title: In vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of pyrazole-based small molecule inhibitors of Mdm2/4-p53 interaction.
abstract: OBJECTIVE
  The interaction of p53 with its negative regulators Mdm2/4 has been widely studied (Khoury and Domling in Curr Pharm Des 18(30):4668-4678, 2012). In p53(+/+) cells, expression of Mdm2/4 leads to p53 turnover, inhibition of downstream transcription, decreasing cell cycle arrest, or apoptosis. We report in vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of YH264, YH263, and WW751, three proposed small molecule inhibitors of the Mdm2/4-p53 interaction.
  
  
  METHODS
  MTT cytotoxicity assays were performed, and alterations in proteins were examined using western blots. Mice were dosed 150 mg/kg YH264 or YH263 IV or PO QDx5. Mice were IV dosed 88, 57, or 39 mg/kg WW751 for 3, 5, or 5 days. YH264, YH263, and WW751 and metabolites were quantitated by LC-MS/MS.
  
  
  RESULTS
  IC50 values for YH264, YH263, and WW751 against p53 wild-type HCT 116 cells after 72 h of incubation were 18.3 ± 2.3, 8.9 ± 0.6, and 3.1 ± 0.2 μM, respectively. Only YH264 appeared to affect p53 expression in vitro. None of the compounds affected the growth of HCT 116 xenografts in C.B-17 SCID mice. YH264 plasma half-life was 147 min; YH263 plasma half-life was 263 min; and WW751 plasma half-life was less than 120 min.
  
  
  CONCLUSIONS
  Despite dosing the mice at the maximum soluble doses, we could not achieve tumor concentrations equivalent to the intracellular concentrations required to inhibit cell growth in vitro. YH263 and WW751 do not appear to affect p53/Mdm2, and none of the three were active in a subcutaneous HCT 116 p53(+/+) xenograft model.
authors.affiliation: Cancer Therapeutics Program, Hillman Cancer Center, The University of Pittsburgh Cancer Institute, Room G27b, 5117 Centre Ave, Pittsburgh, PA, 15213, USA.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Christner
authors.forename: Dana M
authors.initials: DM
authors.lastname: Clausen
authors.forename: Jan H
authors.initials: JH
authors.lastname: Beumer
authors.forename: Robert A
authors.initials: RA
authors.lastname: Parise
authors.forename: Jianxia
authors.initials: J
authors.lastname: Guo
authors.forename: Yijun
authors.initials: Y
authors.lastname: Huang
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Dömling
authors.forename: Julie L
authors.initials: JL
authors.lastname: Eiseman
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor; D005260:Female; D045325:HCT116 Cells; D064593:Heterografts; D006801:Humans; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism*; D011720:Pyrazoles / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; C461497:Mdm4 protein, mouse; D011518:Proto-Oncogene Proteins; D011720:Pyrazoles; D016159:Tumor Suppressor Protein p53; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases