Technological and biological evaluation of tablets containing different strains of lactobacilli for vaginal administration

Abstract

Ten strains of lactobacilli were evaluated for the administration of viable microorganisms to restore the normal indigenous flora in the treatment of urogenital tract infections (UTI) in women. As the strains considered are facultative anaerobes, optimization of the production process was particularly critical to preserve bacterial viability. The microorganisms were formulated in single- and double-layer vaginal tablets. The two layers were characterized by different release properties: one is an effervescent composition that ensures a rapid and complete distribution of the active ingredient over the whole vaginal surface; while the second is a sustained release composition capable of releasing the lactobacilli over a longer period of time. Three different retarding polymers were tested, and all the formulations and tablets were evaluated in terms of technological processability, bacterial viability and stability, and cell adhesion properties of the microorganisms. From the results obtained, three out of ten strains appear particularly suitable for their application in the treatment of UTI. A larger batch of tablets made with a mixture of the three strains was then evaluated, confirming the feasibility of their industrial production and a good bacterial viability in the final dosage form.

Introduction

Recently, a new approach for therapy and prevention of urogenital tract infections (UTI) in the female population is directed towards the restoration of the normal vaginal flora which is competitive to an extensive colonization of pathogens [1], [2].

The vaginal flora of healthy women is dominated by different species of lactobacilli which produce large amounts of lactic acid, lowering the vaginal pH (3.5–4.5) [3]. In women suffering from vaginal and urinary tract infections, several pathogens are observed [3], [4]; in bacterial vaginosis, an increase in the pH values (>5.0) of the vaginal environment underlines the alteration of the microbial balance [5].

Lactobacilli dominate a healthy flora, but they coexist with a multitude of other species, including potential pathogens [6]. A disruption of this balance could be a factor that leads to infections, while the presence of lactobacilli in the vagina seems to be essential to prevent overgrowth of other bacteria [4], [7], [8], [9].

Several microbial products, including hydrogen peroxide (H₂O₂), bacteriocins and lactic acid, among others, have been considered to have a protective effect against intra-vaginal invasion by potential pathogenic bacteria. H₂O₂-generating lactobacilli are bactericidal to Gardnerella vaginalis in vitro [10], and their presence in the vagina has been shown to be related to decreased frequencies of bacterial vaginosis and trichomoniasis [11], [12].

The possibility of utilizing lactobacilli in the maintenance of a healthy state in the human female urogenital tract is based on the capacity of these probiotic microorganisms to produce a barrier population. Lactobacilli could inhibit the adhesion of pathogenic microorganisms to vaginal epithelial cells by a competitive exclusion process. This mechanism may involve both steric hindrance and competition for receptors [8].

Particular attention should be given to the design and production of pharmaceutical dosage forms intended for the administration of living cells, since bacterial suspensions show poor stability and are quickly cleared after vaginal instillation [4], [13]. In continuation to previous studies, we designed a new pharmaceutical formulation for the administration of viable microorganisms in the treatment and prevention of UTI in women. This dosage form was designed to enhance the adhesion of the microorganisms to the mucosa and a proper colonization of the vaginal epithelium. It consists of a double-layer vaginal tablet which combines an effervescent layer for the immediate release of a fraction of the dose and a slow-release layer to increase the residence time of the microorganism preparation in the vaginal environment [13], [14]. To verify the efficiency of the two formulations separately, single-layer tablets were also produced and tested.

To obtain the microbial preparation in a powdered form that can be used in tablet production, the bacterial suspensions were freeze-dried. The lyophilized powders were characterized both from technological and biological viewpoints to select the most suitable product for activity, processability and stability requirements.