Karen A. Maubach

An inverse agonist selective for alpha5 subunit-containing GABAA receptors enhances cognition.

α5IA is a compound that binds with equivalent subnanomolar affinity to the benzodiazepine (BZ) site of GABAA receptors containing an α1, α2, α3, or α5 subunit but has inverse agonist efficacy selective for the α5 subtype. As a consequence, the in vitro and in vivo effects of this compound are mediated primarily via GABAA receptors containing an α5 subunit. In a mouse hippocampal slice model, α5IA significantly enhanced the θ burst-induced long-term potentiation of the excitatory postsynaptic potential in the CA1 region but did not cause an increase in the paroxysmal burst discharges that are characteristic of convulsant and proconvulsant drugs. These in vitro data suggesting that α5IA may enhance cognition without being proconvulsant were confirmed in in vivo rodent models. Hence, α5IA significantly enhanced performance in a rat hippocampal-dependent test of learning and memory, the delayed-matching-to-position version of the Morris water maze, with a minimum effective oral dose of 0.3 mg/kg, which corresponded to a BZ site occupancy of 25%. However, in mice α5IA was not convulsant in its own right nor did it potentiate the effects of pentylenetetrazole acutely or produce kindling upon chronic dosing even at doses producing greater than 90% occupancy. Finally, α5IA was not anxiogenic-like in the rat elevated plus maze nor did it impair performance in the mouse rotarod assay. Together, these data suggest that the GABAA α5-subtype provides a novel target for the development of selective inverse agonists with utility in the treatment of disorders associated with a cognitive deficit.

Chronic substance P (NK1) receptor antagonist and conventional antidepressant treatment increases burst firing of monoamine neurones in the locus coeruleus

The mechanism of action of conventional antidepressants (e.g. imipramine) has been linked to modulation of central monoamine systems. Substance P (NK1) receptor antagonists may have antidepressant and anxiolytic effects in patients with major depressive disorder and high anxiety but, unlike conventional antidepressants, are independent of activity at monoamine reuptake sites, transporters, receptors, or monoamine oxidase. To investigate the possibility that substance P receptor antagonists influence central monoamine systems indirectly, we have compared the effects of chronic administration of imipramine with that of the substance P receptor antagonist L-760735 on the spontaneous firing activity of locus coeruleus neurones. Electrophysiological recordings were made from brain slices prepared from guinea-pigs that had been dosed orally every day for 4 weeks with either L-760735 (3 mg/kg), imipramine (10 mg/kg), or vehicle (water), or naive animals. Chronic, but not acute, treatment with the substance P receptor antagonist L-760735, induced burst firing of neurones in the locus coeruleus. This effect resembles that of the conventional antidepressant imipramine. However, their effects are dissociable since, in contrast to chronic imipramine treatment, chronic L-760735 treatment does not cause functional desensitisation of somatic alpha2 adrenoceptors. The mechanism by which chronic substance P receptor antagonist or conventional antidepressant treatment influences the pattern of firing activity of norepinephrine neurones remains to be elucidated. However, an indirect action in the periphery or distant brain nuclei has been excluded by the use of the in vitro slice preparation, suggesting a local site of action in the locus coeruleus.

Identification of amino acid residues responsible for the α5 subunit binding selectivity of L‐655,708, a benzodiazepine binding site ligand at the GABAA receptor

L‐655,708 is a ligand for the benzodiazepine site of the γ‐aminobutyric acid type A (GABAA) receptor that exhibits a 100‐fold higher affinity for α5‐containing receptors compared with α1‐containing receptors. Molecular biology approaches have been used to determine which residues in the α5 subunit are responsible for this selectivity. Two amino acids have been identified, α5Thr208 and α5Ile215, each of which individually confer approximately 10‐fold binding selectivity for the ligand and which together account for the 100‐fold higher affinity of this ligand at α5‐containing receptors. L‐655,708 is a partial inverse agonist at the GABAA receptor which exhibited no functional selectivity between α1‐ and α5‐containing receptors and showed no change in efficacy at receptors containing α1 subunits where amino acids at both of the sites had been altered to their α5 counterparts (α1ΔSer205‐Thr,Val212‐Ile). In addition to determining the binding selectivity of L‐655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABAA receptor, and further delineate a region just N‐terminal to the first transmembrane domain of the receptor α subunit that contributes to this binding site.

Novel strategies for pharmacotherapy of depression

Modulating monoamine activity as a therapeutic strategy continues to dominate antidepressant research, with a recent emphasis on agents with multiple targets, including combined serotonin/noradrenaline re-uptake inhibitors and numerous serotonin receptor ligands. An important new development has been the emergence of potential novel mechanisms of action, notably modulation of the activity of neuropeptides substance P and corticotrophin-releasing factor, and the intracellular messenger cyclic adenosine monophosphate. Efforts in this area have recently been rewarded by the demonstration of antidepressant efficacy of the substance P receptor antagonist MK-0869.

Substance P stimulates inhibitory synaptic transmission in the guinea pig basolateral amygdala in vitro

To determine the physiological role of tachykinin NK1 receptors in the basolateral nucleus of the amygdala (BLN) we have studied the electrophysiological effects of substance P (SP) in the absence and presence of selective tachykinin receptor antagonists in guinea pig brain slices. Recordings were made from two populations of neurones; spiny pyramidal and stellate neurones, both thought to be projection neurones. Activation of NK1 receptors with SP increased the frequency of spontaneous inhibitory postsynaptic potentials in the majority of cells. This effect was blocked by bicuculline or tetrodotoxin, but not ionotropic glutamate receptor antagonists. The enhanced synaptic activity induced by SP was antagonised by the NK1 receptor antagonist L-760,735 but not by the less active enantiomer L-781,773 or the NK3 receptor antagonist L-769,927. Thus in the basolateral nucleus of the guinea pig amygdala, NK1 receptor activation preferentially stimulates inhibitory synaptic activity. Consistent with this observation, immunohistochemistry revealed NK1 receptor immunoreactivity to be largely restricted to a subset of GABA interneurones. These studies support a physiological role for SP in the regulation of pathways involved in the control of emotional behaviour.

In vitro and in vivo properties of 3-tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d]-[1,2,4]triazine (MRK-016), a GABAA receptor alpha5 subtype-selective inverse agonist

3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human α1-, α2-, α3-, and α5-containing GABAA receptors. It has inverse agonist efficacy selective for the α5 subtype, and this α5 inverse agonism is greater than that of the prototypic α5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (α5IA). Consistent with its greater α5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than α5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC50 value of 15 ng/ml that was similar to the rhesus monkey plasma EC50 value of 21 ng/ml obtained using [11C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3–0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species (∼3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.

Role of the histidine residue at position 105 in the human α5 containing GABAA receptor on the affinity and efficacy of benzodiazepine site ligands

A histidine residue in the N‐terminal extracellular region of α1,2,3,5 subunits of the human GABAA receptor, which is replaced by an arginine in α4 and α6 subunits, is a major determinant for high affinity binding of classical benzodiazepine (BZ)‐site ligands. The effect of mutating this histidine at position 105 in the α5 subunit to an arginine (α5H105R) on BZ‐site pharmacology has been investigated using radioligand binding on HEK293 and L(tk‐) cells and two electrode voltage clamp recording on Xenopus oocytes in which GABAA receptors of subtypes α5, α5H105R, α4 and α6 were co‐expressed with β3γ2s. The classical BZs, diazepam and flunitrazepam (full agonists on the α5 receptor) showed negligible affinity and therefore negligible efficacy on α5H105R receptors. The β‐carbolines DMCM and βCCE (inverse agonists on the α5 receptor) retained some affinity but did not exhibit inverse agonist efficacy at α5H105R receptors. Therefore, the α5H105R mutation confers an α4/α6‐like pharmacology to the classical BZs and β‐carbolines. Ro15‐4513, flumazenil, bretazenil and FG8094, which share a common imidazobenzodiazepine core structure, retained high affinity and were higher efficacy agonists on α5H105R receptors than would be predicted from an α4/α6 pharmacological profile. This effect was antagonized by DMCM, which competes for the BZ‐site and therefore is likely to be mediated via the BZ‐site. These data indicate that the conserved histidine residue in the α subunit is not only a key determinant in the affinity of BZ‐site ligands on α5 containing GABAA receptors, but also influences ligand efficacy.

Triazolam suppresses the induction of hippocampal long-term potentiation.

Benzodiazepines are sedative hypnotics that produce marked anterograde amnesia in humans. These pharmacological properties are thought to result from the potentiation of GABA-A receptor function and subsequent attenuation of long-term potentiation (LTP), however many reports have suggested this is not the case for triazolam. Using electrophysiological recordings in a cell line expressing recombinant GABA-A receptors, we confirm that triazolam is an efficacious positive allosteric modulator of GABA-A receptors. Triazolam also slowed the decay of spontaneous inhibitory synaptic currents, reduced the amplitude of fEPSPs elicited during a theta burst and reduced the magnitude of LTP in hippocampal CA1 neurones in vitro. These data show that triazolam modifies LTP induction consistent with an enhancement of GABA-A receptor function via activation of the allosteric benzodiazepine-site.

Psychiatric drug discovery and development.

This new conference on Psychiatric Drug Research was organised by the Strategic Research Institute and was chaired by P McGonigle (Wyeth Research, USA) and D Schoepp (Eli Lilly, USA). The 2-day meeting featured presentations from an international assembly of industrial and academic experts who have significantly contributed to the current body of knowledge in the field of psychotherapeutics. D Weinberger (NIMH, USA) gave an elegant keynote lecture on the application of genomics in psychopharmacology. Other presentations covered the latest technological advances, animal models and mechanistic approaches utilised in drug discovery for neuropsychiatric disorders and reviewed the current status of numerous novel targets resulting from these strategies.