Use of a Biophysical-Kinetic Model to Understand the Roles of Protein Binding and Membrane Partitioning on Passive Diffusion of Highly Lipophilic Molecules Across Cellular Barriers

Abstract

The novel antioxidants U-78517F and U-74006F, or lazaroids, are highly lipophilic organic molecules with poor brain uptake. To understand this paradoxical behavior better, continuous monolayers of Madin-Darby canine kidney (MDCK) epithelial cells with distinct apical (AP) and basolateral (BL) plasma membrane domains grown on polycarbonate membrane filters and plastic were used to examine the mechanism of transcellular diffusion. Independent kinetic experiments were used to quantify AP to BL flux, efflux from the AP and BL membranes and AP membrane partitioning as functions of bovine serum albumin (BSA) concentration. Fluxes were appropriately reduced to permeability coefficients (Pe) for the membrane, aqueous boundary layer (ABL) and filter, BSA-drug binding constants, and effective (Ke) and intrinsic (Kintr) membrane partition coefficients in the absence of metabolism. Both Pe and Ke decreased exponentially with increased BSA concentration and a concomitant decrease in free drug concentration. Uptake was ABL-controlled under the conditions used and its Pe was 1,000-fold faster than that for efflux due to a large Kintr. Therefore, diffusion across the cellular barrier was limited kinetically by the equilibrium between protein-bound drug and free drug partitioned into the cell membrane and the rate-limiting desorption of drug from the cell membrane into the aqueous receiver. This suggests that brain uptake of these lipophilic antioxidants is limited by interactions with plasma proteins and, possibly, by unfavorable partitioning from the endothelium into the underlying tissue. The present biophysical kinetic model is proposed as generally useful in studying the penetrative ability of other membrane interacting molecules.

In Vitro Stability and In Vivo Pharmacokinetic Studies of a Model Opioid Peptide, H-Tyr-d-Ala-Gly-Phe-d-Leu-OH (DADLE), and Its Cyclic Prodrugs

“…1, II and III) arising from CA-based and OMCA-based cyclic prodrugs seem to also be a significant problem with these linkers, modified linkers having more rapid rates of conversion of the intermediate(s) to DADLE are also being developed in our laboratory. Plasma protein binding can exert a significant influence on the pharmacokinetic properties and biological activities of a drug, especially for brain-targeted compounds (Raub et al, 1993). For DADLE and its prodrugs, more lipophilic compounds had higher fractions bound in the order CA-DADLE Ͼ OMCA-DADLE Ͼ AOA-DADLE Ͼ DADLE, but none of the prodrugs had protein-binding values high enough to substantially affect the distribution of the compounds.…”

Lazaroids: Mechanisms of Action and Implications for Disorders of the CNS

“…After intravenous administration, this happens to be the vascular endothelial cell. Indeed, tirilazad has bcen shown to have high affinity for vascular endothelial cells in vitro (26) and to penetrate the intact blood-brain banicr (BBB) very poorly in vivo (27). Although a high degree of binding of tirilazad to impermeable plasma proteins contributes to the limited BBB penetration, further in vitro studies with canine kidney epithclial monolayers (which display diffusional characteristics similar to brain endothclium) have confirmed that the compound has limited transcellular pcrmcability and tcnds to bccome highly concentrated in.…”

Pharmacology of Traumatic Brain Injury: Where Is the “Golden Bullet”?

“…Tirilazad mesylate is a 21-aminosteroid that has been shown to inhibit lipid peroxidation in experimental animals (14). Tirilazad mesylate penetrates the intact bloodbrain-barrier poorly but has a strong affinity for the vascular endothelium (21). One randomized clinical study investigating the effect of tiralazad mesylate in the setting of TBI has been reported (22).…”