GABA(B(1)) splice variant mRNAs are differentially affected by electroshock induced seizure in rats.

Receptor autoradiography with the high affinity antagonist radioligand [3H]CGP62349 and in situ hybridization with radiolabelled oligonucleotides were used to investigate GABA(B) receptor protein expression, and GABA(B(1)) mRNA splice variant (GABA(B(1a)) and GABA(B(1b)) levels, in brain sections from rats 4 h following a single electroshock-induced generalized seizure. Densitometric analysis indicated that GABA(B(1a)) mRNA levels were not significantly altered by an acute electroshock seizure, but that GABA(B(1b)) mRNA levels were significantly increased throughout the brain. GABA(B) receptor expression at this time point was unaffected by the seizure. The observed up-regulation of GABA(B(1b)) mRNA levels may imply increased importance of this splice variant in the regulation of further seizure activity.

Evaluation of a series of anticonvulsant 1,2,3,4-tetrahydroisoquinolinyl-benzamides.

SAR studies around a series of N-(tetrahydroisoquinolinyl)-2-methoxybenzamides, identified by high-throughput screening at the novel SB-204269 binding site, have provided compounds such as 13, 29-33 with high affinity and excellent anticonvulsant activity in animal models.

Identification of (-)-cis-6-acetyl-4S-(3-chloro-4-fluoro-benzoylamino)- 3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3S-ol as a potential antimigraine agent.

Optimisation of novel cis- and trans-4-(substituted-amido)benzopyran-3-ol derivatives has led to the identification of SB-220453 20 with an in vivo pre-clinical CNS profile predictive of potential antimigraine activity.

Identification of a series of 1,2,3,4-tetrahydroisoquinolinyl-benzamides with potential anticonvulsant activity.

A series of N-(tetrahydroisoquinolinyl)-2-methoxybenzamides was identified by high-throughput screening at the novel SB-204269 binding site. SAR studies have provided compounds 4 and 14 with high affinity and good anticonvulsant activity in animal models.

Profile of SB-204269, a mechanistically novel anticonvulsant drug, in rat models of focal and generalized epileptic seizures.

1. Earlier optimization of structure-activity relationships in a novel series of 4-(benzoylamino)-benzopyrans, led to the discovery of SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol, hemihydrate), a potent orally-active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test. 2. Studies have now been undertaken to determine the effects of SB-204269 in a range of seizure models and tests of neurological deficits in rats. In addition, the compound has been evaluated in a series of in vitro mechanistic assays. 3. SB-204269 proved to be an orally-effective anticonvulsant agent, at doses (0.1-30 mg Kg-1) devoid of overt behavioural depressant properties, in models of both electrically (MEST and maximal electroshock (MEST)) and chemically (i.v. pentylenetetrazol (PTZ) infusion)-evoked tonic extension seizures. However, the compound did not inhibit PTZ-induced myoclonic seizures at doses up to 30 mg kg-1, p.o. 4. SB-204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K+ rat hippocampal slice model at concentrations (0.1-10 microM) that had no effect on normal synaptic activity and neuronal excitability. 5. In all of these seizure models, SB-204269 was equivalent or better than the clinically established antiepileptic drugs carbamazepine and lamotrigine, in terms of anticonvulsant potency and efficacy. 6. Unlike SB-204269, the corresponding trans 3S,4R enantiomer, SB-204268, did not produce marked anticonvulsant effects, an observation in accord with previous findings for other related pairs of trans enantiomers in the benzopyran series. 7. In the rat accelerating rotarod test, a sensitive paradigm for the detection of neurological deficits such as sedation and motor incoordination, SB-204269 was inactive even at doses as high as 200 mg kg-1, p.o. This was reflected in the excellent therapeutic index (minimum significantly effective dose in the rotarod test/ED50 in the MES test) for SB-204269 of > 31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively. 8. At concentrations (> or = 10 microM) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 microM in brain), SB-204269 did not interact with many of the well known mechanistic targets for established antiepileptic drugs (e.g. Na+ channels or GABAergic neurotransmission). Subsequent studies have shown that the anticonvulsant properties of SB-204269 are likely to be mediated by a novel stereospecific binding site present in the CNS. 9. The overall efficacy profile in rodent seizure models, together with a minimal liability for inducing neurological impairment and an apparently unique mechanism of action, highlight the therapeutic potential of SB-204269 for the treatment of refractory partial and generalized tonic-clonic seizures.

Characterization of the binding of [3H]-SB-204269, a radiolabelled form of the new anticonvulsant SB-204269, to a novel binding site in rat brain membranes.

1. SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3, 4-dihydro-2,2-dimethyl-2H-benzol[b]pyran-3R-ol, hemihydrate) shows potent anticonvulsant activity in a range of animal seizure models, with a lack of neurological or cardiovascular side-effects. The profile of the compound suggests that it may have a novel mechanism of action. This study describes the characteristics of a binding site for [3H]-SB-204269 in rat forebrain membranes. 2. Specific [3H]-SB-204269 binding was saturable and analysis indicated binding to a homogenoeous population of non-interacting binding sites with a dissociation constant (KD) of 32 +/- 1 nM and a maximum binding capacity (Bmax) of 253 +/- 18 fmol mg-1 protein. Kinetic studies indicated monophasic association and dissociation. Binding was similar in HEPES or Tris-HCl buffers and was unaffected by Na+, K+, Ca2+ or Mg2+ ions. Specific binding was widely distributed in brain, but was minimal in a range of peripheral tissues. 3. Specific [3H]-SB-204269 binding was highly stereoselective, with a 1000 fold difference between the affinities of SB-204269 and its enantiomer SB-204268 for the binding site. The affinities of analogues of SB-204269 for binding can be related to their activities in the mouse maximal electroshock seizure threshold (MEST) test of anticonvulsant action. 4. None of the standard anticonvulsant drugs, phenobarbitone, phenytoin, sodium valproate, carbamazepine, diazepam and ethosuximide, or the newer anticonvulsants, lamotrigine, vigabatrin, gabapentin and levetiracetam, showed any affinity for the [3H]-SB-204269 binding site. A wide range of drugs active at amino acid receptors, Na+ or K+ channels or various other receptors did not demonstrate any affinity for the binding site. 5. These studies indicate that SB-204269 possesses a specific CNS binding site which may mediate its anticonvulsant activity. This binding site does not appear to be directly related to the sites of action of other known anticonvulsant agents, but may have an important role in regulating neuronal excitability.