Clinical Pharmacokinetic and Bioequivalence Studies of Two Brands of Cephradine in Healthy Korean Using HPLC Method

The goal of our research was to compare the pharmacokinetics and evaluate the bioequivalence of two brands of cephradine 500 mg capsules in 24 normal Korean volunteers. The plasma samples were acquired at 13 time points for 8 h after administration. The concentrations of cephradine in human plasma were measured by a high-performance liquid chromatography (HPLC). Isocratic mobile phase which consisted of acetonitrile, methanol, and 20 mM potassium phosphate (15/5/80, v/v/v, pH 3.48) was used to separate the analytical column cosmosil cholester (250 × 4.6 mm, 3 μm). Analytes were detected in ultraviolet (260 nm). The novel analytical method was described as simple sample preparation, a short retention time (less than 6 min) and making it suitable for use in clinical trials. Pharmacokinetic parameters, such as AUC0-t (20.54 vs 18.42 μg·h/mL), AUC0-infinity (21.22 vs 19.14 μg·h/mL), Cmax (12.69 vs 12.81 μg/mL), Tmax (1.22 vs 0.92 h), half-life (1.02 vs 1.13 h), extrapolation (3.22% vs 3.75%), and Ke (0.73 vs 0.69 h–1) were determined for the reference and test drugs in plasma. Pharmacokinetic parameters with a 90% confidence interval were 87% - 95% for AUC0-t and 91% - 115% for Cmax. They were satisfied within the bioequivalence range 80% - 125% of the KFDA guidelines. Therefore, our HPLC method was well applied in a bioequivalence and pharmacokinetic study of two formulations in normal subjects.

Anti-esophagus cancer activity and mechanism of DN3,a novel natural diterpenoid derivative,as a dual inhibitor of glycolysis and oxidative phosphorylation

OBJECTIVE To probe into the anti-esophagus cancer activity and mechanisms of DN3,a novel natural diterpenoid derivative.METHODS The anti-tumor activity in vitro of DN3 was evaluated by MTT,and by using human esophageal carcinoma cells xenografted into athymic mice model in vivo.The specific mechanisms of DN3,as a dual inhibitor of glycolysis and oxidative phos.phorylation(OXPHOS) were explored through cell and molecular biology techniques.For instance,the manner of cancer cell death induced by DN3 was characterized by hoechst33342,FITC-Annexin V/PI staining and flow cytometric analysis,then these changes of glucose consumption,glucose uptake and lactate production in glycolysis,as well as oxygen consumption rate(OCR) and ATP content in OXPHOS caused by DN3 were performed separately through related kits and SeahorseBioscience XF24 Extra.cellular Flux Analyzer.Furthermore,in order to obtain a clear understanding of the inhibition of DN3 to glycolysis and OXPHOS,these regulatory factors were investigated by Western blot,such as PI3K/AKT,c-Myc and p53 of glycolysis,Bax and HK2 of mitochondrial function.RESULTS DN3 inhibited the growth of esophagus cancer cell EC9706,EC109 and EC1 cells in a dose and time dependent manner,but showed no significant effects on human esophageal epithelial cells(HEECs).DN3 caused significant G2/M arrest of esophagus cancer cell lines and induced apoptosis of these cell lines,which indicated DN3 inhibited the growth of esophagus cancer cell through blocking cell cycle and inducing apoptosis in a dose and time-dependent manner.Importantly,8 μM DN3 decreased the extracellular acidification rate(ECAR) by 45% in EC109,which indicated glycolysis was inhibited by DN3.Mean.while,DN3 decreased the oxygen consumption rate(OCR) and the OCR linked to intracellular ATP production in EC109 cells,but that was not obvious in HEECs,so which indicated that DN3 could selec.tively block OXPHOS of cancer cells.In addition,the accumulation of reactive oxygen species(ROS)and the drop of mitochondrial membrane potential(MMP) were also observed in EC109 incubated by DN3,which suggested mitochondrial biological function was disturbed.Furthermore,the expression of PI3K/AKT,c-Myc and HK2 related to glycolysis were down-regulated by DN3,but the p53 and Bax were up-regulated in esophageal carcinoma cells.The changes of these enzymes accounted for the decreased glycolysisand OXPHOS in esophageal carcinoma cells treated by DN3.CONCLUSION The new compound DN3 has a strong anti-esophageal carcinoma activity,and it is tolerable that DN3 is seen as a dual inhibitor of glycolysis and oxidative phosphorylation.

Metabolic heterogeneity of gastric cancer cell lines

OBJECTIVE Gastric cancer is one of the most common malignant tumors,and the incidence rate is the highest in all kinds of tumors in China.However,it remains unclear that how signifi.cantly gastric cells are dependent on glycolysis,and which type of gastric cells are sensitive to glycolysis inhibition.In this study,several kind of gastric cancer cell lines were used as the research object,and the metabolic characteristics of different cell lines were systematically analyzed to provide theoretical support for the accurate treatment of gastric cancer.METHODS We examined the energy metabolism of four gastric cancer cell lines(MGC-803,SGC-7901,HGC-27 and BGC-823) by using glycolysis inhibitor,2-deoxy-D-glucose(2-DG) and inhibitor of oxidative phosphorylation,oligomycin.Oxygen consumption rates(OCR) and L-lactate were also measured with an XF96 Analyzer(Seahorse Biosciences) to deter.mine the significance of metabolism of oxidative phosphorylation and aerobic glycolysisin gastric cells.In addition,western blot was used to detect the contribution of AMP-activated protein kinase(AMPK),and anti-apoptotic proteins(Bcl-2 and survivin) to clarify the mechanism of death or survival of gastric cancer cells treated by 2-DG or oligomycin.RESULTS In this study,it was shown that the growth of gastric cell lines were suppressed by 2-DG.However,the sensitivity to 2-DG was quite different among cell lines:IC 50 of 2-DG was from 3.28 mmol·L^(-1)(MGC-803) to 15.57 mmol·L^(-1)(BGC-823).MGC-803 was relatively sensitive to 2-DG(IC 50:3.28 mmol·L^(-1)),consumed more glucose and produced more lactate(waste product of glycolysis) than the three other cell lines.Consequently,MGC-803 could be more dependent on glycolysis than other cell lines,which was further confirmed by the fact that glucose(+) FCS(-) medium showed more growth and survival than glucose(-) FCS(+) medium.Alternatively,BGC-823,most resistant to 2-DG(IC50:15.57 mmol · L-1),was most sensitive to oligomycin,and showed more growth and survival in glucose(-) FCS(+) medium than in glucose(+) FCS(-) medium.Thus,we had reasons to think BGC-823 cells depended on oxidative phosphorylation for energy production.In BGC-823,AMPK,which is activated when ATP becomes limiting,was rapidly phosphorylated by 2-DG,and expression of Bcl-2 was augmented,which might result in resistance to 2-DG.Interestingly,AMPK phosphorylation and augmentation of Bcl-2 expression by 2-DG were not observed in MGC-803,which is 2-DG sensitive.CONCLUSION There is a large metabolic difference between gastric cancer cell lines,which will facilitate the future gastric cancer therapy by targeting metabolic pathways.

Antitumor activityand mechanism of JD20,a newly synthetic analog of natural ent-kaurane diterpenoid

OBJECTIVE To study the anti-tumor activity and molecular mechanism of natural diter.pene derivative JD20 in vitro.METHODS Screening the sensitive of gastric carcinoma cell lines to JD20 by cytotoxicity test for 24 h.Cell morphology was evaluated by using DAPI.After staining of can.cer cells with PI or annexin V-FITC/PI respectively,the cell cycle and apoptosis induced by JD20 were detectded by flow cytometry.The change in cell membrane potential was detected by JC-1 test kit.Western blot method was used to detect the apoptosis-related protein.RESULTS The novel natural kaurane diterpene derivative JD20 had a significant inhibitory effect on tumor cells and was particularly active on gastric cancer cell lines HGC-27(IC50=4.72±1.37 μmol·L-1) and MGC-803(IC50=7.36±0.86 μmol·L^(-1)).Further studies found that JD20 resulted in thecell cycle arrest in the G2/M phase,and induced a significant apoptosis in HGC-27.In addition,JD20 also caused the drop of mitochondrial membrane potential of HGC-27 within a short time(3 h).Furthermore,the Western blotting analysis showed that JD20 could induce the up-regulation of p53,Bax and Bim protein expression in gastric can.cer cells,and the releasing of cytochrome c from the mitochondria into the cytoplasm,as well as the ac.tivation of casepase-9/3.CONCLUSION The natural kaurane diterpene derivative JD20 can inhibit the proliferation of various human cancer cells by blocking the cell cycle and inducing apoptosis,and its mechanism of inducing apoptosis may be related to the mitochondria-mediated apoptosis pathway.

Osthole decreases collagen Ⅰ/III contents and their ratio in TGF-β1-overexpressed mouse cardiac fibroblasts through regulating the TGF-β/Smad signaling pathway

The present study was designed to elucidate whether the mechanism by which osthole decreases collagenⅠ/III contents and their ratio is regulating the TGF-β/Smad signaling pathway in TGF-β1-overexpressed mouse cardiac fibroblasts(CFs). These CFs were cultured and treated with different concentrations of osthole. Our results showed that the TGF-β1 expression in the CFs transfected with that the recombinant expression plasmids pc DNA3.1(+)-TGF-β1 was significantly enhanced. After the CFs were treated with 1.25-5 μg·m L^(-1) of osthole for 24 h, the m RNA and protein expression levels of collagensⅠand III were reduced. The collagen Ⅰ/III ratio was also reduced. The m RNA and protein expression levels of TGF-β1, TβRⅠ, Smad2/3, P-Smad2/3, Smad4, and α-SMA were decreased, whereas the expression level of Smad7 was increased. These effects suggested that osthole could inhibit collagen Ⅰ and III expression and reduce their ratio via the TGF-β/Smad signaling pathway in TGF-β1 overexpressed CFs. These effects of osthole may play beneficial roles in the prevention and treatment of myocardial fibrosis.

Comparison of the inhibition potentials of icotinib and erlotinib against human UDP-glucuronosyltransferase 1A1

UDP-glucuronosyltransferase 1A1(UGT1A1) plays a key role in detoxification of many potentially harmful compounds and drugs. UGT1A1 inhibition may bring risks of drug–drug interactions(DDIs), hyperbilirubinemia and drug-induced liver injury. This study aimed to investigate and compare the inhibitory effects of icotinib and erlotinib against UGT1A1, as well as to evaluate their potential DDI risks via UGT1A1 inhibition. The results demonstrated that both icotinib and erlotinib are UGT1A1 inhibitors, but the inhibitory effect of icotinib on UGT1A1 is weaker than that of erlotinib. The IC_(50) values of icotinib and erlotinib against UGT1A1-mediated NCHN-O-glucuronidation in human liver microsomes(HLMs) were 5.15 and 0.68 μmol/L, respectively. Inhibition kinetic analyses demonstrated that both icotinib and erlotinib were non-competitive inhibitors against UGT1A1-mediated glucuronidation of NCHN in HLMs, with the Kivalues of 8.55 and 1.23 μmol/L, respectively. Furthermore, their potential DDI risks via UGT1A1 inhibition were quantitatively predicted by the ratio of the areas under the concentration–time curve(AUC) of NCHN. These findings are helpful for the medicinal chemists todesign and develop next generation tyrosine kinase inhibitors with improved safety, as well as to guide reasonable applications of icotinib and erlotinib in clinic, especially for avoiding their potential DDI risks via UGT1A1 inhibition.