Claus Braestrup

Some properties of brain specific benzodiazepine receptors: New evidence for multiple receptors

Several new lines of evidence suggest the existence of two or more distinct types of benzodiazepine receptors, in contrast to earlier results suggesting the presence of only one class of receptors. Appropriate thermoinactivation experiments indicate two receptors with different thermostabilities. Several triazolopyridazines, with some of the pharmacological properties of anxiolytics have recently been shown to displace 3H-diazepam and 3H-flunitrazepam with Ki values in the 6 to 100 nanomolar range. These new substances are active in conflict tests in rats and monkeys and prevent metrazol induced seizures in vivo, but strikingly lack the ataxia and sedative properties of the benzodiazepines. Hill analyses of dose-response curves for some of these substances yields Hill coefficients in the range of 0.4--0.6, suggesting that these compounds may be able to discriminate between several types of benzodiazepine receptors.

[3H]Propyl β-Carboline-3-Carboxylate as a Selective Radioligand for the BZ1 Benzodiazepine Receptor Subclass

Abstract: Ethyl β‐carboline‐β‐carboxylate (β‐CCE) is a mixed‐type inhibitor of [3H]flunitrazepam ([3H]FNM) binding to benzodiazepine receptors in noncerebellar regions of rat brain. These findings may represent the presence of either receptor multiplicity or negative cooperativity among benzodiazepine receptors. [3H]Propyl β‐carboline‐3‐carboxylate ([3H]PrCC) has previously been shown to bind specifically to benzodiazepine receptors of rat cerebellum. In the present study we found no indication of the presence of true negative cooperativity among benzodiazepine receptors when [3H]PrCC was used as radioligand. However, we observed that [3H]PrCC labelled only 57% of [3H]FNM binding sites in rat hippocampus (Bmax values) and 71% in rat cerebral cortex, whereas the number of receptors labelled by both ligands was equal in the cerebellum. Hofstee analyses of the shallow inhibition curves seen in hippocampus and cerebral cortex when [3H]FNM binding was inhibited by β‐CCE indicate that β‐CCE and some other β‐carboline‐3‐carboxylate derivatives interact preferentially with a subclass of receptors, and that the percentage of this subclass is equivalent to the number of receptors labelled by [3H]PrCC. We conclude that [3H]PrCC at low concentration (0.3–0.4 × 10‐9 M) labels a subclass of benzodiazepine receptors, BZ1, while another class, BZ2 receptors, are not labelled by [3H]PrCC when filtration assays are used. By parallel determinations of the proportion between [3H]FNM and [3H]PrCC binding we calculated the percentage of BZ1 receptors in several regions of rat, guinea pig and calf brain and in mouse forebrain. The values ranged from approximately 50% in hippocampus to 90% in the guinea pig pons.

(R)-N-[4,4-Bis(3-Methyl-2-Thienyl)but-3-en-1-yl]Nipecotic Acid Binds with High Affinity to the Brain γ-Aminobutyric Acid Uptake Carrier

(R)‐N‐[4,4‐Bis(3‐methyl‐2‐thienyl)but‐3‐en‐l‐yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of γ‐[3H]aminobutyric acid ([3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50= 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of [3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of [3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5‐hydroxytryptaminelA receptors was inhibited by NO 328 at 5—30 μM, whereas several other receptors and uptake sites were unaffected. [3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCI. The specific binding of [3H]NO 328 was inhibited by known inhibitors of [3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl‐dependent [3H]NO 328 binding followed that of synaptosomal [3H]GABA uptake. It is concluded that NO 328 is a potent and selective inhibitor of neuronal and glial GABA uptake and that [3H]NO 328 is a useful radioligand for labeling the GABA uptake carrier in brain membranes. In the mouse brain in vivo, [3H]NO 328 likewise showed saturable and reversible binding that could be displaced by analogues of NO 328. Further studies are needed to demonstrate whether the uptake carrier is indeed labeled by [3H]NO 328 in vivo.

Characterization of tiagabine (NO-328), a new potent and selective GABA uptake inhibitor.

Tiagabine (NO-328) (R(-)-N-[4,4-bis(3-methylthien-2-yl)but-3-enyl]nipecotic acid, hydrochloride) is a new centrally acting GABA uptake inhibitor. The anticonvulsant activity of tiagabine was evaluated against seizures induced by methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), pentylenetetrazol, bicuculline, maximal electrostimulation (MES), or high intensity sound. The sedative actions of tiagabine were evaluated in tests for traction, rotarod performance and exploratory behavior. Finally, interoceptive properties of tiagabine were assessed using diazepam-, CGS 9896-, pentylenetetrazol-, or amphetamine-discriminating rats. Tiagabine was an effective anticonvulsant in doses which did not produce sedation or motor debilitation, although it was not potent against MES. In a manner similar to other anti-epileptic drugs, tiagabine potentiated dopaminergic function (methylphenidate-induced gnawing in mice) although it did not substitute for amphetamine in amphetamine-trained animals. Furthermore, although tiagabine antagonized DMCM-induced convulsions, it exhibited neither CGS 9896 or diazepam-like interoceptive effects, nor did it block (or potentiate) pentylenetetrazol-discrimination. Thus, GABA uptake inhibition represents a novel rationale for a valproate-like anticonvulsant drug therapy.

Evaluation of the β-carboline ZK 93 426 as a benzodiazepine receptor antagonist

We describe here biochemical and pharmacological effects of the β-carboline ZK 93426, a new and potent benzodiazepine (BZ) receptor antagonist. ZK 93426 was compared with Ro 15-1788 and CGS 8216, two compounds previously described as BZ receptor antagonists. Certain effects of ZK 93426, Ro 15-1788 and CGS 8216 were quite similar (e.g., 3H-FNM displacement, “GABA ratio”, “photo-shift”). In most pharmacological tests ZK 93426 and Ro 15-1788 lacked overt effects; Ro 15-1788 was a weak agonist in some paradigms, while ZK 93426 exhibited a potent proconflict effect but also a weak anticonvulsant effect. This interesting finding with ZK 93426 suggests that BZ receptor ligands may possess differential efficacy at BZ receptor subtypes. In contrast, CGS 8216 exhibited potent proconvulsant effects in several paradigms in addition to proconflict and pentylenetetrazol generalizing effects. ZK 93426, Ro 15-1788 and CGS 8216 were almost equally potent as antagonists of the effects of BZ receptor agonists, such as diazepam and lorazepam. However, ZK 93426 was the most potent inhibitor of the convulsions produced by the BZ receptor inverse agonist DMCM.

Cocaine: Discussion on the Role of Dopamine in the Biochemical Mechanism of Action

The central stimulant effect of cocaine is generally considered related to its potentiating effect on biogenic amines. However, the individual role and significance of the amines involved in various stimulant effects of cocaine are still a controversial topic. Cocaine is a potent inhibitor of noradrenaline uptake (Hertting, Axelrod, and Whitby, 1961; Ross and Renyi, 1967; Langer and Enero, 1974; Azzaro, Ziance, and Rutledge, 1974), dopamine uptake (Fuxe, Hamberger, and Malmfors, 1967; Ross and Renyi, 1967; Harris and Baldessarini, 1973; Heikkila, Orlansky, Mytilineou, and Cohen, 1975), and serotonin uptake (Ross and Renyi, 1969; Friedman, Gershon, and Rotrosen, 1975). High affinity uptake of tryptophan into synaptosomes is also inhibited (Knapp and Mandell, 1972). In vivo studies have shown that cocaine induces a short-lasting uptake inhibition into brain tissues of noradrenaline (Schanberg and Cook, 1972), dopamine (Fuxe, Ham-berger, and Malmfors, 1967), and serotonin (Ross and Renyi, 1969).

Audiogenic seizures in DBA/2 mice discriminate sensitively between low efficacy benzodazepine receptor agonists and inverse agonists

In experiments with audiogenic seizures in DBA/2 mice, we observed that several socalled benzodiazepine receptor antagonists exhibited either anticonvulsive (Ro 15-1788, PrCC) or proconvulsive (FG 7142, beta-CCE, CGS 8216) effects at high receptor occupancy (17-85%), as compared to benzodiazepines and DMCM which had anticonvulsive and proconvulsive actions, respectively, at very low receptor occupancy (less than 10%). Sensitive distinction between benzodiazepine receptor ligands with low anticonvulsive efficacy (partial agonists) and ligands with low proconvulsive, and maybe anxiogenic, efficacy (partial inverse agonists) can thus be obtained in sound seizure susceptible mice.

The monoamine oxidase B inhibitor deprenyl potentiates phenylethylamine behaviour in rats without inhibition of catecholamine metabolite formation

The drug l-deprenyl has been reported to have antidepressant properties, and in the present study three possible mechanisms of action were investigated in animal experiments. l-Deprenyl, which is a type B monoamine oxidase (MAO) inhibitor, was compared to clorgyline, an MAO A inhibitor with regard to its inhibitory effect on the formation of three major catecholamine metabolites, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylglycol (MOPEG) in the rat brain in vivo. Apart from a difference in dose levels the two drugs showed no difference in the dose--response pattern of all three metabolites. Clorgyline inhibited the formation of HVA, DOPAC and MOPEG with an ED50 of about 0.2 mg/kg s.c. and l-deprenyldopamine and noradrenaline are formed by the same type of monoamine oxidase(s), probably type A, in the rat brain in vivo. Antidepressant properties of l-deprenyl therefore seem to be independent of catecholamine deamination. l-Deprenyl but not clorgyline (2 or 8 mg/kg s.c.) potentiated the stereotyped sniffing behaviour induced by beta-phenylethylamine, a specific substrate for type B monoamine oxidase. This result is discussed in relation to a new hypothesis of phenylethylamine and dopamine involvement in depression. l-Deprenyl was 10,000 times less potent than DMI as inhibitor of noradrenaline uptake in crude synaptosomes from the occipital cortex of rat brain. Inhibition of noradrenaline uptake was therefore excluded as a possible mechanism for the antidepressant action of l-deprenyl.

Methlphenidate-like effects of the new antidepressant drug nomifensine (HOE 984)

Nomifensine (HOE 984) belongs to a chemically new class of drugs with reported antidepressant properties. Nomifensine, like methylphemidate, d-amphetamine and apomorphine, induces strong, intense stereotypes behaviour in the rat. The nomifensine-induced stereotyped behaviour was completely antagonized by pretreatment with reserpine (7.5 mg/kg, 18 h) but not by short-time pretreatment with alpha-methyltyrosine (250 mg/kg, 2 h.) Nomifensine thus differs from d-amphetamine and apomorphine but resembles methylphenidate on stereotyped behaviour. Nominfensine, M1 (8-amino-2-methyl-4-(4-hydroxyphenyl)-1,2,3,4-tetrahydroisoquinoline fumarate) (Hoechst), methylphenidate and d-amphetamine induced a strong increase in the brain level of homovanillec acid (HVA), whereas the dopamine uptake inhibitor benztropine induced no changes in HVA and cocaine induced only a small increase. Nomifensine and the M1 metabolite, like methylphenidate, also increased 3,4-dihydroxyphenylacetic acid (DOPAC) whereas amphetamine, apomorphine, benztropine and cocaine decreased this dopamine metabolite. This suggests that the stereotyped licking and/or biting activities in the rat are related to dopamine releasing properties of nomifensine, methylphenidate and amphetamine. This is further supported by an inverse relationship between the in vitro dopamine uptake inhibitory concentrations and the sterotypy-inducing dose levels of nomifensine and d-amphetamine. Amphetamine caused a strong, and nomifensine and apormorphine a week increase in brain 3-methoxy-4-hydroxyphenylglycol (MOPEG).

ZK 91296, a partial agonist at benzodiazepine receptors

ZK 91296 (ethyl 5-benzyloxy-4-methoxymethyl-β-carboline-3-carboxylate) is a potent and selective ligand for benzodiazepine (BZ) receptors. Biochemical investigations indicate that ZK 91296 may be a partial agonist at BZ receptors. Such partial agonism may explain to some extent why ZK 91296 needs higher BZ receptor occupancy than diazepam for the same effect against chemical convulsants and for behavioural effects. The lack of sedatiye effects, and the very potent inhibition of reflex epilepsy, spontaneous epilepsy and DMCM-induced seizures suggest, furthermore, that ZK 91296 may possess pharmacological selectivity for a particular type of BZ receptor interaction, perhaps including topographic as well as receptor subtype differentiation.