Preclinical Study Of Drug Metabolism Of [¹⁴C]NBP （3-n-butylphthalide）

3-n-Butylphthalide（NBP）possesses considerable neuroprotective effect and is currently used for the treatment of ischemic stroke.NBP was approved for marketing in 2004 and entered the national medical insurance directory in 2009.NBP was listed in the 2010 China Cerebrovascular Disease Prevention and Control Guide.In 2020,the sales volume of NBP reached 6.6 billion yuan.However,information on its metabolism in human and preclinical animal models is insufficient.Although the metabolism of unradiolabeled NBP in human has been reported,the quantitative metabolite profiling（What are the main metabolites?）,blood-to-plasma radioactivity concentration ratio（B/P）,and tissue distribution of this drug remain unclear.We evaluated the pharmacokinetics,tissue distribution,mass balance,and metabolism of NBP in rats after a single oral dose of 60 mg/kg（100μCi/kg）[¹⁴C]NBP and NBP to understand the biotransformation of NBP comprehensively and to provide preclinical drug metabolism data prior to human mass balance studies with[¹⁴C]NBP in the near future.Detailed contents are as follows:（1）Radioactive pharmacokinetics.Blood and plasma were collected at predose and postdose time points,respectively.Blood（oxidative combustion）and plasma were measured with a liquid scintillation counter.NBP absorption was rapid(T_max=0.75 h)and declined with a terminal half-life of 9.73 h.C_max and AUC_0-∞was23051.34 ng eq./m L and 170452.48 h×ng eq./mL,respectively.In rats,the B/P was0.63 during the 48 h postdose period,indicating that drug-related substances tend to be distributed into plasma.（2）Mass balance.Urine samples,fecal samples and bile samples were collected at predose and postdose time points,respectively.Fecal samples（oxidative combustion）,urine samples and bile samples were measured with a liquid scintillation counter.At 168 h after oral administration,the mean cumulative recovered radioactivity was 99.85%of the dose,and was 85.12%in urine and 14.73%in feces.Urine was the main excretion route.Radioactive dose recovery in bile was 24.63%and 46.92%of the dose over 48 h post-dose in male and female rats,respectively.Greater bile excretion than fecal excretion indicates the reabsorption of some drug-related substances in the gut.（3）Tissue distribution.Tissues were collected at postdose time points.After oxidative combustion,the radiation concentration（total concentration of drug-related substances）in each tissue was determined by a liquid scintillation counter.Tissue distribution results showed that drug-related substances were widely distributed throughout the body,but no accumulation was observed in the body.（4）Metabolite radioprofiling and characterization.The plasma was pooled according to Hamilton method,urine and fecal samples were pooled according to the same percentage of volume and weight respectively.The samples were extracted by protein precipitation,and then the supernatants were evaporated and reconstituted.High performance liquid spectrometer was used for metabolite identification in conjunction with radioactive detector and high resolution mass spectrometer.A total of 49 metabolites were identified in rat plasma,urine,and feces.The main metabolic pathways were oxidation,glucuronidation,and sulfation.Overall,NBP was absorbed rapidly,tended to be distributed into plasma,and were widely distributed throughout the body,but didn’t accumulate in tissues.Urine was the main excretion route,and there was reabsorption in the intestine.The main metabolic pathways were oxidation,glucuronidation,and sulfation.In conclusion,NBP was a fully absorbed and fully metabolized drug.