Quantitative and Mechanistic Assessment of Model Lipophilic Drugs in Micellar Solutions in the Transport Kinetics Across MDR1-MDCK Cell Monolayers.

An approach to characterizing P-glycoprotein (Pgp) interaction potential for sparingly water-soluble compounds was developed using bidirectional transport kinetics in MDR1-MDCK cell monolayers. Paclitaxel, solubilized in a dilute polysorbate 80 (PS80) micellar solution, was used as a practical example. Although the passage of paclitaxel across the cell monolayer was initially governed by the thermodynamic activity of the micelle-solubilized drug solution, Pgp inhibition was sustained by the thermodynamic activity (i.e., critical micelle concentration) of the PS80 micellar solution bathing the apical (ap) membrane. The mechanistic understanding of the experimental strategies and treatment of data was supported by a biophysical model expressed in the form of transport events occurring at the ap and basolateral (bl) membranes in series whereas the vectorial directions of the transcellular kinetics were accommodated. The derived equations permitted the stepwise quantitative delineation of the Pgp efflux activity (inhibited and uninhibited by PS80) and the passive permeability coefficient of the ap membrane, the passive permeability at the bl membrane and, finally, the distinct coupling of these with efflux pump activity to identify the rate-determining steps and mechanisms. The Jmax/KM(∗) for paclitaxel was in the order of 10(-4) cm/s and the ap- and bl-membrane passive permeability coefficients were asymmetric, with bl-membrane permeability significantly greater than ap.

Capric Acid Absorption in the Presence of Hydroxypropyl-ß-Cyclodextrin in the Rat Ileum using the In Situ Single-Pass Perfusion Technique.

The purpose of the present study was to gain quantitative mechanistic insight into the role cyclodextrin carriers may play in the intestinal absorption of highly lipophilic molecules. The physical model approach was employed to investigate capric acid absorption in the rat ileum using the in situ single-pass method with 2-hydroxypropyl-ß-cyclodextrin (HPB) present in the perfusate. Two physical models were examined: the flat surface model in which the intestinal wall was treated as a hollow, smooth, circular cylinder, and the villus model in which the intestinal surface allowed for the presence of villi. Capric acid absorption was found to be essentially 100% aqueous boundary layer controlled at low HPB concentrations and increasingly membrane controlled at the higher HPB concentrations. Theoretical calculations based on the experimental data and model parameters were found to be consistent with: at low HPB concentrations, capric acid was mainly absorbed at the villus tips and there was very little capric acid penetration into the intervillus space; in contrast, at 50 mM HPB, there was considerable capric acid penetration into the intervillus space, this corresponding to around a 4.5-fold increase in the accessible area for absorption when compared with 0 mM HPB.

Transport of a lipophilic ionizable permeant (capric acid) across a lipophilic membrane (silicone polymer membrane) from aqueous buffered solutions in the presence of hydroxypropyl-ß-cyclodextrin.

The present study describes a physical model approach applicable to understanding the transport of highly lipophilic, ionizable drugs across a lipophilic membrane between two aqueous compartments in the presence of a cyclodextrin in the aqueous phase. Model predictions were compared with experimental results of capric acid (HA) transport across a silicone polymer membrane in the presence and in the absence of 2-hydroxypropyl-ß-cyclodextrin (HPB) in the aqueous phase over wide ranges of conditions. Key parameters entering into the physical model calculations were the HA-HPB and the A(-)-HPB binding constants, the unionized and ionized free and the complexed HA species diffusion coefficients, the HA pKa, the HA intrinsic silicone polymer membrane permeability coefficient, and the aqueous boundary layer thickness. All of these key parameters were determined from independent or essentially independent experiments. The agreement between the model predictions and the experiments were generally quite good over the entire ranges of the studied independent variables. The results of this study provide an approach that is useful in the mechanistic understanding of how cyclodextrins may enhance the passive absorption of highly lipophilic, low solubility drug molecules in the intestinal tract.

Systematic studies on the paracellular permeation of model permeants and oligonucleotides in the rat small intestine with chenodeoxycholate as enhancer.

The objective of this study was to mechanistically and quantitatively analyze chenodeoxycholate-enhanced paracellular transport of polar permeants and oligonucleotides in the rat jejunum and ileum. Micellar chenodeoxycholate solutions were used to perturbate the tight junctions. Supporting studies included assessment of the aqueous boundary layer (ABL) with ABL-controlled permeants, measurements of the permeability coefficients and fluxes of the bile acid in dilute and micellar concentrations, and determinations of pore sizes with paracellular probes (urea, mannitol, and raffinose). The paracellular permeability coefficients, P(para), of two model oligonucleotides (ON3 and ON6; 12- and 24-mers with 11 and 23 negative charges, respectively) were determined. The enhanced permeabilities paralleled the increased fluxes of micellar bile salt solutions into mesenteric blood and the opening of the tight junctions as compared to controls. As the pore radius increased from 0.7 nm to a maximum of 2.4 nm in the jejunum and ileum, the absorption of ON3 was enhanced up to sixfold in the jejunum and about 14-fold in the ileum with P(para) values between 0.5 x 10(-6) and 6 x 10(-6) cm/s, whereas ON6 was enhanced up to twofold in the jejunum and fivefold in the ileum with permeabilities between 0.3 x 10(-6) and 2 x 10(-6) cm/s.

Biophysical characterization of a large conductance anion channel in hypodermal membranes of the gastrointestinal nematode, Ascaris suum.

Patch-clamp recordings from muscle- and cuticle-facing hypodermal membranes of the gastrointestinal nematode Ascaris suum reveal a high-conductance, voltage- sensitive Ca(2+) -dependent Cl(-) channel. The hypodermal channel has a conductance of 195 pS in symmetrical 160 mM NaCl. The open probability of the channel is highly voltage-sensitive, and channel activity is not observed when Ca(2+) is reduced to <100 microM. The channel is permeable to organic anions that are major end-products of carbohydrate metabolism in A. suum, including acetate, butyrate and 2-methylvalerate. The conductances and relative permeabilities of these organic anions are inversely related to size, with 2-methylvalerate being only approximately 3% as permeable as Cl(-). The diameter of the channel pore was 12.3+/-0.2 A, calculated from the relative permeability coefficients of Cl(-) and the organic anions. Results of this study are consistent with the hypothesis that the large conductance anion channel in A. suum hypodermal membranes provides a low energy pathway for organic anion excretion from the hypodermal compartment, followed by diffusion across the aqueous channels of the cuticle matrix.

A systematic examination of the in vitro Ussing chamber and the in situ single-pass perfusion model systems in rat ileum permeation of model solutes.

In situ and in vitro intestinal absorption in the rat ileum was systematically studied and mechanistically quantified in terms of permeability coefficients (P) of a series of [(3)H]steroids as model transcellular permeants, [(3)H]taurocholate utilizing the active membrane transport systems to define the aqueous boundary layer (ABL), and [(14)C]urea and [(14)C]mannitol as pore-hindered paracellular diffusants. In situ single-pass perfusion experiments were performed in isolated ileal segments and blood samples were collected from the cannulated mesenteric vein. For the in vitro experiments, an excised, serosal and muscular layer-removed, ileal tissue was mounted in the Ussing chamber diffusion cells. In situ and in vitro P values versus logarithm of the partition coefficient in n-octanol/water (log K) of the steroids were characterized by a sigmoidal-shaped curve in which plateau values were attained for the highly lipophilic steroids with log K greater, similar 2.5. The in situ and in vitro transport barriers in series were viewed as ABL/mucosal epithelium and ABL/mucosal epithelium/submucosal tissue, respectively. Within this framework and the use of experimental strategies and theoretical reasoning, the transport barriers of the steroids were quantitatively delineated and the rate-determining barriers identified. In the plateau region, the analyses indicate that the in situ absorption of the lipophilic steroids was essentially ABL controlled, whereas the in vitro absorption was about equally controlled by diffusion across the ABL and submucosal tissue. The in situ and in vitro pore radii of the paracellular route were 7.2 and 9.2 A, respectively, and the difference was likely the result of perturbation of the tight junctions during the in vitro preparation of the ileal tissue.