Solubility measurements

Freeze-dried CFZ-H⁺Cl^− 11 samples were weighed and 25 mg was added to each of five scintillation vials. Mili-Q water (15 mL) was added to each vial to ensure that CFZ-H⁺Cl⁻ crystals were in great excess. An aliquot of 0.1 M NaOH was added to each vial to achieve the initial equilibration pH measurements as follows: sample vials 1–5 initially contained 0, 40, 80, 120, and 200 µL of 0.1 M NaOH solution, respectively. After a 24-hour equilibration period, 10 µL of 0.1 M NaOH was added each day for a period of five days resulting in pH range of 4.5 to 8.9. The sample vials were placed on a magnetic stirrer plate in a 25 °C water bath. Each sample was allowed to equilibrate for at least 24 hours, after which 500 µL of sample was removed and filtered through a Spin-X centrifuge tube filter (0.45 μm cellulose acetate, 2 mL polypropylene tubes, non-sterile, Costar®, Cat # 8163) for 4 min @ 10,000 rpm. The pH of the filtered sample was determined (UltraBasic pH meter, Denver Instrument, Bohemia, NY), after which the sample was subjected to HPLC analysis (see Supplementary Methods) (Waters Alliance, Separations Module 2695) to determine the total solubility of the drug at the measured pH. For each sample, solubility measurement was performed in triplicate, and the average was used to construct the total drug solubility-pH profile. The standard curve was generated using CFZ-H⁺Cl⁻ crystals that were dissolved in the mobile phase at known concentrations (1–50 µM).