The study was conducted in accordance with the Good Clinical Practice guidelines, locally applicable laws and regulations, and ethical principles originating in or derived from the Declaration of Helsinki. The study protocol was developed per the guideline for Bioequivalence Studies of Generic Products , and the study was conducted at Tokyo Heart Center (Tokyo, Japan). The study protocol, its amendments, and the informed consent form were approved by the 124th Institutional Review Board of the study site on December 16, 2015 (Protocol number: SUN Y7017-G-J101). This study was not registered at any registry databases because at the time it was conducted, the registration of healthy subject trials was not required by the Joint Position on the Disclosure of Clinical Trial Information via Clinical Trial Registries and Databases by the International Federation of Pharmaceutical Manufacturers and Associations. Prior to study execution, all subjects provided written informed consent.
Healthy Japanese males aged 20–40 years with 25.0 kg/m2 > BMIs ≥ 18.5 kg/m2 at screening, were eligible for inclusion. The exclusion criteria included: (1) any medical history of central nervous, cardiovascular, respiratory, gastrointestinal, urinary, or blood/hematopoiesis system disorders, or hepatic/renal impairment, thyroid dysfunction, pituitary malfunction, adrenal dysfunction; (2) any clinically significant deviation from the normal range in a physical examination, vital signs (e.g., blood pressure, heart rate, and body temperature), 12-lead electrocardiogram, or clinical laboratory determinations; (3) history of epilepsy or convulsions; (4) incompletely healed oral cavity wound; (5) hypersensitivity or idiosyncratic reactions to any drug; (6) history of alcohol or drug abuse; (7) presence of an infection; (8) previous participation in a memantine hydrochloride clinical study; (9) current or planned receipt of any medical intervention after screening; (10) whole blood collection of > 800 mL within 1 year, > 400 mL within 84 days, or > 200 mL within 28 days, or blood component collection within 14 days of the study; (11) previous clinical trial participation and investigational drug administration within 120 days of the study; (12) current or planned use of other drugs or supplements 14 days prior to hospitalization for the study and until study completion; (13) consumption of grapefruit (juice or pulp) within 7 days prior to hospitalization; (14) not consenting effective contraception use; and (15) investigator-determined ineligibility. Subjects could withdraw from the study at any time after a request.
Two single-dose, randomized, open-label, two-period, two-group, crossover studies were conducted under two dosing conditions: test product [dry syrup, 2%, 1 g (containing 20 mg of memantine hydrochloride); Daiichi Sankyo, Tokyo, Japan] administration as a suspension in water (Study I) and as granules with water (Study II), because the dry syrup will be marketed to be administered in both conditions. Thirty days prior to treatment commencement, subjects were screened for eligibility. Subjects were confined to the study site for 5 days, from the day before treatment commencement (Day −1) until 72-h post-dosing (Day 4). After discharge, the 120- and 192-h post-dosing procedures were conducted during ambulatory visits to the study site on Days 6 and 9. On Day −1 of each treatment period, the study drugs were administered, with a 20- to 24-day washout period between administrations. On Day −1 of the first treatment period of each study, subjects were randomly assigned (1:1 ratio) to either a test–reference or reference-̄test sequence. The follow-up assessment was conducted after 20–24 days of the last administration in the second treatment period.
Randomization was conducted by the investigators in accordance with a randomization schedule, which was created using a computer-generated randomization scheme by the contract research organization.
Treatment and Subject Restrictions
In both studies, subjects fasted overnight (> 10 h) pre administration, and for 4 h post-administration. Water (150 mL) was used for each administration, and beverages were prohibited until 2 h post-dosing. Treatment administration compliance was assessed by a thorough oral cavity examination by the investigators. Standardized meals were served at appropriate times throughout the study. For 14 days before Day −1 of the first treatment period until study completion, the use of other drugs or supplements was prohibited.
Sample Collection and Bioanalytical Methods
Blood samples (5 mL) were collected in vacuum tubes containing the anti-coagulant, sodium heparin, at pre-dosing, and at 1, 3, 4, 5, 6, 8, 12, 24, 48, 72, 120, and 192 h post-dosing in each treatment period of both studies. Plasma was separated by centrifugation and stored at −20 °C until analysis.
Memantine was extracted from 100-µL plasma samples via liquid–liquid extraction with diethyl ether. The organic layer was transferred and then evaporated under a nitrogen stream at approximately 40 °C. The remaining residue was reconstituted with 600 μL of reconstitution solution [0.76:600:400:2 (ammonium formate/water/methanol/formic acid), v/v/v/v]. The final solution was analyzed using a validated liquid chromatography tandem mass spectrometry method. Chromatographic separation was performed using an Inertsil ODS-SP column (2.1 mm I.D. × 50 mm, 3 µm; GL Sciences, Tokyo, Japan). Detection was performed using an API 4000 or 4000 QTRAP (AB SCIEX, Framingham, MA, USA) tandem mass spectrometer with a TurboIonSpray source by electrospray ionization in the positive ion mode, and multiple-reaction monitoring of memantine (m/z 180–163) and its internal standard (memantine-d6, m/z 186–169). The intra-study assay precisions for 1.60, 8.00, and 80.0 ng/mL memantine were 2.6, 1.4, and 0.4%, respectively. Assay accuracy ranged from −3.1 to −2.5%, with a lower limit of quantification of 0.500 ng/mL.
PK parameters were calculated from the plasma concentration–time data of memantine of each subject by a non-compartment analysis using Phoenix WinNonlin (v.6.3; Certara G.K, Tokyo, Japan).
The primary PK parameters were Cmax and area under the concentration–time curve (AUC) up to the last sampling time (AUCall), whereas the secondary PK parameters were the observed time to Cmax (Tmax), T½, and AUC up to infinity (AUCinf).
Logarithmically-transformed values of the primary PK parameters (Cmax and AUCall) were compared for the two dosage formulations, and the test product versus the reference product. A mixed-effect model that included treatment group, treatment period, and formulation as fixed effects, and subject within sequence as random effect, was used for all comparisons. The ratios of geometric least-squares means (GLSM) of the primary PK parameters and their 90% confidence intervals (CIs) were calculated. Subjects whose AUCall could not be properly calculated were excluded from the calculation of GLSM and their 90% CIs. Bioequivalence of the test and reference products was concluded if the 90% CIs of the ratios of GLSM of the primary PK parameters were within the predefined acceptance range of 0.80–1.25. Statistical analyses were performed using SAS® (v.9.2; SAS Institute Japan, Tokyo, Japan).
The sample size for this study was calculated by the reported approach  using previous memantine studies (data on file). The intra-individual geometric coefficient of variations of Cmax and AUCall of memantine were approximately 10%; thus, 18 evaluable subjects were necessary to provide a 90% power to demonstrate bioequivalence of the two dosage formulations in each study. To ensure that we obtained the PK data of ≥ 18 subjects at study procedure completion, 24 subjects were enrolled in each study.
Drug safety was assessed using data of adverse events (AEs), vital signs (body temperature, blood pressure, and pulse rate), body weight, 12-lead electrocardiograms, and laboratory tests (including hematology, serum chemistry, and urinalysis). The intensity, duration, relationship to the study drugs, outcome, and severity of all AEs that occurred after the first administration of study drugs until study completion were recorded.