Martti Vaara

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 C_max 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 C_max 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.

Introduction: Antibacterial photodynamic therapy (aPDT) and antibacterial blue light (aBL) are emerging treatment methods auxiliary to mechanical debridement for periodontitis. APDT provided with a near infrared (NIR) light in conjunction with an indocyanine green (ICG) photosensitizer has shown efficacy in several dental in-office-treatment protocols. In this study, we testedStreptococcus mutansbiofilm sensitivity to either single-light (aPDT or aBL) or dual-light aPDT (simultaneous aPDT and aBL) exposure.Materials and Methods: Biofilm was cultured by pipeting dilutedStreptococcus mutanssuspension with growth medium on the bottom of well plates. Either a single-light aPDT (810-nm aPDT or 405-nm aBL) or a dual-light aPDT (simultaneous 810-nm aPDT and 405nm aBL) was applied, in both cases together with the ICG photosensitizer, while keeping the total light energy constant at 100J/cm². Single-dose light exposures were given after one-day, four-day or fourteen-day biofilm incubations. Also, a repeated daily dose of the same light energy was applied during biofilm incubations on four-day and fourteen-day biofilms. Finally, antibacterial action of the dual-light aPDT with different relative ratios of 810 nm and 405 nm of light energy was examined on the single-day and four-day protocols. Biofilms were scraped, diluted into ratios between 1:1 to 1:100 000, and plated. After re-incubation, colony-forming units (CFUs) were counted, and confocal 3D biofilm imaging was performed.Results: On a one-day biofilm, dual-light aPDT was significantly more efficient than single-light aBL or aPDT, although all modalities were bactericidal. On a four-day maturated biofilm, a single exposure of aPDT or dual-light aPDT was more efficient than aBL, resulting in a four logarithmic scale reduction in bacterial counts. Surprisingly, when the same amount of aPDT was repeated with a daily dosing on a four-day or a fourteen-day biofilm, bacterial viability improved significantly. A similar but milder response of improved bacterial viability was seen after repetitive aBL application. The viability improvement was eliminated when dual-light aPDT was applied. By changing the relative light energy ratios in dual-light aPDT, a relative increase in aBL improved the antibacterial action of dual-light aPDT when the biofilm was older.Conclusion: When aPDT is administered repeatedly toS. mutansbiofilm, a single wavelength-based aBL or aPDT leads to a significant biofilm adaptation and increasedS. mutansviability. The combined use of aBL light in synchrony with aPDT arrests the adaptation and provides significantly improved and sustained antibacterial efficacy.

Corrigendum: Polymyxins and Their Potential Next Generation as Therapeutic Antibiotics

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.

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 B and colistin (polymyxin E) are bactericidal pentacationic lipopeptides that act specifically on Gram‐negative bacteria, first by disrupting their outermost permeability barrier, the outer membrane (OM), and then damaging the cytoplasmic membrane. The discovery of both polymyxin B and colistin was published independently by three laboratories as early as in 1947. They were subsequently used in intravenous therapy. Unfortunately, they also exhibit significant and dose‐limiting nephrotoxicity. Therefore, polymyxins were reserved as agents of last‐line defense. The emergence of extremely multiresistant strains has now forced clinicians to reinstate polymyxins in the therapy of severe infections. However, the current dosage regimens lead to insufficient drug concentrations in serum and clinicians have been advised to use larger doses, which further increases the risk of nephrotoxicity. Very recently, the interest in developing better tolerated and more effective polymyxins has grown. This review focuses on describing four development programs that have yielded novel derivatives that are more effective than the old polymyxins in animal infection models. Compounds from three programs are superior to the old polymyxins in the rodent lung infection model with Acinetobacter baumannii and/or Pseudomonas aeruginosa. One of them is also more effective than polymyxin B in A. baumannii mouse thigh infection. The fourth program includes compounds that are approximately tenfold more effective in Escherichia coli murine pyelonephritis than polymyxin B.

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. 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. 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. NAB739 and NAB815 were unequivocally more effective than polymyxin B in the murine pyelonephritis model.