Elena K. Schneider

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. Copyright

Can Cystic Fibrosis Patients Finally Catch a Breath With Lumacaftor/Ivacaftor?

Cystic fibrosis (CF) is a life‐limiting disease caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) activity. The recent US Food and Drug Administration (FDA) approval of lumacaftor combined with ivacaftor (Orkambi) targets patients with the F508del‐CFTR. The question remains: Is this breakthrough combination therapy the “magic‐bullet” cure for the vast majority of patients with CF? This review covers the contemporary clinical and scientific knowledge‐base for lumacaftor/ivacaftor and highlights the emerging issues from recent conflicting literature reports.

Deficiency in outer dense fiber 1 is a marker and potential driver of idiopathic male infertility

Globally, ∼1 in 15 men of reproductive age are infertile, yet the precise mechanisms underlying their gamete failure are unknown. Although a semen analysis is performed to determine fertilizing potential, the diagnostic suitability of this analysis has been questioned in several reports, as many men, classified as infertile according to their semen analysis, subsequently turn out to be fertile. Herein, we have used a quantitative (phospho)-proteomic analysis, using enrichment on titanium dioxide followed by ion-trap mass spectrometry (LC-MS/MS), to compare the semen of infertile versus fertile males. One protein, namely outer dense fiber 1 (ODF1), was dramatically reduced in infertile males. Using specific antibodies, we then screened the gametes of a cohort of suspected infertile men and demonstrated a reduction in the amount of ODF1 compared with fertile controls. Stress treatment of sperm deficient in ODF1 caused the head to decapitate, suggesting why these gametes fail to initiate fertilization. Interestingly, electron micrographs of ODF1-deficient spermatozoa revealed an abnormal connecting piece, indicating several developmental defects with both the implantation plate and the thin laminated fibers. In some cases, the implantation plate appeared to be reduced in size or was overburdened by granular material near the connecting piece. Hence, a strong reduction ODF1 is a marker of idiopathic male infertility and a potential driver of this condition.

Cationic acrylate oligomers comprising amino acid mimic moieties demonstrate improved antibacterial killing efficiency

Cu(0)-mediated polymerization was employed to synthesize a library of structurally varied cationic polymers and their application as antibacterial peptide mimics was assessed. Eight platform polymers were first synthesized with low degrees of polymerization (DP) using (2-Boc-amino)ethyl acrylate as the monomer and either ethyl α-bromoisobutyrate or dodecyl 2-bromoisobutyrate as the initiator (thus providing hydrocarbon chain termini of C2 or C12, respectively). A two-step modification strategy was then employed to generate the final sixteen-member polymer library. Specifically, an initial deprotection was employed to reveal the primary amine cationic polymers, followed by guanylation. The biocidal activity of these cationic polymers was assessed against various strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. Polymers having a short segment of guanidine units and a C12 hydrophobic terminus were shown to provide the broadest antimicrobial activity against the panel of isolates studied, with MIC values approaching those for Gram-positive targeting antibacterial peptides: daptomycin and vancomycin. The C12-terminated guanidine functional polymers were assayed against human red blood cells, and a concomitant increase in haemolysis was observed with decreasing DP. Cytotoxicity was tested against HEK293 and HepG2 cells, with the lowest DP C12-terminated polymer exhibiting minimal toxicity over the concentrations examined, except at the highest concentration. Membrane disruption was identified as the most probable mechanism of bacteria cell killing, as elucidated by membrane permeability testing against E. coli.

Aminoglycoside concentrations required for synergy with carbapenems against Pseudomonas aeruginosa determined via mechanistic studies and modeling

ABSTRACT This study aimed to systematically identify the aminoglycoside concentrations required for synergy with a carbapenem and characterize the permeabilizing effect of aminoglycosides on the outer membrane of Pseudomonas aeruginosa. Monotherapies and combinations of four aminoglycosides and three carbapenems were studied for activity against P. aeruginosa strain AH298-GFP in 48-h static-concentration time-kill studies (SCTK) (inoculum: 107.6 CFU/ml). The outer membrane-permeabilizing effect of tobramycin alone and in combination with imipenem was characterized via electron microscopy, confocal imaging, and the nitrocefin assay. A mechanism-based model (MBM) was developed to simultaneously describe the time course of bacterial killing and prevention of regrowth by imipenem combined with each of the four aminoglycosides. Notably, 0.25 mg/liter of tobramycin, which was inactive in monotherapy, achieved synergy (i.e., ≥2-log10 more killing than the most active monotherapy at 24 h) combined with imipenem. Electron micrographs, confocal image analyses, and the nitrocefin uptake data showed distinct outer membrane damage by tobramycin, which was more extensive for the combination with imipenem. The MBM indicated that aminoglycosides enhanced the imipenem target site concentration up to 4.27-fold. Tobramycin was the most potent aminoglycoside to permeabilize the outer membrane; tobramycin (0.216 mg/liter), gentamicin (0.739 mg/liter), amikacin (1.70 mg/liter), or streptomycin (5.19 mg/liter) was required for half-maximal permeabilization. In summary, our SCTK, mechanistic studies and MBM indicated that tobramycin was highly synergistic and displayed the maximum outer membrane disruption potential among the tested aminoglycosides. These findings support the optimization of highly promising antibiotic combination dosage regimens for critically ill patients.

A Novel Chemical Biology Approach for Mapping of Polymyxin Lipopeptide Antibody Binding Epitopes

Polymyxins B and E (i.e., colistin) are a family of naturally occurring lipopeptide antibiotics that are our last line of defense against multidrug resistant (MDR) Gram-negative pathogens. Unfortunately, nephrotoxicity is a dose-limiting factor for polymyxins that limits their clinical utility. Our recent studies demonstrate that polymyxin-induced nephrotoxicity is a result of their extensive accumulation in renal tubular cells. The design and development of safer, novel polymyxin lipopeptides is hampered by our limited understanding of their complex structure-nephrotoxicity relationships. This is the first study to employ a novel targeted chemical biology approach to map the polymyxin recognition epitope of a commercially available polymyxin mAb and demonstrate its utility for mapping the kidney distribution of a novel, less nephrotoxic polymyxin lipopeptide. Eighteen novel polymyxin lipopeptide analogues were synthesized with modifications in the polymyxin core domains, namely, the N-terminal fatty acyl region, tripeptide linear segment, and cyclic heptapeptide. Surface plasmon resonance epitope mapping revealed that the monoclonal antibody (mAb) recognition epitope consisted of the hydrophobic domain (N-terminal fatty acyl and position 6/7) and diaminobutyric acid (Dab) residues at positions 3, 5, 8, and 9 of the polymyxin molecule. Structural diversity within the hydrophobic domains and Dab 3 position are tolerated. Enlightened with an understating of the structure-binding relationships between the polymyxin mAb and the core polymyxin scaffold, we can now rationally employ the mAb to probe the kidney distribution of novel polymyxin lipopeptides. This information will be vital in the design of novel, safer polymyxins through chemical tailoring of the core scaffold and exploration of the elusive/complex polymyxin structure-nephrotoxicity relationships.

Development of HPLC and LC-MS/MS methods for the analysis of ivacaftor, its major metabolites and lumacaftor in plasma and sputum of cystic fibrosis patients treated with ORKAMBI or KALYDECO.

ORKAMBI (ivacaftor-lumacaftor [LUMA]) and KALYDECO (ivacaftor; IVA) are two new breakthrough cystic fibrosis (CF) drugs that directly modulate the activity and trafficking of the defective CFTR underlying the CF disease state. Currently, no therapeutic drug monitoring assays exist for these very expensive, albeit, important drugs. In this study, for the first time HPLC and LC-MS methods were developed and validated for rapid detection and quantification of IVA and its major metabolites hydroxymethyl-IVA M1 (active) and IVA-carboxylate M6 (inactive); and LUMA in the plasma and sputum of CF patients. With a mobile phase consisting of acetonitrile/water:0.1% formic acid (60:40v/v) at a flow rate of 1mL/min, a linear correlation was observed over a concentration range from 0.01 to 10μg/mL in human plasma (IVA R2>0.999, IVA M1 R2>0.9961, IVA M6 R2>0.9898, LUMA R2>0.9954). The assay was successfully utilized to quantify the concentration of LUMA, IVA, M1 and M6 in the plasma and sputum of CF patients undergoing therapy with KALYDECO (IVA 150mg/q12h) or ORKAMBI (200mg/q12h LUMA-125mg/q12h IVA). The KALYDECO patient exhibited an IVA plasma concentration of 0.97μg/mL at 2.5h post dosage. M1 and M6 plasma concentrations were 0.50μg/mL and 0.16μg/mL, respectively. Surprisingly, the ORKAMBI patient displayed very low plasma concentrations of IVA (0.06μg/mL) and M1 (0.07μg/mL). The M6 concentrations (0.15μg/mL) were comparable to those of the KALYDECO patient. However, we observed a relatively high plasma concentration of LUMA (4.42μg/mL). This reliable and novel method offers a simple and sensitive approach for therapeutic drug monitoring of KALYDECO and ORKAMBI in plasma and sputum. The introduction of the assay into the clinical setting will facilitate pharmacokinetics/pharmacodynamic analysis and assist clinicians to develop more cost effective and efficacious dosage regimens for these breakthrough CF drugs.

A traceless reversible polymeric colistin prodrug to combat multidrug-resistant (MDR) gram-negative bacteria

ABSTRACT Colistin methanesulfonate (CMS) is the only prodrug of colistin available for clinical use for the treatment of infections caused by multidrug‐resistant (MDR) Gram‐negative bacteria. Owing to its slow and variable release, an alternative is urgently required to improve effectiveness. Herein we describe a PEGylated colistin prodrug whereby the PEG is attached via a cleavable linker (col‐aaPEG) introducing an acetic acid terminated poly (ethylene glycol) methyl ether (aaPEG) onto the Thr residue of colistin. Due to the labile ester containing link, this prodrug is converted back into active colistin in vitro within 24 h. Compared to CMS, it showed a similar or better antimicrobial performance against two MDR isolates of Pseudomonas aeruginosa and Acinetobacter baumannii through in vitro disk diffusion, broth dilution and time‐kill studies. In a mouse infection model, col‐aaPEG displayed acceptable bacterial killing against P. aeruginosa ATCC 27853 and no nephrotoxicity was found after systemic administration, suggesting it to be a potential alternative for CMS. Graphical abstract Figure. No caption available.

Molecular Characterisation of the Haemagglutinin Glycan-Binding Specificity of Egg-Adapted Vaccine Strains of the Pandemic 2009 H1N1 Swine Influenza A Virus

The haemagglutinin (HA) glycan binding selectivity of H1N1 influenza viruses is an important determinant for the host range of the virus and egg-adaption during vaccine production. This study integrates glycan binding data with structure-recognition models to examine the impact of the K123N, D225G and Q226R mutations (as seen in the HA of vaccine strains of the pandemic 2009 H1N1 swine influenza A virus). The glycan-binding selectivity of three A/California/07/09 vaccine production strains, and purified recombinant A/California/07/09 HAs harboring these mutations was examined via a solid-phase ELISA assay. Wild-type A/California/07/09 recombinant HA bound specifically to α2,6-linked sialyl-glycans, with no affinity for the α2,3-linked sialyl-glycans in the array. In contrast, the vaccine virus strains and recombinant HA harboring the Q226R HA mutation displayed a comparable pattern of highly specific binding to α2,3-linked sialyl-glycans, with a negligible affinity for α2,6-linked sialyl-glycans. The D225G A/California/07/09 recombinant HA displayed an enhanced binding affinity for both α2,6- and α2,3-linked sialyl-glycans in the array. Notably its α2,6-glycan affinity was generally higher compared to its α2,3-glycan affinity, which may explain why the double mutant was not naturally selected during egg-adaption of the virus. The K123N mutation which introduces a glycosylation site proximal to the receptor binding site, did not impact the α2,3/α2,6 glycan selectivity, however, it lowered the overall glycan binding affinity of the HA; suggesting glycosylation may interfere with receptor binding. Docking models and ‘per residues’ scoring were employed to provide a structure-recognition rational for the experimental glycan binding data. Collectively, the glycan binding data inform future vaccine design strategies to introduce the D225G or Q226R amino acid substitutions into recombinant H1N1 viruses.

Rapamycin confers neuroprotection against colistin-induced oxidative stress, mitochondria dysfunction and apoptosis through the activation of autophagy and mTOR/Akt/CREB signaling pathways

Our previous studies showed that colistin-induced neurotoxicity involves apoptosis and oxidative damage. The present study demonstrates a neuroprotective effect of rapamycin against colistin-induced neurotoxicity in vitro and in vivo. In a mouse model, colistin treatment (18 mg/kg/d; 14 days) produced marked neuronal mitochondria damage in the cerebral cortex and increased activation of caspase-9 and -3. Rapamycin cotreatment (2.5 mg/kg/d) effectively reduced this neurotoxic effect. In an in vitro mouse neuroblastoma-2a (N2a) cell culture model, rapamycin pretreatment (500 nM) reduced colistin (200 μM) induced cell death from ∼50% to 72%. Moreover, rapamycin showed a marked neuroprotective effect in the N2a cells by decreasing intracellular reactive oxygen species (ROS) production and by up-regulating the activities of the anti-ROS enzymes superoxide dismutase and catalase and recovering glutathione (GSH) levels to normal. Moreover, rapamycin pretreatment protected against colistin-induced mitochondrial dysfunction, caspase activation, and subsequent apoptosis by up-regulating autophagy and activating the Akt/CREB, NGF, and Nrf2 pathways, while inhibiting p53 signaling. Taken together, this is the first study to demonstrate that rapamycin protects against colistin-induced neurotoxicity by activating autophagy, inhibiting oxidative stress, mitochondria dysfunction, and apoptosis. Our data highlight that regulating autophagy to rescue neurons from apoptosis may become a new targeted therapy to relieve the adverse neurotoxic effects associated with colistin therapy.