DNA topoisomerase I--targeted chemotherapy of human colon cancer in xenografts.

Drug development is needed to improve chemotherapy of patients with locally advanced or metastatic colon carcinoma, who otherwise have an unfavorable prognosis. DNA topoisomerase I, a nuclear enzyme important for solving topological problems arising during DNA replication and for other cellular functions, has been identified as a principal target of a plant alkaloid 20(S)-camptothecin. Significantly increased concentrations of this enzyme, compared to that in normal colonic mucosa, were found in advanced stages of human colon adenocarcinoma and in xenografts of colon cancer carried by immunodeficient mice. Several synthetic analogs of camptothecin, selected by tests with the purified enzyme and tissue-culture screens, were evaluated in the xenograft model. Unlike other anticancer drugs tested, 20(RS)-9-amino-camptothecin (9-AC) induced disease-free remissions. The overall drug toxicity was low and allowed for repeated courses of treatment.

Adriamycin damage: in vivo repair by lymphoma cells.

The in vivo repair of ADR-induced cell damage was investigated in the DBA3 transplantable mouse lymphoma. After a single injection of 5 or 15 mg ADR/kg body weight into DBA/2J mice, the survival fraction of clonogens showed a 2.2- to 4.4-fold decrease at 12 or 18 hours post injection and returned to pretreatment levels within 6 hours. These changes were accompanied by the appearance and disappearance of DNA crosslinks and breaks. Because cell division and/or cell loss could not explain the return of clonogens to pretreatment level, the results strongly suggest repair of ADR damage in tumor cells in situ. Such an efficient repair mechanism, responding to a high toxic dose of ADR, constitutes a therapeutically unfavorable event that may contribute to drug resistance.

Identification and kinetics of G1 phase-confined cells in experimental mammary carcinomas.

Three lines of mouse mammary carcinoma growing in isogenic hosts and differing in degree of histological differentiation and rates of proliferation were used to study parenchymal cells with various types of nucleoli. The relative number of cells possessing trabeculate or ring-shaped nucleoli or nucleolar fragments was closely related to the growth rate and degree of differentiation of tumor lines tested. All three subpopulations increased with increasing age and with decelerated tumor growth. In some cells in late telophase, either trabeculate or ring-shaped nucleoli could be distinguished in mitotic poles. This demonstrated that cells with these nucleoli are detected at the beginning of G1 phase. Even low levels of DNA synthesis, which would indicate that some cells with trabeculate or ring-shaped nucleoli or possessing nucleolar fragments were in S phase, could not be demonstrated. Microfluorometric measurements have indicated that cells with trabeculate and ring-shaped nucleoli have a DNA content close to 2c, whereas cells with dense nucleoli have a DNA content corresponding to either 2c, 2 to 4c, 4c, or greater than 4c. On the basis of these observations, it is concluded that cells with trabeculate and ring-shaped nucleoli and cells with nucleolar fragments either proceed slowly through G1 or are arrested in this phase. Cells with trabeculate nucleoli were replaced steadily, having a transit time of no longer than 84 hr. These cells constituted a "fast" component of cell renewal of G1-confined cells. A "slow" component, cells bearing ring-shaped nucleoli or nucleolar fragments, were replaced after a lag of 24 to 48 hr. with residency time for some of these cells being in excess of 84 hr.

Frequent homozygous deletions of D13S218 on 13q14 in B-cell chronic lymphocytic leukemia independent of disease stage and retinoblastoma gene inactivation.

A correlative study has been performed to delineate further the role of the Rb gene in the disease B-cell chronic lymphocytic leukemia (B-CLL). First, we examined DNAs from B cells from 140 B-CLL patients representing all Rai stages of disease for the loss of 13q14 using two microsatellite markers mapping distal to the Rb locus. Loss of heterozygosity (LOH) of D13S133 was infrequent, occurring in 5 of 140 (4%) patients. The frequency for LOH of D13S218 was 33 of 140 (24%) samples and was independent of Rai stage of disease. Rb protein was detected in 19 of 23 (83%) samples. Of 4 patients lacking detectable Rb gene expression, only one showed LOH of D13S218. Rb protein levels varied from undetectable to high in samples with or without LOH for D13S218, and the levels were also independent of Rai stage of disease. Our findings support the role of DBM on 13q14, rather than Rb, as the candidate tumor suppressor gene that is frequently targeted for deletion in B-CLL. In addition, the data suggest that other mechanism(s) contribute to altered Rb expression detected in one-fourth of B-CLL B-cells.

Effects of new N-alkyl analogues of adriamycin on in vitro survival and cell cycle progression of L1210 cells.

The effects of N-benzyladriamycin-14-valerate (AD198) and N,N-dimethyladriamycin-14-valerate (AD199), two novel lipophilic N-alkyl derivatives of Adriamycin (ADR), on cell growth and cell cycle distribution were investigated in L1210 cells grown in suspension. Following a 1-h exposure to the drug levels selected, growth inhibition was noticeable in all cultures for most or all of the observation period of 96 h. With flow cytometry, an asynchronous cell population was measured with respect to cellular DNA, RNA, and light scatter (size) properties following a 1-h incubation with the various ADR analogues. In addition, flow cytometric techniques were utilized to determine whether drug treatment altered the sensitivity of DNA in situ to acid-induced denaturation or to binding by small DNA-intercalating dyes. Unlike the parent compound ADR or its DNA-nonbinding derivative N-trifluoroacetyladriamycin-14-O-hemiadipate (AD143), the N-alkyl derivatives AD198 and AD199 only slightly affected L1210 cell cycle traverse over the first 5 h posttreatment. However, by 24 h, AD199 (0.62 micrograms/ml) caused an S- and G2 + M-phase accumulation which became more dramatic at 48 and 72 h. AD198 (3.27 micrograms/ml) also caused an accumulation of cells predominantly in G2 + M phase at longer culture times (48 to 96 h). The two half-substituted congeners N-benzyladriamycin (AD288) and N,N-dimethyladriamycin (AD280) affected L1210 cell cycle traverse over a similar time scale at concentrations of 12.3 and 4.17 micrograms/ml, respectively. AD280 blocked cells in G1 and G2 + M whereas AD288 caused predominantly a G2 + M accumulation. While neither ADR nor AD143 interfered appreciably with binding and fluorescence of the intercalating dye acridine orange, all of the N-alkyl analogues tested reduced the fluorescence signal of acridine orange-stained L1210 cells by 26 to 60%. This effect lasted, with decreasing intensity, for at least 48 h following a 1-h exposure to the drugs. In addition, while ADR appeared to stabilize DNA in situ against acid-induced denaturation, all N-alkyl derivatives, to varying degrees, tended to increase DNA denaturability. Thus alkylation at the glycoside amine combined with the lipophilic 14-valerate side chain function accounts for several new biochemical and biological properties of AD198 and AD199, relative to ADR.(ABSTRACT TRUNCATED AT 400 WORDS)

Complete growth inhibition of human cancer xenografts in nude mice by treatment with 20-(S)-camptothecin.

20-(S)-Camptothecin (CAM), a plant alkaloid, was tested against 13 human cancer xenograft lines carried by immunodeficient (nude) mice. The drug, formulated in 20% intralipid and given i.m., was more effective than any other clinically available drug tested. It was found that: (a) CAM, at nontoxic doses, suppressed growth and induced regression of cancer of the colon (3 lines), lung (4 lines), breast (2 lines), stomach (1 line), ovary (1 line), and malignant melanoma (2 lines); (b) the drug was equally effective administered i.m. or p.o. Both routes are significantly better than i.v. administration; (c) CAM is substantially more effective and less toxic than its sodium salt, which was unsuccessfully tested in cancer patients. CAM should be further tested against responsive cancers as a drug which is easy to isolate and formulate for large-scale studies.

Growth inhibition of human cancer metastases by camptothecins in newly developed xenograft models.

Several metastatic models have been developed using clonal selection of human malignant cells metastasizing into a specific organ in NIH-I Swiss immunodeficient mice. The organs of choice were the central nervous system (CNS), targeted by metastases of malignant melanoma, and the liver, with metastases of colon adenocarcinoma. Additional models of adrenal metastases by malignant melanoma, and CNS involvement by implanted human lung squamous carcinoma or lymphoblastoid cells, are also available. Organ metastases, as well as the effects of treatment, were confirmed by autopsies and histological examination of the tissues or by a surgical inspection of the liver. The treatment end points were established as the increases in survival times of treated mice relative to placebo-treated controls. Camptothecins injected i.m. or delivered via gastrointestinal tract inhibit the growth of CNS metastases and increase the survival of treated animals. 9-Amino-20(S)-camptothecin was effective in the CNS model and in the model of liver metastases. The drug increased 3.3- and 5.7-fold the survival rates relative to untreated controls with metastases of colon adenocarcinoma to the liver, and all camptothecins were significantly more effective than 5-fluorouracil, currently a drug of choice in treatment of this disease. The xenograft models of metastases are available for studies of drug passage through the blood-brain barrier optimization of drug delivery to the liver, and for the development of new camptothecin-based treatment strategies.

Camptothecin overcomes MDR1-mediated resistance in human KB carcinoma cells.

In order to understand the high efficacy of camptothecin derivatives against human colon tumor xenografts in nude mice, we have studied the transport properties of camptothecin derivatives across cellular membranes of MDR1-overexpressing cells. MDR1 overexpression was shown to have little effect on camptothecin cytotoxicity; camptothecin was equally cytotoxic to both the drug-sensitive parental cell line, KB 3-1, and its multidrug-resistant derivative, KB V1. The ability of camptothecin to overcome MDR1-mediated resistance is most likely due to unimpaired accumulation of camptothecin in MDR1 cells as suggested from the following experiments: (a) cytotoxicity of camptothecin against KB V1 cells was not altered by the known MDR1-reversing agent, verapamil; (b) camptothecin was ineffective as compared with vinblastine in competing with [3H]azidopine for photoaffinity labeling of MDR1; (c) camptothecin was equally efficient in trapping cellular topoisomerase I molecules on chromosomal DNA in the form of cleavable complexes in both KB 3-1 and KB V1 cells. The mechanism by which camptothecin overcomes MDR1-mediated resistance has been further studied using a number of uncharged and charged camptothecin derivatives. In contrast to the uncharged camptothecin derivatives, such as 9-amino-camptothecin and 10,11-methylenedioxy-camptothecin, the charged camptothecin derivative, topotecan, showed reduced cytotoxicity against MDR1-overexpressing KB V1 cells. The reduced cytotoxicity of topotecan in KB V1 cells was due to the overexpression of MDR1 in KB V1 cells since verapamil restored both topotecan accumulation and cytotoxicity. These results suggest that the charge on camptothecin can affect the drug's sensitivity to MDR1. The possible effect of membrane permeability in determining drug selectivity of MDR1 is discussed.

Protein-associated DNA breaks and DNA-protein cross-links caused by DNA nonbinding derivatives of adriamycin in L1210 cells.

The effects of Adriamycin derivatives on L1210 mouse leukemia cells were studied with the DNA alkaline elution assay. The exposure of exponentially growing cells to approximately equitoxic concentrations of N-trifluoroacetyladriamycin-14-valerate (13.8 microM) and its metabolites, N-trifluoroacetyladriamycin (9.0 microM) and N-trifluoroacetyladriamycinol (43.7 microM), for 1 hr in vitro resulted in a high frequency of protein-associated DNA breaks and DNA-protein cross-links. These effects were comparable to those observed with Adriamycin (2.8 microM) and with adriamycinol (26.9 microM). In contrast to Adriamycin and its metabolite adriamycinol, N-trifluoroacetyladriamycin-14-valerate and its two major metabolites do not bind to DNA. Despite the absence of this direct interaction, N-trifluoroacetyladriamycin-14-valerate and its metabolites produce alterations in DNA comparable with the effects of intercalating agents. No evidence for conversion of N-trifluoroacetyladriamycin-14-valerate to Adriamycin or adriamycinol was found in L1210 cells. The similar effects on DNA macromolecules, observed between intercalating and non-DNA-binding anthracyclines, are consistent with the concept that mechanisms other than direct interaction with DNA play a role in the toxic effects of these compounds.

Lack of drug-induced DNA cross-links in chlorambucil-resistant Chinese hamster ovary cells.

Chlorambucil (CLB) is an alkylating agent commonly used in the treatment of several neoplastic disorders. The mechanisms underlying resistance to this drug are not fully defined. We used the DNA alkaline elution technique to study cross-link formation in the wild type (K1) and a CLB-resistant (ChlR) Chinese hamster ovary cell line. [14C]CLB was used to measure drug uptake. The CLB-resistant cells were found to have negligible DNA cross-link formation compared to K1 cells at all time points tested. There was a correlation between the resistance to CLB and the decreased ability of resistant cells to form DNA cross-links. Results of drug uptake experiments excluded altered CLB accumulation as the basis for these findings. Assays of O6-alkylguanine transferase and topoisomerase. II provide evidence against a role of these enzymes in CLB resistance. These studies suggest that the mechanism of CLB cytotoxicity involves the formation of DNA cross-links. Reduced cross-link formation may confer resistance to CLB.

Relationship of adriamycin concentrations to the DNA lesions induced in hypoxic and euoxic L1210 cells.

Exponentially growing L1210 mouse leukemia cells were incubated with Adriamycin (ADR) under hypoxic (95% N2:5% CO2) or euoxic conditions (95% air:5% CO2) for 1 hr at 37 degrees at a drug concentration ranging from 2.8 X 10(-8) to 2.8 X 10(-4) M, i.e., from levels attained clinically by bolus delivery to the high levels used as an i.p. drug dwell or experimentally, in in vitro conditions. High-pressure liquid chromatography analyses showed diminishing efficiency in drug uptake by the cells as the dose was increased. There were no significant differences between hypoxic and euoxic cells in drug uptake and metabolism. The frequency of DNA protein-associated single-strand breaks and DNA-protein cross-links per 10(6) nucleotides, detected by the alkaline elution technique, increased with the dose in the range of 2.8 X 10(-8) to 2.8 X 10(-6) M in both euoxic and hypoxic cells and declined thereafter. However, the number of DNA lesions relative to a normalized drug level declined steadily, starting with the 2.8 X 10(-7) M concentration. Concentrations greater than 2.8 X 10(-6) M of ADR induced still another type of lesion, direct DNA strand breaks, only in euoxic cells. The results indicate that a common mechanism of interaction between drug and DNA is present in hypoxic and in euoxic cells at low ADR, while an O2-dependent mechanism becomes operational in euoxic cells at high ADR levels.

DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues.

20(S)-Camptothecin, the 20(S)-camptothecin sodium salt, and 12 analogues with substituents on the A ring differ widely in their effectiveness in the treatment of murine L1210 lymphoblastic leukemia in vivo. The drugs were screened in the following systems: System 1, the cleavage of DNA in the presence of purified topoisomerase I; System 2, drug-induced trapping of topoisomerase I in a covalent complex with DNA; and System 3, the induction of protein-associated DNA breaks in drug-treated L1210 leukemia cells. 9-Amino-20(S), 10-amino-20(RS), and 10,11-methylenedioxy-20(RS), drugs effective against murine L1210 leukemia in vivo, stabilize topoisomerase I-DNA cleavable complexes in a purified system and in cultured L1210 cells. Other analogues, inactive against L1210 leukemia in vivo, were totally ineffective in topoisomerase I-directed screens. The rest of the analogues were intermediate in terms of their antitumor and topoisomerase I-directed activities. The study shows that the drug-induced accumulation of enzyme-DNA cleavable complexes is directly proportional to drug cytotoxicity and antitumor activity.

DNA topoisomerase II-mediated interaction of doxorubicin and daunorubicin congeners with DNA.

Three groups of doxorubicin and daunorubicin analogues, differing by their substituents on the chromophore and sugar moieties, were used in this study. The 3'-N-unsubstituted (Group 1), 3'-N-acyl (Group 2), and 3'-N-alkyl (Group 3) analogues were tested for: (a) in vivo antitumor activity and in vitro cytotoxicity; (b) cellular or tissue uptake and metabolic conversion; (c) strength of DNA intercalation; and (d) interaction with DNA topoisomerase II (topo-II). Compounds of Group 1 were cytotoxic, were strongly intercalative, and, except for those with C-14 side chain substitution, induced the formation of topo-II-DNA cleavable complexes. As shown previously, esterolysis of C-14-acyl substituents was required to yield a metabolite which can interact with topo-II in the purified system. The C-14-substituted compounds of Group 2 and their C-14-unsubstituted metabolites were cytotoxic. These drugs were weak intercalators, and the C-14-unsubstituted cogeners induced cleavable complex formation in the purified system, but with reduced potency relative to doxorubicin. The type of the 3'-N-position substituent determined whether Group 3 analogues were cytotoxic and strong intercalators, or less active and nonintercalating. Although C-14-unsubstituted intercalators of Group 3 did not form cleavable complexes in the purified system, they were cytotoxic. The study shows that DNA intercalation is required but not sufficient for the activity by topo-II-targeted anthracyclines. In addition to the planar chromophore which is involved in intercalation, two other domains of the anthracycline molecule are important for the interaction with topo-II: (a) substitution of the C-14 position totally inhibits drug activity in the purified system, but enhances cytotoxicity by aiding drug uptake and presumably acting on other cellular targets; and (b) substitutions on the 3'-N position of the sugar ring can, depending on the nature of the substituent, inhibit intercalation and/or topo-II-targeting activity. These findings may provide guidance for the synthesis and development of new active analogues.

Resistance of human leukemic and normal lymphocytes to drug-induced DNA cleavage and low levels of DNA topoisomerase II.

Adriamycin, amsacrine, and etoposide produce protein-associated DNA breaks in numerous cell types. However, in vitro exposure to Adriamycin (0.1-50.0 micrograms/ml) resulted in no detectable DNA cleavage in lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL) or in either B- or T-lymphocytes from normal donors. In contrast, DNA cleavage was observed in T-cells from CLL patients. Exposure to amsacrine or etoposide caused at least 50-fold less DNA cleavage in CLL and normal lymphocytes as compared to L1210 cells. These findings cannot be accounted for by differences in drug uptake. An attempt was made to explain the relative resistance of human lymphocytes to drug-induced DNA cleavage. DNA topoisomerase II, an intracellular target of tested drugs, was assayed in CLL and normal human blood lymphocytes by immunoblotting. The enzyme was detected neither in unfractionated lymphocytes nor in the enriched B- and T-cells from 28 untreated patients with CLL (Stage 0-IV) and from seven normal donors. Exponentially growing L1210 cells had approximately 7 x 10(5) enzyme copies per cell, suggesting a 100-fold higher content than that of CLL or normal lymphocytes. There were, however, detectable levels of DNA topoisomerase II in cells obtained from patients with diffuse histiocytic, nodular poorly differentiated and nodular mixed lymphomas, in Burkitt's lymphoma, acute lymphoblastic leukemia and CLL with prolymphocytic transformation. DNA topoisomerase I, a potential target for anticancer chemotherapy, was detectable in CLL and normal lymphocytes, as well as in cells of other malignancies tested. The above results may offer an explanation for the ineffectiveness of Adriamycin in the treatment of CLL. It could be suggested that low levels of DNA topoisomerase II contribute to drug resistance operating in human malignancies with a large compartment of nonproliferating cells.

Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance.

We investigated the relationship among chemosensitivity to drug-induced apoptosis in vitro, the presence of p53 gene mutations, and the expression of bcl-2 and bax proteins in B-cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. Apoptosis was induced with a camptothecin analogue, 9-amino-20(s)-camptothecin, or a purine analogue, fludarabine. Cell death was monitored by propidium iodide staining and FACS analysis. Drug-induced apoptosis in B-CLL cells was p53-independent. Immunoblot analysis of bcl-2 and bax expression revealed a correlation between drug-induced apoptosis and the ratio of endogenous levels of bcl-2 to bax proteins. B-CLL cells with none to low bcl-2/bax ratios were drug-sensitive as compared to cells with intermediate to high ratios that were drug-resistant (P = 0.015). Prior to drug treatment, bax protein migrated as a single species of 21 kDa. Following drug-induced apoptosis, anti-bax specific protein complexes of 36-42 kDa were up-regulated. Using two-dimensional gel electrophoresis, bax complexes were disrupted under reducing conditions to reveal homo- and heterodimers of 18 and 21 kDa suggesting that disulfide interactions were required for complex formation. The de novo appearance of the 18 kDa anti-bax specific protein together with its increased expression in drug-sensitive B-CLL B-cells undergoing cell death suggests a role for this protein in the regulation of apoptosis.

Antiangiogenic effects of camptothecin analogues 9-amino-20(S)-camptothecin, topotecan, and CPT-11 studied in the mouse cornea model.

Angiogenesis has been correlated with increased invasion and metastases in a variety of human neoplasms. Inadequate inhibition of the growth of tumor microvessels by anticancer agents may result in treatment failure, rated clinically as progressive or stable disease. We have investigated the antiangiogenic properties of three camptothecin analogues, 9-amino-20(S)-camptothecin, topotecan, and camptosar (CPT-11), currently under investigation in clinical settings. Angiogenesis was induced by basic fibroblast growth factor in the cornea of inbred Swiss-Webster mice, with the aim of exploring the suppression of neovascularization by the analogues injected into the mice daily over a period of 6 days. The dose range chosen is known to inhibit, in the mouse model, the growth of various human tumor xenografts or murine tumors. The statistical analysis evaluated the association between the area of neoangiogenesis and the dose of the drugs tested and correlated the effects with observed drug toxicity. It was established that, as the drug doses increased, the area of neovascularization decreased, appearing to approximate a negative exponential curve. 9-Amino-20(S)-camptothecin at 6.89 and 8.26 micromol/kg (2.5 and 3.0 mg/kg) and topotecan at 8.31 micromol/kg (3.5 mg/kg), both drugs being delivered over a 6-day period, had statistically significant reduction (47.2-72.5%) of neoangiogenesis and acceptable toxicity. At higher doses of the two analogues, toxic body-weight losses and deaths were observed. CPT-11 showed statistically significant reduction of neoangiogenesis at a dose of 359 micromol/kg (210 mg/kg) delivered over a 6-day course. Unlike camptothecin analogues, the nontoxic dose of vincristine did not induce a statistically significant inhibition of angiogenesis, and there was no dose-dependent escalation of antiangiogenic effects. The results indicate that camptothecins are most likely cytotoxic against two tumor compartments: in addition to tumor cells of epithelial origin, the drugs act against endothelial cells and prevent the growth of the tumor microvessels. We have hypothesized that treatment failure in some patients is due to incomplete or inadequate inhibition of the microvessel growth by camptothecins. Presumably, an intensive inhibition of the remaining tumor microvasculature in such patients could be achieved by combining a camptothecin with another antiangiogenic anticancer agent or with a highly selective angiogenic inhibitor exerting minimal dose-limiting toxicity. Such treatment by a camptothecin plus a less toxic inhibitor of angiogenesis can improve antitumor efficacy. To validate this concept, preclinical studies followed by clinical trials are planned.

Pharmacodynamics of topoisomerase I inhibition: Western blot determination of topoisomerase I and cleavable complex in patients with upper gastrointestinal malignancies treated with topotecan.

Analogues of camptothecins are specific inhibitors of eukaryotic DNA topoisomerase I (topo I) that lead to DNA damage and, eventually, cellular cytotoxicity. Camptothecin analogues bind to this target enzyme in the course of its normal function and stabilize the DNA-enzyme adduct to form a "cleavable complex." Preclinical experiments using Western blot analyses have shown cleavable complex formation to be the key intermediate step in topo I inhibition. In this series of experiments, it was our goal to convert this laboratory technique into a useful clinical assay, allowing measurement of the target enzyme and detection of the key intermediate in clinical specimens taken from patients being treated with the topo I inhibitor topotecan. Because available antibodies were not sufficiently sensitive at the start of this project, we identified a highly specific human SCL-70 antibody from a patient with scleroderma, which allowed quantitative determination of topo I copy number in HeLa and HT-29 cell lines. Additional refinements of the Western blot technique were accomplished to improve signal:noise ratio. In surgical tumor specimens, we found the median topo I level to be 30.1 x 10(5) copies/cell for gastric adenocarcinomas, compared to 18.4 x 10(5) copies/cell for normal gastric mucosae in the same samples. For lung adenocarcinoma, the median protein level was 21.5 x 10(5) copies/cell, compared with the normal tissue counterpart protein level of 12.7 x 10(5) copies/cell. The median tumor:normal ratios from paired samples of these tumor types were 1.51 and 1.84, respectively. As part of a Phase II study evaluating the efficacy of topotecan (1.5-2.0 mg/m2 daily for 5 days) in upper gastrointestinal malignancies, we obtained tumor and normal mucosa biopsies in 11 patients with gastric or esophageal cancer, 30 min after administration on day 4 or 5. Three patients with gastric adenocarcinoma had stable disease as their best response, with the remainder of patients progressing. Improvement in Western blotting methodology allowed the quantitation of topo I levels in these gastric and esophageal cancer biopsies, which could be augmented by brief heating to release complexed topo I. We were also able to directly visualize high molecular weight topo I-containing bands, which were shown to be cleavable complexes by heat reversal, with restoration of the topo I Mr 100,000 band. Using this heat reversal technique, we determined the presence of cleavable complex in a total of 7 of 11 patient biopsy samples (5 tumors and 2 normal mucosae). In patients treated with topotecan on this dose and schedule, we determined that a median of 73% of the total tumor topo I was involved in cleavable complex (range, 18.3-91%). The intensity of the Mr 100,000 topo I band in biopsy specimens of patients receiving topotecan represented "free" or noncomplexed topo I. The median copy number for the residual, noncomplexed topo I (n = 11) was 7.36 x 10(5) copies/cell, significantly less than the median of 30.1 x 10(5) copies/cell for random tumor specimens from patients with gastric adenocarcinomas (P < 0.001). Pharmacodynamic analysis demonstrated a negative correlation between the noncomplexed topo I copy number and topotecan area under the curve (Spearman rank test: r(s) = -0.81, P = 0.003). Nonlinear regression analyses of these data were best fit with an inhibitory maximum effect model, yielding parameter estimates for Emax and EC50 of 29.3 x 10(5) copies/cell (coefficient of variation = 22%) and 43.1 ng x h/ml (coefficient of variation = 27%), respectively. Through a series of careful modifications and refinements, we have improved the Western blot assay for topo I for use in clinical monitoring. We have demonstrated the ability to directly visualize cleavable complex in patients being treated with topo I inhibitor therapy and have directly quantitated free topo I, as well as the key topo I intermediate (cleavable complex), in biopsy specimens obtained from pat

Two mechanisms of adriamycin-DNA interaction in L1210 cells.

Among the effects exerted by adriamycin (ADR), interaction with DNA is closely related to cytotoxicity. The interaction results in the formation of protein-associated DNA single-strand breaks (PA-SSB) and, at drug levels greater than or equal to 2.8 X 10(-6) M, also in "direct" (nonenzymatic) DNA single-strand breaks (D-SSB). To characterize the two types of DNA lesions, euoxic mouse leukemia L1210 cells were treated with various antioxidant agents in the presence of 2.8 X 10(-6), X 10(-5), or X 10(-4) M concentrations of ADR. The enzymes superoxide dismutase (200 micrograms/ml) or catalase (250 micrograms/ml), the OH' scavengers dimethyl sulfoxide (70 mM) or ethanol (70 mM), and an inhibitor of superoxide production, 2-deoxy-glucose (1 and 10 mM), reduced the frequency of D-SSB to 18.3 to 68.2% of its level in ADR-treated controls, while the frequency of PA-SSB remained unchanged. These observations seem to indicate that ADR-mediated free radicals cause discernible DNA damage in euoxic cells only at very high drug concentrations, greater than the peak plasma level achievable clinically following i.v. bolus. At lower ADR levels, relevant to clinical use, another type of interaction between the drug and DNA prevails, which apparently does not involve a free-radical mechanism.

Effects of N-trifluoroacetyladriamycin-14-O-hemiadipate and radiation on L1210 cells.

N-Trifluoroacetyladriamycin-14-O-hemiadipate (AD 143) is the most active among the 14-O-hemiester adriamycin-trifluoroacetamide derivatives and has been selected for preclinical studies. We now report its ability to enhance the kill by ionizing radiation of murine leukemic cells in culture. A 1-h exposure to either 1.28-12.8 micrograms/ml of AD 143 or to 0.16-1.62 micrograms/ml of adriamycin (ADR) was followed at 0 h by graded doses (0-1 Krad) of radiation, and cell viability was assessed by soft agar cloning technique. Regression analyses of the dose-response curve have shown that both compounds, at the concentrations employed, decrease the reciprocal of the slope D0 from 97 rad for radiation alone, to 66-56 rad for AD 143 (1.28-5.12 micrograms/ml) plus radiation, or to 85-61 rad for ADR (0.16-0.65 micrograms/ml) when used with radiation. ADR, however, had a significant "shoulder"-modifying effect. The Dq remained essentially unchanged after AD 143 pretreatment. Quantitation of synergism (superadditivity), additivity, and antagonism was performed by isobologram analysis and by a computerized method based on the "median effect principle." Both approaches have shown that synergism of AD 143 or ADR with radiation becomes apparent with dose escalation. This effect is discernible at significantly lower levels of AD 143 than of ADR, corresponding to less than LD50 measured by the clonogenic assay.

Intraperitoneal topoisomerase-I inhibitors. Preliminary findings with 9-aminocamptothecin.

The i.p. administration of topoisomerase I (Topo I) inhibitors has a pharmacologic advantage over intravenous application, including preservation of the biologically active lactone form. In our ongoing study, patients have received 9-amino-20(S)-camptothecin (9-AC) i.p. on days 1, 3, 5, 8, 10, and 12, repeated every 4 weeks. The daily dose has been escalated to level IV of 1.5 mg/m2 (9.0 mg/m2 per course), median of 3 cycles, range 1-4, with a reversible Grade 3 neutropenia in one patient. Responses included one CR (resolution of a pleural effusion), two patients without progressive disease (PD), two not evaluable, and two patients too early for evaluation. The area under the curve (AUC)i.p./AUCpl ratio (pharmacologic advantage) ranged from 7.6 to 16.5 on average, and, using nonlinear modeling, the pharmacologic decay data were fit to one- or two-compartmental models. Overall, a 9-AC i.p. application is well tolerated and anticipated to be an active regimen against i.p. malignancies, particularly those known to be sensitive to systemic Topo-I inhibitors.