ABSTRACT
The in vivo occupancy of brain benzodiazepine binding sites by compounds A and B was measured using a [(3)H]Ro 15-1788 binding assay and related to plasma and brain drug concentrations. The plasma concentration associated with 50% occupancy was higher for compound A than compound B (73 and 3.7 nM, respectively), however, there was little difference in the brain concentrations required (73 and 63 nM). Both compounds showed a non-linear relationship between plasma and brain concentrations such that above brain concentrations of approximately 100 nM increasing plasma concentrations did not result in a concomitant increase in brain concentrations. This is consistent with brain concentrations being dependent on a saturable compartment which was postulated to be the benzodiazepine binding site-containing GABA(A) receptors. This hypothesis was tested in alpha1H101R mice, in which the alpha1 subunit of the GABA(A) receptor is rendered insensitive to benzodiazepine binding resulting in an approximate 50% reduction in the total benzodiazepine-containing GABA(A) receptor population. It was shown that the Occ(50) brain concentrations in the alpha1H101R animals was lower (17 nM) than in wild type mice (63 nM), as was the plateau concentration in the brain (105 and 195 nM, respectively). These data suggest measured concentrations of compounds A and B in brain tissue are dependent on receptor expression with a minimal contribution from unbound and non-specifically bound compound.
Subject(s)
Benzodiazepines/metabolism , Brain/metabolism , Ligands , Receptors, GABA-A/metabolism , Administration, Oral , Animals , Anti-Anxiety Agents/administration & dosage , Anti-Anxiety Agents/metabolism , Anti-Anxiety Agents/pharmacokinetics , Anticonvulsants/administration & dosage , Anticonvulsants/metabolism , Anticonvulsants/pharmacokinetics , Benzodiazepines/administration & dosage , Benzodiazepines/pharmacokinetics , Benzodiazepinones/administration & dosage , Benzodiazepinones/metabolism , Benzodiazepinones/pharmacokinetics , Binding Sites , Binding, Competitive/drug effects , Brain/drug effects , Brain Chemistry , Dose-Response Relationship, Drug , Flumazenil/administration & dosage , Flumazenil/metabolism , Flumazenil/pharmacokinetics , Flunitrazepam/administration & dosage , Flunitrazepam/metabolism , Flunitrazepam/pharmacokinetics , GABA Modulators/administration & dosage , GABA Modulators/metabolism , GABA Modulators/pharmacokinetics , GABA-A Receptor Agonists , Injections, Intravenous , Male , Mice , Mice, Mutant Strains , Pyridazines/administration & dosage , Pyridazines/metabolism , Pyridazines/pharmacokinetics , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/genetics , Tissue Distribution , Triazines/administration & dosage , Triazines/metabolism , Triazines/pharmacokineticsABSTRACT
In the present study, the occupancy of flumazenil (Ro 15-1788; 1-30mg/kg p.o.) at the benzodiazepine site of rat brain GABA(A) receptors was compared using in vivo and ex vivo binding methodologies with [(3)H]flumazenil as the radioligand. Animals either received tracer quantities of [(3)H]flumazenil 3min before being killed for the in vivo binding, or were killed and brain homogenates incubated with 1.8nM [(3)H]flumazenil. The flumazenil dose required to inhibit in vivo binding of [(3)H]flumazenil by 50% (ID(50)) was 2.0mg/kg, which represents the most accurate measure of benzodiazepine site occupancy by flumazenil in vivo. Occupancy measured in crude brain homogenates using the ex vivo method was time dependent with a 3mg/kg dose giving occupancies of 77% and 12% using 0.5 or 60min ex vivo incubations times, respectively, presumably due to dissociation from the binding site during the ex vivo incubation. When incubation time was minimised (0.5min), and despite being under non-equilibrium conditions, the ex vivo method gave an ID(50) of 1.5mg/kg which was not too dissimilar from that observed using in vivo binding (2.0mg/kg). As expected, ex vivo binding can give an underestimation of receptor occupancy but this can be minimised by careful attention to the kinetics of unlabelled drug and radioligand.
Subject(s)
Brain Chemistry/drug effects , Flumazenil/metabolism , GABA Modulators/metabolism , Radiopharmaceuticals/metabolism , Receptors, GABA-A/drug effects , Receptors, GABA-A/metabolism , Animals , Binding Sites/drug effects , Brain/drug effects , Brain/metabolism , Dose-Response Relationship, Drug , Flumazenil/pharmacokinetics , GABA Modulators/pharmacokinetics , In Vitro Techniques , Male , Rats , Rats, Sprague-DawleyABSTRACT
The development of a series of GABA(A) alpha2/alpha3 subtype selective pyridazine based benzodiazepine site agonists as anxiolytic agents with reduced sedative/ataxic potential is described, including the discovery of 16, a remarkably alpha3-selective compound ideal for in vivo study. These ligands are antagonists at the alpha1 subtype, with good CNS penetration and receptor occupancy, and excellent oral bioavailability.
Subject(s)
Anti-Anxiety Agents/pharmacology , Anxiety/drug therapy , GABA Agonists/pharmacology , GABA-A Receptor Agonists , Pyridazines/pharmacology , Animals , Anti-Anxiety Agents/administration & dosage , Anti-Anxiety Agents/chemical synthesis , Binding Sites , GABA Agonists/administration & dosage , GABA Agonists/chemical synthesis , Humans , Ligands , Molecular Structure , Pyridazines/administration & dosage , Pyridazines/chemical synthesis , Rats , Recombinant Proteins/agonists , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The identification of a series of imidazo[1,2-b][1,2,4]triazines with high affinity and functional selectivity for the GABA(A) alpha3-containing receptor subtype is described, leading to the identification of a clinical candidate, 11. Compound 11 shows good bioavailability and half-life in preclinical species, and it is a nonsedating anxiolytic in both rat and squirrel monkey behavioral models.
Subject(s)
Anti-Anxiety Agents/chemical synthesis , GABA-A Receptor Agonists , Imidazoles/chemical synthesis , Triazines/chemical synthesis , Animals , Anti-Anxiety Agents/chemistry , Anti-Anxiety Agents/pharmacology , Biological Availability , Half-Life , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Patch-Clamp Techniques , Radioligand Assay , Rats , Receptors, GABA-A/physiology , Saimiri , Structure-Activity Relationship , Triazines/chemistry , Triazines/pharmacologyABSTRACT
The pharmacokinetic and pharmacodynamic (i.e., receptor occupancy) properties of L-655,708, a compound with selectivity for alpha5-over alpha1-, alpha2-, and alpha3-containing GABA(A) receptors, were examined in rats with the aim of developing a formulation that would give sustained (up to 6 h) and selective occupancy of alpha5-containing GABA(A) receptors suitable for behavioral studies. Standard rat pharmacokinetic analyses showed that L-655,708 has a relatively short half-life with kinetics in the brain mirroring those in the plasma. In vivo binding experiments showed that plasma concentrations of around 100 ng/ml gave relatively selective in vivo occupancy of rat brain alpha5-versus alpha1-, alpha2-, and alpha3-containing GABA(A) receptors. Therefore, this plasma concentration was chosen as a target to achieve relatively selective occupancy of alpha5-containing receptors using s.c. implantations of L-655,708 (0.4, 1.5, or 2.0 mg) formulated into tablets of various size (20 or 60 mg) containing different amounts of L-655,708 and combinations of low and high viscosity hydroxypropyl methylcellulose (LV- and HV-HPMC). The optimum formulation, 1.5 mg of L-655,708 compressed into a 60-mg tablet with 100% HV-HPMC, resulted in relatively constant plasma concentrations being maintained for at least 6 h with very little difference between C(max) concentrations (125-150 ng/ml) and plateau concentrations (100-125 ng/ml). In vivo binding experiments confirmed the selective occupancy of rat brain alpha5-over alpha1-, alpha2-, and alpha3-containing GABA(A) receptors.
Subject(s)
GABA Antagonists/pharmacology , GABA Antagonists/pharmacokinetics , Imidazoles/pharmacology , Imidazoles/pharmacokinetics , Receptors, GABA-A/metabolism , Administration, Oral , Animals , Chemistry, Pharmaceutical , Delayed-Action Preparations , Half-Life , Injections, Intravenous , Injections, Subcutaneous , Male , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/drug effects , TabletsABSTRACT
Imidazo[1,2-a]pyrimidines and imidazo[1,2-b][1,2,4]triazines are ligands for the benzodiazepine binding site of GABA(A) receptors that are functionally selective for the alpha2/alpha3 subtypes over the alpha1 subtype. SAR studies to optimise this functional selectivity, pharmacokinetic and behavioural data are described.
Subject(s)
Anxiety Disorders/drug therapy , GABA-A Receptor Agonists , Imidazoles/pharmacology , Protein Subunits/agonists , Triazines/pharmacology , Administration, Oral , Animals , Ataxia/drug therapy , Binding Sites , Biological Availability , Disease Models, Animal , Dogs , Female , GABA Agonists/pharmacokinetics , GABA Agonists/pharmacology , Imidazoles/pharmacokinetics , Male , Maze Learning/drug effects , Molecular Structure , Protein Subunits/metabolism , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/metabolism , Saimiri , Structure-Activity Relationship , Triazines/pharmacokineticsABSTRACT
A series of high-affinity GABA(A) agonists with good oral bioavailability in rat and dog and functional selectivity for the GABA(A)alpha2 and -alpha3 subtypes is reported. The 7-trifluoromethylimidazopyrimidine 14g and the 7-propan-2-olimidazopyrimidine 14k are anxiolytic in both conditioned and unconditioned animal models of anxiety with minimal sedation observed at full BZ binding site occupancy.
Subject(s)
Anxiety Disorders/drug therapy , GABA-A Receptor Agonists , Pyrimidines/pharmacology , Administration, Oral , Animals , Binding Sites , Biological Availability , Cell Line , Disease Models, Animal , Dogs , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Humans , Molecular Structure , Patch-Clamp Techniques , Pyrimidines/chemical synthesis , Pyrimidines/pharmacokinetics , Rats , Receptors, GABA-A , Structure-Activity RelationshipABSTRACT
The cyclopyrrolone pagoclone binds with roughly equivalent high affinity (0.7-9.1nM) to the benzodiazepine binding site of human recombinant GABA(A) receptors containing either an alpha1, alpha2, alpha3 or alpha5 subunit. However, whereas it was a partial agonist at alpha1-, alpha2- and alpha5-containing GABA(A) receptors, pagoclone was a full agonist at receptors containing an alpha3 subunit. In the rat elevated plus maze assay pagoclone (3mg/kg) had significant anxiolytic-like activity but at all three doses tested (0.3, 1 and 3mg/kg p.o.) it produced a significant reduction in the total distance travelled. This sedative-like effect was confirmed in rat chain-pulling and spontaneous locomotor assays. Surprisingly, in the plasma and brain samples derived from the elevated plus maze assay, the major metabolite of pagoclone, 5'-hydroxy pagoclone, was present at 10-20-fold higher concentrations relative to the parent compound. In order to establish whether this metabolite might have pharmacological activity, we measured its affinity and efficacy profile and found that both were comparable to those of pagoclone with the exception that efficacy at the alpha1 subtype was considerably greater for 5'-hydroxy pagoclone compared with the parent. This metabolite had significant anxiolytic-like activity in the elevated plus maze but at these same doses (0.3-3mg/kg p.o.) also produced sedation. It is therefore likely that in rats 5'-hydroxy pagoclone mediates the majority of the pharmacological actions following pagoclone administration.
Subject(s)
Behavior, Animal/drug effects , Naphthyridines/pharmacology , Animals , Binding Sites/drug effects , Brain/metabolism , Cell Line , Dose-Response Relationship, Drug , Fibroblasts , Flumazenil/pharmacology , GABA Modulators/pharmacology , Humans , Indoles/pharmacokinetics , Isoindoles , Isomerism , Male , Maze Learning/drug effects , Mice , Motor Activity/drug effects , Naphthyridines/blood , Naphthyridines/chemistry , Naphthyridines/pharmacokinetics , Protein Subunits/metabolism , Psychomotor Performance/drug effects , Rats , Rats, Sprague-Dawley , Receptors, GABA-A/chemistry , Receptors, GABA-A/drug effects , Time Factors , Tritium/pharmacokineticsABSTRACT
7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) is a triazolopyridazine that binds with equivalent high (subnanomolar) affinity to the benzodiazepine binding site of recombinant human GABA(A) receptors containing an alpha1, alpha2, alpha3, or alpha5 subunit but has partial agonist efficacy at the alpha2 and alpha3 subtypes and essentially antagonist efficacy at the alpha1 and alpha5 subtypes. In rats, TPA023 gave time- and dose-dependent occupancy after oral dosing, with 50% occupancy corresponding to a dose of 0.42 mg/kg. It has anxiolytic-like activity in unconditioned (elevated plus maze) and conditioned (fear-potentiated startle and conditioned suppression of drinking) rat models of anxiety with minimum effective doses (MED; 1-3 mg/kg) corresponding to 70 to 88% occupancy. However, there was no appreciable sedation in a response sensitivity (chain-pulling) assay at a dose of 30 mg/kg, resulting in 99% occupancy. Similarly, TPA023 was robustly anxiolytic in the squirrel monkey conditioned emotional response assay, with a MED of 0.3 mg/kg, but did not produce any sedation in a lever-pressing test of sedation even at 10 mg/kg. TPA023 produced no impairment in performance in the mouse Rotarod assay, and there was only a mild interaction with ethanol. In addition to anxiolytic-like efficacy, TPA023 had anticonvulsant activity in a mouse pentylenetetrazole seizure model. Finally, TPA023 did not cause precipitated withdrawal in mice treated for 7 days with the nonselective agonist triazolam, nor did N-methyl-beta-carboline-3-carboxamide (FG 7142) precipitate withdrawal in mice treated for 7 days with TPA023. In summary, the novel alpha2/alpha3-selective efficacy profile of TPA023 translates into a nonsedating anxiolytic profile that is distinct from nonselective agonists.
Subject(s)
Adrenergic alpha-Agonists/pharmacology , Anti-Anxiety Agents , GABA-A Receptor Agonists , Pyridazines/pharmacology , Triazoles/pharmacology , Acoustic Stimulation , Adrenergic alpha-1 Receptor Antagonists , Animals , Anticonvulsants/pharmacology , Autoradiography , Conditioning, Operant/drug effects , Convulsants/pharmacology , Drinking/drug effects , Emotions/drug effects , Fear/drug effects , Flumazenil/metabolism , GABA Modulators/metabolism , Humans , Male , Mice , Pentylenetetrazole/antagonists & inhibitors , Postural Balance/drug effects , Protein Binding , Radioligand Assay , Rats , Rats, Sprague-Dawley , Receptors, GABA-A , Recombinant Proteins/metabolism , Reflex, Startle/drug effects , Saimiri , Substance Withdrawal Syndrome/psychologyABSTRACT
(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 (1) was recently identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors and enhances performance in animal models of cognition. The routes of metabolism of this compound in vivo in rat have been well characterised, the identities of the major metabolites are confirmed by synthesis and their biological profiles were evaluated. An unusual oxidation of the pyrazolo[1,5-d][1,2,4]triazine core to the corresponding pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one scaffold by aldehyde oxidase has been observed.
Subject(s)
GABA Agonists/metabolism , GABA Agonists/pharmacokinetics , GABA-A Receptor Agonists , Isoxazoles/metabolism , Isoxazoles/pharmacokinetics , Protein Subunits/agonists , Triazines/metabolism , Triazines/pharmacokinetics , Animals , Dogs , Dose-Response Relationship, Drug , GABA Agonists/chemical synthesis , Hydrolysis , Isoxazoles/chemical synthesis , Macaca mulatta , Mice , Molecular Conformation , Rats , Stereoisomerism , Structure-Activity Relationship , Tissue Distribution , Triazines/chemical synthesisABSTRACT
The GABA(A) receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that alpha2-rather than alpha3-containing GABA(A) receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an alpha3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2'-difluoro-5'-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an alpha3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders alpha2-containing receptors BZ insensitive (alpha2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of alpha3-containing GABA(A) receptors is sufficient to produce the anxiolytic effects of BZs and that alpha2 potentiation may not be necessary.
Subject(s)
Anti-Anxiety Agents/therapeutic use , Benzodiazepines/therapeutic use , Protein Subunits/physiology , Receptors, GABA-A/physiology , Animals , Anti-Anxiety Agents/pharmacology , Anxiety/drug therapy , Anxiety/metabolism , Benzodiazepines/pharmacology , Dose-Response Relationship, Drug , GABA-A Receptor Agonists , Humans , Male , Mice , Mice, Transgenic , Protein Binding/physiology , Rats , Rats, Sprague-Dawley , SaimiriABSTRACT
There is increasing evidence that compounds with selectivity for gamma-aminobutyric acid(A) (GABA(A)) alpha2- and/or alpha3-subtypes may retain the desirable anxiolytic activity of nonselective benzodiazepines but possess an improved side effect profile. Herein we describe a novel series of GABA(A) alpha2/alpha3 subtype-selective agonists leading to the identification of the development candidate 17, a nonsedating anxiolytic in preclinical animal assays.
Subject(s)
Anti-Anxiety Agents/chemical synthesis , GABA-A Receptor Agonists , Hypnotics and Sedatives/chemical synthesis , Pyridazines/chemical synthesis , Triazoles/chemical synthesis , Animals , Anti-Anxiety Agents/chemistry , Anti-Anxiety Agents/pharmacology , Binding, Competitive , Cell Line , Dogs , GABA-A Receptor Antagonists , Half-Life , Humans , Hypnotics and Sedatives/chemistry , Hypnotics and Sedatives/pharmacology , Mice , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Primates , Pyridazines/chemistry , Pyridazines/pharmacology , Radioligand Assay , Rats , Receptors, GABA-A/physiology , Recombinant Proteins/agonists , Structure-Activity Relationship , Triazoles/chemistry , Triazoles/pharmacology , XenopusABSTRACT
Classical benzodiazepines (BZs), such as diazepam, bind to GABAA receptors containing alpha1, alpha2, alpha3 or alpha5 subunits that are therefore described as diazepam-sensitive (DS) receptors. However, the corresponding binding site of GABAA receptors containing either an alpha4 or alpha6 subunit do not bind the classical BZs and are therefore diazepam-insensitive (DIS) receptors; a difference attributable to a single amino acid (histidine in alpha1, alpha2, alpha3 and alpha5 subunits and arginine in alpha4 and alpha6). Unlike classical BZs, the imidazobenzodiazepines Ro 15-4513 and bretazenil bind to both DS and DIS populations of GABAA receptors. In the present study, an in vivo assay was developed using lorazepam to fully occupy DS receptors such that [3H]Ro 15-4513 was then only able to bind to DIS receptors. When dosed i.v., [3H]Ro 15-4513 rapidly entered and was cleared from the brain, with approximately 70% of brain radioactivity being membrane-bound. Essentially all membrane binding to DS+DIS receptors could be displaced by unlabelled Ro 15-4513 or bretazenil, with respective ID50 values of 0.35 and 1.2 mg kg(-1). A dose of 30 mg kg(-1) lorazepam was used to block all DS receptors in a [3H]Ro 15-1788 in vivo binding assay. When predosed in a [3H]Ro 15-4513 binding assay, lorazepam blocked [3H]Ro 15-4513 binding to DS receptors, with the remaining binding to DIS receptors accounting for 5 and 23% of the total (DS plus DIS) receptors in the forebrain and cerebellum, respectively. The in vivo binding of [3H]Ro 15-4513 to DIS receptors in the presence of lorazepam was confirmed using alpha1H101R knock-in mice, in which alpha1-containing GABAA receptors are rendered diazepam insensitive by mutation of the histidine that confers diazepam sensitivity to arginine. In these mice, and in the presence of lorazepam, there was an increase of in vivo [3H]Ro 15-4513 binding in the forebrain and cerebellum from 4 and 15% to 36 and 59% of the total (i.e. DS plus DIS) [3H]Ro 15-4513 binding observed in the absence of lorazepam.
Subject(s)
Azides/pharmacology , Benzodiazepines/pharmacology , Receptors, GABA-A/metabolism , Animals , Azides/metabolism , Benzodiazepines/metabolism , Benzodiazepinones/metabolism , Benzodiazepinones/pharmacology , Binding, Competitive/drug effects , Cerebellum/drug effects , Cerebellum/metabolism , Flumazenil/metabolism , Flumazenil/pharmacology , Humans , Isotope Labeling , Ligands , Lorazepam/pharmacology , Male , Mice , Mice, Transgenic , Prosencephalon/drug effects , Prosencephalon/metabolism , Receptors, GABA-A/genetics , Receptors, GABA-B/metabolism , TritiumABSTRACT
L-655,708 is an imidazobenzodiazepine possessing 30-70-fold selectivity for the benzodiazepine binding site of GABA(A) receptors containing an alpha5 rather than alpha1, alpha2 or alpha3 subunit. In the present study, [(3)H]L-655,708 was used to label mouse brain benzodiazepine binding sites in vivo. When compared to inhibition of in vivo binding of the non-selective ligand [(3)H]Ro 15-1788, the pharmacology of mouse in vivo [(3)H]L-655,708 binding was consistent with selective in vivo labelling of alpha5 subunit-containing GABA(A) receptors. Thus, diazepam was equipotent at inhibiting in vivo [(3)H]L-655,708 and [(3)H]Ro 15-1788 binding; zolpidem, which has very low affinity for alpha5-containing GABA(A) receptors, gave no inhibition of in vivo [(3)H]L-655,708 binding despite inhibiting in vivo [(3)H]Ro 15-1788 binding; and L-655,708 was more potent at inhibiting the in vivo binding of [(3)H]L-655,708 compared to [(3)H]Ro 15-1788. This pharmacological specificity of in vivo [(3)H]L-655,708 binding was confirmed autoradiographically. Hence, the anatomical distribution of in vivo [(3)H]L-655,708 binding was comparable to the distribution of alpha5-containing GABA(A) receptors identified in vitro. Moreover, this distribution was distinct from that identified using [(3)H]Ro 15-1788. These data therefore suggest that [(3)H]L-655,708 can be used to identify alpha5-containing GABA(A) receptors in vivo and that this ligand can be used to measure receptor occupancy of alpha5-selective ligands.
Subject(s)
Imidazoles/pharmacology , Receptors, GABA-A/metabolism , Animals , Benzodiazepines/metabolism , Binding Sites , Binding, Competitive , Brain/drug effects , Dose-Response Relationship, Drug , Flumazenil/pharmacology , GABA Agonists/pharmacology , GABA Antagonists/pharmacology , GABA Modulators/pharmacology , Macromolecular Substances , Male , Mice , Protein Subunits/drug effects , Radioligand Assay/methods , Receptors, GABA-A/chemistry , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Spinal Cord/drug effects , Time Factors , TritiumABSTRACT
We have previously identified the 7,8,9,10-tetrahydro-7,10-ethano-1,2,4-triazolo[3,4-a]phthalazine (1) as a potent partial agonist for the alpha(3) receptor subtype with 5-fold selectivity in binding affinity over alpha(1). This paper describes a detailed investigation of the substituents on this core structure at both the 3- and 6-positions. Despite evaluating a wide range of groups, the maximum selectivity that could be achieved in terms of affinity for the alpha(3) subtype over the alpha(1) subtype was 12-fold (for 57). Although most analogues showed no selectivity in terms of efficacy, some did show partial agonism at alpha(1) and antagonism at alpha(3) (e.g., 25 and 75). However, two analogues tested (93 and 96), both with triazole substituents in the 6-position, showed significantly higher efficacy for the alpha(3) subtype over the alpha(1) subtype. This was the first indication that selectivity in efficacy in the required direction could be achieved in this series.
Subject(s)
GABA-A Receptor Agonists , Phthalazines/chemical synthesis , Triazoles/chemical synthesis , Animals , Binding Sites , Cell Line , Female , Humans , Models, Molecular , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Phthalazines/chemistry , Phthalazines/pharmacology , Protein Subunits/agonists , Protein Subunits/physiology , Radioligand Assay , Receptors, GABA-A/physiology , Structure-Activity Relationship , Triazoles/chemistry , Triazoles/pharmacology , Xenopus laevisABSTRACT
Alpha3IA (6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABA(A) receptors containing an alpha3 rather than an alpha1, alpha2 or alpha5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of alpha3IA are most probably mediated by the alpha3 subtype. Alpha3IA has good CNS penetration in rats and mice as measured using a [(3)H]Ro 15-1788 in vivo binding assay. At doses in rats that produce relatively low levels of occupancy (12%) in the cerebellum (i.e. alpha1-containing receptors), alpha3IA (30 mg kg(-1) i.p.), like the nonselective partial inverse agonist N-methyl-beta-carboline-3-carboxamide (FG 7142), not only caused behavioural disruption in an operant, chain-pulling assay but was also anxiogenic in the elevated plus maze, an anxiogenic-like effect that could be blocked with the benzodiazepine antagonist Ro 15-1788 (flumazenil). Neurochemically, alpha3IA (30 mg kg(-1) i.p.) as well as FG 7142 (15 mg kg(-1) i.p.) increased the concentration of the dopamine metabolite 3,4-dihydroxyphenylacetic acid in rat medial prefrontal cortex by 74 and 68%, respectively, relative to vehicle-treated animals, a response that mimicked that seen following immobilisation stress. Taken together, these data demonstrate that an inverse agonist selective for GABA(A) receptors containing an alpha3 subunit is anxiogenic, and suggest that since alpha3-containing GABA(A) receptors play a role in anxiety, then agonists selective for this subtype should be anxiolytic.
Subject(s)
Anxiety/chemically induced , GABA Agonists/pharmacology , Receptors, GABA-A/drug effects , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Anxiety/psychology , Brain Chemistry/drug effects , Carbolines/pharmacology , Cerebellum/drug effects , Cerebellum/metabolism , Fibroblasts/metabolism , GABA Agonists/metabolism , Humans , Male , Mice , Patch-Clamp Techniques , Prefrontal Cortex/drug effects , Prefrontal Cortex/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Proteins , Spinal Cord/drug effects , Spinal Cord/metabolismABSTRACT
(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 (13) has been identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors. 13 is orally bioavailable, readily penetrates the CNS, and enhances performance in animal models of cognition. It does not exhibit the convulsant, proconvulsant, or anxiogenic activity associated with nonselective GABA(A) inverse agonists.
Subject(s)
Cognition/drug effects , GABA-A Receptor Agonists , Isoxazoles/chemical synthesis , Nootropic Agents/chemical synthesis , Triazines/chemical synthesis , Administration, Oral , Animals , Binding Sites , Biological Availability , Dogs , Isoxazoles/adverse effects , Isoxazoles/pharmacology , Maze Learning/drug effects , Mice , Nootropic Agents/adverse effects , Nootropic Agents/pharmacology , Patch-Clamp Techniques , Protein Subunits , Radioligand Assay , Rats , Receptors, GABA-A/metabolism , Triazines/adverse effects , Triazines/pharmacologyABSTRACT
A series of substituted 3,4-dihydronaphthalen-1(2H)-ones with high binding affinity for the benzodiazepine site of GABAA receptors containing the alpha5-subunit has been identified. These compounds have consistently higher binding affinity for the GABAA alpha5 receptor subtype over the other benzodiazepine-sensitive GABAA receptor subtypes (alpha1, alpha2 and alpha3). Compounds with a range of efficacies for the benzodiazepine site of alpha5-containing GABAA receptors were identified, including the alpha5 inverse agonist 3,3-dimethyl-8-methylthio-5-(pyridin-2-yl)-3,4-dihydronaphthalen-1(2H)-one 22 and the alpha5 agonist 8-ethylthio-3-methyl-5-(1-oxidopyridin-2-yl)-3,4-dihydronaphthalen-1(2H)-one 19.
Subject(s)
Naphthalenes/pharmacology , Receptors, GABA-A/drug effects , Allosteric Site , Benzodiazepines/antagonists & inhibitors , Binding Sites , GABA-A Receptor Agonists , GABA-A Receptor Antagonists , Humans , Ligands , Naphthalenes/chemical synthesis , Protein Binding , Protein Subunits , Structure-Activity RelationshipABSTRACT
A series of tricyclic pyridones has been evaluated as benzodiazepine site ligands with functional selectivity for the alpha(3) over the alpha(1) containing subtype of the human GABA(A) receptor ion channel. This investigation led to the identification of a high affinity, functionally selective, orally bioavailable benzodiazepine site ligand that demonstrated activity in rodent anxiolysis models and reduced sedation relative to diazepam.
Subject(s)
Protein Subunits/metabolism , Pyridones/chemistry , Pyridones/metabolism , Receptors, GABA-A/metabolism , Humans , Ion Channel Gating/physiology , Ligands , Protein BindingABSTRACT
Studies with our screening lead 5 and the literature compound 6 led to the identification of 6-benzyloxy-3-(4-methoxy)phenyl-1,2,4-triazolo[3,4-a]phthalazine 8 as a ligand with binding selectivity for the gamma-aminobutyric acid-A (GABA-A) alpha 3- and alpha 5-containing receptor subtypes over the GABA-A alpha 1 subtype (K(i): alpha 2 = 850 nM, alpha 3 = 170 nM, alpha 5 = 72 nM, alpha 1 = 1400 nM). Early optimization studies identified the close analogue 10 (K(i): alpha 2 = 16 nM, alpha 3 = 41 nM, alpha 5 = 38 nM, alpha 1 = 280 nM) as a suitable lead for further study. High-affinity ligands were identified by replacing the 6-benzyloxy group of compound 10 with 2-pyridylmethoxy (compound 29), but binding selectivity was not enhanced (K(i): alpha 2 = 1.7 nM, alpha 3 = 0.71 nM, alpha 5 = 0.33 nM, alpha 1 = 2.7 nM). Furthermore, on evaluation in xenopus oocytes,(22) 29 was discovered to be a weak to moderate inverse agonist at all four receptor subtypes (alpha 1, -7%; alpha 2, -5%; alpha 3, -16%; alpha 5, -5%). Replacement of the 3-phenyl group of 29 with alternatives led to reduced affinity, and smaller 3-substituents led to reduced efficacy. Methyl substitution of the benzo-fused ring of 29 at the 7-, 8-, and 10-positions resulted in increased efficacy although selectivity was abolished. Increased efficacy and retention of selectivity for alpha 3 over alpha 1 was achieved with the 7,8,9,10-tetrahydro-(7,10-ethano)-phthalazine 62. Compound 62 is currently one of the most binding selective GABA-A alpha 3-benzodiazepine-site partial agonists known, and although its selectivity is limited, its good pharmacokinetic profile in the rat (33% oral bioavailability after a 3 mg/kg dose, reaching a peak plasma concentration of 179 ng/mL; half-life of 1 h) made it a useful pharmacological tool to explore the effect of a GABA-A alpha 2/alpha 3 agonist in vivo.