Inhalable antibiotic prodrug therapies for the treatment of intracellular pulmonary infections

Abstract

One hundred years after the discovery of antimicrobials and antibiotics, lower respiratory infections remain one of the leading causes of death worldwide. Infectious agents such as Francisella tularensis and Burkholderia pseudomallei contribute to this burden as the causative agents of pulmonary tularemia and melioidosis, respectively. These pathogens cause substantial morbidity and mortality and due to their aerosolizability are weaponizable pathogens for bio-warfare. As such, the Centers for Disease Control and Prevention classify them as Tier 1 threat agents. Current care of these intracellular lung infections relies solely on weeks to months of intravenous and/or oral antibiotic delivery. Yet, 10-20% of patients die following treatment and another 5-10% relapse. These clinical failures are due to poor drug biodistribution within the lungs and low bioavailability, with potential off-target side effects due to systemic delivery. Inhalable delivery platforms aim to overcome these problems through direct delivery of antibiotics to the sight of infection. Systems such as inhalable free drug dispersions and antibiotics encapsulated within liposomal formulations are under investigation, however these systems fail to control drug pharmacokinetics often delivery a burst release, and often require complex formulations hampering large scale production and regulatory approval. Previous work in the Stayton lab, including work presented in this thesis, helped lay the foundation of a macromolecular inhalable prodrug platform utilizing RAFT polymerization of a ciprofloxacin (cipro) prodrug monomer towards the treatment of pulmonary tularemia. Initial co-monomer investigations were performed with polyethylene glycol methacrylate (PEGMA), carboxybetaine methacrylate, and mannose methacrylate monomers. These co-monomers were investigated for their ability to aid in drug solubility, loading, stability, biocompatibility, and in the case of the mannose monomer, for it’s targeting capabilities to surface receptors on alveolar macrophage. This foundational work lead to PEGMA-cipro polymers capable of prolonging survival in a lethal murine F. novicida infection model in 70% of mice to the experimental endpoint at 14 days post infection over untreated mice with 0% survival at just 4 days post infection following a 3 day treatment dosing schedule. The carboxybetaine co-monomer was only ever investigated in vitro, but was shown to be nontoxic, produce excellent drug solubility and loading, and increase cellular internalization over similar PEGMA polymers. However, the mannose co-monomer unexpectedly outperformed both the PEGMA and carboxybetaine co-monomers. The Mannose-Cipro polymers were shown to increase animal survival to 90% at 16 days post infection, compared to 0% survival in untreated mice at 8-10 days post infection following a 3 day treatment dosing schedule. Additionally, the mannose monomer was shown to increase cellular uptake by alveolar macrophage via receptor-mediated endocytosis, and provide excellent drug solubility, loading, and biocompatibility. Following this strong foundation, a second antibiotic prodrug monomer was synthesized from meropenem. Meropenem is the drug of choice in the treatment of melioidosis and is currently not available in any format other than injectable. This novel meropenem monomer was synthesized utilizing the same hydrolysable linker as the ciprofloxacin monomer for it’s optimal pharmacokinetic profile. The meropenem monomer was copolymerized via RAFT with the mannose-methacrylate co-monomer, and much of its in vitro characterization has been completed. This novel polymer is completely nontoxic in culture to the highest concentration evaluated (10 mg/mL), a dose at which the mannose-cipro polymer causes 50% cell death, indicating excellent biocompatibility. The polymer is also highly bactericidal in an intracellular coculture infection assay against the model bacterium B. thailandensis, with similar activity as the mannose-cipro polymer. Additionally, a terpolymer has been synthesized comprised of mannose, meropenem, and ciprofloxacin that is equally nontoxic but with enhanced bactericidal activity in culture. While drug combination analysis between meropenem and ciprofloxacin is so far inconclusive, the terpolymer activity suggests meropenem and ciprofloxacin have synergistic mechanisms of action against B. thailandensis and may be highly effective at combating pulmonary melioidosis in the form of combination therapies.