Tae-Cheon Jeong

Progress in the Studies on Rutaecarpine

Rutaecarpine is an indolopyridoquinazolinone alkaloid isolated from Evodia rutaecarpa and related herbs, which has shown a variety of intriguing biological properties such as anti-thrombotic, anticancer, anti-inflammatory and analgesic, anti-obesity and thermoregulatory, vasorelaxing activity, as well as effects on the cardiovascular and endocrine systems. Recent progress in the studies on the isolation, synthesis, structure-activity relationship studies, biological activities and metabolism of rutaecarpine are reviewed.

Characterization of the Phase II metabolites of rutaecarpine in rat by liquid chromatography-electrospray ionization-tandem mass spectrometry

From the authors’ previous studies on the Phase I metabolism of rutaecarpine, nine metabolites formed were identified as products of hydroxylation on the aromatic rings in rat liver microsomes. In order to determine the possible metabolic fate of rutaecarpine, the Phase II metabolites of rutaecarpine were characterized in the present study by using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS). When male Sprague–Dawley rats were treated intravenously with 4u2009mgu2009kg−1 rutaecarpine, 16 different Phase I and II metabolites were identified in urine including four sulfate and four glucuronide conjugates. Phase I metabolites of rutaecarpine were identified as four mono-hydroxylated metabolites (M2–5) and four isobaric di-hydroxylated metabolites (M6–9). These metabolites were identical to the in vitro metabolites except one, which was hydroxylated in the aliphatic moiety. In addition, Phase II metabolites were identified as conjugated with sulfate (S1–4) and glucuronide (G1–4). In faeces, 11 different metabolites were identified. The metabolites M8 and glucuronide conjugated (G1–4) were not detected. Structures of all metabolites were confirmed with CID fragmentation spectra of MS2, MS3 and retention times by LC/ESI-MS.

Identification of glutathione conjugates and mercapturic acids of 1,2-dibromopropane in female BALB/c mice by liquid chromatography-electrospray ionization tandem mass spectrometry.

Based on recent results that 1,2-dibromopropane (1,2-DBP) causes hepatotoxicity and immunotoxicity in female BALB/c mice as well as a reduction of hepatic glutathione levels, the possible formation of glutathione conjugates and mercapturic acids of 1,2-DBP was investigated in vivo in the present studies. The following four metabolites were identified in the liver at 12u2009h after treatment with 1,2-DBP, by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS): M1, 2-hydroxypropylglutathione; M2, 2-oxopropylglutathione; M3, N-acetyl-S-(2-hydroxypropyl)-L-cysteine; and M4, N-acetyl-S-(2-oxopropyl)-L-cysteine. Ions of individual conjugates were observed at m/z 366, 364, 222 and 220, respectively. Characteristic product ions at m/z 237, 217, 204 and 202 for the identification of M1, M2, M3 and M4 were observed, respectively. In the sera isolated from the same animals, only mercapturic acids (M3 and M4) were observed by LC-ESI/MS. When female BALB/c mice were treated orally with 1,2-DBP at doses of 150, 300 and 600u2009mgu2009kg−1 once for 12u2009h, the production of glutathione conjugates and mercapturic acids in liver was apparently dose dependent, as were the concentrations of them in sera. When the production of metabolites from 1,2-DBP was investigated in liver following oral treatment with 600u2009mgu2009kg−1 1,2-DBP for 6, 12, 24 and 48u2009h, metabolite concentrations were greatest at the first time point (6u2009h). The results explain the authors’ previous studies that oral treatment with 1,2-DBP reduces the hepatic content of glutathione.

Enantioselective pharmacokinetics of sibutramine in rat

Racemic sibutramine is widely used to treat obesity owing to its inhibition of serotonin and noradrenaline reuptake in synapses. Although the enantioselective effects of sibutramine and its two active desmethyl-metabolites, monodesmethylsibutramine (MDS) and didesmethylsibutramine (DDS), on anorexia and energy expenditure have been elucidated, the enantioselective pharmacokinetics of sibutramine are still unclear. Therefore, we aimed to characterize the enantioselective pharmacokinetics of sibutramine and its metabolites in plasma and urine following an intravenous and a single oral administration of sibutramine in rats. The absolute bioavailability of sibutramine was only about 7%. The pharmacologically less effective S-isomer of DDS was predominant in the plasma: the Cmax and the AUCinf were 28 and 30 times higher than those of the R-isomer, respectively (p<0.001). In the urine, the concentrations of the R-isomers of hydroxylated DDS and hydroxylated and carbamoylglucuronized MDS and DDS appeared to be 11.3-, 5.1-, and 5.3-times the concentrations of the respective S-isomers. Thus, regardless of increased potency than the S-enantiomers, the R-enantiomers of the sibutramine metabolites MDS and DDS were present at lower concentrations, owing to their rapid biotransformation to hydroxylated and/or carbamoylglucuronized forms and their faster excretion in the urine. The present study is the first to elucidate the enantioselective pharmacokinetics of sibutramine in rats.

Simultaneous Determination of Glimepiride and its Metabolites in Human Plasma by Liquid Chromatography Coupled to a Tandem Mass Spectrometry

Glimepiride, a second-generation sulfonylurea, is a glucose-lowering agent widely used to treat diabetes mellitus. It is converted into metabolite M1 by CYP2C9, and M1 is then transformed into the carboxyl derivative M2 by cytosolic enzymes. In this study, we introduce a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determining glimepiride, M1, and M2 in human plasma. After simple protein precipitation with acetonitrile, the analytes were chromatographed on a reversed-phase CN column with a mobile phase of 10 mM ammonium acetate aqueous solution and acetonitrile (1:1, v/v). The accuracy and precision of the assay were in accordance with FDA regulations for the validation of bioanalytical methods. This method was used to measure the concentrations of glimepiride, M1, and M2 in plasma after a single oral 2-mg dose of glimepiride in volunteers.

Prevalidation trial for a novel in vitro eye irritation test using the reconstructed human cornea-like epithelial model, MCTT HCE™.

Here, we report the results of a prevalidation trial for an in vitro eye irritation test (EIT) using the reconstructed human cornea-like epithelium, MCTT HCE™. The optimal cutoff to determine irritation in the prediction model was established at 35% with the receiver operation characteristics(ROC) curve for 126 substances. Within-lab(WL) and between-lab(BL) reproducibility was tested for 20 reference substances by 3 participating laboratories. Viability data described by mean±SD or ±1/2 difference between duplicate wells, and scatter plots, demonstrated the WL/BL consistency. WL/BL concordance with the binary decision, whether non-irritant or irritant was estimated to be 85-95% and 95%, respectively. WL/BL reproducibility of viability data was further supported by a strong correlation(ICC, r>0.9). WL/BL agreement of binary decisions was also examined by Fleiss Kappa statistics, which showed a strong level of agreement (>0.78), nevertheless weaker than the reproducibility of the viability. The EIT with MCTT HCE™ exhibited a sensitivity of 82.2% (60/73), a specificity of 81.1% (43/53), and an accuracy of 81.8% (103/126) for 126 reference substances (for liquids; a sensitivity of 100% (47/47), a specificity of 70.6% (24/34), and an accuracy of 87.7% (71/81), and for solids, a sensitivity of 50% (13/26), a specificity of 100% (19/19), and an accuracy of 71.1% (32/45), suggesting that the accuracy is satisfactory but the sensitivity needs improvement, which shall be addressed through correcting the poor sensitivity for solid substances in future full validation trials.

Predictive capacity of a non-radioisotopic local lymph node assay using flow cytometry, LLNA:BrdU–FCM: Comparison of a cutoff approach and inferential statistics

In order for a novel test method to be applied for regulatory purposes, its reliability and relevance, i.e., reproducibility and predictive capacity, must be demonstrated. Here, we examine the predictive capacity of a novel non-radioisotopic local lymph node assay, LLNA:BrdU-FCM (5-bromo-2-deoxyuridine-flow cytometry), with a cutoff approach and inferential statistics as a prediction model. 22 reference substances in OECD TG429 were tested with a concurrent positive control, hexylcinnamaldehyde 25%(PC), and the stimulation index (SI) representing the fold increase in lymph node cells over the vehicle control was obtained. The optimal cutoff SI (2.7≤cutoff <3.5), with respect to predictive capacity, was obtained by a receiver operating characteristic curve, which produced 90.9% accuracy for the 22 substances. To address the inter-test variability in responsiveness, SI values standardized with PC were employed to obtain the optimal percentage cutoff (42.6≤cutoff <57.3% of PC), which produced 86.4% accuracy. A test substance may be diagnosed as a sensitizer if a statistically significant increase in SI is elicited. The parametric one-sided t-test and non-parametric Wilcoxon rank-sum test produced 77.3% accuracy. Similarly, a test substance could be defined as a sensitizer if the SI means of the vehicle control, and of the low, middle, and high concentrations were statistically significantly different, which was tested using ANOVA or Kruskal-Wallis, with post hoc analysis, Dunnett, or DSCF (Dwass-Steel-Critchlow-Fligner), respectively, depending on the equal variance test, producing 81.8% accuracy. The absolute SI-based cutoff approach produced the best predictive capacity, however the discordant decisions between prediction models need to be examined further.