| Objective In this study,liquid chromatography tandem mass spectrometry(LC-MS/MS)method was developed for sensitive determination of Q808 in different tissue homogenate buffer and excretion samples.All of the methods were validated and applied for non-clinical pharmacokinetics study in Wistar rats.The content of Q808 in different tissues and different excretions could provide reliable bases for the evaluation of Q808.The results of the induction study of Q808 on rat liver microsome and the inhibition study of Q808 on recombinant human metabolic enzymes could provide a reference about drug-drug interaction.Result(1)Tissue distribution in rats:Healthy rats were administrated at the dose of 28 mg/kg.Then different tissues were obtained,and then were made into tissue homogenate buffer for determination.According to the results,there was a certain extent of drug distribution in the tissues such as brain,ulterus,testis,small intestine,large intestine,stomache,kidney,spleen,liver and heart.The concentration of Q808 in pancreas,fat,and ovarian was high.On the contrary,the content of Q808 in muscle was low.The phenomenon might be related to the small polarity of Q808 which can result to better distribution in tissues of high lipid solubility.All above results can tell us that during long term toxicity test we should focus on the study of toxicity in reproductive organs.The concentration of brain was about 70 percents of Cmax,it meaned that there was better distribution of Q808 in brain.(2)The excretion in feces,urine and bile:Healthy rats were administrated at the dose of 28 mg/kg,and feces,urine and bile were obtained and then the contents of Q808 in different excretions were determined.The accumulated excretion ratio of feces,urine and bile could be calculated.After dosing of 28 mg/kg,compared with different excretion ratio,the result was that:feces(6.69 ± 0.859%)>bile(0.0316 ± 0.0101%)>urine(0.0142 ± 0.00449%).It meaned that the main metabolic route of Q808 in rats was through feces.But all of the three excretion ratio was no more than 10%.According to the result of distribution study,there was no accumulation of Q808 in tissues,therefore,we could infer that elimination was the main metabolic pathway,and the metabolite transformed from Q808 instead of Q808 was eliminated.(3)Difference in metabolic species:Q808 was incubated with human,rat,dog and monkey liver microsomes,respectively.In accordance with the results,we got some relevant information about metabolites of human,rat,dog and monkey.Metabolite 1 is the metabolic product of the reductive dechlorination reaction on Q808,only exists in dogs.Metabolite 2 is the metabolic product of single hydroxylation reaction on Q808,which had relative high signal in dogs and low signals in rats and human.Above mentioned results indicated that the metabolic reaction of Q808 in dog liver microsomes was different from others such as human,rat,monkey liver microsomes.As a result,the animals we chose were rats(Wistar rats in this study)and monkeys(rhesus monkeys in this study).(4)Rat plasma protein binding ratio:Standard solution of Q808 was mixed with fresh plasma from healthy Wistar rats to get the plasma samples with the concentration of 500,1500,2400 ng/mL,respectively.The plasma samples were put into equilibrium dialysis bags with oscillation for 24 h in 37 ? water bath.The samples of inside and outside dialysis bags were determined with LC-MS/MS,respectively.The plasma protein binding ratio calculated of three different concentration of rat plasma were 97.4 ± 2.97%,94.9 ± 0.914%,94.5 ±1.53%,respectively.The results indicated that the plasma protein binding ratio of Q808 was relative high,and Q808 was almost existed in the form of binding protein.The study told that we should focus on the influence of the drug-drug interaction on Q808 efficacy.(5)The influence of Q808 on rat liver microsomes:The rat liver microsomes with made from healthy Wistar rats,the contents of protein and CYP450 in rat liver microsomes were determined.The drug probes were incubated with rat liver microsomes in vitro.The activity of different CYP450 subtypes in control and experimental groups were calculated.The results showed that there was no significant difference of activities among control and experimental groups.All above mentioned indicated that there was no obvious induction and inhibition.As a result,there was no potential metabolic interaction of Q808.(6)The influence of Q808 on recombinant human metabolic enzymes:Q808 was incubated with five CYP450 subtypes(CYP1A2,CYP2D6,CYP3A4,CYP2C9,CYP2C19),respectively.Phenacetin,debrisoquine,midazolam,tolbutamide,mephenytoin were related specific substrates.According to the determination of the specific metabolites,the activities of different subtype enzymes were calculated.The results indicated that there was no obvious difference between the control and experimental groups.It meaned that there was no inhibition influence of Q808 on the recombinant human metabolic enzymes.We could infer that there was no inhibition of Q808 when clinical use. |
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