Mechanism of action and preclinical antitumor activity of the novel hypoxia-activated DNA cross-linking agent PR-104.
OBJECTIVE
Hypoxia is a characteristic of solid tumors and a potentially important therapeutic target. Here, we characterize the mechanism of action and preclinical antitumor activity of a novel hypoxia-activated prodrug, the 3,5-dinitrobenzamide nitrogen mustard PR-104, which has recently entered clinical trials.


METHODS
Cytotoxicity in vitro was evaluated using 10 human tumor cell lines. SiHa cells were used to characterize metabolism under hypoxia, by liquid chromatography-mass spectrometry, and DNA damage by comet assay and gammaH2AX formation. Antitumor activity was evaluated in multiple xenograft models (PR-104 +/- radiation or chemotherapy) by clonogenic assay 18 h after treatment or by tumor growth delay.


RESULTS
The phosphate ester "pre-prodrug" PR-104 was well tolerated in mice and converted rapidly to the corresponding prodrug PR-104A. The cytotoxicity of PR-104A was increased 10- to 100-fold by hypoxia in vitro. Reduction to the major intracellular metabolite, hydroxylamine PR-104H, resulted in DNA cross-linking selectively under hypoxia. Reaction of PR-104H with chloride ion gave lipophilic cytotoxic metabolites potentially able to provide bystander effects. In tumor excision assays, PR-104 provided greater killing of hypoxic (radioresistant) and aerobic cells in xenografts (HT29, SiHa, and H460) than tirapazamine or conventional mustards at equivalent host toxicity. PR-104 showed single-agent activity in six of eight xenograft models and greater than additive antitumor activity in combination with drugs likely to spare hypoxic cells (gemcitabine with Panc-01 pancreatic tumors and docetaxel with 22RV1 prostate tumors).


CONCLUSIONS
PR-104 is a novel hypoxia-activated DNA cross-linking agent with marked activity against human tumor xenografts, both as monotherapy and combined with radiotherapy and chemotherapy.
Patterson
Adam V
AV
Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Auckland, New Zealand.
Ferry
Dianne M
DM
Edmunds
Shelley J
SJ
Gu
Yongchuan
Y
Singleton
Rachelle S
RS
Patel
Kashyap
K
Pullen
Susan M
SM
Hicks
Kevin O
KO
Syddall
Sophie P
SP
Atwell
Graham J
GJ
Yang
Shangjin
S
Denny
William A
WA
Wilson
William R
WR
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D020552:Comet Assay; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry*; D004249:DNA Damage; D004354:Drug Screening Assays, Antitumor; D006657:Histones / Q000378:metabolism; D006801:Humans; D000860:Hypoxia; D051379:Mice; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000188:drug therapy*; D009588:Nitrogen Mustard Compounds / Q000737:chemistry / Q000494:pharmacology*; D010710:Phosphates / Q000494:pharmacology*
D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D006657:Histones; D009588:Nitrogen Mustard Compounds; C522381:PR-104; D010710:Phosphates; C475961:gamma-H2AX protein, mouse; D004247:DNA