Oxidative Metabolism of Combretastatin A-1 Produces Quinone Intermediates with the Potential To Bind to Nucleophiles and To Enhance Oxidative Stress via Free Radicals

Abstract

Combretastatins are stilbene-based, tubulin depolymerization agents with selective activity against the tumor vasculature; two variants (A-1 and A-4) are currently undergoing clinical trials. Combretastatin A-1 (CA1) has a greater antitumor effect than combretastatin A-4 (CA4). We hypothesized that this reflects the enhanced reactivity conferred by the second (ortho) phenolic moiety in CA1. Oxidation of CA1 by peroxidase, tyrosinase, or Fe(III) generates a species with mass characteristics of the corresponding ortho-quinone Q1. After administration of CA1-bis(phosphate) to mice, the hydroquinone-thioether conjugate Q1H2-SG, formed from the nucleophilic addition of GSH to Q1, was detected in liver. In competition, electrocyclic ring closure of Q1, over a few minutes at pH 7.4, leads to a second ortho-quinone product Q2, characterized by exact mass and NMR. This product was also generated by human promyelocytic leukemia (HL-60) cells in vitro, provided that superoxide dismutase was added. Q2 is highly reactive toward glutathione (GSH) and ascorbate, stimulating oxygen consumption in a catalytic manner. Free radical intermediates formed during autoxidation of CA1 were characterized by EPR, and the effects of GSH and ascorbate on the signals were studied. Pulse radiolysis was used to initiate selective one-electron oxidation or reduction and provided further evidence, from the differing absorption spectra of the radicals formed on oxidation of CA1 or reduction of Q2, that two different quinones were formed on oxidation of CA1. The results demonstrate fundamental differences between the pharmacological properties of CA1 and CA4 that provide two possible explanations for their differential activities in vivo: oxidative activation to a quinone intermediate likely to bind to protein thiols and possibly to nucleic acids and stimulation of oxidative stress by enhancing superoxide/hydrogen peroxide production. The observation of the GSH conjugate Q1H2-SG in vivo provides a new marker for oxidative metabolism of relevance to current clinical trials of CA1-bis(phosphate) (OXi4503).

Nitric Oxide Synthase Inhibition Enhances the Tumor Vascular-Damaging Effects of Combretastatin A-4 3-O-Phosphate at Clinically Relevant Doses

“…High vascular permeability is considered a predisposing factor for response to CA-4-P (36), and chronic NOS inhibition may impact on vascular permeability by encouraging leukocyteendothelial interactions (37). Induction of reactive oxygen species by CA-4-P is likely to constitute at least part of the mechanism of action of the drug (38,39), and leukocyte-endothelial interactions and reduced levels of NO may direct reactive oxygen species induction along a more damaging pathway under chronic NOS inhibition. NOS inhibition has also been shown to increase RhoA activity (40), a pathway activated by CA-4-P, at least in vitro (41).…”

Tubulin-interactive stilbene derivatives as anticancer agents

“…It remains unexplained why the trans-isomer of compound 6 affects tubulin polymerization in the same way as methoxystilbenes in the cis configuration, which share high structural similarity with CA-4. The tubulininterfering activity of trans-isomers that leads to cell cycle arrest [56,99] still needs to be elucidated. Cai et al [62] synthesized six new stilbene derivatives and screened for cytotoxicity in different human tumor cell lines.…”

“…Most recently, it was shown that the cis isomer of CA-1P significantly impaired tumor blood flow leading to secondary tumor cell death and more than 95% tumor necrosis 24 h post drug exposure. It was suggested that CA-1P may be metabolized to orthoquinone intermediates leading to the formation of cytotoxic free radicals [99]. Treatment with the trans-isomer had no effect on the blood flow parameters.…”

Phase I Clinical and Pharmacokinetic Evaluation of the Vascular-Disrupting Agent OXi4503 in Patients with Advanced Solid Tumors

“…It is a synthetic, phosphorylated prodrug of combretastatin A1 (OXi4500), a naturally occurring derivative from the bark of the South African bush willow tree, Combretum caffrum, that reversibly binds to the b-subunit at the colchicine-binding site of tubulin to inhibit microtubule assembly (3,4). It is metabolized to a reactive orthoquinone species that is also assumed to be directly cytotoxic in tumor cells (5,6) because of the production of a quinone metabolite that could bind to nucleic acids and also produces free radicals leading to the enhancement of oxidative stress (7).…”