Aqueous Humour Ofloxacin Concentration after Topical Instillation in Patients with Dry Eye Disease.

Background and Objectives: A prospective, randomized clinical trial was conducted to evaluate the concentration of ofloxacin in the aqueous humour (AqH) of patients suffering from dry eye disease (DED) after topical instillation. Materials and Methods: Ninety-one (91) cataract patients scheduled for phacoemulsification were categorized into three groups according to DED severity. Group I (n = 17) was comprised of subjects without DED, patients in group II (n = 37) were evaluated as having non-severe DED, while group III (n = 37) consisted of patients suffering from severe DED. Preoperatively, patients received 4 drops of 0.3% of ofloxacin at 15 min intervals. One hour after the last instillation, aqueous samples were collected intraoperatively. Results: The median AqH concentration of ofloxacin in group I was 199.9 ng/mL (range 92.2−442.8 ng/mL), while in group II it was 530.5 ng/mL (range 283.7−1004.9 ng/mL), and 719.2 ng/mL (range 358.0−1512.4 ng/mL) in Group III, p < 0.001 (Kruskal-Wallis tests). Pairwise tests (two-tailed with Bonferroni corrections) between groups resulted in a p-value of 0.001 when group II was compared to group I and group III was compared to group I, and a p-value of 0.020 when group II was compared to group III. The severity of DED, across groups I, II, and III, and the levels of ofloxacin revealed a strong positive correlation (r = 0.639, p < 0.001). Conclusions: Ofloxacin concentration in the AqH after topical drop instillation may be affected by the degree of ocular surface inflammation in patients suffering from DED.

In vivo quantification and pharmacokinetic studies of cotinine in mice after smoke exposure by LC-MS/MS.

A sensitive analytical method was developed and validated for the quantification of cotinine in mouse plasma after exposure to smoke of 0.5, 1.0, and 1.5 commercially available cigarettes, using liquid chromatography tandem mass spectrometry. The method was validated over a linear concentration range of 0.075-20.0 ng/mL with the R2 value being higher than 0.99. Both the precision (coefficient of variation; %) and accuracy (relative error; %) were within acceptable criteria of <15%. The lower limit of quantification (LLOQ) for cotinine was 0.075 ng/mL with sufficient specificity, accuracy, and precision. Following exposure to 0.5, 1.0, and 1.5 cigarette smoke, it was observed that the AUC and the Cmax increased linearly as the doses increased. The pharmacokinetics of cotinine was found linear for the range of 0.5-1.5 commercial cigarette smoke. The quantification of the concentration of cotinine in mouse plasma after smoke exposure will facilitate future behavioral and toxicological experiments in animals and may prove useful in predicting cotinine levels in humans during smoking.

Development and validation of analytical methodologies for the quantification of PCK3145 and PEG-PCK3145 in mice.

PCK3145 is an anti-metastatic synthetic peptide against prostate cancer. The objective of the study is to develop and validate novel and sensitive methods for the determination of PCK3145 and Pegylated PCK3145 in mouse plasma. An LC-MS/MS method was developed and validated for the determination of PCK3145 giving high sensitivity and linearity in the range of 0.125-4.0 µg/mL. PCK3145 characterised by short half-life, therefore, it was conjugated with the poly ethylene glycol (PEG). However, LC-MS/MS has been more difficult to apply for the quantitative analysis of PEGylated peptides due to the large size. A UHPLC-UV method was developed and validated for the determination of PEG-PCK3145, with linearity of 0.05-2.0 mg/mL. In order to further improve the sensitivity for the detection of PEG-PCK3145, an indirect ELISA method was used. It was found that this method was capable of detecting PCK3145 through the quantification of PEG with excellent sensitivity found at 0.132 ng/mL. The in vitro proteolytic stability of PCK3145 and PEG-PCK3145 in mouse plasma and whole blood was studied by LC-MS/MS and UHPLC, respectively. The LC-MS/MS and ELISA methods can be applied for monitoring levels of PCK3145 in mouse plasma for in vivo pharmacokinetic and bioavailability animal studies.

Development and validation of a UHPLC-UV method for the determination of a prostate secretory protein 94-derived synthetic peptide (PCK3145) in human plasma and assessment of its stability in human plasma.

PCK3145 is a synthetic peptide, derived from the Prostate Secreted Protein 94 (PSP94), with promising in vitro and animal in vivo results in prostate cancer. The aim of the present study was to develop and validate a fast and robust ultra-high-performance liquid chromatography with ultraviolet detection for the determination of PCK3145 in human plasma which would be suitable for the assessment of PCK3145 stability to proteolytic degradation. Following protein precipitation, chromatographic separation was carried out on an Aeris Peptide C18 column with mobile phase consisting of acetonitrile-water at a flow-rate of 0.50 mL/min. The calibration curve was linear over the range 0.50-20.00 µg/mL. Intra- and inter-day percentage relative standard deviation and relative error were ≤10%. The limit of detection and the lower limit of quantification were 0.15 and 0.50 µg/mL, respectively. Recovery of PCK3145 from human plasma was ≥96%. The peptide presented high stability in whole blood and in human plasma (>98% intact peptide after 24 h incubation at 37°C in human plasma), which represents a distinctive advantage in the therapeutic use of the compound. This is the first validated UHPLC method for the determination of PCK3145 reported, and it was successfully applied in the study of the proteolytic stability of PCK3145 in human plasma ex vivo. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of Exposure to Tobacco Cigarette, Electronic Cigarette and Heated Tobacco Product on Adipocyte Survival and Differentiation In Vitro.

Cigarette smoking (CS) causes significant morbidity worldwide, attributed to the numerous toxicants generated by tobacco combustion. Electronic cigarettes (ECIG) and heated tobacco products (HTP) are considered alternative smoking/vaping products that deliver nicotine through an inhaled aerosol and emit fewer harmful constituents than CS. However, their long-term impacts on human health are not well established. Nicotine exposure has been linked to lipolysis and body weight loss, while smoking has been associated with insulin resistance and hyperinsulinemia. Enhanced function of beige (thermogenic) adipocytes has been proposed as a means to reduce obesity and metabolic disorders. In this study, we compared the effect of extract-enriched media via exposure of culture medium to CS, HTP aerosol, and ECIG aerosol on the viability and the differentiation of 3T3-L1 pre-adipocytes to beige adipocytes. Only CS extract caused a decrease in cell viability in a dose- and time-dependent manner. Furthermore, relative lipid accumulation and expression levels of the adipocyte markers Pgc-1α, Ppar-γ and Resistin were significantly decreased in cells exposed to CS extract. Our results demonstrate that CS extract, in contrast to HTP and ECIG extracts, significantly impairs differentiation of pre-adipocytes to beige adipocytes and may therefore impact significantly adipose tissue metabolic function.

Development and validation of simple step protein precipitation UHPLC-MS/MS methods for quantitation of temozolomide in cancer patient plasma samples.

Temozolomide (TEMODAL™) (TMZ) is an antineoplastic agent that is primarily used for the treatment of glioblastoma and anaplastic gliomas, two aggressive forms of brain cancer. Due to the poor prognosis of brain tumour patients, there is an increasing body of research into improving the stability and delivery of TMZ past the blood brain barrier using carrier molecules. These require accurate determination of TMZ levels for biodistribution and pharmacokinetic evaluation. Unfortunately, current methodologies for the determination of TMZ in human plasma suffer from low reproducibility, recovery, sensitivity or cost ineffective procedures associated with extensive sample cleaning. To surpass these disadvantages, we developed two bioanalytical methods with high sensitivity and excellent recovery for the determination of TMZ in human plasma at minimum cost. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used and both methods were validated under US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) guidelines. The two methods had minor differences in the sample pre-treatment and each method was developed and applied in separate laboratories. Theophylline was selected as internal standard (IS). Calibration curves were linear over the range of 10-500 ng/mL with extraction recovery ranging from 77.3 to 97.3% while all validation parameters met the acceptance criteria and proved the methods' reliability. The validated methods were successfully applied to plasma samples donated from cancer patient following treatment with temozolomide.

Development and validation of analytical methodology for the quantification of aldehydes in e-cigarette aerosols using UHPLC-UV.

Aldehydes are produced in e-cigarette aerosols, as a result of the thermal decomposition of vegetable glycerin, propylene glycerol and flavorings in the atomizer. These aldehydes were collected with derivatization into 2,4-dinitrophenylhydrazine using impinger trapping. A new methodology for simultaneous quantitative analysis of aldehydes in base liquids was developed and validated. Chromatographic separation was carried out on a Jupiter Proteo 90A column, with the mobile phase consisting of 0.1% formic acid in water and acetonitrile, at a flow rate of 0.4 mL/min. Linearity was demonstrated over the range of concentrations 0.025-10 µg/mL, with correlation of determination ≥ 0.999. Intra- and inter-day % relative standard deviation and relative error were ≤ 10%. The lower limit of detection and quantification were 0.008 and 0.025 µg/mL, respectively. This method was further used for the quantification of aldehydes emitted by different base liquids. It has been shown that vegetable glycerin produces the highest percentage of aldehydes after thermal decomposition compared to propylene glycerol. We propose that it can be used for future e-liquids emissions studies.

Development of a novel conjugatable sunitinib analogue validated through in vitro and in vivo preclinical settings.

Sunitinib is an oral FDA/EMEA approved multi-targeted tyrosine kinase inhibitor. It possesses anti-angiogenic and antitumor activity against a variety of advanced solid tumors. However, its chemical core does not allow a potential linkage to tumor-homing elements that could eventually enhance its potency. Therefore, a novel linkable sunitinib derivative, designated SB1, was rationally designed and synthesized. The pharmaceutical profile of SB1 was explored both in vitro and in vivo. Mass spectrometry and NMR spectroscopy were utilized for characterization, while MTT assays and LC-MS/MS validated protocols were used to explore its antiproliferative effect and stability, respectively. Cytotoxicity evaluation in three glioma cells showed that SB1 preserved the antiproliferative effect of sunitinib. SB1 was stable in vitro after 24 h incubation in mouse plasma, while both agents exhibited bioequivalent pharmacokinetic characteristics after i.v. administration in Balb/c mice. To evaluate the levels of SB1 in mouse plasma, a novel analytical method was developed and validated in accordance to the US FDA and the EU EMA guidelines. We formulated a novel linkable sunitinib analog exhibiting similar antiproliferative and apoptotic properties with native sunitinib in glioma cell lines. Both SB1 and native sunitinib showed identical in vitro stability in mouse plasma and pharmacokinetics after i.v. administration in Balb/c mice.