**3. Results**

The solubility values obtained for furosemide at 37 ◦C and at room temperature (25 ◦C) are summarized in Table 1, as well as the corresponding dose number (D0). Furosemide showed pH-dependent solubility, in accordance with its acidic nature. It can be seen that, while, at pH 7.5, furosemide has suitable solubility (as evident by D0 lower than 1), at the lower pH values, 1.0 and 4.0, it is poorly soluble. When taking 80 mg as the highest dose strength, although D0 < 1 was obtained at pH 7.5, at pH 1.0 and 4.0, the D0 is higher than 1; hence, furosemide was found to be a low-solubility compound according to the BCS.

**Table 1.** Furosemide solubility values (μg/mL) at the tree pH values 1.0, 4.0, and 7.5, at 37 ◦C (upper panel), and at room temperature (25 ◦C; lower panel), as well as the corresponding dose number (D0) calculated for an 80-mg dose. Data presented as mean ± SD; *n* = 6.


Octanol-bu ffer partition coe fficient values of furosemide and metoprolol at the three pH values 6.5, 7.0, and 7.5 (representing the conditions throughout the small intestine) are presented in Figure 1. Both drugs presented a clear pH-dependent Log D values across the studied pH range, with opposite trends; while furosemide's partitioning decreases as the pH rises, metoprolol shows higher partitioning into octanol at higher pH (metoprolol is the acceptable reference drug for the low/high permeability class boundary). In addition, furosemide's Log D at pH 6.5 was higher than that of metoprolol at the same pH; this is a surprising finding since Log D may sometimes be used as a surrogate for passive

permeability. Indeed, at higher pH values (7.0 and 7.5), metoprolol Log D increases, while furosemide decreases, and metoprolol Log D becomes higher than furosemide.

**Figure 1.** The octanol-buffer partition coefficients, Log D, for furosemide and metoprolol at the three pH values 6.5, 7.0, and 7.5. Data are presented as the mean ± S.D.; *n* = 6 in each experimental group.

Furosemide and metoprolol physicochemical properties are presented in Table 2. Figure 2 presents furosemide versus metoprolol theoretical fraction unionized (fu) and fraction extracted into octanol (fe) as a function of pH. The plots have a standard sigmoidal shape, with opposite trends for furosemide vs. metoprolol. The fe vs. pH plot follows the same pattern to the fu plot, only with a shift to the right (higher pH values) for acidic drug (furosemide), and to the left (lower pH values) for basic (metoprolol) drugs. The shift magnitude in both cases equals Log(P − 1) at the midpoint of the fe and fu curves [25,26]. The experimental drug octanol-buffer partitioning at the three pH values (6.5, 7.0, and 7.5) are illustrated in Figure 2, as well, and it can be seen that they were in excellent agreemen<sup>t</sup> with the theoretical plots.


**Table 2.** Physicochemical parameters and chemical structure of furosemide and metoprolol.

**Figure 2.** The theoretical fraction unionized (fu) and fraction extracted into octanol (fe) plots as a function of pH for furosemide and metoprolol, as well as experimental buffer-octanol partitioning of the drugs in the three pH values 6.5, 7.0, and 7.5 (*n* = 5).

The effective permeability coefficient (Peff, cm/sec) values of furosemide and metoprolol determined using the single-pass intestinal perfusion (SPIP) rat model, in three intestinal segments, namely proximal jejunum (pH 6.5), mid small intestine (pH 7.0), and distal ileum (pH 7.5), are presented in Figure 3. It can be seen that significant regional-dependent permeability of furosemide throughout the small intestine was evident: the permeability of furosemide gradually decreases, while the permeability of metoprolol gradually increases, as the SI segments become more distal.

**Figure 3.** Effective permeability values (Peff; cm/s) obtained for furosemide and metoprolol after in-situ single pass perfusion to the rat proximal jejunum at pH 6.5, mid-small intestine at pH 7.0, and to the distal ileum at pH 7.5. Mean ± S.D.; *n* = 4 in each experimental group; \*\* *p* < 0.01, \*\*\* *p* < 0.001.

The input data regarding drug physicochemical and pharmacokinetic properties, used for in-silico simulations, are presented in Table 3. The simulated furosemide plasma concentration profile following p.o. administration is depicted in Figure 4, along with the mean profiles observed in the in-vivo studies. In addition, the observed and model predicted pharmacokinetic parameters are compared in Table 4. The presented data demonstrate that the generated model adequately describes furosemide absorption and disposition. The course of the predicted plasma profile fairly resembles the observed

data. However, certain variations are observed between the mean in-vivo data from di fferent studies referring to the same drug dose (Figure 4, Table 4). Indeed, it has been reported that furosemide oral absorption is highly variable between individuals, e.g., Cmax varied three-fold, and tmax varied five-fold [36,37,41]; moreover, individual AUC values for 40 mg furosemide oral dose varied between 1.57 and 3.76 <sup>μ</sup>g·h/mL (more than two-fold) [36,37,41], and even larger AUC values were observed in another study with the same drug dose (2.23–6.10 <sup>μ</sup>g·h/mL) [42], indicating that, regardless of the high PE(%) values in Table 4, the model predicted value of 3.66 <sup>μ</sup>g·h/mL is not an overestimate of the extent of drug absorption. In addition, extensive intrasubject variability was observed for orally dosed furosemide, and these variations were attributed to the absorption process (i.e., day to day variations in physiological factors) since the repeated i.v. doses showed only marginal intrasubject variability [36,37,41]. Considering pronounced inter- and intraindividual variability in furosemide oral absorption, the simulated profile can be seen as a reasonable estimate (Figure 4). Moreover, the predicted fraction of oral drug absorption (cc. 52%) is in accordance with the values reported in the literature [36,37].


**Table 3.** The selected input parameters for furosemide absorption GastroPlus ® simulation.

**Figure 4.** GastroPlus® simulated (line) versus mean observed (markers) plasma concentration profiles following p.o. administration of furosemide. Mean observed values represent 40 mg immediate-release (IR) tablet profile I [43] and 40 mg IR tablet profile II [37].

**Table 4.** Comparison between GastroPlus® simulated and in-vivo observed furosemide pharmacokinetic parameters following p.o. drug administration.


a Refers to the mean plasma profile from [43] (40 mg IR tablet); b refers to the mean plasma profile from [37] (40 mg IR tablet); NA, not available/not applicable.

The predicted furosemide dissolution and absorption profiles following an IR oral formulation (IR tablet) are illustrated in Figure 5. The generated profiles clearly indicate that drug permeability is the limiting factor for absorption under fasted state GIT conditions. Namely, although furosemide is a low-solubility drug, due to ionization at the elevated pH conditions in the proximal SI, drug dissolution from an IR formulation is expected to be fast (>85% in 30 min). Therefore, furosemide absorption from an IR formulation is mainly governed by poor permeability. The predicted regional-dependent absorption distribution (Figure 6) further highlights the role of furosemide segmental absorption on the overall drug bioavailability. As implied by the regional-dependent permeability data, but also considering the surface area available for absorption, furosemide absorption predominantly happens in the proximal parts of the SI (76.6% of the total amount absorbed into the enterocytes), and only a minor fraction of drug (23.2% of the total amount absorbed into the enterocytes) passes into systemic circulation through mid and distal GIT regions.

**Figure 5.** GastroPlus® simulated dissolution and absorption profiles following p.o. administration of 40 mg furosemide dose (dissolution profile was simulated using the software default Johnson equation).

**Figure 6.** GastroPlus® simulated regional absorption of furosemide following p.o. administration of 40 mg drug dose (the simulated values refer to the fraction of drug dose that entered into the enterocytes).

The prediction results corresponding to various dissolution scenarios are presented in Figure 7b–d and Table 5. According to the simulated data, furosemide release rate from an oral formulation highly impacts the concomitant absorption process, whereas prolonged drug release rate leads to marked delay in the rate and extent of drug absorption. The estimated pharmacokinetic parameters (Table 5) indicate that furosemide bioavailability would show more than a 10-fold decrease in case the complete drug dissolution is achieved within 24 h in comparison to 15 min. A similar trend is observed for Cmax and AUC values (17.75- and 17.38-fold decrease, respectively), while tmax would be prolonged (about two-fold). It is interesting to note that tmax increases with decrease in drug dissolution up to some point, but further decrease in drug dissolution (e.g., 85% in more than 6 h) would not cause additional delay in peak plasma concentration. This is because, after cc. 2 h, the drug leaves proximal parts of the intestine, where majority of furosemide absorption takes place, and, later on, in mid and especially distal intestine, only a small fraction of drug can be absorbed, as illustrated in Figure 7d.

**Figure 7.** GastroPlus® simulated furosemide dissolution profiles (**a**); and (**b**) the corresponding simulated plasma profiles; (**c**) absorption profiles; and (**d**) regional absorption distribution.


