Pharmacokinetic Evaluation Of BRAF^V600E Inhibitor ¹¹C-CEP-32496

Purpose:BRAF mutation has been found in approximately 7%of human cancers,and the main form of mutation is valine-to-glutamate（V600E）,which is correlated with increased malignancy and decreased response to chemotherapy,representing an important target for personalized diagnosis and treatment of cancers.CEP-32496,a novel orally active BRAF^V600E600E inhibitor,has attracted much attention in the treatment of human cancers in preclinical trials.However,no studies in the world reported the pharmacokinetic characteristics of this BRAF^V600E inhibitor comprehensively.Here,we developed ¹¹C-CEP-32496 as a positron emission tomography（PET）radiotracer to evaluate its pharmacokinetics and explore its potential for in vivo imaging in BRAF^V600E-induced cancers.Method:To investigate and establish the methods of ¹¹C-CEP-32496.The identities of these compounds were confirmed by high-resolution mass spectra（HRMS）,and radiochemical purity and stability was detected by high performance liquid chromatography（HPLC）.BRAF^V600E kinase binding assays of ¹¹C-CEP-32496determined the binding ability of CEP-32496 and BRAF^V600E kinase.To investigate in vivo distribution and metabolize of ¹¹C-CEP-32496,microPET scans were performed from 0 to 60 min after ¹¹C-CEP-32496 injection via tail vein,and biodistribution studies were performed at 30 min and 60 min after ¹¹C-CEP-32496injection on subcutaneous A375 melanoma-bearing nude mice.After A375 tumor specimens are prepared,the in vitro binding ability of ¹¹C-CEP-32496 and A375tumor specimens are detected by autoradiography.Result:¹¹C-CEP-32496wassynthesizedbyradiolabelingof5-（1,1,1-trifluoro-2-methylpropan-2-yl）isoxazol-3-amine hydrochloride（1·HCl）with¹¹C-phosgene,followedbyreactionwith3-（6,7-dimethoxyquinozolin-4-yloxy）aniline.The radiochemical purity remained>98%at room temperature for 90 min.¹¹C-CEP-32496 showed high binding affinities with BRAF^V600E kinase.Half maximal inhibitory concentration（IC50）of CEP-32496 and sorafenib was 10.30uM and 6.41uM,respectively.MicroPET imaging showed that ¹¹C-CEP-32496 flowed through blood,lung and heart rapidly,and distributed throughout the whole body after ¹¹C-CEP-32496injection.The radioactivity uptake in liver decreased gradually and low radioactivity uptake was found in kidney.The radioactivity uptake in intestine increased from 12min after injection.No significant radioactive uptake was found in other tissues and organs.Sustained low radioactivity（0.56±0.03%ID/g）accumulated in A375melanoma over time,and no significant difference was found in the radioactivity uptake of A375 melanoma at 5 min pre-injection of GF-120918（0.61±0.00%ID/g,p=0.069）.Biodistribution results revealed that the radioactivity mainly accumulated in live（19.08±0.71%ID/g,25.23±0.57%ID/g）and small intestine（33.79±1.23%ID/g,22.30±3.18%ID/g）at 30 min and 60 min after ¹¹C-CEP-32496 injection,and minimum radioactivity was observed in other organs and tissues including tumors（1.79±0.07%ID/g,1.85±0.09%ID/g）.Autoradiography revealed that dense accumulation in A375 melanoma tissue sections,which can be blocked by sorafenib and unlabeled CEP-32496（p<0.01）.Conclusion:This study indicated that ¹¹C-CEP-32496 has excellent specificity and affinity for BRAF^V600E mutation in vitro,which permitted to noninvasively track and quantify in vivo action of ¹¹C-CEP-32496.¹¹C-CEP-32496 has appreciated pharmacokinetic properties,and it was excreted by intestine and kidney rapidly,which would be helpful for facilitating the development of CEP-32496 in the treatment of BRAF^V600E-positive tumors.