Abstract 185: Development of a novel non-diuretic brain-penetrating ethacrynic acid analog and demonstration of its potent efficacy in orthotopic glioblastoma (GBM) models
Authors: Punganuru Surendra R. , Madala Hanumantha Rao , Srivenugopal Kalkunte S. 
Published: July 2017

Abstract
The incidence of pediatric and adult brain tumors has continued to rise and temozolomide (TMZ) has remained the sole choice for chemotherapy of these cancers. A number of factors including the inefficient drug entry through the BBB, intratumoral heterogeneity, overexpression of MGMT repair protein and bone marrow toxicity due to alkylating regimens have impeded successful therapy of these cancers. There is, therefore, an urgent need to design new, more effective brain-penetrating drugs that can effectively eliminate brain tumors. We are highly interested in exploiting the elevated oxidative stress present in gliomas and have synthesized a hydrophobic, non-diuretic analog of ethacrynic acid (EA) called KSS72 [1-(2,3-dichloro-4-methoxyphenyl)-2-methylenebutan-1-one] by removing the carboxylate side chain. KSS72 was selectively cytotoxic to cancer cells including gliomas. Compared to EA which does not cross the BBB, Pharmacokinetics following intravenous or oral administrations in mice showed its excellent penetrance through the blood brain-barrier, KSS72 accumulating at levels equivalent to TMZ (25% of plasma levels) in the brain In vitro assays measuring protein carbonyl content, GSH content, ROS generation, GSTpi enzyme activity and others showed that KSS72 triggers a redox imbalance by inhibiting GSTpi and by lowering the antioxidant (GSH) and reducing equivalent (NADPH) levels, leading to a significant elevation of ROS levels. We also showed the upregulation of endoplasmic reticulum (ER) stress-responsive proteins, activation of MAPK, autophagy and apoptotic pathways by KSS72 in several cell lines. KSS72-induced autophagy was a post event of redox perturbation that displayed a wide array of characteristic features including double membranous vacuoles with entrapped organelles, acidic vesicular organelles, and increased expression of LC3-II and beclin-1. In view of its excellent brain penetrance and multiple cytocidal events mediated by ROS and autophagy, we tested the antitumor efficacy of KSS72 in human GBM cell lines and intracranial GBM xenografts. SF-188 human GBM cells expressing firefly luciferase were injected into the brain just left of bregma for the development of orthotopic GBM in nude mice. These mice were given 25 mg/kg/day of KSS72 intraperitoneally for 2 weeks. The animals when imaged by IVIS after luciferin injections showed a complete lack of intracranial tumors in all KSS72 administered animals. H&E staining of mouse brain sections confirmed the total elimination of GBM by KSS72. KSS72 did not exert any toxicity on host tissues and serum ALT and AST levels were not altered. In summary, we conclude that KSS72 acting through multiple pathways of oxidative stress is a hugely promising non-toxic anti-glioma drug with potential to enter clinical trials (supported by CPRIT grants RP130266 and RP170207 to KSS).
Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. Development of a novel non-diuretic brain-penetrating ethacrynic acid analog and demonstration of its potent efficacy in orthotopic glioblastoma (GBM) models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 185. doi:10.1158/1538-7445.AM2017-185