Effects of Eptazocine, a Novel Analgesic, on KCN-Induced Changes in the Cerebral Contents of Glycolytic Metabolites and High-Energy Phosphates in Mice

Tomoaki TAMURA, Toshietsu TANIGUCHI, Tetsuo MIYAMOTO, Masaru AOKI and Isami WAKI

Central Research Laboratories, Nihon lyakuhin Kogyo Co., Ltd., 205-1 Shimoumezawa, Namerikawa, Toyama 936, Japan

Accepted July 10, 1990

Abstract-Effects of eptazocine on cerebral metabolic changes due to a sublethal dose of KCN were investigated in mice. KCN (2 mg/kg, i.v.) induced a temporary loss of consciousness being moderated by eptazocine (1-10 mg/kg) in a dose dependent manner. The KCN injection decreased the contents of phosphocreatine (PCr), ATP and glucose and increased the contents of AMP and lactate, resulting in a 34% decrease in energy charge potential (ECP) and an increase in lactate/

pyruvate (L/P) ratio. Such changes were improved by eptazocine (10 mg/kg) and EKC (3 mg/kg), but not by pentazocine (10 mg/kg) and morphine (3 mg/kg), and the improving effect of eptazocine was completely inhibited by MR-2266 (3 mg/kg), a relatively selective opioid ƒÈ-receptor antagonist. On the other hand, eptazocine (3, 10 mg/kg) was found to increase the glucose content in normal mice, but not to give significant changes in the contents of glycolytic metabolites and high-energy phosphates. These results suggest that eptazocine may improve anoxic changes in cerebral energy metabolism.

Eptazocine (/-1,4-dimethyl-10-hydroxy may improve anoxic changes in cerebral 2,3,4,5,6,7 hexahydro 1,6 methano -1 H 4 energy metabolism through opioid rc-recep benzazonine) has been regarded as such an tors. In the present study, effect of eptazocine opioid /l-antagonist-r-agonist, like penta on cerebral metabolic changes due to an in zocine (1-3). In our previous study (4), jection of a sublethal dose of KCN (2 mg/kg, analgesic doses of eptazocine significantly i.v.) were investigated in comparison with prolonged the survival time of mice subjected those of typical opioid compounds. to either hypobaric hypoxia or KCN-induced cytotoxic anoxia in a dose-dependent manner; Materials and Methods and the effect was inhibited by MR-2266, a Animals: Male mice of the ddY strain relatively selective opioid 'c-receptor an (Nihon SLC Co., Ltd., Shizuoka), weighing tagonist, more potently than by naloxone, 26-32 g, were used. They were housed in an suggesting that eptazocine may elicit its anti air-conditioned room at 23±1 °C and were anoxic action via its binding with opioid tc given standard diet (MF, Oriental Yeast Co., receptors. Beneficial effects of opioid tc Ltd., Tokyo) and tap water ad libitum before agonists against ischemic neurological de being used. ficits in experimental animals were reported Drugs: Eptazocine hydrobromide, ethyl (5-7). Actions of these compounds, however, ketocyclazocine methanesulfonate (EKC) and on cerebral energy metabolism, which is ex (-) -2 (3-furylmethyl) 5,9 diethyl 2' hy tremely sensitive to anoxic and ischemic droxy-6,7-benzomorphan (MR-2266) were treatments, have not been elucidated. It is of synthetized in our laboratories. Pentazocine great interest to determine whether eptazocine hydrochloride and morphine hydrochloride were purchased from Sankyo Co., Ltd. scribed by Lowry et al. (9); and G6P, FDP, (Tokyo) ; glucose-6-phosphate (G6P), ADP and AMP were determined by the UV fructose l,6-diphosphate (FDP), hexokinase, methods of Bergmeyer (10-12). The energy G6P dehydrogenase, pyruvate kinase, myo state of each tissue was expressed as the kinase, amyloglucosidase, adenine dinucleo energy charge potential (ECP) of the adenine tides and adenosine phosphates were from nucleotide pool according to Atkinson (13): Sigma Chemical Co. (St. Louis, MO, U.S.A.); ECP=[(ATP)+0.5(ADP)]/[(ATP)+(ADP)+ and lactate dehydrogenase and creatine (AMP)]. kinase were from Boehringer Mannheim Statistical analysis: Values were repre Yamanouchi Co., Ltd. (Tokyo). Other chemi sented as the mean+S.E., and one-way cals were obtained from Wako Pure Chemical analysis of variance followed by Student's t Industries, Ltd. (Osaka). MR-2266 was dis test or the Aspin-Welch t-test were carried solved in 100 mM methanesu Iforate and out. diluted with distilled water. Other test drugs were dissolved in physiological saline. Results Loss of consciousness due to a KCN in Effect of eptazocine on loss of conscious jection: Mice pretreated with test drugs were ness due to a KCN injection: An injection of injected with KCN (2 mg/10 ml/kg) at the KCN (2 mg/kg, i.v.) to saline-treated mice rate of 0.6 ml/10 sec via the tail veins, and immediately induced clonic convulsion, fol durations of loss of the righting reflex and lowed by loss of the righting reflex with a spontaneous movement were measured, re respiratory failure; and about 342 sec later, spectively. the mice had spontaneous movement again. Determination of glycolytic metabolites Effects of eptazocine (1-10 mg/kg, i.p.) on and high-energy phosphates: Mice pretreated KCN-induced loss of the righting reflex and with test drugs and/or KCN (2 mg/kg, i.v.) spontaneous movement were examined, and were sacrificed by microwave irradiation the results are shown in Table 1. Eptazocine (TMW-6402; 5 kW, 1.4 sec, Toshiba). The (10 mg/kg) shortened the duration of loss of whole brains were taken out after the heads consciousness. were cooled in ice-cold water. The brains Effects of eptazocine and opioid com were weighed, and homogenized in 3.5 ml of pounds on changes in the contents of cerebral 2 N perchloric acid. After each portion of the metabolites due to the KCN injection: The homogenate was taken for glycogen analysis preliminary experiments showed that the (8), the residuals were allowed to stand in cerebral ATP content in mice decreased to ice-cold water for 30 min and then cen 82, 55 and 74% of the normal value 45, 90 trifuged at 20,000 x g for 15 min. The superna and 180 sec after the KCN injection, respec tants were neutralized with 6 N K2CO3, cen tively. Therefore, the contents of cerebral trifuged at 1,000 x g for 10 min, and used for metabolites, ECP and L/P ratio were deter the assays. PCr, ATP, glucose, pyruvate and mined 90 sec after the KCN injection (Tables lactate were analyzed according to the 2 and 3). The cerebral contents of PCr and enzymatic fluorometric techniques as de ATP were decreased, and the AMP content

Table 1. Effect of eptazocine on KCN-induced loss of consciousness in mice 181 Fig. 1. Antagonistic effect of MR-2266 on the improving effect of eptazocine against KCN-induced cerebral metabolic changes in mice. Each column with vertical bars represents the mean+S.E. (n=6). *P<0 .05, **P<0.01, compared with each KCN alone value. #P<0.05, ##P<0.01, compared with each (KCN plus eptazocine) value. Eptazocine and MR-2266 were administered i.p. at 20 and 30 min before a KCN (2 mg/kg, i.v.) injection, respectively. Mice received microwave irradiation (5 kW, 1.4 sec) 90

was increased, resulting in a decrease in ECP. in the contents of glycogen, G6P, FDP, PCr, The cerebral anoxic treatment also showed a ATP, lactate and pyruvate. An increase in the decrease in the glucose content and increases glucose content was also shown in the case in the lactate content and L/P ratio. These of EKC (3 mg/kg) and morphine (3 mg/kg). KCN-induced metabolic changes were pre vented by a 30 min-pretreatment with epta Discussion zocine (10 mg/kg) or EKC (3 mg/kg), Cyanide inhibits the terminal oxidase whereas morphine (3 mg/kg) and penta enzyme cytochrome oxidase in the cerebral zocine (10 mg/kg) did not show such effects. tissue, resulting in disorder of the mito MR-2266 was used to determine whether chondria) energy-producing systems (14-16), such effects of eptazocine were mediated via and such a cyanide-induced acute cerebral opioid /c-receptors or not. MR-2266 of 3 mg/ anoxia in animals has been widely used for the kg, which showed no significant effect on the evaluation of cerebral protective drugs (17 cerebral energy metabolism, completely in 19). In the previous report (4), we showed hibited the effects of eptazocine (Fig. 1 ). that eptazocine as well as EKC, a typical Effects of eptazocine and opioid com opioid /c-agonist, prolonged the survival time pounds on the contents of cerebral glycolic of mice subjected to a lethal dose of KCN (3 metabolites in normal mice: We attempted to mg/kg, i.v.). In this study, a sublethal dose of determine whether the above-mentioned KCN (2 mg/kg, i.v.) was tested and KCN effects of eptazocine are shown in normal induced temporary loss of consciousness was mice. Tables 4 and 5 show that a 30-min pre shown to be overcome by the pretreatment treatment with eptazocine induced an in with eptazocine. 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