PHARMACOKINETIC DYNAMIC RELATIONSHIPS Effect of opicapone multiple-dose regimens on levodopa pharmacokinetics

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1 British Journal of Clinical Pharmacology Br J Clin Pharmacol (2017) PHARMACOKINETIC DYNAMIC RELATIONSHIPS Effect of opicapone multiple-dose regimens on levodopa pharmacokinetics Correspondence Patrício Soares-da-Silva, MD, PhD, Department of Research & Development, BIAL Portela & Cª, S.A., À Av. da Siderurgia Nacional, S. Mamede do Coronado, Portugal. Tel.: ; Fax: ; psoares.silva@bial.com Received 19 May 2016; Revised 24 September 2016; Accepted 2 October 2016 José-Francisco Rocha 1,ÉricSicard 2,NicolasFauchoux 3, Amílcar Falcão 4, Ana Santos 1,AnaI.Loureiro 1, Roberto Pinto 1,5, Maria João Bonifácio 1, Teresa Nunes 1,LuísAlmeida 5,6 and Patrício Soares-da-Silva 1,5,6 1 Dept. Research & Development, BIAL Portela & Cª S.A., S. Mamede do Coronado, Portugal, 2 Algorithme Pharma Inc., Quebec, Canada, 3 Biotrial, Rennes, France, 4 Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal, 5 Dept. Pharmacology & Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal, and 6 MedInUP - Center for Drug Discovery and Innovative Medicines, University of Porto, Porto, Portugal Keywords benserazide, carbidopa, catechol-o-methyltransferase inhibitors, levodopa, opicapone, pharmacokinetics AIMS To compare the levodopa/carbidopa (LC) and levodopa/benserazide (LB) pharmacokinetic profiles following repeated doses of opicapone (OPC) administered apart from levodopa. METHODS Two randomized, double blind, sex-balanced, placebo-controlled studies in four groups of 12 or 18 healthy subjects each. In each group, enrolled subjects received a once-daily morning (5, 15 and 30 mg) or evening (5, 15 and 50 mg) administration of OPC or placebo for up to 28 days. On the morning of Day 11, 12 h after the OPC or placebo evening dose, or the morning of Day 21, 1 h after the OPC or placebo dose, a single dose of immediate-release 100/25 mg LC was administered. Similarly, on Day 18 morning, 12 h after the OPC or placebo evening dose, or Day 28 morning, 1 h after the OPC or placebo dose, a single dose of immediaterelease 100/25 mg LB was administered. RESULTS All OPC treatments, in relation to the placebo group, presented a higher extent of exposure (AUC) to levodopa following either LC or LB doses. A relevant but not dose-dependent increase in the levodopa AUC occurred with all OPC dose groups in relation to placebo. All active treatments significantly inhibited both peak (E max ) and extent (AUEC) of the catechol-o-methyltransferase activity in relation to placebo. The tolerability profile was favourable. CONCLUSION Opicapone, as once-daily oral evening regimen and/or 1 h apart from levodopa therapy, increases the bioavailability of levodopa associated with its pronounced, long-lasting and sustained catechol-o-methyltransferase inhibition. The tolerability profile was favourable and similar between OPC and placebo. DOI: /bcp The British Pharmacological Society

2 Opicapone effect on levodopa PK WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT Currently available catechol-o-methyltransferase (COMT) inhibitors used as adjunctive therapy in levodopa-treated Parkinson s disease patients have clinical limitations. Due to liver toxicity, the use of tolcapone requires liver function monitoring. Entacapone is considered to be safe, but its efficacy is limited and requires frequent dosing. Opicapone is a novel third generation COMT inhibitor designed to provide high COMT inhibitory potency and avoid cell toxicity. WHAT THIS STUDY ADDS Opicapone, as a once-daily oral evening regimen and/or 1 h apart from levodopa therapy, increases the bioavailability of levodopa associated with its pronounced, long-lasting and sustained COMT inhibition. It is anticipated that evening opicapone adjunct therapy, potentially at bedtime separated 1 h from last daily levodopa dose, could provide higher flexibility of daily levodopa adjustment by enhancing levodopa s optimization. Tables of Links TARGETS Enzymes [2] LIGANDS Opicapone Levodopa COMT Carbidopa Benserazide Protein kinase A Entacapone Tolcapone These Tables list key protein targets and ligands in this article that are hyperlinked to corresponding entries in the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY [1], and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 [2]. Introduction Despite decades of clinical use, levodopa still remains the most effective symptomatic treatment in Parkinson s disease [3 6]. The therapeutic effect of levodopa depends on its biotransformation to dopamine in the brain. However, levodopa undergoes rapid and extensive metabolism by peripheral aromatic L-amino acid decarboxylase (AADC) and catechol-omethyltransferase (COMT) and only 1% of an oral dose of levodopa actually reaches the brain [7, 8]. Therefore, levodopa is usually coadministered with an AADC inhibitor (AADCi, either carbidopa or benserazide), which increases levodopa bioavailability. However, when using levodopa combined with an AADCi approximately 90% of a levodopa dose undergoes conversion by COMT to 3-O-methyldopa, which competes with levodopa at the level of the blood brain barrier for transport [9 12]. Thus, an additional strategy to further prevent the peripheral levodopa metabolism and increase the delivery of levodopa to the brain is based on the administration of a COMT inhibitor [13 15]. Only two COMT inhibitors (tolcapone and entacapone) are currently available for clinical use, and both have some clinical limitations. Tolcapone requires liver function monitoring and thus is limited to fluctuating patients poorly controlled with other therapies[16].entacaponeisconsideredtobesafe[17],butits efficacy is limited and requires frequent dosing [7]. Therefore, there is a need for more efficacious and safer COMT inhibitors. Opicapone (OPC: 2,5-dichloro-3-[5-(3,4-dihydroxy-5-nitrophenyl)-1,2,4-oxadiazol-3-yl]-4,6-dimethylpyridine 1- oxide, also known as BIA ) is a novel third-generation COMT inhibitor currently under phase III clinical trials by BIAL Portela & Cª, S.A. (S. Mamede do Coronado, Portugal) for use as adjunctive therapy in levodopa-treated Parkinson s diseasepatients.opcwasdesignedasahydrophilic 1,2,4-oxadiazole analogue with a pyridine N-oxide residue at position 3, providing high COMT inhibitory potency and avoiding cell toxicity [18, 19]. OPC is endowed with an exceptionally high binding affinity (subpicomolar K d )[20] that translates into a slow complex dissociation rate constant and a long duration of action in vivo [21]. In liver and brain homogenates from rats administered with OPC, tolcapone and entacapone by gastric tube, OPC showed a stronger and more sustained COMT inhibitory effect than tolcapone and entacapone. One h after administration, COMT inhibition was 99% with OPC vs. 82% with tolcapone and 68% with entacapone. Nine hours after administration, entacapone showed no COMT inhibition and tolcapone produced minimal inhibitory effect (16%), whereas OPC continued to inhibit COMT activity by 91% [21, 22]. In an entry-into-man study in healthy male volunteers, single doses of OPC ranging from 10 to 1200 mg were well tolerated. The adverse event (AE) profile did not differ from that of placebo and the clinical safety tests showed no sign of concern. The extent of systemic exposure to OPC increased in an approximately dose-proportional manner and, despite the short half-life (t 1/2 : h), a dose-dependent and long-lasting COMT inhibitory effect was observed with a maximum S-COMT inhibition (E max )rangingfrom 34.5% (10 mg) to 100% (1200 mg), and an inhibition of % remained 24 h postdose [23, 24]. Following an 8-day once-daily multiple-dose regimen up to 30 mg OPC, sulfated OPC was the predominant metabolite and the bile is likely to be the main route of OPC excretion [24]. Maximum S-COMT inhibition (E max ) ranged from 69.9% to 98.0% following the last dose of OPC [24]. A study in healthy subjects that compared the levodopa pharmacokinetic (PK) profile throughout a day driven by the COMT inhibition Br J Clin Pharmacol (2017)

3 J.-F. Rocha et al. either following repeated doses of OPC or concomitant administration with entacapone, demonstrated superiority of OPC over entacapone upon the bioavailability of levodopa in association with more pronounced, long-lasting, and sustained COMT inhibition [25]. Studies in healthy subjects with concomitant administration of OPC and levodopa/aadci (either carbidopa or benserazide) demonstrated that OPC was well tolerated, inhibited the COMT in a potent manner, but showed a certain degree of interaction during the absorption phase between levodopa/aadci and OPC (data on file), which was considered to be minimized by separating 1 h both administrations. A phase IIa exploratory pilot study in Parkinson s disease patients with motor fluctuations showed that 30 and 15 mg OPC inhibited COMT activity, increased the levodopa bioavailability, and significantly improved various motor outcomes, including a dose-dependent change in absolute OFF-time, and was well tolerated when administered with standard release 100/25 mg levodopa/carbidopa or levodopa/benserazide [26]. The efficacy and safety profile of OPC based on pooled efficacy data from two pivotal, multinational, double-blind, randomized, parallel-group, 14 week, placebo- and activecontrolled studies in patients with Parkinson s diseaseand motor fluctuations, including a total of 758 patients (placebo n = 255,OPC 25 mg n = 241, OPC 50 mg n = 262) has been recently presented [27, 28]. OPC (50 mg) provided a mean reduction in OFF-time of 60.8 min vs. placebo as an adjunct to levodopa in patients with Parkinson s disease with endof-dose motor fluctuations and is, therefore, the only oncedaily COMT inhibitor to provide a mean reduction in time in the OFF state that is clinically relevant [29]. In addition, a higher proportion of patients in the OPC 50 mg group than in the entacapone group had improved on both the clinician s andpatient s global impression of change [29]. OPC was well tolerated in studies in several animal species (data on file; BIAL Portela & Cª, S.A.), healthy volunteers [23, 25, 30 32] and Parkinson s disease patients [27 29, 33]. The present studies aimed to investigate the effect of repeated, once-daily, OPC doses upon COMT activity and levodopa PK using a regimen in which OPC and levodopa/aadci administrations were separated by at least 1 h and, additionally, foreseeing that daily therapeutic regimens of levodopa could be enhanced and better optimized with an OPC oncedaily evening regimen. OPC (5, 15 and 50 mg) or placebo for up to 18 days. The levodopa/carbidopa(lc)daywasday21(study1)morning, 1 h after the OPC or placebo dose or Day 11 (Study 2) morning, 12 h after the OPC or placebo evening dose, where a single dose of immediate-release 100/25 mg LC was administered. Similarly, the levodopa/benserazide (LB) day was Day 28 (Study 1) morning, 1 h after the OPC or placebo dose, or Day18(Study2)morning,12haftertheOPCorplaceboevening dose, a single dose of immediate-release 100/25 mg LB was administered (Table 1). The clinical part from both studies consisted of three inhouse and two ambulatory periods. The first OPC or placebo dose was administered on Day 1 morning (Study 1) or evening (Study 2) and subjects remained in the clinical unit until approximately 6 (Study 1) or 12 (Study 2) h postdose. In Study 1, from Day 2 to Day 20, subjects returned to the clinical unit every morning for OPC or placebo administrations, followedbyanin-houseperiodof1.5daysfromday20evening to Day 22 morning (i.e. 24 h after administration of LC).OnDay22morning,subjectsreceivedadoseofLC1h after OPC or placebo administration. From Day 23 to Day 27, subjects returned again to the clinical unit every morning for OPC or placebo administrations, followed by last in-house period of 1.5 days from Day 27 evening to Day 29 morning (i.e. 24 h after administration of LB). On Day 28 morning, subjects received a dose of LB 1 h after OPC or placebo administration. In Study 2, from Day 2 to Day 9, subjects returned to the clinical unit every evening for OPC or placebo administrations, followed by an in-house period of 1.5 days from Day10eveningtoDay12morning(i.e.24hafteradministration of LC). On Day 11 morning, subjects receive a dose of LC 12 h after OPC or placebo previous administration (Day 10 evening). From Day 12 to Day 16, subjects returned again to the clinical unit every evening for OPC or placebo administrations, followed by last in-house period of 1.5 days from Day17eveningtoDay19morning(i.e.24hafteradministration of LB). On Day 18 morning, subjects receive a dose of LB 12 h after OPC or placebo previous administration (Day 17 evening). Table 1 Summary of clinical trial design Methods Study designs We conducted two randomized, double-blind, sex-balanced and placebo-controlled studies. The first study (Study 1; trial registration NCT ) was a single centre (Algorithme PharmaInc.,Quebec,Canada)phase1study,infourgroups of 12 healthy subjects each (six male and six female) who received a once-daily morning administration of OPC (5, 15 and 30 mg) or placebo for up to 28 days. The second study (Study 2; trial registration EudraCT No ) was also a single centre (Biotrial, Rennes, France) in four groups of 18 healthy subjects each (nine male and nine female) that received a once-daily evening administration of Study 1 Days Treatment Time after last OPC dose 1 28 OPC or placebo at 8 am 21 LC or placebo at 9 am 1 h 28 LB or placebo at 9 am 1 h Study 2 Days Treatment Time after last OPC 1 18 OPC or placebo at 9 pm 11 LC or placebo at 9 am 12 h 18 LB or placebo at 9 am 12 h OPC, opicapone; LC, levodopa/carbidopa; LB, levodopa/ benserazide 542 Br J Clin Pharmacol (2017)

4 Opicapone effect on levodopa PK Potential subjects were screened for eligibility within 28 days of admission. Screening consisted of discussion of informed consent, medical history, physical examination, vital signs, 12-lead electrocardiography (ECG), clinical laboratory tests (haematology, plasma biochemistry, coagulation, urinalysis, viral serology, drugs of abuse screen and pregnancy test) and review of the selection criteria. Subjects were to be aged years (25 45 years for Study 1), within kg m 2 (19 30 kg m 2 for Study 1) of body mass index (BMI) and nonsmokers or ex-smokers; women of childbearing potential had to use a medically acceptable form of contraception. No medication other than the study drugs or necessary for the treatment of AEs was allowed after the first dosing until the end of the study. On each dosing day, subjects were fasting from all food for a minimum of 4 h (8 h for Study 1) before dosing and remained fasted until at least 2 h (4 h for Study 1) postdose. On LC and LB dosing days, subjects had an overnight fast for at least 8 h before the levodopa administration and remained fasted until at least 3 h post-dose. Water drinking was allowed as desired except for 1 h before and after each dosing. At least from 48 h before the first dosing and during the treatment period, subjects were requested to abstain from smoking, drinking alcohol, coffee, tea or beverages containing xanthines. The consumption of food or beverages containing grapefruit was not allowed starting from one week before dosing until end of clinical phase. The study medication was OPC (manufactured by BIAL Portela & Cª, S.A) administered as capsules of 5 and 25 mg OPC, placebo (manufactured by BIAL Portela & Cª, S.A) as capsules, LC (Sinemet, by Bristol-Myers Squibb, Canada, acquired as commercial product for Study 1 and by Merck Sharp & Dohme acquired as commercial product for Study 2) as tablets of 100/25 mg LC and LB [Prolopa (Study 1) and Madopar (Study 2) by F. Hoffmann-La Roche, acquired as commercial product] as capsules of 100/25 mg LB. Placebo and OPC capsules were identical in terms of size, appearance and taste. Sample size estimation was based on a primary levodopa parameter (AUC 0 ).Inapreviousstudy[34]awithin-subject variability (CV%) of 27.5% was observed for levodopa AUC 0. Assuming an alpha error of 5% (two-tailed) and a within-subject CV of 27.5%, 48 subjects (12 subjects per treatment group) provided 80% probability (power) to detect a 30% difference in the geometric means of levodopa AUC 0. In Study 2, 72 subjects (18 subjects per treatment group) were to be enrolled. The comparison between treatment arms was done according to the bioequivalence reference interval of %. The reference interval (bioequivalence testing), although extensively used for PK evaluations within the compoundand/orformulations,wasusedinthepresentstudy for evaluation between treatment arms, even different, but with related endpoints, i.e., PK and pharmacodynamics (PD). Subjects who withdrew or dropped-out from the study had to be replaced to allow the completion of the necessary subjects. The replacement subject was assigned to the same treatment group as the replaced subject. Both studies were conducted according to the Helsinki Declaration, ICH Good Clinical Practice recommendations and applicable local regulations. Study 1 was approved by an Independent Ethics Committee (ETHIPRO, Comité d Éthique de la Recherché Independent, Canada) and by the Canadian Agency (Health Canada Therapeutic Products Directorate). Study 2 was approved by an Independent Ethics Committee (CPP - Comité de Protection des Personnes, Ouest VI, Brest, France) and by the French Medicines Agency (ANSM, formerly AFSSAPS). Written informed consent was obtained from each study participant. Safety assessments Safety and tolerability assessments included routine laboratory tests (blood chemistries, haematological profile, coagulation and urinalysis), physical examination, ECG, and vital signs. Any undesirable sign, symptom, or medical condition occurring after starting the study, whether reported spontaneously or when prompted, was recorded regardless of suspected relation to the study medication. Blood sampling for PK and PD assays Blood samples (3 ml for Study 1 and 6 ml for Study 2) for PK analyses of OPC were drawn by direct venipuncture or intravenous catheter into potassium ethylenediaminetetraacetic acid tubes, at the following time points on each LC or LB dosing days: pre-lc/lb dose and then at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 16 and 24 h post-lc/lb dose. Blood samples were also collected before OPC or placebo on Days 1, 4, 7, 10, 13 (Study 2 only), 14 (Study 1 only), 15 (Study 2 only), 17, 19 (Study 1 only), 24 (Study 1 only) and 26 (Study 1 only). After collection, blood samples were centrifuged at approximately 1500 g for 10 min at 4 C, and the resulting plasma was then separated into two aliquots of 0.5 ml which were stored at 70 C until required for analysis. Blood samples (3-ml) for PK analyses of levodopa and 3-Omethyldopa (3-OMD) were drawn by direct venipuncture or intravenous catheter into potassium ethylenediaminetetraacetic acid tubes, at the following time points on each LC or LBdosingdays:pre-LC/LBdoseandthenat0.5,1,1.5,2,3, 4,6,8,12,16(Study1only)and24(Study1only)hpost- LC/LB dose. After collection, blood samples were centrifuged at approximately 1500 g for 10 min at 4 C, and the resulting plasma was then separated into two aliquots of 0.5 ml which were stored at 70 C until required for analysis. Blood samples collected for OPC PK assays served for the PD assessments. After centrifugation and removal of plasma, the supernatant (uppermost erythrocyte layer) was removed and the tubes containing the erythrocytes were placed in ice. Then, a volume of cold 0.9% NaCl solution equal to double that of erythrocytes was added. The erythrocytes were centrifuged (at 4 C and approximately 1500 g for 10 min) and the washing procedure was repeated three times. Then, two aliquots accurately pipetted of 0.5 ml washed erythrocytes were prepared and stored at 70 C until required for analysis. S-COMTassay Determination of S-COMT activity was carried out in compliance with good laboratory practice (GLP) at BIAL s Pharmacological Laboratory (S. Mamede do Coronado, Portugal) according to a validated method [23, 24]. In brief, prewashed erythrocytes samples were haemolyzed with three Br J Clin Pharmacol (2017)

5 J.-F. Rocha et al. volumes of ice-cold water. After vortexing, the samples were left standing on ice for 10 min and then centrifuged for 20 min at 2 8 C at g. The supernatant was used for S-COMT assay, which was carried out immediately after sample preparation. The incubation mixture contained 300 μlenzyme preparation, 375 μ incubation medium and 75 μl of 5 mmol l 1 adrenaline as S-COMT substrate. The samples were incubated in a water bath at 37 C for 60 min. The tubes were transferred to ice and the reaction was stopped by adding 75 μlofice-cold4moll 1 perchloric acid. The samples were incubated in a refrigerator at 2 8 C for 2 h and then centrifuged at 2 8 C (9000 g,4min)and600μl aliquots of the supernatant, filtered on 0.22-μm poresizespin-xfilter tubes (Costar), were centrifuged at 2 8 C (1400 g, 4 min) and 150 μl used for the assay of metanephrine by means of highpressure liquid chromatography with electrochemical detection, as described elsewhere [35]. S-COMT activity was expressed as the amount of metanephrine (in pmol) formed by the action of erythrocyte S-COMT upon epinephrine, per mg of haemoglobin in the sample, per h (pmol mg Hb 1 h 1 ). Bioanalysis of analytes Determination of plasma concentrations of levodopa and 3- OMD was carried out in compliance with GLP at Nuvisan GmbH (Neu-Ulm, Germany) by liquid chromatography with electrochemical detection using a validated method with a lower limit of quantification (LLOQ) of 25 ng ml 1. The assay accuracy was %; the precision (%CV) was <7.3%. Determination of plasma concentrations of OPC in Study 1 was carried out in compliance with GLP at Algorithme Table 2 Summary of demographic data Study 1 Placebo 5 mg OPC 15 mg OPC 30 mg OPC Total Sex Male/female Race White/other Age Years Height cm Weight kg n 7/7 6/7 6/7 6/6 25/27 % 50/50 46/54 46/54 50/50 48/52 n 13/1 12/1 13/0 10/2 48/4 % 93/7 92/8 100/0 83/17 92/8 n Mean (SD) 32 (6) 36 (8) 34 (7) 35 (6) 34 (7) Range n Mean (SD) 172 (10) 168 (9) 168 (11) 170 (10) 170 (10) Range n Mean (SD) 73 (11) 66 (13) 67 (12) 74 (9) 70 (12) Range BMI n kg m -2 Mean (SD) 25 (3) 23 (3) 24 (3) 26 (3) 24 (3) Range Study 2 n 5 mg OPC 15 mg OPC 50 mg OPC Total Sex Male/female Race White/other Age Years Height cm Weight kg n 9/9 9/10 9/10 9/9 36/38 % 50/50 47/53 47/53 50/50 49/51 n 17/1 19/0 18/1 17/1 71/3 % 94/ 6 100/0 95/5 94/ 6 96/4 n Mean (SD) 31 (11) 32 (9) 31 (8) 30 (10) 31 (9) Range n Mean (SD) 172 (8) 170 (9) 170 (11) 170 (10) 170 (9) Range n Mean (SD) 71 (13) 66 (11) 71 (10) 67 (129) 69 (12) Range BMI n kg m -2 Mean (SD) 24 (3) 23 (2) 25 (2) 23 (3) 24 (3) Range OPC, opicapone; BMI, body mass index 544 Br J Clin Pharmacol (2017)

6 Opicapone effect on levodopa PK Pharma Inc. (Quebec, Canada) by liquid chromatography with tandem mass spectrometry detection using a validated method with a LLOQ of 10.0 ng ml 1.Theassayaccuracy was %; the precision (%CV) was <8.6%. Determination of plasma concentrations of OPC and BIA in Study 2 was carried out in compliance with GLP at SwissBioAnalytics AG (Birsfelden, Switzerland) by LC MS/MS using a validated method with a LLOQ of 10.0 ng ml 1.The assayaccuracywas %; the precision (%CV) was <7.0%. Analyses PD analysis. The following PD parameters were derived from the individual S-COMT activity profiles: maximum inhibition of COMT activity post dose (E max ), time to E max (t Emax ), and area under the effect-time curve (AUEC). The value observed before the first dose was taken as the baseline value (E 0 ). Descriptive statistics were prepared for each parameter. E max and AUEC were compared between treatment groups using a one-way analysis of variance with treatment as fixed effect. Point estimates of the arithmetic Table 3 Mean ± standard deviation pharmacodynamic parameters of S-catechol-O-methyltransferase (S-COMT) activity, including ANOVA results, on Days 11 or 21 and 18 or 28, following once-daily oral administrations of opicapone (OPC) or placebo E 0 E max t Emax [(E 0 -E max )/E 0 ].100 AUEC 0 24 (pmol mg Hb 1 h 1 ) (pmol mg Hb 1 h 1 ) (h) (%) (pmol mg Hb 1 h 1 h) Study 1 Study 2 Day 21 Levodopa/carbidopa Placebo (n =14) 42.9± ± ± ± ±276 5mgOPC(n =13) 35.3± ± ± ± ± mg OPC (n =12) 44.0± ± ± ± ± mg OPC (n =12) 37.8± ± ± ± ±177 Day 28 Levodopa/benserazide Placebo (n =14) 42.2± ± ± ± ±233 5mgOPC(n =13) 35.3± ± ± ± ± mg OPC (n =12) 44.0± ± ± ± ± mg OPC (n =12) 37.8± ± ± ± ±171 Day 11 Levodopa/carbidopa Placebo (n =18) 45.9± ± ± ± ±407 5mgOPC(n =19) 38.7± ± ± ± ± mg OPC (n =19) 38.6± ± ± ± ± mg OPC (n =18) 37.2± ± ± ± ±121 Day 18 Levodopa/benserazide Placebo (n =18) 45.9± ± ± ± ±374 5mgOPC(n =19) 38.7± ± ± ± ± mg OPC (n =19) 38.6± ± ± ± ± mg OPC (n =18) 37.2± ± ± ± ±105 E max % (90%CI) AUEC 0 24 % (90%CI) Study 1 Study 2 Day 21 comparison 5 mg OPC/placebo (31.48; 66.06) (36.33; 63.98) 15 mg OPC/placebo (12.54; 47.11) (21.38; 49.02) 30 mg OPC/placebo ( 3.19; 31.38) (11.12; 38.77) Day 28 comparison 5 mg OPC/placebo (29.26; 60.28) (35.60; 61.31) 15 mg OPC/placebo (10.79; 41.81) (21.46; 47.17) 30 mg OPC/placebo ( 2.76; 28.26) (11.87; 37.58) Day 11 comparison 5 mg OPC/placebo (28.60;56.82) (32.23;59.27) 15 mg OPC/placebo ( 0.867;27.35) (11.56;38.60) 50 mg OPC/placebo 6.11 ( 8.20;20.43) ( 1.62;25.82) Day 18 comparison 5 mg OPC/placebo (27.11;54.50) (31.03;58.15) 15 mg OPC/placebo ( 0.052;27.34) (10.89;38.01) 50 mg OPC/placebo 2.84 ( 11.06;16.74) ( 2.67;24.84) E 0, baseline (prefirst dose) value; E max, maximum observed effect on S-COMT activity; t Emax, time of occurrence of E max ; [(E 0 -E max )/E 0 ] 100, maximum percent inhibition of S-COMT activity; AUEC, area under the effect-time curve Br J Clin Pharmacol (2017)

7 J.-F. Rocha et al. mean ratios and corresponding 90% confidence intervals (CIs) for each parameter and for the ratios OPC/placebo were presented and compared to % reference interval. This reference interval (bioequivalence testing) is extensively used as standard tests for bioequivalence between treatment arms, but can also be considered for other related endpoints. In this study, it was decided to test COMT inhibition in the same bioequivalence test as performed for PK to allow a comparison between OPC plasma exposure and COMT inhibition. PK analysis. The PK parameters were derived, where appropriate, from the individual plasma concentration time profiles of each analyte and included the maximum observed plasma concentration (C max ), time at which C max was observed (t max ), area under the plasma concentration time curve (AUC) calculated using the trapezoidal method from zero to the last quantifiable drug concentration (AUC 0 t ) and from zero to infinity (AUC 0 ), elimination rate constant (λ z ) and the apparent terminal half-life (t ½ ). Summary statistics were prepared for each parameter. For levodopa and 3-OMD in plasma, C max and AUC were compared between treatment groups using ANOVA with treatment as fixed effect. Point estimates of the geometric mean ratios and the corresponding 90%CIs for each parameter and for the ratios OPC/placebo were obtained andcomparedtothe80 125% reference interval. AEs. AEs were coded according to the Medical Dictionary for Regulatory Activities (MedDRA, version 12.0). For the Figure 1 Mean S-catechol-O-methyltransferase activity (metanephrine formed) vs. time following once-daily oral administrations of opicapone (OPC) or placebo for 18 or 28 days (A: Days 1 28; B: Days 1 18) and on (C) Day 21 or (D) Day 11 levodopa/carbidopa, and on (E) Day 28 or (F) Day 18 (levodopa/benserazide,; n =12 for Study 1 and n = 18 for Study 2). The arrow indicates the time of OPC administration (~8:00 am in Study 1 and ~9:00 pm in Study 2). Hb, haemoglobin 546 Br J Clin Pharmacol (2017)

8 Opicapone effect on levodopa PK laboratory safety data, clinically significantly abnormal values were considered as AEs. Statistical analysis. The PK and PD parameters were calculated by using WinNonlin (Version 5.2; Pharsight Co, Palo Alto, CA, USA). Statistical analysis used SAS software release (SAS Institute Inc., Cary, NC, USA). Results Populations InStudy1,atotalof52subjects(25maleand27female)were enrolled and 48 subjects completed the study. Subjects with a mean age (range) of 34 (25 45) years, height 170 ( ) cm and had a BMI of 24 (19 29) kg m 2 (Table 2). Four subjects were withdrawn from study participation. One subject was discontinued by the Principal Investigator due to an AE (darker skin below eyes followed by photosensitivity). Two subjects withdrew consent due to personal reasons. One subject was discontinued by the Principal Investigator due to a protocol violation (took penicillin due to an AE [tonsillitis] prescribed by the Principal Investigator). Therefore, all 52 subjects enrolled constituted the safety population set and the 48 subjects who completed the study constituted the PK population set. In Study 2, a total of 74 subjects (36 male and 38 female) were enrolled and 72 completed the study. The mean age (range) was 31 (18 45) years, height of 170 ( ) cm and BMI of 24 (18 29) kg m 2 (Table 2). One subject (5 mg OPC group, on Day 4) withdrew consent due to personal reasons and another (15 mg OPC group, on Day 16) was withdrawn due to an AE (joint sprain) and both were replaced. Therefore, all 74 subjects enrolled constituted the safety population set and the 72 subjects who completed the study constituted the PK population set. PD Table 3 displays the PD parameters of S-COMT. Statistical comparisons of OPC in relation to placebo group are also provided in Table 3. In both studies, a sustained inhibitory effect of OPC upon S-COMT activity was observed throughout the entire studies (Figure 1A and Figure 1D, for Study 1 and 2, respectively). The S-COMT activity over time on each LC and LB dosing day are depicted, respectively, in Figures 1B and 1C for Study 1, and in Figures 1E and 1F for Study 2. The sustained and dose-dependent inhibitory effect of OPC upon S-COMT activity is also observed on each LC and LB dosing day but in Study 2 (Figures 1E and 1F) an additional effect is perceived after 12 h postlevodopa dose but this effect is due to the daily administration of OPC, which happened 12 h after each LC and LB dose. Maximum S-COMT inhibition (E max )wasbetween2.2h(30mgopc,study1)and13.4h (15 mg OPC, Study 2) post-opc dose (t Emax ), and ranged from 55% (5 mg OPC, Study 1) to 98% (50 mg OPC, Study 2; Table 3). All OPC treatments significantly inhibited, in a dose-dependent manner, both peak (as assessed by E max ) and extent (as assessed by AUEC) of S-COMT activity in relation to placebo (Table 3). PK OPC. Table 4 presents the main PK parameters of plasma OPC. PK results for OPC were consistent with those obtained in previous studies [23, 24]. In Study 2, as blood samples were collected in relation to LC/LB administrations, last OPC administration was 12 h before each LC/LB administrations and due to its very short half-life, limited plasma data was available for OPC. Therefore, no PK parameters were calculated for Study 2. Table 4 Mean (coefficient of variation, %) plasma pharmacokinetic parameters of opicapone (OPC), on Days 21 and 28, following once-daily oral administrations of OPC (5, 15 and 30 mg) or placebo in Study 1 C max (ng ml 1 ) t max (h) AUC 0 t (nghml 1 ) AUC 0 (ng h ml 1 ) t 1/2 (h) Day 21 Levodopa/carbidopa Placebo (n = 14) NA NA NA NA NA 5mgOPC(n = 13) 75.0 (52.1) 1.5 ( ) 223 (26.1) 328 (33.9) 1.92 (59.1) 15 mg OPC (n = 13) 263 (31.3) 3.0 ( ) 872 (47.3) 1000 (49.0) 1.49 (35.1) 30 mg OPC (n = 12) 310 (37.1) 4.0 ( ) 1101 (47.7) 1258 (40.8) 1.39 (31.6) Day 28 Levodopa/benserazide Placebo (n = 14) NA NA NA NC NC 5mgOPC(n = 13) 95.5 (43.0) 3.0 ( ) 232 (35.5) 381 (21.3) 1.88 (55.3) 15 mg OPC (n = 13) 281 (54.6) 2.5 ( ) 836 (59.5) 918 (66.1) 1.39 (38.1) 30 mg OPC (n = 12) 370 (49.1) 3.0 ( ) 1185 (47.4) 1224 (46.1) 1.28 (46.2) C max, maximum observed plasma concentration; t max, time taken to reach C max (t max values are median with range values in parentheses); t 1/2, terminal plasma half-life; AUC 0 t, area under the concentration-time curve (AUC) from prior to each LC/LB dose to the last sampling time at which concentrations were at or above the limit of quantification; AUC 0, AUC from prior to each LC/LB dose to infinity; NA, not available; NC, not calculated Br J Clin Pharmacol (2017)

9 J.-F. Rocha et al. BIA In Study 1, there was no plasma determination of BIA Therefore, no PK parameters were calculated for BIA in Study 1. Systemic exposure to BIA in Study 2 increased in a dose-dependent manner and, apparently, by visual inspection, the steady-state is reached by Day 18. PK results for BIA were consistent with those obtained in a previous study [24]. Levodopa. The plasma levodopa concentration time profiles on each LC and LB dosing day are depicted, respectively, in Figures 2A and 2B for Study 1, and in Figures 2C and 2D for Study 2. Table 5 presents the main PK parameters of plasma levodopa. Statistical comparisons of OPC in relation to control group (placebo) are also provided in Table 5. The levodopa C max was usually similar following OPC when compared to placebo, namely when considering the known variability of levodopa absorption [36]. All OPC treatments, in relation to the placebo group, presented a higher extent of exposure (AUC) to levodopa following either LC or LB doses (Table 5). A relevant increase, although not dose-dependent in Study 1, in the levodopa AUC occurred with all OPC dose groups in relation to placebo. This is in line with the observed increase of up to 2 h of the levodopa t 1/2 with OPC when compared to the placebo group. Of note, plasma levodopa C max and AUC were considerably higher after LB compared with LC in both studies (Table 5). 3-OMD. The plasma 3-OMD concentration time profiles on each LC and LB dosing day are depicted, respectively, in Figures 3A and 3B for Study 1, and in Figures 3C and 3D for Figure 2 Mean plasma levodopa concentration-time profiles following once-daily oral administrations of opicapone (OPC) or placebo on (A) Day 21 or (B) Day 28 (levodopa/carbidopa), and on (C) Day 11 or (D) Day 18 (levodopa/benserazide; n =12forStudy1andn =18forStudy2).Thearrow indicates the time of levodopa administration (~9:00 am in Study 1 and Study 2) 548 Br J Clin Pharmacol (2017)

10 Opicapone effect on levodopa PK Table 5 Mean (coefficient of variation, %) plasma pharmacokinetic parameters of levodopa, including ANOVA results, on Days 21 and 28 (Study 1) or Days 11 and 18 (Study 2), following once-daily oral administrations of opicapone (OPC) or placebo C max (ng ml 1 ) t max (h) AUC 0 t (ng h ml 1 ) AUC 0 (nghml 1 ) t 1/2 (h) Study1 Study 2 Day21 Levodopa/carbidopa Placebo 985 (29.4) 0.5 ( ) 1837 (32.3) 1932 (30.2) 1.71 (23.3) 5 mg OPC 1245 (42.1) 0.75 ( ) 3386 (42.8) 3535 (40.9) 2.19 (16.9) 15 mg OPC 1200 (50.6) 0.5 ( ) 2952 (34.0) 3121 (31.0) 2.75 (18.1) 30 mg OPC 944 (27.1) 1.0 ( ) 2753 (32.2) 2909 (29.6) 2.88 (23.5) Day 28 Levodopa/benserazide Placebo 1704 (40.0) 1.0 ( ) 2438 (29.2) 2518 (28.3) 1.59 (24.5) 5 mg OPC 2100 (43.2) 1.0 ( ) 4115 (37.6) 4253 (36.3) 2.45 (34.0) 15 mg OPC 1727 (55.4) 1.0 ( ) 3442 (35.6) 3608 (33.1) 2.97 (23.8) 30 mg OPC 1795 (43.9) 1.0 ( ) 4056 (25.9) 4242 (23.8) 3.10 (32.4) Day 11 Levodopa/carbidopa Placebo (32) 0.5 (0.5 2) (26) (25) 1.4 (16) 5 mg OPC (43) 0.75 (0.5 4) (28) (27) 1.9 (17) 15 mg OPC (32) 0.5 (0.5 2) (31) (28) 2.3 (18) 50 mg OPC (28) 1.0 (0.5 2) (27) (26) 2.7 (16) Day 18 Levodopa/benserazide Placebo (42) 0.75 (0.5 2) (32) (31) 1.5 (47) 5 mg OPC (44) 1.0 (0.5 2) (23) (22) 1.9 (31) 15 mg OPC (27) 0.5 ( ) (16) (15) 2.2 (35) 50 mg OPC (45) 1.0 (0.5 3) (36) (35) 2.5 (19) C max %(90%CI) AUC 0 t %(90%CI) AUC 0 % (90%CI) Study 1 Day 21 comparison 5 mg OPC/placebo 123 (98; 155) 178 (140; 227) 177 (141; 222) 15 mg OPC/placebo 115 (92; 145) 165 (126; 205) 162 (129; 203) 30 mg OPC/placebo 90 (76; 121) 149 (117; 190) 150 (120; 188) Day 28 comparison 5 mg OPC/placebo 125 (90; 173) 167 (134; 208) 167 (136; 206) 15 mg OPC/placebo 97 (70; 134) 139 (112; 174) 142 (115; 175) 30 mg OPC/placebo 109 (79; 151) 170 (136; 212) 172 (136; 212) Study 2 Day 11 comparison 5 mg OPC/placebo 103 (85; 125) 124 (106; 144) 125 (108; 145) 15 mg OPC/placebo 113 (94; 135) 143 (122; 168) 146 (126; 170) 50 mg OPC/placebo 106 (90; 126) 165 (139; 195) 170 (144; 199) Day 18 comparison 5 mg OPC/placebo 78 (60; 101) 114 (98; 134) 115 (98; 134) 15 mg OPC/placebo 127 (103; 157) 160 (139; 184) 160 (140; 184) 50 mg OPC/placebo 102 (78; 132) 165 (136; 201) 166 (137; 201) C max, maximum observed plasma concentration; t max, time taken to reach C max (t max values are median with range values in parentheses); t 1/2, terminal plasma half-life; AUC 0 t, area under the concentration-time curve (AUC) from prior to each LC/LB dose to the last sampling time at which concentrations were at or above the limit of quantification; AUC 0, AUC from prior to each LC/LB dose to infinity; CI, confidence interval Study 2. Table 6 presents the main PK parameters of plasma 3-OMD. Statistical comparisons of OPC in relation to control group (placebo) are also provided in Table 6. All OPC treatments, in relation to the placebo group, resulted in marked decreases in the extent of exposure (AUC) to 3-OMD following either LC or LB doses (Table 6). Furthermore, OPC decreased 3-OMD C max and AUC in a dose-dependent manner. Tolerability The tolerability profile of all treatments in both studies was favourable. Overall, in Study 1, a total of 135 treatment emergent (TEAEs) were reported by 40 (76.9%) subjects with no dose-effect relationship. The majority of TEAEs were of mild intensity. Majority (111) of TEAEs were considered at least possibly related to the study products. Among these 111 TEAEs, following the treatment groups of placebo, 5, 15 mg Br J Clin Pharmacol (2017)

11 J.-F. Rocha et al. Figure 3 Mean plasma 3-O-methyldopa concentration-time profiles following once-daily oral administrations of opicapone (OPC) or placebo on (A) Day 21 or (B) Day 28 (levodopa/carbidopa), and on (C) Day 11 or (D) Day 18 (levodopa/benserazide; n =12forStudy1andn =18forStudy2). The arrow indicates the time of levodopa administration (~9:00 am in Study 1 and Study 2) and 30 mg OPC, the most frequent adverse events were somnolence (29 cases reported by 19 [36.5%] subjects: seven cases by five [35.7%], five cases by three [23.1%], nine cases by five [38.5%] and eight cases by six [50.0%] subjects, respectively) and headache (10 cases reported by seven [13.5%] subjects: three cases by two [14.3%], one case by one [7.7%], one case by one [7.7%] and five cases by three [25.0%] subjects, respectively). Four subjects were withdrawn from study participation. One subject was discontinued (Day 17 of the 15 mg OPC group) by the Principal Investigator due to an AE (darker skin below the eyes followed by photosensitivity), which was considered possibly related to treatment. Two subjects withdrew their consent due to personal reasons. One subject was discontinued (Day 17 of the placebo group) by the Principal Investigator due to a protocol violation (took penicillin due to an AE [tonsillitis] prescribed by the Principal Investigator). One subject presented a clinical significant increase in plasma potassium levels at the follow-up visit. This potassium increase, which was considered possibly related to treatment by the investigator, was resolved without any sequelae. This subject already presented a clinically relevant abnormal plasma potassium levels at screening; a subsquent test was performed 5 days after and the result was normal (4.7 meq l 1 ). One subject presented clinically relevant aspartate aminotransferase and alanine aminotransferase levels (175 and 135 IU l 1, respectively) at discharge of the treatment period. These increases, which were considered possibly related to treatment by the investigator, were resolved without any sequelae at the follow-up visit. There were no serious AEs or deaths during this study. Neither trends nor relevant changes from baseline were observed in vital signs or ECG parameters. In Study 2, a total of 44 AEs were reported by 29 (39.2%) subjects. Among these, 41 AEs were considered treatment emergent (TEAEs) with no dose-effect relationship. The majority of TEAEs were of mild intensity. Twenty-four (24) TEAEs were considered at least possibly related to the study products. Among these 41 TEAEs, the most common were 550 Br J Clin Pharmacol (2017)

12 Opicapone effect on levodopa PK Table 6 Mean (coefficient of variation, %) plasma PK parameters of 3-O-methyldopa, including ANOVA results, on Days 21 and 28 (Study 1) or Days 11 and 18 (Study 2), following once-daily oral administrations of OPC or placebo C max (ng ml 1 ) t max (h) AUC 0 t (ng h ml 1 ) AUC 0 (ng h ml 1 ) t 1/2 (h) Study1 Study 2 Day21 Levodopa/carbidopa Placebo 456 (28.6) 4.0 ( ) 7631 (23.4) (21.4) 15.6 (24.5) 5 mg OPC 307 (34.4) 8.0 ( ) 5147 (29.8) 7942 (26.2) 14.2 (16.9) 15 mg OPC 167 (49.4) 6.0 ( ) 2836 (52.7) 4499 (56.4) 13.9 (28.3) 30 mg OPC 115 (47.4) 8.0 ( ) 1751 (56.4) 2571 (50.6) 11.7 (22.3) Day 28 Levodopa/benserazide Placebo 688 (33.4) 4.0 ( ) (32.6) (29.6) 15.4 (20.0) 5 mg OPC 360 (26.7) 8.0 ( ) 6205 (23.5) (26.0) 15.9 (29.9) 15 mg OPC 206 (53.8) 6.0 ( ) 3473 (56.5) 5575 (65.0) 14.6 (29.4) 30 mg OPC 160 (38.7) 4.0 ( ) 2623 (40.6) 3915 (45.4) 13.3 (22.2) Day 11 Levodopa/carbidopa Placebo (33) 6.0 (3 6) (26) NC NC 5 mg OPC (25) 6.0 (4 8) (25) NC NC 15 mg OPC (33) 6.0 (4 8) (30) NC NC 50 mg OPC 88.2 (29) 6.0 (4 12) (35) NC NC Day 18 Levodopa/benserazide Placebo 644 (30) 4.0 (1 6) (30) (40) 13.8 (33) 5 mg OPC (26) 6.0 (3 8) (27) NC NC 15 mg OPC (25) 6.0 (3 8) (25) NC NC 50 mg OPC (55) 6.0 (3 12) (38) NC NC C max %(90%CI) AUC 0 t %(90%CI) AUC 0 % (90%CI) Study 1 Study 2 Day 21 comparison 5 mg OPC/placebo 67 (51; 88) 67 (49; 90) 67 (51; 88) 15 mg OPC/placebo 34 (26; 45) 34 (25; 45) 34 (26; 45) 30 mg OPC/placebo 24 (18; 32) 20 (15; 27) 24 (18; 32) Day 28 comparison 5 mg OPC/placebo 54 (41; 71) 56 (43; 74) 54 (41; 71) 15 mg OPC/placebo 28 (21; 37) 28 (21; 37) 28 (21; 37) 30 mg OPC/placebo 23 (18; 31) 23 (17; 30) 23 (18; 31) Day 11 comparison 5 mg OPC/placebo 62 (53; 72) 61 (53; 70) 15 mg OPC/placebo 35 (29; 42) 35 (30; 41) 50 mg OPC/placebo 19 (16; 23) 17 (14; 22) Day 18 comparison 5 mg OPC/placebo 46 (39; 53) 44 (38; 52) 15 mg OPC/placebo 29 (25; 34) 30 (25; 35) 50 mg OPC/placebo 17 (13; 21) 16 (13; 19) OPC, opicapone; C max, maximum observed plasma concentration; t max, time taken to reach C max (t max values are median with range values in parentheses); t 1/2, terminal plasma half-life; AUC 0 t, area under the concentration time curve (AUC) from prior to each LC/LB dose to the last sampling time at which concentrations were at or above the limit of quantification; AUC 0, AUC from prior to each LC/LB dose to infinity; CI, confidence interval headaches (eight TEAEs reported by eight subjects), nauseas (five TEAEs by five subjects), back pains (two TEAEs by two subjects) and diarrhoea (two TEAEs by two subjects). Twenty-four TEAEs were considered at least possibly related to study drug: four in the placebo group (one palpitation, one disturbance in attention, one blepharospasm and 1 headache), eight in 5 mg OPC (two diarrhoeas, one vomiting, four headaches and one somnolence), five in 15 mg OPC (four nauseas and one paraesthesia), and seven in 50 mg OPC (one asthenia, one headache, one insomnia, one nausea, one dizziness, one pruritus and one orthostatic hypotension). One subject (5 mg OPC group, on Day 4) withdrew the consent due to personal reasons and another (15 mg OPCgroup,onDay16)waswithdrawnduetoanAE(joint sprain). There was one serious AE (facial paralysis), which occurred 4 days after last investigational medicinal product Br J Clin Pharmacol (2017)

13 J.-F. Rocha et al. (IMP) administration (i.e. LB under placebo) and who had already experienced such an event 12 years ago. Neither trends nor relevant changes from baseline were observed in vital signs, ECG parameters, physical examination, or in any laboratory parameters assessed. Discussion The purpose of these studies was to compare the effect of OPC vs. placebo upon COMT activity and the PK profiles of a single oral dose of LC or LB following either a once-daily OPC morning or evening regimen apart at least 1 h from levodopa therapy. In both studies, the extent of systemic exposure to levodopa,asmeasuredbyauc,insubjectswhoreceivedopc was considerably greater than that in placebo-treated individuals.instudy1,theincreaseinlevodopaaucwassignificant for both LC and LB at all OPC doses, and significant for LC at all OPC doses but only at both OPC 15 and 50 mg for LB in Study 2. Moreover, this increase it was not dose related in Study 1, and this apparent lack of correlation between dose level of OPC and the increase in levodopa AUC has been previously observed in our studies (data on file). This interaction was also reported for other COMT inhibitors and explanations that have been considered include degradation of levodopa by other metabolic pathways, while inhibition of COMT becomes more marked [37], and/or competition between the COMT inhibitors and levodopa for the saturable transport of large neutral amino acids at the proximal small intestine level [38 40]. Worth mentioning is that, plasma levodopa C max and AUC were considerably higher after LC compared with LC in both studies. This finding has been previously observed in our studies (data on file) and recently reported by others [41]. When compared to placebo, OPC increases the levodopa extent of exposure (AUC), accompanied by a prolonged half-life (t 1/2 ), which is believed to be associated with its pronounced, long-lasting and sustained COMT inhibition. Peak (E max ) and extent (AUEC) of S-COMT inhibition with OPC followed a dose-dependent manner, although this was not entirely proportional to the dose administered. These observations fit well with the findings on the dosedependent effects upon 3-OMD availability and exposure. In fact, the formation of 3-OMD from levodopa is dependent on the COMT activity, particularly at the intestinal level, the main site of O-methylation of levodopa. As previously reported, dose-proportionality in C max and AUC was observed for OPC [23, 24]. OPC shows a long duration of COMT inhibition in human erythrocytes. Despite the low plasma exposure and short clearance half-life of the inhibitor, the levels of erythrocyte COMT inhibition are sustained far beyond the observable point of clearance of circulating drug, which is due to the long residence time of the reversible COMT-OPC complex [23, 24]. The tolerability profile was favourable and similar between placebo and OPC. Studies in healthy subjects with concomitant administrations of OPC and levodopa/aadci (either carbidopa or benserazide) demonstrated that OPC was well tolerated, inhibited the COMT in a potent manner, but showed a certain degree of interaction during the absorption phase between levodopa/aadci and OPC (data on file). On the basis of the results obtained in these studies, evening OPC regimens, potentially a once-daily bedtime administration 1 h after the last daily levodopa/aadci, are considered to provide higher flexibility to the daily levodopa adjustment by enhancing levodopa s optimization and avoiding any interaction during the levodopa absorption phase. This interaction between levodopa/aadci and OPC during the absorption phase was in fact found to be minimized when both administrations were separated by 1 h; this is also supported by evidence on the PK characteristics observed for levodopa and OPC, namely the half-life of the former (~1.5 h) and the t max for the latter ( h). In conclusion, OPC, as once-daily oral evening regimen and 1 h apart from levodopa therapy, increases the bioavailability of levodopa associated with its pronounced, longlasting and sustained COMT inhibition. Competing Interests All authors have completed the Unified Competing Interest form at (available on request from the corresponding author) and declare: J.F.R., A.S., A.I.L., R.P., M.J.B., T.N., L.A. and P.S.S. were employees of BIAL Portela & Cª, S.A. in the previous 3 years; A.F. has received grants from BIAL Portela & Cª S.A. in the previous 3 years; E.S. and N.F. were principal investigators at each clinical research organization subcontracted by BIAL to conducted the study. BIAL Portela&Cª,S.A.supportedthisstudy. References 1 Southan C, Sharman JL, Benson HE, Faccenda E, Pawson AJ, Alexander SP, et al. The IUPHAR/BPS Guide to PHARMACOLOGY in 2016: towards curated quantitative interactions between 1300 protein targets and 6000 ligands. Nucl Acids Res 2016; 44: D1054 D Alexander SPH, Fabbro D, Kelly E, Marrion N, Peters JA, Benson HE, et al. TheConciseGuidetoPHARMACOLOGY2015/16: Enzymes. Br J Pharmacol 2015; 172: Morgan JC, Sethi KD. Emerging drugs for Parkinson s disease. Expert Opin Emerg Drugs 2006; 11: SchapiraAH,EmreM,JennerP,PoeweW.Levodopainthe treatment of Parkinson s disease. Eur J Neurol 2009; 16: Fahn S, Poewe W. Levodopa: 50 years of a revolutionary drug for Parkinson disease. Mov Disord 2015; 30: Poewe W, Antonini A. Novel formulations and modes of delivery of levodopa. Mov Disord 2015; 30: Dingemanse J. Issues important for rational COMT inhibition. Neurology 2000; 55: S24 S27. 8 Palma PN, Bonifácio MJ, Almeida L, Soares-Da-Silva P. Restoring dopamine levels. In: Protein Misfolding in Neurodegenerative Diseases: Mechanisms and Therapeutic Strategies, eds Smith HJ, Simons C, Sewell RDE. Boca Raton: CRC Press, 2007; Gomes P, Soares-da-Silva P. Interaction between L-DOPA and 3-Omethyl-L-DOPA for transport in immortalised rat capillary cerebral endothelial cells. Neuropharmacology 1999; 38: Br J Clin Pharmacol (2017)