Abstract 5474: Optimization of a microdialysis method for evaluation of CP-868,596 penetration in glioblastoma murine models using a sensitive liquid chromatography mass spectrometry (LC-MS/MS) method

Abstract

Abstract Focal amplification and gene rearrangement of the platelet derived growth factor receptor alpha (PDGFRA) gene, resulting in constitutively active signaling, has been identified in a subset of cases of glioblastoma multiforme (GBM). Thus, inhibiting PDGFR signaling represents a valid treatment strategy for these tumors. CP-868,596, a potent inhibitor of the PDGFR kinase, is currently undergoing preclinical testing for the treatment of GBM. CP-868,596 inhibits the intracellular ATP-binding site of PDGFR, thereby attenuating downstream signaling events. To characterize CP-868,596 penetration in murine high grade glioma, we used a cerebral microdialysis approach. This approach would allow us to measure unbound CP-868,596 concentrations in the tumor extracellular fluid (ECF) using microdialysis under steady-state conditions. To accurately measure the potentially low concentrations of CP-868,596 in the dialysates, we developed and validated a highly sensitive LC-MS/MS method for analysis of CP-868,596. The parent drug CP-868,596 and its deuterated form as an internal standard (ISTD) were separated on a Pursuit 3µ C18 column (50 × 2.0 mm) with mobile phase consisting of 85% acetonitrile and 15% 10mM HCOONH4. The ion transitions at m/z 444.4→301.1 for CP-868,596 and at m/z 448.2→374.2 for the ISTD were selected. Samples were processed using liquid-liquid extraction (LLE) with t-butyl methyl ether. The extracted samples were dried by nitrogen gas and reconstituted with 40% acetonitrile. 10 µLs were injected for analysis. Both within-day and between-day precision (%CV) were ≤ 7% and accuracy ranged from 93.7% to 102.5%. Next, we optimized the microdialysis conditions for CP-868,596. Initial in vitro microdialysis experiments using artificial cerebrospinal-fluid (aCSF) as a perfusate demonstrated low CP-868,596 recovery (8.24 ± 1.26, mean ± SD). Addition of 10% hydroxyl-propyl beta cyclodextrin to the perfusate increased recovery to 91.2% ± 4.6%. Several approaches to estimate probe recovery were tested including retrodialysis and Zero-Flow Rate (ZFR) methods. The ZFR method estimated stock concentrations with > 85% accuracy; hence, this method will be used to estimate recovery under in vivo steady-state conditions. To achieve steady-state plasma concentrations, CP-868,596-loaded miniosmotic pump (Alzet model 2001D) was s.c. implanted in wild-type FVB mice (n=6). Analysis showed that steady-state plasma levels of CP-868,596 were maintained for 30 hours (516.8 ± 92.9 ng/ml, mean ± SD). We predict that our sensitive analytical method (LLOQ 0.1 ng/mL) will allow us to measure the low CP-868,596 concentrations expected during in vivo cerebral microdialysis experiments. Studies measuring CP-868,596 tumor concentrations using the cerebral microdialysis approach in several murine GBM models are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5474. doi:10.1158/1538-7445.AM2011-5474