| Background:According to our early experiments of liver microsomal, We made a conclusionthat CYP3A4is the main metabolic enzyme of osthole. Meanwhile, CYP2D6andCYP2C9may also be involved in its metabolism. Our research established metabolicmodels of human liver microsomes and of in vivo rats, studied the activity effect ofosthole to three sub-enzymes, which participate in the metabolism of osthole.Objective:We are aimed at studying the effects of osthole to the three main sub-enzymeprobe drugs MDZ, DM and DIC, which could affect osthole metabolism, throughvitro research method of human hepatomicrosome, as well as in vivo experiment ofrats in order to study the activity effects of osthole to these three sub-enzymes. So thatthe pharmacological study of osthole could be expended, which could providetheoretical foundation of clinical rational use of osthole and its chinese herbology,and simultaneously provide datum for carrying out the effects of coumarin analogs todrug metabolism enzyme.Methods:1. Establish the HPLC-UV detection methods of rat plasma between MDZ, DMand DIC in human hepatomicrosome.2. Through the studying of hatch time, protein concentration and substrateconcentration, we find out the best reaction method of DM, DIC and MDZ in humanliver microsomes, respectively.3. Study the activity effects of osthole to human hepatomicrosome CYP3A4,CYP2C9and CYP2D6.4. In vivo pharmacokinetic effects were studied through giving different dose ofosthole via intraperitoneal injection, followed by injecting the same dose of MDZ viathe vein of rat tail.Results: 1. Establish the HPLC detection methods of rat plasma between MDZ, DM andDIC in human hepatomicrosome.2. DM was incubated in the human liver microsomes under the condition of37℃and120r/min.5-40min: DM presenting linear eliminated,40-80min: rate sloweddown,80-160min: almost no elimination, so the optimal incubation time is80min.Within the scope of0.1to0.5mg/ml, elimination of DM is accelerated with theincrease of protein concentration. So the optimal incubation protein concentration is0.5mg/ml. Within the scope of the concentration of10-100μM, metabolic rate of DMchanges most with the increase of the substrate concentration. Therefore, theoptimum reaction conditions were determined:80min,0.5mg/ml,100μM.The samemethod was used to optimize the reaction condition of DIC and MDZ. Their optimumreaction conditions were96min,0.4mg/ml,80μM and30min,0.2mg/ml,5μM.3. There were no significant differences (P>0.05) of enzyme activity to CYP2D6,CYP2C9, CYP3A4in vitro human liver microsomal, when the concentration ofosthole is5,10μM, when the concentration of osthole are20,40and80μM, themean metabolism of DM reduced30.8%(P<0.05),34.7%(P<0.01) and54.4%(P<0.01), respectively; the mean metabolism of DIC reduced17.7%(P<0.05),20.0%(P<0.05) and51.7%(P<0.01), respectively. But when the concentration of osthole is20μM, the metabolism of MDZ has no significant difference, compared with thecontrol group and when the concentrations are40,80μM, the mean metabolism ofMDZ was reduced by19.7%(P<0.05),61.2%(P<0.01), respectively. ThroughGraghpadPrisrn5.0software, we calculated the inhibition IC50values of osthole, toCYP2D6, CYP2C9and CYP3A which were69.78μM,71.68μM,68.45μM,respectively. Main pharmacokinetic parameters of the four MDZ groups: controlgroup AUC(0-t)(256.502mg·L·min-1), AUC(0-∞)(300.76mg·L·min-1), MRT(0-t）(46.579min), MRT(0-∞)(72.193min), t1/2z(52.869min), CLz(0.014L·min-1·kg-1), Vz (1.007L·kg-1); low dose group AUC(0-t)(325.742mg·L·min-1), AUC(0-∞)(384.122mg·L·min-1), MRT(0-t）(47.081min), MRT(0-∞)(73.678min), t1/2z(54.784min),CLz(0.01Lmin-1·kg-1), Vz(0.82L·kg-1); middle dose group AUC(0-t)(482.665mg·L·min-1),AUC(0-∞)(626.279mg·L·min-1), MRT(0-t)(81.253min), MRT(0-∞)(147.581min), t1/2z(110.381min), CLz (0.006L·min-1·kg-1), Vz(1.02L·kg-1); high dose group AUC(0-t) (1012.791mg·L·min-1), AUC(0-∞)(1168.722mg·L·min-1), MRT(0-t)(95.022min), MRT(0-∞)(140.381min), t1/2z(100.741min), CLz(0.003L·min-1·kg-1), Vz(0.498L·kg-1). Themedicine-time curve area of MDZ is significantly higher than single drug group,which indicates that osthole had significantly influence on MDZ, and the higherconcentration the strong influences. CLz and Vz are much lower comparing withcontrol group.Conclusions:1. We had established sensitive reliable HPLC-UV methods of rat plasma andMDZ, DM and DIC in human hepatomicrosome.2. Osthole has feeblish inhibition to CYPYP2D6, CYP2C9, CYP3A.3. Osthole has significantly inhibition on CYP3Ain rat. |
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