Development and validation of an HPLC method for the quantitation of 1,3-dihydroxy-2-methylxanthone in biodegradable nanoparticles

A rapid and simple high-performance liquid chromatographic method for the analysis of 1,3-dihydroxy-2-methylxanthone (DHMXAN) in biodegradable poly(D,L-lactide-co-glycolide) (PLGA) nanosphere and nanocapsule formulations is developed and validated. The method does not require any complex sample extraction procedure. Chromatographic separation is made with a reversed-phase C18 column, using methanol-water (90:10, v/v) containing 1% (v/v) acetic acid as a mobile phase at a flow rate of 1 mL/min. Identification is made by UV detection at 237 nm. The isocratic system operates at ambient temperature and requires 7.5 min of chromatographic time. The developed method is statistically validated according to ICH guidelines and USP 29 for its specificity, linearity, accuracy, and precision. The assay method proposed in this study is specific for DHMXAN in the presence of nanosphere and nanocapsule excipients. Diode-array analyses confirm the purity of DHMXAN peak in stress conditions (> 99.0%). The method is shown to be linear (r 0.999) over the concentration range of 0.25-3.0 g/mL. Recovery ranges from 99.0% to 102.7% (RSD: 1.49%) and from 98.3% to 101.6% (RSD: 1.07%) for nanospheres and nanocapsules, respectively. Repeatability (intraassay precision) and intermediate precision is acceptable with RSD values ranging from 0.6% to 1.9% and from 0.3% to 2.0%, respectively. The method is shown to be suitable for the evaluation of DHMXAN content entrapped in PLGA nanoparticles

1-Hydr­oxy-3-(3-methyl­but-2-en­yloxy)xanthone

In the title compound, C18H16O4, a monoprenylated xanthone, the xanthone skeleton exhibits an essentially planar conformation (r.m.s. deviation 0.0072 Å) and the isoprenyl side chain remains approximately in the mean plane of the xanthone unit, making a dihedral angle of 4.5 (2)°. The hydroxyl group forms an intra­molecular O—H⋯O hydrogen bond. Moreover, there is a weak inter­molecular C—H⋯O inter­action between a ring C atom and the xanthene O atom. In the crystal structure, there are no inter­molecular hydrogen bonds and the crystallographic packing is governed by van der Waals forces, leading to an arrangement in which the mol­ecules assemble with their planes parallel to each other, having a separation of 3.6 (3) Å

Structure and ligand-based design of P-glycoprotein inhibitors: a historical perspective

Computer-assisted drug design (CADD) is a valuable approach for the discovery of new chemical entities in the field of cancer therapy. There is a pressing need to design and develop new, selective, and safe drugs for the treatment of multidrug resistance (MDR) cancer forms, specifically active against P-glycoprotein (P-gp). Recently, a crystallographic structure for mouse P-gp was obtained. However, for decades the design of new P-gp inhibitors employed mainly ligand-based approaches (SAR, QSAR, 3D-QSAR and phar macophore studies), and structure-based studies used P-gp homology models. However, some of those results are still the pillars used as a starting point for the design of potential P-gp inhibitors. Here, pharmacophore mapping, (Q)SAR, 3D-QSAR and homology modeling, for the discovery of P-gp inhibitors are reviewed. The importance of these methods for understanding mechanisms of drug resistance at a molecular level, and design P-gp inhibitors drug candidates are discussed. The examples mentioned in the review could provide insights into the wide range of possibilities of using CADD methodologies for the discovery of efficient P-gp inhibitors.info:eu-repo/semantics/publishedVersio

Multidrug resistance reversal effects of aminated thioxanthones and interaction with cytochrome P450 3A4

Aminated thioxanthones have recently been described as dual-acting agents: growth inhibitors of leukemia cell lines and P-glycoprotein (P-gp) inhibitors. To evaluate the selectivity profile of thioxanthones as inhibitors of multidrug resistance (MDR), their interaction with other ABC transporters, which were found to have a strong correlation with multidrug resistance, such as multidrug resistant proteins 1 (MRP1), 2 (MRP2) and 3 (MRP3) and breast cancer resistance protein (BCRP) was also evaluated. The interaction of thioxanthones with cytochrome P450 3A4 (CYP3A4) together with the prediction of their binding conformations and metabolism sites was also investigated. Methods. The UIC2 monoclonal antibody-labelling assay was performed using P-gp overexpressing leukemia cells, K562Dox, incubated with eight thioxanthonic derivatives, in order to confirm their P-gp inhibitory activity. A colorimetric-based ATPase assay using membrane vesicles from mammalian cells overexpressing a selected human ABC transporter protein (P-gp, MRP1, MRP2, MRP3, or BCRP) was performed. To verify if some of the thioxanthonic derivatives were substrates or inhibitors of CYP3A4, a luciferin-based luminescence assay was performed. Finally, the in silico prediction of the most probable metabolism sites and docking studies of thioxanthones on CYP3A4 binding site were investigated. Results. Thioxanthones interacted not only with P-gp but also with MRP and BCRP transporters. These compounds also interfere with CYP3A4 activity in vitro, in accordance with the in silico prediction. Conclusion. Thioxanthonic derivatives are multi-target compounds. A better characterization of the interactions of these compounds with classical resistance mechanisms may possibly identify improved treatment applications.info:eu-repo/semantics/publishedVersio

New uses for old drugs: pharmacophore‐based screening for the discovery of P‐glycoprotein inhibitors

P-glycoprotein (P-gp) is one of the best character ized transporters responsible for the multidrug resistance phenotype exhibited by cancer cells. Therefore, there is widespread interest in eluci dating whether existing drugs are candidate P-gp substrates or inhibitors. With this aim, a pharma cophore model was created based on known P-gp inhibitors and it was used to screen a database of existing drugs. The P-gp modulatory activity of the best hits was evaluated by several methods such as the rhodamine-123 accumulation assay using K562Dox cell line, and a P-gp ATPase activ ity assay. The ability of these compounds to enhance the cytotoxicity of doxorubicin was assessed with the sulphorhodamine-B assay. Of the 21 hit compounds selected in silico, 12 were found to significantly increase the intracellular accumulation of Rhodamine-123, a P-gp substrate. In addition, amoxapine and loxapine, two tetracy clic antidepressant drugs, were discovered to be potent non-competitive inhibitors of P-gp, causing a 3.5-fold decrease in the doxorubicin GI50 in K562Dox cell line. The overall results provide important clues for the non-label use of known drugs as inhibitors of P-gp. Potent inhibitors with a dibenzoxazepine scaffold emerged from this study and they will be further investigated in order to develop new P-gp inhibitors.info:eu-repo/semantics/publishedVersio

Dual inhibitors of P-glycoprotein and tumor cell growth: (re)discovering thioxanthones

For many pathologies, there is a crescent effort to design multiple ligands that interact with a wide variety of targets. 1-Aminated thioxanthone derivatives were synthesized and assayed for their in vitro dual activity as antitumor agents and P-glycoprotein (P-gp) inhibitors. The approach was based on molecular hybridization of a thioxanthone scaffold, present in known antitumor drugs, and an amine, described as an important pharmacophoric feature for P-gp inhibition. A rational approach using homology modeling and docking was used, to select the molecules to be synthesized by conventional or microwave-assisted Ullmann C–N cross-coupling reaction. The obtained aminated thioxanthones were highly effective at inhibiting P-gp and/or causing growth inhibition in a chronic myelogenous leukemia cell line, K562. Six of the aminated thioxanthones had GI50 values in the K562 cell line below 10 mM and 1-{[2-(diethylamino)ethyl]amino}-4-propoxy-9H-thioxanthen-9-one (37) had a GI50 concentration (1.90 mM) 6-fold lower than doxorubicin (11.89 mM) in the K562Dox cell line. The best P-gp inhibitor found was 1-[2-(1H-benzimidazol-2-yl)ethanamine]-4-propoxy-9H-thioxanthen-9-one (45), which caused an accumulation rate of rhodamine-123 similar to that caused by verapamil in the K562Dox resistant cell line, and a decrease in ATP consumption by P-gp. At a concentration of 10 mM, compound 45 caused a decrease of 12.5-fold in the GI50 value of doxorubicin in the K562Dox cell line, being 2-fold more potent than verapamil. From the overall results, the aminated thioxanthones represent a new class of P-gp inhibitors with improved efficacy in sensitizing a resistant P-gp overexpressing cell line (K562Dox) to doxorubicin.info:eu-repo/semantics/publishedVersio

Chemical study and biological activity evaluation of two Azorean Macroalgae: Ulva rigida and Gelidium microdon

Copyright: © 2013 Silva M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credite.The green macroalga Ulva rigida C. Agardh (Chlorophyta) and the red macroalga Gelidium microdon Kützing (Rhodophyta), collected from the Azorean archipelago, were investigated for their secondary metabolites and their in vitro growth inhibitory effect on three human tumor cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma), as well as for their antifungal and antibacterial activities. The methanol extract of U. rigida furnished isofucosterol (1), 7(E)-3β-hydroxy-5α,6α-epoxymegastigmane (2) and (+)-dehydrovomifoliol (3) while the methanol extract of G. microdon yielded cholesterol (4) and lumichrome (5). The crude extracts of both macroalgae were found to be moderately active against the three cell lines whereas compound 1 showed a weak effect and compound 2 was inactive. The crude extracts of the two macroalgae were found to be moderately active against some fungi and bacteria while compounds 1 and 2 were inactive against all microorganisms tested

Design and synthesis of new inhibitors of p53–MDM2 interaction with a chalcone scaffold

The virtual screening of a library of chalcone derivatives led us to the identification of potential new MDM2 ligands. The chalcones with the best docking scores obeying the Lipinski rule of five were subsequently prepared by base-catalyzed aldol reactions. The activity of these compounds as inhibitors of p53–MDM2 interaction was investigated using a yeast-based screening assay. Using this approach two chalcones (3 and 4) were identified as putative small molecule inhibitors of p53–MDM2 interaction. The activity of both chalcones was further investigated in a panel of human tumor cells. Chalcones 3 and 4 revealed a pronounced tumor cell growth inhibitory effect on tumor cell lines. Additionally, chalcone 4 caused alterations in the cell cycle profile, induced apoptosis and increased the levels of p53, p21 and PUMA proteins in NCI-H460 cells. Computational docking studies allowed to predict that, like nutlin-3A (a well-known small-molecule inhibitor of p53–MDM2 interaction), chalcones 3 and 4 bind to the p53-binding site of MDM2. The results here presented will be valuable for the structure-based design of novel and potent p53–MDM2 inhibitors.This research was partially supported by the Strategic Funding UID/Multi/04423/2013 , ERDF , COMPETE , and FCT under the projects PTDC/MAR-BIO/4694/2014, and INNOVMAR – Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035 , Research Line NOVELMAR . This work also received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/QUI/50006/2013 and the FCT project PTDC/DTP-FTO/1981/2014, “PEst-C/SAU/LA0003/2013”, “NORTE-07-0162-FEDER-00018 – Contributos para o reforço da capacidade do IPATIMUP enquanto actor do sistema regional de inovação” and NORTE-07-0162-FEDER-000067 – Reforço e consolidação da capacidade infraestrutural do IPATIMUP para o sistema regional de inovação”, both supported by ON.2 – O Novo Norte, through FEDER funds under the QREN. IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT. The authors also thank FCT for the grants of R.T. Lima ( SFRH/BPD/68787/2010 ), J. Soares ( SFRH/BD/78971/2011 ), and S. Gomes ( SFRH/BD/96189/2013 ; Doctoral Programme BiotechHealth), L. Raimundo ( PD/BI/113926/2015 , Doctoral Programme BiotechHealth)

Spatial Analysis of Wildlife Tuberculosis Based on a Serologic Survey Using Dried Blood Spots, Portugal

We investigated the spatial epidemiology of bovine tuberculosis (TB) in wildlife in a multihost system. We surveyed bovine TB in Portugal by serologic analysis of elutes of dried blood spots obtained from hunted wild boar. We modeled spatial disease risk by using areal generalized linear mixed models with conditional autoregressive priors. Antibodies against Mycobaterium bovis were detected in 2.4% (95% CI 1.5%-3.8%) of 678 wild boar in 2 geographic clusters, and the predicted risk fits well with independent reports of M. bovis culture. Results show that elutes are an almost perfect substitute for serum (Cohen unweighted κ = 0.818), indicating that serologic tests coupled with dried blood spots are an effective strategy for large-scale bovine TB surveys, using wild boar as sentinel species. Results also show that bovine TB is an emerging wildlife disease and stress the need to prevent further geographic spread and prevalence increase.Plan Nacional (grant CGL2017-89866 from the Ministry of Economy and Competitiveness, Spain, and Fondo Europeo de Desarollo Regional) and Programa Operacional Regional do Norte (grant ON.2 O Novo Norte), Quadro de Referência Estratégico Nacional through the Fundo Europeu de Desenvolvimento Regional. N.S. was supported by PhD grant SFRH/BD/69390/2010 from Fundação para a Ciência e Tecnologiainfo:eu-repo/semantics/publishedVersio

Psoralen analogues: synthesis, inhibitory activity of growth of human tumor cell lines and computational studies

Eight psoralens have been evaluated for their ability to inhibit the in vitro growth of three human turner cell lines representing different tumor types, MCF-7 (breast cancer), NCI-14460 (non-small cell lung cancer) and SF-268 (CNS cancer). The synthesis of four new psoralens (benzofur-ocournarins) is presented as well as the results of the ab initio calculations to find the parameters that relate the structure with the antitumor activity. This work provides supplementary information that could allow the development of new psoralen analogues with this type of biological activity.For financial support from Fundação para a Ciência e Tecnologia, Portugal (I&D no. 226/9 and to CQ-UM), POCTI and FEDER; Ph.D. grants from FCT: Ana M.A.G. Oliveira (PRAXIS XXI/BD/19707/99), Madalena Pedro (SFRH/BD/1456/2000) and César Portela (SFRH/BD/3036/2000). For financial support and research grants to FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) and CNPq (Conselho Nacional do Desenvolvimento Científico e Tecnológico, Brazil). The authors thank National Cancer Institute, Bethesda, MD (USA) for kindly providing the tumor cell lines and Elisa Pinto for obtaining 1H-NMR and 13C-NMR spectra and elemental analyses.POCTI