Interactions Between Artemisinin Drugs And Drug-metabolizing Enzyme

Objective: Artemisinin derivatives are a kind of sesquiterpene lactone with endoperoxide bridge. In recent years, studies have showed that this kind of medicine had the rapid but incomplete oral absorption and auto-induction metabolism. The auto-induction metabolism of artemisinin is mediated primarily by CYP2B6 (cytochrome P450, family 2, subfamily B, polypeptide 6) with probable secondary contribution of CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4) in individuals with low CYP2B6 expression. Meanwhile it can also induce CYP2C19 (cytochrome P450, family 2, subfamily C, polypeptide 19). The nuclear receptors including pregnane X receptor (PXR, NR1â… 2) and conatitutive androstane receptor (CAR, NR1â… 3) are the key regulation factors of CYP450-induction. It is not clear that whether the induction of CYP450s by artemisinin drugs is regulated by activate nuclear receptor PXR or CAR. In this study, the parent compound artemisinin of artemisinin derivatives and active metabolite dihydroartemisinin were chosen as study objects, and fluorescence spectroscopy and synchronous fluorescence spectrometry methods were used for investigating the interaction between artemisinin drugs and enzyme or protein. Bovine serum albumin (BSA), human serum albumin (HSA), rat liver microsomes (RLM), human liver microsomes (HLM), recombinant drug metabolizing enzymes CYP2B6, CYP2C19, CYP3A4 were used as protein systems for research. And real-time quantitative PCR method was used to investigate the mRNA levels of PXR, CAR and CYP2B, CYP3A, CYP2C isozyme of the small intestines in rats, which were induced by multiple doses of oral or intravenous administration of artemisinin drugs. The possible auto-induction of artemisinin drugs to rat intestinal CYP450s was assessed according to the results.Methods: (1) Fluorescence spectroscopy was used to investigate interactions between artemisinin drugs and drug-metabolizing enzyme. When the concentration of BSA, HSA, RLM, HLM, CYP2B6, CYP2C19 and CYP3A4 were kept invariable, a certain volumes of ART or the DHA solution were gradually added, and the fluorescence spectroscopy and synchronous fluorescence spectroscopy were scanned. According to Stern - Volmer equation, binding constant and the thermodynamic equation, the binding parameters and thermodynamic parameters were calculated. The fluorescence quenching mechanism of the reaction, the major force types, and effects of drugs on protein conformation were evaluated.(2) PCR method was used to investigate the effect of artemisinin drugs on the mRNA levels of both nuclear receptors and CYP450s from small intestines in rats. SD rats were randomly divided into eight groups (six rats per group), oral administration of ART group (80 mg/kg/d), oral administration of DHA group (10 mg/kg/d), oral solvent group (oil, 1 mL/d), intravenous injection of ART group (16 mg/kg/d), intravenous injection of DHA group (5 mg/kg/d), intravenous injection solvent of ART group (50% acetonitrile, 0.2 mL/d), intravenous injection solvent of DHA group (50% ethanol, 0.2 mL/d), blank group. The drugs were administrated once a day for five days. All rats were killed at 24 h after the last dose and their small intestine was taken. The total RNA was isolated from rat small intestine using Trizol one-stage process. mRNA levels of PXR, CAR, CYP2B1, CYP2C6 and CYP3A1 were determined by SYBR green real-time quantitative PCR. 2^{-ΔΔCt} method was used to calculate relative quantitation results and SPSS software was used for statistical analysis.Results: (1) The interaction of ART - BSA, DHA - BSA, ART - HSA and DHA - HSA were static quenching effects. At temperature of 310 K, the binding constants were 5.54×10³, 7.31×10², 1.18×10³ and 2.57×10³ L·mol^-1, respectively. All of the reactions were spontaneous thermodynamics reactions, and the major driving forces were hydrogen bond and van der Waals force. ART and DHA might alter the conformation of BSA and HSA.(2) Both the interaction of ART - RLM and DHA - RLM were static quenching effects. At temperature of 310 K, the binding constants were 1.90×10⁴, 4.07×10¹ L·mol^-1, respectively. The interaction of ART - HLM and DHA - HLM are mainly static quenching effect. At temperature of 310 K, when the level of ART≤10.88 10^-6 mol·L^-1 and≥10.88 10^-6 mol·L^-1, the binding constants of ART - HLM was 1.05×10⁴ L·mol^-1 and 1.55×10⁵ L·mol^-1, respectively. And DHA - HLM binding constant was 1.34×10³ L·mol^-1. All of the four reactions were spontaneous thermodynamics reactions. The major driving forces were hydrogen bond and van der Waals force.(3) For both reactions of ART and DHA to CYP2B6, the main quenching mechanisms were static quenchings. When the temperature rising, the mechanisms changed tostatic mixed with dynamic quenchings. At physiological temperature 310K, the bonding capability for DHA to CYP2B6 was stronger than ART to CYP2B6. All of the reactions were spontaneous thermodynamics reactions. The main force of ART to CYP2B6 system was hydrogen bond and van der waals force at the temperature range of 296K 303K and changed to hydrophobic force when temperature stepped up to 303K 310K. However, the main force of DHA to CYP2B6 was electrostatic force at 296K 303K and change to hydrogen bond and van der Waals force when temperature grew to 303K 310K. All of the reactions were spontaneous thermodynamics reactions. Meanwhile, both ART and DHA had less influence on the conformation of CYP2B6 at the same temperature. (4) The interactions of ART - CYP2C19, DHA - CYP2C19, ART - CYP3A4 and DHA - CYP3A4 are static quenching effects. At temperature of 310 K, the binding constants were 4.76×10¹, 2.43×10³, 6.55×10² and 5.46×10³ L·mol^-1, respectively. All of the reactions were spontaneous thermodynamics reactions, and the major driving forces were hydrogen bond and van der Waals force. When gradually adding a certain volume of ART or DHA solution, microenvironment around the tryptophan residue changed hydrophilic, and tyrosine residues changed to hydrophobic microenvironment, for both CYP2C19 and CYP3A4.(5) Compared with the control group and vehicle control groups, the mRNA levels of PXR, CAR, CYP2B, CYP2C, CYP3A in oral ART or DHA treatment groups and intravenous ART or DHA treatment group did not show significantly differences (P> 0.05).Conclusion: There were binding reactions between ART or DHA and BSA, HSA, RLM, HLM, CYP2B6, CYP2C19 or CYP3A4. Meanwhile, some species differences were found in the reactions. ART and DHA could bind with these proteins at different levels of combinations and by diferent typies of driving force. And the two drugs exhibited different effects on the conformation of these proteins. Real time quantitative PCR results indicated that, ART and DHA did not induce nuclear receptor CAR and PXR, as well as CYP2B1, CYP2C6, CYP3A1 in rat small intestine. The relationship of auto-induction metabolism of artemisinin drugs with intestinal first-pass metabolism need further investigation to evaluate.