Development of Highly Sensitive Fluorescent Sensors for Separation-Free Detection and Quantitation Systems of Pepsin Enzyme Applying a Structure-Guided Approach.

Two fluorescent molecularly imprinted polymers (MIPs) were developed for pepsin enzyme utilising fluorescein and rhodamine b. The main difference between both dyes is the presence of two (diethylamino) groups in the structure of rhodamine b. Consequently, we wanted to investigate the effect of these functional groups on the selectivity and sensitivity of the resulting MIPs. Therefore, two silica-based MIPs for pepsin enzyme were developed using 3-aminopropyltriethoxysilane as a functional monomer and tetraethyl orthosilicate as a crosslinker to achieve a one-pot synthesis. Results of our study revealed that rhodamine b dyed MIPs (RMIPs) showed stronger binding, indicated by a higher binding capacity value of 256 mg g-1 compared to 217 mg g-1 for fluorescein dyed MIPs (FMIPs). Moreover, RMIPs showed superior sensitivity in the detection and quantitation of pepsin with a linear range from 0.28 to 42.85 µmol L-1 and a limit of detection (LOD) as low as 0.11 µmol L-1. In contrast, FMIPs covered a narrower range from 0.71 to 35.71 µmol L-1, and the LOD value reached 0.34 µmol L-1, which is three times less sensitive than RMIPs. Finally, the developed FMIPs and RMIPs were applied to a separation-free quantification system for pepsin in saliva samples without interference from any cross-reactors.

Anti-cancer drug waste disposal practices and wastewater management in hospitals: A Lebanese survey.

INTRODUCTION: To achieve continuous environmental sustainability and protect the population's health, healthcare waste (in liquid or solid form) needs appropriate management and suitable treatment strategies before its final disposal in the environment in order to reduce its adverse impacts. This study aims to identify disparities in the waste management of anti-cancer drugs and the wastewater generated in Lebanese hospitals. METHODS: Three questionnaires were designed to evaluate the level of knowledge, awareness and experience of hospital personnel regardless of their job levels. Data was collected in December 2019 from three departments of each participating hospital: pharmacy, oncology and maintenance departments. A descriptive analysis was conducted to summarise the survey results. RESULTS: The results revealed a lack of transparency and awareness of the participants, with a high frequency of 'prefer not to say' responses when asked about the disposal methods of anti-cancer drugs and with only 5.7% of the participants in the pharmacy department sharing their disposal procedures. The same perception was deduced regarding hospitals' wastewater treatment, where responses were often contradicting, preventing making assumptions about the fate of hospital wastewater. CONCLUSION: The results of this survey support the need to establish a more comprehensive waste management programme in Lebanon that would be maintained through regular training and supervision.

Aquatic ecotoxicology of anticancer drugs: A systematic review.

Anticancer drugs in the aquatic environment have drawn a lot of attention in the last decade. Since wastewater treatment plants are inefficient at fully eliminating trace concentrations of anticancer drugs, these compounds are continuously discharged into the aquatic environment. Subsequently, non-target organisms such as the aquatic biota are directly exposed to a variety of anticancer drugs. To understand the potential impact on the aquatic organisms, a systematic review was conducted in compliance with the PRISMA guidelines. The results acquired from the 152 included studies were analysed and sorted into four categories: the impact of each included anticancer drug, the effect of metabolites, the effect of a mixture of drugs, and risk assessment. Findings showed that risk to the aquatic biota was unlikely to occur as the concentrations needed to induce effects were much higher than those detected in the environment. However, these data were based on acute toxicity and included only basic toxicity endpoints. The concentrations that produced significant effects were much lower when tested in the long-term or in multi-generational studies. Heterogeneity in results was also observed; this depended on the organism tested, the assessment adopted, and the endpoints selected. In this systematic review, an overall view of the research studies was generated by which all the variability factors to be considered were reported and recommendations to guide future studies were proposed.

Occurrence of anticancer drugs in the aquatic environment: a systematic review.

Water contamination with pharmaceutical products is a well-studied problem. Numerous studies have demonstrated the presence of anticancer drugs in different water resources that failed to be eliminated by conventional wastewater treatment plants. The purpose of this report was to conduct a systematic review of anticancer drugs in the aquatic environment. The methodology adopted was carried out in compliance with the PRISMA guidelines. From the 75 studies that met the specific requirements for inclusion, data extracted showed that the most common anticancer drugs studied are cyclophosphamide, tamoxifen, ifosfamide and methotrexate with concentrations measured ranging between 0.01 and 86,200 ng/L. There was significant variation in the methodologies employed due to lack of available guidelines to address sampling techniques, seasonal variability and analytical strategy. The most routinely used technique for quantitative determination was found to be solid-phase extraction followed by LC-MS analysis. The lowest reported recovery percentage was 11%, and the highest limit of detection was 1700 ng/L. This indicated the inadequacy of some methods to analyse anticancer drugs and the failure to obtain reliable results. The significant heterogeneity within methodologies made it difficult to compare results and draw conclusions, nevertheless, this study aids in the extrapolation of proposed recommendations to guide future studies and reviews. Graphical abstract.

Labelling of electronic cigarettes: regulations and current practice.

BACKGROUND: Over the past decade e-cigarettes have established themselves in the global market. E-cigarettes triggered much interest in relation to their content and efficacy as smoking cessation tools, but less attention has been paid to users and environmental safety warnings and guidance. Several regulations have been introduced to promote their safe handling and disposal. From May 2016, liquids and cartridges will be regulated by European Community Directives (ECDs) 2001/83/EC and 93/42/EEC, or 2014/40/EU if marketed as tobacco-related products. Currently, manufacturers and distributors must abide by the Chemical (Hazard Information and Packaging for Supply) Regulations 2009 (CHIP) or Classification, Labelling and Packaging Regulations (CLP), the latter replacing CHIP in June 2015. OBJECTIVE: In this work, the compliance of marketed e-liquids and e-cigarettes with current European Union and UK legislations is assessed. RESULTS: E-liquids and e-cigarettes (21 and 9 brands, respectively) were evaluated. Evidence of non-compliance was found in relation to the CHIP/CLP toxic (13%) and environmental (37%) pictograms, tactile warning (23%), nominal amount of solution (30%), supplier contact telephone number and address (40%). None of the evaluated e-cigarettes displayed information on the correct disposal/recycling of batteries in line with the ECD 2006/66/EC. CONCLUSIONS: More stringent enforcement of regulations is needed to ensure not only the user's safety and awareness, but also the safeguarding of the environment.

HPLC estimation of iothalamate to measure glomerular filtration rate in humans.

Glomerular filtration rate (GFR) is usually determined by estimation of iothalamate (IOT) clearance. We have developed and validated an accurate and robust method for the analysis of IOT in human plasma and urine. The mobile phase consisted of methanol and 50 mM sodium phosphate (10:90; v/v). Flow rate was 1.2 mL/min on a C18 reverse phase column, Synergi-hydro (250 × 4.6 mm) 4 µm 80 Å, with an ultraviolet detector set to 254 nm. Acetonitrile was used for the deproteination and extraction of IOT from human plasma and urine. Precision and accuracy were within 15% for IOT in both plasma and urine. The recoveries of IOT in urine and plasma ranged between 93.14% and 114.74 and 96.04-118.38%, respectively. The linear range for urine and plasma assays were 25-1500 and 1-150 µg/mL respectively. The lower limits of detection were 0.5 µg/mL for both urine and plasma, with no interference from plasma and urine matices. This method has been fully validated according to FDA guidelines and the new HPLC assay has been applied to a new formulation of IOT (Conray™ 43), to calculate GFR in healthy volunteers. The new method is simple, less expensive and it would be instrumental in future clinical and pharmacokinetic studies of iothalamate in kidney patients.

Determination of diclofenac concentrations in human plasma using a sensitive gas chromatography mass spectrometry method.

BACKGROUND: A gas chromatography mass spectrometry (GCMS) method for the determination of diclofenac in human plasma has been developed and validated. RESULTS: This method utilizes hexane which is a relatively less toxic extraction solvent compared to heptane and benzene. In addition, phosphoric acid and acetone were added to the samples as deproteination agents, which increased the recovery of diclofenac. These revised processes allow clean extraction and near-quantitative recovery of analyte (approx. 89-95 %). Separation was achieved on a BP-1 column with helium as carrier gas. The molecular ion peaks of the indolinone derivatives of diclofenac ion (m/z 277) and the internal standard, 4-hydroxydiclofenac ion (m/z 439) were monitored by a mass-selective detector using selected ion monitoring (SIM) mode. The linear range for the newly developed and highly sensitive assay was between 0.25-50 ng/mL. The detection and lower quantifiable limits were 0.125 and 0.25 ng/mL, respectively. The inter-day and intra-day coefficients of variation for high, medium and low quality control concentrations were less than 9 %. The robustness and efficacy of this sensitive GCMS method was further demonstrated by using it for a pharmacokinetic study of an oral dosage form of diclofenac, 100 mg of modified-release capsules (Rhumalgan XL), in human plasma. CONCLUSIONS: This method is rapid, sensitive, specific, reproducible and robust, and offers improved sensitivity over previous methods. This method has considerable potential to be used for detailed pharmacokinetics, pharmacodynamics and bioequivalence studies of diclofenac in humans.

A novel steroidal saponin glycoside from Fagonia indica induces cell-selective apoptosis or necrosis in cancer cells.

Fagonia indica is a small spiny shrub of great ethnopharmacological importance in folk medicine. The aqueous decoction of aerial parts is a popular remedy against various skin lesions, including cancer. We used a biological activity-guided fractionation approach to isolate the most potent fraction of the crude extract on three cancer cell lines: MCF-7 oestrogen-dependent breast cancer, MDA-MB-468 oestrogen-independent breast cancer, and Caco-2 colon cancer cells. A series of chromatographic and spectroscopic procedures were utilised on the EtOAc fraction, which resulted in the isolation of a new steroidal saponin glycoside. The cytotoxic activity of the saponin glycoside was determined in cancer cells using the MTT and neutral red uptake assays. After 24h treatment, the observed IC(50) values of the saponin glycoside were 12.5 µM on MDA-MB-468 and Caco-2 cells, but 100 µM on MCF-7 cells. Several lines of evidence: PARP cleavage, caspase-3 cleavage, DNA ladder assays, and reversal of growth inhibition with the pan-caspase inhibitor Z-VAD-fmk, suggested stimulation of apoptosis in MDA-MB-468 and Caco-2 cells, but not in MCF-7 cells, which do not express caspase-3. The haemolytic activity of the saponin glycoside was confirmed in sheep red blood cells, with cell lysis observed at >100 µM, suggesting that, at this concentration, the saponin glycoside caused necrosis through cell lysis in MCF-7 cells. Using the DNA ladder assay, the saponin glycoside (12.5 µM) was not toxic to HUVEC (human umbilical vein endothelial cells) or U937 cells, indicating some selectivity between malignant and normal cells. We conclude that the steroidal saponin glycoside isolated from F. indica is able to induce apoptosis or necrosis in cancer cells depending on the cell type.

Novel acylated steroidal glycosides from Caralluma tuberculata induce caspase-dependent apoptosis in cancer cells.

AIM OF THE STUDY: Pregnane glycosides are potent cytotoxic agents which may represent new leads in the development of anti-tumour drugs, particularly in the treatment of breast cancer, because of the structural similarity to estrogenic agonists. Caralluma species are natural sources of a wide variety of pregnane glycosides. The aim of the study was to isolate, using an activity-guided fractionation approach, novel pregnane glycosides for testing on breast cancer and other tumour lines. MATERIALS AND METHODS: The effect of crude extracts, specific organic fractions and isolated compounds from Caralluma tuberculata was tested on the growth and viability of MCF-7 estrogen-dependent, and MDA-MB-468 estrogen-independent breast cancer cells, Caco-2 human colonic cells, HUVECs and U937 cells. Neutral red uptake and MTT assays were used. Apoptosis was detected by Western blot of poly-(ADP ribose) polymerase (PARP) as were other markers of nuclear fragmentation (DNA ladder assay, staining of cells with nuclear dye DAPI). The involvement of caspases was investigated using the pan-caspase inhibitor Z-VAD-FMK. RESULTS: The ethyl acetate fraction of Caralluma tuberculata was found to be the most potent anti-proliferative fraction against all three cancer cell lines. Two novel steroidal glycosides were isolated from the active fraction after a series of chromatographic experiments. The structure of the isolated compounds was elucidated solely based on 2D-NMR (HMBC, HETCOR, DQF-COSY) and MS spectral analysis as compound 1: 12-O-benzoyl-20-O-acetyl-3ß,12ß,14ß,20ß-tetrahydroxy-pregnan-3-ylO-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1 → 4)-3-methoxy-ß-D-ribopyranoside, and as compound 2: 7-O-acetyl-12-O-benzoyl-3ß,7ß,12ß,14ß-tetrahydroxy-17ß-(3-methylbutyl-O-acetyl-1-yl)-androstan-3-ylO-ß-D-glucopyranosyl-(1 → 4)-6-deoxy-ß-D-allopyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymapyranosyl-(1→ 4)-ß-D-cymaropyranoside. Compound 1 (pregnane glycoside) and compound 2 (androstan glycoside) induced apoptosis at <25 µM after 48 h as assessed by cell shrinkage, PARP cleavage, DNA fragmentation, and reversal with the caspase inhibitor. CONCLUSIONS: Two novel steroid glycosides isolated from Caralluma tuberculata possess moderate, micromolar cytotoxic activity on breast cancer and other cells in vitro, which may indicate a source of activity in vivo of interest to future drug design.