Protective Effect of DM-9384, a Novel Pyrrolidone Derivative, Against Experimental Cerebral Anoxia
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In the present study, we examined the cerebral protective effect of DM-9384 using various anoxia models in comparison with those of piracetam, aniracetam, pramiracetam and bifemelane. Additionally, the effect of DM-9384 on the cerebral energy metabolism was also investigated to elucidate the mech anism(s) of the anti-anoxic effect of this drug. Protective Effect of DM-9384, a Novel Pyrrolidone Derivative, against Experimental Cerebral Anoxia Takeo SAKURAI, Satoshi HATANAKA, Satoru TANAKA, Terukiyo YAMASAKI, Hiroshi KOJIMA and Akira AKASHI Research Institute, Daiichi Pharmaceutical Co., Ltd., 16-13, Kita-Kasai 1-chome, Edogawa-ku, Tokyo 134, Japan Accepted June 1, 1990 Abstract-The protective effects of DM-9384 [N-(2,6-dimethylphenyl)-2-(2-oxo- 1-pyrrolidinyl) acetamide] against cerebral anoxia were investigated using various animal models. Oral administration of DM-9384 resulted in a significant prolonga tion of survival time in mice and rats subjected to the normobaric hypoxia; its minimal effective doses were 30 and 10 mg/kg, respectively. A significant protection by this drug against hypobaric hypoxia, histotoxic anoxia and cerebral ischemia also occurred in mice at a dose of 100 mg/kg, p.o. Bifemelane (100-300 mg/kg, p.o.) was protective against these models except for hypobaric hypoxia, and the effects of piracetam, aniracetam and pramiracetam (1000 mg/kg, p.o.) were variable de pending on the type of anoxia model used. DM-9384 (100 mg/kg and lower) at tenuated the hypolocomotion and the disturbance of cerebral energy metabolism such as a decrease in ATP, an increase in lactate and lactate/pyruvate ratio induced by hypoxia in rats. The spontaneous motor activity, uptake and utilization of brain glucose in normal animals, however, were not influenced by this drug. Based on these results, DM-9384 is characterized as a broad spectrum anti-anoxic drug with negligible CNS depression, and the cerebral protective effect of this drug may be, at least in part, attributable to its ability to improve the cerebral energy metabolic disturbance. It is well-known that a decrease in the considered to be a cognition-enhancing agent oxygen content (hypoxia) depresses brain like piracetam (8, 9), aniracetam (10) and function in experimental animals and humans. pramiracetam (11 ). The cerebral hypoxic and ischemic models are widely used in the preclinical evaluation of drugs for the treatment of cerebrovascular disorders (1-4). For example, bifemelane, which was recently developed as a new cerebroacting drug, was reported to prolong the duration of respiratory activity in normo baric hypoxic, KCN-induced anoxic and de capitation-induced cerebral ischemic animals (5). DM-9384 is a new pyrrolidone derivative. Materials and Methods It has been shown to improve electrocon Animals: Male ddY mice weighing 22-28 vulsive shock-, scopolamine and GABA g, male Wistar rats weighing 170-290 g and antagonist-induced amnesia, and to ac male Sprague-Dawley (SD) rats weighing celerate acquisition of learning both nega 310-420 g were purchased from Japan SLC, tively and positively reinforced tasks in Inc. The animals were housed in rooms con rodents (6, 7). Accordingly, DM-9384 is trolled at 23±2'C and 55±15% of relative humidity on a 12-hr light-dark cycle (7:00 time (14). 19:00). Locomotor activity in normal and hypoxic Drugs: DM-9384 (Daiichi Pharmaceutical rats: One hour after the drug administration, Co., Ltd.). Piracetam, aniracetam, pramirace locomotor activity of Wistar rats was recorded tam sulfate and bifemelane hydrochloride every 10 min for 30 min with an Animex used as reference drugs were synthesized at (Farad Electronics). Hypoxia was induced by our Research Institute. DM-9384, piracetam, lowering the concentration of oxygen in the aniracetam and pramiracetam were suspended chamber (22.5 x 30.0x 18.5 cm) to about in 0.5% sodium carboxymethyl cellulose solu 3.6% (Pocket oxygen alarm, Yokogawa tion, and bifemelane was dissolved in distilled Hokushin Electric) for a 9-min period by water. Doses of pramiracetam and bifemelane blowing pure nitrogen at the flow rate of 10 were expressed in terms of their salts. These I/min for 110 sec in the chamber , just before drugs were administered orally. [14C] -2 the locomotion test (15). Deoxy-D-glucose (14C-2-DG, specific ac Brain glucose uptake in mice: The pro tivity: 2.04 MBq/mmol) was obtained from cedure used was that of Shibota et al. (16). New England Nuclear Corp. (Boston, MA). The mice were injected with 14C-2-DG (185 Hypobaric hypoxia in mice: Mice were put KBq/kg) intravenously at 60 min after drug into a chamber (3-I desiccator), and the administration. Ten minutes later, the animals inside pressure of this chamber was lowered were decapitated and the brain was removed. to 200 mmHg in approximately 25-26 sec The blood samples were centrifuged, and with a vacuum pump. Survival time was plasma levels of 14C-2-DG and glucose were defined as the time interval between the start determined by a liquid scintillation counter, of hypoxia and the cessation of respiratory and the glucose oxidase technique, respec movements, and the survival time of longer tively. Whole brain was combusted with an than 10 min was assigned the value of 10 min automatic sample combustion system (Aloka in the calculations (12). Drugs were ad Co., Ltd.), and the 14C02 formed by com ministered 1 hr before the hypoxia. bustion was assayed by liquid scintillation Normobaric hypoxia in mice: Mice were counting. The brain glucose uptake (mg/g/10 put into a 500-ml glass container after 1 -hr min) was calculated using the following medication. Normobaric hypoxia was pro equation (16): duced by introducing pure nitrogen gas into brain glucose uptake the container at the rate of 5 I/min. The time =brain 14C activity (dpm/g) when the respiratory arrest occurred was re corded as the survival time. X plasma glucose level (mg/dl) x 1 Histotoxic anoxia in mice: Histotoxic plasma 14C activity (dpm/ml) 100 anoxia was produced by i.v. injection of 4 mg/ Local cerebral glucose utilization (LCGU) kg KCN. The survival time was measured with in rats: LCGU in the fully conscious rat was a limit of 180 sec (13). Drugs were adminis measured by the 14C-2-deoxyglucose method tered 1 hr before the KCN injection. described by Sokoloff et al. (17) with some Complete ischemia in mice: Cerebral isch modification. SD rats, fasted for approximately emia was produced by intersection between 16 hr, were anesthetized with halothane, and the medulla and the spinal cord. The time polyethylene catheters (PE-50, Clay-Adams) between decapitation and the last gasp was were implanted into the femoral artery and recorded as gasping duration (13). Drugs vein for timed sampling of arterial blood and were administered 1 hr before the decapita for the injection of 14C-2-DG, respectively. tion. Halothane was discontinued, the animals Normobaric hypoxia in rats: Wistar rats were allowed to recover for at least 1 hr, and were put into a 3.5-I plastic chamber after 1 hr then the drug was administered. Sixty minutes medication. Hypoxia was produced by intro later, 14C-2-DG (740 KBq/kg) was injected, ducing pure nitrogen gas into the chamber at and timed arterial blood samples were then the rate of 20 I/min. The time when respiratory withdrawn during the following 45 min arrest occurred was recorded as the survival (baseline, 15 and 30 sec, and 1, 2, 3, 5,110, 15, 20, 30 and 45 min). At the end of the 45 min of the brain were measured by the same sampling period, the rats were sacrificed with method as described in the brain glucose an overdose of sodium pentobarbital (50 mg, uptake test. LCGU (R) was calculated by the i.v.), and the brain was removed as rapidly as following equation and the constant reported possible and frozen. The plasma 14C or by Sokoloff et al. (17) using a computer glucose level and 14C activity in each region (FACOM 6658K). = LCi*(r)-k1 *e ckz*+k3*)xf.Cp*e(kz*+k3*)t dt R c Lfo(Cp*/Cp)dt_e-(k2*+k3')Txfo (Cp*/Cp)e(kz*+k3*)tdt] Where Ci* (r)=the local cerebral tissue con centrifuged (10000 rpm for 15 min, at 4°C). centration of 14C; Cp*=the time courses of The protein-free acid extract was used as the plasma radioactivities; Cp=the time samples of energy metabolites assay. ATP, courses of the plasma glucose concentra pyruvate and lactate levels were determined tions; k1*, k2* and k3*=kinetic constants for according to the enzymatic methods using phosphorylation of deoxyglucose; and Lc= reagent kits (Boehringer Mannheim Bioche the lumped constant (0.464). micals). Cerebral energy metabolites in hypoxic Statistical analysis: The results are expres rats: Wistar rats were used; the procedures of sed as the mean±S.E.M. Statistical signifi drug administration and the load of hypoxia cance was assessed using one-way analysis were the same as described in the locomotor of variance and multiple comparison by test. Immediately after the hypoxia, the rats Dunnett. were sacrificed by focussed microwave irradi ation (4.8 kW for 1.2 sec; Muromachi Kikai Results Co., Ltd.). The microwaved brains were imme Effect on hypobaric hypoxia in mice: The diately removed, weighed, and homogenized protective effects of DM-9384 and reference with 7 vol. of 0.6 N perchloric acid and then drugs against hypobaric hypoxia are shown in Table 1. Effects of DM-9384 and reference drugs on the hypobaric hypoxia in mice Table 1. Oral treatment of the hypoxic mice and pramiracetam (100-1000 mg/kg, p.o.) with DM-9384 resulted in a significant pro were not significant.