Faculdade de Farmacia

The objective of this work was to evaluate the use of vitamin C as a biomarker in the inflammatory phase of the rat adjuvant arthritis and to correlate it with other parameters used for disease evaluation. Paw swelling was used for physical evaluation and the levels of ascorbate and dehydroascorbate in the serum of male rats, before and after adjuvant arthritis induction, were quantified by a high-performance liquid chromatography method (HPLC). The optimised HPLC assay enabled the quantification of both forms of the vitamin in rat sera, with the same extraction method and using different detectors, instead of obtaining dehydroascorbate by subtraction of the total ascorbate measurement. This method was used to follow the severity of adjuvant arthritis and the results were correlated with other already established disease activity parameters. A decrease of ascorbic acid and dehydroascorbic acid was observed with the increase of right paw circumference during the course of adjuvant arthritis. The disease associated changes in the serum concentrations of ascorbic acid, from biosynthesis and from recycling, can be evaluated by the direct quantification of dehydroascorbic acid. This provides some evidence for the potential of the quantification of these biomarkers to study the disease activity, and as a tool for the establishment of therapeutic protocols, to evaluate the anti-inflammatory effect of new drugs or formulations.

A strategy not usually used to improve carrier-mediated delivery of therapeutic enzymes is the attachment of the enzymes to the outer surface of liposomes. The aim of our work was to design a new type of enzymosomes with a sufficient surface-exposed enzyme load while preserving the structural integrity of the liposomal particles and activity of the enzyme. The therapeutic antioxidant enzyme superoxide dismutase (SOD) was covalently attached to the distal terminus of polyethylene glycol (PEG) polymer chains, located at the surface of lipid vesicles, to obtain SOD-enzymosomes. The in vivo fate of the optimized SOD-enzymosomes showed that SOD attachment at the end of the activated PEG slightly reduced the residence time of the liposome particles in the bloodstream after IV administration. The biodistribution studies showed that SOD-enzymosomes had a similar organ distribution profile to liposomes with SOD encapsulated in their aqueous interior (SOD-liposomes). SOD-enzymosomes showed earlier therapeutic activity than both SOD-liposomes and free SOD in rat adjuvant arthritis. SOD-enzymosomes, unlike SOD-liposomes, have a therapeutic effect, decreasing liver damage in a rat liver ischemia/reperfusion model. SOD-enzymosomes were shown to be a new and successful therapeutic approach to oxidative stress-associated inflammatory situations/diseases.

The aim of this study was to evaluate the effect of the oral dual ET(A)/ET(B) receptor antagonist bosentan on different surrogate markers in patients with pulmonary arterial hypertension (PAH). Prospective, open label, uncontrolled study in a University Hospital in Brazil. Fifteen PAH patients (11 females) with mean age of 40 +/- 11 years (5 in WHO functional class II, 10 in class III). All patients were investigated at baseline and after 16 weeks of bosentan treatment. We used the following surrogate markers for patients' evaluation: 6-min walk test, quality of life questionnaire (Short Form SF-36) and N-terminal proBNP (B type natriuretic peptide) fraction levels in blood. Between the evaluation at baseline and week 16, the 6-min walk test distance changed from 396 +/- 135 to 434 +/- 137 m (p < 0.05). Each of the eight domains of the SF-36 was significantly improved. Mean NT-proBNP levels were decreased from a mean of 1670 pg/mL to 1010 pg/mL (p = 0.01). The study suggests that bosentan treatment results in the improvement of different kinds of surrogate markers independently of their specificity to reflect functional capacity, quality of life and myocardial stress. It is concluded that the combined use of these different markers may be an alternative endpoint for future short duration clinical trials.

It has long been suggested that superoxide dismutase (SOD) be used for antioxidant therapy on the basis of its ability to catalyze the dismutation of superoxide radicals involved in the pathogenesis of several inflammatory disorders such as rheumatoid arthritis. However, the administration of SOD in free form has some disadvantages, most importantly, the low accumulation of SOD in inflamed areas due to its reduced half-life in the bloodstream and its rapid renal excretion. To overcome this, SOD can be incorporated either in highly loaded conventional liposomes (SA-liposomes) or long circulating liposomes (PEG-liposomes). After an appropriate formulation of SOD in SA-liposomes, the therapeutic effect is strongly increased, as indicated by a reduction of about 40% of inflammation edema compared with treatment with nonencapsulated enzyme. Compared with SA-liposomes, PEG-liposomes show superior therapeutic activity. A second approach consists of the construction of a hydrophobic SOD derivative (Ac-SOD) that can be partially inserted within the lipid matrix of liposomes and that expresses enzymatic activity to the external medium. This hydrophobic enzyme, Ac-SOD, associated with liposomes (so called Ac-SOD-enzymosomes), is able to exert its therapeutic activity while circulating in the organism, regardless of the integrity of the liposomes. Ac-SOD-enzymosomes have a more rapid antiinflammatory effect than SOD liposomes, confirming that the release of Ac-SOD from liposomes is no longer required to achieve dismutation. Different methodologies for the preparation of SOD and Ac-SOD liposomal formulations (conventional and long circulating) have been established and are described in detail here.