155: The Human Metabolism of Lufotrelvir, a Prodrug for the Treatment of SARS-COV2, Investigated By LC+AMS

Statement of Purpose, Innovation or Hypothesis: A microdosing/microtracer approach in combination with accelerator mass spectrometry (AMS) enables the generation of human absorption, distribution, metabolism and excretion data early during drug development. This allows improvement of the clinical development plan and shortening of the overall drug development program. An intraveneous human microtracer study was used for the clinical evaluation for lufotrelvir. Lufotrelvir is a novel phosphate prodrug of PF-00835231 for the treatment of COVID-19. The pandemic emphasized the importance of this accelerated approach because of the potential global need for this drug.

Description of Methods & Materials: Five healthy participants received a single 24-hour constant-rate, continuous intravenous infusion of 500 mg lufotrelvir containing ~420 nCi [¹⁴C]-lufotrelvir. Blood, urine and fecal samples were collected and analyzed for total ¹⁴C by AMS on a 1MV multi-element instrument (HVE. Model 4110; The Netherlands). Prior to analysis fecal samples were homogenized 1:3 w/w feces/MilliQ. Excreta samples were pooled across sampling times to capture >90% of the carbon-14 in that matrix for each subject. Equal volumes of the matrices from the five subjects were pooled for quantitative metabolite profiling. Hamilton pools for plasma were prepared for 0–24 hours (during infusion) and 24–216 hours (post infusion). Plasma and fecal homogenates were extracted with acetonitrile (plasma twice with 1:5 and fecal homogenates three times 1:4). The combined supernatants were lyophilized, and reconstituted. Urine was processed by adding acetonitrile and formic acid. The samples were analyzed on an Acquity UPLC system (Waters) coupled to a Q-Exactive mass spectrometer (Thermo). The column effluent was split to the fraction collector and mass spectrometer. AMS analysis was done off-line on the fractions of the eluent. The pellet remaining after plasma and fecal homogenate extraction was pronase digested. Subsequently the amino acids were derivatized using an AccQ-Tag Ultra derivatization kit (Waters). The processed samples were analyzed by UHPLC-HRMS with fraction collection for off-line AMS analysis.

Data & Results: On average only 63% of the activity administered was recovered in urine and feces after 10 days. Plasma total ¹⁴C analysis indicated a terminal phase half-life >200 hours. The metabolite profiles showed complete conversion of lufotrelvir into the active drug PF-00835231 which was subsequently cleared by hydrolysis, hydroxylation, ketoreduction, epimerization, renal clearance, and secretion into the feces. Also a significant amount of radioactivity was not extractable from plasma and feces and remained in the pellet.

Interpretation, Conclusion or Significance: After plasma and feces extraction the majority of the activity remained in the pellet. The ¹⁴C-label in lufotrelvir was at the leucine carbonyl position. With hydrolysis as an important metabolizing route, it was hypothesized that ¹⁴C-leucine was released and subsequently incorporated into macromolecules. This was confirmed after r investigation of the pellets using pronase digestion in combination with amino acid derivatisation. UHPLC-HRMS and AMS. The presence of ¹⁴C-leucine in the pellet was shown, also explaining the low mass balance recovery. Using a modern AMS approach, human mass balance and metabolite profiling data can be available as early as Phase 1. In this particular study AMS accommodated the very tight timelines for development.

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Additional Information/Authors: corresponding author: esther.vanduijn@tno.nl