Evaluation of the topoisomerase II-inactive bisdioxopiperazine ICRF-161 as a protectant against doxorubicin-induced cardiomyopathy.

Anthracycline-induced cardiomyopathy is a major problem in anti-cancer therapy. The only approved agent for alleviating this serious dose limiting side effect is ICRF-187 (dexrazoxane). The current thinking is that the ring-opened hydrolysis product of this agent, ADR-925, which is formed inside cardiomyocytes, removes iron from its complexes with anthracyclines, hereby reducing the concentration of highly toxic iron-anthracycline complexes that damage cardiomyocytes by semiquinone redox recycling and the production of free radicals. However, the 2 carbon linker ICRF-187 is also is a catalytic inhibitor of topoisomerase II, resulting in the risk of additional myelosuppression in patients receiving ICRF-187 as a cardioprotectant in combination with doxorubicin. The development of a topoisomerase II-inactive iron chelating compound thus appeared attractive. In the present paper we evaluate the topoisomerase II-inactive 3 carbon linker bisdioxopiperazine analog ICRF-161 as a cardioprotectant. We demonstrate that this compound does chelate iron and protects against doxorubicin-induced LDH release from primary rat cardiomyocytes in vitro, similarly to ICRF-187. The compound does not target topoisomerase II in vitro or in cells, it is well tolerated and shows similar exposure to ICRF-187 in rodents, and it does not induce myelosuppression when given at high doses to mice as opposed to ICRF-187. However, when tested in a model of chronic anthracycline-induced cardiomyopathy in spontaneously hypertensive rats, ICRF-161 was not capable of protecting against the cardiotoxic effects of doxorubicin. Modulation of the activity of the beta isoform of the topoisomerase II enzyme by ICRF-187 has recently been proposed as the mechanism behind its cardioprotection. This concept is thus supported by the present study in that iron chelation alone does not appear to be sufficient for protection against anthracycline-induced cardiomyopathy.

Dexrazoxane protects against myelosuppression from the DNA cleavage-enhancing drugs etoposide and daunorubicin but not doxorubicin.

PURPOSE: The anthracyclines daunorubicin and doxorubicin and the epipodophyllotoxin etoposide are potent DNA cleavage-enhancing drugs that are widely used in clinical oncology; however, myelosuppression and cardiac toxicity limit their use. Dexrazoxane (ICRF-187) is recommended for protection against anthracycline-induced cardiotoxicity. EXPERIMENTAL DESIGN: Because of their widespread use, the hematologic toxicity following coadministration of dexrazoxane and these three structurally different DNA cleavage enhancers was investigated: Sensitivity of human and murine blood progenitor cells to etoposide, daunorubicin, and doxorubicin +/- dexrazoxane was determined in granulocyte-macrophage colony forming assays. Likewise, in vivo, B6D2F1 mice were treated with etoposide, daunorubicin, and doxorubicin, with or without dexrazoxane over a wide range of doses: posttreatment, a full hematologic evaluation was done. RESULTS: Nontoxic doses of dexrazoxane reduced myelosuppression and weight loss from daunorubicin and etoposide in mice and antagonized their antiproliferative effects in the colony assay; however, dexrazoxane neither reduced myelosuppression, weight loss, nor the in vitro cytotoxicity from doxorubicin. CONCLUSION: Although our findings support the observation that dexrazoxane reduces neither hematologic activity nor antitumor activity from doxorubicin clinically, the potent antagonism of daunorubicin activity raises concern; a possible interference with anticancer efficacy certainly would call for renewed attention. Our data also suggest that significant etoposide dose escalation is perhaps possible by the use of dexrazoxane. Clinical trials in patients with brain metastases combining dexrazoxane and high doses of etoposide is ongoing with the aim of improving efficacy without aggravating hematologic toxicity. If successful, this represents an exciting mechanism for pharmacologic regulation of side effects from cytotoxic chemotherapy.

Combining etoposide and dexrazoxane synergizes with radiotherapy and improves survival in mice with central nervous system tumors.

PURPOSE: The treatment of patients with brain metastases is presently ineffective, but cerebral chemoradiotherapy using radiosensitizing agents seems promising. Etoposide targets topoisomerase II, resulting in lethal DNA breaks; such lesions may increase the effect of irradiation, which also depends on DNA damage. Coadministration of the topoisomerase II catalytic inhibitor dexrazoxane in mice allows for more than 3-fold higher dosing of etoposide. We hypothesized that dexrazoxane combined with escalated etoposide doses might improve the efficacy of cerebral radiotherapy. EXPERIMENTAL DESIGN: Mice with cerebrally inoculated Ehrlich ascites tumor (EHR2) cells were treated with combinations of etoposide + dexrazoxane + cerebral radiotherapy. Similar chemotherapy and radiation combinations were investigated by clonogenic assays using EHR2 cells, and by DNA double-strand break assay through quantification of phosphorylated histone H2AX (gammaH2AX). RESULTS: Escalated etoposide dosing (90 mg/kg) combined with dexrazoxane (125 mg/kg) and cerebral radiotherapy (10 Gy x 1) increased the median survival by 60% (P = 0.001) without increased toxicity, suggesting that escalated etoposide levels may indeed represent a new strategy for improving radiotherapy. Interestingly, 125 mg/kg dexrazoxane combined with normal etoposide doses (34 mg/kg) also increased survival from radiotherapy, but only by 27% (P = 0.002). This indicates a direct dexrazoxane modulation of the combined effects of etoposide and radiation in brain tumors. Further, in vitro, concurrent dexrazoxane, etoposide, and irradiation significantly increased DNA double-strand breaks. CONCLUSION: Combining etoposide (high or normal doses) and dexrazoxane synergizes with cerebral radiotherapy and significantly improves survival in mice with central nervous system tumors. This regimen may thus improve radiation therapy of central nervous system tumors.

A mouse model for studying the interaction of bisdioxopiperazines with topoisomerase IIalpha in vivo.

The bisdioxopiperazines such as (+)-(S)-4,4'-propylenedi-2,6-piperazinedione (dexrazoxane; ICRF-187), 1,2-bis(3,5-dioxopiperazin-1-yl)ethane (ICRF-154), and 4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione (ICRF-193) are agents that inhibit eukaryotic topoisomerase II, whereas their ring-opened hydrolysis products are strong iron chelator. The clinically approved analog ICRF-187 is a pharmacological modulator of topoisomerase II poisons such as etoposide in preclinical animal models. ICRF-187 is also used to protect against anthracycline-induced cardiomyopathy and has recently been approved as an antidote for alleviating tissue damage and necrosis after accidental anthracycline extravasation. This dual modality of bisdioxopiperazines, including ICRF-187, raises the question of whether their pharmacological in vivo effects are mediated through interaction with topoisomerase II or via their intracellular iron chelating activity. In an attempt to distinguish between these possibilities, we here present a transgenic mouse model aimed at identifying the contribution of topoisomerase IIalpha to the effects of bisdioxopiperazines. A tyrosine 165 to serine mutation (Y165S) in topoisomerase IIalpha, demonstrated previously to render the human ortholog of this enzyme highly resistant toward bisdioxopiperazines, was introduced at the TOP2A locus in mouse embryonic stem cells by targeted homologous recombination. These cells were used for the generation of transgenic TOP2A(Y165S/+) mice, which were demonstrated to be resistant toward the general toxicity of both ICRF-187 and ICRF-193. Hematological measurements indicate that this is most likely caused by a decreased ability of these agents to induce myelosuppression in TOP2A(Y165S/+) mice, highlighting the role of topoisomerase IIalpha in this process. The biological and pharmacological implications of these findings are discussed, and areas for further investigations are proposed.