Abstract 2749: Gemcitabine (GEM), a drug with clinical activity against pediatric solid tumors, demonstrates satisfactory CNS penetration in a preclinical murine medulloblastoma (MB) model.
Authors: Turner David C. , Freeman Burgess B. , Jacus Megan O. , Morfouace Marie , Throm Stacy L. , Vuppala Pradeep K. , Roussel Martine , Gajjar Amar , Stewart Clinton F. 
Published: August 2014

Abstract
GEM, a fluorinated nucleoside analog, was identified by in vitro high-throughput screening as a potent inhibitor of murine tumor cell lines derived from a preclinical model of a highly-aggressive subgroup of pediatric MB (MYC-driven Group 3). To support the potential preclinical-to-clinical transition of GEM for this indication, we determined the pharmacokinetic (PK) disposition and central nervous system (CNS) penetration of GEM in a murine Group 3 MB model. A serial sacrifice plasma PK study (single sample per mouse) was performed in tumor-bearing mice to obtain initial GEM plasma PK parameter estimates. These estimates were used to inform a D-optimal, limited sampling strategy for microdialysis experiments. Cerebral microdialysis was applied in CD-1 nude mice bearing orthotopic allograft Group 3 mouse MB tumor lines (SJCRH 2416/2417), which permitted repeated in situ measurements of GEM tumor extracellular fluid (tECF) concentrations. Microdialysis probes (BASi; 1 mm membrane) were introduced into the tumor through cannulae inserted during tumor cell implantation. After microdialysis probe equilibration, 5 mice were each dosed with 60 mg/kg of GEM via tail vein injection. In each mouse, serial plasma samples were collected at 0.083, 1.5, and 6 hrs after dosing, and tECF dialysate fractions were collected over 60 min intervals for up to 6 hrs post-dose. To measure GEM in both plasma and tECF, a robust, sensitive LC-MS/MS method was developed and validated. Both within-day and between-day precision (%CV) were ≤ 7% and accuracy ranged from 95.3% to 103%. GEM concentration-time plasma and tECF data were accurately described using a unified population PK model, consisting of a central plasma compartment, a peripheral compartment, and a perfusion-limited “well-stirred” tumor compartment. For modeling purposes, both the plasma flow to the brain and the volume of the brain ECF were fixed to published values. The PK analysis showed that plasma elimination of GEM followed bi-exponential kinetics, with an initial rapid decay (mean ± SD) t1/2,alpha of 8.2 ± 3.3 min followed by a relatively slower beta phase t1/2,beta of 1.6 ± 0.3 hrs. The (mean ± SD) area under the concentration-time curve (AUC0-∞) for GEM in plasma was 544 ± 199 μg/ml*min, consistent with previously published AUC values in pediatric patients at tolerable dosages (1,200 mg/m2). On average, GEM CNS penetration (calculated as the tECF/plasma GEM AUC0-8hrs ratio from the simulated concentration-time curves) was 0.21 ± 0.16. The peak tECF concentrations ranged from 2.7 to 76 μM, and simulated tECF concentrations exceeded in vitro cell line IC50 values for approximately 6 hours. Overall, these data provide convincing rationale to prioritize GEM efficacy evaluations in additional MB models within our preclinical brain tumor drug development program.
Citation Format: David C. Turner, Burgess B. Freeman, Megan O. Jacus, Marie Morfouace, Stacy L. Throm, Pradeep K. Vuppala, Martine Roussel, Amar Gajjar, Clinton F. Stewart. Gemcitabine (GEM), a drug with clinical activity against pediatric solid tumors, demonstrates satisfactory CNS penetration in a preclinical murine medulloblastoma (MB) model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2749. doi:10.1158/1538-7445.AM2013-2749