A Phase 1 Microtracer Study Evaluating the Mass Balance, Excretion, and Pharmacokinetics of Osivelotor in Healthy Participants

ABSTRACT

Osivelotor (GBT021601) is an orally bioavailable, small molecule hemoglobin polymerization inhibitor in clinical development for the treatment of sickle cell disease (SCD). In this Phase 1 study, the disposition of osivelotor was assessed using accelerator mass spectrometry (AMS) after a single oral 200‐mg dose containing a microtracer amount of [¹⁴C]‐labeled osivelotor (~74 kBq [~2 μCi]) was received by nine healthy volunteers. Due to a long half‐life (27–30 days), continuous and non‐continuous sample collection was needed over a nearly 7‐month study. The results indicate an average 87.6% (47.2% in urine; 40.4% in feces) recovery of the dose in excreta. Comparison of osivelotor and total radioactivity (TRA) systemic concentration versus time profiles in whole blood and plasma suggests the absence of a long‐lived metabolite. Osivelotor was the main circulating drug‐related material in plasma (68.1% of [¹⁴C]). Also observed in plasma was a glucuronide metabolite (19.7% of [¹⁴C]). In whole blood, the effect compartment, only osivelotor (98.3% of [¹⁴C]) was present and no metabolites were observed. The single oral 200‐mg dose of osivelotor was well tolerated. The results of this unique study design provide insights into the absorption, metabolism, and excretion (ADME) of osivelotor and support its continued development for SCD.

Trial Registration: ClinicalTrials.gov NCT05718687

Study Highlights

• What is the current knowledge on the topic?

  • ○
    Osivelotor is an orally bioavailable, small molecule hemoglobin modifier in development for the treatment of SCD. This is the first publication to describe the mass balance of osivelotor and identify circulating metabolites.
• What question did this study address?

  • ○
    This study explains how osivelotor is processed in healthy participants, which is required to fulfill regulatory requirements of a New Drug Application. This study also provided human metabolite data for the interpretation of preclinical, species‐specific metabolic profiles assessed in longer‐term toxicity studies and for adequate toxicology coverage for the safe development of the drug in patients.
• What does this study add to our knowledge?

  • ○
    Assessment of osivelotor mass balance recovery, osivelotor and TRA pharmacokinetics, and metabolite profiling following a single dose of [¹⁴C]‐osivelotor showed balanced excretion between feces and urine. No metabolites were observed in the effect (whole blood) compartment, and the only major metabolite observed in plasma was the aliphatic glucuronide.
• How might this change clinical pharmacology or translational science?

  • ○
    This study provides information on the disposition of osivelotor, which is being used to guide clinical development for the treatment of SCD. The design of clinical ADME studies to assess mass balance for compounds with a long PK half‐life is a challenge, requiring a balance of operational and practical considerations intended to limit inpatient stays while obtaining complete recovery of [¹⁴C] in excreta. Keys to success include microtracer dosing, continuous and non‐continuous collection of excreta, and use of AMS to measure recovery. Successful design and execution yielded a recovery of 87.6% of the dose. This unique study design can be applied to assess the mass balance of other medicines with a long PK half‐life.

1. Introduction

Polymerization of hemoglobin (Hb) is the pathophysiologic cause of sickle cell disease (SCD), resulting in hemolytic anemia, vaso‐occlusion, and progressive end organ damage with a clinical course characterized by life‐long disability and early death [1, 2, 3]. Osivelotor (GBT021601) is an orally bioavailable, small molecule, next‐generation Hb polymerization inhibitor in clinical development for treatment of SCD [4]. By binding the alpha chain of the Hb molecule, osivelotor increases Hb‐oxygen affinity and improves red blood cell (RBC) health [5]. Osivelotor has demonstrated a relatively safe and tolerable profile in a first‐in‐human Phase 1 single‐ and multiple‐ascending dose study [6]. Further, in a Phase 1b study of adult patients with SCD, multiple‐dose osivelotor treatment increased Hb concentration, reduced markers of hemolysis, and reduced the formation of sickle RBCs [6].

Preclinical studies in multiple species showed osivelotor pharmacokinetic (PK) disposition is characterized by a high degree of partitioning to RBCs [5, 7]. Volume of distribution at steady state was relatively small in whole blood and much larger in plasma, indicating that RBCs are a reservoir for osivelotor, which was expected considering osivelotor was designed to bind hemoglobin. Osivelotor's terminal elimination half‐life was long, reaching many days, and similar between whole blood and plasma.

Osivelotor was metabolically stable in mouse, rat, dog, cynomolgus monkey, and human hepatocytes, with a long half‐life and low intrinsic clearance [5]. Preliminary results of the cytochrome P450 (CYP450) phenotyping study in human liver microsomes and using human recombinant enzymes suggested that osivelotor metabolism was mediated by CYP3A4/5, and to a lesser extent CYP1A2, and uridine 5′‐diphospho‐glucuronosyltransferase (UGT) enzymes (data on file). Further, osivelotor was also shown to have a very low potential to inhibit or induce CYP enzymes [7]. In rats, unchanged osivelotor was the most abundant drug‐related material in plasma and blood accounting for 38% and 66% of total ¹⁴C following oral dosing of 10 mg/kg [¹⁴C]‐osivelotor, respectively.

Following single‐ and multiple‐ascending dose studies in healthy participants, osivelotor PK was characterized by a high blood‐to‐plasma ratio and low systemic clearance due to slow dissociation from Hb [6]. In particular, following a single 50–3000 mg dose, the shape of the plasma concentration–time curve showed relatively fast absorption. After reaching peak concentrations, osivelotor concentrations appeared to decline in a monophasic manner during the terminal elimination phase for all dose levels. The whole blood concentration–time curve was similar in shape, although a slower rise to C_max was observed compared with plasma, suggesting rapid absorption into the central compartment (i.e., plasma) followed by distribution into whole blood/RBCs. Osivelotor had a generally long terminal elimination half‐life (t _1/2), with estimates of approximately 30 and 27 days in plasma and whole blood, respectively, following a 200‐mg single dose. Exposure estimates (AUC_inf and C_max) rose with increasing dose in a generally linear manner. High blood partitioning was observed with blood‐to‐plasma ratios (based on AUC_inf) ranging from 146 to 218.

This absorption, disposition, metabolism, and excretion (ADME) study was conducted to measure the mass balance and excretion properties of osivelotor, a small molecule with a relatively long elimination half‐life. The primary objectives were to assess mass balance and provide blood, plasma, urine, and fecal samples for metabolite profiling. Additionally, the routes and rates of [¹⁴C]‐labeled osivelotor elimination were determined. Any metabolite(s) accounting for > 10% of circulating total radioactivity (TRA) were structurally identified. Safety and tolerability information for osivelotor was also collected.

2. Methods

This Phase 1, open‐label, single‐site ADME study (NCT05718687; EudraCT no: 2022–003108‐34) of osivelotor was conducted at an ICON Clinical Site (Groningen, The Netherlands) between December 22, 2022, and August 10, 2023. The study was conducted in compliance with Good Clinical Practice and enrolled healthy men and women aged 18–55 years who had a body mass index between 18.0 and 27.0 kg/m² and weighed ≥ 50 kg. Additional inclusion and exclusion criteria are listed in Table S1. Notably, the total amount of radioactivity administered to each participant was no more than 72.9 kBq (1.97 μCi). The associated radiation exposure is not anticipated to lead to a radiation burden for a human being above Category I of the International Commission on Radiological Protection [8]. This low amount is exempted from the requirement to submit human dosimetry calculations. The study protocol was approved by the local institutional review board and ethics committees, and all participants provided written informed consent before starting the study.

The study included a screening visit, which occurred between Day −28 and Day −2, a 30‐day inpatient stay period, and a follow‐up period of up to 7 months, which included up to 11 24‐h outpatient visits (Figure 1). After an overnight fast of ≥ 10 h, participants received a single oral 200‐mg dose of osivelotor containing a microtracer amount of [¹⁴C]‐labeled osivelotor (~74 kBq [~2 μCi]) on Day 1 of the inpatient stay. The [¹⁴C] label was nonspecifically distributed through the carbons of the hydroxybenzaldehyde (Figure S1). The clinically relevant 200‐mg dose was selected because the dose level was considered sufficient and necessary to allow for successful metabolite identification. Participants continued to fast for ≥ 4 h postdose.

FIGURE 1.

Study design overview. ^aParticipants received a single oral 200‐mg dose of osivelotor containing ~74 kBq [¹⁴C]‐osivelotor on Day 1 following a ≥ 10‐h overnight fast. ^bBlood samples for measurement of osivelotor PK and TRA and urine and feces samples for measurement of TRA were collected as described in the Methods section. Timeline not to scale.

Whole blood and plasma samples were collected for PK assessment of osivelotor and TRA analyses predose and postdose at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24 (Day 2), 48 (Day 3), 72 (Day 4), 96 (Day 5), 144 (Day 7), 168 (Day 8), 312 (Day 14), 480 (Day 21), and 648 h (Day 28) during the in‐patient period. Whole blood and plasma were also collected for PK analysis during the outpatient visits on Days 35, 42, 56, 70, 84, 98, 112, 136, 150, 178, and 206. Samples were also collected for metabolite identification at predose and 1, 2, 8, 12 h postdose, and at 24 (Day 2), 48 (Day 3), 72 (Day 4), 96 (Day 5), 120 (Day 6), 144 (Day 7), 168 (Day 8), 312 (Day 14), 480 (Day 21), and 648 h (Day 28) postdose during the in‐patient period. Urine samples for TRA analysis and metabolite identification were collected prior to dosing (Day −1) and after dosing from 0 to 6 h, 6 to 12 h, 12 to 24 h postdose, and thereafter at 24‐h intervals up to Day 29. Fecal samples for TRA analysis and metabolite identification were collected prior to dosing (Day −1) and from 0 to 24 h postdose, and thereafter at 24‐h intervals up to Day 29. Urine and fecal samples were also collected at 24‐h intervals starting on Days 35, 42, 56, 70, 84, 98, 112, 136, 150, 178, and 206.

Criteria for ending the collection of blood and excreta in a participant after Day 29 were: (1) ≥ 90% of the radioactive dose administered was recovered in excreta collected to date, or (2) radioactivity was undetectable in urine and feces during 2 consecutive 24‐h collection periods, or (3) the Day 206 visit was reached.

Participants were monitored for adverse events (AEs) throughout the study. Clinical safety laboratory assessments were obtained at screening and on Days −1, 7, 28, 84, and 206. Vital signs were measured at screening, predose on Day 1, and at 1, 2, 8, 12, and 24 h postdose, and on Days 35, 84, and 206. Twelve‐lead electrocardiograms (ECGs) were conducted on Day −1 and on Day 1 at predose and at 1 and 2 h postdose.

2.1. Bioanalytical Methods

The assessment of osivelotor PK in whole blood and plasma samples was performed at PPD Bioanalytical Laboratories (Middleton, US) using validated liquid chromatography with tandem mass spectrometry (LC–MS/MS) assays with analytical ranges of 500–500,000 ng/mL for whole blood and 10–10,000 ng/mL for plasma. The analysis of TRA in whole blood, plasma, urine, and fecal samples was performed by TNO (Zeist, The Netherlands) using an accelerator mass spectrometry (AMS) assay. AMS is an ultra‐sensitive analytical technique that enables the use of much lower radioactive doses (100–1000 times less than conventional methods) in human ADME studies. In this study, the sensitivity of AMS, typically in the attomole carbon range, was also leveraged to quantitate small amounts of excreted [¹⁴C]‐labeled osivelotor over a long period of time to help achieve mass balance. Additionally, the sensitivity of AMS was used to develop reconstructed [¹⁴C] chromatograms to quantitatively define the abundance of metabolites and osivelotor in various matrices [9, 10, 11]. The AMS isotope data was converted to ng‐eq/mL of whole blood, plasma, urine, or fecal homogenate. Further information on quality assurance measures for chromatograms, AMS methodology for TRA measurement, and conversion of units for AMS isotope data is provided in Supporting Information and Table S2. The TRA concentrations in urine and feces were used to determine the percentage of dose recovered in urine and feces based upon the radioactive dose administered to each participant. Metabolite identification, including the structural identification of selected metabolites derived from [¹⁴C]‐osivelotor, in whole blood and plasma samples was performed by TNO (Zeist, The Netherlands) using ultra‐performance liquid chromatography with in‐line MS, MS/MS and fraction collection. Fractions were collected at intervals varying from 6.1 to 18 s. Each resulting fraction was subsequently submitted for [¹⁴C] analysis using AMS.

2.2. Pharmacokinetics and Statistical Analysis

No formal sample size calculations were performed; the sample size of 8–10 participants was based on a conventional number considered sufficient to achieve the study objectives while accounting for potential dropouts given the length of the study. PK analyses were conducted in the PK population, which consisted of all study participants who received the single dose of osivelotor and provided ≥ 1 measurable postdose PK concentration for osivelotor or TRA in whole blood or plasma. PK exposure for osivelotor and TRA was estimated using noncompartmental methods in Phoenix WinNonlin (Certara, Princeton, NJ, US). PK endpoints for plasma and whole blood included maximum concentration (C_max), time to maximum concentration (T_max), area under the time‐concentration curve from time zero to infinity (AUC_0‐∞) and to last measurable concentration (AUC_0‐t), terminal half‐life (t_1/2), apparent total clearance (CL/F), and volume of distribution based on terminal phase (V_z/F). Statistical analysis was performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, US) and was summarized with descriptive statistics including mean, median, or geometric mean (geometric coefficient of variation [CV]). PK data were not imputed with the exception that PK concentrations below the quantifiable limit were set to zero for summarization. In addition, TRA amounts excreted were interpolated between confinement and the Day 35 visit and between subsequent visits. Urinary and fecal PK parameters were calculated using SAS and/or WNL. Excretions in mg equivalent (mg eq) for each interval were derived from the urinary and fecal concentrations and the sample collection interval volumes or weights, respectively, as described in Supporting Information.

Safety was assessed in the safety population, which consisted of all study participants who received the single dose of osivelotor. Safety was monitored throughout the study using standard measures: physical examinations, vital signs, 12‐lead electrocardiograms (ECGs), laboratory assessments, concomitant medication usage, and monitoring treatment‐emergent adverse events (TEAEs; AEs that occurred or worsened after the dose of study drug during the on‐treatment period through a minimum of Day 57 or study discontinuation). TEAEs were reviewed for their relatedness to osivelotor and classified according to the Medical Dictionary for Regulatory Activities version 25.1 and graded according to the Common Terminology Criteria for Adverse Events (CTCAE).

3. Results

3.1. Participants

Nine participants enrolled in the study and received a single oral 201.6‐mg dose of osivelotor containing 72.9 kBq (1.97 μCi) of [¹⁴C]‐osivelotor (note: the nominal dose of 200 mg/~2 μCi is referred to throughout). There were no protocol deviations reported for any participants that may have affected the mass balance outcomes. All 9 participants received a dose of osivelotor and had ≥ 1 measurable postdose PK concentration and were included in the safety and PK populations. The first participant was enrolled on December 22, 2022, and the last participant completed the study on August 10, 2023. Six participants completed the study and 3 withdrew early for personal reasons on Study Days 74, 151, and 179, respectively.

Participants were predominately male (77.8%) and white (88.9%) and ranged in age from 21 to 46 years (Table 1). Median weight was 73.7 kg (range: 53.0 to 91.2 kg). No participants reported taking any prescription medications within 14 days (or 5 half‐life) prior to dosing or any over‐the‐counter medication or herbal remedies within 7 days prior to dosing. Two participants received concomitant medications (paracetamol) for AEs of headache, and one participant received levonorgestrel for contraception during the study. No concomitant medication was anticipated to affect mass balance assessment.

TABLE 1.

Summary of demographic characteristics in the safety population (N = 9).

Parameter
Male, n (%)	7 (77.8)
Race, n (%)
White	8 (88.9)
American Indian or Alaska Native	1 (11.1)
Not Hispanic or Latino	9 (100)
Age, years
Mean (SD)	33.6 (7.42)
Median (range)	31.0 (21–46)
Height, cm
Mean (SD)	174.6 (9.81)
Median (range)	178.0 (156–185)
Weight, kg
Mean (SD)	75.18 (12.266)
Median (range)	73.70 (53.0–91.2)
BMI, kg/m2
Mean (SD)	24.54 (2.354)
Median (range)	25.40 (19.5–26.6)

Abbreviations: BMI, body mass index; kg, kilogram; m, meter; n, number of subjects; SD, standard deviation.

3.2. Mass Balance

The total recovery of radioactivity (mean [range]) following administration of a single oral 200‐mg (~2 μCi) dose of [¹⁴C]‐osivelotor over a 4920 h postdose period was 87.6% (77.0%–93.8%), with 47.2% (39.1%–55.4%) in urine and 40.4% (30.5%–48.4%) in feces (Table 2). Individual urine, feces, and total recovery of radioactivity are shown in Table S3. Mean cumulative excretion of radioactivity over time in urine and feces, and total (urine + feces) recovery, following oral administration of 200 mg (~2 μCi) [¹⁴C]‐osivelotor is illustrated in Figure 2.

TABLE 2.

Arithmetic mean (range) cumulative recovery of total radioactivity in urine, feces, and total (urine and feces combined) following a single oral 200‐mg dose of [¹⁴C]‐osivelotor over 4920 h.

	Urine %Fe N = 9	Feces %Fe N = 9	Total %Fe N = 9
Mean, %	47.2	40.4	87.6
Range, %	39.1–55.4	30.5–48.4	77.0–93.8

Abbreviation: Fe, fraction (%) of the administered radioactive dose excreted.

FIGURE 2.

Arithmetic mean (±SD) cumulative total radioactivity recovery following oral administration of [¹⁴C]‐osivelotor 200 mg (~2 μCi) in urine, feces, and total (urine and feces combined).

3.3. Pharmacokinetics of Osivelotor

3.3.1. Disposition of Osivelotor and TRA in Whole Blood

Following a single oral 200‐mg (~2 μCi) dose of [¹⁴C]‐osivelotor, osivelotor and TRA whole blood concentrations reached peak values with a median (range) T_max of 24.00 h (8.00 to 72.03 h) and 24.00 h (4.00 to 48.00 h), respectively (Table 3). Subsequently, concentrations declined in an apparent monophasic manner during the terminal elimination phase with detectable levels up to 4920 h postdose. The concentration versus time osivelotor and TRA profiles had similar overall shapes (Figure 3A [linear scale] and B [semi‐logarithmic scale]).

TABLE 3.

Pharmacokinetic parameters for osivelotor and total radioactivity in whole blood and plasma following a single oral 200‐mg dose of [¹⁴C]‐osivelotor.

	Osivelotor whole blood	TRA whole blood	Osivelotor plasma	TRA plasma
	N = 9	N = 9	N = 9	N = 9
Cmax, μg/mL, GM (CV%) a	40.06 (27.1)	34.05 (20.8)	1.468 (14.0)	2.950 (12.5)
Tmax, h, median (range)	24.00 (8.00–72.03)	24.00 (4.00–48.00)	1.000 (0.50–1.00)	1.000 (0.50–1.00)
AUC0–t, h* μg/mL, GM (CV%) b	31481.7 (20.9)	27609.6 (18.5)	130.35 (16.7)	165.7 (13.1)
AUC0–∞, h* μg/mL, GM (CV%) b , c	35290.5 (12.1)	30636.1 (10.5)	150.7 (10.8)	179.1 (8.8)
t1/2, h, arithmetic mean (range)	647.8 (526–775)	654.7 (548–834)	707.1 (574–946)	588.9 (438–793)
CL/F, L/h, GM (CV%) c	0.005713 (12.1)	0.006580 (10.5)	1.338 (10.8)	1.1258 (8.8)
Vz/F, L, GM (CV%) c	5.175 (18.3)	5.972 (15.9)	1300.6 (15.5)	906.1 (14.4)

Note: Geometric mean and geometric CV% are shown unless otherwise indicated.

Abbreviations: AUC_0‐∞, area under the concentration–time curve from time zero to infinity; AUC_0‐t, area under the concentration–time curve from time zero to last measurable concentration; CL/F, apparent total clearance; C_max, maximum observed concentration; CV%, coefficient of variance as percentage; Eq, equivalent; GM, geometric mean, SD, standard deviation; t_1/2, terminal half‐life; TRA, total radioactivity; T_max, time to C_max; V_z/F, volume of distribution based on terminal phase.

^a μgEq/mL for total radioactivity.

^b h_* μgEq/mL for total radioactivity.

^c n = 8.

FIGURE 3.

Arithmetic mean (±SD) concentration–time profiles of osivelotor and [¹⁴C]‐osivelotor following a single 200‐mg dose in healthy participants: (A) Linear scale and (B) semi‐logarithmic scale in whole blood and (C) linear scale and (D) semi‐logarithmic scale in plasma (PK population). The linear scale plots are truncated to 24 h postdose for observation of the absorption phase of the concentration versus time curve. Abbreviations: PK, pharmacokinetics; SD, standard deviation.

The geometric mean (geometric CV%) C_max for osivelotor and TRA in whole blood were 40.06 (27.1%) μg/mL and 34.05 (20.8%) μgEq/mL respectively; while the geometric mean (geometric CV%) AUC_0–∞ was 35290.5 (12.1) h_* μg/mL and 30636.1 (10.5) h_* μgEq/mL, respectively. The geometric mean (geometric CV%) V_z/F values for osivelotor and TRA were 5.175 (18.3%) and 5.972 (15.9%) L, respectively, and the geometric mean (geometric CV%) values for CL/F were 0.005713 (12.1%) and 0.006580 (10.5%) L/h, respectively. The arithmetic mean (range) t_1/2 for osivelotor and TRA in whole blood was 647.8 (526–775) and 654.7 (548–834) h, respectively. In general, the variability of exposure parameters was low, based on geometric CV%. Osivelotor accounted for approximately 115% of TRA in whole blood based on AUC_0–∞.

3.3.2. Disposition of Osivelotor and Total Radioactivity in Plasma

Arithmetic mean (SD) concentration versus time profiles for osivelotor and TRA in plasma are shown in Figure 3C (linear scale) and D (semi‐logarithmic scale). The geometric mean (geometric CV%) C_max for osivelotor and TRA in plasma were 1.468 (14.0%) μg/mL and 2.950 (12.5%) h_* μgEq/mL, respectively; while the geometric mean (geometric CV%) AUC_0‐∞ was 150.7 (10.8%) h_* μg/mL and 179.1 (8.8) h_* μgEq/mL, respectively (Table 3). The geometric mean (geometric CV%) V_z/F values for osivelotor and TRA were 1300.6 (15.5%) and 906.1 (14.4%) L, respectively, and geometric mean (geometric CV%) values for CL/F were 1.338 (10.8%) and 1.1258 (8.85%) L/h, respectively. The arithmetic mean (range) t_1/2 for osivelotor and TRA in plasma was 707.1 (574–946) and 588.9 (438–793) h, respectively. In general, the variability of exposure parameters was low, based on geometric CV%. Osivelotor accounted for approximately 84% of TRA in plasma based on AUC_0–∞.

3.4. Circulating Metabolite Profiling and Identification

In whole blood, no metabolites were observed above the threshold for identification, with osivelotor accounting for 98.3% of circulating [¹⁴C] (Table 4 and Figure S1). In plasma, metabolite profiling identified one metabolite above the threshold for identification (those representing ≥ 10% of the circulating radioactivity [“AUC” pool]). This metabolite, eluting at a nominal retention time of 18.7 min, was identified as the aliphatic glucuronide (PF‐08054154) and accounted for approximately 19.7% of [¹⁴C] in plasma (Table 4 and Figure S1). The structure of the glucuronide metabolite was confirmed by comparison of clinical samples with a metabolite standard that was structurally characterized using MS/MS and NMR. Osivelotor, eluting at a nominal retention time of 23.2 min, accounted for 68.1% of the circulating [¹⁴C] in plasma. The remaining radioactivity was distributed in chromatographic peaks that were less than 2% of the total chromatographic radioactivity observed, and therefore did not reach the threshold for identification (Table 4 and Figure S1).

TABLE 4.

Summary of major human biotransformation identified in whole blood and plasma following a single oral 200‐mg dose of [¹⁴C]‐osivelotor.

Component	Monoisotopic molecular weight, Da	Molecular formula	Retention time, min	Whole blood % of TRA (0–648 h postdose) a	Plasma % of TRA (0–648 h postdose) a
PF‐0805414 (glucuronide)	562.1799	C26H30N2O12	18.7	ND	19.7
Osivelotor	386.1478	C20H22N2O6	23.2	98.3	68.1

Abbreviations: ND, not detected; TRA, total radioactivity.

^a Represents ~55% whole blood and plasma TRA area under the curve. Multiple trace level radioactive peaks were unknown and accounted for 12.2% in total.

3.5. Safety and Tolerability

No deaths, serious TEAEs, or TEAEs leading to discontinuation were reported. A total of 7 TEAEs occurred in 4 (44.4%) participants; 1 participant reported nausea and headache that were considered related to treatment by the investigator. All TEAEs were mild severity/grade 1 events (Table S4). The only TEAE reported for more than one participant was headache. No clinically significant out‐of‐range laboratory values (for clinical chemistry, hematology, coagulation, and urinalysis) or clinically significant vital signs, ECG, or physical examination findings were identified during the study.

4. Discussion

Microtracer ADME studies, which pair a low [¹⁴C]‐labeled dose with high‐sensitivity detection AMS methodology, have been successfully applied to track drug metabolism and characterize mass balance [12]. Since such a low amount of radioactive material is used, the microtracer ADME/AMS approach allows for extended PK collection and incorporation of outpatient visits to fully characterize ADME properties for compounds with a long PK half‐life [13, 14, 15, 16]. An extended collection schedule may be necessary to achieve complete recovery of radioactivity for assessment of mass balance or, in the case of enterohepatic recirculation, obtaining additional excreta collections to assess biliary excretion [14, 16, 17]. Thus, using a microtracer ADME/AMS approach, a practical and patient‐centric study design can be created while generating a robust data capture to address key ADME study objectives.

Here, we report the results of an integrated microtracer ADME/AMS study that describes the mass balance, disposition, and circulating metabolites following a single oral administration of osivelotor, a compound with low clearance and a long PK half‐life, attributable to its high affinity for hemoglobin, and a disposition that is partly influenced by the life cycle of RBCs [5]. A clinically relevant dose of osivelotor (200 mg) was administered to healthy participants with a low dose of [¹⁴C]‐labeled osivelotor (~2 μCi) and high‐sensitivity detection using AMS methodology was employed to determine low‐level radioactivity. To achieve sufficient excreta collection, a nearly 7‐month study was designed with both continuous and non‐continuous collection periods. The approximately 1‐month inpatient period was selected to enable continuous collection of excreta over approximately 1 half‐life of osivelotor, while balancing the challenges associated with the clinical conduct of a long‐term inpatient study. The subsequent outpatient follow‐up visits enabled further non‐continuous collection of excreta until stopping criteria were met or maximum study duration was achieved. As whole blood is the site of action for this Hb‐seeking compound, measurement of osivelotor, TRA, and circulating metabolites were assessed in both whole blood and plasma matrices.

This unique study design enabled complete characterization of the mass balance of osivelotor. Recovery during the inpatient collection period was approximately 60%, which is consistent with the long half‐life of osivelotor and the absence of a long‐lived metabolite. The interpolation of results during the outpatient collection period estimated recovery of 87.6% of total radioactivity, which is consistent with complete recovery. Osivelotor was excreted in urine and feces to a similar extent, with 47.2% and 40.4% recovery, respectively. Of note, the inclusion of data for three participants who terminated the study early was not considered to meaningfully impact mass balance results and conclusions.

Following administration of a single oral dose of osivelotor, the median T_max values were similar for osivelotor and TRA in whole blood (the site of drug action), as were the estimated mean t_1/2 values (647.8 and 654.7 h, respectively, or approximately 27 days), which indicated an approximate parallel decrease in unchanged osivelotor and its metabolites. Similar data were observed for plasma. The estimated t_1/2 of osivelotor in whole blood and plasma was similar to that observed in other studies in healthy participants [6]. Parallel osivelotor and TRA concentration–time profiles suggest absence of a long‐lived metabolite(s) and support the translatability of single dose ADME findings to a once‐daily dosing scenario, where notable accumulation can occur due to a long PK half‐life [6]. Additionally, in vitro characterization has not identified any induction mechanisms that impact osivelotor, reducing the concern that osivelotor disposition would be impacted by multiple dosing.

Osivelotor was the only drug‐related component in whole blood (98.3%), the effect compartment, and no metabolites were observed. Therefore, metabolites were not contributing to the pharmacological activity following oral dosing of osivelotor. The geometric mean AUC_0‐inf for osivelotor was approximately 115% of the AUC_0‐inf for total drug‐related material in whole blood (35,291 h_* μg/mL for osivelotor vs. 30,636 h_* μgEq/mL for TRA), which is consistent with the metabolite identification results indicating limited potential for osivelotor metabolites in whole blood. Further, no active metabolites were detected in plasma. The primary metabolite observed in circulation was the aliphatic glucuronide conjugate. Glucuronide conjugates are typically less biologically active than the parent compound. Geometric mean AUC_0‐inf for osivelotor was approximately 84% of the AUC_0‐inf for total drug‐related material in plasma (150.7 h_* μg/mL for osivelotor vs. 179.1 h_* μgEq/mL for TRA), corresponding with the presence of a glucuronide metabolite in plasma.

The single oral dose of osivelotor administered in this study was well tolerated in healthy adult participants. All reported TEAEs were mild in severity. No deaths or serious TEAEs or TEAEs leading to study discontinuation were reported, and no clinically significant laboratory, ECGs, or vital sign findings were observed.

Osivelotor displays disease‐dependent PK when comparing profiles from healthy participants with those from patients with SCD. These differences are considered to be attributed to the lower abundance of Hb (the drug target) and altered health of RBCs in SCD, rather than due to differences in ADME capacity. In SCD, anemia and lower RBC lifespan are associated with reduced PK half‐life and systemic exposure relative to healthy participants [6]. The participation of patients with SCD poses ethical concerns due to the blood sampling burden and lack of clinical benefit from a single dose. Therefore, healthy participants were chosen for this study. Notably, this approach is consistent with a mass balance study of voxelotor, a compound with a similar mechanism of action [18].

In conclusion, a unique clinical study design, employing an approximately 1‐month inpatient and 6‐month additional outpatient collection, incorporating microtracer dosing and analysis by AMS was used in the ADME study. Complete recovery was achieved and the results of this study provide insights into the absorption, metabolism, and excretion of osivelotor and support its continued development for the treatment of SCD. Similar to previous examples of microtracer ADME integrated with AMS to study long PK half‐life compounds, incorporation of continuous and non‐continuous sample collection using inpatient and outpatient visits was essential to achieve complete recovery [14, 15]. Our study provides another successful application of this strategy, and with a total study duration (upwards of 206 days) this is the longest ADME study reported, to the authors' knowledge.

This design was necessary due to the long half‐life of osivelotor and balances inpatient and outpatient visits. This approach may be considered when planning ADME studies for compounds with a long PK half‐life, enabling sufficient data capture to characterize mass balance and excretion while sensitive to the practical and operational aspects of a healthy participant Phase 1 clinical trial. Future analyses will include identification of metabolic pathways and reaction phenotyping to determine enzymes responsible for metabolism.

Author Contributions

E.I.Z., J.V., A.A., C.Z. and K.D. designed the research; E.I.Z., E.C., G.S.W., R.S., J.V., A.A., K.S., K.D. and G.K. performed the research; E.I.Z., E.C., J.V., A.A., K.S. and G.K. analyzed the data; E.I.Z., E.C., G.S.W., R.S. and G.K. wrote the manuscript.

Funding

This study was sponsored by Pfizer, New York, NY, USA.

Disclosure

Prior Presentation: Presented in part at the American Society for Clinical Pharmacology and Therapeutics Conference, May 28–31, 2025, Washington, DC.

Conflicts of Interest

Eric I. Zimmerman: current employee of Pfizer; stock or stock options for Pfizer. Ernesto Callegari: current employee of Pfizer; stock or stock options for Pfizer. Raman Sharma: current employee of Pfizer; stock or stock options for Pfizer. Adeyemi Adenola: past employee of Pfizer; stock or stock options for Pfizer. Jessica Volpe: current employee of Pfizer; stock or stock options for Pfizer. Karthik Seetharaman: current employee of Pfizer; stock or stock options for Pfizer. Chenghong Zhang: current employee of Vividion Therapeutics. Kenneth Duchin: consultant for Global Blood Therapeutics Inc., which was acquired by Pfizer Inc. on October 5, 2022. Gregory S. Walker: current employee of Pfizer; stock or stock options for Pfizer.

Supporting information

Table S1: Eligibility criteria.

Table S2: Determination of total [¹⁴C] concentrations by AMS analysis in human plasma, urine, whole blood and fecal samples.

Table S3: Individual cumulative recovery of total radioactivity in urine, feces, and total (urine and feces combined) following a single oral 200 mg dose of [¹⁴C]‐osivelotor over 4920 h.

Table S4: Participants with treatment‐emergent adverse events.

Figure S1: Representative reconstructed ultra high‐performance liquid chromatography‐accelerator mass spectrometry chromatograms of (A) pooled plasma and (B) pooled blood extracts from healthy volunteers following oral dosing of [¹⁴C]‐osivelotor at 0–648 h. The numbers in parentheses indicate the amount of ¹⁴C in a specific chromatographic peak as a percentage of the ¹⁴C observed in the total chromatogram.

Data Availability 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 participant data. See https://www.pfizer.com/science/clinical‐trials/trial‐data‐and‐results for more information.