| Drug metabolism is an important part in new drug development and the bioavailability of oral drugs and the drug-drug interaction are the pivotal guideline to evaluate the pharmacokinetics. The drug-drug interaction may occur when they combine the same CYP isozyme competitively which leads to enhancing plasma concentration and serious adverse reaction subsequently. In the other hand, drugs can induce and/or inhibit certain CYP450 in expression or activity levels when they enter the body, thereby modify the biotransformation of themselves and other drugs and change the security and curative effect. Therefore, the illumination of the metabolic pathway and the induction/inhibition on metabolic enzymes of new drugs plays an important role to investigate the difference of individual metabolism and the pharmacodynamics of some metabolites for exploration ulteriorly in the preclinical phase of drug development.P-glycoprotein (P-gp) was identified as an ATP-dependent transporter. It has been found in intestinal mucosa, blood-brain-barrier, hepatic cell, kidney and testicle etc. P-gp can pump the toxic compound and numerous drugs out of these tissues and impact the absorption, disposition, metabolism and elimination. The modification of drugs to P-gp may lead to the nonmetabolism drug-drug interaction. So the connection between new drugs and P-gp should be noticed during the developing stage.Moreover, previous studies have shown that, second to liver, intestine and kidney express several metabolic enzymes and transporters which are involved in the drug metabolism.Buagafuran is a synthetic derivative of agarofuran, which showed significant antianxiety activity in several animal models, with higher potency and lower toxicity compared with diazepam and buspirone. The possible antianxiety mechanism of buagafuran was related to the modulation of central monoamine neurotransmitters. The preclinical pharmacokinetics studies indicated that the absorption of buagafuran was extremely poor with an absolute bioavailability below 9.5%. The highest radioactivity of 3H-buagafuran was found in gastrointestinal tract, followed by liver and kidney (unpublished results), but less in brain (target organ). Several rats liver CYP enzymes and intestine P-glycoprotein involved the biotransformation of buagafuran, but it has not been demonstrated that the type of metabolites, the CYP isoforms involved in the biotransformation of buagafuran, the interaction of buagafuran between CYP and P-gp. So, in this thesis the following study results were reported:1. Investigate the CYP isoforms and metabolites of buagafuran with rat and human liver microsomes, recombination human supersomes and CG-MS analysis.2. Evaluate the effect of buagafuran on the liver CYPs in mRNA, protein and activity levels.3. Inspect the impact of buagafuran on the intestinal CYP3A/P-gp and renal CYP2C11,2E1 and P-gp in mRNA, protein and activity levels.4. Using the intact animal and in situ brain perfusion to study the interaction of buagafuran and P-gp in brain.5. According to the results of regulation of buagafuran to CYPs and P-gp, we chose the drugs which may be prescribed combined with buagafuran to study the possible impacts of buagafuran on other drugs from the view of pharmacokinetics in vivo.The results were shown as follows:1. The identification and comparison of the metabolites of buagafuran in rat and human liver microsomes. 1.1 The GC-MS analysis method for the buagafuran and its metabolites in biological samples showed good sensitivity, and high specialization and appropriate for study in microsomes incubation system.1.2 Buagafuran could be transformed to one hydroxyl-, one carbonyl-and two hydroxyl-metabolism in rat liver microsomes. The main metabolites in human liver microsomes were similar as in rats, but the number was less and the rate of production was slower.1.3 Ketoconazole and disulfiram are the selective inhibitors to CYP3A4 and CYP2E1, they could reduce the elimination rate of buagafuran and the generation of the metabolites, and buagafuran could be metabolized in the CYP3A4 supersome. Summarization above results, the CYP3A4 was the main CYP isoform involved in the catalysis of translation of buagafuran into one hydroxyl-and two hydroxyl-metabolites.2. The regulation of buagafuran to rat liver microsomes CYP450s2.1 Buagafuran (4,16,64mg/kg) administered by gavage for 7 continuous days significant increased the activity of CYP1A2 and CYP2E1 in a dose dependent manner (1.56-,1.89-,3.07-fold and 1.4-,1.48-,1.79-fold), while the mRNA and protein levels of CYP 1A2 and 2E1 were elevated in certain extents.2.2 CYP2C6, CYP2C11 were also slightly induced by buagafuran, while buagafuran had no effect on liver CYP3 A2 in rats.2.3 The metabolic rate of buagafuran in self-treated rat liver microsomes was faster than control group, which indicated that the induction of self-metabolism might be related to the up-regulation of liver CYPs.2.4 Buagafuran in 1-10μM showed inhibition on rat liver CYPs in different extents, but the 0.5μM of buagafuran has no inhibit effect in vitro.3. The regulation of buagafuran on CYP450s and P-gp in rat intestine and kidney.3.1 Buagafuran could increase activity and expression of CYP3A in rat intestine and induce expression of CYP3A4 protein in LS-174T. The induction of P-gp expression was found in rat intestine by buagafuran. According the result of digoxin transfer experiments performed in Caco-2 transwell model, buagafuran showed induction/inhibition on function of P-gp.3.2 Multiple oral administration of buagafuran could up-regulate the CYP2C11,2E1 and P-gp in rat kidney, and the extent was more than in liver.4. The interaction between buagafuran and P-gp in rat brain4.1 The efflux of buagafuran in brain was studied in rat in situ brain perfusion model and the result demonstrated that the P-gp inhibitor could enhance the penetration of buagafuran from blood to brain.4.2 The effect of buagafuran on brain P-gp was measured by RT-PCT, western-blotting and the blood-brain ratio of rodanmin123 in rat. The result improved the induction of buagafuran on brain P-gp in mRNA, protein and function levels.5. PK effect of buagafuran on clinical drugs5.1 Phenacetin is a common used antipyretic analgesic and can be transformed to acetaminophen mediated by CYP1A2. After single administration of buagafuran, the PK profiles exhibited the enhenced AUC, Cmax and reduced CL/F. After multiple administration buagafuran the AUC and MRT decreased. The concentration of acetaminophen in plasma was reduced by multiple and increased by single administration of buagafuran. The result above was correlation with the induction and inhibition of buagafuran on liver CYP1A2.5.2 Chlorzoxazone, a centrally acting muscle relaxant, was mainly metabolized by CYP2E1 in vivo. Because of the significant induction on CYP2E1 after multiple doses of buagafuran, the PK interaction of chlorzoxazone and buagafuran coadministration was studied in vivo. The result demonstrated that multiple dosages could induce the metabolism of chlorzoxazone by changing AUC, MRT and CL/F and enhance the 6-hydroxylchlorzoxazone, while single dose had no obvious effect on chlorzoxazone PK profile. The result suggested that multiple doses of buagafuran could accelerate chlorzoxazone metabolism through induction of CYP2E1 activity and protein expression.5.3 It is reported that CYP3A is mainly involved in the hepatic metabolism of a short acting benzodiazepine derivative agent midazolam. The PK interaction between midazolam and buagafuran was investigated just because of the inhibition on CYP3A in vitro. The results showed the inhibition on the midazolam metabolism by single dosage appearing as the increasing plasma concentration, which was consisitant with the inhibition study. Multiple dosages of buagafuran could decrease the AUC, Cmax of midazolam as well as 1'-hydroxyl midazolam. The results might be due to the involvement of P-gp in the biotransformation of midazolam.5.4 Digoxin, an intermediate-acting cardiac glycoside, was a substrate of P-gp. Because of its narrow therapeutic window and unexpected toxic effect, blood drug concentration should be monitored in clinical application. Previous study showed that buagafuran could induce/inhibit the intestinal P-gp, under the above condition, the P-gp mediated interaction between them may happen in all probability. Our result showed that the blood concentration of digoxin was elevated significantly (Cmax increased by 25%, AUC increased by 85%) by single dosing of buagafuran and multiple dosages could reduce the Cmax and AUC of digoxin to 70% and 80% compare with control group which magnified the possibility of adverse effect. Thus, coadministration interaction should be concerned between those drugs, especially the drugs with narrow therapeutic window, and pharmaceutics from buagafuran. Comprehensive analysis should be given to personalize drug therapy.In conclusion, this report included the studies of the induction/inhibition of buagafuran on main CYP450 and P-gp in liver, intestine, kidney and brain systematically by various subcellular fractions, cell lines, intact animal and in situ animal models. The pharmacokinetics was performed on buagafuran and coadministration drugs in order to provide reliable references for prediction of clinical drug-drug interaction. The present study may demonstrate the molecular mechanisms of the interaction between buagafuran and CYPs/P-gp, likewise provide the valuable data for clinical application and structure reformation of buagafuran.
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