Mechanism of Action and Safety of Second Generation Analogs of SDX-101 (R-Etodolac). (2004)

Abstract SDX-101 (R-etodolac), which is currently being evaluated in clinical trials for treatment of chronic lymphocytic leukemia, down regulates the activity of the β-catenin pathway and inhibits the growth of non-Hodgkin’s Lymphoma Daudi tumor xenografts in vivo when dosed orally (AACR PROC 2004 Abs# 2061 and #4574). Initial co-immunoprecipitation experiments conducted on cell nuclear fractions identified a heteromeric nuclear protein complex containing β-catenin and PPAR-γ. Furthermore, we have demonstrated that SDX-101 treatment reduces nuclear β-catenin in the immunoprecipitated complex, indicating that this complex may represent a target of SDX-101 (AACR PROC 2004 Abs# 3672). We recently reported evaluation of novel structural analogs of SDX-101 and have shown that these analogs, whose structures were not disclosed, are 5–10 fold more potent in in vitro cytotoxicity assays than SDX-101 and that they are orally efficacious in vivo (NCI/EORTC 2004 Abs #383). Our current studies further characterize the mechanism of action and safety of these analogs and identify the structures of selected analogs. Novel functional assays were developed to test and compare SDX-101 and the analogs at 4 hours post-treatment, a time before appreciable loss of viability was detected. Best results were obtained using a functional assay co-transfecting a β-catenin-dependent reporter construct (TOPFLASH) and β-catenin and RXR expression vectors. The average IC50 of analogs in this β-catenin reporter system ranged from 50 to 160 μM. These values were approximately five- to ten- fold lower than the IC50 for SDX-101 (~700 μM). Similar results were obtained assessing the inhibition of PPAR-γ-mediated transcription, using a PPAR-dependent reporter and co-transfection with PPAR-γ and RXR expression vectors. The average IC50s of the analogs ranged from 50–150 μM in this functional assay, demonstrating an approximately 10-fold increase in potency of the analogs when compared to SDX-101 (~1000 μM). No effect was observed at the 4 hour time point using a constitutive SV40-based control reporter vector. These results suggest that the primary target for these compounds may be a nuclear complex containing β-catenin, PPAR-γ and RXR, supporting a hypothesis developed upon evaluation of earlier results generated with SDX-101. To evaluate the safety of two SDX-101 analogs in vivo, normal mice were administered each analog at 240, 120 and 60 mg/kg/d (M-F) for four weeks. Mortality, morbidity, clinical signs, hematology/chemistry were monitored. There were no mortalities, overt toxicities or abnormal observations at necropsy with either of the analogs at any of the tested dose levels. There was a transient body weight loss (<5%) and a mild dose-independent increase in platelets and a reversible decrease in total bilirubin. Results of the histopathological examination of critical organs are pending. These results suggest, when given at doses previously shown to be efficacious in a DAUDI murine lymphoma model, these analogs were well tolerated. In conclusion, these data demonstrate that the second generation analogs of SDX-101 display more potent in vitro and in vivo activity while retaining a mechanism of action similar to that of SDX-101.