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Role of Cytochrome P-450 from the Human CYP3A Gene Family in the Potentiation of Morpholino Doxorubicin by Human Liver Microsomes¹

Oncology Division, Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305-5306 [A. D. L., D. H. M. L., G. E. D., B. I. S.], and Imperial Cancer Research Fund, Molecular Pharmacology Group Edinburgh, United Kingdom [C. R. W.]

The cytotoxicity of the morpholino derivative of doxorubicin (MRA) can be potentiated 50- to 100-fold by human liver microsomes and NADPH (J. Natl. Cancer Inst., 81: 1034, 1989). This metabolic potentiation is inhibited by carbon monoxide or hypoxia, indicating that it is cytochrome P-450-dependent. The potentiation is also inhibited by the cytochrome P-450 inhibitors, SKF-525A and cimetidine. The metabolism by the microsomes is substrate-specific, varying markedly with alterations of either the morpholino or anthracycline ring substituents. No potentiation occurred with doxorubicin itself, or the cyanomorpholinyl, methoxypiperidinyl, N-hydroxyethyl or the O-bridged cyanomorpholinyl analogues of doxorubicin. We utilized a panel of human liver microsomes and cytochrome P-450 type-specific antibodies to further identify the isoform(s) of cytochrome P-450 that potentiated the cytotoxicity of MRA. The potentiation correlates well with the benzyloxyresorufin assay (r² = 0.98) and aflatoxin B₁ metabolism (r² = 0.98), both assays that are relatively specific for CYP3A proteins. Correlations were also observed for the expression of protein(s) cross-reacting with an antibody against rat cytochrome P-450 CYP3A1 (r² = 0.97) and MRA metabolism. This antibody against the rat cytochrome P-450 CYP3A isoform(s) inhibited more than 90% of the potentiation of the cytotoxicity by human liver microsomes. Antibodies against the CYP1A2, CYP2C6, and CYP2B2 isoforms produced no inhibition, nor did their expression by Western blotting correlate with MRA potentiation. Complete inhibition of the potentiation of MRA by human liver microsomes was found when the CYP3A substrates cyclosporin A and erythromycin were used in the reaction system. These data indicate that the CYP3A isoform(s) of cytochrome P-450 play a major role in the metabolism of MRA in vitro to a more active species.

¹ Supported in part by NIH Grants CA 42417 and CA 52168, American Cancer Society Grant CH-411A, and Farmitalia Carlo Erba.

² To whom requests for reprints should be addressed, at Oncology Division, Room M-211, Stanford University Medical Center, Stanford, CA 94305-5306.

Received 10/18/91. Accepted 6/ 2/92.

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