Auto-induction Metabolism Mechanism Of Artemisinin Drugs In Rats

Auto-induction metabolism mechanism of artemisinin drugs in ratsObjects:Artemisinin derivatives are a kind of sesquiterpene lactone compounds with endoperoxide and applied well in antimalarial treatment. In recent years, it was reported that this kind of medicine exhibited auto-induction metabolism. It was also known that artemisinin drugs are metabolized by CYP2B6 and CYP3A4 in human and can induce CYP2C19. The nuclear acceptor superfamily is the key regulation factors of CYP450-induction. Mainly there are the orphan nuclear acceptor including pregnane X receptor (PXR, NR1Ⅰ2) and conatitutive androstane receptor (CAR, NR1Ⅰ3). It is not clear the mechanisim involved in artemisinin auto-induction. In this work the parent compound artemisinin (ART) and active metabolite dihydroartemisinin (DHA) of this kind derivatives were chosen as goal medicines, CYP2B, CYP3A and CYP2C were selected as investigation aims, CYP450s induction molecular mechanism of this kind of medicine were investigated in rats by using molecular biology methods and drug metabolism methods.Method:SD rats were randomly divided into eight groups (six rats per group):ART orally treated group (80mg/kg/d); DHA orally treated group (10 mg/kg/d); oral vhicle control group; ART intravenously treated group (6mg/kg/d); DHA intravenously treated group (5mg/kg/d); intravenous vhicle (ART) control group; intravenous vhicle (DHA) control group; blank control group. The drugs were administrated once daily for five consecutive days. All rats were killed and their livers were prepared 24 h after the last dose.Results:(1) mRNA determination:Ribonucleic acid (RNA) was isolated from rat liver using Trizol one-stage process and mRNA levels of PXR, CAR, CYP3A1, CYP2B1 and CYP2C6 were determined by SYBR green real-time quantitative PCR.2-ΔΔCt method was used to calculate relative quantitation results. Compared to vehicle control groups and blank control group, CYP2B1 mRNA levels of ART and DHA orally treated groups increased significantly, CAR mRNA levels of ART orally treated groups increased significantly, yet other nuclear acceptor or CYP450 mRNA levels of ART or DHA intravenously treated groups have no significant difference among groups.(2) Activity of enzyme determination:The liver microsomes of rats were prepared by applying ultracentrifugation approach. Ultraviolet spectrophotometry (UV) was used to determine the contents of protein and cytochrome P450 (CYP450). CYP450 content in ART orally treated group (1.45nmol/mg protein), CYP450 content in DHA orally treated group (1.38nmol/mg protein), CYP450 content in oral vehicle control group (0.81nmol/mg protein), CYP450 content in blank control group (0.79nmol/mg protein), CYP450 content in ART intravenously treated group (0.75nmol/mg protein), CYP450 content in intravenous vhicle (ART) control group (0.78nmol/mg protein), CYP450 content in DHA intravenously treated group (0.79nmol/mg protein), CYP450 content in intravenous vhicle (DHA) control group (0.84nmol/mg protein). Compared to vehicle control groups and blank control group, CYP450 content in ART and DHA orally treated groups increased significantly. The enzymic activity of CYP3A1 was detected by Nash method. Generation rate of HCHO in ART orally treated group (2.29μmol/mg/min), generation rate of HCHO in DHA orally treated group (1.75μmol/mg/min), generation rate of HCHO in orall vehicle control group (0.82μmol/mg/min), generation rate of HCHO in blank control group (0.75μmol/mg/min), generation rate of HCHO in ART intravenously treated group (0.72μmol/mg/min), generation rate of HCHO in intravenous vhicle (ART) control group (0.75μmol/mg/min), generation rate of HCHO in DHA intravenously treated group (0.79μmol/mg/min), generation rate of HCHO in intravenous vehicle (DHA) control group (0.85μmol/mg/min). Compared to vehicle control groups and blank control group, CYP3A1 activity in ART and DHA orally treated groups were more higher. The enzymic activity of CYP2B1 was detected by fluorospectrophotometry. Generation rate of resorufin in ART orally treated group (10.30×10-3μmol/mg/min), generation rate of resorufin in DHA orally treated group (8.19×10-3μmol/mg/min), generation rate of resorufin in oral vehicle control group (5.68×10-3μmol/mg/min), generation rate of resorufin in blank control group (5.41×10-3μmol/mg/min), generation rate of resorufin in ART intravenously treated group (5.24×10-3μmol/mg/min), generation rate of resorufin in intravenous vehicle (ART) control group (5.26×10-3μmol/mg/min), generation rate of resorufin in DHA intravenously treated group (5.25×10-3μmol/mg/min), generation rate of resorufin in intravenous vehicle (DHA) control group (5.74×10-3μmol/mg/min). Compared to vehicle control groups and blank control group, CYP2B1 activity in ART and DHA orally treated groups were more higher. The enzymic activity of CYP2C6 was detected by high-performance liquid chromatographic (HPLC) methods. Metabolic rate of diclofenac in ART orally treated group (31.20%), metabolic rate of diclofenac in DHA orally treated group (18.02%), metabolic rate of diclofenac in oral vehicle control group (16.54%), metabolic rate of diclofenac in blank control group (16.37%), metabolic rate of diclofenac in ART intravenously treated group (15.59%), metabolic rate of diclofenac in intravenous vehicle (ART) control group (16.02%), metabolic rate of diclofenac in DHA intravenously treated group (16.41%), metabolic rate of diclofenac in intravenous vehicle (DHA) control group (17.93%). Compared to vehicle control groups and blank control group, CYP2C6 activity in ART orally treated group were more higher. The results showed that the activity of could be induced by multiple oral administrations of artemisinin or dihydroartemisinin.(3) Protein determination:The liver protein of rats was prepared for protein determination. Western blot was used to determine protein levels of PXR or CAR. Compared to control group, experiment groups has no statistically significant differences (p>0.05). Prepared liver microsomes were used to determine protein levels of CYP3A1, CYP2B1 and CYP2C6. There was no significant differences among ART or DHA-treated groups and control groups.Conclusion:The above results suggeste that artemisinin can activate rat CAR and induce CYP2bl mRNA expression. And dihydroartemisinin can induce CYP2b1 mRNA expression. Artemisinin drugs existed up-regulation effects on drug metabolism likely via CAR. On the other hand, artemisinin and dihydroartemisinin enhanced CYP450 enzyme activity. This kind of medicine induced the CYP450 enzyme activity after multiple oral doses. While for nuclear acceptor or CYP450s mRNA level and enzyme activity, there is no significant differents after multiple intravenous doses. The results suggested the induction effects on CYP450s of ART drugs might be related to oral first pass effects.