2.3. Hydroxychloroquine Biological Activity
Besides the antiviral effects, HCQ possesses several other demonstrated biological activities (Figure 6).
Most of all, it showed immunosuppressive properties, allowing its employment (alone or in combination) in the first-line treatment of several auto-immune diseases, like rheumatoid arthritis, lupus erythematosus, primary Sjögren’s syndrome, and anti-phospholipid syndrome. Moreover, HCQ was revealed to be effective in preclinical and clinical trials to dampening autoimmune disease-dependent cardiovascular complications (Figure 7), as well as in the amelioration of disease-independent hyperglycemia, hyperlipidemia, and gastrointestinal complaints.
HCQ exerts anticancer effects by acting synergistically with common chemotherapic drugs. Although it has a well-defined and favorable toxicity profile, the necessity of increasing the dose, in some cases, limits the utilization, due to the toxicity, mainly at cardiac and ocular levels. A two-compartment model, with first-order absorption and a lag time, describes the pharmacokinetics of HCQ. The long-terminal half-life prolongs the time to reach steady-state concentrations, then delays the therapeutic effects. The next-generation formulations allow modulating the pharmacokinetics of HCQ. Avoiding systemic absorption, then liver first-pass metabolism, HCQ may be used in site-specific inflammation, without toxicity [72,73,74]. The selected studies’ primary outcomes are the extent to which HCQ may limit disease progression or exacerbations (Table 2).

2.3.1. Rheumatoid Arthritis
Rheumatoid arthritis is a systemic autoimmune disease, characterized by chronic inflammation and damage to the joints. Immune dysregulation underlies the pathogenesis of rheumatoid arthritis, leading to uncontrolled production of antibodies, mainly rheumatoid factor and citrullinate, involved in the autoreactivity against cartilage and bone [117]. It is estimated that the prevalence of rheumatoid arthritis is around 1%, mainly in women [118]. The immunosuppressive effects of HCQ are due to the ability to modulate T-cell and B-cell hyperactivity, resulting in a reduction of pro-inflammatory cytokine gene expression. As the involvement of neutrophils in this disease, Jancinova, Pazourekova, Lucova, Perecko, Mihalova, Bauerova, Nosal and Drabikova [75] investigated the impact of oral HCQ administration on these cells, in rats with adjuvant arthritis. At doses of 40 mg/kg daily, per oral administration (p.o.), it strongly decreased the blood concentration of neutrophil-derived oxidants, involved in the tissue damage and the onset of chronic inflammation [75].
A 36-week randomized, double-blind, placebo-controlled trial has evaluated the effect of the administration of HCQ in a dose of up to 7 mg/kg per day (maximum 400 mg/day) in patients with rheumatoid arthritis. Within 36 weeks and during the study, HCQ showed statistically significant benefits on physical function, mainly on synovitis and joint pain, without side effects with respect to the control. Moreover, HCQ was associated with a decrement of corticosteroid injections. By contrast, no improvements in psychological function have been demonstrated [76]. Two comparative double-blind, randomized trials, involving 60 patients each, with sulfasalazine and HCQ have demonstrated that HCQ showed no significant differences among overall clinical effects respect to sulfasalazine, but presented a later onset. In the first study, patients treated with HCQ (400 mg/day for 6 months and 200 mg/day for the next 6 months) experienced time-dependent statistically significant improvements in morning stiffness, pain, swollen joints, together with a decrease in blood levels of immunoglobulin M and erythrocyte sedimentation rate [77], while, in the second study, the same treatment was associated to no erosions in the 12% of the patients [78].
In patients with rheumatoid arthritis, the cardiovascular risk is more than doubled, compared to the healthy population. Chronic inflammatory status leads to an intensification of the atherosclerotic process, resulting in a higher susceptibility to hypertension, obesity, and metabolic syndrome. The overproduction of IL-6 is strictly related to lipid profile alterations, given its role in adipose tissue lipolysis. The inflammation and endothelial damage are exacerbated by leptin production. Batun-Garrido, Salas-Magana and Juarez-Rojop [79] found a significant correlation between HCQ treatment and lower IL-6 and leptin levels. The positive effect of HCQ on dyslipidemia was also confirmed by Morris, Wasko, Antohe, Sartorius, Kirchner, Dancea and Bili [80] in a cohort study involving 706 rheumatoid arthritis diagnosed patients, finding a significant and stable cholesterol-lowering, mainly Low-Density Lipoprotein (LDL), and triglyceride decrease associated with HCQ intake (6.5 mg/kg/day). A small but statistically significant amelioration in total cholesterol and LDL under HCQ treatment at the same dosage was also highlighted by a randomized, double-blind cross-over trial on patients with rheumatoid arthritis [81]. The correlation between HCQ and cardiovascular risk was also assessed in a cross-sectional observational study involving 177 women with rheumatoid arthritis. At doses of 200 mg/kg/day, HCQ usage led to lower fasting glucose in women, arising as a valuable tool to enhance glycemic control [82]. However, apparent different outcomes were derived from a randomized double-blind crossover trial, recruiting 23 non-diabetic subjects with stable rheumatoid arthritis to receive 6.5 mg/kg/day of HCQ for eight weeks. For these patients, no significant changes in insulin resistance were observable [81]. This study evaluated only insulin sensitivity, by Homeostatic Model Assessment (HOMA) index, without considering insulin metabolism. Thus, inconsistency should be explained by this factor, together with the short duration of treatment. The anti-diabetic properties of HCQ have been also assessed in patients without arthritis, as reported in the next paragraphs.

2.3.2. Lupus Erythematosus
Systemic lupus erythematosus is a multisystemic autoimmune chronic inflammatory disorder that mainly involves joints, mucosae, skin, and endothelial vessels [90]. For a long time, HCQ has played a marginal role in the overall management of the disease. Since the 90s, the first evidence of HCQ effectiveness in controlling lupus erythematosus manifestations allowed its employment as a first-line medicament. Although it was not recommended in single therapy, the immunomodulating properties of HCQ seem to play an important role in the disease pathogenesis. It is associated with a decrement of exacerbation events, as well as protective effects towards vascular and thrombotic events [85]. Indeed, a 24-week randomized, double-blind placebo-controlled trial demonstrated that the discontinuing of HCQ treatment (100–400 mg/kg/day for at least six months) increased the risk of exacerbations by 2.5 times in patients with quiescent lupus. People who interrupted the therapy exhibited constitutional symptoms of the disease, as well as skin rashes, arthritis, and ulcers [83]. A long-term randomized study evaluated the withdrawal effects of HCQ on quiescent lupus erythematosus patients, revealing that a reduction by up to 57% is associated with HCQ maintaining treatment (272 mg/day) [84]. A case-control study was carried out in order to define the role of HCQ in the survival of individuals affected by lupus. The positive correlation between HCQ and survival led to the consideration of this drug as a great therapeutic option at the proper dose (6.5 mg/kg/bw) in lupus management [85]. If these clinical trials demonstrated the advantages of keeping up the therapy with HCQ in preventing disease exacerbations, doubts persisted about the efficacy of this treatment in the control of more severe clinical forms [83]. As the important role of the imbalance between immune cell populations, several preclinical and clinical studies have evaluated the role of HCQ in restoring this equilibrium. In particular, elevated levels of effector lymphocyte T (Th17) that mediate the autoimmune answer and decreased levels of regulatory lymphocyte T (Treg) that guarantees the immune homeostasis, may be observed in autoimmune diseases, in particular in lupus. Lately, autophagy had risen among the emerging strategies to reestablish the immune balance. As HCQ is a well-known autophagy inhibitor, An, N. et al. [86] evaluated the influence of HCQ intake (100 mg/kg/day) in MRL/lpr mice with the lupus-like disease. After four weeks of treatment, HCQ clearly restored the immune balance, by both inhibiting Th17 response and enhancing Treg immunosuppressive effects. The levels of autoantibodies and the expression of inflammatory cytokines, mainly in Th17 cells, were remarkably lowered, due to the inhibition of the activated autophagy, as demonstrated by the increase of autophagic flux marker expression in Th17 and Treg, compared with controls. This randomized trial further evidenced that HCQ treatment also remarkably attenuated kidney inflammation, by limiting the migration of lymphoplasmacytic cells into renal tissues [86]. Preclinical outcomes have been confirmed by a prospective cohort study, involving 41 patients with a diagnosis of lupus treated with 400 mg/day of HCQ. Dysregulated cytokine and autoantibody production, deriving from high autoreactivity that characterizes lupus disease, has been restored by two months of HCQ administration, demonstrating its ability in modulating inflammatory response, with normalized complement activity and reduced levels of pro-inflammatory cytokines, mainly IL-6 and TNF-α [87]. In a multiethnic US cohort on 35 lupus patients, HCQ treatment resulted in significant clinical benefits towards disease progression, probably due to the inhibition of toll-like receptor activation, resulting in down-regulation of IFN-α, which plays a pivotal role in lupus pathogenesis [88].
Infiltrating cells, most of all mast cells, could involve skin tissues, causing one of the most common signs of lupus, skin rashes. The consequences of HCQ intake on skin lesions have been investigated on a MRL/lpr murine model of lupus at low (4 mg/kg/day) and high (40 mg/kg/day) oral doses. The number of mast cells decreased with respect to the drinking-water control (from 81 to 50 in low-dose and 12 in high dose), while the mortality rate decreased by up to three times in both treated groups with respect to the control. These in vivo results, together with a significant histopathological alteration regression, suggest that HCQ is a good tool against skin injury in lupus erythematosus [89].
The chronic inflammatory status that features lupus erythematosus leads to a higher susceptibility to cardiovascular complications. Lupus, indeed, is often characterized by endothelial dysfunction, the earliest marker of cardiovascular disease, as well as hypertension and renal damage. In a NZB/W F1 mice model of lupus, oral HCQ gavage of 10 mg/kg/day for five weeks reduced the incidence of thromboembolic events. Moreover, improvements in hypertension, renal damage, and heart hypertrophy occurred, probably due to the normalized endothelium response and reduction of Reactive Oxygen Species (ROS) attributable to HCQ intake [90]. The same effect of normalizing nitric oxide and ROS production have been confirmed in a NZB/W F1 murine model at different times. HCQ at 3 mg/kg/day p.o. protected vascular endothelium, with a strong improvement of endothelial dysfunction [91]. The benefits on atherosclerosis also pass through the lipid-lowering power of HCQ. A clinical study on 155 autoimmune patients revealed a statistically significant association between HCQ (400 mg/day) and a lessening of triglyceride and cholesterol levels, mainly LDL, while no HDL changes were observed [92]. By contrast, HCQ in patients with a mild or inactive condition had no significant effects on lipid profile. A survey involving 65 Chinese lupus patients, treated with HCQ (244 ± 86 mg/day), demonstrated that only triglycerides tended to be lowered, while no statistically significant changes are observable in cholesterol levels [119]. The use of HCQ may be helpful in minimizing cardiovascular risk by improving glycemic homeostasis in lupus patients. A cross-sectional study performed between 2000 and 2005 on 149 nondiabetic women affected by lupus estimated that a mean dose of 400 mg of HCQ affects insulin sensitivity and resistance, as assessed by HOMA index, as well as fasting glucose levels [82].
HCQ remains a worthwhile primary or additional therapy in lupus patients, considering the low cost and its safety profile, also in pregnancy. A randomized double-blind study reported the safety of HCQ during pregnancy, correlating this drug with less disease activity and a lower required dose of prednisone [93]. A 5-year prospective study evaluated the effect of HCQ discontinuation on lupus progression in pregnant women. As it occurs in no pregnant people, interruption of HCQ treatment is linked to an exacerbation of the disease. Moreover, there are no statistically significant differences regarding pregnant complications with respect to the control, showing no fetal toxicity at a dose of 6.5 mg/kg/day in breast milk [94]. Fetal safety has been also assessed in women with lupus nephritis by a multicenter study, reporting a reduction of 85% of the possibility of having a small for gestational age baby in patients under HCQ. Moreover, it exerted protective effects on fetal growth [120].

2.3.3. Antiphospholipid Syndrome
Antiphospholipid syndrome is an autoimmune disorder characterized by antiphospholipid autoantibodies production. If it is not associated with other autoimmune diseases, it is called primary [97]. The incidence of the pathology is greater in young women of reproductive age and it often has a negative impact on pregnancy, with unfortunate outcomes due to the development of placental ischemic pathologies. Resonance spectroscopy, indeed, revealed that the fetal brain and placenta are the main targets of autoantibodies localization [95]. As complement activation plays a central role in the occurrence of the disease, many studies have evaluated the role of HCQ in inhibiting complement activation. In a mouse model of obstetric antiphospholipid syndrome, HCQ, administered in a daily dose of 200 μg per mouse limited placental abnormalities, with an increase of fetal survival, by inhibiting complement activation [95]. A case report on the use of HCQ on a pregnant woman with recurrent venous thromboembolism confirmed the efficacy of HCQ also in clinical practice. The addition of 400 mg daily of HCQ to a common therapeutic regimen of aspirin and heparin dramatically reduced the episodes of vascular thrombosis [96], showing great antithrombotic properties. Given these results, the mechanisms underlying the use of HCQ in thromboprophylaxis were assessed. Two similar preclinical studies, using one-week treatment with 12 μg/g/day of HCQ and three-weeks treatment with 20 mg/kg/day of HCQ, respectively, were in accordance to assess that the overall amelioration of thrombotic status in mice models of antiphospholipid syndrome was linked to endothelial function improvement by modulating the expression of nitric oxide synthase [97,98]. Moreover, the efficacy of HCQ in antithrombotic therapy may lie in the interference in the coagulation cascade. HCQ, indeed, was revealed to decrease the levels of soluble tissue factor, a key initiator of the process, in patients with antiphospholipid syndrome after three months of therapy with a daily dose of HCQ of 200 mg [99].

2.3.4. Sjögren Syndrome
Sjögren syndrome is an autoimmune disease with a strong negative impact on the quality of life of affected people. The main features of the disease are lymphocytic inflammation and alterations in major salivary glands, causing xerostomia [103]. Even if preliminary results about HCQ use in Sjögren syndrome were not encouraging, to date it arises as one of the first-line drugs in disease treatment. Indeed, an earlier prospective, a two-year double-blind crossover trial on 19 subjects correlated an annual intake of a dose of 400 mg/day with no significant improvements in clinical symptoms and signs of pathology, including tear and salivary gland activity, respect to the placebo [121]. However, a few years later, the first evidence of HCQ effectiveness in Sjögren syndrome treatment was reported. Annual treatment with a dose of 200 mg/day of HCQ showed anti-lymphoproliferative and anti-inflammatory effects, with a reduction of IgG and IgA immunoglobulins, anti-Sjögren autoantibodies, and erythrocyte sedimentation rates [100]. Moreover, the salivary flow rate increased in Sjögren syndrome women who received a daily dose of 400 mg of HCQ for 30 weeks [101], while eye dryness was alleviated by HCQ administration (6.5 mg/kg), as demonstrated by a prospective study on 32 patients [102]. Hypo-salivation deriving from acinar atrophy and fibrosis of salivary glands is often associated with over-expression of TGF-β (transforming growth factor-β). Treatment with HCQ downregulated TGF-β levels in a randomized trial on NOD mice exposed to doses of 50 mg/kg/day intragastrically (i.g.) for 16 weeks, with significant results in delaying loss of saliva secretory function. Moreover, HCQ intake was also accompanied by a decrease in autoantibody production and a lower lymphocytic infiltration [103]. These findings were confirmed by Wu, Pu, Yu and Li [104], showing that 8-weeks treatment with HCQ administered at a dose of 60 mg/kg i.g. in 40 randomized NOD mice led to lower lymphocytic infiltration, with a significant improvement in pathological changes in submandibular gland morphology.

2.3.5. Diabetes
As has already been demonstrated for autoimmune patients, HCQ demonstrated great anti-diabetic properties. The first proofs of the role of HCQ in glucose and insulin homeostasis date back to 1999, when Emami, Gerstein, Pasutto, and Jamali [105] demonstrated that diabetic rats treated with oral doses of 80, 120, and 160 mg/kg/day of HCQ exhibited a dose-dependent increase in insulin blood levels, with a consequent reduction of glucose concentration. Higher doses of HCQ (200 mg/kg/day) were tested by Abdel-Hamid, A.A. and El-Firgany Ael, D. [106] on diabetic rats, finding an HCQ-mediated decrease in the pancreas, as the mechanism underlying the improvement of the metabolic profile in diabetic rats. The same authors associated the beneficial impact of HCQ on insulin resistance in diabetic rats with its ability to restore adipokine balance and reduce endothelial stress markers [113]. Given the positive outcomes deriving from preclinical studies, the therapeutic potential of HCQ was also assessed in several clinical trials. Included in a randomized, double-blinded study of 18 months with 300 mg of HCQ twice a day were 135 diabetic obese patients. HCQ treatment improved glycemic control, as demonstrated by the decrease of glycated hemoglobin by up to 1% respect to the placebo, without any side effects [107]. An open-label longitudinal study engaging 13 obese non-diabetic individuals examined the effects of a dose of 6.5 mg/kg/day of HCQ for six weeks, demonstrating a significant reduction in insulin resistance, assessed by HOMA index [108]. In a randomized, double-blinded, controlled trial on 39 prediabetic subjects, the effect of 12-week treatment with 6.5 mg/kg/day of HCQ on glycemic status and lipidic profile was evaluated. Results reported a significant increase in insulin levels, demonstrating the potential use of HCQ to counteract the risk of developing diabetes [109]. A randomized double-blind study involving 267 type-2 diabetic patients compared the efficacy of HCQ (400 mg/day) and pioglitazone, a common anti-diabetic drug, in the control of glycemic and lipidic profiles. No statistically significant differences emerged between the two medicines in terms of glycated hemoglobin and glucose levels, although both drugs produced an improvement in glycemic parameters. Regarding lipidic status, total cholesterol and LDL levels were reduced more by HCQ than pioglitazone. Given the good tolerability of the treatment, HCQ may arise as a therapeutic alternative in diabetes management [110].

2.3.6. Others (Cancer, Inflammation, Cardiovascular Diseases)
Given the well-recognized properties of HCQ against inflammation, it is easily intuitable that this agent could possess interesting insights into cancer treatment. Chronic intestinal inflammation predisposes to the risk of colitis-associated colorectal cancer. In vivo, HCQ was demonstrated to interfere with cancer growth at different stages of development, both preventing tumorigenesis in the early phases and inhibiting tumor growth in the late phases in mice treated with azoxymethane and dextran sodium sulfate to induce cancer. In terms of animal survival, 120 days treatment with 50 mg/kg of HCQ intraperitoneal injection (i.p.) almost restored the survival rate to pre-treatment values and reduced the size of the tumor. The therapeutic effects of HCQ may be attributed to the significant inhibition of pro-tumorigenic and pro-inflammatory cytokines, which not only limited the tumor progression by reducing inflammation of lamina propria, but also decreased the ROS production in macrophages [111]. Many others are the mechanisms by which HCQ exerts anticancer effects, mainly in synergism with conventional chemotherapic drugs, as discussed later.
Regarding the cardioprotective effect, this review has already focused on the positive impact of HCQ on cardiovascular issues in autoimmune patients. A protective effect of HCQ on neonatal rat cardiomyocytes was proven by Bourke, McCormick, Taylor, Pericleous, Blanchet, Costedoat-Chalumeau, Stuckey, Lythgoe, Stephanou and Ioannou [112] in ischemia-reperfusion animal models. The pharmacological preconditioning with HCQ seems to be a good strategy to protect from ischemia-reperfusion injury. The pretreatment with daily gavage of 200 mg/kg of HCQ, indeed, reduced the cardiac infarct size by 47%. The mechanism underlying this effect is linked to the inhibition of apoptosis and total cell death in neonatal rat cardiomyocytes. The atherosclerotic process contributes to increasing the risk of heart failure. The etiology of this condition is still not clear. A hypothesis supposes that the accumulation of lipids in vessels caused the formation of atherosclerotic plaques that are responsible for vessel narrowing, shear stress, and platelet aggregation. HCQ decreased free-fatty acids, triglycerides, total cholesterol, and LDL levels in diabetic rats under doses of 200 mg/kg/day [106]. Moreover, HCQ (10 mg/kg/day) was demonstrated to exhibit functional and structural protection in 40 high-fat diet mice, by reducing atherosclerotic area by 60% with respect to the control [114]. These favorable effects at the metabolic level might be due to its anti-inflammatory power that influences many other biological activities. In gastrointestinal inflammations, mainly in inflammatory bowel disease, HCQ suppressed pro-inflammatory cytokines and enhanced the expression of ILs involved in anti-inflammatory processes. In mouse models of colitis, the HCQ methacryloylated form (30 mg/kg) avoided systemic absorption, accumulating in the gastrointestinal tract, where alterations in the immune homeostasis of the intestinal mucosa had a positive impact on the disease [74]. Inflammation, together with alterations in the immune system, are at the basis of pulmonary hypertension. The ability of HCQ to interfere with the production of pro-inflammatory cytokines from monocytes and lymphocytes might underlie the observed improvements in systolic pressure and ventricular hypertrophy, in rats with pulmonary hypertension treated with 50 mg/kg/day i.p. of HCQ for 20 days [115]. Likewise, in endometriosis, the abnormal presence of endometrium in other organs leads to a chronic inflammatory status that could be affected by HCQ intervention. Ruiz, Rockfield, Taran, Haller, Engelman, Flores, Panina-Bordignon and Nanjundan [116] observed an increment of peritoneal macrophages in mouse models of endometriosis under HCQ (60 mg/kg i.p.). In their role of scavengers, abnormalities in these cell populations may lead to an accumulation of endometrial cells, with impairment of the disease. Moreover, histopathologic improvement of lesions was observed, probably due to the inhibition of autophagy by HCQ that alters anoikis response of endometrial cells [116].