Bioreductive Therapies
Involvement of human cytochromes P450 (CYP) in the reductive metabolism of AQ4N, a hypoxia activated anthraquinone DI-N-oxide prodrug

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Abstract

Purpose: To establish the role of the human cytochromes P450 (CYPs) in the reductive metabolism of the novel anthraquinone di-N-oxide prodrug AQ4N.

Methods and Materials: Metabolism of AQ4N was conducted in a panel of 17 human phenotyped liver microsomes. AQ4N and metabolites were detected by reverse phase isocratic HPLC. CYP inhibitors and Spearman rank correlation were used to determine the significance of AQ4N metabolism versus specific CYP activity and/or expression.

Results: Anaerobic metabolism of AQ4N to the 2-electron reduction product, AQM, and the 4-electron reduced tertiary amine, AQ4, occurred in all 17 human liver microsome preparations. The range (± SE) for total AQ4N turnover was 14.26 ± 1.43 nmol/incubate (highest) to 3.65 ± 1.05 nmol/incubate (lowest). Metabolism was not detected in the absence of NADPH or microsomes. In aerobic incubates, AQM was less than 4% of anaerobic values whereas AQ4 was undetectable. CYP-mediated metabolism of AQ4N was inhibited completely by ketoconazole (KET) and carbon monoxide (CO), two global inhibitors of CYP-mediated metabolism. AQ4N metabolism correlated significantly with probes for CYP 3A, specifically benzoxylresorufin O-dealkylation [r(s) = 0.70, p <0.01] and tamoxifen N-demethylation (r(s) = 0.85, p < 0.01), but not with probes for CYPs 2C, 2D, and 1A. CYP 3A involvement was confirmed by the use of the CYP 3A specific inhibitor, triacetyloleandomycin (TAO), which repressed the formation of AQM to 13% of the uninhibited value and abolished completely the formation of AQ4. Alpha-naphthoflavone (ANF), an inhibitor of CYP 2C and 1A, had no significant effect on AQ4N metabolism.

Conclusions: These data suggest that the human CYP 3A enzymes can contribute to the reductive metabolism of AQ4N. CYP 3A enzymes are highly expressed in a broad spectrum of human cancers. The results show that AQ4N requires anaerobic conditions for CYP 3A-mediated reduction and hence this subfamily of enzymes is likely to selectively activate AQ4N in hypoxic tumors.

Introduction

The poorly defined vascular network of many solid tumors creates a subpopulation of hypoxic cells which can be resistant to chemotherapy and radiotherapy 1, 2. Bioreductively activated prodrugs specifically target hypoxic cells by becoming selectively activated to cytotoxic agents under low oxygen tension 3, 4. AQ4N is a di-N-oxide prodrug which is metabolized to AQ4 (Fig. 1) , a DNA binding agent and topoisomerase II inhibitor 5, 6, 7. In a number of murine tumors, AQ4N has impressive activity both as a chemosensitizer and radiation enhancer 8, 9, 10, 11. To date, detailed studies on the enzymology of AQ4N metabolism have not been described. The involvement of cytochrome P450 (CYP) in the reduction of tertiary amine-N-oxides has been described and there is preliminary evidence that rodent CYP can reduce AQ4N (12). Here we have used a panel of human liver microsomes phenotyped with respect to their CYP expression and activities to investigate the involvement of human CYP enzymes in the bioactivation of AQ4N.

Section snippets

Chemicals

All standard laboratory reagents were purchased as the highest commercially available grade and obtained from BDH Ltd, Poole, UK. The metabolites of AQ4N (1,4,-bis-{{2-(dimethylamino-N-oxide)ethyl]amino}-5,8- dihydroxyanthracene-9,10 dione), namely 1-{[2-(dimethylamino-N-oxide) ethyl]amino}-4-{[2-[di-methylamino] ethyl]amino}-5,8-dihydroxyanthra-cene-9,10-dione (AQM) and 1,4-bis-{{2-(dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene- 9,10-dione (AQ4) were synthesized in house and shown to be

Results and discussion

The anaerobic metabolism of AQ4N to AQM and AQ4 was observed in all 17 human livers assayed (Fig. 2). The range (± SE) for total substrate turnover was 14. 26 ± 1.43 nmol/incubate (highest) to 3.65 ± 1.05 nmol/incubate (lowest). Anaerobic metabolism of AQ4N was not detected in the absence of NADPH or microsomes. Under aerobic conditions, formation of AQ4 was undetectable and the production of AQM was inhibited by >96%. Figure 3 shows that the metabolism of AQ4N correlated significantly

Acknowledgements

The authors wish to thank Dr. R. Weaver, Mary Maley, Duncan Webster, and Ketan Ruparelia for their technical assistance. The work was supported by the Cancer Research Campaign, UK.

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