Funding
The authors received no funding for the current work. AO acknowledges research funding from EPSRC (EP/R024804/1; EP/S012265/1), NIH (R01AI134091; R24AI118397), European Commission (761104) and Unitaid (project LONGEVITY). GAB acknowledges support from the Medical Research Council (MR/S00467X/1). GA acknowledges funding from the MRC Skills Development Fellowship.

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a global pandemic and urgent treatment and prevention strategies are needed. Nitazoxanide, an anthelmintic drug has been shown to exhibit in vitro activity against SARS-CoV-2. The present study used physiologically-based pharmacokinetic (PBPK) modelling to inform optimal doses of nitazoxanide capable of maintaining plasma and lung tizoxanide exposures above the reported nitazoxanide SARS-CoV-2 EC90.
Methods: A whole-body PBPK model was validated against available pharmacokinetic data for healthy individuals receiving single and multiple doses between 500–4000 mg with and without food. The validated model was used to predict doses expected to maintain tizoxanide plasma and lung concentrations above the nitazoxanide EC90 in >90% of the simulated population. PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials.
Results: The PBPK model was successfully validated against the reported human pharmacokinetics. The model predicted optimal doses of 1200 mg QID, 1600 mg TID, 2900 mg BID in the fasted state and 700 mg QID, 900 mg TID and 1400 mg BID when given with food. For BID regimens an optimal sparse sampling strategy of 0.25, 1, 3 and 12h post dose was estimated.
Conclusion: The PBPK model predicted tizoxanide concentrations within doses of nitazoxanide already given to humans previously. The reported dosing strategies provide a rational basis for design of clinical trials with nitazoxanide for the treatment or prevention of SARS-CoV-2 infection.

Introduction

COVID-19 is a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with noticeable symptoms such as fever, dry cough, and difficulty in breathing. There are currently no effective treatment or prevention options and it has become a global health problem with more than 3.1 million cases and over 217,000 deaths as of 29th April 2020 [1]. Urgent strategies are required to manage the pandemic and the repurposing of already approved medicines is likely to bring options forward more quickly than full development of potent and specific antivirals. Antiviral drugs may have application prior to or during early infection, but may be secondary to immunological interventions in later stages of severe disease [2].
Although new chemical entities are likely to have high potency and specificity for SARS-CoV-2, full development is time consuming, costly and attrition in drug development is high [3, 4]. Drug repurposing, where existing or investigational drugs could be used outside the scope of their original indication may present a rapid alternative to new drug development. Several examples of successful repurposing exist, including the use of the anti-angiogenic drug thalidomide for cancer and the use of mifepristone for Cushing’s disease after initially being approved for termination of early pregnancy [5, 6].
SARS-CoV-2 targets the angiotensin-converting enzyme 2 (ACE2) receptors that are present in high density on the outer surface of lung cells. Lungs are the primary site of SARS-CoV-2 replication and infection is usually initiated in the upper respiratory tract [7]. Symptoms that result in neurological, renal and hepatic dysfunction are also emerging due to the expression of ACE2 receptors in these organs [8-11]. Therefore, therapeutic concentrations of antiviral drugs are likely to be needed in the upper airways for treatment and prevention of infection, but sufficient concentrations are also likely to be required systemically for therapy to target the virus in other organs and tissues.
The scale at which antiviral activity of existing medicines is being studied for potential repurposing against SARS-CoV-2 is unprecedented [12]. The authors recently reported a holistic analysis which benchmarked reported in vitro activity of tested drugs against previously published pharmacokinetic exposures achievable with their licenced doses [13]. Importantly, this analysis demonstrated that the majority of drugs that have been studied for anti-SARS-CoV-2 activity are unlikely to achieve the necessary concentrations in the plasma after administration of their approved doses. While this analysis is highly influenced by the drugs selected for analysis to date and highly sensitive to the accuracy of the reported antiviral activity data, a number of candidate agents were identified with plasma exposures above the reported EC50/EC90 against SARS-CoV-2.
One such drug, nitazoxanide, is a thiazolide antiparasitic medicine used for the treatment of cryptosporidiosis and giardiasis that cause diarrhoea [14, 15], and also has reported activity against anaerobic bacteria, protozoa and other viruses [16]. Importantly, rapid deacetylation of nitazoxanide in blood means that the major systemic species of the drug in vivo is tizoxanide, which has not yet been studied for anti-SARS-CoV-2 activity. Notwithstanding, tizoxanide has been shown to exhibit similar in vitro inhibitory activity to nitazoxanide for rotaviruses [17], hepatitis B and C viruses [18, 19], other coronaviruses, noroviruses [20] and influenza viruses [21, 22]. As another respiratory virus, previous work on influenza may be useful to gain insight into the expected impact of nitazoxanide for SARS-CoV-2. Accordingly, the drug has been shown to selectively block the maturation of the influenza haemagglutinin glycoprotein at the post-translational stage [22, 23] and a previous phase 2b/3 trial demonstrated a reduction in symptoms and viral shedding at a dose of 600 mg BID compared to placebo in patients with uncomplicated influenza [24]. Other potential benefits of nitazoxanide in COVID-19 may derive from its impact upon the innate immune response that potentiates the production of type 1 interferons [25, 26] and bronchodilation of the airways through inhibition of TMEM16A ion channels [27]. A clinical trial (NCT04341493) started on 6th April 2020 and aims to evaluate the activity of 500 mg BID nitazoxanide alone or in combination with the 4-aminoquinoline hydroxychloroquine against SARS-CoV-2 [28]. However, there are currently no data within the public domain to support this dose selection for COVID-19. Nitazoxanide is relatively safe in humans and studies showed tolerability of single oral doses up to 4 g with minimal gastrointestinal side effects. Plasma concentrations of tizoxanide have demonstrated dose proportionality, but administration in the fed state increases the plasma exposure [29]. Thus, the drug is recommended for administration with food.
Physiologically based pharmacokinetic (PBPK) modelling is a computational tool that integrates human physiology and drug disposition kinetics using mathematical equations to inform the pharmacokinetic exposure using in vitro and drug physicochemical data [30]. The aim of this study was to validate a PBPK model for tizoxanide following administration of nitazoxanide. Once validated, this model was first used to assess the plasma and lung exposures estimated to be achieved during a previous trial for uncomplicated influenza. Next, different nitazoxanide doses and schedules were simulated to identify those expected to provide tizoxanide plasma and lung trough concentrations (Ctrough) above the reported nitazoxanide SARS-CoV-2 EC90 in the majority (>90%) of patients.

Methods

A whole-body PBPK model consisting of compartments to represent select organs and tissues was developed. Nitazoxanide physiochemical and drug-specific parameters used in the PBPK model were obtained from literature sources as outlined in Table 1. The PBPK model was assumed to be blood-flow limited, with instant and uniform distribution in each tissue or organ and no reabsorption from the large intestine. Since the data are computer generated, no ethics approval was required for this study.

Model development

One hundred virtual healthy adults (50% women, aged 20–60 years between 40–120 kg) were simulated. Patient demographics such as weight, BMI and height were obtained from CDC charts [31]. Organ weight/volumes and blood flow rates in humans were obtained from published literature sources [32, 33]. Transit from the stomach and small intestine was divided into seven compartments to capture effective absorption kinetics as previously described [34]. Tissue to plasma partition ratio of drug and drug disposition across various tissues and organs were described using published mathematical equations [35-37]. Effective permeability (Peff) in humans was scaled from apparent permeability (Papp) in HT29-19A cells (due to lack of available data, it was assumed the same in Caco-2 cells) using the following equations to compute the rate of absorption (Ka in h-1) from the small intestine.
\({\operatorname{}P}_{\text{eff}}\ =\ 0.6836\times\operatorname{}P_{\text{app}}-0.5579\)[38]
\(K_{a}\ =\frac{2\times P_{\text{eff}}\times 60\times 60}{r}\)[39]

Model validation

The PBPK model was validated against available clinical data in healthy individuals in the fed and fasted state for various single oral doses of nitazoxanide ranging from 500 mg to 4000 mg [29, 40], and for multiple dosing at 500 mg and 1000 mg BID with food. Nitazoxanide absorption was considered using the available apparent permeability data (shown in Table 1) and tizoxanide was assumed to form as soon as the drug reached systemic circulation as metabolic studies have shown it takes just 6 minutes for complete conversion into the active circulating metabolite, with no trace of nitazoxanide detected in plasma [41]. Therefore, tizoxanide parameters were used to define drug disposition. The model was assumed to be validated if: 1) the absolute average fold error (AAFE) between the observed and the simulated plasma concentrations–time curve of tizoxanide was less than two; and 2) the simulated pharmacokinetic parameters–maximum concentration (Cmax), area under the plasma concentration–time curve (AUC) and Ctrough (trough concentration at the end of the dosing interval) were less than 2-fold from the mean observed values.

Model simulations

The pharmacokinetics following administration of 600 mg BID as reported in a previous phase 2b/3 clinical trial of nitazoxanide in uncomplicated influenza [24] were first simulated and plotted relative to the average of previously reported influenza EC90s [42, 43] for strains (as shown in Supplementary Table 1) included in the previous trials. This was done to assess the exposure relative toin vitro activity for an indication where clinical benefit was already demonstrated.
For potential SARS-CoV-2 applications, several oral dosing regimens were simulated for BID, TID or QID administration in the fasted state. Antiviral activity data from Wang et al. [44] were digitised using Web Plot Digitiser® software and used to calculate a nitazoxanide EC90 for SARS-CoV-2 of 4.64 µM (1.43 mg/L). Optimal doses were identified such that the concentration at 12 h post-first dose (C12) for BID, 8 h post-first dose (C8) for TID, or 6 h post-first dose (C6) for QID administration were over the recalculated EC90 for nitazoxanide. Plasma and lung tizoxanide exposures at these doses and schedules are reported in addition to plasma–time curves. The doses were optimised using tizoxanide parameters and pharmacokinetics; however, the doses were reported for nitazoxanide.