Comparison of Typical and Atypical Benzodiazepines on the Central and Peripheral Benzodiazepine Receptors

Yasuo WATANABE', Takeshi SHIBUYA'°2, Salahadine KHATAMI2 and Bernard SALAFSKY2 ' Department of Pharmacology , Tokyo Medical College, Tokyo 160, Japan 2 Department of Biochemical Sciences , University of Illinois College of Medicine at Rockford, Rockford IL 61107-1897, U.S.A.

Accepted June 21, 1986

Abstract-The affinities of typical and atypical benzodiazepines (BZs) for 3H flunitrazepam (FLU) and 3H-Ro 5-4864, "peripheral" BZ, binding sites in the central nervous system (CNS) were compared. Each dissociation constant (Kd) value of the 3H-FLU binding sites in the cerebellum, cortex, hippocampus and spinal cord showed the similar results, although the binding maximum (BmaX) for the cortex and cerebellum yielded the largest value and the smallest BmaXappeared in the spinal cord. In contrast, B,,,.. for 3H-Ro 5-4864 binding sites were about three-fold higher in the spinal cord than in the cerebellum. Among the drugs tested, brotizolam demonstrated the highest affinity for 3H-FLU binding sites in the four regions. Typical and atypical BZs did not normally show different affinities for the two BZ receptor subtypes, except for CL 218,872 and Ro 5-4864. A series of compounds including Ro 5-4864, PK 11195, diazepam and brotizolam had high affinity for the 3H-Bo 5-4864 binding sites in the cerebellum and spinal cord. Other BZs did not show affinity for "peripheral" BZ binding sites in the CNS. In conclusion, brotizolam (or atypical BZ) had the binding affinity to both "central" and peripheral" BZ receptors, like diazepam (a typical BZ).

It has been demonstrated that the phar CL 218,872 has a preferential affinity for the macological potencies of some benzo so-called BZ type 1 receptors in vitro and also diazepines (BZs) highly correlate with their may possess antianxiety properties with little binding affinities for 3H-BZ sites in the brain sedation (5, 9, 10). This drug has also higher (1-4). In particular, anticonvulsant activity, affinity binding sites in the cerebellum than antiaggressive activity and muscle-relaxant in the cortex (3, 8, 9). However, most activity were closely correlated with the normal BZ derivatives do not show definite inhibition of 3H-diazepam binding (2). differentiation of their pharmacological These correlations, however, do not always characteristics with respect to different brain demonstrate the mechanisms of all typical regions (e.g., cerebellum, cortex, hip and atypical BZ-induced pharmacological pocampus and corpus striatum). effects. Recently, several papers have Previously, Braestrup and Squires (11) described the existence of multiple BZ found specific binding of 3H-diazepam in receptors (5-8). Furthermore, the distribution some peripheral organs, although they noted of BZ receptor subtypes in the central nervous that clonazepam, a potent anxiolytic BZ, system (CNS) is heterogeneous and the lacked the capability to displace 3H-diazepam proportion of BZ receptor subtypes does binding in some peripheral tissues, unlike differentially localize in some brain regions Ro 5-4864, a "peripheral" BZ receptor (PBS) (see 9). This heterogeneity in distribution may ligand. More recently, the existence of PBS explain the multiple BZ pharmacological in the CNS has been documented (9, 12, 13). effects. For example, the triazolopyridazine For example, the localization of PBS in the CNS has been demonstrated in the olfactory vol. of 0.32 M sucrose. After centrifuging, at bulbs, choroid plexus, pituitary and spinal 1000xg for 10 min, the resulting membrane cord, although some of these 3H-Ro 5-4864 pellet was resuspended in ice-cold 50 mM binding sites (i.e., PBS) may also be non Tris HCI buffer (pH 7.7) and centrifuged at neuronal binding sites, or so-called 30,000xg for 30 min. This operation was " acceptor" sites (9). However, recent reports repeated three times; then the membrane suggest that Ro 5-4864 may be anxiety suspension was used for the binding assay on producing and also produce seizures in mice the same day. and rats in the 30 mg/kg range, and this PBS Radio receptor assay: 3H-FLU (84 Ci/ in the olfactory bulbs may be an important mmol, Amersham, IL) and 3H-Ro 5-4864 key site of the Ro 5-4864-induced con (82.7 Ci/mmol, NEN, MA) were used for vulsion (9, 13). The results of all these determining the BZ type 1 /2 receptors studies appear to suggest that many typical and "peripheral" / "central" BZ receptors, and/or atypical BZs may be classified on the respectively. The characteristics of 3H-FLU basis of localization of the BZ receptor and 3H-Ro 5-4864 were measured using heterogeneity in the CNS, including the PBS. 8 to 12 different concentrations (0.1-20 nM) In this paper, we compared the binding of each ligand; 1 iiM of clonazepam (Sumi characteristics of typical and atypical BZs to tomo Chemical, Japan) and Ro 5-4864 3H-flunitrazepam (FLU) or 3H-Ro 5-4864 (Dr. S.J. Cooper, Univ. of Birmingham, U.K.) sites in the several regions of the CNS. were used as displacers of each radioligand, Zopiclone, suriclone (pi parazinecarboxylate respectively. The dissociation constant value derivatives) (14), brotizolam (a triazolo (Kd) and binding maximum (Borax) were benzodiazepine) and CL 218,872 were used analyzed by our previous method (20). To as atypical BZs, and both diazepam and determine the K; values of zopiclone, nitrazepam were used as the typical BZs. In suriclone (Rhone-Poulenc, France), CL the comparison of PBS in the CNS and the 218,872 (Stuart Pharmaceuticals, Delaware), " central" BZ receptors, Ro 5-4864 and PK brotizolam (Nippon Boehringer Ingeiheim, 11195 (15) were used as selective Japan), diazepam, nitrazepam (Nippon peripheral" BZs. Additionally, because Roche, Japan) Ro 5-4864 and PK 11195 recent studies indicate that the characteristics (Dr. S.J. Cooper, Univ. of Birmingham, U.K.), of BZ receptor subtypes in the rat spinal IC50 values of the affinities of each drug for cord predominantly appeared to be those of 3H-FLU or 3H-Ro 5-4864 binding sites in the type 2 receptor (CL 218,872 insensitive the three brain regions and the spinal cord sites) (16, 17), we chose the cerebellum and were measured using our previous method cortex for type 1 receptor sites, and the spinal (4), and K; values were calculated by the cord and hippocampus for type 2 receptor following equation: K;=IC50/(1 +C/Kd), sites (8, 17). where C=concentration of 3H-FLU or 3H Ro 5-4864 and Kd is the binding affinity of Materials and Methods 3H-FLU or 3H-Ro 5-4864 . The ligands were Membrane preparation: Sprague-Dawley dissolved in methanol (as 10-3 M) and stored rats (male, 300-350 g) were used. After at -20°C. Dilutions for the binding assay decapitation of rats, each of the brain were made with 50 mM Tris buffer (pH 7.7). regions was dissected as described previously The maximum final concentrations of these (18), and spinal cords were immediately ligands for binding assays were 10-5 M, removed using the method of de Sousa and since methanol may affect the 3H-FLU or Horrocks (19). These tissues were stored at 3H-Ro 5-4864 binding sites in tissues . -80°C before preparing the membrane fraction within one month. Membrane Results fractions of these tissues were prepared using Table 1 shows the characteristics of 3H our previous method (20). Briefly, each brain FLU or 3H-Ro 5-4864 binding sites in the region (cortex, cerebellum and hippocampus) cortex, cerebellum, hippocampus and spinal and the spinal cord were homogenized in 10 cord. The Kdvalues of 3H-FLU in each region Table 1. Binding characteristics (binding affinity: K,i=nM, binding capacity; Bni x=fmol/mg protein) of 3H-flunitrazepam (FLU) and 3H-Ro 5-4864 in various rat brain regions

Values are expressed as the mean±S.E., with the number of experiments included in parentheses. no experiments. Bmax value is statistically different from B„n-; of 3H-FLU in the cortex; P<0.05, Student's t-test. '': Bmat value is statistically different from Binas of 3H-Ro 5-4864 in the cerebellum; P<0.05, Student's t-test. (There is no statistical difference between the Kd's of the same radioligand in any tissue.) did not demonstrate any significant differ times less than in the cortex, hippocampus ences. However, the binding capacities of and spinal cord, respectively. However, the each regions did show different values. The K; values of other BZs to 3H-FLU did not Bmax of both the cortex and cerebellum were appear to correlate with the different pro similar and higher than that of the hip portion of BZ-type 1 and 2 receptors in each pocampus. In the spinal cord, the binding region, unlike CL 218,872. The typical BZs, capacity was about 8 and 10 times less than except for Ro 5-4864, showed similar results that of the cerebellum and cortex, respec in each region. tively. In contrast, the Bmax of 3H-Ro 5 In the group of atypical BZs, the K; value 4864 binding sites in the spinal cord was of each drug did not demonstrate definite more than 2 times of that in the cerebellum, differences between the four regions except although the Kd values of 3H-Ro 5-4864 for CL 218,872. However, the K; values of binding sites in both the regions did not zopiclone in the cerebellum was about two appear to show significant differences. times lower than that in the hippocampus. Additionally, the Kd values of 3H-FLU Between the cortex and spinal cord, the binding were significantly different from that affinities of suriclone showed about a two of 3H-Ro 5-4864 in both the cerebellum and fold difference. In addition, Ro 5-4864, a the spinal cord. In both regions, 3H-FLU peripheral type BZ, demonstrated a very high binding sites had much higher affinity than K; value (about 4 iiM) in the spinal cord, 2H-Ro 5-4864 binding sites . although this drug had no affinity for 3H-FLU Kd values were used for measuring K; binding sites in the cerebellum, cortex and values of each BZ to both 3H-ligands in hippocampus. Among the drugs tested, each region (Tables 2 and 3). The inhibitory brotizolam showed the highest affinity in effects of typical and atypical BZs on the each brain region, and these affinities were BZ-type 1 and/or 2 receptors are shown in about one tenth of those of diazepam. As Table 2. In agreement with previous reports, shown in Fig. 1, the displacement curve of all the proportion of BZ type 1 /2 in the drugs tested to 3H-FLU binding sites demon cerebellum, cortex, hippocampus and spinal strated the same shape, comparing the cord were observed at the ratios of 90/10, cerebellum and spinal cord. Similar results 60/40, 40/60 and 10/90, respectively (9, were shown in other brain regions (i.e., 16, 17, 21). The K; value of CL 218,872, hippocampus and cortex), although the which acts preferentially on the BZ-type 1 inhibitory curve of suriclone to 3H-FLU receptor in each region supports these ratios. binding in the cortex appeared much steeper The lowest K; value of CL 218,872 to 3H-FLU between 5 nM and 10 nM than in the spinal was demonstrated in the cerebellum, and cord (these data are not shown). this K; value was about four, nine and fifteen Table 3 demonstrates the K; values of Y. Watanabe et al. Table 3. Relative affinities (kt) of several drugs to 3H-Ro 5-4864 binding sites in cerebellum and spinal cord

Values are expressed as the mean±S.E., with the number of experiments included in parentheses. Kt=1C50/[1 +(C/Ka)], where C=concentration of 3H-Ro 5-4864, and Kt is the dissociation constant of 3H-Ro 5-4864 . Significantly different from Ki value of Ro 5-4864 in the cerebellum; P<0.01. Student's t-test.

Fig. 1. Displacement of 3H-FLU binding sites in rat brain cerebellum (upper side) and spinal cord (lower level) by several drugs. Competition curves were determined as described in Materials and Methods. Specific binding of tritiated ligand was measured in the presence of various concentrations of each unlabelled drug and also in the absence of unlabelled drug in each experiment. Decrease in amount of radioligand bound at each concentration of unlabelled drug is expressed as percent inhibition. Each point represents the mean of 4-6 separate determinations, each run in triplicate. The Hill coefficient numbers were between 0.89 and 0.98. °3: Ratio of the K; value of 3H-FLU in the spinal cord (mostly Type II) to the K; value of 3H-FLU in the cerebellum (mostly Type I). The classification of Type I and Type II was decided by the method of Niehoff et al. (7). '1: Ratio of the K; value of 3H-FLU in the spinal cord (central) to the K; value of 3H-Ro 5-4864 in the spinal cord (peripheral) . N.D.=Not determined (one or both K; values are >10000). typical and atypical BZs to 3H-Ro 5-4864 binding sites in the spinal cord and cere Discussion bellum. In both regions, the K; values of Some typical and atypical BZs were suriclone, zopiclone, nitrazepam and CL compared using the radio receptor assay. The 218,872 did not occur at a drug concentration characteristics of most BZs tested did not less than 10 iM. Ro 5-4864 and PK 11195 show definitive differences between BZ had the highest affinity for 3H-Ro 5-4864 type 1 and 2 receptors, except for CL 218,872. binding sites in the cerebellum and spinal However, diazepam and brotizolam had high cord. Ro 5-4864 significantly demonstrated affinities for not only 3H-FLU but also 3H a higher affinity in the spinal cord than in the Ro 5-4864 binding sites, unlike other BZs. cerebellum. With respect to "central" type Ro 5-4864 had almost 5000 times less BZs, diazepam, in addition to brotizolam, affinity to "central" type receptors than demonstrated a high affinity to PBS. These K; peripheral" type receptors. values were almost in the same range and did To compare typical and atypical BZs, not appear to be different between the spinal nitrazepam, diazepam, brotizolam, zopiclone, cord and cerebellum. suriclone, CL 218,872 and two "peripheral" All tested drugs were classified according type BZs were used. Nitrazepam is well to their selectivity for the BZ-type 1 and/or 2 known as a sleep-inducer. Zopiclone, receptor and the "central" / "peripheral" type brotizolam and suriclone exert similar effects (Table 4). In separating the BZ-type 1 and 2 to nitrazepam (22-24). Nevertheless, their receptors, the K; values of each drug in the structures are quite different from the "classical" BZs (e cerebellum and spinal cord were chosen. K; .g., diazepam or nil values to 3H-FLU and the 3H-Ro 5-4864 trazepam). Additionally, Lippa et al. (3) and binding sites in the spinal cord were used to Dubnick et al. (10) reported on a novel select "central" and "peripheral" type BZ triazolopyridazine which has selective anti receptor, respectively. CL 218,872 showed punishment and anti-convulsant activity in about a 15-fold difference between BZ-type animals without the attendant motor de 1 and 2 receptors. However, other BZs had pression and ataxia associated with BZs, the same affinity for BZ-type 1 and 2 and it also has characteristics of a preferential receptors. I n spite of the fact that the K; BZ-type 1 receptor in in vitro experiments. values of diazepam and brotizolam to 3H-Ro Recently, Chweh et al. (25) proposed that 5-4864 in both cerebellum and spinal cord there was no correlation between the basal were in 10 nM range, both drugs showed the receptor binding affinities of the BZs and characteristics of "central" type BZ. their hypnotic potencies using the 3H-FLU Additionally, Ro 5-4864 demonstrated receptor binding assay in mice forebrain peripheral" type characteristics. membrane fractions and the righting reflex. They suggested that the inhibitory potency of similar affinities to both BZ subtype receptors. CL 218,872 in 3H-FLU binding was However, the slope of CL 218,872 and increased by muscimol, and CL 218,872 also suriclone in the cerebellum was much steeper showed the hypnotic action in mice, although than in the spinal cord. CL 218,872 is well the ED50 for the hypnotic dose of this drug known as a preferential type 1 ligand. About was three times greater than that of 90% of the BZ receptors have characteristics flurazepam. They concluded that the hypnotic of BZ-type 1 in the cerebellum, but the action of BZs involves a supramolecular proportion appears to be completely the structure composed of both BZ and GABA opposite in the spinal cord (17, 21). In receptors, although they failed to demonstrate addition, the affinity value of 3H-suriclone in the hypnotic effects of nitrazepam in mice. the spinal cord was two times lower than in As described in the introduction, it is well the cerebellum, although these results are documented that the anti-convulsant ac not shown in this paper (T. Shibuya et al., tivities of BZs seemed to correlate well with unpublished data). These results suggest their receptor binding affinities, unlike the that suriclone may have higher affinity to BZ relation between hypnotic action and binding type 2. On the different characteristics affinities (1-3, 25). These different obser between suriclone and zopiclone, Trifiletti vations suggest that the hypnotic and anti and Snyder (28) compared the effects of convulsant potencies of BZs do not share the zopiclone and suriclone on the 3H-FLU and same mechanism (see 25). Our results also 3H-Ro 15-1788 (a benzodiazepine an failed to show the correlation between tagonist) binding sites in three brain regions. hypnotic drugs and BZ-type 1 /2 selectivity, Their results on the 3H-FLU binding sites although each drug appeared to show a were about the same as ours. Additionally, different potency to inhibit the 3H-FLU they noted that the binding of 3H-suriclone binding sites in each brain region (Table 2). was not modulated by GABA, pentobarbital According to our previous results, brotizolam or tracazolate, although Skerritt and Mac had much higher affinity to BZ-type 2 than Donald (29) suggested that zopiclone was a BZ-type 1 (26). However, the earlier report weak enhancer of GABA response at high is not in agreement with our current results. nanomolar concentrations. This discrepancy cannot be explained in The number of PBS was about 2.2-fold more detail at present time, but it might be greater in the spinal cord than in the caused by the different tissues used (i.e., cerebellum, while the B,,,ax of 3H-FLU neonatal rat cortex and adult spinal cord). binding sites ("central" type BZ receptors) The number of BZ-type 2 receptors was about was about 7-fold less in the spinal cord two times greater in the neonatal rat than in than in the cerebellum (Table 1). PBS the adult spinal cord (27). It may also be seems to be predominantly associated with more difficult to detect small alterations in non-neuronal sites (e.g., neuroglia), but PBS adult spinal cord than in neonatal rat cortex. in the olfactory bulb may influence the However, as in our previous study (4), convulsant actions (1, 13). The functional brotizolam showed the highest affinity to 3H mechanisms of PBS in the spinal cord are FLU binding sites in the tested drugs. not known, but their existence is well Additionally, the Hill coefficient (data are documented (30). not show) and the slope of the displacement In our studies of binding inhibition, ni curve of brotizolam (Fig. 1) did not show a trazepam (typical BZ), CL 218,872, suriclone significant difference between the BZ-type 1 and zopiclone (atypical BZs) had no affinity preferential site (cerebellum) and the BZ to PBS in both the cerebellum and spinal type 2 preferential site (spinal cord). Other cord. However, Ro 5-4864, diazepam (typical BZs tested (except for suriclone and CL BZs), PK 11195 and brotizolam (atypical 218,872) did not show a difference in either BZs) did inhibit the 3H-Ro 5-4864 binding the coefficient or slope between both sites. sites in the cerebellum and spinal cord. The These results demonstrate that brotizolam, binding affinity of brotizolam to PBS has not zopiclone, diazepam and nitrazepam have previously been reported. Brotizolam had the highest affinity for the "central" BZs receptor drugs. We also thank Ms. H. Tsuji for her secretarial in three brain regions and the spinal cord assistance in the preparation of this manuscript. (Table 2), compared to several "classical" and "new" BZs (4). It is difficult to explain References the function of this drug in relation to PBS 1 Mohler, H. and Okada, T.: Benzodiazepine in the spinal cord and cerebellum at present. receptor: Demonstration in the central nervous Brotizolam seems to have some effects system. Science 198, 849-851 (1977) similar to Ro 5-4864, PK 11195 and diazepam 2 Mackerer, C.R., Kochman, R.L., Bierschenk, such as the enhancement of 3H-methyl B.A. and Bremner, S.S.: The binding of [3H] group incorporation into phosphatidylcholine diazepam to rat brain homogenates. J. Pharmacol. and/or the antior pro-convulsive actions of Exp. Ther. 206, 405-413 (1978) maximum electroshock-induced seizure (6, 3 Lippa, A.S., Critchett, D., Sano, M.C., Klepner, C.A., Greenblatt, EX, Coupet, J. and Beer, B.: 31). In our results, Ro 5-4864 there was Benzodiazepine receptors: Cellular and behavior significantly more binding of Ro 5-4864 in characteristics. Pharmacol. Biochem. Behav. 10, the spinal cord than in the cerebellum. These 831-843 (1979) results support the previous reports on the 4 Shibuya, T., Field, R., Watanabe, Y., Sato, K. and different localization of "PBS" in not only Salafsky, B.: Structure-affinity relationships the brain but also in the spinal cord. between serveral new benzodiazepine derivaties Our results suggest the following con and 3H-diazepam receptor sites. Japan. J. clusions: 1) With respect to the ratio of the Pharmacol. 34, 435-440 (1983) K; value of 3H-FLU in the spinal cord (type 5 Squires, R. E., Benson, D. L, Braestrup, C., 2/type 1 =90/10) to the K, value of 3H-FLU Coupet, J., Klepner, C.A., Myers, V. and Beer, B.: in the cerebellum (type 2/type 1 =10/90), Some properties of brain specific benzodiazepire CL 218,872 appears to be a selective ligand receptors: New evidence for multiple receptors. of the BZ type 1 receptor. Other BZs, Pharmacol. Behav. 10, 825-830 (1979) however, appeared to have similar affinities 6 Gee, K.W., Yamamura, S.H., Roeske, W.R. and for both BZ receptor subtypes (Fig. 4). 2) Yamamura, H. L: Benzodiazepine receptor Not only Ro 5-4864 but diazepam and heterogeneity: Possible molecular basis and functional significance. Fed. Proc. 43, 2767 brotizolam demonstrated the characteristics 2772 (1984) of "peripheral" type BZ ligands. The K; 7 Niehoff, D.L., Marshal, R.D. and Kuhar, M.J.: value of brotizolam to the 3H-Ro 5-4864 Benzodiazepine receptors: Preferential stimu binding sites in the spinal cord showed lation of type 1 receptors by pentobarbital. Eur. results similar to that of diazepam, but the J. Pharmacol. 92, 131-134 (1983) ratio of "central" type BZ receptors to the 8 Watanabe, Y., Hyde, L., Khatami, S., Salafsky, B. peripheral" type of brotizolam was 10 times and Shibuya, T.: The neonatal development of less than that of diazepam (Fig. 4). 3) In the benzodiazepine receptor subtypes in the rat typical BZs, nitrazepam was evaluated like cerebral cortex and cerebellum. Japan. J. diazepam, to have the same affinity for both Psychopharmacol. 5, 335-342 (1985) (in BZ type 1 and 2 receptors, but it had no Japanese) affinity for PBS. 4) The inhibitory effect of 9 Richards, J.G. and Mohler, H.: Benzodiazepine suriclone, an atypical BZ, to 3H-Ro 5-4864 receptors. Neuropharmacology 23, 233-242 binding sites was not determined, although (1984) this drug has the strong affinity to BZ type 10 Dubnick, B., Lippa, A.S., Klepner, C.A., Coupet, 2 receptors. J., Greenblatt, E.N. and Beer, B.: The separation of 3H-benzodiazepine binding sites in brain and Acknowledgements: We would like to thank Dr. of benzodiazepine pharmacological properties. D.H. McCurdy (Stuart Pharmaceuticals) for donation Pharmacol. Biochem. Behav. 18, 311-318 of CL 218,872, Prof. Klupp (Boehringer Ingelheim) (1983) for donation of brotizolam and Rhone-Poulenc 11 Braestrup, C. and Squires, R.F.: Specific Yakuhin K.K.(Japan) for providing us with a supply benzodiazepine receptors in rat brain charac of suriclone and zopiclone. We are indebted to Dr. terized by high-affinity [3H]diazepam binding. S.J. Cooper (Univ. of Birmingham, U.K.) for his Pro. Natl. Acad. Sci. 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