**1. Introduction**

The biopharmaceutical classification system (BCS) developed by Amidon et al. revealed that the solubility/dissolution of the drug and its intestinal permeability are the two key factors that dictate drug absorption following oral administration [1,2]. Drug solubility in the gastrointestinal milieu may change in di fferent intestinal segments, e.g., due to pH changes, in a fairly predictable

manner; depending on the pKa, the solubility of acidic drugs may increase as the luminal pH rises in more distal regions of the small intestine, and vice versa for basic drugs [3–5]. On the other hand, time- and segmental-dependent intestinal permeability is more complicated and harder to predict [1]. Mechanisms contributing to segmental-dependent permeability throughout the gastrointestinal tract (GIT) include di fferent morphology along the GIT, variable intestinal mucosal cell di fferentiation, changes in the drug concentration (in case of carrier-mediated transport), modulation of tight junction permeability, and luminal contents and properties, e.g., pH, transporter expression, variability in the structure/composition of the intestinal membrane itself, and more [6–11].

The four BCS classes highlight the limiting factors of the absorption process: (1) Class I, high-solubility high-permeability drugs, indicate the easier and straightforward development process, and complete absorption is expected; (2) Class II, low-solubility high-permeability drugs, indicate that a solubility/dissolution limitation is expected; (3) Class III, high-solubility low-permeability drugs, indicate that the intestinal absorption of this class of drugs will be limited by the permeability rate; and (4) Class IV, low-solubility low-permeability drugs [12]. Since Class IV drugs su ffer from inadequate solubility and permeability, they have very poor oral bioavailability and are inclined to exhibit very large inter- and intrasubject variability. Therefore, unless the drug dose is very low, they are generally poor oral drug candidates. Yet, according to some estimates, ~5% of the world's top oral drugs belong to this class [13–15]. In some cases, this is due to the absorption window, which is often critical for the success or failure of a certain drug. In order to gather information about the drug absorption window, extensive work and thorough analysis of luminal conditions and drug absorption is needed, within di fferent locations throughout the GIT. Here, we present such analysis for BCS class IV drug, furosemide [16].

Furosemide is a powerful loop diuretic and is indicated for treating edematous conditions associated with heart, renal, and hepatic failure, as well as for the treatment of hypertension [17,18]. Drug therapy with furosemide is often complex, due to apparent erratic oral systemic availability and unpredictable responses to an administered dose [19]. Even though furosemide is a class IV drug, it is a very common and widely prescribed drug on the market.

In this work, we aimed to investigate the reason for apparent success of furosemide as a marketed product, despite its poor biopharmaceutical properties, and classification as BCS class IV drug, in order to allow development of future class IV compounds. We posit that segmental-dependent permeability of furosemide may contribute to its absorption complexity and provide a certain absorption window in which the drug has suitable permeability and, hence, gets absorbed. For this reason, we investigated the in-vivo intestinal permeability of furosemide throughout di fferent segments of the small intestine. Solubility studies, as well as theoretical physicochemical analysis of furosemide and advanced modern in-silico GastroPlus ® simulations, were performed, in order to elucidate the mechanistic reasons behind the experimental results. Furosemide data were compared to the β-blocker metoprolol, the Food and Drug Administration (FDA) reference drug for the low/high permeability class boundary. Overall, this experimental setup allowed us to reveal important insights on the performance of furosemide, despite its unfavorable drug-like properties, and discuss extrapolation of these insights to other BCS class IV drug candidates.
