Original Paper Pharmacology 2013;92:207–216 DOI: 10.1159/000354805
Received: July 26, 2013 Accepted: July 31, 2013 Published online: October 11, 2013
Disposition and Metabolism of Safinamide, a Novel Drug for Parkinson’s Disease, in Healthy Male Volunteers Chiara Leuratti Marco Sardina Paolo Ventura Alessandro Assandri Markus Müller Martin Brunner Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria
Abstract Background/Aims: Absorption, biotransformation and elimination of safinamide, an enantiomeric α-aminoamide derivative developed as an add-on therapy for Parkinson’s disease patients, were studied in healthy volunteers administered a single oral dose of 400 mg 14C safinamide methanesulphonate, labelled in metabolically stable positions. Methods: Pharmacokinetics of the parent compound were investigated up to 96 h, of 14C radioactivity up to 192/200 h post-dose. Results/Conclusions: Maximum concentration was achieved at 1 h (plasma, median Tmax) for parent drug and at 7 and 1.5 h for plasma and whole blood 14C radioactivity, respectively. Terminal half-lives were about 22 h for unchanged safinamide and 80 h for radioactivity. Safinamide deaminated acid and the N-dealkylated acid were identified as major metabolites in urine and plasma. In urine, the β-glucuronide of the N-dealkylated acid and the monohydroxy safinamide were also characterized. In addition, the glycine conjugate of the N-dealkylated acid and 2-[4-hydroxybenzylamino]propanamide were tentatively identified as minor urinary metabolites. © 2013 S. Karger AG, Basel
© 2013 S. Karger AG, Basel 0031–7012/13/0924–0207$38.00/0 E-Mail [email protected] www.karger.com/pha
Introduction
The basic, lipophilic, enantiomeric α-aminoamide derivative safinamide [(S)-(+)-2-[4-(3-fluorobenzyloxy) benzylamino]propanamide] marketed as methanesulphonate salt is an original anticonvulsant and antiparkinson agent presently developed as an add-on therapy to L-dopa or dopamine agonists for patients with Parkinson’s disease [1–3]. Safinamide uniquely combines potent, selective and reversible inhibition of monoamine oxidase B (MAO-B) with blockade of voltage-dependent Na+ and Ca2+ channels and inhibition of glutamate release [4–8], which are of potential benefit in Parkinson’s disease [9], thus showing a novel mode of action, targeting both dopaminergic and glutaminergic systems [10]. Data from phase III clinical studies gave evidence that safinamide 50 or 100 mg/day improves the motor function in Parkinson’s disease when prescribed as add-on therapy to dopamine agonists or L-dopa [9, 11, 12]. Studies in healthy volunteers [13–15] demonstrated that safinamide does not affect oral tyramine metabolism mostly mediated by intestinal MAO-A and confirm that it can be administered without tyramine-related dietary restrictions. Chiara Leuratti, PhD CROSS S.A. via L. Lavizzari 18 CH–6850 Mendrisio (Switzerland) E-Mail chiara.leuratti @ croalliance.com
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Key Words Safinamide · Monoamine oxidase B inhibitor · Pharmacokinetics · Metabolism
Materials and Methods Investigational Product Safinamide salt powder was supplied by Newron Pharmaceuticals S.p.A., Bresso, Italy (purity 99.4%). 14C safinamide salt, i.e. (S)-(+)-2-[4-(3-fluorobenzyloxy) [ring-U-14C] benzylamino] propanamide methanesulphonate (fig. 1), with a radiochemical purity of 98.8%, was provided by Amersham Pharmacia Biotech (UK) as a powder. The study drug was prepared at RCC Ltd., Switzerland, by mixing labelled and unlabelled test items, at a specific activity of 0.2075 μgCi/mg (as free safinamide) and with a radiochemical purity of 98.8%. For use in humans, weighed aliquots of 14C-labelled study drug were prepared into subjects’ dose vials and reconstituted in water (nominal dose: 303.5 mg free safinamide). The mean ± SD effective dose was 303.1 ± 0.15 mg free safinamide corresponding to a radioactive dose of 63.1 ± 0.03 μCi (99%. The radioactive dose was established according to EURATOM directives 80/836, 84/467, 84/466, 89/618, 90/641 and 92/3 and in compliance with the WHO general guidelines. Study Design This single-centre, single-dose, open-label study was approved by the Ethics Committee of the Medical University of Vienna, Austria, and was conducted in accordance with the Declaration of Helsinki and the Good Clinical Practice Guideline of the European Commission (EC-GCP guideline). Study Population and Experimental Procedures Six male, non-smoking, healthy Caucasian volunteers, aged 28.7 ± 5.2 years, were included in the study. Their mean body weight was 82.3 ± 10.4 kg and their body mass index 24.7 ± 1.5. All the subjects received a detailed description of the study and gave their written informed consent before enrolment. Eligibility was assessed through full physical examination, electrocardiogram recording, vital signs measurement and clinical laboratory assays including virology and drug screening. Exclusion criteria included drug treatment with cytochrome CYP3A inhibitors or inducers (1
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Pharmacology 2013;92:207–216 DOI: 10.1159/000354805
*
14 C safinamide methanesulphonate. An asterisk indicates a 14C-uniformly labelled ring.
Fig. 1. Structural formula of
month before study inclusion), general anaesthesia (3 months), diagnostic examinations involving the use of radiation (3 months) and blood donations (1 month). Consumption of alcohol and grapefruit juice was not allowed from 24 h before drug administration and for the whole study period. Volunteers were confined at the study site for a minimum of 8 days, starting from the evening preceding drug administration until radioactivity concentrations in urine were less than 0.5% of the 14C dose. On the administration day, volunteers received standardized lunch and dinner at 4 and 11 h post-dose. The investigational product dissolved in water (10 ml) was administered as a single dose, orally, after a 12-hour fasting period. The administration vial was rinsed with 10 ml of water. The study volunteers drank the rinse, in order to avoid any product loss, and then a further 50 ml of water. Venous blood samples for determination of 14C radioactivity in plasma and whole blood were collected at pre-dose, and 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168 and 192 h post-dose. Plasma parent drug concentrations were determined in samples collected up to 96 h postdose. 14C radioactivity was determined in urine (0–4, 4–8, 8–12, 12–24, 24–48, 48–72, 72–96, 96–120, 120–144, 144–168 and 168– 192 h post-dose) and faeces (0–24, 24–48, 48–72, 72–96, 96–120, 120–144, 144–168 and 168–192 h post-dose). The last collection for radioactivity measurements in urine was extended up to 200 h for 2 subjects, since the previous urine sampling period revealed more than 0.5% of the dose. Safety was monitored by physical examinations, electrocardiogram, clinical laboratory tests (including routine haematology, blood chemistry and urine analysis) at screening and final visit, and vital sign check at each visit. Adverse events reported spontaneously or upon questioning by the investigator were recorded during the entire study. Final assessment and evaluation took place when the radioactivity concentration in urine was less than 0.5% of the dose. 14C
Radioactivity Measurements Measurements of total radioactivity levels in plasma, urine and faeces were performed at RCC Ltd., Switzerland, by liquid scintillation counting using a Packard liquid scintillation counter equipped with dpm and luminescence options (Tri-Carb 2500 TR), provided with automatic external standard quench correction. Plasma (duplicate 300-μl aliquots, weighed) and urine samples (duplicate 1,000-μl aliquots) were counted after mixing with liquid scintillation cocktail. Lyophilized faeces samples were combusted using an automatic sample oxidizer (model 306 Tri-carb Sample Oxidizer, for Liquid Scintillation, Packard Instrument Co., USA) and 14CO2 radioactivity counted.
Leuratti/Sardina/Ventura/Assandri/ Müller/Brunner
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Safety and pharmacokinetics after single- and multiple-dose administrations have been described previously in healthy subjects [16]. At single ascending oral doses ranging from 2.5 to 10.0 mg/kg, safinamide was absorbed in a linear and dose-proportional fashion. Peak plasma levels were obtained on average at 1.8–2.8 h post-dose. Concentrations declined with a terminal half-life of 20– 23 h. A complete long-lasting inhibition of platelet MAOB activity was observed at all doses tested. Food intake prolonged the rate but did not affect the extent of safinamide absorption [16]. The present study was performed to investigate the absorption, disposition, elimination and metabolic pathways of safinamide after single-dose administration of 14C safinamide, uniformly labelled in metabolically stable positions.
Safinamide Analysis Plasma samples were analysed at RCC Ltd. for the content of the parent drug [(S)-enantiomer] and of its (R)-enantiomer using a validated enantioselective liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method with a limit of quantification of 10 ng/ml. The equipment consisted of a Merck L-7100 LC pump, with Merck L-7200 injector, and a CHIRA GROM 4, 2.0 × 60-mm, 8-μm column. Mobile phase: 50 mmol/l ammonium acetate (H2O, native pH)/acetronitrile (70 + 30, v/V); flow rate: 0.110 ml/min. The detector was a Finnigan TSQ3 MS (ionization mode: atmospheric pressure chemical ionization, positive ions). Analysis of Metabolites Metabolite analysis in urine and plasma was performed at Biodynamics, UK. Reference standards of safinamide, 14C safinamide, N-dealkylated acid of safinamide (NiK-14134), the glycine conjugate of N-dealkylated acid (NiK-15009), 2-[4-hydroxybenzylamino]propanamide (NW-1199), p-hydroxybenzoic acid and hippuric acid were provided by Newron S.p.A., Italy. Sample Preparation Selected urine samples from all subjects (0–12, 12–24 and 24– 48 h) and plasma extracts (1-, 2-, 6-, 12- and 24-hour samples) were analysed directly (untreated samples), and after overnight incubation at 37.5 ° C with an equivalent volume of β-glucuronidase/sulphatase (type H1 from Helix pomatia, Sigma-Aldrich, UK), pH 5.
Metabolite Pattern Analysis Metabolite pattern analysis was performed using a high-performance liquid chromatography (HPLC) system Hitachi LaChrom (L-7100) equipped with a Hitachi LaChrom L-7200 autosampler, an L-7400 UV detector and LabLogic beta-RAM radiodetector (LabLogic, Sheffield, UK). The LC column was a Prodigy ODS3, 250 × 4.6 mm, 5 μm (Phenomenex). The analytes were eluted with a gradient of 10 mmol/l ammonium acetate (mobile phase A) and 100 mmol/l ammonium acetate:acetonitrile, 10: 90 (mobile phase B) in 50 min, at a flow rate of 1 ml/min. Metabolite Characterization (LC-MS/MS) Metabolites were characterized by LC-MS/MS analysis using a Finnigam TSQ Quantum Ultra AM (Thermo Electron, Hemel Hempstead, UK), with electrospray ionization mode; positive/ negative mode; spray voltage 4,500 V/–3,000 V; capillary temperature 350 ° C. The LC system consisted of a Surveyor MS pump
(Thermo Electron), a Prodigy ODS3, 25 cm × 4.6 mm i.d., 55-μm column, protected by a guard column (Security Guard Cartridge C18, 4.0 × 2 mm) and a Surveyor autosampler. Mobile phases A and B were as described above for the metabolite pattern analysis. Flow rate was 1 ml/min (approximately 4: 1 split, with approximately 200 μl/min flow to MS). LC-MS/MS full scan and product ion mass spectra were obtained for safinamide (MW 302) in positive ion mode. Product ion spectra were obtained for the reference standards. For urine samples, full scan and product ion mass spectra were obtained to confirm the identity of any drug-related components identified from screening runs (performed by constant neutral loss scans, precursor ion scans and full scan analyses). For plasma (selected 6-, 12- and 24-hour extracts), due to the low concentration of drug-related material, a minimum of three characteristic fragment ions were selected for each component of interest based on the product ion spectra already obtained. The selected transitions were used to create specific multiple reaction monitoring methods in positive and negative ion modes for structure confirmation. Pharmacokinetic Analysis The following pharmacokinetic parameters were measured or calculated for parent safinamide and 14C radioactivity using KineticaTM version 4.1 (Thermo Electron Corp., USA): maximum plasma/blood concentration (Cmax), time to attain Cmax (Tmax), area under the plasma/blood concentration-time curve (AUC0–t), calculated using the linear trapezoidal method from administration (time point 0) to the last quantifiable concentration time, area under the plasma/blood concentration-time curve extrapolated to infinity [AUC0–∞, with AUC0–∞ = (AUC0–t + Ct/λz), where Ct is the last measurable drug concentration], terminal half-life (t1/2), total (systemic) oral body clearance, renal clearance and apparent oral volume of distribution (Vd/F). Mean parameter values were calculated as the mean of the individual parameter values.
Results
Safinamide and Total Radioactivity Pharmacokinetics After single oral administration of 14C safinamide methanesulphonate, circulating concentrations of the parent drug (enantiomer S, free safinamide) and 14C radioactivity rapidly increased (fig. 2). Peak plasma concentration of safinamide was reached at a median time of 1 h (range 0.5–1.5 h), whereas later Tmax values were observed for 14C radioactivity in plasma (median Tmax: 7 h; range 2–12 h) and whole blood (median Tmax: 1.5 h; range 1–8 h). The range of Tmax for 14C radioactivity was quite wide both in plasma and in whole blood, giving rise to two apparent peaks: the first one at approximately the same time as unchanged safinamide (i.e. 1 h), the second one at 7–8 h post-dose. No (R)-enantiomer was detected, suggesting that no in vivo transformation of the active (S)-enantiomer to the (R)-enantiomer occurred.
14C Safinamide Disposition and Biotransformation
Pharmacology 2013;92:207–216 DOI: 10.1159/000354805
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Total radioactivity in whole blood was determined at the Department of Clinical Pharmacology, Medical University of Vienna, Austria, using a Wallac 1410 liquid scintillation counter, with automatic external standard quench correction. Additionally, urinary radioactivity recovery was checked immediately after each sample collection at the clinical site. For whole blood (250-μl sample), hydrogen peroxide (30%, 100 μl) and then a mixture of Soluene-350 and isopropyl alcohol (1: 1) were added to the sample, which was incubated at 60 ° C for 2 h and then returned to room temperature. Hydrogen peroxide (30%, 100 μl) was added again while stirring; the samples were left at room temperature for 15–30 min, incubated at 60 ° C for a further 30 min and finally cooled before counting. Results are expressed as safinamide free base equivalents (μgEq/ml; specific activity 0.2075 μCi/mg).
5.0
5
4.5 4 Concentrations (μg/ml or μgEq/ml)
4.0 3
3.5
2
3.0
1
2.5
0
2.0
0
3
6
9
12 Time (h)
1.5
15
18
21
24
1.0 0.5 0 0
12
24
36
48
60
72
84
96 108 Time (h)
120
132
144
156
168
180
192
Fig. 2. Concentration-versus-time curves of total 14C radioactivity (μgEq/ml) in plasma (diamond), total 14C ra-
dioactivity (μgEq/ml) in whole blood (square) and unchanged safinamide (μg/ml) in plasma (circle) after administration of a single oral dose of 14C safinamide methanesulphonate to 6 male healthy volunteers (mean ± SD). The concentration-versus-time profile from administration up to 24 h post-dose is represented in the insert figure.
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ml/h × kg × F for unchanged safinamide and 17.53 ± 2.71 ml/h × kg × F for 14C radioactivity, in agreement with a terminal half-life of about 80 h. 14C radioactivity renal clearance was 14.72 ± 3.19 ml/h × kg × F, thus representing about 84% of total 14C radioactivity clearance. 14
C Radioactivity Excretion in Urine and Faeces The 14C radioactivity was excreted mainly in urine (mean ± SD: 76.1 ± 9.7%), with individual recovery ranging from 58.1 to 86.3% of the administered dose. 14C radioactivity faecal elimination was quite low, accounting for only the 1.51 ± 0.36% of the dose. Total balance of 14C radioactivity was 77.6 ± 9.6% of the administered dose (range 59.8–87.9%). One subject showed a total balance of 59.8%, which was possibly to be ascribed to incomplete urine sample collection. Metabolite Pattern in Urine Samples Representative radiochromatograms of untreated and enzyme-treated urine samples are shown in figure 3. In untreated samples three metabolite peaks (U1, U2 and Leuratti/Sardina/Ventura/Assandri/ Müller/Brunner
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Main pharmacokinetic parameters of safinamide (free base) in plasma and 14C radioactivity in plasma and whole blood are shown in table 1. Plasma concentrations of the parent compound were lower than plasma radioactivity concentrations. In particular, the ratio of plasma concentrations of 14C-radioactivity/safinamide gradually increased from 1.4 at 0.5 h to 5.3 at 96 h post-dose. 14 C radioactivity in plasma and whole blood declined in a biphasic manner, with terminal half-lives (t1/2) of 77.4 ± 24.6 and 81.1 ± 33.0 h, respectively, whereas the parent compound decreased mono-exponentially with a t1/2 of 22.2 ± 1.4 h. Blood/plasma 14C radioactivity distribution ratio, based on mean Cmax and AUC values, was approximately 1.2:1 and indicated a free exchange of radioactivity between plasma and red blood cells. Average distribution volumes for safinamide and 14C radioactivity per kilogram of body weight were 1,813.3 ± 204.3 and 1,944.6 ± 673.1 ml/kg × F, respectively. Total oral clearance per kilogram of body weight was 57.0 ± 8.5
80.0 Deaminated safinamide acid U3
70.0 60.0 N-dealkylated glucuronide
40.0
U2 U1
30.0 20.0
Safinamide
U4
Bkg 1
10.0
Bkg 2
cps
50.0
0 10:00
20:00
35.0 N-dealkylated acid + deaminated safinamide acid
30.0
30:00
mm:ss
40:00
50:00
U2a+U3
0:00
a
Bkg 2
Safinamide
U5 Region 4
5.0
U3
10.0
Region 2
15.0
Bkg 1 Region 1 U1a U1 U1b
cps
20.0
Region 5
U4
25.0
0
Fig. 3. Representative HPLC radioactivity profile of untreated (a) and deconjugated (b) urine samples (12–24 h). U = Urinary
b
0:00
10:00
20:00
mm:ss
30:00
40:00
50:00
peak.
Table 1. Main pharmacokinetic parameters (mean ± SD) of plasma 14C radioactivity, whole blood 14C radioactivity and plasma parent safinamide
AUC0–t μg × h/ml
AUC0–∞ μg × h/ml
Terminal t1/2, h
Vd/F ml/kg
197.8 ± 28.3
215.9 ± 33.4
77.4 ± 24.6
1,944.6 ± 673.1 17.53 ± 3.40
1.5 (1 – 8) 244.9 ± 40.0 1 (0.5 – 1.5) 62.7 ± 4.3
277.9 ± 55.3 65.9 ± 4.8
81.1 ± 33.0 22.2 ± 1.4
– 1,813.3 ± 204.3
Cmax, μg/ml Tmax, h or μgEq/ml 14C
radioactivity in plasma 3.72 ± 0.44 radioactivity in whole blood 4.5 ± 0.42 Safinamide in plasma 2.37 ± 0.38
7 (2 – 12)
Cl/F ml/h × kg
14C
– 57.0 ± 8.5
14C Safinamide Disposition and Biotransformation
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For Tmax median (min–max) values are displayed.
Metabolite Pattern in Plasma Samples Plasma extracts were analysed by HPLC using off-line fraction collection and liquid scintillation counting, due to low concentrations of radioactivity. At all time points analysed, the N-dealkylated metabolite (NiK-14134) and safinamide represented the majority of the sample radioactivity and co-chromatographed with authentic standards. The deaminated acid metabolite was also observed. Characterization of Metabolite Structures Metabolite structures were characterized by LC-MS/ MS and confirmed by comparison of retention times and fragment spectra with authentic reference standards, when available. Proposed structures are listed in table 2. The N-dealkylated acid metabolite of safinamide (MW 246) was identified as a major component in deconjugated urine and in plasma. In addition, the presence of the N-dealkylated safinamide glucuronide (MW 422; probably four isomers, table 2) in urine and of the deaminated safinamide acid metabolite (MW 303) in urine and plasma was confirmed. Monohydroxy safinamide metabolites (MW318; four isomers) were also identified in urine samples by LC-MS/ MS, but did not correspond to any of the radioactivity peaks obtained in the HPLC profiling work. Mass fragmentation indicated that hydroxylation occurred on the fluorobenzene moiety of safinamide. Finally, the glycine conjugate of N-dealkylated safinamide (MW 303, NiK-15009) and 2-[4-hydroxybenzylamino]propanamide (NW-1199, MW194) were also tentatively identified as minor urinary metabolites. 212
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Safety Safinamide was well tolerated. Four adverse events of mild severity were reported by 3 subjects during the study. One of the reported adverse events, i.e. headache, was judged as possibly related to the investigational product. The other three adverse events (i.e. headache, pain sensation in both eyes and neuralgia from left arm to thumb) were not related to treatment and all resolved by the study end. No clinically significant effects of treatment on laboratory parameters, vital signs or electrocardiogram parameters were observed throughout the study.
Discussion
After a single oral dose of 14C safinamide as methanesulphonate salt to 6 healthy male volunteers, parent safinamide was promptly absorbed, reaching mean peak plasma concentrations at about 1 h post-dose. For 14C radioactivity in plasma and whole blood, two main peaks appeared to be present. The first peak (1–1.5 h) was achieved at a similar Tmax as unchanged safinamide, whereas the second peak, observed at 7–8 h post-dose, could reflect a delayed release of a mixture of 14C-safinamide-related substances with different kinetics into the blood from some peripheral compartment(s). The presence of an enterohepatic circulation occurring postprandial following lunch, however, cannot be completely excluded. Plasma concentrations of the parent compound were lower than plasma radioactivity concentrations (1.4- to 5-fold), in particular at later time points, indicating extensive safinamide metabolization. No chiral inversion of safinamide was observed. Whole blood to plasma ratios for Cmax and AUC were only about 1.2:1, suggesting that radioactivity may freely enter and leave the blood cells. Average distribution volumes per kilogram (Vd/F × kg) of BW for plasma safinamide and 14C radioactivity were 1.8 and 1.9 litres/kg × F, respectively, indicating a total distribution volume (Vd/F) of 150–160 litres for adults of 82 kg (mean value). These values (>100 litres independently of the F value) exceed the actual subjects’ BWs and indicate an extensive organ(s)/tissue(s) distribution of the drug. For the parent compound, the organ/tissue drug is in equilibrium with the blood drug according to a one-compartment open model, corroborated by the observed monoexponential elimination of unchanged safinamide (t1/2 22 h), confirming results obtained in previous clinical studies [17]. Differently, 14C radioactivity decreased in a biphasic manner (terminal t1/2 80 h), indicating a multiLeuratti/Sardina/Ventura/Assandri/ Müller/Brunner
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U3) and safinamide (U4) were observed. Peak U2 was identified by LC-MS/MS (see below) as the β-glucuronide conjugate of the N-dealkylated acid. This peak was present as a major component and disappeared upon complete deconjugation, giving rise to the free N-dealkylated safinamide acid, which co-eluted with the authentic standard (NiK-14134; peak U2a, fig. 3). Peak U3, further identified as deaminated acid of safinamide, was present in untreated and enzyme-treated samples, and in the latter it co-eluted with U2a. Component U1 deconjugated into two other subcomponents, i.e. U1a and U1b. These metabolites did not co-elute with any reference standards and remained unidentified. The stability of safinamide and its metabolites under incubation conditions was investigated: the HPLC analysis did not indicate any deviation from the characteristic profiles of untreated samples.
Table 2. Components characterized in human urine and plasma after single oral dose of 14C safinamide
Component
Identified in
Safinamide MW: 302 Rt: 33.5
urine plasma
Proposed structure and characteristic fragment ions/product ions F
m/z 215
m/z 109
CH 3
NH O
NH2 O
Positive ion mode
N-Dealkylated acid (NiK-14134) MW: 246 Rt: 24.3
deconjugated urine plasma
F
m/z 201 OH O O OH
O
O
m/z 92
O
m/z 136
Negative ion mode
Glucuronide of N-dealkylated acid MW: 422 Rt: 23.9–25.4
urine
F
m/z 245 O
Glucuronide
O O m/z 201 OH O O
m/z 136
Negative ion mode
Acid metabolite MW: 303 Rt: 24.9
urine deconjugated urine plasma
F
m/z 215
m/z 109
CH 3
NH O
OH O
Positive ion mode m/z 106
m/z 148 NH CH 2
m/z 120
CH 2 O
O
O
O
O
Negative ion mode
Monohydroxy safinamide (several isomers) MW: 318 Rt: 18.4, 19.3, 24.8, 25.9
urine
Glycine conjugate of N-dealkylated acid (NiK-15009) MW: 303 Rt: 23.7 (tentative)
urine deconjugated urine
F
m/z 231
m/z 125
NH2 O
HO
Positive ion mode
F
O
m/z 201
OH NH
m/z 258
O m/z 149
m/z 120 O
m/z 92
O
O
m/z 148 NH CH 2 O
urine deconjugated urine
Negative ion mode
O
O
2-[4-hydroxybenzylamino]propanamide (NW-1199) MW: 194 Rt: 8.3 (tentative)
CH 3
NH O
m/z 89 m/z 107
+2H NH
HO
CH 3
m/z 44
NH2 O
Positive ion mode
14C Safinamide Disposition and Biotransformation
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The quoted molecular weights (MW) are for unlabelled components. Retention times (Rt) are taken from 14C radiochromatogram traces where possible, otherwise from product ion analyses. The reference standard code is given in parentheses, when available.
NH2
N H
F
O
O
Safinamide MW 302
F
HO CH3
NH O
NH2
O
Alpha-oxidation by MAO (with intermediate formation)
Monohydroxy safinamide MW 318 CH3 NH HO
NH2
O
2-[4-hydroxybenzylamino]propanamide MW 194
O OH
N H F
O
OH
F
O
O
N-dealkylated acid MW 246
Deaminated acid metabolite MW 303
O
F
OH NH O O
N-dealkylated glycine conjugate MW 303
N-dealkylated acid glucuronide MW 422
Fig. 4. Proposed biotransformation path-
component and, probably, multi-compartmental model. The differences in t1/2 observed between parent safinamide and 14C radioactivity in plasma are explained by the presence of a varying mixture of metabolites, cleared from the body compartments at different rates. Total oral clearance of unchanged safinamide was 57.0 ± 8.5 ml/h × kg × F. The process comprises not only renal excretion but also loss of the parent molecule by metabolism. In contrast, total oral clearance of plasma 14C radioactivity, which accounts for parent safinamide as well as metabolites, was on average only 17.53 ± 2.71 ml/h × kg × F, in agreement with a terminal half-life of about 80 h. 214
Pharmacology 2013;92:207–216 DOI: 10.1159/000354805
Renal clearance of 14C radioactivity (14.72 ± 3.19 ml/h × kg × F) represented about 84% of the total 14C radioactivity clearance. This result is confirmed by the urinary concentrations of 14C radioactivity, which gave cumulative amounts of 230.7 ± 29.4 mgEq and consequently urinary outputs of 76.11 ± 9.68% of the administered dose. On the other hand, 14C radioactivity faecal elimination was almost negligible, suggesting that possibly about 1.5% of the administered dose had not been absorbed or was eliminated by biliary secretion after systemic absorption. This very low faecal excretion is indicative of a substantially complete intestinal absorption of the drug. Leuratti/Sardina/Ventura/Assandri/ Müller/Brunner
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ways for safinamide in man.
At the end of the study (200 h post-dose), total balance of radioactivity accounted for approximately 80% (range 59.78–87.90%) of the dose, although for 1 subject urine sample collection appeared not to be complete, slightly underestimating final urinary output. The major metabolite of safinamide identified in deconjugated urine samples and in plasma was the N-dealkylated acid of safinamide (MW 246). The presence of this metabolite indicates N-dealkylation as a major pathway of safinamide metabolism, characterized by safinamide oxidation by MAO followed by further oxidation to the acid. This metabolic pathway is characteristic of other MAO-B inhibitors, a typical example being milacemide, an anticonvulsant drug which is metabolized by MAO-B [17, 18], giving rise to the N-dealkylated pentanoic acid plus glycinamide, the latter then transformed into glycine. It is possible that N-dealkylation of safinamide might lead to the formation of alaninamide (and then alanine) in the same way. N-dealkylation was postulated as a major metabolic pathway also for ralfinamide, an α-aminoamide derivative analogue of safinamide [19]. In untreated urine samples, the glucuronide conjugate of N-dealkylated acid (MW 422) was identified as a major component, which disappeared upon complete deconjugation giving rise to the free N-dealkylated metabolite. Another major metabolite observed in urine and plasma samples was the deaminated safinamide acid (MW 303), a product of hydrolytic deamination, by amide hydrolase, of the parent molecule. This component was shown to remain constantly high (about 31–38%) in urine up to 48 h post-dose. Monohydroxy safinamide (MW 318, four possible isomers) was identified in urine samples by LCMS/MS but did not correspond to any of the radio-peaks obtained in the HPLC profiling. Monohydroxy safinamide metabolites were also identified during in vitro
studies using rat, dog, monkey and human microsomes [unpublished data]. In the present work, 2-[4-hydroxybenzylamino]propanamide and the glycine conjugate of the N-dealkylated acid were also tentatively identified as minor safinamide metabolism products. On the basis of the results obtained, safinamide metabolic pathways represented in figure 4 are proposed. Previously described tentative intermediates [20] were not experimentally confirmed. In conclusion, the present study allowed for an indepth characterization of safinamide disposition in vivo and identified previously unknown metabolites.
Acknowledgements The authors would like to acknowledge RCC Ltd., Environmental Chemistry and Pharmanalytics Division, Itingen, Switzerland, and BioDynamics Research Ltd., Rushden, UK, for the analytical part of this work and CROSS Alliance Group, Switzerland, for the pharmacokinetic analysis. Newron S.p.A., Italy, funded the clinical study. Newron S.p.A. has licensed safinamide to Zambon Farmaceutici S.p.A.
Disclosure Statement The relationship between the sponsors (Newron S.p.A., Zambon S.p.A., Italy) and CROSS S.A. (Switzerland) was regulated by financial agreements. C.L. is an employee of CROSS S.A., was the study coordinator and is the author of the clinical study report and of the manuscript; M.S. is an employee of Zambon S.p.A., Italy; P.V. is a consultant in Pharmacokinetics and Metabolism, Piacenza, Italy. A.A. is the Scientific Director at CROSS S.A.; M.M. and M.B. are employees of the Department of Clinical Pharmacology, Medical University of Vienna, Austria: M.M. was the principal investigator of the study, M.B. was the co-investigator at the clinical site. All authors reviewed and approved the final draft of the manuscript.
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14C Safinamide Disposition and Biotransformation
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Received: July 26, 2013 Accepted: July 31, 2013 Published online: October 11, 2013
Disposition and Metabolism of Safinamide, a Novel Drug for Parkinson’s Disease, in Healthy Male Volunteers Chiara Leuratti Marco Sardina Paolo Ventura Alessandro Assandri Markus Müller Martin Brunner Department of Clinical Pharmacology, Medical University of Vienna, Vienna, Austria
Abstract Background/Aims: Absorption, biotransformation and elimination of safinamide, an enantiomeric α-aminoamide derivative developed as an add-on therapy for Parkinson’s disease patients, were studied in healthy volunteers administered a single oral dose of 400 mg 14C safinamide methanesulphonate, labelled in metabolically stable positions. Methods: Pharmacokinetics of the parent compound were investigated up to 96 h, of 14C radioactivity up to 192/200 h post-dose. Results/Conclusions: Maximum concentration was achieved at 1 h (plasma, median Tmax) for parent drug and at 7 and 1.5 h for plasma and whole blood 14C radioactivity, respectively. Terminal half-lives were about 22 h for unchanged safinamide and 80 h for radioactivity. Safinamide deaminated acid and the N-dealkylated acid were identified as major metabolites in urine and plasma. In urine, the β-glucuronide of the N-dealkylated acid and the monohydroxy safinamide were also characterized. In addition, the glycine conjugate of the N-dealkylated acid and 2-[4-hydroxybenzylamino]propanamide were tentatively identified as minor urinary metabolites. © 2013 S. Karger AG, Basel
© 2013 S. Karger AG, Basel 0031–7012/13/0924–0207$38.00/0 E-Mail [email protected] www.karger.com/pha
Introduction
The basic, lipophilic, enantiomeric α-aminoamide derivative safinamide [(S)-(+)-2-[4-(3-fluorobenzyloxy) benzylamino]propanamide] marketed as methanesulphonate salt is an original anticonvulsant and antiparkinson agent presently developed as an add-on therapy to L-dopa or dopamine agonists for patients with Parkinson’s disease [1–3]. Safinamide uniquely combines potent, selective and reversible inhibition of monoamine oxidase B (MAO-B) with blockade of voltage-dependent Na+ and Ca2+ channels and inhibition of glutamate release [4–8], which are of potential benefit in Parkinson’s disease [9], thus showing a novel mode of action, targeting both dopaminergic and glutaminergic systems [10]. Data from phase III clinical studies gave evidence that safinamide 50 or 100 mg/day improves the motor function in Parkinson’s disease when prescribed as add-on therapy to dopamine agonists or L-dopa [9, 11, 12]. Studies in healthy volunteers [13–15] demonstrated that safinamide does not affect oral tyramine metabolism mostly mediated by intestinal MAO-A and confirm that it can be administered without tyramine-related dietary restrictions. Chiara Leuratti, PhD CROSS S.A. via L. Lavizzari 18 CH–6850 Mendrisio (Switzerland) E-Mail chiara.leuratti @ croalliance.com
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Key Words Safinamide · Monoamine oxidase B inhibitor · Pharmacokinetics · Metabolism
Materials and Methods Investigational Product Safinamide salt powder was supplied by Newron Pharmaceuticals S.p.A., Bresso, Italy (purity 99.4%). 14C safinamide salt, i.e. (S)-(+)-2-[4-(3-fluorobenzyloxy) [ring-U-14C] benzylamino] propanamide methanesulphonate (fig. 1), with a radiochemical purity of 98.8%, was provided by Amersham Pharmacia Biotech (UK) as a powder. The study drug was prepared at RCC Ltd., Switzerland, by mixing labelled and unlabelled test items, at a specific activity of 0.2075 μgCi/mg (as free safinamide) and with a radiochemical purity of 98.8%. For use in humans, weighed aliquots of 14C-labelled study drug were prepared into subjects’ dose vials and reconstituted in water (nominal dose: 303.5 mg free safinamide). The mean ± SD effective dose was 303.1 ± 0.15 mg free safinamide corresponding to a radioactive dose of 63.1 ± 0.03 μCi (99%. The radioactive dose was established according to EURATOM directives 80/836, 84/467, 84/466, 89/618, 90/641 and 92/3 and in compliance with the WHO general guidelines. Study Design This single-centre, single-dose, open-label study was approved by the Ethics Committee of the Medical University of Vienna, Austria, and was conducted in accordance with the Declaration of Helsinki and the Good Clinical Practice Guideline of the European Commission (EC-GCP guideline). Study Population and Experimental Procedures Six male, non-smoking, healthy Caucasian volunteers, aged 28.7 ± 5.2 years, were included in the study. Their mean body weight was 82.3 ± 10.4 kg and their body mass index 24.7 ± 1.5. All the subjects received a detailed description of the study and gave their written informed consent before enrolment. Eligibility was assessed through full physical examination, electrocardiogram recording, vital signs measurement and clinical laboratory assays including virology and drug screening. Exclusion criteria included drug treatment with cytochrome CYP3A inhibitors or inducers (1
208
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*
14 C safinamide methanesulphonate. An asterisk indicates a 14C-uniformly labelled ring.
Fig. 1. Structural formula of
month before study inclusion), general anaesthesia (3 months), diagnostic examinations involving the use of radiation (3 months) and blood donations (1 month). Consumption of alcohol and grapefruit juice was not allowed from 24 h before drug administration and for the whole study period. Volunteers were confined at the study site for a minimum of 8 days, starting from the evening preceding drug administration until radioactivity concentrations in urine were less than 0.5% of the 14C dose. On the administration day, volunteers received standardized lunch and dinner at 4 and 11 h post-dose. The investigational product dissolved in water (10 ml) was administered as a single dose, orally, after a 12-hour fasting period. The administration vial was rinsed with 10 ml of water. The study volunteers drank the rinse, in order to avoid any product loss, and then a further 50 ml of water. Venous blood samples for determination of 14C radioactivity in plasma and whole blood were collected at pre-dose, and 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168 and 192 h post-dose. Plasma parent drug concentrations were determined in samples collected up to 96 h postdose. 14C radioactivity was determined in urine (0–4, 4–8, 8–12, 12–24, 24–48, 48–72, 72–96, 96–120, 120–144, 144–168 and 168– 192 h post-dose) and faeces (0–24, 24–48, 48–72, 72–96, 96–120, 120–144, 144–168 and 168–192 h post-dose). The last collection for radioactivity measurements in urine was extended up to 200 h for 2 subjects, since the previous urine sampling period revealed more than 0.5% of the dose. Safety was monitored by physical examinations, electrocardiogram, clinical laboratory tests (including routine haematology, blood chemistry and urine analysis) at screening and final visit, and vital sign check at each visit. Adverse events reported spontaneously or upon questioning by the investigator were recorded during the entire study. Final assessment and evaluation took place when the radioactivity concentration in urine was less than 0.5% of the dose. 14C
Radioactivity Measurements Measurements of total radioactivity levels in plasma, urine and faeces were performed at RCC Ltd., Switzerland, by liquid scintillation counting using a Packard liquid scintillation counter equipped with dpm and luminescence options (Tri-Carb 2500 TR), provided with automatic external standard quench correction. Plasma (duplicate 300-μl aliquots, weighed) and urine samples (duplicate 1,000-μl aliquots) were counted after mixing with liquid scintillation cocktail. Lyophilized faeces samples were combusted using an automatic sample oxidizer (model 306 Tri-carb Sample Oxidizer, for Liquid Scintillation, Packard Instrument Co., USA) and 14CO2 radioactivity counted.
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Safety and pharmacokinetics after single- and multiple-dose administrations have been described previously in healthy subjects [16]. At single ascending oral doses ranging from 2.5 to 10.0 mg/kg, safinamide was absorbed in a linear and dose-proportional fashion. Peak plasma levels were obtained on average at 1.8–2.8 h post-dose. Concentrations declined with a terminal half-life of 20– 23 h. A complete long-lasting inhibition of platelet MAOB activity was observed at all doses tested. Food intake prolonged the rate but did not affect the extent of safinamide absorption [16]. The present study was performed to investigate the absorption, disposition, elimination and metabolic pathways of safinamide after single-dose administration of 14C safinamide, uniformly labelled in metabolically stable positions.
Safinamide Analysis Plasma samples were analysed at RCC Ltd. for the content of the parent drug [(S)-enantiomer] and of its (R)-enantiomer using a validated enantioselective liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method with a limit of quantification of 10 ng/ml. The equipment consisted of a Merck L-7100 LC pump, with Merck L-7200 injector, and a CHIRA GROM 4, 2.0 × 60-mm, 8-μm column. Mobile phase: 50 mmol/l ammonium acetate (H2O, native pH)/acetronitrile (70 + 30, v/V); flow rate: 0.110 ml/min. The detector was a Finnigan TSQ3 MS (ionization mode: atmospheric pressure chemical ionization, positive ions). Analysis of Metabolites Metabolite analysis in urine and plasma was performed at Biodynamics, UK. Reference standards of safinamide, 14C safinamide, N-dealkylated acid of safinamide (NiK-14134), the glycine conjugate of N-dealkylated acid (NiK-15009), 2-[4-hydroxybenzylamino]propanamide (NW-1199), p-hydroxybenzoic acid and hippuric acid were provided by Newron S.p.A., Italy. Sample Preparation Selected urine samples from all subjects (0–12, 12–24 and 24– 48 h) and plasma extracts (1-, 2-, 6-, 12- and 24-hour samples) were analysed directly (untreated samples), and after overnight incubation at 37.5 ° C with an equivalent volume of β-glucuronidase/sulphatase (type H1 from Helix pomatia, Sigma-Aldrich, UK), pH 5.
Metabolite Pattern Analysis Metabolite pattern analysis was performed using a high-performance liquid chromatography (HPLC) system Hitachi LaChrom (L-7100) equipped with a Hitachi LaChrom L-7200 autosampler, an L-7400 UV detector and LabLogic beta-RAM radiodetector (LabLogic, Sheffield, UK). The LC column was a Prodigy ODS3, 250 × 4.6 mm, 5 μm (Phenomenex). The analytes were eluted with a gradient of 10 mmol/l ammonium acetate (mobile phase A) and 100 mmol/l ammonium acetate:acetonitrile, 10: 90 (mobile phase B) in 50 min, at a flow rate of 1 ml/min. Metabolite Characterization (LC-MS/MS) Metabolites were characterized by LC-MS/MS analysis using a Finnigam TSQ Quantum Ultra AM (Thermo Electron, Hemel Hempstead, UK), with electrospray ionization mode; positive/ negative mode; spray voltage 4,500 V/–3,000 V; capillary temperature 350 ° C. The LC system consisted of a Surveyor MS pump
(Thermo Electron), a Prodigy ODS3, 25 cm × 4.6 mm i.d., 55-μm column, protected by a guard column (Security Guard Cartridge C18, 4.0 × 2 mm) and a Surveyor autosampler. Mobile phases A and B were as described above for the metabolite pattern analysis. Flow rate was 1 ml/min (approximately 4: 1 split, with approximately 200 μl/min flow to MS). LC-MS/MS full scan and product ion mass spectra were obtained for safinamide (MW 302) in positive ion mode. Product ion spectra were obtained for the reference standards. For urine samples, full scan and product ion mass spectra were obtained to confirm the identity of any drug-related components identified from screening runs (performed by constant neutral loss scans, precursor ion scans and full scan analyses). For plasma (selected 6-, 12- and 24-hour extracts), due to the low concentration of drug-related material, a minimum of three characteristic fragment ions were selected for each component of interest based on the product ion spectra already obtained. The selected transitions were used to create specific multiple reaction monitoring methods in positive and negative ion modes for structure confirmation. Pharmacokinetic Analysis The following pharmacokinetic parameters were measured or calculated for parent safinamide and 14C radioactivity using KineticaTM version 4.1 (Thermo Electron Corp., USA): maximum plasma/blood concentration (Cmax), time to attain Cmax (Tmax), area under the plasma/blood concentration-time curve (AUC0–t), calculated using the linear trapezoidal method from administration (time point 0) to the last quantifiable concentration time, area under the plasma/blood concentration-time curve extrapolated to infinity [AUC0–∞, with AUC0–∞ = (AUC0–t + Ct/λz), where Ct is the last measurable drug concentration], terminal half-life (t1/2), total (systemic) oral body clearance, renal clearance and apparent oral volume of distribution (Vd/F). Mean parameter values were calculated as the mean of the individual parameter values.
Results
Safinamide and Total Radioactivity Pharmacokinetics After single oral administration of 14C safinamide methanesulphonate, circulating concentrations of the parent drug (enantiomer S, free safinamide) and 14C radioactivity rapidly increased (fig. 2). Peak plasma concentration of safinamide was reached at a median time of 1 h (range 0.5–1.5 h), whereas later Tmax values were observed for 14C radioactivity in plasma (median Tmax: 7 h; range 2–12 h) and whole blood (median Tmax: 1.5 h; range 1–8 h). The range of Tmax for 14C radioactivity was quite wide both in plasma and in whole blood, giving rise to two apparent peaks: the first one at approximately the same time as unchanged safinamide (i.e. 1 h), the second one at 7–8 h post-dose. No (R)-enantiomer was detected, suggesting that no in vivo transformation of the active (S)-enantiomer to the (R)-enantiomer occurred.
14C Safinamide Disposition and Biotransformation
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Total radioactivity in whole blood was determined at the Department of Clinical Pharmacology, Medical University of Vienna, Austria, using a Wallac 1410 liquid scintillation counter, with automatic external standard quench correction. Additionally, urinary radioactivity recovery was checked immediately after each sample collection at the clinical site. For whole blood (250-μl sample), hydrogen peroxide (30%, 100 μl) and then a mixture of Soluene-350 and isopropyl alcohol (1: 1) were added to the sample, which was incubated at 60 ° C for 2 h and then returned to room temperature. Hydrogen peroxide (30%, 100 μl) was added again while stirring; the samples were left at room temperature for 15–30 min, incubated at 60 ° C for a further 30 min and finally cooled before counting. Results are expressed as safinamide free base equivalents (μgEq/ml; specific activity 0.2075 μCi/mg).
5.0
5
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Fig. 2. Concentration-versus-time curves of total 14C radioactivity (μgEq/ml) in plasma (diamond), total 14C ra-
dioactivity (μgEq/ml) in whole blood (square) and unchanged safinamide (μg/ml) in plasma (circle) after administration of a single oral dose of 14C safinamide methanesulphonate to 6 male healthy volunteers (mean ± SD). The concentration-versus-time profile from administration up to 24 h post-dose is represented in the insert figure.
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ml/h × kg × F for unchanged safinamide and 17.53 ± 2.71 ml/h × kg × F for 14C radioactivity, in agreement with a terminal half-life of about 80 h. 14C radioactivity renal clearance was 14.72 ± 3.19 ml/h × kg × F, thus representing about 84% of total 14C radioactivity clearance. 14
C Radioactivity Excretion in Urine and Faeces The 14C radioactivity was excreted mainly in urine (mean ± SD: 76.1 ± 9.7%), with individual recovery ranging from 58.1 to 86.3% of the administered dose. 14C radioactivity faecal elimination was quite low, accounting for only the 1.51 ± 0.36% of the dose. Total balance of 14C radioactivity was 77.6 ± 9.6% of the administered dose (range 59.8–87.9%). One subject showed a total balance of 59.8%, which was possibly to be ascribed to incomplete urine sample collection. Metabolite Pattern in Urine Samples Representative radiochromatograms of untreated and enzyme-treated urine samples are shown in figure 3. In untreated samples three metabolite peaks (U1, U2 and Leuratti/Sardina/Ventura/Assandri/ Müller/Brunner
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Main pharmacokinetic parameters of safinamide (free base) in plasma and 14C radioactivity in plasma and whole blood are shown in table 1. Plasma concentrations of the parent compound were lower than plasma radioactivity concentrations. In particular, the ratio of plasma concentrations of 14C-radioactivity/safinamide gradually increased from 1.4 at 0.5 h to 5.3 at 96 h post-dose. 14 C radioactivity in plasma and whole blood declined in a biphasic manner, with terminal half-lives (t1/2) of 77.4 ± 24.6 and 81.1 ± 33.0 h, respectively, whereas the parent compound decreased mono-exponentially with a t1/2 of 22.2 ± 1.4 h. Blood/plasma 14C radioactivity distribution ratio, based on mean Cmax and AUC values, was approximately 1.2:1 and indicated a free exchange of radioactivity between plasma and red blood cells. Average distribution volumes for safinamide and 14C radioactivity per kilogram of body weight were 1,813.3 ± 204.3 and 1,944.6 ± 673.1 ml/kg × F, respectively. Total oral clearance per kilogram of body weight was 57.0 ± 8.5
80.0 Deaminated safinamide acid U3
70.0 60.0 N-dealkylated glucuronide
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U4
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Fig. 3. Representative HPLC radioactivity profile of untreated (a) and deconjugated (b) urine samples (12–24 h). U = Urinary
b
0:00
10:00
20:00
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Table 1. Main pharmacokinetic parameters (mean ± SD) of plasma 14C radioactivity, whole blood 14C radioactivity and plasma parent safinamide
AUC0–t μg × h/ml
AUC0–∞ μg × h/ml
Terminal t1/2, h
Vd/F ml/kg
197.8 ± 28.3
215.9 ± 33.4
77.4 ± 24.6
1,944.6 ± 673.1 17.53 ± 3.40
1.5 (1 – 8) 244.9 ± 40.0 1 (0.5 – 1.5) 62.7 ± 4.3
277.9 ± 55.3 65.9 ± 4.8
81.1 ± 33.0 22.2 ± 1.4
– 1,813.3 ± 204.3
Cmax, μg/ml Tmax, h or μgEq/ml 14C
radioactivity in plasma 3.72 ± 0.44 radioactivity in whole blood 4.5 ± 0.42 Safinamide in plasma 2.37 ± 0.38
7 (2 – 12)
Cl/F ml/h × kg
14C
– 57.0 ± 8.5
14C Safinamide Disposition and Biotransformation
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For Tmax median (min–max) values are displayed.
Metabolite Pattern in Plasma Samples Plasma extracts were analysed by HPLC using off-line fraction collection and liquid scintillation counting, due to low concentrations of radioactivity. At all time points analysed, the N-dealkylated metabolite (NiK-14134) and safinamide represented the majority of the sample radioactivity and co-chromatographed with authentic standards. The deaminated acid metabolite was also observed. Characterization of Metabolite Structures Metabolite structures were characterized by LC-MS/ MS and confirmed by comparison of retention times and fragment spectra with authentic reference standards, when available. Proposed structures are listed in table 2. The N-dealkylated acid metabolite of safinamide (MW 246) was identified as a major component in deconjugated urine and in plasma. In addition, the presence of the N-dealkylated safinamide glucuronide (MW 422; probably four isomers, table 2) in urine and of the deaminated safinamide acid metabolite (MW 303) in urine and plasma was confirmed. Monohydroxy safinamide metabolites (MW318; four isomers) were also identified in urine samples by LC-MS/ MS, but did not correspond to any of the radioactivity peaks obtained in the HPLC profiling work. Mass fragmentation indicated that hydroxylation occurred on the fluorobenzene moiety of safinamide. Finally, the glycine conjugate of N-dealkylated safinamide (MW 303, NiK-15009) and 2-[4-hydroxybenzylamino]propanamide (NW-1199, MW194) were also tentatively identified as minor urinary metabolites. 212
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Safety Safinamide was well tolerated. Four adverse events of mild severity were reported by 3 subjects during the study. One of the reported adverse events, i.e. headache, was judged as possibly related to the investigational product. The other three adverse events (i.e. headache, pain sensation in both eyes and neuralgia from left arm to thumb) were not related to treatment and all resolved by the study end. No clinically significant effects of treatment on laboratory parameters, vital signs or electrocardiogram parameters were observed throughout the study.
Discussion
After a single oral dose of 14C safinamide as methanesulphonate salt to 6 healthy male volunteers, parent safinamide was promptly absorbed, reaching mean peak plasma concentrations at about 1 h post-dose. For 14C radioactivity in plasma and whole blood, two main peaks appeared to be present. The first peak (1–1.5 h) was achieved at a similar Tmax as unchanged safinamide, whereas the second peak, observed at 7–8 h post-dose, could reflect a delayed release of a mixture of 14C-safinamide-related substances with different kinetics into the blood from some peripheral compartment(s). The presence of an enterohepatic circulation occurring postprandial following lunch, however, cannot be completely excluded. Plasma concentrations of the parent compound were lower than plasma radioactivity concentrations (1.4- to 5-fold), in particular at later time points, indicating extensive safinamide metabolization. No chiral inversion of safinamide was observed. Whole blood to plasma ratios for Cmax and AUC were only about 1.2:1, suggesting that radioactivity may freely enter and leave the blood cells. Average distribution volumes per kilogram (Vd/F × kg) of BW for plasma safinamide and 14C radioactivity were 1.8 and 1.9 litres/kg × F, respectively, indicating a total distribution volume (Vd/F) of 150–160 litres for adults of 82 kg (mean value). These values (>100 litres independently of the F value) exceed the actual subjects’ BWs and indicate an extensive organ(s)/tissue(s) distribution of the drug. For the parent compound, the organ/tissue drug is in equilibrium with the blood drug according to a one-compartment open model, corroborated by the observed monoexponential elimination of unchanged safinamide (t1/2 22 h), confirming results obtained in previous clinical studies [17]. Differently, 14C radioactivity decreased in a biphasic manner (terminal t1/2 80 h), indicating a multiLeuratti/Sardina/Ventura/Assandri/ Müller/Brunner
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U3) and safinamide (U4) were observed. Peak U2 was identified by LC-MS/MS (see below) as the β-glucuronide conjugate of the N-dealkylated acid. This peak was present as a major component and disappeared upon complete deconjugation, giving rise to the free N-dealkylated safinamide acid, which co-eluted with the authentic standard (NiK-14134; peak U2a, fig. 3). Peak U3, further identified as deaminated acid of safinamide, was present in untreated and enzyme-treated samples, and in the latter it co-eluted with U2a. Component U1 deconjugated into two other subcomponents, i.e. U1a and U1b. These metabolites did not co-elute with any reference standards and remained unidentified. The stability of safinamide and its metabolites under incubation conditions was investigated: the HPLC analysis did not indicate any deviation from the characteristic profiles of untreated samples.
Table 2. Components characterized in human urine and plasma after single oral dose of 14C safinamide
Component
Identified in
Safinamide MW: 302 Rt: 33.5
urine plasma
Proposed structure and characteristic fragment ions/product ions F
m/z 215
m/z 109
CH 3
NH O
NH2 O
Positive ion mode
N-Dealkylated acid (NiK-14134) MW: 246 Rt: 24.3
deconjugated urine plasma
F
m/z 201 OH O O OH
O
O
m/z 92
O
m/z 136
Negative ion mode
Glucuronide of N-dealkylated acid MW: 422 Rt: 23.9–25.4
urine
F
m/z 245 O
Glucuronide
O O m/z 201 OH O O
m/z 136
Negative ion mode
Acid metabolite MW: 303 Rt: 24.9
urine deconjugated urine plasma
F
m/z 215
m/z 109
CH 3
NH O
OH O
Positive ion mode m/z 106
m/z 148 NH CH 2
m/z 120
CH 2 O
O
O
O
O
Negative ion mode
Monohydroxy safinamide (several isomers) MW: 318 Rt: 18.4, 19.3, 24.8, 25.9
urine
Glycine conjugate of N-dealkylated acid (NiK-15009) MW: 303 Rt: 23.7 (tentative)
urine deconjugated urine
F
m/z 231
m/z 125
NH2 O
HO
Positive ion mode
F
O
m/z 201
OH NH
m/z 258
O m/z 149
m/z 120 O
m/z 92
O
O
m/z 148 NH CH 2 O
urine deconjugated urine
Negative ion mode
O
O
2-[4-hydroxybenzylamino]propanamide (NW-1199) MW: 194 Rt: 8.3 (tentative)
CH 3
NH O
m/z 89 m/z 107
+2H NH
HO
CH 3
m/z 44
NH2 O
Positive ion mode
14C Safinamide Disposition and Biotransformation
Pharmacology 2013;92:207–216 DOI: 10.1159/000354805
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The quoted molecular weights (MW) are for unlabelled components. Retention times (Rt) are taken from 14C radiochromatogram traces where possible, otherwise from product ion analyses. The reference standard code is given in parentheses, when available.
NH2
N H
F
O
O
Safinamide MW 302
F
HO CH3
NH O
NH2
O
Alpha-oxidation by MAO (with intermediate formation)
Monohydroxy safinamide MW 318 CH3 NH HO
NH2
O
2-[4-hydroxybenzylamino]propanamide MW 194
O OH
N H F
O
OH
F
O
O
N-dealkylated acid MW 246
Deaminated acid metabolite MW 303
O
F
OH NH O O
N-dealkylated glycine conjugate MW 303
N-dealkylated acid glucuronide MW 422
Fig. 4. Proposed biotransformation path-
component and, probably, multi-compartmental model. The differences in t1/2 observed between parent safinamide and 14C radioactivity in plasma are explained by the presence of a varying mixture of metabolites, cleared from the body compartments at different rates. Total oral clearance of unchanged safinamide was 57.0 ± 8.5 ml/h × kg × F. The process comprises not only renal excretion but also loss of the parent molecule by metabolism. In contrast, total oral clearance of plasma 14C radioactivity, which accounts for parent safinamide as well as metabolites, was on average only 17.53 ± 2.71 ml/h × kg × F, in agreement with a terminal half-life of about 80 h. 214
Pharmacology 2013;92:207–216 DOI: 10.1159/000354805
Renal clearance of 14C radioactivity (14.72 ± 3.19 ml/h × kg × F) represented about 84% of the total 14C radioactivity clearance. This result is confirmed by the urinary concentrations of 14C radioactivity, which gave cumulative amounts of 230.7 ± 29.4 mgEq and consequently urinary outputs of 76.11 ± 9.68% of the administered dose. On the other hand, 14C radioactivity faecal elimination was almost negligible, suggesting that possibly about 1.5% of the administered dose had not been absorbed or was eliminated by biliary secretion after systemic absorption. This very low faecal excretion is indicative of a substantially complete intestinal absorption of the drug. Leuratti/Sardina/Ventura/Assandri/ Müller/Brunner
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ways for safinamide in man.
At the end of the study (200 h post-dose), total balance of radioactivity accounted for approximately 80% (range 59.78–87.90%) of the dose, although for 1 subject urine sample collection appeared not to be complete, slightly underestimating final urinary output. The major metabolite of safinamide identified in deconjugated urine samples and in plasma was the N-dealkylated acid of safinamide (MW 246). The presence of this metabolite indicates N-dealkylation as a major pathway of safinamide metabolism, characterized by safinamide oxidation by MAO followed by further oxidation to the acid. This metabolic pathway is characteristic of other MAO-B inhibitors, a typical example being milacemide, an anticonvulsant drug which is metabolized by MAO-B [17, 18], giving rise to the N-dealkylated pentanoic acid plus glycinamide, the latter then transformed into glycine. It is possible that N-dealkylation of safinamide might lead to the formation of alaninamide (and then alanine) in the same way. N-dealkylation was postulated as a major metabolic pathway also for ralfinamide, an α-aminoamide derivative analogue of safinamide [19]. In untreated urine samples, the glucuronide conjugate of N-dealkylated acid (MW 422) was identified as a major component, which disappeared upon complete deconjugation giving rise to the free N-dealkylated metabolite. Another major metabolite observed in urine and plasma samples was the deaminated safinamide acid (MW 303), a product of hydrolytic deamination, by amide hydrolase, of the parent molecule. This component was shown to remain constantly high (about 31–38%) in urine up to 48 h post-dose. Monohydroxy safinamide (MW 318, four possible isomers) was identified in urine samples by LCMS/MS but did not correspond to any of the radio-peaks obtained in the HPLC profiling. Monohydroxy safinamide metabolites were also identified during in vitro
studies using rat, dog, monkey and human microsomes [unpublished data]. In the present work, 2-[4-hydroxybenzylamino]propanamide and the glycine conjugate of the N-dealkylated acid were also tentatively identified as minor safinamide metabolism products. On the basis of the results obtained, safinamide metabolic pathways represented in figure 4 are proposed. Previously described tentative intermediates [20] were not experimentally confirmed. In conclusion, the present study allowed for an indepth characterization of safinamide disposition in vivo and identified previously unknown metabolites.
Acknowledgements The authors would like to acknowledge RCC Ltd., Environmental Chemistry and Pharmanalytics Division, Itingen, Switzerland, and BioDynamics Research Ltd., Rushden, UK, for the analytical part of this work and CROSS Alliance Group, Switzerland, for the pharmacokinetic analysis. Newron S.p.A., Italy, funded the clinical study. Newron S.p.A. has licensed safinamide to Zambon Farmaceutici S.p.A.
Disclosure Statement The relationship between the sponsors (Newron S.p.A., Zambon S.p.A., Italy) and CROSS S.A. (Switzerland) was regulated by financial agreements. C.L. is an employee of CROSS S.A., was the study coordinator and is the author of the clinical study report and of the manuscript; M.S. is an employee of Zambon S.p.A., Italy; P.V. is a consultant in Pharmacokinetics and Metabolism, Piacenza, Italy. A.A. is the Scientific Director at CROSS S.A.; M.M. and M.B. are employees of the Department of Clinical Pharmacology, Medical University of Vienna, Austria: M.M. was the principal investigator of the study, M.B. was the co-investigator at the clinical site. All authors reviewed and approved the final draft of the manuscript.
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