Publications

Abstract Tolcapone is an orally active catechol-O-methyltransferase (COMT) inhibitor used as adjuvant therapy in Parkinson's disease. However, it has a highly hepatotoxic profile, as recognized by the U.S. Food and Drug Administration. As a possible solution, nanoscience brought us several tools in the development of new functional nanomaterials with tunable physicochemical properties, which can be part of a solution to solve several drawbacks, including drug's short half-life and toxicity. This work aims to use PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a stable carrier with lower hydrodynamic size and polydispersity to encapsulate tolcapone in order to overcome its therapeutic drawbacks. Using the nanoprecipitation method, tolcapone-loaded nanoparticles with a DLC% of 5.7% were obtained (EE% of 47.0%) and subjected to a lyophilization optimization process to obtain a final shelf-stable formulation. Six different cryoprotectants in concentrations up to 10% (w/v) were tested. A formulation of PLGA nanoparticles with 3% hydroxypropyl-beta-cyclodextrin (HP beta CD) as a cryoprotectant (PLGA-HP@Tolc), presenting sub-200 nm sizes and low polydispersity (PdI < 0.200) was selected. Cytotoxicity assays, namely, MTT and SRB, were used to study the metabolic activity and cell density of tolcapone and PLGA-HP@Tolc-treated cells. In both assays, a hepatocarcinoma cell line (HepG2) growing in glucose or glucose-free media (galactose-supplemented medium) was used. The results demonstrated that the treatment with the PLGA-HP@Tolc formulation led to a decrease in cytotoxicity in comparison to free tolcapone-treated cells in both media tested. Moreover, the elected formulation also counteracted ATP-depletion and excessive ROS production induced by tolcapone. The results suggest that HP beta CD might have a dual function in the formulation: cryoprotectant and anticytotoxic agent, protecting cells from tolcapone-induced damage. Using an in vitro COMT inhibition assay, the PLGA-HP@Tolc formulation demonstrated to inhibit COMT as efficiently as free tolcapone. Overall, the results suggest that tolcapone-loaded PLGA NPs could be an interesting alternative to free tolcapone, demonstrating the same in vitro efficacy in inhibiting COMT but with a safer cytotoxic profile.

Abstract Nitrocatechols are the standard pharmacophore to develop potent tight-binding inhibitors of catechol O-methyltransferase (COMT), which can be used as coadjuvant drugs to manage Parkinson's disease. Tolcapone is the most potent drug of this class, but it has raised safety concerns due to its potential to induce liver damage. Tolcapone-induced hepatotoxicity has been attributed to the nitrocatechol moiety; however, other nitrocatechol-based COMT inhibitors, such as entacapone, are safe and do not damage the liver. There is a knowledge gap concerning which mechanisms and chemical properties govern the toxicity of nitrocatechol-based COMT inhibitors. Using a vast array of cell-based assays, we found that tolcapone-induced toxicity is caused by direct interference with mitochondria that does not depend on bioactivation by P450. Our findings also suggest that (a) lipophilicity is a key property in the toxic potential of nitrocatechols; (b) the presence of a carbonyl group directly attached to the nitrocatechol ring seems to increase the reactivity of the molecule, and (c) the presence of cyano moiety in double bond stabilizes the reactivity decreasing the cytotoxicity. Altogether, the fine balance between lipophilicity and the chemical nature of the C1 substituents of the nitrocatechol ring may explain the difference in the toxicological behavior observed between tolcapone and entacapone.

Abstract The discovery of effective drugs for the treatment of neurodegenerative disorders (NDs) is a deadlock. Due to their complex etiology and high heterogeneity, progresses in the development of novel NDs therapies have been slow, raising social/economic and medical concerns. Nanotechnology and nanomedicine evolved exponentially in recent years and presented a panoply of tools projected to improve diagnosis and treatment. Drug-loaded nanosystems, particularly nanoparticles (NPs), were successfully used to address numerous drug glitches, such as efficacy, bioavailability and safety. Polymeric nanoparticles (PNPs), mainly based on polylactic-co-glycolic acid (PLGA), have been already validated and approved for the treatment of cancer, neurologic dysfunctions and hormonal-related diseases. Despite promising no PNPs-based therapy for neurodegenerative disorders is available up to date. To stimulate the research in the area the studies performed so far with polylactic-co-glycolic acid (PLGA) nanoparticles as well as the techniques aimed to improve PNPs BBB permeability and drug targeting were revised. Bearing in mind NDs pharmacological therapy landscape huge efforts must be done in finding new therapeutic solutions along with the translation of the most promising results to the clinic, which hopefully will converge in the development of effective drugs in a foreseeable future.

Abstract Osteoporosis therapies leveraging bisphosphonates and mineral components (e.g., magnesium, calcium, and strontium) have been raising attention because of their potential for managing this ever-growing disease. The administration of multicomponent therapeutics (combined therapy) in elderly patients is complex and suffers from low patient adherence. Herein, we report an all-in-one combination of four antiosteoporotic components into a new family of coordination complexes: [M-2(H(4)alen)(4)(H2O)(2)]center dot 1.5H(2)O [where M2+ = Mg2+ (1), (Mg0.535Ca0.465)(2+) (2) and (Mg0.505Ca0.450Sr0.045)(2+) (3)]. These solid-state complexes were prepared, for the first time, through microwave-assisted synthesis. It is demonstrated that the compounds are capable of releasing their antiosteoporotic components, both in conditions that mimic the path along the gastrointestinal tract and in long periods under physiological conditions (pH similar to 7.4). More importantly, when administered in low concentrations, the compounds did not elicit a cytotoxic effect toward liver, kidney, and osteoblast-like cell lines. Besides, it is important to highlight the unique coordination complex with four bone therapeutic components, [(Mg0.505Ca0.450Sr0.045)(2)(H(4)alen)(4)(H2O)(2)]center dot 1.5H(2)O (3), which significantly promoted osteoblast metabolic activity up to ca. 1.4-fold versus the control group. These findings bring this type of compounds one-step closer to be considered as an all-in-one and more effective treatment for managing chronic bone diseases, prompting further research on their therapeutic properties.

Abstract Oxidative stress is characterized by an imbalanced redox state that is principally related with a plethora of radical reactions skilled to trigger several pathophysiological mechanisms. So, the antioxidant therapy based on uptake of exogenous dietary antioxidants emerged as a useful approach to modulate the progression of oxidative stress-related events. Among dietary antioxidants, promising results have been obtained in in vitro cell-free and cell-based assays for huperzine A, α-lipoic acid, vitamins C and E, resveratrol, curcumin, epigallocatechin-3-gallate, and phenolic acids. However, the lack of drug-like pharmacokinetic properties precludes their advance in pre-and clinical trials. Although some dietary antioxidants, such as huperzine A, α-lipoic acid, resveratrol, and quercetin, were described to be able to surpass the blood-brain barrier, they have not been approved yet. Thus, new strategies have emerged by means of central chemical modifications or the use of nanoplatforms, along with new administration routes, which can facilitate their delivery across the blood-brain barrier.

Abstract Central nervous system (CNS) diseases, such neurodegenerative disorders (NDs) are increasing over the last years as a consequence of a continuous growing of the life expectancy and aging of population. The molecular mechanisms underlying the pathophysiology of NDs are still not fully understood. Neurodegeneration seems to be multifactorial and associated to a complex set of events comprising oxidative stress, inflammation, protein dysfunction and aggregation, among others, that at the end lead to the demise of neurons. It has been advocated that oxidative stress induced by imbalanced redox states, involving either overproduction of reactive species or dysfunction of the antioxidant system, and the higher levels of iron in the brain has a crucial role in NDs progression. Therefore, antioxidant therapy emerged as a useful approach to modulate oxidative stress events. However, despite the promising results obtained in in vitro cell-free and cell-based assays, the pharmacokinetics/pharmacodynamics properties of the majority of antioxidants preclude their advance in pre- and clinical trials. In this chapter the nonenzymatic endogenous and exogenous antioxidants (single and combined antioxidant therapy) currently in clinical trials, as drugs or supplements, for treatment of NDs will be highlighted. From the data one can conclude that the clinical trials performed so far related with single or combined antioxidants are still in a very early stage. So, a huge research space in the antioxidant field remains to be explored that hopefully will yield new and effective neuroprotective agents in a foreseeable future.

Abstract A series of piperic acid esters and amides were synthesized using a two-step strategy. Mass spectrometry and unidimensional (H-1 NMR, C-13 NMR, DEPT) and bidimensional (COSY, HSQC, HMBC) NMR methodologies were used to study the molecular structure of the final compounds, resulting in the assignment of all detected signals. Preliminary biological data of piperine and the derivatives thereof showed that piperic acid 2 and amides 3 and 4 did not display significant inhibitory activity at 10 tiM against both hMAO-A and hMAO-B. In contrast, the esters 5 and 6 displayed relevant hMAO inhibitory activities, with IC50 values for hMAO-B lower than piperine and within the nanomolar range (compound 1: IC50 = 1.05 ttM; compound 6: IC50 =169 nM; compound 5: IC50 =156 nM). Moreover, X-ray crystallographic analyses showed relevant differences between amides 3 and 4 and esters 5 and 6 constituting a valuable information to understand the ligand-target interactions. Additional studies are warranted for a systematic lead optimization process that can lead in the future to a more potent and selective hMAO-B inhibitor based on piperine scaffold.

Abstract The uptake and transport of dietary antioxidants remains the most important setback for their application in therapy. To overcome the limitations, a PEGylated-based platform was developed to improve the delivery properties of two dietary hydroxycinnamic (HCA) antioxidants caffeic and ferulic acids. The antioxidant properties of the new polymer antioxidant conjugates (PEGAntiOxs), prepared by linking poly(ethylene glycol) (PEG) to the cinnamic acids by a one-step Knovenagel condensation reaction, were evaluated. PEGAntiOxs present a higher lipophilicity than the parent compounds (caffeic and ferulic acids) and similar, or higher, antioxidant properties. PEGAntiOxs were not cytotoxic at the tested concentrations in SH-SYSY, Caco-2, and hCMEC/D3 cells. By contrast, cytotoxic effects in hCMEC/D3 and SH-SYSY cells were observed, at 50 and 100/3M, for caffeic and ferulic acids. PEGAntiOxs operate as antioxidants against several oxidative stress-cellular inducers in a neuronal cell-based model, and were able to inhibit glycoprotein-P in Caco-2 cells. PEGAntiOxs can cross hCMEC/D3 mono layer cells, a model of the blood-brain barrier (BBB) endothelial membrane. In summary, PEGAntiOxs are valid antioxidant prototypes that can uphold the antioxidant properties of HCAs, reduce their cytotoxicity, and improve their BBB permeability. PEGAntiOxs can be used in the near future as drug candidates to prevent or slow oxidative stress associated with neurodegenerative diseases.