The Effect of Safinamide, a Novel Drug for Parkinson s Disease, on Pressor Response to Oral Tyramine: A Randomized, Double-Blind,

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1 nature publishing group The Effect of Safinamide, a Novel Drug for Parkinson s Disease, on Pressor Response to Oral Tyramine: A Randomized, Double-Blind, Clinical Trial A Marquet 1, K Kupas 2, A Johne 3, B Astruc 4, A Patat 4, S Krösser 3 and A Kovar 3 This randomized, double-blind, placebo-, comparator (selegiline 10 mg/day)-, and positive (phenelzine 30 mg/day)- controlled study investigated the pressor response to oral tyramine under fasting conditions after the administration of safinamide at therapeutic (100 mg/day) and supratherapeutic (350 mg/day) dosing regimens in healthy volunteers for the purpose of assessing the need for dietary restrictions. Pressor response was characterized by Tyr30, defined as the tyramine dose that triggers a sustained increase in systolic blood pressure (SBP) of 30 mm Hg as compared with baseline SBP. The primary end point was the tyramine sensitivity factor (TSF), defined as the ratio of Tyr30 at screening to Tyr30 under treatment. Safinamide induced a mild increase in TSF; however, the effect at each of the doses was numerically lower than those of the comparators (geometric mean TSFs: placebo, 1.52; safinamide 100 mg, 2.15; safinamide 350 mg, 2.74; selegiline, 3.12; phenelzine, 9.98). This study confirms that safinamide is a highly selective monoamine oxidase-b inhibitor, even at supratherapeutic doses, and suggests that it can be administered without tyramine-related dietary restrictions. Safinamide, an α-aminoamide derivative, is currently in phase III development as add-on therapy to dopamine agonists for early Parkinson s disease (PD) and to levodopa for mid- to latestage PD. Safinamide has both dopaminergic and nondopaminergic pharmacologic activities, and may have beneficial effects on both motor and nonmotor symptoms of PD. 1 5 The principal dopaminergic activity of safinamide has been defined as a potent (IC 50, i.e., half maximal inhibitory concentration: 79 nm in the human brain), highly selective, and reversible inhibition of monoamine oxidase B (MAO-B), 2,6 8 resulting in an increased dopamine availability at nerve terminals. The nondopaminergic effects of safinamide include inhibition of glutamate release 2,6,9 by means of a mechanism that is thought to be mediated via inhibition of sodium channels. 9 Monoamine oxidase enzymes exist in two functional isoforms, MAO-A and MAO-B, which have different substrate specificities and tissue distributions. 10,11 MAO-A, the predominant form in the gastrointestinal tract, is crucial for the metabolism of the sympathomimetic amine tyramine, in the intestine; by contrast, MAO-B is predominant in the brain, where it deaminates serotonin, dopamine, and other amines. 10,11 The clinical use of first-generation, nonselective, and irreversible MAO inhibitors has been limited by the risk of hypertensive crises in association with consumption of tyramine-rich foods (the so-called cheese effect ). On the contrary, irreversible but more selective secondgeneration MAO-B inhibitors such as rasagiline and selegiline do not appear to enhance the pressor effect of oral tyramine at therapeutic doses. 10,12 14 The selectivity of safinamide for MAO-B is ~1,000 times higher than for MAO-A. 8 Safinamide is about 50 times more selective for MAO-B than selegiline and rasagiline. 7 Two previous studies indicated that concomitant safinamide treatment does not appear to affect the pressor effect of intravenous and oral tyramine. 15,16 The relevance of these studies, however, may be questioned: intravenous administration of tyramine 15 does not represent the clinical situation of tyraminecontaining food metabolized and/or absorbed in the GI tract (where interaction with MAO inhibitors may occur), and the oral tyramine doses used in the study by Di Stefano and Rusca 16 were too small for the primary end point (i.e., an increase in systolic blood pressure (SBP) by >30 mm Hg) to be reached, such that response to oral tyramine in subjects receiving safinamide was not appropriately characterized. 1 Global Exploratory Medicine, Merck Serono S.A., Geneva, Switzerland; 2 Global Biostatistics, Merck KGaA, Darmstadt, Germany; 3 Global Exploratory Medicine, Merck KGaA, Darmstadt, Germany; 4 Biotrial S.A., Rennes, France. Correspondence: A Marquet (anne.marquet@merckgroup.com) Received 23 April 2012; accepted 27 June 2012; advance online publication 5 September doi: /clpt VOLUME 92 NUMBER 4 october

2 The objectives of our double-blind, placebo-, comparatorand positive-controlled study were to confirm previous study results using a thorough design and to evaluate the effect of safinamide on the tyramine pressor response at the highest foreseen therapeutic dosing regimen (100 mg once daily) and at a supratherapeutic dosing regimen (350 mg once daily). The irreversible, non-selective MAO inhibitor phenelzine (an antidepressant) was used as the positive control at a dosage of 30 mg/ day; this dosage, which is lower than the recommended starting dosage of 15 mg three times a day, 17 was selected because of the drug s high potency and the risk of severe hypertension and possible complications at higher doses. The irreversible MAO-B inhibitor selegiline was selected as a comparator at a dosage of 10 mg/day, which is the approved dosage as adjunct treatment for late-stage PD without any tyramine dietary restrictions. 18 Results Demographics and baseline characteristics Of the 172 volunteers who were screened for the study, 2 failed to meet the Tyr30 inclusion criterion (requiring that only those showing a sustained increase in SBP of 30 mm Hg as compared with baseline SBP in response to oral tyramine doses of 200 mg but 700 mg could be enrolled). A total of 89 subjects (45 male and 44 female) were randomized to the various study groups; 88 subjects completed the treatment period (one subject in the safinamide 350 mg group was withdrawn after an adverse event (AE)). Overall, demographic and baseline characteristics, particularly the age distribution, were similar across treatment groups (Table 1). The only slight imbalance observed was that there was a relatively higher proportion of black subjects in the phenelzine and placebo groups. Among the 89 randomized subjects, 68 responded to tyramine doses between 300 and 500 mg at screening (Table 2), a finding in line with the data in the literature 10,11,19 and one that confirmed that the selected tyramine dose range was representative for the general population. Pharmacodynamic outcome measures The primary outcome measure was the tyramine sensitivity factor (TSF), which was defined as the ratio of Tyr30 at screening to Tyr30 under treatment. The geometric mean values of TSFs across the different groups could be ranked in increasing order as follows: 1.52 for placebo, 2.15 for safinamide 100 mg, 2.74 for safinamide 350 mg, 3.12 for selegiline 10 mg, and 9.98 for phenelzine 30 mg. For all the groups the 90% confidence interval (CI) limits Table 1 Demographics and baseline characteristics Characteristic (n = 17) Total (n = 89) Age (years): mean (range) 39.8 (20 70) 41.2 (24 62) 40.6 (24 59) 43.2 (20 68) 38.8 (21 55) 40.7 (20 70) Age in class: 45 years old/ >45 years old, n 10/8 11/7 11/6 9/9 12/6 53/36 Sex: male/female, n 9/9 9/9 9/8 9/9 9/9 45/44 Race, n (%) White 17 (94.4) 16 (88.9) 13 (76.5) 16 (88.9) 15 (83.3) 77 (86.5) Black 0 (0.0) 2 (11.1) 3 (17.6) 1 (5.6) 3 (16.7) 9 (10.1) Asian 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Other 1 (5.6) 0 (0.0) 1 (5.9) 1 (5.6) 0 (0.0) 3 (3.4) BMI (kg/m²): mean (range) ( ) ( ) ( ) ( ) ( ) ( ) BMI, body mass index. Table 2 Tyr30 at screening, Tyr30 under treatment, and TSF Parameter Tyr30 at screening (mg) Tyr30 under treatment (mg) Statistics (n = 17) Mean % CI (L, U) Minimum, maximum (371.24, ) 200, 700 (364.70, ) 300, 500 (339.17, ) 200, 700 (384.17, ) 300, 700 (421.89, ) 200, 700 Mean % CI (L, U) Minimum, maximum (178.08, ) 25, 700 (157.31, ) 25, 500 (34.04, 66.69) 6.25, 150 (129.76, ) 25, 300 (294.59, ) 25, 700 TSF Geometric mean % CI (L, U) Median (1.56, 2.96) 1.83 (1.72, 4.37) 2.50 (7.15, 13.93) (2.44, 3.98) 2.67 (1.07, 2.14) 1.00 Minimum, maximum 0.80, , , , , CI, confidence interval; L, lower limit of 90% CI; TSF, tyramine sensitivity factor; U, upper limit of 90% CI. Clinical pharmacology & Therapeutics VOLUME 92 NUMBER 4 October

3 for the geometric mean TSFs were wide, with an overlap across all groups except the phenelzine group (Table 2). The back-transformed analysis of variance results for TSF were consistent with these results: the geometric least-squares mean ratios of TSF for the treatment groups vs. placebo followed the same ranking of treatment effect (Table 3). Safinamide 100 and 350 mg potentiated the pressor effect of tyramine 1.6-fold and 1.8- fold, respectively (relative to placebo); as compared with 2.2-fold for selegiline 10 mg and sixfold for phenelzine 30 mg. Similar results were also observed with values of the median effective dose (ED50), i.e., the dose at which a sustained increase in SBP of >30 mm Hg occurs in 50% of the individuals. The ED50 for safinamide 100 mg and for safinamide 350 mg was 200 mg, as compared with 150 mg for selegiline, 37.5 mg for phenelzine, and 400 mg for placebo; therefore, the ratios of ED50 values for placebo and study drug were 2.00 for both safinamide groups, 2.67 for selegiline, and for phenelzine. These results are illustrated in Figure 1, which shows the cumulative frequency distribution of Tyr30 by treatment group, with a dotted line indicating the ED50. Pharmacokinetic outcome measures The concentration time profiles of safinamide and its metabolites were similar to previously reported pharmacokinetic data. 20,21 Mean (SD) predose safinamide concentrations on study day (SD) 6 were (184.53) ng/ml in the safinamide 100 mg group and 2,661.7 (762.55) ng/ml in the safinamide 350 mg group, representing a 3.5-fold increase and indicating dose-proportionality of safinamide concentrations. Predose concentrations were stable from SD6 onward, indicating that steady state had been reached at the time when tyramine challenges were started (SD7). On the tyramine challenge days, peak plasma concentrations of safinamide were reached ~120 min after safinamide administration, corresponding to 30 min after tyramine administration. Peak tyramine plasma concentrations were reached close to the 30 min post-tyramine time point, nearly coinciding with the time when the maximum safinamide plasma concentrations were reached. Safety Overall, the safety profile of safinamide was good. A total of 51% of the safety population reported AEs. The nature and Table 3 Back-transformed analysis of variance results for TSF in original scale Contrasts Ratio (%) 90% CI of ratio (L) 90% CI of ratio (U) vs. placebo vs. placebo vs. placebo vs. placebo CI, confidence interval; L, lower limit of 90% CI; TSF, tyramine sensitivity factor; U, upper limit of 90% CI. frequency of AEs among the treatment groups were similar; however, differences were evident when considering the study periods. Although AEs were reported throughout the study, central nervous system AEs (particularly headaches) appeared more frequently during the periods when subjects received tyramine; likewise, cardiac AEs (specifically palpitations) occurred only during these periods, and were considered by the investigator as being likely to be associated with tyramine administration (Table 4). By contrast, GI AEs such as nausea, diarrhea, and constipation were observed during the period when the study drugs (i.e., safinamide, selegiline, or phenelzine) were administered alone (SD1 to SD6). Only two significant AEs were reported during the study. One subject showed an isolated, moderate (3.35-fold) increase in alanine transaminase on SD12, i.e., after 10 days of treatment with safinamide 350 mg, leading to withdrawal of the subject from the study. The alanine transaminase increase fully reversed during the follow-up period and was assessed as possibly related to the study drug. The other significant AE was a serious AE in a subject who received phenelzine 30 mg; the subject had a severe fracture of the fibula in a traffic accident 17 days after the last administration of the study drug. This serious AE was assessed as unrelated to treatment. Mean changes from baseline in hematology, blood chemistry, coagulation, and electrocardiogram parameters were similar among the treatment groups at all time points. No clinically relevant changes were observed. Discussion This study was designed in accordance with specific recommendations for adequately assessing potential pharmacodynamic interactions between safinamide and tyramine, and to provide guidance on intake of tyramine-containing foods for patients concomitantly on safinamide treatment. Accordingly, the effect of safinamide on pressor response to oral tyramine was compared with the effects of positive (phenelzine), comparator (selegiline), % of subjects responding ED Tyr30 dose (mg) Figure 1 Cumulative frequency distribution of Tyr30 values by treatment group. 452 VOLUME 92 NUMBER 4 october

4 Table 4 Summary of treatment-emergent adverse events by preferred term Preferred term Tyramine alone (screening period) (n = 17) Number of subjects/number of events Subjects with events and total events 8/11 2/3 2/2 6/9 5/8 Palpitations 5/6 1/1 1/1 3/3 2/3 Nausea 0 1/1 0 1/1 0 Headache 2/ /3 3/3 Dizziness 1/ /1 0 Hot flush 0 1/1 0 1/1 1/1 Investigational medicinal product alone (day 1 to day 6) Subjects with events and total events 4/5 3/7 2/4 3/4 8/12 Nausea 0 0 1/1 1/1 2/2 Diarrhea 0 1/2 0 1/1 0 Constipation 2/ Headache 1/1 1/1 1/1 1/1 3/3 Insomnia 1/ /3 Pollakiuria 1/ /1 0 Investigational medicinal product + tyramine (day 7 to follow-up) Subjects with events and total events 8/21 7/10 4/11 5/16 6/13 Palpitations 3/3 1/1 2/2 2/2 0 Nausea 1/1 1/1 1/1 1/5 0 Abdominal pain 1/ /2 0 Diarrhea 1/1 0 1/1 0 1/1 Vomiting 0 0 1/1 1/2 0 Chest pain 1/ /1 Gastroenteritis 0 0 1/1 1/1 1/1 Pharyngitis 0 1/1 0 1/1 0 Headache 5/9 1/1 2/2 0 4/7 Paraesthesia 1/ /2 0 Dizziness 2/ Only preferred terms for adverse events occurring in more than one subject are displayed in this table. and placebo controls. The study was conducted in healthy volunteers; tyramine capsules were administered under fasting conditions so that response to tyramine was evaluated in the absence of any confounding factors such as disease and possible differences in the bioavailability of tyramine from different tyramine-containing foods. 12,22 To our knowledge, no differences in tyramine pressor response have been shown between PD patients and healthy volunteers; in fact, one study has shown that the tyramine pressor response is similar between PD patients with neurogenic orthostatic hypotension (due to sympathetic denervation) and healthy volunteers. 23 In contrast with a recent study on another MAO-B inhibitor, rasagiline, 10 our study used a double-dummy approach to blind all active treatments (including phenelzine) as well as tyramine capsules and was designed to provide a more robust estimate of the placebo effect by having a separate placebo treatment group instead of 2:1 randomization of active/placebo in each active treatment group. 10 This study was designed under the assumption, supported by nonclinical and clinical data, 6,8,20 that safinamide does not lose MAO-B selectivity with increasing doses. Therefore, only the highest foreseen therapeutic dose and a supratherapeutic dose were tested instead of evaluating a range of safinamide doses, in a dosing approach similar to that applied in thorough QT studies. According to available data, the average steadystate concentration of safinamide at the supratherapeutic dosing regimen of 350 mg/day in healthy volunteers is 3.5- fold and 2-fold higher than the average and highest observed concentrations, respectively, in patients with PD receiving the therapeutic dosing regimen of 100 mg/day. 20,21 Accordingly, the concentrations reached after administration of the supratherapeutic dose in our study account for such unexpected clinical scenarios as double dosing of safinamide, low body weight, and hepatic impairment (which is the only known factor to increase safinamide concentrations; maximum plasma Clinical pharmacology & Therapeutics VOLUME 92 NUMBER 4 October

5 concentration at steady state, C max, is predicted to increase by 60% in subjects with moderate hepatic impairment). In addition, descriptive analysis was supported by an analysis of variance model that corrected for potential effects such as gender and site of study. To our knowledge, this is the first reported study with these unique and thorough design features that enable better assessment of the magnitude and relevance of the influence of a therapeutic agent on the tyramine pressor effect relative to appropriate controls. A small placebo effect (geometric mean TSF of 1.52 in the placebo group) was observed. Such a slight potentiation of the tyramine pressor effect is in line with the literature 10 and may be due to several factors, including increased sympathetic reactivity related to study conditions, coadministration of several tablets, and a possible time effect. Given that all comparisons were done relative to placebo, this effect had no impact on the results for the treatment groups. Both active comparators selegiline 10 mg and phenelzine 30 mg showed treatment effects relative to placebo, in line with those reported in the literature 10,13,14,24,25 thereby confirming assay sensitivity. The dose of phenelzine used in this study was lower than the recommended therapeutic dose for this drug. However, the TSF of ~9.98 for phenelzine 30 mg/ day was in line with literature showing a TSF of for phenelzine 60 mg/day, with dose-dependent increase in tyramine sensitivity The geometric mean TSF of 3.12 for selegiline in this study was also in line with previous studies. 13,14 The high interindividual variability observed in this study in response to tyramine was similar to those reported in previous tyramine challenge studies. 10,11,13,14 A mild increase in TSF was observed after the administration of safinamide 100 and 350 mg. This effect appeared to be slightly dose-dependent; however, although the 90% CI for the geometric mean values of all treatment groups (except phenelzine) were overlapping, the geometric mean TSFs for safinamide at both doses remained lower than that of the comparator, selegiline 10 mg. These results differentiate safinamide from the selective, irreversible MAO-B inhibitor rasagiline, which, at a supratherapeutic dosage of 4 mg/day (fourfold higher than the approved therapeutic dosage of 1 mg/day), had a greater effect than selegiline 10 mg. 10 For rasagiline, a clear dose-dependent effect was observed which is related to its lower specificity for MAO-B with increasing doses, and is reflected in its product labeling with the mention of a risk for hypertensive crisis if used above the recommended dose. 28 In interpreting the results of this study, an important consideration is the fact that the pressor effect of tyramine after ingestion of tyramine-containing food is about half of the effect observed when tyramine is administered under fasting conditions. 29 This difference appears to be related mostly to reduced bioavailability (by ~50%) of tyramine from food as compared with being ingested in fasting conditions. 11,30,31 Considering this difference, the ED50 value of 200 mg for safinamide 100 mg and 350 mg implies that, in more than half of the population, at least 400 mg of tyramine would have to be ingested as a food ingredient to observe a similar increase in SBP to that seen in this study when taken together with safinamide. This situation appears unlikely, given that the common content of a European-style, five-course meal, which includes several potentially tyramine-rich foods, may contain as much as 36 mg of tyramine, whereas a typical meal contains only ~4 mg of tyramine. 32,33 However, as previously reported, 24,29 some individuals have a high sensitivity to tyramine, corresponding to a Tyr30 of 25 mg. We observed this in our study, irrespective of the treatment group. A maximum TSF of 16 was observed in the placebo group, which is equal to or even higher than the maximum TSFs observed in the safinamide 100 mg (TSF of 12), selegiline (TSF of 12), and safinamide 350 mg (TSF of 16) treatment groups. In contrast, the positive control showed a maximum TSF of 64. Although no differences are expected to exist between healthy volunteers and patients with PD with respect to drug-induced tyramine pressor response, 23 it is noteworthy that no case of drug-related hypertensive crisis or any serious AEs related to increase in blood pressure have been observed to date in safinamide clinical trials, including phase III studies in which more than 1,000 PD patients received safinamide at doses of mg/day. Therefore, in this respect, the available data from patients with PD appear to be in line with those from healthy volunteers. The results of this study confirm that safinamide, whether administered at the expected therapeutic dosing regimen of 100 mg once daily or at a supratherapeutic dosing regimen of 350 mg once daily, does not increase the pressor effect of oral tyramine to a clinically relevant extent. The average effect of safinamide at each of the doses was lower than that of selegiline, which can be safely administered without any dietary restrictions at the dosing regimen investigated. Accordingly, and in line with available safety data in PD patients, these results confirm previous data that showed very high MAO-B specificity of safinamide at high doses. In conclusion, the risk of clinically relevant increases in blood pressure in patients receiving safinamide appears to be extremely low, suggesting that safinamide can be safely administered without any dietary restrictions related to tyramine-containing foods or beverages. Methods Study population. The study was approved by an independent ethics committee and was conducted at Biotrial in Rennes and Rueil- Malmaison, France, in accordance with the Helsinki Declaration of The trial is registered with EudraCT ( ). All subjects provided written informed consent. The study population consisted of healthy male and female subjects years of age with a body mass index of kg/m 2. The study eligibility criteria included normal 12-lead electrocardiogram at screening; normal vital signs, including SBP of mm Hg and diastolic blood pressure of mm Hg measured after 5 min of rest in the supine position; and response to tyramine doses (i.e., showing a screening Tyr30 of 200 to 700 mg). Exclusion criteria were the use of any medications such as over-the-counter drugs and herbal remedies within the 21 days preceding the first administration of the study drug (with the exception of oral contraceptives and occasional use of paracetamol 454 VOLUME 92 NUMBER 4 october

6 or ibuprofen); the use of MAO inhibitors within 90 days or fluoxetine within 5 weeks preceding the first administration of the study drug; a history of any serious drug allergies or allergic reactions in general; a contraindication to any of the study medications; a history of migraine; a positive test result for hepatitis B or C or HIV; and a history of primary or secondary hypertension. Study design. The study was a randomized, double-blind, parallelgroup, placebo comparator and positive-controlled study, consisting of a 21-day screening period, a study period of up to 17 days, and an end-of-study visit (4 weeks after the last dose of study medication) (Figure 2). Blinding was ensured by applying a double-dummy technique, with each active treatment group receiving a combination of an active drug and placebo. The following active study medications were used: round, film-coated safinamide 50- and 100-mg tablets (Merck, Darmstadt, Germany), phenelzine (NARDIL 15 mg tablets; Archimedes Pharma UK, Reading, UK), selegiline (STADA 5 mg tablets; STADApharm, Bad Vilbel, Germany), and tyramine capsules of different strengths (6.25, 25, 100, and 200 mg) (Aptuit, Edinburgh, United Kingdom). s were produced to match each of these medications in appearance: round, filmcoated placebo tablets to match safinamide 50 and 100 mg and NARDILand STADA-matching placebo tablets were manufactured by Merck; tyramine-like capsules filled with an inactive matrix were manufactured by Aptuit. To exclude subjects with very low or very high sensitivity to tyramine, oral tyramine pressor tests were performed after the initial screening visit as part of the screening assessments to determine the Tyr30 of each subject in the absence of any study drug treatment. All the tests were conducted after the subjects had fasted for 8 h. These tests, which were later repeated in the treatment period, consisted of a daily administration of escalating doses of tyramine (100, 200, 300, 400, 500, 600, and 700 mg during the screening period) until a sustained SBP increase of 30 mm Hg was observed relative to the daily-defined baseline values. Sustained increase was determined by means of three consecutive SBP measurements within 4 h after tyramine administration, whereas the daily-defined baseline SBP value was the average of three SBP measurements after an initial 30-min rest in the supine position and within 30 min prior to tyramine administration; the calculations were made only if the SBP was considered stable. The subjects had to stay in the clinic during the screening tyramine pressor challenges (up to 8 days, depending on the time point at which Tyr30 was reached). The eligible subjects were randomized to one of the following five treatment groups: (i) safinamide 100 mg once daily, (ii) safinamide 350 mg once daily, (iii) phenelzine 15 mg twice daily (positive control), (iv) selegiline 5 mg twice daily (comparator), or (v) placebo. Randomization was stratified by gender (1:1 ratio for each treatment group). Thereafter, subjects received the respective treatments in a double-blind manner until SD7. At this time point, the daily oral tyramine challenge tests were initiated until Tyr30 (SD16 at the latest) was reached. Subjects in the safinamide, selegiline, and placebo groups received daily escalating doses of tyramine of 25, 50, 100, 150, 200, 300, 400, 500, 600, and 700 mg. Given the high potency of phenelzine and its potential to potentiate tyramine pressor effects to a significant extent, the daily tyramine doses in the phenelzine treatment group were adjusted to 6.25, 12.5, 25, 37.5, 50, 75, 100, 125, 150, and 200 mg. The subjects remained in the clinic from the afternoon of SD1 until the day after Tyr30 had been reached (or SD17 at the latest). The subjects were instructed not to consume food or fluids containing tyramine for 48 h prior to the screening tyramine pressor challenge and again from 48 h prior to SD1 until 2 weeks after the last dose of trial medication. During each period of stay at the clinic, meals were fully controlled, standardized, and identical for all subjects; in particular, all the meals during the screening tyramine pressor challenges, and from the evening of SD1 until the day after Tyr30 was reached, were tyramine-free. Tyramine was administered 90 min after the morning administration of the study drug. SBP, diastolic BP, and heart rate were monitored with the subjects in the supine position, for at least 4 h after the dose until baseline values were restored; measurements were taken using an automatic device, a Dinamap (Model Pro Series 100, Pro Series 200, or Procare 100; GE Healthcare, Chalfont St. Giles, UK), at 5-min intervals for 2 h after tyramine administration and at 15-min intervals from 2 to 4 h after tyramine administration. However, if a single increase in SBP of 30 mm Hg was observed during the 2- to 4-h period, SBP measurements were performed again at 5-min intervals. Additional safety measurements were to be repeated as often as needed at the discretion of the investigator. All tyramine challenge tests were performed under direct medical supervision after an overnight fast. Sample size. The sample size for the study was based on a survey of the literature on tyramine studies and an assessment (using nquery version 6.01, Statistical Solutions, Saugers, MA) of the estimated precision of the geometric mean ratio of TSF for experimental treatments vs. placebo. It was determined that a minimum sample size of 90 randomized subjects (18 subjects per treatment group), ensuring generation of data with respect to at least 80 subjects (16 subjects per treatment group), would be sufficient to provide important Special diet from 48 h before pressor test Resident over 8 days Screening visit Screening tyramine pressor tests (daily up to 7 consecutive days) Tyr30 <200 mg or >700 mg 200 mg Tyr mg Discontinue Proceed Randomization All treatments blinded Tyramine challenges Daily tyramine challenge tests from SD7 to SD16 unless Tyr30 is reached End of treatment Follow-up SD 21 SD 20 SD 1SD1 SD7 SD16 SD17 SD45 Screening period SD 21 to SD 1 Resident from SD 1 until the day after Tyr30 or SD17 Special diet from 48 h prior to SD 1 Figure 2 Overview of study design. Clinical pharmacology & Therapeutics VOLUME 92 NUMBER 4 October

7 by-treatment group information with regard to TSF and to estimate TSF mean ratio across treatment groups with reasonable precision. This sample size was similar to, or larger than, those in other tyramine interaction studies. 10,34,35 Outcome measures. The primary outcome measure was TSF, which was defined as the ratio of Tyr30 at screening to Tyr30 during treatment. Sensitivity to the tyramine pressor effect in each treatment group was also measured in terms of ED50, i.e., the dose of tyramine resulting in a sustained increase in SBP of >30 mm Hg in 50% of the subjects. The respective ED50 ratios (ED50 for placebo/ed50 for study drug) were calculated for all the treatment groups. Plasma tyramine concentrations, predose and also at 30 min and 60 min after tyramine administration, were analyzed so as to continue appropriate exposure. The concentrations of safinamide and its metabolites in plasma were determined predose and also at 2 h and 2.5 h after safinamide administration, corresponding to the time point of safinamide C max as reported in the available pharmacokinetics data. In addition, predose samples were taken from SD1 to SD6 to confirm steady-state exposure. Safety assessments included incidence of AEs, 12-lead ECGs, laboratory tests (including biochemistry, hematology, coagulation, and urinalysis), and full physical examinations. Statistical methods. The primary end point, TSF, was summarized descriptively according to treatment group so as to facilitate comparisons between experimental treatments and placebo. In addition, an analysis of variance of log-transformed TSF was conducted with treatment, site, and gender as fixed effects, to assess possible covariate effects. Point estimates and 90% CIs of geometric means for the treatment groups and geometric mean ratios for the treatment groups vs. placebo were determined. Other secondary pharmacodynamic and pharmacokinetic end points were summarized descriptively according to treatment group. Safety data were evaluated descriptively. All AEs occurring between the date when the written informed consent was obtained and the date of the post-study visit were evaluated and assigned to different trial periods as follows: AEs that occurred between the pressor test (after tyramine administration) and SD1 (before first dosing of study drug) were assigned to tyramine alone AEs that occurred between SD1 (after study drug administration) and SD7 (before tyramine dosing) were assigned to study drug alone AEs that occurred between SD7 (after tyramine dosing) and follow-up were assigned to study drug plus tyramine All analyses were conducted primarily on data from the safety population, which included all subjects who had received at least one dose of the study drug and had undergone a subsequent safety assessment. Acknowledgments The authors thank the Curry Rockefeller Group, Tarrytown, NY, for editorial assistance in the preparation of the manuscript. This study was supported by Merck Serono S.A., Geneva, Switzerland, a branch of Merck Serono S.A., Coinsins, Switzerland, an affiliate of Merck, Darmstadt, Germany. The study was conducted by Biotrial S.A., which was funded by Merck Serono S.A., Geneva, Switzerland. Author Contributions A.M. wrote the manuscript; A.M., K.K., and S.K. designed the research; A.M. and B.A. performed the research; A.M., K.K., A.J., B.A., A.P., S.K., and A.K. analyzed the data. Conflict of Interest A.M. is an employee of Merck Serono S.A., Geneva, Switzerland; K.K. is an employee of Merck; A.J. is an employee of Merck; S.K. is an employee of Merck; A.K. is an employee of Merck; B.A. is an employee of Biotrial S.A.; A.P. is an employee of Biotrial S.A. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? Pharmacodynamic interactions with tyramine are a typical class effect of MAO inhibitors. These interactions need to be adequately investigated so as to guide clinical use and ensure patient safety with respect to possible blood pressure increases consequent to the intake of tyramine-containing foods or beverages. What question did this study address? This study was conducted to evaluate this class effect with respect to safinamide, a novel molecule in development for the treatment of PD. The study had a unique, double-blind, double-dummy, randomized, placebo-, positive-, and comparator-controlled design and was conducted in healthy volunteers. What this study adds to our knowledge By applying a unique design offering significantly improved methods for assessing the magnitude of the effect vs. appropriate controls, the study was able to confirm the high specificity of safinamide for MAO-B inhibition, thereby providing evidence that it is safe to administer to PD patients without any dietary restrictions related to tyramine-containing food or beverages. 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