In Vitro And In Vivo Metabolic Studies Of Anaphylactone And Isoimperatorin

Imperatorin（IMP） and isoimperatorin（ISOIMP） are the bioactive furanocoumarins compounds naturally occurring in many umbelliferae medicinal plants, such as Radix Angelica dahurica, Radix Angelica pubescentis and Radix Glehniae. IMP and ISOIMP exhibit extensive pharmacological effects, such as analgesia, anti-bacterial, anti-tumor and anti-viral effects. They are also the important bioactive substances in a variety of traditional Chinese medicine preparations e.g. “Huoxiang Zhengqi Liquid”, “Longkui Yinxiao Tablet” and “Yuanhu Zhitong Tablet”, and are widely used in clinic.In this study, a rapid and selective method using liquid chromatography with tandem mass spectrometry（LC-MS/MS） was developed and validated for separation and simultaneous quantitation of IMP, ISOIMP and their metabolites heraclenin（HER）, xanthotoxol（XANT）, oxypeucedanin（OPE） and oxypeucedanin hydrate（OPEH） in biological matrices. The method was successfully used to determine the plasma pharmacokinetics, tissue distribution and excretion（urine, feces and bile） of IMP and ISOIMP. The formation kinetics and excretion of metabolites were also investigated. In addition, the metabolic stabilities, enzyme kinetics and reaction phenotyping were studied in the incubation system with liver microsomes or human recombinant CYP enzyme. The study provides important data to support further development of these two active components and to guide clinical use of IMP and ISOIMP containing herb drugs.In the LC-MS/MS method, chromatographic separation was performed on a CAPCELL PAK MG Ⅱ C18 column that was eluted with a mobile phase of acetonitrile（0.1% formic acid）-water（0.1% formic acid and 5 m M ammonium formate） at flow rate of 0.3 m L/min. The detection was accomplished by multiple-reaction monitoring（MRM） scanning via electrospray ionization（ESI） source, operating in the positive ionization mode. The sensitivity, precision, accuracy, recovery and stability of the method were validated to meet the quantitative requirement. Simple sample preparation procedure with one-step protein precipitation and short running time allowed a high through-put analysis of a large volume of biological samples.In rat pahrmacokinetic study, the peak time of the parent drug in plasma was detected at 1 h after a single oral administration of IMP（30 mg/kg）, and the parameters of t_1/2 and area under concentration-time curve（AUC） were calculated to be 1.5 h and 5078±3512 ng·h·m L^-1, respectively. The metabolites HER and XANT were also detected in plasma. IMP was eliminated rapidly after a single intravenous dose（2 mg/kg）, with t_1/2 and AUC being 0.99 h and 731.2±67.3 ng·h·m L^-1. HER was the onlt metabolite detected in plasma. The absolute bioavailability of IMP in rats was calculated to be 46.3%.The plasma concentration of parent drug reached its peak at 0.8 h after oral administration of ISOIMP（10 mg/kg）, with t_1/2 and AUC being 1.7 h and 396.3±200.5 ng·h·m L^-1. ISOIMP was also eliminated rapidly with t_1/2 at 0.59 h after a single intravenous dose（2 mg/kg）. OPE and OPEH were the metabolites detected in the plasma. The absolute bioavailability of ISOIMP in rat was 18.4%. After oral administration of IMP, the 72 h accumulative excretion of the parent drug in urine, feces and bile were 0.018%, 1.16%, 0.0085%, respectively. The excretion of HER was very low in urine, bile and feces. And the metabolite XANT was excreted as free, glucuronide conjugate and sulfuric acid conjugate forms, with the 72 h accumulative excretion of 0.98% and 5.75% in urine and bile. The accumulative excretion of ISOIMP was 34.7% in feces in 72 h, while its excretion in the urine and bile was very low. Only trace amount of OPE was detected in urine, feces and bile. The metabolites OPEH were detected in free, glucuronide conjugate and sulfuric acid conjugate, and the urinary accumulative excretion of OPEH were 0.033%, 0.054% and 0.68%, respectively.The rat tissues were collected to quantify the drug concentration at the time points of 0.5 h, 2 h and 6 h after an oral dose of IMP（10 mg/kg）. The IMP rapidly distributed to the tissues. The IMP concentrations reached the peak level at 0.5 h post-dose in the most tissues, with the order of liver>kidney>heart>brain>lung>spleen>plasma. The concentration of IMP in the liver was significantly higher than that in the other tissues. ISOIMP also underwent a rapid tissue distribution after an oral dose. The maximum tissue levele reached the peak at 2h for the most of the detected tissues. The order of ISOIMP concentration in the tissues was as follows: liver>kidney>heart>brain>spleen>lung>plasma. The concentration of ISOIMP in the liver was significantly higher than that in the other tissues. The metabolic stability of IMP and ISOIMP were investigated in the incubation with HLM or RLM. The conmpouds were eliminated in the presence of NADPH. The metabolic rates of IMP for 30 min incubation with HLM and RLM were 69.7% and 94.5%, and the t_1/2 were 18.9±0.6 min and 2.85±0.37 min, respectively. The extrapolated hepatic clearance parameters（CLh） were 16.9±0.1 and 51.9±0.4 m L·min-1·kg-1. The Michaelism-Menten parameters Km were 3.6±0.16 and 14±0.24 μmol/L, and maximum velocity Vmax were 2928±96 and 8434±27 nmol·min-1·g-1. The eliminated rates of ISOIMP in 10 min incubation with HLM and RLM were 82.7% and 96.6%, and the t_1/2 were 4.94±0.31 min and 2.27±0.25 min, respectively. The CLh were 19.6±0.1 and 52.6±0.1 m L·min-1·kg-1. The Km were 15.2±0.6 and 16.7±0.4 μmol/L, and the Vmax were 3677±105 and 11423±371 nmol·min-1·g-1. Both IMP and ISOIMP were metabolically eliminated rapidly in HLM and RLM. However, the compounds showed higher velocity in RLM than in HLM. When compared between the two compounds, the metabolic elimination of IMP was faster than ISOIMP in both HLM and RLM.The results of CYP phenotyping indicated that CYP1A2, 2B6, 2C19, and 3A4 were the CYP isoforms responsible for the metabolism of IMP and ISOIMP. Their individual contributions assessed using the method of the total normalized rate were 20.4%, 7.3%, 10.5% and 61.8% for IMP, while 34.8%, 18.3%, 13.6% and 33.4% for ISOIMP, respectively. CYP3A4 and 1A2 were identified as the major isoforms that significant contributed to the metabolism of the two components.In summary, IMP and ISOIMP were absorbed rapidly in rat after an oral dose. The exposure of the two compounds in liver was significantly higher than that in other tissues and plasma. The IMP and ISOIMP were metabolically eliminated in HLM and RLM in the presence of NADPH. It is suggested to improve the metabolic stability in futher drug development. Also, based on the current results and the the previous result of CYP inhibition by IMP and ISOIMP, it is recommended to monitor the possible herb-drug interaction when IMP and ISOIMP containing herbs are used with substrates or inhibitors of CYP1A2 and CYP3A4.