Parallel Artificial Membrane Permeability Assay Research Articles

Comparison of in vitro membrane permeabilities of diverse environmental chemicals with in silico predictions

The parallel artificial membrane permeability assay (PAMPA) is widely used for estimating biomembrane permeabilities of experimental drugs in pharmaceutical research. However, there are few reports of studies using PAMPA to measure membrane permeabilities of chemicals of environmental concern (CECs) outside the pharmaceutical domain, many of which differ substantially from drugs in their physicochemical properties. We applied PAMPA methods simulating gastrointestinal (PAMPA-GIT) and blood-brain barrier (PAMPA-BBB) membranes under consistent conditions to 51 CECs, including some pharmaceuticals. A backward stepwise multivariate linear regression was implemented to explore the correlation between the differences of measured permeabilities from PAMPA-GIT and PAMPA-BBB and Abraham solute descriptors. In addition, a previously reported in silico model was evaluated by comparing predicted and measured permeability results. PAMPA-GIT and PAMPA-BBB experimental permeability results agreed relatively well. The backward stepwise multivariate linear regression identified excess molar refraction and polarizability to be significant at the 0.10 level in predicting the differences between PAMPA-GIT and PAMPA-BBB. The in silico model performed well – with predicted permeability of most compounds within two-fold of experimentally measured values. We found that CECs pose experimental challenges to the PAMPA method in terms of having lower solubility and lower stability compared to most drugs.

[Evaluation of Solubility and Membrane Permeability of Middle-Molecule Compounds Using Artificial Membranes and Living Cells].

In contrast to small molecules, middle molecules present a promising therapeutic modality owing to their elevated specificity, minimal adverse effects, capacity to target protein-protein interactions, and, unlike antibody-based drugs, their suitability for oral administration and intracellular target engagement. Post-oral administration, the paramount considerations encompass solubility and membrane permeability during the initial phase until the drug attains systemic circulation. Furthermore, penetration of the cell membrane is essential to accessing intracellular targets. We evaluated the solubility and membrane permeability of 965 compounds sourced from middle molecule libraries affiliated with Hokkaido University, Kitasato University, and the University of Tokyo. To gauge membrane permeability, we employed both the parallel artificial membrane permeability assay (PAMPA) and Caco-2 cell monolayers. Notably, while membrane permeability in Caco-2 cells exhibited an approximate threefold increase in comparison to PAMPA measurements, certain compounds demonstrated permeability levels less than one-third of those observed in Caco-2 cells. Recognizing the potential involvement of efflux transporters expressed in Caco-2 cells in these variations, we conducted additional assessments involving directional transport in the presence of a transporter inhibitor. Our findings suggest that nearly 80% of these compounds serve as substrates for efflux transporters. Considering the relevance of intracellular targets, we shifted our focus from membrane permeation to intracellular uptake, conducting simulations tailored to assess cellular uptake.

Enhanced bioavailability of curcumin amorphous nanocomposite prepared by a green process using modified starch

Curcumin (Cur), a bioactive compound extracted from plants, has attracted widespread attention due to its multiple pharmacological activities. However, the low bioavailability due to the inherent limitations in water solubility, chemical stability, and permeability poses great challenges for realizing its clinical potentials. In the current study, octenyl succinic anhydride-modified starch (OSAS), a renewable and biodegradable biopolymer, was employed to fabricate Cur amorphous composite nanoparticles (Cur/OSA-S NPs) through a solvent-free pH-driven method with the aim to enhance Cur's bioavailability by improving its solubility and stability. Cur/OSA-S NPs, with mean sizes of about 128.9 ± 8.6 nm, encapsulation efficiencies of about 90.0 %, and the drug loading capacities around 51.0 ± 0.2 %, were successfully prepared. Cur was found to be dispersed within the composite nanoparticles in amorphous state as confirmed by the XRD and DSC characterizations. In addition, Cur/OSA-S NPs offers excellent storage, thermal and light stability, excellent re-dispersibility, and approximately 92 times better solubility than the original Cur. Furthermore, studies of dissolution and the parallel artificial membrane permeability assay (PAMPA) confirmed enhanced dissolution rates and in vitro permeabilities of Cur/OSA-S NPs. Cancer cell viability and uptake experiments revealed that Cur/OSA-S NPs possessed more potent inhibitory effects on cancer cell proliferation compared to the raw Cur. The results obtained from the current study demonstrated the effectiveness of OSA-S for manufacturing Cur amorphous composite nanoparticles with enhanced solubility, stability, and permeability, which might be valuable for further development of Cur based products for treatment of various diseases.

Synthesis and Neuroprotective Evaluation of Substituted Indanone/Benzofuranone and Piperidine Hybrids.

Based on the neuroprotection of butylphthalide and donepezil, a series of indanone/benzofuranone and piperidine hybrids were designed and synthesized for assessment of their neuroprotective activities, aiming to enhance the bioavailability and therapeutic efficacy of natural phthalide analogues. Within this study, it was observed that most indanone derivatives bearing 1-methylpiperidine in the tail segment demonstrated superior neuroprotective effects on the oxygen glucose deprivation/reperfusion (OGD/R)-induced rat primary neuronal cell injury model in vitro compared to benzofuranone compounds. Among the synthesized compounds, 11 (4, 14, 15, 22, 26, 35, 36, 37, 48, 49, and 52) displayed robust cell viabilities in the OGD/R model, along with favorable blood-brain barrier permeability as confirmed by the parallel artificial membrane permeability assay. Notably, compound 4 showed significant neuronal cell viabilities within the concentration range of 3.125 to 100 μM, without inducing cytotoxicity. Further results from in vivo middle cerebral artery occlusion/R experiments revealed that 4 effectively ameliorated ischemia-reperfusion injury, reducing the infarct volume to 18.45% at a dose of 40 mg/kg. This outcome suggested a superior neuroprotective effect compared to edaravone at 20 mg/kg, further highlighting the potential therapeutic efficacy of compound 4 in addressing neurological disorders.

Application of parallel artificial membrane permeability assay technique and chemometric modeling for blood-brain barrier permeability prediction of protein kinase inhibitors.

Aim: This study aims to investigate the passive diffusion of protein kinase inhibitors through the blood-brain barrier (BBB) and to develop a model for their permeability prediction. Materials & methods: We used the parallel artificial membrane permeability assay to obtain logPe values of each of 34 compounds and calculated descriptors for these structures to perform quantitative structure-property relationship modeling, creating different regression models. Results: The logPe values have been calculated for all 34 compounds. Support vector machine regression was considered the most reliable, and CATS2D_09_DA, CATS2D_04_AA, B04[N-S]and F07[C-N] descriptors were identified as the most influential to passive BBB permeability. Conclusion: The quantitative structure-property relationship-support vector machine regression model that has been generated can serve as an efficient method for preliminary screening of BBB permeability of new analogs.

Structure-Guided Design, Synthesis, and Biological Evaluation of Peripheral Anionic Site Selective and Brain Permeable Novel Oxadiazole-Piperazine Conjugates against Alzheimer's Disease with Antioxidant Potential.

Alzheimer's disease (AD) is a multifactorial and emerging neurological disorder, which has invoked researchers to develop multitargeted ligands. Herein, hybrid conjugates of 5-phenyl-1,3,4-oxadiazole and piperazines were rationally designed, synthesized, and pharmacologically evaluated against hAChE, hBChE, and hBACE-1 enzymes for the management of AD. Among the series, compound 5AD comprising pyridyl substitution at terminal nitrogen of piperazine contemplated as a paramount lead compound (hAChE, IC50 = 0.103 ± 0.0172 μM, hBChE, IC50 ≥ 10 μM, and hBACE-1, IC50 = 1.342 ± 0.078 μM). Compound 5AD showed mixed-type enzyme inhibition in enzyme kinetic studies against the hAChE enzyme. In addition, compound 5AD revealed a significant displacement of propidium iodide from the peripheral anionic site (PAS) of hAChE and excellent blood-brain barrier (BBB) permeability in a parallel artificial membrane permeation assay (PAMPA). Besides, 5AD also exhibited anti-Aβ aggregation activity in self- and AChE-induced thioflavin T assay. Further, compound 5AD has shown significant improvement in learning and memory (p < 0.001) against the in vivo scopolamine-induced cognitive dysfunction mice model. The ex vivo study implied that after treatment with compound 5AD, there was a decrease in AChE and malonaldehyde (MDA) levels with an increase in catalase (CAT, oxidative biomarkers) in the hippocampal brain homogenate. Hence, compound 5AD could be regarded as a lead compound and further be explored in the treatment of AD.

Synthesis and biological evaluation of Halogen-Substituted novel α-Ketoamides as potential protein aggregation modulators in Alzheimer’s disease

The escalating prevalence of Alzheimer's disease (AD) has prompted extensive research into potential therapeutic interventions, with a specific focus on molecular targets such as amyloid beta (Aβ) and tau protein aggregation. In this study, a series of α-ketoamide derivatives was synthesized from β,γ-unsaturated α-keto thioesters, achieving high purity and good yield. Thioflavin T based amyloid beta aggregation assay identified four promising compounds (BD19, BD23, BD24, and BD27) that demonstrated significant inhibitory effects on Aβ aggregation. BD23, selected for its better solubility (0.045 ± 0.0012 mg/ml), was further subjected to in vitro Parallel Artificial Membrane Permeability Assay to determine the Blood-Brain-Barrier permeability and c emerged as BBB permeable with permeability rate (Pe) of 10.66 ± 8.11 × 10 − 6 cm/s. In addition to its Aβ inhibitory properties, BD23 exhibited significant inhibition of heparin-induced tau aggregation and demonstrated non-toxicity in SHSY5Y cell lines. Subsequent in vivo assays were conducted, administering compound BD23 to an Aβ induced mouse model at various doses (1, 2, & 5 mg/kg). The results revealed a noteworthy enhancement in cognitive functions, particularly when BD23 was administered at a dosage of 5 mg/kg, comparable to the effects observed with the standard dose of Donepezil (DNP). In silico investigations, including molecular docking, molecular dynamics simulations, and Density Functional Theory calculations, provided insights into BD23′s interactions with the targets and electronic properties. These analyses contribute to the understanding of the therapeutic potential of the lead compounds BD23 which further pave the way for further exploration of its therapeutic potential in the context of Alzheimer's disease.

Abstract 7264: OPN-9840, a non-covalent potent pan-TEAD inhibitor, exhibits single agent efficacy in preclinical malignant mesothelioma models

Abstract The Hippo pathway regulates critical cellular processes during development. In ~10% of human cancers, Hippo pathway dysregulation results in YAP/TAZ overactivation and increased TEAD-dependent transcription that promotes tumor growth and therapy resistance - often by supporting drug-tolerant persister survival. In 40% of malignant mesotheliomas (MM), neurofibromatosis 2 (NF2) gene mutations inactivate the Merlin protein, an upstream negative regulator of TEAD. Preclinical studies indicate high TEAD dependency in NF2-mutant MM. We have discovered an array of potent covalent and non-covalent small molecule inhibitors that occupy the palmitate-binding pocket of TEAD proteins, with varying paralog selectivity profiles. OPN-9840 is an oral non-covalent pan-TEAD inhibitor displaying dose-dependent and on-target in vitro and in vivo efficacy for NCI-H226, an NF2-mutant MM cell line. OPN-9840 showed equivalent binding in thermal shift assays (10°C delta Tm) for depalmitoylated TEAD1-4 proteins, and blocked TEAD reporter activity (IC50 100nM) and NCI-H226 cell growth (IC50 400nM). In an NCI-H226 xenograft model, OPN-9840 dosed orally for 14 days exhibited dose-dependent tumor growth inhibition (TGI) from 5 to 50 mg/kg PO (88 to &amp;gt;100% TGI) with tumor regression in the 15 (2/8 mice) and 50 mg/kg (4/8 mice) dose groups. qPCR of OPN-9840-treated tumors confirmed 50-70% downregulation of canonical TEAD targets, CTGF and CYR61. OPN-9840 shows a favorable safety profile, including a lack of cytotoxicity (IC50&amp;gt;50µM) in HEK293T and HepG2 cells. Additionally, OPN-9840 caused no body weight loss in the NCI-H226 xenograft model following 14 days of dosing. In vitro evaluation of OPN-9840 in the blood-brain barrier (BBB) specific parallel artificial membrane permeability assay exhibited high BBB penetration potential (-Log Pe = 4.7). High oral exposure was achieved at all dose levels (AUC0-24 &amp;gt; 100,000 h•ng/mL) after 14 days in the NCI-H226 study. We next investigated synergy between TEAD inhibition and targeted therapy in MM. Combined treatment of one of our pan-TEAD covalent compounds, OPN-9652 (from a separate chemical series to OPN-9840), at 50 mg/kg with 1 mg/kg trametinib resulted in NCI-H226 xenograft tumor regression (130% TGI) whereas trametinib alone only led to decreased tumor growth (74% TGI). RNA-Seq of treated tumors revealed that OPN-9652 and trametinib synergistically inhibited TEAD downstream target genes, including ANKRD1 and CTGF. OPN-9652 as a single agent caused significant downregulation of E2F targets, such as MYC and TP53, suggesting decreased proliferation. In summary, OPN-9840 is a potent pan-TEAD inhibitor that shows monotherapy efficacy in MM. As we observed synergy between another pan-TEAD inhibitor, OPN-9652, with trametinib, we are exploring synergy of OPN-9840 with additional targeted therapies in tumor types beyond MM. Citation Format: Pan-Yu Chen, Bernice Matusow, James Tsai, Peipei Li, Hoa Nguyen, Gaston Habets, Gideon Bollag, Somenath Chowdhury. OPN-9840, a non-covalent potent pan-TEAD inhibitor, exhibits single agent efficacy in preclinical malignant mesothelioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7264.

Accessible luminal interface of bovine rectal organoids generated from cryopreserved biopsy tissues.

Developing precise species-specific in vitro models that closely resemble in vivo intestinal tissues is essential for advancing our understanding of gastrointestinal physiology and associated diseases. This is especially crucial in examining host-pathogen interactions, particularly in bovines, a known reservoir for microbes and pathogens posing substantial public health threats. This research investigated the viability of producing bovine rectal organoids from cryopreserved tissues. We compared two cryopreservation methods with a traditional technique using fresh tissues, evaluating their effectiveness through growth rates, long-term viability, and comprehensive structural, cellular, and genetic analyses. These assessments utilized phase-contrast imaging, immunofluorescence imaging, and RT-qPCR assays. Additionally, the study developed a sophisticated method for forming a functional epithelial barrier from organoid-derived bovine rectal monolayers, incorporating a wide range of epithelial cells. This methodology employed transepithelial electrical resistance (TEER), parallel artificial membrane permeability assay (Papp), confocal microscopy, and advanced imaging techniques like scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Our findings decisively show that bovine rectal organoids can be effectively generated from cryopreserved biopsy tissues. Moreover, we formulated a robust and optimized protocol for creating functional rectal monolayers from these organoids. This significant progress is particularly relevant given the susceptibility of the bovine rectum to various enteric pathogens of public health concern, marking a vital step forward in veterinary and biomedical research. The creation of accurate species specific in vitro models that faithfully mimic in vivo intestinal tissues is critical for enhancing our understanding of gut physiology and related pathologies. This is particularly relevant in studying the interactions between hosts and microbes or pathogens with significant public health risks where bovine can be the major reservoir.

Insights on Structure-Passive Permeability Relationship in Pyrrole and Furan-Containing Macrocycles.

Macrocycles have recognized therapeutic potential, but their limited cellular permeability can hinder their development as oral drugs. To better understand the structure-permeability relationship of heterocycle-containing, semipeptidic macrocycles, a library was synthesized. These compounds were created by developing two novel reactions described herein: the reduction of activated oximes by LiBH4 and the aqueous reductive mono-N-alkylation of aldehydes using catalytic SmI2 and stoichiometric Zn. The permeability of the macrocycles was evaluated through a parallel artificial membrane permeability assay (PAMPA), and the results indicated that macrocycles with a furan incorporated into the structure have better passive permeability than those with a pyrrole moiety. Compounds bearing a 2,5-disubstituted pyrrole (endo orientation) were shown to be implicated in intramolecular H-bonds, enhancing their permeability. This study highlighted the impact of heterocycles moieties in semipeptides, creating highly permeable macrocycles, thus showing promising avenues for passive diffusion of drugs beyond the rule-of-five chemical space.

Effect of N-o-nitrobenzylation on conformation and membrane permeability of linear peptides

In this study, we explored the potential of the photoremovable o-nitrobenzyl (oNB) group as a tool to manipulate the membrane permeability and regulate the conformation of linear peptides by means of experimental and computational studies. We found that the introduction of one or more oNB groups markedly increased the permeability and altered the conformation, as compared to the corresponding unmodified peptides. We thoroughly investigated the impact of peptide length, number of oNB group, oNB insertion position, and introduction of N- and C-terminal protecting groups on the passive membrane permeability by means of parallel artificial membrane permeability assay (PAMPA). Photoreaction of peptides containing one or two oNB groups proceeded cleanly in moderate to high yields, releasing the unprotected parent linear peptide. The oNB-modified peptides showed a cis/trans conformational equilibrium, while after photolysis, the unprotected linear peptides showed only the trans-amide conformation. Furthermore, a comprehensive comparison of oNB-modified peptides and N-methylated peptides was conducted, encompassing conformational analysis and physicochemical properties. N-Substituted peptides favored a folded-like structure, which may contribute to the improvement in permeability.

Influence of Bile Acids on Clindamycin Hydrochloride Skin Permeability: InVitro and InSilico Preliminary Study.

Topical clindamycin formulations are widely used in clinical practice, but poor bioavailability and restricted skin penetration considerably limit their therapeutic efficacy. Penetration enhancement represents a promising and rational strategy to overcome the drawbacks of conventional topical pharmaceutical formulations. We aim to assess the influence of cholic acid (CA) and deoxycholic acid (DCA) on the permeability of clindamycin hydrochloride by performing the in vitro skin parallel artificial membrane permeability assay (skin-PAMPA) at two relevant pH values (5.5 and 6.5) and the interactions of tested substances with skin ATP-binding cassette (ABC) transporters in silico. After the incubation period, the clindamycin hydrochloride concentrations in both compartments were determined spectrophotometrically, and the apparent permeability coefficients (Papp) were calculated. Vienna LiverTox web service was used to predict the interactions of clindamycin and bile acids with potential drug transporters located in human skin. Both CA and DCA at the highest studied concentration of 100μM in the tested solutions increased the skin-PAMPA membrane permeability of clindamycin hydrochloride. This effect was more pronounced for CA and at a higher studied pH value of 6.5, which is characteristic of most dermatological indications treated with topical clindamycin preparations. Clindamycin transport may also be mediated by ABC transporters located in skin and facilitated in the presence of bile acids. The results of this study provide a solid foundation for further research directed at the improvement of topical formulations using bile acids as penetration-enhancing excipients, as well as the therapeutic efficacy of clindamycin hydrochloride.

In Vitro Assessment of Cortisol Release Inhibition, Bioaccessibility and Bioavailability of a Chemically Characterized Scutellaria lateriflora L. Hydroethanolic Extract.

Excess cortisol release is associated with numerous health concerns, including psychiatric issues (i.e., anxiety, insomnia, and depression) and nonpsychiatric issues (i.e., osteoporosis). The aim of this study was to assess the in vitro inhibition of cortisol release, bioaccessibility, and bioavailability exerted by a chemically characterized Scutellaria lateriflora L. extract (SLE). The treatment of H295R cells with SLE at increasing, noncytotoxic, concentrations (5-30 ng/mL) showed significant inhibition of cortisol release ranging from 58 to 91%. The in vitro simulated gastric, duodenal, and gastroduodenal digestions, induced statistically significant reductions (p < 0.0001) in the bioactive polyphenolic compounds that most represented SLE. Bioavailability studies on duodenal digested SLE, using Caco-2 cells grown on transwell inserts and a parallel artificial membrane permeability assay, indicated oroxylin A glucuronide and oroxylin A were the only bioactive compounds able to cross the Caco-2 cell membrane and the artificial lipid membrane, respectively. The results suggest possible applications of SLE as a food supplement ingredient against cortisol-mediated stress response and the use of gastroresistant oral dosage forms to partially prevent the degradation of SLE bioactive compounds. In vivo studies and clinical trials remain necessary to draw a conclusion on the efficacy and tolerability of this plant extract.

Isatin-tethered halogen-containing acylhydrazone derivatives as monoamine oxidase inhibitor with neuroprotective effect

Sixteen isatin-based hydrazone derivatives (IS1–IS16) were synthesized and assessed for their ability to inhibit monoamine oxidases (MAOs). All the molecules showed improved inhibitory MAO-B activity compared to MAO-A. Compound IS7 most potently inhibited MAO-B with an IC50 value of 0.082 μM, followed by IS13 and IS6 (IC50 = 0.104 and 0.124 μM, respectively). Compound IS15 most potently inhibited MAO-A with an IC50 value of 1.852 μM, followed by IS3 (IC50 = 2.385 μM). Compound IS6 had the highest selectivity index (SI) value of 263.80, followed by IS7 and IS13 (233.85 and 212.57, respectively). In the kinetic study, the Ki values of IS6, IS7, and IS13 for MAO-B were 0.068 ± 0.022, 0.044 ± 0.002, and 0.061 ± 0.001 μM, respectively, and that of IS15 for MAO-A was 1.004 ± 0.171 μM, and the compounds were reversible-type inhibitors. The lead compounds were central nervous system (CNS) permeable, as per parallel artificial membrane permeability assay (PAMPA) test results. The lead compounds were examined for their cytotoxicity and potential neuroprotective benefits in hazardous lipopolysaccharide (LPS)-exposed SH-SY5Y neuroblastoma cells. Pre-treatment with lead compounds enhanced anti-oxidant levels (SOD, CAT, GSH, and GPx) and decreased ROS and pro-inflammatory cytokine (IL-6, TNF-alpha, and NF-kB) production in LPS-intoxicated SH-SY5Y cells. To confirm the promising effects of the compound, molecular docking, dynamics, and MM-GBSA binding energy were used to examine the molecular basis of the IS7-MAO-B interaction. Our findings indicate that lead compounds are potential therapeutic agents to treat neurological illnesses, such as Parkinson's disease.

Favipiravir vs. Deferiprone: Tautomeric, photophysical, in vitro biological studies, and binding interactions with SARS-Cov-2-MPro/ACE2

Coronavirus disease 2019 (COVID-19) still remains the most disastrous infection continuously affecting millions of people worldwide. Herein, we performed a comparative study between the anti-influenza drug favipiravir (FAV) and the anti-thalassemia drug deferiprone (DFP) in order to examine their potential as basic scaffolds for the generation of most effective and structurally novel antivirals. To conduct the initial molecular modelling and virtual screening steps, our recently proposed single crystal X-ray diffraction (SCXRD)/HYdrogen DEssolvation (HYDE) technology platform has been used. This platform allows molecular design, interactive prioritization and virtual evaluation of newly designed molecules, simultaneously affecting two COVID-related targets, including angiotensin-converting enzyme 2 (ACE2) as a host-cellular receptor (host-based approach) and the main protease (Mpro) enzyme of the spike glycoprotein of SARS-Cov-2 (virus-based approach). Based on the molecular docking results, DFP has shown higher binding affinity (Ki HYDE values) over FAV towards both biological targets. The tautomeric, physicochemical, and biological properties of FAV and DFP have been studied both experimentally and theoretically using molecular spectroscopy (UV–VIS absorption), parallel artificial membrane permeability assay, and cell biology (PAMPA and MTT assay), as well as DFT quantum chemical calculations. According to the obtained results, the enol tautomers of both compounds are considerably more stable in different organic solvents. However, the keto tautomer of FAV was estimated to be most preferable under physiological conditions, which is in good agreement with the molecular docking studies. The isolated crystal structure of DFP is in an excellent agreement with the computation in respect of the most stable tautomer. Combined single X-ray/molecular modeling studies including HYDE analyses provided not only insights into the protein–ligand interactions within the binding site of SARS-Cov-2-ACE2 and SARS-Cov-2-Mpro, but also a valuable information regarding the most stable enol tautomeric form of DFP that contributes to its estimated higher potency against these targets.

Pleiotropic Potential of Evernia prunastri Extracts and Their Main Compounds Evernic Acid and Atranorin: In Vitro and In Silico Studies.

Evernia prunastri is a lichen widely distributed in the Northern Hemisphere. Its biological properties still need to be discovered. Therefore, our paper focuses on studies of E. prunastri extracts, including its main metabolites evernic acid (EA) or atranorin (ATR). Phytochemical profiles using chromatographic analysis were confirmed. The antioxidant activity was evaluated using in vitro chemical tests and in vitro enzymatic cells-free tests, namely superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and catalase (CAT). The anti-inflammatory potential using cyclooxygenase-2 (COX-2) and hyaluronidase were determined. The neuroprotective potential using acetylcholinesterase, (AChE), butyrylcholinesterase (BChE), and tyrosinase (Tyr) was estimated. The hypoglycemic activity was also confirmed (α-glucosidase). Principal component analysis was performed to determine the relationship between the biological activity of extracts. The inhibitory effect of EA and ATR on COX-2 AChE, BChE, Tyr, and α-glucosidase was evaluated using molecular docking techniques and confirmed for EA and ATR (besides α-glucosidase). The penetration of EA and ATR from extracts through the blood-brain barrier was confirmed using the parallel artificial membrane permeability assay blood-brain barrier test. In conclusion, depending on chemical surroundings and the concentration, the E. prunastri extracts, EA or ATR, showed attractive pleiotropic properties, which should be further investigated.

Structural optimization of a lysine demethylase 5 inhibitor for improvement of its cellular activity

Lysine demethylase 5 (KDM5) subfamily proteins are important in epigenetic gene regulation. They are involved in the growth and drug resistance of cancer cells. Therefore, KDM5s are potential cancer therapeutic targets, and their inhibitors hold promise as anti-cancer drugs. Several KDM5 inhibitors, including KDM5-C49 (2a), have exhibited potent KDM5-inhibitory activities in in vitro enzyme assays. However, they do not show enough cellular activity despite being converted to their prodrugs. We hypothesized that their poor lipophilicity should prevent them from sufficiently penetrating the cell membrane, and introducing more lipophilic groups should improve cellular activities. In this study, we investigated 2a and KDM5-C70 (3a), a prodrug of 2a, and attempted to improve its cellular activity by replacing the N,N-dimethyl amino group of 3a with more lipophilic groups. N-Butyl, N-methyl amino compound 2e exhibited potent and selective KDM5-inhibitory activity equal to that of 2a. Furthermore, the cell membrane permeability of 3e, an ethyl ester prodrug of 2e, was six times higher than that of 3a in a parallel artificial membrane permeation assay. In addition, western blot analysis indicated that treating human lung cancer A549 cells with 3e increased histone methylation levels more strongly than that with 3a. Thus, we identified compound 3e as a more cell-active KDM5 inhibitor that has sufficient cell membrane permeability.

From waste to a resource? Hemicelluloses enhance the dissolution and stability of the amorphous state of carbamazepine

Both synthetic and natural polymers are used as excipients in various pharmaceutical preparations. Natural polymers offer advantages, such as abundant availability, cost-effectiveness, non-toxicity, biocompatibility, and biodegradability. Hemicelluloses are highly promising natural polymers for pharmaceutical applications due to their advantageous colloidal properties, but their ability to solubilize poorly water-soluble drugs and stabilize their amorphous form is currently virtually unexplored. In this study, we showed the applicability of birch, spruce and wheat straw hemicelluloses as tablet excipients and their ability to enhance dissolution and stability of a model drug, carbamazepine (CBZ), in amorphous solid dispersions. The hemicelluloses formed sufficiently strong tablets by direct-compression, although addition of CBZ weakened the tablets. The hemicelluloses did not modify drug release from the tablets when compared to plain CBZ powder. Solid dispersions with CBZ were identified as amorphous one-phase mixtures, with hydrogen bonding between the hemicelluloses and CBZ. The mixtures remained stable for at least nine months under 40 °C/23 % RH. Dissolution of the amorphous solid dispersions decreased with increasing hemicellulose molar mass. Up to 7.6-fold CBZ supersaturation was achieved, although it did not improve CBZ permeation through a PAMPA (parallel artificial membrane permeability assay) barrier when compared to the corresponding physical mixtures. In summary, birch, spruce, and wheat straw hemicelluloses formed direct-compressed tablets and stable amorphous solid dispersions that may improve dissolution properties of drugs. Differences exist between hemicelluloses from different plant sources and thus more research with structurally different drug substances is needed in the future.

Encapsulation of Olive Leaf Polyphenol-Rich Extract in Polymeric Micelles to Improve Its Intestinal Permeability.

In the present study, polymeric micelles were developed to improve the intestinal permeability of an extract of Olea europaea L. leaf with a high content of total polyphenols (49% w/w), with 41% w/w corresponding to the oleuropein amount. A pre-formulation study was conducted to obtain a stable formulation with a high loading capacity for extract. The freeze-drying process was considered to improve the stability of the formulation during storage. Micelles were characterized in terms of physical and chemical properties, encapsulation efficiency, stability, and in vitro release. The optimized system consisted of 15 mg/mL of extract, 20 mg/mL of Pluronic L121, 20 mg/mL of Pluronic F68, and 10 mg/mL of D-α-tocopheryl polyethylene glycol succinate (TPGS), with dimensions of 14.21 ± 0.14 nm, a polydisersity index (PdI) of 0.19 ± 0.05 and an encapsulation efficiency of 66.21 ± 1.11%. The influence of the micelles on polyphenol permeability was evaluated using both Parallel Artificial Membrane Permeability Assay (PAMPA) and the Caco-2 cell monolayer. In both assays, the polymeric micelles improved the permeation of polyphenols, as demonstrated by the increase in Pe and Papp values.

Development and validation of PAMPA-BBB QSAR model to predict brain penetration potential of novel drug candidates.

Efficiently circumventing the blood-brain barrier (BBB) poses a major hurdle in the development of drugs that target the central nervous system. Although there are several methods to determine BBB permeability of small molecules, the Parallel Artificial Membrane Permeability Assay (PAMPA) is one of the most common assays in drug discovery due to its robust and high-throughput nature. Drug discovery is a long and costly venture, thus, any advances to streamline this process are beneficial. In this study, ∼2,000 compounds from over 60 NCATS projects were screened in the PAMPA-BBB assay to develop a quantitative structure-activity relationship model to predict BBB permeability of small molecules. After analyzing both state-of-the-art and latest machine learning methods, we found that random forest based on RDKit descriptors as additional features provided the best training balanced accuracy (0.70 ± 0.015) and a message-passing variant of graph convolutional neural network that uses RDKit descriptors provided the highest balanced accuracy (0.72) on a prospective validation set. Finally, we correlated in vitro PAMPA-BBB data with in vivo brain permeation data in rodents to observe a categorical correlation of 77%, suggesting that models developed using data from PAMPA-BBB can forecast in vivo brain permeability. Given that majority of prior research has relied on in vitro or in vivo data for assessing BBB permeability, our model, developed using the largest PAMPA-BBB dataset to date, offers an orthogonal means to estimate BBB permeability of small molecules. We deposited a subset of our data into PubChem bioassay database (AID: 1845228) and deployed the best performing model on the NCATS Open Data ADME portal (https://opendata.ncats.nih.gov/adme/). These initiatives were undertaken with the aim of providing valuable resources for the drug discovery community.