Search

Development of multidrug resistance due to overexpression of P-glycoprotein (Pgp), a cell membrane drug efflux pump, occurs commonly during in vitro selections with adriamycin (Adr). Pgp-mediated drug resistance can be overcome by the calcium channel blocker verapamil (Vp), which acts as a competitive inhibitor of drug binding and efflux. In order to identify other mechanisms of Adr resistance, we isolated an Adr-resistant subline by selecting the human breast cancer cell line MCF-7 with incremental increases of Adr in the presence of 10 microgram/ml verapamil. The resultant MCF-7/AdrVp subline is 900-fold resistant to Adr, does not overexpress Pgp, and does not exhibit a decrease in Adr accumulation. It exhibits a unique cross-resistance pattern: high cross-resistance to the potent Adr analogue 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin, lower cross-resistance to the alkylating agent melphalan, and a sensitivity similar to the parental cell line to vinblastine. The levels of glutathione and glutathione S-transferase are similar in the parental line and the Adr-resistant subline. Topoisomerase II-DNA complexes measured by the potassium-sodium dodecyl sulfate precipitation method shows a 2-3 fold decrease in the resistant subline. The MCF-7/AdrVp cells overexpress a novel membrane protein with an apparent molecular mass of 95 kDa. Polyclonal antibodies raised against the P-95 protein demonstrate a correaltion between the level of expression and Adr resistance. Removal of Adr but not verapamil from the selection media results in a decline in P-95 protein levels that parallels a restoration of sensitivity to Adr. Immunohistochemistry demonstrates localization of the P-95 protein on the cell surface. The demonstration of high levels of the protein in clinical samples obtained from patients refractory to Adr suggests that this protein may play a role in clinical drug resistance.

The potential mechanisms of the extremely potent anthracycline analogue 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin (MRA-CN) have been compared with those of doxorubicin (DOX) by examination of drug effects on colony formation, macromolecular synthesis, DNA integrity, and ultrastructure of human leukemia cells in vitro. Following a 1-h exposure, MRA-CN was found to be 1400-fold more cytocidal than DOX which correlated with the drugs' inhibitory effects on DNA and total RNA synthesis. Treatment with MRA-CN resulted in a dose-dependent production of DNA interstrand cross-links as quantified by alkaline elution. One-h treatments with DOX or 3'-deamino-3'-(4-morpholinyl) doxorubicin (the non-cyano-containing analogue of MRA-CN) produced no DNA-DNA cross-links; rather they produced protein-concealed DNA single-strand breaks. After removal of MRA-CN, the DNA of KBM-3 cells displayed time-dependent fragmentation as indicated by rapid DNA filter elution during the pH 10 lysis step which preceded pH 12 elution. Within 4 h of MRA-CN exposure (10 nM, 1 h), 50% of the cellular DNA was in the lysis fraction. By 24 h, all the cellular DNA was in this fraction. MRA-CN (10 nM), 3'-deamino-3'-(4-morpholinyl)doxorubicin (1 microM), and actinomycin D (1 microM), but not DOX (3 mircroM), each produced distinctive nucleolar macrosegregation, indicating an effect on rRNA synthesis. The alpha-CN substituent on the morpholinyl moiety of MRA-CN appears to be responsible for the unique antitumor potency of this anthracycline. Nucleolar macrosegregation is probably associated with the morpholinyl moiety and is independent of the alpha-CN substituent.

The highly lipophilic cyanomorpholinyl adriamycin (CMA) is the most potent antineoplastic anthracycline yet described. CNS distribution and toxicity were examined after i.v. administration of CMA to mice. At doses greater than or equal to 0.1 mg/kg, a neurotoxic syndrome including ataxia, hypokinesia, and tremors appeared. At doses of less than or equal to 0.05 mg/kg, which have been reported to be antineoplastic, no neurotoxicity was observed. On histopathologic examination, no changes were observed in the brain, spinal cord, or dorsal root ganglia. Unlike adriamycin (ADR), which rapidly appears in the nuclei of several tissues, CMA showed no fluorescence, suggesting a different cellular microcompartmentalization. The i.d. injection of CMA disclosed a 200-fold increase in toxicity compared with that of adriamycin. In comparisons of CMA and ADR, neurotoxicity and cardiotoxicity occurred equally only at higher doses; however, the dermatotoxicity and antineoplastic activity of CMA were increased several hundred-fold.

Previously we reported that reductive alkylation of doxorubicin with 2,2'-oxybis[acetaldehyde] and NaBH3CN to form the 4''-morpholinyl derivative also gave the intensely potent 3''-cyano-4''-morpholinyl as a byproduct, by addition of CN- to an iminium intermediate in place of hydride. We now find that sugar 4'-OH is a third nucleophile that can add to the iminium intermediate in this reaction. Bridging of the 4'-OH to the morpholine ring at C.5'' formed a novel byproduct with an oxazolidino ring fused to the sugar and morpholine. The new product was minor at neutral pH but predominant at an acidic pH. When tested against tumors in mice it was 4-6 times more potent than doxorubicin. Hence, in comparison with the 3''-cyano-4''-morpholinyl, potency was reduced up to 100-fold by the O bridge. Analytical HPLC showed the presence of three of the four possible diastereoisomers, and two were isolated. The diastereoisomers appeared to differ in stability. In vitro tests suggested that biological potency varied inversely with stability.

The search for new congeners of the leading anticancer drug doxorubicin has led to an analog that is approximately 1000 times more potent, noncardiotoxic at therapeutic dose levels, and non-cross-resistant with doxorubicin. The new anthracycline, 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin (MRA-CN), is produced by incorporation of the 3' amino group of doxorubicin in a new cyanomorpholinyl ring. The marked increase in potency was observed against human ovarian and breast carcinomas in vitro; it was not accompanied by an increase in cardiotoxicity in fetal mouse heart cultures. Doxorubicin and MRA-CN both produced typical cardiac ultrastructural and biochemical changes, but at equimolar concentrations. In addition, MRA-CN was not cross-resistant with doxorubicin in a variant of the human sarcoma cell line MES-SA selected for resistance to doxorubicin. Thus antitumor efficacy was dissociated from both cardiotoxicity and cross-resistance by this modification of anthracycline structure.

Several simple test methods that revealed alterations in mechanisms of action proved to be useful in the selection of three new anthracycline analogues that are currently in preclinical development. 5-Iminodaunorubicin is a quinone-modified analogue, and the resultant suppression of quinone redox cycling appears to correlate with diminished cardiotoxicity rather than with any effect on antitumour activity. N,N-Dibenzyldaunorubicin is an inactive prodrug requiring metabolic activation, a process that appears to give some selectivity of action leading to improved activity. The cyanomorpholino derivative of doxorubicin shows an intense potency against tumours that is not encountered in other closely-related analogues, indicating a highly specific mode of action as yet unidentified. Results with these examples suggest that simple tests related to mechanisms of action may be more useful for analogue selection than extended tests to define antitumour activity.

Plasma and tissue levels of doxorubicin (DXR) and doxorubicinol (DXR-OL) were measured fluorometrically after high-pressure liquid chromatography at 1, 3, and 24 h following one, nine, and 24 doses of 1.0 mg DXR/kg or one and eight doses of 4.0 mg DXR/kg, IP, to rats. Comparison of plasma levels of DXR found following single and multiple doses suggests significant build-up of DXR at 1 h with successive doses, but not at 3 h. Liver exhibited substantially higher levels of DXR (on a per gram of protein basis) than did plasma, and multiple doses did not produce higher levels than did a single dose. In contrast, the heart accumulated DXR slowly, attaining levels after multiple dosing in excess of those found in the liver. Skeletal muscle exhibited dose-related levels similar to those for heart but the absolute levels of DXR in muscle were only about one-tenth of those observed in heart. DXR-OL was at very low levels of less than or equal to 4% of the DXR levels in the tissues; it was, however, a major circulatory metabolite, attaining levels in the plasma as high as 85% of the concentration of DXR.

A series of quinone- and sugar-modified analogs of adriamycin have been tested for growth inhibition of adriamycin-sensitive (P388/S) and -resistant (P388/ADR) sublines of P388 murine leukemia cells in vitro. P388/ADR is less resistant to analogs of adriamycin containing either a 3'-deamino-3'-(4"-morpholinyl) group, MRA; or a -(3"-cyano-4"-morpholinyl) group, MRA-CN, than to adriamycin. However, MRA-CN was the most potent growth inhibitor of either subline. This potency is reduced by either modification of the quinone unit with a 5-imino substituent or restriction of the cyano-morpholinyl ring by an oxygen bridge to the daunosamine sugar. The calcium antagonist verapamil substantially increases the cytotoxicity of adriamycin to P388/ADR but has no appreciable effect on the cytotoxicity of either MRA or MRA-CN. The results suggest that increased uptake and retention by both MRA and MRA-CN may contribute to their increased cytotoxicity, but that the intense potency of the cyano-morpholinyl analogs must be due to other unique properties of these compounds.

The new adriamycin (ADR) derivatives, 3'-deamino-3'-(4"-morpholinyl)adriamycin (MRA) and 3'-deamino-3'-(3"-cyano-4"-morpholinyl)-adriamycin (MRA-CN), were studied, in comparison with ADR, for their inhibitory effects on DNA and RNA syntheses in vitro using isolated DNA and RNA polymerases from both Escherichia coli and chicken (myeloblastosis) leukemia cells. Under standard assay conditions, MRA and ADR demonstrated a similar inhibitory effect on the enzymes, whereas MRA-CN showed a slightly greater inhibitory activity than ADR or MRA at low drug concentrations, but with the inhibitory effect plateauing when drug concentration reached 10 microM. Both the A and B forms of the MRA-CN diastereoisomers were effective as inhibitors. Kinetic studies of the inhibition showed that unlike ADR, MRA-CN inhibition of DNA or RNA synthesis could not be reversed by increasing DNA-template concentration in the reaction mixture. Whereas ADR or MRA relaxed completely 1 microgram of pBR322 supercoiled DNA at a drug concentration of 15 microM, MRA-CN, at 60 microM, produced a mixture of intermediate relaxation forms of the DNA. A complete relaxation was achieved at 300 microM MRA-CN. Both DNA relaxation and fluorescence spectroscopic studies indicated that DNA-drug interactions occurred in the following order: ADR (or MRA) greater than MRA-CN greater than N-trifluoroacetyl-ADR-14-O-hemiadipate (a DNA-nonbinding anthracycline).

The susceptibility of 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin to hydrolysis at pH 7, 4, and 2 has been compared with that of the typically stable morpholine analogue. At pH 7, 74% of the cyanomorpholine was unchanged after 24 h at room temperature, but at pH 2 only 10% remained. Products identified were aglycon (8%) and N-(2-hydroxyethyl)doxorubicin (7%). Most of the losses were to unidentified polar products not eluted from HPLC. Authentic hydroxyethyl was synthesized from doxorubicin by reductive alkylation with glycolaldehyde. Antitumor potency was comparable to that of doxorubicin rather than of cyanomorpholine.

We have evaluated the dose-, schedule-, and time-dependent effects of doxorubicin (DXR) on the rat electrocardiogram (ECG) and have related ECG alterations to cellular transmembrane potential (TMP) changes and ultrastructural changes in preparations isolated from DXR-treated animals. DXR was administered intraperitoneally at 1, 2, and 4 mg/kg (all five times per week) or 5 mg/kg once per week up to a cumulative dose of 20 mg/kg, or 2 mg/kg daily for 5 days for a cumulative dose of 10 mg/kg. Posttreatment deaths were due to acute toxicity (bone marrow suppression) or congestive cardiomyopathy (8-14 weeks after the end of dosing). The most consistent ECG changes observed with DXR treatment (greater than 10 mg/kg cumulative dose) were a reversible prolongation of the QRS complex and a progressive lengthening of the Q alpha T interval; changes in R-, S-, and T-wave voltages were more variable. ECG toxicity was more pronounced when the drug was given on a weekly schedule. On the cellular level, DXR treatment led to a decrease in Vmax, with a slight increase or no change in resting potential, and a marked prolongation in action potential duration at the 50% and 75% repolarization levels. The ECG and TMP changes were accompanied by ultrastructural changes that increased in severity during the posttreatment period. Our data further support the usefulness of the rat ECG in anthracycline cardiotoxicity studies.

3'-Deamino-3'-(3-cyano-4-morpholinyl)doxorubicin is a new analogue that is 100 to 1000 times more potent than doxorubicin against tumors in cell culture or in mice, that is active by intraperitoneal, intravenous, or oral dosing, and that does not produce chronic myocardial lesions in mice. This analogue was encountered in studies on the reductive alkylation of doxorubicin and daunorubicin with 2,2' - oxybis [acetaldehyde], which constructs a morpholino ring incorporating the amino N. The morpholinyl nitrile byproducts are separated by virtue of their nonbasicity from the expected morpholino derivatives. The 13-dihydro and 5-imino derivatives are also described in this important new class of anthracyclines.