Characterization of the absorption, metabolism, excretion, and mass balance of gefapixant in humans

Abstract Gefapixant (MK‐7264) is a first‐in‐class, selective antagonist of the P2X3 purinergic receptor currently being investigated as a therapeutic agent for the treatment of refractory or unexplained chronic cough. In non‐clinical studies, gefapixant was eliminated primarily by renal excretion of the parent drug. The objective of this study was to assess the disposition of gefapixant in humans. The absorption, metabolism, and excretion profiles of gefapixant were assessed after oral administration of a single dose of [14C]gefapixant to six healthy adult males. Following a single‐oral [14C]gefapixant dose to healthy adult males, the mass balance was achieved, with 98.9% of the administered radioactivity recovered in urine and feces. Elimination of gefapixant occurred primarily via renal excretion of the intact drug (64%); metabolism was a minor pathway of elimination of gefapixant (12% and 2% recovered in urine and feces, respectively). Single‐dose administration of [14C]gefapixant 50 mg was generally well tolerated in healthy adult males. The fraction of the anticipated therapeutic oral dose of gefapixant absorbed is estimated to be at least 78%. Gefapixant is expected to be the major circulating drug‐related material in plasma, and the majority of the dosed drug will be excreted unchanged in urine.

gefapixant 50 mg was generally well tolerated in healthy adult males. The fraction of the anticipated therapeutic oral dose of gefapixant absorbed is estimated to be at least 78%. Gefapixant is expected to be the major circulating drug-related material in plasma, and the majority of the dosed drug will be excreted unchanged in urine.

K E Y W O R D S
AF-219, antitussives, chronic cough, purinergic receptor, RO4926219

| INTRODUC TI ON
The global prevalence of chronic cough (a cough persisting longer than 8 weeks) has been estimated to be approximately 10%. 1 Although the cough reflex is typically protective, chronic cough can negatively impact both the physical and emotional well-being of affected individuals. 2 Gefapixant (MK-7264) is a first-in-class, selective antagonist of the P2X3 purinergic receptor that is currently under investigation as a therapeutic agent for the treatment of refractory or unexplained chronic cough. 3 The P2X3 receptor is an adenosine triphosphate (ATP)-gated ion channel that can be found on sensory nerves. 4 ATP released by damaged, stressed, or inflamed tissues in airways stimulates C fiber sensory nerves through P2X3, initiating a cough reflex. 5 Blockade of extracellular ATP signaling through P2X3 receptors reduces sensory-nerve activation and cough in pre-clinical models, [6][7][8] and data from clinical trials suggest gefapixant can have a similar effect in patients. [9][10][11] In non-clinical studies in animals, gefapixant was eliminated primarily by renal excretion of the parent drug. Here, we report the results of a Phase 1 clinical trial designed to assess the disposition of gefapixant in humans. In this study, the absorption, metabolism, and excretion profiles of gefapixant were assessed after oral administration of a single dose of [ 14 C]gefapixant to healthy adult males.

| MATERIAL S AND ME THODS
The study reported here was approved by Chesapeake Research Review, Inc., MA, USA and conducted at Celerion Inc., Lincoln, NE, USA.
The study was conducted in accordance with Good Clinical Practices guidelines and the ethical principles set forth by the Declaration of Helsinki. All participants gave written informed consent.

| Participants
Eligible study participants were male, 19 to 45 years of age, with a body mass index (BMI) ≥18.5 and ≤32.0 kg/m², and in good health based on medical history, physical examination, laboratory safety tests, vital sign measurements, and electrocardiogram (ECG) assessments. Individuals with a history or presence of hypersensitivity or idiosyncratic reaction to the study drug or related compounds were excluded from the trial.

| Study design
This was a single-center, single-group assignment, open-label, study.
Following an overnight fast, participants received a single oral dose of 50 mg [ 14 C]gefapixant, equivalent to approximately 200 μCi, a dose selected to deliver sufficient radioactivity for analysis and to be within the linear range of the gefapixant dose-exposure relationship. Blood, urine, and fecal samples were collected for radioactivity and pharmacokinetic assessments. Safety was monitored throughout, including collection and evaluation of adverse events (AEs), vital signs, ECGs, and clinical laboratory evaluations (hematology, chemistry, and urinalysis). Participants were discharged within 7 days postdose, a timepoint at which ≥90% of the administered radioactivity had been recovered in urine and feces, and ≤1% of the administered radioactivity was found in each of 2 samples from consecutive 24-h urine and fecal collections.

| Gefapixant quantification
Blood and urine samples were processed and assayed as previously described for gefapixant quantification. 12 Plasma and urine gefapixant concentrations were determined by inVentiv Health Clinique (Quebec, Quebec, Canada) using a validated highperformance liquid chromatographic tandem mass spectrometric

SIGNIFICANCE STATEMENT
[ 14 C]-labeled gefapixant elimination occurs primarily via renal excretion of the intact drug, and metabolism is a minor pathway of elimination. By combining the fractions eliminated by different routes, the fraction of an oral dose of gefapixant that is absorbed is estimated to be at least 78%. The findings in this study enable a targeted approach to further characterization of gefapixant clinical pharmacology, including an estimation of the absorbed fraction without an intravenous dose. method, with a lower limit of quantitation (LLOQ) of 10.00 ng/mL and analytical range of 10.00 to 10000.00 ng/mL for plasma and an LLOQ of 1.00 μg/mL and analytical range of 1.00 to 1000.00 μg/ mL for urine. 12

| Radioactivity measurements
Total radioactivity concentration equivalents in plasma, urine, and feces were determined by Celerion (Lincoln, Nebraska, USA) using liquid scintillation counting (LSC). Radioactivity in urine and plasma was analyzed by direct counting of triplicate sample aliquots in vials containing liquid scintillation cocktail (Ultima Gold XR, PerkinElmer, Boston, MA, USA. Product No. 6013119). The aliquot mass for plasma was 0.25 g. The aliquot mass for urine was 1 g. The aliquot mass for fecal samples was 0.5 g. Fecal homogenate aliquots were dried and oxidized before assaying for [ 14 C] content. The LLOQ for total radioactivity in plasma, urine, and feces were 19.2 ng eq/g, 4.99 ng eq/g, and 11.5 ng eq/g, respectively, LLOQ = {[(average background dpm × 2.5) -average background dpm]/grams average aliquot weight for all aliquots for all participants}/specific activity (dpm/gram). Further details of radioactivity analysis can be found in Supplemental Text.

| Pharmacokinetic analysis
Any gefapixant or radioactivity concentrations from any source that was below the limits of quantitation were replaced with a value of 0 in all analyses. Total radioactivity concentration equivalents in urine and feces, and the urine sample and fecal homogenate weights from individual collection intervals, were used to calculate the urine and fecal radioactivity Ae, Cum Ae (the cumulative sum of Ae), %Dose (Ae/administered dose × 100) and Cum%Dose (cumulative sum of the %Dose) of radioactivity in urine and feces.

| Analyses for metabolites
The percent of dose represented by each of the metabolites was calculated using the radioactivity concentration-equivalent data combined with the metabolite profiling data. The percentage of each identified metabolite to total radioactivity in the plasma was estimated based on plasma metabolite profiling data.

| Statistical analyses
To estimate the proportion of total radioactivity accounted for by When N was ≤2, SD was not calculated.

| Safety
Safety and tolerability were evaluated by clinical assessment of AEs, vital signs, 12-lead ECGs, and laboratory safety tests (hematology, chemistry, and urinalysis) throughout the study.

| Participant demographics and disposition
The trial was initiated July 14, 2017 and completed August 9, 2017. Six healthy adult males were enrolled in the study and five completed the study per protocol. One participant was

| Metabolite profiles
The results of metabolite profiling indicated that parent molecule  (Table 2). Overall, metabolites detected in urine and feces collectively accounted for <15% of the dose (Table 2). In plasma, M1, M5, M8, M11, and M13 were detected as minor metabolites (Figure 3). Each circulating metabolite accounted for less than 10% of the total radioactivity detected ( Table 2).

| Safety
Single   Table 1) and by the analysis of metabolite profiling in plasma (87% of parent in samples pooled from 0.5 to 24 h, Table 2). This observation further supports that metabolism plays a minor role in the elimination of gefapixant, and the exposure of each of the circulating metabolites was less than 10% of the total drug-related exposure.
All metabolites detected in human excreta and plasma have been observed previously in either rats and/or dogs.
The design of the study carries some limitations. Only singledose administration at a single dose level was assessed; however, the PK properties of gefapixant are not time dependent and exposures are dose proportional in this range; therefore, single-dose PK can be used to estimate steady-state PK. The observed safety, while limited by the open-label design of this study, is consistent with previous studies of gefapixant indicating that it is generally well tolerated.
In conclusion, following a single oral [ 14 C]gefapixant dose to healthy adult males, the mass balance was achieved, with 98.9% of the administered radioactivity recovered in urine and feces. In healthy male adults, elimination of gefapixant occurs primarily via renal excretion of the intact drug (64%); metabolism is a minor pathway of elimination of gefapixant (12% and 2% recovered in urine and feces, respectively). Based on these values, the fraction of drug absorbed is estimated to be at least 78%. Gefapixant is the major circulating drug-related material in plasma. Single-dose administration of [ 14 C]gefapixant 50 mg was generally well tolerated in healthy adult males.

ACK N OWLED G EM ENTS
The authors wish to thank the study participants and study staff.  Note: Values represent the percentage of radioactivity in the radiochromatogram associated with parent compound and metabolites. Numbers in parentheses represent the percentage of the dose. Metabolites that were detected in trace amounts were detected by high-resolution mass spectrometry only.