2.4. Hydroxychloroquine and Synergic Effect
The choice of HCQ as an additive therapy in many medical regimens is due to the synergistic effect that enhances the efficacy of other drugs in the treatment of several diseases that have been frequently demonstrated as follows.

2.4.1. Autoimmune Diseases
HCQ belongs to the group of the disease-modifying antirheumatic drug (DMARD), which comprises drugs that are not chemically relted, sharing the same efficacy in dampening the progression of rheumatoid arthritis [122]. It often occurs also that glucocorticoids or natural antioxidant substances are included in the coadjutant therapy of rheumatoid arthritis [123]. A multicenter, randomized clinical trial analyzed the tolerability and the efficacy of combined therapy, including HCQ, sulphasalazine, MXT, and PRD with respect to the use of a single antirheumatic drug in the caring of early rheumatoid arthritis for two years. A total of 195 patients were equally divided into two groups to follow the assigned therapeutic protocol. The primary aim of clinical remission was achieved after one year by 24 of the 97 patients under combinatory therapy, while only by 11 of 98 single-drug therapy users, but this trend was further confirmed during the second year. After one year, 75% of subjects under combinatory therapy and 60% of those under single-therapy reached clinical improvement, intended as 50% clinical response. In terms of tolerability, the cotreatment resulted not to be more dangerous with respect to the single drug [123]. In a prospective trial, patients with the diagnosis of rheumatoid arthritis were scheduled to receive cotreatment of sulphasalazine (1–3 g/day), MXT (7.5–15 mg/week), and HCQ (200 mg/day) for six months. Significant improvements in clinical parameters revealed the efficacy of the cotreatment in counteracting endothelial injury. Indeed, the blood concentrations of endothelial injury markers, mainly soluble E selectin and thrombomodulin, experienced a significant drop after the cotreatment [124]. Likewise, a single-blinded clinical trial on 281 patients confirmed the better therapeutic efficiency of cotreatment (25 mg/week MXT, 2 g/day of sulphasalazine, and 400 mg/day HCQ p.o. plus intramuscular injection (i.m.) doses of 120 mg of methylprednisolone or 80 mg of triamcinolone) with respect to single therapy after three months, without significant differences in side effects [125]. Proofs of the better antirheumatic potential of the combination of drugs with respect to single therapy derived from an observational study that evaluated the higher improvement of quality of patients’ life after one year of coadministration of MXT, HCQ, and corticosteroids with respect to single MXT, or HCQ, or their combination with corticosteroids [126]. Great insights in disease remission were provided by a clinical trial involving 17 patients with active rheumatoid arthritis where the chronic inflammatory status of joints was evaluated through the 18F-FDG PET method. It was found that cotreatment with 7.5–15 mg/week of MXT, 2 g/day of sulphasalazine, 5 mg/kg/day of HCQ and a low dose of oral PRD (under 10 mg/day) is associated with a reduction of 30% in 18F-FDG uptake measures on PET imaging in 81% of patients after four weeks [127]. Although HCQ is effective and well-tolerated, other therapeutic alternatives have emerged in recent years. Among them, monoclonal antibodies are the most promising. In a multicenter open-label clinical trial, performed on 60 patients with juvenile idiopathic arthritis for 54 weeks, the combination of infliximab monoclonal antibodies with a conventional antirheumatic drug provided the better response in terms of disease inactivation with respect to DMARD administration [128].
In addition to rheumatoid arthritis patients, HCQ is also used in lupus erythematosus, another autoimmune disease. Regarding lupus erythematosus, HCQ is widely used. In vivo studies on a NZB/W F1 murine model of lupus showed that HCQ in combination with PRD (2.5 mg/kg/day and 1 mg/kg/day p.o.) decreased autoantibody production, as well as being able to inhibit toll-like receptor activation, resulting in the down-regulation of type I interferon (IFN-α) which is deeply implicated in lupus pathogenesis. The efficacy of treatment is due to the ability of drugs to alter the expression of urinary and immune cell micro RNA that contribute to lupus progression by post-transcriptional modulation of genes involved in the immune response, pro-inflammatory cytokines production and toll-like receptor pathways [129]. In association with low-dose aspirin, HCQ is also indicated for thromboprophylaxis in patients with lupus. The occurrence of thrombotic events was recorded for 13 years in 189 patients, showing that the cardiovascular complications were more frequent in patients treated only with aspirin, while HCQ (up to 600 mg) was associated with a stronger thrombo-protective effect in patients with lupus [130].
Thanks to its immunomodulatory properties, HCQ (20 mg/kg) was revealed to be useful in graft-versus-host disease, in combination with cyclosporine A. They synergistically suppressed T cell response, allowing the reduction of the dose of drugs in mice [131].

2.4.2. Cardiovascular Risk Management
HCQ revealed the great potential in the management of cardiovascular risk by controlling glucose homeostasis and lipidic profile. Until now, in this review, we discussed the effect of HCQ alone to counteract cardiovascular complications, mostly in autoimmune patients. Here, we reviewed the multiple pleiotropic actions of HCQ in combination with conventional medication in the most common cardiovascular diseases. The cardioprotective effects of one week of treatment with 50 mg/kg of HCQ i.g. against ischemia-reperfusion injury in type-2 diabetic mice were assessed in combination with the phosphodiesterase-5 inhibitor, tadalafil. The synergistic effect reduced the myocardial infarct size by up to 20% and improved insulin secretion and sensitivity [132]. Moreover, low-dose HCQ (3.4 mg/kg/day) prevented cardiomyocyte apoptosis in the periinfarct myocardium, dampening ischemic cardiomyopathy, and cardiac stroke, as demonstrated by Jalal, et al. [133] in rat models. The role of HCQ in the inhibition of platelet aggregation was evaluated in healthy volunteers in comparison with aspirin or clopidogrel. The addition of 400 mg/day of HCQ to aspirin resulted in a significant increase in aggregation inhibition (31%). This inhibition was passed by reducing fibrinogen and inflammatory status by interfering with the arachidonic acid cascade [134].

2.4.3. Anticancer
HCQ explains its antitumor activity thanks to its ability to inhibit autophagy. HCQ is, indeed, an FDA-approved drug inhibiting autophagy [135]. Several types of tumors develop chemoresistance by enhancing autophagic flux. Autophagy consists in the sequestration of materials in autophagic vesicles to be eliminated through lysosomal fusion and allows cells to overcome metabolic and therapeutic stresses. By recycling intracellular components, cells may maintain an energy balance and increase their growth. If it occurs in cancer cells, resistance mechanisms may establish. One of the mechanisms responsible for drug resistance is related to increased drug efflux by ATP-binding cassette (ABC) transporters [136,137]. It has been observed that HCQ is significantly reduced the increase in P-gp (ABCB1) expression and, in combination with several anticancer drugs, induced higher cytotoxicity in refractory cancers by inhibiting autophagic activity [138]. However, the role of autophagy in cancer is controversial and depends on genotype and tumor stage development [139]. Many clinical trials examined the synergistic effects of the addition of HCQ to conventional chemotherapic drugs, finding that the role of autophagy is complex and is influenced by several factors. Depending on genetic concomitant alterations, autophagy may possess both pro-tumorigenic and tumor-suppressive roles. It has been confirmed in murine models of pancreatic ductal adenocarcinoma, a type of cancer with a high mortality rate, due to its refractoriness to therapies. Mice presenting activated oncogenic KRAS and normal expression of p53 oncosuppressor experienced a critical regression of tumor developing under HCQ (60 mg/kg/day i.p.). By contrast, in those with a deficiency of p53, the inhibition of autophagy by HCQ increased the tumor progression, demonstrating that autophagy’s role in tumorigenesis is strictly related to the expression of p53 [140]. The expression of p53 is often altered in cancer, so as to be found mutated or absent in the 75% of pancreatic ductal adenocarcinomas [141]. This issue highlights the necessity to carefully evaluate the use of HCQ in certain tumor types. Different outcomes have been previously described, it has been found that inhibition of autophagy by HCQ might arise as a valuable adjuvant in pancreatic ductal adenocarcinoma chemotherapy, regardless of p53 status [142,143]. Given the same doses and route of administration, these inconsistencies between the two reported studies could probably derive from the use of p53 homozygous and heterozygous models of mice, respectively. Regarding KRAS oncoprotein, its downstream pathway is one of the major players of pancreatic carcinogenesis. The inhibition of this pathway by cytotoxic drugs, as well as trametinib, is often associated with an increase in autophagy. For this reason, Drucker and Rosen [144] performed an off-label trial with an association of 400–1200 mg of HCQ and a constant dose of trametinib, observing a partial response with a general reduction of tumor lesion size, circulating tumor markers and cancer-associated pain.
In other cancer types, such as ovarian, prostatic, and human breast cancer, the anticancer or pro-tumorigenic effects of HCQ are determined by tumor stage. In the early stages of the disease, the inhibition of autophagy results in an inhibition of tumorigenesis, while in the advanced phase, it enhances cancer survival [145]. Then, it is important in assessing the contextual role of HCQ in cancer resistance mechanisms. Epirubicin in triple-negative breast cancer therapy often lost efficacy, due to chemoresistance acquiring. It has been shown that this cytotoxic agent induced autophagic flux, increasing cancer cell survival. The combination with HCQ (120 mg/kg by i.p.), thanks to the anti-autophagic properties, significantly suppressed tumor growth by up to 50% with respect to the monotherapy [142]. In addition, estrogen receptor-positive breast cancers developed resistance to treatment with tamoxifen, due to the enhancement of autophagy. The coadministration of HCQ (1–2 mg/day/mice in drinking water) restored the susceptibility of cancer cells to tamoxifen [146]. In mice with thyroid gland xenograft carcinoma, HCQ (150 mg/kg/day p.o. for two weeks) did not provide significant results on tumor growth, while the combination of HCQ with the chemotherapic agent vemurafenib potentiated the anticancer properties of both drugs [147]. Similarly, the two weeks coadministration of HCQ (65 mg/kg) and CCI-779 resulted in a synergism that significantly enhanced their in vivo activity against melanoma tumor growth, in terms of tumor size, with respect to their single treatment [148]. HCQ was revealed also to be active against chemoresistant lung cancer. In this type of cancer, the hypoxic conditions led to lesser susceptibility of cancer cells towards lymphocyte T-mediated cytolysis, thanks to the activation of autophagy. HCQ intake, at doses of 30 mg/kg/day i.p. for 10 days, sensitized tumor cells to lysis and allowed, together with conventional treatment, the eradication of the tumor [149].
Together with autophagy, glycolysis plays a pivotal role in satisfying the increased energetic demand. The dual targeting of the processes may provide a new therapeutic approach in cancer cells. Emonet, et al. [150] performed a randomized preclinical study on Earlic ascites hepatoma-bearing mice, showing that the coadministration of HCQ (60 mg/kg i.p.) and the antiglycolytic inhibitor 3-bromopyruvate possessed a synergistic effect on tumor growth inhibition. Moreover, this treatment is associated with an improvement of oxidative status in hepatic tissue, with a decrement in the number of cancer cells, without affecting the total cell count [151].
Resistance mechanisms also involved alterations in β-Cell Lymphoma (Bcl) Bcl-2 and Bcl-xL and anti-apoptotic gene expression. To evaluate the validity of the dual approach, targeting both apoptosis and autophagy, HCQ (50 mg/kg i.p.) and an apoptosis inhibitor, ABT-737, were administered to prostatic cancer xenograft mice for 15 days. Tumor growth was significantly suppressed by a combination of drugs, with respect to HCQ or ABT-737 alone [152]. In the same way, Fenollar, et al. [153] demonstrated the efficacy of Obatoclax, a pan-Bcl-2 inhibitor, used in association with HCQ (3–60 mg/kg) or conventional in neuroblastoma-bearing mice. Positive outcomes regarded the diminution of tumor size and the complete absence of metastases in cotreated mice with respect to Obatoclax alone or with respect to control [154]. Apoptosis is also at the base of the anticancer activity of interferon-alpha, but the cancer treatment with this drug alone often leads to chemoresistance. It has been demonstrated that autophagy is in the main responsible for chemoresistance, thus the combination of interferon-alpha with inhibitors of autophagic flux may be a useful therapeutic approach. In 30 xenograft mice with head and neck squamous carcinoma, the combination of interferon-alpha with HCQ (60 mg/kg/day i.g.) and wortmannin synergistically promoted apoptosis and inhibited tumor growth [155]. In a similar fashion, Le Goff, et al. [156] investigated the potential synergic role of HCQ (30 mg/kg) in enhancing the anticancer activity of melatonin on tongue squamous cell carcinoma mouse models. The anticancer activity of melatonin depends on its pro-apoptotic effects. Nevertheless, this activity is accompanied by a pro-autophagic activity that caused chemoresistance. The coadministration of the autophagy inhibitor HCQ strongly enhanced melatonin anticancer efficacy, resulting in a smaller tumor size and weight. The effect of inhibition of autophagy on tumor growth may be enhanced if the inhibition of autophagic flux occurs when the process of autophagy is quite completed. This hypothesis has been evaluated by Brönnimann, et al. [157], administering by intravenously TAT–Beclin 1 peptide and HCQ (65 mg/kg) in murine models of breast cancer. Initially, the first agent induced the autophagic flux with the production of autophagosomes, while in the final phase of the process, the second stopped the autophagy by deacidification of lysosomes, causing the accumulation of autophagic vesicles and tumor death. HCQ was administered as HCQ-loaded liposomes, to modulate the onset of autophagy inhibition [158]. This formulation allows us to overcome the limits of HCQ usage, related to the high doses required, which is often unachievable in humans. Relatively high doses of HCQ were loaded in nanoparticles, together with the cytotoxic drug chlorambucil, demonstrating it to be safe and efficient in killing leukemia/lymphoma cancer cells in a human-mouse model of Burkitt’s lymphoma. Eight injections of nanoparticles containing 400 mg of HCQ and chlorambucil led to the overall survival of mice. These concentrations of free drugs are inapplicable, due to their high toxicity [159]. As demonstrated by Naso, Wong, Wong, Chen and Hoang [72], HCQ liposomes (60 mg/kg), together with a pH-sensitive targeting peptide that delivered HCQ into the tumor cells and lysosomes, enhanced the chemotherapic effect of conventional anticancer drug doxorubicin in animal models of melanoma. Likewise, Vayssade, et al. [160] conceived a nanogel (CA4-FeAlg/HCQ) for co-addressing vascular blocker CA4 and anti-autophagic agent HCQ (30 mg/kg) in tumor blood vessels, to synergistically treat A549 lung cancer in mice. Firstly, the release of CA4 exerted anti-angiogenic effects in the vascular site, then FeAlg/HCQ were released into small nanogels and entered in the tumor, where HCQ inhibited autophagy and iron generated ROS with a synergic antitumor effect [161]. Similarly, De Jong, et al. [162] evaluated the response of an animal model of pancreatic cancer to HCQ (5 mg/kg) and paclitaxel administration, loaded in liposomes, modified with an acid environmental sensitive peptide that is responsible for site-specific delivery. Tumor weight, together with the number of liver metastases, was significantly reduced. The administration of HCQ is associated with the inhibition of autophagy and the reduction of IL-6 that is responsible for cross-talking between cancer cells and fibroblasts. All these events avoided the formation of stroma fibrosis, allowing paclitaxel to easily reach the tumor site [104]. The synergism results are essential for HCQ activity in pancreatic cancer. In monotherapy, indeed, HCQ (800–1200 mg/day) did not achieve significant autophagy. This resulted in negligible therapeutic effects in patients with already-treated metastatic pancreatic cancer, of which only 10% were without the progressive disease after two months of therapy [163].
Therefore, the use of modified formulations, such as liposomes, nanogels, etc., may be a precious tools for drug codelivery at the tumor site, enhancing efficacy and reducing side effects. Moreover, the availability of HCQ in those formulations encouraged the use of this molecule in brain tumors, as this formulation highly improved the penetration of this drug in the brain–blood barrier. The co-encapsulation of HCQ with a tyrosine kinase inhibitor, ZD6474, exhibited a synergistic effect, increasing the survival of glioma-bearing mice by two-times with respect to free ZD6474. Those synergistic effects are attributable to significant inhibition of autophagy exerted by HCQ and might provide a valuable therapeutic tool in glioma treatment [164].
The anticancer effect of free HCQ at 200–800 mg/day p.o. has been evaluated in two similar clinical trials on glioblastoma patients in concomitant temozolomide drugs and radiotherapy. Although a dose-dependent significant increase of autophagy markers, no significant effects on tumor suppression were recorded in both studies, as the dose-limiting toxicity was not allowed to achieve higher doses of HCQ [162,165]. The maximum tolerated dose is the dose over which at least one patient from six experienced dose-limiting toxicity, including myelosuppression, anorexia, fatigue, or nausea. Moreover, it has been proved that HCQ severely altered the organization of the Golgi apparatus and the endolysosomal system in C57BL/6JolaHsd mice under 60 mg/kg/day of HCQ i.p. [166]. However, different from Rosenfeld’s studies, no maximum tolerated dose was reached for HCQ in combination with chemotherapic temsirolimus, allowing us to perform a dose-escalation study on 27 patients with solid tumors and 12 with a melanoma diagnosis. In both cases, the standard intravenous dose of temsirolimus with 1200 mg of oral HCQ was considered safe and tolerated and inhibited tumor growth [167]. The same authors further assessed the HCQ anticancer properties and dose-limiting toxicity on 40 patients with metastatic melanoma, by administering a dose intense regimen of temozolomide and escalating doses of HCQ (200–1200 mg/day p.o.). Patients well tolerated the treatment, showing a positive response in the 14% of cases and stability of disease in 27%, due to the modulation of autophagy. No maximum tolerated dose was reached, although common toxicities were manifested [168]. According to the results of Rangwala, a phase I trial on 25 patients with myeloma demonstrated that the recommended dose of HCQ for a phase II trial is 600 mg twice a day. Among eligible patients, 14% experienced a very good response, 14% minor responses, and 45% a period of stability in the disease when the association of HCQ and bortezomib were provided. The synergic effect on myeloma was probably due to the combination of inhibition of HCQ on autophagy and bortezomib on proteasomal degradation, leading to the accumulation of misfolded proteins and autophagic vacuoles in cancer cells [169]. Likewise, doses of 600 mg of HCQ twice a day are not associated with toxicity and its usage as adjuvant therapy with everolimus was well tolerated and produced disease control in 67% of the metastatic clear-cell renal cell carcinoma patients and achieved the rate of six month progression-free survival in 45% of patients [170].

2.4.4. Bacterial Infections
HCQ is known to exert an antibacterial effect through the alkalinization of infected organelles, inhibiting bacterial replication. In clinical practice, HCQ is not used in monotherapy but in combination with antibiotics, like doxycycline, to improve its bactericidal effects on two main bacteria: Coxiella burnetii and Tropheryma whipplei.
C. burnetii is an obligate intraleukocytic Gram-negative bacterium responsible for query fever (Q fever). The infection is mainly caused by direct contact with infected animals, although cases of human transmission have also been described. Q fever diagnosis is primally founded on serological examination and based on a different evolution, acute and chronic infection can be distinguished. In 50% of cases, the acute phase is asymptomatic, but when the acute phase is symptomatic, it is characterized by a febrile illness, myalgia, headache, chills, atypical hepatitis, and pneumonia [122,123]. Approximately 2–5% of C. burnetii infections can develop into the chronic phase, leading to endocarditis and vascular infection. The risk of developing chronic fever is higher in patients with pre-existing vascular disorders or valvulopathies [123,124]. C. burnetii is known to replicate in an intracellular phagolysosome with a pH range of 4–5. However, at this pH, antibiotics, like doxycycline (DXC), exert only a bacteriostatic activity. Therefore, a combination of DXC with a lysosomotropic agent, such as HCQ, was suggested. In fact, HCQ was shown to increase the phagolysosomal compartment’s pH by improving the bactericidal activity of doxycycline [125,126]. The first successful results concerning the treatment of Q fever endocarditis combined with DXC and HCQ date back to 1993 [127]. These results were later confirmed by a case report of a young infected girl, where the treatment with 200 mg/day of DXC and 600 mg/day of HCQ led to a reduction in serum C. burnetii antibodies within 48 h [128]. Furthermore, in a 1999 clinical study, the administration of 100 mg DXC twice daily plus 200 mg HCQ three times daily for at least 18 months led to a short duration of therapy and a reduction in recurrences compared to alternative treatments including DXC plus 200 mg ofloxacin three times daily [129]. Since this moment, all infected subjects have been treated with DXC plus HCQ, as demonstrated by several case reports where this regimen results in an improvement of C. burnetii-related disease [130,131,132,133,134,135,139,140]. Furthermore, in patients with valvulopathy and diagnosticated acute Q fever (serologic criteria of a phase II IgG titer ≥ 200 and a phase II IgM titer ≥ 50) the administration as prophylaxis of DCX plus HCQ for at least 12 months resulted to be efficient in preventing Q fever endocarditis. Contrarily, shorter regimes are associated with a failure of antibiotics prophylaxis [141]. When Q fever endocarditis occurs, the optimal treatment duration with DXC and HCQ seems to be 18 months for native valve patients and 24 months for subjects with prosthetic valves [142]. This duration should only be extended in the absence of favorable serological results. However, long-term treatment with DXC and HCQ is not without important complications, since both can cause photosensitivity [144], abnormal weight gain [145], severe erythroderma, and impaired visual field [142]. Besides, it can be said that while the acute phase of the infection can be treated with only 200 mg/day DXC, the chronic phase is more difficult to treat and therapy with 100 mg DXC twice daily with 200 mg HCQ three times daily for 18–24 months was recommended [146]. Serological titers are used to follow the disease and determine the duration of therapy.
On the other hand, T. whipplei is a Gram-positive bacterium responsible for Whipple’s disease. The natural niche of T. whipplei is the human intestine since, in the intestinal mucosa, the bacterium is taken by macrophages, where it replicates [147]. This bacterial infection is primally characterized by digestive tract disorders such as diarrhea (75% of cases), malabsorption, and weight loss (80–90% of cases). Joint disease may appear more than six years before the diagnosis and occur in more than 80% of patients [148]. Furthermore, neurological and cardiac disorders can also be frequently associated with Whipple’s disease. For years the standard treatment for T. whipplei has included a combination of trimethoprim and sulfamethoxazole; however, relapses were not uncommon [149,150]. Later, in vitro studies, demonstrated that trimethoprim was inactive on this bacterium [154], while sulfamethoxazole induced bacterial resistance, making the co-administration completely ineffective [154,156]. Based on the good results of treating C. burnetii infections, it was decided to test in vitro the association DCX/HCQ on T. whipplei, obtaining good results [154]. DCX/HCQ efficacy on T. whipplei diseases was demonstrated in a clinical trial dated 2014. This study showed that the administration of 200 mg/day DCX and 600 mg/day HCQ to 13 patients results in better outcomes (0/13 failures) even after 1 year of treatment, compared to standard antibiotics regimens [155]. To date, several case reports available in the literature supported a therapy consisting of a combination of HCQ (600 mg/day) and DCX (200 mg/day) for a lifetime or at least one year, followed by a maintenance dosage of DXC used alone [156,157,158]. In some cases, prophylaxis of intravenous ceftriaxone (2g/day) for the first two weeks followed by HCQ/DXC for at least 12–18 months has been recommended [72,159,160,161].
Although HCQ was revealed to be effective against bacterial infections, in the last few years, in light of the current epidemiological situation, the research attention has shifted toward HCQ application as an antiviral agent, as it could be seen in the bubble map (Figure 8). This visual map is obtained by VOSviewer software, analyzing recurring items from all keywords [171].