The pharmaceutical quality of freeze-dried tablets containing therapeutic bacteriophages against Pseudomonas aeruginosa and Staphylococcus aureus

Abstract

The preparation of a solid dosage form containing bacteriophages, which meets pharmaceutical requirements and ensures long-term stability of the phage effect, is significant for implementing phage therapy in practice. A commonly used method for processing phages into a solid form is freeze-drying into a (so-called) freeze-dried cake; however, to date there have been no studies examining the pharmacopeial parameters of freeze-dried tablets with bacteriophages.

Highlights

• A freeze-dried tablet is suitable for long-term preservation of the phage cocktail.
• The preparation process does not lead to a significant decrease in phage titer.
• The final tablets with bacteriophages pass the basic pharmacopoeial requirements.
• The phage potency in tablets is maintained for three months at 5 and 25 °C.

In this study, we describe the preparation and properties of freeze-dried tablets containing a cocktail of purified pseudomonal bacteriophage DSM 33593 from the genus Pbunavirus and staphylococcal bacteriophage DSM 33473 from the genus Kayvirus (10⁸ PFU/tablet) as the active ingredient. Maltodextrin was used as a tablet filler, and D-mannitol was used as a cryoprotectant. The tablet preparation process resulted in a decrease in phage titer by no more than 1 log PFU/mL. For Pbunavirus, the titer values in tablet and liquid form were comparable. Kayvirus was more stable in tablet form than in liquid form after six months of storage at 25 °C (a decrease of 1.9 ± 0.8 log PFU/mL and 3.8 ± 0.7 log PFU/mL, respectively).

The uniformity of mass of single-dose preparations, uniformity of content of single-dose preparations, and their disintegration complied with pharmacopeial requirements. The uniformity of dosage units of the tablets was maintained over three months. A microscopic examination of the internal part of the tablet revealed a heterogeneous structure, which does not affect the required pharmacopeial properties of the tablets. This study highlights the potential of freeze-dried tablets for long-term preservation of the phage effect at room temperature.

Introduction

Drug-resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus are among the so-called ESKAPE pathogens (Rice, 2008), which cause serious co-infections such as pneumonia in patients with cystic fibrosis (Peters et al., 2012) and wound infections (DeLeon et al., 2014). Both pathogens are also implicated in gastrointestinal infections in immunocompromised individuals such as enterocolitis (Chuang et al., 2014, Wei et al., 2015). One promising option for eradicating multidrug-resistant strains of these bacteria is phage therapy (Peters et al., 2012), evidenced by the creation of a new pharmacopeial monograph on Phage therapy medicinal products in the European Pharmacopoeia (EDQM, 2023), which sets requirements for phage therapy and allows its safe implementation in practice. The challenge for this therapeutic approach remains the preparation of dosage forms containing bacteriophages that maintain phage efficacy during their preparation and subsequent storage (Jonczyk-Matysiak et al., 2019, Malik, 2021).

Currently, there are numerous studies focused on processing bacteriophages into liquid dosage forms containing purified phages for topical (Chang et al., 2022), parenteral (Ferry et al., 2022), nasal (Morello et al., 2011, Uyttebroek et al., 2023), otic (Wright et al., 2009), or ophthalmic administration (Fukuda et al., 2012), as well as into semi-solid forms such as gels (Chang et al., 2021, Jokar et al., 2024), creams (Brown et al., 2016) and ointments (Brown et al., 2017). In the case of liquid dosage forms the oral application has been studied in terms of the safety, bioavailability and clarity of this route of administration (Górski et al., 2007, Weber-Dąbrowska et al., 2003). However, liquid dosage forms are susceptible to microbial contamination and have more problematic dosing and application. These disadvantages are largely eliminated in one-dose solid dosage forms, which can be easily administered.

For the preparation of solid dosage forms with bacteriophages, spray drying (Gonzalez-Menendez et al., 2018, Chang et al., 2017, Tabare et al., 2023) and freeze-drying (Puapermpoonsiri et al., 2010, Puapermpoonsiri et al., 2009) are the most commonly used methods. Using spray drying and compression, Khanal et al. (2021) prepared tablets containing bacteriophages that meet pharmacopeial requirements. Available studies indicate good stability of phage potency in the form of a so-called freeze-dried cake (Gonzalez-Menendez et al., 2018, Merabishvili et al., 2013, Puapermpoonsiri et al., 2010) or nasal insert (Alfadhel et al., 2011). For oral tablets, information is available for a few commercial products, such as Georgian Septaphage® compressed tablets (Biochimpharm, 2024).

In this study, we focused on monitoring the parameters of freeze-dried tablets containing a cocktail of a pseudomonal phage of the Pbunavirus genus and a staphylococcal phage of the Kayvirus genus, which are considered therapeutically promising bacteriophages (Leskinen et al., 2017, Merabishvili et al., 2009). Pbunavirus DSM 33593 and Kayvirus DSM 33473, which are included in the therapeutic preparation Duofag® that is currently undergoing clinical trials (ClinicalTrials.gov, 2024). The resulting tablets meet the definition of the oral lyophilisates according to the European Pharmacopoeia as solid preparations intended either to be placed in the mouth or dispersed in water before administration. Consequently, we performed selected Pharmacopoeia tests for tablets, category oral lyophilisates (European Pharmacopoeia, 2023a). Based on this monitoring of the tablet properties and long-term stability of phage potency over six months, we assessed the potential of this dosage form for the possible application of bacteriophages in clinical practice.

Download the full article as PDF here: The pharmaceutical quality of freeze-dried tablets containing therapeutic bacteriophages against Pseudomonas aeruginosa and Staphylococcus aureus

or read it here

Marie Komárková, Martin Benešík, Eva Černá, Lucie Sedláčková, Marek Moša, Lucy Vojtová, Aleš Franc, Roman Pantůček, The pharmaceutical quality of freeze-dried tablets containing therapeutic bacteriophages against Pseudomonas aeruginosa and Staphylococcus aureus, International Journal of Pharmaceutics, 2025, 125199, ISSN 0378-5173, https://doi.org/10.1016/j.ijpharm.2025.125199.

Read also our introduction article on Mannitol here: