António Parada

Decrease of adenosine A1 receptor density and of adenosine neuromodulation in the hippocampus of kindled rats.

Adenosine is a neuromodulator that has been proposed to be a major endogenous anticonvulsant acting via A1 receptors. We tested if implementation of kindling through stimulation of the amygdala affected A1 receptor‐mediated neuromodulation in hippocampal slices taken from rats 4 weeks after the last stage 5 seizure. The A1 receptor agonist, N6‐cyclopentyladenosine (CPA) (6–100 nm), inhibited field excitatory postsynaptic potential (fEPSP) slope with an EC50 of 19.1–19.5 nm in control and sham‐operated rats, but was less potent in kindled rats (EC50 = 42.7 nm). This might result from a decreased number of A1 receptors in hippocampal nerve terminal membranes, because A1 receptor immunoreactivity decreased by 28 ± 3% and the binding density of the A1 receptor agonist [3H]R‐PIA decreased from 1702 ± 64 to 962 ± 78 fmol/mg protein in kindled compared with control rats. The tonic inhibition of hippocampal synaptic transmission by endogenous adenosine was also lower in kindled rats, because A1 receptor blockade with 50 nm 1,3‐dipropyl‐8‐cyclopentyladenosine (DPCPX) enhanced fEPSP slope by 23 ± 3% and θ‐burst‐induced long‐term potentiation by 94 ± 4% in control rats but was virtually devoid of effects in kindled rats. The evoked release of adenosine from hippocampal slices or nerve terminals was 56–71% lower in kindled rats probably due to the combined decrease in the capacity of adenosine transporters and decreased release of adenosine 5′‐triphosphate (ATP), which was partially compensated by a higher extracellular catabolism of ATP into adenosine in kindled rats. These results indicate that, although adenosine might inhibit the onset of epileptogenesis, once kindling is installed, the efficiency of the adenosine inhibitory system is impaired.

Interaction of the Novel Anticonvulsant, BIA 2‐093, with Voltage‐Gated Sodium Channels: Comparison with Carbamazepine

Summary:  Purpose: BIA 2‐093 [(S)‐(‐)‐10‐acetoxy‐10,11‐dihydro‐5H‐dibenz/b,f/azepine‐5‐carboxamide] is endowed with an anticonvulsant potency similar to that of carbamazepine (CBZ), but produces less cognitive and motor impairment. This study evaluated whether voltage‐gated sodium channels (VGSCs) are a primary locus for the action of BIA 2‐093.

The novel anticonvulsant BIA 2-093 inhibits transmitter release during opening of voltage-gated sodium channels: a comparison with carbamazepine and oxcarbazepine.

(S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz/b,f/azepine-5-carboxamide (BIA 2-093) is endowed with high anticonvulsant activity and shares with carbamazepine (CBZ) and oxcarbazepine (OXC) the capability to inhibit voltage-gated sodium channels (VGSC). The present study was aimed to compare the effects of BIA 2-093, CBZ and OXC on the release of glutamate, aspartate, gamma-aminobutyric acid (GABA) and dopamine from striatal slices induced by the VGSC opener veratrine. The release of glutamate, aspartate, GABA and aspartate by veratrine from rat striatal slices was a concentration and time dependent process. All the three dibenzazepine carboxamide derivatives, BIA 2-093, CBZ and OXC inhibited in a concentration dependent manner (from 30 to 300 microM) the veratrine-induced release of glutamate, aspartate, GABA and dopamine. CBZ, OXC and BIA 2-093 were endowed with similar potencies in inhibiting veratrine-induced transmitter release. It is concluded that BIA 2-093, CBZ and OXC inhibit veratrine-induced transmitter release, which is in agreement with their capability to block VGSC. This property may be of importance for the anticonvulsant effects of BIA 2-093.

BIA 3-202, a novel catechol-O-methyltransferase inhibitor, enhances the availability of l-DOPA to the brain and reduces its O-methylation

1-[3,4-Dihydroxy-5-nitrophenyl]-2-phenyl-ethanone (BIA 3-202) is a new long-acting catechol-O-methyltransferase (COMT) inhibitor with limited access to the brain. The present study evaluated the interference of BIA 3-202 upon levels of L-3,4-dihydroxyphenylalanine (L-DOPA) and metabolites in plasma (3-O-methyl-L-DOPA) and brain [3-O-methyl-L-DOPA, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] in rats orally treated with L-DOPA (20 mg/kg) plus benserazide (30 mg/kg). At different time points (1, 3 and 6 h) after the administration of BIA 3-202 (0, 3, 10 and 30 mg/kg) or L-DOPA plus benserazide, rats were sacrificed and the right striatum was quickly dissected out and stored for the assay of L-DOPA, 3-O-methyl-L-DOPA, dopamine and amine metabolites. Levels of L-DOPA, 3-O-methyl-L-DOPA, dopamine, DOPAC and HVA in the striatum in L-DOPA plus benserazide-treated rats were higher than in vehicle-treated rats. However, this increase in striatal L-DOPA, dopamine, DOPAC and HVA was, in a dose- and time-dependent manner, even higher (P<0.05) in rats given BIA 3-202 (3, 10 and 30 mg/kg). This effect was accompanied by a marked decrease in 3-O-methyl-L-DOPA levels in the striatum of L-DOPA plus benserazide-treated rats. Increases in levels of L-DOPA and decreases in 3-O-methyl-L-DOPA levels in plasma also accompanied the administration of BIA 3-202. BIA 3-202 did not significantly affect levels of DOPAC and HVA in the striatum in vehicle-treated rats. It is concluded that administration of BIA 3-202 enhances the availability of L-DOPA to the brain by reducing its O-methylation in the periphery, which may prove beneficial in parkinsonian patients treated with L-DOPA plus an aromatic amino acid decarboxylase inhibitor.

Synthesis, anticonvulsant properties and pharmacokinetic profile of novel 10,11-dihydro-10-oxo-5H-dibenz/b,f/azepine-5-carboxamide derivatives.

A series of novel derivatives of oxcarbazepine (5), 10,11-dihydro-10-oxo-5H-dibenz/b,f/azepine-5-carboxamide was synthesised and evaluated for their anticonvulsant activity and sodium channel blocking properties. The oxime 8 was found to be the most active compound from this series, displaying greater potency than its geometric isomer 9 and exhibiting also the highest protective index value. Importantly, the metabolic profile of 8 differs from the already established dibenz/b,f/azepine-5-carboxamide drugs such as 1 and 5 which undergo rapid and complete conversion in vivo to several biologically active metabolites. In contrast 8 is metabolised to only a very minor extent leading to the conclusion that the observed anti-convulsant effect is solely attributable to 8. It is concluded that 8 may be as effective as 1 and 5 at controlling seizures and that the low toxicity and consequently high protective index should provide the compound with an improved side-effect profile.

The O-methylated derivative of L-DOPA, 3-O-methyl-L-DOPA, fails to inhibit neuronal and non-neuronal aromatic L-amino acid decarboxylase.

The present study examined whether the O-methylated derivative of L-DOPA, 3-O-methyl-L-DOPA (3-OM-L-DOPA), inhibits neuronal (brain) and non-neuronal (liver and kidney) aromatic L-amino acid decarboxylase (AADC) activity. The incubation of brain, liver and kidney homogenates with 3-OM-L-DOPA (5 mM) did not result in the formation of 3-methoxytyramine, the compound expected to result from the decarboxylation of 3-OM-L-DOPA. Incubation of tissue homogenates with L-DOPA resulted in a concentration-dependent formation of dopamine, revealing K(m) values (in mM) of similar magnitude for brain (0.8), liver (1.6) and kidney (1.0). Both benserazide and L-5-hydroxytryptophan (L-5-HTP) were found to produce concentration dependent decreases in AADC activity with K(i) values in the microM range. By contrast, 3-OM-L95% reduction) in liver and kidney AADC activity accompanied by a marked decrease (49% reduction) in brain AADC activity. By contrast, the administration of 30 mg/kg (p.o.) 3-OM-L-DOPA, which generates levels in brain, liver and kidney six-fold those in L-DOPA-treated rats, was found to change neither neuronal nor non-neuronal AADC activity. In conclusion, 3-OM-L-DOPA fails to interact with neuronal and non-neuronal AADC, either as substrate or inhibitor.

BIA 3-202, a Novel Catechol-O-Methyltransferase Inhibitor, Reduces the Peripheral O-Methylation of L-DOPA and Enhances Its Availability to the Brain

The present study aimes at determining the effects of the catechol-O-methyltransferase (COMT) inhibitor BIA 3-202 [1-(3,4-dihydroxy-5-nitrophenyl)-2-phenyl-ethanone] upon levels of L-3,4-dihydroxyphenylalanine (L-DOPA) and metabolites in peripheral circulation (jugular vein), whole brain, and striatal microdialysates in rats orally treated with L-DOPA plus benserazide. A low dose (3 mg/kg) of BIA 3-202 was relatively selective to inhibit liver COMT, being devoid of major significant inhibitory effects upon brain COMT. By contrast, a high dose (30 mg/kg) of BIA 3-202 markedly inhibited liver and brain COMT. BIA 3-202 (3 and 30 mg/kg) reduced the L-DOPA-induced rise of 3-O-methyl-L-DOPA in the peripheral circulation (jugular vein), brain tissue, and striatal dialysate, but failed to increase the levels of dopamine in striatal dialysates despite the increase in dopamine brain levels. However, the changes in brain levels of L-DOPA, 3-O-methyl-L-DOPA, and dopamine and in striatal dialysate levels of L-DOPA and 3-O-methyl-L-DOPA, obtained with 3 mg/kg BIA 3-202, were not different from those obtained with 30 mg/kg BIA 3-202. In conclusion, inhibition of peripheral COMT by BIA 3-202 may suffice to improve the availability of L-DOPA to the brain.

Metabolism of 10,11-dihydro-10-hydroxyimino-5H-dibenz/b, f/azepine-5-carboxamide, a potent anti-epileptic drug

1. 10,11-Dihydro-10-hydroxyimino-5H-dibenz/b, ƒ/azepine-5-carboxamide (BIA 2-024) is a new anti-epileptic drug similar to oxcarbazepine (OXC) in structure and efficacy, but with a preferred pharmacodynamic profile. It possesses high in vitro activity, but since oximes are usually metabolized to their corresponding ketones, it is important to know whether its in vivo potency is a result of acting as a prodrug of OXC or if it is acting on its own. 2. The drug was given orally to rats, mice and rabbits, the metabolites identified and pharmacokinetic profiles compared between those species. Furthermore, the pharmacokinetic profile of the main metabolite was established in the rat. The results were compared to in vitro metabolism studies with liver microsomes from different mammalian species and humans. 3. In an atypical reaction for oximes, BIA 2-024 in rats was rapidly (tmax = 2 h) metabolized to the non-active 10-nitro-derivative (BIA 2-254), whereas rabbits and particularly mice oxidized the oxime moiety to a much lower extent. BIA 2-254 was then transformed to OXC and subsequently to the 10-hydroxy derivative and other minor metabolites. 4. In vitro data showed a very similar cross-species behaviour as the in vivo results; human liver microsomes catalysed the oxidation of BIA 2-024 to the nitro metabolite only at a low rate, and the same was observed for the subsequent metabolism to OXC. 5. The results allow prediction of the in vivo metabolism of BIA 2-024 in humans, where this drug is most likely absorbed efficiently and excreted mainly as the parent compound with a relatively low hepatic clearance. With the exception of rat, BIA 2-024 does not act as a prodrug of OXC.

The dopamine antagonist sch 23390 reverses dizocilpine-induced blockade of cocaine sensitization.

The present work examined the effects of pre-treatment with Sch 23390, a selective D(1) receptor antagonist, on the dizocilpine-induced blockade of sensitization to the locomotor-stimulating effect of cocaine. Rats were given either cocaine [15mgkg(-1)day(-1), intraperitoneally (i.p.)] from day 1 to day 5 (cocaine-experienced rats) or vehicle (cocaine-naïve rats). From day 6 to day 15, animals remained drug-free in their home cages. On day 16 rats received a challenge injection of cocaine (15mgkg(-1)) or vehicle, and were tested for sensitization to the locomotor-stimulating effect of cocaine. In cocaine-naïve rats the acute effect of cocaine was a 1.5 times increase in locomotor activity. In cocaine-experienced rats, the acute effects of cocaine were considerably more pronounced than in cocaine-naïve rats; the stimulating effect of cocaine in these animals was a doubling in locomotor activity. In cocaine-naïve rats, pre-treatment with dizocilpine (100microgkg(-1)), Sch 23390 (100microgkg(-1)) or a combination of the two drugs from day 1 to day 5 changed neither spontaneous locomotor activity nor cocaine stimulant activity. By contrast, cocaine-experienced animals that had been given 100microgkg(-1) dizocilpine from day 1 to day 5 failed to show the increase in locomotor activity when challenged with cocaine on day 16. Pre-treatment with Sch 23390 (100microgkg(-1)day(-1), i.p.) from day 1 to day 5 was found to prevent completely the cocaine anti-sensitization properties of 100microgkg(-1) dizocilpine, but failed to prevent cocaine sensitization. On the other hand, horizontal activity in cocaine-experienced rats that had been given dizocilpine (100microgkg(-1)) 15min before cocaine challenge on day 16 was higher than in corresponding controls. It is concluded that prevention of cocaine sensitization by dizocilpine may be related to the events set into motion by the NMDA antagonist at the level of dopaminergic transmission involving D(1) receptors.

Rat Liver Catechol-O-Methyltransferase Kinetics and Assay Methodology

In mammals, catechol-O-methyltransferase (COMT) is distributed throughout various organs, the highest activities being found in the liver and kidney. However, comparisons of the kinetic parameters are difficult to perform, since the experimental procedures in the enzyme assay vary quite considerably. The present work was aimed at studying the optimal liver COMT assay conditions for determining the kinetics of the enzyme. The COMT assay was performed with liver homogenates from 60 days old male Wistar rats with adrenaline (AD) as the substrate. Time course experiments using 100 microM S-adenosyl-L-methionine (SAMe) and 300 microM AD showed linearity of O-methylation reaction upto 10 min. Using 100 microM SAMe, Vmax (nmol mg protein-1 h-1) and Km (microM) values progressively decreased respectively from 22.1 and 104.8 at 5 min down to 5.8 and 24.62 at 60 min incubation periods. This decrease was not due to end-product inhibition. Using 2500 microM AD, Km values (microM) for the methyl donor SAMe increased progressively from 174 at 5 min upto 1192.5 at 60 min; upto 30 min of incubation Vmax values did not change. When a 5 min incubation period and 500 microM SAMe were used, Vmax and Km values for liver COMT were 63.4 nmol mg protein-1 h-1 and 261.1 microM, respectively. It is concluded that an incubation period of 5 min and a SAMe concentration of 500 microM provide optimal conditions for the liver homogenate COMT assay.