Novel small molecule inhibitors of MDM2/4-p53 interaction, YH264 and its ethyl ester YH263:Preclinical evaluation

Introduction: In p53+/+ cells, expression of MDM2/4 leads to turnover of p53 and inhibition of downstream gene transcription decreasing cell cycle arrest or apoptosis. Prevention of MDM2/4-p53 interaction is a promising therapeutic strategy. Two in-house developed small molecule inhibitors of MDM2/4-p53 binding, YH264 and YH263, had nM activity in protein binding assays, were characterized by co-crystal structures, and had low μM activity in the NCI 60 cell screen. We evaluated in vitro cytotoxicity in HCT 116, human p53+/+ colon cancer cells as well as efficacy, pharmacokinetics, and metabolism in mice bearing HCT 116 xenografts. Methods: Cytotoxicity against HCT 116 cells was assessed by 72 h MTT assay. C.B-17 SCID mice bearing HCT 116 xenografts (5 mice/group) were dosed with either YH264 or YH263 (150 mg/kg IV or PO), or vehicle QDx5 and tumors were measured twice weekly for at least 1 week post dosing. For pharmacokinetic studies, mice bearing HCT 116 xenografts were treated with 150 mg/kg YH264 or YH263 IV and PO. Plasma and tumor were collected between 5 min and 24 h. Urine was collected between 0-6 h and 6-24 h. YH264 and YH263 in cells from the in vitro study and tissues were quantitated with an LC-MS/MS assay. Pharmacokinetic parameters were calculated non-compartmentally. Results: IC50 values of YH264 and YH263 were 18.6 (9 μg/ml) and 8.9 μM (4 μg/ml), respectively. When mice were dosed IV or PO for QDx5 with YH264 or YH263, no decrease in body weights or significant decrease in xenograft volume was observed when compared to vehicle treated mice. Plasma elimination of IV YH264 was biphasic. Plasma and tumor parameters were respectively: Cmax 1,451 μg/ml and 44 μg/g; AUC0-t 125,000 μg·min/ml and 38,000 μg·min/g. Tumor concentrations remained above 17 μg/g out to 24 h. Plasma t1/2 was 147 min and clearance was 1.2 ml/min/kg. Metabolites included hydroxylated and glucuronidated products. After PO dosing, bioavailability was 18% and YH264 was undetectable in tumor. IV plasma elimination of the ethyl-ester YH263 was biphasic as well. Plasma and tumor parameters were respectively: Cmax 995 μg/ml and 21 μg/g; AUC0-t 45,477 μg·min/ml and 18,950 μg·min/g. Tumor concentrations remained above 7 μg/g for 24 h. Plasma t1/2 was 263 min and clearance was 3.3 ml/min/kg. 2% of the YH263 dose was converted to YH264 and there were multiple hydroxylated and glucuronidated metabolites. After PO dosing, bioavailability was 4.8%. In culture YH264 and YH263 cellular concentrations were 60- and 200-fold higher than medium concentrations. Conclusions: At the IC50 medium concentration, the cells accumulated 200-fold higher concentrations: of YH263 than medium. Despite dosing the mice at the maximum soluble dose, we could not achieve tumor concentrations (800 μg/g) equivalent to those required to inhibit cells in vitro. This may explain the absence of efficacy on the current schedule