Study On Interactions Between Imatinib And Betaine Plasma Concentrations And The Possible Mechanism

Background:Imatinib is used for the treatment of Gastrointestinal stromal tumor（GIST）patients by targeting the inhibition of KIT and PDGFR activity,there are still unresolved problems in its individualized medication,and routine use of therapeutic drug monitoring（TDM）has not yet been adopted in clinic.Betaine is an important nutrient ingested mainly through diet.It is mainly involved in the synthesis of methionine as an active methyl donor and is involved in the regulation of renal osmotic pressure.Carnitine/Organic cation transporter 2（OCTN2/SLC22A5）is a multispecific transporter that is widely expressed in liver,kidney,heart and other tissues,which is involved in the transmembrane transport of endogenous compounds such as L-carnitine and imatinib.L-carnitine can be used as the precursor of betaine to generate betaine under the action of L-carnitine dehydrogenase.Our previous experiments showed that mice fed with 2%betaine containing water showed reduced OCTN2 expression in the liver,suggesting possible influence of betaine or L-carnitine on imatinib in vivo disposition by regulating OCTN2 expression.Aims:To explore the causal relationship between betaine and imatinib concentration in vivo and the mechanism,and to determine whether betaine supplementation reverses the influence of imatinib on plasma betaine concentration.The study is supposed to provide new solutions for the individualized use of imatinib and prevention of the adverse reactions.Methods:C57BL/6J mice were treated with 2%betaine containing drinking water for 4 weeks,OCTN2 m RNA and protein expression in the liver were detected by q RT-PCR and Western blot.A total of 80 GIST patients treated with imatinib were recruited in the First Affiliated Hospital of Zhengzhou University.Plasma imatinib C_ss-min and betaine concentrations were determined.Correlation between plasma betaine and imatinib concentrations for the patients were analyzed.Hepatocyte cell line L02 was cultured and treated with series concentrations of betaine,choline chloride,and L-carnitine,OCTN2 m RNA and proteins expressions were determined by q RT-PCR and Western blot,respectively.A two-stage animal study was carried.In the first stage,Wistar rats were fed with free drinking water containing 1%betaine for 2 weeks.On the 14th day,a single gavage ingestion of 30 mg/kg imatinib was given,and tail vein blood samples were drawn at before and series time points within 48 h pose-drug,and the liver,small intestine and kidney tissues of the rats were also collected.In the second stage,SD rats were divided into 4 groups:free drinking water+gavage water（the control group）,free drinking water+gavage 30 mg/kg/day imatinib（imatinib group）,free drinking with 1%betaine containing water+gavage 30 mg/kg imatinib（betaine+imatinib group）,free drinking with 1%betaine containing water+gavage water（betaine group）for 32 days.On the 1st,10th,and 31st days,the tail vein blood samples were drawn before the drug（0 h）and 1h,2h,3h,4h,6h,7.5h,and 24 h,respectively,after the imatinib ingestion.On the 32nd day,the rats were sacrificed,and the liver,kidney,and heart tissues were obtained.The plasma concentrations of imatinib and betaine were detected by LC-MS/MS,differences in the pharmacokinetic parameters of imatinib and plasma betaine concentrations among the groups were analyzed.The m RNA and protein expression of OCTN2 in the liver,small intestine,and kidney were detected by q RT-PCR and Western blot,respectively.Results:（1）OCTN2 protein expression in the liver of C57BL/6J mice fed with 2%betaine containing drinking water for 4 weeks was increased significantly.（2）Normal distribution of betaine concentrations and skewed distribution of imatinib concentrations were observed in the 80 patients.Patients,with imatinib concentrations reached the effective threshold tended to show lower plasma betaine levels（p=0.054）.Plasma concentrations of betaine and imatinib C_ss-min showed a trend of negative correlation in the overall patients（Spearman’s r=-0.1889,p=0.0934,n=80）,correlated negatively with dosage adjusted imatinib C_ss-min in overall patients（Spearman’s r=-0.2860,p=0.0101;n=80）and in patients treated with a standard dose of 400 mg QD imatinib（Spearman’s r=-0.4338,p=0.0041,n=42）.（3）In cultured hepatocytes L02,treatment with betaine,choline chloride,and L-carnitine had no effect on the expression of OCTN2.（4）Fed of rats with 1%betaine containing drinking water for 2weeks had no effect on the expression of OCTN2 in the liver,small intestines,or kidneys in Wistar rats,nor did it affect the pharmacokinetics of a single dose imatinib.（5）As compared with SD rats in the control group,plasma betaine concentration in the imatinib treated group was decreased significantly.No difference in plasma concentration of betaine in the betaine treated group and betaine+imatinib treated group was observed.（6）As compared with the imatinib treated group,area under the curve（AUC）value of imatinib was decreased significantly while the CL/F value was increased significantly in the betaine+imatinib group.（7）Rhythms of plasma betaine concentrations was observed in the rats and was maintained during the study,with lowest plasma concentrations observed at about 6 h-7.5 h after imatinib gavage on each time.（8）No difference in the pharmacokinetic parameters of imatinib was observed in the imatinib group on differernt days.Conclusion:（1）Plasma imatinib C_ss-min and betaine concentrations correlated negatively in GIST patients;（2）Imatinib treatment could decrease plasma betaine concentration,which could be reversed by dietary betaine supplementation,but betaine supplementation also decreased the plasma concentration of imatinib.（3）There was a rhythm in daily plasma concentration of betaine in rats,neither imatinib nor betaine supplementation affected this rhythm.（4）Continuous administration of imatinib once daily did not affect the pharmacokinetics of imatinib in rats.