Lamivudine (3TC) is an antiviral drug used to treat Acquired Immunodeficiency Syndrome (AIDS) in humans with a vastly demonstrated clinical efficacy. However the rapid emergence of resistant viral strains limits its efficacy, thus several strategies, such as the design of prodrugs, have been widely applied to optimize its pharmacotherapeutic properties. In this work we deal with the study of the intestinal permeation of 3TC and two novel prodrugs of 3TC, namely 3TC-Etha and 3TC-Buta, by using rat intestinal segments and applying the gut sac in vitro technique. An adequate bioanalytical method (sample preparation and quantitative analysis) was fully developed and validated for the quantitation of these three compounds. A low permeability coefficient (Papp 0.408 ± 0.049 x10-4 cm/min) was found for 3TC, with no statistically significant difference in its apical-to-basal and. basal-to-apical permeability. We concluded that 3TC permeates through the intestinal tissue by passive diffusion, with no intervention of efflux mechanism on the apical side of the enterocyte. Regarding the prodrugs, both 3TC-Etha and 3TC-Buta were able to increase 3TC permeability (2 and 10 times, respectively). However none of these compounds were able to cross the intestinal tissue in their intact form. In the case of 3TC-Etha, the permeability of the intact compound is impaired by a P-glycoprotein (P-gp) mediated efflux, evidenced by the high permeability coefficient (2.170 ± 0.122 x10-4 cm/min) determined in the presence of verapamil on the apical side of the enterocyte. On the other hand, 3TC-Buta was not subjected to efflux mechanisms on the apical side of the enterocyte, but is efficiently subjected to enzymatic hydrolysis during the permeation process. In light of these results, molecular modeling (docking and molecular dynamics) procedures were applied to further study the structural features that confers this different behavior of these two compound respect to P-gp mediated efflux, stressing the potential of combining experimental and theoretical studies for the design of 3TC prodrugs with high oral bioavailabilities.Fil: Gualdesi, María Soledad. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Briñon, Margarita Cristina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; ArgentinaFil: Quevedo, Mario Alfredo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Farmacia; Argentina