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\begin{document}

\title{Dose Prediction for Repurposing Nitazoxanide in SARS-CoV-2 Treatment or
Chemoprophylaxis}



\author[1]{Rajith Rajoli}%
\author[1]{Henry Pertinez}%
\author[1]{Usman Arshad}%
\author[1]{Helen Box}%
\author[1]{Lee Tatham}%
\author[1]{Paul Curley}%
\author[1]{Megan Neary}%
\author[1]{Joanne Sharp}%
\author[1]{Neill Liptrott }%
\author[1]{Anthony Valentijn}%
\author[1]{Christopher David}%
\author[1]{Steven Rannard}%
\author[2]{Ghaith Aljayyoussi}%
\author[2]{Shaun Pennington}%
\author[1]{Andrew Hill}%
\author[3]{Marta Boffito}%
\author[2]{Steve Ward}%
\author[1]{Saye Khoo}%
\author[4]{Patrick Bray}%
\author[1]{Paul O'Neill}%
\author[1]{W. Hong}%
\author[2]{Giancarlo Biagini}%
\author[1]{Andrew Owen}%
\affil[1]{University of Liverpool}%
\affil[2]{Liverpool School of Tropical Medicine}%
\affil[3]{Chelsea and Westminster Hospital NHS Foundation Trust}%
\affil[4]{Pat Bray Electrical}%


\vspace{-1em}



  
  \date{May 19, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{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 \textgreater{}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.%
\end{abstract}%



\sloppy


\textbf{Dose Prediction for Repurposing Nitazoxanide in SARS-CoV-2
Treatment or Chemoprophylaxis}

Rajith K. R. Rajoli\textsuperscript{1}, Henry
Pertinez\textsuperscript{1}, Usman Arshad\textsuperscript{1}, Helen
Box\textsuperscript{1}, Lee Tatham\textsuperscript{1}, Paul
Curley\textsuperscript{1}, Megan Neary\textsuperscript{1}, Joanne
Sharp\textsuperscript{1}, Neill J. Liptrott\textsuperscript{1}, Anthony
Valentijn\textsuperscript{1}, Christopher David\textsuperscript{1},
Steve P. Rannard\textsuperscript{2}, Ghaith
Aljayyoussi\textsuperscript{3}, Shaun H. Pennington\textsuperscript{3},
Andrew Hill\textsuperscript{1}, Marta Boffito\textsuperscript{4,5},
Stephen A. Ward\textsuperscript{3}, Saye H. Khoo\textsuperscript{1},
Patrick G. Bray\textsuperscript{6}, Paul M. O'Neill\textsuperscript{2},
W. Dave Hong\textsuperscript{2}, Giancarlo Biagini\textsuperscript{3},
Andrew Owen\textsuperscript{1}.

\textsuperscript{1}Department of Molecular and Clinical Pharmacology,
Materials Innovation Factory, University of Liverpool, Liverpool, L7
3NY, UK

\textsuperscript{2}Department of Chemistry, University of Liverpool,
Liverpool, L69 3BX, UK

\textsuperscript{3}Centre for Drugs and Diagnostics, and Department of
Tropical Disease Biology, Liverpool School of Tropical Medicine,
Liverpool L3 5QA, UK

\textsuperscript{4}Chelsea and Westminster NHS Foundation Trust and St
Stephen's AIDS Trust 4th Floor, Chelsea and Westminster Hospital, 369
Fulham Road, London, SW10 9NH, UK

\textsuperscript{5}Jefferiss Research Trust Laboratories, Department of
Medicine, Imperial College, London, W2 1PG, UK

\textsuperscript{6}Pat Bray Electrical, 260D Orrell Road, Orrell, Wigan,
WN5 8QZ, UK

\textbf{Author for correspondence:}

Professor Andrew Owen

Department of Molecular and Clinical Pharmacology

Materials Innovation Factory

University of Liverpool

51 Oxford Street,

Liverpool L7 3NY

United Kingdom

Email - aowen@liverpool.ac.uk

Phone - +44 (0)151 795 7129

\textbf{Key words:} COVID-19, SARS-CoV-2, Coronavirus, Pharmacokinetics,
Lung

\textbf{Conflicts of interest statement}

AO and SPR are Directors of Tandem Nano Ltd. AO has received research
funding from ViiV, Merck, Janssen and consultancy from Gilead, ViiV and
Merck not related to the current paper. PON is currently engaged in a
collaboration with Romark LLC but this interaction did not influence the
prioritisation or conclusions in the current manuscript. No other
conflicts are declared by the authors.

\textbf{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.

\subsection*{Abstract}

{\label{abstract}}

\textbf{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 \emph{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 EC\textsubscript{90}.

\textbf{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
EC\textsubscript{90} in \textgreater{}90\% of the simulated population.
PopDes was used to estimate an optimal sparse sampling strategy for
future clinical trials.

\textbf{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.

\textbf{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.\emph{}

\subsection*{Introduction}

{\label{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 29\textsuperscript{th} 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 \emph{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 EC\textsubscript{50}/EC\textsubscript{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 {[}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 \emph{in vivo} is tizoxanide, which has not yet been
studied for anti-SARS-CoV-2 activity. Notwithstanding, tizoxanide has
been shown to exhibit similar \emph{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 6\textsuperscript{th} 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 \emph{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 (C\textsubscript{trough}) above the reported nitazoxanide
SARS-CoV-2 EC\textsubscript{90} in the majority (\textgreater{}90\%) of
patients.

\subsection*{}

{\label{section}}

\subsection*{Methods}

{\label{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.

\subsubsection*{Model development}

{\label{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 (P\textsubscript{eff}) in humans was
scaled from apparent permeability (P\textsubscript{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 compute the rate of absorption
(K\textsubscript{a} in h\textsuperscript{-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{]}

\subsubsection*{Model validation}

{\label{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
(C\textsubscript{max}), area under the plasma concentration--time curve
(AUC) and C\textsubscript{trough} (trough concentration at the end of
the dosing interval) were less than 2-fold from the mean observed
values.

\subsubsection*{Model simulations}

{\label{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 EC\textsubscript{90}s {[}42,
43{]} for strains (as shown in Supplementary Table 1) included in the
previous trials. This was done to assess the exposure relative
to\emph{in 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\selectlanguage{ngerman}® software and used to calculate a nitazoxanide
EC\textsubscript{90} for SARS-CoV-2 of 4.64 \selectlanguage{greek}µ\selectlanguage{english}M (1.43 mg/L). Optimal
doses were identified such that the concentration at 12 h post-first
dose (C\textsubscript{12}) for BID, 8 h post-first dose
(C\textsubscript{8}) for TID, or 6 h post-first dose
(C\textsubscript{6}) for QID administration were over the recalculated
EC\textsubscript{90} 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.

\textbf{Optimal pharmacokinetic sampling}

Clinical trials should incorporate pharmacokinetic sampling to confirm
tizoxanide plasma exposures, and further validate the predictions from
the PBPK model. Optimal sparse pharmacokinetic timepoint selection
(assuming four blood samples per patient, and 40 patients in the study)
was made on the basis of the prior fed pharmacokinetic data of Stockis
et al. {[}29, 40{]}. Tizoxanide plasma pharmacokinetic data in fed
patients from Stockis et al. was fitted with an empirical
one-compartment disposition model, with first-order absorption and
absorption transit compartment, and the parameters from this fitting
were used (with nominal \%CV interindividual variability in the
pharmacokinetic parameters of 30\%) in the optimal design software
PopDes (University of Manchester Version 4.0) to generate the suggested
optimal sampling timepoints {[}45, 46{]}.

\emph{}

\subsection*{Results}

{\label{results}}

\subsubsection*{Model validation}

{\label{model-validation-1}}

The PBPK model validation against various fasted oral doses is shown in
Figure S1 and the validation against single and multiple doses in the
fed state is shown in Figure S2. The corresponding pharmacokinetic
parameters (AUC, C\textsubscript{max} and C\textsubscript{trough}) are
presented in Table 2 and 3. The AAFE values for the validated doses
ranged between 1.01--1.55 for fasted state and between 1.1--1.58 for fed
state indicating a close match between observed and simulated data. The
ratio between the simulated and the observed pharmacokinetic parameters
-- AUC, C\textsubscript{max} and C\textsubscript{trough} -- was between
0.81--1.54 (Table 2) for fasted state and between 0.67--2.15 for fed
state. The PBPK model simulated tizoxanide plasma concentrations were
within acceptable ranges and therefore the PBPK model was assumed to be
validated.

\subsubsection*{Model simulations}

{\label{model-simulations-1}}

Figure 1a and 1b show the simulated plasma and lung exposures relative
to the average influenza EC\textsubscript{90} after administration of
600 mg BID dose of nitazoxanide with food as reported in the previous
phase 2b/3 trial in uncomplicated influenza {[}24{]}. These simulations
indicate that all patients were predicted to have plasma and lung
tizoxanide C\textsubscript{trough}(C\textsubscript{12}) concentrations
below the average EC\textsubscript{90} (8.4 mg/L, Table S1) {[}43{]},
but that 71\% and 14\% were predicted to have plasma and lung
C\textsubscript{max}concentrations, respectively, above the average
EC\textsubscript{90} for influenza, respectively.

Figure 2a and 2b shows the prediction of trough concentrations in plasma
and lung for the different simulated doses and schedules in healthy
individuals for fasted and fed states, respectively. Doses and schedules
estimated to provide plasma C\textsubscript{trough} concentrations over
1.43 mg/L for at least 50\% of the simulated population were identified.
However, lower doses in each schedule (i.e. 800 mg QID, 1300 mg TID and
1800 mg BID in fasted state and 500 mg QID, 700 mg TID and 1100 mg BID
in fed state) were predicted to result in \textgreater{}40\% of the
simulated population having lung C\textsubscript{trough} below the
SARS-CoV-2 EC\textsubscript{90}. Optimal doses for SARS-CoV-2 in the
fasted state were predicted to be 1200 mg QID, 1600 mg TID and 2900 mg
BID, and in the fed state were 700 mg QID, 900 mg TID and 1400 mg BID.
Figure 3 shows the plasma and lung concentrations for the optimal doses
and schedules in fed state and Figure S3 shows the plasma
concentration--time profile of optimal doses in fasted state. Tizoxanide
concentrations in lung and plasma were predicted to reach steady state
in \textless{}48 h, both in the fasted and fed state.

\subsubsection*{Optimal sparse sampling
design}

{\label{optimal-sparse-sampling-design}}

Results from PopDes optimal design procedure indicate pharmacokinetic
sampling timepoints at 0.25, 1, 3 and 12 h post dose for BID regimens,
and 0.25, 1, 2 and 8 h post dose for TID regimens.\emph{}

\subsection*{Discussion}

{\label{discussion}}

Treatment of SARS-CoV-2 has become a major global healthcare challenge
with no well-defined therapeutic agents to either treat or prevent the
spread of the infection. Short-term treatment options are urgently
required but many ongoing trials are not based upon a rational selection
of candidates in the context of safe achievable drug exposures. In the
absence of a vaccine, there is also an urgent need for chemo
preventative strategies to protect those at high risk such as healthcare
staff, key workers and household contacts who are more vulnerable to
infection. Nitazoxanide has emerged as a potential candidate for
repurposing for COVID-19. The PBPK model presented herein was validated
with an acceptable variation in AAFE and simulated/observed ratio (close
to 1), which provides confidence in the presented predictions. The
present study aimed to define the optimal doses and schedules for
maintaining tizoxanide plasma and lung concentrations above the reported
nitazoxanide EC\textsubscript{90} for the duration of the dosing
interval.

Nitazoxanide was assessed in a double-blind, randomised,
placebo-controlled, phase 2b/3 trial (NCT01227421) of uncomplicated
influenza in 74 primary care clinics in the USA between 27 December 2010
and 30 April 2011 {[}47{]}. The median duration of symptoms for patients
receiving placebo was 117 h compared with 96 h in patients receiving 600
mg BID nitazoxanide with food. Importantly, virus titre in
nasopharyngeal swabs in 39 patients receiving nitazoxanide 600 mg BID
was also lower than in 41 patients receiving placebo. The average of
reported tizoxanide EC\textsubscript{90}s for influenza A and B {[}48{]}
was calculated to be 8.4 mg/L, which is higher than the one reported
EC\textsubscript{90} for nitazoxanide against SARS-CoV-2 {[}44{]}. The
PBPK model was used to simulate plasma and lung exposures after
administration of 600 mg BID for 5 days, and while only plasma
C\textsubscript{max} exceeded the average influenza
EC\textsubscript{90}in the majority of patients, the
C\textsubscript{trough} values did not. The modelling data suggest that
the moderate effects of nitazoxanide seen in influenza could be a
function of underdosing. Taken collectively, these data are encouraging
for the application of nitazoxanide in COVID-19, assuming that
tizoxanide displays anti-SARS-CoV-2 activity comparable to that reported
for nitazoxanide. Moreover, these simulations indicate that higher doses
may be optimal for maximal suppression of pulmonary viruses.

In some cases, food intake may be difficult in patients with COVID-19 so
drugs that can be given without regard for food may be preferred.
However, the presented predictions indicate that optimal plasma and lung
exposures would require 1200 mg QID, 1600 mg TID or 2900 mg BID in the
fasted state. Conversely, the PBPK models predict that doses of 700 mg
QID, 900 mg TID or 1400 mg BID with food provide tizoxanide
concentrations in plasma and lung above the EC\textsubscript{90} value
for nitazoxanide for the entire dosing interval in at least 90\% of the
simulated population. Single doses up to 4000 mg have been administered
to humans previously {[}29{]} but the drug is usually administered at
500 mg BID. The PBPK model simulations indicate a high BID dose of 1400
mg (fed) and caution may be needed for gastrointestinal intolerance at
this dose. The simulations indicate that lower TID and 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.

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 {[}22, 48, 49{]}. Since the SARS-CoV-2
spike protein is also heavily glycosylated {[}50{]} with similar
cellular targets in the upper respiratory tract, a similar mechanism of
action may be expected {[}7, 51{]}. An ongoing trial in Mexico is being
conducted with 500 mg BID nitazoxanide with food {[}28{]} 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 {[}52{]}, and this would not have been
captured in the \emph{in vitro} antiviral activity that informed the
target concentrations for this dose prediction. The simulated population
used in this modelling consisted of healthy individuals up to 60 years
old, but many patients requiring therapy may be older and have
underlying comorbidities. To best knowledge, the impact of renal and
hepatic impairment on pharmacokinetics of this drug have not been
assessed and may impact the pharmacokinetics. Although the current PBPK
model is validated against various single doses in the fasted state and
few multiple doses when given with food, the model may predict with less
accuracy for multiple doses due to the unavailability of clinical data
for multiple dosing over 1000 mg. The presented models were validated
using BID doses only, and confidence in the predictions for TID and QID
doses may be lower. The clinical studies used for model validation were
performed in a limited number of patients {[}29{]} and thus may
underrepresent real inter-subject variability. 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 \emph{in vitro} activity of nitazoxanide against
SARS-CoV-2 {[}44{]} and no data are available for tizoxanide.
Reported\emph{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 {[}17-19, 21, 22{]}.
Finally, none of the reported EC\textsubscript{90} values for influenza
or SARS-CoV-2 were protein binding-adjusted {[}44{]} and tizoxanide is
known to be highly protein bound (\textgreater{}99\%) in plasma
{[}53{]}. Therefore, while the protein binding was used to estimate drug
penetration into the lung, data were not available to correct
the\emph{in vitro} activity.

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.

\textbf{Study} \textbf{Highlights}

\textbf{What is the current knowledge on the topic?}

COVID-19, an acute respiratory infection caused by SARS-CoV-2, has been
declared as a pandemic by the World Health Organization. Several
repurposed drugs are being evaluated but currently there are no robustly
validated treatment or preventative medicines or regimens. Nitazoxanide
has shown \emph{in vitro} activity against SARS-CoV-2, influenza and
several other animal and human RNA viruses.

\textbf{What question did this study address?}

The manuscript describes the pharmacokinetics of tizoxanide, the active
metabolite of nitazoxanide using a physiologically based pharmacokinetic
(PBPK) model. The validated PBPK model was used to estimate the optimal
doses required for SARS-CoV-2 treatment or prevention such that
\textgreater{}90\% of the simulated population would have tizoxanide
concentrations in the plasma and lung above the reported 90\% effective
concentration (EC\textsubscript{90}) value of nitazoxanide for the
entire dosing interval.

\textbf{What does this study add to our knowledge?}

There are no reported studies that identify treatment regimens of
nitazoxanide for SARS-CoV-2 treatment or prevention. The current study
provides evidence-based alternative dosing regimens of nitazoxanide for
SARS-CoV-2 treatment and prevention that exceeds antiviral
EC\textsubscript{90}s in key tissues and organs for the duration of the
dosing interval.

\textbf{How might this change clinical pharmacology or translational
science?}

Efficacy of nitazoxanide has been demonstrated for uncomplicated
influenza and the presented predictions may help inform nitazoxanide
dose selection for COVID-19 clinical trials.

\subsection*{Acknowledgements}

{\label{acknowledgements}}

The authors acknowledge Prof. David Back from the University of
Liverpool for his advice and feedback on this manuscript. The authors
also thank Articulate Science for publication support.

\subsection*{Author Contributions}

{\label{author-contributions}}

All authors wrote the manuscript. A.O. and R.R. designed the research.
U.A., H.B., L.T., H.P. and A.O. performed the research. R.R. and H.P.
analysed the data.

\subsection*{Data availability
statement}

{\label{data-availability-statement}}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

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\textbf{Figure Legends Figure 1} Stimulated plasma (\textbf{a} ) and
lung (\textbf{b} ) concentration for nitazoxanide 600 mg BID for 5 days
with food relative to the average reported tizoxanide
EC\textsubscript{90} value for influenza strains (Table S1) similar to
those in a previous phase 2b/3 trial in uncomplicated influenza
{[}24{]}.

\textbf{Figure 2a} Predicted tizoxanide C\textsubscript{trough} (BID --
12 h, TID -- 8 h, QID -- 6 h) for various dosing regimens of
nitazoxanide in fasted state at the end of the first dose. Data are
presented as mean and error bars represent standard deviation. The
percentages adjacent to the bar chart indicate the percentages of
simulated population over EC\textsubscript{90} of nitazoxanide for
SARS-CoV-2.

\textbf{Figure 2b} Predicted tizoxanide C\textsubscript{trough} (BID --
12 h, TID -- 8 h, QID -- 6 h) for various dosing regimens of
nitazoxanide in fed state at the end of the first dose. Data are
presented as mean and error bars represent standard deviation. The
percentages adjacent to the bar chart indicate the percentages of
simulated population over EC\textsubscript{90} of nitazoxanide for
SARS-CoV-2.

\textbf{Figure 3} Predicted plasma and lung concentrations for optimal
doses during the fed state at different regimens reaching steady state
-- (\textbf{a} ) 1400 mg BID, (\textbf{b} ) 900 mg TID and (\textbf{c} )
700 mg QID. TIZ -- tizoxanide, SD -- standard deviation, solid red line
indicates clinical C\textsubscript{max} of 1 g single dose at fed state
{[}29{]}, solid green line represents clinical C\textsubscript{max} of
500 mg single dose {[}54{]} at fed state and the dotted red line
represents the EC\textsubscript{90} of nitazoxanide for SARS-CoV-2
{[}44{]}.

\textbf{Table Legends}

\textbf{Table 1} Nitazoxanide input parameters for the PBPK model

\textbf{Table 2} Tizoxanide validation against observed data for various
single oral doses in the fasted state

\textbf{Table 3} Tizoxanide validation against observed data for various
single and multiple oral doses when given with food

\subsection*{Table S1 Legend}

{\label{table-s1-legend}}

\textbf{Table S1} Nitazoxanide 50\% and 90 \% effective concentrations
against various influenza virus strains

\subsection*{Figure S1 Legend}

{\label{figure-s1-legend}}

\textbf{Figure S1} Comparison of simulated and observed plasma
concentration--time curve of tizoxanide (TIZ) at fasted state.
(\textbf{a} ) 500 mg, (\textbf{b} ) 1000 mg, (\textbf{c} ) 2000 mg,
(\textbf{d} ) 3000 mg and (\textbf{e} ) 4000 mg.

\subsection*{Figure S2 Legend}

{\label{figure-s2-legend}}

\textbf{Figure S2} Comparison of simulated and observed plasma
concentration--time curve of tizoxanide (TIZ) for single and multiple
dosing regimen (where available) at fed state. (\textbf{a} ) 500 mg,
(\textbf{b} ) 1000 mg, (\textbf{c} ) 2000 mg, (\textbf{d} ) 3000 mg and
(\textbf{e} ) 4000 mg

\subsection*{Figure S3 Legend}

{\label{figure-s3-legend}}

\textbf{Figure S3} Predicted plasma and lung concentrations for optimal
doses during fasted state at different regimens reaching steady state --
(\textbf{a} ) 2900 mg BID, (\textbf{b} ) 1600 mg TID and (\textbf{c} )
1200 mg QID. TIZ -- tizoxanide, SD -- standard deviation, solid red line
indicates clinical C\textsubscript{max} of 1 g single dose at fasted
state {[}29{]}, solid green line represents clinical
C\textsubscript{max} of 500 mg single dose {[}55{]} at fasted state and
the dotted red line represents the EC\textsubscript{90} of nitazoxanide
for SARS-CoV-2 {[}44{]}.\selectlanguage{english}
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