Biopharmaceutical study of therapeutic efficacy of nanostructured formulations

Resumen

This research work was aimed at the characterization in vitro, ex vivo and in vivo of nanostructured systems which independently contain a natural flavanone extracted from Eysenhardtia platycarpa and another four flavanones obtained through semi-synthesis of the first flavanone with the objective of providing evidence of their efficaciousness as anti-inflammatory cutaneous agents. Initially the flavanone was isolated from its natural source, and following on from this, the derivatives were obtained through chemical reactions of acetylation, methylation, cyclization and vinyl cyclization. Calculations were made in silico using computational programs like Molinspiration and PASS Online. These gave the theoretical physio-chemical properties of the flavanones and estimated the profile of their probable anti-inflammatory activity. An analytical methodology was used for the quantification of the flavanones with High Performance Liquid Chromatography (HPLC) in samples that crossed human skin in an ex vivo study. The objective was to demonstrate that an analytical method had been selected that did not cause any interference from the biological components due to the tissue with which we worked. The results showed that the method was lineal, exact and precise in the assayed concentrations interval (1.56 - 200 µg/mL). Afterwards, nano-structured formulations were prepared individually: they contained each flavanone at 0.5 % and the excipients were: Labrasol®, Labrafac® lipophile, Propylene glycol and Plurol Oleic®. These formulations were morphologically and physio-chemically characterized. The results obtained revealed that the flavanones formulations (FF) were suitable for topical administration. An in vitro assay was carried out of the liberation of the flavanones from their individual formulation, using a dialysis membrane with a system of Franz type cells to guarantee that the formulation liberated the flavanones and allowed there to be a sufficient quantity of each component susceptible to being permeated in human skin. Immediately afterwards, ex vivo studies were realized using human skin with the objective of evaluating the permeation profile of the flavanones contained in dissolution individually and in the formulations. The study demonstrated that the quantity of flavanone permeated and retained in the skin was different, depending on the flavanone assayed. This was probably due to the different molecular interactions of the functional groups with the tissue components. The flavanones derived were retained in the skin in greater quantity than natural flavanone. Finally, an in vivo assay was carried out on the anti-inflammatory efficaciousness in a model of rat’s auricular edema induced by arachidonic acid. The results demonstrated that the flavanones were capable of reducing the edema (swelling) and the formulation excipients did not influence in the biological activity. The formulations turned out to be more effective than the reference pharmaceutical drug used in this study (sodium diclofenac gel). It was shown that the structural modification of the natural flavanone improved the therapeutic activity in which the derived methylated and cyclized vinyl stood out. These results are in concordance with the results obtained in the evaluation of the cytokines expression (IL-1β, IL-6 y TNF-α) carried out, and moreover allowed the advantage of the use of nano-structured systems in making the flavanones more effective to be shown in comparison with flavanones assayed in dissolution.