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Giorgio Minotti - One of the best experts on this subject based on the ideXlab platform.
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RUNNING TITLE: Doxorubicin and the cardiac safety of pixantrone
2016Co-Authors: Emanuela Salvatorelli, Giorgio Minotti, Pierantonio Menna, Odalys Gonzalez Paz, Massimo Chello, Elvio Covino, Jack W Singer, At AAbstract:The novel anthracenedione, pixantrone, lacks redox activity and inhibits Doxorubicinol formation in human myocardium: Insight to explain the cardiac safety of pixantrone in doxorubicin treated patient
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The novel anthracenedione, pixantrone, lacks redox activity and inhibits Doxorubicinol formation in human myocardium: insight to explain the cardiac safety of pixantrone in doxorubicin-treated patients
2016Co-Authors: Emanuela Salvatorelli, Pierantonio Menna, Odalys Gonzalez Paz, Massimo Chello, Elvio Covino, Jack W Singer, Giorgio MinottiAbstract:Cardiotoxicity from the antitumor anthracycline doxorubicin correlates with doxorubicin cardiac levels, redox activation to superoxide anion (O2._) and hydrogen peroxide (H2O2), and formation of the long-lived secondary alcohol metabolite Doxorubicinol. Cardiotoxicity may first manifest during salvage therapy with other drugs, such as the anthracenedione mito-xantrone. Minimal evidence for cardiotoxicity in anthracycline-pretreated patients with refractory-relapsed non-Hodgkin lymphoma was observed with the novel anthracenedi-one pixantrone. We characterized whether pixantrone and mitoxantrone caused different effects on doxorubicin levels, redox activation, and Doxorubicinol formation. Pixantrone and mitoxantrone were probed in a validated ex vivo human myocardial strip model that was either doxorubicin-naïve o
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the novel anthracenedione pixantrone lacks redox activity and inhibits Doxorubicinol formation in human myocardium insight to explain the cardiac safety of pixantrone in doxorubicin treated patients
Journal of Pharmacology and Experimental Therapeutics, 2013Co-Authors: Emanuela Salvatorelli, Pierantonio Menna, Odalys Gonzalez Paz, Massimo Chello, Elvio Covino, Jack W Singer, Giorgio MinottiAbstract:Cardiotoxicity from the antitumor anthracycline, doxorubicin, correlates with doxorubicin cardiac levels, redox activation to superoxide anion (O2._) and hydrogen peroxide (H2O2), formation of the long-lived secondary alcohol metabolite, Doxorubicinol. Cardiotoxicity may first manifest during salvage therapy with other drugs, such as the anthracenedione mitoxantrone. Minimal evidence for cardiotoxicity in anthracycline-pretreated patients with refractory-relapsed non Hodgkin’s lymphoma was observed with the novel anthracenedione, pixantrone. We characterized whether pixantrone and mitoxantrone caused different effects on doxorubicin levels, redox activation, Doxorubicinol formation. Pixantrone and mitoxantrone were probed in a validated ex vivo human myocardial strip model that was either doxorubicin-naive or preliminarily subjected to doxorubicin loading and washouts to mimic doxorubicin treatment and elimination in the clinical setting. In doxorubicin-naive strips, pixantrone showed higher uptake than mitoxantrone; however, neither drug formed O2._ or H2O2. In doxorubicin-pretreated strips, pixantrone or mitoxantrone did not alter the distribution and clearance of residual doxorubicin. Mitoxantrone showed an unchanged uptake, lacked effects on doxorubicin levels, but synergized with doxorubicin to form more O2._ and H2O2, as evidenced by O2._ -dependent inactivation of mitochondrial aconitase or mitoxantrone oxidation by H2O2 -activated peroxidases. In contrast, pixantrone uptake was reduced by prior doxorubicin exposure; moreover, pixantrone lacked redox synergism with doxorubicin, and formed an N-dealkylated product that inhibited metabolism of residual doxorubicin to Doxorubicinol. Redox inactivity and inhibition of Doxorubicinol formation correlate with the cardiac safety of pixantrone in doxorubicin-pretreated patients. Redox inactivity in the face of high cardiac uptake suggests that pixantrone might be safe also in doxorubicin-naive patients.
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paclitaxel and docetaxel stimulation of Doxorubicinol formation in the human heart implications for cardiotoxicity of doxorubicin taxane chemotherapies
Journal of Pharmacology and Experimental Therapeutics, 2006Co-Authors: Emanuela Salvatorelli, Giovanni Liberi, Antonio Maria Calafiore, Pierantonio Menna, Sabrina Cascegna, Luca Gianni, Giorgio MinottiAbstract:Antitumor therapy with the anthracycline doxorubicin is limited by a dose-related cardiotoxicity that is aggravated by a concomitant administration of the taxane paclitaxel. Previous limited studies with isolated human heart cytosol showed that paclitaxel was able to stimulate an NADPH-dependent reduction of doxorubicin to its toxic secondary alcohol metabolite Doxorubicinol. Here we characterized that 0.25 to 2.5 μM paclitaxel caused allosteric effects that increased Doxorubicinol formation in human heart cytosol, whereas 5 to 10 μM paclitaxel decreased Doxorubicinol formation. The closely related taxane docetaxel caused similar effects. Basal or taxane-stimulated Doxorubicinol formation was blunted by 2,7-difluorospirofluorene-9,5′-imidazolidine-2′,4′-dione (AL1576), a specific inhibitor of aldehyde reductases. Doxorubicinol was measured also in the cytosol of human myocardial strips incubated in plasma and exposed to doxorubicin in the absence or presence of paclitaxel or docetaxel and their clinical vehicles Cremophor EL or polysorbate 80. Low concentrations of taxanes stimulated Doxorubicinol formation, whereas high concentrations decreased it. Doxorubicinol formation reached its maximum on adding plasma with 6 μM paclitaxel or docetaxel; this corresponded to the partitioning of 1.5 to 2.5 μM taxanes in the cytosol of the strips. Taxane-stimulated Doxorubicinol formation was not mediated by vehicles, nor was it caused by increased doxorubicin uptake or de novo protein synthesis; however, Doxorubicinol formation was blunted by AL1576. These results show that allosteric interactions with cytoplasmic aldehyde reductases enable paclitaxel or docetaxel to stimulate Doxorubicinol formation in human heart. This information serves metabolic insights into the risk of cardiotoxicity induced by doxorubicin-taxane therapies.
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Defective Taxane Stimulation of Epirubicinol Formation in the Human Heart: Insight into the Cardiac Tolerability of
2006Co-Authors: Epirubicin-taxane Chemotherapies, Emanuela Salvatorelli, Pierantonio Menna, Luca Gianni, Giorgio MinottiAbstract:The antitumor anthracycline doxorubicin induces a dose-re-lated cardiotoxicity that correlates with the myocardial levels of its secondary alcohol metabolite Doxorubicinol. Combining doxorubicin with taxanes such as paclitaxel or docetaxel may aggravate cardiotoxicity, presumably because the taxanes cause an allosteric-like stimulation of cytoplasmic aldehyde reductases that convert doxorubicin to Doxorubicinol in the heart. A less severe aggravation of cardiotoxicity was observed on combining taxanes with epirubicin, a closely related analog of doxorubicin; therefore, we characterized whether the cardiac tolerability of epirubicin-taxane therapies could be due to a defective taxane stimulation of the conversion of epirubicin to its secondary alcohol metabolite epirubicinol. Comparisons be-tween doxorubicin and epirubicin in isolated human heart cy
Bruce G. Charles - One of the best experts on this subject based on the ideXlab platform.
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Population pharmacokinetic modelling of doxorubicin and Doxorubicinol in children with cancer: is there a relationship with cardiac troponin profiles?
Cancer Chemotherapy and Pharmacology, 2017Co-Authors: Kuhan Kunarajah, Bruce G. Charles, Stefanie Hennig, Michael Lobb, Ross Pinkerton, Ross L. G. Norris, Andrew S MooreAbstract:Purpose Anthracyclines are a mainstay of the treatment of several childhood malignancies, but their utility is limited by dose-related cardiotoxicity. This study is aimed to explore the link between exposure of paediatric cancer patients to doxorubicin and its metabolite Doxorubicinol, and cardiac troponin I (cTnI). Methods In a prospective pilot study plasma doxorubicin, Doxorubicinol, and cTnI concentrations were measured in samples from children undergoing cancer chemotherapy. A mixed-effects population pharmacokinetic model for doxorubicin and Doxorubicinol and in combination with a turn-over model for cTnI were developed. Results Seventeen patients, aged 3.4–14.7 year, treated for a variety of cancers had 99 doxorubicin and 119 Doxorubicinol concentrations analysed from samples drawn between 0.5 and 336 h after the start of the infusion. Eleven patients had received previous doses of anthracyclines, with a median cumulative prior dose of 90 mg/m^2 (range 0–225 mg/m^2). The median administered doxorubicin dose was 30 mg/m^2 (range 25–75 mg/m^2). Doxorubicin disposition was described by a three-compartment model with first-order elimination and metabolism to Doxorubicinol. Body surface area was related to all clearance and distribution parameters and age further influenced clearance (CL, 58.7 L/h/1.8 m^2 for an average 8.4-year-old patient). Combined doxorubicin and metabolite exposure stimulated a temporary increase in cTnI in plasma, with a concentration of 11.8 µg/L required to achieve half-maximal effect. Prior cumulative anthracycline dosage received by patients was predictive of an increased cTnI baseline prior to a new doxorubicin dose. Conclusion Prior anthracycline exposure increased baseline cTnI in a dose-dependent manner, consistent with the known cumulative risk of anthracycline exposure-induced cardiotoxicity.
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Toxicokinetics of the active doxorubicin metabolite, Doxorubicinol, in sulphur-crested cockatoos (Cacatua galerita).
Research in veterinary science, 2007Co-Authors: C. M. Gilbert, Lucio J. Filippich, Ross P. Mcgeary, Bruce G. CharlesAbstract:The pharmacokinetics of Doxorubicinol, a cytotoxic metabolite of the anticancer drug, doxorubicin, were studied in four healthy sulphur-crested cockatoos (Cacatua galerita) after a 20 min intravenous infusion of 2 mg/kg. Plasma Doxorubicinol concentrations were measured by HPLC. The pharmacokinetic parameters were estimated using a non-compartmental method. The mean (±SD) peak concentration was 8341 ± 3132 μg/L at 17.5 ± 5.0 min after the start of the infusion, and Doxorubicinol concentrations declined biexponentially to 154.3 ± 34.5 μg/L, 40 min after the end of the infusion. Systemic clearance was 0.940 ± 0.473 L/h/kg, mean residence time was 0.165 ± 0.133 h, and steady-state volume of distribution was 0.123 ± 0.0526 L/kg. The terminal half-life was 0.660 ± 0.611 h. Detectible but unquantifiable concentrations of Doxorubicinol were present in the plasma ultrafiltrate of two birds during the infusion, indicating very extensive plasma protein binding. Physiological, haematological and biochemical monitoring over 3 weeks showed that Doxorubicinol at a single infused dose of 2 mg/kg caused no toxicities of major concern.
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Doxorubicin pharmacokinetics following a single-dose infusion to sulphur-crested cockatoos (Cacatua galerita).
Australian veterinary journal, 2004Co-Authors: C. M. Gilbert, Lucio J. Filippich, Bruce G. CharlesAbstract:Objective To determine the pharmacokinetics of doxorubicin in sulphur-crested cockatoos, so that its use in clinical studies in birds can be considered. Design A pharmacokinetic study of doxorubicin, following a single intravenous (IV) infusion over 20 min, was performed in four healthy sulphur-crested cockatoos (Cacatua galerita). Procedure Birds were anaesthetised and both jugular veins were cannulated, one for doxorubicin infusion and the other for blood collection. Doxorubicin hydrochloride (2 mg/kg) in normal saline was infused IV over 20 min at a constant rate. Serial blood samples were collected for 96 h after initiation of the infusion. Plasma doxorubicin concentrations were assayed using an HPLC method involving ethyl acetate extraction, reverse-phase chromatography and fluorescence detection. The limit of quantification was 20 ng/mL. Established non-parametric methods were used for the analysis of plasma doxorubicin data. Results During the infusion the mean +/- SD for the C-max of doxorubicin was 4037 +/- 2577 ng/mL. Plasma concentrations declined biexponentially immediately after the infusion was ceased. There was considerable intersubject variability in all pharmacokinetic variables. The terminal (beta-phase) half-life was 41.4 +/- 18.5 min, the systemic clearance (Cl) was 45.7 +/- 18.0 mL/min/kg, the mean residence time (MRT) was 4.8 +/- 1.4 min, and the volume of distribution at steady state (V-SS) was 238 131 mL/kg. The extrapolated area under the curve (AUC(0-infinity)) was 950 +/- 677 ng/mL.h. The reduced metabolite, Doxorubicinol, was detected in the plasma of all four parrots but could be quantified in only one bird with the profile suggesting formation rate-limited pharmacokinetics of Doxorubicinol. Conclusions and clinical relevance Doxorubicin infusion in sulphur-crested cockatoos produced mild, transient inappetence. The volume of distribution per kilogram and terminal half-life were considerably smaller, but the clearance per kilogram was similar to or larger than reported in the dog, rat and humans. Traces of Doxorubicinol, a metabolite of doxorubicin, were detected in the plasma.
Gary F. Merrill - One of the best experts on this subject based on the ideXlab platform.
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carbonyl reductase 1 plays a significant role in converting doxorubicin to cardiotoxic Doxorubicinol in mouse liver but the majority of the Doxorubicinol forming activity remains unidentified
Drug Metabolism and Disposition, 2020Co-Authors: Daniel H Breysse, Ryan M Boone, Cameron M Long, Miranda Merrill, Christopher M Schaupp, Edward E Schmidt, Collin C White, Terrance J Kavanagh, Gary F. MerrillAbstract:Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, Doxorubicinol. Identification of the enzymes responsible for Doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to Doxorubicinol formation were evaluated: carbonyl reductase 1 (Cbr1), carbonyl reductase 3 (Cbr3), and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed Doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas Doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to Doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total Doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to Doxorubicinol formation in mouse liver, but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that Doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the Doxorubicinol-forming activity in mouse liver cytosol but that the majority of the Doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT Earlier genetic and drug inhibition results suggested Cbr1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic Doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the Doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes – Cbr3 and Tr1 – play no role, and that the majority of the activity remains unidentified. The results suggest that targeting Cbr1 is necessary but not sufficient to eliminate Doxorubicinol-associated cardiotoxicity; identification and targeting of the additional Doxorubicinol-forming activity is an important next challenge.
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carbonyl reductase 1 plays a significant role in converting doxorubicin to cardiotoxic Doxorubicinol in mouse liver but the majority of the Doxorubicinol forming activity remains unidentified
Drug Metabolism and Disposition, 2020Co-Authors: Daniel H Breysse, Ryan M Boone, Cameron M Long, Miranda Merrill, Christopher M Schaupp, Edward E Schmidt, Terrance J Kavanagh, Charles L White, Gary F. MerrillAbstract:Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, Doxorubicinol. Identification of the enzymes responsible for Doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to Doxorubicinol formation were evaluated: carbonyl reductase (Cbr) 1, Cbr3, and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed Doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas Doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to Doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total Doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to Doxorubicinol formation in mouse liver but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that Doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the Doxorubicinol-forming activity in mouse liver cytosol but that the majority of the Doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT: Earlier studies suggested carbonyl reductase (Cbr) 1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic Doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the Doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes-Cbr3 and thioredoxin reductase 1-play no role, and that the majority of the activity remains unidentified. Thus, targeting Cbr1 is necessary but not sufficient to eliminate Doxorubicinol-associated cardiotoxicity; identification of the additional Doxorubicinol-forming activity is an important next challenge.
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metabolism of doxorubicin to the cardiotoxic metabolite Doxorubicinol is increased in a mouse model of chronic glutathione deficiency a potential role for carbonyl reductase 3
Chemico-Biological Interactions, 2015Co-Authors: Christopher M Schaupp, Gary F. Merrill, Collin C White, Terrance J KavanaghAbstract:Abstract Doxorubicin is highly effective at inducing DNA double-strand breaks in rapidly dividing cells, which has led to it being a widely used cancer chemotherapeutic. However, clinical administration of doxorubicin is limited by off-target cardiotoxicity, which is thought to be mediated by Doxorubicinol, the primary alcohol metabolite of doxorubicin. Carbonyl reductase 1 (CBR1), a well-characterized monomeric enzyme present at high basal levels in the liver, is known to exhibit activity toward doxorubicin. Little is known about a closely related enzyme, carbonyl reductase 3 (CBR3), which is present in the liver at low basal levels but is highly inducible by the transcription factor Nrf2. Genetic polymorphisms in CBR3, but not CBR1, are associated with differential cardiac outcomes in doxorubicin treated pediatric patients. Cbr3 mRNA and CBR3 protein are highly expressed in the livers of Gclm−/− mice (a mouse model of glutathione deficiency) relative to wild type mice. In the present study, we first investigated the ability of CBR3 to metabolize doxorubicin. Incubations of doxorubicin and purified recombinant murine CBR3 (mCBR3) were analyzed for Doxorubicinol formation using HPLC, revealing for the first time that doxorubicin is a substrate of mCBR3. Moreover, hepatocytes from Gclm−/− mice produced more Doxorubicinol than Gclm+/+ hepatocytes. In addition, differentiated rat myoblasts (C2C12 cells) co-cultured with primary Gclm−/− murine hepatocytes were more sensitive to doxorubicin-induced cytostasis/cytotoxicity than incubations with Gclm+/+ hepatocytes. Our results indicate a potentially important role for CBR3 in doxorubicin-induced cardiotoxicity. Because there is likely to be variability in hepatic CBR3 activity in humans (due to either genetic or epigenetic influences on its expression), these data also suggest that inhibition of CBR3 may provide protection from Doxorubicinol cardiotoxicity.
Terrance J Kavanagh - One of the best experts on this subject based on the ideXlab platform.
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carbonyl reductase 1 plays a significant role in converting doxorubicin to cardiotoxic Doxorubicinol in mouse liver but the majority of the Doxorubicinol forming activity remains unidentified
Drug Metabolism and Disposition, 2020Co-Authors: Daniel H Breysse, Ryan M Boone, Cameron M Long, Miranda Merrill, Christopher M Schaupp, Edward E Schmidt, Collin C White, Terrance J Kavanagh, Gary F. MerrillAbstract:Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, Doxorubicinol. Identification of the enzymes responsible for Doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to Doxorubicinol formation were evaluated: carbonyl reductase 1 (Cbr1), carbonyl reductase 3 (Cbr3), and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed Doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas Doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to Doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total Doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to Doxorubicinol formation in mouse liver, but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that Doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the Doxorubicinol-forming activity in mouse liver cytosol but that the majority of the Doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT Earlier genetic and drug inhibition results suggested Cbr1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic Doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the Doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes – Cbr3 and Tr1 – play no role, and that the majority of the activity remains unidentified. The results suggest that targeting Cbr1 is necessary but not sufficient to eliminate Doxorubicinol-associated cardiotoxicity; identification and targeting of the additional Doxorubicinol-forming activity is an important next challenge.
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carbonyl reductase 1 plays a significant role in converting doxorubicin to cardiotoxic Doxorubicinol in mouse liver but the majority of the Doxorubicinol forming activity remains unidentified
Drug Metabolism and Disposition, 2020Co-Authors: Daniel H Breysse, Ryan M Boone, Cameron M Long, Miranda Merrill, Christopher M Schaupp, Edward E Schmidt, Terrance J Kavanagh, Charles L White, Gary F. MerrillAbstract:Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, Doxorubicinol. Identification of the enzymes responsible for Doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to Doxorubicinol formation were evaluated: carbonyl reductase (Cbr) 1, Cbr3, and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed Doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas Doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to Doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total Doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to Doxorubicinol formation in mouse liver but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that Doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the Doxorubicinol-forming activity in mouse liver cytosol but that the majority of the Doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT: Earlier studies suggested carbonyl reductase (Cbr) 1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic Doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the Doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes-Cbr3 and thioredoxin reductase 1-play no role, and that the majority of the activity remains unidentified. Thus, targeting Cbr1 is necessary but not sufficient to eliminate Doxorubicinol-associated cardiotoxicity; identification of the additional Doxorubicinol-forming activity is an important next challenge.
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metabolism of doxorubicin to the cardiotoxic metabolite Doxorubicinol is increased in a mouse model of chronic glutathione deficiency a potential role for carbonyl reductase 3
Chemico-Biological Interactions, 2015Co-Authors: Christopher M Schaupp, Gary F. Merrill, Collin C White, Terrance J KavanaghAbstract:Abstract Doxorubicin is highly effective at inducing DNA double-strand breaks in rapidly dividing cells, which has led to it being a widely used cancer chemotherapeutic. However, clinical administration of doxorubicin is limited by off-target cardiotoxicity, which is thought to be mediated by Doxorubicinol, the primary alcohol metabolite of doxorubicin. Carbonyl reductase 1 (CBR1), a well-characterized monomeric enzyme present at high basal levels in the liver, is known to exhibit activity toward doxorubicin. Little is known about a closely related enzyme, carbonyl reductase 3 (CBR3), which is present in the liver at low basal levels but is highly inducible by the transcription factor Nrf2. Genetic polymorphisms in CBR3, but not CBR1, are associated with differential cardiac outcomes in doxorubicin treated pediatric patients. Cbr3 mRNA and CBR3 protein are highly expressed in the livers of Gclm−/− mice (a mouse model of glutathione deficiency) relative to wild type mice. In the present study, we first investigated the ability of CBR3 to metabolize doxorubicin. Incubations of doxorubicin and purified recombinant murine CBR3 (mCBR3) were analyzed for Doxorubicinol formation using HPLC, revealing for the first time that doxorubicin is a substrate of mCBR3. Moreover, hepatocytes from Gclm−/− mice produced more Doxorubicinol than Gclm+/+ hepatocytes. In addition, differentiated rat myoblasts (C2C12 cells) co-cultured with primary Gclm−/− murine hepatocytes were more sensitive to doxorubicin-induced cytostasis/cytotoxicity than incubations with Gclm+/+ hepatocytes. Our results indicate a potentially important role for CBR3 in doxorubicin-induced cardiotoxicity. Because there is likely to be variability in hepatic CBR3 activity in humans (due to either genetic or epigenetic influences on its expression), these data also suggest that inhibition of CBR3 may provide protection from Doxorubicinol cardiotoxicity.
Christopher M Schaupp - One of the best experts on this subject based on the ideXlab platform.
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carbonyl reductase 1 plays a significant role in converting doxorubicin to cardiotoxic Doxorubicinol in mouse liver but the majority of the Doxorubicinol forming activity remains unidentified
Drug Metabolism and Disposition, 2020Co-Authors: Daniel H Breysse, Ryan M Boone, Cameron M Long, Miranda Merrill, Christopher M Schaupp, Edward E Schmidt, Collin C White, Terrance J Kavanagh, Gary F. MerrillAbstract:Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, Doxorubicinol. Identification of the enzymes responsible for Doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to Doxorubicinol formation were evaluated: carbonyl reductase 1 (Cbr1), carbonyl reductase 3 (Cbr3), and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed Doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas Doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to Doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total Doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to Doxorubicinol formation in mouse liver, but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that Doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the Doxorubicinol-forming activity in mouse liver cytosol but that the majority of the Doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT Earlier genetic and drug inhibition results suggested Cbr1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic Doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the Doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes – Cbr3 and Tr1 – play no role, and that the majority of the activity remains unidentified. The results suggest that targeting Cbr1 is necessary but not sufficient to eliminate Doxorubicinol-associated cardiotoxicity; identification and targeting of the additional Doxorubicinol-forming activity is an important next challenge.
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carbonyl reductase 1 plays a significant role in converting doxorubicin to cardiotoxic Doxorubicinol in mouse liver but the majority of the Doxorubicinol forming activity remains unidentified
Drug Metabolism and Disposition, 2020Co-Authors: Daniel H Breysse, Ryan M Boone, Cameron M Long, Miranda Merrill, Christopher M Schaupp, Edward E Schmidt, Terrance J Kavanagh, Charles L White, Gary F. MerrillAbstract:Doxorubicin is a widely used cancer therapeutic, but its effectiveness is limited by cardiotoxic side effects. Evidence suggests cardiotoxicity is due not to doxorubicin, but rather its metabolite, Doxorubicinol. Identification of the enzymes responsible for Doxorubicinol formation is important in developing strategies to prevent cardiotoxicity. In this study, the contributions of three murine candidate enzymes to Doxorubicinol formation were evaluated: carbonyl reductase (Cbr) 1, Cbr3, and thioredoxin reductase 1 (Tr1). Analyses with purified proteins revealed that all three enzymes catalyzed doxorubicin-dependent NADPH oxidation, but only Cbr1 and Cbr3 catalyzed Doxorubicinol formation. Doxorubicin-dependent NADPH oxidation by Tr1 was likely due to redox cycling. Subcellular fractionation results showed that doxorubicin-dependent redox cycling activity was primarily microsomal, whereas Doxorubicinol-forming activity was exclusively cytosolic, as were all three enzymes. An immunoclearing approach was used to assess the contributions of the three enzymes to Doxorubicinol formation in the complex milieu of the cytosol. Immunoclearing Cbr1 eliminated 25% of the total Doxorubicinol-forming activity in cytosol, but immunoclearing Cbr3 had no effect, even in Tr1 null livers that overexpressed Cbr3. The immunoclearing results constituted strong evidence that Cbr1 contributed to Doxorubicinol formation in mouse liver but that enzymes other than Cbr1 also played a role, a conclusion supported by ammonium sulfate fractionation results, which showed that Doxorubicinol-forming activity was found in fractions that contained little Cbr1. In conclusion, the results show that Cbr1 accounts for 25% of the Doxorubicinol-forming activity in mouse liver cytosol but that the majority of the Doxorubicinol-forming activity remains unidentified. SIGNIFICANCE STATEMENT: Earlier studies suggested carbonyl reductase (Cbr) 1 plays a dominant role in converting chemotherapeutic doxorubicin to cardiotoxic Doxorubicinol, but a new immunoclearing approach described herein shows that Cbr1 accounts for only 25% of the Doxorubicinol-forming activity in mouse liver cytosol, that two other candidate enzymes-Cbr3 and thioredoxin reductase 1-play no role, and that the majority of the activity remains unidentified. Thus, targeting Cbr1 is necessary but not sufficient to eliminate Doxorubicinol-associated cardiotoxicity; identification of the additional Doxorubicinol-forming activity is an important next challenge.
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metabolism of doxorubicin to the cardiotoxic metabolite Doxorubicinol is increased in a mouse model of chronic glutathione deficiency a potential role for carbonyl reductase 3
Chemico-Biological Interactions, 2015Co-Authors: Christopher M Schaupp, Gary F. Merrill, Collin C White, Terrance J KavanaghAbstract:Abstract Doxorubicin is highly effective at inducing DNA double-strand breaks in rapidly dividing cells, which has led to it being a widely used cancer chemotherapeutic. However, clinical administration of doxorubicin is limited by off-target cardiotoxicity, which is thought to be mediated by Doxorubicinol, the primary alcohol metabolite of doxorubicin. Carbonyl reductase 1 (CBR1), a well-characterized monomeric enzyme present at high basal levels in the liver, is known to exhibit activity toward doxorubicin. Little is known about a closely related enzyme, carbonyl reductase 3 (CBR3), which is present in the liver at low basal levels but is highly inducible by the transcription factor Nrf2. Genetic polymorphisms in CBR3, but not CBR1, are associated with differential cardiac outcomes in doxorubicin treated pediatric patients. Cbr3 mRNA and CBR3 protein are highly expressed in the livers of Gclm−/− mice (a mouse model of glutathione deficiency) relative to wild type mice. In the present study, we first investigated the ability of CBR3 to metabolize doxorubicin. Incubations of doxorubicin and purified recombinant murine CBR3 (mCBR3) were analyzed for Doxorubicinol formation using HPLC, revealing for the first time that doxorubicin is a substrate of mCBR3. Moreover, hepatocytes from Gclm−/− mice produced more Doxorubicinol than Gclm+/+ hepatocytes. In addition, differentiated rat myoblasts (C2C12 cells) co-cultured with primary Gclm−/− murine hepatocytes were more sensitive to doxorubicin-induced cytostasis/cytotoxicity than incubations with Gclm+/+ hepatocytes. Our results indicate a potentially important role for CBR3 in doxorubicin-induced cardiotoxicity. Because there is likely to be variability in hepatic CBR3 activity in humans (due to either genetic or epigenetic influences on its expression), these data also suggest that inhibition of CBR3 may provide protection from Doxorubicinol cardiotoxicity.