AZ31

Antitumor effects of AZ31 + SN38 are cytostatic rather than cytotoxic^[1].

Pharmacokinetic investigation of AZ31 as a single agent and in combination with irinotecan revealed that plasma concentrations of AZ31 were highest 1-hour after administration followed by a stepwise decrease at 3, 6 and 16 hour in the combination sensitive CRC098^[1].

• ATM enzyme assay

  Compounds in 100% DMSO were added to assay plates by acoustic dispensing. ATM enzyme was added in a Hepes buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 10 mM, MnCl2 1 mM, DTT, 5% v/v Glycerol, 0.05% v/v Tween 20) and allowed to preincubate with compound for 30 minutes prior to addition of substrate solution containing p53 and ATP. The enzyme reaction was stopped after 2 hours by the addition of detection reagent (33 mM HEPES pH 7.4, 20 mM EDTA, 0.1 M KF, 0.1 mg/mL BSA, 13 nM D2 Anti-GST antibody (Cisbio) and 0.5 nM Eu3+ Anti-p53phosphoS15 antibody) and incubated overnight before reading on a Pherastar Instrument with a standard HTRF filter block method. The final concentrations of DMSO, ATP and p53 in the assay were 1%, 5 µM, and 50 nM respectively. IC50 values (concentrations of test compound that inhibited 50% of enzyme activity) were determined using a four parameter fit method (smart fitting model) in the data analysis software.

• Cell lines

  CRC (metastatic colorectal cancer) cell lines: HCT15, HCT116, RKO, CaCo2, LS123 and LOVO

• Concentrations

  1.25, 2.5 or 5 μmol/L

• Incubation Time

  72 hours

• Method

  Six CRC cell lines were treated with AZ31 (dose 1.25, 2.5 or 5 μmol/L), SN38 (0.3125 -20 nM) or AZ31 + SN38 and proliferation was determined by an SRB assay.

• Animal Models

  CRC PDX models (Four-to-six week-old female athymic nude mice with implanted patient-derived colorectal adenocarcinoma tumor)

• Dosages

  100 mg/kg-daily × 3

• Administration

  by oral gavage