Human disposition, metabolism and excretion of etamicastat, a reversible, peripherally selective dopamine β-hydroxylase inhibitor

Abstract

Aims

Etamicastat is a reversible dopamine-β-hydroxylase inhibitor that decreases noradrenaline levels in sympathetically innervated tissues and slows down sympathetic nervous system drive. In this study, the disposition, metabolism and excretion of etamicastat were evaluated following [¹⁴C]-etamicastat dosing.

Methods

Healthy Caucasian males (n = 4) were enrolled in this single-dose, open-label study. Subjects were administered 600 mg of unlabelled etamicastat and 98 µCi weighing 0.623 mg [¹⁴C]-etamicastat. Blood samples, urine and faeces were collected to characterize the disposition, excretion and metabolites of etamicastat.

Results

Eleven days after administration, 94.0% of the administered radioactivity had been excreted; 33.3 and 58.5% of the administered dose was found in the faeces and urine, respectively. Renal excretion of unchanged etamicastat and its N-acetylated metabolite (BIA 5-961) accounted for 20.0 and 10.7% of the dose, respectively. Etamicastat and BIA 5-961 accounted for most of the circulating radioactivity, with a BIA 5-961/etamicastat ratio that was highly variable both for the maximal plasma concentration (19.68–226.28%) and for the area under the plasma concentration–time curve from time zero to the last sampling time at which the concentration was above the limit of quantification (15.82– 281.71%). Alongside N-acetylation, metabolism of etamicastat also occurs through oxidative deamination of the aminoethyl moiety, alkyl oxidation, desulfation and glucuronidation.

Conclusions

Etamicastat is rapidly absorbed, primarily excreted via urine, and its biotransformation occurs mainly via N-acetylation (N-acetyltransferase type 2), although glucuronidation, oxidation, oxidative deamination and desulfation also take place.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

• Etamicastat is a novel, peripherally selective dopamine β-hydroxylase inhibitor that is well tolerated in healthy volunteers up to 1200 mg day⁻¹.

• A high interindividual variability of pharmacokinetic parameters of etamicastat and its acetylated metabolite is associated with N-acetyltransferase type 2 rapid or slow N-acetylating ability.

WHAT THIS STUDY ADDS

• Etamicastat is primarily excreted via urine and its biotransformation occurs mainly via N-acetylation, although glucuronidation, oxidation, oxidative deamination and desulfation also take place.

Introduction

Etamicastat, (R)-5-(2-aminoethyl)-1-(6,8-difluorochroman-3-yl)-1,3-dihydroimidazole-2-thione hydrochloride (Figure 1), is a novel dopamine-β-hydroxylase (DBH; EC 1.14.17.1) inhibitor 1, currently in clinical development for the treatment of cardiovascular disorders 2–4. Etamicastat slows down the drive of sympathetic nervous system by reducing the biosynthesis of noradrenaline via inhibition of DBH 1,5,6, the enzyme that catalyses the conversion of dopamine to noradrenaline in sympathetic nerves 7. This approach also increases dopamine levels, which can improve renal function parameters, such as renal vasodilatation, diuresis and natriuresis 8,9.

Figure 1.

Proposed metabolic pathway of etamicastat in humans after a single oral dose of [¹⁴C]-etamicastat. The structure of metabolites was characterized by mass spectrometry

Etamicastat was specifically designed not to cross the blood–brain barrier and to act as a reversible inhibitor of peripheral DBH 1. Following oral doses of etamicastat, a blood pressure-lowering effect was observed in studies performed in the spontaneously hypertensive rat. Both the systolic and diastolic blood pressure (but not the heart rate) were decreased in spontaneously hypertensive rats in a dose-dependent manner, an effect not observed in normotensive (Wistar–Kyoto) control rats 10; this difference between Wistar–Kyoto and spontaneously hypertensive rats in response to etamicastat may be related to the high sympathetic drive in the spontaneously hypertensive rats. In addition to their antihypertensive effect, DBH inhibitors were also considered drug-development candidates for the treatment of chronic heart failure. Etamicastat increased survival rates in male cardiomyopathic hamsters (Bio TO-2 dilated strains) with advanced congestive heart failure 5. Several inhibitors of DBH have been thus far reported 11–13; however, both first- and second-generation inhibitors were found to be of low potency and poor selectivity for DBH and to have several adverse effects 1,5. Nepicastat, a third-generation inhibitor, is a highly potent DBH inhibitor, but penetrates the blood–brain barrier and thereby causes undesired central nervous adverse effects 14.

Clinical studies with etamicastat have shown that it was well tolerated after repeated administration (25–600 mg) for 10 days 3. Etamicastat has biphasic elimination, characterized by a first short early elimination half-life followed by a longer elimination phase of 16–20 h for etamicastat doses of 100 mg and above 3,4. In humans, etamicastat undergoes N-acetylation, which is markedly influenced by N-acetyltransferase type 2 (NAT2) phenotype. The high interindividual variability of pharmacokinetic parameters observed for etamicastat and its acetylated metabolite was associated with NAT2 phenotyping (rapid or slow N-acetylating ability). In NAT2 poor acetylators, the area under the plasma concentration–time curve from time zero to the last sampling time at which concentrations were at or above the limit of quantification (AUC_0–t) of etamicastat was twice that observed in rapid acetylators. Consistent with that finding, the AUC_0–t of the N-acetylated metabolite was markedly higher in NAT2 rapid acetylators compared with poor acetylators 4. Furthermore, in vitro studies have shown that etamicastat is a preferential substrate for NAT2 15.

Here, we report a study in healthy male subjects after a single oral dose of 600 mg etamicastat containing [¹⁴C]-etamicastat (98 μCi) ([¹⁴C]-etamicastat structure is shown in Figure S1). The purpose of the study was to characterize the disposition, metabolism and excretion of etamicastat and to elucidate the metabolic pathway and the structure of metabolites.

Methods

Subjects

Healthy Caucasian males (n = 4) aged between 40 and 55 years and with a body mass index of 18–28 kg m⁻² were enrolled in this single-dose, open-label study (BIA-5453-103/SPC388-6). Subjects were healthy on the basis of medical history and a prestudy physical examination, electrocardiogram and clinical tests. Safety measurements (12-lead electrocardiogram, vital signs, blood chemistry and haematology) were conducted before and after the study, and adverse events were monitored throughout the study. The diastolic and systolic blood pressure of the subjects, measured in the supine position, during the screening phase ranged between 71 and 82 and between 115 and 133 mmHg, respectively. The heart rate ranged between 63 and 65 beats min⁻¹.

The clinical part of the study was conducted at Covance Clinical Research Unit AG (formerly Swiss Pharma Contract Ltd, Basel, Switzerland) in accordance with Good Clinical Practices and with the Declaration of Helsinki, and an Independent Ethics Committee approved the protocol. All participating subjects gave their written informed consent before participation.

Study design

The study was a single-centre, open, nonplacebo-controlled, single-group, single-dose study. Subjects were hospitalized from the day before the administration until 264 h thereafter. Subjects fasted for at least 10 h before orally dosing with 600 mg of unlabelled etamicastat plus 98 μCi (weighing 0.623 mg) of [¹⁴C]-etamicastat administered with 240 ml of tap water. Four hours after dosing, the subjects received the first meal. The glass used for the administration of the solution containing [¹⁴C]-etamicastat was rinsed afterwards twice with 50 ml of tap water that was swallowed by the subjects. The vials and the glassware used for preparation were stored for radioactivity counting in order to correct the administered dose. Approximately 435 ml of blood was collected from each subject during the entire study, including 35 ml for safety assessments, 20 ml for NAT genotyping and 380 ml for pharmacokinetic (PK) measures.

Genotyping

Subjects were genotyped for NAT1 and NAT2 at baseline. DNA was extracted from venous blood using a QIAamp DNA Blood kit (Qiagen, Hilden, Germany). The NAT2 genotypes were analysed essentially by PCR/restriction fragment length polymorphism, identifying six coding single nucleotide polymorphisms (191G>A, 282C>T, 341T>C, 481C>T, 590G>A and 803A>G) corresponding to NAT2*4, *5A, *5B, *5C, *6A, *12A and *14B. The associated phenotypes were defined according to the literature. The NAT1 variants 190C>T (NAT1*17) were determined by pyrosequencing technology using a PSQ-HS 96A (Biotage, Westborough, MA, USA). Further single nucleotide polymorphisms were analysed, namely 559C>T, 560G>A, 640T>G, 752A>T, D9 1065–1090, 1088C>T and 1095C>A, corresponding to NAT1*4, *10, *11, *14, *15, *17 and *22.

Pharmacokinetic assessment

Whole blood samples (2 ml) for total radioactivity analysis, plasma samples (1.5 ml) for total radioactivity analysis and plasma samples (7 ml) for analysis of etamicastat and its metabolites were collected through in dwelling catheters before dosing and at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, 72, 120, 168, 216 and 264 h postdose. Urine samples were prepared from urine collected over the following intervals: 0–4, 4–8, 8–24, 24–48, 48–72, 72–120, 120–168, 168–216 and 216–264 h postdose. Aliquots of each sample were taken for liquid scintillation counting and others were used for determination of the parent drug and metabolite patterns. Baseline faecal samples were obtained during the screening or baseline period. Following dose, each faecal sample was collected in a separate container during the 264 h postdosing. Exhaled air samples for total radioactivity analysis were collected at baseline and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 h postdose.

Determination of radioactivity

Radioactivity in plasma, whole blood, urine and faeces was measured using a scintillation counter (TriCarb 2800TR; PerkinElmer Life and Analytical Sciences, Downers Grove, IL, USA). The measurements were performed for a counting time of 10 min. The predose plasma and whole blood sample from each subject were measured to evaluate the background radiation. Blood (0.75 ml) was incubated for 1 h at 50°C with isopropanol and tissue solubilizer. After cooling at room temperature, 0.5 ml of hydrogen peroxide (30%) was added and the reaction incubated for 15 min. Thereafter, the samples were incubated for 30 min in a water-bath at 50°C. After cooling at room temperature, 0.25 ml of HCl (1.0 mol l⁻¹) was added. An aqueous-based solubilizer scintillation cocktail (Ultima Gold, PerkinElmer) was added and the samples were allowed to adapt to room temperature and light for 1 h before counting in triplicate. Plasma samples (300 μl) were mixed with 1 ml of water and then 10 ml of scintillation cocktail was added. Each plasma sample was analysed in duplicate.

For urine, two aliquots of 300 μl of urine samples were mixed with 10 ml of scintillation cocktail before counting.

Faecal samples were freeze dried for approximately 48–72 h, and each sample was weighed. The dried sample was homogenized and four aliquots were transferred to a scintillation vial. Then, 300 μl of water and 1 ml of tissue solubilizer (Soluene 350®, PerkinElmer) was added and incubated for 1 h at 50°C followed by addition of 0.5 ml of isopropanol and a further incubation for 2 h at 50°C. After cooling at room temperature, 500 μl of hydrogen peroxide was added and the solution allowed to stand for at least 30 min at 50°C to complete the reaction. A volume of 0.25 ml HCl and 10 ml of an organic-based solubilizer scintillation cocktail (Hionic Fluor, PerkinElmer) were added. The samples were incubated for >12 h in the dark before radioactivity measurements.

To analyse the exhaled air, subjects blew gently through a plastic tube into vials containing a suitable base to trap 1 mmol of carbon dioxide. The end-point was reached when phenolphthalein indicator had consistently bleached. The expired air sample was obtained after the corresponding blood sample had been drawn. The samples were capped immediately and kept at 2–8°C until being assayed.

The quality control samples were prepared by adding small volumes of [¹⁴C]-stearic acid solution to blank human whole blood, plasma, urine, air and faecal samples following the work-up described above in order to have samples with a radioactivity level of 5000 d.p.m. The mean and relative standard deviation (%CV) of quality control samples were calculated within the batches and were within 15% of their respective nominal value for the accepted runs.

Determination of etamicastat and its metabolites BIA 5-961 and BIA 5-998 in plasma

Etamicastat and its major metabolites, BIA 5-961 and BIA 5-998 (Figure 1), were determined in plasma (50 μl), following protein precipitation with acetonitrile (100 μl), by liquid chromatography–mass spectrometry (LC-MS/MS) using a TSQ Quantum, equipped with an APCI interface (Thermo Fisher Scientific, San Jose, CA, USA). Separation was performed on a Reprosil-Pur 100 basic–C18 3.0 μm, 2.0 mm × 50 mm column (Dr Maisch HPLC GmbH, Ammerbuch, Germany) using water containing 5 mm ammonium acetate (solution A) and methanol containing 5 mm ammonium acetate (B), as the mobile phase. The analytical pump gradient conditions were as follows: 0 min, 98% of A and 2% of B; 0.5 min, 98% of A and 2% of B; 1.8 min, 20% of A and 80% of B; 3.5 min, 5% of A and 95% of B; 3.6 min, 98% of A and 2% of B; and 5 min, 98% of A and 2% of B, with a flow rate of 0.4 ml min⁻¹. Detection of compounds was performed by multiple reaction monitoring, monitoring the transitions of m/z 312.1 precursor ion to m/z 283.0 product ion for etamicastat, the transitions of m/z 354.1 precursor ion to m/z 127.0 product ion for BIA 5-961, the transitions of m/z 326.1 precursor ion to m/z 158.0 product ion for BIA 5-998, and the transitions of m/z 402.1 precursor ion to m/z 120.0 product ion for the internal standard.

The method was fully validated in accordance of existing bio-analytical guidelines. The overall precision of the method, assessed by the coefficient of variation (CV%), for the analysis of etamicastat in plasma was in the range of 4.3–10.3%, and the accuracy ranged from 98.6 to 110.0%. For BIA 5-961, the precision ranged from 2.6 to 7.6% and the accuracy from 94.0 to 100.4%. For BIA 5-998, the precision ranged from 1.6 to 5.5% and the accuracy from 98.2 to 102.0%. The concentration range of 5–1000 ng ml⁻¹ was linear for all three analytes. Samples above the limit of quantification were diluted with blank plasma to be quantified in the analytical range validated.

Metabolic profiling in plasma, urine and faeces

Pooled plasma samples prepared by combining fixed volumes from all four subjects for each time point were vortex mixed and centrifuged for 20 min at approximately 3360g. To one aliquot of each sample, the same volume of acetonitrile was added. After protein precipitation at room temperature, plasma samples were centrifuged for 10 min at approximately 50 000g and 8°C, and the supernatant was injected directly into LC-MS/MS precolumn. Composite urine samples were prepared across subjects for each time point. Aliquots of each urine pool were analysed without further work-up. Each composite faecal pool was extracted with 10-fold excess (1/10, w/v) of a mixture of acetonitrile/water (50/50, v/v). The samples were vortex mixed, sonicated and allowed to stand. The samples were centrifuged for 10 min at 50 000g and 8°C and supernatants injected into the LC-MS/MS.

The analysis of the extracted samples was performed using reversed-phase chromatography followed by detection with triple-stage quadruple MS/MS or an ion trap mass spectrometer in different scan modes (Thermo Fisher Scientific). Separation was performed using a column and the mobile phases described above, with analytical pump gradient conditions as follows: 0 min, 98% of A and 2% of B; 1 min, 98% of A and 2% of B; 45 min, 50% of A and 50% of B; 50 min, 20% of A and 80% of B; 55 min, 5% of A and 95% of B; 55.1 min, 98% of A and 2% of B; and 60 min, 98% of A and 2% of B, with a flow rate of 0.15 ml min⁻¹. The metabolites were identified using a full scan, neutral loss and precursor ion scan. The samples were analysed by high-performance liquid chromatography (HPLC), and fractions were collected for radioactivity counting. The recovery for the accounted signals in samples compared with the total counted fractionated signals was in the range of 66.7–96.6%.

The structure of metabolites, where possible, was supported by comparison of their retention time, on HPLC and mass spectra, with those of synthetic standards. The confirmation of the additional metabolites was performed by comparison of retention times obtained from the fraction collection and scintillation counting experiments, and the mass spectrometric information, such as the fragmentation pattern, was used as a means of tentative identification.

Safety assessments

Adverse events were monitored throughout the study. Safety assessments included clinical laboratory testing (including haematology and clinical chemistry), physical examination, blood pressure, pulse rate and electrocardiogram data were conducted at appropriate intervals throughout the studies.

Statistical and pharmacokinetic analysis

Pharmacokinetic parameters were calculated using noncompartmental analysis from the concentration–time profiles using WinNonlin (version 4.1; Pharsight Corporation, Mountain View, CA, USA). Results are given as the median and range. The AUC_0–t values were calculated from time zero to the last sampling time at which the concentration was at or above the limit of quantification using the linear trapezoidal rule. The AUC was extrapolated to infinity, calculated as AUC_0–∞ = AUC_0–t + C_last/λ_z where C_last is the last measurable concentration and λ_z is the elimination rate constant, calculated by log-linear regression of the terminal segment of the plasma concentration–time curve. The terminal half-life (t_1/2) was calculated from ln(2)/λ_z.

Results

Subjects

Four healthy Caucasian male subjects, aged between 40 and 55 years (mean, 49.5 years) with body mass index of 18–28 kg m⁻² (mean, 24.1 kg m⁻²), received the study drug and completed the study. Etamicastat was well tolerated; no serious adverse events occurred, and no subject discontinued the study because of an adverse event. Only mild to moderate adverse events were reported, mostly affecting the digestive tract. The vital sign analyses indicated a trend towards slightly decreased diastolic blood pressure, as expected from the pharmacodynamic features of the study drug; ranging from the median value of 75 (53–84) mmHg at predose to 60.5 (54–75) mmHg at 8 h postdose (Table S2). The pulse rate measured predose had a median value of 60 (53–65) beats min⁻¹ and ranged from 47.0 to 89.0 beats min⁻¹ during the study. The body temperature was normal under treatment and until the end of study examinations. Electrocardiograms (12 lead) were normal or showed borderline deviations from the normal range of no clinical concern at screening and at the end of the study. Physical examinations did not reveal any change from baseline to end-of-study assessments.

Genotyping

The genotyping showed that subject 1 had a NAT1*4/*14A or *10/*10B genotype, subjects 2 and 3 had a NAT1*4/*4 genotype and subject 4 had NAT1*4/*10 genotype, indicating that subject 1 was a slightly poor acetylator and subjects 2, 3 and 4 were NAT1 rapid acetylators. For NAT2, subject 1 had a NAT2*4/*5B genotype, subjects 2 and 3 had a NAT2*5B/*5B genotype and subject 4 had NAT2*6B/*7B genotype, indicating that subject 1 was a NAT2 rapid acetylator and subjects 2, 3 and 4 were NAT2 poor acetylators.

Pharmacokinetics of etamicastat and its metabolites in plasma

The mean plasma concentration–time profile of etamicastat and its metabolites and the pharmacokinetic parameters of total radioactivity are shown in Figure 2 and Table 1 (individual profiles are presented in Figure S2).

Figure 2.

Mean plasma concentration profiles of etamicastat, BIA 5-961 and total radioactivity in healthy male subjects (n = 4) after a single 600 mg oral dose of unlabelled etamicastat and 0.623 mg [¹⁴C]-etamicastat. The limit of quantification for etamicastat and BIA 5-961 was 5 ng ml⁻¹, and total radioactivity concentration was below the limit of quantification for 91.83 ng-eq ml⁻¹. •, total radioactivity; ▪, etamicastat; ○, BIA 5-961

Table 1.

Pharmacokinetic parameters of etamicastat, its metabolites and total radioactivity in plasma following a single 600 mg oral dose of unlabelled etamicastat and 0.623 mg [¹⁴C]-etamicastat

Parameters	Total radioactivity [14C]	Etamicastat	BIA 5-961	BIA 5-998
AUC0–∞ (h μg ml−1)†	97.4 (86.2–154.2)	6.5 (5.1–8.9)	4.2 (1.5–14.1)	0.6*
%AUC (%)†	20.5 (15.6–31.1)	4.0 (2.1–5.4)	3.8 (1.9–5.7)	0.07*
AUC0–t (h μg ml−1)†	75.5 (62.4–130.1)	6.2 (4.8–8.7)	4.1 (1.4–13.9)	0.03 (0.003–0.05)
Cmax†	2.6 (1.8–4.9)	0.6 (0.4–0.7)	0.5 (0.1–1.4)	0.006 (0.005–0.04)
t1/2 (h)	136.9 (113.7–169.8)	22.9 (14.7–35.1)	13.6 (8.3–24.0)	0.001*
tmax (h)	3.5 (3.0–4.0)	2.0 (1.5–3.0)	3.0 (3.0–6.0)	4.5 (3.0–8.0)

Data are medians (range) obtained from four healthy subjects. Abbreviations are as follows: %AUC, percentage of extrapolated plasma AUC; AUC_0–∞, area under the plasma concentration–time curve from time zero to infinity; AUC_0–t, area under the plasma concentration–time curve from time zero to the last sampling time at which the concentration was above the limit of quantification; C_max, maximal plasma concentration; t_1/2, terminal half-life; and t_max, time to maximal plasma concentration.

^*Data available for a single subject.

^†For total radioactivity, the units for C_max and AUC were microgram-equivalents per millilitre and microgram-equivalents hour per millilitre, respectively.

The concentration–time profile of etamicastat-associated radioactivity suggests a relatively quick absorption of etamicastat, with a time to reach the C_max (t_max) ranging from 3.0 to 4.0 h. The peak of radioactivity was followed thereafter by a relatively quick decline and a slow terminal phase of 136.9 (113.7–169.8) h. The AUC_0–t values showed a 2-fold difference between the lowest and highest value, with a median value of 75.5 (62.4–130.1) h μg-equiv ml⁻¹. The C_max values had a 2.7-fold difference between the lowest and highest value, with a median value of 2.6 (1.8–4.9) μg-equiv ml⁻¹.

The unlabelled etamicastat concentration–time profile also showed a relatively quick absorption, with a median t_max of 2.0 (1.5–3.0) h and a moderate to long terminal phase, with a median t_1/2 of 22.9 (14.7–35.1) h. As it was observed for the total radioactivity, the AUC_0–t and C_max values obtained for the unlabelled etamicastat were highly variable (approximately 2-fold difference between the lowest and highest value), with a median value of 6.2 (4.8–8.7) h μg ml⁻¹ and 0.6 (0.4–0.7) μg ml⁻¹, respectively. The etamicastat AUC_0–t values for the different subjects were 4.8, 6.3, 6.1 and 8.7 h μg ml⁻¹ for subjects 1, 2, 3 and 4, respectively. Subject 1 had the lowest AUC_0–t values for the parent drug.

The slightly delayed peak (t_max ranging from 3.0 to 4.0 h) and longer t_1/2 of etamicastat-associated radioactivity, compared with those for parent etamicastat, together with the smaller C_max and smaller AUC_0–t of parent etamicastat, suggest that metabolites accounted for most of the circulating etamicastat-derived moieties. The percentage of unchanged compound vs. total radioactivity in plasma was usually <10%, indicating that unchanged compounds accounted only for a small part of total radioactivity in plasma.

The pharmacokinetics of BIA 5-961, the N-acetylated metabolite of etamicastat, were characterized by a relatively early plasma appearance, with a median t_max of 3.0 (3.0–6.0) h and a t_1/2 with a median value of 13.6 (8.3–24.0) h. The AUC and the C_max values showed a relatively high variability (9.8- and 14-fold difference between the lowest and highest value, respectively), with a median AUC_0–t of 4.1 (1.4–13.9) h μg ml⁻¹ and C_max of 0.6 (0.1–1.4) μg ml⁻¹. The individual AUC_0–t values for BIA 5-961 were 13.8, 4.5, 3.6, 1.4 and h μg ml⁻¹ for subjects 1, 2, 3 and 4, respectively. The BIA 5-961/etamicastat AUC_0–t and C_max ratios (corrected for molecular weight) suggest that BIA 5-961 might be a major metabolite of etamicastat. For subjects 1, 2, 3 and 4, BIA 5-961/etamicastat AUC_0–t ratios were 281.7, 70.9, 58.6 and 15.8%, respectively, and C_max ratios were 226.3, 84.3, 69.4 and 19.7%, respectively. Subject 1 had the highest ratio values of AUC_0–t and C_max, which was more than 3-fold higher than the next highest value for AUC_0–t and more than 2-fold higher than the next highest value for C_max.

The pharmacokinetics of the etamicastat metabolite BIA 5-998, an alkyl oxidation of etamicastat, showed a relatively early plasma appearance, with a t_max of 4.5 (3.0–8.0) h in comparison to the value of the parent drug of 2.0 (1.5–3.0) h. The BIA 5-998/etamicastat AUC_0–t and C_max ratios suggest that BIA 5-998 might be a minor metabolite, corresponding to <1.4% of etamicastat C_max and <0.4% of AUC_0–t.

Expired air, urinary and faecal excretion

The excreted amounts in expired air samples were not considered for calculations because the total recovery of radioactivity in expired air was approximately 1% in all subjects. The individual cumulative recoveries of radioactivity in urine and faeces over 0–264 h are depicted in Figure 3. At day 11 after dosing, on average 94.0 (90.4–97.6)% of the administered dose had been excreted, with a median value of 58.5 (55.9–64.9)% of the radiolabelled material excreted in urine and 33.3 (29.4–37.2)% excreted in faeces. Unchanged drug excreted in urine accounted for 11.6, 20.6, 19.9 and 27.2% of the administered dose, for subjects 1, 2, 3 and 4, respectively.

Figure 3.

Individual cumulative urinary and faecal excretion of total radioactivity in healthy male subjects (n = 4) after a single oral dose of [¹⁴C]- etamicastat. •, subject 1; ○, subject 2; ▪, subject 3; □, subject 4

The recovery of etamicastat as N-acetylated metabolite BIA 5-961 accounted for 26.8, 11.3, 10.1 and 5.0% of the administered dose, for subjects 1, 2, 3 and 4, respectively. Subject 1 had the highest urine recovery of the N-acetylated metabolite, BIA 5-961 and was the only subject for whom BIA 5-961, rather than parent etamicastat was the most abundant moiety in urine. Furthermore, subject 1 was the only rapid NAT2 metabolizer while being a slightly slow NAT1 metabolizer, supporting the idea that NAT2 is the main enzyme mediating etamicastat biotransformation to BIA 5-961. BIA 5-998 accounted for <0.5% of the administered dose.

Etamicastat-related radioactivity in blood and plasma

The characteristics of the blood concentration–time profile of etamicastat-associated radioactivity suggest a relatively quick absorption of etamicastat, as evidenced by a relatively early peak of radioactivity (t_max median of 3.0 h), followed by a relatively fast decline and a relatively slow terminal phase (Figure 4). The t_1/2 tended to be long, with a median value of 19.5 (2.3–35.3) h, but it was clearly shorter than those found in plasma radioactivity. The C_max and AUC values of the blood etamicastat-associated radioactivity were widely variable, with AUC_0–t median values of 20.3 (12.8–40.5) h μg-equiv ml⁻¹ and C_max of 2.3 (1.7–2.9) μg-equiv ml⁻¹. The distribution of etamicastat-related radioactivity between human blood and plasma, determined using the whole blood/plasma C_max and AUC_0–t ratio, was 0.9 (0.6–1.0) for C_max and 0.3 (0.1–0.5) for AUC_0–t, indicating that the radioactivity in blood was mainly distributed to plasma.

Figure 4.

Mean plasma and whole blood total radioactivity in healthy male subjects (n = 4) after a single oral dose of [¹⁴C]-etamicastat. The total radioactivity concentration was below the limit of quantification for 91.83 ng equiv ml⁻¹. □, plasma; ▪, whole blood

Metabolite identification

Metabolism of etamicastat was assessed by profiling plasma, urine and faeces using pooled matrices. The structure of the metabolites identified, the molecular ions and the characteristic product ions for etamicastat and metabolites in plasma, urine and faeces are presented in Table S1, and the proposed metabolic pathway is shown in Figure 1. The main fragmentation pathway of etamicastat was the cleavage between the chromene ring and the imidazole ring, resulting in a fragment ion of m/z 169 (Figure S3) or a neutral loss of 168 Da, which is common in nearly all identified metabolites. The protonated 6,8-difluoro-4H-chromene ion with m/z of 169 was used for parent ion scans and assignment of possible metabolites. Etamicastat was metabolized into a range of metabolites, although high levels of unchanged compound were detected in urine, plasma and faeces. Besides the parent compound, metabolites M1–M25 were identified in urine, M6–M10, M14 and M18–M25 were identified in plasma, and M1, M2, M3, M5–M10, M14 and M15 were identified in faeces by LC-MS/MS.

Identification of M2, M7, M8, M9, M10, M14, M21 and M25 was performed by comparison with the retention time and fragmentation behaviours of authentic standards (synthesized in the Department of Chemistry, BIAL – Portela & Cª, S.A.) by overlap plots from samples and the authentic standards; BIA 5-961 (M14), a N-acetylated metabolite of etamicastat, BIA 5-998 (M9), an alkyl oxidation of etamicastat, BIA 5-965 (M10), an alkyl oxidation and deamination of etamicastat, BIA 5-2311 (M7) a deamination and oxidation of etamicastat and BIA 5-2320 (M8), a desulfation and N-acetylation of etamicastat were identified in plasma, urine and faeces.

The glucurono-conjugate of etamicastat (M19 and M20), and the glucurono-conjugate of BIA 5-961 (BIA 5-2351; M25) and of BIA 5-965 (M23 and M24) were identified in urine and plasma. The glucuronidation of BIA 5-2311 in two different positions, BIA 5-2362 (M21) and M22, were also identified in urine and plasma. Other two metabolites with no identified structure, M17 and M18, were detected with the neutral loss of 176, suggesting a glucuronidation of etamicastat metabolites with molecular ion m/z at 281 and 299.

BIA 5-960 (M2), a desulfated metabolite of etamicastat, and M11–M13 were identified only in urine. M11 and M12 molecular ions and their characteristic product ions indicate a desulfation and oxidation in the sulfate position of BIA 5-961. M5 molecular ion and its MS/MS spectra may indicate an oxidative deamination and oxidation of BIA 5-960. The metabolite without sulfate may also be deaminated and oxidated (M3) or could form an aldehyde (M1). M5, M3 and M1 were detected only in faeces and urine. Three unknown etamicastat metabolites were identified in urine, with protonated molecular ions of m/z 294.1 (M4), 368.2 (M15) and 410.2 (M16).

Metabolic profiling

The radioactivity profiles for etamicastat and its metabolites in plasma, urine and faeces are shown in Figure S4. In plasma, the main radioactivity was observed for the acetylated metabolite BIA 5-961 (m/z 354) followed by the parent. Significant amounts of etamicastat glucurono-conjugated derivatives were also detected. The highest amounts of radioactivity were found in the interval 2–3 h, followed by the 4–6 h pool.

In urine, the main radioactivity in most collection periods corresponded to a fraction containing both etamicastat and desulfated BIA 5-961, but from the MS/MS results, the parent compound accounted for most of this fraction. In the 4–8 h collection period, the main radioactivity corresponded to BIA 5-961, the N-acetylated derivative. Other main urinary moieties included the M10 and the glucuronide of M7, which peaked in the 4–8 h collection period. At the same collection period, a peak of other glucurono-conjugates, including etamicastat, the N-acetylated derivative and a glucuronide of a metabolite with m/z of 299, were also detected. BIA 5-998 was present in relatively small amounts, not detected in radioactivity profiling.

In faeces, the main radioactivity was observed for parent etamicastat in pools collected up to 72 h after dosing and for BIA 5-961 in the later pools. Main radioactivity was determined in the collection time from 0 to 144 h. The maximal radioactivity was found in the first 24 h, with decreasing amounts in the successive pools.

Discussion

The aim of the present study was to characterize the disposition, metabolism and excretion of etamicastat in humans and to elucidate the metabolic pathways and the structure of metabolites. Eleven days after administration of [¹⁴C]-etamicastat (98 μCi) to healthy male subjects, 90.4– 97.6% of the administered radioactivity had been excreted. Despite the limited number of subjects (n = 4) included in study, the total radioactivity recovered strongly suggests no accumulation of etamicastat in the body. The majority of the radioactivity was recovered in the urine (55.9–64.9% of the administered dose), and the proportion of etamicastat-associated radioactivity excreted in faeces ranged from 29.4 to 37.3%. From all radioactivity excreted in urine, 11.7–27.2% of the administered dose corresponded to unchanged etamicastat and 5.0–26.8% to the N-acetylated metabolite (BIA 5-961).

In agreement with the results obtained in plasma, the urinary recovery of etamicastat and its N-acetylated metabolite showed high variability between the four subjects included in study. The subject with the highest fraction of dose recovered as the N-acetylated form had the lowest fraction of parent compound. N-Acetylation is one of the major hepatic phase II metabolic pathways involved in drug metabolism. In humans, this reaction is performed by two functional NAT isoforms, NAT1 and NAT2, which are highly polymorphic, with more than 25 alleles identified in each locus 16–18. Both ‘poor acetylators’ and ‘rapid acetylators’ of both forms NAT1 and NAT2 19 have been identified in the study population, which may explain the high variability in etamicastat N-acetylation by the four individuals enrolled in the study. The significantly higher N-acetylation of etamicastat by subject 1, an NAT2 rapid acetylator, in comparison with subjects 2, 3 and 4, NAT2 poor acetylators and NAT1 rapid acetylators, suggests that etamicastat N-acetylation is mainly performed by NAT2. This is in line with evidence recently made available that the large interindividual variability of etamicastat and its N-acetylation in humans is dependent on the NAT2 acetylator status, indicating that systemic exposure to etamicastat is higher and systemic exposure to BIA 5-961 lower in NAT2 poor metabolizers compared with rapid metabolizers 2,4.

The early (3.0–4.0 h) peak and fast decline of total radioactivity in plasma suggest that the N-acetylated metabolite and parent account for most of the circulating etamicastat-derived moieties. The later phase of the plasma profiles, however, appears to be dominated by other metabolites, because the t_1/2 values for the total radioactivity were much higher than those obtained for the parent compound and the N-acetylated metabolite. Metabolites dominating in later phases of the profile may bind to the blood cells less than the moieties dominating in the earlier parts of the profile, because the blood concentrations of etamicastat-associated radioactivity declined faster than their plasma counterparts. In addition, the whole blood/plasma ratios for etamicastat-associated radioactivity C_max and AUC_0–t suggest that the penetration and binding of etamicastat-associated radioactivity to erythrocytes and other blood cells is clearly lower than in circulating plasma.

The proposed scheme for the etamicastat metabolic pathway in humans is illustrated in Figure 1. A total of 25 metabolites were identified in humans after administration of etamicastat. The major route of metabolism was the N-acetylation of the aminoethyl moiety of etamicastat to M14, by the N-acetyl transferases. The N-acetyl metabolite was then glucurono-conjugated at the sulfur moiety (M25), which is abundant in both plasma and urine. A further N-acetylated derivative without the sulfur moiety (M8) was also detected together with the parent compound and may contribute to later circulating derivatives of etamicastat. The glucurono-conjugates of unknown metabolites (M17 and M18) and of etamicastat (M19 and M20) were also detected, mainly in urine, contributing to the excretion of etamicastat-related compounds. Other glucurono-conjugated derivatives from phase I metabolic reactions, such as the oxidative deamination of etamicastat (M21 and M22) and BIA 5-998 (M23 and M24), were detected. The fragmentation behaviour of some of these less abundant metabolites suggests that these compounds are formed at the ethyl amine or hydroxyl ethyl group as N- or O-glucuronides. However, the major glucurono-conjugates are probably N- or S-glucuronides of the mercaptoimidazole group. Significant amounts of the N-acetylated metabolite without the thiol moiety and further oxidation derivatives were also identified. Other conjugations, desulfation, oxidation and oxidative deamination reactions played a minor role in the biotransformation of etamicastat, and some of these metabolites were detected only in urine. All metabolites detected in faeces were also detected in urine and may arise from biliary excretion or were actively excreted in the intestine 20. Also, the parent compound detected in the intestine may arise from unabsorbed drug.

In conclusion, following oral administration, etamicastat and its metabolites are primarily eliminated via the urinary route. Although glucuronidation, oxidative deamination and desulfation are also involved in etamicastat metabolism, the main metabolic pathway is etamicastat N-acetylation, mostly driven by NAT2.

Competing Interests

All authors have completed the Unified Competing Interest form at http://www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and declare: AIL, JFR, CFL, TN, LCW, LA and PSS were employees of BIAL – Portela & Cª, S.A. in the previous 3 years; no other relationships or activities that could appear to have influenced the submitted work.

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