optimized-lc-msms-method-for-high-throughput-analysis-clinical

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Monash University

Thermosensitive macroporous scaffolds of poly(N-isopropylacrylamide) (polyNIPA) loaded with chitosan/bemiparin nanoparticles are prepared by the free radical polymerization in cryogenic conditions. Chitosan/bemiparin nanoparticles of 102 ± 6.5 nm diameter are prepared by complex coacervation and loaded into polyNIPA cryogels. SEM image reveal the highly porous structure of cryogels and the integration of nanoparticles into the macroporous system. Volume phase transition temperature (VPT) and total freezing water content of cryogels are established by differential scanning calorimetry, and their porosity is determined by image-NMR. Swelling of cryogels (above and below the VPT) is highly dependent on nanoparticles concentration. In vitro release profile of bemiparin from cryogel is highly modulated by the presence of chitosan. Bemiparin released from nanoparticles preserves its biological activity, as shown by the BaF32 cell proliferation assay. Cryogels are not cytotoxic for the human fibroblast cells and present excellent properties for application on tissue engineering and controlled release of heparin.

Thermosensitive macroporous cryogels functionalized with bioactive chitosan/bemiparin nanoparticles.

The article deals with the design, preparation, and evaluation of a new bilayered dressing for application in the healing of compromised wounds. The system is based on the sequential release of two complementary bioactive components to enhance the activation of the regeneration of dermal tissue. The internal layer is a highly hydrophilic and biodegradable film of gelatin and hyaluronic acid (HG), crosslinked with the natural compound genipin, which reacts with the amine groups of gelatin. This film is loaded with the proangiogenic, anti-inflammatory, and antibacterial peptide, proadrenomedullin N-terminal 20 peptide (PAMP), that is released slowly in the wound site. The external layer, more stable and less hydrophilic, is constituted by a biodegradable polyurethane derived from poly(caprolactone) and pluronic L61. This layer is loaded with resorbable nanoparticles of bemiparin (a fractionated low molecular weight heparin), which promotes the activation of growth factors, FGF and VEGF, and provides a good biomechanical stability and controlled permeability of the bilayered dressing. Experiments carried out in mice demonstrate the excellent angiogenic effect of the HG film in the dermal tissue. Application of the bilayered dressing in the wound healing rabbit ear model shows an improved cicatrization of the wound in both ischemic and non-ischemic defects, favoring epithelialization and reducing noticeably the contraction and the inflammation.

Bioactive bilayered dressing for compromised epidermal tissue regeneration with sequential activity of complementary agents.

Natural products using plants have received considerable attention because of their potential to treat various diseases. Arrabidaea chica (Humb. & Bonpl.) B. Verlot is a native tropical American vine with healing properties employed in folk medicine for wound healing, inflammation, and gastrointestinal colic. Applying nanotechnology to plant extracts has revealed an advantageous strategy for herbal drugs considering the numerous features that nanostructured systems offer, including solubility, bioavailability, and pharmacological activity enhancement. The present study reports the preparation and characterization of chitosan-sodium tripolyphosphate nanoparticles (NPs) charged with A. chica standardized extract (AcE). Particle size and zeta potential were measured using a Zetasizer Nano ZS. The NP morphological characteristics were observed using scanning electron microscopy. Our studies indicated that the chitosan/sodium tripolyphosphate mass ratio of 5 and volume ratio of 10 were found to be the best condition to achieve the lowest NP sizes, with an average hydrodynamic diameter of 150±13 nm and a zeta potential of +45±2 mV. Particle size decreased with AcE addition (60±10.2 nm), suggesting an interaction between the extract's composition and polymers. The NP biocompatibility was evaluated using human skin fibroblasts. AcE-NP demonstrated capability of maintaining cell viability at the lowest concentrations tested, stimulating cell proliferation at higher concentrations. Antiulcerogenic activity of AcE-NP was also evaluated with an acute gastric ulcer experimental model induced by ethanol and indomethacin. NPs loaded with A. chica extract reduced the ulcerative lesion index using lower doses compared with the free extract, suggesting that extract encapsulation in chitosan NPs allowed for a dose reduction for a gastroprotective effect. The AcE encapsulation offers an approach for further application of the A. chica extract that could be considered a potential candidate for ulcer-healing pharmaceutical systems.

Chitosan-tripolyphosphate nanoparticles as Arrabidaea chica standardized extract carrier: synthesis, characterization, biocompatibility, and antiulcerogenic activity.

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 R(2)>0.999, IVA M1 R(2)>0.9961, IVA M6 R(2)>0.9898, LUMA R(2)>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.

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.

Novel therapeutic approaches are urgently needed to combat nosocomial infections caused by extremely drug-resistant (XDR) "superbugs." This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with selective estrogen receptor modulators (SERMs) against problematic Gram-negative pathogens. In vitro synergistic antibacterial activity of polymyxin B and the SERMs tamoxifen, raloxifene, and toremifene was assessed using the microdilution checkerboard and static time-kill assays against a panel of Gram-negative isolates. Polymyxin B and the SERMs were ineffective when used as monotherapy against polymyxin-resistant minimum inhibitory concentration ([MIC] ≥8 mg/L) Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. However, when used in combination, clinically relevant concentrations of polymyxin B and SERMs displayed synergistic killing against the polymyxin-resistant P. aeruginosa, K. pneumoniae, and A. baumannii isolates as demonstrated by a ≥2-3 log10 decrease in bacterial count (CFU/ml) after 24 hours. The combination of polymyxin B with toremifene demonstrated very potent antibacterial activity against P. aeruginosa biofilms in an artificial sputum media assay. Moreover, polymyxin B combined with toremifene synergistically induced cytosolic green fluorescence protein release, cytoplasmic membrane depolarization, permeabilizing activity in a nitrocefin assay, and an increase of cellular reactive oxygen species from P. aeruginosa cells. In addition, scanning and transmission electron micrographs showed that polymyxin B in combination with toremifene causes distinctive damage to the outer membrane of P. aeruginosa cells, compared with treatments with each compound per se. In conclusion, the combination of polymyxin B and SERMs illustrated a synergistic activity against XDR Gram-negative pathogens, including highly polymyxin-resistant P. aeruginosa isolates, and represents a novel combination therapy strategy for the treatment of infections because of problematic XDR Gram-negative pathogens.

From Breast Cancer to Antimicrobial: Combating Extremely Resistant Gram-Negative "Superbugs" Using Novel Combinations of Polymyxin B with Selective Estrogen Receptor Modulators.

The emergence of antimicrobial resistance of Gram-negative pathogens has become a worldwide crisis. The status quo for combating resistance is to employ synergistic combinations of antibiotics. Faced with this fast-approaching post-antibiotic era, it is critical that we devise strategies to prolong and maximize the clinical efficacy of existing antibiotics. Unfortunately, reports of extremely drug-resistant (XDR) Gram-negative pathogens have become more common. Combining antibiotics such as polymyxin B or the broad-spectrum tetracycline and minocycline with various FDA-approved non-antibiotic drugs have emerged as a novel combination strategy against otherwise untreatable XDR pathogens. This review surveys the available literature on the potential benefits of employing antibiotic-non-antibiotic drug combination therapy. The apex of this review highlights the clinical utility of this novel therapeutic strategy for combating infections caused by 'superbugs'.

Article	Year
Thermosensitive macroporous cryogels functionalized with bioactive chitosan/bemiparin nanoparticles. Macromolecular bioscience\| PubMed ID: 23956200	2013
Bioactive bilayered dressing for compromised epidermal tissue regeneration with sequential activity of complementary agents. Acta biomaterialia\| PubMed ID: 26022967	2015
Chitosan-tripolyphosphate nanoparticles as Arrabidaea chica standardized extract carrier: synthesis, characterization, biocompatibility, and antiulcerogenic activity. International journal of nanomedicine\| PubMed ID: 26089666	2015
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... Journal of chromatography. B, Analytical technologies in the biomedical and life sciences\| PubMed ID: 27792891	2016
From Breast Cancer to Antimicrobial: Combating Extremely Resistant Gram-Negative "Superbugs" Using Novel Combinations of Polymyxin B with Selective Estrogen Receptor Modulators. Microbial drug resistance (Larchmont, N.Y.)\| PubMed ID: 27935770	2017
Antibiotic-non-antibiotic combinations for combating extremely drug-resistant Gram-negative 'superbugs'. Essays in biochemistry\| PubMed ID: 28258235	2017

Felisa Reyes-Ortega

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