Biotransformation of letrozole in rat liver microsomes: effects of gender and tamoxifen.

The in vitro metabolic kinetics of letrozole were investigated by incubating letrozole (10-500 microM) in female or male rat liver microsomes to assess the effect of gender and to predict the in vivo biotransformation characteristics of letrozole in rats. The effects of tamoxifen (TAM) on the metabolic kinetics of letrozole were also examined by incubating letrozole in female rat liver microsomes in the presence or absence of TAM. The effects of chronic pretreatment of female rats with TAM (0.5, 1.0, 5.0 mg/kg/day, i.p. for 7 consecutive days) on liver microsomal protein content and metabolic activity were also examined. The formation rate of the carbinol metabolite of letrozole, CGP44 645, was significantly higher (p<0.05) in male rat liver microsomes in comparison to female. The V(max)/K(m) ratio for letrozole metabolism in female rat liver microsomes did not change significantly (p>0.05) in the presence of TAM. After chronic pretreatment of female rats with TAM (up to a dose of 1.0mg/kg/day), the hepatic microsomal protein content was significantly increased but the formation rate of CGP44 645, when normalized for protein content, did not change significantly. These results suggest that there is a marked gender difference in letrozole metabolism in rats. It also appears that acute treatment of female rat liver microsomes with TAM produces negligible inhibitory effect on the CYP mediated metabolic clearance of letrozole. However, chronic pretreatment of female rats with TAM appear to induce CYPs, but does not significantly impact the metabolic activities of the enzymes associated with the formation of the carbinol metabolite of letrozole.

Differential tramadol and O-desmethyl metabolite levels in brain vs. plasma of mice and rats administered tramadol hydrochloride orally.

OBJECTIVE: To investigate a possible differential brain uptake of tramadol vs. its major metabolite (O-desmethyl tramadol; M1) in mice and rats. METHODS: An extraction and measurement technique (gas chromatograph equipped with a nitrogen phosphorus detector) was used to measure plasma and brain levels of tramadol and M1 at intervals 10-300 min after oral dosing of tramadol hydrochloride to mice and rats. RESULTS: For all doses of tramadol administered (5, 10, 20, or 40 mg/kg), tramadol and M1 plasma levels were greatest 10 min after dosing: in mice, peak tramadol plasma levels were 47.75-736.72 ng/mL and peak M1 levels were 75.30-1084.92 ng/mL; in rats, peak tramadol plasma levels were 185.03-455.81 ng/mL and peak M1 levels were 106.74-455.70 ng/mL. Tramadol brain levels were also greatest 10 min after dosing. In mice, peak tramadol brain levels were 226.42-1847.46 ng/g. Peak M1 levels (72.17-572.97 ng/g) occurred 20-60 min after dosing. In rats, peak tramadol brain levels were 258.50-1777.37 ng/g and peak M1 levels were 80.35-289.60 ng/g. In mice, the ratio of tramadol/M1 in plasma was 0.5-1.0 throughout the measurements, whereas the ratio in brain was about 10 at 10 min and about 2 from 20 to 50 min. In rats, the ratio of tramadol/M1 in plasma was 0.5-1.5, whereas the ratio in brain was about 15 at 10 min and about 4-7 thereafter. CONCLUSION: In mice and rats, there appears to be preferential brain vs. plasma distribution of tramadol over M1.

Gas chromatographic method using nitrogen-phosphorus detection for the measurement of tramadol and its O-desmethyl metabolite in plasma and brain tissue of mice and rats.

A method that allows the measurement of plasma and brain levels of the centrally-acting analgesic tramadol and its major metabolite (O-desmethyl tramadol) in mice and rats was developed using gas chromatography equipped with nitrogen-phosphorus detection (GC-NPD). Plasma samples were extracted with methyl tert.-butyl ether (MTBE) and were injected directly into the GC system. Brain tissue homogenates were precipitated with methanol, the resulting supernatant was dried then acidified with hydrochloric acid. The aqueous solution was washed with MTBE twice, alkalinized, and extracted with MTBE. The MTBE layer was dried, reconstituted and injected into the GC system. The GC assay used a DB-1 capillary column with an oven temperature ramp (135 to 179 degrees C at 4 degrees C/min). Dextromethorphan was used as the internal standard. The calibration curves for tramadol and O-desmethyl tramadol in plasma and brain tissue were linear in the range of 10 to 10000 ng/ml (plasma) and ng/g (brain). Assay accuracy and precision of back calculated standards were within +/- 15%.

Determination of the adsorption of tramadol hydrochloride by activated charcoal in vitro and in vivo.

Although tramadol is one of the most widely used centrally acting analgesics worldwide, no literature is available regarding adsorption of tramadol HCl powder or tablets (Ultram; 50 mg tramadol HCl per tablet) by activated charcoal (AC) for use as potential adjunct treatment of overdose. The present study incorporated a novel combination of in vitro and in vivo methods to investigate this question. Based on a binding curve of tramadol UV absorbance (UV(a); 225 nm) plotted against the amount of AC, the ratio of amount of tramadol completely adsorbed by AC was 0.05 mg/mg. Also based on UV(a), no tramadol was detected in filtrate of slurries in which up to 62 tablets of Ultram were mixed with 50 g AC; 4.6% of unbound tramadol was detected when 100 tablets of Ultram were mixed with AC. The ratio of amount of tramadol completely adsorbed by AC in this test was 0.10. In vivo, co-administration of 0.1 g/ml of AC produced a 13- to 14-fold rightward shift in tramadol's antinociceptive dose-response curve and a 1.6-fold rightward shift in tramadol's lethality dose-response curve.

Sensitive capillary gas chromatographic-mass spectrometric-selected-ion monitoring method for the determination of diclofenac concentrations in human plasma.

We have modified and validated a capillary GC-MS method reported by Kadowaki et al. [J. Chromatogr., 308 (1984) 329] for the determination of diclofenac in human plasma by using heptane rather than benzene as an extraction agent. In addition, acetone was added to the samples as a deproteination agent which increased the recovery of diclofenac. These revised processes allowed clean extraction and near-quantitative recovery of analyte (> 95%). Separation was achieved on an HP-1 column with helium as carrier gas. The parent ion peaks of diclofenac (m/z 277) and the internal standard, 4'-methoxydiclofenac (m/z 307), were monitored by a mass-selective detector using the selected-ion monitoring mode. The linear range for the routine assay was from 5 to 2000 ng/ml. The detection and lower quantifiable limits were 0.2 and 1 ng/ml, respectively, with no interference from plasma. The within-day and between-day coefficients of variation for high and medium concentrations were less than 5% and were less than 13% for low concentrations (10 ng/ml). This GC-MS assay method has been used for pharmacokinetic and drug interaction studies in humans.

Anticonvulsant activity of indanylspirosuccinimide Mannich bases.

A series of Mannich bases derived from spiro [indan-1,3'pyrrolidine-2',5'-dione] were evaluated for anticonvulsant activity. This activity varied as a function of the amine substituent and several compounds showed protective effects in both the maximal electroshock (MES) and subcutaneous pentylenetetrazol (scMet) assays. In addition, fluorenyl- and cyclopentyl-derived spirosuccinimides, as well as extended chain analogues, were tested and proved to be inactive. A time versus effect study was conducted employing the MES assay and the hydroxyethylpiperazine-derived Mannich base of spiro [indan-1,3 pyrrolidine-2',5',dione]. This compound acted rapidly and its protective half-life was increased as larger doses were administered.