Inwardly rectifying potassium channels mediate polymyxin-induced nephrotoxicity.

Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.

Design, synthesis and antimicrobial studies of some polymyxin analogues.

To find novel polymycin analogues with high antimicrobial activities and low toxicity, 36 novel polymyxin analogues were synthesized, and in which TZ40-J and TZ40-K were evaluated for their antimicrobial activities using broth microdilution method and for their haemolytic toxicity with sterile sheep blood. Preliminary safety assessments of those two compounds were carried out via the MTT cell viability assay in vitro and acute toxicity assay in vivo. Experimental data demonstrated that TZ40-J and TZ40-K were less toxic and indicate higher activities against Pseudomonas aeruginosa than polymyxin B.

Toxicity in Patients.

Polymyxin toxicity remains a significant concern that limits the clinical utility of this class of antibacterials for patient care. The most notable adverse event is the dose- and treatment-limiting nephrotoxicity that occurs in roughly 30-60% of patients receiving a systemic polymyxin. This chapter focuses on this adverse event with a detailed assessment of the incidence of, and risk factors for, polymyxin-associated nephrotoxicity. In particular, the text focuses on the impact of dose, serum concentrations, and polymyxin selection on nephrotoxicity. Additionally, less common, but clinically important adverse events are discussed.

Mechanisms of Polymyxin-Induced Nephrotoxicity.

Polymyxin-induced nephrotoxicity is the major dose-limiting factor and can occur in up to 60% of patients after intravenous administration. This chapter reviews the latest literature on the mechanisms of polymyxin-induced nephrotoxicity and its amelioration. After filtration by glomeruli, polymyxins substantially accumulate in renal proximal tubules via receptor-mediated endocytosis mainly by megalin and PEPT2. It is believed that subsequently, a cascade of interconnected events occur, including the activation of death receptor and mitochondrial apoptotic pathways, mitochondrial damage, endoplasmic reticulum stress, oxidative stress and cell cycle arrest. The current literature shows that oxidative stress plays a key role in polymyxin-induced kidney damage. Use of antioxidants have a potential in the attenuation of polymyxin-induced nephrotoxicity, thereby widening the therapeutic window. Mechanistic findings on polymyxin-induced nephrotoxicity are critical for the optimization of their use in the clinic and the discovery of safer polymyxin-like antibiotics.

Molecular Mechanisms of Neurotoxicity Induced by Polymyxins and Chemoprevention.

Neurotoxicity is one major unwanted side-effects associated with polymyxin (i.e., colistin and polymyxin B) therapy. Clinically, colistin neurotoxicity is characterized by neurological symptoms including dizziness, visual disturbances, vertigo, confusion, hallucinations, seizures, ataxia, and facial and peripheral paresthesias. Pathologically, colistin-induced neurotoxicity is characterized by cell injury and death in neuronal cell. This Review covers our current understanding of polymyxin-induced neurotoxicity, its underlying mechanisms, and the discovery of novel neuroprotective agents to limit this neurotoxicity. In recent years, an increasing body of literature supports the notion that polymyxin-induced nerve damage is largely related to oxidative stress and mitochondrial dysfunction. P53, PI3K/Akt, and MAPK pathways are also involved in colistin-induced neuronal cell death. The activation of the redox homeostasis pathways such as Nrf2/HO-1 and autophagy have also been shown to play protective roles against polymyxin-induced neurotoxicity. These pathways have been demonstrated to be upregulated by neuroprotective agents including curcumin, rapamycin and minocycline. Further research is needed toward the development of novel polymyxin formulations in combination with neuroprotective agents to ameliorate this unwanted adverse effect during polymyxins therapy in patients.

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.

Synthesis and Bioactivity Investigation of the Individual Components of Cyclic Lipopeptide Antibiotics.

In this paper, 26 natural polymyxin components and a new derivative S2 were synthesized, and their differences in efficacy and toxicity have been investigated. Almost all of the synthesized components showed strong activity against both susceptible and resistant strains of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii. The toxicities were obviously different between the components. Only some of the components were tested for toxicity in vivo. Compounds E2, E2-Val, A2, M2, D2, and S2 showed obviously lower renal cytotoxicity and acute toxicity than polymyxins B and E. The in vivo nephrotoxicity of E2, M2, and S2 was similar to that of polymyxin E. Compound S2, with four positive charges, was especially interesting as it possessed both increased efficacy and decreased toxicity. The SAR and toxicity studies indicated that further structural modification could concentrate on polymyxin S. The results also indicated that S2 could be a new drug candidate.

Polymyxins: a new hope in combating Gram-negative superbugs?

Polymyxins have emerged as an important last-line of defense against Gram-negative 'superbugs'. Unfortunately, the effective use of polymyxins in the clinic has been hampered by their nephrotoxic side effects. Over the last 10 years various industry and academic groups across the globe have been trying to develop new polymyxins that are safer and more efficacious than the currently approved polymyxin B and colistin. However these drug discovery programs are yet to deliver a new and improved polymyxin drug into the clinic. In this piece we provide an overview of the current state of these polymyxin drug discovery programs from a medicinal chemistry perspective as well as some thoughts on how future drug discovery efforts may ultimately find success.

Investigating nephrotoxicity of polymyxin derivatives by mapping renal distribution using mass spectrometry imaging.

Colistin and polymyxin B are effective treatment options for Gram-negative resistant bacteria but are used as last-line therapy due to their dose-limiting nephrotoxicity. A critical factor in developing safer polymyxin analogues is understanding accumulation of the drugs and their metabolites, which is currently limited due to the lack of effective techniques for analysis of these challenging molecules. Mass spectrometry imaging (MSI) allows direct detection of targets (drugs, metabolites, and endogenous compounds) from tissue sections. The presented study exemplifies the utility of MSI by measuring the distribution of polymyxin B1, colistin, and polymyxin B nonapeptide (PMBN) within dosed rat kidney tissue sections. The label-free MSI analysis revealed that the nephrotoxic compounds (polymyxin B1 and colistin) preferentially accumulated in the renal cortical region. The less nephrotoxic analogue, polymyxin B nonapeptide, was more uniformly distributed throughout the kidney. In addition, metabolites of the dosed compounds were detected by MSI. Kidney homogenates were analyzed using LC/MS/MS to determine total drug exposure and for metabolite identification. To our knowledge, this is the first time such techniques have been utilized to measure the distribution of polymyxin drugs and their metabolites. By simultaneously detecting the distribution of drug and drug metabolites, MSI offers a powerful alternative to tissue homogenization analysis and label or antibody-based imaging.

Major pathways of polymyxin-induced apoptosis in rat kidney proximal tubular cells.

Identifying the pathways involved in the apoptotic cell death that is associated with polymyxin-induced nephrotoxicity is crucial for the development of strategies to ameliorate this dose-limiting side effect and for the development of novel safer polymyxins. The primary aim of this study was to identify the major pathways which lead to polymyxin-induced apoptosis in cultured rat kidney proximal tubular cells (NRK-52E). Caspase-3, -8, and -9 were activated by polymyxin B treatment in a concentration-dependent manner. Concentration- and time-dependent expression of FasL and deformation of mitochondrial morphology were revealed following polymyxin B treatment. The proportion of cells with filamentous mitochondria (regular morphology) following an 8-h treatment with 1.0 mM polymyxin B was 56.2% ± 9.7% (n = 3). This was decreased to 30.7% ± 7.5% when the polymyxin B concentration was increased to 2.0 mM. The mitochondrial membrane potential (Δψm) decreased to 14.1% ± 2.9% in the cells treated with 1.0 mM polymyxin B for 24 h (n = 3) compared to that in the untreated control group. Concomitantly, concentration- and time-dependent production of mitochondrial superoxide was also observed. This study is the first to have demonstrated that polymyxin-induced apoptosis is mediated through both the death receptor and mitochondrial pathways in cultured renal tubular cells. It provides key information not only for the amelioration of polymyxin-induced nephrotoxicity but also for the discovery of novel safer polymyxin-like antibiotics against Gram-negative "superbugs."

Discovery of Dap-3 polymyxin analogues for the treatment of multidrug-resistant Gram-negative nosocomial infections.

We report novel polymyxin analogues with improved antibacterial in vitro potency against polymyxin resistant recent clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa . In addition, a human renal cell in vitro assay (hRPTEC) was used to inform structure-toxicity relationships and further differentiate analogues. Replacement of the Dab-3 residue with a Dap-3 in combination with a relatively polar 6-oxo-1-phenyl-1,6-dihydropyridine-3-carbonyl side chain as a fatty acyl replacement yielded analogue 5x, which demonstrated an improved in vitro antimicrobial and renal cytotoxicity profiles relative to polymyxin B (PMB). However, in vivo PK/PD comparison of 5x and PMB in a murine neutropenic thigh model against P. aeruginosa strains with matched MICs showed that 5x was inferior to PMB in vivo, suggesting a lack of improved therapeutic index in spite of apparent in vitro advantages.

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.

Impact of a new biopesticide produced by Paenibacillus sp. strain B2 on the genetic structure and density of soil bacterial communities.

The effect of paenimyxin, a new biopesticide produced by Paenibacillus sp. strain B2, on the density of soil bacterial communities was assessed by colony counting and by 16S rDNA and nirK quantitative polymerase chain reaction (PCR). Paenimyxin had a negative effect on the bacterial colony-forming unit (CFU) number, which was significantly reduced 2 and 4 days after treatment. The effect of paenimyxin on cultivatable bacteria was negligible 7 days after treatment. Approximately 10(7) 16S rDNA sequences per gram of soil (dry weight) were detected by quantitative PCR in all samples. Paenimyxin did not affect the quantification of 16S rDNA or of the denitrifying bacterial community. In addition, RISA fingerprinting showed that the genetic structure of the bacterial communities was significantly modified 2 days after paenimyxin application at 50 microM and 4 days after treatment at lower concentrations (0.5 and 5 microM). The impact of paenimyxin treatment on the genetic structure of soil bacterial communities was transient, as no effect could be observed after 7, 14 and 28 days when compared with the untreated control.

Round window membrane permeability during experimental purulent otitis media: altered Cortisporin ototoxicity.

Round window membrane (RWM) permeability is the most critical factor influencing cochlear function following otitis media. Because otic drops are frequently used during purulent otitis media (POM), we investigated RWM permeability and ototoxicity of Cortisporin otic suspension after inducing experimental POM. Unilateral POM was induced in eight chinchillas by inoculating type 7F Streptococcus pneumoniae into the right ears. Left ears were inoculated with phosphate-buffered saline (control). When POM resolved, the animals were divided into two groups. The round window niches of group 1 were covered with Cortisporin otic suspension. Compound action potentials were measured before and after drug application. The RWM permeability was measured in group 2 by use of tetraethylammonium (TEA) ions as tracers, and the arrival time of TEA and the slope of the potassium-selective microelectrode response were measured. Animals with otitis media exhibited less susceptibility to ototoxicity of Cortisporin otic suspension and reduced RWM permeability to the medium-sized molecule TEA.

Effect of topical fosfomycin on polymyxin B ototoxicity.

Fosfomycin is an antibiotic that has been found to reduce the ototoxicity of aminoglycoside antibiotics and cisplatin when systemically coadministered. Polymyxin B, an antibiotic frequently used in ototopical preparations, has been shown to be ototoxic in experimental studies. To investigate the effect of fosfomycin on polymyxin B ototoxicity, topical administration of the two agents into the middle ear cavity was performed. Two groups of chinchillas were used. One group received applications of polymyxin B alone, and the second group received polymyxin B combined with fosfomycin. It was found that application of polymyxin B produces severe damage to the cochlea. However, when polymyxin B was given in combination with fosfomycin, cochlear damage was dramatically reduced. It is likely that in clinical use, a combination of polymyxin B and fosfomycin would demonstrate reduced risk of ototoxicity.

Target sites of polymyxin B ototoxicity.

The present study was undertaken to determine the target sites of polymyxin B ototoxicity. This drug, at the concentration of 1 mM, was perfused through the scala tympani of the guinea pig cochlea, and cochlear microphonics and endocochlear potentials were monitored. Both cochlear potentials altered but in an independent manner. These findings indicated that not only the organ of Corti but also another tissue is involved in the ototoxicity produced. The best locus for this is the vascular stria.

Purification, toxicity, and antiendotoxin activity of polymyxin B nonapeptide.

Polymyxin B, a relatively toxic antibiotic, has potent endotoxin-neutralizing properties that may be beneficial as adjunctive therapy in gram-negative sepsis. Polymyxin B nonapeptide (deacylated polymyxin B) is devoid of antibiotic activity but retains the capacity to disorganize the outer membrane of gram-negative bacteria. To evaluate the potential therapeutic usefulness of this derivative, we produced purified polymyxin B nonapeptide, tested its in vivo toxicity in animals, and evaluated its in vitro antiendotoxin activity. Effectiveness as an antiendotoxin agent was assessed by examining the ability of polymyxin B nonapeptide to block the enhanced release of toxic oxygen radicals induced by lipopolysaccharide in human neutrophils (priming). In vivo, at doses of 1.5 and 3.0 mg/kg, polymyxin B nonapeptide did not exhibit the neuromuscular blocking, neurotoxic, or nephrotoxic effects that were observed with polymyxin B sulfate. Both polymyxin B and polymyxin B nonapeptide inhibited lipopolysaccharide-induced neutrophil priming in a concentration-dependent manner, but the parent compound, polymyxin B, was 63 times more effective on a weight basis. The inhibitory activity of both compounds, however, diminished rapidly when they were added after the start of the lipopolysaccharide-neutrophil incubation. We conclude that polymyxin B nonapeptide is less toxic than polymyxin B and, at the doses tested, lacks the neurotoxicity and nephrotoxicity of the parent compound. Polymyxin B nonapeptide retains the antiendotoxin activity of polymyxin B but is much less potent. The findings suggest that these compounds block an early step in the neutrophil priming process, possibly lipopolysaccharide attachment to or insertion into the neutrophil membrane.

Inflammatory effects of topical antibiotic suspensions containing propylene glycol in chinchilla middle ears.

This study examines the occurrence of inflammatory changes and cholesteatoma in the middle ears of seven chinchillas after the application of topical antibiotic suspensions containing two different concentrations of propylene glycol. The preparations used were Cortisporin otic suspension, which contains neomycin, polymyxin B, hydrocortisone, and 10.5% propylene glycol, and Cortisporin ophthalmic suspension, containing the same ingredients, but only a 2% concentration of propylene glycol. Six weeks after the administration of the Cortisporin preparations, applied to the middle ear through a transbulla approach, no cholesteatomas were found in the seven ears treated with the ophthalmic suspension. Evidence of mild inflammation was present in only two of these ears. In the seven contralateral ears treated with the otic suspension, middle ear adhesions were found in six, cholesteatoma was present in four, serous effusions were found in three, and one had a large tympanic membrane perforation. The ears that showed cholesteatomas also had histologic evidence of squamous metaplasia, granulation tissue, and erosion of the underlying bone. We submit that the pathologic responses of the middle ear mucosa treated with the otic suspension, were due to an inflammatory response to the higher concentration of propylene glycol compared to that of the ophthalmic suspension.

Ototoxicity of neomycin and polymyxin B following middle ear application in the chinchilla and baboon.

Previous experimental studies have demonstrated structural damage of the organ of Corti and stria vascularis following application of combination antibiotic otic drops to the middle ear. In this investigation the ototoxic effects of neomycin and polymyxin B (two antibiotics often used together in ototopical preparations) were separately evaluated after administration of each agent to the middle ear cavities of chinchillas and baboons. The antibiotics were administered in saline solution at the same concentrations used in Cortisporin Otic Suspension (3.5 mg/ml neomycin base, 10,000 units/ml polymyxin B). In both the rodent and primate, polymyxin B consistently produced greater cochlear damage than did neomycin. In fact, the extent of hair cell loss and strial injury produced by polymyxin B alone was, in many cases, comparable to that previously observed after application of Cortisporin Otic Suspension itself. Hair cell loss in the baboon was markedly less severe than in the chinchilla. It is believed that differences in position and structure of the round window membrane are important factors in the differing levels of ototoxicity observed in the rodent and primate.