Membrane Permeability Assay Research Articles

Synergistic antibacterial effect of ginsenoside Rh2 and calcium hydroxide on Enterococcus faecalis.

Treatment of root canal infections becomes more challenging due to the extremely high tolerance of Enterococcus faecalis (E. faecalis) to calcium hydroxide (Ca(OH)2). Ginsenoside is a Chinese herbal extract that has been proven to have antimicrobial properties and synergistic activities. And this study evaluated the antibacterial activity of ginsenoside Rh2 in combination with Ca(OH)2 against E. faecalis and its preliminary mechanism of action. Broth microdilution method, checkerboard dilution method, time-inhibition curve, drug resistance assays, scanning electron microscopy, and biofilm inhibition and removal assays indicated that Rh2 in combination with Ca(OH)2 exhibited potent antibacterial activity against E. faecalis. Rh2 exerted significant in vitro antibacterial activity against E. faecalis, with a minimum inhibitory concentration (MIC) of 3.125μg/mL and minimum bactericidal concentration (MBC) of 6.25μg/mL, and significantly enhanced the susceptibility of E. faecalis to Ca(OH)2 (FICI = 0.5). Furthermore, cell membrane permeability assays, surface hydrophobicity assays, ATPase activity assays, and intra-biofilm extracellular polysaccharides (EPS) assays revealed that Rh2 and Ca(OH)2 synergistically inhibit bacteria mainly by increasing membrane permeability. Ultimately, cytotoxicity assays showed that Rh2 exhibited only low toxicity, the half maximal inhibitory concentration (IC50) of Rh2 was 19.75μg/mL. This study confirmed the synergistic antibacterial activities of Rh2 and Ca(OH)2 against E. faecalis. Our findings indicate that the Rh2 and Ca(OH)2 combination may be a promising alternative approach to treating root canal infections.

Estimation of passive gastrointestinal absorption of new dual DNA gyrase and topoisomerase IV inhibitors using PAMPA and biopartitioning micellar chromatography and quantitative structure-retention relationship analysis

DNA gyrase and topoisomerase IV play significant role in maintaining the correct structure of DNA during replication and they have been identified as validated targets in antibacterial drug discovery. Inadequate pharmacokinetic properties are responsible for many failures during drug discovery and their estimation in the early phase of this process maximizes the chance of getting useful drug candidates. Passive gastrointestinal absorption of a selected group of thirteen dual DNA gyrase and topoisomerase IV inhibitors was estimated using two in vitro tests – parallel artificial membrane permeability assay (PAMPA) and biopartitioning micellar chromatography (BMC). Due to good correlation between obtained results, passive gastrointestinal absorption of remaining ten compounds was estimated using only BMC. With this experimental setup, it was possible to identify compounds with high values of retention factors (k) and highest expected passive gastrointestinal absorption, and compounds with low values of k for which low passive gastrointestinal absorption is predicted. Quantitative structure-retention relationship (QSRR) modelling was performed by creating multiple linear regression (MLR), partial least squares (PLS) and support vector machines (SVM) models. Descriptors with the highest influence on retention factor were identified and their interpretation can be used for the design of new compounds with improved passive gastrointestinal absorption.

Examining dietary supplements containing Euterpe oleracea Mart. (açaí) for potential botanical-drug interactions involving CYP3A4 inhibition

BackgroundBotanical dietary supplements (BDS) are consumed more by cancer patients than otherwise healthier patients to alleviate side effects of chemotherapy drugs or increase quality of life. The enzyme responsible for the metabolism of most anticancer drugs is CYP3A4, an enzyme predominantly located in the liver. HypothesisOur research team examined the potential risk between CYP3A4-interactive cancer drugs and Euterpe oleracea Mart. (açaí) berry and found that the compounds that passively and non-passively diffused from methanol açaí extract displayed significant inhibition of liver CYP3A4 – suggesting the potential for interactions between açaí and drugs metabolized by CYP3A4 if used synchronously. This study aims to identify the compounds responsible for the CYP3A4 inhibition using the newly automated Bioactivity – Global Natural Product Social Molecular Networking (GNPS) tool. MethodsDietary supplement capsules from two manufacturers (F1 and F2) containing aqueous extracts of açaí fruits were extracted separately by methanol (ME) and acidic methanol (AC) and standardized to cyanidin-3-O-glucoside (CG) concentration at a human-relevant dose. After solvent removal, a parallel artificial membrane permeability assay (PAMPA) was utilized to filter intestinal passive diffusion of the four extract constituents so that compounds could be tested for inhibition of hepatic CYP3A4. Passively and non-passively diffused constituents of extracts from PAMPA assays were injected into the liquid chromatography-mass spectrometry (LC–MS) and characterized for visualization and comparison of chemical entities. To elucidate the non-passively diffused compounds responsible for significant CYP3A4 inhibition, the Bioactivity – GNPS tool was used to hypothesize which compounds within the formulation extract were potentially active to accelerate their identification. ResultsThere was no significant inhibition of CYP3A4 by passively diffused constituents for any extract. There was, however, significant inhibition by non-passively diffused constituents of both F1ME and F1AC extracts with 50 % CYP3A4 inhibition at the donor compartment concentrations 5.764 and 15.58 ng/ml CG, respectively. Six compounds were predicted to be inhibitors of CYP3A4 from methanol F1 extract, including betaine and 5 unknown compounds. ConclusionThis indicates that BDS containing açaí may not inhibit CYP3A4 through passively diffused compounds. Compounds from açaí BDS that inhibit CYP3A4 may be absorbed through other mechanisms, such as transporters.

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.

Genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 carbapenem-resistant Klebsiella pneumoniae

ABSTRACT ST11 is the most common lineage among carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in Asia. Diverse morphotypes resulting from genetic mutations are associated with significant differences in microbial characteristics among K. pneumoniae isolates. Here, we investigated the genetic determinants and critical characteristics associated with distinct morphotypes of ST11 CRKP. An ST11-KL47 CRKP isolate carrying a pLVPK-like virulence plasmid was isolated from a patient with a bloodstream infection; the isolate had the “mcsw” morphotype. Two distinct morphotypes (“ntrd” and “msdw”) were derived from this strain during in vitro passage. Whole genome sequencing was used to identify mutations that cause the distinct morphotypes of ST11 CRKP. Transmission electron microscopy, antimicrobial susceptibility tests, growth assays, biofilm formation, virulence assays, membrane permeability assays, and RNA-seq analysis were used to investigate the specific characteristics associated with different morphotypes of ST11 CRKP. Compared with the parental mcsw morphotype, the ntrd morphotype resulted from mutation of genes involved in capsular polysaccharide biosynthesis (wza, wzc, and wbaP), a result validated by gene knockout experiments. This morphotype showed capsule deficiency and lower virulence potential, but higher biofilm production. By contrast, the msdw morphotype displayed competition deficiency and increased susceptibility to chlorhexidine and polymyxin B. Further analyses indicated that these characteristics were caused by interruption of the sigma factor gene rpoN by insertion mutations and deletion of the rpoN gene, which attenuated membrane integrity presumably by downregulating the phage shock protein operon. These data expand current understanding of genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 CRKP.

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 (OSA-S), 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.

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.

Globospiramine from Voacanga globosa Exerts Robust Cytotoxic and Antiproliferative Activities on Cancer Cells by Inducing Caspase-Dependent Apoptosis in A549 Cells and Inhibiting MAPK14 (p38α): In Vitro and Computational Investigations.

Bisindole alkaloids are a source of inspiration for the design and discovery of new-generation anticancer agents. In this study, we investigated the cytotoxic and antiproliferative activities of three spirobisindole alkaloids from the traditional anticancer Philippine medicinal plant Voacanga globosa, along with their mechanisms of action. Thus, the alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) showed in vitro cytotoxicity and antiproliferative activities against the tested cell lines (L929, KB3.1, A431, MCF-7, A549, PC-3, and SKOV-3) using MTT and CellTiter-Blue assays. Globospiramine (1) was also screened against a panel of breast cancer cell lines using the sulforhodamine B (SRB) assay and showed moderate cytotoxicity. It also promoted the activation of apoptotic effector caspases 3 and 7 using Caspase-Glo 3/7 and CellEvent-3/7 apoptosis assays. Increased expressions of cleaved caspase 3 and PARP in A549 cells treated with 1 were also observed. Apoptotic activity was also confirmed when globospiramine (1) failed to promote the rapid loss of membrane integrity according to the HeLa cell membrane permeability assay. Network pharmacology analysis, molecular docking, and molecular dynamics simulations identified MAPK14 (p38α), a pharmacological target leading to cancer cell apoptosis, as a putative target. Low toxicity risks and favorable drug-likeness were also predicted for 1. Overall, our study demonstrated the anticancer potentials and apoptotic mechanisms of globospiramine (1), validating the traditional medicinal use of Voacanga globosa.

Enhanced stability and dissolution of curcumin nanocrystals stabilized by octenyl succinic anhydride modified starch

Curcumin (CUR) has known to have poor oral bioavailability due to its poor aqueous solubility and stability in gastrointestinal fluid condition as well as its limited permeability which greatly limits its applications in food and medical fields. The present study reported the development of CUR nanocrystal suspension (CUR-NS) for enhanced bioavailability, stabilized with octenyl succinic anhydride modified starch (OSA-S), a naturally renewable and biocompatible biomolecule. This preparation was achieved via an antisolvent precipitation combined with ultrasonic dispersion method, following which the suspension was freeze-dried to form powder CUR nanocrystals (CUR-NCs). Physicochemical properties of the prepared CUR-NS and CUR-NCs powders were extensively characterized. The particle size of the nanocrystals was found to be around 322.8 ± 15.7 nm with a small polydispersity index (PDI) of about 0.230 ± 0.039. Rod like crystal shape was observed by the SEM. Results from powder X-ray diffraction (pXRD) and differential scanning calorimetry (DSC) indicated that the crystal form of CUR in CUR-NCs was a monoclinic polymorph Form 2. Stability results indicated that CUR-NCs possessed an excellent storage-, thermal-, and photo-stabilities. CUR-NCs had a significantly higher dissolution rate, a 68-fold increase in its solubility than that of raw CUR. The cytotoxicity assay demonstrated that CUR-NCs exhibited lower toxicity towards normal human kidney epithelial cells compared to raw CURs. Parallel artificial membrane permeability assay (PAMPA) characterization confirmed that the flux of CUR was significantly enhanced after forming nanocrystals with OSA-S, probably resulting from its increased solubility and dissolution rate. The study showed that OSA-S could effectively stabilize CUR-NCs, and CUR-NCs developed in the current work exhibited significant enhancement in the stability and dissolution rate leading to an improved bioavailability which might be helpful for further development of CUR based products.

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.

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 Aβ 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 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 of AD 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 AD.

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 >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>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 > 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.

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.

Promising antibacterial efficacy of arenicin peptides against the emerging opportunistic pathogen Mycobacterium abscessus

BackgroundMycobacterium abscessus, a fast-growing non-tuberculous mycobacterium, is an emerging opportunistic pathogen responsible for chronic bronchopulmonary infections in people with respiratory diseases such as cystic fibrosis (CF). Due to its intrinsic polyresistance to a wide range of antibiotics, most treatments for M. abscessus pulmonary infections are poorly effective. In this context, antimicrobial peptides (AMPs) active against bacterial strains and less prompt to cause resistance, represent a good alternative to conventional antibiotics. Herein, we evaluated the effect of three arenicin isoforms, possessing two or four Cysteines involved in one (Ar-1, Ar-2) or two disulfide bonds (Ar-3), on the in vitro growth of M. abscessus.MethodsThe respective disulfide-free AMPs, were built by replacing the Cysteines with alpha-amino-n-butyric acid (Abu) residue. We evaluated the efficiency of the eight arenicin derivatives through their antimicrobial activity against M. abscessus strains, their cytotoxicity towards human cell lines, and their hemolytic activity on human erythrocytes. The mechanism of action of the Ar-1 peptide was further investigated through membrane permeabilization assay, electron microscopy, lipid insertion assay via surface pressure measurement, and the induction of resistance assay.ResultsOur results demonstrated that Ar-1 was the safest peptide with no toxicity towards human cells and no hemolytic activity, and the most active against M. abscessus growth. Ar-1 acts by insertion into mycobacterial lipids, resulting in a rapid membranolytic effect that kills M. abscessus without induction of resistance.ConclusionOverall, the present study emphasized Ar-1 as a potential new alternative to conventional antibiotics in the treatment of CF-associated bacterial infection related to M. abscessus.

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.