Excretion of the Polymyxin Derivative NAB739 in Murine Urine.

Extremely multiresistant strains of Enterobacteriaceae are emerging and spreading at a worrisome pace. Polymyxins are used as the last-resort therapy against such strains, in spite of their nephrotoxicity. We have previously shown that novel polymyxin derivatives NAB739 and NAB815 are less nephrotoxic in cynomolgus monkeys than polymyxin B and are therapeutic in murine Escherichia coli pyelonephritis at doses only one-tenth of that needed for polymyxin B. Here we evaluated whether the increased efficacy is due to increased excretion of NAB739 in urine. Mice were treated with NAB739 and polymyxin B four times subcutaneously at doses of 0.25, 0.5, 1, 2, and 4 mg/kg. In plasma, a clear dose-response relationship was observed. The linearity of Cmax with the dose was 0.9987 for NAB739 and 0.975 for polymyxin B. After administration of NAB739 at a dose of 0.25 mg/kg, its plasma concentrations at all tested time points were above 0.5 µg/mL while after administration at a dose of 0.5 mg/kg its plasma concentrations exceeded 1 µg/mL. The Cmax of NAB739 in plasma was up to 1.5-times higher after single (first) administration and up to two-times higher after the last administration when compared to polymyxin B. Polymyxin B was not detected in urine samples even when administered at 4 mg/kg. In contrast, the concentration of NAB739 in urine after single administration at a dose of 0.25 mg/kg was above 1 µg/mL and after administration of 0.5 mg/kg its average urine concentration exceeded 2 µg/mL. At the NAB739 dose of 4 mg/kg, the urinary concentrations were higher than 35 µg/mL. These differences explain our previous finding that NAB739 is much more efficacious than polymyxin B in the therapy of murine E. coli pyelonephritis.

The polymyxin derivative NAB739 is synergistic with several antibiotics against polymyxin-resistant strains of Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii.

The antibiotic crisis has reinstated polymyxins, once abandoned because of their toxicity. Now, preclinical studies have revealed better tolerated and more effective derivatives of polymyxins such as NAB739. Simultaneously, polymyxin-resistant (PMR) strains such as the mcr-1 strains have received lots of justified publicity, even though they are still very rare. Here we show that NAB739 sensitizes the PMR strains to rifampin, a classic "anti-Gram-positive" antibiotic excluded by the intact outer membrane (OM) permeability barrier, as well as to retapamulin, the surrogate of lefamulin, an antibiotic under development against Gram-positive bacteria. Polymyxin B was used as a comparator. The combination of NAB739 and rifampin was synergistic against ten out of eleven PMR strains of Escherichia coli (Fractional Synergy Indices, FICs, 0.14-0.19) and that of NAB739 and retapamulin against all the tested eleven strains (FICs 0.19-0.25). Against PMR Klebsiella pneumoniae (n = 7), the FICs were 0.13-0.27 for NAB739 + rifampin and 0.14-0.28 for NAB739+retapamulin. Against Acinetobacter baumannii (n = 2), the combination of NAB739 and rifampin had the FIC of 0.09-0.19. Furthermore, NAB739 and meropenem were synergistic (FICs 0.25-0.50) against four out of five PMR strains that were simultaneously resistant to meropenem.

Human kidney on a chip assessment of polymyxin antibiotic nephrotoxicity.

Drug-induced kidney injury, largely caused by proximal tubular intoxicants, limits development and clinical use of new and approved drugs. Assessing preclinical nephrotoxicity relies on animal models that are frequently insensitive; thus, potentially novel techniques - including human microphysiological systems, or "organs on chips" - are proposed to accelerate drug development and predict safety. Polymyxins are potent antibiotics against multidrug-resistant microorganisms; however, clinical use remains restricted because of high risk of nephrotoxicity and limited understanding of toxicological mechanisms. To mitigate risks, structural analogs of polymyxins (NAB739 and NAB741) are currently in clinical development. Using a microphysiological system to model human kidney proximal tubule, we exposed cells to polymyxin B (PMB) and observed significant increases of injury signals, including kidney injury molecule-1 KIM-1and a panel of injury-associated miRNAs (each P < 0.001). Surprisingly, transcriptional profiling identified cholesterol biosynthesis as the primary cellular pathway induced by PMB (P = 1.22 ×10-16), and effluent cholesterol concentrations were significantly increased after exposure (P < 0.01). Additionally, we observed no upregulation of the nuclear factor (erythroid derived-2)-like 2 pathway, despite this being a common pathway upregulated in response to proximal tubule toxicants. In contrast with PMB exposure, minimal changes in gene expression, injury biomarkers, and cholesterol concentrations were observed in response to NAB739 and NAB741. Our findings demonstrate the preclinical safety of NAB739 and NAB741 and reveal cholesterol biosynthesis as a potentially novel pathway for PMB-induced injury. To our knowledge, this is the first demonstration of a human-on-chip platform used for simultaneous safety testing of new chemical entities and defining unique toxicological pathway responses of an FDA-approved molecule.

Polymyxin derivatives NAB739 and NAB815 are more effective than polymyxin B in murine Escherichia coli pyelonephritis.

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.

Structure-activity studies on polymyxin derivatives carrying three positive charges only reveal a new class of compounds with strong antibacterial activity.

Recent years have brought in an increased interest to develop improved polymyxins. The currently used polymyxins, i.e. polymyxin B and colistin (polymyxin E) are pentacationic lipopeptides that possess a cyclic heptapeptide part with three positive charges, a linear "panhandle" part with two positive charges, and a fatty acyl tail. Unfortunately, their clinical use is shadowed by their notable nephrotoxicity. We have previously developed a polymyxin derivative NAB739 which lacks the positive charges in the linear part. This derivative is better tolerated than polymyxin B in cynomolgus monkeys and is, in contrast to polymyxin B, excreted into urine in monkeys and rats. Here we have conducted further structure-activity relationship (SAR) studies on 17 derivatives with three positive charges only. We discovered a remarkably antibacterial class, as exemplified by NAB815, that carries two positive charges only in the cyclic part.

Antimicrobial activity of the novel polymyxin derivative NAB739 tested against Gram-negative pathogens.

OBJECTIVES: In spite of reported nephrotoxicity, polymyxins have been reinstated as the last-line therapy to treat infections caused by Gram-negative bacterial strains that are resistant to other agents. NAB739 has a cyclic portion identical to that of polymyxin B, but its linear peptide portion consists of threonyl-d-serinyl instead of diaminobutyryl-threonyl-diaminobutyryl. Therefore, NAB739 lacks both of the positive charges present in the linear part of polymyxin B. Here, we compare the antibacterial activity of NAB739 with that of polymyxin B against a representative collection of contemporary Gram-negative bacteria. METHODS: NAB739 and polymyxin B MIC values were determined for 310 clinical isolates by the reference broth microdilution method according to CLSI document M07-A9 (2012). RESULTS: MIC(90)s of NAB739 for the subset consisting of polymyxin-susceptible (MIC, ≤ 2 mg/L) clinical isolates of Escherichia coli (n=51), Klebsiella pneumoniae (n=50), Acinetobacter spp. (n=49) and Pseudomonas aeruginosa (n=49) were 2, 2, 8 and 16 mg/L, respectively. For polymyxin-non-susceptible strains of E. coli (n=12), K. pneumoniae (n=11), Acinetobacter spp. (n=11) and P. aeruginosa (n=14) the NAB739 MIC(90) was ≥ 64 mg/L. CONCLUSIONS: The MIC(90) of NAB739 for polymyxin-susceptible strains of E. coli and K. pneumoniae was identical to and 2-fold higher than that of polymyxin B, respectively. For polymyxin-susceptible strains of Acinetobacter spp. and P. aeruginosa, the MIC(90) of NAB739 was 4-fold and 8-fold higher than that of polymyxin B, respectively. For polymyxin-non-susceptible strains of all these species, the MIC(90) values of NAB739 were high and 2- to 4-fold higher than those of polymyxin B.

Novel polymyxin derivatives are less cytotoxic than polymyxin B to renal proximal tubular cells.

The emergence of very multiresistant Gram-negative bacterial strains has reinstated polymyxins (polymyxin B, colistin), pentacationic lipopeptides, in the therapy, in spite of their nephrotoxicity. Extensive tubular reabsorption concentrates polymyxin in proximal tubular cells. The novel polymyxin derivatives NAB739, NAB7061 and NAB741 have their cyclic part identical to that of polymyxin B, but their side chain consists of uncharged octanoyl-threonyl-d-serinyl, octanoyl-threonyl-aminobutyryl, and acetyl-threonyl-D-serinyl respectively. In this study, we compared the toxicities of NAB739, NAB7061 and NAB741 with that of polymyxin B by using the porcine renal proximal tubular cell line LLC-PK1 electroporated or incubated with the selected compound. Both the ability to cause cell necrosis (quantified as the leakage of lactate dehydrogenase) and the ability to cause apoptosis (as quantified by counting apoptotic nuclei) were assessed. In electroporated cells, polymyxin B induced total (>85%) necrosis of the cells at 0.016 mM, whereas an approx. 8-fold concentration of NAB739 and NAB7961 and an approx. 32-fold concentration of NAB741 was required for the same effect. In cells treated without electroporation (incubated), polymyxin B elicited a marked degree (approx. 50%) of necrosis at 0.5mM, whereas the NAB compounds were inert even at 1mM. Neither polymyxin B nor the NAB compounds induced apoptosis.

Structure-activity studies on novel polymyxin derivatives that carry only three positive charges.

Polymyxin B and colistin are pentacationic lipopeptides that possess a cyclic heptapeptide portion, a linear tripeptide portion, and a fatty acyl tail. They are used, in spite of nephrotoxicity, to treat infections caused by extremely multiresistant Gram-negative bacteria. We have recently developed novel derivatives, that carry three cationic charges only. Some of them, including NAB739, are directly antibacterial whereas others, including NAB7061, lack the direct activity but sensitize bacteria to other antibiotics. NAB739 and NAB7061 differ from the old polymyxins in their renal handling and have reduced affinity to kidney brush border membrane. To further study the structure-activity relationships, we here synthesized eight additional derivatives and tested their antibacterial activity. NAB751 carries methylheptanoyl as the fatty acyl instead of octanoyl in NAB739 and was as active as NAB739, whereas NAB750 with dodecanoyl was less active. NAB781 and NAB782 with the linear peptide portion Ser-DSer and Ser-Ser-DSer, respectively, were less active than NAB739 that carries Thr-DSer. NAB771 with Thr at position 8 in the cyclic portion (instead of Dab in NAB7061) and Thr-Dab as the linear peptide portion (instead of Thr-Abu in NAB7061), resembled NAB7061 in its activity. However, replacement of two Dab residues in the cyclic portion with Thr greatly decreased the activity, even though the loss of the cationic charges was compensated by introducing two Dab residues in the linear portion. These findings reveal that subtle structural modifications have a major effect on the antibacterial activity and that it is possible to design numerous tricationic polymyxin derivatives that are antibacterial.

A novel polymyxin derivative that lacks the fatty acid tail and carries only three positive charges has strong synergism with agents excluded by the intact outer membrane.

Polymyxins are cationic lipopeptides (five cationic charges) and the last resort for the treatment of serious Gram-negative infections caused by multiresistant strains. NAB741 has a cyclic peptide portion identical to that of polymyxin B but carries in the linear peptide portion a threonyl-D-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). At the N terminus of the peptide, NAB741 carries an acetyl group instead of a mixture of methyl octanoyl and methyl heptanoyl residues. NAB741 sensitized Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, and Acinetobacter baumannii to antibiotics against which the intact outer membrane is an effective permeability barrier. When tested by using Etest strips on plates containing increasing concentrations of NAB741, the fractional inhibition concentration index (FICI) of the combination of NAB741 with rifampin ranged from

Novel polymyxin derivatives are effective in treating experimental Escherichia coli peritoneal infection in mice.

OBJECTIVES: Novel synthetic polymyxin derivatives including NAB737 and NAB739 are as effective as polymyxin B in vitro against the common opportunistic pathogen Escherichia coli. Another derivative, NAB7061, lacks direct antibacterial action but sensitizes E. coli to several other antibacterial agents including macrolides. The renal handling of NAB739 and NAB7061 in rats differs from that of polymyxin B. Furthermore, the affinities of NAB739 and NAB7061 for isolated rat kidney brush border membrane are significantly lower than that of polymyxin B. Here we investigate the in vivo antibacterial effect of these compounds. METHODS: The polymyxin derivatives were evaluated in an experimental murine peritonitis model. Immunocompetent mice were infected intraperitoneally with E. coli IH3080 and were subcutaneously treated with NAB737, NAB739 or NAB7061. RESULTS: A >4.0 log(10) reduction in bacterial load compared with saline control was achieved 6 h after initiation of treatment with 1 mg/kg of NAB739 twice or 4 mg/kg of NAB737 twice. Combination therapy with NAB7061 (5 mg/kg) twice and erythromycin (10 mg/kg) resulted during the same time course in a >2.0 log(10) reduction in bacterial load compared with saline control. Neither NAB7061 nor erythromycin was effective as monotherapy. Together with the ability to reduce bacterial load, the NAB compounds also improved the clinical status of the mice. CONCLUSIONS: We found that the three novel synthetic polymyxin B derivatives had a potent in vivo bactericidal effect against E. coli.

Susceptibility of carbapenemase-producing strains of Klebsiella pneumoniae and Escherichia coli to the direct antibacterial activity of NAB739 and to the synergistic activity of NAB7061 with rifampicin and clarithromycin.

OBJECTIVES: To determine the susceptibility of carbapenemase-producing strains of Klebsiella pneumoniae and Escherichia coli to the direct antibacterial activity of NAB739 and to the synergistic activity of NAB7061 with rifampicin and clarithromycin. NAB739 and NAB7061 are novel polymyxin derivatives that lack the cationic charges in the linear peptide portion of polymyxin B and have pharmacokinetic properties different from those of polymyxin B. METHODS: MIC determinations were performed by the agar dilution method using CLSI guidelines. Polymyxin B was used as a comparison. Synergism studies measured fractional inhibitory concentration indices (FICIs) by using increasing concentrations of the compounds in Mueller-Hinton agar and Etests. RESULTS: The MICs of NAB739 for all nine polymyxin-susceptible, carbapenemase-producing strains were identical or very close to those determined for E. coli ATCC 25922, for K. pneumoniae ATCC 13883, as well as for 18 clinical carbapenem-susceptible isolates. At a concentration of 4 mg/L, NAB7061 decreased the MIC of rifampicin and clarithromycin for all carbapenemase strains by factors ranging from 6 to 500. The polymyxin-resistant strain K. pneumoniae CL5762B was sensitized by a factor of 24 to rifampicin (FICI, 0.167) and by a factor of 12 to clarithromycin (FICI, 0.208). CONCLUSIONS: Polymyxin-susceptible, carbapenemase-producing strains are as susceptible to NAB739 as are the carbapenem-susceptible clinical isolates. In addition, NAB7061 has notable synergism with rifampicin and clarithromycin against all the carbapenemase-producing strains tested, including the polymyxin-resistant K. pneumoniae strain.

Pharmacokinetics of novel antimicrobial cationic peptides NAB 7061 and NAB 739 in rats following intravenous administration.

OBJECTIVES: To determine the disposition of novel antimicrobial cationic peptides NAB 7061 and NAB 739 following intravenous administration in rats. METHODS: Sprague-Dawley rats received a single intravenous bolus of 1.0 mg/kg NAB 7061 or NAB 739. Plasma concentrations of NAB 7061 or NAB 739 were determined by HPLC or liquid chromatography-mass spectrometry. The pharmacokinetic parameters of NAB 7061 and NAB 739 were calculated using non-compartmental analysis. RESULTS: Corresponding total body clearance, volume of distribution at steady state and terminal half-life of NAB 7061 and NAB 739 averaged 3.84 and 2.63 mL/min/kg, 339 and 222 mL/kg, and 66.2 and 69.0 min, respectively. Approximately 7.16% and 19.4% of the dose was eliminated in an unchanged form via the urine in 24 h for NAB 7061 and NAB 739, respectively. CONCLUSIONS: While both compounds had generally similar pharmacokinetics to colistin, even minor alterations in the chemical structures appear to have an impact on their pharmacokinetics, especially on their clearance by the kidney. There are also substantial differences in relation to the relative contributions of renal and non-renal clearance to overall elimination from the body.

Novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents.

The lack of novel antibiotics against gram-negative bacteria has reinstated polymyxins as the drugs of last resort to treat serious infections caused by extremely multiresistant gram-negative organisms. However, polymyxins are nephrotoxic, and this feature may complicate therapy or even require its discontinuation. Like that of aminoglycosides, the nephrotoxicity of polymyxins might be related to the highly cationic nature of the molecule. Colistin and polymyxin B carry five positive charges. Here we show that novel polymyxin derivatives carrying only three positive charges are effective antibacterial agents. NAB739 has a cyclic peptide portion identical to that of polymyxin B, but in the linear portion of the peptide, it carries the threonyl-D-serinyl residue (no cationic charges) instead of the diaminobutyryl-threonyl-diaminobutyryl residue (two cationic charges). The MICs of NAB739 for 17 strains of Escherichia coli were identical, or very close, to those of polymyxin B. Furthermore, NAB739 was effective against other polymyxin-susceptible strains of Enterobacteriaceae and against Acinetobacter baumannii. At subinhibitory concentrations, it dramatically sensitized A. baumannii to low concentrations of antibiotics such as rifampin, clarithromycin, vancomycin, fusidic acid, and meropenem. NAB739 methanesulfonate was a prodrug analogous to colistin methanesulfonate. NAB740 was the most active derivative against Pseudomonas aeruginosa. NAB7061 (linear portion of the peptide, threonyl-aminobutyryl) lacked direct antibacterial activity but sensitized the targets to hydrophobic antibiotics by factors up to 2,000. The affinities of the NAB compounds for isolated rat kidney brush border membrane were significantly lower than that of polymyxin B.