Studies On The Disposition Of Tribendimidine In Healthy Chinese Volunteers

Objective: 1. To investigate the stability of tribendimidine in artificial gastric juice and intestinal juice, to identify where tribenimidine decomposes and the format to absorb. 2. To study the metabolite of tribendimidine in human body, to illuminate its disposotion process. 3. To establish a sensitive HPLC-MS method for determination of p-(1-dimethylamino ethylimino) aniline and acetylizad p-(l-dimethylamino ethylimino) aniline product in human plsma; To establish HPLC-UV methods to determine p-(1-dimethylamino ethylimino) aniline, acetylizad p-(1-dimethylamino ethylimino) aniline and terephthalic acid in human urine. 4. To study the pharmacokinetics of p-(1-dimethylamino ethylimino) aniline, acetylizad p-(1-dimethylamino ethylimino) aniline and terephthalic acid in healthy Chinese volunteers after 400mg oral doses of tribendimidine enteric-coated tablets.Methods: 1. Tribenimidine was incubated in inartificial intestinal juice and gastric juice for different time, and then a HPLC-UV method was used to determine the prototype and decomposed products, to investigate the stability. 2. The plasma and urine samples, collected after administrating 400mg tribendimidine enteric-coated tablets, were analyzed by a HPLC-MS/MS method to study the metabolite, including full scan, SIM, product ion, precusor ion, neutral loss procedure. 3. P-(1- dimethyl-amino ethylimino) aniline and acetylizad p-(1-dimethylamino ethylimino) aniline in plasma were detected by HPLC-MS method. The plasma samples were analyzed and separated on a Ultimate C₁₈ column (150mm×4.6mm, 5μm), eluted with the mobile phase of acetonitrile-0.01M ammonium acetate (including 0.3% ammonia water) (80:20,V/V) at a flow rate of 0.8 mL·min^-1. The temperature of column was 25℃. 10μL was injected to analyze. Atmospheric pressure electronic spray ionization (AP-ESI) and ion mass spectrum (m/z) of 178, 220 and 370 for p-(1-dimethylamino ethylimino) aniline, acetylizad p-(1-dimethylamino ethylimino) aniline and amisulpride (IS), respectively, were selected. The pressure of spray gas (Nitrogen gas) was 50 psi. The flow rate and temperature of drying gas (Nitrogen gas) were 11L·min^-1 and 350℃, respectively. The fragmentor were all 110V. 4. Urine samples were diluted and then anlyzed on an Agilent Extend C₁₈ (4.6×250mm,5μm) with the mobile phase of acetonitrile-water-triethylamine (60:40: 0.2,V/V/V) at a flow rate of 0.7mL·min^-1 for the determination of p-(1-dimethylamino ethylimino) aniline and acetylizad p-(1-dimethylamino ethylimino) aniline at 249nm. Internal standard was bicalutamide. A Ultimate XB C₁₈ collumn (4.6×150mm,5μm) with the mobile phase of methanol-water-acetic acid (35:65:0.55, V/V/V) at the flow rate of 0.8 mL·min^-1 were applied to detect the terephthalic acid in urine samples at 240nm. Ferulaic acid was selected as the internal standard. 5. 8 male healthy volunteers were enrolled and orally given 400mg tribendimidine enteric-coated tablets. The plasma and urine samples were collected as scheduled and p-(1-dimethylamino ethylimino) aniline and acetylizad p-(1-dimethylamino ethylimino) aniline in plasma were analyzed by HPLC-MS method, p-(1-dimethylamino ethylimino) aniline, acetylizad p-(1-dimethylamino ethylimino) aniline and terephthalic acid in urine were deteced by HPLC-UV methods. The pharmacokinetic parameters were calculated by DAS 2.0. The adverse reactions, if any, were recorded at the same time.Results: 1. Tribendimidine was so instable in artificial (NE) gastric juice that it decomposed completely at Omin. After incubated in artificial NE intestinal juice for 15min, all the tribendimidine decomposed. No prototype of tribendimidine was detected in artificial intestinal juice after incubated for 5min. The decomposed product were p-(1-dimethylamino ethylimino) aniline and terephalaldehyde detected by HPLC method. 2. The plasma and urine samples of the volunteers were analyzed by HPLC-MS/MS method. Two main metabolites of were found: acetylizad p-(1-dimethylamino ethylimino) aniline and terephthalic acid. 3. Good linearity of p-(1- dimethylamino ethylimino) aniline in plasma was observed in the range of 1-800ng·mL^-1 and 0.5-500 ng·mL^-1 for p-(1- dimethylamino ethylimino) aniline and acetylizad p-(1- dimethylamino ethylimino) aniline in plasma, respectively. The low limit of quantitation (LLOQ) was 1ng·mL^-1 and 0.5ng·mL^-1. The absolute recoveries were 81.47%-84.05% and 83.64%-86.71%. The relative recoveries were 100.89%-101.41% and 97.88%-110.68%. Intra-batch and inter-batch RSD were both less than 8%. p-(1- dimethylamino ethylimino) aniline and acetylizad p-(1- dimethylamino ethylimino) aniline in plasma frozen at -20°C for 24 hours and seven days and after two freeze-thawing cycles was stable, with the RSD of less than 8%. 4. The linear range of p-(1- dimethylamino ethylimino) aniline and acetylizad p-(1- dimethylamino ethylimino) aniline in urine detected by HPLC-UV method were 0.5-500μg·mL^-1 and 0.1-200μg·mL^-1, respectively. The low limit of quantitation (LLOQ) was 0.5μg·mL^-1 and 0.1μg·mL^-1. The absolute recoveries were 94.78%-95.23% and 91.26%-96.99%. The relative recoveries were 98.37%- 101.44% and 99.26%-102.06%. Intra-batch and inter-batch RSD were both less than 7%.p-(1- dimethylamino ethylimino) aniline and acetylizad p-(1- dimethylamino ethylimino) aniline in urine frozen at -20℃for 24 hours and seven days and after two freeze-thawing cycles was stable, with the RSD of less than 7%. 5. The linear range of terephthalic acid in urine detected by HPLC-UV method was 0.2-200μg·mL^-1. The low limit of quantitation (LLOQ) was 0.2μg·mL^-1. The absolute and relative recoveries were 93.18%-94.36% and 99.58%-101.23%. Intra-batch and inter-batch RSD were both less than 5%. Terephthalic acid in urine frozen at -20℃for 24 hours and seven days and after two freeze-thawing cycles was stable, with the RSD of less than 8%. 6. The main pharmacokinetic parameters of p-(1-dimethylamino ethylimino) aniline and acetylizad p-(1-dimethylamino ethylimino) aniline after single oral dose of 400mg tribendimidine enteric-coated tablets were as follows: t_1/2 5.382±2.145h and 7.087±2.060h; Tmax 5.250±1.254h and 7.125±2.416h; Cmax 449.382±136.409ng·mL^-1 and 148.304±117.131 ng·mL^-1; AUC_0-36 4655.038±1113.240ng·mL^-1·h and 1904.929±976.805ng·mL^-1·h; AUC_0-∞ 4768.901±1158.375ng·mL^-1·h and 1989.491±1014.418 ng·mL^-1·h, respectively. 7. Urine accumulated excretion rates within 48h of p-(1- dimethylamino ethylimino) aniline, acetylizad p-(1- dimethylamino ethylimino) aniline and terephthalic acid were 51.50±15.17%, 15.58±8.48% and 53.07±14.66%, respectively. The total accumulated excretion rates of p-(1- dimethylamino ethylimino) aniline and acetylizad p-(1- dimethylamino ethylimino) aniline was 70.82±9.83%. The pharmacokinetic parameters of p-(1- dimethylamino ethylimino) aniline, acetylizad p-(1- dimethylamino ethylimino) aniline and terephthalic acid in urine calculated by DAS 2.0 were as follows: t_1/2 3.459±0.734h, 4.213±0.431h and 3.375±0.565h; Ke 0.213±0.068h^-1, 0.166±0.017h^-1 and 0.210±0.030h^-1.Conclusion: 1. Tribendimidine was unstable in artificial gastric juice and intestinal juice, and decomposed to p-(1- dimethylamino ethylimino) aniline and terephalald-ehyde,which could prove tribendimidine was not absorbed with the format of prototype. 2. Tribendimidine could be metabolized in human body, and the main metabolite were acetylizad p-(1- dimethylamino ethylimino) aniline and terephthalic acid. 3. The pharmacokinetic process of p-(1- dimethylamino ethylimino) aniline and acetylizad p-(1- dimethylamino ethylimino) aniline in healthy volunteers after orally administrated 400mg tribendimidine enteric-coated tablets both fitted to one-compartment model with weight coefficient of 1/cc. They could be absorbed and distributed rapidly. Most of the drug excreted within 48h in the format of p-(1-dimethylamino ethylimino) aniline and acetylizad p-(1- dimethylamino ethylimino) aniline in urine. 4. The trial was well done and all volunteers were in good compliance. No adverse reactions were observed during the whole study.