Artificial Membrane Permeability Assay Research Articles

Discovery of water-soluble semicarbazide-containing sulfonamide derivatives possessing favorable anti-glaucoma effect in vivo and drug-like properties

In order to obtain topical and non-irritating anti-glaucoma drugs, novel semicarbazide-containing sulfonamide derivatives were designed and synthetized by sugar tail method in this study. The hydrophilic monosaccharides were expected to form interaction with the hydrophilic site of hCA II meanwhile the linker semicarbazides are used to further enhance water solubility, and more importantly, regulate the pH values of the target compounds in aqueous solution. First, all target compounds were synthesized and evaluated for their CA inhibitory activities. The results showed our target compounds demonstrated comparable activity to the positive control drug acetazolamide. The best derivative 11d exhibits an IC50 value of 14 nM for hCA II and 2086-fold selectivity over CA I. Subsequently, physicochemical properties study showed that the target compounds displayed very good water solubility (up to 3 %) and neutral pH value in solutions. Meanwhile, the artificial membrane permeability assay was performed to verify that the target compound could also pass through the membrane structure despite their strong water solubility. In the glaucomatous rabbit eye model, the applied topically representative compounds showed strongly lowered intraocular pressure (IOP), as 1 % or 2 % water solutions. Subsequent drug-like evaluation showed our target compounds possessed low hemolysis effect and low cytotoxicity toward human corneal epithelial cell line. Also, it was not found that these target compounds had significant inhibition of hERG and CYP. In addition, these novel analogs also displayed good liver microsomal metabolic stability and plasma stability. Finally, docking studies provided the rational binding modes of representative compounds in complex with hCA II. Taken together, these results suggested that compound 11d may be a promising hCA II inhibitor deserving further development.

The Effect of Isoniazid–Maltitol Solid Dispersions on Aqueous Solubility and Permeability

Maltitol (MAL) is a well-known polyol with potential pharmaceutical applications. Unlike other polyols, its utilization as a carrier for solid dispersions (SDs) has not been adequately investigated. This research studied the feasibility of MAL as an SD carrier to enhance the biopharmaceutical properties of a BCS class I/III drug, isoniazid (INH). SDs of INH–MAL were prepared by the fusion method, and physicochemical characteristics were investigated to determine the solid-state habit, solubility and permeation enhancement of INH. Fourier-transform infrared (FT-IR) spectroscopy demonstrated significant peak broadening for the SDs consisting of a higher MAL concentration. Powder X-ray diffraction indicated a decrease in degree of crystallinity with increasing MAL concentration. Hot-stage microscopy (HSM) and scanning electron microscopy (SEM) revealed that INH–MAL molar ratios affect the type of SD prepared via the fusion method. Results from the equilibrium solubility studies indicated significant INH solubility improvement (p < 0.05) with SDs in comparison with the pure drug and physical mixtures. The artificial membrane permeation assay (PAMPA) of INH was positively affected by the presence of MAL. The results of the study indicated the potential for MAL as a carrier in the preparation of SDs for the solubility and/or permeability enhancement of drugs.

Lessons in Transcellular Membrane Transport Re-Learned

A brief note on the studies we have conducted on total and free drug concentration of thousands of drug discovery compounds in HeLa cells as measured by an approach inspired by the work of Professor Per Arturrsson. We conclude that the familiar QSAR equations of Corwin Hansch which were modelled as a bell shape by using logP and -logP2 terms can be similarly seen in our results and this can be interpreted with the aid of chromatographic Immobilised Artificial Membrane measurements. We also point out the differences between our measurements and those widely used based on Artificial Membrane Permeability Assays.

Solid dispersions with carbamazepine: Optimization of formulation, characterization and examination of long-term stability

Solid dispersions are defined as dispersions of one or more active pharmaceutical ingredients in inert solid-state carriers. They are made with the aim to increase solubility and the dissolution rate of low solubility active pharmaceutical ingredients, with the subsequent increase in their bioavailability. The aim of this study was the development and optimization of solid dispersion formulations with carbamazepine, using D-optimal experimental design, in order to increase the dissolution rate of the selected model drug. By using the method of experimental mixture design, solid dispersions were formulated by varying the ratio of carbamazepine (30-50 %), Gelucire? 44/14 (20-40 %) and Soluplus? polymer (30-50 %) (input parameters). Sixteen formulations were made and used for in vitro testing of the carbamazepine dissolution rate. The observed output parameters were the percentages of carbamazepine released after 10, 20, 30, 45, and 60 minutes. After the data analysis, three test formulations were chosen from different parts of the optimization area. They were prepared and the carbamazepine dissolution rate was determined, followed by stability assessment for 24 months under ambient conditions (25?C, 40 % RH). The highest dissolution rate of carbamazepine from solid dispersions (more than 80 % in 30 minutes) was achieved at the carbamazepine mass fraction of about 40 %, Soluplus? of about 45 % and Gelucire? 44/14 of about 25 %. Comparing the predicted and the experimental obtained release rate profiles of carbamazepine from the three prepared optimized formulations, a significant compliance of the results was observed (f1<15; f2>50). The application of the PAMPA (Parallel Artificial-Membrane Permeability Assay) test has shown that carbamazepine premeability was maintained and mildly increased in two out of the three tested optimzed solid state formulations. Raman spectroscopy, FT-IR and DSC analyes showed that in the three optimized solid dispersions, after preparation and 24 months of storage, interactions between carbamazepine and the excipients were not present and that carbamazepine remained in the single pharmacologically active crystal polymorph form III. Proper selection of solid dispersion proportions of carbamazepine, Gelucire? 44/14 and Soluplus? may significantly increase the dissolution rate of the active substance, and the method of experimental mixture design can be successfully used for optimization of these formulations.

Transport mechanisms of flavanone aglycones across Caco-2 cell monolayers and artificial PAMPA membranes

We recently reported that flavanone aglycones (hesperetin, naringenin and eriodictyol) are efficiently absorbed via proton-coupled active transport, in addition to transcellular passive diffusion, in Caco-2 cells. Here, we aimed to evaluate in detail the absorption mechanisms of these flavanones, as well as homoeriodictyol and sakuranetin. We evaluated the absorption mechanisms of the above compounds by means of in vitro studies in Caco-2 cells in parallel with an artificial membrane permeation assay (PAMPA) under pH-gradient and iso-pH conditions. Comparison of the permeability characteristics of flavanones in Caco-2 cells and in PAMPA under these conditions, as well as a consideration of the physicochemical properties, indicated that hesperetin, naringenin, eriodictyol and homoeriodictyol were efficiently transported by passive diffusion according to the pH-partition hypothesis, except in the case of sakuranetin. However, transport of all flavanones were remarkably temperature-dependent, and was significantly reduced when Caco-2 cells were treated with amino acid-modifying reagents. Our data confirm that both passive diffusion and an active transport mechanism contribute to flavanone absorption through human intestinal epithelium.

A novel screening strategy to identify ABCB1 substrates and inhibitors

We tested the hypothesis whether data on ABCB1 ATPase activity and passive permeability can be used in combination to identify ABCB1 substrates and inhibitors. We determined passive permeability using an artificial membrane permeability assay (HDM-PAMPA) and ABCB1 function, i.e., vanadate-sensitive ATPase activity for a training set (40 INN drugs) and a validation set (26 development compounds). In parallel experiments, we determined ABCB1 function, i.e., vectorial transport in a Caco-2 cell monolayer, and ABCB1 inhibition, i.e., calcein AM extrusion out of K562-MDR cells, to cross-validate the results with cellular assays. We found that compounds that did not modulate ABCB1-ATPase did also not affect calcein AM extrusion and were not actively transported by ABCB1 in Caco-2 cell monolayers. The results corroborated the effect of passive permeability as an important covariate of active transport: active transport in Caco-2 monolayer was only apparent for compounds showing low passive permeability (<5.0 cmx10(-6)/s) in the HDM-PAMPA assay whereas compounds with high passive permeability (>50 cmx10(-6)/s) were shown to inhibit calcein AM efflux with IC50 values close to their respective Km value obtained for ABCB1-ATPase. The use of HDM-PAMPA in combination with ABCB1-ATPase offers a simple, inexpensive experimental approach capable of identifying ABCB1 inhibitors as well as transported substrates.