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Chemotherapeutic treatment with combinations of drugs is front-line therapy for many types of cancer. Combining drugs which target different signaling pathways often lessens adverse side effects while increasing the efficacy of treatment and reducing patient morbidity. A defined scheduling protocol is described by which histone deacetylase inhibitors (HDIs) facilitate the cytotoxic effectiveness of the topoisomerase I inhibitor camptothecin in the killing of tumor cells. Breast and lung cancer cell lines were treated with camptothecin and sodium butyrate (NaB) or suberoylanilide hydroxamic acid on the day of, the day before, or the day after camptothecin addition. Depending on the time of addition, NaBtreated cells displayed a spectrum of responses from protection to sensitization, indicating the critical nature of timing in the use of HDIs. The IC 80 (72-hour assay) dose of 100 nmol/L camptothecin could be lowered to 15 nmol/L camptothecin while maintaining or surpassing cell killing of the single agent if combined with an HDI added 24 to 48 hours after camptothecin. Experiments determined that cells arrested in G 2 -M by camptothecin were most sensitive to subsequent HDI addition. Western blot analysis indicated that in camptothecin-arrested cells, NaB decreases cyclin B levels, as well as the levels of the antiapoptotic proteins XIAP and survivin. These findings suggest that reducing the levels of these critical antiapoptotic factors may increase the efficacy of topoisomerase I inhibitors in the clinical setting if given in a sequence that does not prevent or inhibit tumor cell progression through the S phase. (Cancer Res 2005; 65(15): 6957-66)

The α-amino acid ester prodrugs of the antitumor agent camptothecin and a more potent, lipophilic silatecan analog, DB-67, have been shown by NMR spectroscopy and quantitative kinetic analyses to undergo quantitative conversion to their pharmacologically active lactones via a non-enzymatic mechanism that at pH 7.4 is favored over direct hydrolysis. The alternate pathway involves the reversible intramolecular nucleophilic amine attack at the camptothecin E-ring carbonyl to generate a lactam (I) followed by a second intramolecular reaction to produce a bicyclic hemiorthoester (I′). The intermediates were isolated and shown to exist in an apparent equilibrium dominated by the hemiorthoester in DMSO using NMR spectroscopy. The conversion of prodrugs of camptothecin or DB-67 containing either α-NH 2 or α-NHCH 3 and their corresponding hemiorthoesters were monitored versus time in aqueous buffer (pH 3.0 and 7.4) at 37°C and the kinetic data were fit to a model based on the proposed mechanism. The results indicated that while the prodrugs are relatively stable at pH 3, facile lactone release occurs from both the prodrugs and their corresponding hemiorthoester intermediates under physiological conditions (pH 7.4). The glycinate esters and their hemiorthoesters were found to be more cytotoxic than the N-methylglycinates or their corresponding hemiorthoester intermediates in vitro using a human breast cancer cell line (MDA-MB-435S), consistent with their more rapid conversion to active lactone. The pH dependence of the nonenzymatic pathway for conversion of these α-amino acid ester prodrugs suggests that they may be useful for tumor-targeting via liposomes, as they can be stabilized in an acidic environment in the core of liposomes and readily convert to the active lactone following their intratumoral release.

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Increased expression of the reduced folate carrier confers sensitivity to the antifolate drug methotrexate because it results in increased cellular uptake of this drug, and increased resistance to trimetrexate, a lipid-soluble antifolate drug, because it enables cells to take up exogenous folates that rescue cells from antifolate cytotoxicity. We therefore hypothesized that the reduced folate carrier could act as a suicide gene after treatment with methotrexate and as a selectable marker after exposure to trimetrexate. To test this hypothesis, we constructed replication-defective retroviruses containing the murine-reduced folate carrier (mRFC). Murine bone marrow cells transduced with the mRFC-containing retrovirus showed increased sensitivity to methotrexate and increased resistance to trimetrexate compared to empty vector-transduced controls in colony forming assays. Furthermore, colonies surviving trimetrexate and methotrexate treatment showed an enrichment of the mRFC gene after exposure to trimetrexate and a decrease after exposure to methotrexate. Lethally irradiated mice transplanted with bone marrow cells transduced with the mRFC-retrovirus and treated with the antifolate drugs after hematopoietic recovery demonstrated a relative increase in the number of cells containing the mRFC transgene after trimetrexate treatment and a decrease after methotrexate treatment. Therefore, these studies demonstrate the potential of the reduced folate carrier gene to play a dual role in gene therapy applications.

Synthesis and Evaluation of a Novel E-Ring Modified α-HydroxyKeto Ether Analogue of Camptothecin.-Two novel non-lactone E-ring analogues of camptothecin, (XIX) and (XX), are prepared by the cascade radical annulation route. Keto ether (XIX) is an isomer of homocamptothecin with the extra E-ring methylene group and the carbonyl group reversed, lacking a lactone. Despite these similarities, keto ether (XIX) as well as the isomeric keto ether side product (XX) are inactive in cell assays. -(DU, WU; CURRAN, DENNIS P.; BEVINS, ROBERT L.; ZIMMER, STEPHEN G.; ZHANG, JUNHONG; BURKE, THOMAS G.; Bioorg. Med. Chem. 10 (2002) 1, 103-110; Dep. Chem., Univ. Pittsburgh, Pittsburgh, PA 15260, USA; EN)