Camptothecin analogs with enhanced activity against human breast cancer cells. II. Impact of the tumor pH gradient.

Human breast tumors often exist in an acidic and hypoxic microenvironment, which can promote resistance to radiation and chemotherapies. A tumor-selective pH gradient arises in these tumors which favors uptake and retention of drugs like camptothecin that are weak acids. We evaluated the effect of alkyl substitutions at the 7 position in seven CPTs with varying groups at the 10 position on modulation by acidic extracellular pH in three human breast cancer cell lines. Growth inhibition was assessed by propidium iodide staining of nucleic acids in human breast cancer cells cultured at either extracellular pH 6.8 or 7.4 that were (1) hormone-sensitive (MCF-7/wt), (2) hormone insensitive (MDA-MB-231), or (3) alkylator-resistant (MCF-7/4-hc). Over 10-fold pH modulation was observed in 7-halomethyl analogs of methylenedioxy-CPT and in 7-alkyl analogs of 10-amino-CPT. Of 39 analogs tested, the overall pattern of activity across breast tumor cell lines was similar with some notable exceptions. For example, 7-propyl-10-amino-CPT was modulated 16- to 20-fold by acidic extracellular pH in the MCF-7 cell lines, but only 6-fold in MDA-MB-231 cells. One mechanism that can contribute to pH modulation is enhanced cellular drug uptake and retention. In MCF-7/wt cells, uptake of 10-amino-CPT increased 4-fold, while retention increased over 10-fold at acidic extracellular pH. In addition, gene expression analysis of MCF-7/wt cells indicated that expression of a number of genes changed under acidic culture conditions, including down-regulation of the CPT efflux protein pump breast cancer resistance protein (BCRP). Interestingly, expression of topoisomerase I, the molecular target of CPT, was not affected by acidic growth conditions. These results highlight the importance of maintaining key features of tumor physiology in cell culture models used to study cancer biology and to discover and develop new anticancer drugs. While several substitutions at the 7 and 10 positions enhance potency, 7-halomethyl and 10-amino CPT analogs show selective activity at the acidic pH common to the microenvironment of most solid tumors.

Camptothecin analogs with enhanced activity against human breast cancer cells. I. Correlation of potency with lipophilicity and persistence in the cleavage complex.

The effect of 7-alkyl substitutions on growth inhibition in seven Camptothecin (CPT) ring systems with various groups at the ten position was evaluated in three human breast cancer cell lines that model (1) hormone-sensitive (MCF-7/wt), (2) hormone insensitive (MDA-MB-231), or (3) alkylator-resistant (MCF-7/4-hc) forms of disease. To assess the impact of persistence of cleavage complexes on antiproliferative activity, a post-exposure recovery period in drug-free medium was incorporated into the growth inhibition assay. This modification produced on average a twofold reduction in the growth inhibition endpoint (the IC50), suggesting a greater apoptotic response. The results further revealed a three log range in potency from a mean IC50 of 2 nM (7-butyl-10,11-methylenedioxy-CPT) to 2.5 microM (7-bromomethyl-10-hydryoxy-CPT). Increasing 7-alkyl chain length in six of the ten-substituted CPTs enhanced potency, which was directly correlated with persistence of topoisomerase I-induced DNA cleavage complexes in 10-hydroxy, 10-methoxy, and 10,11-methylenedioxy substituted CPTs. Modeling of the binding mode of 7-butyl-10-amino-CPT revealed a direct hydrogen bond contact for the 10-amino to the side chain of Glu-356 of Core Subdomain I of top1 in addition to known contacts found for other camptothecins. More important, residues 350-356 and 425-431 of Core Subdomain I may provide induced fit stabilization to the lipophilic alkyl moiety at the seven position.

Preclinical evaluation of gemcitabine combination regimens for application in acute myeloid leukemia.

The DNA antimetabolite gemcitabine is an anticancer agent with shown preclinical and clinical utility and a low toxicity profile. In this study, we sought to identify and optimize drug partners for binary and tertiary combinations with gemcitabine for use in the treatment of acute myelogenous leukemia (AML). Drug interaction was assessed by growth inhibition assay with metabolic end points. The combination index method was used to evaluate combinations of gemcitabine with fludarabine, paclitaxel, chlorambucil, doxorubicin, mitoxantrone, and SN-38 in U937 human AML cells. A three-dimensional method was used to determine the effect of dose ratio and schedule on drug interaction. Mechanisms underlying interactions related to cell cycle effects and apoptosis were assessed by flow cytometric and caspase-3 and -7 assays, respectively. The most synergistic binary combination was gemcitabine + fludarabine. The most synergistic tertiary combination was gemcitabine + fludarabine + paclitaxel, where the interaction was sequence dependent with paclitaxel given before gemcitabine + fludarabine, producing a 2-fold increase in synergy. Cell cycle analysis did not reveal a significant G(2)-M arrest, suggesting that the synergistic effect of paclitaxel in this combination, which produced the greatest caspase activation, might be independent of microtubule stabilization. In contrast, the gemcitabine + fludarabine + mitoxantrone combination was synergistic and schedule independent. Moreover, few ratios of gemcitabine + fludarabine to mitoxantrone were antagonistic, which could be important for clinical translation. In conclusion, synergistic interactions with gemcitabine occurred with several drugs, the most promising being gemcitabine + fludarabine, gemcitabine + fludarabine + paclitaxel, and gemcitabine + fludarabine + mitoxantrone. These findings provided a rationale for clinical trials of gemcitabine + fludarabine and gemcitabine + mitoxantrone where responses were observed in heavily pretreated AML patients.

The activity of camptothecin analogues is enhanced in histocultures of human tumors and human tumor xenografts by modulation of extracellular pH.

BACKGROUND: Most solid human tumors exist in an acidic microenvironment, due in part to inefficient vasculature and a higher intrinsic rate of glycolysis. This leads to a tumor-selective pH gradient, which can be exploited therapeutically with antitumor agents such as the camptothecins (CPTs). Previous work in this laboratory has shown that camptothecin activity is enhanced 40- to 60-fold in monolayer cell culture by reducing the extracellular pH to 6.8. Three-dimensional histoculture has been shown to be a technique that allows human tumor tissue to grow in an in vivo-like way with maintenance of tissue histology and function and drug sensitivity for long periods of time. PURPOSE: In the current study, we utilized these features of histoculture to study new analogues of camptothecin that have superior pharmacological properties. METHODS: We evaluated six CPT analogues in histocultures of human brain, neuroblastoma, breast, colon, and prostate tumors. Fragments were exposed to 10,11-methylenedioxy-CPT (MDC), 7-chloromethyl-MDC, SN-38, topotecan (TPT), 9-amino-CPT, 10-amino-CPT, paclitaxel, 5-fluorouracil, 4-hydroperoxycyclophosphamide and doxorubicin, and antitumor activity was assessed. For in vivo tumor outgrowth studies, fragments were treated in parallel, implanted into nude mice, and monitored for development of tumors. RESULTS. Against 15 of 16 tumor xenografts and all primary tumor samples tested, all compounds were cytotoxic at pH 7.4 (IC(50) range 13-921 microM). MDC, SN-38, TPT, and 9-amino-CPT achieved an average 5-fold increase in activity (range 3-14) at pH 6.8, while 7-chloromethyl-MDC was enhanced 8-fold (range 6-14). The most potentiated analogue was 10-amino-CPT at 27-fold (range 17-49). In contrast, the other agents were active against one or more tumor types but were not enhanced by acidic pH. Importantly, the toxicity of MDC in histoculture of D54 glioma xenografts strongly correlated with the outgrowth of treated fragments subsequently implanted in vivo. CONCLUSION: Evaluation of anticancer drug activity in native-state histoculture supports the concept that pH modulation may be an important approach to improve the selectivity and antitumor effectiveness of camptothecin-based chemotherapy.

BACPTDP: a water-soluble camptothecin pro-drug with enhanced activity in hypoxic/acidic tumors.

UNLABELLED: Hypoxia is a common feature of solid tumors. Up-regulation of hypoxia-inducing factor-1 (HIF-1) occurs in the majority of primary malignant tumors and in two-thirds of metastases, while most normal tissues are negative. HIF-1 induces the glycolytic phenotype, which creates an acidic extracellular microenvironment and associated pH gradient such that drugs that are weak acids are selectively taken up and retained in acidic tumors. 7-Butyl-10-amino-camptothecin (BACPT) is a prime example of an agent that can exploit the tumor pH gradient for enhanced selectivity. PURPOSE: This study profiles the antitumor activity of BACPT in vitro and its water-soluble dipeptide ester, BACPTDP, in vivo. METHODS: Antitumor activity was evaluated by proliferation assays in cancer cell lines and in murine xenograft models for human neuroblastoma (IMR-32), colon (HT29), ovarian (SK-OV-3), pancreatic (Panc-1), glioma (SF-295) and non-small-cell lung (NCI-H460) cancers. RESULTS: BACPT had superior antiproliferative activity compared to established drugs in monolayer cultures of human neuroblastoma and pancreatic tumor cell lines and in 3-dimensional histocultures of colon and primary ovarian cancer. Antitumor activity of BACPTDP was comparable to irinotecan in IMR-32, HT29, SF-295 and NCI-H460 xenografts, significantly greater in SK-OV-3 and in Panc-1 where complete regressions were observed. Combination of BACPT with gemcitabine produced additive to synergistic interactions in Panc-1 cells that were independent of drug ratio and optimal when gemcitabine was administered 24 h prior to BACPT. CONCLUSIONS: BACPTDP is a water-soluble camptothecin pro-drug that spontaneously generates the lipid-soluble active agent, BACPT. This topoisomerase inhibitor exploits solid tumor physiology for improved selectivity and activity against multiple tumor types with particular promise for use in treating pediatric neuroblastoma and pancreatic carcinoma.

1,3- vs 1,5-intramolecular alkylation reactions in isophosphoramide and phosphoramide mustards.

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.