The Inhibition And Induction Of CYP450 And The Effect Of Enrofloxacin Metabiolism By Traditional Chinese Medicine In Crucian Crap

This paper was investigated the inhibitory effect of Chinese medicine on CYP450 and on enrofloxacin metabolism. The thesis is divided into four, The main research results were as follows:1.Inhibition of Chinese Herbal on CYP1A and CYP3A of hepatopancreas microsome from Crucian Carp (Carassius auratus gibelio)This study focuses on the inhibition of five kinds of Chinese Herbal (Berberine, Scutellaria, Forsythia, Evodia, Chrysanthemum and Bupleurum) on CYP1A and CYP3A of Crucian Carp (Carassius auratus gibelio) liver microsomal. Determination of IC₅₀ and kinetic parameters in vitro administration, Results shows that incubation system did not interfere with endogenous substances, the level of inhibition of six kinds of Chinese herbs on CYP1A was Berberine>Scutellaria> Evodia> Forsythia> Chrysanthemum> Bupleurum; the IC₅₀ were 0.0038, 0.27,2.88,7.62,16.20 and 16.42 mg/mL, Ki were 0.00028, 0.13,0.38,3.39,1.74 and 70 mg/mL respectively. V_max and K_m were 83.33 pmol/(min.mg) and 3.05±0.89μM respectively. Berberine, Scutellaria, Forsythia and Bupleuru have a weak inhibition of CYP3A, the IC₅₀ were 0.68, 90.9, 70.8, 43.5 mg/mL, Inhibition of CYP1A than CYP3A was significant, The types of inhibition of these herbs on CYP1A were quite different: Scutellaria, Berberine, Scutellaria, Chrysanthemum and Bupleurum were competitive inhibitors, and Evodia and Forsythia were uncompetitive inhibitor.2.The inhibition of CYP450 1A and 3A by berberine in crucian carpThe effects of berberine on microsomal cytochrome P4501A (CYP1A) and CYP3A in crucian carp were investigated. Injecting different concentrations of berberine (0, 5, 25, 50, and 100 mg/kg) suppressed the CYP1A mRNA expression, thereby inhibiting further the P4501A-related ethoxyresorufin-O-deethylase (EROD) activity. Furthermore, both CYP1A expression and EROD activity were further inhibited with increasing concentration of berberine. Meanwhile, the expressions of CYP3A at both the mRNA and protein levels could be down-regμLated by high concentrations of berberine. The CYP3A-related Erythromycin N-demethylase (ERND) catalytic activity could also be inhibited by berberine with a dose of no less than 25 mg/kg. Moreover, at a berberine concentration of above 25 mg/kg, the higher the concentration was, the stronger the inhibition on CYP3A expression and ERND activity became. Subsequently, the in vitro experiments were performed, and the results showed that berberine could competitively inhibit the EROD activity possessing the IC₅₀ of 11.5μM when it was pre-incubated with the crucian carp microsome. However, the ERND activity was only inhibited slightly by berberine with the IC₅₀ of 204.3μM. These results of these metabolic activites and of western-blot and of RNA blot analyses demonstrate the berberine is efficacious in suppressing CYP1A and CYP3A expression and effective in changing the pharmacokinetics of durg metabolized by these isoenzymes, therefore, more care is needed when using the co-administered prescription medicine.3.The effect of Baicalin and glycyrrhizic acid on enrofloxacin in vivo of crucian carpCytochromes P450 (CYPs) play key roles in drug metabolism which are widely distributed in liver in aquatic organisms. CYP(s) mainly catalyzed the N-deethylation reaction of enrofloxacin (EF) biotransfor-mation to ciprofloxacin (CF). However, limited information is available about CYP investigation in fish. This study investigated the metabolism of EF by oral administrated baicalin (100mg/kg) and glycyrrhizic acid (100mg/kg) in crucian carp, and detected the enzymatic activites of cytochrome P450 1A (CYP1A) and 3A (CYP3A) in Liver microsomes. The vary of pharmacokinetics of EF was measured continuous blood samples with each animal. The results were showed follow, (1) The absorption of EF have significant decline, C_max and AUC were deceased. (2) AUC and t_1/2z have a conspicuous reduced, while CL was inceased after oral administrated with baicalin and glycyrrhizic acid. Thes indicated that elimination of EF was accelerated. (3) The C_max CF/C_max EF of BL group and GZ group were 1.48% and 2.22% respectively, but the control was 0.95%; The AUCCF/AUCEF of BL group and GZ group were 2.16%,1.76%, respectively, but the control was 1.7%. there can be reveal BL and GZ were contributed to induce N-deethylation of EF. (4) Pre-treated with BL and GZ can caused significant inceases of ethoxyresorufin-O-deethylation (EROD) and Erythromycin-N-demethylation (ERND), which were the specific probes of CYP1A and CYP3A respectively (p<0.05). Gaven these data, it appears that BL and GZ accelerate the elimination of EF and the production of its metabolite was frequent related with the induced of CYP1A and CYP3A enzymatic activities.4. The role of CYP450 in the enrofloxacin N-deethylationCytochromes P450 (CYPs) play key roles in drug metabolism which are widely distributed in liver in aquatic organisms. CYP(s) mainly catalyzed the N-deethylation reaction of Enrofloxacin (EF) biotransfor-mation to Ciprofloxacin (CF). However, limited information is available about CYPs investigation in fish. In order to supply useful information on CYP(s) characterization for EF metabolition , the present study assessed the effects of fish potent CYP inducers and inhibitors on EF metabolition in liver of crucian carp (Carassius auratus gibelio) by reversed-phase high-performance liquid chromatography (RP-HPLC). Results demonstrated as follow, (1)The pharmacokinetics of EF and CF were investigated given EF alone (10mg/kg, i.p.) after oral administration of rifampicin (RIF) andβ-naphthoflavone (BNF) (12 mg/kg b.w./day×d), the results show that t_1/2z and AUC of EF and CF were decreased, while the CLz/F were increased. This demonstrate RIF and BNF have been attributed to promote the elimination of EF and CF; (2)The peak concentrations of CF after oral administration of RIF and BNF were 0.435±0.138 and 0.048±0.007mg/L, the control group was 0.099±0.029 mg/L, RIF and BNF of AUCCF/AUCEF ratios were 3.69% and 1.84%, while the control group was 1.76%, Integrated CF peak concentration and the ratio of AUCCF/AUCEF, RIF has been attributed to EF N-deethylation; (3) The activites of CYP1A-related ethoxyresorufin-O-deethylase (EROD) and CYP3A-related erythromycin N-demethylase (ERND) were investigated. The results demonstrated that EROD of BNF-treated and ERND of RIF-treated activites were induced (p<0.05). As well as the research of in vitro suggested that CF production rate was increased after the RIF- treated microsome incubated with enrofloxacin (p<0.05), and CF production rate was decreased after the ketoconazole-treated microsome incubated with enrofloxacin (p<0.05). However, CF production rate have no significant change after the BNF-treated microsome incubated with EF (p>0.05). Conbined with in vivo and in vitro data, we suggest that CYP3A may be responsible for EF N-deethylation in liver.