Dispersive liquid-liquid microextraction,an effective tool for the determination of synthetic cannabinoids in oral fluid by liquid chromatography–tandem mass spectrometry

In the present work, dispersive liquid-liquid microextraction (DLLME) was used to extract six synthetic cannabinoids (JWH-018, JWH-019, JWH-073, JWH-200, or WIN 55,225, JWH-250, and AM-694) from oral fluids. A rapid baseline separation of the analytes was achieved on a bidentate octadecyl silica hydride phase (Cogent Bidentate C₁₈; 4.6 mm × 250 mm, 4 μm) maintained at 37 °C, by eluting in isocratic conditions (water:acetonitrile (25:75, V/V)). Detection was performed using positive electrospray ionization–tandem mass spectrometry. The parameters affecting DLLME (pH and ionic strength of the aqueous phase, type and volume of the extractant and dispersive solvent, vortex and centrifugation time) were optimized for maximizing yields. In particular, using 0.5 mL of oral fluid, acetonitrile (1 mL), was identified as the best option, both as a solvent to precipitate proteins and as a dispersing solvent in the DLLME procedure. To select an extraction solvent, a low transition temperature mixture (LTTM; composed of sesamol and chlorine chloride with a molar ratio of 1:3) and dichloromethane were compared; the latter (100 μL) was proved to be a better extractant, with recoveries ranging from 73% to 101 % by vortexing for 2 min. The method was validated according to the guidelines of Food and Drug Administration bioanalytical methods: intra-day and inter-day precisions ranged between 4 % and 18 % depending on the spike level and analyte; limits of detection spanned from 2 to 18 ng/mL; matrix-matched calibration curves were characterized by determination coefficients greater than 0.9914. Finally, the extraction procedure was compared with previous methods and with innovative techniques, presenting superior reliability, rapidity, simplicity, inexpensiveness, and efficiency.     

Recombinant cathepsin S propeptide attenuates cell invasion by inhibition of cathepsin L-like proteases in tumor microenvironment

Human cathepsin L along with cathepsin S, K, and V are collectively known as cathepsin L-like proteases due to their high homology. The overexpression and aberrant activity of each of these proteases has been implicated in tumorigenesis. These proteases contain propeptide domains that can potently inhibit both their cognate protease and other proteases within the cathepsin L-like subfamily. In this investigation, we have produced the cathepsin S propeptide recombinantly and have shown that it is a potent inhibitor of the peptidolytic, elastinolytic, and gelatinolytic activities of the cathepsin L-like proteases. In addition, we show that this peptide is capable of significantly attenuating tumor cell invasion in a panel of human cancer cell lines. Furthermore, fusion of an IgG Fc-domain to the COOH terminus of the propeptide resulted in a chimeric protein with significantly enhanced ability to block tumor cell invasion. This Fc fusion protein exhibited enhanced stability in cell-based assays in comparison with the unmodified propeptide species. This approach for the combined inhibition of the cathepsin L-like proteases may prove useful for the further study in cancer and other conditions where their aberrant activity has been implicated. Furthermore, this strategy for simultaneous inhibition of multiple cysteine cathepsins may represent the basis for novel therapeutics to attenuate tumorigenesis.     

Maintenance immunotherapy in recurrent or metastatic squamous cell carcinoma of the head and neck

The purpose of this study was to determine the efficacy and safety of a maintenance immunotherapy regimen administered to patients with recurrent/metastatic squamous cell carcinoma of the head and neck (RMHN) who showed clinical benefit from docetaxel, ifosfamide, and cisplatin chemotherapy (DIP). Every 4 weeks, patients with RMHN received 60 mg/m docetaxel on day 1, and 1200 mg/m ifosfamide and 20 mg/m cisplatin on days 1 to 4. Low-dose subcutaneous interleukin-2 and oral 13-cis-retinoic acid were administered as maintenance immunotherapy to patients who showed a clinical benefit (complete or partial response, disease stability). The primary end point was response; secondary end points were progression-free survival, overall survival, toxicity, and evaluations of lymphocytes, natural killer cells, and serum vascular endothelial growth factor (VEGF). After a median follow-up of 22 months, 263 courses of chemotherapy were administered to the 54 patients. The overall response rate was 59%. Forty-two patients (78%) had a clinical benefit and received 185 courses of maintenance immunotherapy. Median progression-free survival and overall survival were 11.1 and 21.8 months, respectively. Statistically significant, progressive increases in lymphocytes and natural killer cells and a decrease in VEGF were observed in patients treated with maintenance immunotherapy. The toxicity was relatively well tolerated and caused no death. Outpatient administration of DIP, followed by low-dose interleukin-2 and 13-cis-retinoic acid, was generally well tolerated and showed promising activity against RMHN. Longitudinal changes in lymphocytes, natural killer cells, and VEGF might be useful biomarkers for response and survival.     

Blinding techniques in randomized controlled trials of laser therapy: an overview and possible solution

Low-level laser therapy has evidence accumulating about its effectiveness in a variety of medical conditions. We reviewed 51 double blind randomized controlled trials (RCTs) of laser treatment. Analysis revealed 58% of trials showed benefit of laser over placebo. However, less than 5% of the trials had addressed beam disguise or allocation concealment in the laser machines used. Many of the trials used blinding methods that rely on staff cooperation and are therefore open to interference or bias. This indicates significant deficiencies in laser trial methodology. We report the development and preliminary testing of a novel laser machine that can blind both patient and operator to treatment allocation without staff participation. The new laser machine combines sealed preset and non-bypassable randomization codes, decoy lights and sound, and a conical perspex tip to overcome laser diode glow detection.     

Use of carbon monoxide as a therapeutic agent: promises and challenges

As a by-product of heme catabolism by the heme oxygenase system, carbon monoxide (CO) has been neglected for many years, and only recently has its role as an essential signaling molecule been appreciated. In the past decade, the use of CO gas in pre-clinical experimental models of disease has produced some remarkable data indicating that its therapeutic delivery to mammals could alleviate inflammatory processes and cardiovascular disorders. However, the inherent toxic nature of CO cannot be ignored, knowing that inhalation of uncontrolled amounts of this gas can ultimately lead to serious systemic complications and neuronal derangements. From a clinical perspective, a key question is whether a safe and therapeutically effective threshold of CO can be reached locally in organs and tissues without delivering potentially toxic amounts through the lung. The advent of CO-releasing molecules (CO-RMs), a group of compounds capable of carrying and liberating controlled quantities of CO in cellular systems, appears a plausible alternative in the attempt to overcome the limitations of CO gas. Although in its infancy and far from being used for clinical applications, the CO-RMs technology is supported by very encouraging biological results and reflected by the chemical versatility of these compounds and their endless potential to be transformed into CO-based pharmaceuticals. This article is discussed in the editorial available at: .