Pharmacokinetics of nitazoxanide in plasma separation cards from patients with SARS‐CoV‐2: AGILE CST‐3a/b

27 Background: Nitazoxanide (NTZ) is an anti-parasitic which has demonstrated inhibitory activity against a range of viruses, including in vitro inhibition of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Liquid plasma (L-PL) collection in resource-limited settings (RLS), where high-consequence infections are endemic, can be challenging. HemaSep dried blood spots (HS-DBS) offer a useful alternative to L-PL, separating plasma upon contact as a distinct outer ring (HS-PL) and can be shipped at ambient temperatures. HS-DBS were collected alongside L-PL in the AGILE CST-3a/3b clinical trials to evaluate the pharmacokinetics of single and repeat doses of NTZ in healthy participants (CST-3a) and patients with SARS-CoV-2 (CST-3b). Materials and methods: AGILE-CST-3a and 3b were open-label single-arm phase Ia/b trials with the aim of determining the optimal dose for treatment of symptomatic SARS-CoV-2 patients (CST-3a; n = 14, CST-3b; n = 8). Participants in CST-3b, with confirmed SARS-CoV-2 infection, were randomized 2:1 to receive NTZ or standard of care. NTZ was administered twice daily (BD) at 1500 mg for 5 days. Paired L-PL and HS-DBS were collected over 0-8 hr post-dose (CST-3a) and 0–4 h post-dose (CST-3b) following administration of single (day 1) and multiple doses (day 5). About 100 μL of whole blood was applied to HemaSep cards. HS-PL (entire outer plasma ring) and L-PL samples were extracted with 0.1% formic acid in acetonitrile. Concentrations of NTZ metabolite tizoxanide (TZN) were quantified using LC–MS (HS-PL: 2.35–1960 ng/sample, L-PL: 10–10 000 ng/mL). HS-PL values were normalized to ng/mL and pharmacokinetic parameters (Cmax, AUClast) derived using non-compartmental analysis. Linear regression and Bland Altman plots were used to compare methods. Results: TZN L-PL geometric mean Cmax increased with repeated dosing (Day 1|Day 5) - CST-3a: 14860.8 ng/mL |20342.8 ng/mL; CST-3b: 10946.5 ng/mL |17678.3 ng/mL). HS-PL Cmax also displayed similar increases in concentration - CST-3a: 18206 ng/mL |25479.1 ng/mL; CST-3b: 12073.2 ng/mL|23434.9 ng/mL. Geometric mean ratios (GMR [95% CI]) indicate that differences between Days 1 and 5 were significant for both L-PL (CST3a: (0.68 [0.53, 0.85]), CST3b: (0.74 [0.64, 0.85]) and HS-PL (CST3a: (0.68[0.50, 0.93]), CST3b: (0.68 [0.58, 0.79]). The mean difference between HS-PL and L-PL TZN concentrations was as follows: CST-3a; 20.4% (−48.5%–83.9%; n = 116)|CST-3b; 2.65% (−185.8%–71.2%; n = 102). Intersubject variabilities for Cmax and AUClast were <60% (CST-3a) and <58% (CST-3b). L-PL and HS-PL concentrations and AUClast (CST-3a – r = 0.8343; p = <0.001; n = 24, CST-3b – r = 0.8045; p = <0.001; n = 16) were highly correlated with a mean difference of 5.53 ng/mL (CST-3a) and 1.01 ng/mL (CST-3b) between methods. In both CST-3a and CST-3b, all HS-PL and L-PL concentrations remained above the EC90 target (1430 ng/mL) at pre-dose on day 5. Conclusion: HS-DBS are a viable alternative to L-PL for clinical sample collection and transport for studies of high-consequence infections in RLS, as TZN concentrations were strongly correlated with respective L-PL values, with a minimal mean difference between methods. Generally, HS-PL concentrations were greater than respective L-PL values, likely due to variable plasma ring volumes.