Produtos Farmacêuticos de Administração Vaginal: desenvolvimento, otimização e análise das metodologias de caracterização.

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Preclinical performance of vaginal semisolid products: technological and safety evaluations assuming physiologic parameters

Publication . Machado, Rita Solange Monteiro; Oliveira, José António Martinez Souto de; Oliveira, Rita Manuela Palmeira de

Vaginal semisolid products preclinical evaluations, when performed considering the particularities of the target organ, may represent key tools to predict in vivo performance. Before heading to clinical phases, vaginal semisolids must demonstrate to have an adequate technological and safety profile, in order to achieve higher success rates in the human testing stage. Traditional characterization methods currently used for vaginal semisolids do not undertake an integrative approach, since they do not address, for example, vaginal pH, fluid and temperature. Moreover, early safety assessment methods are largely described and validated not only on scientific literature, but also by regulatory agencies, although they are still mainly focused on cellular-based models. This safety profile of products can be further improved by combining toxicity testing, with drug release and permeation studies. Indeed, the vaginal administration route allows for local and systemic delivery of drugs, depending on the therapeutic purpose. Consequently, the drug should be confined to the chosen location of administration, to obtain maximum efficacy while avoiding side effects. The aim of this work was to develop a full set of assessment methods for characterization of vaginal semisolid products. Commercialized formulations were used to establish new methodological approaches that could be applied in new products development and characterization. Therefore, antimicrobials, Gino-canesten®, Sertopic®, Dermofix®, Gyno-pevaryl®, Lomexin®, Gino Travogen®, Dalacin V®; oestrogens, Ovestin®, Blissel® and Colpotrophine®; and, two reference formulations, Universal Placebo and Replens® were extensively evaluated. Technologically, they were tested in terms of pH, pH-buffering capacity, osmolality, textural parameters and viscosity, using a physiologic standpoint that considered the body temperature and dilution in a physiologic volume of vaginal fluid simulant (VFS); and even an ex vivo porcine model to infer bioadhesion and rheology on an after-administration environment. In terms of safety investigation, cellular toxicity was disclosed on VK2 E6/E7, HeLa and HEC-1A cell-lines, using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and NRU (Neutral Red Uptake) assays. Tissue explants, collected from the ex vivo porcine vaginal model, were also tested concerning toxicity, through MTT and histological analysis. Moreover, to include an advanced in vitro toxicity evaluation, the HET-CAM (Hen's Egg Test – Chorioallantoic Membrane), already in validation for eye irritation testing, was applied to vaginal irritation. A HPLC-DAD (High Performance Liquid Chromatography with Diode Array Detector) quantification method for the molecules present in semisolids included in this work, was developed and validated according to FDA (Food and Drug Administration - USA), EMA (European Medicines Agency) and ICH (International Conference for Harmonization) requirements. Further, this method was applied in drug quantification on in vitro drug release and ex vivo drug permeation experiments. These two techniques were performed using dynamic vertical Franz diffusion cells, having all experimental setting being specifically designed and optimized concerning the molecules in study (estriol, clotrimazole, econazole, isoconazole, sertaconazole and fenticonazole). Concerning technological characteristics, antimicrobial formulations exhibited lower pH than topical oestrogens. Buffering capacity in a vaginal fluid simulant conducted to better predictions of what happens in vivo. Characterization was performed also for those less acidic products to assess their ability to gain physiologic pH after mixing with simulated vaginal fluids. Products osmolality after dilution in VFS were below the upper limit advised by the World Health Organization (WHO). The antimicrobials had similar textural behaviours, while topical oestrogens varied in textural parameters. A slight decrease in viscosity was observed after application of dilution and temperature factors, showing the influence of the surrogate vaginal environment, while maintaining their pseudoplastic behaviour. However, each formulation had its own profile, possibly driven by their composition. Formulations’ viscosity was higher when tested using the ex vivo administration model than when only diluted in VFS at 37ºC. Concerning the in vitro models, VK2 E6/E7, presented relatively higher viabilities than HeLa and HEC-1A cells over the tested product concentrations. Tissue viability results were much higher than those obtained for the in vitro cellular models, revealing that this model could be more robust and closer to the in vivo situation. Across models, antimicrobials showed concentration-dependent viabilities. While oestrogens presented odd profiles, depending on the formulation and concentration tested. Reference products led to the most stable and higher viability profiles across concentrations. On ex vivo permeation studies we have investigated if there were differences in performing ex vivo permeation studies using the porcine vaginal model, when collecting a proximal or a distal tissue within the vaginal tube. No extensive significant differences between these tissues were found, but the caudal vagina could be more suitable for vaginal permeation experiments since it conducted to more reproducible and consistent results. Furthermore, it was observed that drug permeation is not directly dependent on drug release from the formulation. To sum up, the conduction of this integrative preclinical assessment for vaginal semisolids can be a valuable approach in new products development or characterisation, since it could optimize cost-efficiency of new formulations development by predicting in vivo efficacy and safety profiles. In addition, these methodologies have great potential not only to be applied in cosmetics, medical devices and medicines industry, but also in academical research.

2018-01Doctoral thesis

Open access