Francisco Orallo

Effects of cis-resveratrol on inflammatory murine macrophages: antioxidant activity and down-regulation of inflammatory genes.

This study investigated for the first time the effects of the cis isomer of resveratrol (c‐RESV) on the responses of inflammatory murine peritoneal macrophages, namely on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) during the respiratory burst; on the biosynthesis of other mediators of inflammation such prostaglandins; and on the expression of inflammatory genes such as inducible nitric oxide synthase (NOS)‐2 and inducible cyclooxygenase (COX)‐2. Treatment with 1–100 μM c‐RESV significantly inhibited intracellular and extracellular ROS production, and c‐RESV at 10–100 μM significantly reduced RNS production. c‐RESV at 1–100 μM was ineffective for scavenging superoxide radicals (O2•−), generated enzymatically by a hypoxanthine (HX)/xanthine oxidase (XO) system and/or for inhibiting XO activity. However, c‐RESV at 10–100 μM decreased nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase activity in macrophage homogenates. c‐RESV at 100 μM decreased NOS‐2 and COX‐2 mRNA levels in lipopolysaccharide (LPS) interferon gamma (IFN‐γ)‐treated macrophages. At 10–100 μM, c‐RESV also significantly inhibited NOS‐2 and COX‐2 protein synthesis and decreased prostaglandin E2 (PGE2) production. These results indicate that c‐RESV at micromolar concentrations significantly attenuates several components of the macrophage response to proinflammatory stimuli (notably, production of O2•− and of the proinflammatory mediators NO• and PGE2).

Design of New Formulations for Topical Ocular Administration: Polymeric Nanocapsules Containing Metipranolol

To investigate the potential of polymeric nanocapsules for ocular delivery of beta-blockers, several formulations of polyisobutylcyanoacrylate and polyepsiloncaprolactone nanocapsules containing metipranolol base were developed. These formulations differed in the polymer forming the coating and in the type and volume of the oil encapsulated. Analysis of particle-size distribution, electrophoretic mobility, and loading efficiency of the nanocapsules revealed that the type of oil is the most important factor influencing these properties. From the in vitro release studies, we concluded that drug diffusion through a dialysis membrane is delayed as a consequence of the encapsulation process. However, the release profiles were not influenced by the polymeric coating, suggesting that drug release from these systems is governed mainly by the partition of the drug between the oily core and the aqueous release medium. Nevertheless, despite the inability of the polymer coat to control the release of the drug, its contribution to the stabilization of the emulsion was noted. Finally, the suitability of these formulations for ophthalmic administration was investigated. Although the pharmacologic response was not affected by the encapsulated metipranolol compared with the commercial eye drops, a drastic reduction of the drug’s systemic side effects was observed.

In vitro effects of mangiferin on superoxide concentrations and expression of the inducible nitric oxide synthase, tumour necrosis factor-α and transforming growth factor-β genes

This study investigated the effects of the natural polyphenol mangiferin (MA) on superoxide anion (O(2)(-)) production, xanthine oxidase (XO) activity, vascular contractility, inducible nitric oxide synthase (iNOS) mRNA levels, tumour necrosis factor-alpha (TNF-alpha) mRNA levels, and tumour growth factor-beta (TGF-beta) mRNA levels. O(2)(-) was generated by the hypoxanthine-xanthine oxidase (HX-XO) and phenazine methosulphate (PMS)-NADH systems. XO activity was determined by measurement of uric acid production with xanthine as substrate. Vascular contraction experiments were performed with intact rat aortic rings. iNOS, TNF-alpha and TGF-beta gene expression in rat macrophages stimulated in vivo with 3% thioglycollate and in vitro with 100 ng/mL lipopolysaccharide and 10U/mL of interferon-gamma were evaluated semiquantitatively by the retrotranscriptase-polymerase chain reaction. MA at 10-100 microM, like the known O(2)(-) scavenger superoxide dismutase (1U/mL), scavenged O(2)(-) produced by the HX/XO and PMS-NADH systems. By contrast MA at 1-100 microM, unlike allopurinol (10 microM), was unable to inhibit XO activity. MA at 1-100 microM did not modify resting tone or the contractile responses elicited by 1 microM phenylephrine or 1 microM phorbol 12-myristate 13-acetate in rat aorta. MA at 1-100 microM, like dexamethasone (100 microM), decreased iNOS mRNA levels in activated macrophages. At 100 microM, MA also reduced TNF-alpha mRNA levels, but increased TGF-beta mRNA levels. These results thus indicate that MA is an O(2)(-) scavenger and that it inhibits expression of the iNOS and TNF-alpha genes, suggesting that it may be of potential value in the treatment of inflammatory and/or neurodegenerative disorders. In addition, the finding that MA enhances TGF-beta gene expression suggests that this polyphenol might also be of value in the prevention of cancer, autoimmune disorders, atherosclerosis and coronary heart disease.

Chalcones: A valid scaffold for monoamine oxidases inhibitors

A large series of substituted chalcones have been synthesized and tested in vitro for their ability to inhibit human monoamine oxidases A and B (hMAO-A and hMAO-B). While all the compounds showed hMAO-B selective activity in the micro- and nanomolar ranges, the best results were obtained in the presence of chlorine and hydroxyl or methoxyl substituents. To better understand the enzyme-inhibitor interaction and to explain the selectivity of the most active compounds toward hMAO-B, molecular modeling studies were carried out on new, high resolution, hMAO-B crystallographic structures. For the only compound that also showed activity against hMAO-A as well as low selectivity, the molecular modeling study was also performed on the hMAO-A crystallographic structure. The docking technique provided new insight on the inhibition mechanism and the rational drug design of more potent/selective hMAO inhibitors based on the chalcone scaffold.

A new series of 3-phenylcoumarins as potent and selective MAO-B inhibitors

6-Methyl-3-phenylcoumarins 3-6 were designed, synthesized and evaluated as monoamine oxidase A and B (MAO-A and MAO-B) inhibitors. The synthesis of these new compounds (resveratrol-coumarin hybrids) was carried out with good yield by a Perkin reaction, from the 5-methylsalicylaldehyde and the corresponding phenylacetic acid. They show high selectivity to the MAO-B isoenzyme, with IC(50) values in the nanomolar range. Compound 5 is the most active compound and is several times more potent and selective than the reference compound, R-(-)-deprenyl.

Regulation of cytosolic calcium levels in vascular smooth muscle.

Ca2+ plays an important role in the contraction of skeletal, cardiac, and smooth muscle, as well as in a number of important processes, such as secretion and neuronal activity. In this review, I focus on the various mechanisms by which cytosolic Ca2+ concentration is regulated in vascular smooth muscle, in the resting state and during activation. Particular attention is paid to the calcium pumps of the plasmalemma and the sarcoplasmic reticulum, to the inositol 1,4,5-trisphosphate- and ryanodine-sensitive calcium channels of the sarcoplasmic reticulum, and to voltage-dependent and voltage-independent calcium channels of the plasmalemma.

Quantitative structure-activity relationship and complex network approach to monoamine oxidase A and B inhibitors.

The work provides a new model for the prediction of the MAO-A and -B inhibitor activity by the use of combined complex networks and QSAR methodologies. On the basis of the obtained model, we prepared and assayed 33 coumarin derivatives, and the theoretical prediction was compared with the experimental activity data. The model correctly predicted 27 compounds, and most of the active derivatives showed IC 50 values in the muM-nM range against both the MAO-A and MAO-B isoforms. Compound 14 shows the same MAO-A inhibitory activity (IC 50 = 7.2 nM), as clorgyline used as a reference inhibitor and has the highest MAO-A specificity (1000-fold higher compared to MAO-B). On the other hand, compounds 24 (IC 50 = 1.2 nM) and 28 (IC 50 = 1.5 nM) show higher activity than selegiline (IC 50 = 19.6 nM) and high MAO-B selectivity with 100-fold and 1600-fold inhibition levels, with respect to the MAO-A isoform.

Synthesis, Stereochemical Identification, and Selective Inhibitory Activity against Human Monoamine Oxidase-B of 2-Methylcyclohexylidene-(4-arylthiazol-2-yl)hydrazones

A series of 2-methylcyclohexylidene-(4-arylthiazol-2-yl)hydrazones have been investigated for their ability to inhibit selectively the activity of the human A and B isoforms of monoamine oxidase (MAO). The target compounds, which present a stereogenic center on the cyclohexane ring, were obtained as pure (R) and (S) enantiomers by enantioselective HPLC. The absolute configuration of homochiral forms isolated on a semipreparative scale was obtained by a combined strategy based on chemical correlation and single-crystal X-ray diffraction. All compounds showed higher activity against the human MAO-B isoform with IC50 values ranging between 26.81 +/- 2.74 microM and 14.20 +/- 0.26 nM, and the assays carried out on the pure enantiomers showed higher activity for the (R) form. A computational study was performed by molecular mechanics, DFT-based quantomechanics, and docking techniques on the most active and human MAO-B selective inhibitor 8.

Synthesis and evaluation of 6-methyl-3-phenylcoumarins as potent and selective MAO-B inhibitors.

A series of 6-methyl-3-phenylcoumarins 3-6 were synthesized and evaluated as monoamine oxidase A and B (MAO-A and MAO-B) inhibitors. A comparative study between the three possible mono methoxy 3-phenyl derivatives and the p-hydroxy analogue is reported. The synthesis of these new resveratrol-coumarin hybrids was carried out by a Perkin reaction between the 5-methylsalicylaldehyde and the corresponding phenylacetic acids. The p-methoxy substituted compound 3 was hydrolyzed to 6 by a traditional reaction with hydriodic acid. The prepared compounds show high selectivity to the MAO-B isoenzyme, some of them with IC(50) values in the low nanomolar range. Compound 4, with the methoxy group in meta position, is the most active of this series, with an IC(50) against MAO-B of 0.80 nM, and is several times more potent and MAO-B selective than the R-(-)-deprenyl (reference compound).

Study of the mechanisms involved in the vasorelaxation induced by (−)-epigallocatechin-3-gallate in rat aorta

This study investigated several mechanisms involved in the vasorelaxant effects of (−)‐epigallocatechin‐3‐gallate (EGCG). EGCG (1 μM–1 mM) concentration dependently relaxed, after a transient increase in tension, contractions induced by noradrenaline (NA, 1 μM), high extracellular KCl (60 mM), or phorbol 12‐myristate 13‐acetate (PMA, 1 μM) in intact rat aortic rings. In a Ca2+‐free solution, EGCG (1 μM–1 mM) relaxed 1 μM PMA‐induced contractions, without previous transient contraction. However, EGCG (1 μM–1 mM) did not affect the 1 μM okadaic acid‐induced contractions. Removal of endothelium and/or pretreatment with glibenclamide (10 μM), tetraethylammonium (2 mM) or charybdotoxin (100 nM) plus apamin (500 nM) did not modify the vasorelaxant effects of EGCG. In addition, EGCG noncompetitively antagonized the contractions induced by NA (in 1.5 mM Ca2+‐containing solution) and Ca2+ (in depolarizing Ca2+‐free high KCl 60 mM solution). In rat aortic smooth muscle cells (RASMC), EGCG (100 μM) reduced increases in cytosolic free Ca2+ concentration ([Ca2+]i) induced by angiotensin II (ANG II, 100 nM) and KCl (60 mM) in 1.5 mM CaCl2‐containing solution and by ANG II (100 nM) in the absence of extracellular Ca2+. In RASMC, EGCG (100 μM) did not modify basal generation of cAMP or cGMP, but significantly reversed the inhibitory effects of NA (1 μM) and high KCl (60 mM) on cAMP and cGMP production. EGCG inhibited the enzymatic activity of all the cyclic nucleotide PDE isoenzymes present in vascular tissue, being more effective on PDE2 (IC50∼17) and on PDE1 (IC50∼25). Our results suggest that the vasorelaxant effects of EGCG in rat aorta are mediated, at least in part, by an inhibition of PDE activity, and the subsequent increase in cyclic nucleotide levels in RASMC, which, in turn, can reduce agonist‐ or high KCl concentration‐induced increases in [Ca2+]i.