Polymyxins (polymyxin B (PMB) and polymyxin E (colistin)) are cyclic lipodecapeptide antibiotics, highly basic due to five free amino groups, and rapidly bactericidal against Gram-negative bacteria, such as the majority of Enterobacteriaceae as well as Acinetobacter baumannii and Pseudomonas aeruginosa. Their clinical use was abandoned in the 1960s because of nephrotoxicity and because better-tolerated drugs belonging to other antibiotic classes were introduced. Now, due to the global dissemination of extremely-drug resistant Gram-negative bacterial strains, polymyxins have resurged as the last-line drugs against those strains. Novel derivatives that are less toxic and/or more effective at tolerable doses are currently under preclinical development and their properties have recently been described in several extensive reviews. Other derivatives lack any direct bactericidal activity but damage the outermost permeability barrier, the outer membrane, of the target bacteria and make it more permeable to many other antibiotics. This review describes the properties of three thus far best-characterized “permeabilizer” derivatives, i.e., the classic permeabilizer polymyxin B nonapeptide (PMBN), NAB7061, and SPR741/NAB741, a compound that recently successfully passed the clinical phase 1. Also, a few other permeabilizer compounds are brought up.

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.

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 sensitizedEscherichia coli,Klebsiella pneumoniae,Enterobacter cloacae, andAcinetobacter baumanniito 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 ≤0.111 to 0.158 and that with clarithromycin from ≤0.094 to 0.292. When tested by the checkerboard method, the corresponding FICI values againstE. coliATCC 25922 were ≤0.141 to ≤0.155 with rifampin and 0.094 with clarithromycin. In addition, at 4 μg/ml, NAB741 decreased the MICs of azithromycin, mupirocin, fusidic acid, and vancomycin forE. colistrains andE. cloacaeby factors ranging from 8 to 200. A sister peptide, NAB752, carrying a threonyl-aminobutyryl residue as the linear peptide portion, was inactive. Furthermore, NAB741 sensitizedE. colito the bactericidal activity of fresh guinea pig serum. The renal clearance of NAB741 was approximately 400-fold, 16-fold, and 8-fold higher than those measured for colistin, NAB7061, and NAB739, respectively.

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.