**4. Discussion**

BCS class IV drugs (e.g., sulfamethoxazole, ritonavir, paclitaxel, and furosemide) exhibit numerous unfavorable characteristics (low solubility and permeability, high presystemic metabolism, efflux transport), which make their oral drug delivery challenging. In addition to this, class IV drugs often demonstrate inter/intra-subject variability. Indeed, following oral administration, the absorption and bioavailability of furosemide are highly variable (37–51%) [35,41]. It has been suggested that this variability is highly depend on the absorption process [41], which in turn is dependent on drug aqueous solubility and intestinal permeability following oral administration [1,44]. It has also been hypothesized that variable gastric/intestinal first-pass metabolism can be a factor in causing incomplete and irregular furosemide absorption in humans [45]. Despite the unfavorable class IV drug characteristics, furosemide was shown to be exceptionally useful and successful marketed drug product for the treatment of edema [17]. For this reason, we decided to investigate furosemide's solubility and in-vivo regional-dependent permeability throughout the GIT, as main parameters that guide absorption of oral drugs.

It was shown that a correlation between human Peff in the jejunum and physicochemical parameters advocates that there is a high pH-dependent influence on the passive intestinal permeability in-vivo [46]. Indeed, furosemide in-vivo permeability data demonstrate a downward trend towards the distal intestinal segments as the pH gradually increases, a trend that can be expected for acidic drugs, since the pH in the intestinal lumen gradually increases towards distal SI regions (Figure 3). Many BCS class IV drugs are substrates for e fflux transporters [47]. There is some evidence that furosemide might be a substrate for e fflux transporters [48,49]; thus, such permeability trend could also be influenced by the P-glycoprotein (P-gp) transporter in which expression levels are increased from proximal to distal SI segments [6,50–52]. Since metoprolol's intestinal permeability is passive and does not involve carrier-mediated absorption, it exhibited pH-dependent intestinal permeability, with reverse tendency compared to furosemide; as a basic drug, metoprolol showed upward increase in permeability towards distal SI segments with rising pH values (Figure 3). At any point throughout the SI, furosemide exhibited significantly lower permeability than the benchmark (metoprolol's jejunum permeability), which confirms its BCS low-permeability classification and incomplete absorption. Despite the fact that furosemide is a low-permeability drug, the higher permeability in the proximal intestinal regions provides a window for furosemide absorption, and we posit that this is one of the main reasons for furosemide's su fficient bioavailability and success as a marketed drug. Theoretical fu and fe as a function of pH were found to be in excellent correlation to these in-vivo data. In addition, in-silico modeling indicated that furosemide dissolution from an IR formulation would be fairly complete before the drug leaves proximal SI (Figure 5), although the drug is generally classified as low-soluble, enabling timely delivery of the dissolved drug to the distinct absorption site. Complete furosemide dissolution under physiological conditions is also confirmed by the experimental solubility results (Table 1).

Furosemide Log D studies showed higher partition coe fficient in comparison to metoprolol at pH 6.5, whereas, in the in-vivo intestinal perfusion experiment, furosemide showed significantly lower jejunum permeability than metoprolol (Figure 1). A possible reason for this di fference in the partitioning and in-vivo permeability can be the polar surface area (PSA) of both drugs [53]. A sigmoidal relationship between the fraction absorbed following oral administration and the dynamic polar surface area was reported in the past [54–56]. It was shown that orally administered drugs with large PSA (>120) are hardly absorbed by the passive transcellular route, while drugs with a small PSA (<60) are almost completely absorbed [55,56]. This is in agreemen<sup>t</sup> with our results, as furosemide has much higher PSA (127.7) than metoprolol (53.2) [54,55]. Another reason for the di fference in the partitioning and in-vivo permeability may be the presence of active e fflux transport involved in the intestinal permeability. The influence of e fflux transport at pH 6.5 (proximal intestinal segments) could decrease furosemide's permeability in-vivo, which was not accounted for in the octanol partitioning studies.

The Log P value of furosemide (2.3) is in the close proximity to that of metoprolol (2.2), pointing to high permeability (Table 2). However, the Log P calculation is based on the unionized drug fraction, and, since furosemide has acidic nature it is likely that, once it passes the acidic stomach environment, it will mostly be in ionized form (the pH throughout the GIT varies from 5.9–6.3 in the proximal SI to 7.4–7.8 in distal SI segments; pH in the colon is fluctuating between pH 5–8 [57]); therefore the high furosemide Log P is not in correspondence with permeability in-vivo. Thus, we posit that no single parameter can be used for measuring the drug absorption process, but rather, a combination of physicochemical parameters and in-vitro and in-vivo findings, as well as careful consideration of inclusion criteria prior to making decisions. Despite the high Log P value for furosemide, it was indeed confirmed that furosemide is a BCS class IV drug, based on both the solubility data (Table 1) and the intestinal permeability (Figure 3).

Suitable formulation is the main approach to create an e fficacious drug product for the administration of BCS class IV drugs [47]. Absorption windows in the proximal intestinal segments can restrict the oral drug bioavailability and can be a significant limitation for the development of CR drug formulation. The underlying reasons are mechanistically explained by our in-silico results (Figure 7). As mentioned, furosemide permeability results revealed acceptable permeability in the

proximal segments of the SI, which is presumably the reason why furosemide has appropriate drug bioavailability, despite being a BCS class IV drug. However, since CR products release the drug over 12–24 h, mostly in the colon, (transit time throughout the small intestine is 3–4 h [58]), the fact that furosemide is mainly absorbed from proximal SI segments, (with decreased permeability at distant GIT segments) prevents the formulation of furosemide as a CR product, as shown previously [21,59,60]. However, we believe that formulations based on gastro-retentive dosage forms (GRDF) can be shown as prosperous for furosemide [61]. There are several similar examples in the literature where absorption window occurs in the upper GI, and this has been used to create GDRF formulations to improve the drug absorption, such as riboflavin [62] and levodopa [59,63].

Several types of bariatric surgeries (specifically Roux-en-Y gastric bypass and mini bypass) result in bypassing the upper SI. In cases where the absorption window is indeed in this upper SI region, the absorption following the bariatric surgery can be hampered vastly, since the actual segmen<sup>t</sup> responsible for the majority of absorption is bypassed [64–66].
