Pharmacokinetic and Safety Study of Single and Multiple Oral Doses of Safinamide in Healthy Chinese Volunteers

This randomized, parallel‐group study evaluated the plasma pharmacokinetic profile of safinamide in 24 healthy Chinese men and women, randomly assigned to receive 50 or 100 mg of safinamide as a single dose, followed, after a 7‐day washout, by multiple doses once daily for 7 days. Plasma safinamide was determined up to 96 h after the first single dose (day 1) and the last multiple dose (day 14), and up to 24 h after the first multiple dose (day 8). Following single‐ and multiple‐dose administration, peak concentrations were achieved at a median time of 1.5–2 h. Plasma exposure increased in a dose‐proportional manner. After single dose, mean half‐life was 23–24 h. Area under the concentration‐time curve (AUC) from time zero extrapolated to infinity was only slightly higher than AUC from time zero to the last quantifiable concentration, corresponding for the 2 parameters, respectively, to 12,380 and 11,560 ng • h/mL for the 50 mg and to 22,030 and 20,790 ng • h/mL for the 100‐mg dose. AUC in the dosing interval at steady state was 13,150 and 23,100 ng • h/mL for 50 and 100 mg of safinamide. Steady state was reached in 6 days, accumulation was approximately twofold, and the pharmacokinetics were time independent. The plasma safinamide pharmacokinetic profile observed in this study is in line with the published results in both Chinese and non‐Asian populations.

Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by dopamine deficiency resulting from progressive loss of nigrostriatal dopaminergic cells, which leads to a classical set of motor and nonmotor symptoms. 1,2][5] Levodopa (L-dopa) remains the most effective drug for alleviating the motor symptoms of PD, but its longterm use is associated with motor complications, such as fluctuations and dyskinesia. 6In addition, as the disease progresses, nondopaminergic pathways are also involved, 7 and patients benefit from add-on therapy to improve motor fluctuations without exacerbating dyskinesia.
Safinamide, an orally administered α-aminoamide derivative, combines potent, selective, and reversible inhibition of monoamine oxidase (MAO)-B, with blockade of voltage-dependent Na + and Ca 2+ channels and inhibition of glutamate release, [8][9][10][11] thus targeting both dopaminergic and glutaminergic systems. 12,13][25][26] The efficacy of safinamide in reducing motor fluctuations and improving motor symptoms and quality of life has also been demonstrated in a phase III randomized, double-blind, placebo-controlled trial conducted in Chinese patients with idiopathic PD orally administered 100 mg of safinamide once daily for 16 weeks as add-on therapy to L-dopa. 27The safety of the study treatment was good and comparable to that observed in the previous studies.
9][30][31] Orally administered safinamide has linear pharmacokinetics after single and multiple doses, is quickly absorbed (time to maximum concentration [t max ], 1-2.8 h in fasting conditions), and has a high absolute bioavailability (95%), indicating that it is almost completely absorbed after oral administration and that first-pass metabolism is negligible.Food delays safinamide absorption without affecting its bioavailability.1][32] Total clearance is 4.6 L/h. 17 The distribution volume of safinamide is approximately 165 L, 28 indicating extensive extravascular distribution.Safinamide undergoes extensive biotransformation with only 7% and 1.5% excreted unchanged in urine and feces, respectively. 12,29Metabolism occurs primarily via amide hydrolytic oxidation, producing the metabolite safinamide acid.Other pathways include ether bond oxidation (producing O-debenzylated safinamide) and oxidative cleavage of safinamide or safinamide acid to N-dealkylated acid. 17,29None of the metabolites display pharmacological activity. 30,32In urine, the main metabolites were the beta-glucuronide of the N-dealkylated acid and monohydroxy-safinamide. 29,31 In addition, the glycine conjugate of the N-dealkylated acid and 2-[4-hydroxybenzylamino]propenamide were tentatively identified as minor urine metabolites. 29limination is predominantly via the urine (76%) and little via the feces (1.5%).][35] Clinically relevant systemic concentrations of safinamide do not appear to inhibit or induce enzymes significantly. 17Data from in vitro experiments did not find any relevant interactions in oxidative metabolic pathways controlled by cytochrome P450 (CYP) 2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4. 28Moreover, relevant concentrations of safinamide did not inhibit or induce the activity of key CYP enzymes, including CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A3/5, to any meaningful extent in vitro. 17,36In drug interaction studies, the pharmacokinetics of caffeine (CYP1A2 substrate), midazolam (CYP3A4 substrate), and Ldopa were not altered to any clinically relevant extent by safinamide, 17 and the CYP3A4 inhibitor ketoconazole had no clinically relevant impact on safinamide pharmacokinetics. 31In patients with PD receiving L-dopa and/or dopamine agonists with adjunctive safinamide, safinamide clearance was not affected. 17,37afinamide is not a substrate for breast cancer resistance protein, the organic anion transporter 1B1 or 1B3, or the organic anion-transporting polypeptide 1A2 or 2A1 in vitro. 17Age, sex, renal function, and exposure to L-dopa do not have an impact on the pharmacokinetics of safinamide. 38 previous pharmacokinetic study conducted on healthy Chinese volunteers 39 who received safinamide as a 100-mg single dose of a test and a reference safinamide tablet established the bioequivalence of the 2 formulations in terms of both safinamide rate and extent of absorption under both fasting and fed conditions.In addition, as previously observed in non-Asian subjects, 28,30 high-fat food delayed the absorption of safinamide but did not affect its final bioavailability.
In the present study, the pharmacokinetic profile and the safety of safinamide after an oral single dose and at steady state were investigated in healthy Chinese male and female volunteers.

Study Design
The trial was a phase
Subjects were randomized to two study cohorts to receive single and multiple doses of 50-mg safinamide (Cohort 1) or single and multiple doses of 100-mg safinamide (Cohort 2), according to the randomized, parallel-group study design.The randomization list was computer-generated using SAS software (SAS Institute).
Depending on the study cohort, one 50-or 100-mg film-coated tablet was administered on day 1 (Period 1) followed by one tablet once daily (50 or 100 mg) from day 8 to day 14, for a total of seven doses in Period 2. Washout between the single dose of Period 1 and the first dose of Period 2 was 7 days.
The investigational products were orally administered at 8:00 a.m. ± 1 h, under fasting conditions, with 240 mL (total volume) of still mineral water.
During the study, the subjects were confined at the clinical center from the evening preceding the first investigational product administration up to day 18.The subject fasted for about 10 h before investigational product administration of days 1, 8, and 14.On these days, standardized lunch and dinner were provided at about 5 and 13 h after dosing.On the other days of confinement, standardized breakfast, lunch, and dinner were served.
Water was allowed as desired, except for 1 h before and 1 h after administration.Coffee, tea, or food containing xanthines, alcohol, and grapefruit were forbidden from 48 h before the first administration until the end of the study.Smoking was not allowed for the whole study duration.

Subjects
Healthy male and female volunteers of Chinese ethnicity, aged 18-45 years, with a body weight of 50 kg or greater and a body mass index of 19.0-26.0kg/m 2 , were enrolled in the study.All volunteers were in good physical health, as assessed through full physical examination, electrocardiogram (ECG) recording, vital signs measurement, and clinical laboratory assays, according to the study inclusion criteria.Subjects were excluded if on abnormal diets or vegetarians or had a history of drug, alcohol, caffeine, or tobacco abuse.Exclusion criteria also included history or presence of significant renal, hepatic, gastrointestinal, cardiovascular, respiratory, skin, hematological, endocrine, or neurological diseases that could interfere with the aim of the study; positive result to HIV, hepatitis B (except for vaccination), or hepatitis C; retinal degeneration, uveitis, inherited retinopathy, or severe progressive retinopathy; history of hypersensitivity or allergic reactions to the active principal and/or formulations' ingredients.
Medications, including over-the-counter medications, herbal remedies, and traditional Chinese remedies, were not allowed for 2 weeks before and during the entire study.The following treatments were forbidden: statins and 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors in the 2 weeks before the screening visit; medicinal products that are breast cancer resistance protein substrates; treatment with morphine or other similar opioids whose concomitant use with MAO-B inhibitors is contraindicated, selective serotonin reuptake inhibitors, serotonin-norepinephrine reuptake inhibitors, tri-or tetracyclic antidepressant, tramadol, pethidine, dextromethorphan, MAO inhibitors (e.g., selegiline), meperidine derivatives, and antiepileptic drugs in the 4 weeks before the screening visit; and treatment with any known enzyme-inhibiting or -inducing agent within 4 weeks preceding the screening visit.Hormonal contraceptives for women were allowed.Subjects were not enrolled if they had participated in other clinical trials or donated blood in the past 3 months.

Blood Sampling
Venous blood samples (up to 10 mL) for plasma safinamide determination were collected from a forearm vein before dosing (0 h) and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, 24, 36, 48, 72, and 96 h after dosing following the first dose (day 1) and the last dose (day 14); before dosing on days 10-13 and before dosing (0 h) and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16, and 24 h after dosing following the first multiple dose (day 8).Blood samples for pharmacokinetic analysis were collected using an indwelling catheter with switch valve.After each sampling, the cannula was rinsed with about 1 mL of sterile saline solution.At each collection time, the first 2 mL of blood were discarded, and the remaining blood was collected from the catheter and transferred into heparinized tubes (Li-heparin).The samples were centrifuged at 4°C for 10 min at 1300 × g to obtain plasma.Each plasma sample was immediately transferred into appropriate tubes and stored at −70°C or less until analyses.

Bioanalytical Assay
The concentration of safinamide in plasma samples was determined at United Power Pharma Tech Co., Ltd.(UP Pharma), China.
The analyte and the internal standard (safinamide-d3) were processed with a protein precipitation method and analyzed using liquid chromatography-tandem mass spectrometry with a lower quantification limit of 5.00 ng/mL.The system included high-performance liquid chromatography (LC-20AD, Shimadzu), highperformance liquid chromatography column (CAP-CELL PAK C18 MG II 3 μm 2.0 × 50 mm), and mass spectrometer (API 5000).
For liquid chromatography separation, mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile, with a flow of 0.6 mL/min.
Calibration standards in the 5.00-1000 ng/mL range and quality control samples at the levels limit of quantification (5.0 ng/mL), low (15.0 ng/mL), medium (300 ng/mL), and high (750 ng/mL) were prepared using safinamide reference standard (Shanghai ZZBIO Co., Ltd.) and stored at −10 o to −30°C.Accuracy and precision were within 7.2% and 2.2% for all levels of quality control samples.

Pharmacokinetic Parameters
The pharmacokinetic parameters were measured and/or calculated with a noncompartmental analysis, using Phoenix WinNonLin Version 8.0 (Certara, Inc) or derived using SAS Version 9.3.
For days 1 and 8 (single dose), the main pharmacokinetic parameters were safinamide maximum plasma concentration (C max ), t max , last quantifiable concentration (Ct), area under the concentration-time curve up to 24 h after dosing (dosing interval; AUC 0-24 ), and AUC up to the last quantifiable concentration (AUC 0-t ).In addition, for day 1 AUC extrapolated to infinity (AUC 0-∞ ), terminal half-life (t 1/2 ), and apparent clearance (CL/F) were calculated.
For day 14 (last multiple dose), the main pharmacokinetic parameters were safinamide C max at steady state (C max,ss ), time to C max,ss ( max,ss ), minimum observed concentration at steady state (C min,ss ), AUC over the dosing interval (24 h) at steady state (AUC 0-τ ,ss ), AUC 0-t at steady state (AUC 0-t,ss ), CL/F at steady state (Cl/F,ss), average concentration at steady state (C ave,ss ), and peak-through fluctuation, defined as the ratio of C max,ss and C min,ss over the dosing interval.
Safinamide accumulation upon multiple dosing was calculated as accumulation ratio (R acc ) based on AUC 0-τ ,ss /AUC 0-24 after day 1 single dose (R acc,AUC ) and as R acc based on C max,ss /C max after day 1 single dose (R acc ,C max ).

Safety
The safety and tolerability of the study treatment was assessed by treatment-emergent adverse events (TEAEs), physical examination, vital signs, ECG, and laboratory tests.Adverse events were assessed throughout the study and were coded using the Medical Dictionary for Regulatory Activities Version 23.1.

Sample Size
Twelve subjects per cohort (24 in total) were included in the study to have at least 8 completers per cohort.Discontinued subjects were not replaced.Since no hypothesis was tested, no formal sample size calculation was performed.The sample size of this study was determined in agreement with the guidance issued by the Chinese National Medical Product Administration for clinical pharmacokinetic studies.

Data Analysis
Study data were summarized by descriptive statistics and frequencies, as applicable.The statistical analyses were performed using SAS and Phoenix WinNonLin Version 8.0.

Subjects
Overall, 24 subjects (12 in each cohort) satisfied the study inclusion/exclusion criteria, were randomized in the study as planned, and received the study treatment.Twenty-three of them (11 in Cohort 1 and all 12 in Cohort 2) completed the study per protocol and were included in the safety and pharmacokinetic analysis.One subject in the 50-mg safinamide cohort was discontinued prematurely for a major protocol deviation (intake of one dose of 100-mg safinamide on day 14).This subject was included in the safety set only.Demographic characteristics of the analyzed subjects are presented in Table S1.

Pharmacokinetics
Safinamide plasma concentrations after single dose (day 1), first multiple dose (day 8), and last multiple dose (day 14) are shown in Figures 1 and 2 for 50-mg and 100-mg safinamide, respectively.
Single Dose (Days 1 and 8).Main plasma safinamide pharmacokinetic parameters for days 1 and 8 are presented in Table 1.After single oral administration of 50 and 100 mg of safinamide, plasma concentrations of the drug rapidly increased, peaked at a median t max of approximately 1.5-2 h, and declined slowly in a multiphasic manner, with a mean terminal half-life of 23-24 h.After the first single dose (day 1), safinamide was quantifiable in all subjects up to the last sampling point (i.e., 96 h after dosing) in both treatment groups.
Within each dose group, C max and AUC 0-24 were very similar at day 1 and day 8.After the first single dose (day 1), AUC 0-∞ was only slightly higher than AUC 0-t .The safinamide rate (C max ) and extent (AUC) of absorption appeared to increase proportionally  following the single doses of 50-and 100-mg safinamide.CL/F was 4.2 and 4.6 L/h, for the low and high safinamide dose, respectively.
Multiple Dose (Day 14).During the multiple-dose regimen, steady state was reached after 6 days of treatment with both the 50-mg and 100-mg safinamide doses (Table 2).Pharmacokinetic parameters obtained after the last multiple dose (day 14) are shown in Table 3.
Peak safinamide concentrations were reached at median time to C max,ss of 2.0 and 1.5 h for the 50-mg and 100mg dose groups, respectively.
Following multiple-dose administration daily from day 8 to day 14, plasma exposure increased in a dose proportional manner as indicated by the C max,ss , AUC 0-t,ss , AUC 0-τ ,ss and average concentration at steady state values.Mean C min,ss was 358.3 and  583.2 ng/mL for the two dose groups, and the peaktrough fluctuation ratio appeared to be slightly lower following multiple doses of the 50-mg than the 100mg dose (77.8% and 95.2%, respectively).The clearance of safinamide at steady state remained low, with mean CL/F, ss values of 3.9 and 4.4 L/h following administration of 50 and 100 mg safinamide, respectively.The degree of accumulation upon multiple dosing, measured as the ratio between AUC 0-τ ,ss /AUC 0-24 after single dose (R acc,AUC ), was approximately twofold and was consistent across the 50-mg and 100-mg dose groups.An accumulation factor of approximately 2 was also observed for both dose groups when comparing C max,ss and C max after single dose.
Comparison of Results.Results obtained in the present study with the single 50-and 100-mg doses were descriptively compared to results of two previous studies in non-Asian populations of healthy volunteers under the same dosing regimens. 30,31As shown in Table 4, pharmacokinetic parameters were similar between the different populations within each dose.Peak concentration was achieved at a median time of 1.5-2.5 h and 1.5-2 h after single dose of 50 and 100 mg, respectively.
A descriptive comparison of the pharmacokinetic parameters obtained at steady state in this study and in previous studies is presented in Table S2.

Safety
The investigational treatment was safe and well tolerated.No serious adverse event occurred, and no subject withdrew from the study for an adverse event.A total of 16 of 24 subjects experienced at least one TEAE, and all reported TEAEs were deemed related to study treatment.The most common TEAE was somnolence (8 [33.3%] subjects in the single dose period and 2 [8.3%] subjects in the multiple-dose period), followed by diarrhea and weight loss (both reported in 2 [8.3%] subjects in the single-dose period); hypertension (2 [8.3%] subjects in the multiple-dose period); insomnia (one [4.2%]  subject in the single-dose period); and increased blood triglycerides, increased blood uric acid, decreased neutrophil count, bone pain, pain in extremity, leukopenia, constipation, decreased libido, and maculopapular rash (1 subject each [4.2%] in the multiple-dose period).All adverse events were mild or moderate and resolved before study end.No abnormal findings that could give rise to a safety concern were observed in laboratory analysis results, vital signs, physical examinations, and ECGs during the study.

Discussion
In the present study, the pharmacokinetic profile of single and multiple doses of 50-and 100-mg safinamide was characterized in healthy Chinese volunteers.
After the first single dose (day 1) and the first multiple dose (day 8), safinamide concentration-time curves were nearly superimposable in the dosing interval of 24 h.In addition, the safinamide pharmacokinetic profile was similar in shape after both single and multiple administrations at both investigated doses, with a median t max of approximately 1.5-2 h and a relatively long half-life (approximately 23-24 h).In addition, a low plasma clearance was observed.
Similar to the findings of previous studies, 28 steady state was reached in 6 days, accumulation was negligible (approximately twofold) and the pharmacokinetics following multiple-dose administration were time independent.In addition, as also previously reported, a low intersubject variability upon multiple dosing was observed.
Results of the present pharmacokinetic study support previous findings 28 that safinamide pharmacokinetics are dose proportional.In fact, after singledose administration, both rate and extent of absorption appeared to increase proportionally between the two doses, as clearly indicated by the C max and AUC values.Also, following multiple administration of 50 and 100 mg of safinamide daily for 7 days, plasma exposure increased in a dose-proportional manner, as demonstrated by C max,ss , AUC 0-t,ss , and AUC τ ,ss values.
Plasma safinamide concentrations and pharmacokinetic data of this study are in line with the results of published data in both Chinese and non-Asian populations.In the study by Li et al, 39 for the reference safinamide product, which corresponded to the investigational product of the present study and was administered as a single 100-mg dose under fasting conditions, mean C max value was 820.2 ng/mL, that is, very similar to the C max value for the same dose regimen obtained in the present study (792.1 ng/mL at day 1 and 825.3 ng/mL at day 8).In the two studies, C max was obtained at a median time of 1.5-1.8h.
Extent of exposure was nearly identical for the previous and present Chinese trial, with AUC 0-∞ values of 22,500 and 22,030 ng • h/mL, respectively.CL/F corresponded to approximately 4.6 L/h, confirming the low clearance also observed in previous studies conducted in non-Asian populations.
In the study by Seithel-Keuth et al 30 performed on 14 healthy Caucasian male and female volunteers administered a single dose of 50-mg safinamide, an AUC 0-t value of 10,110 ng • h/mL was observed.The reported safinamide extent of exposure value is very similar to the one observed at the same single dose of 50 mg in healthy male and female Chinese subjects in the present study, that is, 11,560 ng • h/mL, also when considering the range of values observed.In addition, results obtained in the present study with the 100-mg single dose of safinamide were comparable to those obtained with the same dose regimen in a previous study in healthy volunteers, 31 with geomean values for AUC 0-t of 16,786 and 20,570 ng • h/mL for the 2 studies, confirming that the bioavailability of the product is similar in Chinese and non-Chinese populations.It was previously demonstrated that metabolism of safinamide is not dependent on CYP enzymes 13,31 and that the pharmacokinetics of safinamide are not influenced by intrinsic factors, such as age, sex, or ethnicity. 379][20][21] This gives additional evidence that the bioavailability of safinamide results In conclusion, the present study thoroughly investigated the pharmacokinetic profile of safinamide in healthy Chinese volunteers administered single and multiple doses of 50 and 100 mg of the drug.Results of the study show that the bioavailability of safinamide is similar in healthy male and female Chinese and non-Chinese subjects.This finding is corroborated by the results of phase III studies, showing a similar efficacy in different populations of patients with mid and late PD.
The safety data collected during this study are in line with the known safety profile of the product and did not raise any safety concern.

Table 1 .
Safinamide pharmacokinetic parameters after single dose (day 1) and first multiple dose (day 8) of 50-mg and 100-mg safinamide AUC 0-24 , area under curve over the dosing interval (24 h); AUC 0-∞ , area under curve from time zero extrapolated to infinity; AUC 0-t , area under curve from time zero to the last quantifiable concentration; CL/F, apparent clearance following oral administration; C max , maximum observed concentration; Ct, last quantifiable concentration; NA, not applicable; SD, standard deviation; t 1/2 , apparent terminal elimination half-life; t max , time to C max .a AUC 0-t for Day 8 was calculated in the dosing interval, that is, up to 24 h after dosing.

Table 4 .
Safinamide pharmacokinetic parameters after single 50-mg and 100-mg oral dose in previous studies and in the present study Safinamide 50 mg a Safinamide 100 mg b in a very similar efficacy outcome in Chinese and non-Chinese subjects.