Study On The Possible Mechanism Of Adverse Reactions Of Duloxetine Based On The High Exposure Under The Diabetic Condition And Drug-drug Plasma Protein Binding Interactions

Objestive:To compare the pharmacokinetics in normal and diabetes mellitus rat models.And to survey the effects of duloxetine with fluoxetine,duloxetine with agomelatine on human plasma protein binding.The goal is to explore the possible mechanism of adverse reactions and provide a theoretical basis for the clinical rational drug use.Methods:?1?To compare the concentration of duloxetine in plasma between normal rat and diabetes mellitus rat models.To establish a liquid chromatography-tandem mass spectrometric?LC-MS/MS?method for the determination of the duloxetine concentration in rat plasma.Diazepam was used as internal standard.The separation was achieved on a WATERS Xterra^?RP18column?4.6 mm×100 mm,3.5?m?with a mobile phase consisting of methanol-0.3%formic acid containing 5 mmol·L^-1 ammonium acetate?75:25?at the flow rate of0.6mL·min^-1.Electrospray ionization source was applied and operated in the positive multiple reaction monitoring mode.The analytes was detected under the following condition:m/z 298.2?154.0 for duloxetine,m/z 285.5?154.0 for diazepam.Plasma samples were pretreated by methanol precipitation.The rat models of diabetes mellitus were established by intraperitoneal injection of streptozotocin.Duloxetine was given by intragastric administration to the normal and diabetic rats 40 mg·kg^-1,both in the single-dose experiment and in the multiple-dose experiment.Blood samples were collected from the orbital venous plexus to determinate duloxetine concentration in the plasma.The pharmacokinetic parameters were calculated by DAS 3.2.3 software.Statistical analysis was performed by SPSS 22 software.?2?To compare the concentration of duloxetine in tissue homogenate between normal rat and diabetes mellitus rat models.To use the LC-MS/MS method of rat plasma to determinate the duloxetine concentration in rat tissue homogenate?including brain,heart,liver,spleen,lung,kidney,stomach,small intestine,large intestine,testis,muscle,fat?.Duloxetine was given by intragastric administration to the normal and diabetic rats 40mg·kg^-1.The brain,heart,liver,spleen,lung,kidney,stomach,small intestine,large intestine,testis,muscle and fat separated and homogenated for determination of the concentration of duloxetine.Statistical analysis was performed between the normal and diabetes group by SPSS 22.?3?To identify the interaction of duloxetine with fluoxetine,duloxetine with agomelatine based on human plasma protein binding.To establish a LC-MS/MS method for the simultaneous determination the concentration of duloxetine,fluoxetine and agomelatine in human plasma and phosphate buffer solution?PBS?.The separation was achieved on a WATERS Xterra^?RP18 column?4.6 mm×100 mm,3.5?m?with a mobile phase consisting of methanol?A?-0.1%formic acid containing 5 mmol·L^-1 ammonium acetate?B?at the flow rate of 0.5 mL·min^-1 under gradient elution?0⁰.1 min,65%A;0.1².5 min,65%-80%A;2.5⁴.5 min,80%A;4.6¹0 min,65%A?.Electrospray ionization source was applied and operated in the positive multiple reaction monitoring mode.Diazepam was used as the internal standard and the analytes were detected under the following condition:m/z 298.2?154.0 for duloxetine,m/z310.1?148.2 for fluoxetine,m/z 244.1?185.0 for agomelatine,m/z285.5?154.0 for diazepam.Control group and experimental group was estabolished.And the control group was as follow:duloxetine group,fluoxetine group and agomelatine group.The experimental group was as follow:duloxetine with fluoxetine group,duloxetine with agomelatine group.Each group contains low,medium and high concentration gradient.The plasma protein binding of each drug was determined by equilibration dialysis method.The plasma protein binding of the drug in the experimental group was compared with that of the control group at the same drug concentration level.Results:?1?To compare the concentration of duloxetine in plasma between normal rat and diabetes mellitus rat models.To establish the LC-MS/MS method for the determination of the duloxetine concentration in rat plasma.The methodological evaluation showed that the endogenous substances would not interfere with the determination of duloxetine and internal standard in rat plasma.And the methods had good specificity.Duloxetine had a good linearity in the concentration range of 10⁵000 ng·mL^-1 in rat plasma.The precision of intra-day and inter-day are both within the acceptable range,The extration recovery was high and reproducible.The stability was well,and the matrix effect would not affect the determination.In the single-dose experiment,the major pharmacokinetic parameters of diabetes group were as follows:C_max?1185±190.0?ng·mL^-1;AUC_0-??8398±1835?ng·mL^-1·h;t_max?1.6±0.4?h;t_1/2z?3.6±0.9?h.The major pharmacokinetic parameters of normal group in the single-dose experiment were as follows:C_max?368.1±40.7?ng·mL^-1;AUC_0-??4145±640.1?ng·mL^-1·h;t_max?1.6±0.3?h;t_1/2z?4.1±0.8?h.In the multiple-dose experiment,the major pharmacokinetic parameters of diabetes group were as follows:C_max?2597.5±969.5?ng·mL^-1,AUC_0-??24597±13413?ng·mL^-1·h,t_max?1.8±0.3?h,t_1/2z?5.9±1.6?h.The major pharmacokinetic parameters of normal group in the multiple-dose experiment were as follows:C_max?998.4±279.6?ng·mL^-1,AUC_0-??9143±2442?ng·mL^-1·h,t_max?1.9±0.2?h?t_1/2z?5.4±1.8?h.Both in the single-dose experiment and in the multiple-dose experiment,duloxetine exposure(C_max and AUC)in the diabetes group were significantly higher than those in the normal group.The results showed that the condition of diabetes would increase the exposure of duloxetine,and that could be due to increased absorption or bioavailability.?2?To compare the concentration of duloxetine in tissue homogenate between normal rat and diabetes mellitus rat models.The methodological evaluation of rat tissue homogenate showed that the endogenous substances would not interfere with the determination of duloxetine and internal standard in tissue homogenate.And the methods had good specificity.Duloxetine had a good linearity in the concentration range of 0.1¹00?g·mL^-1in those tissue homogenate.The precision of intra-day and inter-day were both within the acceptable range,The extration recovery was high and reproducible.The stability was well,and the matrix effect would not affect the determination.The tissue distributions of duloxetine between the two groups is basically similar.In addition to the gastrointestinal tract,duloxetine mainly distributed in the tissues with faster blood flow or more abundant body fluid exchange,such as liver,lung,spleen and kidney.The drug content in the brain of the diabetes group was twice higher than that of the normal group(15.71±6.82vs8.431±4.79?g·g^-1),even though there was no significant difference in the statistics.The results suggested that the content of duloxetine in the brain,which was the pharmacodynamic target tissue,had an increasing tendency in the diabetic condition.?3?To identify the interaction of duloxetine with fluoxetine,duloxetine with agomelatine based on human plasma protein binding.To establish a LC-MS/MS method for the simultaneous determination the concentration of duloxetine,fluoxetine and agomelatine in human plasma and PBS.Both in plasma and PBS,endogenous substances would not interfere with the determination of the analyte and internal standard.The specificity,precision,extraction recovery,matrix effect,and stability of the three analytes were all within the acceptable range.The plasma protein binding of duloxetine,fluoxetine and agomelatine were?96.59±0.47?%,?94.54±0.22?%and?95.61±1.11?%respectively,which was not dependent on concentration.There was no significant difference in plasma protein binding between the experiment group compared with the control group.The results indicated that duloxetine with fluoxetine,duloxetine with agomelatine did not show plasma protein binding interaction.Conclusions:?1?To compare the pharmacokinetics of duloxetine between normal rat and diabetes mellitus rat models,in the diabetes group,the duloxetine exposure was significantly increased,and the duloxetine content in the brain also had an increasing tendency.These may be caused by the increased oral bioavailability of duloxetine in diabetes condition.The adverse reactions of duloxetine may be caused by the higher exposure under the diabetes condition.?2?To identify the interaction of duloxetine with fluoxetine,duloxetine with agomelatine based on human plasma protein binding.The results suggested that the increase in drug interactions and in the adverse reactions,when duloxetine co-administered with fluoxetine or agomelatine,was not caused by the plasma protein binding interaction.In the future study,the interaction based on pharmacological mechanism could be further considered.