Quantitative determination of duloxetine and its metabolite in rat plasma by HPLC-MS/MS.

The major metabolite of duloxetine is a glucuronide conjugate of 4-hydroxy duloxetine (4-HD). However, interestingly, there have been no reports determining concentrations of 4-HD and no fully validated method has been established for measuring duloxetine and 4-HD in rat plasma. We developed a method for the simultaneous quantification of duloxetine and its metabolite in rat plasma using high-performance liquid chromatography tandem mass spectrometry. Duloxetine and 4-HD were analyzed on a reverse-phase C18 analytical column after protein precipitation of the plasma sample with methanol, using carbamazepine as an internal standard. The isocratic mobile phase of 5 mm ammonium acetate-methanol (4:6, v/v) was eluted at 0.4 mL/min. Quantification was performed on a triple-quadrupole mass spectrometer using electrospray ionization, and the ion transition monitored in selective reaction monitoring mode. The coefficient of variation for assay precision was <18.0%, and the accuracy was 84.0-118.0%. This method was successfully used to measure the concentrations of duloxetine and its metabolite in plasma following the oral administration of a single 40 mg/kg dose in rats.

Capsaicin induced apoptosis of B16-F10 melanoma cells through down-regulation of Bcl-2.

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a pungent ingredient of hot chili peppers, has been reported to possess substantial anticarcinogenic and antimutagenic activities. In the present study, we investigated the effect of capsaicin on induction of apoptosis in highly metastatic B16-F10 murine melanoma cells. Capsaicin inhibited growth of B16-F10 cells in a concentration-dependent manner. Proapoptotic effect of capsaicin was evidenced by nuclear condensation, internucleosomal DNA fragmentation, in situ terminal nick-end labeling of fragmented DNA (TUNEL), and an increased sub G1 fraction. Treatment of B16-F10 cells with capsaicin caused release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase in a dose-dependent manner. Furthermore, Bcl-2 expression in the B16-F10 cells was slightly down-regulated by capsaicin treatment. In contrast, there were no alterations in the levels of Bax in capsaicin-treated cells. Collectively, these findings indicate that capsaicin-induces apoptosis of B16-F10 melanoma cells via down-regulation the Bcl-2.

Stability Evaluation of National Reference Standards for Blood Products in Korea.

National reference standards (NRSs) for biologics are established through potency estimation by a multicenter joint study of standard materials used in the approval process for national lot release and quality control of vaccines, blood products, and other biologics. In this study, a stability evaluation was conducted to determine whether the potency of NRSs for six blood products was being maintained at a consistent level in Korea. The present study conducted real-time stability tests via in-vivo/in-vitro bioassay on NRSs for blood coagulation factor VIII concentrate (2nd standard), antithrombin concentrate, prekallikrein activator, anti-hepatitis B immunoglobulin, blood coagulation factor IX concentrate, and anti-tetanus human immunoglobulin, as well as a trend analysis using cumulative annual results. The real-time stability test results showed that the mean potency of six NRSs was all within the control limit. In the trend analysis, the potency of NRS for blood coagulation factor VIII concentrate (2nd standard) showed a decreasing trend, while the potency of all other products had been stably maintained. The present study confirmed that the mean potency of NRSs for six blood products had been stably maintained in Korea. The findings of the present study establish a foundation that can ensure the quality of NRSs for biologics in Korea, and it is expected to make a major contribution to the supply of high-quality biologics.

Characterization of changes in global gene expression in the brain of neuron-specific enolase/human Tau23 transgenic mice in response to overexpression of Tau protein.

Tau is a neuronal phosphoprotein responsible for the formation of the neurofibrillary tangles in Alzheimer's disease. To characterize the changes in global gene expression in the brain of transgenic mice that overexpress human Tau23 protein in response to the increase of Tau23 phosphorylation, total RNA extracted from the hippocampus of 12-month-old transgenic and wild-type mice was converted to cDNA, labeled with biotin and hybridized to oligonucleotide microarrays. The microarray results were confirmed by real-time RT-PCR and Western blotting method. It was determined that 43 genes were up-regulated and 8 genes were down-regulated by Tau23 in transgenic mice compared to controls, based on the arbitrary difference in the 2-fold change. Among the up-regulated transcripts, those encoding for transporter and oxidoreductase were dramatically over-represented, followed by those related to regulatory molecule, cytoskeletal protein, signaling molecule, and extracellular matrix protein. Genes encoding for transcription factor, regulatory molecule, miscellaneous function, and chaperone were significantly reduced in the down-regulated group. The major genes in the up-regulated categories included Ecrg4, Folr1, Defb11, Aqp1 and Soctdc1. The major genes in the down-regulated categories were Ncor1, Gpm6a, and Hspd1. These results indicate that the microarray analysis identifies several gene functional groups and individual genes that respond to a sustained increase in Tau23 phosphorylation levels in the brain of transgenic mice. In addition, the results suggest the microarray test is a useful tool for increased understanding of the role of Tau23 protein in regulating neurodegenerative disorders.