Development and Validation of LC-MS/MS Method for Determination of Cytisine in Human Serum and Saliva.

Cytisine (CYT) is a quinolizidine alkaloid used for nicotine addiction treatment. Recent clinical trial data regarding cytisine confirm its high effectiveness and safety as a smoking cessation treatment. CYT's popularity is growing due to its increased availability and licensing in more countries worldwide. This increased use by smokers has also resulted in an urgent need for continued drug research, including developing appropriate analytical methods for analyzing the drug in biological samples. In this study, a simple, fast, and reliable method combining hydrophilic interaction liquid chromatography and electrospray ionization quadrupole time of flight mass spectrometry (HILIC/ESI-QTOF-MS) for the determination of CYT in human serum and saliva was developed and validated. This was undertaken after the previous pre-treatment of the sample using solid-phase extraction (SPE). A hydrophilic interaction liquid chromatography (HILIC) column with a silica stationary phase was used for chromatographic analysis. In a linear gradient, the mobile phase consisted of acetonitrile (ACN) and formate buffer at pH 4.0. The proposed method was fully validated and demonstrated its sensitivity, selectivity, precision, and accuracy. The method was successfully applied to determine CYT in serum and, for the first time, in saliva. The findings indicate that saliva could be a promising non-invasive alternative to measure the free concentration of CYT.

Interaction of Varenicline with Classic Antiseizure Medications in the Mouse Maximal Electroshock-Induced Seizure Model.

Varenicline (VAR) is a partial agonist of brain α4ß2 nicotinic acetylcholine receptors recommended as a first line pharmacotherapy for smoking cessation. The aim of this study was to examine whether VAR affects the protective activity of four classic antiseizure medications, i.e., carbamazepine (CBZ), phenobarbital (PB), phenytoin (PHT), and valproate (VPA) on maximal electroshock (MES)-induced seizures, which may serve as an experimental model of human-generalized tonic-clonic seizures in mice. VAR administered intraperitoneally (i.p.) at a subthreshold dose of 0.5 mg/kg decreased the protective activity of CBZ against MES-induced convulsions, increasing its median effective dose (ED50) from 10.92 ± 1.0 to 18.15 ± 1.73 mg/kg (p < 0.01). The effect of VAR was dose-dependent because a lower dose of VAR (0.25 mg/kg) failed to antagonize the protective activity of CBZ. VAR administered at the subthreshold dose of 0.5 mg/kg had no impact on the protective activity of PB, PHT, and VPA in the mouse MES model. The inhibitory effect of VAR on the protective activity of CBZ against tonic-clonic convulsions most likely resulted from the pharmacodynamic mechanism(s) and was not associated with the changes in total brain concentrations of CBZ. VAR-evoked alterations in the anticonvulsive activity of CBZ may be of serious concern for epileptic tobacco smokers.

Effect of Cytisine vs Varenicline on Smoking Cessation: A Randomized Clinical Trial.

Importance: Cytisine is more effective than placebo and nicotine replacement therapy for smoking cessation. However, cytisine has not been tested against the most effective smoking cessation medication, varenicline, which is associated with adverse events known to lead to discontinuation of therapy. Objective: To examine whether standard cytisine treatment (25 days) was at least as effective as standard varenicline treatment (84 days) for smoking cessation. Design, Setting, and Participants: This noninferiority, open-label randomized clinical trial with allocation concealment and blinded outcome assessment was undertaken in Australia from November 2017 through May 2019; follow-up was completed in January 2020. A total of 1452 Australian adult daily smokers willing to make a quit attempt were included. Data collection was conducted primarily by computer-assisted telephone interview, but there was an in-person visit to validate the primary outcome. Interventions: Treatments were provided in accordance with the manufacturers' recommended dosage: cytisine (n = 725), 1.5-mg capsules taken 6 times daily initially then gradually reduced over the 25-day course; varenicline (n = 727), 0.5-mg tablets titrated to 1 mg twice daily for 84 days (12 weeks). All participants were offered referral to standard telephone behavioral support. Main Outcomes and Measures: The primary outcome was 6-month continuous abstinence verified using a carbon monoxide breath test at 7-month follow-up. The noninferiority margin was set at 5% and the 1-sided significance threshold was set at .025. Results: Among 1452 participants who were randomized (mean [SD] age, 42.9 [12.7] years; 742 [51.1%] women), 1108 (76.3%) completed the trial. Verified 6-month continuous abstinence rates were 11.7% for the cytisine group and 13.3% for the varenicline group (risk difference, -1.62% [1-sided 97.5% CI, -5.02% to ∞]; P = .03 for noninferiority). Self-reported adverse events occurred less frequently in the cytisine group (997 events among 482 participants) compared with the varenicline group (1206 events among 510 participants) and the incident rate ratio was 0.88 (95% CI, 0.81 to 0.95; P = .002). Conclusions and Relevance: Among daily smokers willing to quit, cytisine treatment for 25 days, compared with varenicline treatment for 84 days, failed to demonstrate noninferiority regarding smoking cessation. Trial Registration: anzctr.org.au Identifier: ACTRN12616001654448.

Comparison of Various Chromatographic Systems for Analysis of Cytisine in Human Serum, Saliva and Pharmaceutical Formulation by HPLC with Diode Array, Fluorescence or Mass Spectrometry Detection.

Background: Identification and quantitative determination of cytisine, especially in biological samples and pharmaceutical formulations, is still a difficult analytical task. Cytisine is an alkaloid with a small and very polar molecule. For this reason, it is very weakly retained on reversed phase (RP) stationary phases, such as commonly used alkyl-bonded phases. The very weak retention of cytisine causes it to be eluted together with the components of biological matrices. Objective: Comparison and evaluation of various chromatographic systems for analysis of cytisine in different matrices-serum, saliva and pharmaceutical formulation-by high performance liquid chromatography (HPLC) with diode array (DAD), fluorescence (FLD) and mass spectrometry (MS) detection. Methods: The analyses were performed using HPLC in reversed phase (RP), hydrophilic interaction liquid chromatography (HILIC) and ion exchange chromatography (IEC) modes. Different sample pre-treatment methods were tested: Protein precipitation (with acetone, methanol (MeOH) or acetonitrile (ACN), and solid phase extraction (SPE) using cartridges with octadecyl (C18), hydrophilic-lipophilic balanced copolymer (HLB) or strong cation exchange sorbents (Strata X-C). Conclusion: Significant differences were observed in retention parameters with a change of the used chromatographic system. The various properties of stationary phases resulted in differences in analyte retention, peaks' shape and systems' efficiency. The weakest retention was observed using RP systems; however, the use of the Polar RP phase can be an alternative for application in green chromatography. In the strongest retention was observed using a strong cation exchange (SCX) phase. The most optimal systems were chosen for the analysis of cytisine in the pharmaceutical preparation, serum and saliva after sample pre-treatment with the new SPE procedure. Due to the sensitivity, the use of HPLC-DAD or HPLC-FLD is the most optimal for drug analysis in pharmaceutical preparations, whereas HPLC-MS is suitable for analysis of cytisine in biological samples.

[Caffeinol: a neuroprotective action in ischemic brain damage]. / Kafeinol: dzialanie neuroprotekcyjne w udarze niedokrwiennym mózgu.

Caffeinol--a combination of ethanol and caffeine in appropriate concentrations--exerts neuroprotective and anticonvulsive action. Research conducted on rats in models of ischemic brain damage have shown that caffeinol decreases the size of cortical damage by about 80%, improves motional coordination and memory. The sooner caffeinol was administered, the better were beneficial therapeutic effects. What is more, the medicine may be safely combined with other methods used in stroke treatment, such as hypothermia and thrombolysis, what additionally increases its neuroprotective influence. Research on people have shown that caffeinol is less effective as neuroprotective agent in patients abusing alcohol, while chronic intake of caffeine does not influence its activity. Mechanism of its activity is not known yet, however, it is assumed that it bases on an antagonism of NMDA receptors. Regarding the fact that the most of strokes in humans concern subcortical areas, it is justified to conduct further research on caffeinol, which would involve other brain structures, thus allowing to define its use in clinical practice.

Effects of WIN 55,212-2 (a non-selective cannabinoid CB1 and CB 2 receptor agonist) on the protective action of various classical antiepileptic drugs in the mouse 6 Hz psychomotor seizure model.

The aim of this study was to characterize the influence of WIN 55,212-2 (WIN--a non-selective cannabinoid CB1 and CB2 receptor agonist) on the anticonvulsant effects of various classical antiepileptic drugs (clobazam, clonazepam, phenobarbital and valproate) in the mouse 6 Hz-induced psychomotor seizure model. Limbic (psychomotor) seizure activity was evoked in albino Swiss mice by a current (32 mA, 6 Hz, 3 s stimulus duration) delivered via ocular electrodes. Drug-related adverse effects were ascertained by use of the chimney test (evaluating motor performance), step-through passive avoidance task (assessing learning) and grip-strength test (evaluating skeletal muscular strength). Total brain concentrations of antiepileptic drugs were measured by fluorescence polarization immunoassay to ascertain any pharmacokinetic contribution to the observed antiseizure effect. Results indicate that WIN (5 mg/kg, administered intraperitoneally) significantly enhanced the anticonvulsant action of clonazepam (P < 0.001), phenobarbital (P < 0.05) and valproate (P < 0.05), but not that of clobazam in the mouse 6 Hz model. Moreover, WIN (2.5 mg/kg) significantly potentiated the anticonvulsant action of clonazepam (P < 0.01), but not that of clobazam, phenobarbital or valproate in the 6 Hz test in mice. None of the investigated combinations of WIN with antiepileptic drugs was associated with any concurrent adverse effects with regard to motor performance, learning or muscular strength. Pharmacokinetic experiments revealed that WIN had no impact on total brain concentrations of antiepileptic drugs in mice. These preclinical data would suggest that WIN in combination with clonazepam, phenobarbital and valproate is associated with beneficial anticonvulsant pharmacodynamic interactions in the mouse 6 Hz-induced psychomotor seizure test.

Measurements of cadmium levels in relation to tobacco dependence and as a function of cytisine administration.

Cigarette smoking delivers a number of heavy metals, including cadmium (Cd), into the body. Bioaccumulation may result in an increase in pathological consequences over time. The assessment of changes in serum Cd concentrations during the treatment of cigarette dependence with cytisine was performed for the first time. Parameters assessing smoking habits, strength of addiction, and effectiveness of therapy were analyzed. Cd was determined before, during, and after the end of treatment. The serum Cd levels were significantly higher in the smokers than in the nonsmokers. Significant differences in Cd concentrations between sampling times in smokers were observed. Individuals who stopped smoking had significantly lower Cd concentrations compared to baseline. A significant positive correlation between the serum Cd before treatment and smoking urges was also obtained. Additionally, salivary Cd determinations were performed before treatment to evaluate the use of this method to assess cigarette addiction. Our findings indicate that Cd can be used as a biomarker of smoking addiction, and provide an alternative assessment of tobacco smoke exposure to other methods. The results provide new knowledge related to Cd concentrations in human body fluids and may play a role in monitoring and assessing the efficacy of cytisine for smoking cessation.

Heat capacity of cytisine - the drug for smoking cessation.

The characterization of cytisine (CYT) and its blends with poly(lactic acid) was performed using thermal analysis, elemental analysis, infrared spectroscopy, and powder X-ray diffractometry. The heat capacities, total enthalpy, and phase transitions of CYT were established from 1.8 to 448.15 K (-271.35 - 175 °C) by advanced thermal analysis. Data were obtained using a Quantum Design Physical Property Measurement System (PPMS) and a differential scanning calorimetry (DSC). The low-temperature heat capacity of the crystalline CYT in the range of 1.8 to 300 K (-271.35 - 26.86 °C) was measured by PPMS and fitted to a theoretical model in the low temperature region below 11 K (-262.15 °C), to orthogonal polynomials in the middle range 5 K < T < 60 K (-268.15 °C < t < -213.15 °C) and to the Debye and Einstein functions in the high range of temperature above 60 K (-213.15 °C). The liquid heat capacity was calculated based on the approximated linear regression data above the molten state of the experimental heat capacity of CYT obtained by the standard DSC measurements, and it was expressed as Cpliquid = 0.0838T + 346.78 J·K-1·mol-1. The calculated heat capacity in the solid state was extended to a higher temperature and was used, together with liquid heat capacity, as the reference baselines for the advanced thermal analysis of CYT. The PPMS and DSC/TMDSC methods are complementary methods for thermal analysis of cytisine. The PPMS method allowed determination of the equilibrium heat capacity in the solid state, which together with the equilibrium heat capacity in the liquid state allowed to analyze of the experimental apparent heat capacity of cytisine obtained based on DSC. The melting temperature and the total heat of fusion of crystalline material were established as 431.8 K (158.65 °C) and 26.5 kJ·mol-1, respectively. The solid and liquid heat capacities and transition parameters of CYT were applied to calculate total enthalpies for fully amorphous and crystalline states. Analyses of DSC and X-ray confirmed the presence of the solid-solid transition linking with not so far described a polymorphism phenomenon of CYT. Based on the thermogravimetric analysis the temperature of degradation of CYT was determined as 460.5 K (187.35 °C). Also, a preliminary thermal analysis of the blends of cytisine and poly(lactic acid) as a new candidate for drug delivery system was presented.

The cytisine-enriched poly(3-hydroxybutyrate) fibers for sustained-release dosage form.

The polymeric cytisine-enriched fibers based on poly(3-hydroxybutyrate) were obtained using electrospinning method. The biocompatibility study, advanced thermal analysis and release of cytisine from the poly(3-hydroxybutyrate) fibers were carried out. The nanofibers' morphology was evaluated by scanning electron microscopy. The formation and description of phases during the thermal processes of fibers by the advanced thermal analysis were examined. The new quantitative thermal analysis of polymeric fibers with cytisine phases based on vibrational, solid and liquid heat capacities was presented. The apparent heat capacity of fibers was measured using the standard differential scanning calorimetry. The quantitative analysis allowed for the study of the glass transition and melting/crystallization process. The mobile amorphous fraction, degree of crystallinity and rigid amorphous fraction were determined depending on the thermal history of semicrystalline polymeric fibers. Furthermore, the cytisine dissolution behaviour was studied. It was observed that the kinetic of the release from polymeric nanofiber is delayed than for the marketed product. The immunosafety of the tested polymeric nanofibers with cytisine was confirmed by the Food and Drug Agency Guidance as well as the European Medicines Agency. The polymeric matrix with cytisine seems to be a promising candidate for the prolonged release formulation.

Effects of cigarette smoking, metabolic syndrome and dehydroepiandrosterone deficiency on intima-media thickness and endothelial function in hypertensive postmenopausal women.

BACKGROUND: Cigarette smoking is a major risk factor of atherosclerosis. The aim of this study was to assess the relationship between smoking and arterial hypertension as well as endothelial dysfunction in postmenopausal women without clinically manifested symptoms of atherosclerosis. MATERIAL/METHODS: The study groups consisted of 35 current smokers and 45 nonsmokers. The thickness of intima-media complex (IMT), a marker of atherosclerosis, was measured in carotid arteries. Plasma concentrations of fasting glucose, insulin, lipoproteins, inflammatory markers (tumor necrosis factor-alpha, intercellular adhesion molecule-1), matrix metalloproteinases (metalloproteinase-9, tissue inhibitor of metalloproteinase-1), insulin, and dehydroepiandrosterone sulfate (DHEA-S) were measured. RESULTS: Smokers compared with nonsmokers showed lower fasting glucose levels in blood (87.0±10.9 and 93.2±13.6 mg/dl, p<0.05), higher mean systolic (131.1±15.9 vs. 123.0±10.9 mm Hg, p<0.05) and diastolic (81.7±11.4 vs. 75.2±9.2 mm Hg, p<0.05) blood pressure during daytime, and higher average heart rate during the daytime (78.2±9.3/min vs. 71.5±9.5/min, p<0.01) and at night (67.2±10.6/min vs. 61.7±7.7/min, p<0.05), respectively. The IMT in the right carotid artery was significantly higher in smokers than in nonsmokers (0.96±0.16 mm vs. 0.82±0.21, p<0.05) and was positively correlated with smoking intensity (R=0.36) and habit duration (R=0.35). The comparison of inflammatory markers, metalloproteinases, and DHEA-S concentrations in plasma did not reveal significant differences between the 2 groups. A significant negative correlation between DHEA-S concentration in plasma and IMT in right carotid artery was found in smokers. CONCLUSIONS: Smoking in hypertensive postmenopausal women is associated with lower fasting blood glucose and BMI values, but higher arterial pressure and heart rate, and increases in IMT in right carotid artery.

Effectiveness, safety and cost-effectiveness of vaporized nicotine products versus nicotine replacement therapy for tobacco smoking cessation in a low-socioeconomic status Australian population: a study protocol for a randomized controlled trial.

BACKGROUND: In Australia, tobacco smoking rates have declined but inequalities remain with significantly higher smoking prevalence among low-socioeconomic populations. Clinical trial data suggest vaporized nicotine products (VNPs) aid smoking cessation. Most VNP trials have used refillable tank systems, but newer generation (pod) devices now comprise the largest market share yet have limited clinical trial evidence on safety and effectiveness. This study evaluates the effectiveness, safety and cost-effectiveness of VNPs (pod and tank device) compared with nicotine replacement therapy ([NRT]-gum or lozenge) for smoking cessation. METHODS: This is a two-arm, open-label, superiority, parallel group, randomized controlled trial (RCT) with allocation concealment and blinded outcome assessment. The RCT is conducted at the National Drug and Alcohol Research Centre at the University of New South Wales, Sydney, Australia. Participants are people who smoke daily, are interested in quitting and receive a government pension or allowance (N = 1058). Participants will be randomized (1:1 ratio) to receive 8 weeks of free: VNPs, with pod (40 mg/mL nicotine salt) and tank device (18 mg/mL freebase nicotine) in mixed flavours; or NRT (gum or lozenge; 4 mg). All participants will receive daily text message behavioural support for 5 weeks. Assessments will be undertaken by telephone at baseline, with three follow-up calls (two check-in calls within the first month and final follow-up at 7 months post randomization) to ascertain smoking status, treatment adherence and adverse events. The primary outcome is 6-month continuous abstinence verified by carbon monoxide breath test of ≤5ppm at 7-month follow-up. Safety and cost-effectiveness of VNPs versus NRT will also be evaluated. DISCUSSION: Further data are required to strengthen certainty of evidence for VNPs aiding smoking cessation, particularly for newer generation pod devices. To our knowledge, this trial is the first to offer choice of VNPs and no comparative effectiveness trial data exists for new pod devices. If effective, the findings can inform wider implementation of VNPs to aid smoking cessation in a priority group. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12621000076875. Registered on 29 January 2021.  https://www.anzctr.org.au.

Hyperglycemia Potentiates Prothrombotic Effect of Aldosterone in a Rat Arterial Thrombosis Model.

We investigated the role of aldosterone (ALDO) in the development of arterial thrombosis in streptozotocin-induced diabetic rats. To evaluate the effect of endogenous ALDO, the rats underwent adrenalectomy (ADX). ADX reduced the development of arterial thrombosis. A 1 h infusion of ALDO (30 µg/kg/h) enhanced thrombosis in adrenalectomized rats, while this effect was potentiated in diabetic rats. ALDO shortened bleeding time, increased plasma levels of tissue factor (TF) and plasminogen activator inhibitor, decreased plasma level of nitric oxide (NO) metabolites, and increased oxidative stress. Moreover, 2 h incubation of human umbilical vein endothelial cells (HUVECs) with ALDO (10-7 M) disrupted hemostatic balance in endothelial cells in normoglycemia (glucose 5.5 mM), and this effect was more pronounced in hyperglycemia (glucose 30 mM). We demonstrated that the acute ALDO infusion enhances arterial thrombosis in rats and hyperglycemia potentiates this prothrombotic effect. The mechanism of ALDO action was partially mediated by mineralocorticoid (MR) and glucocorticoid (GR) receptors and related to impact of the hormone on primary hemostasis, TF-dependent coagulation cascade, fibrinolysis, NO bioavailability, and oxidative stress balance. Our in vitro study confirmed that ALDO induces prothrombotic phenotype in the endothelium, particularly under hyperglycemic conditions.

[Androgens and epilepsy]. / Androgeny a padaczka.

Nowadays there are increasing experimental and clinical data indicating an important role of an endocrine system (especially its neuroendocrine part and sex hormones) in the pathogenesis of epilepsy. The relationships between patomechanisms of epilepsy and activity of hypothalamo-pituitary-ovarian axis in animals and humans are quite well recognized but the role of male sex hormones, i.e androgens, in seizure susceptibility processes is less known. Epidemiological data clearly show that adrogens-related disorders occur more frequently in epileptic men than in general male population. Usually, they appear in the form of hypogonadism associated with low levels of plasma free testosterone and with low excretion of its 17-ketosteroid metabolites in the urine. Reproductive and sexual disorders can be attributed to hypogonadism. Androgen abnormalities in epileptics men are often affected by chronically used anti-epileptic drugs. Antiepileptic drugs, particularly classical ones, substantially modify bioavailability of androgens and can inhibit the activity of hypothalamo-pituitary-testicular axis, and--in a consequence--aggravate hypogonadism. Since neuroactive androgens cross the blood-brain barrier and modify seizure susceptibility, changes in their plasma concentrations can affect the course and clinical outcome of epilepsy. Effects of testosterone on seizures seem to depend on its different metabolic pathways. Aromatization of testosterone leads to formation of 17beta-estradiol that is believed to have proconvulsive activity. Activation of 5alpha-reductase pathway leads to formation of ketosteroid metabolites, primarily andosterone and etiocholanolone that demonstrate the ability to prevent convulsions in majority of animal studies. Recently, it has been shown that androsterone enhances the antiepileptic activity of phenobarbital, carbamazepine, and gabapentin in animal model of epilepsy. Antiepileptic activity of testosterone and its metabolites encourage further investigation of androgens as promising candidates for treatment of epilepsy in men with androgens-related disorders.

Determination of Cytisine and N-Methylcytisine from Selected Plant Extracts by High-Performance Liquid Chromatography and Comparison of Their Cytotoxic Activity.

Quinolizidine alkaloids exhibit various forms of biological activity. A lot of them were found in the Leguminosae family, including Laburnum and Genista. The aim of the study was the optimization of a chromatographic system for the analysis of cytisine and N-methylcytisine in various plant extracts as well as an investigation of the cytotoxic activities of selected alkaloids and plant extracts obtained from Laburnum anagyroides, Laburnum anagyroides L. quercifolium, Laburnum alpinum, Laburnum watereri, Genista germanica, and Genista tinctoria against various cancer cell lines. The determination of investigated compounds was performed by High Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD), while High Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight-Mass Spectrometry (HPLC-QTOF-MS) was applied for the qualitative analysis of plant extracts. The retention, separation selectivity, peaks shape, and systems efficiency obtained for cytisine and N-methylcytisine in different chromatographic systems were compared. The application of columns with alkylbonded and phenyl stationary phases led to a very weak retention of cytisine and N-methylcytisine, even when the mobile phases containing only 5% of organic modifiers were used. The strongest retention was observed when hydrophilic interaction chromatography (HILIC) or especially when ion exchange chromatography (IEC) were applied. The most optimal system in terms of alkaloid retention, peak shape, and system efficiency containing an strong cation exchange (SCX) stationary phase and a mobile phase consisted of 25% acetonitrile and formic buffer at pH 4.0 was applied for investigating alkaloids analysis in plant extracts. Cytotoxic properties of the investigated plant extracts as well as cytisine and N-methylcytisine were examined using human tongue squamous carcinoma cells (SCC-25), human pharyngeal squamous carcinoma cells (FaDu), human triple-negative breast adenocarcinoma cell line (MDA-MB-231), and human breast adenocarcinoma cell line (MCF-7). The highest cytotoxic activity against FaDu, MCF-7, and MDA-MB cancer cell lines was observed after applying the Genista germanica leaves extract. In contrast, the highest cytotoxic activity against SCC-25 cell line was obtained after treating with the seed extract of Laburnum watereri. The investigated plant extracts exhibit significant cytotoxicity against the tested human cancer cell lines and seem to be promising for further research on its anticancer activity.

Modification of NO-cGMP Pathway Differentially Affects Diazepam- and Flunitrazepam-Induced Spatial and Recognition Memory Impairments in Rodents.

This study investigated the influence of sildenafil and methylene blue (MB), two modulators of the nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway on amnesic effects of two benzodiazepines (BZs) (diazepam (DZ) and flunitrazepam (FNZ)), in rodents-mice and rats. In the modified elevated plus maze (mEPM) and novel object recognition (NOR) tests, MB given ip at a dose of 5 mg/kg 5 min prior to DZ administration (0.25 or 1 mg/kg, sc) enhanced/induced memory impairment caused by DZ. When MB (2.5, 5, and 10 mg/kg) was applied 5 min prior to FNZ administration (0.05 and 0.1 mg/kg), an effect was opposite and memory impairment induced by FNZ was reduced. When sildenafil (2.5 and 5 mg/kg, ip) was applied 5 min prior to DZ, we observed a reduction of DZ-induced memory deficiency in the mEPM test. A similar effect of sildenafil was shown in the NOR test when the drug was applied at doses of 1.25, 2.5, and 5 mg/kg prior to DZ. In the mEPM test, sildenafil at abovementioned doses had no effects on FNZ-induced memory impairment. In turns, sildenafil administered at doses of 2.5 and 5 mg/kg increased the effect of FNZ on memory impairment in the NOR test. In conclusion, the NO-cGMP pathway is involved differentially into BZs-induced spatial and recognition memory impairments assessed using the NOR and mEPM tests. Modulators of the NO-cGMP pathway affect animal behavior in these tests in a different way depending on what benzodiazepine is applied.

Stepwise Glucoheptoamidation of Poly(Amidoamine) Dendrimer G3 to Tune Physicochemical Properties of the Potential Drug Carrier: In Vitro Tests for Cytisine Conjugates.

Third-generation poly(amidoamine) dendrimer (PAMAM) was modified by stepwise primary amine group amidation with d-glucoheptono-1,4-lactone. The physicochemical properties of the conjugates-size, ζ potential in lysosomal pH 5 and in neutral aqueous solutions, as well as intramolecular dynamics by differential scanning calorimetry-were determined. Internalization and toxicity of the conjugates against normal human fibroblasts BJ were monitored in vitro in order to select an appropriate carrier for a drug delivery system. It was found that initial glucoheptoamidation (up to 1/3 of amine groups of neat dendrimers available) resulted in increase of conjugate size and ζ potential. Native or low substituted dendrimer conjugates accumulated efficiently in fibroblast cells at nontoxic 1 µM concentration. Further substitution of dendrimer caused consistent decrease of size and ζ potential, cell accumulation, and toxicity. All dendrimers are amorphous at 36.6 °C as determined by differential scanning calorimetry (DSC). The optimized dendrimer, half-filled with glucoheptoamide substituents, was applied as carrier bearing two covalently attached cytisine molecules: a rigid and hydrophobic alkaloid. The conjugate with 2 cytisine and 16 glucoheptoamide substituents showed fast accumulation and no toxicity up to 200 µM concentration. The half-glucoheptoamidated PAMAM dendrimer was selected as a promising anticancer drug carrier for further applications.

Effects of androsterone on convulsions in various seizure models in mice.

It is believed that a deficiency of androgens, including free testosterone, may promote the development of convulsions. The present study revealed differences in the action of androsterone (AND), a major excreted metabolite of testosterone and a neurosteroid, in three commonly used seizure models in mice. AND administered intraperitoneally exhibited dose-dependent protection against tonic-clonic convulsions caused by maximal electroshock (MES) with ED(50) (effective dose(50)) of 227 mg/kg. The compound also inhibited the convulsive action of pentylenetetrazole (PTZ), increasing its CD(50) (convulsive dose(50)) for clonic convulsions from 77.2 (PTZ + saline) to 93.9 (p < 0.05) for PTZ + AND 40 mg/kg and 113.9 mg/kg (p < 0.001) for PTZ + AND 60 mg/kg. In mice pretreated with 60 mg/kg AND, the CD(50) for PTZ-induced tonic convulsions increased from 102 to 127.6 mg/kg (p < 0.01). Surprisingly, doses of 50 and 100 mg/kg AND lowered the CD(50) for kainate (KA)-induced convulsions from 40.8 to 28.7 (p < 0.05) and 25.4 mg/kg (p < 0.001), respectively. In summary, for two of the mouse seizure models, our findings confirmed previous studies that demonstrated protective activity of AND. However, the potentiation of KA-induced convulsions by AND was somewhat unexpected and suggested that AND may also possess proconvulsant activity.

Cytisine for nicotine addiction treatment: a review of pharmacology, therapeutics and an update of clinical trial evidence for smoking cessation.

AIMS: To review cytisine's history of use, pre-clinical evidence, clinical pharmacokinetics, efficacy, adverse reactions (ARs) and safety for smoking cessation. METHODS: A synoptic review of the use of cytisine as a smoking cessation medication, mechanism of action, pharmacokinetics and safety. Relevant literature on data included in these sections were identified through a search of 11 databases with additional literature obtained from reports and monographs. Three databases (PubMed, EMBASE and www.elibrary.ru) were systematically searched for studies published from 2012 to August 2018 in any language to provide an updated meta-analysis of cytisine's efficacy and ARs for smoking cessation compared with placebo. We pooled the relative risks (RR) of abstinence in the efficacy analysis and RR of ARs, either reported by the authors or calculated from the reports. RESULTS: Cytisine has been in use since 1964 and is currently marketed in 18 countries. Systemic bioavailability from oral ingestion is high and clearance is primarily renal, with minimal or no metabolism. Brain uptake in animal models is moderate. The plasma half-life averages 4.8 hours. Eight studies were included for meta-analysis of efficacy. With heterogeneous results, the overall RR versus placebo of successful continuous abstinence at the longest follow-up was 1.74 [95% confidence interval (CI) = 1.38-2.19]. Nausea, vomiting, dyspepsia, upper abdominal pain and dry mouth that were mild or moderate were the most common ARs, with RR versus placebo 1.10 (95% CI = 0.95-1.28). The cost of cytisine in eastern and central Europe is several-fold less than that of other smoking cessation medications. CONCLUSIONS: Cytisine is a low-cost medication found to increase the likelihood of smoking cessation. The most frequently reported ARs of cytisine involve gastrointestinal symptoms that are mostly reported as either mild or moderate in severity.

Effects of androsterone on the protective action of various antiepileptic drugs against maximal electroshock-induced seizures in mice.

This study evaluated the effect of androsterone (AND), a metabolite of testosterone, on the ability of selected classical and novel antiepileptic drugs to prevent seizures caused by maximal electroshock (MES), which may serve as an experimental model of human generalized tonic-clonic seizures in mice. Single intraperitoneal (i.p.) administration of AND (80 mg kg-1) significantly raised the threshold for convulsions in the MES seizure threshold test. Lower doses of AND (5, 10, 20, and 40 mg kg-1) failed to change the threshold. AND at a subthreshold dose of 40 mg kg-1 significantly enhanced the protective activity of carbamazepine, gabapentin, and phenobarbital against MES-induced seizures decreasing their median effective doses (ED50) values ± SEM from 8.59 ± 0.76 to 6.05 ± 0.81 mg kg-1 (p = 0.0308) for carbamazepine, from 419.9 ± 120.6 to 111.5 ± 41.1 mg kg-1 (p = 0.0405) for gabapentin, and from 20.86 ± 1.64 to 10.0 ± 1.21 mg kg-1 (p = 0.0007) for phenobarbital. There were no significant changes in total brain concentrations of carbamazepine, gabapentin, and phenobarbital following AND administration. This suggests that the enhancing effects of AND on the protective activity of these antiepileptic drugs are not related to pharmacokinetic factors. A lower dose of AND (20 mg kg-1) had no effect on the protective activity of carbamazepine, gabapentin, and phenobarbital. AND administered at a dose of 40 mg kg-1 failed to change the anticonvulsant activity of lamotrigine, oxcarbazepine, phenytoin, topiramate, and valproate in the MES test. In the chimney test, AND given at a dose enhancing the protective activity of carbamazepine, gabapentin, and phenobarbital (which alone was without effect on motor performance of mice) did not affect impairment of motor coordination produced by the antiepileptics. Our findings recommend further preclinical and clinical research on AND in respect of its use as adjuvant therapy in the management of epilepsy in men with deficiency of androgens.