Production of highly stable spray dried phage formulations for treatment of Pseudomonas aeruginosa lung infection

Abstract

The potential of bacteriophage therapy for the treatment of pulmonary infections caused by antibiotic-resistant bacteria has been well recognised. The purpose of this study was to investigate the effect of excipients on stabilisation and aerosolisation of spray dried powders of morphologically different phages – PEV podovirus and PEV myovirus. Seven anti-pseudomonal phages were screened against 90 clinical strains of bacterial hosts and three of the phages were selected for formulation study based on the host range. Design of experiments was utilised to assess the effect of different excipients on the stabilisation and aerosolisation of spray dried phages. Both podovirus and myovirus phages were stable in spray dried formulations containing trehalose or lactose and leucine as excipients with less than 1-log₁₀ titre reduction during spray drying, with lactose providing superior phage protection over trehalose. Furthermore, the spray dried phage formulations dispersed in an Osmohaler at 85 L/min produced a high fine particle fraction of over 50%. The results showed that the phages in this study can form respirable dry powder phage formulations using the same excipient composition. Spray dried various types of lytic phages hold significant potential for the treatment of pulmonary infections.

Introduction

The emergence of multiple drug resistance (MDR) bacteria has become a critical problem in the treatment of respiratory infections. Cystic fibrosis patients suffer from chronic bacterial infections by Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenza, and Burkholderia cepacia complex [1]. Chronic lung infection, especially P. aeruginosa shortens the life span of cystic fibrosis patients [2]. Many of these bacteria are intrinsically resistant or have developed resistance to many currently used antibiotics [3], [4]. Additionally, immunocompromised patients in hospital intensive care units are at risk of respiratory infection from MDR bacteria. Despite the efforts to develop new antibiotics to fight emerging antibiotic-resistant bacterial infection, drying pipeline of new antibiotics urges the need for an alternative therapy [5].

Bacteriophage (phage) therapy has been gaining renewed interests for its ability to eradicate MDR bacteria [6]. Phage therapy exploits the lytic life cycle of phages, which causes bacteriolysis followed by subsequent release of progenies. The released phage progenies then target nearby bacteria and the cycle is repeated. Potential advantages of phage therapy over conventional antibiotic treatment are due to the facts that phages are (i) naturally occurring antibacterials with low inherent toxicity, (ii) self-amplifying agents, and (iii) highly specific limiting unnecessary damage to non-targeted bacteria [7], [8]. The activity of phages against MDR bacteria has been shown in in vitro studies [9], [10] as well as their efficacy in animals [10], [11], [12] and humans [13], [14], [15]. A recent study used intranasal administration of phages to reduce the infective burden and inflammation in a P. aeruginosa lung infection model in mice [11]. In addition, bacterial load and inflammatory response reduction were observed with prophylactic treatment. Other in vitro studies have demonstrated the ability of phages to penetrate and disrupt bacterial biofilms [16], and the synergistic effect of using combinational therapy with antibiotics [17], [18]. The potential use of phages for the treatment of infectious disease is being extensively discussed.

Over the past decade, great efforts have been put into delivering therapeutic dosage forms of phage for treatment of respiratory infections. Successful phage therapy requires phages to remain viable during the production and delivery in aerosolised form so that clinically significant dose can reach the lower airways. Nebulisation has been utilised by earlier studies in Europe for inhalation of liquid phage formulation [19]. More recent studies have confirmed that, depending on the types of nebuliser, phages can withstand the stress and remain viable during nebulisation process with high titre reaching the lower respiratory tract [20], [21], [22].

Powder formulations have the potential to provide easy storage, transport and administration with long shelf-life over liquid formulations. Several studies have shown processing liquid phage formulation into dry powders using lyophilisation [23], spray drying [24], [25], [26] and spray-freeze drying [25]. Vandenheuvel and colleagues [26] produced highly stable spray dried anti-Pseudomonal Podoviridae phage powders using trehalose as an excipient with less than 1 log₁₀ unit reduction in phage titre. On the other hand, powders containing anti-Staphylococcal phage from Myoviridae family resulted in 2.5 log₁₀ loss. This apparent difference in titre reduction seems to confirm that phage stabilisation is phage dependent. Matinkhoo et al. [24] reported that myovirus phages spray dried with trehalose, leucine and a surfactant resulted in less than 1 log₁₀ unit titre reduction with superior aerosol performance. A more recent study by Leung et al. [25] compared the performance of spray drying with spray freeze drying in producing stable anti-Pseudomonal Podoviridae phage powders using trehalose, mannitol and leucine. Spray drying provided better phage protection over spray freeze drying; however, the three-excipient combination failed to give less than 1 log₁₀ unit titre reduction which is generally considered as a desirable loss incurred during the inhalation process.

For the commercialisation of sprayed dried phage formulations for pulmonary infections, the physical stability of inhalable powders and biological viability of phages are the critical parameters. This study aimed to explore the effect of various excipients on stabilisation and aerosolisation of PEV podovirus and myovirus in spray dried powders, using comprehensive formulation screening, followed by further refinement of excipient composition and ratios within.