Absorption, metabolism and excretion of opicapone in human healthy volunteers

Aims The absorption, metabolism and excretion of opicapone (2,5‐dichloro‐3‐(5‐[3,4‐dihydroxy‐5‐nitrophenyl]‐1,2,4‐oxadiazol‐3‐yl)‐4,6‐dimethylpyridine 1‐oxide), a selective catechol‐O‐methyltransferase inhibitor, were investigated. Methods Plasma, urine and faeces were collected from healthy male subjects following a single oral dose of 100 mg [14C]‐opicapone. The mass balance of [14C]‐opicapone and metabolic profile were evaluated. Results The recovery of total administered radioactivity averaged >90% after 144 hours. Faeces were the major route of elimination, representing 70% of the administered dose; 5% and 20% were excreted in urine and expired air, respectively. The C max of total radioactivity matched that of unchanged opicapone, whereas the total radioactivity remained quantifiable for a longer period, attributed to the contribution of opicapone metabolites, involving primarily 3‐O‐sulfate conjugation (58.6% of total circulating radioactivity) at the nitrocatechol ring. Other circulating metabolites, accounting for <10% of the radioactivity exposure, were formed by glucuronidation, methylation, N‐oxide reduction and gluthatione conjugation. Additionally, various other metabolites resulting from combinations with the opicapone N‐oxide reduced form at the 2,5‐dichloro‐4,6‐dimethylpyridine 1‐oxide moiety, including nitro reduction and N‐acetylation, reductive opening and cleavage of the 1,2,4‐oxadiazole ring and the subsequent hydrolysis products were identified, but only in faeces, suggesting the involvement of gut bacteria. Conclusion [14C]‐opicapone was fully excreted through multiple metabolic pathways. The main route of excretion was in faeces, where opicapone may be further metabolized via reductive metabolism involving the 1,2,4‐oxadiazole ring‐opening and subsequent hydrolysis.


| INTRODUCTION
Opicapone (also known as BIA 9-1067) is a reversible, peripherally selective high binding affinity and long-acting third-generation nitrocatechol catechol-O-methyltransferase (COMT) inhibitor 1,2 that has recently been granted marketing authorization under the trade name Ongentys ® by, among other regulatory agencies, the European Medicines Agency, the US Food and Drug Administration and the Japan Pharmaceutical and Medical Decices Agency as adjunctive therapy to the combination levodopa plus dopa decarboxylase inhibitor for the treatment of endof-dose motor fluctuations in Parkinson's disease patients. [3][4][5][6][7][8][9][10][11] Opicapone was found not to induce cytotoxicity. 12 In humans, despite opicapone's short apparent terminal elimination half-life (t 1/2 ) of 0.8-3.2 hours, the half-life of erythrocyte COMT inhibition is in the range of 60-130 hours, 13,14 far beyond the observable point of plasma opicapone clearance (6-10 hours), making it suitable for a once-daily regimen. 13,14 Previous investigations have demonstrated that the pharmacokinetic profile of opicapone is dose-proportional up to 1200 mg and 3-O-sulfate-opicapone (BIA 9-1103) appeared to be the main metabolite of opicapone, with a relatively long t 1/2 , ranging from 25.1 to 26.9 hours following a single dose and from 98.5 to 112.0 hours following repeated dosing. 13,14 Along with BIA 9-1103 other opicapone metabolites were quantified in clinical studies, such as the N-oxide reduced form of opicapone (BIA 9-1079), the 3-O-glucuronide-opicapone (BIA 9-1106) and the 3-O-methyl-opicapone (BIA 9-1100). However, the definitive information concerning quantitative and comprehensive metabolic profiles of opicapone in human plasma and excreta is only provided by a clinical study with radiolabeled opicapone. 15 This type of study is considered one of the most critical steps in drug development where the primary mechanism(s) of clearance, excretion, mass balance and metabolism are evaluated, 15 contributing to the pharmacokinetics (PK) characterisation of a drug candidate. The further characterization of metabolites, such as their profile, potential for on-target pharmacological activity and drug-drug interactions (DDIs), are essential to better understand the safety of therapeutic agents. Previously, in an absorption, distribution, metabolism and excretion study with [ 14 C]-opicapone in rats, we demonstrated that opicapone was rapidly absorbed after oral dosing and that the drug-derived radioactivity was excreted mainly into faeces. 16 Furthermore, we found that opicapone underwent extensive metabolism via diverse pathways and various metabolites were detected in plasma and excreta. Although O-glucuronidation, -sulfation and -methylation of the nitrocatechol moiety were the principal metabolic pathways, s small amount of the N-acetyl derivative was detected as a result of reduction of the nitro group and subsequent conjugation. Other metabolic transformations include N-oxide reduction to the pyridine derivative and reductive cleavage of the 1,2,4-oxadiazole ring followed by further conjugation reactions. The pharmacological activity of some of those metabolites was also evaluated. 16 In this study the absorption, metabolism and excretion of opicapone-related radioactivity were evaluated in healthy male subjects after oral administration of 100 mg of [ 14 C]-opicapone. The total radioactivity and metabolic profile in urine and faeces were measured using the conventional liquid scintillation counting method; in plasma, due to the low amount of radioactivity, the metabolic profile was evaluated by accelarator mass spectrometry (AMS), an extremely sensitive method for the detection and determination of isotopic ratios. 17 2 | MATERIALS AND METHODS

| Reagents
The unlabelled opicapone was manufactured by Patheon (formerly

| Clinical study design
This was an open-label, single-dose, single-period study designed to assess the mass balance recovery, metabolite profile and metabolite and no additional residency period was required. All subjects attended four further return visits for PK sampling on day 28, day 42 (±3 days), day 56 (±3 days) and day 77 (±3 days).

| Sample collection and storage
Venous blood samples were collected at predose and then at regular intervals from 0.5 to 648 hours post-oral [ 14 C]-opicapone administration. Whole blood was collected at predose and up to 96 hours post-dosing; Urine, faeces and expired air were collected at predose and then at regular intervals until 504 hours post-oral [ 14 C]-opicapone dosing (additional information on collection timepoints is given in the Supporting Information).

| Measurement of total radioactivity
The residual radioactivity in the dosing bottles was quantified and the recovery of total radioactivity was less than 0.0333%. Total radioactivity in urine and plasma was measured after pooling and mixed with phosphate-buffered saline.
Total radioactivity in the faecal samples was determined by the combustion method using an automatic sample oxidiser (Model 307, Perkin Elmer, Waltham, MA, USA). The combustion products were absorbed into CarboSorb E and mixed with the scintillator cocktail PermaFluor E + for measurement of radioactivity. The same procedure was applied to whole blood. Total radioactivity in expired air was measured in air-trapping solution obtained from individual expired air. Measurements of radioactivity in urine, faeces, whole blood and plasma were performed in duplicate (single measurement for expired air) using liquid scintillation counting (2300TR, 2900TR or 3100TR Tri-Carb, Scintillation Counter, Perkin Elmer) (additional information on total radioactivity measurements is given in the Supporting Information).

| Quantitation of concentrations in plasma samples by liquid chromatography-tandem mass spectrometry
Concentrations of opicapone and its metabolites, BIA 9-1103, BIA 9-1079, BIA 9-1106 and BIA 9-4588, in plasma were measured using a validated liquid chromatography-tandem mass spectrometry. Briefly, plasma samples (100 mL) were mixed with 200 mL of acetonitrile and internal standards. The samples were vortex and centrifuged, and the supernatant was diluted with water to be injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) TSQ Quantum (Thermo Fisher Scientific, Waltham, MA, USA). The limit of quantification for all metabolites was 10 ng/mL. Calibration curves and a set of quality control (QC) samples were analyzed with each batch of study samples. The analytical method was demonstrated to be precise (<15%, coefficient of variation) and accurate (±15%) over the analytical range.

| Pharmacokinetic and statistical analyses
The PK of total radioactivity in plasma and blood, opicapone and its metabolites in plasma were analyzed using WinNonlin (version 6.3; Certara USA, Inc., USA) for concentration versus time for each subject. Pharmacokinetic parameters were calculated by noncompartmental analysis (additional information on pharmacokinetic parameters is given in the Supporting Information).

| Extraction and column recovery
The recovery of radioactivity following extraction and reconstitution was determined for all samples by comparing the radioactivity in the initial sample pool to that recovered in the supernatant and in the reconstituted extract (additional information on recovery is given in the Supporting Information).

| 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 PHARMA-COLOGY, and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20). 19,20 3 | RESULTS  was 138 hours (range 133-143 h). As shown in Table 1, low levels of BIA 9-1106 were quantifed and BIA 9-1079 was below the limit of quantification (BLQ, <10 ng/mL) in almost all time points.

| Metabolite profiling and identification in plasma, urine and faeces
Plasma AUC sample radiochromatograms were generated using AMS radiodetection. A representative radiochromatogram for plasma is shown in Figure 4. Structural assignment and MS data are summarized in Table 2. The proposed metabolic pathway is shown in Figure 5.

| Plasma
As presented in Figure 4 and Table 2  a Product ion spectra were generated for the authentic reference standard that matched the elemental formula and retention time with the radio and MS peak.
represented an average of 5.49% of circulating radioactivity.
Although the elemental formula and fragmentation are consistent with a dicysteine conjugate of either BIA 9-1104 or BIA 9-1100, the exact location of the conjugation is difficult to ascertain. These

| Urine
Only trace amounts of unchanged parent were observed in urine, indicating minimal renal clearance of opicapone. The most abundant metabolite observed in urine was BIA 9-1106, which represented on average 1.72% of the administered radioactivity.

| Faeces
The majority of drug-related radioactivity was excreted in faeces,

| Extraction and column recovery
The overall recovery of radioactivity from the t max plasma was 91.3% and from AUC plasma extracts was 59.3%. The mean recovery from the urine samples following concentration was 94.7% and from faeces was 42.7%. A column recovery experiment performed for human urine was determined to be 97.2%. LC-AMS column recovery was between 80% and 120% of the total radioactivity injected onto the system (additional information on recovery results is given in the Supporting Information).

| DISCUSSION
The mass balance, excretion and metabolism of opicapone were by liver enzymes. 25,26 Furthermore the ring-opening reaction and in some cases ring-cleaving reactions were also described for 1,2,4-oxadiazole rings. 27,28 There is evidence that the ring-opening reaction is enhanced by anaerobic conditions in human hepatocytes and in rat liver but enzymes including P450 might not contribute to this unique reaction, 28 [29][30][31] There is also evidence that drugs containing a nitro or azo group are chemical targets for metabolic modification, undergoing reductive reactions in anaerobic conditions by human gut bacteria. 30 Furthermore, the involvement of microbiome in the generation of 14 CO 2 , corresponding to $20% of total radioactivity, cannot be excluded. Indeed, BIA 9-3752, an aromatic carboxylic acid metabolite observed in faeces but not in plasma, represents a likely intermediate from which 14 CO 2 could be generated. BIA 9-3752 could potentially be further metabolized by the microbiome to release CO 2 in the colon, which would then be absorbed and excreted in expired air. A recent publication on the metabolism of ozanimod, which shares the oxadiazol ring system with a 14 C label in the same position as in [ 14 C]-opicapone, showed that the loss of radioactivity and release of 14 CO 2 does occur in human faecal homogenates after in vitro incubation for 96 hours under anaerobic conditions. 32 In the presence of antibiotics, this loss was prevented, implicating the role of anaerobic bacterial metabolism leading to loss of 14 CO 2 . In addition, the corresponding product was formed only in incubations without antibiotics and not in control incubations or with antibiotics, showing that anaerobic bacterial metabolism leads to loss of CO 2. 32 Although the reductive metabolic pathway, including N-oxide reduction, nitro group reduction and reductive opening of the 1, 2, 4-oxadiazole moiety followed by a hydrolytic cleavage, was also identified in rats, 16 the percentage of detected CO 2 in rats was significantly lower, which may suggest a difference in the extension of this metabolic reaction in both species.

| CONCLUSION
The total radioactivity is fully excreted after

CONSENT TO PARTICIPATE
Each patient provided written informed consent before participation.
Each investigator received prospective approval of the study protocol, protocol amendments, informed consent documents and all other relevant documents from their individual institution's review board or ethics committee.

DATA AVAILABILITY STATEMENT
Due to the nature of this research, participants of this study did not agree for their data to be shared publicly, so supporting data is not available.