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.