Abstract 1475: Localization of mitochondrial targeted GS-nitroxide JP4-039 BODIPY FL conjugate during radioprotection and mitigation in vivo

Abstract

Abstract JP4-039 is a mitochondrial targeted GS nitroxide, which protects and mitigates against ionizing irradiation damage. Using a JP4-039 BODOIPY FL conjugate (BODIPYJP4-039) we have demonstrated that BODIPYJP4-039 co-localizes with MitoTracker stained mitochondria in cells in vitro and that when delivered by swallow in F15 emulsion, JP4-039 protects the esophagus from single dose or fractionated irradiation and is rapidly detected in excised esophagus. In vitro testing demonstrated that addition of BODIPY FL to JP4-039 did not alter radioprotection or mitigation of 32Dcl3 murine hematopoietic cells in clonogenic survival curve assays and that mitochondrial targeting of BODIPY JP4-039 was preserved in co-localization experiments with MitoTracker dye. In vitro irradiation survival curves were performed comparing JP4-039 to BODIPY JP4-039. 32D cells were incubated with 10 µM JP4-039 or BODIPY JP4-039 either pre or post irradiation and irradiated to doses from 0 to 8 Gy and plated in methylcellulose-containing medium. Colonies of greater than 50 cells were counted on day 7. The use of JP4-039 or BODIPY JP4-039 before irradiation resulted in an increase in Do and ñ from 1.3 ± 0.1 to 2.3 ± 0.3 for JP4-039 and to 2.6 + 0.3 Gy for BODIPY JP4-039 (p = 0.0433 and 0.0254, respectively). When used post irradiation, there was also an increase in the ñ from 1.1 + 0.1 to 3.5 + 0.2 for JP4-039 and to 4.5 + 1.1 for BODIPYJP4-039 (p = 0.0001 and 0.0258, respectively). Co-localization of BODIPY JP4-039 and MitoTracker demonstrated that drug targeted mitochondria. BODIPYJP4-039 in F15 liposomes was administered by swallow to C57BL/6NHsd mice. Ten minutes post swallow, the esophagus, liver, lungs, and brain removed and frozen sections observed using fluorescence microscopy. In other experiments relative fluorescent units (RFUs) per mg of tissue were measured in excised organs (esophagus, liver, lung, brain) and in isolated mitochondria. The mitochondria had a 6 fold RFU increase over control compared to 1.3 for cytosol. Esophagus samples showed a 3 fold RFU increase over control. Additional in vivo experiments esophagus was excised 10 minutes post BODIPY JP4-039 swallow and a single cell suspension prepared for flow cytometric sorting of side population (SP) stem cells. Sorted SP and non-side population (nSP) cells were stained with MitoTracker dye. BODIPY JP4-039 localized to mitochondria in both sorted SP and nSP cells. BODIPYJP4-039 targets mitochondria of stem cell and non stem cell populations in the esophagus in vivo and is rapidly detected. Therefore, BODIPY JP4-039 should be a useful tool for determining the pharmacokinetics of radioprotection by JP4-039. Supported by NIH/UAID U19A168021 and NIH RO1CA83876 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1475. doi:1538-7445.AM2012-1475