James L. Flowers

Camptothecin analogues with enhanced antitumor activity at acidic pH

Background: Camptothecin (CPT) is a specific inhibitor of the nuclear enzyme topoisomerase I, which is involved in cellular DNA replication and transcription. Topoisomerase I is therefore an attractive target for anticancer drug development, and two analogues of CPT, topotecan (TPT) and irinotecan (CPT-11), have demonstrated significant antitumor activity in the clinic. This activity is limited, however, by lability of the CPT E ring lactone, which forms the inactive hydroxy acid at physiological pH. The reaction is reversible at acidic pH, which provides a rationale for selectivity, because many solid tumors create an acidic extracellular environment while maintaining a normal intracellular pH. Purpose: To exploit the tumor-selective pH gradient to improve the efficacy of CPT-based chemotherapy. Methods: CPT analogues were evaluated by growth inhibition assay in three human breast cancer cell lines that had been adapted to in vitro culture at acidic pH versus the respective cells cultured at physiological pH. The MCF-7, MDA-MB-231, and MCF-7/hc cell lines represent the hormone-dependent and hormone-independent stages of the disease, and a MCF-7 variant that is resistant to the alkylating agent 4-hydroperoxycyclophosphamide (4-HC), respectively. Antiproliferative activity of SN-38 (the active metabolite of CPT-11), and TPT was compared to that of CPT and two CPT analogues, 10,11-methylenedioxy-CPT (MDC), and the alkylating derivative, 7-chloromethyl-10,11-MDC (CMMDC). Results: In general, MDC was the most potent and TPT or CPT the least potent analogue, regardless of pH. However, if the comparison was based on magnitude of potentiation by pH, a different rank order emerged. CPT was modulated 4-fold; MDC, SN-38, and TPT were each modulated 5- to 6-fold, while the activity of CMMDC was increased 10- to 11-fold by acidic pH in MCF-7 lines, and 65-fold in MDA-MB-231 cells. Thus MDC was the superior CPT analogue based on potency, but CMMDC was the best candidate for pH modulation. Drug specificity was also observed. While the alkylating agent, 4-HC, was 2- to 3-fold more active at acidic pH, modulation was not observed for 5-fluorouracil, doxorubicin, or paclitaxel. Preliminary mechanism studies indicated that pH modulation of CPT analogues was directly correlated to intracellular levels of glutathione. In addition, protein-associated DNA strand breaks were more rapidly induced at acidic pH. Conclusion: These results suggest that CPT-based drug development and resulting chemotherapy could benefit from evaluation of differential activity at acidic versus physiological pH. Analogues have been identified that could have improved therapeutic indices based on the pH gradient that selectively exists in human tumors.

Evidence for a role of chloroethylaziridine in the cytotoxicity of cyclophosphamide

Abstract A number of investigators have observed that the use of 4-hydroperoxycyclophosphamide (4-HC) in multiwell plate cytotoxicity assays can be associated with toxicity to cells in wells that contain no drug. Previous reports have implicated diffusion of 4-HC decomposition products, and acrolein in particular, as the active species. Purpose: The purpose of this study was to elucidate the species responsible for the airborne cytotoxicity of 4-HC, and to devise ways to minimize such effects in chemosensitivity assays. Methods: To this end, analogues of 4-HC were synthesized to identify the contributions of individual cyclophosphamide metabolites to cytotoxicity. The analogues were then tested for activity against three human breast tumor cell lines (including a line resistant to 4-HC), and one non-small-cell lung carcinoma line. Cytotoxicity was evaluated by assays that quantitate cellular metabolism and nucleic acid content. Results: Didechloro-4-hydroperoxycyclophosphamide, a compound that generates acrolein and a nontoxic analogue of phosphoramide mustard, gave no cross-well toxicity. In contrast, a significant neighboring well effect was observed with phenylketophosphamide, a compound that generates phosphoramide mustard but not acrolein. Addition of authentic chloroethylaziridine reproduced the airborne toxicity patterns generated by 4-HC and phenylketophosphamide. Increasing the buffering capacity of the growth medium and sealing the microtiter plates prevented airborne cytotoxicity. Conclusions: Since it is unlikely that phosphoramide mustard is volatile, these findings implicate chloroethylaziridine rather than acrolein as the volatile metabolite of 4-HC that is responsible for airborne cytotoxicity. The fact that chloroethylaziridine is generated in amounts sufficient to volatilize, diffuse across wells and cause cytotoxicity indicates that it is an important component in the overall cytotoxicity of 4-HC in vitro. Furthermore, these findings suggest that chloroethylaziridine may also contribute to the toxicity of cyclophosphamide in vivo.

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 G2-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.

Pre-clinical evaluation of SN-38 and novel camptothecin analogs against human chronic B-cell lymphocytic leukemia lymphocytes

The topoisomerase I inhibitor camptothecin and its analogs have potent activity against a wide range of solid tumors and several hematologic malignancies. Previous studies with these compounds using the MTT metabolic inhibition assay have shown significant cytotoxicity against lymphocytes from patients with chronic B-cell lymphocytic leukemia (B-CLL). Yet the water soluble analogue, topotecan, which was inhibitory at > 1 microM in vitro, had no clinical activity in vivo. In the present study, we evaluated the in vitro cytotoxicities of SN-38, the active form of irinotecan, and two newer water soluble camptothecin derivatives 10,11-methylenedioxy-20(S)-camptothecin glycinate (MDCG) and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin glycinate (CMMDCG). These two glycinate esters are prodrugs for 10,11-methylenedioxy-20(S)-camptothecin (MDC) and 7-chloromethyl-10,11-methylenedioxy-20(S)-camptothecin (CMMDC), respectively. Effects on cellular metabolism, induction of apoptosis, and overall cell survival were used to evaluate chemosensitivity. We report that the relative cytotoxic potency for these compounds is MDC > or = CMMDC > or = SN-38 >> TPT > CPT-11, where MDC, CMMDC, and SN-38 were over an order of magnitude more cytotoxic than TPT and CPT-11. We also investigated potential mechanisms underlying the unexpected cytotoxicity of these camptothecin derivatives in B-CLL cells that are known to be arrested in G0/G1 of the cell cycle, and found that this class of compounds inhibited [3H]uridine incorporation. We therefore postulate that the inhibition of RNA rather than DNA synthesis may be responsible for the observed cytotoxicity in non-cycling B-CLL cells.

Abstract C223: BACPTDP: A water‐soluble camptothecin pro‐drug that exploits the tumor pH gradient for enhanced activity in hypoxic/acidic tumors

Hypoxia is a common feature of solid tumors. Related 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 (the Warburg Effect). Since tumor cells maintain a normal intracellular pH, the glycolytic phenotype also creates a cellular pH gradient such that drugs that are weak acids are selectively taken up and retained in tumors, while those that are weak bases are excluded. Consequently, one approach to improve selectivity of chemotherapy is to design pH‐sensitive antitumor drugs. Camptothecin (CPT) is unique in this regard, since it contains an E ring lactone that opens to form the inactive hydroxy acid at physiological pH, but remains closed to maintain the active species under acidic conditions. Moreover, camptothecins have recently been shown to directly inhibit HIF‐1 in addition to the traditional genotoxic target, topoisomerase I. Accordingly, we screened for CPT analogs with enhanced activity at acidic pH and discovered 7‐butyl‐10‐amino‐camptothecin (BACPT), an analog that was 10 to 20‐fold more active against breast cancer cell lines cultured at pH 6.8 vs 7.4 (Adams et al., Cancer Chemother Pharmacol, 57:145, 2006). We now report that BACPT has superior antiproliferative activity compared to established drugs in monolayer culture models for human neuroblastoma and pancreatic carcinoma and in 3‐dimensional histoculture models of colon and primary ovarian cancer. Esterification of BACPT at the 20S position with β‐alanine‐lysine dipeptide produced a highly water soluble pro‐drug, BACPTDP, suitable for in vivo use. BACPTDP displayed antitumor activity in murine xenograft flank models for human colon (HT29), ovarian (SK‐OV‐3), pancreatic (Panc‐1), glioma (SF‐295) and non‐small cell lung (NCI‐H460) cancers. Activity of BACPTDP was comparable to irinotecan in HT29, SF‐295 and NCI‐H460, significantly greater in SK‐OV‐3 and remarkably superior in the Panc‐1 model, where complete regressions were observed (T‐C = 60 d; 2/6 LTS vs T‐C = 14 d; 0/6 LTS for irinotecan). Of note, Panc‐1 models advanced aggressive disease, since it is poorly differentiated, expresses mutated p53 and K‐ras, the c‐Src oncogene and is relatively insensitive to gemcitabine and other chemotherapeutic drugs. Based on these data, we evaluated combinations of BACPT with gemcitabine, the standard of care chemotherapy for pancreatic cancer, against Panc‐1 cells in vitro. Median effect analysis revealed additive to synergistic interactions that were independent of drug ratio and optimal when gemcitabine was administered 24 h prior to BACPT. Minimal drug antagonism was seen. Conclusion: BACPTDP is a water‐soluble camptothecin pro‐drug with defined molecular mechanism(s) that can exploit solid tumor physiology for improved selectivity and activity against multiple tumor types with particular promise for pancreatic cancinoma. Supported by SBIR/NCI grant CA125871. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C223.