Pharmacokinetic Evaluation of 4b
The pharmacokinetic study was conducted at Charles River USA. Dose formulation preparation. All dose formulation procedures were conducted under yellow lighting conditions to protect 4b from light. For the subcutaneous dose group, 2.504 mg 4b was weighed, and transferred into a formulation container. NMP (0.05 mL) was added and the contents were vortexed until 4b was completely dissolved. With continuous vortexing after each addition, PEG400 (0.45 mL), Solutol HS-15 (0.25 mL) and finally 1% Lutrol F68 in RODI water (1.75 mL) followed by sonication for 6 min. The formulation was filtered into a sterile serum vial using a 0.22 mm Millex GV PVDF filter. The resultant formulation was a clear colorless solution. For the oral dose group, 4b was weighed (21.138 mg) and transferred to a formulation container. The prepared vehicle (0.5% CMC, 2.11 mL) was transferred into the formulation container, and the contents were stirred and sonicated for a total of 20 min. The resultant formulation was a white homogenous suspension. For sc formulation, quadruplicate retention samples (100 mL each) were collected and placed in polypropylene vials postfiltration. Triplicate samples were stored at 22uC 65uC, protected from light, For po formulation, triplicate retention samples (100 mL each) were collected (1 per strata; top, middle, and bottom) from the dose formulation after vortexing. The retention samples were placed into polypropylene vials and stored at 22uC 65uC, protected from light. The retention samples were analyzed 10 days following preparation. Blood collection and plasma preparation. Whole blood samples (225 mL each ,90 mL plasma) were collected at each time point per animal into tubes containing K2EDTA. Samples were held on wet ice prior to centrifugation. Mice were bled twice; the first bleed was made via the submandibular facial vein, the second bleed was a terminal collection via cardiac puncture. Whole blood was centrifuged at 2200 6 g for 10 min at 5uC 63uC to separate the plasma. The entire volume of the plasma sample was then transferred to individual tubes contained in a 96-well Matrix plate kept chilled in icy water through the entire process until storage at 270uC 610uC. Collected plasma samples were stored at 270uC 610uC until transferred to the analytical laboratory frozen on dry ice for bioanalysis. Tissue collection and preparation. The brain of each animal was collected following CO2 euthanasia and terminal bleeding. Each brain was rinsed with saline once removed from the body (to flush away any blood that may have been present), snap frozen in liquid nitrogen, and stored individually in 15 mL conical tubes at 270uC 610uC.Preparation of brain homogenate prior to bioanalysis. Prior to extraction, acetonitrile: water (3:1, v/v)Metabolite IdentificationIncubations (n = 2) of test compounds (1 mM initial concentration; 0.5 mg protein/mL) or DMSO control were performed in mouse liver microsomes with and without NADPH, as described above. Aliquots were taken for at 0 and 60 min, added to an equal volume of acetonitrile and submitted for analysis. All incubations (including DMSO control incubations) were analysed by LC-MS.
Instrument Conditions
Analytes were separated by UPLC and analyzed by mass spectrometry. Chromatographic separation was achieved with a Waters Acquity UPLC BEH C18 column (2.1650 mm61.7 mm), the injection volume was 2 mL and the flow rate was 0.6 mL/min. Mobile Phase B was held isocratic at 5% B for 0.2 min, and increased linearly to 95% in 1.2 min. The column was washed with 95% B for 0.6 min, and equilibrated to starting conditions (5% B), for 0.2 min. Mobile Phases were A (0.01% formic acid in water) and B (0.01% formic acid in methanol). A Waters XevoTQ mass spectrometer (Waters Ltd, Centennial Park, Hertfordshire) was used for metabolite identification, with an electrospray source at following settings: capillary voltage = 3.5 kV, cone voltage = 35 V, extractor voltage = 1.6 V, source temperature = 150uC, desolvation gas temperature = 500uC, desolvation gas flow = 1000 L/h, cone gas flow = 100 L/h and collision gas flow = 0.15 mL/min.
Analysis
Incubation extracts were scanned over a mass range of 50 to 1000 amu in both positive and negative ionisation modes. Mass chromatograms were generated for ions observed in the extracts from incubated compounds relative to DMSO controls. In addition, single ion recording (SIR) methods were set up for metabolites thought likely on the basis of the structure of the test compounds. These included likely hydrolysis and oxidation products and also for oxidative metabolic products considered likely based on the compound structures. Where chromatographic peaks of greater intensity than controls were confirmed in the incubated samples, daughter (fragmentation) spectra were obtained and structures proposed for putative metabolites based on the fragmentation patterns when compared to those of parent compounds. Daughter (product) ion scans of putative metabolites were performed using four collision energies (10 eV, 20 eV, 40 eV andwas added to each tissue sample at a tissue: solvent ratio of 1:3 (w/ v) or 1:2.536 (w/w), and homogenized using an OMNI tissue homogenizer. The actual volume of extraction buffer or organic solvent added to each tissue was recorded. Samples were kept chilled in icy water through the entire process until processed for 4b extraction. Immediately prior to extraction, tissue homogenates were thoroughly vortexed and aliquoted into 96-well plate(s). The entire process, from tissue homogenization to placing the samples in the tubes for 4b extraction, was conducted within 8 h.
Preparation of external calibration standards to construct a standard curve 4b. primary stock solution wasprepared at 1 mg eq./mL in DMSO. The matrix calibration standards were prepared at 1, 2, 5, 10, 50, 250, 1000, 2500, 5000 and 10000 ng eq./mL in mouse plasma for plasma, and 0.25, 0.5, 1.25, 2.5, 12.5, 62.5, 250, 625, 1250, 2500 ng eq./mL in tissue homogenate for tissue analysis. Plasma samples were quantified against an external calibration curve generated in control plasma from male C57BL/6NCRL mice. Tissue samples were quantified against an external calibration standard curve generated in control brain tissue from male C57BL/6NCRL mice.
