510. Bacteriophages as Drug Delivery Vehicles for Polymyxin B
Authors: Yang Yanxi , Chen Irene 
Published: January 2025

Background

The goal of this project is to develop a safer, more effective formulation of polymyxin B for E. coli bacteremia. Polymyxins are a family of cyclic lipopeptides that display exceptional antibiotic activities toward gram-negative pathogens such as E. coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. The PMB label includes an FDA black box warning for serious nephrotoxicity and neurotoxicity. However, today, with the growing emergence of “superbugs” and a dry antibiotic discovery pipeline, PMB is increasingly used as a last-line therapy to treat systemic infections. Thus, although PMB is a potent antibacterial agent, its toxicity currently prevents its use as an earlier or more widespread treatment.

Transmission electron microscopy (TEM; negative stain) images of anti-LPS phage bound to gram-negative organisms.

Phages were labeled with gold nanoparticles (dark spheres) coated with antibodies against pVIII, allowing phages to be easily identified (red darts). Gold-labeled anti-LPS phage are shown (A) without bacteria; or incubated with (B) E. coli DH5α; (C) E. coli ATCC BAA 1161; (D) A. baumannii ATCC 19606. Phage width is increased by the labeling reagents. Scale bars = 500 nm.

Methods

We engineered M13 to bind the core antigen of lipopolysaccharide (LPS), enabling binding to a variety of top-priority pathogenic gram-negative bacterial species, including E. coli, P. aeruginosa, A. baumannii, Klebsiella pneumoniae, and Burkholderia cepacia. Since the anti-LPS phages are able to bind but do not infect (nonviable), they are not cytotoxic by themselves. However, delivery of PMB by the anti-LPS phage confers antibiotic activity. Thus, in our strategy, PMB molecules are conjugated to the anti-LPS phage capsid, and delivered specifically to gram-negative bacterial cells through the specificity and high affinity of the anti-LPS binding domain. We have shown that the PMB – anti-LPS phage conjugate (termed “PMB–Phage” hereafter) effectively lowers the minimal inhibitory concentration of PMB by multiple orders of magnitude in vitro compared to PMB sulfate.

MIC and MBC of PMB and PMB-Phage, determined in vitro for several gram-negative organisms.

The heat map corresponds to the MIC or MBC values. Equivalent PMB concentrations for PMB-Phage MIC and MBC were calculated from the measured phage concentration and the average amount of PMB loaded per virion. * = resistant. ND = not determined due to technical limitation on phage concentration.

Results

We have already demonstrated following results.A broad-spectrum anti-LPS phage was engineered and verified to bind to several gram-negative bacterial species including E. coli.Polymyxin B was conjugated to the anti-LPS phage, creating PMB-Phage, which was highly antibacterial in vitro.PMB-Phage is non-toxic to mammalian cells in vitro and non-toxic in mice.PMB-Phage is distributed to various organs within 1 hour and has an elimination half-life of approximately 1 day.PMB-Phage appears to be effective in treating E. coli bacteremia in a mouse model.

PMB-Phage Biodistribution

Figure 9: Time course biodistribution of PMB-Phage in various organs, over time in male mice with (red) or without (blue) 1×10^7cfu (non-lethal dose) of E. coli ATCC BAA 1161 injected (n=3). Signals are measured as a percentage of the injected dose (ID) per volume (per cc). Representative biodistribution images (j) of 1 animal.

Conclusion

PMB-Phage will greatly reduce the nephrotoxicity of PMB, enabling safe treatment for a broader patient population.

Liver and kidney blood biomarkers at end point of 7-day toxicity studies.

Animals in experimental groups were injected 100µL of test materials through IV tail vein injection daily for 7 consecutive days. Experimental group animals for each condition (N=3), receiving either PMB or PMB-Phage at different doses show no significant difference comparing to control groups of animals group receiving no treatment (N=5) and 1xPBS buffer (N=3), in light and dark grey columns respectively. The only exception being all animals receiving 72 mg/kg body weight PMB (N=3) did not survive a single dose and died on day 1 (indicated by red cross in figure).

Disclosures

All Authors: No reported disclosures