Materials and Reagents.

Methylphenidate hydrochloride (racemic form) was purchased from Sigma-Aldrich Japan, Inc. (Tokyo, Japan). Cocaine was purchased from Takeda Pharmaceutical Company Limited (Osaka, Japan). Fluorescent-based neurotransmitter transporter uptake assay dye was obtained from Molecular Devices (Sunnyvale, CA). Saline was purchased from Otsuka Pharmaceuticals (Tokyo, Japan). All other reagents and solvents were commercial products of reagent grade.

Animals.

Animal care and all experimental procedures were performed with the approval of the Institutional Animal Care and Use Committee at Shionogi & Co., Ltd., in terms of the replacement/reduction/refinement principles. Male Crlj:WI rats were purchased at 5 weeks of age from Charles River Laboratories Japan, Inc. (Yokohama, Japan). After quarantine for a week, the rats were acclimated for several days in the animal compartment. The rats were used for the experiments at 6 weeks of age. During the acclimation and experimental periods, the rats were placed under the conditions of room temperature at 20–26°C, relative humidity at 30%–70%, and lighting for 12 hours [light (8:00 AM to 8:00 PM)/dark (8:00 Pm to 8:00 AM)], and allowed free access to tap water and solid laboratory food (CE-2; CLEA Japan, Inc.).

Fluorescence-Based Uptake Study.

Methylphenidate was dissolved in assay buffer (Hanks’ balanced salt solution containing 20 mM HEPES, 0.1% bovine serum albumin, pH 7.3) to prepare the methylphenidate solution at 0.0781–2.50 μM. Neurotransmitter assay loading dye reagent (DAT substrate) was dissolved in Hanks’ balanced salt solution containing 20 mM HEPES to prepare the loading buffer. Human embryonic kidney 293 cells stably transfected with human recombinant DAT were seeded at a density of 1 × 10⁵ cells/ml (50 µl/well) in 384-well, clear-bottomed, black-walled plates (Greiner Bio-One, Frickenhausen, Germany) and allowed to proliferate overnight. On the day of the experiment, the culture medium was aspirated from the cell plate using a plate washer followed by addition of 20 µl assay buffer. After the plate was placed in the fluorometric imaging plate reader, the cells were incubated at room temperature for 5 minutes to capture the fluorescence at baseline. Next, both 10 µl of loading buffer and 10 µl of methylphenidate solution were added to the cells cultured on a 384-well plate, and the cells were incubated at room temperature for 90 minutes. During the incubation, fluorescence was measured every 3 seconds for the first 3 minutes and every 10 seconds for the residual 87 minutes (total points were 580 per concentration). To measure fluorescence, the dye was excited using light at a wavelength of 470–495 nm, and emission was collected at 515–575 nm. The same study was performed with cocaine (0.313 and 0.625 μM), which has the potential of DAT inhibition (Gatley et al., 1996), to support the validity of our mathematical model. The fluorescence strengths represented the mean of duplicated data.

Pharmacokinetic Study.

Rats were anesthetized with isoflurane, and a cannula was inserted into the jugular vein 4 days before the pharmacokinetic study. Methylphenidate dissolved in saline was intraperitoneally administered to conscious rats (n = 3–5/dose) at doses of 1, 3, and 6 mg/2 ml/kg using a 1-ml syringe with a 25-gauge needle. Blood (approximately 0.2 ml) was collected from the inserted cannula using a 1-ml syringe with a 23-gauge needle containing heparin and EDTA-2K at 0.033, 0.083, 0.25, 0.5, 1, 2, and 4 hours after administration. To other rats, methylphenidate solution was intraperitoneally administered (n = 3 per dose) at doses of 1, 3, and 6 mg/2 ml/kg to measure the brain and cerebrospinal fluid (CSF) concentrations of methylphenidate. The concentrations in brain were evaluated for brain penetration and the concentrations in CSF were evaluated for unbound concentration in brain. CSF was collected by inserting a 23-gauge needle connected to a syringe through a polyethylene tube into the cisterna magna at 30 minutes after collection of whole blood via the inferior vena cava. The brain was also collected from the same rats. The collected whole blood was centrifuged at 1600g for 10 minutes at 4°C to obtain plasma. The polyethylene tube for CSF sampling was washed with an equal amount of acetonitrile to prevent adsorption of the compound to the tube. Water was added to the brain samples at a ratio of brain/water = 1/3 (v/v), and the samples were homogenized. The obtained plasma, CSF, and brain samples were frozen and stored at −30°C until analysis. Methylphenidate in plasma and brain samples was extracted with acetonitrile and centrifugal separation (5000 rpm for 5 minutes at 8°C), and each supernatant was injected into a high-performance liquid chromatography–tandem mass spectrometry system using API4000 (SCIEX, Foster City, CA). The range of calibration standards was 0.5–5000 ng/ml for plasma and brain samples and 0.15–1500 ng/ml for CSF samples.

In Vivo Microdialysis Study of Dopamine Release in Nucleus Accumbens.

Rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and butorphanol (5 mg/kg, s.c.), and a dialysis probe (EicomCorp., Kyoto, Japan) with a guide cannula was stereotaxically implanted at the NAc shell of each rat (Anterior +1.8 mm, Lateral +0.8 mm, Ventral +6.2 mm, from the bregma and skull) (Paxinos and Watson, 1986). The cannula was cemented in place with dental acrylic, and the animal was kept warm and allowed to recover from anesthesia. Postoperative analgesia was performed by a single injection of buprenorphine (0.02 mg/kg, i.p.) (Ago et al., 2006; Sato et al., 2007). The active probe membranes were 2 mm long. Two or three days after surgery, the probe was perfused with Ringer’s solution (147.2 mM NaCl, 4.0 mM KCl, and 2.2 mM CaCl₂; pH 6.0; Fuso Pharmaceutical Industries, Ltd., Osaka, Japan) at a constant flow rate of 2 μl/min and stabilized for 5 hours. Next, after methylphenidate solution had been intraperitoneally administered to conscious rats (n = 4 per dose) at doses of 1, 3, and 6 mg/2 ml/kg, microdialysis samples (12 μl) were collected every 6 minutes over 240 minutes and immediately injected to high-performance liquid chromatography analysis to determine the dopamine level, as previously reported (Koda et al., 2010; Ago et al., 2011). After the experiments, Evans Blue dye was microinjected through the cannula to histologically verify the position of the probe. Only data from animals with the correct probe placements were used for the analysis.

In Vitro Mathematical Model.

Figure 1 presents the scheme of the in vitro kinetic assay. The concentrations of fluorescent substrates (C_sub) and fluorescence strength (C_flu) profiles were described using the following equations:(1)(2)(3)(4)where, k_on′ is the permeability rate constant of the fluorescent substrates; DAT_free is the unbound fraction of DAT; DAT_bound is the bound fraction of DAT by the DAT inhibitor; k_on and k_off are the association and dissociation rate constants of the DAT inhibitor, respectively; and C_inh is the concentration of DAT inhibitor (micromolars). The initial values of DAT_free and DAT_bound are 1 and 0, respectively.

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Fig. 1.

Scheme of the in vitro study. The experiment in the absence (a) or presence (b) of methylphenidate. To observe the uptake of the DAT substrate, which is composed of masking dye and fluorophore, the masking dye is removed and only the fluorophore is transported into the cell. Therefore, the transport activity of the DAT substrate was investigated by measuring the fluorescence captured from the intracellular fluorophore. To measure fluorescence, the fluorophore was excited with light at a wavelength of 470–495 nm, and emission was collected at 515–575 nm. Each parameter is defined as follows: C_inh, concentration of inhibitor; k_off, dissociation rate constant of methylphenidate for DAT (per minute); k′_on permeability rate constant for fluorescent substrate via DAT (per minute); k_on, association rate constant of methylphenidate for DAT (micromolars per minute).

This analysis was based on two hypotheses: 1) fluorescence strength is equal to the concentration of fluorescent substrates (C_sub) that penetrate through DAT and 2) at steady state, all substrates are transported via DAT (C_sub at t = 0 is considered the maximum fluorescence strength).

In the absence of DAT inhibitor (Fig. 1A) DAT_free was set as 1 constantly because there was no inhibitor for DAT, since fluorescent substrates mainly penetrate via all DATs into the cells and produce fluorescence. Therefore, C_sub and C_flu profiles were described by the eqs. 1 and 2, and both C_sub and k′_on were estimated by a fitting approach based on the observed fluorescence strength profiles.

In the presence of DAT inhibitor (Fig. 1B), fluorescent substrates penetrate via only the free DAT into the cells and produce fluorescence. Therefore, the changes in C_sub, C_flu, and DAT occupancy were described by the eqs. 1–4. The k_on and k_off values of the DAT inhibitor were estimated by a fitting approach based on the observed fluorescence strength profiles.

Mechanism-Based PK-PD Model.

Figure 2 presents the structure of the mechanism-based PK-PD model for dopamine levels in rat brain after intraperitoneal administration of methylphenidate. Our PK-PD model was developed based on the following four points: 1) a two-compartment model for plasma concentration of methylphenidate; 2) a compartment model for CSF concentration of methylphenidate attached to the plasma compartment; 3) a component model for DAT occupancy calculated with the concentration in CSF of methylphenidate, k_on, and k_off; and 4) a model incorporating dopamine biosynthesis, release from a synapse, reuptake via free DAT into the synapse, and elimination from the synapse. The concentrations of methylphenidate in plasma and CSF were described by a linear two-compartment model as follows:(5)(6)(7)(8)(9)where A_ip, A_central, and A_peri are the methylphenidate amounts (milligrams per kilogram) in the abdominal cavity, central, and peripheral compartments, respectively; F is the bioavailability; V_d/F is the distribution volume corrected by F of methylphenidate in the central compartment (liters per kilogram); k_a, k₁₂, k₂₁, and k_e are the first-order rate constants of absorption from the injection site, distribution to a peripheral or central compartment, and elimination from the central compartment (per minute), respectively; MW is the molecular weight of methylphenidate (233 g/mol); C_plasma is the concentration of methylphenidate in plasma (micromolars); K_{p_CSF} is the CSF-to-plasma concentration ratio; and C_CSF is the concentration of methylphenidate in CSF.

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Fig. 2.

The structure of the PK-PD model for dopamine levels in the brain after intraperitoneal administration of methylphenidate. Each parameter is defined as follows: Central, central compartment; CSF, cerebrospinal flow compartment; DAT, DAT occupancy compartment; Ip, administration compartment; k₁₂, first-order rate constant from the plasma to peripheral compartment; k₂₁, first-order rate constant from the peripheral to plasma compartment; k_a, absorption rate constant; k_deg, degradation rate constant of dopamine in the synapse; k_e, elimination rate constant from the central compartment; k_release, first-order rate constant for dopamine release via DAT; k_syn, synthesis rate constant of dopamine in the synapse; k_uptake, first-order rate constant for dopamine reuptake from the synapse; Periph, peripheral compartment.

To analyze the dopamine levels in the brain, DAT occupancy is described by eq. 10 and the dopamine levels in a synapse are described by eqs. 11 and 12:(10)(11)(12)where DAT_RO is the fraction of DAT occupied by methylphenidate in the brain; k_on and k_off are the in vitro association and dissociation rate constants of methylphenidate for DAT, respectively; DA_pre and DA_post are the dopamine levels in the presynapse and extracellular space. Here, DA_{pre_0} and DA_{post_0}, which are the initial values of DA_pre and DA_post, are DA_{post_0} × k_uptake/k_release (= k_uptake/k_release) and 1 (100%), respectively; k_syn is the zero-order rate constant for dopamine biosynthesis, where k_syn = k_deg × DA_{pre_0}; k_release, k_uptake, and k_deg are the first-order rate constants for dopamine release from the presynapse to the extracellular space, dopamine reuptake from extracellular space to the synapse, and degradation from the synapse, respectively; and γ is the Hill coefficient.

Data Analysis.

Pharmacokinetic analysis for the concentration of methylphenidate in plasma was performed based on a noncompartment model with uniform weighting. The area under the plasma concentration-time curve from time zero to infinity (AUC_0–inf) was calculated by the trapezoidal rule. The other pharmacokinetic parameters were as follows: the maximum plasma concentration (C_max) and time to reach the maximum plasma concentration. Pharmacodynamic analysis of dopamine level per time was also performed based on the noncompartment model with uniform weighting. The change in the area under the effect-time curve for the dopamine level from time zero to the last time was calculated by the trapezoidal rule. The maximum dopamine level and time to reach the maximum dopamine level were also calculated.

The k_on and k_off values of the DAT inhibitors were estimated based on the mean data of duplication. The model-dependent pharmacokinetic and PK-PD parameters were estimated by individual data for all doses to obtain the unique estimates for all parameters. For the process of PK-PD modeling, a sequential pharmacokinetic and pharmacodynamic modeling approach was applied, i.e., first the pharmacokinetic modeling was conducted, and then PK-PD modeling was performed using pharmacokinetic parameters estimated from the pharmacokinetic model to find the mean concentrations of methylphenidate in plasma.

Model-independent pharmacokinetic parameters and all model-dependent parameters were estimated using Phoenix WinNonlin (version 6.2.1; Pharsight Corp.). Model-independent pharmacodynamic parameters were calculated using Microsoft Excel 2010. Dose proportionalities were evaluated using SAS (version 9.2, SAS Institute Inc., Cary, NC) and a power model for ln-transformed C_max and AUC_0–inf.