A novel orally active MDM2 inhibitor (MI-219) activates the p53 pathway and is selectively toxic to tumor cells

B271 Introduction: The tumor suppressor p53 is a key regulator of cell cycle, apoptosis, senescence and DNA repair. Due to its important role in oncogenesis, it is not surprising that p53 function is compromised in all human cancers. In half the cancers, p53 gene is directly mutated/deleted, rendering p53 protein inactive, while in the remaining cancers with wild-type p53, p53 function is effectively inhibited by its cellular endogenous inhibitor the minute double minute 2 (MDM2) protein through direct interaction. Thus, targeting the MDM2-p53 interaction by small-molecule inhibitors is a promising approach for the reactivation of p53 function, and is being intensely pursued as a cancer therapeutic strategy. Since MDM2 is an essential regulator of p53 activity, there is a serious concern that unleashing p53 function by small-molecule inhibitors of MDM2 may result in toxicity to normal healthy tissues, casting doubts on the suitability of MDM2 inhibitors as potential cancer therapeutic strategy.
 Methods: We employed multiple human cancer cell line and xenograft tumor models, including SJSA-1 osteosarcoma and LNCaP prostate cancer, and performed following assays/procedures: 1) Binding affinity was determined in fluorescence polarization-based binding assays, 2) cell growth by WST assay, 3) cell cycle by BrdU incorporation, 4) apoptosis by Annexin V staining, 5) MDM2-p53 interaction by co-immunoprecipitation, 6) p53 was downregulated by siRNA oligonucleotide, 7) oral availability by pharmacokinetic (PK) studies, 8) p53 activation by immunohistochemistry and immunoblotting, 9) cell proliferation in tissues by BrdU incorporation, 10) apoptosis in tissues by TUNEL staining, 11) anti-tumor activity in xenograft models, 12) toxicity by animal body weight and necropsy, and 13) normal tissue damage by histopathology. Results: Employing a computational structure-based approach, we have designed a new, highly potent (K i = 5 nM) and selective (>10,000-fold over MDM2 homologue MDMX), orally-available small-molecule MDM2 inhibitor (MI-219) with a distinctively different chemical structure from other known MDM2 inhibitors. MI-219 disrupts intracellular MDM2-p53 interaction in cancer cell lines, leading to accumulation and activation of p53. p53 pathway is activated in both normal and cancer cells with wild-type p53, and leads to cell cycle arrest in both normal and cancer cells with wild-type p53, but selective apoptosis in cancer cells. Induction of cell cycle arrest and apoptosis is strictly p53-dependent. MI-219 has excellent oral bioavailability in mice and rats. A single oral dose of MI-219 induces rapid, but transient, activation of p53 in xenograft tumors and normal mouse tissues, strongly correlating with the plasma levels of MI-219. Multiple oral dosing of MI-219 potently inhibits tumor growth without causing animal weight loss or other signs of toxicity. Importantly, therapeutically effective dose schedules of MI-219 do not result in any damage to normal radio-sensitive and radio-resistant tissues.
 Conclusion: We conclude that activation of p53 by potent and specific small-molecule MDM2 inhibitors is selectively toxic to tumors without causing damage to normal tissues. Our study thus provides a strong rationale to advance MI-219 or its analogues for clinical development as a new and promising cancer therapeutic strategy.