Pharmacology Biochemistry & Behavior, Vol. 17, pp. 367-369, 1982. Printed in the U.S.A. Tofizopam Modulates the Affinity of Benzodiazepine Receptors in the Rat Brain VEIJO SAANO AND ARTO URTTI* Department of Pharmacology and Toxicology and *Department of Pharmacy, University of Kuopio, P.O. Box 138, 70101 Kuopio 10, Finland Received 19 October 1981 SAANO, V. AND A. URTTI. Tofizopam modulates the affinity ofbenzodiazepine receptors in the rat brain. PHARMAC. BIOCHEM. BEHAV. 17(2) 367-369, 1982.--Tofizopam, a 3,4-benzodiazepine, lacks the sedative action common to 1,4-benzodiazepines, but has anxiolytic activity. In this study we administered tofizopam (50 mg/kg) to rats perorally twice a day for six days, and analyzed the binding of [SH]flunitrazepam to benzodiazepine receptors of these drug-treated rats. The effect of tofizopam treatment was compared to that brought about by treatment with diazepam (12 mg/kg twice a day for six days) and to binding in controls treated with vehicle. Compared to the controls, the diazepam group had a marked decrease in binding of [3H]flunitrazepam to benzodiazepine receptors both in the forebrain and in the hindbrain. As a result of the increased affinity of the receptors tofizopam slightly, but statistically significantly, enhanced binding. With both drugs the number of receptors was unaltered. The effect of tofizopam in the hindbrain was similar to that in the forebrain. The results of this study support our earlier finding from single-dose studies that tofizopam acts indirectly on benzodiazepine receptors. Tofizopam Diazepam Benzodiazepine receptors TOFIZOPAM, a 3,4-benzodiazepine, has been shown to have an anxiolytic effect [4, 10, 17]. In contrast to 1,4-ben- zodiazepines (e.g., diazepam, flunitrazepam, etc.), to- fizopam does not cause sedation or muscle relaxation [12, 15, 17]. The therapeutic potency of various 1,4-ben- zodiazepines correlates positively with their ability to displace radiolabeled benzodiazepines from benzodiazepine receptors in the central nervous system [8]. Tofizopam does not displace tritiated flunitrazepam from receptors, but in vitro enhances the receptor-specific binding of flunitrazepam to rat brain and in vivo has been found to enhance this bind- ing in rats after acute treatment [14]. The aim of the present study was to analyze the effects of tofizopam on benzodiaz- epine receptors after prolonged treatment. A small group of rats was treated with diazepam so that we could compare the effects of tofizopam to those caused by a 1,4-benzo- diazepine; diazepam has been shown to occupy benzodiaz- epine receptors during acute and chronic treatment to the extent that is easy to identify in vitro using homogenates of brain tissue from drug-treated animals [6,11]. METHOD Twenty-three male rats of inbred strain (BD IX/Kuo) weighing 220-340 g were housed in opaque plastic boxes under standard laboratory conditions: 10 hr dark/14 hr light cycle, air temperature 20-+0.5°C, relative air humidity 55- 75 ° . Before and during drug treatment, animals had free ac- cess to food and tap water. Tofizopam and diazepam were suspended in 1% solution of Tween 80/water and were administered through a stomach tube in a volume of 5 ml/kg body weight. Drugs (or vehicle to controls) were administered twice a day at 12 hr intervals. Each dose of tofizopam was 50 mg/kg; each dose of di- azepam was 12 mg/kg. [3H]flunitrazepam was used as a ligand; it had a specific activity of 86.4 Ci/mmole and was purchased from New England Nuclear, Boston, MA. To- fizopam, diazepam, and non-radioactive flunitrazepam were generous gifts from the Research Center of Farmos Group, Turku, Finland. Drug treatment continued for six days. Rats were decapi- tated 2 hr after they had received the last dose of drug. After decapitation the brain was removed immediately and the forebrain was separated from the hindbrain by an incision posterior to the occipital lobes and across the brain stem. The hindbrain (cerebellum + pons and medullar oblongata) and the forebrain were homogenized individually in 32 vol- umes of ice-cold Tris-HC1 buffer (50 mM, pH 7.4). Aliquots of homogenate (400/zl) were added to a set of duplicate test tubes containing 6-9 concentrations of [aH[flunitrazepam (range from 0.06 to 8.12 nM). For determination of nonspecific binding another set of tubes also contained 10 /zM concentration of non-radioactive flunitrazepam. The tubes were incubated at 4°C for 30 minutes before the con- tents were filtered through Whatman GF/B filters which were then placed in 6 ml of Aquasol ® scintillation liquid for at least 16 hr, after which the radioactivity was measured. The protein content of each homogenate was determined using the method described by Lowry et al. [7]. Maximum binding and the dissociation constant for [3H]flunitrazepam binding were determined by Scatchard analysis as described by Bennett [2]. Statistical significance of the differences be- tween values from drug-treated rats and those from control rats was tested using Student's t-test. Copyright © 1982 ANKHO International Inc.--0091-3057/82/080367-03503.00/0