*2.6. Dissolution*

Dissolution of the formulations was performed using USP dissolution apparatus 2 (Agilent 708-DS, Santa Clara, CA, USA) equipped with an autosampler (Agilent 850- DS Dissolution Sampling Station) in a 900 mL dissolution media (water containing 1 in 20,000 HPC, pH adjusted to 4.5) at 37 ± 0.5 ◦C and 50 rpm. The samples were withdrawn at 30, 60, 90, and 120 min, and the amount of drug dissolved was determined by the validated HPLC method. Dissolution samples were diluted with SLS solution (1%) in a ratio of 9:1 to ensure drug solubility and to prevent crystallization during analysis. The dissolution experiment was performed in triplicate.

### *2.7. Stability*

Short-term stability of CAD formulation F16 (SAIB) and ASD formulation F17 (HPMC) was performed by packing in an HDPE bottle and storing at 25 ◦C/60% RH and 40 ◦C/75% RH for three months and one month, respectively. The samples were examined for physical and chemical changes by dissolution, FTIR, XRPD, and DSC.

### *2.8. Pharmacokinetics*

This study was carried out to compare the pharmacokinetic profiles of HPMC-based ASD formulation (F17), SAIB-based CAD formulation (F16), and a tablet containing the crystalline form of the drug (F18). Four beagle dogs (2 males and 2 females, 10 ± 2 Kg) were used in this study. The study (IACUC 2019-0241) was approved by the Institutional Animal Care and Use Committee (IACUC) of Texas A&M University. The animals were given F16 (SAIB as a carrier), F17 (HPMC as a carrier), or crystalline drug tablets (F18) containing 5 mg TAC with 15 days washout period between the studies. The animals were fasted overnight (midnight to 7 a.m.) before administration of the dose and 2 h post-dosing. The animals had free access to water during fasting and food and water 2 h post-dosing. Lidocaine cream was applied to the catheterization site before catheterization/needle stick and applied at each puncture site. A bitter apple was applied to the catheter to prevent licking and chewing of the catheter, and E-collar was also used to prevent dogs from reaching the catheter. 3 mL of blood was collected at 0, 0.25, 0.5, 1, 2, 3, 4, 6, and 8 h through the cephalic or saphenous catheter. Blood samples at 12, 24, 36, 48, and 72 h were directly withdrawn from the cephalic or saphenous vein. Blood was immediately transferred to a heparinized tube and stored at −80 ◦C until analysis. The protein precipitation method was used to extract the drug from the sample. Whole blood samples (500 μL) were treated with 0.1 M ZnSO4 (50 μL) to break red blood cells, followed by addition of methanol (900 μL) and internal standard (IS) (100 μL TAC-13C3D2, 50 ng/mL). The samples were vortexed for 2 min and centrifuged at 13,300 rpm and 4 ◦C for 15 min. The supernatant was analyzed for TAC and IS by the UPLC-MS method.

### *2.9. High-Performance Liquid Chromatography*

A reported HPLC method was modified and validated for dissolution and assay analysis of the formulations [30]. The HPLC consisted of Agilent 1260 series (Agilent Technologies, Wilmington, DE, USA) equipped with a quaternary pump, online degasser, column heater, autosampler, and UV/Vis detector. Separation of the analyte was achieved on a 4.6 × 150 mm, 3 μm Luna C18 (Phenomenex, Torrance, CA, USA) column and a C18, 4.6 × 2.5 mm (5 μm packing) Luna C18 guard column (Phenomenex, Torrance, CA). The mobile phase was ACN and 0.05 M phosphoric acid (65:35 *v*/*v*) flowing at 1.0 mL/min. The column and auto-sampler were maintained at 60 ◦C. A sample volume of 200 μL was injected into the system and detected at 210 nm. Two injections per sample were analyzed to demonstrate reproducibility of the method. Data was collected and analyzed using OpenLab software (Agilent Technologies, Wilmington, DE, USA).

### *2.10. Ultra-Performance Liquid Chromatography-Mass Spectroscopy*

UPLC was performed on a Waters Acquity® UPLC system (Waters Corporation, Milford, MA, USA) equipped with Agilent InfinityLab Poroshell 120 EC-C18 (4.6 × 50 mm, 2.7 μm) and maintained at 60 ◦C in the column oven. Separation of the analytes was performed using a mobile phase consisting of 5 mM ammonium acetate adjusted to pH 5 with formic acid and 0.1% formic acid (*v*/*v*) in methanol (5:95, *v*/*v*). The flow rate and run time were 0.5 mL/min, and 3 min, respectively. The retention times of TAC and TAC-13C3D2 were 1.405 and 1.402 min, respectively, and the peaks were well separated from the baseline. Mass spectrometry parameters were electrospray positive ionization (ESI+) mode with 0.8 KV capillary voltage and 15V collision energy. The TAC and TAC-13C3D2 molecular masses were detected at 804 and 806 Dalton by the QDa detector. The calibration range was 4-100 ng/mL. Concentrations of 4, 10, 50, and 100 ng/mL TAC were used in precision and accuracy assessment, and met the requirement of ±15% of nominal concentration. The method was validated per FDA bioanalytical method validation guidance [31].
