*2.1. Materials*

TAC monohydrate >98% and hydroxypropyl cellulose (HPC, MW 100,000) were purchased from Sigma-Aldrich (St Louis, MO, USA). Deuterated TAC-13C3D2 (>85%) and Beagle dog plasma were obtained from Toronto Research Chemicals, Ontario, Canada, and BioChemed Services, Winchester, VA, USA, respectively. BioSustane™ SAIB and microcrystalline cellulose (MCC, Avicel® PH-101) were obtained from Eastman Chemical Company (Kingsport, TN, USA) and FMC Corporation (Princeton, NJ, USA), respectively. Hydroxypropyl methylcellulose (HPMC, 50 cps), magnesium stearate (MGS), croscarmellose sodium (CCS), lactose monohydrate, orthophosphoric acid (OPA), methanol, ammonium acetate, zinc sulfate (ZnSO4) and formic acid were purchased from Fisher Scientific (Asheville, NC, USA). Sodium lauryl sulfate (SLS) was purchased from VWR Chemicals, LLC (Fountain Parkway, OH, USA). All reagents were of analytical grade and used as received. In-house water (18 MΩcm, Millipore Milli-Q Gradient A-10 water purification system) was used in the study.

### *2.2. Preparation of CAD and ASD*

The formulations were prepared by solvent evaporation method as per Table 1. Briefly, the drug and the SAIB or HPMC were dissolved/dispersed in ethanol by sonication, followed by the addition SLS with sonication, and MCC and CCS. Solvents were evaporated under the hood at room temperature with stirring to form a solid mass. The solid mass was crushed and passed through #60 sieve. The physical mixtures (PM) of CAD (F16) were prepared by adding the drug to the placebo formulation. Dried powder equivalent to 5 mg TAC either filled into hard gelatin capsules or mixed with MGS and compressed into tablets using a Mini Press-1 (Globe Pharma, New Brunswick, NJ, USA) 10-station tableting machine with 5-mm concave die and punches (Natoli Engineering Company, Saint Charles, MO, USA). A tablet formulation (F18) compositionally identical to F16 or F17 without SAIB or HPMC, and contained crystalline form of the drug was also prepared for pharmacokinetic study. Various solutions/dispersions of TAC and SAIB were also prepared by heating at 100–120 ◦C. All samples were stored in a desiccator until further analysis.


**Table 1.** Composition of tacrolimus CAD and ASD formulations.

### *2.3. Fourier Transform Infrared Spectroscopy*

The Fourier-transform infrared (FT-IR) spectra of the samples were collected by a modular NicoletTM iS™ 50 system (Thermo Fisher Scientific, Austin, TX, USA). The spectra were obtained in absorbance mode over a wavelength range of 400–4000 cm−<sup>1</sup> with a data resolution of 8 cm−<sup>1</sup> and 100 scans. A small amount of powder was placed on the diamond crystal and pressed with the attached arm to avoid any air entrapment in the sample. OMNIC software, version 9.0 (ThermoFisher Scientific), was used to capture and analyze the spectra.
