Beyond Overton's rule: quantitative modeling of passive permeation through tight cell monolayers.

One of the great challenges in pharmacokinetics is to find a means to optimize the transport across cell barriers. In this work, permeation across a cell monolayer, such as the tight endothelia in the blood-brain barrier, was modeled using a homologous series of amphipatic molecules, 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled alkyl chain amphiphiles (NBD-Cn, n = 2 to 16), to obtain rules that relate permeant structure to permeability. The amphiphile enters the system from the serum, equilibrated with serum albumin and lipoproteins, and its sequestration by serum components, interaction with the endothelium, and accumulation in the tissue is followed over time. The dependence of the permeability coefficient on the number of carbons of the amphiphile's alkyl chain has a parabolic-like shape. After a threshold value, an increase in the hydrophobicity of the amphiphile, along the homologous series, results in a decrease in the characteristic rate of permeation to the tissue. A sensitivity analysis was performed, and the rate limiting steps for permeation of each amphiphile were identified. Sequestration in the serum and rate of interaction with the endothelium, particularly the rate of desorption, were found to be the determinant processes for some amphiphiles, while for others translocation was the rate limiting step. Additionally, for some amphiphiles a single rate limiting step could not be identified, with several steps contributing significantly to the overall permeation. Finally, we derived analytical equations that adequately describe the rate of amphiphile accumulation in the tissue for the cases where permeation is controlled by a single rate limiting step.

Chain length effect on the binding of amphiphiles to serum albumin and to POPC bilayers.

The interaction of small molecules, such as drugs or metabolites, with proteins and biomembranes is of fundamental importance for their bioavailability. The systematic characterization of the binding affinity for structurally related ligands may provide rules that allow its prediction for any other relevant molecule. In this work we have studied a homologous series of fluorescent fatty amines with the fluorescent moiety 7-nitrobenz-2-oxa-1,3-diazol-4-yl covalently bound to the amine group (NBD-C(n); n = 4, 6, 8, 10, 12, 14, and 16) in aqueous solution and associated with BSA or lipid bilayers. We have found a linear dependence with the length of the alkyl chain, up to NBD-C(10), for the Gibb's free energy of partition between the aqueous solution and 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers equal to ΔΔG = -2.5 ± 0.3 kJ/mol per methylene group. Additionally, the amphiphiles interacted efficiently with bovine serum albumin, and it was inhibited by fatty acids indicating that binding occurs to the fatty acids highest affinity binding site. The association of the amphiphiles with BSA and POPC bilayers was performed at different temperatures (15-35 °C) allowing for the calculation of the enthalpic and entropic contributions. A value of ΔH = -15 ± 4 kJ/mol was obtained for all amphiphiles and binding agents. The entropy contribution was always positive and increased with the length of the alkyl chain. The location of the ligand in the biological membrane is also of high relevance, namely because this will determine its effect on biomembrane properties at high ligand concentrations. With this goal, we have measured some photophysical properties of the amphiphiles inserted in POPC bilayers, and we found no significant variation along the series, indicating that the NBD group is located in a region with similar properties regardless of the length of the nonpolar group. An exception was noted for the case of NBD-C(14) whose parameters were somewhat different from the trend observed.