# Efficacy of Inhalable Endolysin Cpl-1 Formulations in Combination with Gentamicin or Endolysin Pal in a Murine Lung Infection Model

## TL;DR

This study shows that inhalable endolysin formulations can reduce bacterial lung infections in mice, with combinations of endolysins being more effective than single treatments.

## Contribution

The study introduces a new method of delivering endolysins via inhalable dry powder and demonstrates the synergy of combining endolysins for treating lung infections.

## Key findings

- A single dose of Cpl-1 in powder or liquid form reduced bacterial load by 1 log10.
- Combining Cpl-1 with Pal in liquid form led to a synergistic 2.0 log10 reduction in bacterial load.
- Cpl-1 retained full enzymatic activity after exposure to BALF ex vivo.

## Abstract

Inhalable liquid formulation of endolysins represents a promising alternative to conventional antibiotics. Dry powder formulations offer improved stability for endolysin pulmonary delivery. This study aimed to evaluate the efficacy of an inhalable dry powder or liquid formulation of endolysin Cpl-1 alone and to compare it with liquid combinations of Cpl-1 with either gentamicin or endolysin Pal in a murine model of S. pneumoniae lung infection.

A dry powder formulation of Cpl-1 was produced via spray drying, while liquid formulations were prepared by dissolving Cpl-1, or in combination with gentamicin or endolysin Pal in liquid. The droplet size distribution of aerosolized formulations was also characterized. Mice were intratracheally infected with S. pneumoniae and treated with either powder or liquid formulations. The bacterial load in respiratory system was assessed 26 h post-infection. The stability and activity of Cpl-1 in BALF were also evaluated ex vivo.

A single dose of Cpl-1 powder formulation or Cpl-1 liquid formulation (40 µg/mouse) reduced pulmonary bacterial load by approximately 1 log10. Importantly, the combination of Cpl-1 and Pal in liquid form resulted in a synergistic 2.0 log10 reduction, significantly greater than either endolysin alone, while combining Cpl-1 with gentamicin did not enhance antibacterial activity. Ex vivo assays confirmed that Cpl-1 retained full enzymatic activity after incubation in BALF.

This proof-of-principle study demonstrated that inhalable endolysin liquid and powder formulations could potentially be used to treat bacterial lung infections. Moreover, the combination of multiple endolysins could increase antimicrobial activity over endolysin monotherapy.