Nicotine Diminishes Alpha2-Adrenergic Receptor-Dependent Protection Against Oxidative Stress in H9c2 Cardiomyocytes.

Introduction: Nicotine is a major component of cigarette smoke with various detrimental cardiovascular effects, including increased oxidative stress in the heart. Agonism of α2-adrenergic receptors (ARs), such as with dexmedetomidine, has been documented to exert cardioprotective effects against oxidative stress and related apoptosis and necroptosis. α2-ARs are membrane-residing G protein-coupled receptors (GPCRs) that primarily activate Gi/o proteins. They are also subjected to GPCR-kinase (GRK)-2-dependent desensitization, which entails phosphorylation of the agonist-activated receptor by GRK2 to induce its decoupling from G proteins, thus terminating α2AR-mediated G protein signaling. Objective: In the present study, we sought to examine the effects of nicotine on α2AR signaling and effects in H9c2 cardiomyocytes exposed to H2O2 to induce oxidative cellular damage. Methods and Results: As expected, treatment of H9c2 cardiomyocytes with H2O2 significantly decreased cell viability and increased oxidative stress, as assessed by reactive oxygen species (ROS)-associated fluorescence levels (DCF assay) and superoxide dismutase activity. Both H2O2 effects were partly rescued by α2AR activation with brimonidine in control cardiomyocytes but not in cells pretreated with nicotine for 24 hours, in which brimonidine was unable to reduce H2O2-induced cell death and oxidative stress. This was due to severe α2AR desensitization, manifested as very low Gi protein activation by brimonidine, and accompanied by GRK2 upregulation in nicotine-treated cardiomyocytes. Finally, pharmacological inhibition of adenylyl cyclase (AC) blocked H2O2-dependent oxidative damage in nicotine-pretreated H9c2 cardiomyocytes, indicating that α2AR activation protects against oxidative injury via its classic coupling to Gai-mediated AC inhibition. Discussion/Conclusions: Nicotine can negate the cardioprotective effects of α2AR agonists against oxidative injury, which may have important implications for patients treated with this class of drugs that are chronic tobacco smokers.

Brimonidine-montmorillonite hybrid formulation for topical drug delivery to the eye.

Brimonidine (BMD) is often prescribed as an eye drop to reduce the intraocular pressure (IOP) for glaucoma treatment. However, eye drops are limited by rapid clearance from the preocular surface, and hence a low ocular drug bioavailability. Therefore, in this study, we propose montmorillonite (MMT), as a delivery carrier, hybridized with BMD (BMD-MMT) for topical drug delivery to the eye. The BMD-MMT hybrid was prepared by intercalating the BMD molecules in the interlayer space of the MMT lattice via ion-exchange reaction; it was then formulated with polyvinyl alcohol (PVA) to produce a dry tablet (i.e., BMD-MMT@PVA). The BMD-MMT@PVA hybrid drug released BMD in a sustained manner for more than 5 h under in vitro conditions. When the hybrid drug was administered to rabbit eyes in vivo, 43% and 18.5% BMD-MMT still remained on the preocular surface for 10 and 60 min after administration, respectively. Thus, the BMD-MMT@PVA hybrid drug exhibited a prolonged decrease in IOP, that is, for 12 h, which was approximately two times longer than that observed with the commercially available BMD eye drop, Alphagan® P.

The evaluation of short- and long-term stability studies for brimonidine in aqueous humor by DPV/BDDE method-possible application for direct assay in native samples.

A novel voltammetric method was developed for brimonidine (BRIM) determination in deproteinized aqueous humor, simplifying preparation of biological samples for analysis for stability studies. The differential pulse voltammetric (DPV) method using boron doped diamond electrode (BDDE), based on characteristic oxidation peaks, was proposed and successfully applied. The linearity range was within 5.0 × 10-6 to 5.0 × 10-5 M of brimonidine, and limit of detection and limit of quantitation were 1.94 × 10-6 M and 6.46 × 10-6 M, respectively. Intra-day and inter-day precision and accuracy were evaluated and all results were in accordance with validation ICH guidelines. The best short-term stability study results were obtained for a concentration level of 3.0 × 10-5 M expressed by deviation of + 1.86% between initial and post storage concentrations. A long-term stability study was performed for two concentrations of 3.0 × 10-5 M and 5.0 × 10-5 M and resulted in deviations of + 1.63% and + 3.56%, respectively. A freeze and thaw stability study indicated that samples might be frozen only once. The enhancement of DPV/BDDE method sensitivity gained by modification, for the analysis of immeasurable BRIM quantities in native, untreated aqueous humor, was reached for quantities of 6 or 12 nmol/0.1 mL aqueous humor with acceptable accuracy (up to + 7.5%). The nature of the process-the irreversible one electron oxidation voltammetric peak of BRIM-limited the sensitivity. Only electrochemical pre-treatment of the BDD electrode before each measurement significantly speeded up the whole procedure. The advantages of the proposed method are simplicity, short-time performance, and good specificity/selectivity, as well as satisfactory accuracy, and no chemical modification of BDDE was necessary.