Predicting the intrinsic membrane permeability of Caco-2/MDCK cells by the solubility-diffusion model.

Membrane permeability is one of the main determinants for the absorption, distribution, metabolism and excretion of compounds and is therefore of crucial importance for successful drug development. Experiments with artificial phospholipid membranes have shown that the intrinsic membrane permeability (P0) of compounds is well-predicted by the solubility-diffusion model (SDM). However, using the solubility-diffusion model to predict the P0 of biological Caco-2 and MDCK cell membranes has proven unreliable so far. Recent publications revealed that many published P0 extracted from Caco-2 and MDCK experiments are incorrect. In this work, we therefore used a small self-generated set as well as a large revised set of experimental Caco-2 and MDCK data from literature to compare experimental and predicted P0. The P0 extracted from Caco-2 and MDCK experiments were systematically lower than the P0 predicted by the solubility-diffusion model. However, using the following correlation: log P0,Caco-2/MDCK = 0.84 log P0,SDM - 1.85, P0 of biological Caco-2 and MDCK cell membranes was well-predicted by the solubility-diffusion model.

Pitfalls in evaluating permeability experiments with Caco-2/MDCK cell monolayers.

When studying the transport of molecules across biological membranes, intrinsic membrane permeability (P0) is more informative than apparent permeability (Papp), because it eliminates external (setup-specific) factors, provides consistency across experiments and mechanistic insight. It is thus an important building block for modeling the total permeability in any given scenario. However, extracting P0 is often difficult, if not impossible, when the membrane is not the dominant transport resistance. In this work, we set out to analyze Papp values measured with Caco-2/MDCK cell monolayers of 69 literature references. We checked the Papp values for a total of 318 different compounds for the extractability of P0, considering possible limitations by aqueous boundary layers, paracellular transport, recovery issues, active transport, a possible proton flux limitation, and sink conditions. Overall, we were able to extract 77 reliable P0 values, which corresponds to about one quarter of the total compounds analyzed, while about half were limited by the diffusion through the aqueous layers. Compared to an existing data set of P0 values published by Avdeef, our approach resulted in a much higher exclusion of compounds. This is a consequence of stricter compound- and reference-specific exclusion criteria, but also because we considered possible concentration-shift effects due to different pH values in the aqueous layers, an effect only recently described in literature. We thus provide a consistent and reliable set of P0, e.g. as a basis for future modeling.

Symptoms of older orthopedic and rheumatic patients : A telephone survey about symptoms, symptom communication, treatment and further support after hospital discharge.

BACKGROUND: In older multimorbid orthopedic and rheumatic patients, data on symptoms besides pain or reduced mobility are rarely published. OBJECTIVE: We investigated patients' perspectives on their symptoms after hospital discharge. MATERIAL AND METHODS: Orthopedic and rheumatic patients aged over 70 years were asked via telephone interviews about (i) their symptoms, (ii) communication, (iii) treatment, and (iv) support. RESULTS: (i) The 60 participants (35 women and 25 men) reported a median of 6 (min-max: 1-14) different symptoms, of which 86% (356 of 415) had existed before hospitalization, (ii) patients did not communicate 28% (117) of symptoms to either healthcare professionals, family or friends and (iii) 52 (87%) patients desired improvement. Of the 280 most impairing symptoms, 19% (52) were not treated at all. (iv) Almost all patients (59; 98%) considered it easy to obtain support. CONCLUSION: Remarkably, many symptoms were not communicated or treated despite the patients having been hospitalized.

Revisiting the pKa-Flux method for determining intrinsic membrane permeability.

Intrinsic membrane permeability is one of several factors that critically determine the intestinal absorption of a chemical. The intrinsic membrane permeability of a chemical is usually extracted from transwell experiments with Caco-2 or MDCK cells, preferably by the pKa-Flux method, which is considered the method of choice when aqueous boundary layer effects need to be excluded. The pKa-Flux method has two variants, the iso-pH method, where apical and basolateral pH are equal, and the gradient-pH method, where apical and basolateral pH are different. The most commonly used method is the gradient-pH method, as it is intended to reflect the pH-conditions in the gastrointestinal tract. However, concentration-shift effects caused by the applied pH-difference between apical and basolateral compartment in the gradient-pH method have not been considered in the evaluation of the experimental data in the past. Consequently, incorrect intrinsic membrane permeabilities have been determined. In this work, we present a revised method for extracting the intrinsic membrane permeability from gradient-pH data that considers concentration-shift effects in the basolateral aqueous boundary layer and filter as well as in the cytosol. Furthermore, we propose the use of the iso-pH method, where only concentration-shift effects in the cytosol need to be considered, as an alternative to the gradient-pH method. We use the five lipophilic bases amantadine, chloroquine, propranolol, venlafaxine and verapamil as examples to compare gradient-pH method and iso-pH method with regard to the extractability of the intrinsic membrane permeability. For lipophilic bases, the iso-pH method proves to be advantageous. All intrinsic membrane permeabilities determined in this work were substantially higher than the intrinsic membrane permeabilities reported in literature.

Impact of cholesterol and sphingomyelin on intrinsic membrane permeability.

Transwell experiments with Caco-2 or MDCK cells are the gold standard for determining the intestinal permeability of chemicals. The intrinsic membrane permeability (P0), that can be extracted from these experiments, might be comparable to P0 measured in black lipid membrane (BLM) experiments and P0 predicted by the solubility-diffusion model. Unfortunately, the overlap between experimental P0,Caco-2/MDCK and P0,BLM data is very small. So far, differences between both approaches have been attributed to the cholesterol and sphingomyelin content of cell membranes, but the database is too sparse to thoroughly test this theory. To create a diverse dataset, we measured P0,BLM of ten chemicals in BLM experiments using DPhPC and DPhPC/cholesterol/sphingomyelin membranes. The results were compared to predicted BLM data and experimental Caco-2/MDCK data obtained from literature. While P0,BLM of all chemicals was well predicted by the solubility-diffusion model, P0,Caco-2/MDCK was only predictable for rather hydrophilic compounds with logarithmic hexadecane/water partition coefficients below -0.5. The effect of cholesterol and sphingomyelin on P0,BLM was negligibly small.