key: cord-0741333-yezho5jh authors: FitzGerald, R.; Dickinson, L.; Else, L.; Fletcher, T.; Hale, C.; Amara, A.; Walker, L.; Dilly Penchala, S.; Lyon, R.; Shaw, V.; Greenhalf, W.; Lavelle-Langham, L.; Reynolds, H.; Painter, W.; Holman, W.; Ewings, S.; Griffiths, G.; Khoo, S. title: Pharmacokinetics of β-d-N4-hydroxycytidine, the active metabolite of prodrug molnupiravir, in non-plasma compartments of patients with SARS-CoV-2 infection date: 2021-12-07 journal: nan DOI: 10.1101/2021.12.06.21267342 sha: d044dd25bc8a82c8af720289c30f5910c3620dce doc_id: 741333 cord_uid: yezho5jh Background: Molnupiravir, an orally administered prodrug of the broadly active, direct-acting antiviral, ribonucleoside analogue {beta}-d-N4-hydroxycytidine (NHC) is a promising COVID-19 drug candidate. We characterised the pharmacokinetics of NHC in saliva, nasal secretions and tears of patients enrolled in the phase I AGILE trial (NCT04746183) to understand its potential in preventing infection and transmission. Methods: Patients with PCR-confirmed SARS-CoV-2 infection, within 5 days of symptom onset with mild-to-moderate disease were randomised to oral molnupiravir 300, 600 or 800 mg twice daily or placebo. Plasma and non-plasma (saliva, nasal secretions and tears) samples were collected at pre-dose, 0.5, 1, 2, and 4 hours post-dose on study days 1 and 5 and molnupiravir and NHC measured by LC/MS with a lower limit of quantification of 2.5 ng/mL in all matrices. Pharmacokinetic parameters were determined by noncompartmental methods and non-plasma:plasma ratios (RNP:P; based on AUC0-4) calculated. Results: Twelve participants (n=4 per dosing arm; 75% female) completed the study. NHC Tmax ranged between 1.00-4.00 hours for saliva (n=21) and nasal swabs (n=22) and 0.50-4.00 hours (n=17) for tears compared to 1.00-2.00 hours for plasma (n=19). Median (range) saliva RNP:P pooled across doses was 0.03 (0.01-0.11); n=16. RNP:P for nasal secretions and tears were 0.21 (0.05-0.73); n=17 and 0.22 (0.09-1.05); n=12, respectively. Non-plasma and plasma concentrations were significantly correlated (p<0.0001). Conclusion: These data provide encouraging information regarding the distribution of NHC at sites of viral transmission and have important implications for prophylactic coverage. There is a need for effective antivirals to treat SARS-CoV-2 infection, which if commenced in a timely manner may prevent the development of severe disease. An extended therapeutic goal of antiviral therapy is the prevention of infection in individuals that have been exposed to an infected person. Viral infection occurs through inhalation or inoculation of virus onto upper respiratory airways and mucosal surfaces. In order to be an effective prophylactic agent in such cases, drug must penetrate into sites of inoculation in sufficient quantities. Molnupiravir (EIDD-2801; MK-4482), a prodrug of the broadly active, direct-acting antiviral, ribonucleoside analogue 14 β-d-N4-hydroxycytidine (NHC), has recently been licensed in the UK for the treatment of symptomatic COVID-19 in adults with at least one risk factor for developing severe disease. Following oral administration, molnupiravir is rapidly hydrolysed by esterases to NHC, which in turn is phosphorylated by host kinases to active metabolite EIDD-1931-5'-triphosphate (EIDD-2061) [1, 2] . AGILE, a UK platform for early-phase trials of novel COVID-19 therapies [3] , has evaluated molnupiravir within its AGILE Candidate-Specific Trial (CST)-2 seamless phase Ib/IIa protocol. We previously reported phase Ib evaluation of molnupiravir across three dosing arms (300, 600 and 800 mg twice daily), establishing that a dose of 800 mg twice daily for 5 days was well-tolerated, achieving plasma concentrations within the target range extrapolated from animal models, and was suitable for progression to phase II [4] , which is currently recruiting. In this study we investigated the pharmacokinetics of molnupiravir and NHC in saliva, nasal secretions and tears, in comparison with concentrations in plasma within the phase Ib component which we report here. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 7, 2021. ; https://doi.org/10.1101/2021.12.06.21267342 doi: medRxiv preprint The pharmacokinetics of molnupiravir were evaluated as part of a phase I dose-escalation study in patients with PCR-confirmed SARS-CoV-2 infection, who were within 5 days of symptom onset and presenting with mild or moderate disease (clinicaltrials.gov registration number NCT04746183). The study design has previously been described [4] . In brief, eligible individuals were randomly assigned to one of three sequential dosing cohorts -molnupiravir dosed orally at 300, 600 and 800 mg twice daily. Within each cohort, patients were randomised at a 2:1 allocation ratio to receive either molnupiravir plus standard-of-care (treatment arms; n=4 per cohort) or standard-of-care alone (control arms; n=2 per cohort). Plasma and non-plasma (saliva, nasal secretions and tears) samples were collected at pre-dose, 0.5, 1, 2, and 4 hours post-dose on study days 1 and 5 in order to characterise the single dose and steady-state pharmacokinetics of the molnupiravir and its active metabolite, NHC within these compartments. Plasma samples were collected as previously described [5] . Saliva samples were collected using Salivette TM tubes (Sarstedt Ltd, UK). The patient was asked to chew on the Salivette . CC-BY 4.0 International license It is made available under a perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 7, 2021. ; https://doi.org/10.1101/2021.12.06.21267342 doi: medRxiv preprint swab for approximately 60 seconds after which the swab was returned to the Salivette and centrifuged (within 30 minutes of collection) to yield liquid saliva. The saliva supernatants (50 µL) were then immediately treated with acetonitrile (acetonitrile:saliva 3:1 v/v) in order to prevent ongoing conversion (via host esterases) of the molnupiravir pro-drug to NHC. Nasal secretions were collected using 2 x Synthetic Absorptive Matrix (SAM) strips (Mucosal Diagnostics, UK). The applicator was removed from the tube and the absorption end of the swab carefully inserted into the patient's nostril (one each side) with the absorbent strip placed flat against the surface of the inferior turbinate for 60 seconds. The swab was then returned to its original tube and screwed in place using the applicator handle. The weight (to the nearest 0.1 mg) of each SAM strip was determined before and after sampling in order to ascertain the approximate weight of fluid absorbed. Tears were collected using 2 x Schirmer Tear Test strips which were inserted under the patient's lower eyelid (one in each eye) for 5 minutes. The approximate volume (to the nearest µL) was recorded immediately after collection using the graduated markings on the strip (1-35 µL) and the strip placed inside a clean labelled 2 mL polypropylene tube. All pharmacokinetic specimens were transported and processed on wet ice and stored at -80°C within 60 minutes of collection. Molnupiravir and NHC concentrations in plasma and saliva were determined at the University of Liverpool Bioanalytical Facility (Liverpool, UK) using a validated LC-MS method, as described previously [5] . Similarly, NHC concentrations in nasal secretions and tears (swab is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted December 7, 2021. ; https://doi.org/10.1101/2021.12.06.21267342 doi: medRxiv preprint samples) were determined using an adaptation of this analytical method. In brief, 2 mL of acetonitrile:1mM ammonium acetate (50:50 v/v) was added directly to the tubes containing the swab (calibrators, quality controls and patient samples) and allowed to stand for 1 hour at room temperature. The tubes were sonicated for 30 minutes and exactly 1.8 mL transferred to clean labelled 5 mL glass tubes containing 20 μL of working internal standard solution (2.5 µg/mL 13 C15N2-N4-hydroxycytidine). Samples were then vortexed, evaporated to dryness under nitrogen at ambient temperature and reconstituted with 200 μL of 1 mM ammonium acetate (adjusted to pH 4.3) ready for injection onto the LC-MS system. Standards and quality controls were freshly prepared on the day of analysis. Working solutions were prepared in 1 x phosphate-buffered saline (PBS) fresh (which served as a surrogate matrix) and 15 µL of each calibrator level pipetted onto a clean SAM or tear strip (in duplicate). The calibration curve ranged between 0.15-75 ng/sample, and was described using a weighted (1/x 2 ) least square linear regression model. Concentrations of NHC in plasma and saliva were measured in ng/mL, whereas nasal and tear swab NHC were quantified using a ng/sample calibration curve, and converted to ng/mL based on swab volume in µL. Following administration of 300, 600 and 800 mg doses of molnupiravir, NHC concentrations on study day 1 and 5 in each sampling matrix, at each nominal time point [pre-dose (0), 0.5, 1, 2, 4 hours post-dose] were described using summary statistics such as geometric mean (90% CI), mean, standard deviation, median and range. Samples below the lower limit of quantification of the assay (LLQ; <2.5 ng/mL) at pre-dose on day 1 were included as 0 ng/mL, whereas those