Publications

Abstract Caffeic acid phenethyl ester (CAPE) is a bioactive polyphenolic compound obtained from propolis extract. Although it has a broad therapeutic potential, the bioavailability of CAPE is limited, due to reduced solubility and poor plasmatic stability. Efforts to reduce these pharmacokinetic drawbacks resulted in the synthesis of caffeic acid phenethyl amide (CAPA). Cyclodextrins have been proved as promising excipients for the formulation of active ingredients. Herein, we report the inclusion complexation behavior and binding ability of CAPE and CAPA with hydroxypropyl-beta-cyclodextrin (HP-beta-CD). The supramolecular interactions were examined through UV and FTIR spectroscopy, DSC, H-1 NMR and 2D ROESY. The CAPE/HP-beta-CD and CAPA/HP-beta-CD inclusion complexes stability constants were determined to be, respectively, 2911.6 and 584.6 M-1 in water and 2866.2 and 700.1 M-1 at physiological pH. The aqueous solubility increased notably, proving that HP-beta-CD can be potentially useful to improve the biological, chemical and physical properties of CAPE and CAPA.

Abstract Despite research efforts to discover new drugs for Parkinson treatment, the majority of candidates fail in preclinical and clinical trials due to inadequate pharmacokinetic properties, namely blood-brain barrier permeability. Within the high demand to introduce new drugs to market, nano technology can be used as a solution. Accordingly, PEGylated PLGA nano particles (NPs) were used as a smart delivery carrier to solve the suboptimal aqueous solubility, which precludes its use in in vivo assays, of a potent, reversible, and selective monoamine oxidase B inhibitor (IMAO-B) (coumarin C75, IC50 = 28.89 +/- 1.18 nM). Long-term stable PLGA@C75 NPs were obtained by nanoprecipitation method, with sizes around 105 nm and a zeta potential of -10.1 mV. The encapsulation efficacy was around 50%, achieving the final C75 concentration of 807 +/- 30 mu M in the nano formulation, which corresponds to a therapeutic concentration 27828-fold higher than its IC50 value. Coumarin C75 showed cytotoxic effects at 50 mu M after 48 and 72 h of exposure in SH-SY5Y, Caco-2, and hCMEC/D3 cell lines. Remarkably, no cytotoxic effects were observed after nanoencapsulation. Furthermore, the data obtained from the P-gp-Glo assay and the cellular uptake studies showed that C75 is a P-glycoprotein (P-gp) substrate having a lower uptake profile in intestinal and brain endothelial cells. Moreover, it was shown that this membrane transporter influences C75 permeability profile in Caco-2 and hCMEC/D3 cells. Interestingly, PLGA NPs inhibited P-gp and were able to cross intestinal and brain membranes allowing the successful transport of C75 through this type of biological barriers. Overall, this work showed that nanotechnology can be used to solve drug discovery related drawbacks.

Abstract Alzheimer's disease (AD) is a multifactorial age-related disease associated with oxidative stress (OS) and impaired cholinergic transmission. Accordingly, targeting mitochondrial OS and restoring cholinergic transmission can be an effective therapeutic strategy toward AD. Herein, we report for the first time dual-target hydroxybenzoic acid (HBAc) derivatives acting as mitochondriotropic antioxidants and cholinesterase (ChE) inhibitors. The studies were performed with twomitochondriotropic antioxidants AntiOxBEN(1) (catechol derivative), and AntiOxBEN(2) (pyrogallol derivative) and compounds 15-18, which have longer spacers. Compounds AntiOxBEN(1) and 15, with a shorter carbon chain spacer (six- and eight-carbon) were shown to be potent antioxidants and BChE inhibitors (IC50 = 85 +/- 5 and 106 +/- 5 nM, respectively), while compounds 17 and 18 with a 10-carbon chain weremore effective AChE inhibitors (IC50 = 7.7 +/- 0.4 and 7.2 +/- 0.5 mu M, respectively). Interestingly, molecular modeling data pointed toward bifunctional ChEs inhibitors. The most promising ChE inhibitors acted by a non-competitive mechanism. In general, with exception of compounds 15 and 17, no cytotoxic effects were observed in differentiated human neuroblastoma (SH-SY5Y) and human hepatocarcinoma (HepG2) cells, while A beta-induced cytotoxicity was significantly prevented by the new dual-target HBAc derivatives. Overall, due to its BChE selectivity, favorable toxicological profile, neuroprotective activity and drug-like properties, which suggested blood-brain barrier (BBB) permeability, themitochondriotropic antioxidant AntiOxBEN(1) is considered a valid lead candidate for the development of dual acting drugs for AD and other mitochondrial OS-related diseases.

Abstract Menadione, also known as vitamin K-3, is a 2-methyl-1,4 naphthoquinone with a potent cytotoxic activity mainly resulting from its quinone redox-cycling with production of reactive oxygen species (ROS). Although increased ROS generation is considered a relevant mechanism in cancer cell death, it may not be sufficiently effective to kill cancer cells due to phenotypic adaptations. Therefore, combining ROS-generating agents with other molecules targeting important cancer cell phenotypes can be an effective therapeutic strategy. As mitochondrial dysfunction has been implicated in many human diseases, including cancer, we describe here the discovery of a mitochondrial-directed agent (MitoK(3)), which was developed by conjugating a TPP cation to the C3 position of the menadione's naphthoquinone ring, increasing its selective accumulation in mitochondria, as well as led to alterations of its redox properties and consequent biological outcome. MitoK(3) disturbed the mitochondrial bioenergetic apparatus, with subsequent loss of mitochondrial ATP production. The combinatory strategy of MitoK3 with anticancer agent doxorubicin (DOX) resulted in a degree of cytotoxicity higher than those of the individual molecules, as the combination triggered tumour apoptotic cell death evident by caspase 3/9 activities, probably through mitochondrial destabilization or by interference with mitochondrial redox processes. The results of this investigation support the importance of drug discovery process in developing molecules that can be use as adjuvant therapy in patients with specific cancer subtypes.

Abstract Pharmacological interventions targeting mitochondria present several barriers for a complete efficacy. Therefore, a new mitochondriotropic antioxidant (AntiOxBEN(3)) based on the dietary antioxidant gallic acid was developed. AntiOxBEN(3) accumulated several thousand-fold inside isolated rat liver mitochondria, without causing disruption of the oxidative phosphorylation apparatus, as seen by the unchanged respiratory control ratio, phosphorylation efficiency, and transmembrane electric potential. AntiOxBEN(3) showed also limited toxicity on human hepatocarcinoma cells. Moreover, AntiOxBEN(3) presented robust iron-chelation and antioxidant properties in both isolated liver mitochondria and cultured rat and human cell lines. Along with its low toxicity profile and high antioxidant activity, AntiOxBEN(3) strongly inhibited the calcium-dependent mitochondrial permeability transition pore (mPTP) opening. From our data, AntiOxBEN(3) can be considered as a lead compound for the development of a new class of mPTP inhibitors and be used as mPTP de-sensitiser for basic research or clinical applications or emerge as a therapeutic application in mitochondria dysfunction-related disorders. [GRAPHICS] .

Abstract Phenolic acids are ubiquitous antioxidants accounting for approximately one third of the phenolic compounds in our diet. Their importance was supported by epidemiological studies that suggest an inverse relationship between dietary intake of phenolic antioxidants and the occurrence of diseases, such as cancer and neurodegenerative disorders. However, until now, most of natural antioxidants have limited therapeutic success a fact that could be related with their limited distribution throughout the body and with the inherent difficulties to attain the target sites. The development of phenolic antioxidants based on a hybrid concept and structurally based on natural hydroxybenzoic (gallic acid) and hydroxycinnamic (caffeic acid) scaffolds seems to be a suitable solution to surpass the mentioned drawbacks. Galloyl cinnamic hybrids were synthesized and their antioxidant activity as well as partition coefficients and redox potentials evaluated. The structure property activity relationship (SPAR) study revealed the existence of a correlation between the redox potentials and antioxidant activity. The galloyl cinnamic acid hybrid stands out as the best antioxidant supplementing the effect of a blend of gallic acid plus caffeic acid endorsing the hypothesis that the whole is greater than the sum of the parts. In addition, some hybrid compounds possess an appropriate lipophilicity allowing their application as chain-breaking antioxidant in biomembranes or other type of lipidic systems. Their predicted ADME properties are also in accordance with the general requirements for drug-like compounds. Accordingly, these phenolic hybrids can be seen as potential antioxidants for tackling the oxidative status linked to the neurodegenerative, inflammatory or cancer processes.

Abstract Hydroxycinnamic acids (such as ferulic, caffeic, sinapic, and p-coumaric acids) are a group of compounds highly abundant in food that may account for about one-third of the phenolic compounds in our diet. Hydroxycinnamic acids have gained an increasing interest in health because they are known to be potent antioxidants. These compounds have been described as chain-breaking antioxidants acting through radical scavenging activity, that is related to their hydrogen or electron donating capacity and to the ability to delocalize/stabilize the resulting phenoxyl radical within their structure. The free radical scavenger ability of antioxidants can be predicted from standard one-electron potentials. Thus, voltammetric methods have often been applied to characterize a diversity of natural and synthetic antioxidants essentially to get an insight into their mechanism and also as an important tool for the rational design of new and potent antioxidants. The structure-property-activity relationships (SPARs) correlations already established for this type of compounds suggest that redox potentials could be considered a good measure of antioxidant activity and an accurate guideline on the drug discovery and development process. Due to its magnitude in the antioxidant field, the electrochemistry of hydroxycinnamic acid-based antioxidants is reviewed highlighting the structure-property-activity relationships (SPARs) obtained so far.

Abstract Tamoxifen is a selective estrogen receptor modulator that is used as an adjuvant and/or chemotherapeutic agent for the treatment of all stages of hormone-dependent breast cancer. Currently there is a deep interest in the study of tamoxifen biotransformation and identification of metabolites since they can significantly contribute to the overall pharmacological or adverse effects of the drug. Accordingly, the study of the electrochemical behavior of tamoxifen in aqueous solution is reported. To clarify the occurring oxidative process and to assess the influence of the functional groups on the oxidation mechanism, the voltammetric assessment was extended to the study of tamoxifen's analogues (E)-tamoxifen and dihydrotamoxifen, and to its main phase I oxidative metabolite, N-desmethyl tamoxifen. The data found shows that the oxidative processes occurring in tamoxifen are essentially related with the two chemical moieties present in the molecule: the substituted aromatic nucleus and the tertiary amine group. Moreover, the results obtained suggest that the ethylenic linkage is not critical for tamoxifen's oxidation although it could play an important role in the course of the oxidation process. These results could contribute to highlight some remaining questions regarding tamoxifen's metabolic behavior and to the development of new analytical strategies, based on electrochemical approaches.