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