2.2.2. Solvent-Shift Method

A total of eight candidate polymers were evaluated to test their ability to maintain NTZ solubilized: Methocel ® MC, Methocel ® 60 HG, HPMC, HPC 80,000, HPC 370,000, Kollisolv ®, Kolliphor ® P 407, and Kollidon ® 25 (see Table S1, for further polymer details). The tests were performed by the modified solvent shift method proposed by Childs et al. in 2013 [24], see Figure S1. Aqueous solutions were prepared containing 0.5% *w*/*v* of each pre-dissolved polymer in pH 7.5 phosphate bu ffer solution (PBS) and also one containing only bu ffer solution. A volume of 3 mL of each medium was placed in a

spectrophotometer quartz cell, followed by a stepwise addition of a total of 13 aliquots of 10 μL each from NTZ stock solution in DMSO (25 mg/mL) at 5 min interval times. The ability of the polymeric compounds to maintain the API dissolved was determined by analysis of the presence or absence of precipitated NTZ in solution, which can be monitored by UV-vis spectrophotometry measured at λ = 550 nm.

### 2.2.3. Powder Dissolution under Non-Sink Conditions

For the powder dissolution experiments under non-sink conditions, 200 mg of pure NTZ or the equivalent of NTZ cocrystal were added to 10 mL of pH 7.5 bu ffer dissolution medium. All NTZ samples were passed through a sieve mesh 200.

The dissolution experiments were carried out at 37 ± 0.5 ◦C with magnetic stirring at 90 rpm (Personal Reaction Station, J-Kem Scientific Inc., St. Louis, MO, USA). Samples of 1 mL were withdrawn and subsequently filtered (Whatman 3), each minute during the first 10 min, then every two minutes until 30 min, and finally at 45, 60, 75 and 90 min. A total of 200 μL samples were then diluted to 5 mL with the corresponding dissolution medium and analyzed by UV-vis spectrophotometry at 435 nm to determine the amount of NTZ dissolved. Dissolution test with solutions containing pre-dissolved HPMC or Methocel ® 60 HG polymer (0.5% *w*/*v*) in pH 7.5 PBS were employed and compared with the results obtained from polymer-free solutions (PBS alone). All experiments were carried out in triplicate. After the dissolution experiments, the solid residues were collected and dried at room temperature for analysis by powder X-ray di ffraction (PXRD).

### 2.2.4. Preparation of Powder Formulations with Methocel ® 60 HG

Powder formulations with NTZ consisted in physical mixtures of the components listed in Table 1. Formulations at three di fferent concentrations (1.0, 2.5 and 5.0% *w*/*w*) of Methocel ® 60 HG were prepared considering 250 mg of nitazoxanide in the powder mix (entries 4 to 12). For comparison, the corresponding control experiments in the absence of Methocel ® 60 HG were also obtained (entries 1, 2 and 3). All NTZ samples were passed through a sieve mesh 200.

**Table 1.** Composition (mg) of powder formulations with and without Methocel ® 60 HG for NTZ, the physical mixture of NTZ and succinic acid (SUC) (2:1), and the NTZ-SUC cocrystal (2:1) a.


a 298 mg of NTZ-SUC (2:1) is the molar equivalent of 250 mg of NTZ. b Percentage (*w*/*w*) of the polymer in the formulation was calculated by taking as reference the average weight of the commercial tablet (ca. 758 mg) containing 500 mg of NTZ. The equivalent weight of the tablet for 250 mg of NTZ corresponds to 379 mg.

### 2.2.5. Powder Dissolution Experiments in the USP 1 (Basket) Apparatus

For the dissolution experiments employing the USP 1 apparatus, the powder formulations shown in Table 1 were added to a volume of 600 mL of pH 7.5 PBS solution, with a rotation speed of 100 rpm at 37 ± 0.5 ◦C. Samples of 4 mL were taken at 2, 5, 10, 15, 30, 60, 120 and 180 min, and an equal volume of fresh medium was added to maintain the dissolution medium volume constant. The NTZ concentration was quantified by UV-vis spectrophotometry at 435 nm (Figures S2 and S3). Each dissolution profile represents the average of three experiments. After the dissolution experiments, the solid residues were collected and dried at room temperature for analysis by PXRD and by scanning electron microscopy (SEM). Finally, a test comparing a powder formulation with Methocel ® 60 HG 5% *w*/*w* containing 500 mg of NTZ as NTZ-SUC cocrystal and a commercially available formulation (500 mg tablet) was performed. The reference tablets of NTZ (Daxon®, 500 mg NTZ) were ground in an agate mortar and passed through a sieve mesh 200. All the dissolution profiles are presented as mg of NTZ dissolved vs. time. The mg of NTZ dissolved was calculated as:

$$\text{mg of drug dissolved } = \text{C}\_{\text{n}} \times V\_{\text{n}} + \sum\_{i=1}^{n-1} \text{C}\_{i} \times V\_{\text{s}}$$

where *Cn* = concentration of drug in sample *n*, *Vn* = volume of dissolution media at the time of taking the sample *n*; *Ci* = concentration of drug in sample *n* − 1 and *Vs* = volume of aliquot due to sampling [47].
