Favipiravir pharmacokinetics in immunocompromised infants and children with chronic RNA viral infections

IntroductionFavipiravir selectively inhibits RNA polymerase responsible for single-stranded viral replication. It is licensed for treating influenza and repurposed to treat other diseases such as Ebola and COVID-19. It is metabolised by hepatic aldehyde oxidase (AO) and is an AO inhibitor with complex pharmacokinetics. We have used favipiravir, in combination with other antivirals, in severely immunocompromised children with life-threatening RNA virus infections. As an unlicensed indication, favipiravir pharmacokinetics were routinely monitored at our institution. Population pharmacokinetic model is used to describe the favipiravir pharmacokinetic properties, drug exposure and sources of variability in these children.MethodsRoutine favipiravir plasma levels of 9 patients (0.8–11yrs, mean age=5.3yrs;median weight=15kg) were analysed retrospectively (62 samples). All patients received favipiravir 200mg or 400mg tds and had at least one plasma level 45min (peak), 3h and 8h (trough) post-dose. Parameter estimation and model simulation properties (visual predictive check) were assessed using R language (v 4.1.2) and RStudio (2022.02.0+443).ResultsA one-compartment model with weight as covariate best describes the data, with (1) elimination clearance=1L/h and volume of distribution=7.54L, both allometric scaled centring at median weight, and (2) estimated t1/2=5.17h with Cmax = 24µg/mL at 200mg and 41µg/mL at 400mg.ConclusionsTo our knowledge this is the first report of favipiravir pharmacokinetic parameters in infants and young children. Weight significantly improves the model fit as a covariate. Reported EC50 for norovirus in vitro was 19–39µg/mL and enterovirus 71 was 23µg/mL, indicating higher favipiravir doses or combination with other antivirals are required.

Antiviral efficacy of favipiravir against Zika and SARS-CoV-2 viruses in non-human primates.

The COVID-19 pandemic has exemplified that rigorous evaluation in large animal models is key for translation from promising in vitro results to successful clinical implementation. Among the drugs that have been largely tested in clinical trials but failed so far to bring clear evidence of clinical efficacy is favipiravir, a nucleoside analogue with large spectrum activity against several RNA viruses in vitro and in small animal models. Here, we evaluate the antiviral activity of favipiravir against Zika or SARS-CoV-2 virus in cynomolgus macaques. In both models, high doses of favipiravir are initiated before infection and viral kinetics are evaluated during 7 to 15 days after infection. Favipiravir leads to a statistically significant reduction in plasma Zika viral load compared to untreated animals. However, favipiravir has no effects on SARS-CoV-2 viral kinetics, and 4 treated animals have to be euthanized due to rapid clinical deterioration, suggesting a potential role of favipiravir in disease worsening in SARS-CoV-2 infected animals. To summarize, favipiravir has an antiviral activity against Zika virus but not against SARS-CoV-2 infection in the cynomolgus macaque model. Our results support the clinical evaluation of favipiravir against Zika virus but they advocate against its use against SARS-CoV-2 infection.

Pre-clinical evaluation of antiviral activity of nitazoxanide against SARS-CoV-2.

BACKGROUND: To address the emergence of SARS-CoV-2, multiple clinical trials in humans were rapidly started, including those involving an oral treatment by nitazoxanide, despite no or limited pre-clinical evidence of antiviral efficacy. METHODS: In this work, we present a complete pre-clinical evaluation of the antiviral activity of nitazoxanide against SARS-CoV-2. FINDINGS: First, we confirmed the in vitro efficacy of nitazoxanide and tizoxanide (its active metabolite) against SARS-CoV-2. Then, we demonstrated nitazoxanide activity in a reconstructed bronchial human airway epithelium model. In a SARS-CoV-2 virus challenge model in hamsters, oral and intranasal treatment with nitazoxanide failed to impair viral replication in commonly affected organs. We hypothesized that this could be due to insufficient diffusion of the drug into organs of interest. Indeed, our pharmacokinetic study confirmed that concentrations of tizoxanide in organs of interest were always below the in vitro EC50. INTERPRETATION: These preclinical results suggest, if directly applicable to humans, that the standard formulation and dosage of nitazoxanide is not effective in providing antiviral therapy for Covid-19. FUNDING: This work was supported by the Fondation de France "call FLASH COVID-19", project TAMAC, by "Institut national de la santé et de la recherche médicale" through the REACTing (REsearch and ACTion targeting emerging infectious diseases), by REACTING/ANRS MIE under the agreement No. 21180 ('Activité des molécules antivirales dans le modèle hamster'), by European Virus Archive Global (EVA 213 GLOBAL) funded by the European Union's Horizon 2020 research and innovation program under grant agreement No. 871029 and DNDi under support by the Wellcome Trust Grant ref: 222489/Z/21/Z through the COVID-19 Therapeutics Accelerator".

Hydroxychloroquine and azithromycin used alone or combined are not effective against SARS-CoV-2 ex vivo and in a hamster model.

Drug repositioning has been used extensively since the beginning of the COVID-19 pandemic in an attempt to identify antiviral molecules for use in human therapeutics. Hydroxychloroquine and azithromycin have shown inhibitory activity against SARS-CoV-2 replication in different cell lines. Based on such in vitro data and despite the weakness of preclinical assessment, many clinical trials were set up using these molecules. In the present study, we show that hydroxychloroquine and azithromycin alone or combined does not block SARS-CoV-2 replication in human bronchial airway epithelia. When tested in a Syrian hamster model, hydroxychloroquine and azithromycin administrated alone or combined displayed no significant effect on viral replication, clinical course of the disease and lung impairments, even at high doses. Hydroxychloroquine quantification in lung tissues confirmed strong exposure to the drug, above in vitro inhibitory concentrations. Overall, this study does not support the use of hydroxychloroquine and azithromycin as antiviral drugs for the treatment of SARS-CoV-2 infections.

Favipiravir antiviral efficacy against SARS-CoV-2 in a hamster model.

Despite no or limited pre-clinical evidence, repurposed drugs are massively evaluated in clinical trials to palliate the lack of antiviral molecules against SARS-CoV-2. Here we use a Syrian hamster model to assess the antiviral efficacy of favipiravir, understand its mechanism of action and determine its pharmacokinetics. When treatment is initiated before or simultaneously to infection, favipiravir has a strong dose effect, leading to reduction of infectious titers in lungs and clinical alleviation of the disease. Antiviral effect of favipiravir correlates with incorporation of a large number of mutations into viral genomes and decrease of viral infectivity. Antiviral efficacy is achieved with plasma drug exposure comparable with those previously found during human clinical trials. Notably, the highest dose of favipiravir tested is associated with signs of toxicity in animals. Thereby, pharmacokinetic and tolerance studies are required to determine whether similar effects can be safely achieved in humans.

Preclinical evaluation of Imatinib does not support its use as an antiviral drug against SARS-CoV-2.

Following the emergence of SARS-CoV-2, the search for an effective and rapidly available treatment was initiated worldwide based on repurposing of available drugs. Previous reports described the antiviral activity of certain tyrosine kinase inhibitors (TKIs) targeting the Abelson kinase 2 against pathogenic coronaviruses. Imatinib, one of them, has more than twenty years of safe utilization for the treatment of hematological malignancies. In this context, Imatinib was rapidly evaluated in clinical trials against Covid-19. Here, we present the pre-clinical evaluation of imatinib in multiple models. Our results indicated that imatinib and another TKI, the masitinib, exhibit an antiviral activity in VeroE6 cells. However, imatinib was inactive in a reconstructed bronchial human airway epithelium model. In vivo, imatinib therapy failed to impair SARS-CoV-2 replication in a golden Syrian hamster model despite high concentrations in plasma and in the lung. Overall, these results do not support the use of imatinib and similar TKIs as antivirals in the treatment of Covid-19.

Development and Validation of a Simple and Rapid Ultrahigh-Performance Liquid Chromatography Tandem Spectrometry Method for the Quantification of Hydroxychloroquine in Plasma and Blood Samples in the Emergency Context of SARS-CoV-2 Pandemic.

ABSTRACT: Therapeutic drug monitoring of hydroxychloroquine (HCQ) has been recommended to optimize the treatment of patients with COVID-19. The authors describe an ultrahigh-performance liquid chromatography tandem spectrometry method developed in a context of emergency, to analyze HCQ in both human plasma and blood samples. After adding the labeled internal standard and simple protein precipitation, plasma samples were analyzed using a C18 column. Blood samples required evaporation before analysis. The total chromatographic run time was 4 minutes (including 1.5 minutes of column equilibration). The assay was linear over the calibration range (r2 > 0.99) and up to 1.50 mcg/mL for the plasma samples (5.00 mcg/mL for the blood matrix). The limit of quantification was 0.0150 mcg/mL for plasma samples (0.05 mcg/mL blood matrix) with accuracy and precision ranging from 91.1% to 112% and from 0.750% to 11.1%, respectively. Intraday and interday precision and accuracy values were within 15.0%. No significant matrix effect was observed in the plasma or blood samples. This method was successfully applied to patients treated for COVID-19 infection. A simple and rapid ultrahigh-performance liquid chromatography tandem spectrometry method adapted to HCQ therapeutic drug monitoring in the context of SARS-CoV-2 infection was successfully developed and validated.

SARS-CoV-2 viral dynamics in non-human primates.

Non-human primates infected with SARS-CoV-2 exhibit mild clinical signs. Here we used a mathematical model to characterize in detail the viral dynamics in 31 cynomolgus macaques for which nasopharyngeal and tracheal viral load were frequently assessed. We identified that infected cells had a large burst size (>104 virus) and a within-host reproductive basic number of approximately 6 and 4 in nasopharyngeal and tracheal compartment, respectively. After peak viral load, infected cells were rapidly lost with a half-life of 9 hours, with no significant association between cytokine elevation and clearance, leading to a median time to viral clearance of 10 days, consistent with observations in mild human infections. Given these parameter estimates, we predict that a prophylactic treatment blocking 90% of viral production or viral infection could prevent viral growth. In conclusion, our results provide estimates of SARS-CoV-2 viral kinetic parameters in an experimental model of mild infection and they provide means to assess the efficacy of future antiviral treatments.

Neglecting Plasma Protein Binding in COVID-19 Patients Leads to a Wrong Interpretation of Lopinavir Overexposure.

Boffito et al. recalled the critical importance to correctly interpret protein binding. Changes of lopinavir pharmacokinetics in coronavirus disease 2019 (COVID-19) are a perfect illustration. Indeed, several studies described that total lopinavir plasma concentrations were considerably higher in patients with severe COVID-19 than those reported in patients with HIV. These findings have led to a reduction of the dose of lopinavir in some patients, hypothesizing an inhibitory effect of inflammation on lopinavir metabolism. Unfortunately, changes in plasma protein binding were never investigated. We performed a retrospective cohort study. Data were collected from the medical records of patients hospitalized for COVID-19 treated with lopinavir/ritonavir in intensive care units or infectious disease departments of Toulouse University Hospital (France). Total and unbound concentrations of lopinavir, C reactive protein, albumin, and alpha-1-acid glycoprotein (AAG) levels were measured during routine care on the same samples. In patients with COVID-19, increased total lopinavir concentration is the result of an increased AAG-bound lopinavir concentration, whereas the unbound concentration remains constant, and insufficient to reduce the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) viral load. Although international guidelines have recently recommended against using lopinavir/ritonavir to treat severe COVID-19, the description of lopinavir pharmacokinetics changes in COVID-19 is a textbook case of the high risk of misinterpretation of a total drug exposure when changes in protein binding are not taken into consideration.

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapidly spread around the globe after its emergence in Wuhan in December 2019. With no specific therapeutic and prophylactic options available, the virus has infected millions of people of which more than half a million succumbed to the viral disease, COVID-19. The urgent need for an effective treatment together with a lack of small animal infection models has led to clinical trials using repurposed drugs without preclinical evidence of their in vivo efficacy. We established an infection model in Syrian hamsters to evaluate the efficacy of small molecules on both infection and transmission. Treatment of SARS-CoV-2-infected hamsters with a low dose of favipiravir or hydroxychloroquine with(out) azithromycin resulted in, respectively, a mild or no reduction in virus levels. However, high doses of favipiravir significantly reduced infectious virus titers in the lungs and markedly improved lung histopathology. Moreover, a high dose of favipiravir decreased virus transmission by direct contact, whereas hydroxychloroquine failed as prophylaxis. Pharmacokinetic modeling of hydroxychloroquine suggested that the total lung exposure to the drug did not cause the failure. Our data on hydroxychloroquine (together with previous reports in macaques and ferrets) thus provide no scientific basis for the use of this drug in COVID-19 patients. In contrast, the results with favipiravir demonstrate that an antiviral drug at nontoxic doses exhibits a marked protective effect against SARS-CoV-2 in a small animal model. Clinical studies are required to assess whether a similar antiviral effect is achievable in humans without toxic effects.

Hydroxychloroquine and azithromycin tolerance in haemodialysis patients during COVID-19 infection.

BACKGROUND: Haemodialysis patients are at risk of developing severe forms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection: coronavirus disease 2019 (COVID-19). In March 2020, hydroxychloroquine (HCQ) and azithromycin (AZI) were proposed as potential treatments of COVID-19, but with warnings concerning their possible toxicity. No data are available regarding the toxicity of this treatment in haemodialysis patients. METHODS: We report the use of HCQ and AZI in a cohort of COVID-19 haemodialysis patients with focus on safety concerns. RESULTS: Twenty-one patients received 200 mg HCQ thrice daily during 10 days, and AZI 500 mg on Day 1, and 250 mg on the four following days. HCQ plasma concentrations were within the recommended range (0.1-1.0 µg/mL) in all patients except one, in which maximum concentration was 1.1 µg/mL. HCQ concentration raised until the third day and remained stable thereafter. No cardiac event occurred in spite of progressive lengthening of corrected QT interval (QTc) during the treatment. One patient experienced a long QTc syndrome (QTc >500 ms) without any arrhythmia episode, although HCQ concentration was in the target range. Five (23.8%) patients experienced hypoglycaemia, a well-known HCQ side-effect. SARS-CoV-2 RNA remained detectable in nasopharyngeal swabs for a long time in haemodialysis patients (mean time 21 days). CONCLUSIONS: HCQ and AZI are safe in haemodialysis patients at these doses but can lead to long QTc syndrome and hypoglycaemia. HCQ concentrations were not correlated with side effects. We recommend monitoring of the QTc length throughout treatment, as well as glycaemia. SARS-CoV-2 could persist for longer in haemodialysis patients than in the general population.

Effects of Hydroxychloroquine on Covid-19 in Intensive Care Unit Patients: Preliminary Results.

During the Covid-19 pandemic, many intensive care unit (ICU) patients received hydroxychloroquine. The primary objective of this study was to assess the effects of hydroxychloroquine according to its plasma concentration in ICU patients. A single-center retrospective study was performed from March to April 2020 in an ICU of a university hospital. All patients admitted to the ICU with confirmed Covid-19 pneumonia and treated with hydroxychloroquine were included. The study compared 17 patients in whom the hydroxychloroquine plasma concentration was in the therapeutic target (on-target) and 12 patients in whom the plasma concentration was below the target (off-target). The follow-up of patients was 15 days. No association was found between hydroxychloroquine plasma concentration and viral load evolution (P = 0.77). There was no significant difference between the two groups for duration of mechanical ventilation, length of ICU stay, in-hospital mortality, and 15-days mortality. These findings indicate that hydroxychloroquine administration for Covid-19 patients hospitalized in ICU is not associated with improved outcomes. Larger multicenter studies are needed to confirm these results.

Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates.

Coronavirus disease 2019 (COVID-19) has rapidly become a global pandemic and no antiviral drug or vaccine is yet available for the treatment of this disease1-3. Several clinical studies are ongoing to evaluate the efficacy of repurposed drugs that have demonstrated antiviral efficacy in vitro. Among these candidates, hydroxychloroquine (HCQ) has been given to thousands of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes COVID-19-worldwide but there is no definitive evidence that HCQ is effective for treating COVID-194-7. Here we evaluated the antiviral activity of HCQ both in vitro and in SARS-CoV-2-infected macaques. HCQ showed antiviral activity in African green monkey kidney cells (Vero E6) but not in a model of reconstituted human airway epithelium. In macaques, we tested different treatment strategies in comparison to a placebo treatment, before and after peak viral load, alone or in combination with azithromycin (AZTH). Neither HCQ nor the combination of HCQ and AZTH showed a significant effect on viral load in any of the analysed tissues. When the drug was used as a pre-exposure prophylaxis treatment, HCQ did not confer protection against infection with SARS-CoV-2. Our findings do not support the use of HCQ, either alone or in combination with AZTH, as an antiviral drug for the treatment of COVID-19 in humans.

Concerns about pharmacokinetic (PK) and pharmacokinetic-pharmacodynamic (PK-PD) studies in the new therapeutic area of COVID-19 infection.

In the context of the COVID-19 pandemic, several drugs have been repurposed as potential candidates for the treatment of COVID-19 infection. While preliminary choices were essentially based on in vitro potency, clinical translation into effective therapies may be challenging due to unfavorable in vivo pharmacokinetic properties at the doses chosen for this new indication of COVID-19 infection. However, available pharmacokinetic and pharmacokinetic-pharmacodynamic studies suffer from severe limitations leading to unreliable conclusions, especially in term of dosing optimization. In this paper we propose to highlight these limitations and to identify some of the major requirements that need to be addressed in designing PK and PK-PD studies in this era of COVID. A special attention should be paid to pre-analytical and analytical requirements and to the proper collection of covariates affecting dose-exposure relationships (co-medications, use of specific organ support techniques and other clinical and para-clinical data). We also promote the development of population PK and PK-PD models specifically dedicated to COVID-19 patients since those previously developed for other diseases (SEL, malaria, HIV) and clinical situations (steady-state, non-ICU patients) are not representative of severe patients. Therefore, implementation of well-designed PK and PD studies targeted to COVID-19 patients is urgently needed. For that purpose we call for multi-institutional collaborative work and involvement of clinical pharmacologists in multidisciplinary research consortia.

Potential drug-drug interactions associated with drugs currently proposed for COVID-19 treatment in patients receiving other treatments.

Patients with COVID-19 are sometimes already being treated for one or more other chronic conditions, especially if they are elderly. Introducing a treatment against COVID-19, either on an outpatient basis or during hospitalization for more severe cases, raises the question of potential drug-drug interactions. Here, we analyzed the potential or proven risk of the co-administration of drugs used for the most common chronic diseases and those currently offered as treatment or undergoing therapeutic trials for COVID-19. Practical recommendations are offered, where possible.