Due to the rapid outbreak of the transmission (~3.64 million positive cases and high mortality as of 5 May 2020), the world is looking for immediate and better therapeutic options. Still, much information is not known, including origin of the disease, complete genomic characterization, mechanism of transmission dynamics, extent of spread, possible genetic predisposition, clinical and biological diagnosis, complete details of disease-induced pathogenicity, and possible therapeutic options. Although several known drugs are already under clinical evaluation with many in repositioning strategies, much attention has been paid to the aminoquinoline derivates, chloroquine (CQ) and hydroxychloroquine (HCQ). These molecules are known regulators of endosomes/lysosomes, which are subcellular organelles central to autophagy processes. By elevating the pH of acidic endosomes/lysosomes, CQ/HCQ inhibit the autophagic process. In this short perspective, we discuss the roles of CQ/HCQ in the treatment of COVID-19 patients and propose new ways of possible treatment for SARS-CoV-2 infection based on the molecules that selectivity target autophagy.
Coronaviridae Orthocoronavirinae Betacoronavirus Sarbecovirus (coronavirus [CoV]) causes infections (mild to severe) in both mammalian and avian species and comes under the category of positive-sense single-stranded RNA viruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also referred to as 2019-novel coronavirus (2019-nCoV), causes coronavirus disease 2019 (COVID-19). Among the CoV family, four viruses (229E, OC43, NL63, and HKU1) and two viruses (severe acute respiratory syndrome coronavirus [SARS-CoV] and Middle East respiratory syndrome-related coronavirus [MERS-CoV]) cause the cold, and severe respiratory diseases, respectively. Although the SARS-CoV-2 surface morphology is known, the complex structure and specific modifications are not documented. It is hypothesized that, like other recent coronaviruses (SARS-CoV and MERS-CoV), SARS-CoV-2 has spread to humans from wild animals (i.e., it is a zoonotic disease). Bats have been identified as a source for the SARS-CoV (bat-SL-CoVZC45, MG772933.1), which has a very high nucleotide similarity (96%) with SARS-CoV-2. In addition, pangolins are also suspected for the zoonotic transmission of this virus into humans; however, speculation of zoonotic transmission of SARS-CoV-2 requires further scientific validation. SARS-CoV-2 was first isolated from the bronchoalveolar-lavage fluids of Chinese adult patients. Later, a positive case was confirmed in the US with a history of recent travel to China. The clinical symptoms, especially signs of a severe pneumonia-like fever, cough and chest/breathing discomfort, have been observed, primarily 7 d after infection, but with symptoms appearing over a span of 2–14 d. SARS-CoV-2 infection in severe cases leads to pneumonia, pulmonary edema, lung damage, and failure of other vital organs, such as liver, kidney, and heart. Patients who are suffering from severe SARS-CoV-2 infection need supportive mechanical ventilator management in intensive care units, with a high risk of other complications resulting from the infection.
CQ and HCQ have long been used to treat malaria (HCQ was first synthesized in 1946). CQ and HCQ exert their anti-malarial activity by inhibiting hemozoin (polymerized crystalline heme) formation in the parasite food vacuoles, which are the main sources for the conversion of host hemoglobin into amino acid residues. Being an acidic environment, the food vacuole is a favorable site for CQ and HCQ entrapment, where they selectively bind to heme and prevent hemozoin formation. If the parasite is not converting the heme to hemozoin, the free heme is available for the oxidation and release of superoxide radicals. This oxidative stress damages the food vacuoles and, as a consequence, the parasite. Also, monobasic forms of CQ and HCQ display inhibitory activity on the heme polymerase enzyme, which helps in the polymerization of heme.
During the current COVID-19 pandemic, CQ and HCQ have been administered as first-line drugs in several studies. Although these clinical trials were done rapidly, in small cohorts of patients, often with no control groups, and were poorly controlled, a few have described some positive effects of CQ and HCQ in SARS-CoV-2-infected patients. A recent initial clinical trial in 100 COVID-19 patients has suggested the efficacy of CQ, which mitigates SARS-CoV-2-induced pneumonia. Another retrospective study involving 568 critically ill COVID-19 patients indicated that patients who received HCQ along with standard antiviral drugs and antibiotics had a significantly lower mortality rate. Furthermore, a multi-center observational study implied the usefulness of CQ (500 mg/once daily) without any significant toxicity for the treatment of COVID-19 [57]. Though other pre-clinical and clinical studies and a meta-analysis did not find clinical benefits of HCQ for COVID-19, the short-term use of CQ/HCQ as an adjunct therapy in COVID-19 infection still seems advisable, particularly during early phase, to block virus replication, shedding and community spread. A total of 148 clinical trials (by mid April 2020) have been planned on CQ/HCQ either alone or in combination for the treatment of COVID-19.
CQ/HCQ has been commonly used as a prominent tool to monitor autophagy via lysosomal acidification inhibition. For in vitro experiments, a dose range of 10–100 μM has been used for the inhibition of autophagy in various cells. For in vivo experiments in mice, a dose of 30–70 mg/kg body weight (not more than 3 doses/week) has been used. However, in the treatment of systemic lupus erythematosus and other autoimmune diseases, and depending on the severity of the disease, patients have received 200–400 mg of CQ/HCQ either by a single daily dose or two divided doses [63]. For the treatment of COVID-19, different clinical trials have explored CQ/HCQ at a dose range of 400–600 mg/d (not more than 10 g/14 d schedule). However, in view of well-known secondary effects, CQ/HCQ have to be used under strict medical vigilance. HCQ analogs with less toxicity are under development. Though inconclusive, based on data from the open-label trials that used combination of HCQ and azithromycin in COVID-19 patients (Zithromax), randomized clinical trials are underway that combine these two molecules. Of note, azithromycin also acts by preventing lysosomal acidification. It will also be of interest to explore other molecules, including peptides that directly affect endo-lysosomes.
Being an autophagy inhibitor, CQ/HCQ might also block the entry (endocytosis), uncoating and exit (exocytosis) process of SARS-CoV-2. During endocytosis and exocytosis, cells produce danger signals that eventually activate the bystander innate immune cells. Moreover, inhibition of autophagy also leads to the reduction of recycled materials, which may serve as a nucleation material for SARS-CoV-2. Nevertheless, these propositions need to be strictly validated in dedicated experiments. It appears that all malaria-infected populations are partly immune to SARS-CoV-2.
In addition to its effect on lysosomal pH, CQ exhibits broad-spectrum antiviral and immunomodulatory properties, which might work in a synergistic manner to benefit COVID-19 patients. CQ inhibits glycosylation of the ACE2 receptor, which is essential for the SARS-CoV-2 S protein to bind and infect the host cells. Computational studies have further determined that CQ and HCQ can interfere with S protein binding of gangliosides. Of note, inflammatory cytokines are responsible for lung damage in SARS-CoV-2 infection. Interestingly, CQ inhibits inflammatory cytokines at the transcription level, independent of the lysosome. CQ/HCQ inhibits the activation of monocytes, macrophages, and T cells, and thereby reduces the release of pro-inflammatory cytokines (IL1B, IL6, TNF, IFNG, and others). However, a recent in vitro study performed in peripheral blood mononuclear cells has revealed that HCQ has minimal immunomodulatory activity; but it must be noted that this study was performed under non-stimulatory conditions and might not mimic inflammatory conditions observed in SARS-CoV-2 infection or other autoimmune and inflammatory diseases.
The therapeutic use of CQ/HCQ is strictly regulated due to their cardiovascular side effects. HCQ, although less toxic than CQ, still displays some well-known undesirable properties. The main concern is with regard to cardiomyopathy and heart failure, and the development of retinopathy due to HCQ affinity to melanin-containing cells, a secondary effect noticed in patients with lupus disease. Therefore, it is recommended that the daily dose of CQ/HCQ in autoimmune patients should not exceed 5 mg/kg/d. Cardiac adverse effects were also noticed in high-dose CQ (12 g for 10 d)-treated severely ill COVID-19 patients or those receiving a combination of HCQ and azithromycin.
Thus, irrespective of conflicting results in COVID-19 patients, many countries are using CQ/HCQ, either alone or in combination, as a first-choice treatment. Based on the current evidence, we speculate that the therapeutic outcome of CQ/HCQ in COVID-19 patients depends on many factors, such as pharmacokinetics (bioavailability), pharmacodynamics, severity of the disease, comorbidity, ethnicity, age and others. Heterogeneity in the treated COVID-19 patients with respect to severity, genetic background, and pre-existing comorbidities are the major confounding factors that contributed to conflicting results on the therapeutic use of CQ/HCQ. In addition, the dose of the drug also matters: many times, CQ with ≥ 500 mg/d (but not more than 600 mg/d) along with azithromycin have shown greater beneficial effects than lower doses. This notion is also supported by mathematical modeling.
Considering the present situation with COVID-19, efforts must be focused on fast-tracking drug development and using the rich knowledge gained during previous SARS-CoV and MERS-CoV outbreaks. Several FDA- or EMA-approved therapeutic molecules are available for treating patients with COVID-19. Based on the current knowledge with CQ/HCQ, molecules that target autophagy pathways might be useful to treat COVID-19 patients. However, randomized controlled clinical trials are urgently needed to validate their therapeutic benefits and safety either as monotherapy or combination therapy with antiviral molecules. This view is also supported by a recent observational study on the use of HCQ in consecutive hospitalized COVID-19 patients. Although the recent randomized clinical studies are not supportive of HCQ therapy for COVID-19 (both therapeutic and postexposure prophylaxis), the other questions such as pre-exposure prophylaxis use of CQ/HCQ, the effect of CQ/HCQ on the degree of disease severity with age, virus load and virus transmission remain for future research. Despite differing results on CQ/HCQ, it is apparent that tailor-made changes on the pharmacophores of aminoquinolines would be an advantage for treating other viral diseases.
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