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      Hydroxychloroquine and Azithromycin to Treat Patients With COVID‐19: Both Friends and Foes?

      1 , 2
      The Journal of Clinical Pharmacology
      Wiley

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          Abstract

          The world is facing a frightening pandemic due to coronavirus disease 2019 (COVID‐19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) with thousands of severe infections and fatalities. Since no therapy has proven effective, an extraordinary race is taking place to identify an effective and safe treatment able to limit the disease progression and severity. Two nonrandomized open‐label trials conducted in France and China 1 , 2 established the effectiveness of hydroxychloroquine alone or combined with azithromycin in decreasing nasopharyngeal viral load and carriage duration in patients with COVID‐19, although evidence to support clinical benefits remained low. Thereafter, many studies, still unpublished in peer‐reviewed journals for the majority, showed contrasting results and revealed potential safety hazards (Table 1). 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 To date, multiple trials aiming at investigating chloroquine or hydroxychloroquine at various dose regimens to treat COVID‐19 (N = 81) or prevent the disease in high‐risk populations (N = 19) are cited on clinicaltrials.gov (accessed April 21, 2020). Interestingly, only 14 trials (17%) will investigate the azithromycin‐hydroxychloroquine combination. Table 1 Emerging List of Studies Investigating Chloroquine/Hydroxychloroquine With or Without Azithromycin to Treat COVID‐19 Authors Country Design N Time in the Disease Course and Infection Severity Groups and Dose Regimen Main Resultsa Gautret et al 1 published France Uncontrolled noncomparative observational study 80 Early (d 5) Mild 1 group, HCQ (200 mg ×3/d, 10 d) + AZ (500 mg on d 1 followed by 250 mg/d, 4 d) Clinical improvement, rapid discharge, rapid fall in nasopharyngeal viral load, negative viral culture on d 5 in almost all patients Chen et al 2 China Randomized open‐label parallel‐group trial 62 Unknown Moderate 2 groups, HCQ (200 mg ×2/d, 5 d) vs no HCQ Significant clinical improvement based on body temperature recovery and cough remission times and increased recovery from pneumonia Chen et al 3 published China Randomized open‐label controlled trial 30 Early (d 6) Mild 2 groups, HCQ (200 mg ×2/d, 5 d) vs no HCQ No reduction in the percentage of negative SARS‐CoV‐2 nucleic acid of throat swabs, the time from hospitalization to virus nucleic acid negative conservation, temperature normalization, and radiological progression Molina et al 4 published France Uncontrolled noncomparative observational study 11 Unknown Moderate 1 group, HCQ (600 mg/d, 10 d) + AZ (500 mg/d, d 1 and 250 mg/d, d 2‐5) Positive SARS‐CoV‐2 RNA in 8/10 patients (80%, 95% confidence interval, 49%‐94%) at d 5‐6 after treatment initiation Magagnoli et al 5 United States Retrospective cohort study 368 Unknown Moderate 3 groups, HCQ vs HCQ+AZ vs no HCQ (dosages not available) No reduction in mechanical ventilation Increased overall mortality in HCQ group Mahévas et al 6 France Retrospective cohort study 181 Early (d 7) Moderate 2 groups, HCQ (600 mg/d within 48 h after admission) vs no HCQ No reduction in ICU transfer or death, death within 7 d and ARDS within 7 d Million et al 7 France Uncontrolled noncomparative observational study 1061 Early (d 6) Mild 1 group, HCQ (200 mg ×3/d, 10 d) + AZ (500 mg on d 1 followed by 250 mg/d, 4 d); analysis of the patients who took HCQ + AZ during at least 3 d Significant reduction in mortality in comparison to patients treated with other regimens in all Marseille public hospitals Barbosa et al 8 United States Retrospective cohort study 63 Unknown Moderate 2 groups, HCQ vs No HCQ (dosages not available) Increased need for escalation of respiratory support and no benefits on mortality, lymphopenia, or neutrophil‐to‐lymphocyte ratio improvement Tang et al 9 China Randomized open‐label controlled trial 150 Delayed (day 16) Mild to moderate 2 groups, HCQ (1200 mg/d for 3 d followed by 800 mg/d; total duration: 2 wks [mild/moderate] or 3 wks [severe]) vs no HCQ No differences in the overall 28‐d negative conversion rate, the negative conversion rate at d 4, 7, 10, 14, or 21, the improvement rate of clinical symptoms within 28 d, the normalization of C‐reactive protein and the blood lymphocyte count within 28 d Increased adverse events Borba et al 10 published Brazil Randomized double‐blinded parallel phase IIb trial Safety‐oriented study 81 Early (d 7) Moderate 2 groups, high‐dose CQ (600 mg ×2/d, f10 d, or total dose 12 g) vs low‐dose CQ (450 mg for 5 d, ×2/d only on the first day, or total dose 2.7 g) No differences in clinical outcome However, high‐dose CQ with potential safety hazards (QTc prolongation), especially when taken concurrently with AZ and oseltamivir Chorin et al 11 United States Retrospective cohort study Safety‐oriented study 84 Unknown Moderate to severe HCQ + AZ (dosages not available) QTc prolongation >500 ms in 11% patients; no torsade de pointes ARDS, acute respiratory distress syndrome; AZ, azithromycin; CQ, chloroquine; HCQ, hydroxychloroquine; ICU, intensive care unit; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2. Results as presented in the study, considering that the majority has not been peer reviewed yet. John Wiley & Sons, Ltd. This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency. Drug 1: Hydroxychloroquine Hydroxychloroquine is a cheap and readily available decades‐old drug with immunomodulatory properties used to treat autoimmune rheumatic diseases. Its multitude of anti‐inflammatory and immune‐regulatory effects continue to puzzle medical experts worldwide. Pharmacological pathways mainly include the blockage of Toll‐like receptor–mediated signaling (Toll‐like receptor‐7 and ‐9, the endosomal innate immune sensor capable of detecting single‐stranded RNA), the modulation of complement‐dependent antigen/antibody reactions, the activation of T‐regulatory cells, and the inhibition of proinflammatory cytokine production such as interleukin‐6, tumor necrosis factor‐α and interferon‐γ. 12 Therefore, hydroxychloroquine immediately appeared attractive to attenuate the inflammatory response directed against SARS‐CoV‐2 that results in the cytokine storm, which is held responsible for severe COVID‐19 presentations. The processes of SARS‐CoV‐2 replication with the resulting pulmonary epithelial and endothelial cell injury and angiotensin‐converting enzyme‐2 (ACE2) downregulation and shedding rapidly result in exuberant inflammatory responses, evidenced by increasing plasma concentrations of various cytokines including interleukin‐6. Hydroxychloroquine‐mediated inhibition of interleukin‐6 production has been well established in vitro on peripheral blood mononuclear cells stimulated by phytohemagglutinin or lipopolysaccharide. 13 Its benefit in counteracting the inflammatory rheumatologic disease activity is well correlated in vivo with the resulting lowering effects on most cytokines and proinflammatory markers. 14 Therefore, since interleukin‐6 plays a key starter role in COVID‐19–related cytokine storm, hydroxychloroquine rapidly appeared, at least theoretically, as a potential immunomodulatory anti–COVID‐19 drug, if administered early enough in the disease time course. The 4‐aminoquinoline compounds are active in vitro against a range of viruses with different suggested mechanisms of action. Recently, hydroxychloroquine‐attributed anti‐SARS‐CoV‐2 activity was established in vitro (50% effective concentration [EC50] = 0.72 μmol/L at a multiplicity of infection of 0.01 [100 PFU/well] in Vero cells for 2 hours) and found more potent than chloroquine (EC50 = 5.47 μmol/L in the same conditions). 15 However, the mechanisms of hydroxychloroquine‐attributed anti–COVID‐19 activity remain presumptive (Figure 1). Figure 1 Suggested mechanisms for the antiviral and immunomodulatory activities of hydroxychloroquine and azithromycin in COVID‐19 highlighting possible synergic effects between the 2 drugs if prescribed in combination (adapted from Savarino et al, 34 with permission). Possible hydroxychloroquine‐attributed effects include (1) interference with ACE2 glycosylation and reduction of viral binding, (2) endosome and lysosome alkalization limiting viral uncoating and assembly, (3) alteration of antigen processing and MHC class II–mediated autoantigen presentation, (4) disruption of RNA interaction with TLRs and nucleic acid sensors, (5) inhibition of proinflammatory genes transcription, (6) inhibition of T‐cell activation, and (7) inhibition of cytokine production. Possible azithromycin‐attributed effects include (1) interference with ACE2 and reduction of viral binding, (2) endosome and lysosome alkalization limiting viral uncoating and assembly, and (3) role of lysosomal P‐glycoprotein that enhances intralysosomal concentrations of azithromycin. ACE2, angiotensin‐converting enzyme 2; AZ, azithromycin; COVID‐19, coronavirus 2019 disease; HCQ, hydroxychloroquine; IL, interleukin; MHC, major histocompatibility complex; P‐gp, P‐glycoprotein; RNA, ribonucleic acid; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; TNF‐α, tumor necrosis factor‐α; TLR, Toll‐like receptor. In addition to the previously reported immunomodulatory action, hydroxychloroquine may alter ACE2 glycosylation, blocking SARS‐CoV‐2 interaction with its membrane receptor and subsequently the virus/host cell membrane fusion. 16 Consistent with the hypothesis of ACE2 interaction, chloroquine was shown to inhibit quinone reductase‐2, a structural neighbor of UDP‐N‐acetylglucosamine‐2‐epimerases, involved in sialic acid biosynthesis. Although deserving deeper investigations, this molecular mechanism is considered to mediate antimalarial drug activity in vitro on various viruses such as HIV, SARS‐CoV‐1, and orthomyxoviruses. 16 , 17 However, when discussing any potential benefits of interacting with the ACE/ACE2 system, we should acknowledge that the clinical effects of ACE inhibitors in patients with COVID‐19 still remain controversial. These drugs may appear attractive to treat cardiovascular diseases by reducing pulmonary inflammation; however, their use may be accompanied by enhanced ACE2 expression that facilitates the viral invasion. 18 Interestingly, as a weak base, hydroxychloroquine increases the intracellular pH, mainly in the acidic organelles such as endosomes/lysosomes (pH 4.5) where it intensively accumulates. Moreover, hydroxychloroquine alkalinizes the phagolysosomes (pH ∼6.5; known as the lysozomotropic activity) and may thus inhibit the viral cleavage mediated by pH‐dependent proteases, disrupt the fusion process and stop the viral replication. These last effects complete the wide pharmacological target spectra of hydroxychloroquine in COVID‐19. Finally, in antigen‐presenting cells, hydroxychloroquine prevents antigen processing and subsequently T‐cell activation, differentiation, and cytokine production. 19 , 20 At the molecular level, hydroxychloroquine acts by (1) altering the digestion pattern of the antigenic peptides, (2) retarding the major histocompatibility complex class II α and β chains from forming a stable compact complex with the antigenic peptide due to the diminished degradation of the nonpolymorphic invariant chain, and (3) altering the recycling of α‐β‐peptide complexes from the cell surface. 19 , 20 To date, there is no definitive evidence that all these potential antiviral activities could be achieved by the usual hydroxychloroquine doses (400‐600 mg daily) that are considered clinically safe, as initially suggested. 15 The exact clinically effective hydroxychloroquine dose is still undetermined. Given the weaknesses of Gautret's 1 and Chen's 2 studies along with the negative results of the recently released US veterans study 5 (Table 1), improving effectiveness by increasing hydroxychloroquine doses has been questioned by different modeling approaches including a physiologically based pharmacokinetic study 15 and a pharmacokinetic/virology/QT model. 21 Both studies concluded that hydroxychloroquine doses >400 mg twice daily for at least 5 days are needed to ensure efficacy on viral load decline and cardiac safety. Moreover, the last study highlighted that suboptimal dosing is not efficient on viral load resulting in wasted time and resources. Several trials like the PATCH study (see clinicaltrials.gov) are currently investigating higher doses up to 600 mg twice daily, mainly for the sickest patients. Another major issue also remains the time in the disease course at which the treatment is initiated since, based on the reported antiviral or immunomodulatory mechanisms of action, expected benefits may be reached only if hydroxychloroquine is started early. Drug 2: Azithromycin Azithromycin is a macrolide antimicrobial agent with established antiviral properties in vitro and anti–SARS‐CoV‐2 activity (EC50 of 2.12 μmol/L). Several mechanisms have been proposed to explain its antiviral effects (see review; Figure 1). 22 Similarly to hydroxychloroquine, azithromycin is highly trapped in the subcellular acidic organelles such as lysosomes, 23 causing even more severe impairment of acidification. Additionally, azithromycin is responsible for a global amplification of the host's interferon pathway‐mediated antiviral responses. Finally, it may alter SARS‐CoV‐2 entry by interfering between its spike protein and host ACE2 receptor. Very recently, an energetics‐based modeling provided high binding affinity for azithromycin at the interaction point between SARS‐CoV‐2 spike and ACE2, whereas hydroxychloroquine appeared ineffective to directly inhibit this interaction. 24 Clinical studies have shown the ability of azithromycin to reduce the viral load with demonstrated benefits on patient outcome including accelerated recovery (influenza A infection), reduced respiratory morbidity (respiratory syncytial virus and SARS infections) and improved mortality (Middle East respiratory syndrome–coronavirus infection). 22 Several trials in addition to those testing the hydroxychloroquine‐azithromycin combination are currently ongoing to investigate the anti–COVID‐19 benefits of azithromycin alone or in combination with other drugs, focusing as end points not only on viral load but also on clinical outcomes. Interestingly, azithromycin is a known substrate of P‐glycoprotein (ABCB1), a member of the adenosine triphosphate–binding cassette transporters superfamily, highly expressed and oriented from the cytosolic to the internal side of the lysosome membrane. 25 Therefore, we hypothesized that azithromycin accumulates more intensively under the ABCB1 trapping effect inside the lysosomes and like hydroxychloroquine, reduces lysosomal acidity and exhibits its antiviral properties. Consistently with our hypotheses, synergistic in vivo and in vitro properties have been attributed to the azithromycin/chloroquine combination, used to protect against malaria and treat sexually transmitted infections. 26 , 27 Similarly, the antiviral synergy of the azithromycin/hydroxychloroquine combination was observed in vitro at concentrations achieved in vivo and detected in pulmonary tissues 28 and was shown to accelerate viral clearance in humans in comparison to hydroxychloroquine alone. 1 Thus, azithromycin ABCB1‐dependent lysosomal sequestration plus hydroxychloroquine is very likely an optimal combination to hamper the low‐pH–dependent steps of viral replication and limit COVID‐19 progression. Future clinical studies investigating the azithromycin/chloroquine combination have to focus on clinical outcome improvement and not only viral load reduction, although this target may also be interesting to limit interhuman contagiousness. Risks‐Benefits of Combining the 2 Drugs Fears may emerge from increased risks of QT interval prolongation (resulting from the human Ether‐à‐go‐go‐Related Gene potassium channel blockage), torsade de pointes, and cardiovascular death. 29 Despite potential risks acknowledged to be limited if either drug is prescribed singly, 30 , 31 drug‐drug interaction resulting from their coadministration as well as patients’ advanced age, preexisting comorbidities, and COVID‐19–related myocardial and kidney injuries represent challenging conditions. Interestingly, a recent multinational, network cohort, and self‐controlled case series study supported the safety of short‐term hydroxychloroquine treatment but highlighted the risks of heart failure and cardiovascular mortality when combining hydroxychloroquine with azithromycin, potentially due to synergistic effects on QT length. 32 Data from recent COVID‐19 trials clearly reported increased adverse events with hydroxychloroquine, especially at high doses and in combination with azithromycin. 9 , 10 , 11 Additionally, extending prescriptions to mildly infected patients is also at risk of misuse and overdose. Clinical toxicologists remember the 1982 suicide outbreak in France following the publication of Suicide: A How‐To Guide, which promoted chloroquine ingestion to complete suicide, 33 resulting in a major crisis of fatalities attributed to chloroquine poisonings. In patients with COVID‐19 treated with the azithromycin/hydroxychloroquine combination, physicians should be cautious when coprescribing QT interval–prolonging drugs (enhanced toxicity) or P‐glycoprotein substrates/inhibitors (reduced effectiveness). Nevertheless, abandoning azithromycin may importantly limit hydroxychloroquine‐attributed effectiveness. Therefore, well‐designed randomized, double‐blind and placebo‐controlled clinical trials are awaited to evaluate the exact synergy/toxicity balance of this potentially lifesaving combination. Appropriate statistical hypotheses should be formulated when designing the study so that the alternative hypothesis can be inferred upon the rejection of the null hypothesis. Observational studies are not sufficient in nature to meet regulatory requirements and decide if a treatment with potentially serious toxicity should be advocated. Pharmacovigilance departments and poison control centers should be alert to collect all useful toxicological exposure data and trends to guide public health response. In conclusion, both hydroxychloroquine and azithromycin are friends and foes, when considering the balance issue between the expected synergistic effectiveness to clear the virus from the body and the safety concerns to avoid possible risks of cardiotoxicity when the 2 drugs are combined. Conflicts of Interest The authors declare no conflicts of interest.

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          In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

          Abstract Background The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) first broke out in Wuhan (China) and subsequently spread worldwide. Chloroquine has been sporadically used in treating SARS-CoV-2 infection. Hydroxychloroquine shares the same mechanism of action as chloroquine, but its more tolerable safety profile makes it the preferred drug to treat malaria and autoimmune conditions. We propose that the immunomodulatory effect of hydroxychloroquine also may be useful in controlling the cytokine storm that occurs late-phase in critically ill SARS-CoV-2 infected patients. Currently, there is no evidence to support the use of hydroxychloroquine in SARS-CoV-2 infection. Methods The pharmacological activity of chloroquine and hydroxychloroquine was tested using SARS-CoV-2 infected Vero cells. Physiologically-based pharmacokinetic models (PBPK) were implemented for both drugs separately by integrating their in vitro data. Using the PBPK models, hydroxychloroquine concentrations in lung fluid were simulated under 5 different dosing regimens to explore the most effective regimen whilst considering the drug’s safety profile. Results Hydroxychloroquine (EC50=0.72 μM) was found to be more potent than chloroquine (EC50=5.47 μM) in vitro. Based on PBPK models results, a loading dose of 400 mg twice daily of hydroxychloroquine sulfate given orally, followed by a maintenance dose of 200 mg given twice daily for 4 days is recommended for SARS-CoV-2 infection, as it reached three times the potency of chloroquine phosphate when given 500 mg twice daily 5 days in advance. Conclusions Hydroxychloroquine was found to be more potent than chloroquine to inhibit SARS-CoV-2 in vitro.
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            Renin–Angiotensin–Aldosterone System Inhibitors in Patients with Covid-19

            The renin–angiotensin–aldosterone system (RAAS) is an elegant cascade of vasoactive peptides that orchestrate key processes in human physiology. Severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and SARS-CoV-2, which have been responsible for the SARS epidemic in 2002 to 2004 and for the more recent coronavirus disease 2019 (Covid-19) pandemic, respectively, interface with the RAAS through angiotensin-converting enzyme 2 (ACE2), an enzyme that physiologically counters RAAS activation but also functions as a receptor for both SARS viruses. 1,2 The interaction between the SARS viruses and ACE2 has been proposed as a potential factor in their infectivity, 3,4 and there are concerns about the use of RAAS inhibitors that may alter ACE2 and whether variation in ACE2 expression may be in part responsible for disease virulence in the ongoing Covid-19 pandemic. 5-8 Indeed, some media sources and health systems have recently called for the discontinuation of ACE inhibitors and angiotensin-receptor blockers (ARBs), both prophylactically and in the context of suspected Covid-19. Given the common use of ACE inhibitors and ARBs worldwide, guidance on the use of these drugs in patients with Covid-19 is urgently needed. Here, we highlight that the data in humans are too limited to support or refute these hypotheses and concerns. Specifically, we discuss the uncertain effects of RAAS blockers on ACE2 levels and activity in humans, and we propose an alternative hypothesis that ACE2 may be beneficial rather than harmful in patients with lung injury. We also explicitly raise the concern that withdrawal of RAAS inhibitors may be harmful in certain high-risk patients with known or suspected Covid-19. Covid-19 and Older Adults with Coexisting Conditions Initial reports 5-8 have called attention to the potential overrepresentation of hypertension among patients with Covid-19. In the largest of several case series from China that have been released during the Covid-19 pandemic (Table S1 in the Supplementary Appendix, available with the full text of this article at NEJM.org), hypertension was the most frequent coexisting condition in 1099 patients, with an estimated prevalence of 15% 9 ; however, this estimate appears to be lower than the estimated prevalence of hypertension seen with other viral infections 10 and in the general population in China. 11,12 Coexisting conditions, including hypertension, have consistently been reported to be more common among patients with Covid-19 who have had severe illness, been admitted to the intensive care unit, received mechanical ventilation, or died than among patients who have had mild illness. There are concerns that medical management of these coexisting conditions, including the use of RAAS inhibitors, may have contributed to the adverse health outcomes observed. However, these conditions appear to track closely with advancing age, 13 which is emerging as the strongest predictor of Covid-19–related death. 14 Unfortunately, reports to date have not rigorously accounted for age or other key factors that contribute to health as potential confounders in risk prediction. With other infective illnesses, coexisting conditions such as hypertension have been key prognostic determinants, 10 and this also appears to be the case with Covid-19. 15 It is important to note that, despite inferences about the use of background RAAS inhibitors, specific details have been lacking in studies (Table S1). Population-based studies have estimated that only 30 to 40% of patients in China who have hypertension are treated with any antihypertensive therapy; RAAS inhibitors are used alone or in combination in 25 to 30% of these treated patients. 11,12 Given such estimates, only a fraction of patients with Covid-19, at least in China, are anticipated to have been previously treated with RAAS inhibitors. Data showing patterns of use of RAAS inhibitors and associated health outcomes that rigorously account for treatment indication and illness severity among patients with Covid-19 are needed. Uncertain Effects of RAAS Inhibitors on ACE2 in Humans Tissue-specific and circulating components of the RAAS make up a complex intersecting network of regulatory and counterregulatory peptides (Figure 1). ACE2 is a key counterregulatory enzyme that degrades angiotensin II to angiotensin-(1–7), thereby attenuating its effects on vasoconstriction, sodium retention, and fibrosis. Although angiotensin II is the primary substrate of ACE2, that enzyme also cleaves angiotensin I to angiotensin-(1–9) and participates in the hydrolysis of other peptides. 16 In studies in humans, tissue samples from 15 organs have shown that ACE2 is expressed broadly, including in the heart and kidneys, as well as on the principal target cells for SARS-CoV-2 (and the site of dominant injury), the lung alveolar epithelial cells. 17 Of interest, the circulating levels of soluble ACE2 are low and the functional role of ACE2 in the lungs appears to be relatively minimal under normal conditions 18 but may be up-regulated in certain clinical states. Because ACE inhibitors and ARBs have different effects on angiotensin II, the primary substrate of ACE2, the effects of these agents on ACE2 levels and activity may be anticipated to differ. Despite substantial structural homology between ACE and ACE2, their enzyme active sites are distinct. As a result, ACE inhibitors in clinical use do not directly affect ACE2 activity. 19 Experimental animal models have shown mixed findings with respect to the effects of ACE inhibitors on ACE2 levels or activity in tissue. 20-25 Similarly, animal models have had inconsistent findings with respect to the effects of ARBs on ACE2, with some showing that ARBs may increase messenger RNA expression or protein levels of ACE2 in tissue 21,26-34 and others showing no effect. 23 In contrast to available animal models, there are few studies in humans regarding the effects of RAAS inhibition on ACE2 expression. In one study, the intravenous administration of ACE inhibitors in patients with coronary artery disease did not influence angiotensin-(1–7) production, a finding that calls into question whether ACE inhibitors have any direct effects on ACE2-directed angiotensin II metabolism. 35 Similarly, in another study, among patients with hypertension, angiotensin-(1–7) levels appeared to be unaffected after initial treatment with the ACE inhibitor captopril; however, with exposure to captopril monotherapy over a period of 6 months, angiotensin-(1–7) levels increased. 36 Furthermore, few studies have examined plasma ACE2 activity or urinary ACE2 levels in patients who have received long-term treatment with RAAS inhibitors. In cross-sectional studies involving patients with heart failure, 37 atrial fibrillation, 38 aortic stenosis, 39 and coronary artery disease, 40 plasma ACE2 activity was not higher among patients who were taking ACE inhibitors or ARBs than among untreated patients. In a longitudinal cohort study involving Japanese patients with hypertension, urinary ACE2 levels were higher among patients who received long-term treatment with the ARB olmesartan than among untreated control patients, but that association was not observed with the ACE inhibitor enalapril or with other ARBs (losartan, candesartan, valsartan, and telmisartan). 41 Previous treatment with ACE inhibitors was associated with increased intestinal messenger RNA levels of ACE2 in one study, but that association was not observed with ARBs 25 ; data are lacking regarding the effects of RAAS inhibitors on lung-specific expression of ACE2. These seemingly conflicting data indicate the complexity underlying RAAS responses to pathway modulators and reinforce the concept that findings from preclinical models may not readily translate to human physiology. Such data do suggest that effects on ACE2 should not be assumed to be uniform across RAAS inhibitors or even in response to therapies within a given drug class. 41 It is important to note that the plasma ACE2 level may not be a reliable indicator of the activity of the full-length membrane-bound form, in part because ACE2 is shed from the membrane, a process that appears to be separately regulated by an endogenous inhibitor. 42 In addition to the degree of expression, the biologic relevance of ACE2 may vary according to tissue and clinical state. Unfortunately, data showing the effects of ACE inhibitors, ARBs, and other RAAS inhibitors on lung-specific expression of ACE2 in experimental animal models and in humans are lacking. Furthermore, even if RAAS inhibitors modify ACE2 levels or activity (or both) in target tissue beds, clinical data are lacking to indicate whether this would in turn facilitate greater engagement and entry of SARS-CoV-2 spike protein. Further mechanistic studies in humans are needed to better define the unique interplay between SARS-CoV-2 and the RAAS network. Potential for Benefit Rather Than Harm of RAAS Blockers in Covid-19 SARS-CoV-2 appears not only to gain initial entry through ACE2 but also to subsequently down-regulate ACE2 expression such that the enzyme is unable to exert protective effects in organs. It has been postulated but unproven that unabated angiotensin II activity may be in part responsible for organ injury in Covid-19. 43,44 After the initial engagement of SARS-CoV-2 spike protein, there is subsequent down-regulation of ACE2 abundance on cell surfaces. 45 Continued viral infection and replication contribute to reduced membrane ACE2 expression, at least in vitro in cultured cells. 46 Down-regulation of ACE2 activity in the lungs facilitates the initial neutrophil infiltration in response to bacterial endotoxin 47 and may result in unopposed angiotensin II accumulation and local RAAS activation. Indeed, in experimental mouse models, exposure to SARS-CoV-1 spike protein induced acute lung injury, which is limited by RAAS blockade. 45 Other mouse models have suggested that dysregulation of ACE2 may mediate acute lung injury that is secondary to virulent strains of influenza 48,49 and respiratory syncytial virus. 50 In a small study, patients with Covid-19 appeared to have elevated levels of plasma angiotensin II, which were in turn correlated with total viral load and degree of lung injury. 44 Restoration of ACE2 through the administration of recombinant ACE2 appeared to reverse this devastating lung-injury process in preclinical models of other viral infections 49,50 and safely reduced angiotensin II levels in a phase 2 trial evaluating acute respiratory distress syndrome in humans. 51 Dysregulated ACE2 may theoretically also attenuate cardioprotection in the context of myocardial involvement and abnormal pulmonary hemodynamics 52,53 in Covid-19. Markers of myocardial injury have been shown to be elevated during the disease course of Covid-19 54 and to increase rapidly with clinical deterioration and preceding death. 14 Many viruses are cardiotropic, and subclinical viral myocarditis is commonly seen in viremia associated with a wide range of infectious agents. ACE2 has a well-recognized role in myocardial recovery and injury response; in one study, ACE2 knockout in animal models contributed to adverse left ventricular remodeling in response to acute injury driven by angiotensin II. 55 In autopsies of patients who died from SARS, 35% of heart samples showed the presence of viral RNA, which in turn was associated with reduced ACE2 protein expression. 56 Administration of recombinant ACE2 normalizes angiotensin II levels in human explanted hearts with dilated cardiomyopathy. 57 These hypotheses have prompted trials to test whether the provision of recombinant ACE2 protein may be beneficial in restoring balance to the RAAS network and potentially preventing organ injury (ClinicalTrials.gov number, NCT04287686). In addition, paired trials of losartan as a treatment for Covid-19 are being conducted among patients who have not previously received treatment with a RAAS inhibitor and are either hospitalized (NCT04312009) or not hospitalized (NCT04311177). Maintenance of RAAS Inhibitors with Known or Suspected Covid-19 Despite these theoretical uncertainties regarding whether pharmacologic regulation of ACE2 may influence the infectivity of SARS-CoV-2, there is clear potential for harm related to the withdrawal of RAAS inhibitors in patients in otherwise stable condition. Covid-19 is particularly severe in patients with underlying cardiovascular diseases, 9 and in many of these patients, active myocardial injury, 14,54,58-60 myocardial stress, 59 and cardiomyopathy 59 develop during the course of illness. RAAS inhibitors have established benefits in protecting the kidney and myocardium, and their withdrawal may risk clinical decompensation in high-risk patients. Although rates of heart failure have been infrequently reported in epidemiologic reports from China to date, the prevalence of heart failure among critically ill patients with Covid-19 in the United States may be high (>40%). 59 In the Quinapril Heart Failure Trial, among patients with chronic symptomatic heart failure, withdrawal of quinapril resulted in a progressive decline in clinical status. 61 In the TRED-HF trial, among asymptomatic patients with heart failure with recovered left ventricular ejection fraction, the phased withdrawal of medical therapy (including RAAS inhibitors) resulted in rapid relapse of dilated cardiomyopathy. 62 In addition, RAAS inhibitors are a cornerstone of therapy after myocardial infarction: maintenance of therapy in the days to weeks after the index event has been shown to reduce early mortality. 63 Among patients with unstable clinical status, myocardial injury associated with Covid-19 may pose even higher early risks after withdrawal of RAAS inhibitors. Withdrawal of RAAS inhibitors that are being administered for the management of hypertension may be less risky than withdrawal of RAAS inhibitors that are being administered for conditions in which they are considered guideline-directed therapy but may be associated with other challenges. Switching from a RAAS inhibitor to another antihypertensive therapy in a stable ambulatory patient may require careful follow-up to avoid rebound increases in blood pressure. In addition, selection of dose-equivalent antihypertensive therapies may be challenging in practice and may be patient-dependent. Even small and short-lived periods of blood pressure instability after a therapeutic change have been associated with excess cardiovascular risk. 64-66 This may be an especially important consideration in patients with Covid-19, which appears to result in a state of RAAS activation, 44 and in settings (e.g., China) where baseline blood-pressure control is infrequently reached at the population level. 11,12 The effects of withdrawing RAAS inhibitors or switching treatments are uncertain among patients with chronic kidney disease. Although reported rates of chronic kidney disease appear to be low among hospitalized patients with Covid-19 in China (1 to 3%) (Table S1), the prevalence may be higher among patients who are critically ill and among those in other geographic regions. 59 Many patients have varying degrees of acute kidney injury during illness. 14,67,68 For these high-risk patients, individualized treatment decisions regarding the maintenance of RAAS inhibitors that are guided by hemodynamic status, renal function, and clinical stability are recommended. On the basis of the available evidence, we think that, despite the theoretical concerns and uncertainty regarding the effect of RAAS inhibitors on ACE2 and the way in which these drugs might affect the propensity for or severity of Covid-19, RAAS inhibitors should be continued in patients in otherwise stable condition who are at risk for, are being evaluated for, or have Covid-19 (see text box), a position now supported by multiple specialty societies (Table S2). Although additional data may further inform the treatment of high-risk patients with Covid-19, clinicians need to be cognizant of the unintended consequences of prematurely discontinuing proven therapies in response to hypothetical concerns that may be based on incomplete experimental evidence. 69 Key Points Related to the Interplay between Covid-19 and the Renin–Angiotensin–Aldosterone System • ACE2, an enzyme that physiologically counters RAAS activation, is the functional receptor to SARS-CoV-2, the virus responsible for the Covid-19 pandemic • Select preclinical studies have suggested that RAAS inhibitors may increase ACE2 expression, raising concerns regarding their safety in patients with Covid-19 • Insufficient data are available to determine whether these observations readily translate to humans, and no studies have evaluated the effects of RAAS inhibitors in Covid-19 • Clinical trials are under way to test the safety and efficacy of RAAS modulators, including recombinant human ACE2 and the ARB losartan in Covid-19 • Abrupt withdrawal of RAAS inhibitors in high-risk patients, including those who have heart failure or have had myocardial infarction, may result in clinical instability and adverse health outcomes • Until further data are available, we think that RAAS inhibitors should be continued in patients in otherwise stable condition who are at risk for, being evaluated for, or with Covid-19
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              No evidence of rapid antiviral clearance or clinical benefit with the combination of hydroxychloroquine and azithromycin in patients with severe COVID-19 infection

              The COVID-19 epidemic is the worst worldwide pandemic in a century with more than 500,000 cases and 25,000 deaths so far. In France, more than 30,000 cases have been reported up to March 27, and nearly 2000 have died. Pending the availability of a vaccine, there is a critical need to identify effective treatments and a number of clinical trials have been implemented worldwide. Chloroquine analogs have been shown to inhibit the acidification of endosomes and to exhibit in vitro a non-specific antiviral activity at high micromolar concentration against a broad range of emerging virus (HIV, dengue, hepatitis C, chikungunya, influenza, Ebola, SARS and MERS viruses) and more recently COVID-19 [1], [2]. In France, following the results of a clinical study in Marseille, there is considerable interest for the use of hydroxychloroquine to treat COVID-19 disease, and the French Ministry of Health recently allowed the use of hydroxychloroquine to treat COVID-19 disease pending the results of ongoing clinical trials [3]. In their study, Gautret et al. reported a 100% viral clearance in nasopharyngeal swabs in 6 patients after 5 and 6 days of the combination of hydroxychloroquine and azithromycin [3]. This rate of viral clearance was lower with hydroxychloroquine alone (57.1%) and was only 12.5% in patients who did not receive hydroxychloroquine (P  < 0.001). Such a rapid and full viral clearance was quite unexpected and we wished to assess in a prospective study virologic and clinical outcomes of 11 consecutive patients hospitalised in our department who received hydroxychloroquine (600 mg/d for 10 days) and azithromycin (500 mg day 1 and 250 mg days 2 to 5) using the same dosing regimen reported by Gautret et al. [3]. There were 7 men and 4 women with a mean age of 58.7 years (range: 20–77), 8 had significant comorbidities associated with poor outcomes (obesity: 2; solid cancer: 3; hematological cancer: 2; HIV-infection: 1). At the time of treatment initiation, 10/11 had fever and received nasal oxygen therapy. Within 5 days, one patient died, two were transferred to the ICU. In one patient, hydroxychloroquine and azithromycin were discontinued after 4 days because of a prolongation of the QT interval from 405 ms before treatment to 460 and 470 ms under the combination. Mean through blood concentration of hydroxychloroquine was 678 ng/mL (range: 381–891) at days 3–7 after treatment initiation. Repeated nasopharyngeal swabs in 10 patients (not done in the patient who died) using a qualitative PCR assay (nucleic acid extraction using Nuclisens Easy Mag®, Biomerieux and amplification with RealStar SARS CoV-2®, Altona), were still positive for SARS-CoV2 RNA in 8/10 patients (80%, 95% confidence interval: 49–94) at days 5 to 6 after treatment initiation. These virologic results stand in contrast with those reported by Gautret et al. and cast doubts about the strong antiviral efficacy of this combination. Furthermore, in their report Gautret et al. also reported one death and three transfers to the ICU among the 26 patients who received hydroxychloroquine, also underlining the poor clinical outcome with this combination. In addition, a recent study from China in individuals with COVID-19 found no difference in the rate of virologic clearance at 7 days with or without 5 days of hydroxychloroquine, and no difference in clinical outcomes (duration of hospitalisation, temperature normalisation, radiological progression) [4]. These results are consistent with the lack of virologic or clinical benefit of chloroquine in a number of viral infections where it was assessed for treatment or prophylaxis with sometimes a deleterious effect on viral replication [5], [6], [7], [8]. In summary, despite a reported antiviral activity of chloroquine against COVID-19 in vitro, we found no evidence of a strong antiviral activity or clinical benefit of the combination of hydroxychloroquine and azithromycin for the treatment of our hospitalised patients with severe COVID-19. Ongoing randomised clinical trials with hydroxychloroquine should provide a definitive answer regarding the alleged efficacy of this combination and will assess its safety. Ethical Approval All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki declaration and its later amendments. Disclosure of interest The authors declare that they have no competing interest.
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                Author and article information

                Journal
                The Journal of Clinical Pharmacology
                The Journal of Clinical Pharmacology
                Wiley
                0091-2700
                1552-4604
                May 20 2020
                Affiliations
                [1 ]Department of Medical and Toxicological Critical CareLariboisière Hospital, Federation of Toxicology APHPUniversity of Paris INSERM UMRS‐1144 Paris France
                [2 ]Faculty of PharmacyUniversity of Paris INSERM UMRS‐1144 Paris France
                Article
                10.1002/jcph.1646
                a112018f-8559-4835-a33a-7922839c0778
                © 2020

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                http://doi.wiley.com/10.1002/tdm_license_1.1

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