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Asymmetrical N, N-bis(alkanol)amine aryl esters (FRA77, GDE6, and GDE19) are potent multidrug resistance (MDR) reversers. Their structures loosely remind that of the Ca antagonist verapamil. Therefore, the aim of this study was to investigate their vascular activity in vitro. Their effects on the mechanical activity of fresh and cultured rat aorta rings on Ca1.2 channel current ( I ) of A7r5 cells and their cytotoxicity on A7r5 and EA.hy926 cells were analyzed. Docking at the rat α subunit of the Ca1.2 channel was simulated in silico. Compounds tested were cytotoxic at concentrations >1 μM (FRA77, GDE6, GDE19) and >10 μM (verapamil) in EA.hy926 cells, or >10 μM (FRA77, GDE6, GDE19) and at 100 μM (verapamil) in A7r5 cells. In fresh rings, the three compounds partly antagonized phenylephrine and 60 mM K (K60)-induced contraction at concentrations ≥1 and ≥3 μM, respectively. On the contrary, verapamil fully relaxed rings pre-contracted with both agents. In cultured rings, 10 μM GDE6, GDE19, FRA77, and verapamil significantly reduced the contractile response to both phenylephrine and K60. Similarly to verapamil, the three compounds docked at the α subunit, interacting with the same amino acids residues. FRA77, GDE6, and GDE19 inhibited I with IC values 1 order of magnitude higher than that of verapamil. FRA77-, GDE6-, and GDE19-induced vascular effects occurred at concentrations that are at least 1 order of magnitude higher than those effectively reverting MDR. Though an unambiguous divergence between MDR reverting and vascular activity is of overwhelming importance, these findings consistently contribute to the design and synthesis of novel and potent chemosensitizers. [ABSTRACT FROM AUTHOR]

In a continuing search for potent P-gp-dependent multidrug-resistant (MDR) reversers we synthesized and studied a new series of N-alkanol-N-cyclohexanol amine aryl esters characterized by the presence of two linkers with different flexibility: a polymethylene chain of variable length and a cyclohexylic scaffold, that gave origin to two geometrical isomers (cis and trans). The reversal activity of the new compounds was evaluated on the K562/DOX cell line by three tests: pirarubicin uptake modulation, doxorubicin cytotoxicity enhancement (reversal fold, RF) and inhibition of P-gp-mediated rhodamine-123 (Rhd 123) efflux tests. The chemical stability of their ester function was evaluated in the experimental conditions utilized (phosphate buffer solution (PBS), bovine serum and in the presence of K562/DOX cells) and in human plasma. The new series of molecules showed very interesting MDR reversing properties; in particular compound 5b (ELF26B), characterized by trans stereochemistry and a 5-methylene chain, presented the best pharmacological profile and is stable in each tested medium. Compound 5b could be an interesting lead for the development of new potent and efficacious P-gp-dependent MDR modulators., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Rationale: A series of drug plasma stability experiments were carried out to evaluate the bioavailability of three multidrug resistance inhibitors. The studied compounds are positional isomers; therefore, a chromatographic separation or taking advantage of specific collisionally activated decomposition pathways, obtained by tandem mass spectrometry (MS/MS) experiments, is necessary in order to resolve them., Methods: A method was developed for quantitative determination of the analytes in plasma using liquid chromatography (LC) coupled with a triple quadrupole mass spectrometer operating in MS/MS mode. Different collisional approaches were employed based on the potentiality of a triple quadrupole system. Aside from the classical product ion spectroscopy, energy-resolved MS/MS experiments and a post-processing mathematical algorithm tool (LEDA) were used to distinguish among different kinds of inhibitors present in the sample batch., Results: The developed LC/MS/MS method showed precision between 1.8-7.9%, accuracy ranging from 92.8 to 99.9% and limit of detection (LOD) values in the range 1.0-1.4 ng mL(-1) for all the analytes. The evaluation of matrix effects demonstrated that the sample preparation procedure did not affect the ionization efficiency or recovery (matrix effects and recovery larger than 88%). Finally, the LEDA tool was able to differentiate among the isomers, ensuring their proper monitoring., Conclusions: The proposed LC/MS/MS method was suitable for evaluating the stability of the analytes in plasma samples, although small concentration variations occurred. Furthermore, the investigation on the energetics of fragmentation pathways allowed the better product ions and optimal abundance ratios to be selected for LEDA application into a multi-component analysis. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

A series of N-alkanol-N-cyclohexanol amine aryl esters cis/trans isomers that showed high efficacy to reverse the acquired resistance of cancer cells during chemotherapeutic therapy (MDR mechanism) was studied. These compounds were two 1,4 cyclohexane cis/trans derivatives (named ELF26A and ELF26B, respectively), and their positional isomers (named ELF34A and ELF34B, respectively) where the aryl-moieties were exchanged. In order to evaluate the behaviour of these compounds during biological tests, a method based on liquid chromatography coupled with mass spectrometry (LC-MS), operating in tandem mass spectrometry (MS/MS) mode, was developed. A unique chromatographic method suitable to separate the two pairs of cis/trans isomers was not achieved and the MS/MS experiments of the different compounds was not always able to characterise the different isomers. Therefore, a system of linear equations of deconvolution analysis (LEDA) tool was proposed to determine the relative proportions of individual cis/trans isomers in the sample. Considering the pharmaceutical interest of the compounds under investigation, the analytical method developed was tested to be effective at the active concentration levels, corresponding to a concentration of ng mL -1 of compound in a processed sample. Precision and accuracy of the LEDA algorithm at three levels of relative concentrations of analytes were checked, i.e. low-level (about 25% in the mixture), mid-level (about 50% in the mixture) and high-level (about 70% in the mixture). Evaluation of performances of the algorithm proved that the accuracy (between 88.3% and 99.9%) and precision (between 2.0% and 3.7%) for simultaneous analysis of the mixtures of the four isomers is feasible. It is worth highlighting that the choice of characteristic product ions and optimal abundance ratios plays an important role in the application of the LEDA approach. Therefore, performing an investigation on the energetics of fragmentation pathway allowed the selection of the better product ions for each analyte in terms of both sensitivity of detection and specificity, i.e. the capability to distinguish between isomeric compounds. Finally, the developed approach was applied to determine the relative proportions of individual cis/trans isomers in spiked human plasma samples. The results obtained confirm the reliability of the proposed method in biological samples as well.

A set of basic aryl-group-containing compounds was synthesized with the aim of developing potent and selective P-glycoprotein (P-gp) modulators that are able to reverse multidrug resistance (MDR). The natures of the spacer (dicyclohexylamine or dialkylamine) and the aryl moieties were modified to investigate selectivity and the mechanism of P-gp interaction. The inhibitory activities of the compounds toward P-gp, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), the most relevant ATP binding cassette (ABC) transporters for MDR, were evaluated. The mechanism of P-gp interaction for each compound was investigated with three biological assays: apparent permeability (Papp ) determination (B→A/A→B) in Caco-2 cell monolayers, ATP cell depletion, and inhibition of Calcein-AM transport in MDCK-MDR1 cells. These assays allowed us to estimate the selectivity of the compounds for the three efflux pumps and to identify the structural requirements that define the P-gp-interaction profile. All dicyclohexylamine derivatives were found to be P-gp substrates, whereas one dialkylamine derivative was shown to be a P-gp inhibitor. The good MRP1 activity of one cis/cis isomer highlighted this as a lead candidate for the development of MRP1 ligands., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A series of 2-phenyl- or 3-phenyl piperazines, structurally related to DM235 and DM232, two potent nootropic agents, have been prepared and tested in the mouse passive-avoidance test, to assess their ability to revert scopolamine-induced amnesia. Although the newly synthesized molecules were less potent than the parent compounds, some useful information has been obtained from structure-activity relationships. A small but significant enantioselectivity has been found for the most potent compound 5a., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

As a continuation of our research on potent and efficacious P-gp-dependent multidrug resistance (MDR) reversers, several new N,N-bis(alkanol)amine aryl esters were designed and synthesized, varying the aromatic moieties or the length of the methylenic chain. The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay, where most of the new compounds were shown to be active. In particular the asymmetrical compounds, characterized by two linkers of different length, generally showed fairly high activities as MDR reversers. Some selected compounds (isomers 15-17) were further studied by evaluating their doxorubicin cytotoxicity enhancement (reversal fold, RF) on the K562/DOX cell line. The results of both pharmacological assays indicate that compounds 16 (GDE6) and 17 (GDE19) could be interesting leads for the development of new P-gp dependent MDR modulators., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and often challenging. The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

After an acute central nervous system injury, axonal regeneration is limited as the result of a lack of neuronal intrinsic competence and the presence of extrinsic inhibitory signals. The injury fragments the myelin neuronal insulating layer, releasing extrinsic inhibitory molecules to signal through the neuronal membrane-bound Nogo receptor (NgR) complex. In this paper, we show that a neuronal transcriptional pathway can interfere with extrinsic inhibitory myelin-dependent signaling, thereby promoting neurite outgrowth. Specifically, retinoic acid (RA), acting through the RA receptor β (RAR-β), inhibited myelin-activated NgR signaling through the transcriptional repression of the NgR complex member Lingo-1. We show that suppression of Lingo-1 was required for RA-RAR-β to counteract extrinsic inhibition of neurite outgrowth. Furthermore, we confirm in vivo that RA treatment after a dorsal column overhemisection injury inhibited Lingo-1 expression, specifically through RAR-β. Our findings identify a novel link between RA-RAR-β-dependent proaxonal outgrowth and inhibitory NgR complex-dependent signaling, potentially allowing for the development of molecular strategies to enhance axonal regeneration after a central nervous system injury.

The aim of the present observational study was to assess the value of the C825T polymorphism of the beta-3 subunit of G proteins (GNB3) as well as of variants in the SLC6A4 gene (5HTTLPR and STin2 VNTR) and DRD2 gene (TaqI A and NcoI) as predictive markers for consistency in headache response to triptans in migraine patients. Consistent responders to triptans were defined as the migraineurs who experienced a > or =2 point reduction in a 4-point scale intensity of pain from 3 (severe) to 0 (absent) 2h after triptan administration, in at least two attacks out of the three. Genotyping was performed by PCR and PCR-RFLP on genomic DNA extracted from peripheral blood. The impact of clinical and biological variables on consistency status of headache response to triptans was evaluated by using a binary logistic regression model with stepwise selection. Forty-three (33%) of the 130 migraine patients included in the study did not consistently respond to triptan administration. In a binary logistic regression model, STin 2.12/12 genotype (OR=3.363, 95% CI: 1.262-8.966, P=0.005) and non-use of migraine prophylactic medications (OR=2.848, 95% CI: 1.019-7.959, P=0.010) were found as significant factors increasing the odds of achieving inconsistent response to triptans. The analysis of classificatory power of the model showed moderate values of sensitivity (0.56), high specificity (0.87), and an overall prediction correctness (0.77). These results support the role of STin2 VNTR polymorphism of serotonin transporter gene as a relevant genetic factor conferring a higher risk of inconsistent response to triptans in migraine patients., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Various reports have demonstrated that the sphingolipids sphingosine and sphingosine-1-phosphate are able to induce Ca2+ release from intracellular stores in a similar way to second messengers. Here, we have used the sea urchin egg homogenate, a model system for the study of intracellular Ca2+ release mechanisms, to investigate the effect of these sphingolipids. While ceramide and sphingosine-1-phosphate did not display the ability to release Ca2+, sphingosine stimulated transient Ca2+ release from thapsigargin-sensitive intracellular stores. This release was inhibited by ryanodine receptor blockers (high concentrations of ryanodine, Mg2+, and procaine) but not by pre-treatment of homogenates with cADPR, 8-bromo-cADPR or blockers of other intracellular Ca2+ channels. However, sphingosine rendered the ryanodine receptor refractory to cADPR. We propose that, in the sea urchin egg, sphingosine is able to activate the ryanodine receptor via a mechanism distinct from that used by cADPR.