Results
Model validation
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, Cmax and Ctrough) 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, Cmax and Ctrough – 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.
Model simulations
Figure 1a and 1b show the simulated plasma and lung exposures relative
to the average influenza EC90 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 Ctrough(C12) concentrations below the average
EC90 (8.4 mg/L, Table S1) [43], but that 71% and
14% were predicted to have plasma and lung Cmaxconcentrations, respectively, above the average EC90 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 Ctrough 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 >40% of the
simulated population having lung Ctrough below the
SARS-CoV-2 EC90. 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 <48 h, both in the fasted and fed state.
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.
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 EC90 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 EC90s for influenza A and B [48]
was calculated to be 8.4 mg/L, which is higher than the one reported
EC90 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
Cmax exceeded the average influenza EC90in the majority of patients, the Ctrough 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 EC90 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 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 in vitro activity of nitazoxanide against
SARS-CoV-2 [44] and no data are available for tizoxanide. Reportedin 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 EC90 values for influenza or
SARS-CoV-2 were protein binding-adjusted [44] and tizoxanide is
known to be highly protein bound (>99%) in plasma
[53]. Therefore, while the protein binding was used to estimate drug
penetration into the lung, data were not available to correct thein 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.
Study Highlights