Preclinical Pharmacokinetics Of DAT-230

DAT-230 is synthetic tubulin-binding VDA candidates,which was identified as a potential lead-based on extremely potent cytotoxicity with good pharmacological properties.It is structurally designed from Combretastain A4,disrupting intracellular microtubule network and inhibit tubulin polymerization through binding to the colchicines-binding site.In this paper,method for concentration assay of this drug in biological matrix was developed,and the pharmacokinetic process of DAT-230 was systematically studied.1.The pharmacokinetics of DAT-230 in rat.We established an UPLC-MS/MS method for the determination of DAT-230 in rat plasma,and apply the method to the pharmacokinetic study of DAT-230 in rats.Plasma samples were pretreated by protein precipitation with methol.Chromatographic separation was performed on an ACQUITY UPLC BEH C18 column with mobile phase consisting of methanol and 0.2%formic acid in water(80:20,v/v).The detection was carried out by multiple reactions monitoring in positive ion mode by monitoring the ion transitions m/z 372.17→m/z 357.17 for DAT-230 and m/z 406.08→m/z 345.16 for COH-203(IS).The method was linear over the range of 0.10～5200 ng/mL,and the intra-and inter-day precision were below 10%.The extraction recovery ranged from 76.1%to 79.4%.The matrix effect was between 91.8%and 98.3%.DAT-230 plasma samples were found to be stable in different conditions investigated in this article.The method was already confirmed by the guildlines on bioanalytical method validation and was successfully applied to the pharmacokinetic study of DAT-230 in ratUPLC-MS/MS method was developed to determine the concentrations of DAT-230 in rat.The method was successfully applied to the estimation of pharmacokinetic parameters of DAT-230 after intravenous doses of 5 mg/kg in rats.The mean(±SD)area under the plasma concentration-time curve and elimination half-life for DAT-230 were 861.5±281.3 ng·h/mL and 1.0 ± 0.4 h,respectively.2.The tissue distribution of DAT-230 in rat.It indicated that DAT-230 could be distributed rapidly and widely,and eliminated rapidly with time,and there was no long-term accumulation,which was in accordance with the variation trend of plasma concentration.It is an advantage that DAT-230 eliminated rapidly in vivo.As we known,anti-cancer drug has a relative high toxicity and DAT-230 could not be an exception.There is no long-term accumulation of DAT-230 in rat tissues and can eliminated rapidly could reduce toxicity for most tissues and cells.In addition,DAT-230 was detectable with low concentration in brain,suggesting that DAT-230 did not efficiently cross the blood-brain barrier.The level of DAT-230 in liver was significantly higher than other tissues,indicating its accumulation in liver.Furthermore,the distribution of analyte in liver,intestine and stomach were thought to be consistent with literature that DAT-230 has high tumor-selective activity on hepatocellular adenocarcinoma cell,colonic adenocarcinoma cell or gastric adenocarcinoma cell in vitro which could be a potential candidate for the treatment.Meanwhile,DAT-230 was found with lower concentration in brain,heart,lung,which implied that the distribution of DAT-230 was not depended on the blood flow or perfusion rate of the organ.The lower level of concentration of DAT-230 in heart and lung can reduce the toxicity.The tissue distribution in rat after intravenous injection of DAT-230 at a dose of 5 mg/kg was investigated.The result showed that DAT-230 could be distributed rapidly and widely,eliminated rapidly with time,and there was no long-term accumulation.In addition,the level of DAT-230 in liver was significantly higher than other tissues,indicating its accumulation in liver and it’s the target tissue3.The excretion of DAT-230 in rat.After an intravenous injection of 5 mg/kg,the cumulative amount of DAT-230 in urine,bile and feces were amounted 0.13%,2.27%and 2.80%,respectively,of the given dosage.DAT-230 was excreted unchanged about 5.3%of administrated dose,indicating that DAT-230 was mainly excreded changed4.The metabolism of DAT-230.Six fungus were used to evaluate the abilities to biotransform DAT-230,Cunninghamella elegans AS 3.2028 gave the best biotransformation yield at 90.3%and produced various metabolites of DAT-230,so it was chosen as the model microorganism for further investigation.In order to isolate major metabolites of DAT-230 in suffieient quantities for structural elucidation,the biotransformation of DAT-230 by Cunninghamella elegans AS 3.2028 was carried out on preparative scale according to the optimization experiments(initial medium pH 7.0,substrate concentration 0.4 mg/mL,biotransformation time 7 d).Three major metabolites were isolated by the semi-preparative HPLC and identified by NMR and MS,which could be used as reference substances in DAT-230 metabolism studiesMetabolites of DAT-230 in microbial model and in rat were investigated by LC-MSn method.A total of 10 metabolites were found which including O-demethylated metabolites,hydroxylated metabolites,acetylated and glucuronide conjugates.And the 4 major metabolite pathways of DAT-230 in microbial model and in rat were O-demethylation,hydroxylation,acetylation and glucuronidation.Secondary metabolism via these pathways was also evidenced.