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Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention.

Nishikawa T, Miyahara E, Kurauchi K, Watanabe E, Ikawa K, Asaba K, Tanabe T, Okamoto Y, Kawano Y - PLoS ONE (2015)

Bottom Line: When treated with ISO or BIO, metabolism of CY was significantly inhibited.Pre-treatment with NAC, however, did not inhibit the metabolism of CY: compared to control samples, we observed no difference in HCY, a significant increase of CEPM, and a significant decrease of acrolein.Furthermore, NAC pre-treatment did not affect intracellular amounts of ROS produced by CYS9.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

ABSTRACT
Observed only after administration of high doses, cardiotoxicity is the dose-limiting effect of cyclophosphamide (CY). We investigated the poorly understood cardiotoxic mechanisms of high-dose CY. A rat cardiac myocardial cell line, H9c2, was exposed to CY metabolized by S9 fraction of rat liver homogenate mixed with co-factors (CYS9). Cytotoxicity was then evaluated by 3-(4,5-dimethyl-2-thiazolyl)¬2,5-diphenyl¬2H-tetrazolium bromide (MTT) assay, lactate dehydrogenase release, production of reactive oxygen species (ROS), and incidence of apoptosis. We also investigated how the myocardial cellular effects of CYS9 were modified by acrolein scavenger N-acetylcysteine (NAC), antioxidant isorhamnetin (ISO), and CYP inhibitor β-ionone (BIO). Quantifying CY and CY metabolites by means of liquid chromatography coupled with electrospray tandem mass spectrometry, we assayed culture supernatants of CYS9 with and without candidate cardioprotectant agents. Assay results for MTT showed that treatment with CY (125-500 μM) did not induce cytotoxicity. CYS9, however, exhibited myocardial cytotoxicity when CY concentration was 250 μM or more. After 250 μM of CY was metabolized in S9 mix for 2 h, the concentration of CY was 73.6 ± 8.0 μM, 4-hydroxy-cyclophosphamide (HCY) 17.6 ± 4.3, o-carboxyethyl-phosphoramide (CEPM) 26.6 ± 5.3 μM, and acrolein 26.7 ± 2.5 μM. Inhibition of CYS9-induced cytotoxicity occurred with NAC, ISO, and BIO. When treated with ISO or BIO, metabolism of CY was significantly inhibited. Pre-treatment with NAC, however, did not inhibit the metabolism of CY: compared to control samples, we observed no difference in HCY, a significant increase of CEPM, and a significant decrease of acrolein. Furthermore, NAC pre-treatment did not affect intracellular amounts of ROS produced by CYS9. Since acrolein seems to be heavily implicated in the onset of cardiotoxicity, any competitive metabolic processing of CY that reduces its transformation to acrolein is likely to be an important mechanism for preventing cardiotoxicity.

No MeSH data available.


Related in: MedlinePlus

Pharmacokinetics of high-dose cyclophosphamide in patients.We measured CY and CY metabolites in blood plasma samples from patients receiving high-dose CY and here present the concentration–time profiles obtained from all three patients for (A) CY, (B) HCY, and (C) CEPM. Underlying diseases were acute mixed lineage leukemia (male, 8 y.o.; open square) and granulocytic sarcoma (male, 17 y.o.; open circle), and acute myeloid leukemia (male, 1 y.o.; open triangle).
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pone.0131394.g003: Pharmacokinetics of high-dose cyclophosphamide in patients.We measured CY and CY metabolites in blood plasma samples from patients receiving high-dose CY and here present the concentration–time profiles obtained from all three patients for (A) CY, (B) HCY, and (C) CEPM. Underlying diseases were acute mixed lineage leukemia (male, 8 y.o.; open square) and granulocytic sarcoma (male, 17 y.o.; open circle), and acute myeloid leukemia (male, 1 y.o.; open triangle).

Mentions: To determine the concentration of CY in this experiment, we measured CY and CY metabolites in blood plasma samples from patients receiving high-dose CY. Fig 3 shows concentration–time profiles obtained from all three patients for CY, HCY, and CEPM. The underlying diseases were acute mixed lineage leukemia (male, 17 y.o.), granulocytic sarcoma (male, 8 y.o.), and acute myeloid leukemia (male, 1 y.o.). The average concentration of CY at 3 hours after administration of high-dose CY therapy was 257 ± 46 μM. Area under the curve (AUC) variability for CY was 2.0×; HCY, 3.8×; CEPM, 1.5×, and HCY/CY 4.9×. No overt cardiac failures nor abnormal ECGs were observed. All three patients underwent engrafting. One patient (acute myeloid leukemia) relapsed after HSCT and died owing to the underlying disease. The other two patients are still alive with no sequelae and have been in continuous remission.


Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention.

Nishikawa T, Miyahara E, Kurauchi K, Watanabe E, Ikawa K, Asaba K, Tanabe T, Okamoto Y, Kawano Y - PLoS ONE (2015)

Pharmacokinetics of high-dose cyclophosphamide in patients.We measured CY and CY metabolites in blood plasma samples from patients receiving high-dose CY and here present the concentration–time profiles obtained from all three patients for (A) CY, (B) HCY, and (C) CEPM. Underlying diseases were acute mixed lineage leukemia (male, 8 y.o.; open square) and granulocytic sarcoma (male, 17 y.o.; open circle), and acute myeloid leukemia (male, 1 y.o.; open triangle).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482695&req=5

pone.0131394.g003: Pharmacokinetics of high-dose cyclophosphamide in patients.We measured CY and CY metabolites in blood plasma samples from patients receiving high-dose CY and here present the concentration–time profiles obtained from all three patients for (A) CY, (B) HCY, and (C) CEPM. Underlying diseases were acute mixed lineage leukemia (male, 8 y.o.; open square) and granulocytic sarcoma (male, 17 y.o.; open circle), and acute myeloid leukemia (male, 1 y.o.; open triangle).
Mentions: To determine the concentration of CY in this experiment, we measured CY and CY metabolites in blood plasma samples from patients receiving high-dose CY. Fig 3 shows concentration–time profiles obtained from all three patients for CY, HCY, and CEPM. The underlying diseases were acute mixed lineage leukemia (male, 17 y.o.), granulocytic sarcoma (male, 8 y.o.), and acute myeloid leukemia (male, 1 y.o.). The average concentration of CY at 3 hours after administration of high-dose CY therapy was 257 ± 46 μM. Area under the curve (AUC) variability for CY was 2.0×; HCY, 3.8×; CEPM, 1.5×, and HCY/CY 4.9×. No overt cardiac failures nor abnormal ECGs were observed. All three patients underwent engrafting. One patient (acute myeloid leukemia) relapsed after HSCT and died owing to the underlying disease. The other two patients are still alive with no sequelae and have been in continuous remission.

Bottom Line: When treated with ISO or BIO, metabolism of CY was significantly inhibited.Pre-treatment with NAC, however, did not inhibit the metabolism of CY: compared to control samples, we observed no difference in HCY, a significant increase of CEPM, and a significant decrease of acrolein.Furthermore, NAC pre-treatment did not affect intracellular amounts of ROS produced by CYS9.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

ABSTRACT
Observed only after administration of high doses, cardiotoxicity is the dose-limiting effect of cyclophosphamide (CY). We investigated the poorly understood cardiotoxic mechanisms of high-dose CY. A rat cardiac myocardial cell line, H9c2, was exposed to CY metabolized by S9 fraction of rat liver homogenate mixed with co-factors (CYS9). Cytotoxicity was then evaluated by 3-(4,5-dimethyl-2-thiazolyl)¬2,5-diphenyl¬2H-tetrazolium bromide (MTT) assay, lactate dehydrogenase release, production of reactive oxygen species (ROS), and incidence of apoptosis. We also investigated how the myocardial cellular effects of CYS9 were modified by acrolein scavenger N-acetylcysteine (NAC), antioxidant isorhamnetin (ISO), and CYP inhibitor β-ionone (BIO). Quantifying CY and CY metabolites by means of liquid chromatography coupled with electrospray tandem mass spectrometry, we assayed culture supernatants of CYS9 with and without candidate cardioprotectant agents. Assay results for MTT showed that treatment with CY (125-500 μM) did not induce cytotoxicity. CYS9, however, exhibited myocardial cytotoxicity when CY concentration was 250 μM or more. After 250 μM of CY was metabolized in S9 mix for 2 h, the concentration of CY was 73.6 ± 8.0 μM, 4-hydroxy-cyclophosphamide (HCY) 17.6 ± 4.3, o-carboxyethyl-phosphoramide (CEPM) 26.6 ± 5.3 μM, and acrolein 26.7 ± 2.5 μM. Inhibition of CYS9-induced cytotoxicity occurred with NAC, ISO, and BIO. When treated with ISO or BIO, metabolism of CY was significantly inhibited. Pre-treatment with NAC, however, did not inhibit the metabolism of CY: compared to control samples, we observed no difference in HCY, a significant increase of CEPM, and a significant decrease of acrolein. Furthermore, NAC pre-treatment did not affect intracellular amounts of ROS produced by CYS9. Since acrolein seems to be heavily implicated in the onset of cardiotoxicity, any competitive metabolic processing of CY that reduces its transformation to acrolein is likely to be an important mechanism for preventing cardiotoxicity.

No MeSH data available.


Related in: MedlinePlus