Can membrane permeability of zwitterionic compounds be predicted by the solubility-diffusion model?

Zwitterions contain both positively and negatively charged functional groups, resulting in an overall net neutral charge. Nevertheless, the membrane permeability of the zwitterionic form of a compound is assumed to be much lower than the permeability of the uncharged neutral form. Although a significant proportion of pharmaceuticals are zwitterionic, it has not been clear so far whether their permeability is dominated by the permeation of the zwitterionic or the neutral form, since neutral fractions are often quite low as compared to the zwitterionic fraction. This complicates the in silico prediction of the permeability of zwitterionic compounds. In this work, we re-evaluated existing in vitro permeability data from literature measured with Caco-2/MDCK cell assays, using more strict exclusion criteria for effects like diffusion limitation by the aqueous boundary layers, paracellular transport, active transport and retention. Using this re-evaluated data set, we show that extracted intrinsic permeabilities of the neutral fraction are well predicted by the solubility-diffusion model (RMSE = 1.21; n = 18) if the permeability of the zwitterionic species is assumed negligible. Our work thus suggests that only the neutral species is relevant for the membrane permeability of zwitterionic compounds, and that membrane permeability of zwitterionic compounds is indeed predictable by the solubility-diffusion model.

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.

Effects of Aqueous Boundary Layers and Paracellular Transport on the Efflux Ratio as a Measure of Active Transport Across Cell Layers.

The efflux ratio (ER), determined by Caco-2/MDCK assays, is the standard in vitro metric to establish qualitatively whether a compound is a substrate of an efflux transporter. However, others have also enabled the utilisation of this metric quantitatively by deriving a relationship that expresses the ER as a function of the intrinsic membrane permeability of the membrane (P0) as well as the permeability of carrier-mediated efflux (Ppgp). As of yet, Ppgp cannot be measured directly from transport experiments or otherwise, but the ER relationship provides easy access to this value if P0 is known. However, previous derivations of this relationship failed to consider the influence of additional transport resistances such as the aqueous boundary layers (ABLs) and the filter on which the monolayer is grown. Since single fluxes in either direction can be heavily affected by these experimental artefacts, it is crucial to consider the potential impact on the ER. We present a model that includes these factors and show both mathematically and experimentally that this simple ER relationship also holds for the more realistic scenario that does not neglect the ABLs/filter. Furthermore, we also show mathematically how paracellular transport affects the ER, and we experimentally confirm that paracellular dominance reduces the ER to unity and can mask potential efflux.

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.

A framework for understanding the bioconcentration of surfactants in fish.

Surfactants are a class of chemicals released in large quantities to water, and therefore bioconcentration in fish is an important component of their safety assessment. Their structural diversity, which encompasses nonionic, anionic, cationic and zwitterionic molecules with a broad range of lipophilicity, makes their evaluation challenging. A strong influence of environmental pH adds a further layer of complexity to their bioconcentration assessment. Here we present a framework that penetrates this complexity. Using simple equations derived from current understanding of the relevant underlying processes, we plot the key bioconcentration parameters (uptake rate constant, elimination rate constant and bioconcentration factor) as a function of its membrane lipid/water distribution ratio and the neutral fraction of the chemical in water at pH 8.1 and at pH 6.1. On this chemical space plot, we indicate boundaries at which four resistance terms (perfusion with water, transcellular, paracellular, and perfusion with blood) limit transport of surfactants across the gills. We then show that the bioconcentration parameters predicted by this framework align well with in vivo measurements of anionic, cationic and nonionic surfactants in fish. In doing so, we demonstrate how the framework can be used to explore expected differences in bioconcentration behavior within a given sub-class of surfactants, to assess how pH will influence bioconcentration, to identify the underlying processes governing bioconcentration of a particular surfactant, and to discover knowledge gaps that require further research. This framework for amphiphilic chemicals may function as a template for improved understanding of the accumulation potential of other ionizable chemicals of environmental concern, such as pharmaceuticals or dyes.

Mechanistic modeling of the bioconcentration of (super)hydrophobic compounds in Hyalella azteca.

Bioconcentration tests using the freshwater amphipod Hyalella azteca as an alternative to conventional fish tests have recently received much attention. An appropriate computational model of H. azteca could help in understanding the mechanisms behind bioconcentration, in comparison to the fish as test organism. We here present the first mechanistic model for H. azteca that considers the single diffusive processes in the gills and gut. The model matches with the experimental data from the literature quite well when appropriate physiological information is used. The implementation of facilitated transport was essential for modeling. Application of the model for superhydrophobic compounds revealed binding to organic matter and the resulting decrease in bioavailable fraction as the main reason for the observed counterintuitive decrease in uptake rate constants with increasing octanol/water partition coefficient. Furthermore, estimations of the time needed to reach steady state indicated that durations of more than a month could be needed for compounds with a log Kow > 8, limiting the experimental applicability of the test. In those cases, model-based bioconcentration predictions could be a preferable approach, which could be combined with in vitro biotransformation measurements. However, our sensitivity analysis showed that the uncertainty in determining the octanol/water partition coefficients is a strong source of error for superhydrophobic compounds.

Modeling of SGLT1 in Reconstituted Systems Reveals Apparent Ion-Dependencies of Glucose Uptake and Strengthens the Notion of Water-Permeable Apo States.

The reconstitution of secondary active transporters into liposomes shed light on their molecular transport mechanism. The latter are either symporters, antiporters or exchangers, which use the energy contained in the electrochemical gradient of ions to fuel concentrative uptake of their cognate substrate. In liposomal preparations, these gradients can be set by the experimenter. However, due to passive diffusion of the ions and solutes through the membrane, the gradients are not stable and little is known on the time course by which they dissipate and how the presence of a transporter affects this process. Gradient dissipation can also generate a transmembrane potential (VM). Because it is the effective ion gradient, which together with VM fuels concentrative uptake, knowledge on how these parameters change within the time frame of the conducted experiment is key to understanding experimental outcomes. Here, we addressed this problem by resorting to a modelling approach. To this end, we mathematically modeled the liposome in the assumed presence and absence of the sodium glucose transporter 1 (SGLT1). We show that 1) the model can prevent us from reaching erroneous conclusions on the driving forces of substrate uptake and we 2) demonstrate utility of the model in the assignment of the states of SGLT1, which harbor a water channel.

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.

Screening of 6000 Compounds for Uncoupling Activity: A Comparison Between a Mechanistic Biophysical Model and the Structural Alert Profiler Mitotox.

Protonophoric uncoupling of phosphorylation is an important factor when assessing chemicals for their toxicity, and has recently moved into focus in pharmaceutical research with respect to the treatment of diseases such as cancer, diabetes, or obesity. Reliably identifying uncoupling activity is thus a valuable goal. To that end, we screened more than 6000 anionic compounds for in vitro uncoupling activity, using a biophysical model based on ab initio COSMO-RS input parameters with the molecular structure as the only external input. We combined these results with a model for baseline toxicity (narcosis). Our model identified more than 1250 possible uncouplers in the screening dataset, and identified possible new uncoupler classes such as thiophosphoric acids. When tested against 423 known uncouplers and 612 known inactive compounds in the dataset, the model reached a sensitivity of 83% and a specificity of 96%. In a direct comparison, it showed a similar specificity than the structural alert profiler Mitotox (97%), but much higher sensitivity than Mitotox (47%). The biophysical model thus allows for a more accurate screening for uncoupling activity than existing structural alert profilers. We propose to use our model as a complementary tool to screen large datasets for protonophoric uncoupling activity in drug development and toxicity assessment.

Exploring the octanol-water partition coefficient dataset using deep learning techniques and data augmentation.

Today more and more data are freely available. Based on these big datasets deep neural networks (DNNs) rapidly gain relevance in computational chemistry. Here, we explore the potential of DNNs to predict chemical properties from chemical structures. We have selected the octanol-water partition coefficient (log P) as an example, which plays an essential role in environmental chemistry and toxicology but also in chemical analysis. The predictive performance of the developed DNN is good with an rmse of 0.47 log units in the test dataset and an rmse of 0.33 for an external dataset from the SAMPL6 challenge. To this end, we trained the DNN using data augmentation considering all potential tautomeric forms of the chemicals. We further demonstrate how DNN models can help in the curation of the log P dataset by identifying potential errors, and address limitations of the dataset itself.

Predicting Uncoupling Toxicity of Organic Acids Based on Their Molecular Structure Using a Biophysical Model.

We present a purely mechanistic model to predict protonophoric uncoupling activity ECw of organic acids. All required input information can be derived from their chemical structure. This makes it a convenient predictive model to gain valuable information on the toxicity of organic chemicals already at an early stage of development of new commercial chemicals (e.g., in agriculture or pharmaceutical industries). A critical component of the model is the consideration of the possible formation of heterodimers from the neutral and anionic monomer, and its permeation through the membrane. The model was tested against literature data measured in chromatophores, submitochondrial particles, isolated mitochondria, and intact green algae cells with good success. It was also possible to reproduce pH-dependencies in isolated mitochondria and intact cells. Besides the prediction of the ECw, the mechanistic nature of the model allows researchers to draw direct conclusions on the impact of single input factors such as pH- and voltage-gradients across the membrane, the anionic and neutral membrane permeability, and the heterodimerization constant. These insights are of importance in drug design or chemical regulation.

COSMOperm: Mechanistic Prediction of Passive Membrane Permeability for Neutral Compounds and Ions and Its pH Dependence.

We present a new and entirely mechanistic COSMOperm method to predict passive membrane permeabilities for neutral compounds, as well as anions and cations. The COSMOperm approach is based on compound-specific free energy profiles within a membrane of interest from COSMO-RS (conductor-like screening model for realistic solvation) calculations. These are combined with membrane layer-specific diffusion coefficients, for example, in the water phase, the polar head groups, and the alkyl tails of biochemical phospholipid bilayers. COSMO-RS utilizes first-principle quantum chemical structures and physically sound intermolecular interactions (electrostatic, hydrogen bond, and van der Waals). For this reason, it is unbiased toward different application scenarios, such as in cosmetics and industrial chemical or pharmaceutical industries. A fully predictive calculation of passive permeation through phospholipid bilayer membranes results in a performance of r2 = 0.92; rmsd = 0.90 log10 units for neutral compounds and anions, as compared to gold standard black lipid membrane experiments. It will be demonstrated that new membrane types can be generated by the related COSMOplex method and directly used for permeability studies by COSMOperm.

Membrane/Water Partitioning and Permeabilities of Perfluoroalkyl Acids and Four of their Alternatives and the Effects on Toxicokinetic Behavior.

The search for alternatives to bioaccumulative perfluoroalkyl acids (PFAAs) is ongoing. New, still highly fluorinated alternatives are produced in hopes of reducing bioaccumulation. To better estimate this bioaccumulative behavior, we performed dialysis experiments and determined membrane/water partition coefficients, Kmem/w, of six perfluoroalkyl carboxylic acids (PFCAs), three perfluoroalkanesulfonic acids, and four alternatives. We also investigated how passive permeation might influence the uptake kinetics into cells, measuring the passive anionic membrane permeability Pion through planar lipid bilayers for six PFAAs and three alternatives. Experimental Kmem/w and Pion were both predicted well by the COSMO-RS theory (log RMSE 0.61 and 0.46, respectively). Kmem/w values were consistent with the literature data, and alternatives showed similar sorption behavior as PFAAs. Experimental Pion values were high enough to explain observed cellular uptake by passive diffusion with no need to postulate the existence of active uptake processes. However, predicted pKa and neutral permeabilities suggest that also the permeation of the neutral species should be significant in case of PFCAs. This can have direct consequences on the steady-state distribution of PFAAs across cell membranes and thus toxicity. Consequently, we propose a model to predict pH-dependent baseline toxicity based on Kmem/w, which considers the permeation of both neutral and anionic species.

[Interdisciplinary Management of Sellar Masses]. / Interdisziplinäres Management von Hypophysentumoren.

Interdisciplinary Management of Sellar Masses Abstract. Sellar masses may present with an impairment of pituitary function (hypopituitarism), hormone hypersecretion (prolactinoma, acromegaly, glucocorticoid excess) or neurological symptoms (visual impairment, headache). An increasing number of them is discovered as an incidentaloma. Among the various entities, benign pituitary adenomas and cystic lesions are most frequently encountered. The work-up includes a laboratory evaluation for hormone hyper- or hyposecretion and an MRI of the pituitary gland. If the optic chiasm is compromised, a visual field examination is mandatory. Except for prolactinomas, symptomatic sellar masses are usually resected via an endoscopic transsphenoidal approach. If a total resection is not feasible because of the invasion of surrounding structures, debulking to relieve pressure from the optic chiasm is the primary goal and radiotherapy may be considered. Residual hormone excess can be treated medically. In the early postoperative period special attention to the development and treatment of disordered body water homeostasis and hypopituitarism is crucial. Interdisciplinary work-up and decision making are of utmost importance and will offer the best management.

Passive Permeability of Planar Lipid Bilayers to Organic Anions.

The membrane permeability P of organic ions was reported to be governed by the structure of the permeating molecule. Thus far, it is unclear whether the ion structure alters membrane partition or translocation proper across the membrane. Here, we obtained P values for 24 anionic compounds (18 concrete values, 6 upper limits) measuring the current that they carry through folded planar lipid bilayers. The P values range over more than 10 log units. Our measured permeability values correlate well (r = 0.95; logRMSE 0.74) with the hexadecane/water partition coefficients of the respective chemicals predicted by the COSMO-RS theory. Other attempts to predict P from the partition coefficient of the neutral molecule and from the solvation energy (Born energy) that opposes transfer into the membrane once the molecule is charged were unsuccessful. The uncertainties in assigning an effective radius to nonspherical molecules were much too large. The observation underlines that the actual structure of the molecules needs to be considered to predict partition and thus P by the solubility-diffusion model.