PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T1	0-98	Sentence	denotes	Recent Clinical and Preclinical Studies of Hydroxychloroquine on RNA Viruses and Chronic Diseases:
T2	99-118	Sentence	denotes	A Systematic Review
T3	120-128	Sentence	denotes	Abstract
T4	129-312	Sentence	denotes	The rapid spread of the new Coronavirus Disease 2019 (COVID-19) has actually become the newest challenge for the healthcare system since, to date, there is not an effective treatment.
T5	313-398	Sentence	denotes	Among all drugs tested, Hydroxychloroquine (HCQ) has attracted significant attention.
T6	399-532	Sentence	denotes	This systematic review aims to analyze preclinical and clinical studies on HCQ potential use in viral infection and chronic diseases.
T7	533-719	Sentence	denotes	A systematic search of Scopus and PubMed databases was performed to identify clinical and preclinical studies on this argument; 2463 papers were identified and 133 studies were included.
T8	720-915	Sentence	denotes	Regarding HCQ activity against COVID-19, it was noticed that despite the first data were promising, the latest outcomes highlighted the ineffectiveness of HCQ in the treatment of viral infection.
T9	916-1060	Sentence	denotes	Several trials have seen that HCQ administration did not improve severe illness and did not prevent the infection outbreak after virus exposure.
T10	1061-1215	Sentence	denotes	By contrast, HCQ arises as a first-line treatment in managing autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, and Sjögren syndrome.
T11	1216-1310	Sentence	denotes	It also improves glucose and lipid homeostasis and reveals significant antibacterial activity.
T12	1312-1314	Sentence	denotes	1.
T13	1315-1327	Sentence	denotes	Introduction
T14	1328-1493	Sentence	denotes	The 4-aminoquinolonehydroxychloroquine (HCQ) was synthesized for the first time in 1946, but its history began as far back as the 1600s thanks to the Incas in Chile.
T15	1494-1716	Sentence	denotes	They introduced the special properties of cinchona bark to the Jesuits and in 1820 quinine and cinchonine were isolated and identified as the main alkaloids responsible for the antimalarial activity attributed to the bark.
T16	1717-1811	Sentence	denotes	For these reasons, in 1900, the Dutch and British transplanted this “miraculous tree” to Java.
T17	1812-1909	Sentence	denotes	The quinine soon began to be also used for the treatment of systemic lupus erythematosus [1,2,3].
T18	1910-2041	Sentence	denotes	The increasing need for quinine, due to malaria diffusion, led the pharmaceutical industry to the creation of a synthetic molecule.
T19	2042-2105	Sentence	denotes	In 1934 Johann (Hans) Andersag and co-workers at the Bayer I.G.
T20	2106-2249	Sentence	denotes	Farbenindustrie in Elberfeld laboratories, Germany, synthesized chloroquine (CQ) for the first time, judged by the Germans to be too toxic [4].
T21	2250-2375	Sentence	denotes	However, the production of natural quinine was blocked by the Japanese army’s occupation of Java during the Second World War.
T22	2376-2474	Sentence	denotes	Therefore, it was necessary to deepen the studies on molecules that could replace natural quinine.
T23	2475-2656	Sentence	denotes	The Americans soon synthesized from CQ the HCQ, which resulted in it being less toxic than its ancestor so that in 1955 the scientific community proposed it as an alternative to CQ.
T24	2657-2734	Sentence	denotes	Figure 1 represents the historical development that led to HCQ synthesis [5].
T25	2735-2990	Sentence	denotes	This molecule, which has always been known as antimalarial, has come back into vogue in recent months due to the ongoing new coronavirus (2019-nCoV, or COVID-19 or 2019-nCo) worldwide pandemic, that causes a severe acute respiratory syndrome (SARS-CoV-2).
T26	2991-3173	Sentence	denotes	In fact, several research groups have evaluated the use of CQ (C18H26ClN3) and in particular its derivative HCQ (C18H26ClN3O), as a promising treatment for COVID-19 patients [6,7,8].
T27	3174-3382	Sentence	denotes	CQ and HCQ have a core structure consisting of two aromatic rings fused, the 4-aminoquinoline nucleus, and an amphiphilic weak basic side chain (represented in green and red colors in Figure 1, respectively).
T28	3383-3519	Sentence	denotes	The two chemical structures differ only for the hydroxyl group at the end of N-ethyl side chain (represented in blue color in Figure 1).
T29	3520-3714	Sentence	denotes	These are water-soluble molecules and, for their chemical nature, can pass the cell membranes; however, the presence of the hydroxyl group in the HCQ makes it more polar and less lipophilic [8].
T30	3715-3867	Sentence	denotes	Moreover, the accumulation of CQ and HCQ in intracellular compartments is due principally to the side chain and both have enantiomers (R and S isomers).
T31	3868-3972	Sentence	denotes	Researches also demonstrated that the R-HCQ is present in the blood at higher concentrations than S-HCQ.
T32	3973-4065	Sentence	denotes	These results suggested the stereoselective processes in the metabolism of the molecule [9].
T33	4066-4272	Sentence	denotes	Studies of structure-activity relationships have demonstrated that the halogen substitutions of different 4-aminoquinolinesderivatives reduced the toxicity, but it also reduced the pharmacological activity.
T34	4273-4376	Sentence	denotes	Instead, the therapeutic ratio is decreased by substitution of the alkyl side chain with an aryl chain.
T35	4377-4517	Sentence	denotes	Nevertheless, the therapeutic ratio decreases and toxicity increases with an increase of the alkyl side-chain length above 5 carbons [7,10].
T36	4518-4660	Sentence	denotes	Usually, CQ and HCQ are administered as phosphate and sulphate salts, respectively, and both drugs are absorbed in the upper intestinal tract.
T37	4661-4747	Sentence	denotes	However, clinically, HCQ is more frequently used than CQ for its lesser toxicity [11].
T38	4748-4938	Sentence	denotes	Both molecules give mild nausea, stomach cramps, gastrointestinal upset, and mild diarrhoea as adverse effects and long-term usage determines loss of retinal function and severe retinopathy.
T39	4939-5131	Sentence	denotes	These drugs are used, to date, in malaria patients and in several inflammatory diseases like rheumatoid arthritis, Sjögren syndrome, dermatomyositis, and systemic lupus erythematosus [7,8,10].
T40	5133-5135	Sentence	denotes	2.
T41	5136-5159	Sentence	denotes	Results and Discussions
T42	5161-5165	Sentence	denotes	2.1.
T43	5166-5180	Sentence	denotes	Study Analysis
T44	5181-5281	Sentence	denotes	The initial survey of the literature identified 2756 reports (2066 from Scopus and 690 from PubMed).
T45	5282-5416	Sentence	denotes	Then, 293 the duplicates between the two databases were eliminated and were considered only one at a time, resulting in 2463 articles.
T46	5417-5573	Sentence	denotes	After the primary screening based on titles and abstracts, 959 manuscripts were excluded since they did not fulfil the inclusion criteria or were off-topic.
T47	5574-5658	Sentence	denotes	Finally, 1504 articles were thoroughly analyzed and among these, 1442 were excluded.
T48	5659-5823	Sentence	denotes	The analysis of the reference lists from some selected items led to the inclusion of an extra 71 appropriate articles, after titles, abstracts, and full-text study.
T49	5824-5879	Sentence	denotes	In total, 133 papers were selected for data extraction.
T50	5880-5959	Sentence	denotes	A flowchart illustrating the steps of the study selection is shown in Figure 2.
T51	5960-6087	Sentence	denotes	In our review, the articles included were also analyzed in relation to the year of publication and the experimental model used.
T52	6088-6189	Sentence	denotes	Regarding the chronology of the publications, most of the articles were published in the last decade.
T53	6190-6448	Sentence	denotes	The high peaks correspond to the proportional number of papers on antiviral activity in 2020; this result is consistent with the actual increasing interest towards the role of HCQ as a possible therapeutic strategy in the current COVID-19 sanitary emergency.
T54	6449-6536	Sentence	denotes	HCQ alone, or in combination with other drugs, was used in various types of infections.
T55	6537-6616	Sentence	denotes	HIV and COVID-19 were the most cited, with 10 and 7 reports each, respectively.
T56	6617-6694	Sentence	denotes	Our systematic review included 49 preclinical studies and 84 clinical trials.
T57	6695-6779	Sentence	denotes	The considered in vivo studies were carried out using allograft or xenograft models.
T58	6780-6862	Sentence	denotes	The most important outcomes of the review are graphically illustrated in Figure 3.
T59	6863-7011	Sentence	denotes	The assessment of bias risk based on a checklist adapted from the Cochrane Handbook for Systematic Reviews of Interventions is reported in Figure 4.
T60	7012-7082	Sentence	denotes	The number of high-risk reports is due to the case reports considered.
T61	7084-7088	Sentence	denotes	2.2.
T62	7089-7128	Sentence	denotes	Hydroxychloroquine and Viral Infections
T63	7129-7237	Sentence	denotes	HCQ has been used mainly as an antimalarial drug, but it has also proven effective against viral infections.
T64	7238-7426	Sentence	denotes	HCQ demonstrated its antiviral efficacy in inhibiting the endosomal-lysosomal acidification, which is essential for the entry, replication, and infection process of different viruses [12].
T65	7427-7558	Sentence	denotes	In particular, HCQ-induced alkalinization processes cause the expansion and vacuolization of lysosomes, inhibiting their functions.
T66	7559-7728	Sentence	denotes	This activity can reduce post-transcriptional protein modification, enzyme release, receptor recycling, activation of cell signaling pathways, and cell membranes repair.
T67	7729-7784	Sentence	denotes	As a result, there is the prevention of cell infection.
T68	7785-7887	Sentence	denotes	Further, HCQ antiviral activity is also related to its anti-inflammatory and immunomodulatory effects.
T69	7888-8163	Sentence	denotes	Different studies have, indeed, demonstrated that viral diseases are caused by a direct viral infection of susceptible cells and also by an impact on the immune response with consequent uncontrolled release of pro-inflammatory chemokines, cytokines, and other mediators [13].
T70	8164-8333	Sentence	denotes	The most studied antiviral activity of HCQ is that exerted against HIV; however, the current spread of the Coronavirus infection has brought attention back to this drug.
T71	8334-8581	Sentence	denotes	In this systematic review, clinical and in vivo studies evaluating HCQ antiviral activity against the Human Immunodeficiency Virus (HIV), Chikungunya Virus (CHIKV), Flavivirus, and Coronavirus have been analyzed (Figure 3B, Figure 5, and Table 1).
T72	8583-8589	Sentence	denotes	2.2.1.
T73	8590-8595	Sentence	denotes	HIV-1
T74	8596-8711	Sentence	denotes	HIV is part of the genus Lentivirus, of the Retroviridae family, and it is divided into two lines: HIV-1 and HIV-2.
T75	8712-8811	Sentence	denotes	The most virulent and infectious is HIV-1, since it causes most of the HIV infections in the world.
T76	8812-8951	Sentence	denotes	The target cells of HIV are those rich in CD4 receptors, such as some lymphocytes called CD4+, which play a crucial role in human immunity.
T77	8952-9082	Sentence	denotes	Indeed, these lymphocytes activate different immune system cells depending on the type of unwanted host they come in contact with.
T78	9083-9194	Sentence	denotes	It seems that acute HIV infection is highly linked to a rapid depletion of CD4+ T cells in gut lymphoid tissue.
T79	9195-9367	Sentence	denotes	This event is related to an alteration of the intestinal mucosa integrity, resulting in bloodstream translocation of microbial products like lipopolysaccharides (LPS) [42].
T80	9368-9545	Sentence	denotes	It has been hypothesized that LPS, through the binding and activation of Toll-like receptor 4 (TLR-4), is responsible for the immune system activation observed in HIV infection.
T81	9546-9760	Sentence	denotes	Although the real HCQ mechanism of action has not been well assessed, it seems that the anti-HIV effects are highly linked to the post-translational modification of glycoprotein 120 (gp120) in monocyte and T cells.
T82	9761-9883	Sentence	denotes	Consequently, there was a modification of gp120 immunogenic properties and a reduction of new virions infectivity [15,16].
T83	9884-10013	Sentence	denotes	The first randomized, double-blind, placebo-controlled clinical trial about the anti-HIV-1 activity of HCQ was published in 1995.
T84	10014-10125	Sentence	denotes	In this study, 40 asymptomatic HIV-infected patients received either 800 mg/day HCQ or placebo for eight weeks.
T85	10126-10315	Sentence	denotes	All patients treated with antiretroviral therapy (HAART = zidovudine (ZDV), 2′,3′-dideoxyinosine, or 2′,3′-dideoxycytidine) and stopped taking it four weeks before the clinical trial start.
T86	10316-10436	Sentence	denotes	Unlike placebo, at the eighth week, HCQ displayed a reduction in HIV-1 RNA total plasma levels in 12 out of 19 patients.
T87	10437-10649	Sentence	denotes	Furthermore, the percentage of CD4+ T cells decreased significantly in the placebo group and remained stable during the treatment with HCQ, indicating a probable stabilization of immune function in the HCQ group.
T88	10650-10750	Sentence	denotes	HCQ administration also induced a decrease in serum interleukin 6 (IL-6) and immunoglobulin G (IgG).
T89	10751-10825	Sentence	denotes	However, no significant changes were found in the IgA and IgM levels [17].
T90	10826-11041	Sentence	denotes	The anti-HIV-1 effect of HCQ was also compared with that of ZDV, a nucleoside reverse transcriptase inhibitor, in a randomized, placebo-controlled clinical trial conducted on 72 HIV-l-infected asymptomatic patients.
T91	11042-11137	Sentence	denotes	All subjects were treated for 16 weeks with 800 mg/day HCQ (n = 35) or 500 mg/day ZDV (n = 37).
T92	11138-11260	Sentence	denotes	As in the previous study, patients who had received HAART stopped taking it four weeks before the clinical trial’s outset.
T93	11261-11461	Sentence	denotes	After 16 weeks, total plasma HIV-1 RNA levels were reduced in both the ZDV group and HCQ group, although the extent of anti-HIV-l activity in HCQ patients was lower than that observed in ZDV subjects.
T94	11462-11626	Sentence	denotes	However, eight subjects in the Azithromycin (AZM) group showed an increase in HIV-1 RNA levels in the 16th week, indicating the rapid emergence of viral resistance.
T95	11627-11832	Sentence	denotes	Contrarily, in the HCQ group, increased antiviral activity was revealed after 16 weeks rather than after 8 weeks, and no subject showed an increase in HIV-1 RNA levels at either 8 or 16 weeks of treatment.
T96	11833-11955	Sentence	denotes	This evidence suggests that no resistance developed under HCQ therapy or that it might develop more slowly than under ZDV.
T97	11956-12128	Sentence	denotes	A reduction in serum p24 antigen levels in both ZDV and HCQ groups was also described, while only in the HCQ group could a decrease in IL-6 and IgG levels be observed [18].
T98	12129-12328	Sentence	denotes	This reduction of IgG levels displayed after HCQ treatment in both studies may be significant since autoantibodies contribute to CD4+ cell depletion and autoimmune diseases observed in HIV infection.
T99	12329-12614	Sentence	denotes	Further, as lymphoid tissue is considered the primary site of HIV reservoirs and a critical site affected by CD4+ T cells depletion, the HCQ concentration was assessed in the plasma and adenoid tissue (At) of 8 HIV-infected patients administrating 400 or 800 mg of HCQ for eight weeks.
T100	12615-12746	Sentence	denotes	After taking these dosages, it was demonstrated that the mean HCQ concentration was significantly higher in At than in plasma [21].
T101	12747-12905	Sentence	denotes	This different drug distribution was also confirmed by an in vivo study using rabbits as an experimental model, receiving 15 mg/kg of HCQ subcutaneously [22].
T102	12906-13056	Sentence	denotes	Thus, the anti-HIV activity of HCQ could be linked to its accumulation in lymphoid tissue, a relevant site for HIV immunopathogenesis and replication.
T103	13057-13192	Sentence	denotes	Since monotherapy is not recommended in treating HIV, HCQ has also been tested in synergy with other drugs commonly used to manage HIV.
T104	13193-13442	Sentence	denotes	In this regard, 400 mg/day of HCQ in a combination regimen with 1000 mg/day hydroxyurea and 250–400 mg/day didanosine (dosed per body weight) was administered for 48 weeks to 22 asymptomatic HIV-1 infected patients naïve to antiretroviral treatment.
T105	13443-13485	Sentence	denotes	Only 16 out of 22 patients were evaluable.
T106	13486-13609	Sentence	denotes	These 16 subjects, at the 12th week, showed a significant reduction in viral load which was maintained until the 48th week.
T107	13610-13737	Sentence	denotes	Furthermore, at week 12, an increase in CD4+ percentage was also shown, and this improvement was kept until the 48th week [19].
T108	13738-13903	Sentence	denotes	To evaluate the long-term efficacy and safety of HCQ, hydroxyurea, and didanosine combination, they were also tested on 17 HIV-infected naïve patients for 144 weeks.
T109	13904-13996	Sentence	denotes	All subjects received 200 mg HCQ, 500 mg hydroxyurea, and 125–200 mg didanosine twice daily.
T110	13997-14083	Sentence	denotes	Of the 17 patients who started treatment, 14 remained until the end of the 144th week.
T111	14084-14341	Sentence	denotes	After 114 weeks, viral load was reduced by 1.6 Log10 copies/mL under baseline (p < 0.001), eight patients (47%) had an unnoticeable viral load (< 400 copies/mL), and two patients (12%) had a measurable viral load, but resistance mutations were not detected.
T112	14342-14582	Sentence	denotes	Four patients (24%) had both detectable viral load and resistance mutation: one with both 62V and 65R and three with both 74V and 184V mutations; the latter three were assessed as didanosine resistant, while no resistance was found for HCQ.
T113	14583-14667	Sentence	denotes	However, in all cases, the viral load remained below the baseline at the 144th week.
T114	14668-14789	Sentence	denotes	The CD4+ cell count had increased significantly, while the percentage of CD8 cells was reduced up to the 144th week [20].
T115	14790-15022	Sentence	denotes	This HCQ noticeable impact on immune activation, thereby increasing CD4+ T cells, was also demonstrated in a prospective study conducted on 20 HIV-infected immunologic no responders treated with standard antiretroviral therapy [23].
T116	15023-15187	Sentence	denotes	These results suggested that the combination of HCQ, hydroxyurea, and didanosine could be a valid alternative to the highly active commercial HAART in HIV-patients.
T117	15188-15326	Sentence	denotes	Nonetheless, these latter studies have some limitations, such as the small number of subjects included and the absence of a control group.
T118	15327-15476	Sentence	denotes	Therefore, it is not possible to determine the contribution made by HCQ to the overall decrease in viral load obtained from the combination of drugs.
T119	15477-15709	Sentence	denotes	Anyway, the potential anti-HIV efficacy of HCQ, when added to existing treatment with an antiretroviral regimen, was also confirmed by a case report about a patient with HLA-B27-associated spondyloarthropathy and HIV infection [43].
T120	15710-15930	Sentence	denotes	In contrast to the results mentioned above has been published a randomized, double-blind, placebo-controlled trial performed on 83 patients to which 400 mg HCQ (n = 42) or placebo (n = 41) were administered for 48 weeks.
T121	15931-16132	Sentence	denotes	All patients were naïve to HAART or had stopped this therapy 22 months before the trial began; 17 subjects in the HCQ group and 8 in the placebo group interrupted study medication before the 48th week.
T122	16133-16327	Sentence	denotes	At the end of treatment, in the HCQ group, compared to placebo, patients showed a reduction in total CD4 cell count and a significant viral load increased from the 12th week above baseline [24].
T123	16328-16554	Sentence	denotes	Hence, based on these results, HCQ did not decrease immune activation in patients with chronic HIV infection who were naïve to HAART, as there was an increase in HIV viral replication and a negative effect on CD4+ cell counts.
T124	16555-16804	Sentence	denotes	In light of these results, there was the need to consider that the first two described clinical trials, which reported the antiviral effect of HCQ, were on short-term treatments (8 or 16 weeks) and that they used an HCQ dosage of 800 mg/day [17,18].
T125	16805-16920	Sentence	denotes	In contrast, the latter used only 400 mg/day [24], corresponding to the maximum recommended dose for long-time use.
T126	16921-17200	Sentence	denotes	Besides, the latter study also enrolled more patients than the other studies, and unlike the trial of Piconi et al. [23], which described significative effects in reducing immune activation after HCQ administration, was conducted in the absence of antiretroviral treatments [24].
T127	17201-17392	Sentence	denotes	Therefore, further clinical trials involving a larger number of subjects would be necessary to assess the real antiviral activity of HCQ in monotherapy and synergy with antiretrovirals drugs.
T128	17393-17606	Sentence	denotes	Finally, assuming that women resistant to HIV infection show a low activation of the immune system at the level of the female genital tract (FGT), HCQ has been investigated as a drug able to prevent HIV infection.
T129	17607-17733	Sentence	denotes	It has been shown that the “immune quiescent” state of HIV-resistant women keeps the immune response against pathogens intact.
T130	17734-17931	Sentence	denotes	For this reason, it was thought to induce pharmacologically, in a rabbit model, this immunological quiescence state through an intravaginal implant capable of providing a controlled release of HCQ.
T131	17932-18202	Sentence	denotes	Considering that a concentration above 6.48 μg/mL of HCQ was able to interfere with the gp120 glycosylation process, the vaginal implant was projected to release HCQ with the longest possible duration at a concentration greater than 4.34 μg/mL but lower than 21.7 μg/mL.
T132	18203-18451	Sentence	denotes	After six days, this implant improved mucosal epithelial integrity, reduced submucosal immune cell recruitment, decreased gene expression and protein of T cell activation markers, and minimized the activation of key pro-inflammatory mediators [25].
T133	18452-18610	Sentence	denotes	Hence, HCQ can be considered a promising drug able to maintain a low baseline level of immune activation and may also play a role in preventing HIV infection.
T134	18612-18618	Sentence	denotes	2.2.2.
T135	18619-18636	Sentence	denotes	Chikungunya Virus
T136	18637-18784	Sentence	denotes	The single-stranded RNA virus, Chikungunya virus (CHIKV), is an alphavirus belonging to the Togaviridae family, spread mainly in America’s regions.
T137	18785-18905	Sentence	denotes	The usual CHIKV vectors are rodents, while humans are infected by Aedes albopictus and Aedes aegypti and mosquito bites.
T138	18906-19460	Sentence	denotes	The first incubation stage can vary between 2 and 12 days, and three phases follow it: (1) the acute viraemic phase, characterized by severe polyarthritis, fever, and a rash, generally resolving in three weeks; (2) the post-acute stage, identified by arthritis with the addition of synovial and periarticular inflammation, neuropathy, neuropsychiatric disorders, and peripheral vascular disorders, usually takes its time at the end of three months; (3) the chronic phase that appears when the symptoms of the previous phase do not end after three months.
T139	19461-19730	Sentence	denotes	Generally, the acute phase of CHIKV infections is treated with non-steroidal anti-inflammatory drugs (NSAIDs), while for the chronic persistent phase, treatments involving HCQ as monotherapy or combined with methotrexate (MTX) and/or sulfasalazine seem to be effective.
T140	19731-19866	Sentence	denotes	HCQ does not appear to affect the initial stage of infection, as demonstrated in a prospective randomized parallel-group study of 2009.
T141	19867-19988	Sentence	denotes	In this trial, combinations of NSAIDs, HCQ, and/or corticosteroids were assessed in patients with classic CHIKV features.
T142	19989-20156	Sentence	denotes	A total of 120 subjects were divided into groups treated with 200 mg/day aceclofenac (ACF), 400 mg/day HCQ, and 10 mg/day prednisolone (PRD) in different combinations.
T143	20157-20223	Sentence	denotes	Only 114 subjects remained until the end of the trial (six weeks).
T144	20224-20489	Sentence	denotes	It was seen that HCQ had no benefit in the early stages of CHIKV infection and also in the reduction of the VAS (used for pain assessment) and in the improvement of Barthel’s indexes (used for instrumental activities of daily living and activities of daily living).
T145	20490-20575	Sentence	denotes	In fact, there was no difference between groups treated with ACF + HCQ and ACF alone.
T146	20576-20677	Sentence	denotes	Similarly, the combination of ACF + HCQ + PRD did not add any additional benefit over ACF + PRD [26].
T147	20678-20759	Sentence	denotes	In contrast, HCQ seems to affect the improvement of CHIKV chronic phase diseases.
T148	20760-20900	Sentence	denotes	In a recent multicenter study, the efficacy of HCQ was evaluated in 39 patients with rheumatic manifestation related to CHIKV chronic phase.
T149	20901-21097	Sentence	denotes	In four subjects, the treatment was interrupted due to the onset of side effects such as nausea, stomatitis, rash, headache, while the evolution of CHIKV disease was evaluated in only 22 patients.
T150	21098-21283	Sentence	denotes	After three months of treatment, evidence of synovitis was disappeared in 10 of 20 subjects (50%) with swollen joins while complete remission was verified in five patients (19.2%) [27].
T151	21284-21430	Sentence	denotes	However, another study demonstrated that the effects of HCQ in combination with MXT and sulfasalazine were superior to those shown in monotherapy.
T152	21431-21640	Sentence	denotes	In particular, in a randomized controlled open-label study, the impact of HCQ in monotherapy or association with MTX and sulfasalazine was assessed in 72 patients with chronic persistent chikungunya arthritis.
T153	21641-21819	Sentence	denotes	In this trial, 400 mg/day HCQ were administrated to 35 subjects, while a combination of 15 mg/day MTX, 1 g/day sulfasalazine, and 400 mg/day HCQ was administrated to 37 patients.
T154	21820-21946	Sentence	denotes	Either treatment lasted 24 weeks and for the first 6 weeks, both groups received an escalated dose of prednisone (7.5 mg/day).
T155	21947-22069	Sentence	denotes	At the end of the 24th week, only the combination of drugs improved disease activity and reduced disability and pain [28].
T156	22070-22332	Sentence	denotes	These results, following those obtained in a previous uncontrolled 16-week study, since a reduction in ACR score was shown after MTX (15–20 mg/weekly) and HCQ (400 mg/day) administration to CHIKV infected patients with persistent inflammatory polyarthritis [29].
T157	22333-22435	Sentence	denotes	The results obtained could be explained with the different characterization of virus infection phases.
T158	22436-22720	Sentence	denotes	It is known that while the acute phase of CHIKV infections is due to the development of viremia, the chronic phase is closely related to an immune-mediated phenomenon, as pro-inflammatory cytokines and chemokines play important roles in the pathogenesis of chikungunya arthritis [44].
T159	22721-23009	Sentence	denotes	Therefore, considering the results obtained from these clinical studies, it is possible to say that although HCQ does not affect viremia reduction, as demonstrated by the lack of activity in the first phase of infection, it can improve the chronic phase diseases by reducing inflammation.
T160	23011-23017	Sentence	denotes	2.2.3.
T161	23018-23030	Sentence	denotes	Flaviviruses
T162	23031-23268	Sentence	denotes	Hepatitis virus, an RNA virus belonging to the Flaviviridae family, is closely related to liver disease, which in 70–80% of patients becomes chronic, resulting in major complications such as cirrhosis and, in the year, also liver cancer.
T163	23269-23365	Sentence	denotes	The antiviral activity of HCQ on the hepatitis C virus (HCV) in monotherapy has not been tested.
T164	23366-23448	Sentence	denotes	However, it seems that this drug increases the antiviral effect of standard drugs.
T165	23449-23691	Sentence	denotes	In a study including 120 Egyptian patients infected by the hepatitis C virus, it was seen that the combination of HCQ with pegylated interferon and ribavirin could improve biochemical and virological responses in chronic hepatitis C subjects.
T166	23692-23949	Sentence	denotes	All patients were randomized and divided into two groups; the control group treated with standard-of-care (SOC) consisted of 160 µg of subcutaneous pegylated interferon and 1000–12000 mg/day of oral ribavirin, and the group treated with 200 mg HCQ plus SOC.
T167	23950-24126	Sentence	denotes	At the end of the treatment (12 weeks), patients of HCQ + SOC group showed a high virological response compared to the control group (54/60 (90%) vs. 43/60 (71.7%); p = 0.011).
T168	24127-24345	Sentence	denotes	Moreover, in the HCQ + SOC group, there was a normalization of ALT levels, as is also demonstrated by the earlier biochemical response (EBR) highlighted in HCQ + SOC group 58/60 (96.7%) than SOC group 42/60 (70%) [30].
T169	24346-24472	Sentence	denotes	These results were confirmed by several case reports regarding patient treatment with Porphyria Cutanea Tarda and Hepatitis C.
T170	24473-24665	Sentence	denotes	It was seen that a low dose of HCQ (100 mg twice weekly) prevented the recurrence of Porphyria Tarda and reduced the viral response during HCV therapy, including ribavirin and interferon [45].
T171	24666-24923	Sentence	denotes	Furthermore, in a patient with chronic hepatitis and rheumatoid arthritis, a low risk for hepatitis virus reactivation was observed after treatment with steroids (< 7.5 mg/day), HCQ or sulfadiazine [46] in combination with antivirals as prophylaxis [47,48].
T172	24924-25092	Sentence	denotes	Another possible antiviral effect of HCQ is exerted on Zika virus (ZIKV), an RNA virus of the Flaviviridae family transmitted by numerous mosquitoes of the genus Aedes.
T173	25093-25280	Sentence	denotes	No clinical studies have been conducted yet, but an in vivo study has demonstrated the ability of HCQ to attenuate vertical transmission of ZIKV by reducing placental and fetal infection.
T174	25281-25414	Sentence	denotes	In this study, pregnant mice were treated with 40 mg/kg/day HCQ via intraperitoneal injection beginning one day after ZIKV infection.
T175	25415-25581	Sentence	denotes	It was seen that HCQ acted as an autophagy inhibitor by increasing p62 levels in trophoblast and thus reducing placental ZIKV infection and fetal growth defects [31].
T176	25582-25693	Sentence	denotes	To date, no clinical trials have been conducted to evaluate HCQ antiviral effects in patients infected by ZIKV.
T177	25695-25701	Sentence	denotes	2.2.4.
T178	25702-25729	Sentence	denotes	Coronavirus Disease of 2019
T179	25730-25940	Sentence	denotes	Coronaviruses, belonging to the Coronaviridae family, are enveloped positive-sense single-stranded RNA viruses highly distributed in humans and vertebrates like bats, which are proposed as their main reservoir.
T180	25941-26123	Sentence	denotes	Specifically, Coronavirus Disease of 2019 (COVID-19) was first identified in December 2019 in Wuhan, the capital city of Hubei (China), and it is caused by the SARS-CoV-2 virus [49].
T181	26124-26298	Sentence	denotes	Since COVID-19 has spread rapidly in many countries, it has quickly become a global pandemic, so it is necessary to develop drugs able to exert antiviral activity against it.
T182	26299-26394	Sentence	denotes	A recent study showed HCQ in vitro antiviral activity against SARS-CoV-2 (EC50 = 0.72 μM) [50].
T183	26395-26596	Sentence	denotes	HCQ seemed to be able to inhibit the first step of the viral replication cycle by interfering with the link between spike (S) viral protein and the angiotensin-converting enzyme 2 (ACE-2) receptor [8].
T184	26597-26756	Sentence	denotes	It would also appear that HCQ was able to induce changes in cell membrane pH resulting in reduced viral entry and inhibition of the last stages of replication.
T185	26757-26879	Sentence	denotes	Moreover, HCQ may abolish the cytokine storm related to the advanced stages of COVID-19 through immunomodulatory activity.
T186	26880-26989	Sentence	denotes	To date, several clinical studies are underway to evaluate the efficacy of HCQ for the treatment of COVID-19.
T187	26990-27136	Sentence	denotes	In a randomized clinical trial conducted in China, 62 patients with COVID-19 were randomly assigned to two groups: the control and the HCQ groups.
T188	27137-27375	Sentence	denotes	Either group received standard treatment including antiviral agents, oxygen therapy, immunoglobulin, and antibacterial agents, with or without corticosteroids, but patients in the HCQ group also received oral HCQ 400 mg/day for five days.
T189	27376-27521	Sentence	denotes	During treatment, clinical characteristics, clinical recovery time (TTCR), and radiological results were assessed to determine the effect of HCQ.
T190	27522-27771	Sentence	denotes	It was seen that in the HCQ group, the recovery time of body temperature was shorter than in the control group and that the cough remission time was also significantly decreased, while only patients in the control group progressed to severe illness.
T191	27772-27868	Sentence	denotes	Furthermore, in the HCQ group, 61.3% of patients showed significant absorption of pneumonia [6].
T192	27869-28001	Sentence	denotes	In another open-label non-randomized French clinical trial, the efficiency of HCQ in reducing the viral count was also demonstrated.
T193	28002-28058	Sentence	denotes	In this study, 36 subjects were divided into two groups:
T194	28059-28154	Sentence	denotes	16 patients for the control group and 20 subjects for HCQ who were administered 600 mg/day HCQ.
T195	28155-28320	Sentence	denotes	Among the HCQ group, six patients also received 500 mg of AZM for the first day, followed by 250 mg/day for the next four days to prevent super bacterial infections.
T196	28321-28466	Sentence	denotes	PCR results from nasopharyngeal samples were negative for 70% of patients treated with HCQ and 12.5% in the control group (p = 0.001) on day six.
T197	28467-28715	Sentence	denotes	Furthermore, when the effect of HCQ as monotherapy was compared to that of HCQ + AZM, it was found that at day six, 100% of HCQ + AZM treated subjects were virologically negative compared with 57.1% of patients treated with HCQ as monotherapy [32].
T198	28716-28933	Sentence	denotes	These results suggest a synergistic effect between HCQ and AZM and are supported by an uncontrolled non-comparative observational study conducted by the same France group, in a cohort of 80 slightly infected patients.
T199	28934-29101	Sentence	denotes	In this study, HCQ + AZM treated people were 83% virologically negative on day 7 and 93% on day 8; after 10 days of treatment, only 2 subjects still remain contagious.
T200	29102-29226	Sentence	denotes	Furthermore, the average duration of hospitalization was found to be 4.6 days after the administration of HCQ plus AZM [33].
T201	29227-29308	Sentence	denotes	However, these last two studies have a limit, as Gautret et al. carried both out.
T202	29309-29484	Sentence	denotes	In particular, in the first study, data are available up to 6 days despite the planned 10 days, and in the second study, 6 patients from the previous study were also included.
T203	29485-29683	Sentence	denotes	Gautret also conducted a retrospective analysis of 1061 cases in which, after treatment with HCQ + AZM, good virological care and clinical outcomes were found in 973 patients (91.7%) within 10 days.
T204	29684-29853	Sentence	denotes	A prolonged viral carriage was observed in only 47 (4.4%) subjects with a high viral load at the moment of hospitalization, but on day 10 the viral culture was negative.
T205	29854-29907	Sentence	denotes	Finally, all but one on day 15 were PCR cleared [51].
T206	29908-30043	Sentence	denotes	By contrast, there are results obtained by several clinical studies that dismiss the possible use of HCQ for the treatment of COVID-19.
T207	30044-30389	Sentence	denotes	In particular, a prospective study on 11 severe COVID-19 infected patients treated with the same dosage used by Gautret et al. (600 mg/day HCQ and 500 mg AZM for the first day followed by 250 mg/day from day 2 to 5) was shown to provide no evidence of clinical benefits or a strong antiviral activity through the combination of HCQ and AZM [34].
T208	30390-30605	Sentence	denotes	The ineffectiveness of HCQ has also been declared in a multicenter, open-label, randomized controlled trial involving 150 hospitalized infected patients (148 with mild or moderate disease and 2 with severe disease).
T209	30606-30804	Sentence	denotes	All subjects were divided into two groups (in a 1:1 ratio): the control group that received only SOC, consisting of antiviral, glucocorticoid, and antiviral, and the group treated with HCQ plus SOC.
T210	30805-30960	Sentence	denotes	The administrated dose of HCQ consisted of 200 mg/day for the first three days, followed by 800 mg/day as maintained dosage until the end of the treatment.
T211	30961-31100	Sentence	denotes	After 28 days of treatment, there was no significant difference in SARS-CoV-2 negative conversion in either the HCQ + SOC or the SOC group.
T212	31101-31291	Sentence	denotes	Similarly, there were no significant differences in the median time to negative conversion and the probability of symptom alleviation within 28 days between HCQ + SOC and the SOC group [35].
T213	31292-31439	Sentence	denotes	Another multicenter, randomized controlled Egyptian trial evaluated, in 194 subjects with COVID-19, the safety and efficacy of HCQ compared to SOC.
T214	31440-31666	Sentence	denotes	In terms of mechanical ventilation need and mortality, the addition of HCQ (400 mg twice daily (in day 1) followed by 200 mg tablets twice daily) to SOC was not associated with an improvement of COVID-19 patients’ health [36].
T215	31667-31791	Sentence	denotes	These results were confirmed by a recent randomized, double-blind, placebo-controlled trial conducted in the USA and Canada.
T216	31792-32026	Sentence	denotes	In this study, 419 early and mild COVID-19 subjects were randomly assigned to two groups, the HCQ group treated with 800 mg HCQ once, followed by 600 mg in 6 to 8 h, then 600 mg daily for 4 more days, and the placebo or control group.
T217	32027-32296	Sentence	denotes	Within 14 days of treatment, there was no change in the severity of symptoms in non-hospitalized patients between the HCQ group and the placebo group (difference in symptom severity: relative, 12%; absolute, −0.27 points (95% CI, −0.61 to 0.07 points); p = 0.117) [37].
T218	32297-32492	Sentence	denotes	Likewise, 2 studies carried out by Mahévas et al. supported the ineffectiveness of HCQ and highlighted that HCQ administration to COVID-10 patients was highly related to ECG modification [38,39].
T219	32493-32695	Sentence	denotes	ECG modification, resulting in QT-interval prolongation, is a characteristic side effect associated with HCQ treatment that has been shown in several clinical studies on patients infected with COVID-19.
T220	32696-32895	Sentence	denotes	In particular, the risk of QT-interval prolongation was increased when HCQ was associated with AZM, as demonstrated in a cohort of 84 patients who received 400 mg twice-daily HCQ plus 500 mg/day AZM.
T221	32896-33071	Sentence	denotes	In these patients, on day 3.6 ± 1.6 of therapy, the EGC showed a QTc prolongation from a baseline average of 435 ± 24 ms (mean ± SD) to a maximal average value of 463 ± 32 ms.
T222	33072-33240	Sentence	denotes	Moreover, in nine subjects (11%) there was a severe prolongation of QTc to > 500 ms (baseline average of 447 ± 30 ms to 527 ± 17 ms (p < 0.01 (one-sample t-test)) [52].
T223	33241-33354	Sentence	denotes	This complication was also confirmed in a retrospective study of 251 COVID-19 patients treated with HCQ/AZM [53].
T224	33355-33482	Sentence	denotes	It seems that a predictor of extreme QTc prolongation was renal failure and that its incidence increased with longer treatment.
T225	33483-33760	Sentence	denotes	The probability of QTc prolongation may also increase in the presence of other factors such as previous cardiovascular diseases, metabolic degeneration (hypoxia, pH, multiorgan system failure, and electrolyte abnormalities), age, and sex (females seem to be more at risk) [54].
T226	33761-34046	Sentence	denotes	Therefore, since QTc prolongation to more than 500 ms is known to be associated with a high risk for malignant arrhythmia and fatal stroke, recent guidance suggests the ECG screening with QTc evaluation in COVID-19-infected patients treated with novel therapies including HCQ/AZM [55].
T227	34047-34349	Sentence	denotes	It has also been suggested that the use of drugs that block late sodium channels (mexiletine or lidocaine) and close attention to serum electrolytes, in addition to the evaluation of heart rate and QT intervals, may allow the administration of HCQ/AZM even in subjects with prolonged QT intervals [54].
T228	34350-34495	Sentence	denotes	Despite the lack of real antiviral evidence related to HCQ administration, this drug has also been investigated as a possible prophylactic agent.
T229	34496-34699	Sentence	denotes	In fact, the pharmacokinetics of HCQ, like its long half-life and the high concentration in the lung (500-times higher than blood concentration), has made it an ideal candidate for prophylactic use [56].
T230	34700-34845	Sentence	denotes	The first study conducted on this line was performed in South Korea on 211 virus-exposed individuals, including 189 patients and 22 care-workers.
T231	34846-35078	Sentence	denotes	The HCQ administration (400 mg day) as post-exposure prophylaxis resulted in the negative follow-up PCR tests after the end of 14 days of quarantine period (only 97.4% of patients and 95.5% of care-workers completed the study) [40].
T232	35079-35247	Sentence	denotes	However, it is necessary to consider that this is a single-center clinical study with a high risk of bias and that a subsequent randomized clinical study has denied it.
T233	35248-35412	Sentence	denotes	In particular, Boulware D.R. et al., in a randomized, double-blind, placebo-controlled trial, tested HCQ as a prophylactic agent within 4 days after virus exposure.
T234	35413-35594	Sentence	denotes	There were 821 asymptomatic participants randomly assigned to receive either placebo or HCQ (800 mg once, followed by 600 mg in 6 to 8 h, then 600 mg per day for 4 additional days).
T235	35595-35890	Sentence	denotes	After 14 days of treatment, it was demonstrated that HCQ did not prevent COVID-19 infection when compared to placebo, since the incidence of illnesses compatible with Covid-19 disease did not differ significantly between subjects receiving HCQ (49 of 414 (11.8%)) or placebo (58 of 407 (14.3%)).
T236	35891-36013	Sentence	denotes	Furthermore, the onset of side effects was more frequent in patients treated with HCQ than placebo (40.1% vs. 16.8%) [41].
T237	36014-36176	Sentence	denotes	To better assess the incidence of side effects linked to HCQ administration as post-exposure prophylaxis, a cross-sectional study was conducted among 140 doctors.
T238	36177-36425	Sentence	denotes	Sixty nine adverse events were documented in 44 subjects (31%); the most common reported symptoms were headache followed by nausea, dizziness, abdominal cramps, and loose stools, while hypoglycemia was seen in only three diabetic participants [57].
T239	36426-36577	Sentence	denotes	Hence, even if the side effects highlighted were not serious, it is recommended to take the utmost care before using HCQ for COVID-19 chemoprophylaxis.
T240	36578-36708	Sentence	denotes	The ineffectiveness of HCQ administration as post-exposure prophylaxis has also been demonstrated by an in vivo study on macaques.
T241	36709-36843	Sentence	denotes	Maisonnasse P. et al. tested different treatment strategies, including HCQ alone or in combination with AZM, in comparison to placebo.
T242	36844-36909	Sentence	denotes	All the treatments were administrated before or after viral load.
T243	36910-37043	Sentence	denotes	It was seen that when HCQ was administrated as pre-exposure prophylaxis, it did not protect against the acquisition of the infection.
T244	37044-37216	Sentence	denotes	Similarly, neither HCQ nor HCQ + AZM had beneficial effects in improving viral infection’s symptoms [58], confirming previously analyzed clinical studies’ negative results.
T245	37217-37404	Sentence	denotes	Several case reports have supported all these results since people already using HCQ for a long time to treat inflammatory diseases also showed severe illness related to COVID-19 [59,60].
T246	37405-37599	Sentence	denotes	In the light of collected data, despite the success of the first clinical trials, the latest studies have shown the ineffectiveness of HCQ for the treatment and prevention of COVID-19 infection.
T247	37600-37620	Sentence	denotes	So that, if the U.S.
T248	37621-37914	Sentence	denotes	Food and Drug Administration (FDA) had initially authorized the use of HCQ in case of emergency [61], in June 2020, the FDA revoked this authorization [62] since the potential HCQ effectiveness in treating COVID-19 was overtaken by severe cardiac adverse events and other serious side effects.
T249	37915-38158	Sentence	denotes	In fact, there is the need to consider that in subjects with severe COVID-19, the abundance of inflammatory molecules like interleukins and tumor necrosis factors generate a cytokine storm, leading to a septic shock and multiple organ failure.
T250	38159-38265	Sentence	denotes	In hepatic and renal dysfunction, HCQ metabolism and clearance were compromised and its safety is altered.
T251	38266-38501	Sentence	denotes	Moreover, recently the Surviving Sepsis Campaign guidelines on the management of critically ill patients with COVID-19 concluded that there was insufficient evidence to recommend the routine use of HCQ in patients admitted in ICU [63].
T252	38502-38629	Sentence	denotes	In the same way, the American College of Physicians practice guidelines do not recommend HCQ for prophylaxis or treatment [64].
T253	38630-38890	Sentence	denotes	International trials like SOLIDARITY (International trial by World Health Organisation) [65], RECOVERY (Randomised Evaluation of Covid-19 Therapy) [66], and DISCOVERY (Trial of Treatments for COVID-19 in Hospitalized Adults) [67] have also stopped the HCQ arm.
T254	38891-39121	Sentence	denotes	In particular, the World Health Organisation (WHO), in the SOLIDARITY trial project, has arrested all arms involving HCQ, as evidence showed that it did not reduce the mortality of hospitalized COVID-19 patients compared with SOC.
T255	39122-39261	Sentence	denotes	However, this decision was not applied to HCQ use or evaluation in pre- or post-exposure prophylaxis for subjects exposed to COVID-19 [68].
T256	39262-39352	Sentence	denotes	To conclude, the HCQ treatment of SARS-CoV-2 infection was not met with its hoped success.
T257	39353-39510	Sentence	denotes	This is probably related to the inability of the dosing regimens currently in use to achieve the blood concentration required for the HCQ antiviral activity.
T258	39511-39749	Sentence	denotes	Initially, based on physiological pharmacokinetic models, Yao et al. recommended for SARS-CoV-2 infection a loading oral HCQ dose of 400 mg twice daily, followed by a maintenance dose of 200 mg administered twice daily for four days [50].
T259	39750-40087	Sentence	denotes	However, this recommended HCQ dosage regimen was based only on the ratio of free lung trough concentration to in vitro EC50 values (the EC50 of HCQ for SARS-CoV-2 ranged between 0.72 and 17.31µM) and did not consider the tendency of HCQ to accumulate within acidic cellular organelles like endosomes, lysosomes, and Golgi apparatus [69].
T260	40088-40243	Sentence	denotes	In fact, it has been demonstrated that HCQ concentration in lysosomes is higher than the extracellular concentration (80 µM vs. 0.5 µM, respectively) [70].
T261	40244-40407	Sentence	denotes	Based on these results, it was considered necessary to compare the EC50 values obtained in vitro with the plasma concentration and not with the lung concentration.
T262	40408-40696	Sentence	denotes	In a study investigating HCQ pharmacokinetics in COVID-19 patients treated with 600 mg/day of HCQ, it was found that the mean blood concentration of HCQ was 0.46 mg/day [32], which was below the lowest estimated levels of 0.48 mg/mL corresponding to the in vitro concentration of 0.72 µM.
T263	40697-40904	Sentence	denotes	Further, a plasma concentration predicted for HCQ antiviral EC50 made by Garcia-Cremades et al. found that it should be 4,7 µM corresponding to 1.58 mg/mL, which is much higher than in vivo plasmatic values.
T264	40905-41095	Sentence	denotes	To reach this plasma concentration, it should be necessary to take an amount of HCQ higher than 400 mg twice a day for five days or more [71], which would increase the onset of side effects.
T265	41096-41349	Sentence	denotes	Thus, the ineffectiveness of HCQ antiviral activity again SARS-CoV-2 can be related to the low current dosing regimens and the impossibility to increase the administered doses due to the increased risk of severe side effects, especially QT prolongation.
T266	41351-41355	Sentence	denotes	2.3.
T267	41356-41394	Sentence	denotes	Hydroxychloroquine Biological Activity
T268	41395-41500	Sentence	denotes	Besides the antiviral effects, HCQ possesses several other demonstrated biological activities (Figure 6).
T269	41501-41772	Sentence	denotes	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.
T270	41773-42051	Sentence	denotes	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.
T271	42052-42140	Sentence	denotes	HCQ exerts anticancer effects by acting synergistically with common chemotherapic drugs.
T272	42141-42338	Sentence	denotes	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.
T273	42339-42446	Sentence	denotes	A two-compartment model, with first-order absorption and a lag time, describes the pharmacokinetics of HCQ.
T274	42447-42567	Sentence	denotes	The long-terminal half-life prolongs the time to reach steady-state concentrations, then delays the therapeutic effects.
T275	42568-42646	Sentence	denotes	The next-generation formulations allow modulating the pharmacokinetics of HCQ.
T276	42647-42786	Sentence	denotes	Avoiding systemic absorption, then liver first-pass metabolism, HCQ may be used in site-specific inflammation, without toxicity [72,73,74].
T277	42787-42911	Sentence	denotes	The selected studies’ primary outcomes are the extent to which HCQ may limit disease progression or exacerbations (Table 2).
T278	42913-42919	Sentence	denotes	2.3.1.
T279	42920-42940	Sentence	denotes	Rheumatoid Arthritis
T280	42941-43059	Sentence	denotes	Rheumatoid arthritis is a systemic autoimmune disease, characterized by chronic inflammation and damage to the joints.
T281	43060-43290	Sentence	denotes	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].
T282	43291-43387	Sentence	denotes	It is estimated that the prevalence of rheumatoid arthritis is around 1%, mainly in women [118].
T283	43388-43563	Sentence	denotes	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.
T284	43564-43800	Sentence	denotes	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.
T285	43801-44012	Sentence	denotes	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].
T286	44013-44222	Sentence	denotes	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.
T287	44223-44414	Sentence	denotes	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.
T288	44415-44490	Sentence	denotes	Moreover, HCQ was associated with a decrement of corticosteroid injections.
T289	44491-44574	Sentence	denotes	By contrast, no improvements in psychological function have been demonstrated [76].
T290	44575-44825	Sentence	denotes	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.
T291	44826-45256	Sentence	denotes	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].
T292	45257-45377	Sentence	denotes	In patients with rheumatoid arthritis, the cardiovascular risk is more than doubled, compared to the healthy population.
T293	45378-45552	Sentence	denotes	Chronic inflammatory status leads to an intensification of the atherosclerotic process, resulting in a higher susceptibility to hypertension, obesity, and metabolic syndrome.
T294	45553-45673	Sentence	denotes	The overproduction of IL-6 is strictly related to lipid profile alterations, given its role in adipose tissue lipolysis.
T295	45674-45751	Sentence	denotes	The inflammation and endothelial damage are exacerbated by leptin production.
T296	45752-45889	Sentence	denotes	Batun-Garrido, Salas-Magana and Juarez-Rojop [79] found a significant correlation between HCQ treatment and lower IL-6 and leptin levels.
T297	45890-46255	Sentence	denotes	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).
T298	46256-46486	Sentence	denotes	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].
T299	46487-46644	Sentence	denotes	The correlation between HCQ and cardiovascular risk was also assessed in a cross-sectional observational study involving 177 women with rheumatoid arthritis.
T300	46645-46781	Sentence	denotes	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].
T301	46782-46997	Sentence	denotes	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.
T302	46998-47084	Sentence	denotes	For these patients, no significant changes in insulin resistance were observable [81].
T303	47085-47217	Sentence	denotes	This study evaluated only insulin sensitivity, by Homeostatic Model Assessment (HOMA) index, without considering insulin metabolism.
T304	47218-47320	Sentence	denotes	Thus, inconsistency should be explained by this factor, together with the short duration of treatment.
T305	47321-47447	Sentence	denotes	The anti-diabetic properties of HCQ have been also assessed in patients without arthritis, as reported in the next paragraphs.
T306	47449-47455	Sentence	denotes	2.3.2.
T307	47456-47475	Sentence	denotes	Lupus Erythematosus
T308	47476-47638	Sentence	denotes	Systemic lupus erythematosus is a multisystemic autoimmune chronic inflammatory disorder that mainly involves joints, mucosae, skin, and endothelial vessels [90].
T309	47639-47728	Sentence	denotes	For a long time, HCQ has played a marginal role in the overall management of the disease.
T310	47729-47884	Sentence	denotes	Since the 90s, the first evidence of HCQ effectiveness in controlling lupus erythematosus manifestations allowed its employment as a first-line medicament.
T311	47885-48034	Sentence	denotes	Although it was not recommended in single therapy, the immunomodulating properties of HCQ seem to play an important role in the disease pathogenesis.
T312	48035-48167	Sentence	denotes	It is associated with a decrement of exacerbation events, as well as protective effects towards vascular and thrombotic events [85].
T313	48168-48415	Sentence	denotes	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.
T314	48416-48552	Sentence	denotes	People who interrupted the therapy exhibited constitutional symptoms of the disease, as well as skin rashes, arthritis, and ulcers [83].
T315	48553-48768	Sentence	denotes	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].
T316	48769-48890	Sentence	denotes	A case-control study was carried out in order to define the role of HCQ in the survival of individuals affected by lupus.
T317	48891-49068	Sentence	denotes	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].
T318	49069-49298	Sentence	denotes	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].
T319	49299-49473	Sentence	denotes	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.
T320	49474-49730	Sentence	denotes	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.
T321	49731-49823	Sentence	denotes	Lately, autophagy had risen among the emerging strategies to reestablish the immune balance.
T322	49824-49985	Sentence	denotes	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.
T323	49986-50136	Sentence	denotes	After four weeks of treatment, HCQ clearly restored the immune balance, by both inhibiting Th17 response and enhancing Treg immunosuppressive effects.
T324	50137-50419	Sentence	denotes	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.
T325	50420-50604	Sentence	denotes	This randomized trial further evidenced that HCQ treatment also remarkably attenuated kidney inflammation, by limiting the migration of lymphoplasmacytic cells into renal tissues [86].
T326	50605-50756	Sentence	denotes	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.
T327	50757-51107	Sentence	denotes	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].
T328	51108-51400	Sentence	denotes	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].
T329	51401-51532	Sentence	denotes	Infiltrating cells, most of all mast cells, could involve skin tissues, causing one of the most common signs of lupus, skin rashes.
T330	51533-51694	Sentence	denotes	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.
T331	51695-51929	Sentence	denotes	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.
T332	51930-52102	Sentence	denotes	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].
T333	52103-52234	Sentence	denotes	The chronic inflammatory status that features lupus erythematosus leads to a higher susceptibility to cardiovascular complications.
T334	52235-52389	Sentence	denotes	Lupus, indeed, is often characterized by endothelial dysfunction, the earliest marker of cardiovascular disease, as well as hypertension and renal damage.
T335	52390-52519	Sentence	denotes	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.
T336	52520-52737	Sentence	denotes	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].
T337	52738-52867	Sentence	denotes	The same effect of normalizing nitric oxide and ROS production have been confirmed in a NZB/W F1 murine model at different times.
T338	52868-52982	Sentence	denotes	HCQ at 3 mg/kg/day p.o. protected vascular endothelium, with a strong improvement of endothelial dysfunction [91].
T339	52983-53065	Sentence	denotes	The benefits on atherosclerosis also pass through the lipid-lowering power of HCQ.
T340	53066-53292	Sentence	denotes	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].
T341	53293-53400	Sentence	denotes	By contrast, HCQ in patients with a mild or inactive condition had no significant effects on lipid profile.
T342	53401-53628	Sentence	denotes	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].
T343	53629-53745	Sentence	denotes	The use of HCQ may be helpful in minimizing cardiovascular risk by improving glycemic homeostasis in lupus patients.
T344	53746-53999	Sentence	denotes	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].
T345	54000-54141	Sentence	denotes	HCQ remains a worthwhile primary or additional therapy in lupus patients, considering the low cost and its safety profile, also in pregnancy.
T346	54142-54313	Sentence	denotes	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].
T347	54314-54424	Sentence	denotes	A 5-year prospective study evaluated the effect of HCQ discontinuation on lupus progression in pregnant women.
T348	54425-54535	Sentence	denotes	As it occurs in no pregnant people, interruption of HCQ treatment is linked to an exacerbation of the disease.
T349	54536-54732	Sentence	denotes	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].
T350	54733-54936	Sentence	denotes	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.
T351	54937-54999	Sentence	denotes	Moreover, it exerted protective effects on fetal growth [120].
T352	55001-55007	Sentence	denotes	2.3.3.
T353	55008-55033	Sentence	denotes	Antiphospholipid Syndrome
T354	55034-55146	Sentence	denotes	Antiphospholipid syndrome is an autoimmune disorder characterized by antiphospholipid autoantibodies production.
T355	55147-55229	Sentence	denotes	If it is not associated with other autoimmune diseases, it is called primary [97].
T356	55230-55439	Sentence	denotes	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.
T357	55440-55572	Sentence	denotes	Resonance spectroscopy, indeed, revealed that the fetal brain and placenta are the main targets of autoantibodies localization [95].
T358	55573-55733	Sentence	denotes	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.
T359	55734-55955	Sentence	denotes	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].
T360	55956-56102	Sentence	denotes	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.
T361	56103-56297	Sentence	denotes	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.
T362	56298-56396	Sentence	denotes	Given these results, the mechanisms underlying the use of HCQ in thromboprophylaxis were assessed.
T363	56397-56778	Sentence	denotes	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].
T364	56779-56890	Sentence	denotes	Moreover, the efficacy of HCQ in antithrombotic therapy may lie in the interference in the coagulation cascade.
T365	56891-57111	Sentence	denotes	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].
T366	57113-57119	Sentence	denotes	2.3.4.
T367	57120-57136	Sentence	denotes	Sjögren Syndrome
T368	57137-57251	Sentence	denotes	Sjögren syndrome is an autoimmune disease with a strong negative impact on the quality of life of affected people.
T369	57252-57381	Sentence	denotes	The main features of the disease are lymphocytic inflammation and alterations in major salivary glands, causing xerostomia [103].
T370	57382-57536	Sentence	denotes	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.
T371	57537-57827	Sentence	denotes	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].
T372	57828-57939	Sentence	denotes	However, a few years later, the first evidence of HCQ effectiveness in Sjögren syndrome treatment was reported.
T373	57940-58173	Sentence	denotes	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].
T374	58174-58434	Sentence	denotes	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].
T375	58435-58593	Sentence	denotes	Hypo-salivation deriving from acinar atrophy and fibrosis of salivary glands is often associated with over-expression of TGF-β (transforming growth factor-β).
T376	58594-58816	Sentence	denotes	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.
T377	58817-58943	Sentence	denotes	Moreover, HCQ intake was also accompanied by a decrease in autoantibody production and a lower lymphocytic infiltration [103].
T378	58944-59236	Sentence	denotes	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.
T379	59238-59244	Sentence	denotes	2.3.5.
T380	59245-59253	Sentence	denotes	Diabetes
T381	59254-59360	Sentence	denotes	As has already been demonstrated for autoimmune patients, HCQ demonstrated great anti-diabetic properties.
T382	59361-59706	Sentence	denotes	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.
T383	59707-59959	Sentence	denotes	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.
T384	59960-60146	Sentence	denotes	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].
T385	60147-60288	Sentence	denotes	Given the positive outcomes deriving from preclinical studies, the therapeutic potential of HCQ was also assessed in several clinical trials.
T386	60289-60413	Sentence	denotes	Included in a randomized, double-blinded study of 18 months with 300 mg of HCQ twice a day were 135 diabetic obese patients.
T387	60414-60577	Sentence	denotes	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].
T388	60578-60814	Sentence	denotes	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].
T389	60815-61004	Sentence	denotes	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.
T390	61005-61155	Sentence	denotes	Results reported a significant increase in insulin levels, demonstrating the potential use of HCQ to counteract the risk of developing diabetes [109].
T391	61156-61364	Sentence	denotes	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.
T392	61365-61559	Sentence	denotes	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.
T393	61560-61662	Sentence	denotes	Regarding lipidic status, total cholesterol and LDL levels were reduced more by HCQ than pioglitazone.
T394	61663-61781	Sentence	denotes	Given the good tolerability of the treatment, HCQ may arise as a therapeutic alternative in diabetes management [110].
T395	61783-61789	Sentence	denotes	2.3.6.
T396	61790-61844	Sentence	denotes	Others (Cancer, Inflammation, Cardiovascular Diseases)
T397	61845-62008	Sentence	denotes	Given the well-recognized properties of HCQ against inflammation, it is easily intuitable that this agent could possess interesting insights into cancer treatment.
T398	62009-62105	Sentence	denotes	Chronic intestinal inflammation predisposes to the risk of colitis-associated colorectal cancer.
T399	62106-62379	Sentence	denotes	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.
T400	62380-62574	Sentence	denotes	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.
T401	62575-62853	Sentence	denotes	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].
T402	62854-63003	Sentence	denotes	Many others are the mechanisms by which HCQ exerts anticancer effects, mainly in synergism with conventional chemotherapic drugs, as discussed later.
T403	63004-63153	Sentence	denotes	Regarding the cardioprotective effect, this review has already focused on the positive impact of HCQ on cardiovascular issues in autoimmune patients.
T404	63154-63380	Sentence	denotes	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.
T405	63381-63498	Sentence	denotes	The pharmacological preconditioning with HCQ seems to be a good strategy to protect from ischemia-reperfusion injury.
T406	63499-63603	Sentence	denotes	The pretreatment with daily gavage of 200 mg/kg of HCQ, indeed, reduced the cardiac infarct size by 47%.
T407	63604-63734	Sentence	denotes	The mechanism underlying this effect is linked to the inhibition of apoptosis and total cell death in neonatal rat cardiomyocytes.
T408	63735-63815	Sentence	denotes	The atherosclerotic process contributes to increasing the risk of heart failure.
T409	63816-63866	Sentence	denotes	The etiology of this condition is still not clear.
T410	63867-64062	Sentence	denotes	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.
T411	64063-64196	Sentence	denotes	HCQ decreased free-fatty acids, triglycerides, total cholesterol, and LDL levels in diabetic rats under doses of 200 mg/kg/day [106].
T412	64197-64391	Sentence	denotes	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].
T413	64392-64532	Sentence	denotes	These favorable effects at the metabolic level might be due to its anti-inflammatory power that influences many other biological activities.
T414	64533-64723	Sentence	denotes	In gastrointestinal inflammations, mainly in inflammatory bowel disease, HCQ suppressed pro-inflammatory cytokines and enhanced the expression of ILs involved in anti-inflammatory processes.
T415	64724-64977	Sentence	denotes	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].
T416	64978-65083	Sentence	denotes	Inflammation, together with alterations in the immune system, are at the basis of pulmonary hypertension.
T417	65084-65380	Sentence	denotes	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].
T418	65381-65544	Sentence	denotes	Likewise, in endometriosis, the abnormal presence of endometrium in other organs leads to a chronic inflammatory status that could be affected by HCQ intervention.
T419	65545-65739	Sentence	denotes	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.).
T420	65740-65890	Sentence	denotes	In their role of scavengers, abnormalities in these cell populations may lead to an accumulation of endometrial cells, with impairment of the disease.
T421	65891-66061	Sentence	denotes	Moreover, histopathologic improvement of lesions was observed, probably due to the inhibition of autophagy by HCQ that alters anoikis response of endometrial cells [116].
T422	66063-66067	Sentence	denotes	2.4.
T423	66068-66106	Sentence	denotes	Hydroxychloroquine and Synergic Effect
T424	66107-66335	Sentence	denotes	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.
T425	66337-66343	Sentence	denotes	2.4.1.
T426	66344-66363	Sentence	denotes	Autoimmune Diseases
T427	66364-66581	Sentence	denotes	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].
T428	66582-66727	Sentence	denotes	It often occurs also that glucocorticoids or natural antioxidant substances are included in the coadjutant therapy of rheumatoid arthritis [123].
T429	66728-66990	Sentence	denotes	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.
T430	66991-67096	Sentence	denotes	A total of 195 patients were equally divided into two groups to follow the assigned therapeutic protocol.
T431	67097-67325	Sentence	denotes	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.
T432	67326-67486	Sentence	denotes	After one year, 75% of subjects under combinatory therapy and 60% of those under single-therapy reached clinical improvement, intended as 50% clinical response.
T433	67487-67601	Sentence	denotes	In terms of tolerability, the cotreatment resulted not to be more dangerous with respect to the single drug [123].
T434	67602-67805	Sentence	denotes	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.
T435	67806-67931	Sentence	denotes	Significant improvements in clinical parameters revealed the efficacy of the cotreatment in counteracting endothelial injury.
T436	67932-68101	Sentence	denotes	Indeed, the blood concentrations of endothelial injury markers, mainly soluble E selectin and thrombomodulin, experienced a significant drop after the cotreatment [124].
T437	68102-68493	Sentence	denotes	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].
T438	68494-68853	Sentence	denotes	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].
T439	68854-69072	Sentence	denotes	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.
T440	69073-69344	Sentence	denotes	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].
T441	69345-69451	Sentence	denotes	Although HCQ is effective and well-tolerated, other therapeutic alternatives have emerged in recent years.
T442	69452-69509	Sentence	denotes	Among them, monoclonal antibodies are the most promising.
T443	69510-69824	Sentence	denotes	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].
T444	69825-69939	Sentence	denotes	In addition to rheumatoid arthritis patients, HCQ is also used in lupus erythematosus, another autoimmune disease.
T445	69940-69990	Sentence	denotes	Regarding lupus erythematosus, HCQ is widely used.
T446	69991-70330	Sentence	denotes	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.
T447	70331-70639	Sentence	denotes	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].
T448	70640-70746	Sentence	denotes	In association with low-dose aspirin, HCQ is also indicated for thromboprophylaxis in patients with lupus.
T449	70747-71041	Sentence	denotes	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].
T450	71042-71191	Sentence	denotes	Thanks to its immunomodulatory properties, HCQ (20 mg/kg) was revealed to be useful in graft-versus-host disease, in combination with cyclosporine A.
T451	71192-71299	Sentence	denotes	They synergistically suppressed T cell response, allowing the reduction of the dose of drugs in mice [131].
T452	71301-71307	Sentence	denotes	2.4.2.
T453	71308-71338	Sentence	denotes	Cardiovascular Risk Management
T454	71339-71468	Sentence	denotes	HCQ revealed the great potential in the management of cardiovascular risk by controlling glucose homeostasis and lipidic profile.
T455	71469-71607	Sentence	denotes	Until now, in this review, we discussed the effect of HCQ alone to counteract cardiovascular complications, mostly in autoimmune patients.
T456	71608-71753	Sentence	denotes	Here, we reviewed the multiple pleiotropic actions of HCQ in combination with conventional medication in the most common cardiovascular diseases.
T457	71754-71973	Sentence	denotes	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.
T458	71974-72099	Sentence	denotes	The synergistic effect reduced the myocardial infarct size by up to 20% and improved insulin secretion and sensitivity [132].
T459	72100-72312	Sentence	denotes	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.
T460	72313-72449	Sentence	denotes	The role of HCQ in the inhibition of platelet aggregation was evaluated in healthy volunteers in comparison with aspirin or clopidogrel.
T461	72450-72562	Sentence	denotes	The addition of 400 mg/day of HCQ to aspirin resulted in a significant increase in aggregation inhibition (31%).
T462	72563-72692	Sentence	denotes	This inhibition was passed by reducing fibrinogen and inflammatory status by interfering with the arachidonic acid cascade [134].
T463	72694-72700	Sentence	denotes	2.4.3.
T464	72701-72711	Sentence	denotes	Anticancer
T465	72712-72791	Sentence	denotes	HCQ explains its antitumor activity thanks to its ability to inhibit autophagy.
T466	72792-72856	Sentence	denotes	HCQ is, indeed, an FDA-approved drug inhibiting autophagy [135].
T467	72857-72934	Sentence	denotes	Several types of tumors develop chemoresistance by enhancing autophagic flux.
T468	72935-73117	Sentence	denotes	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.
T469	73118-73220	Sentence	denotes	By recycling intracellular components, cells may maintain an energy balance and increase their growth.
T470	73221-73287	Sentence	denotes	If it occurs in cancer cells, resistance mechanisms may establish.
T471	73288-73431	Sentence	denotes	One of the mechanisms responsible for drug resistance is related to increased drug efflux by ATP-binding cassette (ABC) transporters [136,137].
T472	73432-73668	Sentence	denotes	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].
T473	73669-73785	Sentence	denotes	However, the role of autophagy in cancer is controversial and depends on genotype and tumor stage development [139].
T474	73786-73983	Sentence	denotes	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.
T475	73984-74101	Sentence	denotes	Depending on genetic concomitant alterations, autophagy may possess both pro-tumorigenic and tumor-suppressive roles.
T476	74102-74262	Sentence	denotes	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.
T477	74263-74432	Sentence	denotes	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.).
T478	74433-74653	Sentence	denotes	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].
T479	74654-74794	Sentence	denotes	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].
T480	74795-74891	Sentence	denotes	This issue highlights the necessity to carefully evaluate the use of HCQ in certain tumor types.
T481	74892-75117	Sentence	denotes	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].
T482	75118-75322	Sentence	denotes	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.
T483	75323-75431	Sentence	denotes	Regarding KRAS oncoprotein, its downstream pathway is one of the major players of pancreatic carcinogenesis.
T484	75432-75556	Sentence	denotes	The inhibition of this pathway by cytotoxic drugs, as well as trametinib, is often associated with an increase in autophagy.
T485	75557-75833	Sentence	denotes	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.
T486	75834-75989	Sentence	denotes	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.
T487	75990-76160	Sentence	denotes	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].
T488	76161-76255	Sentence	denotes	Then, it is important in assessing the contextual role of HCQ in cancer resistance mechanisms.
T489	76256-76362	Sentence	denotes	Epirubicin in triple-negative breast cancer therapy often lost efficacy, due to chemoresistance acquiring.
T490	76363-76464	Sentence	denotes	It has been shown that this cytotoxic agent induced autophagic flux, increasing cancer cell survival.
T491	76465-76642	Sentence	denotes	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].
T492	76643-76784	Sentence	denotes	In addition, estrogen receptor-positive breast cancers developed resistance to treatment with tamoxifen, due to the enhancement of autophagy.
T493	76785-76912	Sentence	denotes	The coadministration of HCQ (1–2 mg/day/mice in drinking water) restored the susceptibility of cancer cells to tamoxifen [146].
T494	76913-77181	Sentence	denotes	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].
T495	77182-77429	Sentence	denotes	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].
T496	77430-77500	Sentence	denotes	HCQ was revealed also to be active against chemoresistant lung cancer.
T497	77501-77672	Sentence	denotes	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.
T498	77673-77846	Sentence	denotes	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].
T499	77847-77949	Sentence	denotes	Together with autophagy, glycolysis plays a pivotal role in satisfying the increased energetic demand.
T500	77950-78041	Sentence	denotes	The dual targeting of the processes may provide a new therapeutic approach in cancer cells.

T1

0-98

Sentence

denotes

Recent Clinical and Preclinical Studies of Hydroxychloroquine on RNA Viruses and Chronic Diseases:

T2

99-118

Sentence

denotes

A Systematic Review

T3

120-128

Sentence

denotes

Abstract

T4

129-312

Sentence

denotes

The rapid spread of the new Coronavirus Disease 2019 (COVID-19) has actually become the newest challenge for the healthcare system since, to date, there is not an effective treatment.

T5

313-398

Sentence

denotes

Among all drugs tested, Hydroxychloroquine (HCQ) has attracted significant attention.

T6

399-532

Sentence

denotes

This systematic review aims to analyze preclinical and clinical studies on HCQ potential use in viral infection and chronic diseases.

T7

533-719

Sentence

denotes

A systematic search of Scopus and PubMed databases was performed to identify clinical and preclinical studies on this argument; 2463 papers were identified and 133 studies were included.

T8

720-915

Sentence

denotes

Regarding HCQ activity against COVID-19, it was noticed that despite the first data were promising, the latest outcomes highlighted the ineffectiveness of HCQ in the treatment of viral infection.

T9

916-1060

Sentence

denotes

Several trials have seen that HCQ administration did not improve severe illness and did not prevent the infection outbreak after virus exposure.

T10

1061-1215

Sentence

denotes

By contrast, HCQ arises as a first-line treatment in managing autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, and Sjögren syndrome.

T11

1216-1310

Sentence

denotes

It also improves glucose and lipid homeostasis and reveals significant antibacterial activity.

T12

1312-1314

Sentence

denotes

1.

T13

1315-1327

Sentence

denotes

Introduction

T14

1328-1493

Sentence

denotes

The 4-aminoquinolonehydroxychloroquine (HCQ) was synthesized for the first time in 1946, but its history began as far back as the 1600s thanks to the Incas in Chile.

T15

1494-1716

Sentence

denotes

They introduced the special properties of cinchona bark to the Jesuits and in 1820 quinine and cinchonine were isolated and identified as the main alkaloids responsible for the antimalarial activity attributed to the bark.

T16

1717-1811

Sentence

denotes

For these reasons, in 1900, the Dutch and British transplanted this “miraculous tree” to Java.

T17

1812-1909

Sentence

denotes

The quinine soon began to be also used for the treatment of systemic lupus erythematosus [1,2,3].

T18

1910-2041

Sentence

denotes

The increasing need for quinine, due to malaria diffusion, led the pharmaceutical industry to the creation of a synthetic molecule.

T19

2042-2105

Sentence

denotes

In 1934 Johann (Hans) Andersag and co-workers at the Bayer I.G.

T20

2106-2249

Sentence

denotes

Farbenindustrie in Elberfeld laboratories, Germany, synthesized chloroquine (CQ) for the first time, judged by the Germans to be too toxic [4].

T21

2250-2375

Sentence

denotes

However, the production of natural quinine was blocked by the Japanese army’s occupation of Java during the Second World War.

T22

2376-2474

Sentence

denotes

Therefore, it was necessary to deepen the studies on molecules that could replace natural quinine.

T23

2475-2656

Sentence

denotes

The Americans soon synthesized from CQ the HCQ, which resulted in it being less toxic than its ancestor so that in 1955 the scientific community proposed it as an alternative to CQ.

T24

2657-2734

Sentence

denotes

Figure 1 represents the historical development that led to HCQ synthesis [5].

T25

2735-2990

Sentence

denotes

This molecule, which has always been known as antimalarial, has come back into vogue in recent months due to the ongoing new coronavirus (2019-nCoV, or COVID-19 or 2019-nCo) worldwide pandemic, that causes a severe acute respiratory syndrome (SARS-CoV-2).

T26

2991-3173

Sentence

denotes

In fact, several research groups have evaluated the use of CQ (C18H26ClN3) and in particular its derivative HCQ (C18H26ClN3O), as a promising treatment for COVID-19 patients [6,7,8].

T27

3174-3382

Sentence

denotes

CQ and HCQ have a core structure consisting of two aromatic rings fused, the 4-aminoquinoline nucleus, and an amphiphilic weak basic side chain (represented in green and red colors in Figure 1, respectively).

T28

3383-3519

Sentence

denotes

The two chemical structures differ only for the hydroxyl group at the end of N-ethyl side chain (represented in blue color in Figure 1).

T29

3520-3714

Sentence

denotes

These are water-soluble molecules and, for their chemical nature, can pass the cell membranes; however, the presence of the hydroxyl group in the HCQ makes it more polar and less lipophilic [8].

T30

3715-3867

Sentence

denotes

Moreover, the accumulation of CQ and HCQ in intracellular compartments is due principally to the side chain and both have enantiomers (R and S isomers).

T31

3868-3972

Sentence

denotes

Researches also demonstrated that the R-HCQ is present in the blood at higher concentrations than S-HCQ.

T32

3973-4065

Sentence

denotes

These results suggested the stereoselective processes in the metabolism of the molecule [9].

T33

4066-4272

Sentence

denotes

Studies of structure-activity relationships have demonstrated that the halogen substitutions of different 4-aminoquinolinesderivatives reduced the toxicity, but it also reduced the pharmacological activity.

T34

4273-4376

Sentence

denotes

Instead, the therapeutic ratio is decreased by substitution of the alkyl side chain with an aryl chain.

T35

4377-4517

Sentence

denotes

Nevertheless, the therapeutic ratio decreases and toxicity increases with an increase of the alkyl side-chain length above 5 carbons [7,10].

T36

4518-4660

Sentence

denotes

Usually, CQ and HCQ are administered as phosphate and sulphate salts, respectively, and both drugs are absorbed in the upper intestinal tract.

T37

4661-4747

Sentence

denotes

However, clinically, HCQ is more frequently used than CQ for its lesser toxicity [11].

T38

4748-4938

Sentence

denotes

Both molecules give mild nausea, stomach cramps, gastrointestinal upset, and mild diarrhoea as adverse effects and long-term usage determines loss of retinal function and severe retinopathy.

T39

4939-5131

Sentence

denotes

These drugs are used, to date, in malaria patients and in several inflammatory diseases like rheumatoid arthritis, Sjögren syndrome, dermatomyositis, and systemic lupus erythematosus [7,8,10].

T40

5133-5135

Sentence

denotes

2.

T41

5136-5159

Sentence

denotes

Results and Discussions

T42

5161-5165

Sentence

denotes

2.1.

T43

5166-5180

Sentence

denotes

Study Analysis

T44

5181-5281

Sentence

denotes

The initial survey of the literature identified 2756 reports (2066 from Scopus and 690 from PubMed).

T45

5282-5416

Sentence

denotes

Then, 293 the duplicates between the two databases were eliminated and were considered only one at a time, resulting in 2463 articles.

T46

5417-5573

Sentence

denotes

After the primary screening based on titles and abstracts, 959 manuscripts were excluded since they did not fulfil the inclusion criteria or were off-topic.

T47

5574-5658

Sentence

denotes

Finally, 1504 articles were thoroughly analyzed and among these, 1442 were excluded.

T48

5659-5823

Sentence

denotes

The analysis of the reference lists from some selected items led to the inclusion of an extra 71 appropriate articles, after titles, abstracts, and full-text study.

T49

5824-5879

Sentence

denotes

In total, 133 papers were selected for data extraction.

T50

5880-5959

Sentence

denotes

A flowchart illustrating the steps of the study selection is shown in Figure 2.

T51

5960-6087

Sentence

denotes

In our review, the articles included were also analyzed in relation to the year of publication and the experimental model used.

T52

6088-6189

Sentence

denotes

Regarding the chronology of the publications, most of the articles were published in the last decade.

T53

6190-6448

Sentence

denotes

The high peaks correspond to the proportional number of papers on antiviral activity in 2020; this result is consistent with the actual increasing interest towards the role of HCQ as a possible therapeutic strategy in the current COVID-19 sanitary emergency.

T54

6449-6536

Sentence

denotes

HCQ alone, or in combination with other drugs, was used in various types of infections.

T55

6537-6616

Sentence

denotes

HIV and COVID-19 were the most cited, with 10 and 7 reports each, respectively.

T56

6617-6694

Sentence

denotes

Our systematic review included 49 preclinical studies and 84 clinical trials.

T57

6695-6779

Sentence

denotes

The considered in vivo studies were carried out using allograft or xenograft models.

T58

6780-6862

Sentence

denotes

The most important outcomes of the review are graphically illustrated in Figure 3.

T59

6863-7011

Sentence

denotes

The assessment of bias risk based on a checklist adapted from the Cochrane Handbook for Systematic Reviews of Interventions is reported in Figure 4.

T60

7012-7082

Sentence

denotes

The number of high-risk reports is due to the case reports considered.

T61

7084-7088

Sentence

denotes

2.2.

T62

7089-7128

Sentence

denotes

Hydroxychloroquine and Viral Infections

T63

7129-7237

Sentence

denotes

HCQ has been used mainly as an antimalarial drug, but it has also proven effective against viral infections.

T64

7238-7426

Sentence

denotes

HCQ demonstrated its antiviral efficacy in inhibiting the endosomal-lysosomal acidification, which is essential for the entry, replication, and infection process of different viruses [12].

T65

7427-7558

Sentence

denotes

In particular, HCQ-induced alkalinization processes cause the expansion and vacuolization of lysosomes, inhibiting their functions.

T66

7559-7728

Sentence

denotes

This activity can reduce post-transcriptional protein modification, enzyme release, receptor recycling, activation of cell signaling pathways, and cell membranes repair.

T67

7729-7784

Sentence

denotes

As a result, there is the prevention of cell infection.

T68

7785-7887

Sentence

denotes

Further, HCQ antiviral activity is also related to its anti-inflammatory and immunomodulatory effects.

T69

7888-8163

Sentence

denotes

Different studies have, indeed, demonstrated that viral diseases are caused by a direct viral infection of susceptible cells and also by an impact on the immune response with consequent uncontrolled release of pro-inflammatory chemokines, cytokines, and other mediators [13].

T70

8164-8333

Sentence

denotes

The most studied antiviral activity of HCQ is that exerted against HIV; however, the current spread of the Coronavirus infection has brought attention back to this drug.

T71

8334-8581

Sentence

denotes

In this systematic review, clinical and in vivo studies evaluating HCQ antiviral activity against the Human Immunodeficiency Virus (HIV), Chikungunya Virus (CHIKV), Flavivirus, and Coronavirus have been analyzed (Figure 3B, Figure 5, and Table 1).

T72

8583-8589

Sentence

denotes

2.2.1.

T73

8590-8595

Sentence

denotes

HIV-1

T74

8596-8711

Sentence

denotes

HIV is part of the genus Lentivirus, of the Retroviridae family, and it is divided into two lines: HIV-1 and HIV-2.

T75

8712-8811

Sentence

denotes

The most virulent and infectious is HIV-1, since it causes most of the HIV infections in the world.

T76

8812-8951

Sentence

denotes

The target cells of HIV are those rich in CD4 receptors, such as some lymphocytes called CD4+, which play a crucial role in human immunity.

T77

8952-9082

Sentence

denotes

Indeed, these lymphocytes activate different immune system cells depending on the type of unwanted host they come in contact with.

T78

9083-9194

Sentence

denotes

It seems that acute HIV infection is highly linked to a rapid depletion of CD4+ T cells in gut lymphoid tissue.

T79

9195-9367

Sentence

denotes

This event is related to an alteration of the intestinal mucosa integrity, resulting in bloodstream translocation of microbial products like lipopolysaccharides (LPS) [42].

T80

9368-9545

Sentence

denotes

It has been hypothesized that LPS, through the binding and activation of Toll-like receptor 4 (TLR-4), is responsible for the immune system activation observed in HIV infection.

T81

9546-9760

Sentence

denotes

Although the real HCQ mechanism of action has not been well assessed, it seems that the anti-HIV effects are highly linked to the post-translational modification of glycoprotein 120 (gp120) in monocyte and T cells.

T82

9761-9883

Sentence

denotes

Consequently, there was a modification of gp120 immunogenic properties and a reduction of new virions infectivity [15,16].

T83

9884-10013

Sentence

denotes

The first randomized, double-blind, placebo-controlled clinical trial about the anti-HIV-1 activity of HCQ was published in 1995.

T84

10014-10125

Sentence

denotes

In this study, 40 asymptomatic HIV-infected patients received either 800 mg/day HCQ or placebo for eight weeks.

T85

10126-10315

Sentence

denotes

All patients treated with antiretroviral therapy (HAART = zidovudine (ZDV), 2′,3′-dideoxyinosine, or 2′,3′-dideoxycytidine) and stopped taking it four weeks before the clinical trial start.

T86

10316-10436

Sentence

denotes

Unlike placebo, at the eighth week, HCQ displayed a reduction in HIV-1 RNA total plasma levels in 12 out of 19 patients.

T87

10437-10649

Sentence

denotes

Furthermore, the percentage of CD4+ T cells decreased significantly in the placebo group and remained stable during the treatment with HCQ, indicating a probable stabilization of immune function in the HCQ group.

T88

10650-10750

Sentence

denotes

HCQ administration also induced a decrease in serum interleukin 6 (IL-6) and immunoglobulin G (IgG).

T89

10751-10825

Sentence

denotes

However, no significant changes were found in the IgA and IgM levels [17].

T90

10826-11041

Sentence

denotes

The anti-HIV-1 effect of HCQ was also compared with that of ZDV, a nucleoside reverse transcriptase inhibitor, in a randomized, placebo-controlled clinical trial conducted on 72 HIV-l-infected asymptomatic patients.

T91

11042-11137

Sentence

denotes

All subjects were treated for 16 weeks with 800 mg/day HCQ (n = 35) or 500 mg/day ZDV (n = 37).

T92

11138-11260

Sentence

denotes

As in the previous study, patients who had received HAART stopped taking it four weeks before the clinical trial’s outset.

T93

11261-11461

Sentence

denotes

After 16 weeks, total plasma HIV-1 RNA levels were reduced in both the ZDV group and HCQ group, although the extent of anti-HIV-l activity in HCQ patients was lower than that observed in ZDV subjects.

T94

11462-11626

Sentence

denotes

However, eight subjects in the Azithromycin (AZM) group showed an increase in HIV-1 RNA levels in the 16th week, indicating the rapid emergence of viral resistance.

T95

11627-11832

Sentence

denotes

Contrarily, in the HCQ group, increased antiviral activity was revealed after 16 weeks rather than after 8 weeks, and no subject showed an increase in HIV-1 RNA levels at either 8 or 16 weeks of treatment.

T96

11833-11955

Sentence

denotes

This evidence suggests that no resistance developed under HCQ therapy or that it might develop more slowly than under ZDV.

T97

11956-12128

Sentence

denotes

A reduction in serum p24 antigen levels in both ZDV and HCQ groups was also described, while only in the HCQ group could a decrease in IL-6 and IgG levels be observed [18].

T98

12129-12328

Sentence

denotes

This reduction of IgG levels displayed after HCQ treatment in both studies may be significant since autoantibodies contribute to CD4+ cell depletion and autoimmune diseases observed in HIV infection.

T99

12329-12614

Sentence

denotes

Further, as lymphoid tissue is considered the primary site of HIV reservoirs and a critical site affected by CD4+ T cells depletion, the HCQ concentration was assessed in the plasma and adenoid tissue (At) of 8 HIV-infected patients administrating 400 or 800 mg of HCQ for eight weeks.

T100

12615-12746

Sentence

denotes

After taking these dosages, it was demonstrated that the mean HCQ concentration was significantly higher in At than in plasma [21].

T101

12747-12905

Sentence

denotes

This different drug distribution was also confirmed by an in vivo study using rabbits as an experimental model, receiving 15 mg/kg of HCQ subcutaneously [22].

T102

12906-13056

Sentence

denotes

Thus, the anti-HIV activity of HCQ could be linked to its accumulation in lymphoid tissue, a relevant site for HIV immunopathogenesis and replication.

T103

13057-13192

Sentence

denotes

Since monotherapy is not recommended in treating HIV, HCQ has also been tested in synergy with other drugs commonly used to manage HIV.

T104

13193-13442

Sentence

denotes

In this regard, 400 mg/day of HCQ in a combination regimen with 1000 mg/day hydroxyurea and 250–400 mg/day didanosine (dosed per body weight) was administered for 48 weeks to 22 asymptomatic HIV-1 infected patients naïve to antiretroviral treatment.

T105

13443-13485

Sentence

denotes

Only 16 out of 22 patients were evaluable.

T106

13486-13609

Sentence

denotes

These 16 subjects, at the 12th week, showed a significant reduction in viral load which was maintained until the 48th week.

T107

13610-13737

Sentence

denotes

Furthermore, at week 12, an increase in CD4+ percentage was also shown, and this improvement was kept until the 48th week [19].

T108

13738-13903

Sentence

denotes

To evaluate the long-term efficacy and safety of HCQ, hydroxyurea, and didanosine combination, they were also tested on 17 HIV-infected naïve patients for 144 weeks.

T109

13904-13996

Sentence

denotes

All subjects received 200 mg HCQ, 500 mg hydroxyurea, and 125–200 mg didanosine twice daily.

T110

13997-14083

Sentence

denotes

Of the 17 patients who started treatment, 14 remained until the end of the 144th week.

T111

14084-14341

Sentence

denotes

After 114 weeks, viral load was reduced by 1.6 Log10 copies/mL under baseline (p < 0.001), eight patients (47%) had an unnoticeable viral load (< 400 copies/mL), and two patients (12%) had a measurable viral load, but resistance mutations were not detected.

T112

14342-14582

Sentence

denotes

Four patients (24%) had both detectable viral load and resistance mutation: one with both 62V and 65R and three with both 74V and 184V mutations; the latter three were assessed as didanosine resistant, while no resistance was found for HCQ.

T113

14583-14667

Sentence

denotes

However, in all cases, the viral load remained below the baseline at the 144th week.

T114

14668-14789

Sentence

denotes

The CD4+ cell count had increased significantly, while the percentage of CD8 cells was reduced up to the 144th week [20].

T115

14790-15022

Sentence

denotes

This HCQ noticeable impact on immune activation, thereby increasing CD4+ T cells, was also demonstrated in a prospective study conducted on 20 HIV-infected immunologic no responders treated with standard antiretroviral therapy [23].

T116

15023-15187

Sentence

denotes

These results suggested that the combination of HCQ, hydroxyurea, and didanosine could be a valid alternative to the highly active commercial HAART in HIV-patients.

T117

15188-15326

Sentence

denotes

Nonetheless, these latter studies have some limitations, such as the small number of subjects included and the absence of a control group.

T118

15327-15476

Sentence

denotes

Therefore, it is not possible to determine the contribution made by HCQ to the overall decrease in viral load obtained from the combination of drugs.

T119

15477-15709

Sentence

denotes

Anyway, the potential anti-HIV efficacy of HCQ, when added to existing treatment with an antiretroviral regimen, was also confirmed by a case report about a patient with HLA-B27-associated spondyloarthropathy and HIV infection [43].

T120

15710-15930

Sentence

denotes

In contrast to the results mentioned above has been published a randomized, double-blind, placebo-controlled trial performed on 83 patients to which 400 mg HCQ (n = 42) or placebo (n = 41) were administered for 48 weeks.

T121

15931-16132

Sentence

denotes

All patients were naïve to HAART or had stopped this therapy 22 months before the trial began; 17 subjects in the HCQ group and 8 in the placebo group interrupted study medication before the 48th week.

T122

16133-16327

Sentence

denotes

At the end of treatment, in the HCQ group, compared to placebo, patients showed a reduction in total CD4 cell count and a significant viral load increased from the 12th week above baseline [24].

T123

16328-16554

Sentence

denotes

Hence, based on these results, HCQ did not decrease immune activation in patients with chronic HIV infection who were naïve to HAART, as there was an increase in HIV viral replication and a negative effect on CD4+ cell counts.

T124

16555-16804

Sentence

denotes

In light of these results, there was the need to consider that the first two described clinical trials, which reported the antiviral effect of HCQ, were on short-term treatments (8 or 16 weeks) and that they used an HCQ dosage of 800 mg/day [17,18].

T125

16805-16920

Sentence

denotes

In contrast, the latter used only 400 mg/day [24], corresponding to the maximum recommended dose for long-time use.

T126

16921-17200

Sentence

denotes

Besides, the latter study also enrolled more patients than the other studies, and unlike the trial of Piconi et al. [23], which described significative effects in reducing immune activation after HCQ administration, was conducted in the absence of antiretroviral treatments [24].

T127

17201-17392

Sentence

denotes

Therefore, further clinical trials involving a larger number of subjects would be necessary to assess the real antiviral activity of HCQ in monotherapy and synergy with antiretrovirals drugs.

T128

17393-17606

Sentence

denotes

Finally, assuming that women resistant to HIV infection show a low activation of the immune system at the level of the female genital tract (FGT), HCQ has been investigated as a drug able to prevent HIV infection.

T129

17607-17733

Sentence

denotes

It has been shown that the “immune quiescent” state of HIV-resistant women keeps the immune response against pathogens intact.

T130

17734-17931

Sentence

denotes

For this reason, it was thought to induce pharmacologically, in a rabbit model, this immunological quiescence state through an intravaginal implant capable of providing a controlled release of HCQ.

T131

17932-18202

Sentence

denotes

Considering that a concentration above 6.48 μg/mL of HCQ was able to interfere with the gp120 glycosylation process, the vaginal implant was projected to release HCQ with the longest possible duration at a concentration greater than 4.34 μg/mL but lower than 21.7 μg/mL.

T132

18203-18451

Sentence

denotes

After six days, this implant improved mucosal epithelial integrity, reduced submucosal immune cell recruitment, decreased gene expression and protein of T cell activation markers, and minimized the activation of key pro-inflammatory mediators [25].

T133

18452-18610

Sentence

denotes

Hence, HCQ can be considered a promising drug able to maintain a low baseline level of immune activation and may also play a role in preventing HIV infection.

T134

18612-18618

Sentence

denotes

2.2.2.

T135

18619-18636

Sentence

denotes

Chikungunya Virus

T136

18637-18784

Sentence

denotes

The single-stranded RNA virus, Chikungunya virus (CHIKV), is an alphavirus belonging to the Togaviridae family, spread mainly in America’s regions.

T137

18785-18905

Sentence

denotes

The usual CHIKV vectors are rodents, while humans are infected by Aedes albopictus and Aedes aegypti and mosquito bites.

T138

18906-19460

Sentence

denotes

The first incubation stage can vary between 2 and 12 days, and three phases follow it: (1) the acute viraemic phase, characterized by severe polyarthritis, fever, and a rash, generally resolving in three weeks; (2) the post-acute stage, identified by arthritis with the addition of synovial and periarticular inflammation, neuropathy, neuropsychiatric disorders, and peripheral vascular disorders, usually takes its time at the end of three months; (3) the chronic phase that appears when the symptoms of the previous phase do not end after three months.

T139

19461-19730

Sentence

denotes

Generally, the acute phase of CHIKV infections is treated with non-steroidal anti-inflammatory drugs (NSAIDs), while for the chronic persistent phase, treatments involving HCQ as monotherapy or combined with methotrexate (MTX) and/or sulfasalazine seem to be effective.

T140

19731-19866

Sentence

denotes

HCQ does not appear to affect the initial stage of infection, as demonstrated in a prospective randomized parallel-group study of 2009.

T141

19867-19988

Sentence

denotes

In this trial, combinations of NSAIDs, HCQ, and/or corticosteroids were assessed in patients with classic CHIKV features.

T142

19989-20156

Sentence

denotes

A total of 120 subjects were divided into groups treated with 200 mg/day aceclofenac (ACF), 400 mg/day HCQ, and 10 mg/day prednisolone (PRD) in different combinations.

T143

20157-20223

Sentence

denotes

Only 114 subjects remained until the end of the trial (six weeks).

T144

20224-20489

Sentence

denotes

It was seen that HCQ had no benefit in the early stages of CHIKV infection and also in the reduction of the VAS (used for pain assessment) and in the improvement of Barthel’s indexes (used for instrumental activities of daily living and activities of daily living).

T145

20490-20575

Sentence

denotes

In fact, there was no difference between groups treated with ACF + HCQ and ACF alone.

T146

20576-20677

Sentence

denotes

Similarly, the combination of ACF + HCQ + PRD did not add any additional benefit over ACF + PRD [26].

T147

20678-20759

Sentence

denotes

In contrast, HCQ seems to affect the improvement of CHIKV chronic phase diseases.

T148

20760-20900

Sentence

denotes

In a recent multicenter study, the efficacy of HCQ was evaluated in 39 patients with rheumatic manifestation related to CHIKV chronic phase.

T149

20901-21097

Sentence

denotes

In four subjects, the treatment was interrupted due to the onset of side effects such as nausea, stomatitis, rash, headache, while the evolution of CHIKV disease was evaluated in only 22 patients.

T150

21098-21283

Sentence

denotes

After three months of treatment, evidence of synovitis was disappeared in 10 of 20 subjects (50%) with swollen joins while complete remission was verified in five patients (19.2%) [27].

T151

21284-21430

Sentence

denotes

However, another study demonstrated that the effects of HCQ in combination with MXT and sulfasalazine were superior to those shown in monotherapy.

T152

21431-21640

Sentence

denotes

In particular, in a randomized controlled open-label study, the impact of HCQ in monotherapy or association with MTX and sulfasalazine was assessed in 72 patients with chronic persistent chikungunya arthritis.

T153

21641-21819

Sentence

denotes

In this trial, 400 mg/day HCQ were administrated to 35 subjects, while a combination of 15 mg/day MTX, 1 g/day sulfasalazine, and 400 mg/day HCQ was administrated to 37 patients.

T154

21820-21946

Sentence

denotes

Either treatment lasted 24 weeks and for the first 6 weeks, both groups received an escalated dose of prednisone (7.5 mg/day).

T155

21947-22069

Sentence

denotes

At the end of the 24th week, only the combination of drugs improved disease activity and reduced disability and pain [28].

T156

22070-22332

Sentence

denotes

These results, following those obtained in a previous uncontrolled 16-week study, since a reduction in ACR score was shown after MTX (15–20 mg/weekly) and HCQ (400 mg/day) administration to CHIKV infected patients with persistent inflammatory polyarthritis [29].

T157

22333-22435

Sentence

denotes

The results obtained could be explained with the different characterization of virus infection phases.

T158

22436-22720

Sentence

denotes

It is known that while the acute phase of CHIKV infections is due to the development of viremia, the chronic phase is closely related to an immune-mediated phenomenon, as pro-inflammatory cytokines and chemokines play important roles in the pathogenesis of chikungunya arthritis [44].

T159

22721-23009

Sentence

denotes

Therefore, considering the results obtained from these clinical studies, it is possible to say that although HCQ does not affect viremia reduction, as demonstrated by the lack of activity in the first phase of infection, it can improve the chronic phase diseases by reducing inflammation.

T160

23011-23017

Sentence

denotes

2.2.3.

T161

23018-23030

Sentence

denotes

Flaviviruses

T162

23031-23268

Sentence

denotes

Hepatitis virus, an RNA virus belonging to the Flaviviridae family, is closely related to liver disease, which in 70–80% of patients becomes chronic, resulting in major complications such as cirrhosis and, in the year, also liver cancer.

T163

23269-23365

Sentence

denotes

The antiviral activity of HCQ on the hepatitis C virus (HCV) in monotherapy has not been tested.

T164

23366-23448

Sentence

denotes

However, it seems that this drug increases the antiviral effect of standard drugs.

T165

23449-23691

Sentence

denotes

In a study including 120 Egyptian patients infected by the hepatitis C virus, it was seen that the combination of HCQ with pegylated interferon and ribavirin could improve biochemical and virological responses in chronic hepatitis C subjects.

T166

23692-23949

Sentence

denotes

All patients were randomized and divided into two groups; the control group treated with standard-of-care (SOC) consisted of 160 µg of subcutaneous pegylated interferon and 1000–12000 mg/day of oral ribavirin, and the group treated with 200 mg HCQ plus SOC.

T167

23950-24126

Sentence

denotes

At the end of the treatment (12 weeks), patients of HCQ + SOC group showed a high virological response compared to the control group (54/60 (90%) vs. 43/60 (71.7%); p = 0.011).

T168

24127-24345

Sentence

denotes

Moreover, in the HCQ + SOC group, there was a normalization of ALT levels, as is also demonstrated by the earlier biochemical response (EBR) highlighted in HCQ + SOC group 58/60 (96.7%) than SOC group 42/60 (70%) [30].

T169

24346-24472

Sentence

denotes

These results were confirmed by several case reports regarding patient treatment with Porphyria Cutanea Tarda and Hepatitis C.

T170

24473-24665

Sentence

denotes

It was seen that a low dose of HCQ (100 mg twice weekly) prevented the recurrence of Porphyria Tarda and reduced the viral response during HCV therapy, including ribavirin and interferon [45].

T171

24666-24923

Sentence

denotes

Furthermore, in a patient with chronic hepatitis and rheumatoid arthritis, a low risk for hepatitis virus reactivation was observed after treatment with steroids (< 7.5 mg/day), HCQ or sulfadiazine [46] in combination with antivirals as prophylaxis [47,48].

T172

24924-25092

Sentence

denotes

Another possible antiviral effect of HCQ is exerted on Zika virus (ZIKV), an RNA virus of the Flaviviridae family transmitted by numerous mosquitoes of the genus Aedes.

T173

25093-25280

Sentence

denotes

No clinical studies have been conducted yet, but an in vivo study has demonstrated the ability of HCQ to attenuate vertical transmission of ZIKV by reducing placental and fetal infection.

T174

25281-25414

Sentence

denotes

In this study, pregnant mice were treated with 40 mg/kg/day HCQ via intraperitoneal injection beginning one day after ZIKV infection.

T175

25415-25581

Sentence

denotes

It was seen that HCQ acted as an autophagy inhibitor by increasing p62 levels in trophoblast and thus reducing placental ZIKV infection and fetal growth defects [31].

T176

25582-25693

Sentence

denotes

To date, no clinical trials have been conducted to evaluate HCQ antiviral effects in patients infected by ZIKV.

T177

25695-25701

Sentence

denotes

2.2.4.

T178

25702-25729

Sentence

denotes

Coronavirus Disease of 2019

T179

25730-25940

Sentence

denotes

Coronaviruses, belonging to the Coronaviridae family, are enveloped positive-sense single-stranded RNA viruses highly distributed in humans and vertebrates like bats, which are proposed as their main reservoir.

T180

25941-26123

Sentence

denotes

Specifically, Coronavirus Disease of 2019 (COVID-19) was first identified in December 2019 in Wuhan, the capital city of Hubei (China), and it is caused by the SARS-CoV-2 virus [49].

T181

26124-26298

Sentence

denotes

Since COVID-19 has spread rapidly in many countries, it has quickly become a global pandemic, so it is necessary to develop drugs able to exert antiviral activity against it.

T182

26299-26394

Sentence

denotes

A recent study showed HCQ in vitro antiviral activity against SARS-CoV-2 (EC50 = 0.72 μM) [50].

T183

26395-26596

Sentence

denotes

HCQ seemed to be able to inhibit the first step of the viral replication cycle by interfering with the link between spike (S) viral protein and the angiotensin-converting enzyme 2 (ACE-2) receptor [8].

T184

26597-26756

Sentence

denotes

It would also appear that HCQ was able to induce changes in cell membrane pH resulting in reduced viral entry and inhibition of the last stages of replication.

T185

26757-26879

Sentence

denotes

Moreover, HCQ may abolish the cytokine storm related to the advanced stages of COVID-19 through immunomodulatory activity.

T186

26880-26989

Sentence

denotes

To date, several clinical studies are underway to evaluate the efficacy of HCQ for the treatment of COVID-19.

T187

26990-27136

Sentence

denotes

In a randomized clinical trial conducted in China, 62 patients with COVID-19 were randomly assigned to two groups: the control and the HCQ groups.

T188

27137-27375

Sentence

denotes

Either group received standard treatment including antiviral agents, oxygen therapy, immunoglobulin, and antibacterial agents, with or without corticosteroids, but patients in the HCQ group also received oral HCQ 400 mg/day for five days.

T189

27376-27521

Sentence

denotes

During treatment, clinical characteristics, clinical recovery time (TTCR), and radiological results were assessed to determine the effect of HCQ.

T190

27522-27771

Sentence

denotes

It was seen that in the HCQ group, the recovery time of body temperature was shorter than in the control group and that the cough remission time was also significantly decreased, while only patients in the control group progressed to severe illness.

T191

27772-27868

Sentence

denotes

Furthermore, in the HCQ group, 61.3% of patients showed significant absorption of pneumonia [6].

T192

27869-28001

Sentence

denotes

In another open-label non-randomized French clinical trial, the efficiency of HCQ in reducing the viral count was also demonstrated.

T193

28002-28058

Sentence

denotes

In this study, 36 subjects were divided into two groups:

T194

28059-28154

Sentence

denotes

16 patients for the control group and 20 subjects for HCQ who were administered 600 mg/day HCQ.

T195

28155-28320

Sentence

denotes

Among the HCQ group, six patients also received 500 mg of AZM for the first day, followed by 250 mg/day for the next four days to prevent super bacterial infections.

T196

28321-28466

Sentence

denotes

PCR results from nasopharyngeal samples were negative for 70% of patients treated with HCQ and 12.5% in the control group (p = 0.001) on day six.

T197

28467-28715

Sentence

denotes

Furthermore, when the effect of HCQ as monotherapy was compared to that of HCQ + AZM, it was found that at day six, 100% of HCQ + AZM treated subjects were virologically negative compared with 57.1% of patients treated with HCQ as monotherapy [32].

T198

28716-28933

Sentence

denotes

These results suggest a synergistic effect between HCQ and AZM and are supported by an uncontrolled non-comparative observational study conducted by the same France group, in a cohort of 80 slightly infected patients.

T199

28934-29101

Sentence

denotes

In this study, HCQ + AZM treated people were 83% virologically negative on day 7 and 93% on day 8; after 10 days of treatment, only 2 subjects still remain contagious.

T200

29102-29226

Sentence

denotes

Furthermore, the average duration of hospitalization was found to be 4.6 days after the administration of HCQ plus AZM [33].

T201

29227-29308

Sentence

denotes

However, these last two studies have a limit, as Gautret et al. carried both out.

T202

29309-29484

Sentence

denotes

In particular, in the first study, data are available up to 6 days despite the planned 10 days, and in the second study, 6 patients from the previous study were also included.

T203

29485-29683

Sentence

denotes

Gautret also conducted a retrospective analysis of 1061 cases in which, after treatment with HCQ + AZM, good virological care and clinical outcomes were found in 973 patients (91.7%) within 10 days.

T204

29684-29853

Sentence

denotes

A prolonged viral carriage was observed in only 47 (4.4%) subjects with a high viral load at the moment of hospitalization, but on day 10 the viral culture was negative.

T205

29854-29907

Sentence

denotes

Finally, all but one on day 15 were PCR cleared [51].

T206

29908-30043

Sentence

denotes

By contrast, there are results obtained by several clinical studies that dismiss the possible use of HCQ for the treatment of COVID-19.

T207

30044-30389

Sentence

denotes

In particular, a prospective study on 11 severe COVID-19 infected patients treated with the same dosage used by Gautret et al. (600 mg/day HCQ and 500 mg AZM for the first day followed by 250 mg/day from day 2 to 5) was shown to provide no evidence of clinical benefits or a strong antiviral activity through the combination of HCQ and AZM [34].

T208

30390-30605

Sentence

denotes

The ineffectiveness of HCQ has also been declared in a multicenter, open-label, randomized controlled trial involving 150 hospitalized infected patients (148 with mild or moderate disease and 2 with severe disease).

T209

30606-30804

Sentence

denotes

All subjects were divided into two groups (in a 1:1 ratio): the control group that received only SOC, consisting of antiviral, glucocorticoid, and antiviral, and the group treated with HCQ plus SOC.

T210

30805-30960

Sentence

denotes

The administrated dose of HCQ consisted of 200 mg/day for the first three days, followed by 800 mg/day as maintained dosage until the end of the treatment.

T211

30961-31100

Sentence

denotes

After 28 days of treatment, there was no significant difference in SARS-CoV-2 negative conversion in either the HCQ + SOC or the SOC group.

T212

31101-31291

Sentence

denotes

Similarly, there were no significant differences in the median time to negative conversion and the probability of symptom alleviation within 28 days between HCQ + SOC and the SOC group [35].

T213

31292-31439

Sentence

denotes

Another multicenter, randomized controlled Egyptian trial evaluated, in 194 subjects with COVID-19, the safety and efficacy of HCQ compared to SOC.

T214

31440-31666

Sentence

denotes

In terms of mechanical ventilation need and mortality, the addition of HCQ (400 mg twice daily (in day 1) followed by 200 mg tablets twice daily) to SOC was not associated with an improvement of COVID-19 patients’ health [36].

T215

31667-31791

Sentence

denotes

These results were confirmed by a recent randomized, double-blind, placebo-controlled trial conducted in the USA and Canada.

T216

31792-32026

Sentence

denotes

In this study, 419 early and mild COVID-19 subjects were randomly assigned to two groups, the HCQ group treated with 800 mg HCQ once, followed by 600 mg in 6 to 8 h, then 600 mg daily for 4 more days, and the placebo or control group.

T217

32027-32296

Sentence

denotes

Within 14 days of treatment, there was no change in the severity of symptoms in non-hospitalized patients between the HCQ group and the placebo group (difference in symptom severity: relative, 12%; absolute, −0.27 points (95% CI, −0.61 to 0.07 points); p = 0.117) [37].

T218

32297-32492

Sentence

denotes

Likewise, 2 studies carried out by Mahévas et al. supported the ineffectiveness of HCQ and highlighted that HCQ administration to COVID-10 patients was highly related to ECG modification [38,39].

T219

32493-32695

Sentence

denotes

ECG modification, resulting in QT-interval prolongation, is a characteristic side effect associated with HCQ treatment that has been shown in several clinical studies on patients infected with COVID-19.

T220

32696-32895

Sentence

denotes

In particular, the risk of QT-interval prolongation was increased when HCQ was associated with AZM, as demonstrated in a cohort of 84 patients who received 400 mg twice-daily HCQ plus 500 mg/day AZM.

T221

32896-33071

Sentence

denotes

In these patients, on day 3.6 ± 1.6 of therapy, the EGC showed a QTc prolongation from a baseline average of 435 ± 24 ms (mean ± SD) to a maximal average value of 463 ± 32 ms.

T222

33072-33240

Sentence

denotes

Moreover, in nine subjects (11%) there was a severe prolongation of QTc to > 500 ms (baseline average of 447 ± 30 ms to 527 ± 17 ms (p < 0.01 (one-sample t-test)) [52].

T223

33241-33354

Sentence

denotes

This complication was also confirmed in a retrospective study of 251 COVID-19 patients treated with HCQ/AZM [53].

T224

33355-33482

Sentence

denotes

It seems that a predictor of extreme QTc prolongation was renal failure and that its incidence increased with longer treatment.

T225

33483-33760

Sentence

denotes

The probability of QTc prolongation may also increase in the presence of other factors such as previous cardiovascular diseases, metabolic degeneration (hypoxia, pH, multiorgan system failure, and electrolyte abnormalities), age, and sex (females seem to be more at risk) [54].

T226

33761-34046

Sentence

denotes

Therefore, since QTc prolongation to more than 500 ms is known to be associated with a high risk for malignant arrhythmia and fatal stroke, recent guidance suggests the ECG screening with QTc evaluation in COVID-19-infected patients treated with novel therapies including HCQ/AZM [55].

T227

34047-34349

Sentence

denotes

It has also been suggested that the use of drugs that block late sodium channels (mexiletine or lidocaine) and close attention to serum electrolytes, in addition to the evaluation of heart rate and QT intervals, may allow the administration of HCQ/AZM even in subjects with prolonged QT intervals [54].

T228

34350-34495

Sentence

denotes

Despite the lack of real antiviral evidence related to HCQ administration, this drug has also been investigated as a possible prophylactic agent.

T229

34496-34699

Sentence

denotes

In fact, the pharmacokinetics of HCQ, like its long half-life and the high concentration in the lung (500-times higher than blood concentration), has made it an ideal candidate for prophylactic use [56].

T230

34700-34845

Sentence

denotes

The first study conducted on this line was performed in South Korea on 211 virus-exposed individuals, including 189 patients and 22 care-workers.

T231

34846-35078

Sentence

denotes

The HCQ administration (400 mg day) as post-exposure prophylaxis resulted in the negative follow-up PCR tests after the end of 14 days of quarantine period (only 97.4% of patients and 95.5% of care-workers completed the study) [40].

T232

35079-35247

Sentence

denotes

However, it is necessary to consider that this is a single-center clinical study with a high risk of bias and that a subsequent randomized clinical study has denied it.

T233

35248-35412

Sentence

denotes

In particular, Boulware D.R. et al., in a randomized, double-blind, placebo-controlled trial, tested HCQ as a prophylactic agent within 4 days after virus exposure.

T234

35413-35594

Sentence

denotes

There were 821 asymptomatic participants randomly assigned to receive either placebo or HCQ (800 mg once, followed by 600 mg in 6 to 8 h, then 600 mg per day for 4 additional days).

T235

35595-35890

Sentence

denotes

After 14 days of treatment, it was demonstrated that HCQ did not prevent COVID-19 infection when compared to placebo, since the incidence of illnesses compatible with Covid-19 disease did not differ significantly between subjects receiving HCQ (49 of 414 (11.8%)) or placebo (58 of 407 (14.3%)).

T236

35891-36013

Sentence

denotes

Furthermore, the onset of side effects was more frequent in patients treated with HCQ than placebo (40.1% vs. 16.8%) [41].

T237

36014-36176

Sentence

denotes

To better assess the incidence of side effects linked to HCQ administration as post-exposure prophylaxis, a cross-sectional study was conducted among 140 doctors.

T238

36177-36425

Sentence

denotes

Sixty nine adverse events were documented in 44 subjects (31%); the most common reported symptoms were headache followed by nausea, dizziness, abdominal cramps, and loose stools, while hypoglycemia was seen in only three diabetic participants [57].

T239

36426-36577

Sentence

denotes

Hence, even if the side effects highlighted were not serious, it is recommended to take the utmost care before using HCQ for COVID-19 chemoprophylaxis.

T240

36578-36708

Sentence

denotes

The ineffectiveness of HCQ administration as post-exposure prophylaxis has also been demonstrated by an in vivo study on macaques.

T241

36709-36843

Sentence

denotes

Maisonnasse P. et al. tested different treatment strategies, including HCQ alone or in combination with AZM, in comparison to placebo.

T242

36844-36909

Sentence

denotes

All the treatments were administrated before or after viral load.

T243

36910-37043

Sentence

denotes

It was seen that when HCQ was administrated as pre-exposure prophylaxis, it did not protect against the acquisition of the infection.

T244

37044-37216

Sentence

denotes

Similarly, neither HCQ nor HCQ + AZM had beneficial effects in improving viral infection’s symptoms [58], confirming previously analyzed clinical studies’ negative results.

T245

37217-37404

Sentence

denotes

Several case reports have supported all these results since people already using HCQ for a long time to treat inflammatory diseases also showed severe illness related to COVID-19 [59,60].

T246

37405-37599

Sentence

denotes

In the light of collected data, despite the success of the first clinical trials, the latest studies have shown the ineffectiveness of HCQ for the treatment and prevention of COVID-19 infection.

T247

37600-37620

Sentence

denotes

So that, if the U.S.

T248

37621-37914

Sentence

denotes

Food and Drug Administration (FDA) had initially authorized the use of HCQ in case of emergency [61], in June 2020, the FDA revoked this authorization [62] since the potential HCQ effectiveness in treating COVID-19 was overtaken by severe cardiac adverse events and other serious side effects.

T249

37915-38158

Sentence

denotes

In fact, there is the need to consider that in subjects with severe COVID-19, the abundance of inflammatory molecules like interleukins and tumor necrosis factors generate a cytokine storm, leading to a septic shock and multiple organ failure.

T250

38159-38265

Sentence

denotes

In hepatic and renal dysfunction, HCQ metabolism and clearance were compromised and its safety is altered.

T251

38266-38501

Sentence

denotes

Moreover, recently the Surviving Sepsis Campaign guidelines on the management of critically ill patients with COVID-19 concluded that there was insufficient evidence to recommend the routine use of HCQ in patients admitted in ICU [63].

T252

38502-38629

Sentence

denotes

In the same way, the American College of Physicians practice guidelines do not recommend HCQ for prophylaxis or treatment [64].

T253

38630-38890

Sentence

denotes

International trials like SOLIDARITY (International trial by World Health Organisation) [65], RECOVERY (Randomised Evaluation of Covid-19 Therapy) [66], and DISCOVERY (Trial of Treatments for COVID-19 in Hospitalized Adults) [67] have also stopped the HCQ arm.

T254

38891-39121

Sentence

denotes

In particular, the World Health Organisation (WHO), in the SOLIDARITY trial project, has arrested all arms involving HCQ, as evidence showed that it did not reduce the mortality of hospitalized COVID-19 patients compared with SOC.

T255

39122-39261

Sentence

denotes

However, this decision was not applied to HCQ use or evaluation in pre- or post-exposure prophylaxis for subjects exposed to COVID-19 [68].

T256

39262-39352

Sentence

denotes

To conclude, the HCQ treatment of SARS-CoV-2 infection was not met with its hoped success.

T257

39353-39510

Sentence

denotes

This is probably related to the inability of the dosing regimens currently in use to achieve the blood concentration required for the HCQ antiviral activity.

T258

39511-39749

Sentence

denotes

Initially, based on physiological pharmacokinetic models, Yao et al. recommended for SARS-CoV-2 infection a loading oral HCQ dose of 400 mg twice daily, followed by a maintenance dose of 200 mg administered twice daily for four days [50].

T259

39750-40087

Sentence

denotes

However, this recommended HCQ dosage regimen was based only on the ratio of free lung trough concentration to in vitro EC50 values (the EC50 of HCQ for SARS-CoV-2 ranged between 0.72 and 17.31µM) and did not consider the tendency of HCQ to accumulate within acidic cellular organelles like endosomes, lysosomes, and Golgi apparatus [69].

T260

40088-40243

Sentence

denotes

In fact, it has been demonstrated that HCQ concentration in lysosomes is higher than the extracellular concentration (80 µM vs. 0.5 µM, respectively) [70].

T261

40244-40407

Sentence

denotes

Based on these results, it was considered necessary to compare the EC50 values obtained in vitro with the plasma concentration and not with the lung concentration.

T262

40408-40696

Sentence

denotes

In a study investigating HCQ pharmacokinetics in COVID-19 patients treated with 600 mg/day of HCQ, it was found that the mean blood concentration of HCQ was 0.46 mg/day [32], which was below the lowest estimated levels of 0.48 mg/mL corresponding to the in vitro concentration of 0.72 µM.

T263

40697-40904

Sentence

denotes

Further, a plasma concentration predicted for HCQ antiviral EC50 made by Garcia-Cremades et al. found that it should be 4,7 µM corresponding to 1.58 mg/mL, which is much higher than in vivo plasmatic values.

T264

40905-41095

Sentence

denotes

To reach this plasma concentration, it should be necessary to take an amount of HCQ higher than 400 mg twice a day for five days or more [71], which would increase the onset of side effects.

T265

41096-41349

Sentence

denotes

Thus, the ineffectiveness of HCQ antiviral activity again SARS-CoV-2 can be related to the low current dosing regimens and the impossibility to increase the administered doses due to the increased risk of severe side effects, especially QT prolongation.

T266

41351-41355

Sentence

denotes

2.3.

T267

41356-41394

Sentence

denotes

Hydroxychloroquine Biological Activity

T268

41395-41500

Sentence

denotes

Besides the antiviral effects, HCQ possesses several other demonstrated biological activities (Figure 6).

T269

41501-41772

Sentence

denotes

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.

T270

41773-42051

Sentence

denotes

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.

T271

42052-42140

Sentence

denotes

HCQ exerts anticancer effects by acting synergistically with common chemotherapic drugs.

T272

42141-42338

Sentence

denotes

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.

T273

42339-42446

Sentence

denotes

A two-compartment model, with first-order absorption and a lag time, describes the pharmacokinetics of HCQ.

T274

42447-42567

Sentence

denotes

The long-terminal half-life prolongs the time to reach steady-state concentrations, then delays the therapeutic effects.

T275

42568-42646

Sentence

denotes

The next-generation formulations allow modulating the pharmacokinetics of HCQ.

T276

42647-42786

Sentence

denotes

Avoiding systemic absorption, then liver first-pass metabolism, HCQ may be used in site-specific inflammation, without toxicity [72,73,74].

T277

42787-42911

Sentence

denotes

The selected studies’ primary outcomes are the extent to which HCQ may limit disease progression or exacerbations (Table 2).

T278

42913-42919

Sentence

denotes

2.3.1.

T279

42920-42940

Sentence

denotes

Rheumatoid Arthritis

T280

42941-43059

Sentence

denotes

Rheumatoid arthritis is a systemic autoimmune disease, characterized by chronic inflammation and damage to the joints.

T281

43060-43290

Sentence

denotes

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].

T282

43291-43387

Sentence

denotes

It is estimated that the prevalence of rheumatoid arthritis is around 1%, mainly in women [118].

T283

43388-43563

Sentence

denotes

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.

T284

43564-43800

Sentence

denotes

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.

T285

43801-44012

Sentence

denotes

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].

T286

44013-44222

Sentence

denotes

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.

T287

44223-44414

Sentence

denotes

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.

T288

44415-44490

Sentence

denotes

Moreover, HCQ was associated with a decrement of corticosteroid injections.

T289

44491-44574

Sentence

denotes

By contrast, no improvements in psychological function have been demonstrated [76].

T290

44575-44825

Sentence

denotes

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.

T291

44826-45256

Sentence

denotes

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].

T292

45257-45377

Sentence

denotes

In patients with rheumatoid arthritis, the cardiovascular risk is more than doubled, compared to the healthy population.

T293

45378-45552

Sentence

denotes

Chronic inflammatory status leads to an intensification of the atherosclerotic process, resulting in a higher susceptibility to hypertension, obesity, and metabolic syndrome.

T294

45553-45673

Sentence

denotes

The overproduction of IL-6 is strictly related to lipid profile alterations, given its role in adipose tissue lipolysis.

T295

45674-45751

Sentence

denotes

The inflammation and endothelial damage are exacerbated by leptin production.

T296

45752-45889

Sentence

denotes

Batun-Garrido, Salas-Magana and Juarez-Rojop [79] found a significant correlation between HCQ treatment and lower IL-6 and leptin levels.

T297

45890-46255

Sentence

denotes

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).

T298

46256-46486

Sentence

denotes

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].

T299

46487-46644

Sentence

denotes

The correlation between HCQ and cardiovascular risk was also assessed in a cross-sectional observational study involving 177 women with rheumatoid arthritis.

T300

46645-46781

Sentence

denotes

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].

T301

46782-46997

Sentence

denotes

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.

T302

46998-47084

Sentence

denotes

For these patients, no significant changes in insulin resistance were observable [81].

T303

47085-47217

Sentence

denotes

This study evaluated only insulin sensitivity, by Homeostatic Model Assessment (HOMA) index, without considering insulin metabolism.

T304

47218-47320

Sentence

denotes

Thus, inconsistency should be explained by this factor, together with the short duration of treatment.

T305

47321-47447

Sentence

denotes

The anti-diabetic properties of HCQ have been also assessed in patients without arthritis, as reported in the next paragraphs.

T306

47449-47455

Sentence

denotes

2.3.2.

T307

47456-47475

Sentence

denotes

Lupus Erythematosus

T308

47476-47638

Sentence

denotes

Systemic lupus erythematosus is a multisystemic autoimmune chronic inflammatory disorder that mainly involves joints, mucosae, skin, and endothelial vessels [90].

T309

47639-47728

Sentence

denotes

For a long time, HCQ has played a marginal role in the overall management of the disease.

T310

47729-47884

Sentence

denotes

Since the 90s, the first evidence of HCQ effectiveness in controlling lupus erythematosus manifestations allowed its employment as a first-line medicament.

T311

47885-48034

Sentence

denotes

Although it was not recommended in single therapy, the immunomodulating properties of HCQ seem to play an important role in the disease pathogenesis.

T312

48035-48167

Sentence

denotes

It is associated with a decrement of exacerbation events, as well as protective effects towards vascular and thrombotic events [85].

T313

48168-48415

Sentence

denotes

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.

T314

48416-48552

Sentence

denotes

People who interrupted the therapy exhibited constitutional symptoms of the disease, as well as skin rashes, arthritis, and ulcers [83].

T315

48553-48768

Sentence

denotes

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].

T316

48769-48890

Sentence

denotes

A case-control study was carried out in order to define the role of HCQ in the survival of individuals affected by lupus.

T317

48891-49068

Sentence

denotes

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].

T318

49069-49298

Sentence

denotes

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].

T319

49299-49473

Sentence

denotes

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.

T320

49474-49730

Sentence

denotes

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.

T321

49731-49823

Sentence

denotes

Lately, autophagy had risen among the emerging strategies to reestablish the immune balance.

T322

49824-49985

Sentence

denotes

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.

T323

49986-50136

Sentence

denotes

After four weeks of treatment, HCQ clearly restored the immune balance, by both inhibiting Th17 response and enhancing Treg immunosuppressive effects.

T324

50137-50419

Sentence

denotes

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.

T325

50420-50604

Sentence

denotes

This randomized trial further evidenced that HCQ treatment also remarkably attenuated kidney inflammation, by limiting the migration of lymphoplasmacytic cells into renal tissues [86].

T326

50605-50756

Sentence

denotes

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.

T327

50757-51107

Sentence

denotes

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].

T328

51108-51400

Sentence

denotes

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].

T329

51401-51532

Sentence

denotes

Infiltrating cells, most of all mast cells, could involve skin tissues, causing one of the most common signs of lupus, skin rashes.

T330

51533-51694

Sentence

denotes

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.

T331

51695-51929

Sentence

denotes

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.

T332

51930-52102

Sentence

denotes

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].

T333

52103-52234

Sentence

denotes

The chronic inflammatory status that features lupus erythematosus leads to a higher susceptibility to cardiovascular complications.

T334

52235-52389

Sentence

denotes

Lupus, indeed, is often characterized by endothelial dysfunction, the earliest marker of cardiovascular disease, as well as hypertension and renal damage.

T335

52390-52519

Sentence

denotes

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.

T336

52520-52737

Sentence

denotes

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].

T337

52738-52867

Sentence

denotes

The same effect of normalizing nitric oxide and ROS production have been confirmed in a NZB/W F1 murine model at different times.

T338

52868-52982

Sentence

denotes

HCQ at 3 mg/kg/day p.o. protected vascular endothelium, with a strong improvement of endothelial dysfunction [91].

T339

52983-53065

Sentence

denotes

The benefits on atherosclerosis also pass through the lipid-lowering power of HCQ.

T340

53066-53292

Sentence

denotes

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].

T341

53293-53400

Sentence

denotes

By contrast, HCQ in patients with a mild or inactive condition had no significant effects on lipid profile.

T342

53401-53628

Sentence

denotes

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].

T343

53629-53745

Sentence

denotes

The use of HCQ may be helpful in minimizing cardiovascular risk by improving glycemic homeostasis in lupus patients.

T344

53746-53999

Sentence

denotes

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].

T345

54000-54141

Sentence

denotes

HCQ remains a worthwhile primary or additional therapy in lupus patients, considering the low cost and its safety profile, also in pregnancy.

T346

54142-54313

Sentence

denotes

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].

T347

54314-54424

Sentence

denotes

A 5-year prospective study evaluated the effect of HCQ discontinuation on lupus progression in pregnant women.

T348

54425-54535

Sentence

denotes

As it occurs in no pregnant people, interruption of HCQ treatment is linked to an exacerbation of the disease.

T349

54536-54732

Sentence

denotes

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].

T350

54733-54936

Sentence

denotes

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.

T351

54937-54999

Sentence

denotes

Moreover, it exerted protective effects on fetal growth [120].

T352

55001-55007

Sentence

denotes

2.3.3.

T353

55008-55033

Sentence

denotes

Antiphospholipid Syndrome

T354

55034-55146

Sentence

denotes

Antiphospholipid syndrome is an autoimmune disorder characterized by antiphospholipid autoantibodies production.

T355

55147-55229

Sentence

denotes

If it is not associated with other autoimmune diseases, it is called primary [97].

T356

55230-55439

Sentence

denotes

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.

T357

55440-55572

Sentence

denotes

Resonance spectroscopy, indeed, revealed that the fetal brain and placenta are the main targets of autoantibodies localization [95].

T358

55573-55733

Sentence

denotes

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.

T359

55734-55955

Sentence

denotes

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].

T360

55956-56102

Sentence

denotes

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.

T361

56103-56297

Sentence

denotes

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.

T362

56298-56396

Sentence

denotes

Given these results, the mechanisms underlying the use of HCQ in thromboprophylaxis were assessed.

T363

56397-56778

Sentence

denotes

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].

T364

56779-56890

Sentence

denotes

Moreover, the efficacy of HCQ in antithrombotic therapy may lie in the interference in the coagulation cascade.

T365

56891-57111

Sentence

denotes

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].

T366

57113-57119

Sentence

denotes

2.3.4.

T367

57120-57136

Sentence

denotes

Sjögren Syndrome

T368

57137-57251

Sentence

denotes

Sjögren syndrome is an autoimmune disease with a strong negative impact on the quality of life of affected people.

T369

57252-57381

Sentence

denotes

The main features of the disease are lymphocytic inflammation and alterations in major salivary glands, causing xerostomia [103].

T370

57382-57536

Sentence

denotes

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.

T371

57537-57827

Sentence

denotes

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].

T372

57828-57939

Sentence

denotes

However, a few years later, the first evidence of HCQ effectiveness in Sjögren syndrome treatment was reported.

T373

57940-58173

Sentence

denotes

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].

T374

58174-58434

Sentence

denotes

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].

T375

58435-58593

Sentence

denotes

Hypo-salivation deriving from acinar atrophy and fibrosis of salivary glands is often associated with over-expression of TGF-β (transforming growth factor-β).

T376

58594-58816

Sentence

denotes

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.

T377

58817-58943

Sentence

denotes

Moreover, HCQ intake was also accompanied by a decrease in autoantibody production and a lower lymphocytic infiltration [103].

T378

58944-59236

Sentence

denotes

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.

T379

59238-59244

Sentence

denotes

2.3.5.

T380

59245-59253

Sentence

denotes

Diabetes

T381

59254-59360

Sentence

denotes

As has already been demonstrated for autoimmune patients, HCQ demonstrated great anti-diabetic properties.

T382

59361-59706

Sentence

denotes

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.

T383

59707-59959

Sentence

denotes

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.

T384

59960-60146

Sentence

denotes

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].

T385

60147-60288

Sentence

denotes

Given the positive outcomes deriving from preclinical studies, the therapeutic potential of HCQ was also assessed in several clinical trials.

T386

60289-60413

Sentence

denotes

Included in a randomized, double-blinded study of 18 months with 300 mg of HCQ twice a day were 135 diabetic obese patients.

T387

60414-60577

Sentence

denotes

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].

T388

60578-60814

Sentence

denotes

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].

T389

60815-61004

Sentence

denotes

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.

T390

61005-61155

Sentence

denotes

Results reported a significant increase in insulin levels, demonstrating the potential use of HCQ to counteract the risk of developing diabetes [109].

T391

61156-61364

Sentence

denotes

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.

T392

61365-61559

Sentence

denotes

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.

T393

61560-61662

Sentence

denotes

Regarding lipidic status, total cholesterol and LDL levels were reduced more by HCQ than pioglitazone.

T394

61663-61781

Sentence

denotes

Given the good tolerability of the treatment, HCQ may arise as a therapeutic alternative in diabetes management [110].

T395

61783-61789

Sentence

denotes

2.3.6.

T396

61790-61844

Sentence

denotes

Others (Cancer, Inflammation, Cardiovascular Diseases)

T397

61845-62008

Sentence

denotes

Given the well-recognized properties of HCQ against inflammation, it is easily intuitable that this agent could possess interesting insights into cancer treatment.

T398

62009-62105

Sentence

denotes

Chronic intestinal inflammation predisposes to the risk of colitis-associated colorectal cancer.

T399

62106-62379

Sentence

denotes

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.

T400

62380-62574

Sentence

denotes

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.

T401

62575-62853

Sentence

denotes

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].

T402

62854-63003

Sentence

denotes

Many others are the mechanisms by which HCQ exerts anticancer effects, mainly in synergism with conventional chemotherapic drugs, as discussed later.

T403

63004-63153

Sentence

denotes

Regarding the cardioprotective effect, this review has already focused on the positive impact of HCQ on cardiovascular issues in autoimmune patients.

T404

63154-63380

Sentence

denotes

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.

T405

63381-63498

Sentence

denotes

The pharmacological preconditioning with HCQ seems to be a good strategy to protect from ischemia-reperfusion injury.

T406

63499-63603

Sentence

denotes

The pretreatment with daily gavage of 200 mg/kg of HCQ, indeed, reduced the cardiac infarct size by 47%.

T407

63604-63734

Sentence

denotes

The mechanism underlying this effect is linked to the inhibition of apoptosis and total cell death in neonatal rat cardiomyocytes.

T408

63735-63815

Sentence

denotes

The atherosclerotic process contributes to increasing the risk of heart failure.

T409

63816-63866

Sentence

denotes

The etiology of this condition is still not clear.

T410

63867-64062

Sentence

denotes

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.

T411

64063-64196

Sentence

denotes

HCQ decreased free-fatty acids, triglycerides, total cholesterol, and LDL levels in diabetic rats under doses of 200 mg/kg/day [106].

T412

64197-64391

Sentence

denotes

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].

T413

64392-64532

Sentence

denotes

These favorable effects at the metabolic level might be due to its anti-inflammatory power that influences many other biological activities.

T414

64533-64723

Sentence

denotes

In gastrointestinal inflammations, mainly in inflammatory bowel disease, HCQ suppressed pro-inflammatory cytokines and enhanced the expression of ILs involved in anti-inflammatory processes.

T415

64724-64977

Sentence

denotes

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].

T416

64978-65083

Sentence

denotes

Inflammation, together with alterations in the immune system, are at the basis of pulmonary hypertension.

T417

65084-65380

Sentence

denotes

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].

T418

65381-65544

Sentence

denotes

Likewise, in endometriosis, the abnormal presence of endometrium in other organs leads to a chronic inflammatory status that could be affected by HCQ intervention.

T419

65545-65739

Sentence

denotes

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.).

T420

65740-65890

Sentence

denotes

In their role of scavengers, abnormalities in these cell populations may lead to an accumulation of endometrial cells, with impairment of the disease.

T421

65891-66061

Sentence

denotes

Moreover, histopathologic improvement of lesions was observed, probably due to the inhibition of autophagy by HCQ that alters anoikis response of endometrial cells [116].

T422

66063-66067

Sentence

denotes

2.4.

T423

66068-66106

Sentence

denotes

Hydroxychloroquine and Synergic Effect

T424

66107-66335

Sentence

denotes

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.

T425

66337-66343

Sentence

denotes

2.4.1.

T426

66344-66363

Sentence

denotes

Autoimmune Diseases

T427

66364-66581

Sentence

denotes

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].

T428

66582-66727

Sentence

denotes

It often occurs also that glucocorticoids or natural antioxidant substances are included in the coadjutant therapy of rheumatoid arthritis [123].

T429

66728-66990

Sentence

denotes

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.

T430

66991-67096

Sentence

denotes

A total of 195 patients were equally divided into two groups to follow the assigned therapeutic protocol.

T431

67097-67325

Sentence

denotes

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.

T432

67326-67486

Sentence

denotes

After one year, 75% of subjects under combinatory therapy and 60% of those under single-therapy reached clinical improvement, intended as 50% clinical response.

T433

67487-67601

Sentence

denotes

In terms of tolerability, the cotreatment resulted not to be more dangerous with respect to the single drug [123].

T434

67602-67805

Sentence

denotes

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.

T435

67806-67931

Sentence

denotes

Significant improvements in clinical parameters revealed the efficacy of the cotreatment in counteracting endothelial injury.

T436

67932-68101

Sentence

denotes

Indeed, the blood concentrations of endothelial injury markers, mainly soluble E selectin and thrombomodulin, experienced a significant drop after the cotreatment [124].

T437

68102-68493

Sentence

denotes

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].

T438

68494-68853

Sentence

denotes

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].

T439

68854-69072

Sentence

denotes

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.

T440

69073-69344

Sentence

denotes

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].

T441

69345-69451

Sentence

denotes

Although HCQ is effective and well-tolerated, other therapeutic alternatives have emerged in recent years.

T442

69452-69509

Sentence

denotes

Among them, monoclonal antibodies are the most promising.

T443

69510-69824

Sentence

denotes

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].

T444

69825-69939

Sentence

denotes

In addition to rheumatoid arthritis patients, HCQ is also used in lupus erythematosus, another autoimmune disease.

T445

69940-69990

Sentence

denotes

Regarding lupus erythematosus, HCQ is widely used.

T446

69991-70330

Sentence

denotes

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.

T447

70331-70639

Sentence

denotes

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].

T448

70640-70746

Sentence

denotes

In association with low-dose aspirin, HCQ is also indicated for thromboprophylaxis in patients with lupus.

T449

70747-71041

Sentence

denotes

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].

T450

71042-71191

Sentence

denotes

Thanks to its immunomodulatory properties, HCQ (20 mg/kg) was revealed to be useful in graft-versus-host disease, in combination with cyclosporine A.

T451

71192-71299

Sentence

denotes

They synergistically suppressed T cell response, allowing the reduction of the dose of drugs in mice [131].

T452

71301-71307

Sentence

denotes

2.4.2.

T453

71308-71338

Sentence

denotes

Cardiovascular Risk Management

T454

71339-71468

Sentence

denotes

HCQ revealed the great potential in the management of cardiovascular risk by controlling glucose homeostasis and lipidic profile.

T455

71469-71607

Sentence

denotes

Until now, in this review, we discussed the effect of HCQ alone to counteract cardiovascular complications, mostly in autoimmune patients.

T456

71608-71753

Sentence

denotes

Here, we reviewed the multiple pleiotropic actions of HCQ in combination with conventional medication in the most common cardiovascular diseases.

T457

71754-71973

Sentence

denotes

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.

T458

71974-72099

Sentence

denotes

The synergistic effect reduced the myocardial infarct size by up to 20% and improved insulin secretion and sensitivity [132].

T459

72100-72312

Sentence

denotes

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.

T460

72313-72449

Sentence

denotes

The role of HCQ in the inhibition of platelet aggregation was evaluated in healthy volunteers in comparison with aspirin or clopidogrel.

T461

72450-72562

Sentence

denotes

The addition of 400 mg/day of HCQ to aspirin resulted in a significant increase in aggregation inhibition (31%).

T462

72563-72692

Sentence

denotes

This inhibition was passed by reducing fibrinogen and inflammatory status by interfering with the arachidonic acid cascade [134].

T463

72694-72700

Sentence

denotes

2.4.3.

T464

72701-72711

Sentence

denotes

Anticancer

T465

72712-72791

Sentence

denotes

HCQ explains its antitumor activity thanks to its ability to inhibit autophagy.

T466

72792-72856

Sentence

denotes

HCQ is, indeed, an FDA-approved drug inhibiting autophagy [135].

T467

72857-72934

Sentence

denotes

Several types of tumors develop chemoresistance by enhancing autophagic flux.

T468

72935-73117

Sentence

denotes

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.

T469

73118-73220

Sentence

denotes

By recycling intracellular components, cells may maintain an energy balance and increase their growth.

T470

73221-73287

Sentence

denotes

If it occurs in cancer cells, resistance mechanisms may establish.

T471

73288-73431

Sentence

denotes

One of the mechanisms responsible for drug resistance is related to increased drug efflux by ATP-binding cassette (ABC) transporters [136,137].

T472

73432-73668

Sentence

denotes

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].

T473

73669-73785

Sentence

denotes

However, the role of autophagy in cancer is controversial and depends on genotype and tumor stage development [139].

T474

73786-73983

Sentence

denotes

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.

T475

73984-74101

Sentence

denotes

Depending on genetic concomitant alterations, autophagy may possess both pro-tumorigenic and tumor-suppressive roles.

T476

74102-74262

Sentence

denotes

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.

T477

74263-74432

Sentence

denotes

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.).

T478

74433-74653

Sentence

denotes

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].

T479

74654-74794

Sentence

denotes

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].

T480

74795-74891

Sentence

denotes

This issue highlights the necessity to carefully evaluate the use of HCQ in certain tumor types.

T481

74892-75117

Sentence

denotes

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].

T482

75118-75322

Sentence

denotes

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.

T483

75323-75431

Sentence

denotes

Regarding KRAS oncoprotein, its downstream pathway is one of the major players of pancreatic carcinogenesis.

T484

75432-75556

Sentence

denotes

The inhibition of this pathway by cytotoxic drugs, as well as trametinib, is often associated with an increase in autophagy.

T485

75557-75833

Sentence

denotes

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.

T486

75834-75989

Sentence

denotes

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.

T487

75990-76160

Sentence

denotes

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].

T488

76161-76255

Sentence

denotes

Then, it is important in assessing the contextual role of HCQ in cancer resistance mechanisms.

T489

76256-76362

Sentence

denotes

Epirubicin in triple-negative breast cancer therapy often lost efficacy, due to chemoresistance acquiring.

T490

76363-76464

Sentence

denotes

It has been shown that this cytotoxic agent induced autophagic flux, increasing cancer cell survival.

T491

76465-76642

Sentence

denotes

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].

T492

76643-76784

Sentence

denotes

In addition, estrogen receptor-positive breast cancers developed resistance to treatment with tamoxifen, due to the enhancement of autophagy.

T493

76785-76912

Sentence

denotes

The coadministration of HCQ (1–2 mg/day/mice in drinking water) restored the susceptibility of cancer cells to tamoxifen [146].

T494

76913-77181

Sentence

denotes

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].

T495

77182-77429

Sentence

denotes

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].

T496

77430-77500

Sentence

denotes

HCQ was revealed also to be active against chemoresistant lung cancer.

T497

77501-77672

Sentence

denotes

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.

T498

77673-77846

Sentence

denotes

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].

T499

77847-77949

Sentence

denotes

Together with autophagy, glycolysis plays a pivotal role in satisfying the increased energetic demand.

T500

77950-78041

Sentence

denotes

The dual targeting of the processes may provide a new therapeutic approach in cancer cells.

T501

78042-78309

Sentence

denotes

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.

T502

78310-78501

Sentence

denotes

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].

T503

78502-78627

Sentence

denotes

Resistance mechanisms also involved alterations in β-Cell Lymphoma (Bcl) Bcl-2 and Bcl-xL and anti-apoptotic gene expression.

T504

78628-78837

Sentence

denotes

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.

T505

78838-78950

Sentence

denotes

Tumor growth was significantly suppressed by a combination of drugs, with respect to HCQ or ABT-737 alone [152].

T506

78951-79142

Sentence

denotes

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.

T507

79143-79321

Sentence

denotes

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].

T508

79322-79477

Sentence

denotes

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.

T509

79478-79679

Sentence

denotes

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.

T510

79680-79888

Sentence

denotes

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].

T511

79889-80089

Sentence

denotes

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.

T512

80090-80164

Sentence

denotes

The anticancer activity of melatonin depends on its pro-apoptotic effects.

T513

80165-80265

Sentence

denotes

Nevertheless, this activity is accompanied by a pro-autophagic activity that caused chemoresistance.

T514

80266-80412

Sentence

denotes

The coadministration of the autophagy inhibitor HCQ strongly enhanced melatonin anticancer efficacy, resulting in a smaller tumor size and weight.

T515

80413-80576

Sentence

denotes

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.

T516

80577-80746

Sentence

denotes

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.

T517

80747-81012

Sentence

denotes

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.

T518

81013-81111

Sentence

denotes

HCQ was administered as HCQ-loaded liposomes, to modulate the onset of autophagy inhibition [158].

T519

81112-81250

Sentence

denotes

This formulation allows us to overcome the limits of HCQ usage, related to the high doses required, which is often unachievable in humans.

T520

81251-81486

Sentence

denotes

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.

T521

81487-81599

Sentence

denotes

Eight injections of nanoparticles containing 400 mg of HCQ and chlorambucil led to the overall survival of mice.

T522

81600-81686

Sentence

denotes

These concentrations of free drugs are inapplicable, due to their high toxicity [159].

T523

81687-81980

Sentence

denotes

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.

T524

81981-82204

Sentence

denotes

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.

T525

82205-82454

Sentence

denotes

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].

T526

82455-82721

Sentence

denotes

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.

T527

82722-82808

Sentence

denotes

Tumor weight, together with the number of liver metastases, was significantly reduced.

T528

82809-82983

Sentence

denotes

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.

T529

82984-83100

Sentence

denotes

All these events avoided the formation of stroma fibrosis, allowing paclitaxel to easily reach the tumor site [104].

T530

83101-83175

Sentence

denotes

The synergism results are essential for HCQ activity in pancreatic cancer.

T531

83176-83260

Sentence

denotes

In monotherapy, indeed, HCQ (800–1200 mg/day) did not achieve significant autophagy.

T532

83261-83461

Sentence

denotes

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].

T533

83462-83650

Sentence

denotes

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.

T534

83651-83852

Sentence

denotes

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.

T535

83853-84043

Sentence

denotes

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.

T536

84044-84213

Sentence

denotes

Those synergistic effects are attributable to significant inhibition of autophagy exerted by HCQ and might provide a valuable therapeutic tool in glioma treatment [164].

T537

84214-84397

Sentence

denotes

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.

T538

84398-84629

Sentence

denotes

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].

T539

84630-84803

Sentence

denotes

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.

T540

84804-84988

Sentence

denotes

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].

T541

84989-85241

Sentence

denotes

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.

T542

85242-85395

Sentence

denotes

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].

T543

85396-85635

Sentence

denotes

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.).

T544

85636-85791

Sentence

denotes

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.

T545

85792-85880

Sentence

denotes

No maximum tolerated dose was reached, although common toxicities were manifested [168].

T546

85881-86052

Sentence

denotes

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.

T547

86053-86239

Sentence

denotes

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.

T548

86240-86480

Sentence

denotes

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].

T549

86481-86809

Sentence

denotes

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].

T550

86811-86817

Sentence

denotes

2.4.4.

T551

86818-86838

Sentence

denotes

Bacterial Infections

T552

86839-86969

Sentence

denotes

HCQ is known to exert an antibacterial effect through the alkalinization of infected organelles, inhibiting bacterial replication.

T553

86970-87135

Sentence

denotes

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:

T554

87136-87178

Sentence

denotes

Coxiella burnetii and Tropheryma whipplei.

T555

87179-87284

Sentence

denotes

C. burnetii is an obligate intraleukocytic Gram-negative bacterium responsible for query fever (Q fever).

T556

87285-87419

Sentence

denotes

The infection is mainly caused by direct contact with infected animals, although cases of human transmission have also been described.

T557

87420-87570

Sentence

denotes

Q fever diagnosis is primally founded on serological examination and based on a different evolution, acute and chronic infection can be distinguished.

T558

87571-87775

Sentence

denotes

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].

T559

87776-87904

Sentence

denotes

Approximately 2–5% of C. burnetii infections can develop into the chronic phase, leading to endocarditis and vascular infection.

T560

87905-88029

Sentence

denotes

The risk of developing chronic fever is higher in patients with pre-existing vascular disorders or valvulopathies [123,124].

T561

88030-88121

Sentence

denotes

C. burnetii is known to replicate in an intracellular phagolysosome with a pH range of 4–5.

T562

88122-88217

Sentence

denotes

However, at this pH, antibiotics, like doxycycline (DXC), exert only a bacteriostatic activity.

T563

88218-88306

Sentence

denotes

Therefore, a combination of DXC with a lysosomotropic agent, such as HCQ, was suggested.

T564

88307-88442

Sentence

denotes

In fact, HCQ was shown to increase the phagolysosomal compartment’s pH by improving the bactericidal activity of doxycycline [125,126].

T565

88443-88571

Sentence

denotes

The first successful results concerning the treatment of Q fever endocarditis combined with DXC and HCQ date back to 1993 [127].

T566

88572-88784

Sentence

denotes

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].

T567

88785-89087

Sentence

denotes

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].

T568

89088-89316

Sentence

denotes

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].

T569

89317-89607

Sentence

denotes

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.

T570

89608-89699

Sentence

denotes

Contrarily, shorter regimes are associated with a failure of antibiotics prophylaxis [141].

T571

89700-89886

Sentence

denotes

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].

T572

89887-89973

Sentence

denotes

This duration should only be extended in the absence of favorable serological results.

T573

89974-90186

Sentence

denotes

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].

T574

90187-90454

Sentence

denotes

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].

T575

90455-90543

Sentence

denotes

Serological titers are used to follow the disease and determine the duration of therapy.

T576

90544-90638

Sentence

denotes

On the other hand, T. whipplei is a Gram-positive bacterium responsible for Whipple’s disease.

T577

90639-90793

Sentence

denotes

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].

T578

90794-90956

Sentence

denotes

This bacterial infection is primally characterized by digestive tract disorders such as diarrhea (75% of cases), malabsorption, and weight loss (80–90% of cases).

T579

90957-91068

Sentence

denotes

Joint disease may appear more than six years before the diagnosis and occur in more than 80% of patients [148].

T580

91069-91174

Sentence

denotes

Furthermore, neurological and cardiac disorders can also be frequently associated with Whipple’s disease.

T581

91175-91335

Sentence

denotes

For years the standard treatment for T. whipplei has included a combination of trimethoprim and sulfamethoxazole; however, relapses were not uncommon [149,150].

T582

91336-91545

Sentence

denotes

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].

T583

91546-91709

Sentence

denotes

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].

T584

91710-91799

Sentence

denotes

DCX/HCQ efficacy on T. whipplei diseases was demonstrated in a clinical trial dated 2014.

T585

91800-92017

Sentence

denotes

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].

T586

92018-92267

Sentence

denotes

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].

T587

92268-92439

Sentence

denotes

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].

T588

92440-92716

Sentence

denotes

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).

T589

92717-92818

Sentence

denotes

This visual map is obtained by VOSviewer software, analyzing recurring items from all keywords [171].

T590

92820-92822

Sentence

denotes

3.

T591

92823-92844

Sentence

denotes

Materials and Methods

T592

92846-92850

Sentence

denotes

3.1.

T593

92851-92866

Sentence

denotes

Search Strategy

T594

92867-93075

Sentence

denotes

According to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a systematic literature search was performed in May 2020 and included all reports published to August 2020.

T595

93076-93566

Sentence

denotes

The search was performed on specialized databases (PubMed and Scopus) using different combinations of HCQ and the following keywords: history, discovery, ethnomedical, synthesis, chemical structure, SAR, RAS, biological activity, approved biological activities, antiviral, antiviral mechanism, COVID-19, Q fever, Whipple’s disease, synergistic effects, synergic effects, toxicological effects, toxic effects, toxicity, animal model and antiviral activity, clinical study, preclinical study.

T596

93567-93674

Sentence

denotes

We did not request full-text to investigators if not available and we did not try to find unpublished data.

T597

93676-93680

Sentence

denotes

3.2.

T598

93681-93696

Sentence

denotes

Study Selection

T599

93697-93950

Sentence

denotes

The manuscript selection was based on the inclusion criteria: preclinical (in vivo) and clinical studies involving the use of HCQ and combinations, only articles published in English and containing keywords in the title or in the abstract were selected.

T600

93951-94218

Sentence

denotes

Other review articles, meta-analysis, retrospective studies, abstracts, conferences, editorials, letters, conference proceedings, manuscripts without full text available or articles that did not meet the inclusion criteria were not included in this systematic review.

T601

94219-94402

Sentence

denotes

For selecting the sources, three independent investigators (I.F., F.L., and M.P.) first selected the articles according to the title and abstract and then by analyzing the full-texts.

T602

94403-94569

Sentence

denotes

In cases of non-consensus, authors tried to resolve any disagreements by discussion or, if necessary, two more independent reviewers were consulted (L.M. and N.D.T.).

T603

94570-94714

Sentence

denotes

The selected articles were carefully reviewed with the aim of identify or exclude the manuscripts that did not fit the criteria described above.

T604

94715-94825

Sentence

denotes

Additional papers were added to this review after the analysis of the bibliography from the included articles.

T605

94827-94831

Sentence

denotes

3.3.

T606

94832-94847

Sentence

denotes

Data extraction

T607

94848-95149

Sentence

denotes

Data were collected and examined by the authors and information from the selected manuscripts on HCQ, as well as study design, experimental models, general mechanisms implicated in antiviral and biological activities, major outcomes doses or concentrations, and route of administration were extracted.

T608

95151-95155

Sentence

denotes

3.4.

T609

95156-95189

Sentence

denotes

Methodological Quality Assessment

T610

95190-95502

Sentence

denotes

The risk of bias and the quality of the preclinical and clinical investigations were assessed independently by the authors, using a checklist adapted from Cochrane Handbook for Systematic Reviews of Interventions, specifically adjusted for animal intervention study (SYRCLE’s) [171,172] and clinical trials [97].

T611

95503-95822

Sentence

denotes

The evaluation of the selected studies’ methodological quality was based on the presence or absence of information regarding the main objectives and findings, randomization of the treatment allocation, blinded drug administration, blinded outcome assessment and outcome measurements, as reported in Table 3 and Table 4.

T612

95823-95954

Sentence

denotes

Only studies that reported a positive judgment in all considered parameters were assessed to be of a higher methodological quality.

T613

95955-96153

Sentence

denotes

In contrast, the studies that did not wholly fulfil the criteria were included in the medium risk of bias, while those that completely lacked this information were deemed to be at high risk of bias.

T614

96155-96157

Sentence

denotes

4.

T615

96158-96169

Sentence

denotes

Conclusions

T616

96170-96265

Sentence

denotes

The bubble map of Figure 8 summarizes the pleiotropic activity of HCQ evaluated in this review.

T617

96266-96426

Sentence

denotes

As highlighted by the colorimetric variation, the research, in the early 2000s, has been focused on the application of HCQ as an antimalarial drug (blue color).

T618

96427-96664

Sentence

denotes

In contrast, in the last few years, scientists have moved their attention to the influence of HCQ on many pathways involved in inflammation, infections, autoimmune diseases, cardiovascular pathologies, and diabetes (blue to green color).

T619

96665-96824

Sentence

denotes

Finally, in the last months, it is evident that the rapid spread of the COVID-19 pandemic has led to the revaluation of HCQ in viral infections (yellow color).

T620

96825-97135

Sentence

denotes

However, the analysis of currently available clinical studies showed that the administration of HCQ to prevent and cure COVID-19 infection is questionable, since results from clinical trials are contrasting, and the last data did not support the use of HCQ for the treatment and prevention of COVID-19 disease.

T621

97136-97275

Sentence

denotes

Despite these results, HCQ is considered to be a safe drug since it is generally better tolerated than other 4-aminoquinolines, such as CQ.

T622

97276-97543

Sentence

denotes

Hence, nowadays, HCQ arises as a first-line treatment in managing autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, and Sjögren syndrome, mainly in association with methotrexate or corticosteroids, showing a synergistic effect on disease control.

T623

97544-97663

Sentence

denotes

It also improves glucose and lipid homeostasis and revealed significant antibacterial activity in combination with DXC.

T624

97664-97828

Sentence

denotes

To better characterize HCQ activity, computational models should be useful for targeting and docking the molecular features responsible for its mechanism of action.

T625

97829-97932

Sentence

denotes

Based on this work, it should be possible to hypothesize future applications of HCQ in medical therapy.

T626

97934-98057

Sentence

denotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

T627

98059-98079

Sentence

denotes

Author Contributions

T628

98080-98550

Sentence

denotes

Conceptualization, L.M. and N.D.T.; methodology, L.M., I.F. and N.D.T.; software, I.F., F.L. and M.P.; validation, L.M. and N.D.T.; formal analysis, I.F., F.L. and M.P.; investigation, I.F., F.L. and M.P.; resources, L.M. and N.D.T.; data curation, I.F., F.L. and M.P.; writing—original draft preparation, I.F., F.L. and M.P.; writing—review and editing, N.D.T.; supervision, L.M. and N.D.T.; project administration, L.M. and N.D.T.; funding acquisition, L.M. and N.D.T.

T629

98551-98627

Sentence

denotes

All authors have read and agreed to the published version of the manuscript.

T630

98629-98636

Sentence

denotes

Funding

T631

98637-98665

Sentence

denotes

This research was funded by:

T632

98666-98929

Sentence

denotes

Regione Basilicata; Project ALIMINTEGRA, GO NUTRIBAS financed on 16.1 PSR Basilicata funding ex D.G.R. n° 312/17 CUP: C31G18000210002; Italian Ministry of the Economic Development “Fondo per la Crescita Sostenibile-Sportello “Agrifood” PON I&C 2014–2020, Prog. n.

T633

98930-98949

Sentence

denotes

F/200099/01-03/X45.

T634

98951-98972

Sentence

denotes

Conflicts of Interest

T635

98973-99017

Sentence

denotes

The authors declare no conflict of interest.

T636

99018-99031

Sentence

denotes

Abbreviations

T637

99032-99048

Sentence

denotes

ACF Aceclofenac

T638

99049-99067

Sentence

denotes

At Adenoid tissue

T639

99068-99085

Sentence

denotes

AZM Azithromycin

T640

99086-99106

Sentence

denotes

Bcl β-Cell Lymphoma

T641

99107-99131

Sentence

denotes

CHIKV Chikungunya Virus

T642

99132-99154

Sentence

denotes

cpm Counts Per Minute

T643

99155-99210

Sentence

denotes

COVID-19 or 2019-nCoV new coronavirus delivery in 2019

T644

99211-99226

Sentence

denotes

CQ Chloroquine

T645

99227-99272

Sentence

denotes

DMARDs Disease-Modifying Antirheumatic Drugs

T646

99273-99289

Sentence

denotes

DXC Doxycycline

T647

99290-99312

Sentence

denotes

ECG Electrocardiogram

T648

99313-99338

Sentence

denotes

FGT Female Genital Tract

T649

99339-99368

Sentence

denotes

HAART Antiretroviral therapy

T650

99369-99392

Sentence

denotes

HCQ Hydroxychloroquine

T651

99393-99426

Sentence

denotes

HIV Human Immunodeficiency Virus

T652

99427-99461

Sentence

denotes

HOMA Homeostatic Model Assessment

T653

99462-99466

Sentence

denotes

i.g.

T654

99468-99484

Sentence

denotes

Intragastrically

T655

99485-99506

Sentence

denotes

IgG Immunoglobulin G

T656

99507-99511

Sentence

denotes

i.m.

T657

99513-99536

Sentence

denotes

Intramuscular injection

T658

99537-99541

Sentence

denotes

i.p.

T659

99543-99568

Sentence

denotes

Intraperitoneal injection

T660

99569-99594

Sentence

denotes

ICU Intensive care units

T661

99595-99619

Sentence

denotes

IFN-α Type I interferon

T662

99620-99635

Sentence

denotes

IL Interleukin

T663

99636-99664

Sentence

denotes

LDL Low-Density Lipoprotein

T664

99665-99682

Sentence

denotes

MXT Methotrexate

T665

99683-99728

Sentence

denotes

NSAIDs Non-steroidal anti-inflammatory drugs

T666

99729-99733

Sentence

denotes

p.o.

T667

99735-99758

Sentence

denotes

Per oral administration

T668

99759-99776

Sentence

denotes

PRD Prednisolone

T669

99777-99785

Sentence

denotes

Q fever.

T670

99787-99798

Sentence

denotes

Query fever

T671

99799-99830

Sentence

denotes

RPE Retinal Pigment Epithelium

T672

99831-99859

Sentence

denotes

ROS Reactive Oxygen Species

T673

99860-99924

Sentence

denotes

SARS-CoV-2 Severe Acute Respiratory Syndrome caused by COVID-19

T674

99925-99946

Sentence

denotes

SOC Standard-of-care

T675

99947-99982

Sentence

denotes

TGF-β Transforming growth factor-β

T676

99983-100010

Sentence

denotes

TLR-4 Toll-like receptor 4

T677

100011-100036

Sentence

denotes

Tregs T-regulatory cells

T678

100037-100066

Sentence

denotes

TNF-α Tumor necrosis factors

T679

100067-100091

Sentence

denotes

VAS Visual Analog Scale

T680

100092-100107

Sentence

denotes

ZDV Zidovudine

T681

100108-100124

Sentence

denotes

ZIKV Zika virus

T682

100126-100197

Sentence

denotes

Figure 1 Historical development of hydroxychloroquine (HCQ) synthesis.

T683

100198-100332

Sentence

denotes

In green is represented the 4-aminoquinoline nucleus, in red is the amphiphilic weak basic side chain and in blue, the hydroxyl group.

T684

100333-100411

Sentence

denotes

Figure 2 Flowchart detailing literature search according to PRISMA statement.

T685

100412-100785

Sentence

denotes

Figure 3 Distribution of the selected studies by year of publication focusing on antiviral activity or other biological activities. (a) Distribution, and the total number of preclinical and clinical studies per virus (b) HIV = herpes virus simple, HCV = hepatitis C virus, ZIKA = Zika virus, CHIKV = Chikungunya virus, COVID-19 = new Coronavirus Disease 2019; n° = number.

T686

100786-100918

Sentence

denotes

Figure 4 The quality assessment is based on a checklist adapted from the Cochrane Handbook for Systematic Reviews of Interventions.

T687

100919-101084

Sentence

denotes

The preclinical and clinical studies have been classified as being of high (red section), medium (yellow section), and low risk (green section) of bias; n° = number.

T688

101085-101187

Sentence

denotes

Figure 5 Proposed mechanism for Hydroxychloroquine (HCQ) antiviral activity against COVID-19 and HIV.

T689

101188-101394

Sentence

denotes

HCQ seems to block the virus’ entry into the cell by preventing the binding of viruses to the cell surface receptor and increasing the phagolysosome pH, thus interrupting the virus fusion to the host cells.

T690

101395-101597

Sentence

denotes

HCQ can also inhibit nucleic acid replication, viral proteins glycosylation, virus assembly, transport of new virus particles, viruses release, and other processes to achieve its antiviral effects [14].

T691

101598-101721

Sentence

denotes

Specifically, the anti-HIV activities are highly linked to the post-translational modification of glycoprotein 120 (gp120).

T692

101722-101821

Sentence

denotes

This leads to the loss of gp120 immunogenic properties and reduces new virions infectivity [15,16].

T693

101822-102049

Sentence

denotes

On the other hand, HCQ antiviral activity against COVID-19 seems to be related to its ability to modify the n-terminal glycosylation of ACE-2, leading to reduced interaction between ACE-2 and Spike and so to cell infection [8].

T694

102050-102154

Sentence

denotes

Figure 6 Major biological activities of Hydroxychloroquine (HCQ) and its relative mechanisms of action.

T695

102155-102264

Sentence

denotes

Figure 7 Effects of Hydroxychloroquine (HCQ) on immune diseases and cardiovascular-associated complications.

T696

102265-102341

Sentence

denotes

Figure 8 Bubble map visualizing items from articles included in the review.

T697

102342-102473

Sentence

denotes

A total of 24 items representing the different fields of action of HCQ have been grouped into clusters, based on their relatedness.

T698

102474-102549

Sentence

denotes

The distance between the two terms represents the relatedness of the terms.

T699

102550-102662

Sentence

denotes

Generally, the smaller the distance between two terms, the stronger the relationship of the terms to each other.

T700

102663-102764

Sentence

denotes

Two items are closer to each other if they co-occurred more frequently in the evaluated publications.

T701

102765-102880

Sentence

denotes

The item size indicates the words’ appearance frequency (multiple appearances in a single manuscript count as one).

T702

102881-102968

Sentence

denotes

As explained in the legend, the time colors indicate the research focus over the years.

T703

102969-103013

Sentence

denotes

Table 1 Antiviral effects of HCQ, outcomes.

T704

103014-103117

Sentence

denotes

Author(Year) Study TypePopulation DosageTime Outcomes Adverse Events Noted Limitation of the Study

T705

103118-103123

Sentence

denotes

HIV-1

T706

103124-103812

Sentence

denotes

Sperber, et al. (1995) [17] Randomized, double-blind, placebo-controlled clinical trial40 asymptomatic HIV-1 infected patients HCQ group - > 800 mg/dayControl group - > placebo8 weeks Total HIV-1 RNA plasma levelssignificantly decreased in the HCQ group (range, 98 to 2517 cpm; mean, 168 ± 144 cpm vs. 311 ± 331 cpm; p = 0.022).CD4+ T cells percentage remained stable in HCQ group (18.1 ± 9.2% before treatment vs. 18.6 ± 10.5% after treatment)Absolute CD4+ has not reported significant changes in both groupsIL-6 and IgG levels decrease in HCQ group (14.3 ± 13.5 U/mL vs. 12.0 f 16.7 U/mL; p = 0.023 and 2563 ± 1352 mg/mL vs. 2307 ± 1372 mg/dL; p = 0.032, respectively) Not reported.

T707

103814-103832

Sentence

denotes

Small sample-size.

T708

103833-103893

Sentence

denotes

All of the patients were asymptomatic with a low viral load.

T709

103894-103923

Sentence

denotes

A short period of study time.

T710

103924-104508

Sentence

denotes

Sperber, et al. (1997) [18] Randomized, placebo-controlled clinical trial72 asymptomatic HIV-1 infected patients 800 mg/d HCQ (n = 35)500 mg/d ZDV (n = 37)16 weeks After 16 weeks total plasma HIV-1 RNA levels were reduced in both ZDV group (42.709 ± 33.050 vs. 11.228 ± 7459 copies/mL; p = 0.001) and HCQ group (39.456 ± 31.000 vs. 16.434 ± 11.373 copies/mL; p = 0.02).No significant change occurred in CD4+ cellsOnly in HCQ group it was a reduction in the levels of IL-6 (12.4 ± 12.9 vs. 6.3 ± 5.4 U/nL; p = 0.03) and Ig-G (1453 ± 453 vs. 395 ± 544 mg/dL; p = 0.02) Not reported.

T711

104510-104528

Sentence

denotes

Small sample-size.

T712

104529-104567

Sentence

denotes

All of the patients were asymptomatic.

T713

104568-104883

Sentence

denotes

Paton, et al. (2002) [19] non-comparative clinical study22 asymptomatic HIV-1 infected patients HCQ (200 mg) + hydroxyurea (500 mg) + didanosine (125–200 mg), taken twice daily.48 weeks In the 12th week there was a significant reduction of 1.3 log10 in viral load and an increase in CD4+ percentage by mean 4.3%.

T714

104884-104933

Sentence

denotes

These values were maintained until the 48th week.

T715

104935-104948

Sentence

denotes

Not reported.

T716

104950-105141

Sentence

denotes

Small sample-size.This is a non-comparative design pilot study which not allow determining the contribution made by HCQalone to the overall decrease in viral load obtained by the combination.

T717

105142-105624

Sentence

denotes

Paton, et al. (2005) [20] open-label, noncomparative stud17 HIV-1 infected patients HCQ (200 mg) + hydroxyurea (500 mg) + didanosine (125–200 mg), taken twice daily.144 weeks Mean viral load was reduced by 1.6 log10 copies/mL below baseline (p = 0.001)CD4+ cell counts were significantly increased by a mean of 3.3 ± 6.9%, p = 0.095 at 144th week.CD8 cells percentage was reduced by 11.5 ± 14% per 48th week (p = 0.005) and remained around 10% until the 144th week Not reported.

T718

105626-105644

Sentence

denotes

Small sample-size.

T719

105645-105672

Sentence

denotes

Absence of a control group.

T720

105673-105792

Sentence

denotes

Aguirre-Cruz, et al. (2010) [21] Randomized clinical study8 HIV-infected adults with adenoid hypertrophywere included.

T721

105794-105929

Sentence

denotes

Group A - > 400 mg/dayGroup B - > 800 mg/day8 days HCQ main concentration was significantly higher in at than in plasma Not reported.

T722

105930-106049

Sentence

denotes

González-Hernández, et al. (2014) [22] In vivo on rabbit model Subcutaneous HCQ injection of 15 mg/kg of body weight.

T723

106051-106113

Sentence

denotes

HCQ had a higher affinity for lymphoid tissues than for blood.

T724

106115-106128

Sentence

denotes

Not reported.

T725

106129-106483

Sentence

denotes

Piconi, et al. (2011) [23] Prospective noncomparativeStudy20 HIV-infected immunologic non-responders 400 mg/day HCQ6 months After 6 months, there was an increase in CD4+ T-cells percentage; a reduction of activation/proliferation in CD4+ T-cells (Ki67+) and CD14+ cells (CD69+); a decrease of plasma LPS levels; a downregulation of TLR-7/8 expression.

T726

106485-106557

Sentence

denotes

One patient reported maculopapular exanthema after 10 days of treatment.

T727

106559-106649

Sentence

denotes

Small sample-size.Patients were taking antiretroviral drugs during the treatment with HCQ.

T728

106650-106999

Sentence

denotes

Paton, et al. (2015) [24] Randomized, double-blind, placebo-controlled trial83 asymptomatic HIV-1 infected patients 400 mg/day HCQ or placebo48 weeks At 48th in HCQ group is revealed a faster decline of CD4+ T-cell counts; no change in activation/proliferation levels in CD8+ and CD4+ T-cells; no change in IL-6 levels; an increase in viral load.

T729

107001-107115

Sentence

denotes

Patients in the HCQ group reported influenza-like illness compared with the placebo group (29% vs. 10%; p = 0.03).

T730

107117-107135

Sentence

denotes

Small sample-size.

T731

107136-107556

Sentence

denotes

Chen, et al. (2018) [25] In vivo on a rabbit model Intravaginal implant designed to release an HCQ concentration above 4.34 µg/mL but below 21.7 µg/mL6 days After 6 days, there was seen an improvement of mucosal epithelial integrity, a reduction in submucosal immune cell recruitment, a decrease of gene expression and T cell activation marker protein, and a minimization of key pro-inflammatory mediators activation.

T732

107558-107571

Sentence

denotes

Not reported.

T733

107573-107669

Sentence

denotes

No clinical study has been designed to test the effectiveness of HCQ in preventing HIV infection

T734

107670-107687

Sentence

denotes

Chikungunya Virus

T735

107688-108025

Sentence

denotes

Padmakumar, et al. (2009) [26] Prospective, randomized, parallel-group study120 patients in the acute phase of CHIKV infection Group A -> 200 mg/day ACFGroup B -> ACF + 400 mg/day HCQGroup C -> ACF + 10 mg/day PRDGroup D -> ACF + HCQ + PDR HCQ did not confer any additional benefit in the treatment of the early stages of chikungunya.

T736

108027-108040

Sentence

denotes

Not reported.

T737

108042-108178

Sentence

denotes

The duration of the study can be considered as a limitation with respect to the efficacy assessment of HCQ, which is a slow-acting drug.

T738

108179-108600

Sentence

denotes

Bouquillar, et al. (2018) [27] Multicenter study39 patients with chronic CHIKV infection 400 mg/day HCQ3 months After three months of treatment, evidence of synovitis was disappeared in 10 of 20 subjects (50%) with swollen joins while complete remission was verified in 5 patients (19.2%) In four subjects, the treatment was interrupted due to the onset of side effects such as nausea, stomatitis, rash, and headache.

T739

108602-108620

Sentence

denotes

Small sample-size.

T740

108621-109115

Sentence

denotes

Ravindran, et al. (2017) [28] Randomized controlled open-label study72 patients with chronic CHIKV infection 400 mg/day HCQ (n = 35)15 mg/day MTX, 1g/day sulfasalazine, and 400 mg/day HCQ (n = 37)34 weeks At the end of the 24th week, only the combination of drugs improved disease activity (mean ± SD DAS28; 3.39 ± 0.87 vs. 4.74 ± 0.65, p < 0.0001) and reduces disability (mean ± SD HAQ; 1.4 ± 0.31 vs. 1.8 ± 80.47, p < 0.0001) and pain (mean ± SD VAS 46 ± 6.13 vs. 60.8 ± 11.6, p < 0.0001).

T741

109117-109178

Sentence

denotes

In the combination group, one patient withdrew due to nausea.

T742

109180-109264

Sentence

denotes

It is not a blinded study and so the bias in reporting improvement could be present.

T743

109265-109497

Sentence

denotes

Pandaya S. (2008) [29] Uncontrolled clinical study305 patients with chronic CHIKV infection 15–20 mg/weekly MTX + 400 mg/day HCQ16 weeks At 16th week a reduction in ACR score was shown Not reported There is not a control group.

T744

109498-109536

Sentence

denotes

Only 114 subjects completed the study.

T745

109537-109621

Sentence

denotes

It is not a blinded study and so the bias in reporting improvement could be present.

T746

109622-109632

Sentence

denotes

Flavivirus

T747

109633-110068

Sentence

denotes

Helal, et al. (2016) [30] Prospective, randomized, controlled, interventional, single-blind study120 patients affected by hepatitis C virus Group 1 -> SOC (160 µg pegylated interferon subcutaneously and 1000–12000 mg/day ribavirin orally)Group 2 -> SOC + 200 mg/day HCQ12 weeks HCQ + SOC group showed a high virological response compared to control group [54/60 (90%) vs. 43/60 (71.7%); p = 0.011] and a normalization of ALT levels.

T748

110070-110180

Sentence

denotes

Both groups showed symptoms such as headache,fatigue, influenza-like illness, and gastrointestinaldisturbance.

T749

110182-110211

Sentence

denotes

A short period of study time.

T750

110212-110336

Sentence

denotes

There was a lack of the rapid virological response (RVR) assessment of defined as HCV RNA negativity at week 4 of treatment.

T751

110337-110548

Sentence

denotes

Cao, et al. (2017) [31] In vivo study on pregnant mice infected with ZIKV 40 mg/kg/day HCQ HCQ attenuated placental and fetal ZIKV infection and ameliorated adverse placental and fetal outcomes Not reported.

T752

110550-110648

Sentence

denotes

No clinical study has been designed to test the effectiveness of HCQ in preventing ZIKV infection.

T753

110649-110657

Sentence

denotes

COVID-19

T754

110658-111184

Sentence

denotes

Chen et al. (2020) [6] Randomized, parallel-group clinical trial62 patients suffering from COVID-19 HCQ group -> 400 mg/day HCQControl group -> SOCDay 5 Body temperature recovery time in the HCQ group was shorter than the control group (2.2 vs. 3.2 days, p = 0.0008).Cough remission time was significantly decreased in the HCQ group (2.0 vs. 3.1 days, p = 0.0016).Improvement of pneumonia in HCQ group (80.6% vs. 54.8%)Pneumonia absorption in HCQ group (61.3%) One patient developed a rash.One patient reported a headache.

T755

111186-111296

Sentence

denotes

Small sample-size.Detail about antiviral and antibacterial agents used in the control group are not available.

T756

111297-111759

Sentence

denotes

Gautret et al. (2020) [32] Open-label non-randomized clinical trial36 patients HCQ group -> 600 mg/day HCQ (n = 14); 600 mg/day HCQ +500 mg AZM on day 1 followed by 250 mg/day for 4 days (n = 6)Control group (n = 16)Day 10 On day 6, 70% of HCQ-treated patients were virologically cured comparing to 12.5% in the control groupOn day 6, 100% of HCQ+AZM treated patients are virologically cured comparing to 57.1% in the HCQ group and 12.5% in the control group.

T757

111761-111900

Sentence

denotes

Gastrointestinal side effects in one patient of HCQ group.One patient of the HCQ group died on day 3 although he was PCR-negative on day 2.

T758

111902-112071

Sentence

denotes

Small sample-size.Dropout of six patients from HCQ group.Data available up to 6 days despite the planned 10 days.Details about control group treatment are not available.

T759

112072-112602

Sentence

denotes

Gautret, et al. (2020) [33] Uncontrolled, non-comparative, observational study80 mildly infected patients 600 mg/day HCQ per 10 days + 500 mg AZM on day 1 followed by 250 mg/day for 4 daysFor patients with pneumonia and NEWS score ≥ 5 ceftriaxone was added to HCQ/AZM treatment On day 7, nasopharyngeal viral load tested by qPCR was negative for 83% of patients and for 93% of patients at day 8.At day 5 in 97.5% of patients, virus cultures of the respiratory sample were negative.After 10 days only 2 patients were contagious.

T760

112604-112722

Sentence

denotes

One patient died.Six patients had GI side effects (2 nausea or vomiting and 4 diarrhea)One patient had blurred vision.

T761

112724-112809

Sentence

denotes

Six patients from previous trials by Gautret et al. were also included in this study.

T762

112810-112951

Sentence

denotes

No analytical approach has been made to take into account possible factors of confusion, including in particular the severity of the disease.

T763

112952-113283

Sentence

denotes

Molina et al. (2020) [34] Prospective, non-comparative study11 severe COVID-19 infected patients 600 mg/day HCQ per 10 days + 500 mg AZM on day 1 followed by 250 mg/day for 4 days On day 5 two patients were transferred to the ICU.At days 5 to 6, after treatment initiation 8 of 10 patients were still positive for SARS-CoV2 RNA.

T764

113285-113374

Sentence

denotes

One patient died.One patient discontinued the treatments due to QT interval prolongation.

T765

113376-113451

Sentence

denotes

Small sample size, 8 of 11 had comorbidities associated with poor outcomes.

T766

113452-114533

Sentence

denotes

Tang et al. (2020) [35] Multicenter, open-label, randomized controlled trial150 mild/moderate or severe COVID-19 infected patients HCQ group -> SOC+ HCQ (200 mg daily for three days followed by a maintained dose of 800 mg daily)Control group-> SOC2 for mild/moderate patients and 3 weeks for severe patients Within 28 days of treatment, the probability of negative conversion of SARS-CoV-2 was 85.4% (95% CI 73.8% to 93.8%) in the HCQ + SOC group and 81.3% (95% CI 71.2% to 89.6%) in the SOC group.No significant differences in the median time to negative conversion were found between the HCQ + SOC group (8 days, 95% CI 5 to 10 days) and SOC group (7 days, 95% CI 5 to 8 days).No difference in PCRnegativity was found between two groups at day 4, 7, 10, 14, or 21.No significant differences in the meantime of clinical symptom alleviation were found between the two groups (19 days for HCQ + SOC vs. 21 days for SOC) Adverse events noted in 30% of the HCQ group compared to 8.8% ofcontrol groupThe most common adverse effect was diarrhea (10%).One patient had blurred vision.

T767

114535-114592

Sentence

denotes

The study is only based on the virus-negative conversion.

T768

114593-114959

Sentence

denotes

Abd-Elsalam, et al. (2020) [36] Multicenter, randomized controlled trial194 COVID-19 infected patients HCQ group -> SOC+ HCQ (400 mg twice daily, on day 1, followed by 200 mg tablets twice daily)Control group -> SOC4 weeks of treatment There was no significant difference between the two groups regarding any laboratory parameters or the baseline characteristics.

T769

114960-115082

Sentence

denotes

Four patients (4.1%) in the HCQ group and 5 (5.2%) patients in the control group needed mechanical ventilation (p = 0.75).

T770

115083-115256

Sentence

denotes

There were no differences in the overall mortality between the two groups, as six patients (6.2%) died in the HCQ group and five (5.2%) died in the control group (p = 0.77).

T771

115258-115271

Sentence

denotes

Not reported.

T772

115273-115375

Sentence

denotes

Small sample size, whichwas not adequately powered for survival endpoints.Lack of long-term follow-up.

T773

115376-116001

Sentence

denotes

Skipper, et al. (2020) [37] Randomized, double-blind, placebo-controlled trial491 symptomatic, non-hospitalized adult patients with early or mild COVID-19 HCQ group -> HCQ 800 mg once, followed by 600 mg in 6 to 8 h, then 600 mg daily for 4 more daysControl group-> masked placebo14 weeks of treatment HCQ did not reduce symptom severity when compared with placebo in non-hospitalized early/mild COVID-19 patients (difference in symptom severity: relative, 12%; absolute, −0.27 points (95% CI, −0.61 to 0.07 points); p = 0.117) With HCQ, the most commonly reported adverse effect was related to gastrointestinal symptoms:

T774

116002-116135

Sentence

denotes

31% (66 of 212) of participants reported upset stomach or nausea, and 24% (50 of 212) reported abdominal pain, vomiting, or diarrhea.

T775

116137-116259

Sentence

denotes

Lack of confirmed SARS-CoV-2 infection in all participants.The use of epidemiologic linkage to enroll symptomatic persons.

T776

116260-116918

Sentence

denotes

Mahévas, et al. (2020) [38] No-randomize clinical study181 COVID-19 infected patients HCQ group -> 600 mg/day for 5 days (n = 84) within 48 h of admission to hospitalControl group (n = 97) Within day 7:20.2% infected patients of the HCQ group and 22.1% in the control group died or were transferred to the ICU;27.4% of the HCQ group and 24.4% of the no-HCQ group shown acute respiratory distress;On day 7 the percentage of death was similar in both HCQ and control group (2.8 vs. 4.8%, 3 vs. 4 events) 7 patients of the HCQ group showed QT interval prolongation.One patient presented first-degreeatrioventricular block after 2 days of HCQ administration.

T777

116920-117003

Sentence

denotes

The study was not randomized.Potential unmeasured confounders may bias the results.

T778

117004-117448

Sentence

denotes

Mahévas, et al. (2020) [39] Observational comparativestudy181 severe COVID-19 infected patients HCQ group -> 600 mg/day (n = 92)Control group -> SOC (n = 89) On day 21:Overall survival was 89% in the HCQ group and 91% in the control group;survival without acute respiratory distress syndrome was 69% in the HCQ group and 74% in the control group;patients who had been weaned from oxygen was 82% in the HCQ group and 76% in the control group.

T779

117450-117597

Sentence

denotes

7 patients of HCQ group showed QT interval prolongationOne patient presented first-degreeatrioventricular block after 2 days of HCQ administration.

T780

117599-117776

Sentence

denotes

Treatment was not randomly assigned and potential unmeasured confounders could bias the results.Patients from previous trials by Mahévas et al. were also included in this study.

T781

117777-117995

Sentence

denotes

Lee, et al. (2020) [40] Single-center clinical study211 individuals exposed to COVID-19 400 mg day of HCQ as post-exposure prophylaxis14 days At the end 14 days of quarantine, there was negative follow-up PRC tests.

T782

117997-118135

Sentence

denotes

The most common side effects were diarrhea or loose stool (9%), skin rash (4.3%), gastrointestinal upset (0.95%) and, bradycardia (0.95%).

T783

118136-118281

Sentence

denotes

In 5 patients (2.7%) post-exposure prophylaxis was discontinued due to bradycardia (2), gastrointestinal upset (2), and the need for fasting (1).

T784

118283-118362

Sentence

denotes

There was not a control group and the study was carried out at a single center.

T785

118363-118494

Sentence

denotes

Boulware, et al. (2020) [41] Randomized, double-blind, placebo-controlled clinical trial 821 asymptomatic participants HCQ group:

T786

118495-118861

Sentence

denotes

800 mg once, followed by 600 mg in 6 to 8 h, then 600 mgdaily for 4 additional daysPlacebo group The incidence of new illness compatible with Covid-19 did not differ significantly between the HCQ group (49 of 414 (11.8%)) and the placebo group (58 of 407 (14.3%)); the absolute difference was −2.4 percentage points (95% confidence interval, −7.0 to 2.2; p = 0.35).

T787

118863-118937

Sentence

denotes

Nausea, loose stools, and abdominal discomfort were the main side effects.

T788

118938-119021

Sentence

denotes

There were no intervention-related severe adverse reactions or cardiac arrhythmias.

T789

119023-119040

Sentence

denotes

Small sample-size

T790

119041-119454

Sentence

denotes

Maissonasse, et al. (2020) [40] In vivo study on macaques Different strategies of treatment were compared with placebo, including HCQ alone or in combination with AZM, administrated either before or after viral load When HCQ was administrated as pre-exposure prophylaxis, it did not protect against infection acquisition.Neither HCQ nor HCQ + AZM had beneficial effects in improving viral infection’s symptoms.

T791

119456-119469

Sentence

denotes

Not reported.

T792

119470-119716

Sentence

denotes

The abbreviations are for Hydroxychloroquine (HCQ), Zidovudine (ZDV), Aceclofenac (ACF), prednisolone (PRD), Methotrexate (MXT), Azithromycin (ZAM), Standard-of-care (SOC), Intensive Care Units (ICU), Chikungunya Virus (CHIKV), Zika virus (ZIKV).

T793

119717-119789

Sentence

denotes

Table 2 Main mechanisms of action underlying biological effects of HCQ.

T794

119790-119859

Sentence

denotes

Disease Experimental Model Dosage Mechanisms of Action References

T795

119860-119962

Sentence

denotes

Rheumatoid arthritis (RA) Preclinical 40 mg/kg/day ↓neutrophil-derived oxidants ↓inflammation [75]

T796

119963-120057

Sentence

denotes

Clinical (randomized double-blind, placebo-controlled trial) 7 mg/kg/day ↓inflammation [76]

T797

120058-120150

Sentence

denotes

Clinical (comparative randomized double-blind trial) 200–400 mg/day ↓inflammation [77,78]

T798

120151-120240

Sentence

denotes

RA-associated cardiovascular disease Clinical n.a. ↓IL-6 and leptin↓dyslipidemia [79]

T799

120241-120322

Sentence

denotes

Clinical (cohort study) 6.5 mg/kg/day ↓triglycerides and LDL↓dyslipidemia [80]

T800

120323-120430

Sentence

denotes

Clinical (randomized double-blind cross-over trial) 6.5 mg/kg/day ↓cholesterol and LDL↓dyslipidemia [81]

T801

120431-120516

Sentence

denotes

Clinical (cross-sectional observational study) 200 mg/kg/day ↓fasting glucose [82]

T802

120517-120674

Sentence

denotes

Systemic lupus erythematosus (SLE) Clinical (randomized double-blind placebo-controlled trial) 100–400 mg/kg/day ↓inflammation↓risk of exacerbations [83]

T803

120675-120767

Sentence

denotes

Clinical (long-term randomized study) 272 mg/day ↓inflammation↓risk of exacerbations [84]

T804

120768-120843

Sentence

denotes

Clinical (case-control study) 6.5 mg/kg/day ↓inflammation↑ survival [85]

T805

120844-120924

Sentence

denotes

Preclinical 100 mg/kg/day ↓Th17 response ↑Treg immunosuppressive effects [86]

T806

120925-121001

Sentence

denotes

Clinical (prospective cohort study) 400 mg/day ↓inflammatory markers [87]

T807

121002-121054

Sentence

denotes

Clinical (multiethnic US cohort) n.a. ↓IFN-α [88]

T808

121055-121115

Sentence

denotes

Preclinical 4–40 mg/kg/day ↓ mast cells↓ skin lesion [89]

T809

121116-121211

Sentence

denotes

SLE-associated cardiovascular disease Preclinical 10 mg/kg/day ↓ROS↓endothelial damage [90]

T810

121212-121285

Sentence

denotes

Preclinical 3 mg/kg/day ↓ROS and nitric oxide↓ endothelial damage [91]

T811

121286-121336

Sentence

denotes

Clinical 400 mg/day ↓triglycerides and LDL [92]

T812

121337-121406

Sentence

denotes

Clinical (cross-sectional study) 400 mg/day ↓ fasting glucose [82]

T813

121407-121508

Sentence

denotes

SLE-associated pregnancy complications Clinical (randomized double-blind) n.a. ↓inflammation [93]

T814

121509-121595

Sentence

denotes

Clinical (prospective study) 6.5 mg/kg/day ↓inflammation↓risk of exacerbations [94]

T815

121596-121713

Sentence

denotes

Antiphospholipid syndrome Preclinical 200 μg/day ↓inflammation↓complement activation↓placental abnormalities [95]

T816

121714-121776

Sentence

denotes

Clinical (case report) 400 mg/day ↓vascular thrombosis [96]

T817

121777-121851

Sentence

denotes

Preclinical 12 μg/g/day ↓endothelial damage ↓nitric oxide synthase [97]

T818

121852-121927

Sentence

denotes

Preclinical 20 mg/kg/day ↓endothelial damage ↓nitric oxide synthase [98]

T819

121928-122051

Sentence

denotes

Clinical (observational prospective study) 200 mg/day ↓thrombotic events in patients ↓soluble tissue factor levels. [99]

T820

122052-122125

Sentence

denotes

Sjögren syndrome Clinical 200 mg/day ↓inflammation ↓IgG and IgA [100]

T821

122126-122186

Sentence

denotes

Clinical (prospective study) 400 mg/day ↓xerostomia [101]

T822

122187-122247

Sentence

denotes

Clinical (prospective study) 6.5 mg/kg ↓eye dryness [102]

T823

122248-122315

Sentence

denotes

Preclinical 50 mg/kg/day ↓ xerostomia ↓ TGF-β↓inflammation [103]

T824

122316-122388

Sentence

denotes

Preclinical 60 mg/kg/day ↓inflammation↓lymphocytic infiltration [104]

T825

122389-122455

Sentence

denotes

Diabetes Preclinical 80–120–160 mg/kg/day ↓blood glucose [105]

T826

122456-122530

Sentence

denotes

Preclinical 200 mg/kg/day ↓inflammatory markers↑metabolic profile [106]

T827

122531-122618

Sentence

denotes

Clinical (randomized, double-blinded study) 2 × 300 mg/kg ↓glycated hemoglobin [107]

T828

122619-122702

Sentence

denotes

Clinical (open-label longitudinal study) 6.5 mg/kg/day ↓insulin resistance [108]

T829

122703-122800

Sentence

denotes

Clinical (randomized, double-blinded controlled trial) 6.5 mg/kg/day ↓insulin resistance [109]

T830

122801-122894

Sentence

denotes

Clinical (randomized, double-blinded trial) 400 mg/day ↑glycemic and lipidic profile [110]

T831

122895-122991

Sentence

denotes

Cancer Preclinical 50 mg/kg ↓tumor size↓pro-tumorigenic and pro-inflammatory cytokines [111]

T832

122992-123076

Sentence

denotes

Cardiovascular diseases Preclinical 200 mg/kg ↓apoptosis in cardiomyocites [112]

T833

123077-123134

Sentence

denotes

Preclinical 200 mg/kg/day ↓triglycerides and LDL [113]

T834

123135-123198

Sentence

denotes

Preclinical 10 mg/kg/day ↓atherosclerosis↓inflammation [114]

T835

123199-123281

Sentence

denotes

Inflammatory bowel disease and colitis Preclinical 30 mg/kg ↓inflammation [74]

T836

123282-123353

Sentence

denotes

Pulmonary hypertension Preclinical 50 mg/kg/day ↓inflammation [115]

T837

123354-123426

Sentence

denotes

Endometriosis Preclinical 60 mg/kg ↓inflammation↓lesion number [116]

T838

123427-123655

Sentence

denotes

LDL: low-density lipoproteins, Th17: effector lymphocyte T, Treg: regulatory lymphocyte T, IFN-α: type I interferon, ROS: radical oxygen species, Ig: immunoglobulin, TGF-β: transforming growth factor-β; n.a.= data not available.

T839

123656-123743

Sentence

denotes

Table 3 Checklist for assessment of the risk of bias in preclinical studies [171,172].

T840

123744-123807

Sentence

denotes

Checklist for Assessment of Risk of Bias in Preclinical Studies

T841

123808-123872

Sentence

denotes

Are the hypothesis and objective of the study clearly described?

T842

123873-123928

Sentence

denotes

Are the main outcomes to be measured clearly described?

T843

123929-123982

Sentence

denotes

Are the main findings of the study clearly described?

T844

123983-124026

Sentence

denotes

Are the samples size calculations reported?

T845

124027-124081

Sentence

denotes

Are the animals randomly housed during the experiment?

T846

124082-124148

Sentence

denotes

Are the investigators blinded from knowledge which treatment used?

T847

124149-124183

Sentence

denotes

Are the outcome assessors blinded?

T848

124184-124249

Sentence

denotes

Is the dose/route of administration of the HCQ properly reported?

T849

124250-124332

Sentence

denotes

Is the dose/route of administration of the drug in co-treatment properly reported?

T850

124333-124385

Sentence

denotes

Is the frequency of treatments adequately described?

T851

124386-124461

Sentence

denotes

Table 4 Checklist for assessment of risk of bias in clinical studies [97].

T852

124462-124525

Sentence

denotes

Checklist for Assessment of Risk of Bias in Preclinical Studies

T853

124526-124590

Sentence

denotes

Are the hypothesis and objective of the study clearly described?

T854

124591-124646

Sentence

denotes

Are the main outcomes to be measured clearly described?

T855

124647-124700

Sentence

denotes

Are the main findings of the study clearly described?

T856

124701-124744

Sentence

denotes

Are the samples size calculations reported?

T857

124745-124799

Sentence

denotes

Are the animals randomly housed during the experiment?

T858

124800-124866

Sentence

denotes

Are the investigators blinded from knowledge which treatment used?

T859

124867-124901

Sentence

denotes

Are the outcome assessors blinded?

T860

124902-124967

Sentence

denotes

Is the dose/route of administration of the HCQ properly reported?

T861

124968-125050

Sentence

denotes

Is the dose/route of administration of the drug in co-treatment properly reported?

T862

125051-125103

Sentence

denotes

Is the frequency of treatments adequately described?

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