Introduction

Without a therapeutic vaccine or specific antiviral drugs, and with a desperate attempt to find a cure against novel Corona Virus Disease 2019 (Covid-19) [1], the limelight was shifted to hydoxychloroquine (derivative of chloroquine that has antimalarial, antiinflammatory, immunosuppressive and antiautophagy activities [2, 3]; upon a tweet by US president Mr. Donald J. Trump. On 21st March, 2020, he tweeted, “Hydroxychloroquine and Azithromycin, taken together, have a real chance to be one of the biggest game changers in the history of medicine” (https://twitter.com/realdonaldtrump/status/1241367239900778501?lang=en).

However, easier said than done, initial studies of Covid-19 patients treated with hydoxychloroquine with or without azithromycin were shown to have mixed results, with more scrutiny being raised over the lack of proper design of these studies. Without clinical trials and robust methodologically correct scientific studies there is skepticism over benefits and efficacy of hydoxychloroquine against Covid-19. Till the writing of this article, as per the information available on NCBI, currently worldwide 1450 clinical trials are going on (https://clinicaltrials.gov/ct2/results?cond=%22wuhan+coronavirus%22) and 180 different compounds are being tested against Covid-19 (https://pubchem.ncbi.nlm.nih.gov/#tab=compound&query=covid-19%20clinicaltrials).

Though the exact mechanisms are still unclear; however, it has been established from scientific literature that at the molecular level, hydroxychloroquine affects the activity of lysosomes and in turn autophagy and disrupts lysosomal membrane stability. Also, it alters the transcriptional activity and signaling pathways. At the cellular level also, the precise mode of action of hydroxychloroquine is yet to be delineated. Based upon current understanding, hydroxychloroquine has been shown to inhibit immune system activation. Suppression of immune system activation by hydroxychloroquine is achieved by reducing cytokine production and Toll-like receptor (TLR) signaling pathways [2, 3].

The main aim of this review is to discuss the mode of action of hydroxychloroquine at cellular and molecular levels, that potentially support the clinical efficacy and few adverse side effects observed in Covid-19 patients treated with hydroxychloroquine, which may further help in improving the clinical outcomes by modifying or altering the drug itself or its restricted use in certain individuals by enforcing strict inclusion and exclusion criteria. The readers are referred to more holistic review for clinical use of hydroxychloroquine in other diseases like rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid syndrome and primary Sjögren syndrome [2, 3].

Covid-19

Towards the end of year 2019 (December, 2019), Chinese Province of Wuhan reported the emergence of Covid-19 in humans characterized with atypical pneumonia, fever, dry cough and progressive dyspnea [4]. Novel coronavirus 2019 (nCoV-2019), officially known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was demonstrated to cause Covid-19 [5]. Within a span of around 3 months, Covid-19 spread worldwide forcing the World Health Organization (WHO) to declare it as pandemic on 11th March 2020. As per the WHO-Covid-19 dashboard available at https://covid19.who.int/, as of 31st May, 2020, there have been 5,939,234 confirmed cases of Covid-19, including 367,255 deaths worldwide. It is now well established that respiratory transmission and direct contact transmission are the main routes for Covid-19 transmission. Previously, Coronaviruses (CoVs) also have been responsible for the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) outbreaks. [6, 7].

Clinical Features and Laboratory Diagnosis of Covid-19

According to Centers for Disease Control and Prevention (CDC), USA (available at https://www.cdc.gov/coronavirus/2019-ncov/symptoms-testing/symptoms.html) and recent literature, Covid-19 infected people demonstrate a wide range of symptoms—ranging from mild symptoms to severe illness. Most common symptoms of confirmed positive Covid-19 patients were fever, cough, shortness of breath, myalgia or fatigue and sore throat; whereas, headache, chills, new loss of taste or smell, sputum production, diarrhea, and vomiting like symptoms may also occur [8,9,10,11,12].

Elderly people and people, who have severe underlying medical conditions, seem to be at higher risk for developing more serious complications from COVID-19 compared to young healthy adults [9, 10, 13, 14]. Most of deaths due to Covid-19 were due to severe respiratory failure, pneumonia and multiple organ failure [10].

Initially, a combination of computed tomography (CT) imaging, electron microscopy, and whole genome sequencing were used to screen and identify SARS-CoV-2 [4, 15,16,17,18,19]. Confirmatory diagnosis of Covid-19 relies on the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method. RNA-dependent RNA polymerase (RdRp) gene of the ORF1ab sequence, E gene, N gene, and S gene of the SARS-CoV-2 genome is the basis of RT-PCR tests for the diagnosis of SARS-CoV-2 [4, 15, 16].

Among the laboratory parameters, most moderate Covid-19 patient’s exhibited leucopenia, lymphopenia, high level of C-reactive protein (CRP) and erythrocyte sedimentation rate [12, 20]. However, severe Covid-19 patients showed frequent lymphopenia, with increased levels of lactate dehydrogenase, CRP, ferritin, alanine aminotransferase, and D-dimer along with grossly higher levels of interleukins (ILs) IL-2R, IL-6, IL-10, and Tumor necrosis factor-α [9, 12, 14, 20]. Acute respiratory distress syndrome (ARDS) and multiple-organ failure in Covid-19 are thought to result from “Cytokine storm” also known as cytokine release syndrome. “Cytokine storm” is an excessive immune response to an external stimulus. “Cytokine storm” in severe Covid-19 cases, if left untreated for a longer time, results in an increased risk of vascular hyperpermeability, multiorgan failure, and eventually death [21, 22]. Due to observance of Cytokine storm in severe Covid-19 cases; some authors have suggested theoretical treatment strategies like use of IL-6 and IL-1 family antagonists, TNF blockers and corticosteroid therapy among other strategies [22, 23].

Hydroxychloroquine: Structure and Its Chemistry

Hydroxychloroquine, (PubChem CID: 3652, https://pubchem.ncbi.nlm.nih.gov/compound/3652, Molecular formula: C18H26ClN3O) also known as Plaquenil, Oxichloroquine, Oxychlorochin, is a derivative of chloroquine exhibiting immunosuppressive, anti-inflammatory, antiautophagy, and antimalarial activities. Due to these properties, hydroxychloroquine is now most often used as an antirheumatologic agent in systemic lupus erythematosis and rheumatoid arthritis. This is the reason that both hydroxychloroquine and chloroquine are classified under Disease-modifying antirheumatic drugs (DMARDs). Hydroxychloroquine is derived from chloroquine, and differ from it by the presence of a hydroxyl group. Both hydroxychloroquine and chloroquine are weak bases, and a half-life of around 50 days. Hydroxychloroquine and chloroquine belong to 4-aminoquinolines drugs class (reviewed in [3].

Pharmacokinetics of Hydroxychloroquine

As chloroquine and hydroxychloroquine have almost similar structure and mode of action, both drugs can be used against SARS-CoV-1 and SARS-CoV-2 [24, 25]. As compared to chloroquine, hydroxychloroquine is a better therapeutic agent in terms of safety index. Various modes of action have been postulated to explain the therapeutic uses of hydroxychloroquine, but most of them are based on in vitro studies (reviewed in [3]). Hydroxychloroquine, which is administered as hydroxychloroquine sulfate, has a lag time of 0–0.85 h for 200 mg salt. It is the time between oral absorption and its measurement in the blood. It is usually absorbed in the upper intestinal region [26] and has an overall bioavailability of 0.7–0.8. The concentration of hydroxychloroquine can vary not only in plasma, blood and serum of individual patient but also among different patients [27]. It is intriguing to note that hydroxychloroquine has a half-life period of approximately 1-2 months and the mean residence time of nearly 1300 h [28].

Mode of Action of Hydroxychloroquine

The precise mechanism of action of hydroxychloroquine is not known till date, and there is lots of research going on the existing knowledge gap using diverse advanced scientific tools like [3] Computational biology [29], Immunology [30, 31], Structural biology [32, 33], Modern molecular medicine [34, 35], Synthetic biology [36, 37], and ‘big data’-driven public health science [38, 39].

Hydroxychloroquine may suppress immune function by interfering with the processing and presentation of antigens, and the production of cytokines. Acidic vesicles, such as endosomes/lysosomes, which are essential for the membrane fusion, act as major locations of action of chloroquine and hydroxychloroquine [2, 3, 40]. Chloroquine accumulates in lysosomes (lysosomotropism), and by increasing their pH, interferes with the glycosylation of cellular receptor of SARS-CoV-2 and thereby inhibits the infection of virus [14, 40]. An important feature of hydroxychloroquine is its capability to inhibit the innate as well as acquired immunity [41]. Hydroxychloroquine may also affect signaling of TLR receptors by changing the endosomal pH [42] as well as by inhibiting TLR–ligand interactions. Further research is required to know the exact mechanism by which this drug affects TLR activation.

Additionally, hydroxychloroquine by interfering the lysosomal activity and autophagy, inhibits the antigen processing and presentation along with MHC-II molecules and subsequently immune system activation. Reduced activation of CD4 + T cells leads to decreased production of proinflammatory markers and cytokines [14, 40]. It has been suggested that autophagy is involved in autoantigen presentation mediated by MHC class II molecules, and activation of T-helper cells [43]. Because of immunomodulatory and anti-inflammatory properties, hydroxychloroquine can prove to be a better remedy for SARS-CoV-2 patients, where cytokine storm has emerged as a potentially fatal condition [12, 22].

As an anti-inflammatory agent, hydroxychloroquine is also used to treat various autoimmune disorders such as rheumatoid arthritis, systemic lupus erythematosus and Sjogren’s syndrome [3, 5]. Hydroxychloroquine also exhibits antibacterial property particularly against intracellular bacteria such as Coxiella burnetii [44]. However, due to strong affinity of hydroxychloroquine for melanin, it can deposit in tissues such as the skin and the eyes (hydroxychloroquine retinopathy) [3].

Hydroxychloroquine is thought to prevent the thrombotic complications by behaving like anticoagulant agents, and hence can prove beneficial for cases with a secondary coagulopathy owing to systemic inflammation [45]. This is done most probably by inhibiting the platelet aggregation [46, 47] or inhibition of binding of antiphospholipid antibody in autoinflammatory diseases [48]. Hydroxychloroquine is found to be safe to use during pregnancy and various studies have shown that it checks the initiation of congenital heart blockage [49, 50], possibly by reducing the expression of a type I interferon (IFN) signature [51].

Hydroxychloroquine interaction with other drugs

During combination therapy, hydroxychloroquine may interfere the functioning of other drugs or vice versa because it is metabolized by the same enzyme cytochrome P450 (CYP), which is responsible for the metabolism of many other drugs [3, 27, 52, 53]. Hydroxychloroquine increases the bioavailability of metoprolol in blood plasma by reducing its metabolism. Although similar effect is not observed for drugs, such as dextromethorphan [54] and methotrexate [55]. On the other hand, bioavailability of hydroxychloroquine may be affected by the medicines that increase the pH of gastric juice [3]. Hydroxychloroquine can also interfere with drugs that affect the QT interval; hence possibility of cardiac arrhythmia should be checked in such patients [56, 57]. Combination therapy of breast cancer using tamoxifen and hydroxychloroquine may cause eye toxicity due to synergistic inhibition of enzymes in lysosomes of retinal epithelial cells [58]. Thus, duration of such therapy should be monitored carefully. Some of the adverse outcomes of hydroxychloroquine usage are gastrointestinal effects, including nausea, vomiting, diarrhea and abdominal discomfort [59]. Moreover, Hydroxychloroquine-associated cardiotoxic effects including rhythm disorders and cardiomyopathy in rheumatic patients have also been reported [57, 60].

Hydroxychloroquine and Covid-19

Hydroxychloroquine has attracted attention over the past few decades as a potential antiviral agent, and currently as a possible treatment for Covid-19 [40, 61, 62]. However, whether hydroxychloroquine works in vivo against Covid-19 remains to be seen conclusively. The word of caution is that in randomized controlled trials against a number of viruses, including influenza, hydroxychloroquine haven’t work at all. Hydroxychloroquine is known to decrease the acidity in endosomes, which in turn might avert the endosome from releasing the virus into the cytoplasm. Biggest limitation is the lack of preclinical data for hydroxychloroquine against Covid-19 and its repeated failure against most viral diseases under in vivo conditions [61].

Another issue with hydroxychloroquine is adverse outcomes in certain patients with comorbid conditions who are on other medications [40, 61, 62]. It has been reported that hydroxychloroquine can also interfere with drugs that affect the QT interval [57]. One concern in the Covid-19 patients receiving hydroxychloroquine/chloroquine ± azithromycin is the prolongation of QT interval, which may possibly increase the risk of Torsade de pointes (TdP; [63]) and sudden cardiac death. A prospective observational study of large cohort of COVID-19 patients treated with chloroquine/hydroxychloroquine ± azithromycin has demonstrated no instances of TdP or arrhythmogenic death. Even though, it was shown that use of these medications resulted in QT prolongation [64].

Due to its cellular and molecular effects as discussed in previous sections, quite a few clinical trials are studying the effectiveness and safety of hydroxychloroquine (also chloroquine) for Covid-19 (https://clinicaltrials.gov/ct2/results?cond=%22wuhan+coronavirus%22). Upon searching for interventional studies among clinical trials against Covid-19, till the writing of this article we (authors) have found 885 interventional studies using various drugs. Among these 885 studies, 51 studies were found to be using hydroxychloroquine alone (Table 1) and 104 studies are using hydroxychloroquine in combination with other antiviral/nutritional supplements/probiotics etc. (Supplementary material 1). Most of the studies for hydroxychloroquine against Covid-19 are focussing on its safety and efficacy as well its use as a prophylaxis measure against Covid-19 (Table 1). Few of these clinical trials are also evaluating optimal hydroxychloroquine dose, and its use in moderate and severe Covid-19 cases, and pregnant Covid-19 positive women (Table 1). Seven clinical trial registries were also found in Chinese Clinical Trial Registry (http://www.chictr.org.cn) for using hydoxychloroquine to treat Covid-19 [5].

Table 1 Clinical trials with hydroxychloroquine alone
Full size table

In a recent significant development (on May 22nd, 2020), an observational study published in Lancet journal reported that hydroxychloroquine or chloroquine, when used alone or with macrolide, on in-hospital outcomes for Covid-19 were associated with decreased in-hospital survival along with an increased frequency of ventricular arrhythmias [65]. Acting swiftly on this report on May 27th, 2020, WHO executive group implemented a temporary pause of the hydroxychloroquine arm within the Solidarity Trial while the safety data is reviewed by the Data Safety Monitoring Board (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/global-research-on-novel-coronavirus-2019-ncov/solidarity-clinical-trial-for-covid-19-treatments). Expressing shock over this move by WHO, various eminent scientists (> 140 scientists) have written an open letter to Editor of Lancet journal and authors of the study about their concerns over data integrity, plenty of loopholes and erroneous conclusions in the study among host of other concerns (https://www.tropicalmedicine.ox.ac.uk/news/an-open-letter-to-richard-horton-editor-of-the-lancet-regarding-mehra-etal).

However, Indian Council of Medical Research (ICMR), New Delhi, India has maintained its earlier guidelines on use of hydroxychloroquine as prophylaxis for SARS-CoV-2 infection (https://www.mohfw.gov.in/pdf/AdvisoryontheuseofHydroxychloroquinasprophylaxisforSARSCoV2infection.pdf) in its revised guidelines too (https://www.icmr.gov.in/pdf/covid/techdoc/V5_Revised_advisory_on_the_use_of_HCQ_SARS_CoV2_infection.pdf). In its revised guidelines published on 22nd May, 2020, ICMR has expanded to include following personnel for use of hydroxychloroquine as prophylaxis for SARS-CoV-2 infection (1) All asymptomatic healthcare workers working in Covid hospital/non-Covid hospitals/non-Covid areas of Covid hospitals/blocks (2) Asymptomatic frontline workers/paramilitary/police personnel working in Covid-19 containment zones, and (3) Asymptomatic household contacts of laboratory confirmed Covid-19 cases.

Summary and Future Directions

Covid-19, caused by SARS-CoV-2, had become a pandemic and public health concern considering health and economy related challenges to combat and contain this disease. Due to sudden emergence of Covid-19, the healthcare system was caught unaware without an effective and safe treatment for this infectious disease. Chloroquine and hydroxychloroquine both are weak bases that tend to accumulate in acidic compartments such as lysosomes and inflamed (acidic) tissues. By raising the intra-lysosomal pH, hydroxychloroquine impairs the autophagic protein degradation [3].

More studies are required to understand the concentration, distribution and effects of hydroxychloroquine in various tissues, as well as interrelationship between given dose and response generated. Identification of exact molecular targets of hydroxychloroquine within the cell as well as on different microbes is the need of hour. More importantly, efficacy of anti-malarial compounds, other than chloroquine and hydroxychloroquine, should be monitored against SARS-CoV including SARS-CoV-2. Newer compounds closely mimicking the hydroxychloroquine like hydroxyferrochloroquine have been synthesized in past, and have shown to exert antiviral effects with some selectivity toward SARS-CoV infection [25]. This approach can be further exploited and/or reinforced to make newer compound with less toxicity and greater selectivity towards SARS-CoV-2.

There are reports of high risk of thrombosis in patients with severe SARS-CoV-2 infection [66] and procoagulant pattern of patients with COVID-19 ARDS justify the clinical reports of thromboembolic complications (pulmonary embolism) during the course of the Covid-19 [67]. Interestingly, hydroxychloroquine has been associated with reduced risk of thrombosis in several in vitro and animal studies. Potential mechanisms by which hydroxychloroquine and chloroquine reduce the procoagulatory state in autoinflammatory diseases include inhibition of antiphospholipid antibody binding [48] or inhibition of platelet aggregation [46, 47, 68]. Notably, in a mouse model of antiphospholipid syndrome, hydroxychloroquine treatment was associated with improvement in endothelial function [69, 70]. The exact molecular mechanisms by which DMARD hydroxychloroquine drug mediate their antithrombotic effects remain largely unknown and lay the platform for future prospective studies.