Evaluation of the in vitro and in vivo activities of robenidine and its analogues against human and veterinary pathogens
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Date
2024
Authors
Pi, Hongfei
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Trott, Darren J.
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Thesis
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Abstract
Multidrug resistant pathogens are a public health threat worldwide. Leading drug-resistant bacterial pathogens (Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa) were responsible for 929,000 deaths due to antimicrobial resistance (Murray et al., 2022) and over 3.5 million deaths associated with antimicrobial resistance in 2019, projected to increase to >10 million/year by 2050 if not addressed, with cumulative economic impact rising to over 100 trillion USD. Therefore, there is an urgent medical need to develop new, broad-spectrum antimicrobials with novel chemistry and mechanisms of action, preventing further cross-resistance to existing drug classes. This thesis aimed to characterise two new antibacterial drug classes (robenidine and its analogues, as well as a repurposed fasciolicide) with novel mechanisms of action as new therapeutic options against multidrug resistant pathogens. The anticoccidial drug robenidine (NCL812) and one of its analogues (NCL195) possessed excellent bactericidal activity against methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE) and Streptococcus pneumoniae, returning minimum inhibitory concentration (MIC) range of 2-8 μg/mL against these bacteria. Additionally, monomeric analogues NCL259 and NCL265 showed the best activity against Gram-negative “KAPE” pathogens (K. pneumoniae, A. baumannii, P. aeruginosa and E. coli), returning MIC range of 2–64 μg/mL for both compounds against these bacteria. Additionally, time-kill kinetics assays showed that both NCL259 and NCL265 were bactericidal against E. coli and K. pneumoniae. Therefore, NCL812 and the three analogues (NCL195, NCL259 and NCL265) were subjected to further investigation. NCL812 in the presence of sub-inhibitory concentrations of Gram-negative outer membrane permeabilizers ethylenediaminetetraacetic acid (EDTA) or polymyxin B nonapeptide (PMBN) displayed a 4- to 256-fold increase in the susceptibility of the tested Gram-negative KAPE pathogens. Mammalian cell toxicity studies showed IC50/MIC ratio ranging from 6-fold (for Gram-negative pathogens) to 3-fold (for Gram-positive pathogens) in the presence of EDTA, and IC50/MIC ratio ranged from approximately 2- to 24-fold against all the cell lines tested in the presence of PMBN. NCL195 demonstrated synergistic activity against P. aeruginosa, E. coli, K. pneumoniae, and Enterobacter spp. strains in the presence of sub-inhibitory concentrations of EDTA, PMBN, or polymyxin B (PMB), and was less toxic than NCL812 against mammalian cell lines tested. In bioluminescent Streptococcus pneumoniae and S. aureus murine sepsis challenge models, mice that received two 50 mg/kg intraperitoneal doses of NCL195 4 or 6 h apart exhibited significantly reduced bacterial loads and longer survival times than untreated mice. NCL259 and NCL265 demonstrated moderate antimicrobial activity against a large range (n = 236) of human and companion animal Gram-negative pathogens, with NCL265 being consistently more active, achieving lower MICs in the range of 2–16 μg/mL. NCL259 and NCL265 in combination with sub-inhibitory concentrations of PMB elicited a synergistic or additive activity against the strains tested, reducing the MIC of NCL259 by 8- to 256- fold and the MIC of NCL265 by 4- to 256- fold. Further testing of strains resistant to NCL259 and NCL265 indicated a significant role for the AcrAB-TolC efflux pump from Enterobacterales in imparting resistance to these robenidine analogues. However, NCL259 and NCL265 had much higher levels of toxicity to a range of human cell lines compared to NCL812, precluding their further development as novel antibiotics against Gram-negative pathogens. The fasciolicide selectively killed methicillin-sensitive (MSSA49775) and methicillinresistant S. aureus (MRSA USA 300 and MRSA 610) and S. pseudintermedius (3 clinical S. pseudintermedius isolates and 10 clinical MRSP isolates) at a MIC range of 2–4 μg/mL, and VRE (VRE35C, VRE60FR and VRE252) at a MIC range of 4–8 μg/mL. The fasciolicide also inhibited key Gram-negative bacteria in the presence of sub-inhibitory concentrations of PMB, returning MIC90 values of 1 μg/mL for E. coli, 8 μg/mL for K. pneumoniae, 2 μg/mL for A. baumannii and 4 μg/mL for P. aeruginosa. Repeated 4-hourly oral treatment of mice with 50 mg/kg fasciolicide after systemic S. aureus challenge resulted in a significant reduction in S. aureus populations in the blood up to 18 h post-infection (compared to untreated mice) but did not clear the infection from the bloodstream, consistent with its in vivo bacteriostatic activity. In conclusion, the promising in vitro and in vivo activity obtained for NCL195 and the fasciolicide indicate that they are viable candidates for future pre-clinical evaluation to treat serious MDR infections either alone or in combination with sub-inhibitory concentrations of Gram-negative outer membrane permeabilizers (e.g., PMB). This aligns with promoted new approaches to combat the emergence of antimicrobial resistance and multidrug resistance (drug repurposing and synergistic combination therapy). Further medicinal chemistry and pharmaceutical improvement of NCL195 and the fasciolicide may enhance their PK/PD parameters, increase potency, solubility and selectivity for advancement for future clinical development.
School/Discipline
School of Animal and Veterinary Science
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Science, 2024
Provenance
This thesis is currently under embargo and not available.