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P.1.g.024 Potential prodrugs with anticonvulsant activity based on calixarene and gamma-aminobutyric acid
S216
P.1.g. Basic and clinical neuroscience − Neuropharmacology
Results: Application of (+)-SKF-38393 10 mM decreased K+-currents amplitude by 14.3±4.3% (n = 6, p < 0.01) with recovery after washing up to 100.0±1.0%. Application of (−)-quinpirole10 mM attenuated K+-currents amplitude in 11 neurons by 5.5±2.5% (p < 0.01), and elevated it in 6 neurons by 13.2±3.0% (p < 0.01) with recovery after washing in both cases up to 100.0±1.0%. In control tests when the physiological saline was applied instead of (−)-quinpirole, the same effects were not observed, K+-currents amplitude was decreased only by 0.49±0.07% (n = 15, p = 0.003), and was increased by 0.65±0.19% (n = 7, p = 0.01). The variation range of (−)-quinpirole effects on potential-activated K+-currents amplitude was 8.4±1.0%, and 0.1±0.1% in control group. (−)-Sulpiride 10 mM completely blocked the effect of (+)-SKF-28393, a D1 dopamine agonist (n = 5; p = 0.2), of (−)-quinpirole, a D2 dopamine agonist (n = 5; p > 0.05) and of dopamine (n = 6; p > 0.05) as well (all compounds in 10 mM). In control, (−)-sulpiride 10 mM did not effect on the potential-activated K+-currents amplitude (n = 16; p > 0.05). At the same time, (−)-sulpiride 10 mM strengthened dopamine effects on pick amplitude of potential-activated Na+-currents in the same neurons, as a result of a dimer complex formation [1]. The time dependence up to the Na+-currents pick under command potential −50 mV (tp ) was 1.5 ms. The time dependence up to the K+currents pick under command potential +40 mV (tp ) was 5 ms. Conclusion: Therefore, the formation of dimer (heteromer) complex of dopamine receptor on the CNS neuronal membrane processed in the period of 1.5 ms. The dimer (heteromer) structures were shown to form from all types of receptors represented in the CNS [2,3]. The ways of impulse activity transmission in the CNS could be observed on the basis of monomer/dimer model of dopamine neurons located in multipolar spinal cord neuron membranes of the lamprey larva [1]. If a physiological neurotransmitter/neuromodulator binds with the second receptor of dimer complex instead of artificial antagonist like used in the paper and any another transmitter/modulator binds with the first receptor, the physiological role of dimer (heteromer) complex formation in the CNS of mammalians will be coding information in form of dual code. References [1] Bukinich, A.A., Shabanov, P.D., 2013 A monomer/dimer model of dopamine membrane receptors for pharmacological investigations. 26th ECNP Congress, 5−9 October 2013, Barcelona, Spain, abstract number CG13P-0127. [2] Franco, R., Casado, V., Cortes, A., Mallol, J., Ciruela, F., Ferre, S., Lluis, C., Canela, E.I., 2008 G-protein-coupled receptor heteromers: function and ligand pharmacology, Brit. J. Pharmacol. 153, S90-S98. [3] Casad´o, V., Ferrada, C., Bonaventura, J., Gracia, E., Mallol, J., Canela, E.I., Llu´ıs, C., Cort´es, A., Franco, R., 2009 Useful pharmacological parameters for G-protein-coupled receptor homodimers obtained from competition experiments. Agonist-antagonist binding modulation. Biochem. Pharmacol. 78(12), 1456−63.
P.1.g.024 Potential prodrugs with anticonvulsant activity based on calixarene and gammaaminobutyric acid M. Nesterkina1 ° , E. Alekseeva2 , I. Kravchenko1 1 Odessa National University, Pharmaceutical chemistry, Odessa, Ukraine; 2 A.V. Bogatsky Physico-Chemical Institute National Academy of Sciences of Ukraine, Molecular structure and chemoinformatics, Odessa, Ukraine g-Aminobutyric acid (GABA), the principal inhibitory transmitter in the cerebral cortex, maintains the inhibitory tone that coun-
terbalances neuronal excitation [1]. Because of its abundance in the brain, ability to produce hyperpolarizing inhibition of almost all neurons and the discovery that many convulsants inhibit its synthesis, GABA has often appeared to be the key to epilepsy [2]. A variety of compounds with properties of a gamma-aminobutyric acid agonist have been investigated in an effort to improve the poor uptake of GABA through blood–brain barrier (BBB) into the central nervous system and to provide potential new treatments for neuropsychiatric disorders such as epilepsy and Huntington’s disease. Low bioavailability of the acid is explained by poor BBB permeability due to a number of physico-chemical properties like its high polarity − under physiological pH values such substances are in the form of bipolar ions whereby not penetrate the barrier formed by capillary endothelial cells. One approach for permeability increasing of hydrophilic drugs across BBB is carriers use applied as a molecular platform. Among the macromolecules the calixarenes are widely used due to their high lipophilicity and limited toxicity. Therefore, we decided to obtain a supramolecular complex (I) based on macrocycle with GABA as a ‘guest’ molecule as well as compound containing GABA (or its ester) covalently attached to calixarene scaffold (II). These compounds were investigated as potential anticonvulsant agents. The anticonvulsant activity of I and II was evaluated by determining the minimum effective dose of convulsant agent Pentylenetetrazol, causing clonico-tonic convulsions (DCTC, %) and tonic extension (DTE, %) in experimental mice upon intravenous infusion. It was shown that complex I demonstrates higher anticonvulsant effect in comparison to the initial amino acid for short time periods (3 and 6 hours). In addition, the complex I possesses prolonged anticonvulsant action which is 217% and 215% for DCTC and DTE, respectively (compared with control) after 24 hours upon oral administration. Previously, it has been shown that GABA esters exceed biological activity of amino acid [3]. These data served as precondition for obtaining a calixarene derivative with residues of GABA methyl ester. Compound II is considered as a prodrug capable of being hydrolyzed in brain tissue by esterases and amidases with GABA release. Using the assay procedure described above the following results were obtained for DCTC and DTE, respectively: GABA − 138 and 139%, GABA methyl ester − 168 and 178%, compound II − 155 and 159%. As seen, compound II demonstrates lower anticonvulsant activity compared with GABA ester that may be explained by the short time period (6 hours) after administration and, as a consequence, low hydrolysis degree of calixarene derivative II. This observation calls for much detailed research aiming to time increase after prodrug administration. This investigation is already under way along with hydrolysis study in vitro. References [1] Treiman David M., 2001. GABAergetic mechanisms in epilepsy. Epilepsia 42, 8−12. [2] Alekseeva E., 2006. Calix[4]arenes containing benzodiazepinone fragments at the lower rim. Russian J. of Gen Chemistry 76, 1464–1467. [3] Jacob J., 1985. Synthesis, brain uptake, and pharmacological properties of lipid esters of g-aminobutyric acid. J. Med. Chem. 28, 106–110.
P.1.g. Basic and clinical neuroscience − Neuropharmacology
Results: Application of (+)-SKF-38393 10 mM decreased K+-currents amplitude by 14.3±4.3% (n = 6, p < 0.01) with recovery after washing up to 100.0±1.0%. Application of (−)-quinpirole10 mM attenuated K+-currents amplitude in 11 neurons by 5.5±2.5% (p < 0.01), and elevated it in 6 neurons by 13.2±3.0% (p < 0.01) with recovery after washing in both cases up to 100.0±1.0%. In control tests when the physiological saline was applied instead of (−)-quinpirole, the same effects were not observed, K+-currents amplitude was decreased only by 0.49±0.07% (n = 15, p = 0.003), and was increased by 0.65±0.19% (n = 7, p = 0.01). The variation range of (−)-quinpirole effects on potential-activated K+-currents amplitude was 8.4±1.0%, and 0.1±0.1% in control group. (−)-Sulpiride 10 mM completely blocked the effect of (+)-SKF-28393, a D1 dopamine agonist (n = 5; p = 0.2), of (−)-quinpirole, a D2 dopamine agonist (n = 5; p > 0.05) and of dopamine (n = 6; p > 0.05) as well (all compounds in 10 mM). In control, (−)-sulpiride 10 mM did not effect on the potential-activated K+-currents amplitude (n = 16; p > 0.05). At the same time, (−)-sulpiride 10 mM strengthened dopamine effects on pick amplitude of potential-activated Na+-currents in the same neurons, as a result of a dimer complex formation [1]. The time dependence up to the Na+-currents pick under command potential −50 mV (tp ) was 1.5 ms. The time dependence up to the K+currents pick under command potential +40 mV (tp ) was 5 ms. Conclusion: Therefore, the formation of dimer (heteromer) complex of dopamine receptor on the CNS neuronal membrane processed in the period of 1.5 ms. The dimer (heteromer) structures were shown to form from all types of receptors represented in the CNS [2,3]. The ways of impulse activity transmission in the CNS could be observed on the basis of monomer/dimer model of dopamine neurons located in multipolar spinal cord neuron membranes of the lamprey larva [1]. If a physiological neurotransmitter/neuromodulator binds with the second receptor of dimer complex instead of artificial antagonist like used in the paper and any another transmitter/modulator binds with the first receptor, the physiological role of dimer (heteromer) complex formation in the CNS of mammalians will be coding information in form of dual code. References [1] Bukinich, A.A., Shabanov, P.D., 2013 A monomer/dimer model of dopamine membrane receptors for pharmacological investigations. 26th ECNP Congress, 5−9 October 2013, Barcelona, Spain, abstract number CG13P-0127. [2] Franco, R., Casado, V., Cortes, A., Mallol, J., Ciruela, F., Ferre, S., Lluis, C., Canela, E.I., 2008 G-protein-coupled receptor heteromers: function and ligand pharmacology, Brit. J. Pharmacol. 153, S90-S98. [3] Casad´o, V., Ferrada, C., Bonaventura, J., Gracia, E., Mallol, J., Canela, E.I., Llu´ıs, C., Cort´es, A., Franco, R., 2009 Useful pharmacological parameters for G-protein-coupled receptor homodimers obtained from competition experiments. Agonist-antagonist binding modulation. Biochem. Pharmacol. 78(12), 1456−63.
P.1.g.024 Potential prodrugs with anticonvulsant activity based on calixarene and gammaaminobutyric acid M. Nesterkina1 ° , E. Alekseeva2 , I. Kravchenko1 1 Odessa National University, Pharmaceutical chemistry, Odessa, Ukraine; 2 A.V. Bogatsky Physico-Chemical Institute National Academy of Sciences of Ukraine, Molecular structure and chemoinformatics, Odessa, Ukraine g-Aminobutyric acid (GABA), the principal inhibitory transmitter in the cerebral cortex, maintains the inhibitory tone that coun-
terbalances neuronal excitation [1]. Because of its abundance in the brain, ability to produce hyperpolarizing inhibition of almost all neurons and the discovery that many convulsants inhibit its synthesis, GABA has often appeared to be the key to epilepsy [2]. A variety of compounds with properties of a gamma-aminobutyric acid agonist have been investigated in an effort to improve the poor uptake of GABA through blood–brain barrier (BBB) into the central nervous system and to provide potential new treatments for neuropsychiatric disorders such as epilepsy and Huntington’s disease. Low bioavailability of the acid is explained by poor BBB permeability due to a number of physico-chemical properties like its high polarity − under physiological pH values such substances are in the form of bipolar ions whereby not penetrate the barrier formed by capillary endothelial cells. One approach for permeability increasing of hydrophilic drugs across BBB is carriers use applied as a molecular platform. Among the macromolecules the calixarenes are widely used due to their high lipophilicity and limited toxicity. Therefore, we decided to obtain a supramolecular complex (I) based on macrocycle with GABA as a ‘guest’ molecule as well as compound containing GABA (or its ester) covalently attached to calixarene scaffold (II). These compounds were investigated as potential anticonvulsant agents. The anticonvulsant activity of I and II was evaluated by determining the minimum effective dose of convulsant agent Pentylenetetrazol, causing clonico-tonic convulsions (DCTC, %) and tonic extension (DTE, %) in experimental mice upon intravenous infusion. It was shown that complex I demonstrates higher anticonvulsant effect in comparison to the initial amino acid for short time periods (3 and 6 hours). In addition, the complex I possesses prolonged anticonvulsant action which is 217% and 215% for DCTC and DTE, respectively (compared with control) after 24 hours upon oral administration. Previously, it has been shown that GABA esters exceed biological activity of amino acid [3]. These data served as precondition for obtaining a calixarene derivative with residues of GABA methyl ester. Compound II is considered as a prodrug capable of being hydrolyzed in brain tissue by esterases and amidases with GABA release. Using the assay procedure described above the following results were obtained for DCTC and DTE, respectively: GABA − 138 and 139%, GABA methyl ester − 168 and 178%, compound II − 155 and 159%. As seen, compound II demonstrates lower anticonvulsant activity compared with GABA ester that may be explained by the short time period (6 hours) after administration and, as a consequence, low hydrolysis degree of calixarene derivative II. This observation calls for much detailed research aiming to time increase after prodrug administration. This investigation is already under way along with hydrolysis study in vitro. References [1] Treiman David M., 2001. GABAergetic mechanisms in epilepsy. Epilepsia 42, 8−12. [2] Alekseeva E., 2006. Calix[4]arenes containing benzodiazepinone fragments at the lower rim. Russian J. of Gen Chemistry 76, 1464–1467. [3] Jacob J., 1985. Synthesis, brain uptake, and pharmacological properties of lipid esters of g-aminobutyric acid. J. Med. Chem. 28, 106–110.