Simultaneous determination of nicotine and its metabolite, cotinine, in rat blood and brain tissue using microdialysis coupled with liquid chromatography: pharmacokinetic application.

To elucidate the disposition of nicotine in the brain is important because the neuropharmacological effects from nicotine exposure are centrally predominated. The aim of the present study was to develop a rapid and simple method for the simultaneous determination of unbound nicotine and its main metabolite, cotinine, in rat blood and brain tissue. We coupled a multiple sites microdialysis sampling technique with HPLC-UV system to characterize the pharmacokinetics of both nicotine and cotinine. Microdialysis probes were inserted into the jugular vein/right atrium and brain striatum of Sprague-Dawley rats, and nicotine (2 mg/kg, i.v.) was administered via the femoral vein. Dialysates were collected every 10 min and injected directly into a HPLC system. Both nicotine and cotinine were separated by a phenyl-hexyl column (150 mm x 4.6 mm) from dialysates within 12 min. The mobile phase consisted of an acetonitrile-methanol-20 mM monosodium phosphate buffer (55:45:900, v/v/v, pH adjusted to 5.1) with a flow-rate of 1 ml/min. The wavelength of the UV detector was set at 260 nm. The limit of quantification for nicotine and cotinine were 0.25 microg/ml and 0.05 microg/ml, respectively. Intra- and inter-day precision and accuracy of both measurements fell well within the predefined limits of acceptability. The blood and brain concentration-time profile of nicotine and cotinine suggests that nicotine is easily to get into the central nervous system and cotinine exhibits a long retention time and accumulates in blood.

Dimemorfan N-demethylation by mouse liver microsomal cytochrome P450 enzymes.

Dimemorfan (d-3-methyl-N-methylmorphinan), an analogue of dextromethorphan, is commonly used as a non-opioid antitussive. To clarify the contribution of cytochrome P450 (P450) in dimemorfan N-demethylation, effects of selective inducers and inhibitors were studied in ICR mice. Phenobarbital (PB)- and dexamethasone (Dex)-treatments caused 5-fold increases of liver microsomal dimemorfan N-demethylation activity. In untreated mouse liver microsomes, demethylation activity was strongly inhibited by a CYP3A inhibitor, ketoconazole. In PB-and Dex-treated mouse liver microsomes, ketoconazole caused strong inhibition, whereas orphenadrine caused a decrease of less than 20%. Pretreatment of control mouse liver microsomes with anti-CYP3A inhibited demethylation activity, whereas pre-treatment with anti-CYP2B had no effect. In PB-and Dex-treated mouse liver microsomes, the demethylation activity was inhibited by both anti-CYP3A and anti-CYP2B. In control mice, the intrinsic clearance of dimemorfan from N-demethylation was 5.8 microl min(-1)mg protein(-1). In PB- and Dex-treated mice, the correlation coefficient of fitting using one-enzyme and two-enzyme models were similar. The intrinsic clearances of induced mouse liver microsomes were similar. These results revealed that CYP3A played a major role in hepatic demethylation in untreated mice. Both CYP3A and CYP2B were involved in this demethylation in PB- and Dex-treated mice.

Prevention of macrophage adhesion molecule-1 (Mac-1)-dependent neutrophil firm adhesion by taxifolin through impairment of protein kinase-dependent NADPH oxidase activation and antagonism of G protein-mediated calcium influx.

Taxifolin has been reported to down-regulate the expression of intercellular adhesion molecule-1 (ICAM-1), a receptor-mediating firm adhesion with beta2 integrin (e.g., Mac-1) expressed on leukocytes. To evaluate whether taxifolin could modulate Mac-1-dependent firm adhesion by neutrophils, and the possible mechanism(s) underlying its anti-inflammatory action, its effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol-12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were studied. Pretreatment with taxifolin (1-100 microM) concentration-dependently diminished fMLP- or (PMA)-induced Mac-1-dependent firm adhesion and upexpression of surface Mac-1. Mobilisation of intracellular calcium and production of reactive oxygen species (ROS) signal the upexpression of Mac-1 and firm adhesion by neutrophils. Taxifolin impeded the calcium influx induced by fMLP (a receptor-mediated activator) or AlF(4)(-) (a G protein-mediated activator). Taxifolin also effectively inhibited the fMLP- or PMA-induced ROS production with 50% inhibitory concentration (IC(50)) less than 10microM, possibly through impairing the activation of NADPH oxidase, a major ROS-generating enzyme in neutrophils, by restricting the activation of p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase C (PKC). In conclusion, we propose that impairment of ROS production by NADPH oxidase through interfering with p38 MAPK- and/or PKC-dependent signals, and antagonism of G protein-mediated calcium influx may account for the inhibition of Mac-1-dependent neutrophil firm adhesion that confers taxifolin the anti-inflammatory activity.

Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata.

We have previously shown that a concentrated ethanol extract of the fruiting bodies of Antrodia camphorata exhibited immunomodulating effects in human leukocytes and fourteen compounds including zhankuic acids A, B, C, and antcin K were identified in the extract. In this study, an acute cellular model in isolated peripheral human neutrophils was established to elucidate the anti-inflammatory effects of these compounds. Reactive oxygen species (ROS) production and firm adhesion by neutrophils display two important responses during inflammation. To evaluate whether these compounds could prevent inflammatory responses by neutrophils, their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were examined. Pretreatment with 1 - 25 microM of zhankuic acids A, B, C, or antcin K concentration-dependently diminished fMLP- or PMA-induced ROS production, as measured by a lucigenin-amplified chemiluminescence, with IC (50) (microM) around 5 - 20 microM. Zhankuic acids A, B, C, or antcin K also effectively inhibited the fMLP- or PMA-induced firm adhesion without interfering with the up-expression of surface Mac-1 (CD11b/CD18), a beta2 integrin mediating the firm adhesion of neutrophils to endothelium. The anti-inflammatory actions of these drugs were not due to cytotoxic effects because no significant difference in cell viability was observed compared to vehicle control. These data suggest that inhibition of both ROS production and firm adhesion by neutrophils has no significant cytotoxic effect that could give these drugs the potential to be anti-inflammatory agents for the clinical treatment.