*2.2. Solubility Studies*

The pH-dependent solubility studies were performed using the shake flask method, as previously reported [20–23]. The equilibrium solubility of furosemide was determined at both 37 ◦C and at room temperature (25 ◦C), in phosphate buffer pH 7.5, acetate buffer pH 4.0, and maleate buffer pH 1.0. Surplus quantity of furosemide was introduced to glass vials holding buffer solutions with different pH; the pH of those solutions was measured following drug addition to the buffers and, consequently, placed in the shaking incubator (100 rpm) at 37 ◦C. The vials were centrifuged (10,000 rpm, 10 min), and the supernatant was instantly analyzed by UPLC. The dose number for furosemide was calculated using the established equation: *D*0 = *M*/*V*0/*C*s; *M* being the highest single-unit dose strength of furosemide (taken as 80 mg [24]), *V*0 is the initial volume of water (250 mL), and *C*s is the solubility at each pH; the drug is considered highly soluble if the *D*0 < 1.

#### *2.3. Evaluation of Octanol-Bu*ff*er Partition Coe*ffi*cients (Log D)*

Furosemide and metoprolol experimental octanol-buffer partition coefficients (Log D) were studied at pH 6.5, 7.0, and 7.5 using the shake-flask method [8,11]. Drug solutions in octanol-saturated phosphate buffer (pH 6.5, 7.0, 7.5) were equilibrated at 37 ◦C for 48 h. The octanol and water phase were divided via centrifugation, and the drug content in the water phase was quantified using UPLC; the furosemide/metoprolol concentration in the octanol phase was determined by mass balance.

### *2.4. Physicochemical Analysis*

The theoretical fraction extracted into octanol (fe) was calculated using the following equation [25,26].

$$\mathbf{f\_e} = \frac{\mathbf{f\_u}\mathbf{P}}{1 + \mathbf{f\_u}\mathbf{P'}} \mathbf{f'} $$

in which P represents the octanol-water partition coefficient of the unionized drug form, and fu is the fraction unionized of the drug at a certain pH. Experimental Log P values were taken from the literature for both furosemide (2.29) [27] and metoprolol (2.19) [28]. The fu versus pH was plotted according to the Henderson-Hasselbalch equation, using the pKa literature values: 9.68 for metoprolol [29] and 3.8 for furosemide [24].

#### *2.5. Rat Single-Pass Intestinal Perfusion*

Effective permeability coefficient (Peff) of furosemide versus metoprolol in various intestinal segments was assessed using the single-pass rat intestinal perfusion (SPIP) in-vivo model. The murine studies were completed according to the approved protocol by Ben-Gurion University of the Negev Animal Use and Care Committee (Protocol IL-08-01-2015). The animals (male Wistar rats weighing 230–260 g, Harlan, Israel) were housed and handled according to Ben-Gurion University of the Negev Unit for Laboratory Animal Medicine Guidelines. All animals were fasted overnight (12–18 h) with free access to water; rats were randomly allocated to different experimental groups. The intestinal perfusion study was performed according to the previous reports [7,9,30–32]. Animals were anesthetized via intramuscular injection of 1 mL/kg ketamine-xylazine solution (9%:1%) and placed on a heated (37 ◦C) surface (Harvard Apparatus Inc., Holliston, MA, USA); the rat abdomen was uncovered via a midline incision (~3 cm). Permeability (Peff) was measured in proximal jejunum (starting 2 cm lower from the ligament of Treitz), mid-small intestine (SI) segmen<sup>t</sup> (isolated between the end of the upper and the beginning of the lower segments), and distal segmen<sup>t</sup> of the ileum (ending 2 cm above the cecum) accounting for the complexity of the entire SI [7]. Intestinal segments were cannulated on both ends and perfused with drug-free buffer. Working solutions containing furosemide (320 μg/mL), metoprolol (400 μg/mL), and phenol red (a non-absorbable marker for water flux measurements) were prepared with potassium phosphate monobasic and sodium phosphate dibasic, to achieve pH of 6.5, 7.0 and 7.5; osmolarity (290 mOsm/L) and ionic strength in all buffers was maintained throughout the study. Drug solutions were incubated in a 37 ◦C water bath. Steady-state environment was ensured by perfusing the drug-containing buffer for 1 h, followed by additional 1 h of perfusion, during which sampling was done every 10 min. The pH of the collected samples was measured in the outlet sample to verify that there was no pH change throughout the perfusion. All samples were assayed by UPLC. The length of each perfused intestinal segmen<sup>t</sup> was measured in the end of the experiment. The effective permeability (Peff; cm/s) through the rat SI wall was calculated according to the following equation:

$$P\_{\rm eff} = \frac{-\text{Qln}\left(\text{C}'\_{\rm out}/\text{C}'\_{\rm in}\right)}{2\pi \text{RL}},$$

in which Q is the perfusion buffer flow rate (0.2 mL/min); Cout/Cin is the ratio of the outlet/inlet drug concentration adjusted for water transport; R is the radius of the intestinal segmen<sup>t</sup> (conventionally used as 0.2 cm); and L is the exact length of the perfused SI segmen<sup>t</sup> as was measured at the experiment endpoint [7,33,34].
