Disposition of orally administered atuliflapon, a novel 5‐lipoxygenase‐activating protein inhibitor in healthy participants

Abstract

In this study, the mass balance, pharmacokinetics (PK) and metabolism of atuliflapon, a novel 5‐lipoxygenase‐activating protein inhibitor, were investigated in healthy male subjects. A single oral dose of 200 mg [¹⁴C]atuliflapon suspension was administered to six healthy male subjects. Mass balance, PK and metabolite profiles of atuliflapon were analyzed using radioactivity monitoring and liquid chromatography with mass spectrometry analysis. The safety of atuliflapon was assessed during the study. Atuliflapon was rapidly absorbed with a median tmax of 1.5 h, followed by a biphasic decline in plasma exposure rendering a terminal half‐life of ~20 h. Unchanged atuliflapon was the predominant radioactive component in plasma, accounting for 40.1% of the total drug‐related exposure (DRE), while a direct N‐glucuronide was the only metabolite exceeding 10% of DRE, accounting for 20.9%. Renal excretion of intact atuliflapon accounted for <1% of the administered dose. In total 85.2% of administered radioactivity was recovered over 312 h with 79.3% and 5.9% in feces and urine, respectively. Parent atuliflapon contributed to approximately 40% of the recovered dose in excreta, while metabolites resulting from phase 1 oxidative pathways accounted for more than 30% of the excreted dose. Overall, a single oral dose of 200 mg [¹⁴C]atuliflapon suspension was well tolerated in healthy male subjects. The human metabolism and disposition data obtained will support future development and submissions of atuliflapon as a potential candidate drug for the treatment of cardiovascular, cardiorenal, and respiratory indications.

Proposed metabolic pathways for atuliflapon in humans. Asterisk (*) designates the site of 14C labelling.

1. INTRODUCTION

Leukotrienes (LTs) are lipid mediators derived from arachidonic acid and are reported to have potent inflammatory and vasoactive properties. ¹ , ² A potential link between LTs and cardiovascular disease was initially proposed following the observation that enhanced vasoconstrictive responses to LTC₄ and LTD₄ occur in coronary arteries that have developed atherosclerotic plaques. ³ This observation has been extended by additional studies elucidating the role of LTs in cardiovascular disease, by expression and activity of the 5‐lipoxygenase (5 LO)‐pathway being associated with atherosclerotic plaque progression and symptoms of plaque instability, that could lead to plaque rupture, thrombosis and myocardial infarct (MI). ⁴ , ⁵ Furthermore, the elevated levels of circulating LTB₄ and urinary LTE₄ were shown in association with symptoms of myocardial ischemia in patients with acute coronary syndrome. ⁶ , ⁷ It has been reported that the first step of the biosynthesis of LTs is via 5‐LO enzymes, and the integral protein 5‐lipoxygenase‐activating protein (FLAP) is a necessary partner for the activation of 5‐LO for this process. ¹ Helgadottir et al showed that genetic haplotypes in the FLAP gene (ALOX5AP) have been significantly associated with the risk of MI. ⁸ The inhibition of FLAP could also have beneficial effects to address the multifactorial nature of cardiorenal disease, as inflammatory drive has been considered associated with diabetic nephropathy, where 5‐LO and FLAP genes are overly expressed in tubulointerstitial tissue from patients with chronic kidney disease (CKD), designating LTs as a potential culprit in CKD. ⁹ In general, FLAP inhibitors are expected to act as broad‐spectrum LT‐modifier drugs resulting in the loss of LT production, and therefore become attractive candidates that may have a wide range of therapeutic applications. ¹⁰

Atuliflapon (previously named as AZD5718) is a novel FLAP inhibitor that binds selectively and reversibly to the FLAP protein, shown in cell assay in vitro, as well as potently suppresses LT (e.g., LTB₄, LTC₄) production in human whole blood. ¹¹ Effective inhibition (>80%) of the LTs have been shown in healthy volunteers, ¹² , ¹³ patients with a recent MI (FLAVOR (NCT03317002)), ¹⁴ and CKD patients (FLAIR (NCT04492722)). ¹⁵ , ¹⁶ In addition, a phase 2 study evaluating safety and efficacy of atuliflapon in patients with asthma (NCT05251259) is ongoing. There is clinical experience with other FLAP inhibitors that have shown reduced LTB₄ production in ex vivo stimulated blood and to lower urinary LTE₄ levels. ² , ¹⁷ , ¹⁸ The 5‐LO inhibitor zileuton (Zyflo®) is approved for treatment of mild‐to‐moderate asthma, ¹ however, atuliflapon is the only FLAP inhibitor in clinical development to the best of our knowledge.

This study was performed in healthy participants following a single oral dose of carbon‐14 labeled atuliflapon to support further clinical development and potential regulatory approval process of atuliflapon, where the assessment of absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetics (PK) are key supportive data. ¹⁹ The primary objectives of this study were: to determine the mass balance including routes and rates of elimination, to characterize metabolite profiles in plasma, urine and feces; and to assess the PK of atuliflapon and total radioactivity. Furthermore, human ADME data is essential to guide the design of clinical drug–drug interaction studies and evaluate exposure to metabolites in human in safety testing. ¹⁹ Secondary objectives in the present study included assessing the extent of distribution of total radioactivity into blood cells, and providing additional information on the safety and tolerability after a single dose of atuliflapon.

2. MATERIALS AND METHODS

2.1. Materials

The unlabeled atuliflapon was manufactured by AstraZeneca UK, and the [¹⁴C]atuliflapon was manufactured at Pharmaron UK Ltd. (Rushden, UK), from which Quotient Sciences (Nottingham, UK) produced the oral [¹⁴C]atuliflapon suspension for drug administration at a dose of 200 mg (8 MBq, 216 μCi). The structure of atuliflapon and the position of C‐14 labelling are shown in Figure 1.

FIGURE 1.

Proposed metabolic pathways for atuliflapon in humans. Asterisk (*) designates the site of ¹⁴C labelling.

2.2. Other chemicals

Methanol (MeOH) and acetonitrile (ACN) (LC/MS grade) were obtained from Fisher Scientific (Loughborough, UK). Ultra‐pure water was prepared by an in‐house water purification system (Milli‐Q, Integral 3, Millipore Co., Solna, Sweden). Ultima Gold liquid scintillation counting (LSC) cocktail was obtained from PerkinElmer (PerkinElmer Inc., MA, USA). All other solvents and reagents were at least of analytical grade and were acquired from commercial suppliers.

2.3. Dosing, subjects, and study design

Single oral doses up to 1200 mg have been shown to be safe and well tolerated in a previous clinical study, ¹² and a dose of 200 mg is expected to yield clinically relevant exposures in the treatment of cardiovascular diseases. Therefore, a single oral dose of 200 mg was selected for this study. The dose of radioactivity was determined following review of human dosimetry calculations provided by Public Health England (PHE). ²⁰ The expected radiation exposure was estimated according to the recommendations of the International Commission on Radiologic Protection. The individual oral [¹⁴C]atuliflapon suspension dose was 8 MBq (216 μCi, 2.3 mSv). In accordance with the “as low as reasonably practicable” principle, this was close to the minimum practicable amount of radioactivity considered sufficient to adequately assess the metabolite profile and achieve the objectives of the study. This exposure fell within World Health Organization (1977) Category II for radioactive dose (0.5 mSv–5 mSv) which is stated to be a risk being “within dose limits for members of the public.”

This was a single center, open‐label, single period study to investigate the ADME after a single oral dose of 200 mg [¹⁴C]atuliflapon in six healthy male subjects between 30 to 65 years old. Informed consent was obtained from each subject in writing before any assessment was performed. This study was conducted at Quotient Sciences, Nottingham, UK, under Good Clinical Practices and in compliance with the Helsinki Declaration. The study protocol and consent form were reviewed and approved by the independent Office for Research Ethics Committees Northern Ireland (ORECNI) and the study was registered (ClinicalTrials.gov: NCT03948451).

Eligible subjects were admitted to the clinical unit in the evening on the day before dosing (Day −1). Each subject received a single oral dose of 200 mg [¹⁴C]atuliflapon suspension in the morning of Day 1 after an 8 h overnight fast, and continuing 4 h post‐dose. Whole blood, and plasma samples were collected at the following time points: pre‐dose, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post‐dose. Urine samples were collected over the following intervals: pre‐dose, 0–6, 6–12, 12–24, 24–48, and 48–72 h post‐dose; and at 24‐h intervals thereafter until the study discharge criteria had been met. Similarly, fecal samples were collected at 24‐h intervals until discharge criteria were met. Subjects remained at the clinical unit for up to 7 days post‐dose and urine and fecal samples were collected consecutively until the accumulated excretion rates of two consecutive collections each had radioactivity levels less than 1.0% of the total administered radioactivity. As the mass balance criteria were not met by all subjects at 168 h post‐dose (Day 8), the residency period was extended to the maximum additional 48 h (Day 10) for further collection of urine and feces for the analysis of total radioactivity and metabolite profiling and identification, as appropriate. Further home collections of feces were requested of two subjects up to 312 h post‐dose at the discretion of the investigator until the stop criteria was met. The tolerability and safety assessments involved evaluation of vital signs, electrocardiogram (ECG), telemetry, routine clinical laboratory examinations, and adverse events. These assessments were taken before and at scheduled times after dosing and when discharged from the clinic. A follow up call was done 7–10 days after discharge from the clinic.

2.4. Total radioactivity measurement

Scintillation cocktail was added directly to plasma and urine samples, whereas samples of whole blood were solubilized, and then decolored, and fecal samples were dried and combusted before the addition of scintillation cocktail and analysis by LSC analysis. Detailed summary of the procedure used for detection of radioactivity is described in the Supplemental Methods. Samples were analyzed in duplicate on a PerkinElmer Tri‐Carb 3100 scintillation counter (PerkinElmer Inc., Waltham, MA) with automatic external standard quench correction to determine total ¹⁴C radioactivity. The limit of quantification using LSC was taken as twice the background disintegrations per minute (dpm) value for samples of the same type.

2.5. Determination of blood‐plasma partitioning

The extent of distribution of total radioactivity into blood cells was evaluated by assessing the total radioactivity in whole blood to plasma ratio.

2.6. Metabolite profiling and identification

2.6.1. Sample preparation of plasma, urine, and feces

Plasma. For each time point across subjects (n = 6), pools of plasma were generated by taking equal volumes for each subject and time point. The pooled samples were then used to generate an area under the curve (AUC) pool in a time proportional manner (0–24 h). ²¹ Thus, one plasma sample, referred to as the AUC_0‐24h pool, represented the average AUC of atuliflapon and its metabolites in six subjects over the first 24 h post‐dose administration. Pooled plasma samples (1000 μL) were protein precipitated with three volumes of ACN:MeOH (1:1, v/v). After vortex mixing each sample was centrifuged at 10000g, 4°C, for 10 min and the supernatant was transferred to a new sample vial and concentrated to approximately 100 μL under a steady flow of nitrogen. An aliquot of 40 μL ACN:MeOH (1:1, v/v) was added to the vial followed by vortex mixing for 1 min. The sample was again centrifuged, and the final supernatant was analyzed by LSC for determination of extraction recovery of radioactivity or injected to the analytical system to generate liquid chromatography‐high‐resolution mass spectrometry (LC‐HRMS) data and off‐line radio chromatograms.

Urine and feces. After total radioactivity measurement, urine and fecal samples excreted between 0 and 24 h and 0–216 h, respectively, were pooled proportionally to the total weight of the eliminated material from each sampling interval. The final pools of urine_0‐24h and faeces_0‐216h across six subjects were analyzed. The urine pools were centrifuged and supernatants were analyzed directly or diluted with one volume of 20% ACN:MeOH (1:1, v/v) before injected into the LC column for LC‐HRMS analysis and off‐line radioactivity monitoring (RAM).

Pooled fecal homogenates (ca. 0.2 g) were weighed and placed in Precellys 2 mL reinforced tubes (Bertin Corp., MD, USA) preloaded with six 3‐mm diameter ceramic balls per tube. An extraction mixture of ACN:MeOH (1:1, v/v) was added to sample tubes at a ratio of 4:1 of organic solvent to fecal homogenate weight. The samples were homogenized and extracted using Precellys24 homogenizer (Bertin Corp., MD, USA). The mixtures were then centrifuged, and the supernatants were transferred to a new sample vial and an aliquot was analyzed by LSC for the determination of extraction recovery. An aliquot of 60 μL of the supernatant was diluted by 90 μL of water before injected into LC column for LC‐HRMS analysis and off‐line radioactivity measurementsRAM.

2.6.2. LC‐HRMS‐RAM analysis

Metabolite profiling and identification were performed using LC‐HRMS‐RAM analysis. RAM was used for recording of metabolite profiles and quantification, and MS for structural elucidation of metabolites. MassLynx and Metabolynx 4.1 were used for LC‐HRMS data analyses and metabolite identification. Details of the applied LC‐HRMS conditions are described in the Supplemental Methods. In brief, sample separation was conducted using reverse phase UPLC chromatography. After sample injection, the LC eluent was split with one part (1/7, v/v) directed into the mass spectrometer and the remaining six parts (6/7, v/v) transferred into 96‐well plates (LumaPlate‐96 Deep‐Well, PerkinElmer, Waltham, MA, USA) for fraction collection throughout the chromatographic run. After the microplates were dried in a ventilated area at room temperature, the radioactivity was determined using a microplate scintillation counter (TopCount NXT, PerkinElmer Inc., MA, USA). Counting results were imported to the software Laura (version 6, LabLogic, Sheffield, UK) to construct radiochromatograms for peak integration after background subtraction.

2.6.3. Data analysis for metabolite quantification and characterization

Parent compound and metabolites were quantified as percent of the eluted radioactivity in the analyzed samples. In combination with the obtained recovered dose in the excreta, the abundance of atuliflapon and metabolites in urine and feces was calculated and expressed as percent of administered dose for the pooled collection intervals. The relative abundance of atuliflapon and metabolites in plasma is reported as % of AUC_0‐24h. HRMS precursor molecular ions of atuliflapon and metabolites were identified at retention times which agreed with peaks in the corresponding radiochromatograms, and was used to propose molecular compositions. Product ion spectra acquired by MS/MS were used to propose metabolite structures. In the positive MS detection mode, parent compound and its metabolites underwent significant in‐source fragmentation resulting in the neutral loss of H₂O (18 Da) from the protonated molecules. Therefore, MS detection was also performed in the negative ionization mode to confirm the molecular weight of the detected metabolites of atuliflapon.

2.7. Pharmacokinetic analysis of atuliflapon

2.7.1. Sample analysis of atuliflapon in plasma and urine

Validated LC–MS analytical methods used in this study to determine atuliflapon concentrations in human plasma and urine have been reported earlier. ¹² , ¹³ Atuliflapon and the stable isotope carbon‐13 labeled internal standard [¹³C₆]AZD5718 were extracted from plasma by protein precipitation and from urine by sample dilution before analysis by liquid chromatography followed by tandem mass spectrometry (LC–MS/MS). The lower limit of quantification (LLOQ) of atuliflapon in plasma and urine was 1 nmol/L and 20 nmol/L, respectively.

2.7.2. Pharmacokinetics assessment

The PK parameters for atuliflapon in plasma and total radioactivity in plasma and whole blood, as well as urine, were estimated for each subject by non‐compartmental methods using Phoenix WinNonlin software (v8.1, Certara USA, Inc., USA). The AUC was calculated by log‐linear trapezoidal rule from time 0 to the time for the last measurable concentration (t_last) plus the extrapolated residual area to infinity. The residual area after t_last was calculated as C_last, pred/λ_Z, where C_last, pred was the predicted concentration at t_last and λ_Z was the terminal rate constant determined by linear regression analysis of ln plasma/blood concentration versus time, using the last concentrations (at least 3 points) from each participant. For the rest of the PK parameters descriptive statistics and graphical visualization of the PK data were considered adequate for a study of this type.

2.8. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, ²² and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24. ²³

3. RESULTS

3.1. Demographics

A total of 23 subjects were enrolled into the study (informed consent received), 17 subjects were screening failures and six subjects received study drug and completed the study. These healthy male subjects were between 39 and 58 years of age and the mean body weight was 80 kg with a mean body mass index (BMI) of 27 kg/m². A summary of the demographic data is presented in Table S1.

3.2. Mass balance and excretion

Of the administered radioactivity, the total mean recovery in urine and feces was 85.2% (range: 80.1–90.4%) in the six subjects, of which 79.3% and 5.9% was recovered in feces and urine, respectively. The majority of the administered radioactivity was recovered within the first 12 and 144 h in urine and feces, respectively. Cumulative recovery of total radioactivity in urine and feces is depicted in Figure 2 and Table S2.

FIGURE 2.

Mean (SD, N = 6) cumulative excretion of total radioactivity following a single oral administration of 200 mg [¹⁴C]atuliflapon suspension.

3.3. Pharmacokinetics

3.3.1. Pharmacokinetics of atuliflapon in plasma and urine

Following oral administration of 200 mg [¹⁴C]atuliflapon, the parent compound was rapidly absorbed with a median time to maximum plasma concentration (t_max) of 1.5 h (range: 1.0 h–2.0 h), Figure 3. The PK properties of atuliflapon and total radioactivity in plasma and whole blood are summarized in Table 1.

FIGURE 3.

Total radioactivity (TR) in whole blood and plasma and atuliflapon plasma over time after a single oral dose of 200 mg [¹⁴C]atuliflapon suspension, N = 6.

TABLE 1.

Geometric mean (geometric CV%) plasma and whole blood pharmacokinetic parameters following a single oral dose of 200 mg [¹⁴C]atuliflapon suspension in healthy participants.

Analyte	Atuliflapon	Total Radioactivity
Matrix	Plasma	Plasma	Whole blood
Parameter	N = 6	N = 6	N = 6
AUClast (h*nmol/L or h*nmol equiv/L)	5023 (17.4%)	10 580 (22.5%)	6988 (10.4%)
AUC0‐inf (h*nmol/L or h*nmol equiv/L)	5080 (17.3%)	11 580 (21.5%)	8131 (11.3%)
Cmax (nmol/L or nmol equiv/L)	1035 (25.2%)	1748 (15.6%)	1134 (11.2%)
tmax a (h)	1.5 (1.0‐2.0)	2.0 (1.0–2.0)	3.0 (1.0–3.0)
t½λz (h)	19.7 (37.0%)	5.6 (43.7%)	3.9 (4.1%)
CL/F (L/h)	93.9 (17.3%)	NC	NC
CLR (L/h)	0.6 (21.2%)	NC	NC
Vz/F (L)	2662 (46.9%)	NC	NC

Abbreviation: NC, not calculated.

^a Median (range).

After maximum plasma concentration (C_max), subsequent plasma concentrations of atuliflapon declined in a biphasic manner and remained quantifiable until 72 h post‐dose in 5 of the 6 subjects and until 144 h post‐dose in one subject. The geometric mean half‐life was 19.7 h (range: 14.1–39.2 h), with geometric mean apparent plasma clearance (CL/F) of 93.9 L/h (geometric coefficient of variation (CV%): 17.3%) and the geometric mean apparent volume of distribution during the terminal phase (V_z/F) was 2662 L (geometric CV%: 46.9%). The inter‐subject variability was moderate for C_max, with a geometric CV% of 25.2%, and low for AUC_last and AUC_0‐inf, with values of 17.4% and 17.3%, respectively. All plasma concentration vs. time profiles were well defined with <2% extrapolated. Renal clearance (CL_R) was low with a geometric mean of 0.6 L/h (geometric CV%: 21.2%).

3.3.2. Pharmacokinetics of total radioactivity in plasma and whole blood

The appearance of total radioactivity in plasma was rapid, with a median t_max of 2.0 h (range: 1.0 h–2.0 h), see Figure 3. Total radioactivity was quantifiable at the first post‐dose time point (i.e., 15 min post‐dose) for all but one subject. After C_max, plasma concentrations of total radioactivity declined in an essentially monophasic manner with a mean apparent half‐life of 5.9 h and remained quantifiable until between 12 h and 24 h post‐dose, Table 1. The plasma total radioactivity vs. time profiles were well defined with <11% extrapolated and the inter‐subject variability was low to moderate for C_max, AUC_last and AUC_0‐inf with geometric CV% of 15.6%, 22.5% and 21.5%, respectively.

The median t_max for appearance of total radioactivity in whole blood was 3.0 h (range: 1.0 h–3.0 h), see Figure 3. After C_max, whole blood concentrations of total radioactivity declined in an essentially monophasic manner with a mean apparent half‐life of 3.9 h (SD: 0.2 h) and remained quantifiable up to 12 h post‐dose that is, to the final PK sampling time point for whole blood. The whole blood total radioactivity vs. time profiles were well defined with <17% extrapolated and the inter‐subject variability was low for C_max, AUC_last and AUC_0‐inf, with geometric CV% of <15% for these parameters.

3.3.3. Blood‐plasma partitioning of total radioactivity

The geometric mean of total radioactivity in whole blood to plasma ratio ranged between 0.74 and 0.88 between 1 h and 12 h post‐dose. The overall range, based on individual subject data was between 0.67 and 1.04 with no apparent trend over time. The data indicate that atuliflapon‐related compounds distribute into red blood cells although slightly more atuliflapon‐related material was circulating in the plasma compartment.

3.4. Metabolite profiling and characterization of atuliflapon

3.4.1. Metabolite profiling

Fourteen metabolite fractions were detected by RAM of which 11 were assigned structures based on MS data. The metabolite fractions to which no MS data could be assigned are denoted MX1‐MX3 and may comprise one or more metabolites each. Quantitative estimates are presented in Table 2 and metabolite profiles are presented in Figure 4.

TABLE 2.

Quantitative estimates (% of dose) of atuliflapon and metabolites in samples collected following a single oral dose of 200 mg [¹⁴C]atuliflapon suspension in healthy participants.

Compound	LC‐RAM Retention tR (min)	Urine0‐24h (% Dose)	Faeces0‐216h (% Dose)	Plasma0‐24h (% AUC0‐24h)
MX1	2.0	NQ a	NQ	4.9
MX2	2.8	0.5	NQ	NQ
M2	3.6	0.6	ND b	ND
M19	7.5	0.2	0.9	ND
M10	8.3	0.3	3.3	NQ
M11	11.3	0.4	1.5	NQ
M7	13.5	1.4	NQ	20.9
M1	14.0	NQ	1.8	NQ
M3/M4	16.2	0.6	19.1	4.9
M5	18.7	NQ	1.7	NQ
M6	21.7	0.2	1.2	NQ
M8	27.7	0.2	NQ	NQ
Atuliflapon	33.6	0.4	38.8	40.1
MX3	37.3	NQ	NQ	7.4
Sum c		4.8	68.3
Total d		5.4	76.3

^a NQ, detected by MS but not quantified due to below the limit of detection (10 cpm) in radiochromatogram.

^b ND, not detected by either RAM or MS.

^c Sum of integrated radioactive peaks in the pooled intervals expressed as percent of administered dose.

^d Total radioactivity in each analyzed sample pool expressed as percent of administered dose.

FIGURE 4.

Metabolite profiles of pooled plasma (0–24 h AUC pool), urine (0–24 h), and feces (0–216 h) following a single oral administration of [¹⁴C]atuliflapon suspension in healthy male participants.

Unchanged atuliflapon contributed to 40.1% of the total radioactivity in the 0–24 h pooled plasma. Eleven metabolite fractions were detected in plasma, of which metabolite M7, a direct N‐glucuronide, was the most abundant component, representing 20.9% of drug‐related exposure (DRE). Metabolite fraction MX3, accounted for 7.4% of DRE. The remaining radioactivity was distributed over several minor peaks or attributed to noisy chromatographic background. None of the minor peaks individually accounted for more than 5% of the radioactivity in the sample.

Approximately 5.9% of the administered dose was excreted in urine within 0–216 h. The sample analyzed for metabolite profiling was a pool across the first 24 h after dosing, representing 5.4% of the dose, with atuliflapon representing 0.4% of the dose in this interval. The radiochromatograms indicated the formation of 10 metabolite fractions in urine, of which metabolite M7 was the most abundant metabolite, representing 1.4% of the dose. The other metabolites detected each accounted for 0.2 to 0.6% of the dose. The remaining radioactivity was attributed to noisy chromatographic background and accounted for less than 0.1% of dose.

Approximately 79.3% of the administered dose was detected in feces within 0–312 h. The sample analyzed for metabolite profiling was a pool across the first 216 h after dosing, representing 76.3% of the dose. Unchanged atuliflapon was the predominant fraction in the pooled feces 0–216 h accounting for 38.8% of the dose and eight metabolite fractions were identified. The second largest fraction constituted two coeluting mono‐oxidized metabolites, M3 and M4, which together accounted for 19.1% of the dose. Metabolite M10 (oxidation & ketone reduction) was the third most abundant metabolite representing 3.3% of the dose. The other five metabolites each accounted for 0.9%–1.8% of the dose, and the remaining radioactivity in the sample was distributed over several minor peaks or attributed to noisy chromatographic background that was less than 1.0% of the administered dose in total.

3.4.2. Metabolite identification

Tentative transformations and structures of metabolites are shown in Figure 1. Several monohydroxylated metabolites (M1, M3‐M6) were identified in urine and feces, as well as their secondary metabolites, in total accounting for over 30% of the DRM in excreta, suggesting that oxidation is a significant phase I biotransformation reaction of atuliflapon. All of the monohydroxylated metabolites were detected in plasma. The MS/MS data suggests that the hydroxylated metabolites M1 and M3‐M5 were diastereomers and the hydroxylation occurred on the cyclohexyl moiety of the molecule. Metabolite M3 and M4 were co‐eluting radiochromatographic peaks, however, they were partially separated by LC–MS detection. The MS/MS data of M6 shows that the hydroxylation occurred on the methylpyrazole moiety of atuliflapon. Metabolite M19 were dihydroxylated metabolite having two hydroxyl groups in the cyclohexyl ring. Ketone reduction of parent atuliflapon formed metabolite M8, that was detected in urine and feces although at low levels. Further oxidation of the hydroxylated metabolite M6 formed the corresponding carboxylic acid M11. Metabolite M10 was formed via the combination of ketone reduction and hydroxylation of the cyclohexyl ring of the parent molecule. Two phase II metabolites of atuliflapon, an N‐glucuronide M7 and a glucuronyl conjugate of a hydroxylated metabolite M2, were identified. The N‐glucuronide M7 was the most abundant metabolite detected in urine and plasma. Three metabolite fractions, MX1‐MX3, were identified by RAM, however no MS data could be assigned that indicated relation to atuliflapon. The relatively large portion of the radioactivity in plasma eluting after atuliflapon, that is, MX3, constituting 7.4% of AUC_0‐24h was found to be attributed to several metabolite fractions. The pooled 2 h plasma sample, containing MX3, was used as a representative sample and analyzed using a shallower LC gradient (Figure S1). The profile thereof clearly indicated that fraction MX3 was composed of several compound‐related products that were separated to several minor peaks, each of which were below the limit of quantification (10 cpm). No further work was performed to characterize their structures.

3.5. Tolerability

Atuliflapon was well tolerated in the six healthy male subjects and there were no serious adverse advents (SAEs) or adverse advents (AEs) leading to subject withdrawal and no new safety signals were detected. One AE of constipation was reported which resolved after 7 days with treatment using lactulose. It was assessed as mild and not related to atuliflapon. There were no significant clinical laboratory or physical examination findings, and all vital signs and ECG were normal or not clinically significant.

4. DISCUSSION

This study was conducted in order to investigate ADME properties of atuliflapon in humans, and to provide further PK and tolerability data. There are several reasons for generating ADME data, thus, timing and design of these studies varies. ²⁴ , ²⁵ , ²⁶ Most commonly, the human ADME study is conducted in healthy subjects with normal physiology to understand the routes of metabolism and elimination and to guide the clinical pharmacology package to fulfill the regulatory requirements of a New Drug Application. Comprehensive knowledge about human metabolites obtained from human ADME study, including identification and quantification of systemic and excreted metabolites and metabolic elimination pathways, enables the assessment of conducting any further safety testing of drug metabolites (MIST) or drug interaction studies according to regulatory guidances. ²⁷ , ²⁸

In this study, the mean half‐life of unchanged atuliflapon after oral administration was determined to approximately 20 h, estimated from the apparent terminal phase which started at 24 h post‐dose or later in all subjects. By contrast, much shorter half‐life values of 5.9 h and 3.9 h, respectively, were determined for total radioactivity in plasma and whole blood. This was attributed to the plasma and whole blood concentrations of total radioactivity not being quantifiable later than 24 h post‐dose; therefore, it is unlikely that the half‐life determined from total radioactivity is representative of the terminal phase and it can be seen from the concentration vs. time profiles, that both atuliflapon and total radioactivity decline in an approximately parallel manner while both were quantifiable. In addition, systemic exposure to atuliflapon was lower than the total radioactivity, with ratios of total radioactivity: atuliflapon of 1.7, 2.1 and 2.3 based upon plasma C_max, AUC_last and AUC_0‐inf, respectively, which showed that metabolites contribute towards the circulating total radioactivity in plasma.

In pooled plasma, 11 metabolite fractions were detected with M7 being the major metabolite accounting for 21% of DRE. The exposures of the remaining metabolites detected were low and each accounted for less than 8% of total exposure of DRE in plasma. M7 was identified as an N‐glucuronide conjugate of atuliflapon. According to the FDA 2020 Regulatory Guidance for Safety testing of Drug Metabolite (Safety Testing of Drug Metabolites Guidance for Industry. 2020), ²⁷ phase 2 metabolites are generally exempt from safety testing because Phase 2 conjugation reactions generally render a compound more water soluble and pharmacologically inactive, thereby eliminating the need for further evaluation.

The overall total recovery of radioactivity was 85.2% over 312 h, which was lower than the ideally 90% recovery as proposed by the recent FDA draft guidance on radiolabeled mass balance studies. ²⁶ In a retrospective analysis of human mass balance studies, Roffey et al. reported 42% of 171 studies had overall recovery values below 90%, and fecal elimination appears to be of greater importance for drugs that yield lower recovery values. ²⁴ The authors suggested the prolonged excretion into feces represents a significant technical barrier due to non‐homogenous samples and irregularity of defecation. In this study 79.3% of the dosed radioactivity was eliminated in feces with the majority recovered up to 144 h post dose. High variations of individual fecal excretion during each collection interval were also observed (Table S3). However, all subjects did not have inconsistent urine recovery or altered pharmacokinetics of circulating parent drug and total DRE (Figure 3). Therefore the observed lower recovery is likely driven by issues related to feces production, collection and preparation, and not related to elimination mechanisms of atuliflapon. Only a small amount of radioactivity was recovered in urine, with <1% of the dose identified as atuliflapon, suggesting that renal impairment is not likely to have a clinically relevant influence on the pharmacokinetics of the parent compound. However, the recovered radioactivity in excreta further indicated the importance of metabolism in the clearance of atuliflapon. It was noted that the unchanged atuliflapon found in feces accounted for 38.8% of the dose. This may be due to incomplete absorption of the oral dose, or intestinal secretion as well as bile excretion of the absorbed dose, which would require further studies to assess. Given that the direct N‐glucuronide metabolite M7 was the major circulating metabolite which was found in low amounts in urine and was not measurable in feces, it cannot be excluded that at least part of the atuliflapon detected in feces originated from the degradation of M7 to the parent atuliflapon by the intestinal or faeacal microflora.

5. CONCLUSION

Following oral administration, the major metabolic pathways of atuliflapon were oxidation and direct N‐glucuronidation. Overall, parent atuliflapon contributed to approximately 40% of the recovered dose in excreta, while metabolites resulting from phase 1 oxidative pathways accounted for more than 30% of the excreted dose. There were no major human circulating metabolites identified that required further safety assessment in animal studies. The single oral dose of 200 mg [¹⁴C]atuliflapon suspension was generally well tolerated in healthy male participants.

AUTHOR CONTRIBUTIONS

Participated in research design: C.A.; H.E; J.K.; K.N.; K.S.; L.W.; M.G.; RA.B; P.G.; S.S; X‐Q. L.; Conducted experiments: K.S.; S.S; Performed data analysis: B.L.; C.A.; H.E.; K.S.; S.S.;X‐Q. L.; Wrote or contributed to the writing of the manuscript: B.L.; C.A.; E‐L. L.; H.E; J.K.; K.N.; K.S.; L.W.; M.H.; M.G.; P.G.; RA.B; S.S.; X‐Q. L.

FUNDING INFORMATION

This study was funded by AstraZeneca.

CONFLICT OF INTEREST STATEMENT

X‐Q. L.; B.L.; C.A.;K.S.; K.S.; L.W.; K.N.; J.K.; M.H.;RA.B.; M.G.; E‐L. L.; P.G.; H.E.; are or former employees of AstraZeneca and may be holding stock in the company.

ETHICS STATEMENT

The study was performed in accordance with ethical principles consistent with the International Conference on Harmonization Good Clinical Practice guidance and the ethical guidelines of the Declaration of Helsinki.

Supporting information

Data S1.

Li X‐Q, Lindmark B, Amilon C, et al. Disposition of orally administered atuliflapon, a novel 5‐lipoxygenase‐activating protein inhibitor in healthy participants. Pharmacol Res Perspect. 2024;12:e70029. doi: 10.1002/prp2.70029

DATA AVAILABILITY STATEMENT

Data underlying the findings described in this article maybe obtained in accordance with AstraZeneca's data sharing policy described at https://astrazenecagrouptrials.pharmacm.com/ST/Submission/Disclosure.