key: cord-291923-jvbehgb7 authors: Rajoli, R. K.; Pertinez, H.; Arshad, U.; Box, H.; Tatham, L.; Curley, P.; Neary, M.; Sharp, J.; Liptrott, N. J.; Valentijn, A.; David, C.; Rannard, S. P.; Aljayyoussi, G.; Pennington, S. H.; Hill, A.; Boffito, M.; Ward, S. A.; Khoo, S. H.; Bray, P. G.; O'Neill, P. M.; Hong, W. D.; Biagini, G.; Owen, A. title: Dose prediction for repurposing nitazoxanide in SARS-CoV-2 treatment or chemoprophylaxis date: 2020-05-06 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2020.05.01.20087130 sha: doc_id: 291923 cord_uid: jvbehgb7 Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared a global pandemic by the World Health Organisation and urgent treatment and prevention strategies are needed. Many clinical trials have been initiated with existing medications, but assessments of the expected plasma and lung exposures at the selected doses have not featured in the prioritisation process. Although no antiviral data is currently available for the major phenolic circulating metabolite of nitazoxanide (known as tizoxanide), the parent ester drug has been shown to exhibit in vitro activity against SARS-CoV-2. Nitazoxanide is an anthelmintic drug and its metabolite tizoxanide has been described to have broad antiviral activity against influenza and other coronaviruses. 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 90% effective concentration (EC90) against SARS-CoV-2. Methods: A whole-body PBPK model was constructed for oral administration of nitazoxanide and validated against available tizoxanide pharmacokinetic data for healthy individuals receiving single doses between 500 mg SARS-CoV-2 4000 mg with and without food. Additional validation against multiple-dose pharmacokinetic data when given with food was conducted. The validated model was then used to predict alternative doses expected to maintain tizoxanide plasma and lung concentrations over the reported nitazoxanide EC90 in >90% of the simulated population. Optimal design software PopDes was used to estimate an optimal sparse sampling strategy for future clinical trials. Results: The PBPK model was validated with AAFE values between 1.01 SARS-CoV-2 1.58 and a difference less than 2-fold between observed and simulated values for all the reported clinical doses. 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, to provide tizoxanide plasma and lung concentrations over the reported in vitro EC90 of nitazoxanide against SARS-CoV-2. For BID regimens an optimal sparse sampling strategy of 0.25, 1, 3 and 12h post dose was estimated. Conclusion: The PBPK model predicted that it was possible to achieve plasma and lung tizoxanide concentrations, using proven safe doses of nitazoxanide, that exceed the EC90 for SARS-CoV-2. The PBPK model describing tizoxanide plasma pharmacokinetics after oral administration of nitazoxanide was successfully validated against clinical data. This dose prediction assumes that the tizoxanide metabolite has activity against SARS-CoV-2 similar to that reported for nitazoxanide, as has been reported for other viruses. The model and the reported dosing strategies provide a rational basis for the design (optimising plasma and lung exposures) of future clinical trials of nitazoxanide in the treatment or prevention of SARS-CoV-2 infection. Introduction 78 79 COVID-19 is a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-80 CoV-2) with noticeable symptoms such as fever, dry cough, and difficulty in breathing. There are 81 currently no effective treatment or prevention options and it has become a global health problem 82 with more than 3.1 million cases and over 217,000 deaths as of 29 th April 2020 [1] . Urgent strategies 83 are required to manage the pandemic and the repurposing of already approved medicines is likely to 84 bring options forward more quickly than full development of potent and specific antivirals. Antiviral 85 drugs may have application prior to or during early infection, but may be secondary to 86 immunological interventions in later stages of severe disease [2] . repurposing exist, including the use of the anti-angiogenic drug thalidomide for cancer and the use 93 of mifepristone for Cushing's disease after initially being approved for termination of early 94 pregnancy [5, 6] . 95 96 SARS-CoV-2 targets the angiotensin-converting enzyme 2 (ACE2) receptors that are present in high 97 density on the outer surface of lung cells. Lungs are the primary site of SARS-CoV-2 replication and 98 infection is usually initiated in the upper respiratory tract [7] . Symptoms that result in neurological, 99 renal and hepatic dysfunction are also emerging due to the expression of ACE2 receptors in these 100 organs [8] [9] [10] [11] . Therefore, therapeutic concentrations of antiviral drugs are likely to be needed in the 101 upper airways for treatment and prevention of infection, but sufficient concentrations are also likely 102 to be required systemically for therapy to target the virus in other organs and tissues. 103 The scale at which antiviral activity of existing medicines are being studied for potential repurposing 105 against SARS-CoV-2 is unprecedented [12] . The authors recently reported a holistic analysis which 106 benchmarked reported in vitro activity of tested drugs against previously published pharmacokinetic 107 exposures achievable with their licenced doses [13] . Importantly, this analysis demonstrated that the 108 majority of drugs that have been studied for anti-SARS-CoV-2 activity are unlikely to achieve the 109 necessary concentrations in the plasma after administration of their approved doses. While this 110 analysis is highly influenced by the drugs selected for analysis to date and highly sensitive to the 111 accuracy of the reported antiviral activity data, a number of candidate agents were identified with 112 plasma exposures above the reported EC50/EC90 against SARS-CoV-2. 113 114 One such drug, nitazoxanide, is a thiazolide antiparasitic medicine used for the treatment of 115 cryptosporidiosis and giardiasis that cause diarrhoea [14, 15] , and also has reported activity against 116 anaerobic bacteria, protozoa and other viruses [16] . Importantly, rapid deacetylation of nitazoxanide 117 in blood means that the major systemic species of the drug in vivo is tizoxanide, which has not yet 118 been studied for anti-SARS-CoV-2 activity. Notwithstanding, tizoxanide has been shown to exhibit 119 similar in vitro inhibitory activity to nitazoxanide for rotaviruses [17], hepatitis B and C viruses [18, 120 19 Model development 160 One hundred virtual healthy adults (50% women, aged 20-60 years between 40-120 kg) were 161 simulated. Patient demographics such as weight, BMI and height were obtained from CDC charts 162 [37] . Organ weight/volumes and blood flow rates in humans were obtained from published 163 literature sources [38, 39] . Transit from the stomach and small intestine was divided into seven 164 compartments to capture effective absorption kinetics as previously described [40] . Tissue to plasma 165 partition ratio of drug and drug disposition across various tissues and organs were described using 166 published mathematical equations [41] [42] [43] . Effective permeability (Peff) in humans was scaled from 167 apparent permeability (Papp) in HT29-19A cells (due to lack of available data, it was assumed the 168 same in Caco-2 cells) using the following equations to compute the rate of absorption (Ka in h -1 ) from 169 the small intestine. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. The corresponding pharmacokinetic parameters (AUC, Cmax and Ctrough) are presented in Table 2a and 222 2b. The AAFE values for the validated doses ranged between 1.01 -1.55 for fasted state and 223 between 1.1 -1.58 for fed state indicating a close match between observed and simulated data. The 224 ratio between the simulated and the observed pharmacokinetic parameters -AUC, Cmax and Ctrough 225 was between 0.81 -1.54 (Table 2a) and schedules estimated to provide plasma Ctrough concentrations over 1.43 mg/L for at least 50% of 239 the simulated population were identified. However, lower doses in each schedule (i.e. 800 mg QID, 240 1300 mg TID and 1800 mg BID in fasted state and 500 mg QID, 700 mg TID, 1100 mg BID in fed state) 241 were predicted to result in >40% of the simulated population having lung Ctrough below the SARS-CoV-242 2 EC90. Optimal doses for SARS-CoV-2 in the fasted state were predicted to be 1200 mg QID, 1600 mg 243 TID, 2900 mg BID and in the fed state were 700 mg QID, 900 mg TID and 1400 mg BID. Figure 3 244 shows the plasma and lung concentrations for the optimal doses and schedules in fed state and 245 supplementary figure 3 shows the plasma concentration -time profile of optimal doses in fasted 246 state. Tizoxanide concentrations in lung and plasma were predicted to reach steady state in <48 247 hours, both in the fasted and fed state. 248 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. * Cmax and AUC0-12h are represented as arithmetic mean ± SD, ^C max and AUC0-24h are represented as geometric mean (mean -SD, mean + SD), † Ctrough is C12 and 256 has been digitised from the pharmacokinetic curve as the geometric mean is not available, arithmetic mean is shown for observed and arithmetic mean 257 (mean -SD, mean + SD) are shown for simulated data, ^C max, AUC0-24h and ^C trough were normalised to a 1000 mg dose. Cmax and AUC are represented as geometric mean (mean -SD, mean + SD) and ^C max and AUC0-24h were normalised to a 1000 mg dose, ^C max and AUC0-24h 260 were normalised to a 1000 mg dose, * AUC is represented as AUC0-∞ after the first dose for single and AUC0-12 on day 7, † Ctrough is C12 has been digitised from 261 the pharmacokinetic curve as the geometric mean is not available, arithmetic mean is shown for observed and arithmetic mean (mean -SD, mean + SD) are 262 shown for simulated data. 263 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020 . . https://doi.org/10.1101 Results from PopDes optimal design procedure indicate pharmacokinetic sampling timepoints at 285 0.25, 1, 3 and 12h post dose for BID regimens, and 0.25, 1, 2 and 8h post dose for TID regimens. 286 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020 . . https://doi.org/10.1101 Discussion 287 Treatment of SARS-CoV-2 has become a major global healthcare challenge with no well-defined 288 therapeutic agents to either treat or prevent the spread of the infection. Short-term treatment 289 options are urgently required but many ongoing trials are not based upon a rational selection of 290 candidates in the context of safe achievable drug exposures. In the absence of a vaccine, there is 291 also an urgent need for chemo preventative strategies to protect those at high risk such as 292 healthcare staff, key workers and household contacts who are more vulnerable to infection. 293 Nitazoxanide has emerged as a potential candidate for repurposing for COVID-19. The PBPK model 294 presented herein was validated with an acceptable variation in AAFE and simulated/observed ratio 295 (close to 1), which provides confidence in the presented predictions. The present study aimed to 296 define the optimal doses and schedules for maintaining tizoxanide plasma and lung concentrations 297 above the reported nitazoxanide EC90 for the duration of the dosing interval. [55] was calculated to be 8.4 mg/L, which is higher than the one reported EC90 for nitazoxanide 306 against SARS-CoV-2 [50]. The PBPK model was used to simulate plasma and lung exposures after 307 administration of 600 mg BID for 5 days, and while only plasma Cmax exceeded the average influenza 308 EC90 in the majority of patients, the Ctrough values did not. The modelling data suggest that the 309 moderate effects of nitazoxanide seen in influenza could be a function of under dosing. Taken 310 collectively, these data are encouraging for the application of nitazoxanide in COVID-19, assuming 311 that tizoxanide displays anti-SARS-CoV-2 activity comparable to that reported for nitazoxanide. 312 Moreover, these simulations indicate that higher doses may be optimal for maximal suppression of 313 pulmonary viruses. 314 In some cases, food intake may be difficult in patients with COVID-19 so drugs that can be given 316 without regard for food may be preferred. However, the presented predictions indicate that optimal 317 plasma and lung exposures would require 1200 mg QID, 1600 mg TID or 2900 mg BID in the fasted 318 state. Conversely, the PBPK models predict that doses of 700 mg QID, 900 mg TID or 1400 mg BID 319 with food provide tizoxanide concentrations in plasma and lung above the EC90 value for 320 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. . https://doi.org/10. 1101 nitazoxanide for the entire dosing interval in at least 90% of the simulated population. Single doses 321 up to 4000 mg have been administered to humans previously [29] but the drug is usually 322 administered at 500 mg BID.. The PBPK model simulations indicate a high BID dose of 1400 mg (fed) 323 and caution may be needed for gastrointestinal intolerance at this dose. The simulations indicate 324 that lower TID and QID dosing regimens may also warrant investigation, and 900 mg TID as well as 325 700 mg QID (both with food) regimen are also predicted to provide optimal exposures for efficacy. 326 Importantly, the overall daily dose was estimated to be comparable between the different optimal 327 schedules and it is unclear whether splitting the dose will provide gastrointestinal benefits. For 328 prevention application where individuals will need to adhere to regimens for longer durations, 329 minimising the frequency of dosing is likely to provide adherence benefits. However, for short term 330 application in therapy, more frequent dosing may be more acceptable to minimise gastrointestinal 331 intolerance. 332 333 Nitazoxanide mechanism of action for SARS-CoV-2 is currently unknown. However, for influenza it 334 has been reported to involve interference with N-glycosylation of hemagglutinin [22, 55, 56] . Since 335 the SARS-CoV-2 spike protein is also heavily glycosylated [57] with similar cellular targets in the 336 upper respiratory tract, a similar mechanism of action may be expected [7, 58] . An ongoing trial in 337 Mexico, is being conducted with 500 mg BID nitazoxanide with food [28] but these doses may not be 338 completely optimal for virus suppression across the entire dosing interval. 339 340 This analysis provides a rational dose optimisation for nitazoxanide for treatment and prevention of 341 COVID-19. However, there are some important limitations that must be considered. PBPK models 342 can be useful in dose prediction but the quality of predictions is only as good as the quality of the 343 available data on which they are based. Furthermore, the mechanism of action for nitazoxanide for 344 other viruses has also been postulated to involve an indirect mechanism through amplification of the 345 host innate immune response [59] , and this would not have been captured in the in vitro antiviral 346 activity that informed the target concentrations for this dose prediction. The simulated population 347 used in this modelling consisted of healthy individuals up to 60 years old, but many patients 348 requiring therapy may be older and have underlying comorbidities. To best knowledge, the impact of 349 renal and hepatic impairment on pharmacokinetics of this drug have not been assessed and may 350 impact the pharmacokinetics. Although the current PBPK model is validated against various single 351 doses in the fasted state and few multiple doses when given with food, the model may predict with 352 less accuracy for multiple doses due to the unavailability of clinical data for multiple dosing over 353 1000 mg. The presented models were validated using BID doses only, and confidence in the 354 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. . predictions for TID and QID doses may be lower. The clinical studies used for model validation were 355 performed in a limited number of patients [29] and thus may underrepresent real inter-subject 356 variability. Also, the disposition parameters (apparent clearance and rate of absorption) obtained for 357 the PBPK model were from a fasted study of 500 mg BID, and the parameters were adjusted to 358 validate the tizoxanide model in the fed state, which may limit confidence in the model at higher 359 doses. Only one manuscript has described the in vitro activity of nitazoxanide against SARS-CoV-2 360 [50] and no data are available for tizoxanide. Reported in vitro data may vary across laboratories and 361 due to this the predicted optimal doses may change. However, the reported comparable activity of 362 nitazoxanide and tizoxanide against a variety of other viruses (including other coronaviruses) does 363 strengthen the rational for investigating this drug for 21, 22] . Finally, none of the 364 reported EC90 values for influenza or SARS-CoV-2 were protein binding-adjusted [50] and tizoxanide 365 is known to be highly protein bound (>99%) in plasma [32] . Therefore, while the protein binding was 366 used to estimate drug penetration into the lung, data were not available to correct the in vitro 367 activity. 368 In summary, the developed PBPK model of nitazoxanide was successfully validated against clinical 370 data and based on currently available data, optimal doses for COVID-19 were estimated to be 700 371 mg QID, 900 mg TID or 1400 mg BID with food. Should nitazoxanide be progressed into clinical 372 evaluation for treatment and prevention of COVID-19, it will be important to further evaluate the 373 pharmacokinetics in these population groups. In treatment trials particularly, intensive 374 pharmacokinetic sampling may be challenging. Therefore, an optimal sparse sampling strategy for 375 BID, TID and QID dosing is also presented. 376 377 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. . https://doi.org/10.1101/2020.05.01.20087130 doi: medRxiv preprint 566 567 C) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 6, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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