| Many drugs used currently in clinic are optically active and the majority of them are marketed as racemates. The endogenous macromolecules such as enzyme, receptorand plasma proteins are also optically active. In the process of absorption, distribution, metabolism and excretion, the enantiomer of racemic drug generally differ in pharmacokinetic properties as a consequence of Stereoselective interaction with endogenous macromolecules and result in different action and adverse reaction. Developing the method of separation and quantitation of chiral drug and its metabolite is extremely valuable to study pharmacokinetics, pharmacodynamics and toxicology of chiral drug.Propranolol (PROP) is a adrenoreceptor antagonist which is used as racemate in clinic. In this study, a sensitive reversed-phase high-performance liquid chromatography was developed to determine directly the propranolol enantiomer glucuronides and to evaluate Stereoselective metabolism of PROP in rat liver microsome induced by phenobarbital (PB). This method can also be used to determine directly the propranolol enantiomer glucuronides in human urine. The concentration of R-(+)- and S-(-)-propranolol glucuronides(PG) in urine samples from 16 healthy volunteers taken 20mg RS-PROP were determined to assay the Stereoselective metabolism of PROP in human.1. Determination of Stereoselective glucuronidation of racemic propranolol in rat liver microsome induced by phenobarbitalPropranolol is a nonselective adrenoreceptor antagonist that is used as racemic mixture of R-(+) and S-(-)- PROP in clinic. An analytical method for determination of propranolol enantiomer glucuronides in rat liver microsomes was developed by RP-HPLC to study the stereoselective metabolism of PROP.Chromatographic Conditions Chromatographic column: ALLTIMA C18 (250 4.6mmID, 5m);mobile phase: 67mmol.L-1 KH2PO4 buffer solution - CH3OH (55:45); pH 3.5; column temperature: 35 ; wavelength of UV detector: 290nm; flow rate: 1.0ml.min-1; injection volume: 20 1.Analytical Procedure R/S-PROP was added as substrate to 0.5ml of microsomal incubate. UDPGA(3mmol/L) was added to start enzymatic reaction after preincubation for 5 min at 37. At designed time, CCl3COOH (2mmol/L) was added to stop the incubation and nitrobenzoic acid was added as internal standard. Sodium hydroxide (2mol/L) was added to change the value of pH to about seven. After centrifugation(8000rpm, 10min), 20 u 1 of the supernant was analyzed by HPLC. Results A good separation for PROP enantiomer glucuronides was achieved and the impurity of microsomal incubate did not interfere the quantitation of R-(+) and S-(-)-PROP glucuronides. The regression equations of the standard curves based on the concentration of R-(+)-PROP glucuronide versus the peak-area of R-(+)-PROP glucuronide were As/Ar = 0.04125C+1.834 10-3 , r = 0.9998. The linear range was from 0.2439 to 73.17 u mol/L. The method affords the average recovery of 99.40+ 1.04%. The relative standard deviation for intra-day was less than 3.32% and was less than 4.52% for inter-day.The differences of kinetic parameters in control group and in PB group were significant. When R-(+)-PROP was used as substrate, the Vmax and Clint in PB group were about 4.3 and 9.8 times greater than those in control group and the Km in PB group was about 0.4 times lower than in control group. When S-(-)-PROP was used substrate, the Vmax and Clint in PB group were about 10.6 and 6.9 times greater than those in control group and the Km in PB group was about 1.5 times greater than that in control group. It shows that the enzyme affinity and catalyzing ability for R-(+)-PROP were significantly enhanced in rat liver microsome induced by PB and the catalyzing ability for S-(-)-PROP was also significantly enhanced, but the enzyme affinity for S-(-)-PROP was abated. The metabolic interaction between enantiomer of PROP would occur, which depended on the substrate concentration.2. Assay of stereoselective glucuronidation of racemic propranolol in humanby RP-HPLCUp till now, the assay of stereosele...
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