| Cajaninstilbene acid(CSA,3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid),an active constituent of pigeonpea leaves,one of important tropical crops,is known for its clinical effects in the treatment of diabetes,hepatitis,measles and potential anti-tumor effect.A rapid and sensitive liquid chromatography tandem mass spectrometry(LC-MS/MS)method was developed and validated for the determination of CSA in rat plasma and various tissues(brain,heart,lung,liver,spleen,small intestine and kidney)of rat for the first time.The effect of the cytochrome P450 isozymes on the activity of CSA was investigated and the metabolites formed by human liver microsomes were identified in the study.We also evaluate the potential effects of CSA,studies were conducted to assess its ability to inhibit the major CYP450 enzymes(CYP1A2,CYP2A6,CYP2E1,CYP2C9,CYP2C19,CYP2D6,and CYP3A4)with human liver microsomes and to induce the CYP450 enzymes expression in primary human hepatocytes.Conclusions were summarized below:1.a simple and highly sensitive LC-MS/MS method was developed for the first time to determine CSA levels in rat plasma samples.Both CSA and ISL(internal standard)were eluted within 10 min with retention times of approximately 9.19 and 3.50 min,respectively.The calibration curve was linear from 10 to 6000 ng/mL(R= 0.9967)for plasma samples and the lower limit of quantification(LLOQ)which was 10 ng/mL.The intra-day accuracy ranged from 99.2%to 103.4%,and the inter-day accuracy ranged from 102.0%to 107.5%.The mean intra-and inter-day precision was between 1.8%and 3.8%and 2.2-6.6%,respectively.The mean recoveries for CSA(10,100 and 1000 ng/mL)were 103.4 ±3.6%,102.7 ±3.0%and 99.1 ± 1.9%,the recovery of the internal standard determined at 400 ng/mL was 95.6± 5.2%.Maximum deviations of short-term stability,freeze/thaw stability,autosampler stability,and long-term stability are lower than 6.7%.For the application to pharmacokinetic studies,Each rat was administered an oral dose of 2 mg/kg CSA.Blood samples were collected at 0,0.083,0.167,0.25,0.5,1,2,4,6,and 8 h.As for intragastric administration,the terminal half-life(t1/2)was 51.40 ± 6.54 min.The area underthe plasma concentration curve(AUC0→∞)of CSA after intragastric administration was 343,372.89±2546.66 ng min/mL.The Tmax and Cmax were 10.7±0.31min and 4500±345.83 ng/mL.In the present study,the Tmax of CSA in plasma was<15 min indicated that CSA was rapidly absorpted.Half-life of CSA in plasma was<1 h indicated that CSA was rapidly eliminated.2.For the first time,a highly sensitive and specific method for the determination of CSA in rats tissues(brain,heart,lung,liver,spleen,small intestine and kidney)was developed using highperformance liquid chromatographic separation with tandem mass spectrometric detection.The calibration curves were showed good linearity in the range 10-6000 ng/mL for CSA.The calibration curves for all matrices showed good linearity(R>0.9974)over the concentration ranges tested.The intra-day accuracy ranged from 93.5%to 104.6%,and the inter-day accuracy ranged from 93.3%to 106.4%.The mean intra-and inter-day precision was in the ranges of 0.6-6.1%and 1.5-5.8%,respectively.The mean recoveries in all tissue samples were above 95.0±4.8%.Maximum deviations of short-term stability,freeze/thaw stability,autosampler stability,and long-term stability for tissue homogenates samples are lower than 7.5%.Tissue distribution of CSA was investigated following a single oral dose of 2 mg/kg CSA to rats.Following 60 min CSA showed substantial disposition in heart,lungs,liver,spleen,small intestine and kidneys.The highest levels were detected in small intestine,followed by liver and kidneys.The highest concentrations were found in small intestine may be attributable mainly attributed to the residual drug content.The high liver concentrations indicate that CSA was possibly absorpted in liver.On the contrary,increasing concentrations of CSA in kidneys over time indicate that renal excretion might be a major elimination route for CSA.Meanwhile,CSA was not found in brain,suggesting that CSA did not efficiently cross the blood-brain barrier.3.Human liver microsomes and recombinant human P450 enzymes were used to investigate the in vitro formation of CSA and identify the CYP450 enzymes involved in the metabolism of CSA.The formation of the metabolite was dependent on the presence of NADPH and the yield of metabolite appeared to be linear with respect to incubation time(up to 30 min).Two hydroxylation metabolites were identified in the study,according to the parent drug fragmentation pattern and the fragmentations of characteristic fragment ions,it can be concluded that the predominated metabolites M1 and M2 were 12-hydroxy cajaninstilbene acid and 6-hydroxy cajaninstilbene acid.The reaction phenotype study showed that CYP3A4,CYP2C9 and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA.Kinetic analysis for the formation of M1 and M2 were also studied with pooled human liver microsomes.The apparent Km values of M1 and M2 catalyzed by human liver microsomes are 28.8,and 23.6μM,respectively.The Vmax(apparent)values were 141.6,and 122.7 pmol/mg/min,respectively.The apparent intrinsic clearances[Clint]of M1 and M2 formation were 4.9 and 5.2 μL/mg protein/min,respectively.4.To evaluate the potential effects of CSA,studies were conducted to assess its ability to inhibit the major CYP450 enzymes with human liver microsomes and to induce the CYP450 enzymes expression in primary human hepatocytes.The effect of CSA inhibition/induction of enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques.CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with an IC50 of 28.3μM and 31.3μM,respectively,while exhibited no inhibition activities to CYP1A2,CYP2A6,CYP2C19,CYP2D6 and CYP2E1.CSA showed a weak effect on CYP450 enzymes in time-dependent manner.CSA did not substantially induce CYP1A2,CYP2A6,CYP2E1,CYP2C9,CYP2C19,CYP2D6 or CYP3A4 at the concentration up to 30μM in primary human hepatocytes.The results of our experiments may be helpful to predict clinically significant drug-drug interactions when other drugs are combined administration with CSA. |
