Pharmacokinetic Study Of A Novel B-Raf Inhibitor LXK4

Ras-Raf-Mek-Erk signaling pathway plays a critical role in regulating cell growth,proliferation and differentiation.The overexpression and mutation of the protein members in the signaling pathway can cause the formation of tumors.B-Raf is one of the Raf kinase family members in the signaling pathway,among which the mutation occurring in the 600 amino acid residues of the active region,namely the glutamate substitution of valine(B-Raf^V600E)is the most common site.B-RafV600 E has become one of the most important targets of human cancers.Drug candidate LXK4 is a novel selective B-RafV600 E inhibitor,which was discovered by Guangzhou Institutes of Biomedicine and Health,Chinese Academy of Science,exhibiting potent inhibitory activity with an IC₅₀ value of 35 nM for B-RafV600 E and also displaying antiproliferative activity against colo205 and HT29 cells with IC₅₀ values of 111 nM and 92 nM,respectively.LXK4 displays good antitumor potency.Therefore,we studied the preclinical pharmacokinetic differences of LXK4 in rats and beagle dogs to evaluate its druggability and assessed the potential risk of drug interactions.The main contents of this research are as follows:?1?A LC-MS/MS method was established for the determination of LXK4 in plasma,which performed good selectivity,and the calibration curves in the range 0.5-1000 ng/m L were liner.The method was sensitive with the lower limit of quantification of 0.5 ng/m L.The intra-and inter day precision and accuracy as well as the stability were all in line with the requirements of the quantitative analysis of biological samples.The extract recovery of the method was higher than 85% and the matrix effect can be negligible under the current conditions.The LC-MS/MS method can be applied to quantify the concentration of LXK4 in plasma for the pharmacokinetic study in rats and dogs.?2?After intravenous injection of LXK4 in rats and beagle dogs,the concentration of LXK4 declined very fast in an initial short period of time,and then declined slowly.After oral administration of LXK4 in rats and beagle dogs,the drug was absorbed quickly with the Tmax of 1 h.The mean t1/2 covered the range of 1.5-2.4 h with no significant difference.The oral bioavailability of LXK4 in rats and beagle dogs were 14.6% and 30.5%,respectively,which indicates some differences between species.?3?The metabolic stability of LXK4 in different species of liver microsome was evaluated using a substrate depletion approach in vitro.The results showed that the depletion of LXK4 was fast in human liver microsome,followed by in rat liver microsome and dog liver microsome.We further researched the enzyme phenotypes which metabolized LXK4 using specific CYP inhibitors and found that LXK4 was mainly metabolized by CYP2C8 and CYP3A4 in the phase I metabolism.?4?Human liver microsome was incubated with specific probe substrates and LXK4 to assess the inhibitory effects of LXK4 on the seven subtypes of P450 s.To varying degrees,LXK4 can inhibited six of these subtypes of P450 s.Specifically,LXK4 inhibited CYP1A2 with IC₅₀ of 5.31 ?M,CYP2C8 with IC₅₀ of 8.77 ?M,CYP2C9 with IC₅₀ of 4.90 ?M,CYP2C19 with IC₅₀ of 6.09 ?M,CYP2D6 with IC₅₀ of 10.1 ?M,CYP3A4 with IC₅₀ of 8.6 ?M?substrate is midazolam?and CYP3A4 with IC₅₀ of 11.2 ?M?substrate is testosterone?,which can provide information for the prediction of the potential drug-drug interaction.