Dose prediction for repurposing nitazoxanide in SARS‐CoV‐2 treatment or chemoprophylaxis

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 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 and 4000 mg with and without food. The validated model was used to predict doses expected to maintain tizoxanide plasma and lung concentrations above the 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 and 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 12 hours 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. A concordant higher dose of nitazoxanide is now planned for investigation in the seamless phase I/IIa AGILE trial.


| 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. 1 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 29 April 2020. 2 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. 3 Although new chemical entities are likely to have high potency and specificity for SARS-CoV-2, full development is time-consuming and costly, and attrition in drug development is high. 4 repurposing exist, including the use of the anti-angiogenic drug thalidomide for cancer, the use of mifepristone for Cushing's disease after initially being approved for termination of early pregnancy and the repurposing of sildenafil from angina to erectile disfunction. 6,7 It should be noted, however, that drug repurposing is considerably faster when the approved dose is successfully repurposed, with additional complexity in clinical development when higher doses are required.
SARS-CoV-2 targets the angiotensin-converting enzyme 2 (ACE2) receptors that are present in high density on the outer surface of lung cells. 1 Lungs are the primary site of SARS-CoV-2 replication and infection is usually initiated in the upper respiratory tract. 8 Symptoms that result in neurological, renal and hepatic dysfunction are also emerging due to the expression of ACE2 receptors in these organs. [9][10][11][12] 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 the antiviral activity of existing medicines is being studied for potential repurposing against SARS-CoV-2 is unprecedented. 13 The authors recently reported an analysis which benchmarked reported in vitro activity of tested drugs against previously published pharmacokinetic exposures achievable with their licenced doses. 14 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 EC 50 /EC 90 against SARS-CoV-2.
One such drug, nitazoxanide, is a thiazolide antiparasitic medicine used for the treatment of cryptosporidiosis and giardiasis that cause diarrhoea, 15,16 and also has reported activity against anaerobic bacteria, protozoa and other viruses. 17 Several reports have confirmed the activity of nitazoxanide against SARS-CoV-2 in different cell types. 18 20,[26][27][28] 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 27,29 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. 30 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 27,31 and bronchodilation of the airways through inhibition of TMEM16A ion channels. 32 As of 9 August 2020, a total of 19 trials were listed as either planned or recruiting on clinicaltrials.gov but all of these studies are focusing on doses of ≤1000 mg BID nitazoxanide either alone or in combination with\ other agents. 33 However, there are currently no data within the public domain to support these doses for COVID-19.
Nitazoxanide is relatively safe in humans and a review of the safety and minimum pricing was recently published. 34 Plasma concentrations of tizoxanide have demonstrated dose proportionality, but administration in the fed state increases the plasma exposure. 35 Thus, the drug is recommended for administration with food.
The prerequisites for successful development of antiviral drugs for SARS-CoV-2 have yet to be elucidated and gaps in knowledge exist in terms of the exposure-response relationship. However, the lung has emerged as a clear site of primary infection, and pulmonary co-morbidities are a key driver of mortality in the sickest patients. [36][37][38] Therefore, in treatment of early disease at least it seems likely that successful antiviral regimens will require drugs to penetrate into the lung at sufficient concentrations to exert their activity. Using HIV as a paradigm for successful chemoprophylactic approaches, antiviral drugs also require penetration into key sites of transmission such as the anal and vaginal mucosa. [39][40][41] Therefore, the authors hypothesise that drugs achieving concentrations in lung that exceed those needed for activity will underpin successful antiviral development.
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. 42 Recently, several international groups have called for a more robust integration of clinical pharmacology principles into COVID-19 drug development. 43,44 Accordingly, 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 (C trough ) above the reported nitazoxanide SARS-CoV-2 EC 90 in the majority (>90%) of patients.

| METHODS
A previously published whole-body PBPK model consisting of compartments to represent select organs and tissues developed in Simbiology (MATLAB R2019a, MathWorks Inc., Natick, MA, USA) was used in this study. 45,46 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 and 120 kg) were simulated. The required duration for successful SARS-CoV-2 antiviral activity has not yet been robustly elucidated but for clarity in presentation, the simulations were conducted over 5 days of dosing. It should be noted that similar exposures would be expected beyond this once the drug has reached steady-state pharmacokinetics. Patient demographics such as weight, body mass index and height were obtained from CDC charts. 47 Organ weight/volumes and blood flow rates in humans were obtained from published literature sources. 48,49 Transit from the stomach and small intestine was divided into seven compartments to capture effective absorption kinetics as previously described. 50 Tissue to plasma partition ratio of drug and drug disposition across various tissues and organs were described using published mathematical equations. [51][52][53] Effective permeability (P eff ) in humans was scaled from apparent permeability (P app ) in HT29-19A cells (due to lack of available data, it was assumed the same in Caco-2 cells) using the following equations to T A B L E 1 Nitazoxanide input parameters for the PBPK model
a Protein binding was considered as 99% for the PBPK model.
compute the rate of absorption (K a in h −1 ) from the small intestine. 54,55 log 10 P eff = 0:6836 × log 10 P app −0:5579 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 to 4000 mg, 35,56 and for multiple dosing at 500 and 1000 mg BID with food.
Nitazoxanide absorption was considered using the available apparent permeability data (shown in Table 1 and C trough (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 30      AUC is represented as AUC 0-∞ after the first dose for single and AUC 0-12 on day 7. b C trough is C 12 and has been digitised from the pharmacokinetic curve as the geometric mean is not available. The arithmetic mean is shown for observed and arithmetic mean (mean -SD, mean + SD) is shown for simulated data. c C max and AUC are represented as geometric mean (mean -SD, mean + SD) and C max and AUC 0-24h were normalised to a 1000 mg dose. C max and AUC 0-12h are represented as arithmetic mean ± SD. b C trough is C 12 and has been digitised from the pharmacokinetic curve as the geometric mean is not available. The arithmetic mean is shown for observed and arithmetic mean (mean -SD, mean + SD) is shown for simulated data. c C max and AUC 0-24h are represented as geometric mean (mean -SD, mean + SD). C max , AUC 0-24h and C trough were normalised to a 1000 mg dose.

| Model simulations
BID dose of nitazoxanide with food as reported in the previous phase 2b/3 trial in uncomplicated influenza. 30 These simulations indicate that all patients were predicted to have plasma and lung tizoxanide C trough (C 12 ) concentrations below the average EC 90 (8.4 mg/L, Supporting Information Table S1), 59 but that 71% and 14% were predicted to have plasma and lung C max concentrations, respectively, above the average EC 90 for influenza, respectively.  Figure 3 shows the plasma and lung concentrations for the optimal doses and schedules in the fed state and Supporting Information Figure S3 shows the plasma concentrationtime profile of optimal doses in the fasted state. Tizoxanide concentrations in lung and plasma were predicted to reach steady state in <48 hours, in both the fasted and fed stated.
F I G U R E 1 Simulated plasma A, and lung B, concentrations for nitazoxanide 600 mg BID for 5 days with food relative to the average reported tizoxanide EC 90 value for influenza strains (Supporting Information Table S1)    QID dosing regimens may also warrant investigation, and 900 mg TID as well as 700 mg QID (both with food) regimens are also predicted to provide optimal exposures for efficacy. Importantly, the overall daily dose was estimated to be comparable between the different optimal schedules and it is unclear whether splitting the dose will provide gastrointestinal benefits. For prevention application where individuals will need to adhere to regimens for longer durations, minimising the frequency of dosing is likely to provide adherence benefits. However, for short-term application in therapy, more frequent dosing may be more acceptable to minimise gastrointestinal intolerance.
The nitazoxanide mechanism of action for SARS-CoV-2 is currently unknown. However, for influenza it has been reported to involve interference with N-glycosylation of haemagglutinin. 27,63,64 Since the SARS-CoV-2 spike protein is also heavily glycosylated 65 with similar cellular targets in the upper respiratory tract, a similar mechanism of action may be expected. 8,66 An ongoing trial in Mexico is being conducted with 500 mg BID nitazoxanide with food, 33 but these doses may not be completely optimal for virus suppression across the entire dosing interval.
This analysis provides a rational dose optimisation for nitazoxanide for treatment and prevention of COVID-19. However, there are some important limitations that must be considered. PBPK models can be useful in dose prediction but the quality of predictions is only as good as the quality of the available data on which they are based. Furthermore, the mechanism of action for nitazoxanide for other viruses has also been postulated to involve an indirect mechanism through amplification of the host innate immune response, 67  Also, the disposition parameters (apparent clearance and rate of absorption) obtained for the PBPK model were from a fasted study of 500 mg BID and the parameters were adjusted to validate the tizoxanide model in the fed state, which may limit confidence in the model at higher doses. Only one manuscript has described the in vitro activity of nitazoxanide against SARS-CoV-2 18 and no data are available for tizoxanide. Reported in vitro data may vary across laboratories and due to this the predicted optimal doses may change.
However, the reported comparable activity of nitazoxanide and tizoxanide against a variety of other viruses (including other coronaviruses) does strengthen the rationale for investigating this drug for COVID-19. [22][23][24]26,27 Recently, several international investigators with experience of protein binding and its application in successful therapy for other viruses initiated a discussion about protein binding to reach consensus on its correct interpretation for SARS-CoV-2. 68 As an outcome of this consensus, care should be taken to neither overnor under-represent its consequences. Unfortunately, assessment of the consequences of protein binding needs to involve empirical determination as part of the in vitro methodology and none of the reported EC 90 values for influenza or SARS-CoV-2 were protein bindingadjusted. 18 Tizoxanide is known to be highly protein bound (>99%) in plasma, 69 but while this was used to estimate drug penetration into the lung, data were not available to correct the in vitro activity to make a robust assessment in relation to the free drug pharmacokinetics.
The doses estimated to be necessary to maintain active tizoxanide concentrations in plasma and lung are considerably higher than the approved dose (500 mg BID) or other multiple dose studies that have been published to date. However, single doses of up to 4000 mg have been given safely to humans previously, and several of the authors recently reviewed the safety of nitazoxanide across the different doses at which it has been studied. 34,35 Nitazoxanide appears to be a remarkably safe drug but the major concerns are likely to relate to gastrointestinal safety. Accordingly, the doses proposed here will require a clinical development pathway that robustly addresses safety. As a first step, Unitaid have recently agreed funding and an independent scientific advisory board has approved inclusion of high-dose nitazoxanide in the seamless phase I/IIa AGILE platform trial (www.agiletrial.net), subject to successful relevant ethical and regulatory approvals.
In summary, the developed PBPK model of nitazoxanide was successfully validated against clinical data and based on currently available data optimal doses for COVID-19 were estimated to be 700 mg QID, 900 mg TID or 1400 mg BID with food. Should nitazoxanide be progressed into clinical evaluation for treatment and prevention of COVID-19, it will be important to further evaluate the pharmacokinetics in these population groups. In treatment trials particularly, intensive pharmacokinetic sampling may be challenging. Therefore, an optimal sparse sampling strategy for BID, TID and QID dosing is also presented.