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Development and stabilization of semi-finished dosage forms for phage-therapy

par Tabare, Emilie
Président du jury Fontaine, Véronique
Promoteur Goole, Jonathan
Publication Non publié, 2023-11-23

Thèse de doctorat

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Résumé :

The dramatic increase in the number of multi-drug resistant bacteria worldwide has created an urgent need for new antibacterial therapies. Different alternatives or additional tools to antibiotics could be used. Phage-therapy is one of these solutions that can be used to treat or decolonize one or more pathogenic bacterial populations. This therapy, which has long been neglected in Western Europe, is making a comeback, and is even becoming the last resort for some patients. In 2016, the Belgian government even decided to consider bacteriophages as an active ingredient in magistral preparations. Despite the many advantages of phage-therapy (e.g., specificity, rare side effects, multiplication at the site of infection, effectiveness even if biofilms are present, etc.), it has certain limitations due to the sensitivity of the bacteriophages. Indeed, in liquid form, the bacteriophages are sensitive to heat and must be kept in the refrigerator for short- and medium-term stability, or in frozen or freeze-dried form for long term stability. However, the latter two techniques are particularly costly and energy consuming. On the other hand, bacteriophages are sensitive to acidity and stomach enzymes, and the easiest and least invasive route of administration, which allows good patient compliance, is the oral route. Therefore, it is important to develop inexpensive dry forms containing bacteriophages that are stable for at least one year at room temperature. In addition, their formulation must allow for a targeted and controlled release in the colon without inactivation. The pharmacist will then be able to use the available formulations to deliver a personalized medicine treatment comprising one or more formulated bacteriophages (cocktail) to the patient. In this doctoral thesis, the research was first focused on the characterization and pre-formulation of different bacteriophages before their formulation. This step is important because it ensures the purity, activity, and safety of the bacteriophages as well as a better understanding of their sensitivities, thus saving precious time during the development of formulations. The spray-drying process was then selected for the stabilization of the bacteriophages and an experimental design was used to study and optimize the formulation and drying parameters for two bacteriophages with different morphologies: a Podoviridae and a Myoviridae. Subsequently, different bacteriophages were spray-dried, and a principal component analysis showed a correlation between the success of spray-drying (low inactivation after the process) and genome size. Then, in addition to these studies, a selective pressure study after several drying cycles was tested on two different bacteriophages, to improve their viability after spray-drying, but no genetic mutation was observed. The hypothesis was developed that phenotypic modification could allow bacteriophages to be more resistant after drying. To ensure gastro-resistance as well as a colonic release of the bacteriophages, Eudragit® FS30D polymer was used for the spray-drying process. The optimized formulation obtained protected bacteriophages from acidity and could be used as tablets, capsules, or sticks. Then to obtain granules that could be used to fill gastro-resistant capsules or to obtain coated tablets containing the bacteriophages, the wet granulation technique was used. This technique is widely used in the pharmaceutical industry and is known to improve the flow and the compressibility of powders. The bacteriophages had to be used in liquid form in this process as their use in spray-dried (amorphous) form showed significant inactivation (6 log10) due to crystallization. In addition, the significance of the manufacturing parameters of the granules on the activity of the bacteriophages was evaluated and no influence was found. However, an influence on the activity of the bacteriophages was observed during post-granulation drying. Indeed, a decrease in activity was observed when the residual humidity decreased. When filling capsules with the granules, no sticking was observed, and mass uniformity was obtained. The granules were also tableted, these mini tablets were coated with a dispersion based on Eudragit® FS30D. The granules were also produced by wet granulation from a twin-screw extruder which resulted in a substantial loss of bacteriophage activity. Moreover, drying further exacerbated this reduction, resulting in an almost complete loss of bacteriophage activity within the granules. This finding highlighted the advantage of the high shear wet granulation method as it generated a lower shear stress and exhibited a more conservative approach for preserving the bacteriophage activity. After, two alternative techniques for obtaining oral dosage forms were tested: multilayering on neutral Cellets® and extrusion followed by spheronization and coating. These techniques did not show a better efficiency regarding the preservation of activity after the process in comparison with the previously used techniques. Finally, the stability, the release profile, and the efficacy, in an in vivo model, of the most promising formulations were evaluated. The bacteriophages in optimized powders obtained after spray-drying were stable for at least 1 year at +40°C/75% RH. This is the first time that spray-dried bacteriophages have been described to be stable at 40°C/75%RH. This is remarkably interesting for regions such as Africa or Asia where the World Health Organisation predicts between 4 and 5 million of deaths per year by 2050. For the evaluated oral dosage forms, bacteriophages encapsulated with Eudragit® FS30D by spray-drying are stable for at least 1 year at +5°C. This was also the first time that bacteriophages encapsulated with Eudragit® FS30D by spray-drying allowed for the stability and the resistance of bacteriophages to acidity for at least 1 year at +5°C. In SHIME® model, this powder also showed a modified release allowing a treatment all along the ileum, cecum, and proximal colon of a patient. Regarding the granules obtained by high shear wet granulation, they were stable for at least 3 months at +5°C and a colonic release profile was demonstrated when filled into colonic capsules or in the form of coated mini tablets. Evaluation of the bacteriophage-containing granules or of the encapsulated bacteriophages in a Galleria mellonella model showed no difference in survival compared to unformulated bacteriophages. Such results demonstrated that these processes were not deleterious for the bacteriophage activity in vivo. The results presented in this thesis discuss different approaches for the stabilization and modified release formulation of bacteriophages and thus present alternatives to conventional antibiotics for the pharmacist at the hospital as well as in compounding pharmacies, and for industrialization as well.