Nipecotic acid and guvacine antagonism on morphine analgesia in rats.
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Synthesis of novel GABA uptake inhibitors. 3. Diaryloxime and diarylvinyl ether derivatives of nipecotic acid and guvacine as anticonvulsant agents.
(3R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-piperidinecarboxylic acid 1 (tiagabine, Gabitril) is a potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor with proven anticonvulsant efficacy in humans. This drug, which has a unique mechanism of action among marketed anticonvulsant agents, has been launched for add-on treatment of partial seizures with or without secondary generalization in patients >12 years of age. Using this new agent as a benchmark, we have designed two series of novel GABA uptake inhibitors of remarkable potency, using a putative new model of ligand interaction at the GABA transporter type 1 (GAT-1) uptake site. This model involves the postulated interaction of an electronegative region in the GABA uptake inhibitor with a positively charged domain in the protein structure of the GAT-1 site. These two novel series of anticonvulsant agents contain diaryloxime or diarylvinyl ether functionalities linked to cyclic amino acid moieties and were derived utilizing the new model, via a series of design steps from the known 4,4-diarylbutenyl GABA uptake inhibitors. The new compounds are potent inhibitors of [(3)H]-GABA uptake in rat brain synaptosomes in vitro, and their antiepileptic potential was demonstrated in vivo by their ability to protect against seizures induced by the benzodiazepine receptor inverse agonist methyl 4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM) in mice. From structure-activity studies of these new GABA uptake inhibitors, we have shown that insertion of an ether oxygen in conjugation with the double bond in tiagabine (K(i) = 67 nM) improves in vitro potency by 5-fold to 14 nM.
Animals↗
Nipecotic acid enhances synaptic transmission in the frog optic tectum by an action independent of GABA uptake inhibition.
In the frog optic tectum maintained in vitro at 7 degrees C to depress gamma-aminobutyric acid (GABA) transport systems the GABA uptake inhibitor nipecotic acid reversibly enhanced field potentials evoked by optic nerve stimulation. This effect was dose dependent and picrotoxin sensitive. Responses to low concentrations of GABA agonists were not affected by nipecotic acid, whereas responses to the maximal muscimol concentration were depressed. It is suggested that in addition to its well known inhibitory action on GABA uptake systems, nipecotic acid can evoke GABA mimetic effects whose precise origin is presently unclear.
Animals↗
Anticonvulsant activity of the nipecotic acid ester, (+/-)-m-nitrophenyl-3-piperidinecarboxylate.
The nipecotic acid ester, (+/-)-m-nitrophenyl-3-piperidinecarboxylate hydrochloride (MNPC) is a potent inhibitor of uptake of GABA in vitro and should be able to penetrate into the brain much more readily than the parent compound nipecotic acid. A study of the effects of MNPC on convulsions induced by chemicals which interfere with GABA-mediated neurotransmission was carried out in the mouse, with MNPC being administered by subcutaneous injection 30, 60 or 90 min prior to challenge with bicuculline. It was found that MNPC protected against convulsions induced by bicuculline with ED50 values for clonic and tonic convulsions of 157.8 and 138.8 mg/kg, respectively, at the time of peak effect of 60 min and MNPC abolished both the clonic and tonic components of isoniazid convulsions with respective ED50 values of 255.3 and 76.7 mg/kg at 1 hr. Picrotoxin and pentylenetrazol-induced seizures were also blocked with corresponding ED50 values for clonic convulsions of 224.9 and 235.9 mg/kg at 1 hr. No serious side effects were observed during the 90 min period after the administration of MNPC in doses up to 600 mg/kg.
Animals↗
[3H]nipecotic acid binding to GABA uptake sites in human brain.
The binding of [3H]nipecotic acid to frozen post-mortem human brain tissue has been characterized. Competition experiments with gamma-aminobutyric acid (GABA), GABA uptake inhibitors, ligands active at post-synaptic GABA receptors and receptors for other neurotransmitter systems, suggest that [3H]nipecotic acid binds to the neuronal (but not glial) GABA uptake site. Competition and kinetic experiments suggest that 85% of the binding is to a high affinity site. The dissociation constants (Kd) measured in kinetic and equilibrium experiments were in the same range (0.5-0.6 microM). The regional distribution was studied in 19 brain regions and the binding was relatively homogenous. It is concluded that [3H]nipecotic acid binding can be used as a marker for neuronal GABA uptake sites in post-mortem human brain tissue.
Aged↗
Syntheses and GABA uptake properties of 6-ether- and 6-enol ether-substituted nipecotic acids.
6-aralkylether- and 6-arylenol-ether-substituted nipecotic acids were synthesized. These analogues are poor GABA uptake inhibitors. The electronegative region concept developed in the N-substituted nipecotic acid series cannot be transferred on the side chain of this series of 6-substituted analogues.
Ethers↗
Assay for nipecotic acid in small blood samples by gas chromatography-mass spectroscopy.
An analytical method for nipecotic acid quantification in rat blood was developed utilizing a stable isotope internal standard and capillary gas chromatography-mass spectroscopy. The method involves a solid phase extraction step followed by a two-step derivatization. The analytes are separated by capillary gas chromatography and detected by selected ion monitoring of their base peaks at 180 and 185 m/z, respectively. The assay has a limit of detection (LOD) of 10 ng/ml and a limit of quantification of 26 ng/ml in 200 microl of rat whole blood. The linear range of the assay covers from 26 to 6500 ng/ml (r2=0.9996, n=9). The coefficient of variation was less than 10% at concentrations of 50, 1000 and 5000 ng/ml. The assay was used to characterize the pharmacokinetics of R-(-)-nipecotic acid in a rat. R-(-)-nipecotic acid clearance was 4.2 ml/min, its half-life was 1.5h and its volume of distribution at steady state was 325 ml.
Animals↗
Uptake of GABA and nipecotic acid in astrocytes and neurons in primary cultures: changes in the sodium coupling ratio during differentiation.
The coupling ratio between sodium and GABA or the GABA analog, (RS)- nipecotic acid, in neuronal and glial uptake of GABA or nipecotic acid was investigated as a function of the morphological differentiation of these cell types in primary cultures. Both in neurons and astrocytes a high-affinity uptake of GABA and nipecotic acid was observed regardless of the degree of differentiation. The values of Km were essentially identical in undifferentiated and differentiated cells. On the other hand, Vmax was significantly increased both in 10-day-old neurons compared to 1- and 3-day-old neurons and in astrocytes treated with dBcAMP compared to untreated cells, i.e., Vmax increased as a function of differentiation in both cell types. In neurons as well as in astrocytes the morphological differentiation resulted in an alteration in the coupling ratio between sodium and GABA. From calculated Hill coefficients it could be deduced that the coupling ratio between sodium and GABA was changed from 1 to 2 during differentiation. Similar results were obtained for (RS)-nipecotic acid.
Animals↗
Determination of m-nitrophenol and nipecotic acid in mouse tissues by high-performance liquid chromatography after administration of the anticonvulsant m-nitrophenyl-3-piperidinecarboxylate hydrochloride.
The nipecotic acid ester m-nitrophenyl-3-piperidinecarboxylate (MNPC) possesses anticonvulsant activity. In the present study, the metabolites m-nitrophenol and nipecotic acid were determined in mouse blood and brain tissue after administration of MNPC. This determination was used as an indication of the distribution of the parent compound MNPC and to provide information regarding the differences in distribution between the enantiomers of MNPC, the times of onset, and effectiveness when (+/-) MNPC was administered by subcutaneous (sc) and intraperitoneal (ip) routes. m-Nitrophenol was determined by a previously reported high-performance liquid chromatography (HPLC) method. There was no significant difference in m-nitrophenol distribution after sc administration of (+)MNPC and (-)MNPC (400 mg/kg each). This similar pattern of distribution is in agreement with the earlier reported equi-effectiveness of the enantiomers of anticonvulsants. Peak m-nitrophenol levels in blood, which occurred at 15 min, were three times greater when (+/-) MNPC was administered by ip injection as compared with sc injection. This significant difference is most likely due to enhanced absorption and the peripheral metabolism of MNPC by the liver when the ip route is employed. A novel HPLC assay for the determination of nipecotic acid in mouse brain was developed, based on a modification of a reported amino acid analysis procedure. The results of the brain distribution studies showed that nipecotic acid brain levels peaked at 30 min after sc administration of (-)MNPC.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
In vivo effects of nipecotic acid on levels of extracellular GABA and taurine, and hippocampal excitability.
The effect of nipecotic acid on the extracellular levels of the putative hippocampal inhibitory transmitters, gamma-aminobutyric acid (GABA) and taurine, and also excitability in the dentate gyrus, were studied in anaesthetised rats using the dialytrode technique. The dentate gyrus was perfused by means of a dialytrode , using Krebs-Ringer bicarbonate or nipecotic acid solutions. Dentate field potentials, evoked by electrical stimulation of the perforant pathway, were recorded. Nipecotic acid drastically elevated the levels of extracellular GABA and taurine and the amplitude of population spikes in the dentate was concomitantly decreased. These results indicate that changes in extracellular levels of endogenous GABA and/or taurine influence the excitability of the hippocampus.
Animals↗
Postsynaptic action of endogenous GABA released by nipecotic acid in the hippocampus.
Intracellular and whole-cell recording from CA1 pyramidal cells and dentate granule cells was used to study the release of endogenous GABA by nipecotic acid. Local application of nipecotic acid produced responses that could be entirely blocked by a combination of the GABAA receptor antagonist picrotoxin and the GABAB receptor antagonist CGP 35348. These responses were due to the heteroexchange release of endogenous GABA because they were blocked by low Na+ which blocks the GABA transporter and by SKF 89976 which is a competitive antagonist of the GABA transporter. Local application of nipecotic acid could, depending on the location, evoke pure GABAA or pure GABAB responses supporting proposals that GABAA and GABAB receptors can be segregated at separate inhibitory synapses.
Animals↗
Kinetic characterization of inhibition of gamma-aminobutyric acid uptake into cultured neurons and astrocytes by 4,4-diphenyl-3-butenyl derivatives of nipecotic acid and guvacine.
The effects of N-(4,4-diphenyl-3-butenyl) derivatives of nipecotic acid (SKF-89976-A and SKF-100844-A) and guvacine (SKF-100330-A) on neuronal and astroglial gamma-aminobutyric acid (GABA) uptake were investigated. In addition, the uptake of SKF-89976-A was studied using the tritiated compound. All of the compounds were found to be competitive inhibitors of GABA uptake irrespective of the cell type, with Ki values similar to or lower than those of the parent amino acids. Moreover, none of the compounds exhibited selectivity with regard to inhibition of neuronal and glial GABA uptake. In spite of the competitive nature of SKF-89976-A, the compound was not transported by the GABA carriers in the two cell types, because no saturable uptake could be demonstrated.
Animals↗
Nipecotic acid: preferential accumulation in the cochlea by GABA uptake systems and selective retrograde transport to brainstem.
[3H]Nipecotic acid was shown to be preferentially accumulated by the same cochlear structures which selectively accumulate [3H]gamma-aminobutyric acid ([3H]-GABA), including the terminals of a subset of olivocochlear neurons. With both amino acids, olivocochlear fibers selectively transported label in a retrograde direction, from cochlea to brainstem. However, only [3H]nipecotic acid produced dense labeling, and labeling of cell bodies in the superior olive, presumably because it is metabolized very slowly. Nipecotic acid appears to provide a selective retrograde tracer, specific to neurons whose terminals exhibit preferential GABA uptake.
Animals↗
Differential effects of nipecotic acid and 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol on extracellular gamma-aminobutyrate levels in rat thalamus.
Using the microdialysis technique and HPLC (high-performance liquid chromatography) determination of amino acids, the extracellular concentrations of gamma-aminobutyrate (GABA), glutamate, aspartate and a number of other amino acids were determined in rat thalamus during infusion through the microdialysis tubing of the GABA transport inhibitors 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) and nipecotic acid. Administration of 5.0 mM THPO led to a 200% increase in the extracellular GABA concentration. Simultaneous infusion of THPO and GABA (50 microM) increased the extracellular GABA concentration to 1200% of the basal level whereas GABA alone was found to increase the GABA level to 500%. If nipecotic acid (0.5 mM) was administered together with GABA (50 microM) the extracellular concentration of GABA was not increased further. While administration of GABA alone or GABA together with nipecotic acid had no effect on the extracellular levels of glutamate and aspartate it was found that GABA plus THPO increased the extracellular concentration of these amino acids. GABA administered alone or together with nipecotic acid or THPO led to relatively small but significant increases in the extracellular concentrations of the amino acids glycine, glutamine, serine and threonine. The results demonstrate that THPO, which preferentially inhibits glial GABA uptake and which is not a substrate for the GABA carriers, was more efficient increasing the extracellular concentration of GABA than nipocotic acid which is a substrate and an inhibitor of both neuronal and glial GABA uptake. This indicates that GABA uptake inhibitors that are not substrates for the carrier and which preferentially inhibit glial GABA uptake may constitute a group of drugs by which the efficacy of GABAergic neurotransmission may be enhanced.
Animals↗
Free amino acids in the pre-retinal vitreous space. Effect of high potassium and nipecotic acid.
The pre-retinal vitreous space of the eye of albino rabbits was perfused in vivo via a dialysis probe in order to measure the concentration of endogenous amino acids. Studies of isotope concentrations in the vitreal effluent following intracarotid injections of 3HOH and [14C]mannitol demonstrated that entrance of 3HOH into the dialysis probe occurred within a few minutes whereas [14C]mannitol was almost completely excluded. Among the amino acids, analysed with high-pressure liquid chromatography (HPLC), glutamine had a concentration similar to that in plasma and cerebrospinal fluid (CSF). Taurine was four times higher than in the CSF. Perfusion with 125 mM KCl media increased particularly the taurine and phosphoethanolamine content. Nipecotic acid, a potent inhibitor of gamma-aminobutyric acid (GABA) uptake, increased GABA concentration over 60 times. In addition, it increased taurine levels by almost 10 times.
Alanine↗
Electrophysiological evidence that nipecotic acid can be used in vivo as a false transmitter.
Dentate gyrus of the rat hippocampal formation was perfused with the potent inhibitor of GABA uptake, nipecotic acid, by means of an implanted dialytrode. Evoked population spikes in dentate gyrus were decreased in amplitude and often abolished during perfusion. However, multiple (2-4) population spikes developed shortly after nipecotic acid withdrawal. This excitability increase, which presented a pattern of repetitive discharge resembling that following blocking of GABAergic transmission was interpreted as electrophysiological evidence that nipecotic acid can act as a false transmitter 'in vivo', as previously postulated from uptake and release 'in vitro' studies.
Animals↗
Actions of nipecotic acid and SKF89976A on GABA transporter in cone-driven horizontal cells dissociated from the catfish retina.
Whole-cell voltage-clamp recordings were made from dissociated horizontal cells of the catfish retina. In the presence of picrotoxin (PTX, 100 microM), GABA evoked a sustained inward current at negative holding potentials. Dose-response measurements were well fitted by a logistic curve with a Hill coefficient of 1.11 and EC50 of 9.76 microM. When external Na+ was replaced with Li+, this GABA-induced current was eliminated. The substitution of methanesulfonate for Cl- also suppressed the current. This current was blocked by either nipecotic acid or SKF89976A. However, the mechanisms by which these drugs suppress the GABA-induced current differ. Intracellularly applied SKF89976A blocked the GABA-induced current, while nipecotic acid intracellularly had no effect. beta-Alanine at concentrations greater than 1 mM exerted a slight inhibitory effect. Extracellularly applied SKF89976A produced no current by itself but suppressed GABA-induced currents. Dose-response curves showed that SKF89976A has an IC50 of 0.93 microM and a Hill coefficient of 2.68. Nipecotic acid evoked a current response, like GABA. A Hill coefficient was 1.64 and an EC50 was 7.69 microM. This nipecotic acid-induced current was blocked by substituting Li+ for Na+ or by the addition of SKF89976A. This result is consistent with other studies indicating that nipecotic acid is transported in place of GABA. Extracellular Na+ was required for the prolonged suppression by extracellularly applied SKF89976A, while the extracellular Cl- depletion has no influence on the suppression. The pharmacological profile of this GABA transporter fits the neuronal rather than the glial type of cloned transporters.
Animals↗
Structure-activity studies on benzhydrol-containing nipecotic acid and guvacine derivatives as potent, orally-active inhibitors of GABA uptake.
The introduction of lipophilic groups onto the ring nitrogen of nipecotic acid and guvacine, two known GABA uptake inhibitors, afforded potent, orally-active anticonvulsant drugs. A series of compounds is reported which explores the structure-activity relationships (SAR) in this series. Among the areas explored: side-chain SAR (aromatic-, heterocyclic-, and tricyclic-containing side chains) and modifications to the tetrahydropyridine ring. The benzhydrol ether-containing side chains afforded the most potent compounds with several exhibiting in vitro IC50 values for GABA uptake of < 1 microM (including 5, Table I; 37, 43, Table IV; and 44, Table V). Compound 44 was selected for extensive evaluation and subsequently progressed to Phase 1 clinical trials with severe adverse effects seen after single dose administration to humans.
Administration, Oral↗