ABSTRACT
CPI-0004Na is a tetrapeptidic extracellularly tumour-activated prodrug of doxorubicin. The tetrapeptide structure ensures blood stability and selective cleavage by unidentified peptidase(s) released by tumour cells. The purpose of this work was to identify the enzyme responsible for the first rate-limiting step of CPI-0004Na activation, initially attributed to a 70 kDa acidic (pI=5.2) metallopeptidase active at neutral pH that was subsequently purified from HeLa cell homogenates. Two electrophoretic bands were isolated and identified by matrix-assisted laser desorption ionisation-time of flight (MALDI-tof) and electrospray ionisation-quadrupole-time of flight (ESI-Q-tof) mass spectrometry as thimet oligopeptidase (TOP). The identity of the CPI-0004Na activating enzyme and TOP was further supported by the similar substrate specificity of the purified enzyme and recombinant TOP, by thiol stimulation of CPI-0004Na cleavage by cancer cell conditioned media (unique characteristic of TOP) and by the inhibition of CPI-0004Na activation by specific inhibitors or immunoprecipitation. Although other enzymes can be involved, TOP clearly appears to be a likely candidate for extracellular activation of the CPI-0004Na prodrug.
Subject(s)
Antineoplastic Agents/therapeutic use , Doxorubicin/analogs & derivatives , Metalloendopeptidases/pharmacology , Neoplasms/drug therapy , Neoplasms/metabolism , Oligopeptides/metabolism , Prodrugs/metabolism , Doxorubicin/metabolism , Doxorubicin/therapeutic use , Drug Interactions , HeLa Cells , Humans , Mass Spectrometry , Oligopeptides/therapeutic use , Prodrugs/therapeutic use , Tumor Cells, CulturedABSTRACT
Intravenous administration of N-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin (4) induces an acute toxic reaction, killing animals in a few minutes. This results from its positive charge at physiological pH combined with its propensity to form large aggregates in aqueous solutions. Negatively charged N-capped versions of 4 such as the succinyl derivative 5 can be administered by the iv route at more than 10 times the LD(50) of doxorubicin without inducing the acute toxic reaction, and they are active in vivo.