*2.6. Di*ff*erential Scanning Calorimetry (DSC) Measurements*

DSC measurements were recorded using a DSC 2920, (TA Instruments, New Castle, DE, USA). The heating processes investigated for the TBA/HIMO and BA/HIMO cocrystals were performed in a mu ffle furnace (SM-2002) manufactured by Czylok (Jastrzebie Zdroj, Poland). The measurements were carried out in temperature range of 0 ◦C–300 ◦C. The heating rate was 10 K/min. The sample mass was 1.7040 mg for TBA/HIMO and 1.8200 mg for BA/HIMO.

### *2.7. Biological Studies of HIMO, TBA*/*HIMO and BA*/*HIMO*

The cytotoxic impact of the tested compounds on the viability of cells after concentration-dependent treatment was determined by standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Experiments were performed with cancer (HeLa–cervical cancer, K562–chronic myelogenous leukemia) and noncancerous (fibroblasts, 293T–derived from human embryonic kidney) human cell lines. A colorimetric assay was used to measure the cell viability through the metabolism of tetrazolium salt into formazan by mitochondrial dehydrogenase in living cells. In brief, the cells were plated into 96-well transparent plates (Nunc) at a density of 7 × 10<sup>3</sup> cells per well in 200 μL of fresh RPMI or DMEM medium (supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin). After overnight incubation (37 ◦C, 5% CO2), the medium was replaced for the control and the wells containing various concentrations (1 μM, 10 μM, 50 μM and 100 μM) of the tested compounds. After a further 48 h of incubation under the same conditions, 25 μL of MTT solution (5 mg/mL) was added to each well. After a subsequent 2 h of incubation, 95 μL of lysis bu ffer (20% SDS, 50% aqueous DMF, pH 4.5) was added. Afterward, the plates were mixed on a microplate shaker (2 h at room temperature) to dissolve the formazan. Then, the optical density (OD) was measured by a microplate reader at 570 nm with a reference wavelength of 630 nm. Percent cell viability relative to the control was calculated as cell viability (%) = (OD treatment/OD untreated control cells) × 100%. The data represent the mean values from five repeats from three independent experiments.

### *2.8. Solubility Measurements of BA, TBA, TBA*/*HIMO and BA*/*HIMO*

Solubility measurements were performed by preparing supersaturated solutions of the tested cocrystals (BA/HIMO, TBA/HIMO) and pure BA and TBA in Milli-Q water (pH 5.7), simulated gastric fluid without pepsin (SGFsp, pH 1.2) and ethanol. SGFsp was prepared by adding 2 g NaCl and 7 mL concentrated HCl to 1000 mL distilled water. The obtained suspensions were stirred at room temperature for 24 h. After filtration through a 0.45 μm PTFE syringe, the filtrates were diluted with water to obtain an appropriate concentration (in the range of UPLC-MS calibration).

The concentration of BA and TBA was determined by an ACQUITY UPLC I-Class chromatography system coupled with SYNAPT G2-Si mass spectrometer equipped with an electrospray source and quadrupole-Time-of-Flight mass analyzer (Waters Corp., Milford, MA, USA). Acquity BEHTM C18 column (100 × 2.1 mm, 1.7 μm), maintained at 45 ◦C for the chromatographic separation of analyte. A gradient was employed with the mobile phase combining solvent A (1% formic acid in water) and solvent B (1% formic acid in acetonitrile) as follows: 3% B (0–0.5 min), 3–97% B (0.5–1.5 min), 97–97% B (1.5–1.8 min), 97–3% B (1.8–1.9 min) and 3–3% B (1.9–3.5 min). The flow rate was 0.40 mL/min, and the injection volume was 2 μL.

For mass spectrometric detection, the electrospray source operated in negative resolution mode. The optimized source parameters were as follows: capillary voltage 2.8 kV; cone voltage 25 V; desolvation gas flow 900 L/h with a temperature of 450 ◦C; nebulizer gas pressure 6.5 bar; and source temperature 110 ◦C. Mass spectra were recorded over an *m*/*z* range of 100 to 1200. Mass spectrometer conditions were optimized by direct infusion of the standard solution. The system was controlled using the MassLynx software (Version 4.1), and data processing (peak area integration, construction of the calibration curve) was performed using TargetLynxTM (Waters Corp., Milford, MA, USA).

The initial stock calibration solutions of BA and TBA were prepared at a concentration of approximately 10 mg/mL in water and stored at 4 ◦C. The stock solutions were diluted (with water) to obtain the target concentrations. The calibration curves were prepared at seven di fferent concentrations of BA and TBA. The calibration curves were linear over a concentration range from 10 ng/mL to 1500 ng/mL with the correlation coe fficients of >0.994 and 0.992, respectively, for BA and TBA.

The concentrations determined for BA and TBA (in pure form and in the tested cocrystals) were reported as the average of two replicated experiments for each sample. The injections for each sample were repeated four times.
