Tosedostat (CHR2797)

Female rats (CBH/cbi) injected i.v. with HOSP.1P cells, male (CBH/cbi) rats injected with HSN LV10 cells, and nude mice (MF1 (nu/nu) inoculated with MDA-MB 435 cells or MDA-MB-468 cells

(S)-cyclopentyl 2-((R)-2-((S)-1-hydroxy-2-(hydroxyamino)-2-oxoethyl)-4-methylpentanamido)-2-phenylacetate

Dissolved in DMSO, final concentration ca.1 mM

ca.100 mg/kg

100 nM, 100 nM, 100 nM, 100 nM, 100 nM, 100 nM

Administration of CHR-2797 (ca.100 mg/kg) decreases tumor volumes in vivo, compared to controls, , in a dose-response manner in the rat HOSP.1 lung colonisation model, the rat HSN LV10 chondrosarcoma liver colonisation model, the human MDA-MB-435 breast cancer spontaneous metastasis model, and the human MDA-MB-468 cell xenograft model. [1]

Aminopeptidase assays, LAP activity is determined by measuring the hydrolysis of the tripeptide, LGG, which is detected by the derivatization reagent OPA in the presence of beta-mercaptoethanol. The assay is carried out in 96-well assay plates. Wells contain diluted CHR-2797 (5 uL), 10 ug/mL LAP (5 uL) and 40 uL of 0.5 mM LGG. The plate is shaken briefly, and incubated for 90 minutes at 37 C. The reaction is terminated by addition of 200 uL of OPA/beta-Mercaptoethanol per well. The plate is read on the Victor Wallac3 plate reader: excitation, 355nm and emission, 460nm. PuSA activity is determined using the fluorogenic substrate Ala-AMC. Incubation mix contains diluted CHR-2797 (20 uL), substrate (125 uM Ala-AMC in 0.125 M Tris-HCl buffer, pH 7.5, 40 uL) and enzyme (40 uL). After incubation for 2 hours at 37 C, the reaction is stopped by addition of 100 uL 3% (v/v) acetic acid. Fluorescence is measured using a SLT Fluostar fluorimeter. Aminopeptidase N is assayed using the fluorogenic substrate, Ala-AMC. Incubation mix contains diluted CHR-2797 (20 uL), substrate (40 uL, final concentration, 60 uM) and enzyme (40 uL, 1:8000 dilution) and is incubated for 60 minutes at 37 C prior to addition of 100 uL 3% (v/v) acetic acid, to stop the reaction. Fluorescence is measured using a SLT Fluostar fluorimeter.

406, 47

DMSO 51 mg/mL, Water <1 mg/mL, Ethanol 5 mg/mL