Stefania Dragoni

Red wine alcohol promotes quercetin absorption and directs its metabolism towards isorhamnetin and tamarixetin in rat intestine in vitro.

Moderate consumption of red wine has been associated with beneficial effects on human health, and this has been attributed to the flavonoid content. Factors that influence the bioavailability of this group of polyphenolic compounds are therefore important. Using the rat cannulated everted jejunal sac technique, we have investigated the effect of alcohol on the intestinal absorption of quercetin and its 3‐O‐glucoside from red wine. Tissue preparations were incubated in whole or dealcoholised red wine, diluted 1 : 1 with Krebs buffer for 20 min at 37°C, after which the mucosa was removed and processed for HPLC analysis. Tissues exposed to red wine had significantly higher amounts of both quercetin (× −3; P<0.001) and quercetin‐3‐O‐glucoside (× −1.5; P<0.01) associated with them, compared with sacs incubated in the dealcoholised equivalent. In addition, both tamarixetin (T) and isorhamnetin (I), in the mucosal tissue from sacs exposed to the whole wine, were significantly elevated approximately two fold (P<0.05; P<0.01, respectively). Similar results were obtained when sacs were incubated in Krebs buffer containing a mixture of pure quercetin and quercetin‐3‐O‐glucoside with or without alcohol, and, although effects on the apparent absorption of Q and Q‐3‐G were not so marked, concentrations of the metabolites quercetin‐3‐O‐glucuronide and I were significantly increased by the presence of alcohol (P<0.01 and P<0.001, respectively). It is therefore plausible that the moderate alcohol content of red wine contributes to its beneficial health effects in humans by both increasing the absorption of quercetin and quercetin‐3‐O‐glucoside and by channelling their metabolism towards O‐methylation to yield compounds (T and I), which have potential protective effects against cancer and cardiovascular diseases.

Interactions of taurine and structurally related analogues with the GABAergic system and taurine binding sites of rabbit brain

The aim of this study was to find taurinergic compounds that do not interact with brain GABA ergic systems. Washed synaptic membranes (SM) from whole rabbit brain were able to bind [3H]muscimol. Saturation experiments of the binding of [3H]GABA to GABAB receptors showed that SM possess two binding components; twice Triton X‐100‐treated SM contained 0.048 mmol endogenous taurine/kg protein and bound [3H]taurine in a saturable manner (Kd=249.0±6.3 nM and Bmax=3.4±1.0 pmol mg−1 prot). Among the 19 structural analogues of taurine, 6‐aminomethyl‐3‐methyl‐4H‐1,2,4‐benzothiadiazine 1,1‐dioxide (TAG), 2‐aminoethylarsonic (AEA), 2‐hydroxyethanesulfonic (ISE) and (±)cis‐2‐aminocyclohexane sulfonic acids (CAHS) displaced [3H]taurine binding (Ki=0.13, 0.13, 13.5 and 4.0 μM, respectively). These analogues did not interact with GABAA and GABAB receptors and did not affect taurine‐ and GABA‐uptake systems and GABA‐transaminase activity. 3‐Aminopropanesulfonic acid (OMO), β‐alanine, pyridine‐3‐sulfonic acid, N,N,N‐trimethyltaurine (TMT), 2‐(guanidino)ethanesulfonic acid (GES), ethanolamine‐O‐sulphate, N,N‐dimethyltaurine (DMT), taurine and (±)piperidine‐3‐sulfonic acid (PSA) inhibited [3H]muscimol binding to GABAA receptors with different affinities (Ki=0.013, 7.9, 24.6, 47.5, 52.0, 91.0, 47.5, 118.1 and 166.3 μM, respectively). Taurine, 2‐aminoethylphosphonic acid, DMT, TMT and OMO inhibited the binding of [3H]GABA to GABAB receptors with Kis in the μM range (0.8, 3.5, 4.4, 11.3 and 5.0, respectively). GES inhibited taurine uptake (IC50=3.72 μM) and PSA GABA transaminase activity (IC50=103.0 μM). In conclusion, AEA, TAG, ISE and CAHS fulfill the criteria for taurinergic agents.

Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 4. Identification of New Potent and Selective Ligands for the Cannabinoid Type 2 Receptor with Diverse Substitution Patterns and Antihyperalgesic Effects in Mice

Experimental evidence suggests that selective CB2 receptor modulators may provide access to antihyperalgesic agents devoid of psychotropic effects. Taking advantage of previous findings on structure-activity/selectivity relationships for a class of 4-quinolone-3-carboxamides, further structural modifications of the heterocyclic scaffold were explored, leading to the discovery of the 8-methoxy derivative 4a endowed with the highest affinity and selectivity ever reported for a CB2 ligand. The compound, evaluated in vivo in the formalin test, behaved as an inverse agonist by reducing at a dose of 6 mg/kg the second phase of the formalin-induced nocifensive response in mice.

Gold nanoparticles uptake and cytotoxicity assessed on rat liver precision-cut slices.

A major obstacle in the field of nanotoxicology is the development of an in vitro model that accurately predicts an in vivo response. To address this concern, rat liver precision-cut slices were used to assess the impact of 5-nm gold nanoparticles (GNPs) coated with polyvinylpyrrolidone (PVP) on the mammalian liver, following exposure to different concentrations and for a duration of up to 24 h. The presence of GNPs inside endocytotic vesicles of hepatocytes was appreciable within 30 min of their addition. After 2 h, GNPs were clearly visualized inside endosome-like vesicles within the slice, not only in hepatocytes but also in endothelial and Kupffer cells located within the first two cellular layers. This uptake did not translate into modifications of either phase I or phase II of 7-ethoxycoumarin metabolism or alter activities of cytochrome P450 toward marker substrates. Furthermore, although the GNPs were rapidly internalized, no overt signs of cytotoxicity, assessed through lactate dehydrogenase release, reduction of methylthiazolyldiphenyl tetrazolium bromide, and glutathione levels, were observed. In conclusion, the use of rat liver slices successfully enhanced nanomaterial screening and determined that PVP-coated 5-nm GNPs were biocompatible with rat liver cells.

Investigations on the 4-Quinolone-3-Carboxylic Acid Motif Part 5: Modulation of the Physicochemical Profile of a Set of Potent and Selective Cannabinoid-2 Receptor Ligands through a Bioisosteric Approach†

Three heterocyclic systems were selected as potential bioisosteres of the amide linker for a series of 1,6‐disubstituted‐4‐quinolone‐3‐carboxamides, which are potent and selective CB2 ligands that exhibit poor water solubility, with the aim of improving their physicochemical profile and also of clarifying properties of importance for amide bond mimicry. Among the newly synthesized compounds, a 1,2,3‐triazole derivative (1‐(adamantan‐1‐yl)‐4‐[6‐(furan‐2‐yl)‐1,4‐dihydro‐4‐oxo‐1‐pentylquinolin‐3‐yl]‐1H‐1,2,3‐triazole) emerged as the most promising in terms of both physicochemical and pharmacodynamic properties. When assayed in vitro, this derivative exhibited inverse agonist activity, whereas, in the formalin test in mice, it produced analgesic effects antagonized by a well‐established inverse agonist. Metabolic studies allowed the identification of a side chain hydroxylated derivative as its only metabolite, which, in its racemic form, still showed appreciable CB2 selectivity, but was 150‐fold less potent than the parent compound.

Biological models for phytochemical research: from cell to human organism

Nutrigenomics represents a shift of nutrition research from epidemiology and physiology to molecular biology and genetics. Nutrigenomics seeks to understand nutrition influences on homeostasis, the mechanism of genetic predispositions for diseases, to identify the genes influencing risk of diet related diseases. This review presents some in vitro models applicable in nutrigenomic studies, and discusses the use of animal models, their advantages and limitations and relevance for human situation. In vitro and in vivo models are suitable for performance of DNA microarrays, proteomic and transcriptomic analyses. In vitro models (intracellular organelles and suborganellar compartments, cell cultures, or tissue samples/cultures) give insight in metabolic pathways and responses to test stimuli on cellular and molecular levels. Animal models allow evaluation of the biological significance of the effects recorded in vitro and testing of the hypothesis on how a specific factor affects specific species under specific circumstances. Therefore, the evaluation of the data in relation to human organism should be done carefully, considering the species differences. The use of in vitro and in vivo models is likely to continue as the effects of nutrition on health and disease cannot be fully explained without understanding of nutrients action at nuclear level and their role in the intra- and intercellular signal transduction. Through advances in cell and molecular biology (including genomic and proteomic), the use of these models should become more predictively accurate. However, this predictive value relies on an underpinning knowledge of the advantages and limitations of the model in nutrigenomic research as in other fields of biomedical research.

DP7, a novel dihydropyridine multidrug resistance reverter, shows only weak inhibitory activity on human CYP3A enzyme(s)

The aim of this study was to investigate the effects of 3,5-dibenzoyl-4-(3-phenoxy-phenyl)-1,4-dihydro-2,6-dimethylpyridine (DP7), a novel multidrug resistance (MDR) reverter, on cytochrome P450 (CYP)-activities by human and rat liver microsomes. Effects of DP7 were assessed with use of selective substrates, markers of CYP activities. With rat microsomes, ethoxyresorufin (ETR) was used as substrate for CYP1A1, penthoxyresorufin (PTR) for 2B, benzyloxyresorufin (BZR) for 1A1/2, 2B, 2C, 3A. CYP3A enzyme activities of rat (3A2) and human (3A4) liver microsomes, were assessed fluorimetrically using either 7-benzyloxy-quinoline (BQ) or [3-[3(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl)-phenyl]furan-2-(5H)-one] (DFB). When rat microsomes were incubated with DP7, concentration-inhibition curves were obtained. DP7 inhibitions gave IC(50) values of 3.8 microM for PTR, 3.8 microM for ETR and 10.4 microM for BZR and were not competitive in nature; moreover, they were reversible. When BQ was used as substrate of rat microsomes, DP7 inhibited its oxidation with an IC(50) value of 4.17 microM, while this oxidation was inhibited by only 25% at the highest DP7 concentration used (75 microM) with human microsomes. On the contrary, when DFB was used as substrate, DP7 showed identical IC(50) values (34.67 microM) with microsomal preparations from either species. The moderate inhibition of CYP isoforms of rat liver microsomes and the weak inhibition of human CYP3A4 enzyme activity operated by DP7, suggest that DP7 in man should not give rise to important, unpredictable pharmacokinetic interactions. This conclusion supports the role of this compound as a lead for the development of novel MDR reverterting dihydropyridines of therapeutic interest.

l -Deprenyl metabolism by the cytochrome P450 system in monkey (Cercopithecus aethiops) liver microsomes

1. The aim was to clarify the kinetic and cytochrome P450 (CYP) enzymes involved in l -deprenyl metabolism by liver microsomal preparations from African green monkeys, an animal model extensively used in the study of Parkinsons disease. 2. CYP levels and monoxygenase activities were similar to those observed in microsomes from other monkey strains. The enzyme kinetics of both l -methamphetamine and l -nordeprenyl formation were characterized by a high- and low-affinity component. For l -methamphetamine, the apparent K m 1 and K m 2 were 1.07 ±0.01 and 350 ±2.7 µM, and V max 1 and V max 2 were 4.70 ±0.01 and 8.9 ±0.02 nmol min <1 mg protein <1, respectively. For l -nordeprenyl, K m 1 and K m 2 were 0.96 ±0.05 and 168 ±15 µM, and V max 1 and V max 2 were 3.34 ±0.02 and 3.91 ±0.02 nmol min <1 mg protein <1, respectively The ratio V max / K m for both metabolites was 2 orders of magnitude higher for the low K m component than for the high K m, suggesting that the former component is the major determinant of l -deprenyl N-dealkylation. At 15 µM l -deprenyl, both ketoconazole and 8-methoxypsoralen significantly inhibited l -methamphetamine and l -nordeprenyl formation, indicating that CYP3A and CYP2A enzymes were involved in both reactions. At 500 µM l -deprenyl, however, inhibition studies suggest the involvement of CYP1A and 2D enzymes. 3. The metabolism of l -deprenyl by monkey liver microsomes is very efficient, indicating that CYP-dependent metabolism is relevant and could contribute to neuroprotection in primate models of Parkinsons disease.

Antioxidant properties of propargylamine derivatives: assessment of their ability to scavenge peroxynitrite.

A series of arylpropargylamines, variously substituted in the hydrogen in p‐position and in the propargyl moiety, were studied as potential peroxynitrite scavengers. The scavenging activity of these compounds was evaluated through peroxynitrite (ONOO−)‐mediated oxidation of dichlorofluorescin and linoleic acid by measuring the dichlorofluorescein formation and oxygen consumption, respectively. Among tested compounds, only 1‐phenylpropargylamine (AP3) promoted concentration‐dependent inhibition of ONOO−‐induced dichlorofluorescin and linoleic acid oxidation with IC50 values of 637 and 63 μm, respectively. The AP3 spectral changes in UV‐visible absorbance properties in the presence of peroxynitrite suggested the formation of a new compound. This was identified by gas‐chromatograph‐mass spectrometer analysis as phenylpropargyl alcohol. Structure—activity relationship analysis indicated that the scavenging activity of AP3 was due to the aminopropargyl moiety and availability of the nitrogen electron pair. This data suggested that AP3 could be considered a lead compound for the synthesis of new ONOO− scavenger derivatives.

Cytochrome P450-dependent metabolism of l-deprenyl in monkey (Cercopithecus aethiops) and C57BL/6 mouse brain microsomal preparations

The aim of the present investigation was to characterize the cytochrome P450 (CYP)‐dependent metabolism of l‐deprenyl by brain microsomal preparations obtained from two different animal models that have been extensively used in Parkinsons disease studies, namely monkey (Cercopithecus aethiops) and C57BL/6 mouse. In monkey brain microsomal fractions, the apparent Km values for methamphetamine formation from l‐deprenyl were 67.8 ± 1.0 and 72.0 ± 1.6 μm, in the cortex and striatum, respectively. Similarly, for nordeprenyl formation from l‐deprenyl, Km values in cortex and striatum were 21.3 ± 3.2 and 27.3 ± 4.0 μm, respectively. Both metabolic pathways appear to be more efficient in the cortex than in the striatum as the Vmax for microsomal preparation was lower in the striatum for the formation of both metabolites. The formation rate of l‐methamphetamine was up to one order of magnitude greater than that of nordeprenyl. Inhibition analysis of both pathways in monkey brain suggested that l‐methamphetamine formation is catalysed by CYP2A and CYP3A, whereas only CYP3A appears to be involved in nordeprenyl formation. With microsomal preparations from whole brain of C57BL/6 mice, the only l‐deprenyl metabolite that could be detected was methamphetamine and the Km and Vmax values were similar to those determined in monkey cortex (53.6 ± 2.9 μm and 33.9 ± 0.4 pmol/min/mg protein, respectively). 4‐Methylpyrazole selectively inhibited methamphetamine formation, suggesting the involvement of CYP2E1. In conclusion, the present study indicates that l‐deprenyl is effectively metabolised by CYP‐dependent oxidases in the brain, giving rise mainly to the formation of methamphetamine, which has been suggested to play a role in the pharmacological effects of the parent drug. The results also demonstrate that there are differences between species in CYP‐dependent metabolism of l‐deprenyl.