Abstract Objectives Extremely multiresistant strains of Enterobacteriaceae, such as those of Escherichia coli and Klebsiella pneumoniae, are emerging and spreading at a worrisome speed. Polymyxins (polymyxin B, colistin) are used as last-line therapy against such strains, in spite of their notable nephrotoxicity that may even require discontinuation of the therapy. We have previously developed polymyxin derivatives NAB739 and NAB815 that are better tolerated in cynomolgus monkeys than polymyxin B and are, in contrast to polymyxin B, excreted in the cynomolgus urine to a very significant degree. Here we have compared the efficacy of these NAB compounds and polymyxin B in the therapy of murine pyelonephritis caused by E. coli. Methods The challenge organism was a uropathogenic E. coli clinical isolate. Mice were inoculated via urethral catheterization with 5 × 108 cfu. All treatment groups consisted of 12 animals. On day 1 and day 2 post-infection, the mice were treated subcutaneously with NAB739, NAB815, polymyxin B or vehicle twice a day and on day 3 post-infection the animals were sacrificed. cfu in the kidney and bladder tissues and in the urine were determined. Results NAB739 reduced the bacterial burden in the kidney, urine and bladder at doses approximately 10-fold lower than those of polymyxin B. In the kidneys, the half-maximal effective dose (ED50) was 9-fold lower for NAB739 than for polymyxin B (0.24 mg/kg versus 2.1 mg/kg, respectively). NAB815 was as effective as NAB739. Conclusions NAB739 and NAB815 were unequivocally more effective than polymyxin B in the murine pyelonephritis model. Introduction The crisis due to emerging extremely multiresistant Gram-negative bacteria is worrisome. Escherichia coli and Klebsiella pneumoniae cause almost 40% of all community-acquired bacteraemias and approximately one-third of all healthcare-associated bacteraemias.1–3 Polymyxins (polymyxin B, colistin) are strongly cationic cyclic lipodecapeptides. They were largely abandoned as an intravenous therapy in the sixties because of their nephrotoxicity and because more effective antibiotics became available. Now, they have been reinstated as last-line therapy of severe Gram-negative infections. The nephrotoxicity of polymyxins complicates the therapy and may require its discontinuation.4,5 The nephrotoxicity rate of polymyxin B and colistin varies from 20% to 60%4 and the risk of nephrotoxicity must be weighed against the beneficial effects on patient survival.5 On the other hand, contemporary data indicate that the current dosage regimens are suboptimal in critically ill patients and lead to too low serum concentrations.6 Clinicians are advised to use larger doses, but this further increases nephrotoxicity. The general interest in polymyxins and in the development of their improved derivatives has increased in recent years, as reviewed by Brown and Dawson,7 Cochrane and Vederas,8 Pirri et al.,9 Vaara10 and Velkov et al.11 Our group has constructed derivatives that carry altogether three positive charges only, instead of five as in polymyxin B and colistin.10,12–14 NAB739 does not carry any positive charges in the linear part and NAB815 carries only one positive charge in the linear part and only two positive charges in the cyclic part.14 Both compounds are less cytotoxic to human kidney proximal tubular cell line HK-2 than polymyxin B and colistin.10,14 Furthermore, they are better tolerated in cynomolgus monkeys than the comparator, polymyxin B, and have half-lives identical to polymyxin B.13 The objective of this paper was to evaluate the efficacy of NAB739 and NAB815 against E. coli in a murine urinary tract infection model15 as compared with that of polymyxin B. Materials and methods Compounds NAB739 sulfate and NAB815 sulfate were custom-made by Bachem AG (Bubendorf, Switzerland). The purity, as estimated by HPLC, was 97.3% for NAB739 and 98.4% for NAB815. Polymyxin B sulfate was from Sigma–Aldrich (St Louis, MO, USA; product number P0972). Bacterial challenge organism The challenge organism was E. coli C175‐94 (serotype O8:K48:H4), a clinical isolate elaborating type 1 fimbriae, previously used for developing and validating the urinary tract infection model.15 The MICs were determined by the agar dilution method according to CLSI protocol M07-A10 by using Mueller–Hinton agar.16 Animals Upon arrival at the Statens Serum Institut animal facility, female OF-1 mice (Charles River Laboratories, France), 27–33 g, were randomized to six mice per cage (type 3 macrolone cages). Bedding was from Tapvei and Enviro-Dri nesting material and cardboard houses (Bio-serv) were offered as enrichment. The temperature was 22 ± 2 °C and the humidity was 55 ± 10%. The number of air changes per hour was 8–12 (70–73 per hour inside racks); light/dark periods were 6 am–6 pm/6 pm–6 am. Mice had free access to domestic quality drinking water and food (Teklad Global diet 2916C-Envigo) and peanuts and sunflower seeds (Køge Korn A/S). All animal experiments were approved by the National Committee of Animal Ethics, Denmark, and adhered to the standards of EU Directive 2010/63/EU. Mice were monitored twice daily for clinical symptoms of infection or discomfort and euthanized if reaching humane endpoints specified in the ethical permissions. Inoculation, treatment and sampling Overnight E. coli colonies were suspended in saline to 109 cfu/mL and mice were inoculated under anaesthesia with Zoletil and Torbugesic via urethral catheterization with 5 × 108 cfu into the bladders as described previously.15 On day 1 and day 2 post-infection, mice were treated subcutaneously with 0.2 mL of NAB739, NAB815 or polymyxin B formulated in 0.9% NaCl or vehicle (0.9% NaCl) at 9 am and 3 pm. Each experiment consisted of eight treatment groups and all treatment groups consisted of 12 animals. Twelve mice is the lowest number that robustly gives significant reduction of the bacterial loads with this strain. On day 1, day 2 and day 3 post-infection urine was sampled for colony counts. On day 1 (the inoculum control group) and day 3 post-infection mice were sacrificed, after urine sampling, by cervical dislocation and bladder and kidneys were collected and stored at −80 °C and later homogenized in saline. All samples were 10-fold diluted in saline and 20 μL spots were applied on agar plates in duplicates. Undiluted samples of urine were spread on a separate agar plate to determine the lowest possible detection level of colony counts. All agar plates were incubated for 18–22 h at 35 °C in ambient air. The half-maximal effective dose (ED50), i.e. the dose of a compound required for a 50% reduction of the infection load, was calculated using GraphPad Prism software (La Jolla, CA, USA). Significance levels (see Table 1) were calculated with one-way GraphPad Prism ANOVA, Dunnett’s multiple comparison test compared with vehicle treatment. Table 1. Efficacy comparison between NAB739 and NAB815 as measured by determining the difference in cfu (log10 scale) from the vehicle treatment control group in urine, kidney and bladder samples NAB739 NAB815 Urine samples 0.25 mg/kg twice a day −3.2*** −1.9 0.5 mg/kg twice a day −3.2*** −3.5*** 1 mg/kg twice a day −5.1**** −5.3**** Kidney samples 0.25 mg/kg twice a day −1.5 −1.8 0.5 mg/kg twice a day −1.0 −1.0 1 mg/kg twice a day −1.8* −1.9* Bladder samples 0.25 mg/kg twice a day −1.3 −1.1 0.5 mg/kg twice a day −1.7 −2.1 1 mg/kg twice a day −2.3*** −2.4*** NAB739 NAB815 Urine samples 0.25 mg/kg twice a day −3.2*** −1.9 0.5 mg/kg twice a day −3.2*** −3.5*** 1 mg/kg twice a day −5.1**** −5.3**** Kidney samples 0.25 mg/kg twice a day −1.5 −1.8 0.5 mg/kg twice a day −1.0 −1.0 1 mg/kg twice a day −1.8* −1.9* Bladder samples 0.25 mg/kg twice a day −1.3 −1.1 0.5 mg/kg twice a day −1.7 −2.1 1 mg/kg twice a day −2.3*** −2.4*** Asterisks indicate statistically significant differences from the vehicle treatment control group. ****, *** and * correspond to P < 0.0001, P < 0.001 and P < 0.05, respectively. Results We employed the well-established and validated E. coli murine urinary tract infection model that uses the uropathogenic strain C175-94.15 This model is relevant for potential clinical settings, since it causes not only cystitis but also pyelonephritis, provided that the challenge dose is sufficient. The MICs of polymyxin B, NAB739 and NAB815 for this strain, as determined according to CLSI by the agar dilution method, were 0.5, 2 and 2 mg/L, respectively. Accordingly, in vitro, polymyxin B was 4-fold more effective than NAB739 and NAB815. We first compared the efficacy of NAB739 with that of polymyxin B (Figure 1). In kidneys, NAB739 at the dose of 0.25 mg/kg was very effective (reduction in bacterial load from that in the vehicle control group of 1.8 log10) while polymyxin B at 2 mg/kg had a subtle effect and at 4 mg/kg reduced the load by 2 log10. Accordingly, the difference in efficacy was more than 8-fold in favour of NAB739. In accordance, the ED50, determined using GraphPad Prism software, was 9-fold lower for NAB739 than for polymyxin B (0.24 mg/kg versus 2.1 mg/kg, respectively). Figure 1. View largeDownload slide Efficacy of NAB739 and polymyxin B at doses of 0.25 mg/kg to 8 mg/kg twice a day subcutaneously in murine urinary tract infection, as measured by quantifying bacterial load (cfu) in urine, bladder and kidney. The mean reduction in log10 (±SEM) from start of treatment is shown. ED50 values, determined using GraphPad Prism software, are also shown. Figure 1. View largeDownload slide Efficacy of NAB739 and polymyxin B at doses of 0.25 mg/kg to 8 mg/kg twice a day subcutaneously in murine urinary tract infection, as measured by quantifying bacterial load (cfu) in urine, bladder and kidney. The mean reduction in log10 (±SEM) from start of treatment is shown. ED50 values, determined using GraphPad Prism software, are also shown. In bladder, treatment with 0.25 mg/kg NAB739 reduced the bacterial load by more than 2 log10, whereas a similar reduction by polymyxin B was achieved at the dose of 4 mg/kg. The resulting ED50 values were 10-fold lower for NAB739 than for polymyxin B. Urine results were in line with the kidney and bladder results. We then compared NAB739 and NAB815 (Table 1). In urine, treatment with NAB739 at 0.25, 0.5 and 1 mg/kg lowered the cfu (as compared with vehicle treatment) by more than 3 log10 (P < 0.001, P < 0.001 and P < 0.0001, respectively). Treatment with 0.25 mg/kg NAB815 lowered the cfu by 2 log10 (no statistical significance) and that with 0.5 and 1 mg/kg by more than 3 log10 (P < 0.001 and P < 0.0001, respectively). In kidneys, treatment with NAB739 and NAB815 at 1 mg/kg lowered the cfu by approximately 2 log10 as compared with vehicle treatment (P < 0.05), whereas at 0.25 and 0.5 mg/kg no statistically significant differences (as compared with vehicle treatment) were reached. Bladder studies gave conclusions that were similar to those of the kidney studies. Minor clinical signs of discomfort were observed in all treatment groups. Discussion NAB739 was found to be remarkably more active than polymyxin B in the treatment of murine urinary tract infection. The difference in the efficacy is approximately 10-fold. NAB815 is equally effective in the kidney and almost as effective in the urine as NAB739. These findings might seem surprising, since the MICs of NAB739 and NAB815 for the E. coli challenge strain were 4-fold higher than that of polymyxin B. However, the NAB compounds are excreted in the urine in cynomolgus monkeys and rats to a very significant degree, whereas polymyxin B and colistin are not.13,17 In patients, only less than 1% of a polymyxin B dose is excreted in urine.18 Accordingly, the plausible explanation for the difference in the efficacy observed in the present study is that in mice, too, the urinary levels of NAB739 and NAB815 are much higher than that of polymyxin B. Preliminary studies on the pharmacokinetics, disposition and excretion of NAB739, NAB815 and polymyxin B in mice, still to be completed, support this explanation. Our findings are significant because complicated urinary tract infections (cUTIs), as mimicked in our mouse model, are clinically truly common. Approx. 6.3 million patients were treated in 2013 in the hospital wards in Europe, the USA and Japan due to cUTIs caused by E. coli and K. pneumoniae.3 These two bacteria cause 80% of all cUTIs treated in hospitals. In the future, many of those infections will be caused by XDR strains.3 Accordingly, compounds such as NAB739 and NAB815 might be very useful in clinical therapy. Further studies on the superiority of the NAB compounds over polymyxins are needed and should include studies where the challenge organism is a polymyxin-resistant strain, such as the K. pneumoniae mgrA strains as well as the enterobacterial mcr-1 through mcr-4 strains.19,20 Acknowledgements We thank Jytte Mark Andersen (Statens Serum Institut) for her excellent and exceptionally flexible technical contribution. We thank ENABLE colleagues and especially Frederik Deroose (Asclepia, Belgium) and Johan Gising (University of Uppsala, Sweden) for their useful activity and comments during the programme. Funding The research has received partial funding [as a part of European Gram-negative Antibacterial Engine (ENABLE) project WP4e] from the Innovative Medicines Initiative (IMI) Joint Undertaking under grant agreement n°115583, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/20072013) and European Federation of Pharmaceutical Industry Associations (EFPIA) companies’ in kind contribution. The ENABLE project is also financially supported by contributions from Academic and SME partners (where SME stands for small and medium-sized enterprises). Transparency declarations M. V. and T. V. are employers and shareholders of Northern Antibiotics Ltd. C. V. L.: none to declare. In ENABLE, NAB739, NAB815 and polymyxin B were code-named as EBL684, EBL685 and EBL691, respectively. References 1 Al-Hasan MN, Eckel-Passow JE, Baddour LM. Impact of healthcare-associated acquisition on community-onset Gram-negative bloodstream infection: a population-based study: healthcare-associated Gram-negative BSI. Eur J Clin Microbiol Infect Dis 2012; 31: 1163– 71. Google Scholar CrossRef Search ADS PubMed 2 Kollef MH, Zilberberg MD, Shorr AF et al. Epidemiology, microbiology and outcomes of healthcare-associated and community-acquired bacteremia: a multicenter cohort study. J Infect 2011; 62: 130– 5. Google Scholar CrossRef Search ADS PubMed 3 Stewart TM, Dorfman K. Pharmacol Inf Dis, Decision Resources 2015; 1– 209. 4 Justo JA, Bosso JA. Adverse reactions associated with systemic polymyxin therapy. Pharmacotherapy 2015; 35: 28– 33. Google Scholar CrossRef Search ADS PubMed 5 Kelesidis T, Falagas ME. The safety of polymyxin antibiotics. Expert Opin Drug Saf 2015; 14: 1687– 701. Google Scholar CrossRef Search ADS PubMed 6 Garonzik SM, Li J, Thamlikitkul V et al. Population pharmacokinetics of colistin methanesulfonate and formed colistin in critically ill patients from a multicenter study provide dosing suggestions for various categories of patients. Antimicrob Agents Chemother 2011; 55: 3284– 94. Google Scholar CrossRef Search ADS PubMed 7 Brown P, Dawson M. Development of new polymyxin derivatives for multi-drug resistant Gram-negative infections. J Antibiot 2017; 70: 386– 94. Google Scholar CrossRef Search ADS PubMed 8 Cochrane SA, Vederas JC. Lipopeptides from Bacillus and Paenibacillus spp.: a gold mine of antibiotic candidates. Med Res Rev 2016; 36: 4– 31. Google Scholar CrossRef Search ADS PubMed 9 Pirri G, Giuliani A, Nicoletto SF et al. Lipopeptides as anti-infectives: a practical perspective. Cent Eur J Biol 2009; 4: 258– 73. 10 Vaara M. Novel derivatives of polymyxins. J Antimicrob Chemother 2013; 68: 1213– 9. Google Scholar CrossRef Search ADS PubMed 11 Velkov T, Roberts KD, Thompson PE, Li J. Polymyxins: a new hope in combating Gram-negative superbugs. Future Med Chem 2016; 8: 1017– 25. Google Scholar CrossRef Search ADS PubMed 12 Vaara M, Fox J, Loidl G et al. Novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. Antimicrob Agents Chemother 2008; 52: 3229– 36. Google Scholar CrossRef Search ADS PubMed 13 Vaara M, Vaara T. Polymyxin Derivative and Uses Thereof . Patent Application, 2016. WO2016113470. 14 Vaara M, Vaara T, Tyrrell J. Structure-activity studies on polymyxin derivatives carrying three positive charges only reveal a new class of compounds with strong antibacterial activity. Peptides 2017; 91: 8– 12. Google Scholar CrossRef Search ADS PubMed 15 Hvidberg H, Struve C, Krogfelt KA et al. Development of a long-term ascending urinary tract infection mouse model for antibiotic treatment studies. Antimicrob Agents Chemother 2000; 44: 156– 63. Google Scholar CrossRef Search ADS PubMed 16 Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Tenth Edition: Approved Standard M07-A10. CLSI, Wayne, PA, USA, 2015. 17 Ali FE, Cao G, Poydal A et al. Pharmacokinetics of novel antimicrobial cationic peptides NAB7061 and NAB739 in rats following intravenous administration. J Antimicrob Chemother 2009; 64: 1067– 70. Google Scholar CrossRef Search ADS PubMed 18 Zavascki AP, Goldani LZ, Cao G et al. Pharmacokinetics of intravenous polymyxin B in critically ill patients. Clin Infect Dis 2008; 47: 1298– 304. Google Scholar CrossRef Search ADS PubMed 19 Poirel L, Jayol A, Nordmann P. Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes. Clin Microbiol Rev 2017; 30: 557– 96. Google Scholar CrossRef Search ADS PubMed 20 Carattoli A, Villa L, Feudi C et al. Novel plasmid-mediated colistin resistance mcr-4 gene in Salmonella and Escherichia coli, Italy 2013, Spain and Belgium, 2015 to 2016. Euro Surveill 2017; 22: pii=30589. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: firstname.lastname@example.org.
Journal of Antimicrobial Chemotherapy – Oxford University Press
Published: Feb 1, 2018
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