| Background: Azathioprine（AZA）was widely used in the treatment of Inflammatory Bowe Disease（IBD）,however,the incidence of AZA efficacy and adverse reactions was significantly different among individuals,and some AZA patients could not tolerate its toxic and side effects,so the treatment was discontinued.In particular,the concentration level of 6-thioguanine nucleotide（6-TGN）,the active metabolite of AZA,can be used as an important marker to determine the efficacy and toxicity of AZA.Therapeutic drug monitoring（TDM）of 6-TGN is a routine work in clinical pharmaceutical care of IBD.Studies have shown that gene polymorphisms of key AZA metabolic enzymes,such as TPMT and NUDT15,are associated with efficacy and toxicity,but they are still insufficient as TDM markers for AZA toxicity prediction.As a metabolic "organ",intestinal flora has great variation in the internal composition characteristics of individuals.The diversity of intestinal flora affects the response of individuals to specific drugs,and then changes the pharmacokinetic characteristics of drugs and their metabolites in vivo.At present,researchers have studied the effects of intestinal flora on drug metabolism with multiple pseudo-asepsis（PGF）model.Bioinformatics studies have shown that intestinal flora has metabolic enzymes required to metabolize thiopurine compounds.In vitro incubation experiments showed that e.coli DH5α could transform thiopurine drugs into thioguanine nucleotides,and the changes of AZA-related enzyme activity in intestinal flora of patients were more obvious than that of host enzymes.In addition,many studies have shown that intestinal flora plays a crucial role in the prediction and treatment of IBD.Therefore,based on in vivo exposure monitoring of AZA metabolites and detection of TPMT and NUDT5 gene polymorphism,further studies on the correlation between intestinal flora diversity and AZA metabolism are expected to provide new ideas for individualized drug treatment of AZA-TDM and IBD.Objective: The purpose of this study was to investigate the role of gut flora composition variation in AZA metabolic differences,and to analyze the effects of gut flora mediated AZA biotransformation on the pharmacokinetics of main metabolites after oral AZA by establishing PGF model and comparing with the control group.Methods:（1）PGF model was established by using mixed antibiotics as drinking water.qPCR was used to detect the variation of 16 S r RNA gene abundance in intestinal flora during model establishment.（2）The changes of TPMT and NUDT15 genotypes in the control group and PGF group were detected by Sanger gene sequencing,and the expressions of XO and TPMT in the host liver were detected by Western Blot.（3）AZA was extracted from rat faecal intestine for in vitro incubation.The incubated samples were qualitatively analyzed by ULTRA-performance liquid chromatography-high resolution mass spectrometry,and the concentrations of 6-TGN and 6-MMP in the incubated samples were quantitatively analyzed by ULTRA-performance liquid chromatography-tandem mass spectrometry.（4）After the successful establishment of PGF model,pharmacokinetics of AZA was studied in vivo,and the whole blood drug concentrations of active metabolites 6-TGN and 6-methylmercaptopurine（6-MMP）of AZA were quantified by ULTRA performance liquid chromatography tandem mass spectrometry.Phoenix Win Nonlin software（Version 8.3.2.116,Certara,US）was used to analyze 6-TGN and 6-MMP blood concentration-time data and to calculate their pharmacokinetic parameters in a non-av model.（5）Taxonomic characteristics were analyzed by fecal 16 S r RNA sequencing,and the correlation between intestinal flora diversity and pharmacokinetic differences was explored.RDA redundancy analysis was performed on the results of enzyme incubation and sequencing of intestinal bacteria in vitro.Results:（1）qPCR results showed that 16 S r RNA gene abundance and species diversity of intestinal flora decreased significantly one week after antibiotic intervention.Continuous AZA administration also resulted in intestinal microbiota disorder.（2）There was no mutation of TPMT and NUDT15 genes in the host in the control group and PGF group,and no statistical difference was found in the expression of TPMT and XO proteins in the liver of the two groups.（3）Intestinal flora is involved in AZA transformation,and the structural variation of intestinal flora in rats can change the single pharmacokinetic characteristics of 6-TGN and 6-MMP.（4）There was a correlation between intestinal flora diversity and AZA metabolism,and it was found that intestinal flora metabolized AZA to generate 6-TGN and 6-MMP related bacterial markers.Conclusion: In this study,we found that intestinal microbiome mediated AZA metabolism can change the in vivo exposure to 6-TGN after oral AZA,and the diversity of intestinal microflora is correlated with the difference in AZA metabolism,and Escherichia coli,Prevotellaceae UCG-001,Bacteroides and Rothberia are expected to be az A-TDM markers.It is suggested that attention should be paid to the status of intestinal flora in individualized AZA treatment strategy. |
