Therapeutic use of chloroquine and hydroxychloroquine in COVID-19 and other viral infections: A narrative review

Abstract

The rapidly spreading Coronavirus Disease (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus (SARS-CoV-2), represents an unprecedented serious challenge to the global public health community. The extremely rapid international spread of the disease with significant morbidity and mortality made finding possible therapeutic interventions a global priority. While approved specific antiviral drugs against SARS-CoV-2 are still lacking, a large number of existing drugs are being explored as a possible treatment for COVID-19 infected patients. Recent publications have re-examined the use of Chloroquine (CQ) and/or Hydroxychloroquine (HCQ) as a potential therapeutic option for these patients. In an attempt to explore the evidence that supports their use in COVID-19 patients, we comprehensively reviewed the previous studies which used CQ or HCQ as an antiviral treatment. Both CQ and HCQ demonstrated promising in vitro results, however, such data have not yet been translated into meaningful in vivo studies. While few clinical trials have suggested some beneficial effects of CQ and HCQ in COVID-19 patients, most of the reported data are still preliminary. Given the current uncertainty, it is worth being mindful of the potential risks and strictly rationalise the use of these drugs in COVID-19 patients until further high quality randomized clinical trials are available to clarify their role in the treatment or prevention of COVID-19.

1. Coronaviruses and the COVID-19 pandemic

Coronaviruses (CoVs) are important human and animal pathogens that have the ability to emerge and cross the species barrier, causing novel and occasionally fatal diseases [1,2]. They belong to the subfamily Coronavirinae of the Coronaviridae family in the order Nidovirales [3]. According to the International Committee on the Taxonomy of Viruses (ICTV), coronaviruses are classified into four genera including, alphacoronavirus, betacoronavirus (contains 4 lineages A, B, C and D), gammacoronavirus and deltacoronavirus [4]. They are large enveloped viruses with a large single-stranded RNA, 5′-capped, non-segmented genome with positive polarity ranging from 26 to 32 kb in size [5]. While CoVs from all genera infect a large number of mammals and birds, bats are proposed to be their natural reservoir [6,7]. In humans, on the other hand, only alpha and beta CoVs have been associated with diseases ranging from mild common cold to fatal severe respiratory infections. Two human alpha CoVs (hCoV-229E and hCoV-NL63) and two beta CoVs (hCoV-OC43 and hCoV-HKU1) are associated with common cold [[8], [9], [10], [11]]. In 2002 and 2012, two novel highly pathogenic beta CoVs known as the severe acute respiratory syndrome-CoV (SARS-CoV) and the Middle East respiratory syndrome-CoV (MERS-CoV) emerged in China and Saudi Arabia, respectively [[12], [13], [14], [15]]. These two viruses have spread widely and were associated with severe respiratory diseases with mild to severe and fatal outcomes. More recently, a novel human CoV known as severe acute respiratory syndrome-CoV-2 (SARS-CoV-2) emerged in December 2019 in Wuhan, the capital city of Hubei province in China as the third known highly pathogenic human beta CoV [16].

Since its emergence, SARS-CoV-2, which causes the Coronavirus Disease (COVID-19), has rapidly spread to more than 214 countries around the world, causing a large-scale global pandemic. Until April 10^th, more than 1.6 million COVID-19 confirmed cases have been reported globally, including more than 100,000 deaths. There are currently no vaccines or specific antiviral drugs for SARS-CoV-2 [17]. The rapid global spread of this virus and the worrisome associated mortality rate encouraged the medical community and policy makers to expediate the process of exploring all available and potential interventions to control and mitigate this outbreak [18]. Several interventional treatment options for COVID-19 have been suggested with unclear efficacy and safety considerations [19]. Recent publications have suggested using chloroquine (CQ), a broadly used antimalarial drug, and its derivative hydroxychloroquine (HCQ) as a treatment for COVID-19 patients [[20], [21], [22]]. In this review, we explore the antiviral activities of CQ and HCQ against CoVs and non-CoVs in the majority of previously published in vitro, in vivo and clinical trial studies with an aim to find evidence that supports their use in COVID-19 patients.

2. Possible mechanisms of CQ and HCQ antiviral activities

Both CQ and HCQ, known antimalarial and antirheumatic drugs, have closely related chemical structures [22]. However, their mechanisms of action are still not fully elucidated. Several studies have revealed that both drugs have antiviral activity in vitro through different mechanisms [[23], [24], [25]]. In particular, CQ has been shown to interfere with different stages of the viral life cycle as shown in Fig. 1 [[26], [27], [28], [29]]. Different studies have reported the ability of CQ to inhibit viral entry [[30], [31], [32]], uncoating [33], assembly and budding [34,35]. One of the suggested mechanisms by which CQ can affect the entry step of viruses is by inhibiting quinone reductase 2 [36], which is required for the biosynthesis of sialic acid [37]. Sialic acid was found to be involved in virus attachment and entry into host cells by several viruses including hCoV-OC43 and MERS-CoV [38,39]. Moreover, CQ was shown to potently inhibit entry of SARS-CoV into cells by interfering with the glycosylation of its cellular receptor angiotensin converting enzyme 2 receptor (ACE2). SARS-CoV-2 also uses ACE2 as a receptor for cell entry, suggesting a possible similar effect of CQ on SARS-CoV-2 at this step of virus replication [40]. CQ can also affect early stage of virus replication by inhibiting virus-endosome fusion, likely via increasing endosomal pH [41]. CoVs such as SARS-CoV were shown to be able to enter target cells via pH-dependent mechanism in which the acidic pH of the lysosome triggers fusion of the viral and endosomal membranes resulting in viral particle uncoating and subsequent release of viral nucleic acid into the cytoplasm [42]. CQ can also impair posttranslational modifications of viral proteins through interfering with proteolytic processes [43] and inhibition of glycosylation via specific interactions with sugar-modifying enzymes or glycosyltransferases [28]. CQ can also hamper lysosomal protein degradation and lysosomal fusion with autophagosomes [[44], [45], [46]]. Moreover, it has been suggested that CQ has the ability to affect the cytotoxic mechanisms and works as anti-autophagy agent in vitro [47]. CQ works as anti-inflammatory agent through reducing tumor necrosis factor (TNFα) release and suppressing TNF receptors on monocytes [26,28].

Fig. 1.

Cellular and molecular possible sites of action of CQ ± HCQ as antiviral agents. (X) Represents the site of inhibition by CQ ± HCQ. (1) CQ and HCQ inhibit virus binding to its cell surface receptor, (2) CQ inhibits sialic acid biosynthesis through suppressing quinone reductase 2 activity which affect ACE2 receptor activity, (3) CQ and HCQ inhibit virus pH-dependent endocytosis through increasing pH, (4) CQ interferes with virus uncoating, (5) CQ interferes with assembly/budding leading to accumulation of viral vesicles within trans-Golgi network, (6) CQ interferes with lysosomal protein degradation and lysosomal fusion with autophagosomes. HCQ can interfere with lysosomal activity and prevent major histocompatibility complex (MHC) class II expression, (7) CQ interferes with TNF release and binding from macrophages and/to monocytes, (8) CQ inhibits phosphorylation of P38 MAPK and caspase in Th1 cells which in turn inhibits pro-inflammatory cytokines production and virus replication, (9) HCQ blocking of MHC expression prevents T cell activation, expression of CD145 and cytokines release, (10) HCQ impairs TLR signaling through increasing endosomal pH and interfering with TLR7 and TLR9 binding to their DNA/RNA ligands thereby inhibiting transcription of pro-inflammatory genes, (11) HCQ inhibits the binding of DNA to the cGAS and therefore reduce cytokines transcription and production. ACE2: Angiotensin converting enzyme 2; MHC: Major histocompatibility complex; TLR: Toll-like receptors; cGAS: Cyclic GMP-AMP synthase; MAPK: Mitogen-activated protein kinase. This figure was created with BioRender.com.

On the other hand, HCQ has similar effects to CQ in interfering with the glycosylation of ACE2, blocking virus/cell fusion and inhibiting lysosomal activity by increasing pH [22]. HCQ can also impede major histocompatibility complex (MCH) class II expression which inhibits T cell activation, expression of CD145 and cytokines release [[48], [49], [50]]. Furthermore, HCQ has been shown to impair Toll-like receptors (TLRs) signaling through increasing endosomal pH and interfering with TLR7 and TLR9 binding to their DNA/RNA ligands thereby inhibiting transcription of pro-inflammatory genes [[51], [52], [53]]. The aforementioned immunomodulatory properties of CQ and HCQ have raised the interest in using these drugs in COVID-19 patients at risk of cytokines release syndrome (CRS) [22].

3. CQ and HCQ pharmacokinetics

The fact that both CQ and HCQ are considered for the management of COVID-19 patients clearly highlights the need to better understand their pharmacokinetics (PK) parameters. However, a full understanding of these parameters has been challenging despite the numerous reported studies. Generally, PK parameters for CQ and HCQ are comparable (Table 1 ) [54,55]. Following oral administration of CQ and HCQ, their bioavailability can reach up to 80% with plasma peak time around 2–4 h [[56], [57], [58]]. Thus, parenteral administration, if available, might be a better route especially that oral administration has shown huge interpatient variability [56,59,60]. The long half-life of both CQ and HCQ which could range from 30 to 60 days is likely attributed to their large volume of distribution (200–800 L/kg) and extensive tissue uptake [[61], [62], [63], [64], [65], [66], [67], [68]]. CQ and HCQ are metabolized via CYP-450 enzymes to other active compounds, which are responsible for the extended pharmacological actions and increased toxicity [61,69]. Up to 60% of CQ and HCQ is primarily excreted renally as unchanged or metabolized forms, and the remaining (40%) is usually cleared through the liver, feces and skin or stored in other lean body tissues [54,[69], [70], [71], [72], [73], [74]]. It's important to note that CQ and HCQ have a chiral center, which produces two enantiomers R(−) or S(+) forms or isomers [75], in which little is known about the differences in their pharmacological activity and their corresponding metabolites. Most clinically used CQ and HCQ exist as a racemic mixture (50:50) of both isomers which complicates the understanding of their PK and associated toxicity as they could behave differently inside the body [57,[75], [76], [77]].

Table 1.

CQ and HCQ pharmacokinetic parameters.

Pharmacokinetic parameters	CQ	HCQ
Bioavailability	89 ± 16%	74 ± 13%
Half-life	30–60 days	30–52 days
Peak plasma time	2–4 h
Metabolism	Liver CYP-450
Excretion	Kidney and liver (40–60%) unchanged or metabolized

4. CQ and HCQ adverse effects and related toxicities

The most common CQ and HCQ adverse effects are gastrointestinal symptoms such as nausea, vomiting and abdominal discomfort [78], and uncommonly worrisome fulminant hepatic failure [79], toxic epidermal necrolysis (TEN) [80] and cardiotoxicity that could manifest with QT abnormality [[81], [82], [83]]. Nevertheless, over the years CQ and HCQ have maintained a good safety profile when used in several chronic diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Despite some animal experiments suggesting that HCQ is probably less toxic than CQ, there is a lack of high quality evidence from clinical trials supporting this claim [74,[84], [85], [86], [87]]. These toxicities could be related to the very long half-life and the large volume of distribution of both drugs. One of the significant toxic effects of CQ and HCQ is the possible ocular pigmentation due to their binding to melanin, which could lead to damage in different parts of the eye including the cornea, ciliary body and retina [88]. Notably, the incidence of such ocular toxicity is usually rare. For instance, it was shown that only 0.5% out of ~400 patients treated with HCQ (≤6.5 mg/kg/day) for 6 years due to RA or SLE had developed ocular related complications [89]. Most studies have shown that such complications might only occur with long term treatment of chronic diseases which extends for more than 5 years with doses above or equal to 6.5 mg/kg/day [90,91]. However, ocular toxicity and changes could still occur with shorter treatments. Other complications such as development of proximal myopathy associated with respiratory failure have also been reported in patients treated with either CQ or HCQ [[92], [93], [94], [95]]. Nonetheless, most of these complications were seen in elderly patients with an average age of 70 years suffering from chronic RA or autoimmune diseases. Both CQ and HCQ were also shown to be associated with rare but life-threatening cardiomyopathy [[96], [97], [98]]. Other less reported CQ and HCQ toxicities include urticaria [99], ototoxicity [100,101] and some neurological effects [102,103].

5. In vitro antiviral activity of CQ and HCQ

The antiviral effects of CQ were suggested at least 50 years ago [23,25]. Since then, several studies have tested the ability of CQ and HCQ to inhibit the replication of a wide range of CoVs and non-CoV viruses in vitro as shown in Table 2, Table 3 , respectively. The majority of these studies have revealed a substantial ability of CQ and HCQ as well as some of their derivatives to inhibit viral replication with no to low toxicity. Specifically, CQ has been shown to inhibit the replication of different CoVs including SARS-CoV, MERS-CoV and SARS-CoV-2 among others in several studies (Table 2) [37,[104], [105], [106], [107], [108], [109], [110], [111], [112], [113], [114], [115]]. Only two studies showed no significant inhibitory effects of CQ on MERS-CoV and mouse hepatitis virus (MHV4) [116,117]. Other CQ derivatives such as amodiaquine (AMD), ferroquine (FQ), hydroxy ferroquine (HFQ) have been also shown to exerts some antiviral activity [105,106]. Interestingly enough, while HCQ does not seem to have a significant effect in reducing SARS-CoV and Feline CoV replication [106], it was recently shown to have a potent in vitro inhibitory effects against SARS-CoV-2 replication [112,116].

Table 2.

In vitro antiviral activity of CQ and its derivatives on CoVs.

Drug	Virus	Cells	EC50 (μM)	SI	Main findings	Year	Ref
CQ	SARS-CoV	Vero E6	8·8 ± 1·2	30	↓ viral replication	2004	[104]
CQ	SARS-CoV	Vero E6	4.4 + 1·0	–	↓ viral replication	2005	[37]
CQ	SARS-CoV	Vero 76	1–5	2–20	↓ viral replication	2006	[105]
CQ	SARS-CoV	Vero	6.5 ± 3.2	>15	↓ viral replication	2006	[106]
CQ	SARS-CoV	Vero E6	4.1 ± 1.0	>31	↓ viral replication	2014	[107]
CQ-MP	SARS-CoV	Vero 76	4–6	3–8	↓ viral replication	2006	[105]
CQ-DP	SARS-CoV	Vero 76	3–8	2–10	↓ viral replication	2006	[105]
AMD	SARS-CoV	Vero 76	3–10	2–10	↓ viral replication	2006	[105]
HCQ	SARS-CoV	Vero	34 ± 5	>3	Ineffective	2006	[106]
FQ	SARS-CoV	Vero	1.4 ± 0.1	15	↓ viral replication	2006	[106]
HFQ	SARS-CoV	Vero	1.9–4.9	4–17	↓ viral replication	2006	[106]
CQ	MERS-CoV	Huh7	3.0 ± 1.1	19.4	↓ viral replication	2014	[107]
CQ	MERS-CoV	Vero E6	6.3	–	Ineffective	2018	[116]
CQ	SARS-CoV-2	Vero E6	1.13	>88.5	↓ viral replication	2020	[113]
CQ	SARS-CoV-2	Vero	5.47	–	↓ viral replication	2020	[112]
CQa	SARS-CoV-2	Vero E6	2.71–7.36	37.12–100.81	↓ viral replication	2020	[114]
HCQ	SARS-CoV-2	Vero	0.72	–	↓ viral replication	2020	[112]
HCQa	SARS-CoV-2	Vero E6	4.06–17.31	14.41–61.45	↓ viral replication	2020	[114]
CQ	HCoV-229E	L132	–	–	↓ viral replication	2008	[109]
CQ	HCoV-229E	Huh7	3.3 ± 1.2	>15	↓ viral replication	2014	[107]
CQ	HCoV-OC43	HRT-18	0.3 ± 0.0	1369	↓ viral replication	2009	[108]
CQ	MHV4	Murine cells	–	–	Ineffective	1991	[117]
CQ	MHV3	Murine MΦ	–	–	↓ viral replication	1966	[115]
CQ	F–CoV	CRFK	>0.8	–	↓ viral replication	2006	[106]
HCQ	F–CoV	CRFK	28 ± 27	–	Ineffective	2006	[106]
FQ	F–CoV	CRFK	2.9 ± 1.2	–	↓ viral replication	2006	[106]
HFQ	F–CoV	CRFK	>4	–	Weak effect	2006	[106]
CQ	FIPV	fcwf-4	–	–	↓ viral replication	2013	[110]
CQ	PHEV	Neuro-2a	–	–	↓ viral replication	2017	[111]

CQ: Chloroquine; CQ-MP: Chloroquine monophosphate; CQ-DP: Chloroquine diphosphate; AMD: Amodiaquine; HCQ: Hydroxychloroquine; FQ: Ferroquine; HFQ: Hydroxy ferroquine; SARS-CoV: Sever acute respiratory syndrome-coronavirus; MERS-CoV: Middle East respiratory syndrome-coronavirus; SARS-CoV-2: Sever acute respiratory syndrome-coronavirus 2; MHV4: Mouse hepatitis virus Type 4; F–CoV: Feline coronavirus; FIPV: Feline infectious peritonitis virus; PHEV: Porcine hemagglutinating encephalomyelitis virus; Vero cells: African green monkey kidney epithelial cells; Huh7 cells: Human hepatocyte-derived carcinoma cells; L132: human epithelial lung cells; HRT-18: Human ileocecal colorectal adenocarcinoma cells; MΦ: macrophages; CRFK cells: Crandell–Reese feline kidney cells; fcwf-4 cells: Felis catuswhole fetus-4 cells; Neuro-2a: murine neuroblastoma cells; EC₅₀: 50% Effective concentration; SI: Selectivity index defined as the ratio of drug efficacy to cytotoxicity.

^aTested at different multiplicities of infections (MOIs) of 0.01–0.8.

Table 3.

In vitro antiviral activity of CQ and its derivatives on non-CoVs.

Drug	Virus	Cells	EC50 (μM)	SI	Main findings	Year	Ref
CQ	HIV-1	HL3Tl	–	–	↑ viral replicationp	1988	[126]
CQ	HIV-1	H-9	–	low toxicity	↓ viral replication	1990	[118]
CQa	HIV-1	H-9	–	No toxicity	↓ viral replication	1998	[119]
CQa	HIV-1	U-937	–	No toxicity	↓ viral replication	1998	[119]
CQb	HIV-1	H-9	0.9	No toxicity	↓ viral replication	1999	[120]
CQb	HIV-1	U-937	0.4	No toxicity	↓ viral replication	1999	[120]
CQb	HIV-1	T cellsk	0.9	No toxicity	↓ viral replication	1999	[120]
CQb	HIV-1	Monocytesk	0.2	No toxicity	↓ viral replication	1999	[120]
CQb	HIV-1	U-1l	0.1	No toxicity	↓ viral replication	1999	[120]
CQb	HIV-1	ACH-2m	1	No toxicity	↓ viral replication	1999	[120]
CQc	HIV-1	U-937	0.4	No toxicity	↓ viral replication	2001	[121]
CQc	HIV-1	H-9	0.9	No toxicity	↓ viral replication	2001	[121]
CQc	HIV-1	T cellsk	0.9	No toxicity	↓ viral replication	2001	[121]
CQc	HIV-1	MΦk	0.2	No toxicity	↓ viral replication	2001	[121]
CQc	HIV-1	U-1l	0.1	No toxicity	↓ viral replication	2001	[121]
CQc	HIV-1	ACH-2m	1	No toxicity	↓ viral replication	2001	[121]
CQd	HIV-1	H-9	1–10	No toxicity	↓ viral replication	2004	[122]
CQ	HIV-1	MT-4	8.86 ± 1.18	6	↓ viral replication	2006	[106] r
HCQ	HIV-1	U-937	1	low toxicity	↓ viral replication	1993	[123]
HCQ	HIV-1	CEM	10	low toxicity	↓ viral replication	1993	[123]
HCQ	HIV-1	63	0.01	No toxicity	↓ viral replication	1996	[124]
HCQ	HIV-1	SP	0.1	No toxicity	↓ viral replication	1996	[124]
HCQ	HIV-1	63HIV	–	–	↓ viral replication	1996	[124]
HCQ	HIV-1	SPH	–	–	↓ viral replication	1996	[124]
HCQ	HIV-1	MT-4	>12	–	Ineffective	2006	[106] r
FQ	HIV-1	MT-4	>2.4	–	Ineffective	2006	[106] r
HFQ	HIV-1	MT-4	2.9 ± 1.1	3	↓ viral replication	2006	[106]
CQd	HIV-2	MT-4	1–10	No toxicity	↓ viral replication	2004	[122]
CQ	IAV H1N1	MDCK	–	–	↓ viral replication	1981	[127]
CQ	IAV H1N1	MDCK	3.60	–	↓ viral replication	2006	[128]
CQ	IAV H1N1	A549	–	–	↓ viral replication	2007	[129]
CQ	IAV H1N1	MDCK	1.26	–	↓ viral replication	2007	[130]
CQ	IAV H3N2	MDCK	0.84	–	↓ viral replication	2006	[128]
CQ	IAV H3N2	MDCK	1.53	–	↓ viral replication	2007	[130]
CQ	IAV H3N2	A549	–	–	↓ viral replication	2007	[129]
CQ	IAV H5N1	A549	–	–	↓ viral replication	2013	[29]
CQ	IAV H5N9	MDCK	14.38	–	↓ viral replication	2007	[130]
CQ	IAV H7N3fh	MDCK	>20	–	Ineffective	2007	[130]
CQ	IAV H7N3gh	MDCK	14.39	–	↓ viral replication	2007	[130]
CQ	Flu B	MDCK	–	–	↓ viral replication	1983	[131]
CQ	DENV-2	BHK	–	–	↓ viral replicationq	1990	[43]
CQ	DENV-2	Vero	–	No toxicity	↓ viral replication	2013	[141]
CQ	DENV-2	C6/36	–	No toxicity	Ineffective	2013	[141]
CQ	DENV-2	U-937	–	No toxicity	↓ viral replication	2014	[140]
CQ	ZIKV	Vero	9.82	No toxicity	↓ viral replication	2016	[135]
CQ	ZIKV	hBMECs	14.20	No toxicity	↓ viral replication	2016	[135]
CQ	ZIKV	NSCs	12.36	No toxicity	↓ viral replication	2016	[135]
CQ	ZIKV	NSs	–	–	↓ viral replication	2016	[135]
CQ	ZIKV	Vero	4.15	–	↓ viral replication	2017	[134]
CQ	ZIKV	Huh7	1.72–2.72	–	↓ viral replication	2017	[134]
CQ	ZIKV	NSs	10	–	↓ viral replication	2017	[136]
AMD	ZIKV	Vero	–	–	↓ viral replication	2017	[134]
CQ	CHIKV	HeLa	–	–	↓ viral replication	2007	[132]
CQ pre	CHIKV	Vero	7.0 ± 1.5	37.14	↓ viral replication	2010	[41]
CQ post	CHIKV	Vero	17.2 ± 2.1	15.29	↓ viral replication	2010	[41]
CQ con	CHIKV	Vero	10.0 ± 1.2	26	↓ viral replication	2010	[41]
CQ	CHIKV	MDMn	–	low toxicity	↓ viral replication	2018	[133]
CQ	CHIKV	Fibroblastsn	–	high toxicity	↓ viral replication	2018	[133]
CQ	EBOVi	HEK 293T	4.7	–	↓ viral replication	2013	[139]
CQ	EBOVj	Vero 76	16	–	↓ viral replication	2013	[139]
HCQ	EBOVi	HEK 293T	9.5	–	↓ viral replication	2013	[139]
HCQ	EBOVj	Vero 76	22	–	↓ viral replication	2013	[139]
AMD	EBOVi	HEK 293T	2.6	–	↓ viral replication	2013	[139]
AMD	EBOVj	Vero 76	8.4	–	↓ viral replication	2013	[139]
AQ	EBOVi	HEK 293T	4.3	–	↓ viral replication	2013	[139]
AQ	EBOVj	Vero 76	21	–	↓ viral replication	2013	[139]
CQ	EBOV	MRC-5	–	low toxicity	↓ viral replication	2015	[137]
CQ	EBOV	Vero E6	1.77o	–	↓ viral replication	2015	[138]
CQ	SINV	BHK-21	–	–	↓ viral replication	1981	[142]
CQ	VSV	BHK-21	–	–	↓ viral replication	1981	[142]
CQ	VSV	B104	–	–	↓ viral replication	2010	[149]
CQ	Rabies	NS-20	–	–	↓ viral replication	1984	[143]
CQ	PICV	BHK-21			↓ viral replication	1989	[147]
CQ	Poliovirus	HeLa	–	–	Ineffective	1991	[151]
CQ	SLE	BHK	–	–	↓ viral replicationq	1990	[43]
CQ	POW	BHK	–	–	↓ viral replicationq	1990	[43]
CQ	NiV	Vero	–	–	↓ viral replication	2009	[150]
CQ	NiV	HeLa	0.62	–	↓ viral replication	2010	[148]
CQ	HeV	Vero	–	–	↓ viral replication	2009	[150]
CQ	HeV	HeLa	0.71	–	↓ viral replication	2010	[148]
CQ	EBV	HH514-16	–	–	↑ viral replication	2017	[125]
CQe	HCV	Huh-7	0.22	–	↓ viral replication	2010	[144]
CQ	DHBV	PDH	–	–	↓ viral replication	1990	[145]
CQ	DHBV	PDH	–	No toxicity	↓ viral replication	1991	[146]
CQ	JEV	B104	–	–	↓ viral replication	2010	[149]
CQ	MARVi	HEK 293T	5.5	–	↓ viral replication	2013	[139]
CQ	MARVj	Vero 76	15	–	↓ viral replication	2013	[139]
HCQ	MARVi	HEK 293T	9.8	–	↓ viral replication	2013	[139]
HCQ	MARVj	Vero 76	18	–	↓ viral replication	2013	[139]
AMD	MARVi	HEK 293T	2.3	–	↓ viral replication	2013	[139]
AMD	MARVj	Vero 76	8.3	–	↓ viral replication	2013	[139]
AQ	MARVi	HEK 293T	4.3	–	↓ viral replication	2013	[139]
AQ	MARVj	Vero 76	42	–	↓ viral replication	2013	[139]
CQ	CCHFV	Vero E6	–	–	↓ viral replication	2015	[150]
CQ	CCHFV	Huh7	–	21.3	↓ viral replication	2015	[150]

CQ: Chloroquine; HCQ: Hydroxychloroquine; FQ: Ferroquine; HFQ: Hydroxy ferroquine; AMD: Amodiaquine; Pre: pre-treatment; Post: post-treatment; Con: concurrent; AQ: Aminoquinoline; HIV: Human immunodeficiency viruses; IAV: Influenza A virus; Flu B: Influenza B virus DENV-2: Dengue virus 2; ZIKV: Zika virus; CHIKV: Chikungunya virus; EBOV: Ebola virus; SINV: Sindbis virus; VSV: Vesicular stomatitis virus; PICV: Pichinde virus; SLE: St. Louis encephalitis virus; POW: Powassan virus; NiV: Nipah virus; HeV: Hendra virus; EBV: Epstein-Barr virus; HCV: Hepatitis C virus; DHBV: Duck hepatitis B virus; JEV: Japanese encephalitis virus; MARV: Marburg virus; CCHFV: Crimean-Congo hemorrhagic virus; HL3Tl: HeLa derivative cells; H-9: Human T lymphocytic cells; U-937: Human promonocytic cells; U-1: Human promonocytic cells; ACH-2: Human T lymphocytic cells; MΦ: macrophages; MT-4 cells: HTLV-I-transformed T-cell line; CEM: Human T lymphoblast cells; 63: Human macrophage hybridoma; SP: T-cell line derived from the pleural fluid of an HIV- 1-infected individual; 63_HIV: 63 cells infected by HIV; SPH: SP cells infected by HIV; MDCK: Madin Darby canine kidney; A549 cells: Human adenocarcinomic alveolar basal epithelial cells; BHK/BHK-21 cells: Syrian golden Syrian golden fibroblast cells; Vero cells: African green monkey kidney epithelial cells; C6/36: Aedes albopictus cell line; hBMEC: Human brain microvascular endothelial cells; NSCs: Neural stem cells; NS: Neurospheres; Huh7 cells: Human hepatocyte-derived carcinoma cells; HeLa: Human epithelial cell line; MDM: Monocyte-derived macrophages; HEK 293T: Human embryonic kidney cells; MRC-5: Human normal lung fibroblasts; MRC-5: Medical Research Council cell strain 5; B104: Rat neuroblastoma cell; NS-20: Murine neuroblastoma; HH514–16: Burkitt lymphoma cell line; PDH: Primary duck hepatocytes; EC₅₀: 50% Effective concentration; SI: selectivity index defined as the ratio of drug efficacy to cytotoxicity (when no SI value was reported, level of toxicity was indicated if available).

^aEither alone or combined with hydroxyurea (HU1) + didanosine (ddI).

^bIn combination with hydroxyurea (HU1) + didanosine (ddI).

^cIn combination with hydroxyurea (HU1) + didanosine (ddI) or with hydroxyurea (HU1) + zidovudine (ZDV).

^dEnhanced inhibition against HIV-1 and HIV-2 in combination with HCQ in H9 and MT-4 cells; and against HIV-1 in combination with indinavir (IDV), saquinavir (SQV) or ritonavir (RTV) in MT-4 cells or peripheral blood mononuclear cells (PBMCs).

^eSynergistic inhibitory effect of CQ with IFN-α.

^fA/Mallard/It/43/01 (H7N3).

^gA/Ty/It/220158/02 (H7N3).

^hThe haemagglutinins (HAs) of the two avian H7N3 strains differ in two amino acid residues (261 in the HA1 subunit and 161 in HA2 subunit) and display different pH requirements.

ⁱViral entry (viral pseudotype assay).

^jViral replication.

^kPrimary cells.

^lCells stimulated with LPS.

^mCells stimulated with PMA.

ⁿPrimary non-human primates derived cells.

^oEC₅₀ in μg/mL.

^pSuggested enhanced replication and protection of tat from proteolytic degradation with CQ.

^qSuggested inhibition of virus replication based on increased prM protein in progeny virions rather than M protein due to inhibition of proteolytic process.

^rCQ, HCQ and FQ showed no significant activity against parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie virus, Punta Toro virus, respiratory syncytial virus (RSV), herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), vaccinia virus, vesicular stomatitis virus (VSV), and influenza A virus (H3N2).

Similarly, these compounds have shown excellent in vitro antiviral activity against several non CoV (mostly RNA viruses) with low toxicity in most cases (Table 3). For instance, HIV was shown to be inhibited by CQ alone or in combination with HCQ, hydroxyurea (HU1), didanosine (ddI), zidovudine (ZDV), indinavir (IDV), saquinavir (SQV) or ritonavir (RTV) [106,[118], [119], [120], [121], [122]]. While other derivatives such as HCQ and HFQ have been also shown to inhibit HIV replication [106,123,124], one study showed no effect of HCQ and FQ on HIV [106]. Similarly, it was found that CQ could enhance Epstein-Barr virus replication [125]. Furthermore, another study has suggested possible enhanced HIV replication with CQ treatment through protection of tat protein from proteolytic degradation [126]. Influenza A and B viruses have also been shown to be inhibited by CQ [27,[127], [128], [129], [130], [131]] although contradicting results have been seen for some subtypes and strains such as avian H7N3 strains (A/Mallard/It/43/01 and A/Ty/It/220158/02) [106,130]. Several other studies have also reported in vitro inhibitory effect of CQ on multiple viruses such as chikungunya virus (CHIKV) [41,132,133], zika virus (ZIKV) [[134], [135], [136]], Ebola virus (EBOV) [[137], [138], [139]], dengue viruses (DENV) in mammalian cells [43,140,141] but not insect cells [141] as well as several others [43,139,[142], [143], [144], [145], [146], [147], [148], [149], [150]]. Nonetheless, some reports failed to observe antiviral activity of CQ, HCQ and FQ on several other viruses including polio virus, reovirus, respiratory syncytial virus (RSV), herpes simplex viruses, coxsackie virus, vesicular stomatitis virus (VSV), vaccinia virus, sindbis virus, parainfluenza-3 virus and Punta Toro virus [106,151].

6. In vivo animal antiviral activity of CQ and HCQ

There are limited studies established to investigate the possible antiviral effects of CQ or HCQ in animal models (Table 4 ). In general, studies showed no significant effect of CQ on CoVs including SARS-CoV and feline infectious peritonitis virus (FIPV) replication or clinical scores in mice and cats, respectively [105,110]. However, it has been found that CQ significantly reduced HCoV-OC43 dissemination and replication in mice central nervous system (CNS) [152] and increased the survival rate of HCoV-OC43 infected newborn mice when their mothers treated by CQ most probably through placental and maternal milk transfer [108].

Table 4.

In vivo animal studies on the antiviral activity of CQ and its derivatives on CoVs and non-CoVs.

	Drug	Virus	Model	Dose (mg/kg)	Route	Main findings	Year	Ref
CoVs	CQ	SARS-CoV	Mice	1–50	i.p or i.n.	Tolerated; ineffective	2006	[105]
	AMD	SARS-CoV	Mice	9.4–75	i.p or i.n.	Tolerated; ineffective	2006	[105]
	CQ	HCoV-OC43	Mice	15 (daily)	s.c.	Effectivec	2009	[108]
	CQ	HCoV-OC43	Mice	30 then 15	s.c.	Effective	2019	[152]
	CQ	FIPV	Cat	10/3 days	s.c.	Not significant effect	2013	[110]
Non CoVs	CQ	IAV H1N1	Mice	12.5 (daily)	i.t. or oral	Toxic; ineffective	2007	[129]
	CQ	IAV H3N2	Mice	12.5–37.5 (daily)	i.t. or oral	Toxic; ineffective	2007	[129]
	CQ	IAV H3N2	Ferrets	10 (daily)	oral	Ineffective	2007	[129]
	CQ	IAV H5N1	Mice	50	i.p.	Effectived	2013	[29]
	CQ	EBOV	GUPI	33.75 (2 daily)	i.v. or oral	Toxic; ineffective	2015	[137]
	CQ	EBOV	Mice	90	i.p.	Toxic; ineffective	2015	[138]
	CQ	EBOV	Hamsters	90	i.p.	Toxic; ineffective	2015	[138]
	CQa	EBOV	Hamsters	50	i.p.	Tolerated; ineffective	2015	[138]
	CQ	EBOV	Mice	90	i.p	Effective	2013	[139]
	CQ	NiV	Ferrets	25 (daily)	–	Ineffective	2009	[153]
	CQb	NiV	Hamsters	50	i.p.	Ineffectivee	2010	[148]
	CQb	HeV	Hamsters	50/2 days	i.p.	Ineffectivee	2010	[148]
	CQ	LASV	Mice	90	i.p	Ineffective	2013	[139]
	CQ	ZIKV	Mice	100	i.g.	Effectivef	2017	[134]
	CQ	ZIKV	Mice	50 (5 days)	oral	Effectiveg	2017	[136]
	CQ	CHIKV	NHP	14 (daily)	s.c.	Toxic; ineffectiveh	2018	[133]
	CQ	SFV	Mice	~10	i.p	Toxic; ineffective	1991	[154]

CQ: Chloroquine; AMD: Amodiaquine; SARS-CoV: Sever acute respiratory syndrome-coronavirus; FIPV: Feline infectious peritonitis virus; IAV: Influenza A virus: EBOV: Ebola virus; NiV: Nipah virus; HeV: Hendra virus; LASV: Lassa fever viruses; ZIKV: Zika virus; CHIKV: Chikungunya virus; SFV: Semliki Forest Virus; GUPI: Guinea pig; NHP: non-human primate; i.p: intraperitoneal; i.n: intranasal; s.c.: subcutaneous; i.t: intratracheal; i.v: intravenous; i.g: intragastric.

^aCombined with doxycycline (2.5 mg/kg) and azithromycin (50 mg/kg).

^bEither alone or combined with ribavirin.

^cDose-dependent protection of infected pups when given to mothers prepartum or postpartum (placental and maternal milk transfer).

^dtherapeutically but not prophylactically.

^eDisease exacerbation.

^fin both wild type and IFNAR deficient mice. Also, protected infected pups from infection and microcephaly when given to mothers.

^gCQ extended the average lifespan of ZIKV-infected AG129 mice, and suppresses vertical transmission from pregnant infected mice.

^hDisease exacerbation correlating with increased type I IFN response and delayed immune response.

On the other hand, CQ administration has shown contradicting outcomes when used against non-CoVs RNA viruses in different animal models. Some studies have demonstrated antiviral efficacy of CQ in influenza A virus H5N1, ZIKV and EBOV infected mice [29,134,139]. Interestingly, CQ was effective against ZIKV in both wild type and IFNAR deficient mice, and protected infected suckling pups from infection and microcephaly when given to their mothers [29,134,136]. However, several other studies showed no significant antiviral effect of CQ against influenza A H1N1 and H3N2 viruses in mice and Ferrets, respectively [129]. Similarly, CQ was ineffective against EBOV in guinea pigs, mice and hamsters [137,138], Nipah virus (NiV) in Ferrets and hamsters [148,153], Hendra virus (HeV) in hamsters [148], CHIKV in cynomolgus macaques [133], Lassa virus (LASV) in mice [139] and Semliki Forest Virus (SIV) in mice [154]. Importantly, most of these previous in vivo studies showed toxicity in animals [129,133,137,138,154]. Furthermore, it was shown that CQ could lead to disease exacerbation correlating with increased type I IFN response and delayed immune responses in CHIKV infected macaques [133], increased mortality rate of SFV-infected mice [154] and NiV or HeV infected hamsters [148].

7. Use of CQ and HCQ as antiviral agents in clinical trials

There are very limited published clinical trials that studied the possible antiviral effects of CQ or HCQ in CoV and non-CoV infected patients (Table 5 ). These published clinical trials have clearly shown no significant benefit of using CQ in the prevention or treatment against influenza, DENV or CHIKV infections in patients [133,[155], [156], [157], [158]]. In fact, in one study, patients treated with CQ were more likely to develop adverse effects such as arthralgia at day 200 post-treatment [157]. On the other hand, few studies have reported that HCQ could decease HIV-1 viremia, stabilize CD4 T cell count and reduce IL-6 and IgG levels in infected patients [159], although others showed contradicting finding of increased HIV RNAemia in HCQ treated patients [160,161]. Interestingly, while few clinical studies have suggested that the use of HCQ alone or with azithromycin (AZT) could be beneficial for COVID-19 patients as it reduces viral shedding and time to clinical recovery [[162], [163], [164]], others have reported no effect in infected patients [165,166]. However, it is important to note that most of these studies have several limitations in study designs with small sample sizes. Nonetheless, around 104 clinical trials are ongoing in different countries to asses and evaluate the therapeutic and prophylactic effects of both CQ and/or HCQ in COVID-19 patients (Table 6 ).

Table 5.

Main findings of clinical trials on the antiviral activity of CQ and its derivatives on CoVs and non-CoVs.

Drug	Virus	Design	Dose mg/day	Total No.	Main Findings	Year Ref
HCQ + AZT	SARS-CoV-2	SAOLS	600 mg/day (10 days)	42	↓ viral loada	2020 [162]
HCQ	SARS-CoV-2	RCT	400 mg/day (5 days)	62	↓ Recovery time	2020 [163]
HCQ	SARS-CoV-2	Pilot	400 mg/day (5 days)	30	Ineffectiveb	2020 [165]
HCQ + AZT	SARS-CoV-2	OS	600 mg/day (10 days)	80	↓ viral load	2020 [164]
HCQ + AZT	SARS-CoV-2	SAOLS	600 mg/day (10 days)	11	Ineffectivec	2020 [166]
CQ	Influenza A/B	RDBPCS	500 mg/day (1 week) Once a week (11 weeks)	1516	Ineffective	2011 [155]
CQ	DENV	RDBPCS	600 mg/day (day 1 and 2) 300 mg/day (day 3)	307	Ineffectived	2010 [156]
CQ	DENV	RDBPCS	500 mg/day BID (3 days)	37	Ineffectivee	2013 [158]
CQ	CHIKV	RDBPCS	600 mg (day 1) 300 mg (BID, days 2 and 3) 300 mg (days 4 and 5)	54	Ineffectivef	2008 [157] 2018 [133]
HCQ	HIV 1	Case report	600 mg/day	2	↓ viral loadg	1996 [169]
HCQ	HIV 1	RDBPCS	800 mg/day (8 weeks)	40	↓ viral load Stable CD4+ level ↓ serum IL-6 & IgG	1995 [170]
HCQ	HIV 1	RDBS	800 mg/day (16 weeks)	72	↓ viral load Stable CD4+ level ↓ serum IL-6 & IgG	1997 [159]
HCQ	HIV	RDBPCS	400 mg/day (42 weeks)	83	Ineffective ↑ viral load ↓ CD4+ level	2012 [161]
CQ – ART	HIV	RDBPCS	250 mg/day (12 weeks)	33	↑ viral replication	2016 [171]
CQ + ART	HIV	RDBPCS	250 mg/day (12 weeks)	37	↓ Immune cell activation	2016 [171]

HCQ: Hydroxychloroquine; AZT: Azithromycin; CQ: Chloroquine; ART: Antiretroviral therapy; SARS-CoV-2: Sever acute respiratory syndrome-coronavirus 2; DENV: Dengue Virus; CHIKV: Chikungunya virus; HIV: Human immunodeficiency virus; SAOLS: Single arm open labelled study; RCT: Randomized clinical trial; OS: Observational study; RDBPCS: Randomized double blind placebo controlled study; RDBS: Randomized double blinded study; BID: Twice per day.

^aSmall sample size study, 1 death and 3 transferred to ICU among 26 patients treated with HCQ + AZT.

^b1 patient developed to sever stage.

^c1 death, 2 transferred to ICU, 1 complained of QT interval prolongation among 11 patients treated with HCQ + AZT.

^dLonger duration of DENV viremia, CQ was associated with a significant reduction in fever clearance time.

^eTemporary improvement in the quality of life.

^fDelayed immune response and more frequent arthralgia in treated group.

^gIn one patient.

Table 6.

Characteristics of ongoing clinical trials studying the efficacy and safety of CQ and HCQ in patients with COVID-19.

Drug	Design	Status	Group(s)	Total No	Primary outcomes	Country Registration No.
HCQ	Interventional ROLCS	Completed	Conventional treatment HCQ	360	Viral clearance	China ChiCTR2000029868
HCQ	Interventional ROLCS	Recruiting	Conventional treatment HCQ	78	Clinical status	China ChiCTR2000029740
HCQ	Interventional RDBS	Recruiting	Placebo HCQ	300	Viral clearance T cell recovery time	China ChiCTR2000029559
HCQ	Retrospective Observational	Not yet recruiting	HCQ	1200	Pneumonia incidence	China ChiCTR2000031782
HCQ	Interventional ROLS	Completed	Conventional treatment HCQ	30	Viral clearance Mortality	China NCT04261517
CQ	Interventional RROLCS	Recruiting	Control CQ	80	Clinical recovery time	China ChiCTR2000030718
CQ	Interventional RCT	Recruiting	Placebo CQ/FAV FAV	150	Improvement or recovery Viral clearance	China ChiCTR2000030987
CQ	Interventional RRSBCS	Recruiting	Placebo CQ	300	Viral clearance	China ChiCTR2000031204
CQ	Interventional ROLCS	Not yet recruiting	HCQ Arbidol	320	No. patients progressed to suspected/confirmed	China ChiCTR2000029803
CQ	Interventional RSBCS	Recruiting	Conventional/CQ CQ	100	Length of hospital stay	China ChiCTR2000029939
CQ	Interventional SAOLS	Recruiting	Conventional/CQ	100	Length of hospital stay	China ChiCTR2000029935
CQ	Interventional OLS	Not yet recruiting	LPV/RTV CQ/LPV/RTV CQ	205	Viral clearance	China ChiCTR2000029609
CQ	Interventional cohort study	Recruiting	Conventional treatment CQ	20	Viral clearance Mortality	China ChiCTR2000029542
CQ	Interventional ROLCS	Recruiting	LPV/RTV CQ	112	Clinical status Mortality Viral clearance	China ChiCTR2000029741
CQ	Interventional OLS	Recruiting	Control CQ	80	Clinical recovery time	China ChiCTR2000029988
CQ	Interventional SAOLS	Not yet recruiting	CQ	10	Viral clearance Mortality	China ChiCTR2000029975
CQ	Interventional RDBPCS	Recruiting	Placebo FAV CQ/FAV	150	Time to and frequency of improvement or recovery Viral clearance	China NCT04319900
CQ	Interventional ROLCS	Not yet recruiting	Carrimycin CQ LPV/RTV Arbidol	520	Fever HRCT Viral clearance	China NCT04286503
HCQ CQ	Interventional ROLCS	Recruiting	CQ HCQ	100	Clinical recovery time	China ChiCTR2000029899
HCQ CQ	Interventional ROLCS	Recruiting	CQ HCQ	100	Clinical recovery time	China ChiCTR2000029898
HCQ CQ	Interventional ROLS	Not yet recruiting	Conventional treatment HCQ CQ	100	Clinical recovery time	China ChiCTR2000030054
HCQ CQ	Interventional ROLS	Not yet recruiting	Conventional treatment CQ HCQ	100	Clinical recovery time Viral clearance	China ChiCTR2000029992
HCQ	Interventional RCT	Not yet recruiting	Placebo HCQ	1600	No. symptomatic confirmed cases	USA NCT04318444
HCQ	Interventional ROLS	Not yet recruiting	Standard of care HCQ AZT HCQ/AZT	500	Clinical status	USA NCT04335552
HCQ	Interventional OLS	Not yet recruiting	HCQ Vit C Vit D Zinc	600	Viral clearance Blood pressure Presence of side effects	USA NCT04335084
HCQ	Interventional RCT	Recruiting	HCQ Vit C	1250	Hospitalization IMV	USA NCT04334967
HCQ	Interventional OLS	Not yet recruiting	HCQ/AZT/Vit C/Vit D/Zinc	60	Symptoms resolution Viral clearance Safety	USA NCT04334512
HCQ	Interventional ROLS	Recruiting	HCQ AZT	1550	Hospital admission	USA NCT04334382
HCQ	Interventional RDBPCS	Recruiting	Placebo HCQ	210	Viral clearance	USA NCT04333654
HCQ	Interventional OLS	Recruiting	HCQ Control	360	Rate of positivity	USA NCT04333225
HCQ	Interventional ROLS	Not yet recruiting	Standard of care HCQ HCQ/AZT	160	Viral clearance	USA NCT04336332
HCQ	Interventional RDBPCS	Recruiting	Placebo HCQ	510	Clinical status	USA NCT04332991
HCQ	Interventional RDBPCS ROLCS	Not yet recruiting	Placebo HCQ	400	Quarantine release rate Hospital discharge rate Infection rate	USA NCT04329923
HCQ	Interventional RCT	Recruiting	Placebo HCQ	3500	Survival/recovery	USA NCT04328467
HCQ	Interventional RDBPCS	Recruiting	Placebo HCQ LPV/RTV LST	4000	Clinical status	USA NCT04328012
HCQ	Interventional ROLS	Recruiting	HCQ AZT	300	Clinical status	USA NCT04329832
HCQ	Interventional RSBS	Not yet recruiting	Ascorbic Acid HCQ	2000	Viral clearance	USA NCT04328961
HCQ CQ	Interventional ROLCS	Recruiting	HCQ HCQ/AZT CQ CQ/AZT	500	Recovery	USA NCT04341727
HCQ	Interventional RCT	Recruiting	Placebo HCQ	3000	Incidence in asymptomatic Severity	USA/Canada NCT04308668
HCQ CQ	Interventional RDBPCS	Not yet recruiting	Placebo CQ HCQ	55000	Disease severity	USA, Australia, Canada, Ireland, South Africa, UK NCT04333732
HCQ	Interventional OLS	Not yet recruiting	Standard of care LPV/RTV HCQ Baricitinib Sarilumab	1000	Clinical status	Canada NCT04321993
HCQ	Interventional RDBPCS	Not yet recruiting	Placebo HCQ	1660	Hospitalization IMV Mortality	Canada NCT04329611
CQ	Interventional ROLCS	Not yet recruiting	Standard of care CQ/AZT	1500	Outpatients: admission or death Inpatients: IMV or death	Canada NCT04324463
HCQ	Interventional RDBPCS	Not yet recruiting	Placebo HCQ LPV/RTV	1200	Confirmed infection in HCW	France NCT04328285
HCQ	Interventional RDBPCS	Recruiting	Placebo HCQ	1300	Mortality IMV	France NCT04325893
HCQ	Interventional ROLS	Recruiting	Standard of care RDV LPV/RTV LPV/RTV/IFβ-1a HCQ	3100	Clinical status	France NCT04315948
HCQ	Interventional ROLCS	Recruiting	RDV LPV/RTV IFβ-1a HCQ	3100	Clinical status	France EudraCT 2020-000936-23
HCQ	–	Recruiting	HCQ	25	Viral clearance	France EudraCT 2020-000890-25
HCQ	Interventional OLCS	Recruiting	Standard of care HCQ/AZT	1000	Incidence Mortality	France EudraCT 2020-001250-21
HCQ	Interventional RDBPCS	Recruiting	Placebo HCQ	1300	IMV Death	France EudraCT 2020-001271-33
HCQ	–	Recruiting	HCQ	50	HCQ pharmacokinetics	France EudraCT 2020-001281-11
CQ	Interventional CSS	Recruiting	Any drug used to treat Covid-19 including CQ	1000	Renal failure	France NCT04314817
CQ	Interventional ROLCS	Recruiting	Standard of care CQ analogue NIVO TCZ	273	Survival rate	France NCT04333914
HCQ	Interventional RDBS	Not yet recruiting	HCQ/LPV/RTV HCQ/LPV/RTV/LEV/BUD/FORM	30	Chest CT-scan Viral clearance	Iran NCT04331470
HCQ	Interventional ROLCS	Recruiting	HCQ/LPV/RTV HCQ/LPV/RTV/IFβ-1b	30	Clinical status Lab/radiological findings Adverse reactions	Iran IRCT20100228003449N27
HCQ	Interventional ROLCS	Recruiting	HCQ/LPV/RTV HCQ/LPV/RTV/IFβ-1a	30	Clinical status Lab/radiological findings Adverse reactions	Iran IRCT20100228003449N28
HCQ	Interventional ROLCS	Recruiting	HCQ/LPV/RTV HCQ/LPV/RTV/SOF/LDV	50	Clinical status Lab/radiological findings Adverse reactions	Iran IRCT20100228003449N29
HCQ	Interventional SAOLS	Recruitment completed	HCQ/OTV/LPV/RTV/IFβ-1a	20	Clinical status	Iran IRCT20151227025726N12
HCQ	Interventional SAOLS	Recruiting	HCQ/LPV/RTV HCQ/ATV/RTV	50	Clinical status Lab/radiological findings Adverse reactions	Iran IRCT20100228003449N30
HCQ	Interventional ROLCS	Not yet recruiting	Standard of care HCQ HCQ/AZT	630	Clinical status	Brazil NCT04322123
HCQ	Interventional ROLS	Recruiting	HCQ HCQ/AZT	440	Clinical status	Brazil NCT04321278
HCQ	Interventional OLS	Not yet recruiting	HCQ/AZT	400	Evolution of ARS, SpO2, hemodynamic stability	Brazil NCT04329572
CQ	Interventional RDBS	Recruiting	Low Dose CQ High Dose CQ	440	Mortality	Brazil NCT04323527
HCQ	Interventional OLCS	Not yet recruiting	RDV HCQ HCQ/RDV	700	Mortality	Norway NCT04321616
HCQ	Interventional ROLCS	Recruiting	Standard of care HCQ	202	Viral clearance	Norway NCT04316377
HCQ	Interventional RCT	Recruiting	Standard of care HCQ/RDV	443	Safety and efficacy	Norway EudraCT 2020-000982-18
HCQ	Interventional ROLCS	Recruiting	Standard of care HCQ	200	Viral clearance	Norway EudraCT 2020-001010-38
HCQ	Interventional ROLCS	Recruiting	Standard measures HCQ/DRV/COBI	3040	Incidence of secondary cases	Spain NCT04304053
HCQ	Interventional RDBPCS	Not yet recruiting	Placebo HCQ TDF/FTC HCQ/TDF/FTC	4000	Confirmed symptomatic infections	Spain NCT04334928
HCQ	Interventional RDBPCS	Recruiting	Placebo HCQ	440	No. confirmed cases	Spain NCT04331834
HCQ	Interventional ROLCS	Recruiting	HCQ/AZT HCQ/AZT/TCZ	276	Mortality IMV	Spain NCT04332094
HCQ	Interventional ROLCS	Recruiting	LPV/RTV Dexamethasone IFβ-1a HCQ	2000	Mortality	UK EudraCT 2020-001113-21a
HCQ	Interventional ROLCS	Recruiting	Standard of care HCQ	350	Change in SpO2/FiO2	UK EudraCT 2020-001270-29
HCQ	Interventional RPCS	Recruiting	Placebo HCQ	3000	Hospital admission Mortality	UK EudraCT 2020-001209-22
HCD CQ	Interventional RDBPCS	Not yet recruiting	Placebo CQ or HCQ	40,000	No. symptoms Severity	UK NCT04303507
HCQ	Interventional ROLCS	Recruiting	No intervention control LPV/RTV HCQ	150	Viral clearance	Korea NCT04307693
HCQ	Interventional ROLCS	Not yet recruiting	Control CIC HCQ/CIC	141	Viral clearance	Korea NCT04330586
HCQ	Interventional RCT	Not yet recruiting	Control HCQ	2486	Incidence	Korea NCT04330144
HCQ	Interventional RPCS	Not yet recruiting	Placebo HCQ	2700	Clinical status	Germany NCT04340544
HCQ	Interventional RPCS	Recruiting	Placebo HCQ	220	Viral clearance	Germany NCT04342221
HCQ	Interventional RPCS	Not yet recruiting	Placebo/HCQ HCQ/COBI	334	Hospital admission	Germany NCT04338906
HCQ	Interventional RDBPCS	Ongoing	Placebo HCQ	220	Viral clearance	Germany EudraCT 2020-001224-33
HCQ	SAOLS	Recruiting	HCQ	150	Dose optimization	Australia ACTRN12620000447954
HCQ	Interventional RCT	Not yet recruiting	Control HCQ LPV/RTV HCQ/LPV/RTV	2500	No. patients not admitted to ICU	Australia ACTRN12620000445976
CQ	Interventional ROLCS	Not yet recruiting	CQ	680	Sick days of HCW	Australia ACTRN12620000417987
HCQ	Interventional RTBCS	Not yet recruiting	Placebo HCQ	400	Infection rate	Mexico NCT04318015
HCQ	Interventional RDBPCS	Not yet recruiting	Placebo HCQ	500	Mortality	Mexico NCT04315896
HCQ	Interventional ROLCS	Not yet recruiting	Control HCQ	1116	Development of severe infection or death	Israel NCT04323631
CQ	Interventional ROLS	Not yet recruiting	Standard of care CQ	210	Viral clearance Clinical status	Israel NCT04333628
HCQ	Observational CCPS	Recruiting	HCQ	80	Protection	Turkey NCT04326725
HCQ	Interventional ROLCS	Not yet recruiting	Convalescent Plasma/HCQ/AZT HCQ/AZT	80	Viral clearance IgM titers IgG titers	Colombia NCT04332835
HCQ	Interventional ROLS	Not yet recruiting	Quarantine/no treatment HCQ/OTV OTV/LPV/RTV HCQ/OTV/DRV/RTV FAV/LPV/RTV HCQ/FAV/DRV/RTV	80	Viral clearance	Thailand NCT04303299
HCQ	Interventional SAOLS	Recruiting	HCQ/LPV/RTV ± OTV	50	CRP level	Japan jRCTs031190227
CQ	Interventional OLS	Recruiting	CQ	60	Symptoms reduction Pneumonia prevention	Greece EudraCT 2020-001345-38
CQ	Interventional ROLCS	Not yet recruiting	Standard of care CQ	250	Viral clearance	Vietnam NCT04328493
HCQ CQ	Interventional RSBCS	Not yet recruiting	Natural Honey LPV/RTV Arbidol HCQ CQ OTV ± AZT	1000	Viral clearance Fever Resolution of lung inflammation	Egypt NCT04323345
HCQ	Interventional RSBCS	Not yet recruiting	Placebo HCQ/AZT HCQ	75	Clinical status	Pakistan NCT04328272
HCQ	Observational Randomized Trial	Not yet recruiting	Control HCQ AZT OTV HCQ/AZT HCQ/OTV AZT/OTV HCQ/AZT/OTV	500	Viral clearance	Pakistan NCT04338698
HCQ CQ	Interventional ROLCS	Not yet recruiting	Standard of care HCQ CQ	950	Disease progression Admission to ICU or death	Netherlands Trial NL8490
HCQ	Interventional RDBPCS	Recruiting	Placebo HCQ/AZT	226	Survival Hospitalization	Denmark NCT04322396
HCQ	Interventional ROLCS	Recruiting	Control HCQ HCQ/LPV/RTV Wide range of drugsb	6800	Mortality Days alive and outside ICU	New Zealand NCT02735707
HCQ	Interventional RDBPCS	Not yet recruiting	Placebo HCQ	440	Viral clearance	Austria NCT04336748
CQ	Interventional ROLCS	Not yet recruiting	CQ/OTV RTV/DRV/OTV LPV/RTV/OTV FAV/LPV/RTV CQ/RTV/DRV/OTV CQ/RTV/DRV/FAV Quarantine	440	Viral clearance	Austria NCT04303299
CQ	Interventional ROLCS	Not yet recruiting	Standard of care CQ	400	Hospitalization or all causes of death	Poland NCT04331600

ROLCS: Randomized open label controlled study; RDBS: Randomized double blind study; ROLS: Randomized open label study; RROLCS: Retrospective randomized open label controlled study; RCT: Randomized clinical trial; RRSBCS: Retrospective randomized single blind controlled study; RSBCS: Randomized single blind controlled study; SAOLS: Single arm open label study; OLS: Open label study; RDBPCS: Randomized double blind placebo controlled study; RSBS: Randomized single blind study; OLCS: Open label controlled study; CSS: Cross-sectional study; RPCS: Randomized placebo controlled study; RTBCS: Randomized triple blind controlled study; CCPS: Case-control prospective study.

HCQ: Hydroxychloroquine; CQ: Chloroquine; FAV: Favipiravir; LPV: Lopinavir; RTV: Ritonavir; AZT: Azithromycin; Vit C: Vitamin C; Vit D: Vitamin D; LST: Losartan; RDV: Remdesivir; IFß-1a: Interferon β-1a; NIVO: Nivolumab; TCZ: Tocilizumab; LEV: Levamisole; BUD: Budesonide; FORM: Formoterol; SOF: Sofosbuvir; LDV: Ledipasvir; OTV: Oseltamivir; ATV: Atazanavir; COBI: Cobicistat; TDF: Tenofovir disoproxil fumarate; FTC: Emtricitabine; CIC: Ciclesonide; DRV: Darunavir.

HRCT: Pulmonary inflammation resolution time, IMV: invasive mechanical ventilation; HCW: Healthcare workers;; ARS: Acute respiratory syndrome; SpO2/FiO2: oxygen saturation/fraction of inspired oxygen ratio; ICU: Intensive Care Unit; CRP: C-reactive protein.

Data were obtained from NIH. U.S. National Library of Medicine (https://www.clinicaltrials.gov/); the Chinese Clinical Trial Registry (http://www.chictr.org.cn/); the European Union Clinical Trials Registry (https://www.clinicaltrialsregister.eu); ISRCTN registry (http://www.isrctn.com/); Netherlands Trial Registry (https://www.trialregister.nl/); Iranian Registry for Clinical Trials (IRCT) (https://en.irct.ir/); Japanese Registry for Clinical Trials (JRCT) (https://jrct.niph.go.jp/) and the Australian New Zealand Clinical trial Registry (ANZCTR) (https://www.anzctr.org.au/).

^aThe same study was registered in ISRCTN registry (registration no. ISRCTN50189673) with a total number of 5000 patients.

^bHydrocortisone, Ceftriaxone, Moxifloxacin or Levofloxacin, Piperacillin-tazobactam, Ceftaroline, Amoxicillin-clavulanate, Macrolide, OTV, IFβ-1a, and Anakinra.

8. Conclusion

The COVID-19 pandemic has spread out of control and has caused considerable morbidity and mortality in several countries. In this unprecedented situation, clinicians have tried all kinds of treatments in an effort to stem the progression of this disease. One treatment that has received huge attention was the empirical use of anti-malarial CQ/HCQ. While there is no strong and enough scientific and clinical data to support their use, several countries have already included CQ/HCQ in COVID-19 treatment protocols [167,168], not only as a treatment option for severely ill patients but also as a prophylactic measure.

In this comprehensive review of the antiviral effects of CQ and HCQ on SARS-CoV-2 as well as other viruses, we show a broad variation in the research outcomes. Both CQ and HCQ demonstrated promising in vitro results, however, such data have not yet been translated into meaningful in vivo studies. While few clinical trials have suggested some beneficial effects of CQ and HCQ in COVID-19 patients, most of the reported data are still preliminary [20,162,163]. Furthermore, at least 7 of the ongoing trials were canceled or stopped and it is not yet clear if this was due to possible adverse effects, ineffectiveness or other reasons.

There are several toxicities associated with these drugs [[78], [79], [80]], the one that is foremost concerning is the possibility of QT prolongation and the risk of Torsades de pointes, which is a potentially life-threatening arrhythmia [[81], [82], [83]]. Nevertheless, while our literature review showed that this is quite rare, it is not yet evident whether there would be any additive or possible synergistic risk when these drugs are combined with other medications such as AZT [83]. In fact, it is challenging to base a treatment decision in the absence of a complete research cycle and a clear vision of drug efficacy and safety. Given the current uncertainty, it is worth being mindful of the potential risks and strictly rational the use of these drugs in COVID-19 patients until further high quality randomized clinical trials are available to clarify their role in the treatment or prevention of COVID-19.

Funding

This work was supported by King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia,

grant number 09-1, which is a part of the Targeted Research Program (TRP).

Declaration of competing interest

None declared.