Correlative proteomics identify the key roles of stress tolerance strategies in Acinetobacter baumannii in response to polymyxin and human macrophages.

The opportunistic pathogen Acinetobacter baumannii possesses stress tolerance strategies against host innate immunity and antibiotic killing. However, how the host-pathogen-antibiotic interaction affects the overall molecular regulation of bacterial pathogenesis and host response remains unexplored. Here, we simultaneously investigate proteomic changes in A. baumannii and macrophages following infection in the absence or presence of the polymyxins. We discover that macrophages and polymyxins exhibit complementary effects to disarm several stress tolerance and survival strategies in A. baumannii, including oxidative stress resistance, copper tolerance, bacterial iron acquisition and stringent response regulation systems. Using the spoT mutant strains, we demonstrate that bacterial cells with defects in stringent response exhibit enhanced susceptibility to polymyxin killing and reduced survival in infected mice, compared to the wild-type strain. Together, our findings highlight that better understanding of host-pathogen-antibiotic interplay is critical for optimization of antibiotic use in patients and the discovery of new antimicrobial strategy to tackle multidrug-resistant bacterial infections.

Rescuing the Last-Line Polymyxins: Achievements and Challenges.

Antibiotic resistance is a major global health challenge and, worryingly, several key Gram negative pathogens can become resistant to most currently available antibiotics. Polymyxins have been revived as a last-line therapeutic option for the treatment of infections caused by multidrug-resistant Gram negative bacteria, in particular Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacterales. Polymyxins were first discovered in the late 1940s but were abandoned soon after their approval in the late 1950s as a result of toxicities (e.g., nephrotoxicity) and the availability of "safer" antibiotics approved at that time. Therefore, knowledge on polymyxins had been scarce until recently, when enormous efforts have been made by several research teams around the world to elucidate the chemical, microbiological, pharmacokinetic/pharmacodynamic, and toxicological properties of polymyxins. One of the major achievements is the development of the first scientifically based dosage regimens for colistin that are crucial to ensure its safe and effective use in patients. Although the guideline has not been developed for polymyxin B, a large clinical trial is currently being conducted to optimize its clinical use. Importantly, several novel, safer polymyxin-like lipopeptides are developed to overcome the nephrotoxicity, poor efficacy against pulmonary infections, and narrow therapeutic windows of the currently used polymyxin B and colistin. This review discusses the latest achievements on polymyxins and highlights the major challenges ahead in optimizing their clinical use and discovering new-generation polymyxins. To save lives from the deadly infections caused by Gram negative "superbugs," every effort must be made to improve the clinical utility of the last-line polymyxins. SIGNIFICANCE STATEMENT: Antimicrobial resistance poses a significant threat to global health. The increasing prevalence of multidrug-resistant (MDR) bacterial infections has been highlighted by leading global health organizations and authorities. Polymyxins are a last-line defense against difficult-to-treat MDR Gram negative pathogens. Unfortunately, the pharmacological information on polymyxins was very limited until recently. This review provides a comprehensive overview on the major achievements and challenges in polymyxin pharmacology and clinical use and how the recent findings have been employed to improve clinical practice worldwide.

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.

Preparation of Free-Flowing Spray-Dried Amorphous Composites Using Neusilin®.

A highly porous additive, Neusilin®, with high adsorption capability is investigated to improve bulk properties, hence processability of spray-dried amorphous solid dispersions (ASDs). Griseofulvin (GF) is applied as a model BCS class 2 drug in ASDs. Two grades of Neusilin®, US2 (coarser) and UFL2 (finer), were used as additives to produce spray-dried amorphous composite (AC) powders, and their performance was compared with the resulting ASDs without added Neusilin®. The resulting AC powders that included Neusilin® had greatly enhanced flowability (flow function coefficient (FFC) > 10) comparable to larger particles (100 µm) yet had finer particle size (< 50 µm), hence retaining the advantage of fast dissolution rate of finer sizes. Dissolution results demonstrated that achieved GF supersaturation for AC powders with Neusilin® was as high as 3 times that of crystalline GF concentration and was achieved within 30 min. In addition, 80% of drug was released within 4 min. The flowability improvement for AC powders with Neusilin® was more significant as compared to spray-dried ASDs without Neusilin®. Thus, the role of Neusilin® in flowability improvement was evident, considering that spray-dried AC with Neusilin® UFL2 has higher FFC than ASDs having a similar size. Lastly, the AC powders retained a fully amorphous state of GF after 3-month ambient storage. The overall results conveyed that the improved flowability and dissolution rate could outweigh the loss of drug loading resulted by addition of Neusilin®.

Multicomponent Droplet Drying Modeling Based on Conservation and Population Balance Equations.

The aim of this work is to present a modeling tool to describe drying kinetics and delineate evolving physical and chemical behavior of multicomponent droplets during drying. Conservation equations coupled with population balance equations (PBE) are used to achieve this goal. Modeling results are gauged with single salt-water droplet drying from literature and show congruent trends. This model is then extended to a more complex system: various droplet sizes containing methanol (solvent), Felodipine (active ingredient), and PVP (polyvinylpyrrolidone as excipient). The FIB-SEM (Focused-Ion Beam Scanning Electron Microscopy) imaging results from spray-dried particles produced with similar formulation and processing conditions are consistent with phase behavior predicted by the model. The results show competing impacts of transport phenomena on the intermittent shell formation process and final particle structure and chemical heterogeneity. Solute diffusion, solvent efflux, and intra-drop flow impact the model system. It is found that shell formation follows a fluctuating profile where the initial precipitation of the dissolved species on the droplet surface is dampened, and nucleated particles become dispersed periodically until the shell becomes strong enough to withstand internal circulations. These internal effects are dependent on droplet size and are pronounced for larger droplets. That is, the particle phase behavior and physical nature are functions of the atomized droplet size. Stemming understating from this study would inform an optimized unit, operating in target design space. This would provide better product quality control and minimize discrepancies observed in process development during the early phase vs. commercial scale.

Improved Aerosolization Stability of Inhalable Tobramycin Powder Formulation by Co-Spray Drying with Colistin.

PURPOSE: Tobramycin shows synergistic antibacterial activity with colistin and can reduce the toxic effects of colistin. The purpose of this study is to prepare pulmonary powder formulations containing both colistin and tobramycin and to assess their in vitro aerosol performance and storage stability. METHODS: The dry powder formulations were manufactured using a lab-scale spray dryer. In vitro aerosol performance was measured using a Next Generation Impactor. The storage stability of the dry powder formulations was measured at 22°C and two relative humidity levels - 20 and 55%. Colistin composition on the particle surface was measured using X-ray photoelectron spectroscopy. RESULTS: Two combination formulations, with 1:1 and 1:5 molar ratios of colistin and tobramycin, showed fine particle fractions (FPF) of 85%, which was significantly higher than that of the spray dried tobramycin (45%). FPF of the tobramycin formulation increased significantly when stored for four weeks at both 20% and 55% RH. In contrast, FPF values of both combination formulations and spray dried colistin remained stable at both humidity levels. Particle surface of each combination was significantly enriched in colistin molecules; 1:5 combination showed 77% by wt. colistin. CONCLUSIONS: The superior aerosol performance and aerosolization stability of 1:1 and 1:5 combination formulations of colistin and tobramycin could be attributed to enrichment of colistin on the co-spray dried particle surface. The observed powder properties may be the result of a surfactant-like assembly of these colistin molecules during spray drying, thus forming a hydrophobic particle surface.

Polymyxin-Induced Metabolic Perturbations in Human Lung Epithelial Cells.

Inhaled polymyxins are associated with toxicity in human lung epithelial cells that involves multiple apoptotic pathways. However, the mechanism of polymyxin-induced pulmonary toxicity remains unclear. This study aims to investigate polymyxin-induced metabolomic perturbations in human lung epithelial A549 cells. A549 cells were treated with 0.5 or 1.0 mM polymyxin B or colistin for 1, 4, and 24 h. Cellular metabolites were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and significantly perturbed metabolites (log2 fold change [log2FC] ≥ 1; false-discovery rate [FDR] ≤ 0.2) and key pathways were identified relative to untreated control samples. At 1 and 4 h, very few significant changes in metabolites were observed relative to the untreated control cells. At 24 h, taurine (log2FC = -1.34 ± 0.64) and hypotaurine (log2FC = -1.20 ± 0.27) were significantly decreased by 1.0 mM polymyxin B. The reduced form of glutathione (GSH) was significantly depleted by 1.0 mM polymyxin B at 24 h (log2FC = -1.80 ± 0.42). Conversely, oxidized glutathione (GSSG) was significantly increased by 1.0 mM both polymyxin B (log2FC = 1.38 ± 0.13 at 4 h and 2.09 ± 0.20 at 24 h) and colistin (log2FC = 1.33 ± 0.24 at 24 h). l-Carnitine was significantly decreased by 1.0 mM of both polymyxins at 24 h, as were several key metabolites involved in biosynthesis and degradation of choline and ethanolamine (log2FC ≤ -1); several phosphatidylserines were also increased (log2FC ≥ 1). Polymyxins perturbed key metabolic pathways that maintain cellular redox balance, mitochondrial ß-oxidation, and membrane lipid biogenesis. These mechanistic findings may assist in developing new pharmacokinetic/pharmacodynamic strategies to attenuate the pulmonary toxicities of inhaled polymyxins and in the discovery of new-generation polymyxins.

Impact of Critical Material Attributes (CMAs)-Particle Shape on Miniature Pharmaceutical Unit Operations.

The U.S. Food and Drug Administration (FDA) emphasizes drug product development by Quality by Design (QbD). Critical material attributes (CMAs) are a QbD element that has an impact on pharmaceutical operations and product quality. Pharmaceutical drugs often crystallize as needle-shaped (a CMA) particles and affect the process due to poor flowability, low bulk density, and high compressibility, and eventually the product performance. In this study, the product obtained from crystallization was needle-shaped Ciprofloxacin HCl (CIPRO), formed lumps during drying, and compacted during processing through feeders. To delump small amounts of materials and break the needles, multiple available devices (mortar-pestle, Krups grinder) and custom-made grinder were assessed before formulation. The processed CIPRO powder was then used to make tablets in the miniature tablet manufacturing unit developed by the team at MIT. The critical quality attributes (CQA) of the tablets, set by the United States Pharmacopeia (USP), were then assessed for the drug powder processed with each of these devices. Powder properties comparable to commercial CIPRO were obtained when the custom MIT-designed grinder was used, leading to tablets that meet the USP criteria, with comparable dissolution profiles of those for marketed CIPRO tablets. This study demonstrates how needle-shaped crystals have an impact on pharmaceutical operations, even if it is on a miniature scale, and how proper shape and subsequent flow properties can be obtained by processing the particles through the MIT team-designed grinder.

Polymyxin-Induced Cell Death of Human Macrophage-Like THP-1 and Neutrophil-Like HL-60 Cells Associated with the Activation of Apoptotic Pathways.

Innate immunity is crucial for the host to defend against infections, and understanding the effect of polymyxins on innate immunity is important for optimizing their clinical use. In this study, we investigated the potential toxicity of polymyxins on human macrophage-like THP-1 and neutrophil-like HL-60 cells. Differentiated THP-1 human macrophages (THP-1-dMs) and HL-60 human neutrophils (HL-60-dNs) were employed. Flow cytometry was used to measure the concentration-dependent effects (100 to 2,500 µM for THP-1-dMs and 5 to 2,500 µM for HL-60-dNs) and time-dependent effects (1,000 µM for THP-1-dMs and 300 µM for HL-60-dNs) of polymyxin B over 24 h. Effects of polymyxin B on mitochondrial activity, activation of caspase-3, caspase-8, and caspase-9, and Fas ligand (FasL) expression in both cell lines were examined using fluorescence imaging, colorimetric, and fluorometric assays. In both cell lines, polymyxin B induced concentration- and time-dependent loss of viability at 24 h with 50% effective concentration (EC50) values of 751.8 µM (95% confidence interval [CI], 692.1 to 816.6 µM; Hill slope, 3.09 to 5.64) for THP-1-dM cells and 175.4 µM (95% CI, 154.8 to 198.7 µM; Hill slope, 1.42 to 2.21) for HL-60-dN cells. A concentration-dependent loss of mitochondrial membrane potential and generation of mitochondrial superoxide was also observed. Polymyxin B-induced apoptosis was associated with concentration-dependent activation of all three tested caspases. The death receptor apoptotic pathway activation was demonstrated by a concentration-dependent increase of FasL expression. For the first time, our results reveal that polymyxin B induced concentration- and time-dependent cell death in human macrophage-like THP-1 and neutrophil-like HL-60 cells associated with mitochondrial and death receptor apoptotic pathways.

Intracellular localization of polymyxins in human alveolar epithelial cells.

Background: Current inhaled polymyxin therapy is empirical and often large doses are administered, which can lead to pulmonary adverse effects. There is a dearth of information on the mechanisms of polymyxin-induced lung toxicity and their intracellular localization in lung epithelial cells. Objectives: To investigate the intracellular localization of polymyxins in human lung epithelial A549 cells. Methods: A549 cells were treated with polymyxin B and intracellular organelles (early and late endosomes, endoplasmic reticulum, mitochondria, lysosomes and autophagosomes), ubiquitin protein and polymyxin B were visualized using immunostaining and confocal microscopy. Fluorescence intensities of the organelles and polymyxin B were quantified and correlated for co-localization using ImageJ and Imaris platforms. Results: Polymyxin B co-localized with early endosomes, lysosomes and ubiquitin at 24 h. Significantly increased lysosomal activity and the autophagic protein LC3A were observed after 0.5 and 1.0 mM polymyxin B treatment at 24 h. Polymyxin B also significantly co-localized with mitochondria (Pearson's R = 0.45) and led to the alteration of mitochondrial morphology from filamentous to fragmented form (n = 3, P < 0.001). These results are in line with the polymyxin-induced activation of the mitochondrial apoptotic pathway observed in A549 cells. Conclusions: Accumulation of polymyxins on mitochondria probably caused mitochondrial toxicity, resulting in increased oxidative stress and cell death. The formation of autophagosomes and lysosomes was likely a cellular response to the polymyxin-induced stress and played a defensive role by disassembling dysfunctional organelles and proteins. Our study provides new mechanistic information on polymyxin-induced lung toxicity, which is vital for optimizing inhaled polymyxins in the clinic.

On-Demand Manufacturing of Direct Compressible Tablets: Can Formulation Be Simplified?

PURPOSE: Oral direct compressible tablets are the most frequently used drug products. Manufacturing of tablets requires design and development of formulations, which need a number of excipients. The choice of excipients depends on the concentration, manufacturability, stability, and bioavailability of the active pharmaceutical ingredients (APIs). At MIT, we developed a miniature platform for on-demand manufacturing of direct compressible tablets. This study investigated how formulations could be simplified to use a small number of excipients for a number of different API's in which long term stability is not required. METHOD: Direct compressible tablets of five pharmaceutical drugs, Diazepam, Diphenhydramine HCl, Doxycycline Monohydrate, Ibuprofen, and Ciprofloxacin HCl, with different drug loadings, were made using direct compression in an automated small scale system.. The critical quality attributes (CQA) of the tablets were assessed for the quality standards set by the United States Pharmacopeia (USP). RESULTS: This miniature system can manufacture tablets - on-demand from crystalline API using the minimum number of excipients required for drug product performance. All drug tablets met USP quality standards after manufacturing and after 2 weeks of accelerated stability test, except for slightly lower drug release for Ibuprofen. CONCLUSIONS: On-demand tablets manufacturing where there is no need for long term stability using a flexible, miniature, automated (integrated) system will simplify pharmaceutical formulation design compared to traditional formulations. This advancement will offer substantial economic benefits by decreasing product time-to-market and enhancing quality.

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.

History, Chemistry and Antibacterial Spectrum.

Polymyxins are naturally occurring cyclic lipopeptides that were discovered more than 60 years ago. They have a narrow antibacterial spectrum, which is mainly against Gram-negative pathogens. The dry antibiotic pipeline, together with the increasing incidence of bacterial resistance in the clinic, has been dubbed 'the perfect storm'. This has forced a re-evaluation of 'old' antibiotics, in particular the polymyxins, which retain activity against many multidrug-resistant (MDR) Gram-negative organisms. As a consequence, polymyxin B and colistin (polymyxin E) are now used as the last therapeutic option for infections caused by 'superbugs' such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. This chapter covers the history, chemistry and antibacterial spectrum of these very important last-line lipopeptide antibiotics.

Methionine Ameliorates Polymyxin-Induced Nephrotoxicity by Attenuating Cellular Oxidative Stress.

Polymyxins are a last line of defense against multidrug-resistant Gram-negative pathogens. Recent pharmacological data show that intravenous polymyxins can cause nephrotoxicity in up to 60% of patients, and the plasma concentrations of polymyxins achieved with the currently recommended dosage regimens are suboptimal in a large proportion of patients. Simply increasing the daily dose of polymyxins is not possible due to nephrotoxicity. This study aimed to examine the protective effect of methionine against polymyxin-induced nephrotoxicity. Methionine (400 mg/kg of body weight), polymyxin B (35 mg/kg), a combination of methionine (100 or 400 mg/kg) and polymyxin B, and saline were administered to mice twice daily over 3.5 days. Kidneys were collected immediately at the end of the experiment for histological examination. The effect of methionine on the pharmacokinetics of polymyxin B was investigated in rats. The attenuation of polymyxin B (0.75 mM)-induced mitochondrial superoxide production by methionine (10.0 mM) was examined in rat kidney (NRK-52E) cells. Histological results revealed that the polymyxin-induced nephrotoxicity in mice was ameliorated by methionine in a dose-dependent manner. The methionine doses were well tolerated in the mice and rats, and the pharmacokinetics of polymyxin B in rats were not affected by methionine. In the group receiving polymyxin B-methionine, the total body clearance of polymyxin B was very similar to that in the group receiving polymyxin B alone (3.71 ± 0.57 versus 3.12 ± 1.66 ml/min/kg, P > 0.05). A substantial attenuation of polymyxin-induced mitochondrial superoxide production in NRK-52E cells was observed following pretreatment with methionine. Our results demonstrate that coadministration of methionine significantly ameliorated polymyxin-induced nephrotoxicity and decreased mitochondrial superoxide production in renal tubular cells.

Potential Toxicity of Polymyxins in Human Lung Epithelial Cells.

Inhaled polymyxins are of considerable utility in achieving optimal exposure in the respiratory tract for the treatment of lung infections caused by multidrug-resistant Gram-negative pathogens. Current inhaled polymyxin therapy is empirical, and often large doses are used that may lead to potential pulmonary adverse effects. This study aimed to investigate the effect of polymyxins on human lung epithelial (A549) cells. The viability of A549 cells was examined after treatment with polymyxins by flow cytometry. Activation of caspases 3, 8, and 9, expression of Fas ligand (FasL), loss of mitochondrial membrane potential, and mitochondrial oxidative stress induced by polymyxin B were evaluated. The concentration of polymyxin B required to induce 50% of maximal cell death was 1.74 mM (95% confidence interval, 1.60 to 1.90 mM). Colistin was at least 2-fold less toxic than polymyxin B, while colistimethate was nontoxic. With 2.0 mM polymyxin B, 30.6% ± 11.5% (mean ± standard deviation) of the cells were apoptotic at 8 h and this increased to 71.3% ± 3.72% at 24 h. Concentration- and time-dependent activation of caspases 3, 8, and 9 was evident, while the activation of caspase 9 was more dramatic. Furthermore, polymyxin B caused concentration- and time-dependent FasL expression, production of mitochondrial reactive oxygen species, and changes in mitochondrial membrane potential. This is the first study to demonstrate that both extrinsic death receptor and intrinsic mitochondrial pathways are involved in polymyxin-induced toxicity in A549 cells. This knowledge base is critical for the development of novel strategies for the safe and effective inhalation therapy of polymyxins against Gram-negative "superbugs."

Gelofusine Ameliorates Colistin-Induced Nephrotoxicity.

Colistin therapy is used as the last line of defense against life-threatening Gram-negative infections. Nephrotoxicity is the major dose-limiting side effect that impedes optimal dosing of patients. This study aims to examine the nephroprotective effect of the plasma volume expander gelofusine against colistin-induced nephrotoxicity. Renal protection was assessed in mice that were subcutaneously injected with colistin sulfate (14 mg/kg of body weight × 6 doses every 2 h; accumulated dose, 84 mg/kg) and simultaneously injected in the intraperitoneal region with gelofusine (75, 150, 300, or 600 mg/kg × 6). At 2 and 20 h after the last colistin dose, mice were euthanized, and the severity of renal alteration was examined histologically. Histological findings in mice revealed that colistin-induced nephrotoxicity was ameliorated by gelofusine in a dose-dependent manner, whereas significant histological abnormalities were detected in the kidneys of mice in the colistin-only group. The impact of coadministered gelofusine on colistin pharmacokinetics was investigated in rats. Rats were administered a single intravenous dose of gelofusine at 400 mg/kg 15 min prior to the intravenous administration of colistin (1 mg/kg). Gelofusine codosing did not alter the pharmacokinetics of colistin in rats; however, gelofusine did significantly lower the accumulation of colistin in the kidney tissue of mice. This is the first study demonstrating the protective effect of gelofusine against colistin-induced nephrotoxicity. These findings highlight the clinical potential of gelofusine as a safe adjunct for ameliorating the nephrotoxicity and increasing the therapeutic index of polymyxins.

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."

Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes.

Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity.

Significant accumulation of polymyxin in single renal tubular cells: a medicinal chemistry and triple correlative microscopy approach.

Polymyxin is the last-line therapy against Gram-negative 'superbugs'; however, dose-limiting nephrotoxicity can occur in up to 60% of patients after intravenous administration. Understanding the accumulation and concentration of polymyxin within renal tubular cells is essential for the development of novel strategies to ameliorate its nephrotoxicity and to develop safer, new polymyxins. We designed and synthesized a novel dual-modality iodine-labeled fluorescent probe for quantitative mapping of polymyxin in kidney proximal tubular cells. Measured by synchrotron X-ray fluorescence microscopy, polymyxin concentrations in single rat (NRK-52E) and human (HK-2) kidney tubular cells were approximately 1930- to 4760-fold higher than extracellular concentrations. Our study is the first to quantitatively measure the significant uptake of polymyxin in renal tubular cells and provides crucial information for the understanding of polymyxin-induced nephrotoxicity. Importantly, our approach represents a significant methodological advancement in determination of drug uptake for single-cell pharmacology.

Drug-drug plasma protein binding interactions of ivacaftor.

Ivacaftor is a novel cystic fibrosis (CF) transmembrane conductance regulator (CFTR) potentiator that improves the pulmonary function for patients with CF bearing a G551D CFTR-protein mutation. Because ivacaftor is highly bound (>97%) to plasma proteins, there is the strong possibility that co-administered CF drugs may compete for the same plasma protein binding sites and impact the free drug concentration. This, in turn, could lead to drastic changes in the in vivo efficacy of ivacaftor and therapeutic outcomes. This biochemical study compares the binding affinity of ivacaftor and co-administered CF drugs for human serum albumin (HSA) and α1 -acid glycoprotein (AGP) using surface plasmon resonance and fluorimetric binding assays that measure the displacement of site-selective probes. Because of their ability to strongly compete for the ivacaftor binding sites on HSA and AGP, drug-drug interactions between ivacaftor are to be expected with ducosate, montelukast, ibuprofen, dicloxacillin, omeprazole, and loratadine. The significance of these plasma protein drug-drug interactions is also interpreted in terms of molecular docking simulations. This in vitro study provides valuable insights into the plasma protein drug-drug interactions of ivacaftor with co-administered CF drugs. The data may prove useful in future clinical trials for a staggered treatment that aims to maximize the effective free drug concentration and clinical efficacy of ivacaftor.