Simple method for determination of the active metabolite of the inotropic drug pimobendan in rat liver microsomes

Abstract Background: Panax notoginseng saponins (PNS) is commonly used in combination with clopidogrel in clinic. This study was to investigate the effect of PNS on the pharmacokinetics of clopidogrel active metabolite CAMD and the effect on the activities of clopidogrel metabolic enzymes in rats. Methods: In pharmacokinetics studies, the rats were divided into clopidogrel and combination groups, and continuously administered for seven days. The concentration of CAMD was determined by LC-MS/MS. In enzymes studies, the rats were divided into control and PNS groups. After administration for seven days, CYP2C19 and CYP3A4 activities were measured by the metabolic rate of the specific substrate in rat liver microsomes. The activity of CES1 enzyme was determined by double antibody sandwich method.Results: PNS significantly increased AUC0-∞ of CAMD from 43.1±11.6 to 72.0±25.1 h·ng/mL (p<0.05). Combination group had lower CL/F and Vd/F than clopidogrel group (p<0.05). PNS significantly decreased the activity of CYP3A4 and CES1 (p<0.01), but no significant effect on CYP2C19.Conclusions: The combination use of PNS and clopidogrel produced a significant increase in the AUC of clopidogrel active metabolite CAMD. PNS could inhibit the activity of CYP3A4 and CES1 enzyme in rat.

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A Direct, Highly Sensitive Fluorometric Assay for a Microsomal Cytochrome P450-Mediated O-Demethylation Using a Novel Coumarin Analog as Substrate

Zeitschrift für Naturforschung C ◽

10.1515/znc-2000-11-1212 ◽

2000 ◽

Vol 55 (11-12) ◽

pp. 915-922 ◽

Cited By ~ 6

Author(s):

René Roser ◽

Helmut Thomas

Keyword(s):

Rat Liver ◽

Liver Microsomes ◽

Fluorometric Assay ◽

Excitation Wavelength ◽

Monooxygenase System ◽

Rat Liver Microsomes ◽

Monooxygenase Activity ◽

Continuous Increase ◽

Highly Sensitive

A highly sensitive fluorometric assay for the determination of monooxygenase activity in liver microsomes is described. The assay is based on the use of 3-chloro-7-methoxy-4-methylcoumarin which is demethylated to 3-chloro-7-hydroxy-4-methylcoumarin. The rate of formation of 3-chloro-7-hydroxy-4-methylcoumarin was recorded as an increase of fluorescence (λA = 380 nm, λF = 480 nm ) with time. When 3-chloro-7-methoxy-4-methylcoumarin was incubated in the presence of MgCl2and NADPH with rat liver microsomes, a continuous increase of the fluorescence could be measured. The reaction proceeded linearly for about 10 min and at least up to a concentration of 0.1 mg/ml of microsomal protein. Besides 3-chloro-7-hydroxy-4-methylcoumarin a hydroxylated derivative of the substrate was formed as a second metabolite during the incubation. Using an excitation wavelength of 480 nm and a fluorescence/em ission w avelength of 480 nm, the fluorescence of this substance (λA = 338 nm, λF = 422 nm ) amounted only to about 1% of the fluorescence of the main product. The use of 3-chloro-7-methoxy-4-methylcoumarin as substrate enables the fluorometric determination of the O-dealkylation activity of a cytochrom e P450-dependent monooxygenase system in rat liver which is inducible by phenobarbital but not by 3-methylcholanthrene.

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