Parallel Artificial Membrane Permeability Research Articles

Rhizochalinin Exhibits Anticancer Activity and Synergizes with EGFR Inhibitors in Glioblastoma In Vitro Models.

Rhizochalinin (Rhiz) is a recently discovered cytotoxic sphingolipid synthesized from the marine natural compound rhizochalin. Previously, Rhiz demonstrated high in vitro and in vivo efficacy in various cancer models. Here, we report Rhiz to be highly active in human glioblastoma cell lines as well as in patient-derived glioma-stem like neurosphere models. Rhiz counteracted glioblastoma cell proliferation by inducing apoptosis, G2/M-phase cell cycle arrest, and inhibition of autophagy. Proteomic profiling followed by bioinformatic analysis suggested suppression of the Akt pathway as one of the major biological effects of Rhiz. Suppression of Akt as well as IGF-1R and MEK1/2 kinase was confirmed in Rhiz-treated GBM cells. In addition, Rhiz pretreatment resulted in a more pronounced inhibitory effect of γ-irradiation on the growth of patient-derived glioma-spheres, an effect to which the Akt inhibition may also contribute decisively. In contrast, EGFR upregulation, observed in all GBM neurospheres under Rhiz treatment, was postulated to be a possible sign of incipient resistance. In line with this, combinational therapy with EGFR-targeted tyrosine kinase inhibitors synergistically increased the efficacy of Rhiz resulting in dramatic inhibition of GBM cell viability as well as a significant reduction of neurosphere size in the case of combination with lapatinib. Preliminary in vitro data generated using a parallel artificial membrane permeability (PAMPA) assay suggested that Rhiz cannot cross the blood brain barrier and therefore alternative drug delivery methods should be used in the further in vivo studies. In conclusion, Rhiz is a promising new candidate for the treatment of human glioblastoma, which should be further developed in combination with EGFR inhibitors.

Evaluation of bottom-up modeling of the blood-brain barrier to improve brain penetration prediction via physiologically based pharmacokinetic modeling.

Predicting the brain penetration of drugs has been notoriously difficult; however, recently, permeability-limited brain models have been constructed. Lead optimization for central nervous system compounds often focuses on compounds that have low transporter efflux, where passive permeability could be a main driver in determining cerebrospinal fluid (CSF)/brain concentrations. The main objective of this study was to evaluate the translatability of passive permeability data generated from different in vitro systems and its impact on the prediction of human CSF/brain concentrations using physiologically-based pharmacokinetic (PBPK) modeling. In vitro data were generated using gMDCK and parallel artificial membrane permeability assay-blood-brain barrier for comparison and predictions using a quantitative structure-activity relationship model were also evaluated. PBPK modeling was then performed for seven compounds with moderate-high permeability and a range of efflux in vitro, and the CSF/brain mass concentrations and Kpuu were reasonably predicted. This work provides the first step of a promising approach using bottom-up PBPK modeling for CSF/brain penetration prediction to support lead optimization and clinical candidate selection.

Preparation of pectin-tannic acid coated core-shell nanoparticle for enhanced bioavailability and antihyperlipidemic activity of curcumin

As a functional polyphenolic compound, curcumin (CUR) has limited application due to its low solubility and bioavailability. In this work, curcumin loaded micelles self-assembled from disodium glycyrrhizin (Na2GA) was coated with pectin and tannic acid to construct a core-shell solid dispersion (SD-CUR). The assembly mechanism of the core-shell nanoparticle was clarified by molecular dynamics simulation. Improved solubility and in vitro release characteristics of SD-CUR in simulated gastric and intestinal juice were achieved. The parallel artificial membrane permeability (PAMPA) test confirmed that the permeability of curcumin in SD-CUR was significantly improved compared with the pure curcumin. Pharmacokinetic studies showed that the bioavailability was increased by 10 times. Intestinal in situ absorption study proved SD-CUR could enhance drug to transport across intestinal epithelial cells. Further hypolipidemic study demonstrated that the SD-CUR medication group could considerably reduce the levels of total cholesterol (TC), triglyceride (TG), and low density lipoprotein (LDL) in hyperlipidemia rats. These findings confirmed that SD-CUR had broad application prospects in the prevention and treatment of hyperlipidemia.

Distribution of Psychotropic Drugs Into Lipoproteins.

The aim of this pilot study was to investigate whether psychotropic drugs frequently analyzed in a routine therapeutic drug monitoring laboratory bind to low-density lipoproteins/very-low-density lipoproteins (LDL/VLDL) in human serum. Drug concentrations in 20 serum sample pools containing one psychotropic drug each, and in the LDL/VLDL fractions extracted from the same samples, were measured by triple quadrupole liquid chromatography tandem mass spectrometry. The membrane permeability of the drugs was measured using a Parallel Artificial Membrane Permeability Assay. Of the 20 antidepressants, antipsychotics, and antiepileptics examined, 7 drugs were detected in both the pooled serum samples and in the LDL/VLDL fraction. Binding of drugs to LDL/VLDL significantly correlated with high octanol: water partition coefficient (logP), high degree of protein binding, and a low polar surface area. The drugs found in LDL/VLDL, with the exception of aripiprazole, were also characterized by high or intermediate membrane permeability. The present results indicate that psychotropic drugs with certain characteristics bind to LDL/VLDL in blood. This further implies that lipoproteins could play an important role in drug transport.

Antinociceptive potency of a fluorinated cyclopeptide Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2

Opioid peptides and opiate drugs such as morphine, mediate their analgesic effects, but also undesired side effects, mostly through activation of the mu opioid receptor. However, delta- and kappa-opioid receptors can also contribute to the analgesic effects of opioids. Recent findings showed that simultaneous activation of multiple opioid receptors may result in additional analgesia with fewer side effects. Here, we evaluated the pharmacological profile of our formerly developed mixed mu/kappa-opioid receptor ligands, Dmt-c[D-Lys-Phe-Phe-Asp]NH2 (C-36) and Dmt-c[D-Lys-Phe-p-CF3-Phe-Asp]NH2 (F-81). The ability of these peptides to cross the blood–brain barrier was tested in the parallel artificial membrane permeability (PAMPA) assay. On the basis of the hot-plate test in mice after central and peripheral administration, analog F-81 was selected for the anti-nociceptive and anti-inflammatory activity assessment after peripheral administration.

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.

Selective and membrane-permeable small molecule inhibitors of nicotinamide N-methyltransferase reverse high fat diet-induced obesity in mice

There is a critical need for new mechanism-of-action drugs that reduce the burden of obesity and associated chronic metabolic comorbidities. A potentially novel target to treat obesity and type 2 diabetes is nicotinamide-N-methyltransferase (NNMT), a cytosolic enzyme with newly identified roles in cellular metabolism and energy homeostasis. To validate NNMT as an anti-obesity drug target, we investigated the permeability, selectivity, mechanistic, and physiological properties of a series of small molecule NNMT inhibitors. Membrane permeability of NNMT inhibitors was characterized using parallel artificial membrane permeability and Caco-2 cell assays. Selectivity was tested against structurally-related methyltransferases and nicotinamide adenine dinucleotide (NAD+) salvage pathway enzymes. Effects of NNMT inhibitors on lipogenesis and intracellular levels of metabolites, including NNMT reaction product 1-methylnicotianamide (1-MNA) were evaluated in cultured adipocytes. Effects of a potent NNMT inhibitor on obesity measures and plasma lipid were assessed in diet-induced obese mice fed a high-fat diet. Methylquinolinium scaffolds with primary amine substitutions displayed high permeability from passive and active transport across membranes. Importantly, methylquinolinium analogues displayed high selectivity, not inhibiting related SAM-dependent methyltransferases or enzymes in the NAD+ salvage pathway. NNMT inhibitors reduced intracellular 1-MNA, increased intracellular NAD+ and S-(5′-adenosyl)-l-methionine (SAM), and suppressed lipogenesis in adipocytes. Treatment of diet-induced obese mice systemically with a potent NNMT inhibitor significantly reduced body weight and white adipose mass, decreased adipocyte size, and lowered plasma total cholesterol levels. Notably, administration of NNMT inhibitors did not impact total food intake nor produce any observable adverse effects. These results support development of small molecule NNMT inhibitors as therapeutics to reverse diet-induced obesity and validate NNMT as a viable target to treat obesity and related metabolic conditions. Increased flux of key cellular energy regulators, including NAD+ and SAM, may potentially define the therapeutic mechanism-of-action of NNMT inhibitors.

Multifunctional Bile Acid Derivatives as Efficient RNA Transporters (Carriers)

Multifunctional Bile Acid Derivatives as Efficient RNA Transporters (Carriers) Abstract RNA interference (RNAi) is an emerging technology that is revolutionizing many strategic approaches to biochemical pathway analysis, drug discovery, and therapy. The siRNA can be used to suppress a specific target. Even though these molecules may have potential and strong utility function, many limitations make their clinical application difficult, including delivery problems, side effects due to off-target actions, disturbance of physiological functions of the cellular machinery involved in gene silencing, and induction of the innate immune response. This paper focus on efficient RNA transports using designed carriers, guanidine steroids. It presents the synthesis of guanidine substituted bile acids (cholic acid, deoxycholic acid and lithocholic acid) by a simple method with moderate yield. The guanidine substituted compounds are shown to be very efficient tools in generating cellular delivery vehicles as determined by in vitro studies using PAMPA (parallel artificial membrane permeability) assay.

Cassette Incubation Followed By Bioanalysis Using High-Resolution Ms For In Vitro Adme Screening Assays

High-resolution MS (HRMS) has recently received a considerable interest in quantitative bioanalysis using full-scan acquisition mode. The benefits include complete elimination of compound-specific MS method development, and simultaneous collection of mass spectral data on both targeted and non-targeted components. One additional advantage that has not been widely discussed is its suitability for simultaneous quantitation of, theoretically, an unlimited number of compounds, which is not possible with selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. We took advantage of this unique bioanalytical capability of HRMS and developed a novel in vitro ADME workflow of cassette incubation of as many as 32 compounds, followed by quantitative bioanalysis using full-scan acquisition on an Orbitrap HRMS. The workflow was evaluated for a serum protein-binding assay and a parallel artificial membrane permeability (PAMPA) assay. The bioanalytical assay displayed acceptable sensitivity, selectivity and linearity for all compounds in the cassettes, and the biological results obtained using this approach were similar to those from discrete incubation and analysis, demonstrating the feasibility of the workflow. Additional benefits of this platform include a saving of analysis time due to the reduced sample numbers from the cassette approach, as well as cost saving due to the reduction in the required assay reagents. Cassette incubation with bioanalysis using HRMS is a feasible approach for high-throughput in vitro ADME assays evaluated in this study.