Mass balance and pharmacokinetic characterization of zavegepant in healthy male subjects

Abstract

Zavegepant, a high‐affinity, selective, small‐molecule calcitonin gene‐related peptide receptor antagonist, is approved as a nasal spray for acute treatment of migraine in adults. This phase I, open‐label, single‐center, single‐period, nonrandomized study in six healthy male subjects assessed mass balance recovery after a single 15‐min intravenous (IV) infusion dose of carbon‐14 ([¹⁴C])‐zavegepant. Blood, urine, and fecal samples were collected over 192 h for analysis of zavegepant in plasma and urine; total radioactivity (TR) in plasma, whole blood, urine, and feces; and zavegepant metabolite profiling and structural identification in plasma, urine, and feces. An average of 96.6% of radioactivity administered was recovered in excreta. Most TR (mean 84.9%) was recovered in the feces, indicating that biliary/fecal elimination was the main route. Volume of distribution of zavegepant based on the terminal phase (129 L) was higher than total body water (42 L), indicating substantial distribution into tissue. Total plasma clearance of zavegepant (220 mL/min) is identical to whole blood clearance given the blood/plasma partition ratio of 1, lower than typical hepatic blood flow (1450 mL/min). The observed plasma terminal half‐life of zavegepant was 6.8 h. Exposure to zavegepant accounted for ~90% of circulating plasma TR, suggesting that very low levels of uncharacterized circulating metabolites were present. Metabolite profiling did not identify any metabolites representing ≥10% of radioactivity in plasma, urine, or feces. A single IV infusion of 5 mg [¹⁴C]‐zavegepant was well tolerated in healthy male subjects. Disposition findings of IV [¹⁴C]‐zavegepant are applicable to the disposition of the approved zavegepant nasal spray.

Study Highlights.

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

Zavegepant is a third‐generation, high‐affinity, selective and structurally unique, small‐molecule calcitonin gene‐related peptide receptor antagonist indicated for the treatment of acute migraine with or without aura in adults. This is the first publication to describe the mass balance recovery for intravenous zavegepant that is also applicable to the disposition of the approved zavegepant nasal spray.

• WHAT QUESTION DID THIS STUDY ADDRESS?

The first question is to provide data to understand how the drug is processed in physiologically normal subjects, because understanding the routes of metabolism and elimination in a healthy population generates the appropriate data to guide the clinical pharmacology package required to fulfill the regulatory requirements of a New Drug Application. The second question is to provide human metabolite data that can be used to interpret the metabolism profiles seen in the preclinical species employed in the longer‐term toxicity studies and to ensure that there is adequate toxicology coverage for the safe development of the drug in patients.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

Assessment of mass balance recovery after a single 15‐min intravenous infusion dose of carbon‐14 ([¹⁴C])‐zavegepant, metabolite profiling, and structural identification showed that biliary/fecal elimination was the main route and that zavegepant undergoes minimal metabolism.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

This study provides clinicians with detailed information on the disposition of single‐dose intravenous zavegepant, which is applicable to the approved zavegepant nasal spray and will permit appropriate dosing in humans.

INTRODUCTION

Migraine is a primary headache disorder characterized by recurrent attacks of head pain and characteristic associated symptoms (e.g., photophobia, phonophobia, and nausea) with an overall prevalence in the United States of ~15% (20.7% in women and 9.7% in men) ¹ , ² that is often associated with significant disability. ² , ³ , ⁴ Traditional migraine treatment (e.g., acetaminophen, nonsteroidal anti‐inflammatory drugs, triptans, antiemetics, and ergot alkaloids) is often limited by insufficient therapy response due to slow speed of effect, recurrence of pain, need for additional medication, intolerance, and cardiovascular‐related contraindications necessitating individualized treatment. ⁵

Calcitonin gene‐related peptide (CGRP) is a neuropeptide and a potent vasodilator that has a fundamental role in migraine pathophysiology ⁶ , ⁷ , ⁸ and is a key target for both the acute and preventive treatment of migraine. ⁷ , ⁹ , ¹⁰ CGRP antagonists are the first class of migraine‐specific medication that does not have vasoconstrictive action. ¹¹

Zavegepant nasal spray (ZAVZPRET™, Pfizer Inc., New York, NY) is a third‐generation, high‐affinity, selective, and structurally unique, small‐molecule CGRP receptor antagonist indicated for acute treatment of migraine with or without aura in adults via the intranasal route. ¹² , ¹³ , ¹⁴ Two randomized, placebo‐controlled phase II/III trials involving ~3000 subjects have demonstrated the efficacy and safety profile of zavegepant nasal spray for the acute treatment of migraine. ¹⁵ , ¹⁶ Notably, zavegepant nasal spray provided rapid onset of pain relief—15 min, the earliest measured timepoint, and sustained benefits through 48 h after a single intranasal dose. ¹⁶

Preclinical studies showed that zavegepant exhibited low permeability and moderate protein binding in plasma, did not preferably distribute into red blood cells, and had negligible distribution to the brain (data on file, Pfizer Inc). In vitro, zavegepant is a substrate of cytochrome P450 (CYP) 3A4 and to a lesser extent CYP2D6; zavegepant is also a substrate for the transporters of organic anion transporting polypeptides 1B3, sodium taurocholate co‐transporting polypeptide, P‐glycoprotein, multidrug and toxin extrusion 1, and multidrug and toxin extrusion 2 K. Across animal species, the total plasma clearance of zavegepant was low. The major route of zavegepant elimination was via the feces, accounting for ~ 63% of the dose, while renal elimination accounted for ~ 25% of the dose.

In humans, after a single 10 mg dose of the nasal spray, the time to peak plasma concentration is ~ 30 min; absolute bioavailability is ~ 5%; mean apparent volume of distribution is ~ 1774 L; plasma protein binding is ~ 90%; effective half‐life is 6.55 h; and mean apparent clearance of intranasal zavegepant is 266 L/h. ¹⁴

This mass balance study was conducted to better understand the pharmacokinetic behavior of zavegepant. The primary objectives were to assess the mass balance recovery after a single IV 5 mg dose of carbon‐14 ([¹⁴C])‐zavegepant and to provide blood, urine, and fecal samples for metabolite profiling and structural identification. The secondary objectives were to explore the IV pharmacokinetics of zavegepant to determine routes and rates of elimination of [¹⁴C]‐zavegepant to identify the chemical structure of each metabolite accounting for >10% of circulating total radioactivity (TR), to evaluate the extent of distribution of TR into blood cells, and to provide additional safety and tolerability information for zavegepant.

METHODS

Study design

This phase I, open‐label, single‐center, single‐period, nonrandomized study evaluated the mass balance, disposition, and metabolic profiling of a single IV dose of 5 mg [¹⁴C]‐zavegepant as a 15‐min infusion in healthy male subjects and was conducted at Quotient Sciences (Nottingham, United Kingdom [UK]) between 17 July 2020 and 28 August 2020. This study involving human subjects, including the protocol, any amendments, and consent form, was reviewed and approved by an Institutional Review Board/Independent Ethics Committee. The study was conducted in accordance with Good Clinical Practice, as defined by the International Council for Harmonization. Written informed consent was obtained from subjects prior to any study‐specific procedures. All analyses were performed according to a predefined protocol.

The study included a screening visit (Day −28 to Day −2) and a single treatment period. During the treatment period, subjects resided in the clinical research unit from the morning of Day −1 (the day before dosing). Following an overnight fast (8 h), subjects were dosed on Day 1 with a single 15‐min IV infusion dose of 5 mg [¹⁴C]‐zavegepant. All subjects were discharged from the unit on Day 9 following the completion of all final assessments.

Based on the intranasal bioavailability in rabbits of ~30% at 100 mg/mL (data on file, Pfizer Inc.), the 5 mg IV dose of zavegepant was selected as it was expected to reach similar exposures to those observed with the 20 mg intranasal dose from the dose‐ranging phase II/III study. ¹⁵ Although 5 mg IV dosing could have higher exposures considering lower human intranasal bioavailability, the zavegepant concentrations after IV dosing in humans were expected to be within safety margins based on preclinical toxicology studies. The dose of radioactivity was determined following review of human dosimetry calculations provided by Public Health England. The total amount of radioactivity administered to each subject was no more than 3.6 MBq (97.2 μCi). The associated radiation exposure fell within International Commission on Radiological Protection (1992) Guidelines for Category IIa studies (0.1–1 millisievert). ¹⁷ The IV route of administration was chosen as a surrogate for an intranasal dose owing to manufacturing difficulties with providing a solution of radiolabeled investigational medicinal product (IMP) that could be instilled via the commercially available devices for administration of intranasal medication and the possibility of loss of radiolabeled IMP due to exhalation into the air after administration, which could have led to inaccuracies in quantification for mass balance.

A blood sample was collected at 0 h, which was immediately followed by the start of the IV infusion of [¹⁴C]‐zavegepant with continued fasting for 4 h postdose. Subjects received IV zavegepant in the fasting state to ensure that food did not confound the pharmacokinetics of IV zavegepant since preclinical studies showed that zavegepant was eliminated through biliary excretion (data on file, Pfizer Inc.). Over a 192‐h sampling period, blood, urine, and fecal samples were collected at various timepoints for analysis of zavegepant in plasma, TR in plasma, whole blood, urine, and feces, metabolite profiling and structural identification in plasma, urine, and feces, and mass balance of zavegepant (Figure S1). Blood samples for clinical laboratory assessments and urine for urinalysis were also collected, and adverse event monitoring, vital signs, electrocardiogram (ECG), Sheehan Suicidality Tracking Scale (S‐STS), ¹⁸ , ¹⁹ and physical examination assessments were performed.

Use of any drugs known to induce or inhibit hepatic drug metabolism within 30 days prior to the start of treatment was prohibited. No prescribed or over‐the‐counter medication or herbal remedies were permitted from 14 days before IMP administration until discharge from the study except for up to 4 g of paracetamol per day and those deemed necessary by the investigator to treat adverse events.

Study population

Healthy male subjects between 30 and 60 years of age with body mass index between 18.0 and 32.0 kg/m², an S‐STS score of 0, and a history of regular bowel movements were eligible. Additional eligibility criteria included nonsmokers and no history of drug abuse.

Subjects with any clinically significant cardiovascular, renal, hepatic, pulmonary, gastrointestinal, hematologic, neoplastic, endocrine, immunological, neurological, or psychiatric disease or disorder, or any clinically significant abnormal biochemistry, hematology, or urinalysis were ineligible for enrollment.

Safety

Subjects were closely monitored for adverse events throughout the study. The incidence of adverse events was tabulated, and all adverse events were reviewed for their relation to zavegepant and clinical importance. Vital signs, ECG, clinical laboratory tests (hematology, clinical chemistry, or urinalysis), S‐STS, and physical examination were assessed at various times from screening through study completion.

Sample collection and processing for zavegepant, total radioactivity, and metabolite concentrations

On Day 1, blood samples were collected for plasma zavegepant concentration, plasma TR, whole blood TR, and metabolite profiling and structural identification analysis over 192 h postdose and transferred into dipotassium ethylenediaminetetraacetic acid (K₂EDTA) tubes. Plasma was separated by centrifugation. Samples were frozen within 60 min of collection and were stored at −80°C or below until they were shipped to the bioanalysis and radioanalysis vendor (Pharmaron UK, Ltd., Rushden, Northamptonshire, UK).

Urine samples for analyses of zavegepant, TR, and metabolite profiling and structural identification were collected over 192 h postdose into individual appropriately sized amber polyethylene containers and weighed. A volume of methanol equivalent to 25% of the urine sample was added immediately following sample collection as per the validated method, with the resultant methanol/urine being 20/80 (volume/volume). Samples were then re‐weighed and stored at 2°C to 8°C prior to shipment.

Fecal samples for analyses of zavegepant, TR, and metabolite profiling and structural identification were collected over 192 h postdose and stored in individual polypropylene containers and weighed. Fecal samples and toilet paper were collected into individual polypropylene containers, weighed, and stored at −20°C prior to shipment.

During the study, other accidental sources of elimination were to be collected as voided (e.g., emesis) and shipped to Pharmaron for analysis of TR.

Bioanalytical methods

Plasma, urine, and fecal zavegepant concentrations were determined using liquid chromatography with tandem mass spectrometry (LC–MS/MS) by Pharmaron. The validated range for zavegepant was 0.400–200 ng/mL in plasma, 0.160–400 ng/mL in urine treated with methanol (in a 4:1 ratio of urine: methanol), and 5.00–5000 ng/mL in feces.

TR concentrations in plasma, whole blood, urine, and feces were determined using liquid scintillation counting by Pharmaron. The lower limit of detection was 1.12 ng equivalents (eq)/mL for plasma, 3.2542 ng eq/mL for whole blood, 0.0761 ng eq/g for urine, and 1.48 ng eq/g for feces.

Metabolite profiling of plasma, urine, and feces was performed using liquid chromatography radio detection with subsequent high‐resolution mass spectrometry where appropriate (positive ion mode accurate mass full scan and product ion analyses). Identification of the chemical structure of each metabolite accounting for >10% of circulating radioactivity in plasma (“AUC pool”) and accounting for >10% of the dose in the urine and feces (from urine pools and feces homogenate pools) was performed by Pharmaron.

Pharmacokinetics and statistical analysis

No formal sample size calculation was performed for this study. The inclusion of 4–6 subjects was deemed sufficient. ²⁰ A formal statistical analysis was not planned for mass balance or pharmacokinetic data.

Mass balance parameters were calculated based on urine and feces samples for analysis of TR and included amount of TR excreted in urine (Ae_Urine), amount of TR excreted in urine expressed as a percentage of radioactive dose administered (%Ae_Urine), cumulative amount of TR excreted in urine (Cumulative Ae_Urine), cumulative amount of TR excreted in urine expressed as a percentage of radioactive dose administered (Cumulative %Ae_Urine), amount of TR excreted in feces (Ae_Feces), amount of TR excreted in feces expressed as a percentage of radioactive dose administered (%Ae_Feces), cumulative amount of TR excreted in feces (Cumulative Ae_Feces), cumulative amount of TR excreted in feces expressed as a percentage of radioactive dose administered (Cumulative %Ae_Feces), amount of TR excreted in urine and feces combined (Ae_Total), amount of TR excreted in urine and feces combined expressed as a percentage of the radioactive dose administered (%Ae_Total), cumulative amount of TR excreted in urine and feces combined (Cumulative Ae_Total), and cumulative amount of TR excreted in urine and feces combined expressed as a percentage of radioactive dose administered (Cumulative %Ae_Total).

Mean plasma concentration versus time curves for zavegepant, TR in plasma, and TR in whole blood were presented as both linear and semilogarithmic scales. Descriptive statistics were presented for the following pharmacokinetic parameters of zavegepant in plasma, TR in plasma, and TR in whole blood and were estimated by standard non‐compartmental methods using validated Phoenix® WinNonlin® (version 8.0, Certara, USA): maximum observed concentration (C _max), time of observed C _max (t _max), area under the concentration–time curve from time zero to time 24 h (AUC_0‐24), area under the concentration–time curve from time zero to last timepoint (AUC_0‐last), area under the concentration–time curve from time zero to infinity (AUC_0‐inf), the area under the concentration–time curve extrapolated beyond the last measurable concentration (AUC_extrap), apparent elimination half‐life (t _1/2), systemic (total body) clearance (CL), renal clearance (CLr), predicted volume of distribution at steady state (V _ss), and volume of distribution based on elimination phase (V _z). All parameters were presented as geometric mean (geometric coefficient of variance [CV%]), except for t _max that is presented as median (minimum, maximum).

RESULTS

Study population

Among 15 subjects who were screened, 6 (40%) were enrolled. All six subjects received a single infusion of [¹⁴C]‐zavegepant as a 15‐min infusion, had evaluable TR concentration (urinary and fecal) data and no protocol deviations that may have affected the mass balance analysis, and had ≥1 plasma pharmacokinetic profile, and were included in the safety and pharmacokinetic populations. The first subject was enrolled on 17 July 2020, and the last subject completed the study on 28 August 2020.

The demographic characteristics of the six male subjects were predominantly white (83.3%) ranging in age from 39 to 60 years; none were current smokers (Table 1). No subject reported taking any drugs known to induce or inhibit hepatic drug metabolism within 30 days prior dosing or any prescribed or over‐the‐counter medication or herbal remedies within 14 days prior to dosing and until discharge from the study.

TABLE 1.

Summary of demographic characteristics: mass balance, pharmacokinetic, and safety populations (N = 6).

Parameter
Age (years), mean (SD)	51.2 (8.2)
Male, n (%)	6 (100)
Race, n (%)
White	5 (83.3)
Not Hispanic or Latino	6 (100)
Weight (kg), mean (SD)	80.10 (8.62)
BMI (kg/m2), mean (SD)	27.33 (3.3)

Abbreviations: BMI, body mass index; SD, standard deviation.

Mass Balance

Over the 192‐h sampling period, 96.6% (range 92.1%–102.4%) of the radioactivity administered was recovered in urine and feces (Table 2, Figure 1). Most of the TR was recovered in the feces (mean 84.9% [range 79.7%–94.0%]) followed by 11.7% (range 8.4%–13.6%) in the urine (Table 2).

TABLE 2.

Mean (range) cumulative recovery of total radioactivity in urine, feces, and total (urine and feces combined) following a single intravenous dose of 5 mg [¹⁴C]‐zavegepant containing ≤3.6 MBq (97.2 μCi) [¹⁴C]: Mass balance population (N = 6).

	Cumulative %AeUrine a	Cumulative %AeFeces a	Cumulative %AeTotal a
Collection interval	(%)	(%)	(%)
(h)	(N = 6)	(N = 6)	(N = 6)
0 – 6	9.4 (7.2–11.0)	—	—
0 – 12	10.1 (7.6–11.3)	—	—
0–24	11.1 (8.0–12.7)	0.8 (0.0–5.0)	11.9 (8.1–17.2)
0–48	11.4 (8.2–13.1)	28.3 (0.0–66.2)	39.7 (8.3–78.9)
0–72	11.5 (8.3–13.3)	70.2 (29.6–85.3)	81.7 (40.1–98.2)
0–96	11.6 (8.3–13.4)	80.9 (74.9–89.9)	92.5 (86.9–98.7)
0–120	11.6 (8.4–13.5)	83.6 (78.4–92.9)	95.2 (90.0–101.2)
0–144	11.7 (8.4–13.5)	84.2 (79.3–93.0)	95.8 (91.3–101.4)
0–168	11.7 (8.4–13.6)	84.6 (79.5–93.9)	96.3 (91.7–102.3)
0–192	11.7 (8.4–13.6)	84.9 (79.7–94.0)	96.6 (92.1–102.4)

^a %Ae_Urine = amount of total radioactivity excreted in urine expressed as a percentage of the radioactive dose administered; %Ae_Feces = amount of total radioactivity excreted in feces expressed as a percentage of the radioactive dose administered; %Ae_Total = amount of total radioactivity excreted in urine and feces combined expressed as a percentage of the radioactive dose administered.

FIGURE 1.

Arithmetic mean (± arithmetic SD) cumulative recovery of total radioactivity in urine, feces, and total (urine and feces combined) following a single intravenous dose of 5 mg [¹⁴C]‐zavegepant containing not more than 3.6 MBq (97.2 μCi) [¹⁴C]: Mass balance population. Mass balance parameter values were entered as zero when a subject failed to void or has no concentration over a particular collection interval. Cumulative %Ae, amount of total radioactivity excreted expressed as a percentage of the radioactive dose administered; SD, standard deviation.

Pharmacokinetics of zavegepant

Disposition of zavegepant and total radioactivity in plasma

Following a single dose of 5 mg [¹⁴C]‐zavegepant containing ≤3.6 MBq (97.2 μCi) [¹⁴C] as a 15‐min IV infusion, increasing concentrations of zavegepant and TR in plasma were observed until the end of infusion, which then subsequently declined in a biphasic manner and with detectable levels up to 36 h postdose. Arithmetic mean linear/linear scales and geometric mean semilogarithmic/linear scales plasma concentration–time profiles of zavegepant and TR are shown in Figure 2.

FIGURE 2.

Arithmetic mean plasma pharmacokinetic zavegepant and total radioactivity concentrations through 36 h following a single intravenous dose of 5 mg [¹⁴C]‐zavegepant (inset – geometric mean). Results truncated at 36 h. For the calculation of summary statistics, concentration values reported as below the limit of quantification or ND have been set to ½ × lower limit of quantitation except for pre‐dose where no imputation was performed. Lower limit of quantitation for total radioactivity in plasma = 1.27 ng eq/mL and for zavegepant in plasma = 0.4 ng/mL. Where ng eq is presented this is the free drug equivalent value.

Plasma pharmacokinetic parameters following a single IV dose of 5 mg [¹⁴C]‐zavegepant are summarized in Table 3. The geometric mean (geometric CV%) C _max and AUC_0‐inf values were 389 ng/mL (18.8%) and 374 ng*h/mL (31.5%), respectively. The geometric mean V _z was 129 L (21.3%). The geometric mean (geometric CV%) CL and CLr values were 220 mL/min (31.5%) and 13.7 mL/min (16.5%), respectively. The mean plasma t _1/2 of zavegepant was 6.8 h (range 4.31 to 8.95 h). An average of ~6.4% of the dose administered was recovered as zavegepant in the urine by the end of the sampling period.

TABLE 3.

Geometric mean (geometric CV%) pharmacokinetic parameters for plasma zavegepant, plasma total radioactivity, and whole blood total radioactivity following a single intravenous dose of 5 mg [¹⁴C]‐zavegepant administered over 15 min.

Parameter	Zavegepant plasma (N = 6)	Total radioactivity plasma (N = 6)	Total radioactivity whole blood (N = 6)
t max (h) a	0.25 (0.25–0.33)	0.50 (0.50–0.52)	0.50 (0.50–0.52)
C max (ng/mL) b	389 (18.8%)	250 (15.8%)	140 (16.0%)
AUC0‐24 (ng*h/mL) c	365 (30.4%)	388 (28.0%)	347 (32.1%)
AUC0‐last (ng*h/mL) c	366 (32.3%)	384 (30.2%)	333 (39.8%)
AUC0‐inf (ng*h/mL) c	374 (31.5%)	416 (32.9%) [n = 5]	355 (266–443) a [n = 2]
AUCextrap (%)	1.91 (34.6%)	5.57 (18.6%) [n = 5]	15.4 (11.3–19.4) a [n = 2]
t 1/2 (h)	6.8 (27.4%)	8.0 (24.4%) [n = 5]	10.5 (70.5%) [n = 4]
CL (mL/min)	220 (31.5%)	NA	NA
CLr (mL/min)	13.7 (16.5%)	NA	NA
V z (L)	129 (21.3%)	NA	NA
V ss (L)	41.6 (12.4%)	NA	NA

Abbreviations: AUC_0‐24, area under the concentration–time curve from time zero to time 24 h; AUC_0‐inf, area under the concentration–time curve from time zero to infinity; AUC_0‐last, area under the concentration–time curve from time zero to last observed concentration; AUC_extrap, area under the concentration–time curve extrapolated beyond the last measurable concentration; CL, systemic (total body) clearance; CLr, renal clearance; C _max, maximum observed concentration; CV%, coefficient of variance; eq, equivalent; NA, not available; t _1/2, apparent elimination half‐life; t _max, time of observed concentration; V _ss, predicted volume of distribution at steady state; V _z, volume of distribution based on elimination phase.

^a Median (range).

^b ng eq/mL for total radioactivity.

^c ng eq*h/mL for total radioactivity.

Exposure to zavegepant accounted for ~90% of circulating plasma TR of the AUC_0‐inf (Figure 2, Table 3).

Disposition of total radioactivity in plasma and whole blood

Mean arithmetic linear/linear scales and geometric mean semilogarithmic/linear scales plasma concentration–time profiles of TR in plasma and whole blood are shown in Figure 3.

FIGURE 3.

Arithmetic mean plasma and whole blood total radioactivity concentrations following a single intravenous dose of 5 mg [¹⁴C]‐zavegepant (inset – geometric mean). Results truncated at 36 h. For the calculation of summary statistics, concentration values reported as below the limit of quantification or ND have been set to ½ × lower limit of quantitation except for pre‐dose where no imputation was performed. Lower limit of quantitation for total radioactivity in plasma = 1.27 ng eq/mL and total radioactivity in whole blood = 3.2542 ng eq/mL. Where ng eq is presented as the free drug equivalent value.

Following a single dose of 5 mg [¹⁴C]‐zavegepant containing ≤3.6 MBq (97.2 μCi) [¹⁴C], maximum plasma TR concentrations occurred between 0.50 and 0.52 h postdose (first measured timepoint) (Table 3). Concentrations then declined in a biphasic manner and remained quantifiable until between 12 and 24 h postdose (Figure 3). Terminal slopes were reliably determined for 5 of the 6 subjects, and the resultant mean plasma TR t _1/2 was 8.2 h. The geometric mean (geometric CV%) C _max and AUC_0‐inf values for plasma TR were 250 ng/mL (15.8%) and 416 ng*h/mL (32.9%), respectively (Table 3).

Whole blood concentrations of TR were quantifiable in all subjects from 0.5 h and remained quantifiable until between 12 and 24 h postdose (Figure 3). Terminal slopes were reliably determined for 4 of the 6 subjects with a mean whole blood TR t _1/2 of 11.9 h. The geometric mean (geometric CV%) C _max and median AUC_0‐inf values for whole blood TR were 140 ng/mL (16.0%) and 355 ng*h/mL (266 to 443 ng*h/mL), respectively (Table 3). The geometric mean (range) whole blood to plasma TR concentration ratios ranged from 0.56 (0.53 to 0.59) at 0.5 h postdose, gradually increasing from 0.66 (0.61 to 0.72) at 1.75 h postdose to 3.18 (2.83–3.58) at 24.25 h postdose.

Metabolite profiling and identification

Metabolite profiling did not identify any metabolites above the threshold for identification (those representing ≥10% of the circulating radioactivity in plasma [“AUC” pool] or accounting for >10% of the dose in urine and feces). Only one component was identified: a component eluting at nominal retention time 37.1 min in plasma, urine, and feces (P1, U1, and F1), which was identified as zavegepant (Table 4). Zavegepant accounted for 94.85% of the TR in the pooled plasma, 11.11% and 80.49% of the dose in urine and feces, respectively.

TABLE 4.

Summary of results for components identified in plasma, urine, and feces following a single intravenous dose of 5 mg [¹⁴C]‐zavegepant.

Component	Molecular weight a (Da)	Molecular formula	Nominal retention time b (min)	Plasma (% ROI)	Urine (% dose)	Feces (% dose)
M638 zavegepant:	638	C36H46N8O3	37.1	P1 c (94.85) d	U1 c (11.11) d	F1 c (80.49) e

Abbreviations: Da, Dalton; ROI, region of interest (% contribution of this component to the total sample radioactivity).

^a Molecular weights quoted are mono‐isotopic and refer to unlabeled components.

^b Retention times are nominal and are taken from accurate mass extracted ion chromatograms for urine sample (subjects 001 to 006, 0 to 6 h postdose).

^c Assigned components corresponding to notable metabolites identified from the metabolite profiling experiments have been designated as, e.g., ‘P1’, where the first character indicates the matrix (‘P/U/F' = plasma/urine/feces), followed by a sequential number.

^d Indicates mean % (subjects 001–006) of circulating radioactivity in plasma. Where a region identifier is shown in BOLD, this indicates >10% circulating radioactivity in at least one plasma sample.

^e Indicates mean % (subjects 001 to 006) of administered dose in urine or feces. Where a region identifier is shown in BOLD, this indicates >10% of the dose in a urine and feces sample.

Safety and tolerability

No deaths, serious adverse events, or adverse events leading to discontinuation were reported. Overall, 3 (50.0%) of the subjects reported five treatment‐emergent adverse events (TEAEs) including diarrhea, back pain, limb discomfort, paresthesia, and miliaria. All TEAEs were mild in intensity, were considered unrelated to the study drug, and resolved by the end of study.

No clinically significant changes or notable trends in laboratory assessments (hematology, clinical chemistry, or urinalysis), vital signs, ECG, S‐STS, or physical examinations were identified.

DISCUSSION

Understanding human pharmacokinetic properties of a new drug is an essential part of drug discovery drug development. The findings of the current phase I study describe the mass balance recovery, disposition, and metabolite profiling and structural identification following administration of a single IV dose of zavegepant. Zavegepant nasal spray was developed and approved for acute treatment of migraine. However, the bioavailability of intranasal zavegepant is low (~5% based on a population pharmacokinetic analysis ¹⁴ ) and the pharmacokinetics variability was high (33.0%–146.0%) following intranasal administration. ²¹ Zavegepant IV administration was chosen as a surrogate for intranasal dosing owing to the possibility of loss of radiolabeled drug due to exhalation into the air after administration, which could have led to inaccuracies in quantification for mass balance. Additionally, similar to that of intranasal administration, the IV route bypasses any potential first pass metabolism. Thus, the disposition findings of IV [¹⁴C]‐zavegepant are applicable to the disposition of the approved zavegepant nasal spray.

In this mass balance study, following a single IV infusion (Day 1) of 5 mg [¹⁴C]‐zavegepant to six healthy male subjects, 96.6% of the TR was recovered in excreta over the 8‐day collection period, which was consistent with expectation of near complete recovery following IV dosing and confirmed the adequacy of the 192‐h sampling duration. The vast majority (84.9%) of the radioactivity administered was recovered in the feces, indicating that biliary/fecal elimination was the main route of excretion.

The concentration–time profiles of zavegepant and TR in plasma were consistent following a single‐dose IV administration of [¹⁴C]‐zavegepant containing NMT 3.6 MBq (97.2 μCi) [¹⁴C]. Maximum plasma zavegepant concentrations were reached by the end of infusion at a median t _max of 0.25 h. Following C _max, concentrations showed a biphasic decline with a mean terminal t _1/2 of 6.8 h, comparable to the pharmacokinetic profiles and t _1/2 observed for zavegepant in other pharmacokinetic studies. ²¹ As expected with the IV route of administration, the inter‐subject variability associated with plasma zavegepant exposure was low and ranged from 12.4% to 32.3% compared with the corresponding variabilities of 33.0% to 146.0% observed following single‐dose intranasal administration. ²¹ The V _z of zavegepant, based on the terminal phase (129 L), was higher than total body water (42 L), ²² suggesting substantial zavegepant distribution into human tissue compartments. Because V _z exceeded V _ss (129 L vs. 41.6 L, respectively), this suggests that the redistribution of the drug from tissue back into the central compartment could be a rate‐limiting process for the terminal elimination phase. Total clearance of zavegepant in plasma (220 mL/min) is identical to whole blood clearance given the blood/plasma partition ratio of 1, <20% lower than the typical hepatic blood flow rate (1450 mL/min), ²² indicating that zavegepant is a low to moderate hepatic clearance drug. Blood clearance of zavegepant is anticipated to be essentially identical, given the measured blood/plasma concentration TR concentration ratio of 0.56. Given the fraction unbound in human plasma (~11%) ¹⁴ and a glomerular filtration rate of 125 mL/min, a CL_r of ~13.75 mL/min could be expected for zavegepant in the absence of any tubular secretion or reabsorption. The observed zavegepant CL_r of 13.7 mL/min in the current study is consistent with the expected CL_r, suggesting that no tubular secretion or reabsorption of zavegepant occurred.

Exposure to zavegepant (AUC_0‐inf) accounted for ~90% of circulating plasma TR, indicating minimal uncharacterized circulating metabolites in plasma following administration of [¹⁴C]‐zavegepant, which is consistent with the metabolite profiling results that indicate that zavegepant comprises nearly 95% of the radioprofile in plasma. Metabolite profiling in feces and urine resulted in 80.5% and 11.1% of zavegepant recoveries, respectively. Comparing to the corresponding TR recoveries of 84.9% and 11.7%, it also suggests that zavegepant is the major component (~95%) in urine and feces. It was noted that the cumulative urinary recovery of zavegepant was 6.4% based on zavegepant urine pharmacokinetic data vs. the 11.1% from the metabolite profiling. This minor discrepancy could be a result of variability in assay methods and sample processes.

The geometric mean whole blood to plasma TR concentration ratios ranged from 0.56 at 0.5 h to 3.18 at 24.25 h postdose. The increase in the ratio of TR in whole blood to plasma over time may be due to a unique radiolabel moiety, perhaps a minor metabolite or impurity, that is slightly more retained in whole blood than parent, such that the radioactivity associated with the cellular component declines marginally slower than the radioactivity in the plasma, and this is manifest in the calculated ratios.

An IV infusion of a single 5 mg [¹⁴C]‐zavegepant dose was well tolerated in the six healthy males subjects. No deaths, serious adverse events, or TEAEs leading to study discontinuation were reported. Furthermore, zavegepant was not associated with any new safety concerns, including ECG, vital signs, and clinical laboratory assessments. These safety findings are similar to those reported following intranasal zavagepant, which is ~5% bioavailable. ¹⁴

Of note, the geometric mean C _max for plasma zavegepant in this study (389 ng/mL) was ~10‐fold the originally projected value (i.e., 37 ng/mL following a 5 mg IV infusion) and ~11‐fold the previously observed highest geometric mean C _max in human clinical trials (i.e., 33.95 ng/mL following 20 mg intranasal administration). ²¹ The geometric mean AUC_0‐inf for plasma zavegepant in this study was ~4‐fold the highest geometric mean AUC_0‐inf in human clinical trials (i.e., 89.10 ng*h/mL following 20 mg intranasal administration). ²¹ However, the higher exposures following IV dosing in humans are still lower than the exposures observed at no‐observed‐adverse‐effect level (NOAEL) doses in preclinical rat and monkey species (data on file, Pfizer Inc.). Consistent with this observation, the observed plasma zavegepant exposures are within the safety margins based on toxicology studies, and no safety issues were noted. This provides further reassurance regarding the safety of high systemic zavegepant exposures such as in instances of overdose.

In conclusion, following administration of 5 mg [¹⁴C]‐zavegepant as an IV infusion to healthy males, an average of 96.6% of the TR administered was recovered in excreta. Although zavegepant was administered IV in this study, most of the TR was recovered in feces (84.9%), indicating that biliary/fecal elimination was the main route and that zavegepant undergoes minimal metabolism. The pharmacokinetic profile of IV zavegepant included a volume of distribution that was higher than total body water, indicating substantial distribution into tissue; an identical total plasma clearance and whole blood clearance lower than typical hepatic blood flow; and a mean elimination plasma t _1/2 of 6.8 h. The mean t _1/2 for plasma TR was only slightly longer than the t _1/2 observed for zavegepant. Metabolite profiling identified unchanged zavegepant as the primary component and no major metabolites in plasma, urine, or feces. A single IV infusion of 5 mg [¹⁴C]‐zavegepant was safe and well tolerated in healthy male subjects. The disposition findings of IV [¹⁴C]‐zavegepant are applicable to the disposition of the approved zavegepant nasal spray.

AUTHOR CONTRIBUTIONS

Ra.B., Ri.B., R.C., and J.L. wrote the manuscript; Ra.B., M.D., J.M., M.S.A., Ri.B., and R.C. designed the research; M.D., J.M., and R.C. performed the research; Ra.B., M.D., J.M., M.S.A., K.M., Ri.B., R.C., and J.L. analyzed the data.

FUNDING INFORMATION

This study was sponsored by Biohaven, which was acquired by Pfizer in October 2022.

CONFLICT OF INTEREST STATEMENT

Rajinder Bhardwaj and Matt S. Anderson are employees of Certara who were paid consultants to Biohaven, acquired by Pfizer in October 2022, for this study. Mary Donohue, Jennifer Madonia, and Richard Bertz are current or former employees of Biohaven, acquired by Pfizer in October 2022, for this study. Robert Croop was an employee of Biohaven Pharmaceuticals, owns stock in Biohaven, Ltd., was an employee of Pfizer, has received research payments from Pfizer, and provides services to Collima, LLC, which has had consulting agreements with Pfizer, Actio Biosciences, Inc., Aptose Biosciences Inc., Biohaven Pharmaceuticals, Inc., Manistee Therapeutics, and Vida Ventures Management Co., LLC. Kyle Matschke and Jing Liu are employed by and hold stock/options in Pfizer Inc.

DATA AVAILABLE STATEMENT

Upon request, and subject to review, Pfizer will provide the data that support the findings of this study. Subject to certain criteria, conditions, and exceptions, Pfizer may also provide access to the related individual de‐identified subject data. See https://www.pfizer.com/science/clinical‐trials/trial‐data‐and‐results for more information.

Supporting information

Figure S1.

Bhardwaj R, Donohue M, Madonia J, et al. Mass balance and pharmacokinetic characterization of zavegepant in healthy male subjects. Clin Transl Sci. 2024;17:e70015. doi: 10.1111/cts.70015