John H. Kehne

Effects of the selective 5-HT2A receptor antagonist MDL 100,907 on MDMA-induced locomotor stimulation in rats

(±)3,4-Methylenedioxymethamphetamine (MDMA) releases dopamine and serotonin in vivo and stimulates locomotor activity. Previous work demonstrated that MDMA-stimulated dopamine release could be reduced by the selective 5-HT2A receptor antagonist [R-(+)-a-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol] (MDL 100,907). In the present study MDL 100,907 significantly reduced MDMA-stimulated locomotion without affecting basal levels of locomotion. Other agents with 5-HT2A antagonist activity (ritanserin, clozapine, MDL 28,133A, or methiothepin), as well as agents that block 5-HT1A-(propranolol), D2-(haloperidol), or D1 receptors (SCH 23390) also reduced MDMA-stimulated locomotion. Intraventricularly administered 5,7-dihydroxytryptamine decreased regional 5-HT levels and attenuated MDMA-stimulated locomotion. These data support the conclusion that serotonin released onto 5-HT2A receptors contributes to MDMA-stimulated locomotion and suggest that MDMA-stimulated locomotion may be useful as an in vivo behavioral measure of 5-HT2A antagonism. The data also support previous reports of contributions of 5-HT1A, D1 and D2 receptors to MDMA-stimulated locomotion. A preliminary time-course analysis indicating time-dependent contributions of different receptors to MDMA-stimulated locomotion suggests the potential utility of this model for characterizing potential atypical antipsychotic compounds.

Electrophysiological, biochemical and behavioral evidence for 5-HT2 and 5-HT3 mediated control of dopaminergic function

Several lines of evidence have suggested a link between serotonergic and dopaminergic systems in the brain. The interpretation of much of these early data needs careful reevaluation in light of the recent understanding of the plethora of serotonin receptor subtypes, their distribution in the brain and the new findings with more selective serotonin antagonists. Electrophysiological, biochemical and behavioral evidence obtained using highly selective antagonists of the 5-HT2 or 5-HT3 receptor subtypes, MDL 100,907 or MDL 73,147EF, respectively, supports the thesis that serotonin modulates the dopaminergic system. This modulation is most evident when the dopaminergic system has been activated.

Reversal of amphetamine-induced behaviours by MDL 100,907, a selective 5-HT2A antagonist

MDL 100,907 is a potent and selective antagonist of the 5-HT2A receptor which, unlike other antagonists at this receptor, has little affinity for the 5-HT2C receptor. We have investigated the antipsychotic potential of MDL 100,907 by examining its ability to antagonise different behavioural effects of amphetamine in rats. MDL 100,907 reversed the locomotor stimulant effects of amphetamine in rats without itself having any effect on locomotor activity. It also antagonised the disruptive effects of amphetamine on the development of latent inhibition. In contrast, MDL 100,907 had no effect on the discriminative stimulus properties of amphetamine, nor did it affect the ability of amphetamine to reduce the threshold required to sustain rewarding brain stimulation in the ventral tegmental area. This profile is different from that of typical and atypical neuroleptics, and also from other 5-HT2 receptor antagonists, which lack the selectivity of MDL 100,907. These results suggest that MDL 100,907 may have a unique interaction with dopaminergic systems and support the further development of selective 5-HT2 receptor antagonists as a novel therapeutic strategy for schizophrenia.

MDL 100,458 and MDL 102,288: two potent and selective glycine receptor antagonists with different functional profiles

Glycine receptor antagonists have been proposed to have multiple therapeutic applications, including the treatment of stroke, epilepsy, and anxiety. The present study compared the biochemical and behavioral profiles of two strychnine-insensitive glycine receptor antagonists, MDL 100,458 (3-(benzoylmethylamino)-6-chloro-1H-indole-2- carboxylic acid) and MDL 102,288 (5,7-dichloro-1,4-dihydro-4-[[[4- [(methoxycarbonyl)amino]phenyl]sulfonyl]imino]-2-quinolinecarboxylic acid monohydrate). Both compounds potently inhibited [3H]glycine binding to rat cortical/hippocampal membranes (Ki = 136, 167 nM, respectively) without showing significant activity in 18 other receptor binding assays. In an in vitro functional assay, both compounds completely antagonized N-methyl-D-aspartate (NMDA)-stimulated cGMP accumulation in rat cerebellar slices. However, in contrast to their equipotency in the glycine receptor assay, MDL 100,458 was approximately 6-fold more potent than MDL 102,288 in the cGMP assay (IC50 values = 1.25, 7.8 microM, respectively). Behavioral tests demonstrated that MDL 102,288 and MDL 100,458 exhibited strikingly different in vivo profiles. MDL 100,458 antagonized audiogenic seizures in DBA/2J mice (ED50 = 20.8 mg/kg i.p.), whereas MDL 102,288 was without effect in the dose range tested (ED50 > 300 mg/kg i.p.). Central nervous system penetration did not appear to account for this difference. For example, MDL 102,288 was not active following direct intracerebroventricular administration (ED50 > 16 micrograms; vs. 0.78 microgram for MDL 100,458). In a test of anxiolytic activity, MDL 102,288 reduced separation-induced ultrasonic vocalizations in rat pups (ED50 = 6.3 mg/kg i.p.) whereas MDL 100,458 was only weakly active (ED50 = 80.8 mg/kg i.p.). Furthermore, the anxiolytic effect of MDL 102,288 was selective in that it occurred at doses that did not produce motoric disruption as measured by an inclined-plane test (ED50 > 160 mg/kg; therapeutic index > 25.4). In contrast, the anxiolytic activity of MDL 100,458 was non-selective in that it occurred at doses that also produced motoric disruption (ED50 = 57.7 mg/kg; therapeutic index = 0.7). Thus, two glycine receptor antagonists which have similar in vitro binding profiles as selective ligands for the strychnine-insensitive glycine receptor, demonstrate different in vitro and in vivo functional profiles. The reason for these differences is not clear, though one possibility could be that the compounds may act on different NMDA receptor subtypes. These data support the possibility that different glycine receptor antagonists may have different therapeutic targets.

MDL 26,479: a potential cognition enhancer with benzodiazepine inverse agonist-like properties.

1 The present study investigated biochemical, electrophysiological and behavioural properties of the novel cognition enhancer, MDL 26,479 (5‐(3‐fluorophenyl)‐2,4,‐dimethyl‐3H‐1,2,4‐triazole‐3‐thione). 2 The 5‐aryl‐1,2,4‐triazole, MDL 26,479, potently (0.22 ± 0.05 mg kg−1) inhibited [3H]‐flumazenil (Ro15–1788) binding in mouse cortex but was ineffective in vitro at displacing radioligand binding to the GABAA receptor complex. 3 Parenteral administration of MDL 26,479 (1 mg kg−1) or the benzodiazepine (BZD) inverse agonist methyl 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM) (0.3 mg kg−1) increased cortical ex vivo binding of [3H]‐hemicholinium‐3 ([3H]‐HC‐3), a marker for cholinergic activation. This effect of MDL 26,479 was blocked by pretreatment with the antagonist flumazenil (1 mg kg−1). 4 MDL 26,479 (20 μm) and DMCM (1 μm) increased excitation in the hippocampal long‐term potentiation (LTP) slice preparation; however, unlike DMCM, the effect of MDL 26,479 was not blocked by flumazenil. 5 In behavioural studies, MDL 26,479 did not exhibit adverse properties characteristic of drugs associated with the GABAA receptor complex. It lacked convulsant, anxiogenic, anxiolytic, or depressant effects. Since MDL 26,479 lacks activity with the BZD receptor in vitro we suggest that it acts via the GABAA receptor complex at another site on this receptor or in an as yet undefined manner or an active metabolite is formed in vivo. 6 Previous work showed that MDL 26,479 enhances learning acquisition in animal models. The present study suggests that at least some of the cognition enhancing properties are due to the enhancement of cortical and hippocampal cholinergic function and LTP.

Mixed D2/5-HT2A Antagonism of Cocaine-Induced Facilitation of Brain Stimulation Reward

Previous behavioral, neurochemical and neurophysiological experiments have shown that selective 5-HT2A and mixed D2/5-HT2A antagonists can attenuate some, but not all, responses to amphetamine. The generality of these findings were determined in the present experiment by assessing the effect of mixed D2/5-HT2A antagonists on cocaine-induced facilitation of ventral tegmental area self-stimulation in rats. Although amphetamine and cocaine influence activity in monoaminergic neurons through different mechanisms, our previous research has shown that selective D2 and 5-HT2A antagonists have similar effects on behavioral responses to these psychostimulants. Therefore, we expected a similar pattern of results using mixed D2/5-HT2A antagonists. As shown previously, cocaine decreased self-stimulation threshold in a dose-dependent manner. Haloperidol and the mixed D2/5-HT2A antagonists risperidone and MDL 28, 133A antagonized cocaine-induced facilitation of self-stimulation, but only at doses that increased baseline self-stimulation threshold. There was a significant correlation (r = 0.87, p < 0.001) between antagonist-induced change in baseline threshold and attenuation of cocaines effect on threshold. Taken together, the results of this and previous experiments support the importance of D2 receptors in the mechanisms of brain stimulation reward. 5-HT2A receptors appear not to be involved in mediation of both brain stimulation reward and amphetamine- and cocaine-induced facilitation of brain stimulation reward.

MDL 27,531 selectively reverses strychnine-induced seizures in mice

1 Strychnine‐sensitive glycine receptors are primarily localized in the brainstem and spinal cord where they are the major mediators of postsynaptic inhibition. A compound which acts functionally like a glycine receptor agonist would be potentially useful as a pharmacological tool and as a therapeutic agent for treating disorders of glycinergic transmission. 2 MDL 27,531 (4‐methyl‐3‐methylsulphonyl‐5‐phenyl‐4H‐1,2,4‐triazole) blocked strychnine‐induced tonic extensor seizures in mice following either intraperitoneal (ED50 = 12.8 mg kg−1; 30 min) or oral (ED50 = 7.3 mg kg−1; 30 min) administration. Time course studies revealed a maximal effect at 30–60 min, though significant activity was still seen after 24 h. 3 MDL 27,531 was selective in antagonizing strychnine seizures and little or no activity was seen against seizures produced by other chemical convulsants (bicuculline; quinolinic acid; mercaptopropionic acid); by electrical stimuli (maximal electroshock); or by sensory stimuli (audiogenic seizure susceptible mice). MDL 27,531 blocked pentylenetetrazol‐induced seizures with an ED50 = 55 mg kg−1. This profile differed from those of the anticonvulsants diazepam, valproic acid, and diphenylhydantoin. 4 The antagonism of strychnine seizures by MDL 27,531 occurred at doses that did not produce signs of sedation (suppression of spontaneous motor activity), motor ataxia (disruption of rotarod performance), muscle relaxation (inhibition of morphine‐induced Straub tail), or CNS depression (potentiation of hexobarbitone sleep time). MDL 27,531 had less side effect potential (as derived from ratios obtained from the above measures) relative to those of the known muscle relaxants diazepam and baclofen. 5 Although MDL 27,531 behaved functionally like a selective agonist at the strychnine‐sensitive glycine receptor, the compound did not alter the in vitro binding of [3H]‐strychnine to mice brainstem/spinal cord membranes at concentrations of up to 100 μm. In further in vitro binding assays, MDL 27,531 at concentrations of up to 100 μm, did not displace the binding of [3H]‐muscimol, [3H]‐flunitrazepam, or [35S]‐t‐butylbicyclophosphorthionate to rat cortical membranes. These ligands bind to the γ‐aminobutyric acid (GABA), benzodiazepine, and picrotoxin‐convulsant binding sites, respectively. 6 MDL 27,531 (10–100 mg kg−1, i.p.) enhanced binding of the benzodiazepine antagonist [3H]‐Ro 15–1788 to mouse cerebral cortex in vivo without directly affecting GABA levels. 7 Ro 15–1788 (16, 32 mg kg−1) significantly blocked the MDL 27,531 antagonism of strychnine‐induced seizures, though this antagonism was not competitive. The same doses of Ro 15–1788 produced parallel rightward shifts in the dose‐response curves for diazepam inhibition of pentylenetetrazol‐induced seizures, consistent with a competitive antagonism. 8 Thus, MDL 27,531 acts functionally like an agonist at the strychnine‐sensitive glycine receptor in its capacity to reverse selectively strychnine‐induced seizures. Though the precise mechanism of action of MDL 27,531 is unknown, MDL 27,531 may act at an allosteric site on the strychnine‐sensitive receptor which produces agonist‐like activity.

MDL 27,531 reduces spontaneous hindlimb contractions in rats with chronic transections of the spinal cord

Disrupted glycinergic inhibition in the brainstem and spinal cord may contribute to some of the alterations in reflex control seen in patients with spastic disorders. MDL 27,531, which acts functionally like a glycine agonist in its capacity to selectively reverse seizures produced by the glycine antagonist strychnine, was evaluated in a model of spinal injury-induced reflex dysfunction. Rats recovering chronically from complete spinal cord transections exhibited intermittent contractions of the paralyzed hindlimbs, as measured with an automated apparatus. MDL 27,531 selectively decreased these hindlimb contractions, as did the clinically demonstrated antispastic agent clonidine. In its therapeutic dose range, clonidine, but not MDL 27,531, produced ataxia in non-transected rats. These data suggest that MDL 27,531 may be a useful therapeutic agent for the treatment of dysfunctions of reflex control seen in spastic disorders of spinal origin, with potentially fewer side effects than are seen with existing drug therapies.