*3.1. Materials*

DAS (MW = 488 g/mol), was purchased from Sigma Aldrich (Milan, Italy). HP-β-CD (hydroxypropil-β-cyclodextrin, MW = 1396 Dalton, substitution degree 0.65) was kindly provided by Roquette (FR). HCl and phosphate salts for the preparation of buffers were purchased from Fluka (Sigma Aldrich, Milan, Italy). Bidistilled water was bought from Carlo Erba (Milan, Italy). The cell counting Kit-8 (CCK-8) used for cytotoxicity studies was purchased from Sigma Aldrich (Milan, Italy). All other products and reagents used in this work were of analytical grade.

### *3.2. Quantitative Analysis of DAS*

The quantitative analysis of DAS was performed by High-performance liquid chromatography (HPLC). In detail, a HPLC station composed of a Agilent 1260 LCVL quaternary pump, a variable wavelength UV-visible detector and a fixed 20 μL loop manual injector was used. The analytical data were processed with the Agilent OpenLab LC software. For the analysis, a C18 Zorbax SB – Aq (4.6 × 150 mm) column was eluted in isocratic mode with a methanol/ammonium acetate pH = 3 60/40 *v*/*v* mixture, continuously monitoring the eluent at 280 nm. In these conditions, the retention time of the drug was about 2.8 min.

Standard calibration curves were prepared at a wavelength of 280 nm using the same analysis conditions and they resulted in a linear plot (*r*2 = 0.999) in the range of tested concentrations (from 2.17 × 10−<sup>4</sup> M and 6.78 × 10−<sup>6</sup> M).

### *3.3. Solubility and Phase-Solubility Studies*

The phase solubility study was conducted in accordance with Higuchi and Connors [21]. In detail, DAS was added in excess to an aqueous solution containing HP-β-CD in the appropriate concentration (0–10% *w*/*v*) until saturation and the suspensions thus obtained were placed in 4 mL vials with screw cap to avoid changes that are caused by evaporation. The obtained mixtures were vortexed for about 5 min and then placed in a thermostat bath at 25 ◦C for three days.

Subsequently, an aliquot of the aqueous phase of each mixture was transferred into a 5 mL glass syringe and filtered through a 0.22 μm cellulose acetate membrane filter (Millipore®, Milan, Italy).

The obtained filtrate was suitably diluted and subjected to subsequent HPLC analysis for the quantification of the drug. All of the determinations were conducted in triplicate.

The obtained data were used to determine the apparent stability 1:1 constant (K1:1) of the DAS/HP-β-CD inclusion complex, using the slope of the phase solubility diagrams straight line, as reported by Higuchi and Connors in the following equation:

$$K\_{1:1} = \frac{slope}{S\_0(1 - slope)}\tag{1}$$

where *S*0 represents DAS solubility in absence of cyclodextrin determined in the same way.

### *3.4. Preparation of Solid DAS /HP-β-CD Inclusion Complex*

The DAS/HP-β-CD inclusion complex was prepared in the solid state by freeze drying [18]. The lyophilized product was prepared by adding DAS and HP-β-CD in water in equimolar amounts. The obtained suspension was vigorously vortexed for about five minutes, left under stirring for two days, filtered through 0.22 μm cellulose acetate filters (Millipore), then frozen, and lyophilized (Lio 5P, Milan, Italy). The obtained product was characterized by DSC and FT-IR.

### *3.5. Determination of DAS Incorporation Degree in the Solid Cyclodextrin Inclusion Complex*

The amount of DAS that is present in the DAS/HP-β-CD solid complex was determined by solubilizing about 5 mg of sample in 5 mL of deionized water. Samples were injected in HPLC after filtration with 0.22 μm cellulose acetate filters (Millipore®). The incorporation degree of DAS into the inclusion complex was determined from the peak areas obtained and expressed as g of DAS per 100 g of complex.

### *3.6. Job's Plot Method*

The stoichiometry of the inclusion complex DAS/HP-β-CD in aqueous solution was determined by the continuous variation method or Job's method [15]. Briefly, equimolar (1.02 × 10−<sup>3</sup> M) CD3OD/D2O (50/50, *v*/*v*) solutions of DAS and HP-β-CD were mixed to a fixed volume by varying the molar ratio from 0 to 1, keeping the total molar concentration of the species constant. After stirring for 1 h, for each solution the 1H-NMR spectra were registered and the chemical shifts of the host's protons were calculated and expressed as ppm. The Δchemical shift was determined as the difference between chemical shifts with and without HP-β-CD. Subsequently, Δppm × [DAS] was plotted versus *r*, where:

$$r = \frac{[DAS]}{[DAS] + [HP - \beta - CD]} \tag{2}$$

### *3.7. 1H-NMR and Heteronuclear Multiple Bond Correlation (HMBC) Spectroscopic Studies*

1H nuclear magnetic resonance (1H-NMR) spectra were recorded using a NMR Agilent Technologies 500/54 Premium Shielded instrument and 1H chemical shifts were referred to DHO as internal standard. For the Heteronuclear Multiple Bond Correlation (HMBC) experiment, an Agilent 500 mHz spectrometer was used. The concentration of the drug was 10 mg/mL in a 5-mm NMR tube. Sample temperature was set to 25 ◦C. The following parameters were used for 2D 1H- 13Cheteronuclear multiple bond correlation (HMBC) experiment: number of scans, 2, number of complex data points (experiments) in F1, 128; number of complex data points in F2, 2048; sweep width in F1 and F2, 222 and 13ppm, respectively; spectrometer offset for 1H and 13C, 6 and 100 ppm, respectively; interscan delay, 1.5 s. Data were processed with the software Topspin. For 2D, the spectrum was zero filled to 512 data apodization function in both dimensions prior to Fourier transform and phase correction. Chemical shifts were expressed in parts per million (ppm) with respect to the DMSO-d6 signal for carbon and H2O2 for proton [27–31].

### *3.8. Fourier Transform Infrared (FT-IR) Spectroscopy*

The FT-IR spectra of DAS, HP-β-CD, and DAS/HP-β-CD solid complex were recorded with a Perkin-Elmer 1600 FTIR spectrophotometer dispersing each sample in KBr for spectroscopy (2 mg of sample in 200 mg of KBr) [32]. The scan range used was 400–4000 cm<sup>−</sup>1, with a resolution of 1 cm<sup>−</sup>1. The instrument was periodically calibrated.

### *3.9. Differential Scanning Calorimetry (DSC) Analysis*

The thermal analysis of DAS and DAS/HP-β-CD solid complex were performed using a Mettler Toledo DSC 822e Stare 202 system (Mettler Toledo, Switzerland) equipped with a thermal analysis automatic program, as described in a previous work [33]. The instrumentation was calibrated periodically, using indium as reference.

### *3.10. Dissolution Studies*

Dissolution experiments were performed at 37 ◦C using a BIODIS USP III apparatus (Varian Inc., Cary North Carolina, CA, USA), equipped with a rod stirrer maintaining a rotational speed of 100 rpm during the test. Samples of DAS or DAS/HP-β-CD solid complex, equivalent to about 2 mg of DAS, were suspended in the dissolution medium (80 mL of 0.05 M phosphate buffer at pH 7.4 or HCl 0.05 M pH = 1.2). The volume of 80 mL was chosen taking into account the HPLC quantification limit for the determination of DAS.

At predetermined time intervals, 600 μL of suspension were collected and, in order to keep constant the initial volume, 600 μL of the same dissolution medium previously thermostated at the same temperature were added. Samples were subsequently filtered using a 0.22 μm membrane filter (Millipore®cellulose acetate), and the filtrates thus obtained were subjected to HPLC analysis after appropriate dilution. For quantitative analysis the calibration curve previously constructed was used and the dissolution profiles shown correspond to the average of three determinations.

### *3.11. Cytotoxicity Studies*

C2C12 myocytes were cultured in DMEM that was supplemented with 10% fetal bovine serum, 1% penicillin, 1% streptomycin and 1% glutamine and were maintained at 37 ◦C in 5% CO2/95% air. Cell viability was evaluated by measuring the succinic dehydrogenases activity in the cell suspension using the cell counting Kit-8 (CCK-8) (Sigma Aldrich), which utilizes a highly water-soluble tetrazolium salt and whose detection sensitivity is higher than other tetrazolium salts [34].

Cells were seeded in 96-well cultures at a density of approximately 4.5 × 10<sup>3</sup> cells per well and then cultured for 16 h. Afterwards, the cells were treated for 5 h with free DAS (in DMSO <0.15% in order to ensure cellular vitality) or complexed with HP-β-CD, but at the same concentration calculated on the basis of the incorporation degree, both being dissolved in DMEM. Following exposure, 10 μL of CCK-8 were added into each well and then the plate was incubated for additional two hours. The absorbance at 450 nm was measured using a spectrophotometer (microplate reader Victor V31420–40; PerkinElmer, Wellesley, Massachusetts). Cell viability (%) is expressed according to the following formula:

$$\text{cell viability} \left( \% \right) = \left[ \left( \text{test value} - \text{blank} \right) / \left( \text{control value} - \text{blank} \right) \times 100 \right] \tag{3}$$

where the blank value represents that of a cell-free wells; the control value represents that of wells of cells do not treated with DAS and the test value represents that of wells of cells treated with DAS. The results are expressed as the percentage of the control and presented as the mean ± SD. Each data is from 24–48 replicates (wells) and 6–9 different culture dishes.

### *3.12. In Vivo Study*

A total of 10 sedentary WT male mice C57Bl/6J (Charles River, Italy for Jackson Laboratories), homogeneous for age and body weight (BW) were divided into 2 groups as follows: 4 WT mice vehicle-treated (HP-β-CD 10%) and 6 WT mice treated with DAS/HP-β-CD inclusion complex at the dose of 15 mg/Kg. Drug and vehicle were administered in drinking water for 1 week. The dose was chosen based on data in literature, in fact, the human dose commonly administered in clinical practice, converted in the appropriate animal equivalent, resulted to be approximately 20 mg/kg per day [35]. Care in animal handling and environment conditions was used to avoid any animal discomfort and stress during the study period. Food intake was monitored, and composition was maintained constant [36]. No abnormal gross findings in animal well-being and no animal deaths were observed during the study period.

### *3.13. Ex vivo Study: Pharmacokinetic Analysis*

The pharmacokinetic (PK) analysis were commissioned to the CRO "XenoGesis Ltd.—Preclinical DMPK & Bioanalysis services, Nottingham, UK. In detail, analysis was performed in quadriceps (Quad) and livers of treated animals. Tissues were individually weighed into a "FastPrep" tube and PBS was added (3:1 ratio). Each tube was placed in the fast prep homogenizer on a predetermined 1min cycle to ensure complete homogenization. 40 μL of each homogenate was aliquoted to a fresh tube and 50 μL of MeOH plus 150 μL of Methanol-containing Internal standard (25 ng/mL Imipramine HCl) was added. Each sample was mixed on a Bioshake for 1 min and then transferred to the freezer at −20 ◦C for at least two hours prior centrifugation at 2500 × *g* for 20 min. The supernatants were then transferred to a 96-well plate for sampling by the LC-MS/MS. A Thermo TSQ Quantiva with Thermo Vanquish UHPLC system was used (Thermo Fisher Scientific Inc, Milan, Italy). Separation was achieved on a ACE-AR C18 (50 × 2.1 mm, 1.7 μm) column, with MilliQwater 0.1% formic acid (solvent A) and methanl-0.1% formic acid (solvent B) at 65 ◦C and at a flow rate of 0.8 mL/min. Positive ion spray voltage and vaporizer temperature were set at 3500 V and 450 ◦C, respectively, while the ion transfer tube temperature was set at 365 ◦C. Finally, sheath gas and auxiliary gas pressures were fixed at 54 and 17 bar, respectively. Detection was performed using a multiple reaction monitoring (MRM) via a positive ESI source spray voltage. Quantitative analysis was conducted by MRM at 232.06 to 401.11 *m*/*z* for DAS inclusion complex and at 86.10 to 193.04 *m*/*z* for the internal standard Imipramine. Mass transitions were combined for each compound to maximize sensitivity.
