Background. The human oral bioavailability (BA) of acamprosate is 11% and its oral absorption is permeability limited (BCS class III). Acamprosate is not metabolized, therefore it’s BA has the same nominal value as its fraction absorbed (fa). It is however controversial whether the intestinal effective permeability of acamprosate, which is fully ionized in vivo (pKa 1.83; molecular weight 181.2 g/mol), is predominantly paracellular (Ppara) or is transcellularly mediated by solute carriers (Ptrans). Aim. The overall aim was to get a better insight on the contribution of Ppara and Ptrans to the overall acamprosate apparent permeability. Methods. Acamprosate apparent permeability (Papp, exp) was determined across Caco-2 monolayers in the apical-to-basolateral transport direction using a buffer pH of 7.4 and several cell passages (N). Acamprosate concentrations were quantified by LC/MS-MS. For the neutral paracellular marker [14C]-mannitol, which has a very similar molecular radius compared to acamprosate, the Papp, exp was measured in parallel. The experimental value for acamprosate was then compared to a mechanistically modelled Caco-2 apparent permeability, Papp, calc. Papp, calc was mathematically accounting for the unstirred boundary layer permeability (PUBL), the filter permeability (Pf), the intrinsic passive transcellular permeability (Ptrans,0) and Ppara (1-3). The mathematical model thereby accounted for (i) the physical-chemical properties of acamprosate and mannitol (molecular weight, aqueous diffusion coefficient, lipophilicity, molecular radius, and ionization state), (ii) the properties of the Caco-2 monolayers (pore radius, porosity, potential drop, villus-fold area expansion factor), and (iii) the experimental settings used (temperature, plate rotation speed, filter support area, thickness, and porosity). Results. The mean (± SD) Papp, exp of acamprosate and [14C]-mannitol across Caco-2 cell monolayers was measured as 0.19 ± 0.07 x 10-6 cm/s (n = 2, N = 3) and 0.35 ± 0.17 x 10-6 cm/s (n = 3, N = 4), respectively. Acamprosate PUBL and Pf were estimated as 200 - 3150 x 10-6 cm/s (for an UBL thickness of 30 - 500 µm, respectively) and 1132 x 10-6 cm/s, respectively. The transcellular permeability for the ionized and unionized acamprosate was estimated at 0 and 0.8 x 10-6 cm/s, respectively. Ppara was estimated (from the experimental [14C]-mannitol permeability, by accounting for the ionization difference) to be 0.20 ± 0.10 x 10-6 cm/s. Conclusion. Acamprosate Pf and PUBL are large compared to the acamprosate Ptrans and Ppara. For this reason, Pf and PUBL have a negligible contribution to the overall acamprosate permeability (< 1%). The Ptrans,0 plays an insignificant role in acamprosate permeability, as only a very low fraction of acamprosate is in the neutral form at pH 7.4. The estimated acamprosate Ppara accounts for nearly 100% of the mathematically determined acamprosate Papp, calc (0.20 ± 0.10 x 10-6 cm/s), which matches well with the experimentally determined Papp, exp (0.19 ± 0.07 x 10-6 cm/s). These results suggest that the paracellular route is the dominating permeation route of acamprosate across Caco-2 monolayers, in the apical-to-basolateral transport direction.Abbreviations:BA – oral bioavailability; BCS III - biopharmaceutical classification system class III; pKa – acid dissociation constant; Ppara – paracellular permeability; Papp, exp – experimentally determined apparent permeability; Papp, calc. – calculated apparent permeability; PUBL - unstirred boundary layer permeability; Pf - polycarbonate filter permeability; Ptrans,0 - intrinsic passive transcellular permeability; LC/MS-MS – liquid chromatography / tandem mass spectrometry; ; N = number of passages; n = number of replicates;References:1.Pade D, Jamei M, Rostami-Hodjegan A, Turner DB. Application of the MechPeff model to predict passive effective intestinal permeability in the different regions of the rodent small intestine and colon. Biopharm Drug Dispos. 2017;38(2):94-114.2.Avdeef A. Leakiness and size exclusion of paracellular channels in cultured epithelial cell monolayers-interlaboratory comparison. Pharm Res. 2010;27(3):480-9.3.Avdeef A. Absorption and Drug Development: Solubility, Permeability, and Charge State - 2nd ed. Hoboken, New Jersey: John Wiley & Sons, Inc.; 2012.