Repurposing of Chloroquine and Its Derivative, Hydroxychloroquine for COVID-19: Implications for People Living with HIV in Africa

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2, has been declared by the World Health Organization (WHO) as a pandemic. Unfortunately, finding a vaccine or developing drugs from the scratch is a timeconsuming luxury given the widespread and high fatality rates of the virus. In the short term, repurposing of drugs already in use seem to be the most rational step to quickly and effectively curb the virus. Several antiviral agents had been proposed as possible remedies, but the 4-aminoquinolines, Chloroquine (CHQ) and hydroxychloroquine (HCHQ) appear to be generating more interest. They are generic, cheaply available and have proven efficacy against malaria parasites in Africa. The human immunodeficiency virus (HIV), on the other hand, targets the immune system thereby reducing the patient’s ability to fight infections. Sadly, 68% of the global HIV burden occur in Africa. It is therefore anticipated that incidence of severe forms of COVID-19 could occur in Africa because of associated endemic conditions that Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 22 April 2020 doi:10.20944/preprints202004.0395.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license. compromise the immune system. With CHQ and HCHQ being considered for clinical use against COVID-19, there is a need to highlight their potential merits and confounding variables in the subgroup of patients with or without HIV.

reporting of cases in the continent and preclude effective surveillance and monitoring [3]. Evidence emanating from the demographics of patients infected with COVID-19 in Wuhan, China indicated that the elderly and patients with comorbid conditions such as hypertension, diabetes, cardiovascular disease, or chronic lung disease were at higher risks of severe outcomes, including death [4][5]. Presently, there is no data on the effect of COVID-19 infection on HIV immune-compromised patients, but it is reasonable to speculate that this sub-group may require special clinical care. As at the time of this report (10 April 2020) there was just a single study on a patient presenting with a co-infection of COVID-19 and HIV in Wuhan, China [6]. Contrariwise, HIV-infected patients receiving standard anti-HIV drugs might not have increased risk for COVID-19, as was the case in Thailand [7]. However, a nation-wide community survey of people living with HIV in China buttressed the need for HIV-specific protective measures during this outbreak [8]. The comprehensive case reports from other high incidence countries such as the USA, Italy and Spain are still being awaited. In the interim, severe pneumonia-like symptoms, caused by the Middle East Respiratory Syndrome Coronavirus (MERS CoV), were observed in a HIV patient [9]. (Shalhoub et al, 2015).
To underscore the need for further evaluation of the association between HIV and COVID-19, a fatal case was reported in a patient presenting with influenza A H5N1 and HIV co-infection [10]. While the search for anti-COVID-19 drugs is ongoing, repurposing of drugs previously used to treat other diseases could be the best short-term solution. A set of guidelines released by the WHO, the Center for Disease Control and Prevention, and the United States Food and Drug Administration have proposed several potential drugs to treat COVID-19 infections. These drugs (with their mechanisms of action) included darunavir, lopinavir, ritonavir, imatinib (inhibit key enzymes involved in virus replication); interferon-α (blocks viral replication); ribavirin (blocks viral mRNA synthesis); remdesivir (inhibits RNAdependent RNA polymerases); azithromycin and tocilizumab (suppress cytokine production), CHQ and HCHQ (inhibit ACE 2 cellular receptors and viral DNA/RNA polymerases [11][12]. CHQ and HCHQ are cheaply available in Africa. The pharmacokinetic and pharmacodynamic profiles of CHQ had been studied extensively, including as an immuno-modulator during HIV infection [13]. This review discusses the anti-viral activities of CHQ and HCHQ and the variables of their use in the treatment of patients with COVID-19, with or without an underlying HIV infection.

The evolvement of CHQ and HCHQ as anti-viral agents
Chloroquine (CHQ), chemically designated as an N4-(7-Chloro-4-quinolinyl)-N1, N1-diethyl-1, 4-pentanediamine, had been in use as an antimalarial drug for several decades. Hydroxychloroquine (HCHQ), with similar pharmacokinetic profile as CHQ, has a β-hydroxyl group attached to the N-di-ethyl substituent of CHQ leading to their slightly different clinical indications. The consideration of CHQ and HCHQ as antiviral agents against the coronavirus 2019 is an example of drug repurposing, a strategy that deploys already existing drugs for new medical indications. Up till the year 2000, CHQ remained the first-line therapy for uncomplicated malaria in sub-Saharan countries. Its use waned as a result of the development of resistance by Plasmodium falciparum strains. Incidentally, CHQ use in sub-Saharan Africa never abated as it had remained accessible to the general population [14][15].
Earlier studies indicated the inhibitory effect of CHQ on the growth of mouse hepatitis virus and other animal viruses such as polio type 1, influenza, Newcastle disease [16][17]. In a specific study, chick embryo cells were exposed to vesicular stomatitis virus and treated with CHQ (12.5 µg/ml), resulting in depleted viral yield [17]. In the past three decades, Africa had been plagued by Lassa fever, Ebola virus, Marburg virus, Rift Valley fever, and the Crimean-Congo hemorrhagic virus (CCHFV) [18]. In the course of the CCHFV epidemic in Africa, researchers proposed the repurposing of CHQ to treat the virus. In a study utilizing Vero E6 and Huh 7 cell lines, the 50% inhibitory concentration values for CHQ against two different CCHFV strains ranged from 28 to 43 μM. Several combinations, including CHQ and ribavirin, also showed synergistic effect against CCHFV [19]. The use of CHQ as an anti-viral agent gained prominence against the Ebola virus, a filovirus that had proved to be re-emerging in sub-Saharan Africa [20]. In an in vitro study in MRC-5 human cell lines, CHQ at the concentrations of 4.7 and 0.47 μM, significantly reduced the cycle threshold of viral RNA levels following post-infection with the Ebola virus. The anti-viral effects of CHQ were linked to its ability to limit the acidification of the endosomes [21]. In yet another study, MT-4 cells infected with X4, R5 or R5/X4 HIV-1 strains from clades A-E and HIV-2 were treated with CHQ concentrations ranging from 0 -12.5 μM. Results showed that CHQ, at the tested concentrations, blocked the HIV-1 post-integration by affecting newly produced viral envelope glycoproteins [22]. Similar successes had been achieved using CHQ against Chikungunya infection in Vero E6 cells. Consistent with the results obtained with MT-4 cells infected with the Ebola virus, the preponderant mechanism of the anti-viral action of CHQ in Vero E6 cells infected with the Chikungunya virus was the modification of the endosomal pH [23]. An earlier review had hypothesized that CHQ could exert direct antiviral effect against coronaviruses by inhibiting their pH-dependent replications [24]. A study demonstrating the inhibitory effect of CHQ against the replication of human coronaviruses strain OC43 corroborated this hypothesis [25]. These workers reported that CHQ inhibited HCoV-OC43 replication in HRT-18 cells, with a 50% effective concentration of 0.306 μM. In a more recent study, CHQ and HCHQ inhibited SARS-CoV-2 infected Vero cells with 50% effective concentrations of 5.47 and 0.72 μM, respectively [26]. Despite its potential, the deployment of CHQ as a treatment strategy for viral infections has been dampened by several in vivo studies indicating a lack of protection. At a dose of 33.75 mg/kg, CHQ administered per os for 8 days, afforded no protection in guinea pigs exposed to the Ebola virus [21]. This is in agreement with another study that showed that the therapeutic intervention with CHQ resulted in toxicity and did not extend the survival of rodents infected with the Ebola virus [27). In clinical trials, the shadow of doubt on the efficacy of CHQ as an antiviral molecule had persisted. In a randomized control trial, 54 patients infected with the Chikungunya virus received 600 mg of CHQ per day for three days and an additional dose of 300 mg for another two days. Results revealed that CHQ treatment did not affect viremia or clinical parameters during the acute stage of the disease [23]. Following the outbreak of COVID-19 in December 2019, anecdotal reports of the antiviral efficacy of CHQ and HCHQ resurfaced. In the face of the global pandemic, the first documented clinical trial of CHQ in COVID-19 infected patients was quickly conducted in China. In a multi-center clinical trial involving over 100 patients, CHQ inhibited COVID-19 associated pneumonia and also improved clearance of virus from the lungs [28]. Another early clinical study, a single-arm protocol human study, was conducted with HCHQ in France. Findings from this study revealed that HCHQ alone and in combination with azithromycin was highly effective in clearing viral nasopharyngeal carriage within six days in COVID-19 subjects [29].
Although these two clinical studies used small sample sizes and not-too-robust protocols, they served as baseline for other studies. For example, the synergistic effect on viremia observed with the combination therapy of HCHQ with azithromycin could be the basis for an ongoing clinical trial in China using CHQ and favipiravir. Currently, there are ongoing clinical intervention studies in several countries testing the efficacy of CHQ and HCHQ as drug candidates against COVID-19 infection. A simple search in two registries, the US-based clinical trials.gov and the Chinese Clinical Trial Register using keywords such as COVID-19, Coronavirus, (Hydroxy) Chloroquine yielded over twenty-three entries from fifteen different countries (Table 1).

COVID-I9 proteins as therapeutic targets for CHQ and HCHQ
The synthesis of proteins and their maturation are necessary for the replication of viruses. These steps could be targeted in the development of antiviral agents [30]. Viral proteins could be classified as structural, nonstructural, regulatory or accessory. Anti-HIV drugs targeting many of the HIV viral proteins and enzymes had been produced. Drugs blocking the reverse transcriptase (Zidovudine, Stavudine, Nevirapine, Etravirine), protease (Saquinavir, Ritonavir, Darunavir) or the integrase (Raltegravir, Dolutegravir) had led to a significant increase in life expectancy of people living with HIV in Africa [31][32].
As of 6 March 2020, a landmark breakthrough was achieved with a report of the first whole genomic sequencing of COVID-19 in Africa.
This research was conducted at the African Centre of Excellence for the Genomics of Infectious Disease (ACEGID), Redeemer's University, Ede, Nigeria in collaboration with the Nigerian Institute of Medical Research [33]. Interestingly, the length of this fully sequenced genome was 29759 bp, comparable with that of SARS-COV (≈29700 bp) [34]. The experience gained from elucidating the mechanisms of action of anti-HIV drugs targeting viral enzymes could prove invaluable in unraveling potential targets to suppress the replication of COVID-19.
Other structural and accessory proteins of COVID-19 and HIV-1 are summarized in Table 2.  [24,26,37,38]. Some of these mechanisms included: inhibition of glycosylation at the virus receptor ACE 2, stabilization of lysosomal membranes, inhibition of new virus particle transport, immunomodulation of cytokines, and inhibition of viral polymerases. CHQ had been reported to reduce viral infectivity in HIV-1 experimental models without interfering with the HIV enzymes, reverse transcriptase, and integrase. This lack of interference had prompted the testing of CHQ and HCHQ in combination with antiretrovirals in clinical intervention studies [39]. It is reasonable to

Repurposing CHQ and HCHQ as antiviral drugs: knotty issues in the African setting
Despite several studies describing the molecular mechanisms of the anti-viral effects of CHQ and HCHQ, a number of issues remain unsolved. One major factor is the paucity of safety data on CHQ and HCHQ, obtained from human studies over a period of time. The majority of the studies addressing the antiviral effects of the 4-aminoquinolines had relied on in vitro studies that were unable to fully capture their biological responses in human disease conditions. Hence, the therapeutic utility of CHQ and HCHQ, and their appropriate clinical doses, in the treatment of COVID-19 needs to be established from the ongoing clinical trials. This will also put to rest the discordant tunes emanating from different regulatory bodies and medical associations. An editorial published in The BMJ on 8 April 2020 cautioned against the use of CHQ and HCHQ for the treatment of COVID-19 because the antimalarial drugs were effective in laboratory experiments but lacked robust data from clinical settings [40]. A news release also reported the cancellation of trials involving CHQ as an anti-COVID-19 drug. Patients had complained of severe headaches, loss of vision and cramps [41]. In the not too distant future, the University of Oxford-anchored Randomised Evaluation of COVID-19 Therapy (Recovery Trial) using HCHQ, and other ongoing studies across the globe, would provide better and properly powered clinical trials that would determine the fate of CHQ and HCHQ as therapeutic interventions against COVID-19. In Africa, where CHQ presents a cheap option to treat the coronavirus 2019, its potential to cause adverse effects in people living with other underlying medical conditions is a source of worry. For example, people with HIV, who are already on lopinavir and ritonavir therapy, might suffer toxicity when exposed to CHQ and HCHQ. Lopinavir and ritonavir are inhibitors of CYP3A, a member of the Cytochrome P450 superfamily that mediates CHQ metabolism. Co-administration of these antiretroviral drugs with CHQ increases the plasma concentration of CHQ due to inhibition of the drug-metabolizing machinery of cytochrome P450. Cardiac arrhythmias are major public health issues in sub-Saharan Africa [42], but drugs such as Amiodarone, Bepridil and Flecainide, used to manage these conditions, are contra-indicated against CHQ and HCHQ (Table 4)  infected patients should be considered. Previous studies had reported CHQ-induced oxidative stress and genotoxicity in the liver of rodents [46][47]. It is plausible that the CHQ and HCHQ regimen being proposed as therapeutic interventions could exacerbate oxidative damage in susceptible organs such as the lungs, kidney, and liver.
Despite the spate of research and publications on COVID-19, researchers are uncertain whether the disease will ultimately cease or remain a deadly infectious disease. Whichever scenario that eventually pans out, the potential for COVID-19 infection in children could become a public health concern in Africa. During the MERS-COV and SARS-COV epidemics, few pediatric cases were observed. This trend, which has continued with COVID-19, gives a false impression that children are not susceptible to the infection. But, a current case series conducted in Shenzhen, China, showed that children presented with COVID-19 symptoms ranging from asymptomatic to mild [48]. Other studies suggested that the levels of expression of ACE2 receptors were not age-discriminatory, meaning that children could equally be at risk as adults [49][50]. The portent for Africa is that asymptomatic or mildly symptomatic children could escalate community transmission of the disease. Glucose-6-phosphate dehydrogenase deficiency (G6PDH) has been implicated as one of the causes of jaundice in children in sub-Saharan Africa. Unfortunately, CHQ and HCHQ are pro-oxidant drugs and their use in children with G6PDH could distort the redox status in red blood cells leading to hemolysis and other potentially fatal conditions. Invariably, policymakers must weigh the pros and cons of using CHQ and HCHQ as a therapeutic drugs for COVID-19 before endorsing their widespread use in Africa.

Beyond CHQ and HCHQ repurposing: The African experience in managing COVID-19
During the COVID-19 pandemic, the European Centre for Disease Prevention and Control (ECDC) published data from all continents of the world. The COVID-19 epidemiological profiles of the ten most populous countries in Africa were obtained from the ECDC on 10 April 2020. Analysis of the data showed that the two North African countries Egypt and Algeria contributed 27.4 % of the total confirmed cases with a combined mortality of 22.1%. Incidence in South Africa was 15.7%, with a mortality rate of 0.93%. The percentage infections and deaths in the East African countries (Ethiopia, Kenya, Tanzania, and Uganda) were 2.6% and 11.5%, respectively. Nigeria had 2.4% of the total COVID-19 cases with a death rate of 2.4% (Table 5). The final aim of this literature review was to stimulate further research and trials on repurposing CHQ and HCHQ as anti-COVID drugs taking into consideration the peculiarities of Africans, especially those with underlying health conditions. The lack of clinical results on the safety and tolerability of CHQ and HCHQ signify that it is yet early times to make definite conclusions about their overall efficacy against COVID-19. Hopefully, the scientific community will see this pandemic as an opportunity to develop broad-spectrum vaccines against the coronaviruses and other pathogenic viruses.

Data Availability
All relevant data are embedded in the manuscript.

Conflicts of Interest
The author declares that there is no conflict of interest regarding the publication of this paper.

Funding
This research did not receive any funding from any organization.