suspected or confirmed covid-19 in Belgium. April 7, 2020. Version 7. Available from: https://covid- 19.sciensano.be/sites/default/files/Covid19/COVID- 19_InterimGuidelines_Treatment_ENG.pdf. Accessed on May 20, 2020.
31. Smith T, Bushek J, LeClaire A, Prosser T. COVID-19 Drug Therapy. Last updated on June 17, 2020. Available from: https://www.elsevier.com/ data/assets/pdf_file/0007/988648/COVID-19-Drug- Therapy_2020-6-17.pdf. Accessed on May 20, 2020.
32. Instruction for medical use of arbidol. Available from: http://www.arbidol.org/dosage.pdf. Accessed on May 20, 2020.
33. Zhang C, Wu Z, Li JW, Zhao H, Wang GQ. Cytokine release syndrome in severe COVID- 19: interleukin-6 receptor antagonist tocilizumab may be the key to reduce mortality. Int J Antimicrob Agents. 2020;55:105954. https://doi.org/10.1016/j.ijantimicag.2020.105954.
34. Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, et al. Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020;30:269-71. https://doi.org/10.1038/s41422-020-0282-0.
35. Kadam RU, Wilson IA. Structural basis of influenza virus fusion inhibition by the antiviral drug Arbidol. Proc Natl Acad Sci U S A. 2017;114:206-14. https://doi.org/10.1073/pnas.1617020114.
36. Warren TK, Jordan R, Lo MK, Ray AS, Mackman RL, Soloveva V, et al. Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Version 2. Nature. 2016;531:381-5. https://doi.org/10.1038/nature17180.
37. Sanders JM, Monogue ML, Jodlowski TZ, Cutrell JB. Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19): A Review. JAMA. 2020. https://doi.org/10.1001/jama.2020.6019.
38. Ministry of Health Serbia. Diagnosis & Treatment Protocol for COVID-19. March, 2020. [Internal Document].
39. Accepted Article
1. Bhimraj A, Morgan RL, Shumaker AH, Lavergne V, Baden L, Cheng VC, et al. Infectious Diseases Society of America Guidelines on the Treatment and Management of Patients with COVID-19. Clin Infect Dis. 2020:ciaa478. 1. https://doi.org/10.1093/cid/ciaa478.
40. Van Ierssel S, Dauby N, Bottieau E. Interim Clinical Guidance for adults with suspected or confirmed covid-19 in Belgium. May 26, 2020. Version 9. Available from: https://covid- 19.sciensano.be/sites/default/files/Covid19/COVID- 19_InterimGuidelines_Treatment_ENG.pdf. Accessed on May 30, 2020.
41. National Institutes of Health: COVID-19 Treatment Guidelines. Coronavirus Disease 2019 (COVID-19) Treatment Guidelines. Last updated on June 25, 2020. Available from: https://www.covid19treatmentguidelines.nih.gov/. Accessed on June 30, 2020.
42. Vinetz JM. Chemotherapy of malaria. In: Brunton LL, editor. Goodman and Gilman’s Pharmacological basis of therapeutics. Thirteenth edition. New York: McGraw-Hill Education; 2018, p. 969-86.
43. Fox RI. Mechanism of action of hydroxychloroquine as an antirheumatic drug. Semin Arthritis Rheum. 1993;23:82-91. https://doi.org/10.1016/s0049-0172(10)80012-5.
44. Savarino A, Boelaert JR, Cassone A, Majori G, Cauda R. Effects of chloroquine on viral infections: an old drug against today’s diseases? Lancet Infect Dis. 2003;3:722-7. https://doi.org/10.1016/s1473-3099(03)00806-5.
45. Blau D, Holmes K. Human Coronavirus HCoV-229E enters susceptible cells via the endocytic pathway. In: The Nidoviruses, Coronaviruses and Arteriviruses. Lavi E, editor. New York: Kluwer; 2001, p. 193-7.
46. Nauwynck HJ, Duan X, Favoreel HW, Van Oostveldt P, Pensaert MB. Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis. J Gen Virol. 1999;80:297-305. https://doi.org/10.1099/0022-1317-80-2-297.
47. Vincent MJ, Bergeron E, Benjannet S, Erickson BR, Rollin PE, Ksiazek TG, et al. Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol J. 2005;2:69. https://doi.org/10.1186/1743-422X-2-69.
48. Keyaerts E, Vijgen L, Maes P, Neyts J, Van Ranst M. In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine. Biochem Biophys Res Commun. 2004;323:264-8. https://doi.org/10.1016/j.bbrc.2004.08.085.

49. Accepted Article
1. Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 2003;426:450-4. 1. https://doi.org/10.1038/nature02145.
50. Xu X, Chen P, Wang J, Feng J, Zhou H, Li X, et al. Evolution of the novel corona virus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. Sci China Life Sci 2020;63:457-60. https://doi.org/10.1007/s11427-020- 1637-5.
51. Mackenzie AH. Dose refinements in long-term therapy of rheumatoid arthritis with antimalarials. Am J Med. 1983;75:40-5. https://doi.org/10.1016/0002-9343(83)91269-x.
52. Yao X, Ye F, Zhang M, Cui C, Huang B, Niu P, et al. In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Clin Infect Dis. 2020:ciaa237. https://doi.org/10.1093/cid/ciaa237.
53. McChesney EW. Animal toxicity and pharmacokinetics of hydroxychloroquine sulfate. Am J Med. 1983;75:11-8. https://doi.org/10.1016/0002-9343(83)91265-2.
54. McChesney EW, Banks WF Jr, Sullivan DJ. Metabolism of chloroquine and hydroxychloroquine in albino and pigmented rats. Toxicol Appl Pharmacol. 1965;7:627-
36. https://doi.org/10.1016/0041-008x(65)90050-5.
55. McChesney EW, McAuliff JP. Laboratory studies of the 4-aminoquinoline antimalarials. I Some biochemical characteristics of chloroquine, hydroxychloroquine and SN-7718. Antibiot Chemother. 1961;11:800-10.
56. Singh AK, Singh A, Shaikh A, Singh R, Misra A. Chloroquine and hydroxychloroquine in the treatment of COVID-19 with or without diabetes: A systematic search and a narrative review with a special reference to India and other developing countries. Diabetes Metab Syndr. 2020;14:241-6. https://doi.org/10.1016/j.dsx.2020.03.011.
57. Marmor MF, Kellner U, Lai TY, Melles RB, Mieler WF; American Academy of Ophthalmology. Recommendations on Screening for Chloroquine and Hydroxychloroquine Retinopathy (2016 Revision). Ophthalmology. 2016;123:1386-94. https://doi.org/10.1016/j.ophtha.2016.01.058.
58. Rainsford KD, Parke AL, Clifford-Rashotte M, Kean WF. Therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus

Accepted Article
erythematosus, rheumatoid arthritis and related diseases. Inflammopharmacology. 2015;23:231-69. https://doi.org/10.1007/s10787-015-0239-y.
59. Perinel S, Launay M, Botelho-Nevers É, Diconne É, Louf-Durier A, Lachand R, et al. Towards Optimization of Hydroxychloroquine Dosing in Intensive Care Unit COVID-19 Patients. Clin Infect Dis. 2020:ciaa394. https://doi.org/10.1093/cid/ciaa394.
60. Gao J, Tian Z, Yang X. Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies. Biosci Trends. 2020;14:72-3. https://doi.org/10.5582/bst.2020.01047.
61. Gautret P, Lagier JC, Parola P, Hoang VT, Meddeb L, Mailhe M, et al. Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open- label non-randomized clinical trial. Int J Antimicrob Agents. 2020:105949. https://doi.org/10.1016/j.ijantimicag.2020.105949.
62. Cortegiani A, Ingoglia G, Ippolito M, Giarratano A, Einav S. A systematic review on the efficacy and safety of chloroquine for the treatment of COVID-19. J Crit Care. 2020;57:279-83. https://doi.org/10.1016/j.jcrc.2020.03.005.
63. Kapoor KM, Kapoor A. Role of Chloroquine and Hydroxychloroquine in the Treatment of COVID-19 Infection- A Systematic Literature Review. https://doi.org/10.1101/2020.03.24.20042366. [MedRxiv- Preprint]. Available from: https://www.medrxiv.org/content/10.1101/2020.03.24.20042366v1. Accessed on June 30, 2020.
64. Multicenter collaboration group of Department of Science and Technology of Guangdong Province and Health Commission of Guangdong Province for chloroquine in the treatment of novel coronavirus pneumonia. [Expert consensus on chloroquine phosphate for the treatment of novel coronavirus pneumonia]. Zhonghua Jie He He Hu Xi Za Zhi. 2020;43:185-8. Chinese. https://doi.org/10.3760/cma.j.issn.1001-0939.2020.03.009.
65. Chen J, Liu D, Liu L, Liu P, Xu Q, Xia L, et al. [A pilot study of hydroxychloroquine in treatment of patients with moderate COVID-19]. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2020;49:215-9. Chinese.
66. Tang W, Cao Z, Han M, Wang Z, Chen J, Sun W, et al. Hydroxychloroquine in patients with mainly mild to moderate coronavirus disease 2019: open label, randomised controlled trial. BMJ. 2020;369:m1849. https://doi.org/10.1136/bmj.m1849.

67. Accepted Article
1. Borba MGS, Val FFA, Sampaio VS, Alexandre MAA, Melo GC, Brito M, et al. Effect of High vs Low Doses of Chloroquine Diphosphate as Adjunctive Therapy for Patients Hospitalized With Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection: A Randomized Clinical Trial. JAMA Netw Open. 2020;3:e208857. https://doi.org/10.1001/jamanetworkopen.2020.8857.
68. Chen Z, Hu J, Zhang Z, Jiang S, Han S, Yan D, et al. Efficacy of hydroxychloroquine in patients with COVID-19: results of a randomized clinical trial. 2020. [MedRxiv- Preprint]. Available from: https://www.medrxiv.org/content/10.1101/2020.03.22.20040758v3. Accessed on June 28, 2020.
69. Medicines & Healthcare products Regulatory Agency. Chloroquine and Hydroxychloroquine not licensed for coronavirus (COVID-19) treatment. March 25, 2020. Available from: https://www.gov.uk/government/news/chloroquine-and- hydroxychloroquine-not-licensed-for-coronavirus-covid-19-treatment. Accessed on May 30, 2020.
70. U.S. Food and Drug Administration. FDA cautions against use of hydroxychloroquine or chloroquine for COVID-19 outside of the hospital setting or a clinical trial due to risk of heart rhythm problems. Available from: https://www.fda.gov/drugs/drug-safety-and- availability/fda-cautions-against-use-hydroxychloroquine-or-chloroquine-covid-19- outside-hospital-setting-or. Accessed on May 30, 2020.
71. European Medicines Agency. Science Medical Health. COVID-19: chloroquine and hydroxychloroquine only to be used in clinical trials or emergency use programmes. April 1, 2020. Available from: https://www.ema.europa.eu/en/news/covid-19-chloroquine- hydroxychloroquine-only-be-used-clinical-trials-emergency-use-programmes. Accessed on May 30, 2020.
72. Mehra MR, Desai SS, Ruschitzka F, Patel AN. RETRACTED: Hydroxychloroquine or chloroquine with or without a macrolide for treatment of COVID-19: a multinational registry analysis. Lancet. 2020:S0140-6736(20)31180-6. https://doi.org/10.1016/S0140- 6736(20)31180-6. Retraction in: Lancet. 2020;null.
73. World Health Organization. “Solidarity” clinical trial for COVID-19 treatments. May 27, 2020. Available from: https://www.who.int/emergencies/diseases/novel-coronavirus- 2019/global-research-on-novel-coronavirus-2019-ncov/solidarity-clinical-trial-for-covid- 19-treatments. Accessed on May 30, 2020.

74. Accepted Article
1. RECOVERY Randomized evaluation of COVID-19 Therapy. Recruitment to the RECOVERY trial continues as planned. May 26, 2020. Available from: 1. https://www.recoverytrial.net/news/recruitment-to-the-recovery-trial-continues-as-planned. Accessed on May 30, 2020.
75. RECOVERY Randomized evaluation of COVID-19 Therapy. Randomized Evaluation of COVID 19 therapy. Available from: https://www.recoverytrial.net/files/recovery-protocol- v6-0-2020-05-14.pdf. Accessed on June 15, 2020.
76. POLITICO. France bans use of hydroxychloroquine as coronavirus treatment. May 27, 2020. Available from: https://www.politico.com/news/2020/05/27/france-bans-use-of- hydroxychloroquine-to-cure-coronavirus-283724. Accessed on June 15, 2020.
77. COVID-19: l’ANSM souhaite suspendre par précaution les essais cliniques évaluant l’hydroxychloroquine dans la prise en charge des patients – Point d’Information. May 26, 2020. Available from: https://www.ansm.sante.fr/S-informer/Points-d-information-Points- d-information/COVID-19-l-ANSM-souhaite-suspendre-par-precaution-les-essais- cliniques-evaluant-l-hydroxychloroquine-dans-la-prise-en-charge-des-patients-Point-d- Information. Accessed on June 1, 2020.
78. National Institutes of Health. NIH halts clinical trial of hydroxychloroquine. June 20, 2020. Available from: https://www.nih.gov/news-events/news-releases/nih-halts-clinical-trial- hydroxychloroquine. Accessed on June 22, 2020.
79. van den Broek MPH, Möhlmann JE, Abeln BGS, Liebregts M, van Dijk VF, van de Garde EMW. Chloroquine-induced QTc prolongation in COVID-19 patients. Neth Heart J. 2020:1-4. https://doi.org/10.1007/s12471-020-01429-7.
80. Geleris J, Sun Y, Platt J, Zucker J, Baldwin M, Hripcsak G, et al. Observational Study of Hydroxychloroquine in Hospitalized Patients with Covid-19. N Engl J Med. 2020;382:2411-8. https://doi.org/10.1056/NEJMoa2012410.
81. Boulware DR, Pullen MF, Bangdiwala AS, Pastick KA, Lofgren SM, Okafor EC, et al. A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19. N Engl J Med. 2020:NEJMoa2016638. https://doi.org/10.1056/NEJMoa2016638.
82. World Health Organization. Guidance for managing ethical issues in infectious disease outbreaks. Spain: World Health Organization; 2016. Available from: https://apps.who.int/iris/handle/10665/250580. Accessed on May 30, 2020.
83. Accepted Article
1. Yazdany J, Kim AHJ. Use of Hydroxychloroquine and Chloroquine During the COVID-19 Pandemic: What Every Clinician Should Know. Ann Intern Med. 2020;172:754-5. 1. https://doi.org/10.7326/M20-1334.