Anticonvulsant properties of two GABA uptake inhibitors NNC 05-2045 and NNC 05-2090, not acting preferentially on GAT-1

doi:10.1016/S0920-1211(97)00033-8

Epilepsy Research

Volume 28, Issue 1, July 1997, Pages 51-61

https://doi.org/10.1016/S0920-1211(97)00033-8 Get rights and content

Abstract

Two novel nipecotic acid derivatives, 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(4-methoxyphenyl)-4-piperidinol (NNC 05-2045) and 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methoxyphenyl)-4-piperidinol (NNC 05-2090) have been tested for inhibition of γ-amino butyric acid (GABA) transporters in synaptosomal preparations of rat cerebral cortex and inferior colliculus and found to differ markedly from gabitril® (tiagabine), a selective GAT-1 inhibitor. IC₅₀ values for inhibition of [³H]GABA uptake into synaptosomes from cerebral cortex for NNC 05-2045 and NNC 05-2090 were 12±2 and 4.4±0.8 μM, respectively. In synaptosomes from inferior colliculus in the presence of 1 μM 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid (NNC 05-0711), a highly potent and selective GAT-1 inhibitor, IC₅₀ values for inhibition of [³H]GABA uptake were 1.0±0.1 and 2.5±0.7 μM, respectively. A receptor profile showed that NNC 05-2045 has binding affinities to sigma-, α₁- and D₂-receptors of 113, 550 and 122 nM, respectively. NNC 05-2090 displayed α₁- and D₂-receptor affinity of 266 and 1632 nM, respectively. The anticonvulsant action of both compounds was tested in four rodent models after intra peritoneal (i.p.) injection. Both NNC 05-2045 and NNC 05-2090 dose-dependently inhibited sound-induced tonic and clonic convulsions in DBA/2 mice with ED₅₀ values of 6 and 19 μmol/kg, respectively. NNC 05-2045 also antagonized sound-induced seizures in genetic epilepsy prone rats (GEP rats) with ED₅₀ values against wild running, clonic and tonic convulsions of 33, 39 and 39 μmol/kg, respectively (NNC 05-2090 was not tested in GEP rats). Both NNC 05-2045 and NNC 05-2090 dose-dependently antagonized tonic hindlimb extension in the maximal electroshock (MES) test with ED₅₀ values of 29 and 73 μmol/kg, respectively. In amygdala kindled rats NNC 05-2045 and NNC 05-2090 significantly (P<0.05) reduced generalized seizure severity (seizure grade 3–5) at highest doses (72–242 μmol/kg) and NNC 05-2090 also significantly reduced afterdischarge duration at these doses (P<0.05). These data show that inhibition of GABA uptake through non-GAT-1 transporters has different anticonvulsant effects than selective GAT-1 inhibitors (e.g. tiagabine) in that enhanced efficacy against MES and reduced efficacy against kindled seizures is observed. Although a contribution of adrenergic agonistic effects cannot be entirely ruled out, it is proposed that inhibition of GAT-3 (mouse GAT4) is primarily responsible for the anticonvulsant action of these two nipecotic acid derivatives in MES, amygdala kindled rats and in sound-induced seizures in GEP-rats and DBA/2 mice.

Introduction

Many reports have indicated a role for the neurotransmitter, γ-aminobutyric acid (GABA) in epilepsy 24, 25. Indeed, several clinically effective anti-epileptic drugs, such as benzodiazepines and vigabatrin, act through potentiation of GABAergic neurotransmission. Another pharmacological approach to enhance GABAergic neurotransmission in vivo is to inhibit the uptake of GABA into neurons and glia. The advantage of GABA-uptake inhibition is that it selectively potentiates endogenously released GABA, which may circumvent the problem of tolerance, seen with direct GABA_A agonists or benzodiazepines [34].

Four different GABA transporters have been identified in the mouse 21, 22and other species 3, 4, 10, 16, 36. In short, the nomenclature of rat and mouse GABA transporters translates as follows: rat GAT-1 is homologous to mouse GAT1, rat GAT-2 is the homologue of mouse GAT3, rat GAT-3 is the homologue of mouse GAT4 and finally mouse GAT2 is homologous to Betaine/GABA transporter 1 (BGT-1) which, however, has not yet been cloned from rats. Except where indicated, in this paper the nomenclature of rat-GABA transporters will be used. Potent and highly selective GABA uptake inhibitors acting at GAT-1 have been known for some time (tiagabine, NNC 05-0711, SK and F 89976-A and CI-966) 4, 6. In vivo, GAT-1 uptake inhibitors are potent anticonvulsants [32]. Specifically, inhibition of GABA-uptake by tiagabine has shown remarkable anticonvulsant effects in several animal models 12, 27, 33and recently also clinical effect against refractory complex partial seizures 1, 31.

The effect of inhibiting GABA uptake mediated by BGT-1, GAT-2 or GAT-3 in relevant animal models is at present unknown due to the lack of in vivo active and selective inhibitors at these transporters, but the potential therapeutic use of such inhibitors could be as anticonvulsants, anxiolytics or/and antidepressants. The expression of GAT-2 in the adult CNS is restricted to the arachnoid membrane [17], which makes it unlikely to play a role in the above mentioned CNS-disorders. However, the expression of GAT-3 in the midbrain and brainstem as well as its high affinity for GABA, have made it an interesting pharmacological target in the search for novel antiepileptic drugs. Furthermore, although BGT-1 has a low affinity for GABA and presumably is responsible for only a small fraction of GABA transport in the brain in comparison with GAT-3 [5], the overall expression of BGT-1 in the brain suggests that drugs acting at this site could have significant effects in CNS disorders.

A triarylnipecotic acid derivative ((S)-1-2-(tris(4-methoxyphenyl)methoxy]ethyl)-3-piperidinecarboxylic acid, 4(S)) (SNAP-5114) was recently reported to be selective for the cloned human GAT-3, homologous to rat GAT-3 [11]. However, no in vivo studies were made with this compound. We recently reported on the sub-type selectivity of tiagabine, SNAP-5114, NNC 05-2045 and NNC 05-2090 in inhibiting [³H]GABA uptake by the cloned mouse GABA-transporters [35].

Here we report on the efficacy of NNC 05-2045 and NNC 05-2090 in inhibiting GABA uptake from rat brain synaptosomes prepared from rat cerebral cortex and inferior colliculus. Potential anticonvulsant properties of NNC 05-2045 and NNC 05-2090 have been assessed in different animal models of epilepsy and epileptic seizures: sound-induced seizures in genetically epilepsy prone rats (GEPR) and DBA/2 mice, amygdala kindled rats and maximal electroshock (MES) in mice.

Section snippets

[³H]GABA uptake into synaptosomal preparations from rat brain

Uptake of [³H]GABA into synaptosomal preparations was performed exactly as described previously [6]. The main component of [³H]GABA uptake into cortical membranes is thought to be mediated mainly via GAT-1 transporters since the potent and highly selective GAT-1 inhibitor, NNC 05-0711 inhibits 95–96% of [³H]GABA uptake 2, 5. The non-GAT-1 mediated component of [³H]GABA uptake is assessed in synaptosome preparations prepared from cortex or inferior colliculus in the presence of 1 μM NNC 05-0711

Receptor profile of NNC 05-2045

NNC 05-2045 displays little or no affinity (IC₅₀>10μM) for the following binding sites: nitrendepine (L-type Ca-channels), TBX (Na-channels), TBPS (GABA-operated Cl−-channel), flunitrazepam and PK11195 (central and peripheral benzodiazepine, respectively), strychnine and glycine (glycine receptors), TCP, CNQX and kainate (NMDA, quisqualate and kainate receptors, respectively), PIA and CGS21680 (adenosine A1- and A2-receptors, respectively), 8-OH-DPAT, ketanserin and GR65630 (serotonin 1A-, 2-

Discussion

GABA is the predominant inhibitory neurotransmitter in the CNS. As GABA clearly plays a role in various CNS-diseases (e.g. epilepsy, depression, anxiety and mania) pharmacological approaches to increasing GABAergic neurotransmission regionally or universally in the CNS have been investigated. The inhibition of GABA uptake by the GAT-1-inhibitor tiagabine increases GABA levels in the brain [14]and is likely to be responsible for the prominent anticonvulsant activity of tiagabine in different

Acknowledgements

The authors would like to acknowledge the skillful technical assistance of Kira Meyhoff-Madsen, Charlotte Halle and Jette Platou.

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γ-Aminobutyric acid transporters are responsible for regulating the GABA level in the synaptic cleft. In this way, they affect GABA-ergic transmission which is important for the proper functioning of the central nervous system. The exact structure of GABA transporters is still unknown, which hinders the design of new, potent and selective inhibitors. For these reasons, we decided to create models of all types of human gamma-aminobutyric acid transporters. They were built based on crystal structures of related proteins from the SLC6 family using homology modeling methods. The reliability of the received models has been confirmed by a number of tools assessing the quality of protein models. To determine the ligand binding mode and indicate the amino acids responsible for selectivity, docking studies and molecular dynamics simulations were performed. The amino acids lining the bottom of the main binding site have a major impact on the selective ligand binding. In addition, an important element is the non-helical fragment of the transmembrane domain 10, and several amino acids within the vestibule of the transporters, which affect its volume. To check whether obtained models are suitable to distinguish active compounds from inactive ones, enrichment plots were prepared. Results suggest that our models may be useful in the search for new inhibitors of GABA transporters of the desired selectivity.
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Citation Excerpt :
Another structural analog, NNC 05–2090, which is a nonselective inhibitor of BGT-1, GAT-1, and GAT-3, showed a very similar profile of anticonvulsant efficacy. NNC 05–2090 reducing the severity and duration of seizures after stimulation with the amygdala of the rats [161]. The EF1502 compound, which inhibits both GAT-1 and BGT-1 transporters, has a broad antiepileptic profile in general and was found to be effective against partial seizures in many animal models [87,162].
γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the nervous system. It plays a crucial role in many physiological processes. Upon release from the presynaptic element, it is removed from the synaptic cleft by reuptake due to the action of GABA transporters (GATs). GATs belong to a large SLC6 protein family whose characteristic feature is sodium-dependent relocation of neurotransmitters through the cell membrane. GABA transporters are characterized in many contexts, but their spatial structure is not fully known. They are divided into four types, which differ in occurrence and role. Herein, the special attention was paid to these transporting proteins. This comprehensive review presents the current knowledge about GABA transporters. Their distribution in the body, physiological functions and possible utilization in the therapy of different diseases were fully discussed. The important structural features were described based on published data, including sequence analysis, mutagenesis studies, and comparison with known SLC6 transporters for leucine (LeuT), dopamine (DAT) and serotonin (SERT). Moreover, the most important inhibitors of GABA transporters of various basic scaffolds, diverse selectivity and potency were presented.
Interactions of aliskiren, a direct renin inhibitor, with antiepileptic drugs in the test of maximal electroshock in mice
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Experimental studies showed that certain angiotensin-converting enzyme inhibitors and angiotensin AT₁ receptor antagonists can decrease seizure severity in rodents. Additionally, some of these blockers of the renin-angiotensin system have been documented to enhance the anticonvulsant activity of antiepileptic drugs against maximal electroshock-induced seizures. The aim of the current study was to investigate the effect of aliskiren, a direct renin inhibitor and a novel antihypertensive drug, on the protective action of numerous antiepileptic drugs (carbamazepine, valproate, clonazepam, phenobarbital, oxcarbazepine, lamotrigine, topiramate and pregabalin) in the test of maximal electroshock in mice. The examined drugs were administered intraperitoneally. Aliskiren up to a dose of 75 mg/kg did not affect the threshold for electroconvulsions, however, aliskiren (75 mg/kg) enhanced the anticonvulsant action of clonazepam and valproate. Following aliskiren treatment, a higher brain concentration of valproate was noted, suggesting a pharmacokinetic interaction. In the rota-rod test, the concomitant treatment with aliskiren (50 or 75 mg/kg) and clonazepam (22.6 mg/kg) impaired motor coordination while clonazepam (22.6 mg/kg) alone showed strong tendency towards this impairment. The combination of aliskiren (75 mg/kg) with phenobarbital (25.5 mg/kg) caused long-term memory deficits in the passive avoidance task. This study shows that there are no negative interactions between aliskiren and the examined antiepileptic drugs as concerns their anticonvulsant activity. Aliskiren even potentiated the anticonvulsant action of clonazepam and valproate against maximal electroshock. The impact of aliskiren alone on seizure activity or on the anticonvulsant and adverse activity of antiepileptic drugs needs further evaluation in other animal models of seizures.
Anticonvulsant active inhibitor of GABA transporter subtype 1, tiagabine, with activity in mouse models of anxiety, pain and depression
2015, Pharmacological Reports
Tiagabine, a selective inhibitor of GABA transporter subtype 1 is used as an add-on therapy of partial seizures in humans but its mechanism of action suggests other potential medical indications for this drug. In this research we assess its pharmacological activity in several screening models of seizures, pain, anxiety and depression in mice.
For pharmacological tests tiagabine was administered intraperitoneally 60 min before the assay. Behavioral tests were performed using models of chemically and electrically induced seizures, thermal acute pain and formalin-induced tonic pain. Anxiolytic-like properties were evaluated using the four plate test and the elevated plus maze test. Antidepressant-like activity was assessed in the forced swim test. In addition, to exclude false positive results in these assays, the influence of tiagabine on animals’ locomotor activity and motor coordination was investigated, too.
Tiagabine demonstrated anticonvulsant properties in chemically induced seizures (pentylenetetrazole and pilocarpine seizures). At the dose of 100 mg/kg it also elevated the seizure threshold for electrically induced seizures by 31.6% (p < 0.01), but it had no activity in the maximal electroshock seizure test. Tiagabine showed anxiolytic-like and antidepressant-like effects. Although it apparently reduced animals’ nociceptive responses in pain tests, these activities rather resulted from its sedative and motor-impairing properties demonstrated in the locomotor activity and the rotarod tests, respectively.
The results obtained in the present study suggest that tiagabine, apart its anticonvulsant effect, has anxiolytic-like, sedative and antidepressant-like properties. In view of this, it can be potentially used in the treatment of anxiety and mood disorders.
Antiallodynic action of 1-(3-(9H-carbazol-9-yl)-1-propyl)-4-(2- methyoxyphenyl)-4-piperidinol (NNC05-2090), a betaine/GABA transporter inhibitor
2014, Journal of Pharmacological Sciences
The GABAergic system in the spinal cord has been shown to participate in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling the synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In the present study, we compared the betaine/GABA transporter (BGT-1) with other GAT subtypes to determine its participation in neuropathic pain using a mouse model of sciatic nerve ligation. 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), an inhibitor that displays moderate selectivity for BGT-1, had an antiallodynic action on model mice treated through both intrathecally and intravenous administration routes. On the other hand, SKF89976A, a selective GAT-1 inhibitor, had a weak antiallodynic action, and (S)-SNAP5114, an inhibitor that displays selectivity for GAT-3, had no antiallodynic action. Systemic analysis of these compounds on GABA uptake in CHO cells stably expressing BGT-1 revealed that NNC05-2090 not only inhibited BGT-1, but also serotonin, noradrenaline, and dopamine transporters, using a substrate uptake assay in CHO cells stably expressing each transporter, with IC₅₀: 5.29, 7.91, and 4.08 μM, respectively. These values were similar to the IC₅₀ value at BGT-1 (10.6 μM). These results suggest that the antiallodynic action of NNC05-2090 is due to the inhibition of both BGT-1 and monoamine transporters.
Evaluation of anxiolytic-like, anticonvulsant, antidepressant-like and antinociceptive properties of new 2-substituted 4-hydroxybutanamides with affinity for GABA transporters in mice
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The inhibition of plasma membrane GABA transporters (GATs) is responsible for anxiolytic-like, anticonvulsant, antinociceptive and antidepressant-like effects in mice. It also influences animals' motor coordination and their sensitivity to ethanol. The aim of this study was to assess the pharmacological activity of two novel 2-substituted 4-hydroxybutanamides (BM 130 and BM 131) in some screening models. An attempt has been made to establish the relationship between the inhibition of GAT subtype and the observed in vivo activity.
The affinity for GAT subtypes was evaluated by means of [³H]GABA uptake assay. It indicated that BM 130 inhibited GAT1 and GAT2, whereas BM 131 inhibited GAT1 and GAT3. In mice anxiolytic-like, antidepressant-like, anticonvulsant and antinociceptive properties of the test compounds were assessed. Their influence on motor coordination, locomotor activity and the ability to potentiate effects of subnarcotic doses of ethanol was also tested.
Both compounds administered intraperitoneally exerted a significant anxiolytic-like effect in the four plate test with ED₅₀ values 3.4 and 7.9 mg/kg, respectively. At 30 mg/kg they reduced duration of immobility in the forced swim test for 33% and 19%, respectively. They had no effect on electroconvulsive threshold or pain reactivity in the hot plate assay but they were antinociceptive in the acetic acid-induced writhing test (ED₅₀ values were 12.7 and 18.6 mg/kg, respectively) and in both phases of the formalin test (ED₅₀ values in the first phase were 10.2 and 2.1 mg/kg for BM 130 and BM 131, respectively). No motor adverse effects were observed in mice pretreated with the test compounds in the rotarod or chimney tests but BM 131 caused a transient but statistically significant decrease of animals' locomotor activity.
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Anticonvulsant properties of two GABA uptake inhibitors NNC 05-2045 and NNC 05-2090, not acting preferentially on GAT-1

Abstract

Introduction

Section snippets

[3H]GABA uptake into synaptosomal preparations from rat brain

Receptor profile of NNC 05-2045

Discussion

Acknowledgements

J Epilepsy

Eur J Pharmacol

J Biol Chem

Neuron

Epilepsy Res

Brain Res

Eur J Pharmacol

Mol Brain Res

J Biol Chem

J Biol Chem

Int Rev Neurobiol

Eur J Pharmacol

Electroencephal Clin Neurophysiol

Epilepsy Res

Eur J Pharmacol

J Biol Chem

Cloning and expression of a betaine/GABA transporter from human brain

J Neurochem

Re-evaluation of GABA transport in neuronal and glial cell cultures: Correlation of pharmacology and mRNA localization

Receptors Channels

[³H]GABA uptake into synaptosomal preparations from rat brain