Metabolic Characteristics Of Propranolol Enantiomers In Human Hepatocytes Treated With Different Inducers

Chirality is a structural characteristics of chemical substance. Almost 75% drugs used in clinical practice exist molecular asymmetry, so called as chiral drugs. Recently, the clinical significance of drug chirality has been closely remarked. The interaction of demic chiral environment and specific enantiomers has resulted in pharmacokinetic or/and pharmacodynamic stereoselectivity differences between various enantiomers. Chiral characteristics of pharmacokinetics are studied much more than of pharmacodynamics. The former is based on the stereoselective processes of drug absorption, distribution, metabolism and excretion. Among them, the incidence rate of metabolic stereoselectivity of drugs is the highest (ca. 40%). However, the researches on drug-interaction were usually dealt with racemics as single compounds owing to ignoring the differences in metabolic stereoselectivity between enantiomers. As a result, the obtained conclusions were probably discordancy in curative effect and adverse reaction, and can not correctly guide rational use of clinical medication.Drugs are mainly metabolized in liver, depending on cytochrome P450 (CYP 450). It is essential enzyme system of drug biotransformation and participates metabolic processes of endogenous and exogenous compounds in vivo. There are generous liver drug enzymes in organelle of liver cells. Thus every drug will be metabolized by various kinds of enzymes. Blood drug level could just figure out total results without detecting metabolic participation of enzyme. There would be side effects or inadequacy of therapeutic effects during drug combination, especially when the drugs could strikingly strengthen or reduce enzyme activity. And enzymes would also show up stereoselectivity to entiomers, altering or even deteriorating the accretion rate of one of them.The chiral drug of this assay is propranolol(PPL). Propranolol is nonselective blocking agent ofβ-adrenoceptor. Cmax after oral administration is 1-3 hours, and t1/2 is 2-5 hours. It is mainly metabolized in liver, with 60%-70% first pass effect and 30% bioavailability. Propranolol is used clinically as racemics with partes aequales of S(-)-PPL and R(+)-PPL, but theβ-adrenoceptor blocking effects of S(-)-PPL is 100 times more than R(+)-PPL. There are various kinds of enzyme participating metabolism of propranolol in CYP 450, especially CYP 2D6 and CYP 1A2. Nevertheless, the previous researches just concentrate on portrait study of them. This essay selected CYP 1A1 and CYP 3A4 to investigate. The contents of CYP 1A1 are very low in liver. But it is very easily induced to create tumors. CYP 3A4 is the major components of CYP 450, occupying 30%-40%. Therefore, it is necessary to study these two enzymes participation in metabolism of propranolol enantiomers.Hepatocytes are suitable for studying in CYP 450 enzyme activity induction and inhibiton. This essay was used human hepatocyte as reaction system, and did the research in metabolic characteristics of the enantiomers of propranolol in the human hepatocytes treated with different inducers, including enzymatics and pharmacokinetics, in order to identify whether the enzymes participate the metabolism of enantiomers.I. The culture of human hepatocytes and the activity detection of CYP 1A1, CYP 3A4Object To observe metabolism with enzyme in hepatocytes. To describe enzyme activity through detection of substrates metabolism by enzyme treated with inducers and define the best induced concentration.Method 1. The culture of human hepatocytes.2. Detection of cells survival rate with MTT.3. Using 7-ER and Rifampicine as specific substrates to determine the activities of CYP1A1 and CYP3A4 and to define the best induced concentration.Result 1. The best induced concentration of BNF is 0.8μmol·L-1. 2. The best induced concentration of RIF is 15μmol·L-1.II. Chiral separation of propranolol enantiomersObject To verificate whether the enantiomers would convert structure after metabolism.Method The enantiomers were reacted with a pre-column chiral derivatization reagent 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl isothiocyanate(GITC) and separated on HPLC.Result The enantiomers could be separated completely with this method.And they don't convert after metabolism. They could be detected without separation.III. HPLC detection of propranolol enantiomers in cell culturesObject To establish a high performance, convenient, fast method for drug detection in medium in vitro.Method 1. Preparation of standard solution and sample pretreatment.2. HPLC detection of propranolol enantiomers and methodology corroboration.Result 1. The regression equation of S(-)-PPL and R(+)-PPL are Y = 0.5627X + 0.0581 and Y = 0.6699X - 0.2068. correlation coefficient are 0.9993 and 0.9994. The minimum quantitated concentration(LOD,S/N≥9)is 0.5μmol·L-12. The extraction are larger than 80% and method recovery are 90%. The intra-day and inter-day precision.3. This method is sensitive, accurate, and could be used as study of medium sample.IV. Metabolic characteristics of the enantiomers of propranololin the human hepatocytesObject To inspect whether the enzyme participate propranolol metabolism.Method R(+) , S(-) propranolol are metabolized by BNF and RIF induced cells. Then the substrate concentration-time curves and enzyme parameters (Km , V max) of carvedilol enantiomers were provided.Result Enantiomers were metabolized faster after induction. Enzyme catalytic abilities had a stereoselectivity to R(-)-PPL in control group and RIF induced group, while there is stereoselectivity to S(-)-PPL in BNF induced group.Conclusion1. Both these two enzymes are partipated in metabolism of propranolol enantiomers.2. their catalytic abilities had a stereoselectivity to R(-)-PPL in control group and RIF induced group, while there is stereoselectivity to S(-)-PPL in BNF induced group.3. Metabolism of R(-)-PPL will be increased when used the drugs which can strikingly strengthen the activity of CYP 3A4, while metabolism of S(+)-PPL will be increased when used the drugs which can sharply increase the activity of CYP 1A1.