Effects Of Cytochrome P450 Enzymes On Gelsemium Alkaloids Metabolism And Its Toxicity

Gelsemium elegans,a small genus of the family Loganiaceae,have been used in traditional Chinese medicine to treat rheumatism pain and neuralgia.Due to its analgesic efficacy without addiction and next to morphine,investigations on Gelsemium elegans are becoming increasing in recent years.Indol alkaloid compounds are a kind of active ingredients and as well as toxicant of Gelsemium elegans,causing poisoning accidents in clinical application at times.Therefore,how to reduce toxicity is the key issue to be solved in applications of Gelsemium elegans.It's noted that cytochrome P450(CYP)has playing an important role in reducing toxicity of drugs,which mediates the mechanism of detoxification in many toxic Chinese medicine.Based on the above,three typical Gelsemium alkaloids,namely koumine(KOU),GA gelsemine(GA),humantenmine(HMT),were selected to be research objects.The present study investigated the relationship between cytochrome P450 enzyme metabolism and toxicity of Gelsemium alkaloids,in order to reveal the scientific connotation of reducing their toxicity,providing reference for improving efficacy as well as safety of drugs.The main research contents are as follows:1.The metabolism of Gelsemium alkaloids by CYP enzymesAfter incubated with liver microsomes,KOU,GA and HMT were transformed into at least four,five and four metabolites,respectively.The metabolic pathways were showed to be similar,including demethylation,dehydrogenation,hydroxylation,demethyl-dehydrogenation and oxidation.Additionally,the results of selective chemical inhibitors and recombinant human cytochrome P450 enzymes experiments demonstrated that CYP3A4/5 was the most important isoenzyme in Gelsemium alkaloids metabolism.The activity of CYP3A4/5 in mice could be induced or inhibited by intraperitoneal injection of dexamethasone or ketoconazole.Accordingly,metabolic activities of Gelsemium alkaloids were significantly increased or decreased after incubation with the liver microsomes from group induction or inhibition.Thus,it proved that CYP3A4/5 was the most efficient isoform in biotransformation of Gelsemium alkaloids in vivo.In order to study the effects of liver microsomes from different species on GA metabolism,we investigated the metabolic rates of GA by using humans,rats and mice liver microsomes on the concentration between 2.5 and 640 ?M.First of all,the urine sample was collected from rats after intragastic administration of GA.GA metabolites were separated and purified,and its major metabolite was identified as 4-N-demethyl GA by nuclear magnetic resonance.The content changes of 4-N-demethyl GA were represented to tell GA metabolic behavior in liver microsomes.Results showed that metabolic behaviors of GA in the three kinds of liver microsomes were fitted to classic Michealis-Menten kinetics.The metabolic rates were ranked as follows:human liver microsomes(705.6±36.2 pmol/mg/min)>mice liver microsomes(632.1 ± 16.0 pmol/mg/min)>rat liver microsomes(229.1 ±15.9 pmol/mg/min).These results suggested that there were significant species differences in the metabolism of GA in vitro.In order to investigate the disposition characteristics of HMT in rats,a sensitive UPLC-MS/MS method was established,and the pharmacokinetic parameters of HMT in rats were calculated.HMT was absorbed rapidly in rats.After oral administration of HMT 12.50±2.74 min,it reached the concentration peak with concentration of 28.49±6.65 nmol/L,and the half-time(T1/2)in plasma was 82.22±35.55 min.We hypothesized that the rapid decrease of plasma concentration was closely related to the drug metabolism by metabolic enzymes.2.Study on the relationship between the metabolism by CYP enzymes and toxicity of Gelsemium alkaloidsFirstly,the proliferation of Gelsemium alkaloids on human normal liver cells,namely L-02 cells,was determined by MTT method.The results showed that KOU and GA had different inhibition on the proliferation of L-02 cells at relatively high concentrations(600-800 ?M)(P<0.001).While HMT had significantly inhibitory effect on the proliferation of L-02 cells and showed dose dependency(P<0.001).Secondly,mice were intraperitoneally injected with ketoconazole(KET)to form the model of the inhibition of CYP3A4/5 enzyme activity in vivo.After 4 days intraperitoneal injection of KET,the mice in experimental group were taken GA by intragastric administration,while mice were directly administrated by the same dose of GA in group control.The results showed that no death was observed in group control mice.However,the survival rates of the mice in experimental group were reduced to 0%(GA)or below 20%(HMT)within 14 days.These indicated that the inhibition of CYP3A4/5 activity significantly increased the toxicity of Gelsemium alkaloids.Finally,in order to further demonstrate the effects of CYP enzymes on the toxicity of GA,zebrafish were exposed to GA and metabolite 4-N-demethyl GA under same concentration.When exposed to GA at 750 ?M,partial death and obvious reaction were observed on embryos of experimental zebrafish.However,no death numbers were recorded for zebrafish exposed to 4-N-demethyl GA at the same concentration.The median lethal concentrations of GA and 4-N-demethyl GA on zebrafish were calculated as 0.81 and 7.42 mg/mL,respectively.The results showed that GA was transformed into a less toxic metabolite by CYP enzymes.In summary,the present study demonstrated the metabolic pathways of Gelsemium alkaloids in human,mice liver microsomes included demethylation,dehydrogenation,hydroxylation,demethyl-dehydrogenation and oxidation.CYP3A4/5 was the most important isoenzyme in Gelsemium alkaloids metabolism.It also proved that toxicity of Gelsemium alkaloids was significantly reduced after metabolized by CYP3A4/5.The research can provide theoretical reference for early warning of toxicity and clinical application of Gelsemium alkaloids.