Isolation of MDCK cells with low expression of mdr1 gene and their use in membrane permeability screening

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P--glycoprotein activity. Clone isolation via the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery.

A Bidirectional Permeability Assay for beyond Rule of 5 Compounds

Bidirectional permeability measurement with cellular models grown on Transwell inserts is widely used in pharmaceutical research since it not only provides information about the passive permeability of a drug, but also about transport proteins involved in the active transport of drug substances across physiological barriers. With the increasing number of investigative drugs coming from chemical space beyond Lipinski’s Rule of 5, it becomes more and more challenging to provide meaningful data with the standard permeability assay. This is exemplified here by the difficulties we encountered with the cyclic depsipeptides emodepside and its close analogs with molecular weight beyond 1000 daltons and cLogP beyond 5. The aim of this study is to identify potential reasons for these challenges and modify the permeability assays accordingly. With the modified assay, intrinsic permeability and in vitro efflux of depsipeptides could be measured reliably. The improved correlation to in vivo bioavailability and tissue distribution data indicated the usefulness of the modified permeability assay for the in vitro screening of compounds beyond the Rule of 5.

Deletion of the Imprinted Phlda2 Gene Increases Placental Passive Permeability in the Mouse

Genomic imprinting, an epigenetic phenomenon that causes the expression of a small set of genes in a parent-of-origin-specific manner, is thought to have co-evolved with placentation. Many imprinted genes are expressed in the placenta, where they play diverse roles related to development and nutrient supply function. However, only a small number of imprinted genes have been functionally tested for a role in nutrient transfer capacity in relation to the structural characteristics of the exchange labyrinthine zone. Here, we examine the transfer capacity in a mouse model deficient for the maternally expressed Phlda2 gene, which results in placental overgrowth and a transient reduction in fetal growth. Using stereology, we show that the morphology of the labyrinthine zone in Phlda2−/+ mutants is normal at E16 and E19. In vivo placental transfer of radiolabeled solutes 14C-methyl-D-glucose and 14C-MeAIB remains unaffected at both gestational time points. However, placental passive permeability, as measured using two inert hydrophilic solutes (14C-mannitol; 14C-inulin), is significantly higher in mutants. Importantly, this increase in passive permeability is associated with fetal catch-up growth. Our findings uncover a key role played by the imprinted Phlda2 gene in modifying placental passive permeability that may be important for determining fetal growth.

The Block Relevance (BR) Analysis Makes the Choice of Methods for Measuring Lipophilicity and Permeability Safer and Speeds Up Drug Candidate Prioritization

The Block Relevance (BR) analysis with its recent implementation in MATLAB is a computational tool that allows deconvoluting the balance of intermolecular interactions governing a given drug discoveryrelated phenomenon described by a QSPR/PLS model. Here we discuss a few applications to show how BR analysis can make faster and more efficient the assessment of the drug-likeness of drug candidates. First, we describe how identifying the best chromatographic system provides reliable log Poct surrogates and log P in apolar environments. Then we focus on permeability and show how BR analysis allows to check the universality of passive permeability among cell types and the identification of the PAMPA method that provides the same picture in terms of balance of intermolecular interactions as cell-based systems.

The Effects of Multiple Exposure Ethanol on Barrier Tightness and Passive Permeability in a Human Stem Cell Derived Blood-Brain Barrier Model.

Background and Hypothesis: Numerous animal studies have shown the negative aspects of ethanol at sustained concentrations as well as the intense depressive effects of multiple ethanol exposures on the central nervous system. Chronic ethanol use as a possible contributor to earlier onset neurocognitive decline has been indicated. A portion of these studies have implicated that ethanol exposure induces blood-brain barrier (BBB) impairment; however, these effects are not completely understood. In humans the BBB serves as a protective barrier that restricts the passage of nutrients, metabolites, and pathogens into the central nervous system from the blood and is essential in protecting the brain tissue from harmful substances. We hypothesize that multiple doses of pathologically-relevant ethanol will cause decreased BBB tightness and increase passive permeability. Experimental Design: In this study, we utilized brain microvascular endothelial cells (BMECs) derived from human induced pluripotent stem cells (iPSCs) to assess the effects ethanol has on barrier tightness and passive permeability through the BBB. BMECs were treated with multiple exposures of 50mM ethanol and transendothelial electrical resistance and sodium fluorescein permeabilities were measured. Trolox, a free radical scavenger, was used to identify if ethanol-induced barrier damage could be salvaged by reducing its oxidative impact. Results: Upon multiple exposure treatment with ethanol, iPSC-derived BMECs displayed diminished transendothelial electrical resistance and elevated sodium fluorescein permeability when compared to non-treated BMECs. Additionally, BMECs that were treated simultaneously with Trolox and ethanol had reduced barrier damage compared to ethanol treatment alone. Conclusion and Potential Impact: From these results, we conclude that multiple ethanol exposure-induced barrier damage in iPSC-derived BMECs, is in part due to elevated oxidative stress. Disruption of the BBB can potentiate a number of negative effects on the brain parenchyma and can lead to earlier onset neurocognitive decline. Alcohol’s impact on the BBB must be studied to ensure we limit these effects.

The Passive Permeability Landscape Around Geometrically Diverse Hexa- and Heptapeptide Macrocycles

Recent advances in DNA and mRNA encoding technologies have enabled the discovery of high-affinity macrocyclic peptides and peptide-like ligands against virtually any protein target of interest. Unfortunately, even the most potent biochemical leads from these screening technologies often have weak cellular activity due to poor absorption. Biasing such libraries towards passive cell permeability in the design phase would facilitate development of leads against intracellular targets. We set out to empirically evaluate the intrinsic permeability of thousands of geometrically diverse hexa- and heptapeptide scaffolds by permuting backbone stereochemistry and N-methylation, and by including peptoid and β-amino acid residues at select positions, with the goals of providing a resource for biasing library-based screening efforts toward passive membrane permeability and studying the effects of the backbone elements introduced on a large number of compounds. Libraries were synthesized via standard split-pool solid phase peptide synthesis, and passive permeability was measured in pools of 150 compounds using a highly multiplexed version of the parallel artificial mem-brane permeability assay (PAMPA) under sink conditions. Compounds were identified using CycLS, a high-resolution mass spectrometry-based method that uses stable isotopes to encode stereochemistry and matches MSMS data to virtual fragment libraries based on the expected macrocyclic products. From the compounds that were identified with high confidence, 823 hexameric and 1330 heptameric scaffolds had PAMPA permeability coefficients greater than 1x10-6 cm/s. The prevalence of high permeability compounds in these two libraries suggests that passive permeability is achievable for hexa- and heptapeptides with highly diverse backbone geometries.