key: cord-0847096-hr95yibz authors: Damle, Bharat; Vourvahis, Manoli; Wang, Erjian; Leaney, Joanne; Corrigan, Brian title: Clinical Pharmacology Perspectives on the Antiviral Activity of Azithromycin and Use in COVID‐19 date: 2020-05-12 journal: Clin Pharmacol Ther DOI: 10.1002/cpt.1857 sha: 0f35c86dcb31e45a49a8da2302a2aecada8ab639 doc_id: 847096 cord_uid: hr95yibz Azithromycin (AZ) is a broad‐spectrum macrolide antibiotic with a long half‐life and a large volume of distribution. It is primarily used for the treatment of respiratory, enteric, and genitourinary bacterial infections. AZ is not approved for the treatment of viral infections, and there is no well‐controlled, prospective, randomized clinical evidence to support AZ therapy in coronavirus disease 2019 (COVID‐19). Nevertheless, there are anecdotal reports that some hospitals have begun to include AZ in combination with hydroxychloroquine or chloroquine (CQ) for treatment of COVID‐19. It is essential that the clinical pharmacology (CP) characteristics of AZ be considered in planning and conducting clinical trials of AZ alone or in combination with other agents, to ensure safe study conduct and to increase the probability of achieving definitive answers regarding efficacy of AZ in the treatment of COVID‐19. The safety profile of AZ used as an antibacterial agent is well established.(1) This work assesses published in vitro and clinical evidence for AZ as an agent with antiviral properties. It also provides basic CP information relevant for planning and initiating COVID‐19 clinical studies with AZ, summarizes safety data from healthy volunteer studies, and safety and efficacy data from phase II and phase II/III studies in patients with uncomplicated malaria, including a phase II/III study in pediatric patients following administration of AZ and CQ in combination. This paper may also serve to facilitate the consideration and use of a priori–defined control groups for future research. A single-arm, nonrandomized study in Marseilles, France suggested that hydroxychloroquine (HCQ) alone or in combination with azithromycin (AZ) reduced viral load in coronavirus disease 2019 (COVID-19) patients. 2 AZ was added to prevent bacterial superinfection in a subset of patients, while untreated patients from another center and those refusing treatment served as unmatched controls. At Day 6, 100% of patients (6/6) treated with HCQ and AZ had negative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nasopharyngeal polymerase chain reaction (PCR) test, compared with 57.1% patients (8/14) treated with HCQ alone, and 12.5% controls (2/16) (P < 0.001). The authors concluded in this study that HCQ was associated with viral load reduction and its effect was complemented by AZ. In a separate report (preprint), a subsequent single-arm study from the same center, 80 COVID-19 patients (including 6 patients from the prior study) received HCQ and AZ. A rapid fall in nasopharyngeal viral load tested by quantitative PCR (qPCR) was noted, with 83% negative at Day 7, and 93% at Day 8. Virus cultures from patient respiratory samples were negative in 97.5% of patients at Day 5, which the authors noted was much earlier than untreated patients in prior cases. 3 The authors concluded that HCQ with AZ was potentially effective in reducing transmission and in the therapy of COVID-19. To help determine the validity of these early clinical findings, it is important to understand if AZ demonstrates antiviral properties in vitro and in vivo, and the activity of AZ and HCQ in combination. AZ is a broad-spectrum macrolide antibiotic primarily used for the treatment of respiratory, enteric, and genitourinary bacterial infections and has a well-established safety profile. 1 AZ is indicated for infections caused by susceptible bacterial pathogens in respiratory tract infections such as bronchitis and pneumonia. The minimum inhibitory concentrations for AZ against most of these bacterial pathogens are ≤2.0 mg/L (2.67 µM). 1 The antibacterial mechanism of action of AZ is the binding to the 23S rRNA of the 50S ribosomal subunit of microorganisms, inhibiting bacterial protein synthesis and impeding the assembly of the 50S ribosomal subunit. 1 AZ is not approved for antiviral therapy but has been studied in vitro and in clinical trials for activity against several viruses. This review was undertaken to assess key AZ published data on in vitro antiviral activity and clinical studies across a variety of viral infections to support the design of future controlled studies. Numerous investigations have reported in vitro antiviral activity of AZ against viral pathogens with 50% inhibitory concentrations ranging from ~ 1-6 µM, with the exception of H1N1 influenza In other studies, the calculated in vitro EC 50 for HCQ against SARS-CoV-2 ranged from 0.72 to 17.31 µM at an MOI from 0.01 to 0.8, measured at 48 hours post infection. 5, 6 The selectivity index for HCQ is high, with a reported 50% cytotoxic concentration of 250 µM. 6 In a separate study (preprint), following a 60-hour incubation period, a synergistic effect with the combination HCQ 2 µM + AZ 10 µM was observed in vitro on SARS-CoV-2 at concentrations expected in human lung, leading to total inhibition of viral replication. 7 Caution should be exercised in comparing the EC 50 values across these studies due to the differences in experimental conditions (e.g., different cell lines, MOI, time of drug addition to culture, incubation times, and analytical methods). The precise mechanism is unknown; however, multiple mechanisms have been proposed for the putative antiviral properties observed with AZ. Endosome maturation and function require an acidic environment. AZ is a weak base and preferentially accumulates intracellularly in endosomal vesicles and lysosomes, which could increase pH levels, and potentially block endocytosis and/or viral genetic shedding from lysosomes, thereby limiting viral replication. 15, 16 An acidic environment is also required for the uncoating of enveloped viruses such as influenza and HIV, 17 and a similar mechanism is plausible for coronaviruses, also enveloped viruses. These mechanisms have also been proposed for the antiviral effect noted with HCQ and chloroquine (CQ); 5,6 in fact, evidence suggests that AZ causes a more severe impairment of acidification than CQ. 15 The putative antiviral effects of AZ may also be mediated by a global amplification of the host's interferon (IFN) pathway-mediated antiviral responses. Data suggest that AZ has the ability to induce pattern recognition receptors, IFNs, and IFNstimulated genes, leading to a reduction of viral replication. 8, 14, 18 In addition, AZ directly acts on bronchial epithelial cells to maintain their function and reduce mucus secretion to facilitate lung function. 19 Specific to SARS-CoV-2, recent quantum mechanical modeling suggests a potential role of AZ in interfering with viral entry via binding interaction between the SARS-CoV-2 spike protein and host receptor ACE2 (angiotensin converting enzyme-2) protein; 20 further experimental work on this is necessary to confirm the model. The pharmacokinetics of AZ are well understood. AZ is rapidly absorbed following oral administration, has a long serum half-life (68 hours), 1 and large volume of distribution (31 L/kg). 21 AZ is taken up by leucocytes at concentrations that are about 300-fold higher than plasma. 22 In infected tissues, AZ concentrations are higher than in plasma, due to recruitment of leucocytes at the site of infection. Numerous studies have shown excellent penetration of AZ in a variety of infected tissues, and selected data pertinent to lung penetration are provided in Table 2 . Excellent tissue penetration in the lung allows for the treatment of respiratory infections for indicated bacterial pathogens for which the pharmacokinetic-pharmacodynamic target is linked to area under the curve / minimum inhibitory concentration. The pharmacokinetic-pharmacodynamic target(s) for the potential antiviral activity of AZ is unknown. Hence, for informational purposes only, calculated ratios for maximum concentration (C max ) vs. reported EC 50 for SARS-CoV-2 are presented in Table 2 . As indicated ( Table 2) , lung tissue homogenates and alveolar macrophages have AZ concentrations well in excess of the EC 50 for SAR-CoV-2, as well as for other respiratory viruses listed in Table 1 , following approved doses of AZ. One limitation of these data is that concentrations in lung homogenates may not represent concentrations in infected cells. Once in the lung, concentrations of AZ persist for several days after plasma concentrations become undetectable. 24, 26 The estimated terminal half-life in lung tissue and bronchial washings were 133 and 74 hours, respectively. 23 It is plausible that due to this unique pharmacokinetic property of AZ, coupled with target tissue concentrations in excess of in vitro EC 50 against several viruses, AZ could play a potential therapeutic role in respiratory viral infections, including SARS-CoV-2. Additional considerations for elderly patients may be applicable for COVID-19 infections. As per the product label, AZ exposures in geriatric patients were shown to be similar to those in young adults. In subjects with mild-to-moderate renal impairment, there was little increase in mean C max (5.1%) and AUC (4.2%) following a single 1 g dose of AZ. 1 Dose adjustment is not considered to be required for geriatric patients with normal renal and hepatic function; however, it should be noted that elderly patients may be more susceptible to the development of torsades de pointes. 1 In subjects with severe renal impairment, the mean AUC and C max increased 35% and 61%, respectively, compared with subjects with normal renal function; thus caution should be exercised when dosing AZ in this population. 1 Clinical studies Table 3 summarizes available clinical data on the efficacy of AZ alone, or in combination with other drugs, against various viral infections. With some exceptions, the studies in Table 2 have been observational, single-arm, nonrandomized studies or retrospective evaluations. Many of these studies have reported clinical observations or conducted post hoc analyses. Studies in COVID-19 patients have mainly focused on viral load as an end point, and detailed evaluation of clinical outcomes has not been reported. Notwithstanding the limitations of these studies, collectively they present preliminary evidence that inclusion of AZ in various treatment regimens can influence the course of viral infection and has the potential to influence clinical outcomes. Confirmatory evidence with randomized controlled trials is essential to understand the role of AZ in the treatment of COVID-19. The safety profile of AZ used as an antibacterial agent is well established, and the risks associated with its use are minimized through provision of relevant information in product labeling 1 to support safe use of the product. There have been numerous studies using dosing regimens of AZ and CQ either coadministered as separate tablets (AZ + CQ) or administered as fixed-dose combination tablets (AZCQ). These studies include three phase I studies in healthy adult subjects; nine safety and efficacy phase II or phase II/III studies in adult patients with uncomplicated malaria; a single phase II/III study in pediatric patients with uncomplicated Plasmodium falciparum; and two phase III studies in asymptomatic pregnant women for intermittent preventative treatment of P. falciparum in pregnancy. Details of some of these studies are presented in Table 4 . From these studies, AZ + CQ at doses up to 2,000 mg AZ and 600 mg CQ (base), administered for up to 3 days, was shown to be generally well tolerated, safe in patients with uncomplicated malaria, and safe to be used in different age groups (age range from 18 to > 75 years), including pediatric patients (age range from 6 months to 12 years) and pregnant women. However, at the higher doses (≥1,500 mg) AZ was less well tolerated due to adverse events (AEs) such as vomiting. In the studies in pregnant women, AZCQ combination therapy was less well tolerated than sulfadoxine-pyrimethamine; AEs such as vomiting, dizziness, headache, and asthenia were reported more frequently in the AZCQ treatment group than the sulfadoxine-pyrimethamine group, and serious AEs and discontinuations due to AEs were more frequent in the AZCQ treatment group. In general, the most frequently reported AEs associated with the treatment of AZCQ or AZ + CQ were generally gastrointestinal in nature and included diarrhea, nausea, vomiting, and abdominal pain. Pruritus was also reported, which was considered to be secondary to CQ. Prolonged cardiac repolarization and QT interval, which may impart a risk of torsade de pointes, has been seen in treatment with macrolides, including AZ; CQ is also known to prolong the QT interval. In the studies presented in this document ( Table 4 ), in a total of > 2,000 subjects exposed to 3-day regimens of AZ and CQ combinations, no relevant cardiovascular serious AEs of concern were reported. Available data on the concomitant use of AZ and CQ in these studies indicated no increased risk of QT prolongation above that observed with CQ alone. During drug development, it is essential to demonstrate robust in vitro evidence of activity prior to further study in humans. Subsequently, for a development candidate to have potential to elicit the desired effect over the necessary period of time in vivo, three fundamental "pillars" need to be demonstrated: 33 1. Exposure at the target site of action over a desired period of time 2. Binding to the pharmacological target as expected for its mechanism of action 3. Expression of pharmacological activity commensurate with the demonstrated target exposure and target binding The in vitro evidence presented here suggests that AZ has antiviral properties, including activity against SARS-CoV-2, at concentrations that are physiologically achievable with doses used to treat bacterial infections in the lung. One plausible mechanism for the antiviral properties is the intracellular sequestration of AZ resulting in an increase in endosomal and/or lysosomal pH. Lack of an (X mg/L × (1 mol/749.0 g) × 1,000)/ 2.12 mg/L. 1. 1,000 mg AZ + 600 mg CQ (N = 114) 2. 500 mg AZ + 600 mg CQ (N = 14) b 3. 1,000 mg A + 400 mg P (A-P) (N = 116) One subject in the 1000 mg AZ + CQ treatment group discontinued due to a treatment-related AE of vomiting. The treatment-related AEs most frequently reported by subjects treated with 500 mg AZ + CQ were pruritus (4 subjects; 28.6%), gastritis (1 subject (7.1%)) and mouth ulceration (1 subject (7.1%)); and with 1,000 mg AZ + CQ were pruritus (28 subjects; 24.6%), diarrhea/ loose stools (8 subjects (7.1%)), and paresthesia (6 subjects (5.3%)). Most events were mild to moderate; three treatment-related AEs were assessed as severe: pruritus (1,000 mg AZ + CQ), gastritis (500 mg AZ + CQ), and abdominal pain (A-P). There were no treatment-related SAEs. The incidence of AEs was higher in the AZ combination treatment groups than in the A-P group and was attributed primarily to the incidence of pruritus which is secondary to CQ treatment. A0661134: A phase II/III, randomized, double-blind, comparative trial of AZ plus CQ vs. mefloquine for the treatment of uncomplicated P. falciparum malaria in Africa Adults, aged 18-63 years (AZ + CQ) and 18-68 years (mefloquine) Three treatment groups: 1. 1,000 mg AZ + 600 mg CQ, once daily for 3 days (N = 114) 2. 500 mg AZ + 600 mg CQ, once daily for 3 days (N = 9) b 3. 750 + 500 mg mefloquine on Day 0 (N = 115) Most frequently reported treatment-related AEs with 500 mg AZ + CQ were pruritus (2 subjects (22.2%)), abdominal pain (1 subject (11.1%)), dyspepsia (1 subject (11.1%)), loose stools (1 subject (11.1%)), and vomiting (1 subject (11.1%)); and with 1,000 mg AZ + CQ were pruritus (58 subjects (50.9%)), vomiting (18 subjects (15.8%)), and headache (15 subjects (13.2%)); the majority of AEs were mild. There was one severe treatment-related AE of vomiting in the 1,000 mg AZ + CQ treatment group, and two subjects from this treatment group discontinued the study due to vomiting and vomiting/ dizziness/tinnitus. There were no SAEs which were considered related to AZ + CQ. open label, noncomparative trial of AZ 2,000 mg plus CQ 600 mg base daily for three days for the treatment of uncomplicated P. falciparum malaria Adults, aged 18-77 years 2,000 mg AZ + 600 mg CQ (N = 110), each administered for 3 days Most frequently reported treatment-related AEs were nausea (30.0%), vomiting (18.2%), and diarrhea (11.8%) which were all mild or moderate with the exception of one severe event of vomiting. There were no SAEs or discontinuations due to AEs. Triplicate ECGs were measured on Days 0 (predose), Days 1 and 2 (predose and postdose) and on Days 3 and 7. Mean increases in QTcF from baseline ranged from 12 msec to 49.9 msec and overall, 30 (29%), 6 (6%), and 2 (2%) subjects met the criteria of absolute QTcF values of 450 to < 480 msec, 480 to < 500 msec, and ≥ 500 msec, respectively. The QTcF prolongation observed was consistent with that reported for CQ alone and for AZ + CQ in previous studies. REVIEW optimal acidic environment in the intracellular milieu potentially attenuates viral replication. This mechanism is similar to that proposed for CQ and HCQ, and could explain how two drugs, both weak bases, can act in a complementary manner to inhibit viral replication. In a companion in vitro study by investigators in Marseilles, France, when AZ was dosed in combination with HCQ, 2,3 a synergistic effect was observed in vitro against SARS-CoV-2; however, no EC 50 was determined. 7 The determination of in vitro EC 50 for agents administered alone and in combination against SARS-CoV-2 under similar experimental conditions is needed to further understand the putative antiviral effect of this combination. Other possible mechanisms including the amplification of the host's IFN pathway-mediated antiviral responses as well as AZ's potential to interfere with viral entry requires further experimental work. Drugs known to interact with AZ, HCQ, or CQ are noted in their respective product labels. 1, 34, 35 In a study designed specifically to evaluate interaction between AZ and CQ, drug-drug interactions were not observed 36 and similar results would be expected with HCQ. CQ and HCQ are both substrates and potential inhibitors of P-glycoprotein (P-gp); 37-39 however, given that AZ is not a sensitive substrate of P-gp, 40 potential inhibition of P-gp by HCQ would not be expected to significantly impact the systemic exposure of AZ as observed in the aforementioned study with CQ. Furthermore, CQ and HCQ are metabolized by multiple cytochrome P450 pathways, including cytochrome P450 3A, which AZ has not been shown to substantially modulate. 1, 41, 42 Although AZ has been shown to be an inhibitor of P-gp, 43 AZ is unlikely to affect the lung penetration of HCQ, given that HCQ is highly permeable; thus P-gp efflux would not be expected to be rate limiting. The lung penetration of HCQ in humans has not been reported; however, data in toxicology studies in albino rats, at human-equivalent plasma exposure, suggests HCQ distributes to the lung at concentrations of approximately 92 µM, 44 which is far in excess of its EC 50 values against SARS-CoV-2. 4 A favorable clinical outcome is unlikely without clearance of the pathogen. However, translating the effect on viral (or bacterial) clearance into a clinical outcome in patients is confounded by the disease, variability in patients, design of the studies, and end points measured. This is apparent in the literature on clinical studies and observations with AZ in a variety of viral infections, which present a mixed picture of the utility of AZ dosed alone or with other drugs in the treatment of viral infection. Nonetheless, RNA-sequencing data from the MORDOR II (Macrolides Oraux pour Réduire les Décès avec un Oeil sur la Résistance) study on the reduction in both alpha-coronavirus and beta-coronavirus burden and from the recent studies in COVID-19 patients 3 provides exploratory evidence on AZ, alone or in combination, against SARS-CoV-2, a novel beta-coronavirus. AZ has been reported to exhibit antiinflammatory activity. 45 ,46 These effects are described as an acute phase inhibition of inflammation and a late phase of resolution of chronic inflammation. HCQ also has antiinflammatory properties and is approved for the treatment of lupus erythematosus and rheumatoid arthritis. 34 These effects, while unlikely to contribute to antiviral activity, could ameliorate the inflammatory processes caused by SARS-CoV-2 infection. Furthermore, as bacterial coinfection has been noted in COVID-19 patients, AZ may have a role in treatment of indicated pathogens. Although not an approved indication, the combination of AZ and CQ was well tolerated in healthy subjects and patients infected with malaria. Available data on the concomitant use of AZ and CQ in these studies indicated no increased risk of QT prolongation above that of CQ alone. In a recent preprint, 47 it was reported that in COVID-19 patients (N = 84), 11% of patients treated with an unspecified dosage regimen of HCQ and AZ had recorded QT intervals > 500 msec and 12% of patients had a change from baseline of > 60 msec; there were no events of torsade de pointes recorded. In conclusion, the literature presented here provides a foundation for the study of AZ combined with HCQ in prospective randomized clinical trials or other control methods defined a priori for the treatment of COVID-19 that evaluate clinical outcomes, in addition to reductions in viral burden. As of April 8, 2020, there are 19 studies listed on clinicaltrials.gov (https://clini caltr ials. gov/) using the search terms "azithromycin" and "COVID-19" that will further examine the use of AZ. Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial Clinical and microbiological effect of a combination of hydroxychloroquine and azithromycin in 80 COVID-19 patients with at least a six-day follow up: an observational study