Cancer Chemother Pharmacol (1987) 19:301-306 ancer _ hemotherapy and LPharmacology © Springer-Verlag 1987 Antitumor activity of new anthracycline analogues in combination with interferon alfa* Michael E. Berens l'**, Toshiaki Saito l, Charles E. Welander 1, and Edward J. Modest 2 1 Section of Gynecologic Oncology, and 2Department of Biochemistry, Bowman Gray School of Medicine, 300 South Hawthorne Road, Winston-Salem, NC 27103, USA Summary. Combinations of recombinant interferon alfa 2 (IFN-c~2) with doxorubicin, 4'-epidoxorubicin, 4'-deoxy- doxorubicin, or 4-demethoxydaunorubicin were tested for antiproliferative activity against a panel of human tumor cell lines in a human tumor clonogenic assay. The histolo- gies of the cell lines were ovary, cervix, breast, and mela- noma. Each of the cytotoxic compounds showed dose-de- pendent antiproliferative effects against each of the cell lines, and the results indicated that doxorubicin deriva- tives were consistently more potent than the parent drug. In all instances, 4-demethoxydaunorubicin was the most potent derivative, requiring 2-20 times less drug to inhibit 70% of tumor colony formation. Combinations of IFN-a2, with doxorubicin or its derivatives may show additive or synergistic antiproliferative activity against certain tumor cell lines. The ovarian carcinoma cell line, BG-1, re- sponded synergistically to each of the four compounds in combination with IFN-c~2. The cervical carcinoma cell line, CaSki, and the breast carcinoma line, MCF-7, re- sponded to the combinations in a manner best described as additive. In the melanoma line, SK-Mel-28, the drugs were found to be subadditive or even antagonistic. While the potency of the anthracycline derivatives ranked con- sistently across the different cell lines, the synergistic inter- action with IFN-a 2 is a cell line-specific phenomenon un- related to sensitivity to either anthracyclines or interferon. Introduction Identification or development of truly new chemothera- peutic agents is extremely slow, prompting structural ma- nipulation of existing drugs or testing of new drug combi- nations to improve efficacy. In this latter respect, we have previously reported in vitro potentiation of the antitumor effect of doxorubicin by alpha interferon (IFN-~) [22]. Optimal scheduling of the agents was found to be either si- multaneous application of the two drugs, or pretreatment of the tumor cells with IFN-~ before doxorubicin expo- sure. * Supported by a grant from Farmitalia Carlo Erba, Milan, Italy ** Current address: Brain Tumor Research Center, HSW 783, University of California at San Francisco, San Francisco, CA 94143, USA Offprint requests to: C. E. Welander Recently, new derivatives of doxorubicin and dauno- rubicin have been synthesized, with the goal of reducing the cardiac toxicity of the anthracycline antibiotic or gene- rating a molecule with more potent antitumor effects. In the present investigation, doxorubicin, two of its synthetic analogues, and a derivative of daunorubicin were tested for antiproliferative activity against a series of four human tumor cell lines in a Soft agarose clonogenic assay. The rel- ative ranking of the efficacies of the analogue against these different cell lines was determined. In addition, the effect of recombinant IFN-ct (IFN-(t2) on the antiproliferative activity of the cytotoxic agents was investigated. We were concerned to determine the degree to which the direct an- tiproliferative effect was associated with additive, syner- gistic, or inhibitory interactions with IFN-tz. Materials and methods Drugs. All anthracyclines and their derivatives were sup- plied by Farmitalia Carlo Erba, Milan, Italy. The com- pounds tested were doxorubicin (DOX), 4'epidoxorubicin (4'-EDX), 4'-deoxydoxorubicin (4'-DDX) and 4-deme- thoxydaunorubicin (4-DDR). Each of these was soluble in water, In all experiments, an appropriate vehicle solvent was included as a control, The drugs were stored as lyophi- lized aliquots in light-protected vials. IFN-CtZb was supplied by Schering Corporation, Bloomfield, NJ. The recombinantly cloned material de- monstrated a specific antiviral activity of 1.9 x l0 s units/ mg protein. Purity was greater than 98%. Aliquots of IFN- ct 2 in phosphate-buffered saline were stored at -20 ° C. Human tumor cell lines. A series of human tumor cell lines was used to evaluate the antiproliferative activity of the drugs. Ovarian carcinoma (BG-1 [22], breast carcinoma (MCF-7 [19]), cervical carcinoma (CaSki [12]), and the melanoma (SK-Mel-28 [3]) cell lines were propagated as monolayers in McCoy's 5A culture media (Gibco Laborat- ories) enriched with 10% fetal calf serum and 0.1% penicil- lin and streptomycin. Cultures were routinely tested for mycoplasma contamination (Hoechst stain) and found to be negative. Cells were seeded in culture flasks and al- lowed to expand in logarithmic phase growth prior to in- itiation in soft agarose clonogenic culture. Soft agarose tumor clonogenie assay. A modified two-layer semisolid agarose assay was used to determine changes in