Effect of tobacco and nicotine replacement therapy on biomarkers associated with endothelial dysfunction

Tobacco use is a major cause of cardiovascular diseases (CVDs), which begin with endothelial dysfunction. In this thesis, we objectified, by the quantification of specific biomarkers, an elevation of inflammation, oxidative stress, and endothelial activation in smokers, associated with an altered lipid profile. All these processes are involved in endothelial dysfunction and therefore, promote CVDs development. We found that these effects could be reversible on quitting. These improvements were already visible at short term. The role of nicotine, especially of nicotine replacement therapy (NRT), in CVDs progression remains controversial in the literature. Therefore, we assessed the influence of NRT on biomarkers and we demonstrated that they did not affect the biological improvements observed with smoking cessation.(BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 202

Development and Validation of a Quantification Method of Cotinine in Urine Using Two Innovative Technologies: Supported Liquid Extraction and QDa Detection

Objective: Cotinine is the best biomarker of tobacco smoke exposure because of its long half-life. Several methods are developed to quantify cotinine in biological fluids, including high performance liquid chromatography. This method requires an extraction step in order to clean-up the sample and concentrate the analyte of interest. The aim of this study was to optimise the extraction step and to develop an easy, reproducible and specific method to measure cotinine in urine. Methods: An extraction of neutrals was chosen and performed by Supported Liquid Extraction (SLE). SLE consists in liquidliquid extraction in the presence of a sorbent enabling efficient extraction with less organic solvent and without any emulsion formation. Urine was basified by treatment with NH4OH in order to neutralise cotinine before loading on SLE plate. After drying, neutrals were eluted with a mix containing dichloromethane and isopropyl alcohol. Solvent was then evaporated and samples were reconstituted with water. Detection of cotinine was performed by mass detection using a QDa detector after UHPLC separation with a C18 column at a flow rate of 0.4 ml/min. A gradient elution of H2O +0.1% NH4OH and CH3CN was used. The method was validated based on linearity, precision, recovery and limits of detection and quantification. Results: The range of linearity was 0.001 μg/ml -5 μg/ml with a determination coefficient of 0.997. The precision was evaluated by the Relative Standard Deviation (RSD) for intra- and interassay and was below 5% and 10% respectively. 3 levels of concentration were tested to assess the recovery rate which was consistent and higher than 96%. Conclusion: Cotinine concentration can be measured in urine by SLE extraction and UHPLC-QDa detection. This method is easy, reproducible and allows quantification of low concentrations. It is a good solution to assess patients’ tobacco smoke exposure in medical laboratory

Development of an Ultra-High Performance Liquid Chromatography method for the simultaneous mass detection of tobacco biomarkers in urine.

The quantification of tobacco exposure biomarkers is relevant to follow the patients' tobacco use. They allow to discriminate between tobacco users, non-users, passive smokers, and nicotine products users, such as in nicotine replacement therapy. The aim of this study was to develop and validate a quantification method of tobacco biomarkers of choice - nicotine, cotinine, trans-3'-hydroxycotinine, anatabine and anabasine - in urine. The challenge was to develop an easy and rapid liquid chromatography method requiring only one extraction step and allowing simultaneous detections. Some methods are described in the literature but need specific investment in terms of instrumentation and users training. Here, the developed method had to be carried out with instrumentation easily accessible for medical laboratories. The extraction of the analytes was performed by Supported Liquid Extraction (SLE), which consists in liquid-liquid extraction but supported by a sorbent. It allows to insure efficient neutrals extraction with less organic solvent and without any emulsion formation. 200 µl of basified urine - analytes of interest are neutral in this condition - were loaded on Novum SLE 96-Well Plates (Phenomenex) and analytes were eluted with 1 % formic acid in dichloromethane/propan-2-ol (95/5). After solvent evaporation, samples were reconstituted with 100 µl of water for injection. A mass detector (QDa, Waters) was used to detect analytes, this pre-optimised quadrupole mass analyser being less expensive and requiring less adjustments than traditional mass spectrometers while benefiting of the reliability of mass spectral data. This detector was integrated after an Ultra-high performance liquid chromatography (UHPLC) separation on a BEH C18 column (Waters) at a flow rate of 0.5 ml/min. A gradient elution of HO (pH 10 with NHOH) and CHCN was used. Finally, the developed method was validated. This new method is conclusive to assess the patients' tobacco exposure and is easy to implement in medical laboratories

Physical instability of an infusion containing ropivacaine, clonidine and adrenaline tartrate in syringes for pre-operative administration.

To the Editor, We investigate the physical stability of an infusion containing ropivacaine, clonidine and adrenaline tartrate, three pharmaceutical compounds usually used in operating room for loco-regional anaesthesia. The awareness of the stability of a ready-to-use mixture containing the three components could allow staff anaesthetists to inject patient only once. Moreover, the preparation in advance by a Centralised IntraVenous Admixtures Service (CIVAS) could be considered in terms of preparation quality and time management. [...

Microwave Freeze-thaw Technique for Injectable Drugs: A Review Updated from 1980 to 2021.

The objective of this review was to collect information and results about the method of the microwave freeze-thaw treatment of injectable drugs and whether the method can support the development of Centralized Intravenous Admixtures Services. A systematic review of the scientific literature about injectable drug stability studies was performed. The data are presented in a table, which describes the name of the drug, producer, final concentration, temperature and time of freezing storage, type of microwave oven, thawing power, method of dosage, and the results after treatment or final long-term storage at 5°C ± 3°C. From 1980 to 2021, 60 drugs were studied by the microwave freeze-thaw treatment, and the results were presented in 49 publications. Forty papers were presented by 8 teams (2 to 18 by team). The temperatures of freezing storage varied from -70°C to -10°C, the time storage from 4 hours to 12 months, and the thaw from low to full power. Drug concentrations were mainly determined by high-performance liquid chromatography. Most of the 59 drugs were stable during and after treatment. Only three teams tested the long-term stability after the microwave freeze-thaw treatment, the first for ganciclovir after 7 days, the second for ceftizoxime after 30 days, and the third for 20 drugs after 11 to 70 days. This review can help Centralized Intravenous Additive Services take charge of the productions of ready-to-use injectable drugs

Novel proposed cutoff values for anatabine and anabasine in differentiating smokers from non-smokers.

Anatabine and anabasine are two tobacco alkaloids used to differentiate between tobacco users and abstainers, including users of nicotine replacement therapy. Cutoff values (>2 ng/mL for both alkaloids) have not been revised since their implementation in 2002. These values may be too high, leading to increased likelihood of misclassification between smokers and abstainers. This results in major consequences, especially adverse outcomes of transplantation when smokers were incorrectly identified as being abstinent. This study proposes that a lower threshold for anatabine and anabasine will better distinguish tobacco users from non-users and thereby improve patients' care. A new and more sensitive analytical method by liquid chromatography-mass detection was developed to allow the quantification of low concentrations. Anatabine and anabasine were measured in urine samples of 116 self-reported daily smokers and 47 long-term non-smokers (confirmed by the analysis of nicotine and its metabolites). The best compromise between sensitivity and specificity allowed us to determine new cutoff values. The thresholds >0.097 ng/mL for anatabine and >0.236 ng/mL for anabasine were associated with a sensitivity of 97% (anatabine) and 89% (anabasine) and a specificity of 98% for both alkaloids. These cutoff values greatly increased the sensitivity given that it dropped to 75% (anatabine) and 47% (anabasine) when using the reference value (>2 ng/mL). The cutoff values >0.097 ng/mL for anatabine and >0.236 ng/mL for anabasine appear to better differentiate tobacco users from abstainers than the current reference threshold (>2 ng/mL for both alkaloids). It may considerably impact patients' care, especially in transplantation settings in which smoking abstinence is essential to avoid adverse outcomes of transplantation

Changes in biomarkers of endothelial function, oxidative stress, inflammation and lipids after smoking cessation: A cohort study.

Tobacco use is known to be involved in the development of cardiovascular diseases, which leads to premature mortality. Endothelial dysfunction, the first step in this process, was shown induced by smoking. It is reported that quitting smoking could reduce the risk of diseases, but the implied mechanisms are still unclear. This study aimed to evaluate the biological markers of endothelial function in smokers when actively smoking and after cessation. Quantification of several biomarkers reflecting inflammation, endothelium activation, oxidative stress, and lipids was performed in 65 smokers when actively smoking and after cessation (median abstinence duration of 70 days). A possible decrease of inflammation was observed through the concentration reduction of a proinflammatory cytokine (interleukine-6) on quitting. A decrease of endothelium activation was visible by the reduced level of the soluble intercellular adhesion molecule. Two antioxidants, uric acid and vitamin C, were found at higher concentration than before the cessation, potentially reflecting the decrease of oxidative stress on quitting. Lipid profile was improved post-quit since HDL level was increased and LDL level was decreased. All these effects were visible at short term with abstinence duration less than 70 days. No sex-specific difference was observed and no additional changes were observed for longer abstinence duration. These observations suggest that some adverse effects of smoking on endothelial function could be reversible on quitting smoking. It could encourage smokers to enter a cessation program to reduce the risk for cardiovascular diseases development

Effect of the nicotine replacement therapy on biomarkers of inflammation, endothelial dysfunction, oxidative stress and lipids in smokers who quit smoking.

Our previous study showed major changes in biomarkers on quitting compared to the smoking state. They reflected a decrease of inflammation, endothelial activation, and oxidative stress, as well as an improved lipid profile. Nicotine replacement therapy (NRT) is effective to increase the rate of successful quitting, but health care professionals may have concerns to prescribe this first line smoking cessation treatment because its effect on inflammation and related processes is controversial. Therefore, the present study assessed the influence of NRT on biomarkers of inflammation, endothelial function, oxidative stress, and lipids, in people who quit smoking. Sixty-five subjects who daily smoke cigarettes were recruited and followed on quitting. Thirty-five quit using NRT and thirty quit without NRT. Biomarkers of inflammation, endothelial function, oxidative stress, and lipids were quantified at baseline when actively smoking and after cessation in the presence of NRT or not. Changes in biomarkers on quitting did not differ according to the treatment used. No difference was found when comparing participants who were exposed to NRT and those who were not. These results may indicate that NRT has no effect on inflammation, endothelial function, oxidative stress, and lipids, when used as a medication aid for quitting smoking. This study provides new evidence to support the safety profile of nicotine replacement therapy products regarding the biomarkers of endothelial function, oxidative stress, inflammation, and lipids

Physical stability of an IV mixture of morphine and clonidine in syringe

BACKGROUND AND AIM Clonidine is frequently combined to opioids for the management of chronic pain. The aim of this study was to evaluate the physical stability of this combination at high and low concentrations in polypropylene syringes at 5±3°C and at 22±3°C. METHODS Five syringes with low concentration of clonidine (0.003 mg/mL) and morphine hydrochloride (0.417 mg/mL) and five syringes with high concentration of clonidine (0.032 mg/mL)and morphine hydrochloride (4.286 mg/mL) were evaluated. The turbidity, the pH and the crystal formation were controlled after the production and until 72h post-production. RESULTS No changes in color or appearance of opacity or turbidity were observed. Spectrophotometric measurements (at 350, 410 and 550 nm) and pH were not affected. The microscopic analysis did not detect any aggregate or crystal formation. DISCUSSION AND CONCLUSION The mixture of clonidine and morphine hydrochloride at low and high concentrations is physically stable for at least 72 hours post-production at 5±3°C and at 22±3°C. These results paved the way for a subsequent chemical stability study