Comparative Study of the Free Radical Scavenging Activities of Original and Generic Edaravone Determined by Electron Spin Resonance

Oxygen free radical species may play detrimental roles in brain ischemia and edema [1-3], and onset and progression of amyotrophic lateral sclerosis (ALS) [4]. Edaravone (3-methyl-1 phenyl-2-pyrazolin5-one, MCI-186, Radicut; developed by Mitsubishi Tanabe pharma corporation, Osaka, Japan), a powerful free radical scavenger, is currently used for the treatment of cerebral infarction and ALS [58]. It was shown that Edaravone scavenged radicals such as hydroxyl radical (HO), peroxyl radicals (LO2), DPPH (1,1-diphenyl-2picrylhydrazyl) radicals, and nitric oxide (NO) directly [5-7,9]. It was demonstrated that the inhibition of lipid peroxidation by Edaravone was probably due to the scavenging of the chain carrying LO2 and that, in combination with vitamin E or C, the antioxidant activity of Edaravone may be enhanced [7]. In clinical studies, Edaravone improved the core neurological deficits, the activities of daily life, and the functional outcome of stroke patients [10,11].

Recently, over 20 generic Edaravone injections have been put on the market in Japan. The generic injections should keep the same active ingredient, however, this rule cannot be applied to the additives. The additive L-cysteine present in the original Edaravone injections is substituted with citric acid, alpha thioglycerin, or glycine in some generic injections. The additives were changed in 10 generic Edaravone injections among 21 generics that have been approved in Japan in 2011 (Table 1). L-cysteine, citric acid, alpha thioglycerin, and glycine possess free radical scavenging activities, but their antioxidant potency is different. Hence, substitution of additives has the potential of changing the antioxidant ability of the original Edaravone injections.
In the present study, we investigated the dissimilarity between the original and generic Edaravone injections by focusing on their free radical scavenging activities determined by electron spin resonance (ESR) spectroscopy.

Reagents
The original Edaravone injection and 6 generic Edaravone Abstract Edaravone, a powerful free radical scavenger, is the drug available in the clinical practice for the treatment of cerebral infarction and amyotrophic lateral sclerosis. Recently, many generic Edaravone injections have been commercialized. Substitution of additives in generic injections has the potential effect of changing the antioxidant ability of the original Edaravone injection. We investigated the dissimilarity between original and generic Edaravone injections focusing on their free radical scavenging activities determined by electron spin resonance spectroscopy. There were no significant differences between the original and generic Edaravone regarding the antioxidant abilities toward the hydroxyl radical. However, the generics in which the additive L-cysteine was substituted with glycerin or citric acid showed significant reduction in their antioxidant activity toward superoxide (p <0.01). Our in vitro findings suggest that the antioxidant ability of generic Edaravone against the hydroxyl radical is equivalent to that of the original Edaravone and that substitution of additives in the generic Edaravone might change its antioxidant activity toward the superoxide.
injections are analyzed in this study. All the generics have the same active ingredient; however, the additives are different. Two generics employ glycine (A) and citric acid (E) instead of L-cysteine, while the other 4 generics have the same additives (Table 1). Solvents and other reagents are of the highest grade commercially available.

Statistical analysis
Statistical comparisons were made using non-repeated measures analysis of variance (ANOVA). Data are expressed as mean ± standard deviation (SD). The statistical significance was set at p < 0.05.

・-
We investigated the antioxidant effect of the original and generic Edaravone on O 2 ・generated by TiO 2 photocatalysis using the ESR spin trapping technique with CYMPO. The significant reduction in the antioxidant activity toward O 2 ・in the case of 2 generic Edaravone injections compared with that of the original drug was measured (p < 0.01). There were no significant differences in the generics in which the additives were equivalent to those of the original injection (generic Edaravone B, C, D and F); however, the generics A and E in which the additive L-cysteine was substituted with glycerin or citric acid respectively showed reduction in their antioxidant activity toward O 2 ・- (Figure 1).

Dissimilarity between the original and generic Edaravone on HO ・
We investigated the antioxidant activity of the original and generic Edaravone on HO ・ generated by Fenton's reaction or ultraviolet irradiation of H 2 O 2 , using ESR spin trapping with CYMPO. There were no significant differences between the original and generic Edaravone injections (Figures 2 and 3).

Discussion
The relationship between ischemic brain injury and lipid peroxidation disorder by free radicals was first described by Flamm et al. in 1978 [2]. Increasing evidence suggests that oxygen free radical species may play detrimental roles in ischemic diseases [1,3,[17][18][19] and ALS [4] and selective free radical scavengers as brain protective agents are considered to be potential drugs against brain ischemia [20] and ALS [8]. Although the world's first clinical use of Edaravone, which has been developed as a neuroprotectant, was approved in Japan in 2001 [11], it remains under clinical investigation in various other countries [21] and its use has not been approved yet in Western countries. However, Edaravone is recommended by the American Heart Association in the guidelines for the early management of adults with acute ischemic stroke [22]. The administration of Edaravone during Alteprase infusion is likely to enhance the recanalization in patients with acute ischemic stroke [23]. The generic Edaravone injections have been approved in   ・-) generated by the TiO 2 photocatalysis reaction calculated using the relative ESR signal intensities. The data are expressed as mean ± standard deviation (SD, n = 9) calculated as 100% with the mean value of distilled water as the control. The additives were substituted from L-cysteine to glycine in A, and to citric acid in E ( Table 1). The significant differences compared to the original injection were recognized in A and E. A-F: Generic Edaravone injections which have been made in each pharmaceutical company, DW: distilled water, cont: control, *: Significantly different (p<0.01) compared to the original injection Japan in 2011 and are now produced in several countries such as Korea, India, and China.
The basic chemical structure of Edaravone (3-Methyl-1-phenyl-2-pyrazolin-5-one) was found to show promising activity as an antioxidative radical scavenger, was capable of quenching HO ・ , which among other oxidative free radicals has the strongest oxidative ability, and could inhibit both HO ・ -dependent and HO ・ -independent lipid peroxidation. Furthermore, additional free radical scavenging and antioxidative actions of Edaravone were identified in LOO ・ -induced peroxidation [7] and peroxynitrite (ONOO -)-induced tyrosine nitration [6,9]. However, this basic chemical structure of Edaravone does not show antioxidant activity for O 2 -・ , which has very low oxidative ability [6]. In our study, the ability of the generic Edaravone as a free radical scavenger for HO ・ was found to be equivalent to that of the original drug, proving the safety of the basic chemical structure of the generic Edaravone. Therefore, the generic Edaravone drugs have achieved the objective of the development of original Edaravone equally.
Whether Edaravone could scavenge O 2 ・or not is still controversial [24]. It is known that the thiol group (-SH) of L-cysteine reacts with the DPPH radical and thus scavenges free radicals [25,26]. Consistently, a significant reduction in the O 2 ・scavenging activity was found only in those generics in which the additive L-cysteine was substituted with glycerin or citric acid. Even though O 2 ・has low oxidative ability, it is the key radical that initiates other free radical chain reactions. The fact that the substitution of additives affected the scavenging activity of Edaravone for O 2 ・showed that L-cysteine plays an important role in scavenging O 2 ・in Edaravone injections. There is a report mentioned that 1mM Edaravone injection suppressed the formation of O 2 ・in the xanthine oxidase-hypoxanthine system [24]. Therefore, the generic Edaravone injections in which additive L-cysteine has been substituted with another additive have the possibility to reduce its own ability as an antioxidative radical scavenger. However, our study is an in vitro analysis, while the additives injected into the veins are immediately diluted and spread by a large amount of blood; therefore, in vivo, the radical scavenging activity of L-cysteine might disappear. Further in vivo studies are necessary to draw the conclusion that additive changes in generic Edaravone involve the antioxidant ability in clinical use.

Conclusion
Our in vitro findings suggest that the antioxidant ability of generic Edaravone against the HO ・ radical is equivalent to that of the original Edaravone and that substitution of additives in the generic Edaravone might change its antioxidant activity toward the O 2  The data are expressed as mean ± standard deviation (SD, n = 9) calculated as 100% with the mean value of distilled water as the control. There were no significant differences between the original and generic injections. A-F: Generic Edaravone injections which have been made in each pharmaceutical company, and the additives were substituted from L-cysteine to glycine in A and to citric acid in E, DW: distilled water, cont: control Figure 3: Antioxidant activity of the original and generic Edaravone injections toward the hydroxyl radical (HO ・ ) generated by the ultraviolet irradiation of H 2 O 2 , calculated using the ESR signal intensities. The data are expressed as mean ± standard deviation (SD, n = 9) calculated as 100% with the mean value of distilled water as the control. There were no significant differences between the original and generic injections. A-F: Generic Edaravone injections which have been made in each pharmaceutical company and the additives were substituted from L-cysteine to glycine in A and to citric acid in E, DW: distilled water, cont: control