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Protection of theanine against doxorubicin-induced acute cardiac toxicity
Biomedicine & Preventive Nutrition 3 (2013) 197–199
Available online at
www.sciencedirect.com
Short communication
Protection of theanine against doxorubicin-induced acute cardiac toxicity Katsuhito Nagai ∗ , Hiroki Konishi Laboratory of Clinical Pharmacy and Therapeutics, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi 584-0066, Japan
a r t i c l e
i n f o
Article history: Received 6 March 2013 Accepted 11 March 2013 Keywords: Anthracycline Theanine Biochemical modulation
a b s t r a c t Doxorubicin (DOX) is a highly potent anti-neoplastic agent, which is widely used in clinical practice; however, its dosage and duration of administration are strictly limited due to dose-related cardiac damage. The present study investigated whether theanine, an amino acid found in green tea leaves, can reduce DOX-induced acute cardiotoxicity by examining changes in serum enzyme activities. The activities of creatine kinase (CK) and lactate dehydrogenase (LDH), biochemical markers of cardiac impairment, in serum were elevated by the administration of 20 mg/kg DOX to mice. Elevations in serum enzyme activities were attenuated by concurrent treatment with theanine, and significant suppression was observed at a dose of 10 mg/kg, which is comparable to daily intake in the form of supplement. Especially, CK activity was recovered by theanine almost to control levels. Body weight loss caused by DOX was significantly suppressed by theanine treatment. These results suggest that theanine can protect against DOX-induced acute cardiac injury. © 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction Anthracycline derivatives, such as doxorubicin (DOX), are some of the most important topoisomerase II-targeting drugs that are widely used for the treatment of a variety of human malignancies including acute leukemia, lymphomas, stomach, breast, and ovarian cancer, Kaposi’s sarcomas, and bone tumors [1]. However, these drugs lead to dose-dependent cardiac myopathy etc., which restricts their clinical usefulness [2]. The proposed principal mechanism of anthracycline cardiotoxicity is an increase in oxidative stress, as evident from increased levels of reactive oxygen species and lipid peroxidation [3]. Because of recent developments in modified or new dosing regimens with anthracycline, the avoidance of severe adverse events has become a critical issue in clinical practice. In this regard, treatment with antioxidants and natural phenolic compounds has been found to be a rational approach to protect against DOX-induced cardiotoxicity [4]. Theanine, a glutamate derivative, is known to contribute to the favorable umami taste of tea and exerts various pharmacologically beneficial effects [5,6]. Theanine promotes relaxation, as seen by the induction of alpha waves on electroencephalograms. In animal studies, theanine decreased blood pressure and improved learning ability. Furthermore, it has been shown that the antitumor efficacy of DOX is enhanced by theanine administration to tumor-bearing mice at a dose of 10 mg/kg. There has been growing interest in theanine as an ingredient for novel functional foods and as a dietary
∗ Corresponding author. Tel.: +81 721 24 9184; fax: +81 721 24 9164. E-mail address: [email protected] (K. Nagai). 2210-5239/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2013.03.011
supplement. Recent reports demonstrated that theanine attenuated -amyloid-induced cognitive dysfunction and alcoholic liver injury by reducing oxidative damage [7,8]. Based on these findings, we hypothesized that DOX-induced cardiotoxicity may be improved by concurrent treatment with theanine. In the present study, we examined the effect of theanine on serum enzyme activities that reflect acute cardiac injury after the administration of DOX to mice. 2. Materials and methods 2.1. Chemicals DOX hydrochloride was obtained from Meiji Seika Kaisha (Tokyo, Japan). Theanine was purchased from Wako Pure Chemical Ind. (Osaka, Japan). DOX hydrochloride and theanine were dissolved in physiological saline and used for injections to mice. All other reagents were of commercial or analytical grade requiring no further purification. 2.2. Animal treatment Male BDF1 mice aged 5 weeks were obtained from Japan SLC, Inc. (Hamamatsu, Japan). Mice were acclimatized for at least 2 days before assignment to their experimental groups, and were housed in a clean room maintained at 23 ± 2 ◦ C with a relative humidity of 55 ± 10% and 12-h light/dark cycle. They were allowed free access to regular animal diet and tap water. Mice were divided into four treatment groups, which were designated as DOX, theanine, DOX plus theanine, and control groups. In the case of the DOX group,
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K. Nagai, H. Konishi / Biomedicine & Preventive Nutrition 3 (2013) 197–199
Fig. 1. Effect of doxorubicin (DOX) on serum enzyme activities. Results are shown as the means ± SD of four mice per group. *: significantly different from the mean value without the injection of DOX (P < 0.05, Dunnett’s test).
the indicated doses of DOX (10–20 mg/kg, i.p.) were injected on the 1st day, and physiological saline (i.p.) was administrated once a day from the 2nd day to the 4th day. In the theanine group, theanine (10 mg/kg, i.p.) was injected once a day for four consecutive days (from the 1st day to the 4th day). In the DOX plus theanine group, a dose of 20 mg/kg DOX was injected on the 1st day, and the indicated doses of theanine (2.5–10 mg/kg, i.p.) were administrated once a day from the 1st day to the 4th day. Control mice received physiological saline only at the same time. The volume of vehicle was fixed at 0.025 mL/g. The body weight of mice was measured every day throughout the experiment period. Experimental protocols and animal care methods in the experiment were approved by the Animal Experiment Committee at Osaka Ohtani University.
3. Results
2.3. Biochemical determination
3.2. Effect of doxorubicin on serum enzyme activities
Blood was collected by cardiac puncture under anesthesia on the 5th day, and the serum fraction was separated by centrifugation. The activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were measured by enzyme-based colorimetric methods using commercial reagent kits run on a biochemistry analyzer (SpotochemTM EZsp-4430 analyzer; ARKRAY Co., Kyoto, Japan).
The toxic effect of DOX on the heart was assessed by examining changes in the activities of CK and LDH in serum. The activities of CK and LDH were significantly increased by the administration of 20 mg/kg DOX, but not by 10 mg/kg DOX (Fig. 1).
2.4. Statistical analysis Data were expressed as means ± S.D. Statistical comparisons among groups were made by means of analysis of variance (ANOVA) followed by Dunnett’s test or Tukey’s test. Differences with a P value of 0.05 or less were considered significant.
3.1. Changes in the body weight of mice treated with doxorubicin and theanine There were no significant differences in the body weights of mice at the time of assignment to each experimental group (data not shown). The rates of changes in the body weight of mice were determined by dividing the weight on the 5th day by that on the 1st day. The rates of changes in the control, DOX, and DOX plus theanine groups were 1.026 ± 0.034, 0.861 ± 0.017 and 1.043 ± 0.004, respectively, which showed that the body weight of mice treated with DOX alone was significantly lower than that on the 1st day. However, there was no decrease in the body weight of the DOX plus theanine group.
3.3. Effect of theanine on doxorubicin-induced changes in serum enzyme activities We evaluated the effect of theanine on the elevated serum enzyme activities observed after DOX treatment. The administration of theanine alone to mice had no effect on enzyme activities (Fig. 2). Changes in serum enzyme activities by DOX were significantly suppressed by the treatment with 10 mg/kg theanine, and CK activity recovered to almost the same level as that of the control group (Fig. 2). Next, the dose-dependence of theanine on
Fig. 2. Effect of theanine on doxorubicin (DOX)-induced cardiac toxicity. Results are shown as the means ± SD of four mice per group. *: significantly different from the mean value without the injection of DOX (P < 0.05, Tukey’s test). : significantly different from the mean value with the injection of DOX (P < 0.05, Tukey’s test).
K. Nagai, H. Konishi / Biomedicine & Preventive Nutrition 3 (2013) 197–199
protection against DOX-induced cardiac toxicity was examined. Elevated enzyme activities showed a tendency toward reduction even when given at doses of 2.5 mg/kg and 5 mg/kg, although their improvements did not reach statistically significant level (Fig. 2). 4. Discussion Theanine has been reported to act as an antioxidant [7,8], and so it is likely that theanine can improve the severity of cardiac injury caused by DOX. In this study, we examined the effect of theanine on DOX-induced acute cardiotoxicity in mice. This is the first report to demonstrate success in preventing DOX-induced acute cardiac damage by theanine treatment. As DOX has been reported to induce cardiac toxicity [2], we constructed a cardiotoxic model in mice by treating them with DOX. DOX significantly increased serum levels of CK and LDH in a dosedependent manner, confirming the successful preparation of model mice with cardiac impairments due to DOX. Elevations in serum enzyme activities by DOX were significantly attenuated by treatment with 10 mg/kg theanine. The absence of loss in body weight was also indicative of a reduction in the direct systemic damage induced by DOX. These findings strongly suggested that concomitant use of theanine was capable of protecting against DOX-induced cardiac toxicity. On the other hand, the administration of 2.5 mg/kg and 5 mg/kg theanine did not cause significant improvements in any enzyme levels, which shows the dose-dependent cardioprotective ability of theanine. Theanine is widely available as a dietary supplement without a prescription, aside from its intake in food and beverages such as green tea. The proposed daily requirement of theanine in currently marketed productions is approximately 600 mg, which is comparable to the dose used in the present study. Theanine is rapidly absorbed from the gastrointestinal tract, and this absorption behavior ensures sufficient systemic exposure of theanine after oral administration [9]. Therefore, considering the lack of significant adverse effects of theanine, it is applicable to routine medication to reduce DOX-induced cardiotoxicity. Biochemical modulation represents either an enhancement in pharmacological properties or reduction in the toxicity of an antitumor agent by another agent, and has been playing an increasingly important role in cancer chemotherapy [10]. The antitumor efficacy of DOX in tumor-bearing mice was enhanced by theanine, which exhibits no anticarcinogenesis activity [5]. In the present study,
199
theanine caused marked recovery from DOX-induced cardiac toxicity, which is the main dose-limiting factor for the clinical use of anthracyclines [2]. Accordingly, theanine can be regarded as a useful modulator when used in combination with DOX, and is greatly supportive of the expanded clinical application of DOX. In conclusion, the present study demonstrated that theanine protected against DOX-induced acute cardiac toxicity. Our findings will be helpful in promoting the appropriate use of anthracyclines for cancer chemotherapy. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement We appreciate Toshie Araki in our laboratory for her assistance in this study. References [1] Blum RH, Carter SK. Adriamycin. A new anticancer drug with significant clinical activity. Ann Intern Med 1974;80:249–59. [2] Zhang Y, Shi J, Li Y, Wei L. Cardiomyocyte death in doxorubicin-induced cardiotoxicity. Arch Immunol Ther Exp (Warsz) 2009;57:435–45. [3] Chatterjee K, Zhang J, Honbo N, Karliner JS. Doxorubicin cardiomyopathy. Cardiology 2010;115:155–62. [4] Bast A, Kaiserova H, den Hartog GI, Haenen GR, van der Vijgh WJ. Protectors against doxorubicin-induced cardiotoxicity: flavonoids. Cell Biol Toxicol 2007;23:39–47. [5] Sadzuka Y, Sugiyama T, Hirota S. The effects of theanine, as a novel biochemical modulator, on the antitumor activity of adriamycin. Cancer Lett 1996;105:203–9. [6] Vuong QV, Stathopoulos CE, Golding JB, Nguyen MH, Roach PD. Optimum conditions for the water extraction of L-theanine from green tea. J Sep Sci 2011;34:2468–74. [7] Kim TI, Lee YK, Park SG, Choi IS, Ban JO, Park HK, et al. L-theanine, an amino acid in green tea, attenuates beta-amyloid-induced cognitive dysfunction and neurotoxicity: reduction in oxidative damage and inactivation of ERK/p38 kinase and NF-kappaB pathways. Free Rad Biol Med 2009;47: 1601–10. [8] Li G, Ye Y, Kang J, Yao X, Zhang Y, Jiang W, et al. L-Theanine prevents alcoholic liver injury through enhancing the antioxidant capability of hepatocytes. Food Chem Toxicol 2012;50:363–72. [9] Desai MJ, Gill MS, Hsu WH, Armstrong DW. Pharmacokinetic of theanine enantiomers in rats. Chirality 2005;17:154–62. [10] O’Connel MJ. A phase III trial of 5-fluorouracil and leucovolin in the treatment of advanced colorectal cancer. Cancer 1998;63:1026–30.
Available online at
www.sciencedirect.com
Short communication
Protection of theanine against doxorubicin-induced acute cardiac toxicity Katsuhito Nagai ∗ , Hiroki Konishi Laboratory of Clinical Pharmacy and Therapeutics, Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi 584-0066, Japan
a r t i c l e
i n f o
Article history: Received 6 March 2013 Accepted 11 March 2013 Keywords: Anthracycline Theanine Biochemical modulation
a b s t r a c t Doxorubicin (DOX) is a highly potent anti-neoplastic agent, which is widely used in clinical practice; however, its dosage and duration of administration are strictly limited due to dose-related cardiac damage. The present study investigated whether theanine, an amino acid found in green tea leaves, can reduce DOX-induced acute cardiotoxicity by examining changes in serum enzyme activities. The activities of creatine kinase (CK) and lactate dehydrogenase (LDH), biochemical markers of cardiac impairment, in serum were elevated by the administration of 20 mg/kg DOX to mice. Elevations in serum enzyme activities were attenuated by concurrent treatment with theanine, and significant suppression was observed at a dose of 10 mg/kg, which is comparable to daily intake in the form of supplement. Especially, CK activity was recovered by theanine almost to control levels. Body weight loss caused by DOX was significantly suppressed by theanine treatment. These results suggest that theanine can protect against DOX-induced acute cardiac injury. © 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction Anthracycline derivatives, such as doxorubicin (DOX), are some of the most important topoisomerase II-targeting drugs that are widely used for the treatment of a variety of human malignancies including acute leukemia, lymphomas, stomach, breast, and ovarian cancer, Kaposi’s sarcomas, and bone tumors [1]. However, these drugs lead to dose-dependent cardiac myopathy etc., which restricts their clinical usefulness [2]. The proposed principal mechanism of anthracycline cardiotoxicity is an increase in oxidative stress, as evident from increased levels of reactive oxygen species and lipid peroxidation [3]. Because of recent developments in modified or new dosing regimens with anthracycline, the avoidance of severe adverse events has become a critical issue in clinical practice. In this regard, treatment with antioxidants and natural phenolic compounds has been found to be a rational approach to protect against DOX-induced cardiotoxicity [4]. Theanine, a glutamate derivative, is known to contribute to the favorable umami taste of tea and exerts various pharmacologically beneficial effects [5,6]. Theanine promotes relaxation, as seen by the induction of alpha waves on electroencephalograms. In animal studies, theanine decreased blood pressure and improved learning ability. Furthermore, it has been shown that the antitumor efficacy of DOX is enhanced by theanine administration to tumor-bearing mice at a dose of 10 mg/kg. There has been growing interest in theanine as an ingredient for novel functional foods and as a dietary
∗ Corresponding author. Tel.: +81 721 24 9184; fax: +81 721 24 9164. E-mail address: [email protected] (K. Nagai). 2210-5239/$ – see front matter © 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.bionut.2013.03.011
supplement. Recent reports demonstrated that theanine attenuated -amyloid-induced cognitive dysfunction and alcoholic liver injury by reducing oxidative damage [7,8]. Based on these findings, we hypothesized that DOX-induced cardiotoxicity may be improved by concurrent treatment with theanine. In the present study, we examined the effect of theanine on serum enzyme activities that reflect acute cardiac injury after the administration of DOX to mice. 2. Materials and methods 2.1. Chemicals DOX hydrochloride was obtained from Meiji Seika Kaisha (Tokyo, Japan). Theanine was purchased from Wako Pure Chemical Ind. (Osaka, Japan). DOX hydrochloride and theanine were dissolved in physiological saline and used for injections to mice. All other reagents were of commercial or analytical grade requiring no further purification. 2.2. Animal treatment Male BDF1 mice aged 5 weeks were obtained from Japan SLC, Inc. (Hamamatsu, Japan). Mice were acclimatized for at least 2 days before assignment to their experimental groups, and were housed in a clean room maintained at 23 ± 2 ◦ C with a relative humidity of 55 ± 10% and 12-h light/dark cycle. They were allowed free access to regular animal diet and tap water. Mice were divided into four treatment groups, which were designated as DOX, theanine, DOX plus theanine, and control groups. In the case of the DOX group,
198
K. Nagai, H. Konishi / Biomedicine & Preventive Nutrition 3 (2013) 197–199
Fig. 1. Effect of doxorubicin (DOX) on serum enzyme activities. Results are shown as the means ± SD of four mice per group. *: significantly different from the mean value without the injection of DOX (P < 0.05, Dunnett’s test).
the indicated doses of DOX (10–20 mg/kg, i.p.) were injected on the 1st day, and physiological saline (i.p.) was administrated once a day from the 2nd day to the 4th day. In the theanine group, theanine (10 mg/kg, i.p.) was injected once a day for four consecutive days (from the 1st day to the 4th day). In the DOX plus theanine group, a dose of 20 mg/kg DOX was injected on the 1st day, and the indicated doses of theanine (2.5–10 mg/kg, i.p.) were administrated once a day from the 1st day to the 4th day. Control mice received physiological saline only at the same time. The volume of vehicle was fixed at 0.025 mL/g. The body weight of mice was measured every day throughout the experiment period. Experimental protocols and animal care methods in the experiment were approved by the Animal Experiment Committee at Osaka Ohtani University.
3. Results
2.3. Biochemical determination
3.2. Effect of doxorubicin on serum enzyme activities
Blood was collected by cardiac puncture under anesthesia on the 5th day, and the serum fraction was separated by centrifugation. The activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were measured by enzyme-based colorimetric methods using commercial reagent kits run on a biochemistry analyzer (SpotochemTM EZsp-4430 analyzer; ARKRAY Co., Kyoto, Japan).
The toxic effect of DOX on the heart was assessed by examining changes in the activities of CK and LDH in serum. The activities of CK and LDH were significantly increased by the administration of 20 mg/kg DOX, but not by 10 mg/kg DOX (Fig. 1).
2.4. Statistical analysis Data were expressed as means ± S.D. Statistical comparisons among groups were made by means of analysis of variance (ANOVA) followed by Dunnett’s test or Tukey’s test. Differences with a P value of 0.05 or less were considered significant.
3.1. Changes in the body weight of mice treated with doxorubicin and theanine There were no significant differences in the body weights of mice at the time of assignment to each experimental group (data not shown). The rates of changes in the body weight of mice were determined by dividing the weight on the 5th day by that on the 1st day. The rates of changes in the control, DOX, and DOX plus theanine groups were 1.026 ± 0.034, 0.861 ± 0.017 and 1.043 ± 0.004, respectively, which showed that the body weight of mice treated with DOX alone was significantly lower than that on the 1st day. However, there was no decrease in the body weight of the DOX plus theanine group.
3.3. Effect of theanine on doxorubicin-induced changes in serum enzyme activities We evaluated the effect of theanine on the elevated serum enzyme activities observed after DOX treatment. The administration of theanine alone to mice had no effect on enzyme activities (Fig. 2). Changes in serum enzyme activities by DOX were significantly suppressed by the treatment with 10 mg/kg theanine, and CK activity recovered to almost the same level as that of the control group (Fig. 2). Next, the dose-dependence of theanine on
Fig. 2. Effect of theanine on doxorubicin (DOX)-induced cardiac toxicity. Results are shown as the means ± SD of four mice per group. *: significantly different from the mean value without the injection of DOX (P < 0.05, Tukey’s test). : significantly different from the mean value with the injection of DOX (P < 0.05, Tukey’s test).
K. Nagai, H. Konishi / Biomedicine & Preventive Nutrition 3 (2013) 197–199
protection against DOX-induced cardiac toxicity was examined. Elevated enzyme activities showed a tendency toward reduction even when given at doses of 2.5 mg/kg and 5 mg/kg, although their improvements did not reach statistically significant level (Fig. 2). 4. Discussion Theanine has been reported to act as an antioxidant [7,8], and so it is likely that theanine can improve the severity of cardiac injury caused by DOX. In this study, we examined the effect of theanine on DOX-induced acute cardiotoxicity in mice. This is the first report to demonstrate success in preventing DOX-induced acute cardiac damage by theanine treatment. As DOX has been reported to induce cardiac toxicity [2], we constructed a cardiotoxic model in mice by treating them with DOX. DOX significantly increased serum levels of CK and LDH in a dosedependent manner, confirming the successful preparation of model mice with cardiac impairments due to DOX. Elevations in serum enzyme activities by DOX were significantly attenuated by treatment with 10 mg/kg theanine. The absence of loss in body weight was also indicative of a reduction in the direct systemic damage induced by DOX. These findings strongly suggested that concomitant use of theanine was capable of protecting against DOX-induced cardiac toxicity. On the other hand, the administration of 2.5 mg/kg and 5 mg/kg theanine did not cause significant improvements in any enzyme levels, which shows the dose-dependent cardioprotective ability of theanine. Theanine is widely available as a dietary supplement without a prescription, aside from its intake in food and beverages such as green tea. The proposed daily requirement of theanine in currently marketed productions is approximately 600 mg, which is comparable to the dose used in the present study. Theanine is rapidly absorbed from the gastrointestinal tract, and this absorption behavior ensures sufficient systemic exposure of theanine after oral administration [9]. Therefore, considering the lack of significant adverse effects of theanine, it is applicable to routine medication to reduce DOX-induced cardiotoxicity. Biochemical modulation represents either an enhancement in pharmacological properties or reduction in the toxicity of an antitumor agent by another agent, and has been playing an increasingly important role in cancer chemotherapy [10]. The antitumor efficacy of DOX in tumor-bearing mice was enhanced by theanine, which exhibits no anticarcinogenesis activity [5]. In the present study,
199
theanine caused marked recovery from DOX-induced cardiac toxicity, which is the main dose-limiting factor for the clinical use of anthracyclines [2]. Accordingly, theanine can be regarded as a useful modulator when used in combination with DOX, and is greatly supportive of the expanded clinical application of DOX. In conclusion, the present study demonstrated that theanine protected against DOX-induced acute cardiac toxicity. Our findings will be helpful in promoting the appropriate use of anthracyclines for cancer chemotherapy. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement We appreciate Toshie Araki in our laboratory for her assistance in this study. References [1] Blum RH, Carter SK. Adriamycin. A new anticancer drug with significant clinical activity. Ann Intern Med 1974;80:249–59. [2] Zhang Y, Shi J, Li Y, Wei L. Cardiomyocyte death in doxorubicin-induced cardiotoxicity. Arch Immunol Ther Exp (Warsz) 2009;57:435–45. [3] Chatterjee K, Zhang J, Honbo N, Karliner JS. Doxorubicin cardiomyopathy. Cardiology 2010;115:155–62. [4] Bast A, Kaiserova H, den Hartog GI, Haenen GR, van der Vijgh WJ. Protectors against doxorubicin-induced cardiotoxicity: flavonoids. Cell Biol Toxicol 2007;23:39–47. [5] Sadzuka Y, Sugiyama T, Hirota S. The effects of theanine, as a novel biochemical modulator, on the antitumor activity of adriamycin. Cancer Lett 1996;105:203–9. [6] Vuong QV, Stathopoulos CE, Golding JB, Nguyen MH, Roach PD. Optimum conditions for the water extraction of L-theanine from green tea. J Sep Sci 2011;34:2468–74. [7] Kim TI, Lee YK, Park SG, Choi IS, Ban JO, Park HK, et al. L-theanine, an amino acid in green tea, attenuates beta-amyloid-induced cognitive dysfunction and neurotoxicity: reduction in oxidative damage and inactivation of ERK/p38 kinase and NF-kappaB pathways. Free Rad Biol Med 2009;47: 1601–10. [8] Li G, Ye Y, Kang J, Yao X, Zhang Y, Jiang W, et al. L-Theanine prevents alcoholic liver injury through enhancing the antioxidant capability of hepatocytes. Food Chem Toxicol 2012;50:363–72. [9] Desai MJ, Gill MS, Hsu WH, Armstrong DW. Pharmacokinetic of theanine enantiomers in rats. Chirality 2005;17:154–62. [10] O’Connel MJ. A phase III trial of 5-fluorouracil and leucovolin in the treatment of advanced colorectal cancer. Cancer 1998;63:1026–30.