UHPLC-MS/MS method for analysis of sobuzoxane, its active form ICRF-154 and metabolite EDTA-diamide and its application to bioactivation study.

Sobuzoxane (MST-16) is an approved anticancer agent, a pro-drug of bisdioxopiperazine analog ICRF-154. Due to the structural similarity of ICRF-154 to dexrazoxane (ICRF-187), MST-16 deserves attention as a cardioprotective drug. This study presents for the first time UHPLC-MS/MS assay of MST-16, ICRF-154 and its metabolite (EDTA-diamide) in cell culture medium, buffer, plasma and cardiac cells and provides data on MST-16 bioactivation under conditions relevant to investigation of cardioprotection of this drug. The analysis of these compounds that differ considerably in their lipophilicity was achieved on the Zorbax SB-Aq column using a mixture of aqueous ammonium formate and methanol as a mobile phase. The biological samples were either diluted or precipitated with methanol, which was followed by acidification for the assay of MST-16. The method was validated for determination of all compounds in the biological materials. The application of the method for analysis of samples from in vitro experiments provided important findings, namely, that (1) MST-16 is quickly decomposed in biological environments, (2) the cardiac cells actively metabolize MST-16, and (3) MST-16 readily penetrates into the cardiac cells and is converted into ICRF-154 and EDTA-diamide. These data are useful for the in-depth examination of the cardioprotective potential of this drug.

The dynamics of DNA topoisomerase IIalpha in living cells.

Here, we describe methods to prepare a mammalian expression plasmid encoding EGFP fused to the amino-terminus of human DNA topoisomerase IIalpha (Topo IIalpha) for use in studying the dynamics of Topo IIalpha in living cells. In previous studies, this plasmid was transfected into LLC-Pk cells, a porcine epithelial cell line that remains relatively flat during mitosis. After selection for stable integration, cells were cloned by serial dilution in microwells and used to grow a stable cell line expressing EGFP-Topo IIalpha; this cell line was termed LPk-GT2. Using photobleaching methods with conventional and patterned photobleaching, LPk-GT2 cells were used to demonstrate the rapid dynamics of Topo IIalpha exchange in both interphase nuclei and mitotic chromosomes. These rapid dynamics are dependent on enzyme activity since ICRF159, a catalytic inhibitor of Topo IIalpha, slows dynamics significantly. The methods utilized in these studies are described herein.

Suppression of mitoxantrone cardiotoxicity in multiple sclerosis patients by dexrazoxane.

OBJECTIVE: To explore the potential of dexrazoxane to suppress subclinical cardiotoxicity in MS patients receiving mitoxantrone. METHODS: An open-label study was performed to evaluate possible subclinical cardiotoxicity in multiple sclerosis patients treated quarterly with mitoxantrone (48 mg/m(2) cumulative), with and without concomitant dexrazoxane, using blinded serial radionucleide ventriculography. RESULTS: No patient experienced symptoms of heart failure. Patients receiving dexrazoxane, which is cardioprotective for anthracyclines, exhibited a significantly lesser decline in left ventricular ejection fraction (mean change, -3.80% vs -8.55%, p < 0.001). INTERPRETATION: These results support a cardioprotective effect of dexrazoxane in mitoxantrone treated multiple sclerosis patients.

Metabolism of the cardioprotective drug dexrazoxane and one of its metabolites by isolated rat myocytes, hepatocytes, and blood.

The metabolism of the antioxidant cardioprotective agent dexrazoxane (ICRF-187) and one of its one-ring open metabolites to its active metal ion binding form N,N'-[(1S)-1-methyl-1,2-ethanediyl-]bis[(N-(2-amino-2-oxoethyl)]glycine (ADR-925) has been investigated in neonatal rat myocyte and adult rat hepatocyte suspensions, and in human and rat blood and plasma with a view to characterizing their hydrolysis-activation. Dexrazoxane is clinically used to reduce the iron-based oxygen free radical-mediated cardiotoxicity of the anticancer drug doxorubicin. Dexrazoxane may act through its hydrolysis product ADR-925 by removing iron from the iron-doxorubicin complex, or binding free iron, thus preventing oxygen radical formation. Our results indicate that dexrazoxane underwent partial uptake and/or hydrolysis by myocytes. A one-ring open metabolite of dexrazoxane underwent nearly complete dihydroorotase-catalyzed metabolism in a myocyte suspension. Hepatocytes that contain both dihydropyrimidinase and dihydroorotase completely hydrolyzed dexrazoxane to ADR-925 and released it into the extracellular medium. Thus, in hepatocytes, the two liver enzymes acted in concert, and sequentially, on dexrazoxane, first to produce the two ring-opened metabolites, and then to produce the metabolite ADR-925. We also showed that the hydrolysis of one of these metabolites was promoted by Ca2+ and Mg2+ in plasma, and thus, further metabolism of these intermediates likely occurs in the plasma after they are released from the liver and kidney. In conclusion, these studies provide a nearly complete description of the metabolism of dexrazoxane by myocytes and hepatocytes to its presumably active form, ADR-925.

The metabolites of the cardioprotective drug dexrazoxane do not protect myocytes from doxorubicin-induced cytotoxicity.

The clinically approved cardioprotective agent dexrazoxane (ICRF-187) and two of its hydrolyzed metabolites (a one-ring open form of dexrazoxane and ADR-925) were examined for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Dexrazoxane may protect against doxorubicin-induced damage to myocytes through its strongly metal-chelating hydrolysis product ADR-925, which could act by displacing iron bound to doxorubicin or chelating free or loosely bound iron, thus preventing site-specific iron-based oxygen radical damage. The results of this study showed that whereas dexrazoxane was able to protect myocytes from doxorubicin-induced lactate dehydrogenase release, neither of the metabolites displayed any protective ability. Dexrazoxane also reduced apoptosis in doxorubicin-treated myocytes. The ability of dexrazoxane and its three metabolites to displace iron from a fluorescence-quenched trapped intracellular iron-calcein complex was also determined to see whether the metabolites were taken up by myocytes. Although ADR-925 was taken up in the absence of calcium in the medium, in the presence of calcium, its uptake was greatly slowed, presumably because it formed a complex with calcium. Both of the one-ring open metabolites were taken up by myocytes and displaced iron from its complex with calcein. These results suggest either that the anionic metabolites do not have the same access to iron pools in critical cellular compartments, that their uptake is slowed in the presence of calcium, or, less likely, that dexrazoxane protects by some other mechanism.

Analysis of bisdioxopiperazine dexrazoxane binding to human DNA topoisomerase II alpha: decreased binding as a mechanism of drug resistance.

Topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double stranded DNA segment and transporting it through another DNA molecule. Despite the extensive use of topoisomerase II-targeted drugs in cancer chemotherapy and the impact of drug resistance on the efficacy of treatment, much remains unknown concerning the interactions between these agents and topoisomerase II. To identify the interaction of the bisdioxopiperazine dexrazoxane (ICRF-187) with topoisomerase II, we developed a rapid gel-filtration assay and characterized the binding of ((3)H)-dexrazoxane to human topoisomerase II alpha. Dexrazoxane binds to human topoisomerase II alpha in the presence of DNA and ATP with an apparent K(d) of 23 microM and a stoichiometry of 1 drug molecule per enzyme dimer. Various N-terminal single amino acid substitutions in human topoisomerase II alpha that were previously shown to confer specific bisdioxopiperazine resistance either totally abolished drug binding or resulted in less efficient binding. The effect of the various mutations on drug binding correlated well with their effect on drug resistance in vivo and in vitro. Interestingly, an altered active site tyrosine mutant of human topoisomerase II alpha, which is incapable of carrying out DNA strand passage, was unable to bind dexrazoxane, which agrees with the drug's proposed mechanism of action late in the topoisomerase II catalytic cycle. The direct correlation between the level of drug binding and dexrazoxane resistance is consistent with a decreased drug binding mechanism of action for these dexrazoxane resistance conferring mutations.

Metabolism of dexrazoxane (ICRF-187) used as a rescue agent in cancer patients treated with high-dose etoposide.

PURPOSE: The study was undertaken to determine the metabolism of dexrazoxane (ICRF-187) to its one-ring open hydrolysis products and its two-rings opened metal-chelating product ADR-925 in cancer patients with brain metastases treated with high-dose etoposide. In this phase I/II trial dexrazoxane was used as a rescue agent to reduce the extracerebral toxicity of etoposide. METHODS: Dexrazoxane and its one-ring open hydrolysis products were determined by HPLC and ADR-925 was determined by a fluorescence flow injection assay. RESULTS: The two one-ring open hydrolysis intermediates of dexrazoxane appeared in the plasma at low levels upon completion of dexrazoxane infusion and then rapidly decreased with half-lives of 0.6 and 2.5 h. A plasma concentration of 10 micro M ADR-925 was also detected at the completion of the dexrazoxane i.v. infusion period, indicating that dexrazoxane was rapidly metabolized in vivo. A plateau level of 30 micro M ADR-925 was maintained for 4 h and then slowly decreased. The pharmacokinetics of dexrazoxane were found to be similar to other reported data in other settings and at lower doses. CONCLUSIONS: The rapid appearance of ADR-925 in plasma may make ADR-925 available to be taken up by heart tissue and bind free iron. These results suggest that the dexrazoxane intermediates are enzymatically metabolized to ADR-925 and provide a pharmacodynamic basis for the antioxidant cardioprotective activity of dexrazoxane.

Dihydroorotase catalyzes the ring opening of the hydrolysis intermediates of the cardioprotective drug dexrazoxane (ICRF-187).

The enzyme kinetics of the hydrolysis of the one-ring open metabolites of the antioxidant cardioprotective agent dexrazoxane [ICRF-187; (+)-1,2-bis(3,5-dioxopiperazin-1-yl)propane] to its active metal ion binding form ADR-925 [N,N'-[(1S)-1-methyl-1,2-ethanediyl]bis[N-(2-amino-2-oxoethyl)glycine] by dihydroorotase (DHOase) has been investigated by high-performance liquid chromatography (HPLC). A spectrophotometric detection HPLC assay for dihydroorotate was also developed. Dexrazoxane is clinically used to reduce the iron-based oxygen free radical-mediated cardiotoxicity of the anticancer drug doxorubicin. DHOase was found to catalyze the ring opening of the metabolites with an apparent V(max) that was 11- and 27-fold greater than its natural substrate dihydroorotate. However, the apparent K(m) for the metabolites was 240- and 550-fold larger than for dihydroorotate. This report is the first that DHOase might be involved in the metabolism of a drug. Furosemide inhibited DHOase, but the neutral 4-chlorobenzenesulfonamide did not. Because dihydroorotate, the one-ring open metabolites, and furosemide all have a carboxylate group, it was concluded that a negative charge on the substrate strengthened binding to the positively charged active site. The presence of DHOase in the heart may explain the cardioprotective effect of dexrazoxane. Thus, dihydropyrimidinase and DHOase acting in succession on dexrazoxane and its metabolites to form ADR-925 provide a mechanism by which dexrazoxane is activated to exert its cardioprotective effects. The ADR-925 thus formed may either remove iron from the iron-doxorubicin complex, or bind free iron, thus preventing oxygen radical formation.

The displacement of iron(III) from its complexes with the anticancer drugs piroxantrone and losoxantrone by the hydrolyzed form of the cardioprotective agent dexrazoxane.

Piroxantrone and losoxantrone are new DNA topoisomerase II-targeting anthrapyrazole antitumor agents that display cardiotoxicity both clinically and in animal models. A study was undertaken to see whether dexrazoxane or its hydrolysis product ADR-925 could remove iron(III) from its complexes with piroxantrone or losoxantrone. Their cardiotoxicity may result from the formation of iron(III) complexes of losoxantrone and piroxantrone. Subsequent reductive activation of their iron(III) complexes likely results in oxygen-free radical-mediated cardiotoxicity. Dexrazoxane is in clinical use as a doxorubicin cardioprotective agent. Dexrazoxane presumably acts through its hydrolyzed metal ion binding form ADR-925 by removing iron(III) from its complex with doxorubicin, or by scavenging free iron(III), thus preventing oxygen-free radical-based oxidative damage to the heart tissue. ADR-925 was able to remove iron(III) from its complexes with piroxantrone and losoxantrone, though not as efficiently or as quickly as it could from its complexes with doxorubicin and other anthracyclines. This study provides a basis for utilizing dexrazoxane for the clinical prevention of anthrapyrazole cardiotoxicity.

Chemistry of dexrazoxane and analogues.

The bisdioxopiperazine dexrazoxane (DEX; ICRF-187) has proven to be clinically effective in reducing the cardiotoxicity of doxorubicin and the toxicity of other anthracyclines. Doxorubicin and the other anthracyclines are thought to exert their toxicity through iron-based oxygen free radical-induced oxidative stress on the relatively unprotected cardiac muscle. On hydrolysis, DEX forms a compound (ADR-925) similar in structure to EDTA, which, like EDTA, is a strong chelator of iron and other metal ions. Dexrazoxane presumably exerts its cardioprotective effects by either binding free or loosely bound iron, or iron complexed to doxorubicin, thus preventing or reducing site-specific oxygen radical production that damages cellular components. The hydrolysis-activation of DEX to ADR-925 can occur through either enzymatic or nonenzymatic routes. Iron(III)-anthracycline complexes are directly able to promote ring-opening hydrolysis of DEX. Both ferrous and ferric ions (as well as several other divalent metal ions) can promote the hydrolysis of the one-ring open intermediates of DEX to ADR-925, which suggests that these intermediates may be pharmacologically active. Paradoxically, the ferric complex of ADR-925 has been shown to be capable of being reductively activated to mediate hydroxyl radical formation. This observation suggests that DEX may be acting through its ability to prevent site-specific oxygen radical damage by iron-anthracycline complexes.

Analysis of a core domain in Drosophila DNA topoisomerase II. Targeting of an antitumor agent ICRF-159.

To investigate the biochemical properties of individual domains of eukaryotic topoisomerase (topo) II, two truncation mutants of Drosophila topo II were generated, ND406 and core domain. Both mutants lack the ATPase domain, corresponding to the N-terminal 406 amino acid residues in Drosophila protein. The core domain also lacks 240 amino acid residues of the hydrophilic C-terminal region. The mutant proteins have lost DNA strand passage activity while retaining the ability to cleave the DNA and the sequence preference in protein/DNA interaction. The cleavage experiments carried out in the presence of several topo II poisons suggest that the core domain is the key target for these drugs. We have used glass-fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both truncation mutant proteins can form a clamp complex in the presence of an antitumor agent, ICRF-159, suggesting that the drug targets the core domain of the enzyme and promotes the intradimeric closure at the N-terminal interface of the core domain. Furthermore, the salt stability of the closed protein clamp induced by ICRF-159 depends on the presence and closure of the N-terminal ATPase domain.

The one-ring open hydrolysis intermediates of the cardioprotective agent dexrazoxane (ICRF-187) do not inhibit the growth of Chinese hamster ovary cells or the catalytic activity of DNA topoisomerase II.

Dexrazoxane (ICRF-187), which is clinically used to reduce doxorubicin-induced cardiotoxicity, has growth inhibitory properties through its ability to inhibit the catalytic activity of DNA topoisomerase II. Because the bisdioxopiperazine dexrazoxane undergoes significant ring-opening hydrolysis under physiological conditions to form two one-ring open hydrolysis intermediates, a study was undertaken to determine if these two intermediates had either any growth inhibitory or topoisomerase II inhibitory effects. Neither of the one-ring open intermediates exhibited growth inhibitory effects towards Chinese hamster ovary cells nor were they able to inhibit topoisomerase II. Thus, it was concluded that only intact dexrazoxane is able to inhibit the catalytic activity of topoisomerase II.

NADPH-cytochrome-P450 reductase promotes hydroxyl radical production by the iron complex of ADR-925, the hydrolysis product of ICRF-187 (dexrazoxane).

ICRF-187 (dexrazoxane) is currently in clinical trials as a cardioprotective agent for the prevention of doxorubicin-induced cardiotoxicity. ICRF-187 likely acts through its strongly metal ion-binding rings-opened hydrolysis product ADR-925 by removing iron from its complex with doxorubicin or by chelating free iron. The ability of NADPH-cytochrome-P450 reductase to promote hydroxyl radical formation by iron complexes of ADR-925 and EDTA was compared by EPR spin trapping. The iron-EDTA complex produced hydroxyl radicals at six times the rate that the iron-ADR-925 complex did. The aerobic oxidation of ferrous complexes of ADR-925, its tetraacid analog, EDTA and DTPA was followed spectrophotometrically. The iron(II)-ADR-925 complex was aerobically oxidized 700 times slower than was the EDTA complex. It is concluded that even though ADR-925 does not completely eliminate iron-based hydroxyl radical production, it likely protects by preventing site-specific hydroxyl radical damage by the iron-doxorubicin complex.

Metabolism of the cardioprotective agents dexrazoxane (ICRF-187) and levrazoxane (ICRF-186) by the isolated hepatocyte.

1. The metabolism of dexrazoxane (ICRF-187) and its optical isomer levrazoxane (ICRF-186) by the isolated rat hepatocyte was studied by hplc. 2. 4-Chlorobenzenesulphonamide, which is a strong inhibitor of dihydropyrimidine amidohydrolase (DHPase), caused 82% inhibition of the loss of dexrazoxane from the hepatocyte suspension. 3. Dexrazoxane was metabolized at an initial rate by isolated hepatocytes that was 1.8 times faster than levrazoxane. This ratio is close to that found for purified DHPase, suggesting that DHPase present in the hepatocyte catalyses the ring-opening hydrolysis of these drugs. 4. The ratios of the rates at which each of the one-ring open intermediates of dexrazoxane and levrazoxane were produced in the hepatocyte suspension are also consistent with DHPase being primarily responsible for the metabolism of dexrazoxane and levrazoxane. 5. Thus, the DHPase-catalysed formation of the one-ring opened intermediates enhances the rate at which the presumably active metal-ion binding forms of dexrazoxane are produced in the hepatocyte. 6. The DHPase content of the hepatocyte was estimated to be 1.2 nmol/kg of total hepatocyte mass, or equivalently 5700 molecules of DHPase per hepatocyte.

Stereoselective hydrolysis of ICRF-187 (dexrazoxane) and ICRF-186 by dihydropyrimidine amidohydrolase.

The enzymatic ring-opening hydrolyses of the doxorubicin cardioprotective agents (+)-(S)-ICRF-187 (dexrazoxane), (-)-(R)-ICRF-186, and rac-ICRF-159 by the enzyme dihydropyrimidine amidohydrolase (DHPase) have been studied. ICRF-187 underwent enzymatic ring-opening hydrolysis by DHPase 4.5 times faster than did ICRF-186. It was also shown that DHPase opens only one ring of ICRF-186 and does not act on this one-ring open hydrolysis product, as has been observed for ICRF-187. Differences in the rates at which the two optical isomers are acted upon by DHPase suggest that they could have differing protective effects.

Pharmacodynamics of the hydrolysis-activation of the cardioprotective agent (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane.

The hydrolysis of the cardioprotective agent ICRF-187 [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane] to its presumed active form under conditions of physiologic pH and temperature were followed by HPLC chromatography. Successful chromatography of all of the hydrolysis products required the use of EDTA in the aqueous eluant to prevent metals in the HPLC flow system from binding to the strongly metal ion-binding product ADR-925. The kinetics of the hydrolysis was followed to approximately 200 h. The ring closest to the methyl group on ICRF-187 was observed to open at about twice the rate of the other ring. This product accumulates in the reaction mixture not only because it is produced more quickly but also because it decays more slowly. ICRF-187 is lost from the reaction mixture with a half-life of 9.3 h, whereas the final hydrolysis product ADR-925 is produced with a half-life of 23.0 h. Rate constants for ring opening to one-ring and two-ring opened hydrolysis products were obtained with a reaction scheme that assumed parallel and consecutive first-order reactions for these steps.

Enzymatic ring-opening reactions of the chiral cardioprotective agent (+) (S)-ICRF-187 and its (-) (R)-enantiomer ICRF-186 by dihydropyrimidine amidohydrolase.

The kinetics of the ring-opening reaction of the cardioprotective agent (+) (S)-ICRF-187 [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane, dexrazoxane] and its (-) (R)-analog ICRF-186 by the enzyme dihydropyrimidine amidohydrolase (DHPase) has been studied by HLLC. Chromatographic separation of the two single-ring opened hydrolysis products allowed an estimation of the Michaelis parameters for the opening of each individual ring on the two optical isomers. Under nonsaturating conditions, DHPase acts on ICRF-187 4 times faster than it does on ICRF-186. However, the single-ring opened hydrolysis products of both ICRF-187 and ICRF-186 are not substrates of DHPase. Because the active form of ICRF-187 is thought to be its rings-opened metal ion chelating form, these results suggest that DHPase-catalyzed hydrolysis occurring in the liver and the kidney may lead to differences in the pharmacological action and effectiveness of these two optical isomers.

The hydrolysis product of ICRF-187 promotes iron-catalysed hydroxyl radical production via the Fenton reaction.

d-1,2-Bis(3,5-dioxopiperazine-1-yl)propane (ICRF-187) (ADR-529) is a drug that ameliorates the cardiotoxicity of Adriamycin. The drug enters cells where hydrolysis leads to its diacid diamide product, dl-N,N'-dicarboxamidomethyl-N,N'-dicarboxymethyl-1,2-diamino propane (ICRF-198) (ADR-925), which is structurally similar to ethylenediaminetetraacetic acid (EDTA). The protective mechanism of ICRF-187 is unknown, but a plausible explanation is that ICRF-198 chelates iron intracellularly to prevent iron-dependent free radical reactions such as hydroxyl radical (.OH) production. We have compared Fe(ICRF-198) with Fe(EDTA) in its ability to promote .OH formation in several Fenton reaction systems. The Fenton reaction was studied with H2O2 and Fe2+ chelates or catalytic amounts of the iron chelates in the presence of Adriamycin radicals, paraquat radicals, superoxide anion radicals (O2-), and ascorbate as reducing species. .OH was detected with deoxyribose and dimethyl sulfoxide. The two methods gave comparable results. Fe(ICRF-198) was 80-100% as effective as Fe(EDTA) at promoting .OH production in the presence of the organic radicals and ascorbate, 30-70% in the presence of O2-, and 150% with non-cycling Fe2+. Fe(EDTA) is a more efficient catalyst of .OH production than physiological chelates such as ADP, ATP and citrate. Therefore, by comparing previous work which examined physiological chelates and Fe(EDTA) with the present work, Fe(ICRF-198) appears to be a better .OH catalyst than the physiological chelates. These results suggest that ICRF-198 generated in vivo from ICRF-187 would not protect against intracellular .OH production. They also imply that .OH production may not be as important in Adriamycin cardiotoxicity as other radical reactions, such as lipid peroxidation and thiol oxidation, that are inhibited by ICRF-198.

Is ICRF-187 [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane] unusually reactive for an imide?

The hydrolysis of ICRF-187 and two model compounds, 4-methylpiperazine-2,6-dione (4-MP) and 3-methylglutarimide (3-MG), was investigated over the neutral to alkaline pH range at 25 degrees C and an ionic strength of 0.5 (sodium chloride). The purpose of the study was to correlate the influence of molecular changes to the reactivity of these imides. Additionally, an improved chromatographic resolution of all the components of the degradation and NMR confirmation of the identity of the degradation products are presented. Based on the study of 4-MP, which is essentially half of an ICRF-187 molecule, and 3-MG, which has a carbon in place of the piperazine nitrogen, several conclusions can be drawn with regard to the stability of ICRF-187. The tertiary piperazine nitrogen/s of 4-MP and ICRF-187 contributed to the base-catalyzed hydrolysis of these compounds above pH 7 and caused a significant decrease in the pKa values of the imide moiety of ICRF-187 and 4-MP compared with 3-MG. One 2,6-piperazinedione ring of ICRF-187 was shown to affect only minimally the rate of hydrolysis of the second ring. ICRF-187 hydrolyzes by parallel consecutive pathways forming two monoacids with one ring opened and, subsequently, the diacid with both rings hydrolyzed.

The enzymatic hydrolysis-activation of the adriamycin cardioprotective agent (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane.

The cardioprotective agent ICRF-187 [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane, ADR-529] has shown a great deal of promise against what may be an iron-based adriamycin-induced cardiotoxicity. ICRF-187 likely exerts its actions through its rings-opened hydrolysis product, which has a structure similar to EDTA and like-wise strongly binds metal ions. The 105,000g soluble supernatant fraction of homogenates of porcine liver and kidney, but not of heart, enzymatically caused a ring-opening hydrolysis of ICRF-187 at the rate of 1.2, 1.4, and less than 0.15 nmol.(mg protein)-1.min-1, respectively. This enzymatic hydrolysis of ICRF-187 was completely abolished by 4-chlorobenzenesulfonamide, which suggested that dihydropyrimidine amidohydrolase (DHPase) might be, in large part, responsible for the ICRF-187 hydrolase activity. Bovine liver DHPase was isolated and was shown to enzymatically hydrolyze ICRF-187 with a Vmax of 14,900 nmol.(mg DHPase)-1.min-1, a value that exceeded that of its natural substrates. The KM for ICRF-187 of 6.7 mM was, however, much larger than that of its natural substrates. The enzyme kinetics were consistent with DHPase acting on ICRF-187 to form one-ring-opened hydrolysis product only. Thus, DHPase is not able to act on the one-ring-opened hydrolysis product to produce two-ring-opened product. The Ki for 4-chlorobenzenesulfonamide inhibition of DHPase was measured to be 26 microM.