Current Medicinal Chemistry - Volume 8, Issue 1, 2001

The proficiency with which anthracyclines and other DNA-binding drugs target certain sequences in eukaryotic promoters offers a potential approach to interfere with the mechanisms that regulate gene expression in tumor cells. An in vitro transcription assay has been used to compare the ability of the bisintercalating anthracycline WP631 and the monointercalating anthracycline daunorubicin in terms of their ability to inhibit initiation of transcription of the adenovirus major late promoter linked to a G-less transcribed DNA template. Both drugs inhibit basal transcription by RNA polymerase II. However, WP631 is ~15 times more efficient at inhibiting transcription initiation from an adenovirus promoter containing an upstream Sp1-protein binding site. The differences in the ability of each drug to inhibit transcription initiation appear to be related to the competition between Sp1 and the anthracyclines for binding to the same site. To see whether WP631's strong effect on transcription can also be observed in cells, we compared the effects of WP631 and other anthracyclines on the transcription of the c-myc gene, which promoter contains Sp1 binding sites. The resulting data suggest that WP631 might circumvent some kinds of tumor resistance at rather low drug concentrations, inhibit c-myc expression in some cell lines, and exert its antitumoral effect by inducing apoptosis.

We designed a screening system for new anthracyclines totally based on human tumor material. In the first step of the system, the relative cytotoxicity versus doxorubicin of all new compounds is investigated in a panel of human tumor cell lines, well characterized for resistance factors and p53 status. Only a few analogs are selected through this step for further evaluation. The second step is aimed to investigate the therapeutic efficacy and the tolerability of the analog, which is compared to doxorubicin in a series of human tumor xenografts selected for presenting natural or acquired (by known mechanisms) resistance to the parent drug. Cardiotoxicity in mice is also studied. Cellular and molecular pharmacology studies are also considered. The results of a series of disaccharide anthracycline analogs screened by the system are presented. An analog of the series, MEN 10755, was selected for clinical investigation and is currently evaluated in Phase I trials. The ability of human tumor xenografts to predict the clinical efficacy of anthracycline analogs is also discussed.

Recent and new results which support a drug-DNA covalent bonding mechanism for cell toxicity of the clinical antitumor drugs, daunorubicin, doxorubicin, and epidoxorubicin, are summarized. The mechanism involves the iron complex of the drugs inducing oxidative stress to yield formaldehyde, which then mediates covalent attachment to G-bases of DNA. At NGC sites the combination of covalent and non-covalent drug interactions serve to virtually crosslink the DNA. Structural data for virtual crosslinks are compared as a function of drug structure. Elucidation of the mechanism led to the synthesis and evaluation of drug formaldehyde conjugates, Daunoform, Doxoform, and Epidoxoform, as improved chemotherapeutics. Drug uptake, nuclear targeting, drug release, and cytotoxicity of the clinical drugs by sensitive and resistant breast and prostate cancer cells are contrasted with those of the corresponding formaldehyde conjugates. Conjugates are taken up better, retained longer, and are more toxic to a wide variety of tumor cells. The kinetics of drug release from Doxoform and Epidoxoform treated MCF-7/Adr cells are biexponential and correlate with the biexponential kinetics of drug release from extracellular DNA. The results of the lead conjugate, Epidoxoform, in the National Cancer Institute 60 human tumor cell screen are presented and discussed in terms of some resistance mechanisms. Epidoxoform shows increased toxicity to all panels relative to doxorubicin and epidoxorubicin, and this enhanced toxicity is especially evident with the more resistant cell lines.

Cellular resistance to anthracyclines is a major limitation of their clinical use in the treatment of human tumors. Resistance to doxorubicin is described as a multifactorial phenomenon involving the overexpression of defense factors and alterations in drug-target interactions. Such changes do not account for all manifestations of drug resistance, in particular intrinsic resistance of solid tumors. Since anthracyclines can induce apoptotic cell death, an alternative promising approach to drug resistance has focused on the study of cellular response to drug-induced DNA damage, with particular reference to the relationship between cytotoxicity/antitumor efficacy and apoptotic response. The evidence that a novel disaccharide analog (MEN 10755), endowed with an improved preclinical activity over doxorubicin, was also more effective as an inducer of apoptosis provided additional insights to better understand the cellular processes that confer sensitivity to anthracyclines. Although the presence or alteration of a single apoptosis-related factor (e.g., p53, bcl-2) is not predictive of the sensitivity/resistance status, the complex interplay among DNA damage-activated pathways is likely an important determinant of tumor cell sensitivity to anthracyclines.

Multidrug resistance may be conferred by P-glycoprotein (Pgp, ABCB1) or the multidrug resistance associated protein (MRP). These membrane proteins are members of the ATP binding cassette transporter superfamily and are responsible for the removal from the cell of several anticancer agents including doxorubicin. Modulators can inhibit these transporters. LY335979 is among the most potent modulators of Pgp with a Ki of 59 nM. LY335979 is selective for Pgp, and does not modulate MRP-mediated resistance by MRP1 (ABCC1) and MRP2 (ABCC2). LY335979 significantly enhanced the survival of mice implanted with Pgp-expressing murine leukemia (P388/ADR) when administered in combination with either daunorubicin, doxorubicin or etoposide. Coadministration of LY335979 with paclitaxel compared to paclitaxel alone significantly reduced the tumor mass of the Pgp-expressing UCLA-P3.003VLB lung carcinoma in a xenograph model and delayed the development of tumors in mice implanted with the parental drug-sensitive UCLA-P3 tumor. LY335979 was without significant effect on the pharmacokinetics of these anticancer agents. This may be due impart to its poor inhibition of four major cytochrome P450 isozymes important in metabolizing doxorubicin and other oncolytics. The selectivity and potency of this modulator allows the clinical evaluation of the role of Pgp in multidrug resistance. LY335979 is currently in clinical trials.

Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins--the 170-kDa P-glycoprotein (P-gp) and the 190-kDa multidrug resistance-associated protein (MRP1)--that pump drugs out of MDR cells. The intracellular level of a drug, which influences the drug's cytotoxic effect, is a function of the amount of drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux). One possible pharmacological approach to overcoming drug resistance is the use of specific inhibitors that enhance the cytotoxicity of known antineoplastic agents. Many compounds have been proven to be very efficient in inhibiting P-gp activity, but only some of them can inhibit MRP1. However, the clinical results obtained so far by this approach have been rather disappointing. The other likely approach is based on the design and synthesis of new non-cross-resistant drugs whose physicochemical properties favor the uptake of such drug by resistant cells. Our recent studies have shown that whereas the P-gp- and MRP1-mediated efflux of different anthracycline-based drugs may not differ considerably, their kinetics of uptake do. Thus, the high uptake of drug by cells may lead to concentrations at the cellular target site high enough to achieve the needed cytotoxicity against MDR cells. Therefore, increased drug lipophilicity might be one factor in improving drug cytotoxicity in MDR cells. In vitro studies have shown that idarubicin, an analogue of daunorub cin, is more effective than daunorubicin and doxorubicin against MDR tumor cell lines and that this increased effectiveness is related in part to the increased lipophilicity of idarubicin. Other studies have also confirmed the strong impact of lipophilicity on the uptake and retention of anthracyclines in MDR cells.