| As the change of environment, resources and population, cardiovascular disease, cancer, viral infections and other major disease research are threatening human health, cardiovascular disease has been a hot area of research around the world. As the development of Marine resources, Marine microorganism is an important part of marine biological. Special environment of ocean makes the marine microorganisms different greatly with terrestrial microorganisms in metabolism and genetic structure and so on. In this paper, we studied the structural identification and fibrinolytic evaluation of FGFC1; The pharmacokinetics and tissue distribution of FGFC1 in Beagle dogs; The absorption and transportation characteristic of FGFC1 in CACO-2 cell monolayer; The Effects of FGFC1 on cytochrome P450 in Vitro and its safety evaluation.In the first chapter, we summarized the research status of thrombolytic drugs at home and abroad. Meanwhile this chapter introduced the methods of pharmacokinetics in vivo, such as isotope tracing methods and Metabonomics, and the methods of pharmacokinetics in vitro.In the second chapter, the structure of FGFC1 was elucidated by 1H NMR, 13 C NMR and MS datas; moreover, it was also evaluated for fibrinolytic activity in vitro and in vivo. The results showed that 0.1-0.4 mmol/L of FGFC1 could stimulate generation of plasmin activity(increased by 2.05-11.44 folds) by measuring Glu-plasminogen and Lys-plasminogen activation in vitro. The experiment of fluorescein isothiocyanate(FITC)-fibrinogen degradation indicated that the effect of FGFC1 on fibrinolytic activity was mediated by plasminogen and scu PA. In addition, FGFC1(10 mg/kg) could dissolve most of pulmonary thrombus of Wistar rat in vivo.In the third chapter, we studied the pharmacokinetics and tissue distribution of FGFC in Beagle dogs. The dog plasma samples was collected at different intervals after intravenous injection of three different doses(7.5, 5.0, 2.5mg·kg-1) of FGFC1, and the concentration of FGFC1 in plasma and tissue was determined by HPLC method for estimating pharmacokinetic parameters and tissue distribution. The result of pharmacokinetics: Its elimination half-life(T1/2β) being 49.035±2.171, 48.422±2.113 and 48.811±2.372 min, respectively; The peak concentration(Cmax) was 56.48±6.23, 48.63±5.53, 13.64±2.76 μg.m L-1, respectively; clearance rate(CL) being 0.0062±0.0004, 0.0071±0.0008 and 0.0092 ±0.0006 L·min-1·kg-1, respectively; mean retention time(MRT) being 28.17±1.16, 26.23±0.35 and 28.66±0.84 min, respectively. it showed a good pharmacokinetic profile. Tissue distribution revealed that FGFC1 could quickly distributed in to the heart, liver, spleen, lung, kidney, intestine, stomach, brain, intestine, testicle, urine and feces. Interestingly, the highest drug(FGFC1) concentration level was detected in the liver.In the fourth chapter, we studied the CACO-2 cell monolayer. The transportation characteristic was evaluated by Papp, efflux ration and the mean total recoveries of FGFC1 with concentration of 0.5~5μmol·L-1 with LC/MS/MS. The total recovery at the APâ†'BL direction is(35.778±1.285)%-(52.336±4.342)%, the total recovery at the BLâ†'AP direction is(25.485±1.131)%-(53.022 ± 2.011)%, Papp APâ†'BL and Papp BLâ†'AP are both below 2.5×10-6 cm·s-1, the efflux rate at the APâ†'BL direction is(8.745 ± 0.155)%-(26.759 ± 1.024)%, the efflux rate at the BLâ†'AP direction is(25.549 ± 1.551)%-(52.421 ± 2.203)%, the outside ratio close to 1.5. Passive diffusion is the major pattern of which absorption of FGFC1 in CACO-2 cell model. Low penetrability of FGFC1 indicated that the intravenous administration is more appropriate than oral.In the fifth chapter, we studied the CYP450. Three specific probe drugs were utilized for co-incubating in rat liver microsomes, including phenacetin(CYP1A2), chlorzoxazone(CYP2E1) and midazolam(CYP3A4).The concentrations of the specific probe drugs’ metabolites and the specific probe drugs were determined by HPLC. Pathomorphology of rat liver was observed with HE staining to describe the potential hepatotoxicity. The ECG of rat and rabbit was measured by Multichannel biological signal acquisition instrument. As a result, the high dose group(41.6 g·kg-1·d-1) had a significant inducing effect on CYP3A4, the pathomorphology of rat liver showed the FGFC1 resulted in minor injury on liver tissue, and in the experiment of ECG, FGFC1 has no significant impact on heart activity of rats and rabbits. The drug-drug interactions as well as hepatic injury might occur when FGFC1 taken in high dose administration.In conclusion, the structure of FGFC1 was elucidated by 1H NMR, 13 C NMR, IR, and MS data,FGFC1 named 2,5 –bis((2R,3R)-2-((E)-4,8 –dimethylnona-3,7-dien-1-yl)-3,5-dihydroxy-2-methyl-7-oxo-3,4,7,9-tetrahydropyrano[2,3-e]isoindol-8(2H)-yl)pentanoic acid; meanwhile, it was also evaluated for fibrinolytic activity in vitro and in vivo. In the Beagle dogs, FGFC1 showed a good pharmacokinetic profile and Tissue distribution. Passive diffusion is the major pattern of which absorption of FGFC1 in CACO-2 cell model. Low penetrability of FGFC1 indicated that the intravenous administration is more appropriate than oral. The ECG of rat and rabbit was measured by Multichannel biological signal acquisition instrument. The drug-drug interactions as well as hepatic injury might occur when FGFC1 taken in high dose administration. |
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