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Ihtesham Ur Rehman - One of the best experts on this subject based on the ideXlab platform.
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synthesis and characterization of a novel fast set Proline Derivative containing glass ionomer cement with enhanced mechanical properties
Acta Biomaterialia, 2009Co-Authors: Alireza Moshaverinia, Nima Roohpour, Ihtesham Ur RehmanAbstract:In this study, a methacryloyl Derivative of l-Proline was synthesized, characterized and incorporated into a conventional glass ionomer cement (GIC) with a polyacid composition. Subsequently, the effects of incorporation of synthesized N-methacryloyl-Proline and terpolymer on the GIC's mechanical and working properties were studied. 1-Methacryloylpyrrolidone-2-carboxylic acid was synthesized and used in a polymerization reaction with acrylic acid and itaconic acid in order to form terpolymer which was used in Fuji II commercial GIC formulations. Chemical structural characterization of the resulting products was performed using (1)H nuclear magnetic resonance and Fourier transform infrared spectroscopy. The viscosity and molecular weight of the terpolymer were also measured. The mechanical strength properties of the modified GICs were evaluated after 24h or 1 week of immersion in distilled water at 37 degrees C. Analysis of variance was used to study the statistical significance of the mechanical strengths and working properties, and to compare them with a control group. Results showed that N-methacryloyl-Proline modified GICs exhibited significantly higher compressive strength (CS; 195-210MPa), higher diametral tensile strength (DTS; 19-26MPa) and higher biaxial flexural strength (38-46MPa) in comparison to Fuji II GIC (161-166MPa in CS, 12-14MPa in DTS and 13-18MPa in biaxial flexural strength). The working properties (setting and working time) of the modified samples showed that the modified cement was a fast-set cement. It was concluded that a novel amino acid-containing GIC has been developed in this study with 27%, 94% and 170% increases in values for compressive, diametral tensile and biaxial flexural strength, respectively, in comparison to commercial Fuji II GIC.
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synthesis and characterization of a novel fast set Proline Derivative containing glass ionomer cement with enhanced mechanical properties
Acta Biomaterialia, 2009Co-Authors: Alireza Moshaverinia, Nima Roohpour, Ihtesham Ur RehmanAbstract:In this study, a methacryloyl Derivative of l-Proline was synthesized, characterized and incorporated into a conventional glass ionomer cement (GIC) with a polyacid composition. Subsequently, the effects of incorporation of synthesized N-methacryloyl-Proline and terpolymer on the GIC's mechanical and working properties were studied. 1-Methacryloylpyrrolidone-2-carboxylic acid was synthesized and used in a polymerization reaction with acrylic acid and itaconic acid in order to form terpolymer which was used in Fuji II commercial GIC formulations. Chemical structural characterization of the resulting products was performed using 1H nuclear magnetic resonance and Fourier transform infrared spectroscopy. The viscosity and molecular weight of the terpolymer were also measured. The mechanical strength properties of the modified GICs were evaluated after 24 h or 1 week of immersion in distilled water at 37 °C. Analysis of variance was used to study the statistical significance of the mechanical strengths and working properties, and to compare them with a control group. Results showed that N-methacryloyl-Proline modified GICs exhibited significantly higher compressive strength (CS; 195-210 MPa), higher diametral tensile strength (DTS; 19-26 MPa) and higher biaxial flexural strength (38-46 MPa) in comparison to Fuji II GIC (161-166 MPa in CS, 12-14 MPa in DTS and 13-18 MPa in biaxial flexural strength). The working properties (setting and working time) of the modified samples showed that the modified cement was a fast-set cement. It was concluded that a novel amino acid-containing GIC has been developed in this study with 27%, 94% and 170% increases in values for compressive, diametral tensile and biaxial flexural strength, respectively, in comparison to commercial Fuji II GIC. © 2008 Acta Materialia Inc.
Annamaria De Luca - One of the best experts on this subject based on the ideXlab platform.
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searching for novel anti myotonic agents pharmacophore requirement for use dependent block of skeletal muscle sodium channels by n benzylated cyclic Derivatives of tocainide
Neuromuscular Disorders, 2012Co-Authors: Annamaria De Luca, Michela De Bellis, Filomena Corbo, Carlo Franchini, Marilena Muraglia, Alessia Catalano, Alessia Carocci, Diana Conte CamerinoAbstract:Drug screening on sodium currents of native myofibers by means of voltage-clamp recordings is predictive of pre-clinical anti-myotonic activity in vivo and ex vivo. By this approach we identified the N-benzylated beta-Proline Derivative of tocainide (To10) as the most potent use-dependent blocker of Nav1.4 so far. We tested novel analogs with modifications on the pharmacophore groups of To10. The substitution of the Proline cycle with less planar piperidine or piperazine rings disclosed the importance of a two carbon atom distance and/or an additional nitrogen atom for potency. Structural changes on the xylididic group corroborated the role of a proper electronic cloud for hydrophobic interactions with the binding site. The N-benzylated moiety lead to a stereoselective behavior only in the rigid alpha-Proline analog To11 vs. To10 and N-benzylated tocainide (To12). The results confirm the strict structural requirements of Nav1.4 blockers and allow to refine the drug design toward novel anti-myotonic drugs.
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increased rigidity of the chiral centre of tocainide favours stereoselectivity and use dependent block of skeletal muscle na channels enhancing the antimyotonic activity in vivo
British Journal of Pharmacology, 2001Co-Authors: S Talon, Jf Desaphy, Annamaria De Luca, Michela De Bellis, Filomena Corbo, Carlo Franchini, Giovanni Lentini, Antonio Scilimati, Paolo Tortorella, Harald JockuschAbstract:Searching for the structural requirements improving the potency and the stereoselectivity of Na+ channel blockers as antimyotonic agents, new Derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid α-Proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers. Their ability to block Na+ currents, elicited from −100 to −20 mV at 0.3 Hz (tonic block) and 2 – 10 Hz (use-dependent block) frequencies, was investigated in vitro on single fibres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method. The α-Proline Derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2- and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. Contrarily to tocainide, all Proline Derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic Derivatives markedly decreased the drug potency with respect to tocainide. Antimyotonic activity, evaluated as the shortening of the time of righting reflexes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more effective for R-To5 than for R-Tocainide. Thus, constraining the chiral centre of tocainide in α-Proline cycle leads to more potent and stereoselective use-dependent Na+ channel blockers with improved therapeutic potential. Keywords: Na+ channel, native frog skeletal muscle fibres, vaseline-gap voltage-clamp, tocainide Derivatives, use-dependent block, stereoselectivity, antimyotonic activity, adr/adr mice Introduction Tocainide is an orally effective lidocaine Derivative, able to block the voltage-gated Na+ channels in a use-dependent manner, a feature that enhances its potency in situations of prolonged depolarization and/or excessive firing of action potentials with respect to a physiological excitability (Catterall, 1987; de luca et al., 1997b; Conte Camerino et al., 2000). Thus, tocainide is actually among the few drugs clinically used for the symptomatic treatment of hyperexcitability of myotonic syndromes, hereditary disorders of skeletal muscle due to genetic mutations on either sodium or chloride channel genes (Streib, 1986; Kwiecinski et al., 1992, Rudel et al., 1994; Lehmann-Horn & Rudel, 1996; Cannon, 1997; Ptacek, 1998). However, its clinical use presents some disadvantages, due to the high doses required for its antimyotonic effectiveness with the risk of a greater incidence of serious side effects, especially at the cardiac, haematopoietic and central nervous system levels (Roden & Woosley, 1986; Puniani & Bertorini, 1991; Rudel et al., 1994). The use-dependent inhibition of Na+ channels by tocainide and other local anaesthetic (LA) drugs relies on the higher affinity of these drugs for their receptor site on the α-subunit of the channel when this latter is open and/or inactivated, and to a slow recovery from inactivation of the drug-bound channels during membrane repolarization (Catterall, 1987; de luca et al., 1997a). At the resting state, the LA interacts with its presumed receptor through hydrophobic interactions with two aromatic amino acid residues at the D4 – S6 segment (Ragsdale et al., 1994; Wright et al., 1998). Gating movements due to the channel opening may then allow further drug interactions with other residues in the pore that could stabilize the drug-bound channel in an inactivated state (Wang et al., 2000; Yarov-Yarovoy et al., 2001). Furthermore, a site in D1 – S6 segment would also be involved in determining the drug stereoselectivity (Nau et al., 1999). This feature is important because tocainide and mexiletine, another LA-like compound, have a chiral carbon atom that contributes to the potency and the stereoselectivity of the drug (Tricarico et al., 1991; de luca et al., 1997a; 2000; Desaphy et al., 1999). In particular, we found that the introduction of an aromatic lipophilic group on the chiral centre of mexiletine highly increases the drug potency suggesting the establishment of a strong two-points interaction with the two hydrophobic pockets of the receptor in D4 – S6 segment (Wright et al., 1998; de luca et al., 2000). Furthermore, the nature of the group on the stereogenic centre is important for the drug stereoselectivity because the introduction of an unbending lipophilic group such as the isopropyl one enhanced the stereoselectivity, likely due to the occurrence of a three-points interaction of this more rigid structure with the receptor (de luca et al., 1997a; 2000). In agreement with previous data, we confirmed that a higher basicity in the molecule determines a greater use-dependent behaviour (de luca et al., 1997a). To go further in the study of the structure-activity relationship and to better evaluate the role of the chiral centre in the potency and/or stereoselectivity of drug for blocking Na+ channels, the effects of the pure enantiomers of a new Derivative of tocainide, in which the asymmetric carbon atom is constrained in a rigid α-Proline cycle (To5, Figure 1) were evaluated on Na+ currents of native frog skeletal muscle fibres. In addition, by introducing methyl groups on one or both of the nitrogen atoms of the Proline Derivative (as in To6, To7 and To8, Figure 1) or by constraining the chiral centre in a pyrrolo-imidazolic cycle (To3 and To4, Figure 1), we have investigated whether a further decrease in the ability of the molecule to transit through various conformations could modify the drug potency and/or stereoselectivity, and consequently the interaction with the receptor. The results indicate that constraining the stereogenic centre in a α-Proline cycle, as in To5, highly improves potency, stereoselectivity and use-dependent behaviour of Na+ channel blockers. Accordingly, an acute in vivo administration of tocainide and To5 in myotonic adr/adr mice showed that To5 was 3 fold more effective than its parent compound in decreasing the time of righting reflex of myotonic mice. On the basis of these data, we can conclude that the newly synthesized Proline Derivative of tocainide, To5, may be considered as a potential therapeutic agent in the treatment of myotonic syndromes. Figure 1 Chemical structure of tocainide and its newly synthesized α-Proline and pyrrolo-imidazolic analogues. Methods Fibre preparation and voltage clamp apparatus Segments of undamaged single muscle fibres (about 1 cm in length) were obtained by microsurgery (plucking procedure) from the ventral branch of the semitendinosus muscle of Rana Esculenta bathed in normal physiological solution at room temperature. The cut-end fibre was then superfused with an internal solution and mounted across three chamber partitions, which delineated the four pools. Three strips of vaseline were applied over the fibre and carefully sealed to the fibre to reduce leakage. The width of the gaps of the central pools (A and B) had been previously set to 70 – 100 μM and 200 μM, respectively. Four KCl/agar bridges electrodes connected the recording chamber to the voltage clamp amplifier based on methods described by Hille & Campbell (1976) and detailed elsewhere (de luca et al., 1995; 1997a). When the solution level was lowered below the vaseline strips, the four pools were physically and electrically independent from each other: this was confirmed by verifying that no leak current was flowing upon increasing the amplifier gain. Then the solution in the pool A was replaced with the external solution and after about 10 min of equilibration the recordings were performed at 10°C. The usual holding potential (h.p.) was −100 mV. Sodium currents were recorded using an amplifier connected via a A/D and D/A Digidata 1200 Interface (Axon Instruments, Foster City, CA., U.S.A.) to a 486 DX2/66 personal computer and stored on the hard disk. The stimulation protocols and data acquisition were driven by the Clampex program (pClamp6 software package; Axon Instruments). The currents flowing in response to depolarizing command voltages were low pass filtered at 10 kHz (Frequency Devices, U.S.A.), visualized on an oscilloscope and sampled at 20 kHz. When necessary, leak and capacities currents were subtracted by P/4 method. The acquired traces were analysed later using Clampfit program (pClamp6 software package; Axon Instruments).
Harald Jockusch - One of the best experts on this subject based on the ideXlab platform.
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increased rigidity of the chiral centre of tocainide favours stereoselectivity and use dependent block of skeletal muscle na channels enhancing the antimyotonic activity in vivo
British Journal of Pharmacology, 2001Co-Authors: S Talon, Jf Desaphy, Annamaria De Luca, Michela De Bellis, Filomena Corbo, Carlo Franchini, Giovanni Lentini, Antonio Scilimati, Paolo Tortorella, Harald JockuschAbstract:Searching for the structural requirements improving the potency and the stereoselectivity of Na+ channel blockers as antimyotonic agents, new Derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid α-Proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers. Their ability to block Na+ currents, elicited from −100 to −20 mV at 0.3 Hz (tonic block) and 2 – 10 Hz (use-dependent block) frequencies, was investigated in vitro on single fibres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method. The α-Proline Derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2- and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. Contrarily to tocainide, all Proline Derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic Derivatives markedly decreased the drug potency with respect to tocainide. Antimyotonic activity, evaluated as the shortening of the time of righting reflexes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more effective for R-To5 than for R-Tocainide. Thus, constraining the chiral centre of tocainide in α-Proline cycle leads to more potent and stereoselective use-dependent Na+ channel blockers with improved therapeutic potential. Keywords: Na+ channel, native frog skeletal muscle fibres, vaseline-gap voltage-clamp, tocainide Derivatives, use-dependent block, stereoselectivity, antimyotonic activity, adr/adr mice Introduction Tocainide is an orally effective lidocaine Derivative, able to block the voltage-gated Na+ channels in a use-dependent manner, a feature that enhances its potency in situations of prolonged depolarization and/or excessive firing of action potentials with respect to a physiological excitability (Catterall, 1987; de luca et al., 1997b; Conte Camerino et al., 2000). Thus, tocainide is actually among the few drugs clinically used for the symptomatic treatment of hyperexcitability of myotonic syndromes, hereditary disorders of skeletal muscle due to genetic mutations on either sodium or chloride channel genes (Streib, 1986; Kwiecinski et al., 1992, Rudel et al., 1994; Lehmann-Horn & Rudel, 1996; Cannon, 1997; Ptacek, 1998). However, its clinical use presents some disadvantages, due to the high doses required for its antimyotonic effectiveness with the risk of a greater incidence of serious side effects, especially at the cardiac, haematopoietic and central nervous system levels (Roden & Woosley, 1986; Puniani & Bertorini, 1991; Rudel et al., 1994). The use-dependent inhibition of Na+ channels by tocainide and other local anaesthetic (LA) drugs relies on the higher affinity of these drugs for their receptor site on the α-subunit of the channel when this latter is open and/or inactivated, and to a slow recovery from inactivation of the drug-bound channels during membrane repolarization (Catterall, 1987; de luca et al., 1997a). At the resting state, the LA interacts with its presumed receptor through hydrophobic interactions with two aromatic amino acid residues at the D4 – S6 segment (Ragsdale et al., 1994; Wright et al., 1998). Gating movements due to the channel opening may then allow further drug interactions with other residues in the pore that could stabilize the drug-bound channel in an inactivated state (Wang et al., 2000; Yarov-Yarovoy et al., 2001). Furthermore, a site in D1 – S6 segment would also be involved in determining the drug stereoselectivity (Nau et al., 1999). This feature is important because tocainide and mexiletine, another LA-like compound, have a chiral carbon atom that contributes to the potency and the stereoselectivity of the drug (Tricarico et al., 1991; de luca et al., 1997a; 2000; Desaphy et al., 1999). In particular, we found that the introduction of an aromatic lipophilic group on the chiral centre of mexiletine highly increases the drug potency suggesting the establishment of a strong two-points interaction with the two hydrophobic pockets of the receptor in D4 – S6 segment (Wright et al., 1998; de luca et al., 2000). Furthermore, the nature of the group on the stereogenic centre is important for the drug stereoselectivity because the introduction of an unbending lipophilic group such as the isopropyl one enhanced the stereoselectivity, likely due to the occurrence of a three-points interaction of this more rigid structure with the receptor (de luca et al., 1997a; 2000). In agreement with previous data, we confirmed that a higher basicity in the molecule determines a greater use-dependent behaviour (de luca et al., 1997a). To go further in the study of the structure-activity relationship and to better evaluate the role of the chiral centre in the potency and/or stereoselectivity of drug for blocking Na+ channels, the effects of the pure enantiomers of a new Derivative of tocainide, in which the asymmetric carbon atom is constrained in a rigid α-Proline cycle (To5, Figure 1) were evaluated on Na+ currents of native frog skeletal muscle fibres. In addition, by introducing methyl groups on one or both of the nitrogen atoms of the Proline Derivative (as in To6, To7 and To8, Figure 1) or by constraining the chiral centre in a pyrrolo-imidazolic cycle (To3 and To4, Figure 1), we have investigated whether a further decrease in the ability of the molecule to transit through various conformations could modify the drug potency and/or stereoselectivity, and consequently the interaction with the receptor. The results indicate that constraining the stereogenic centre in a α-Proline cycle, as in To5, highly improves potency, stereoselectivity and use-dependent behaviour of Na+ channel blockers. Accordingly, an acute in vivo administration of tocainide and To5 in myotonic adr/adr mice showed that To5 was 3 fold more effective than its parent compound in decreasing the time of righting reflex of myotonic mice. On the basis of these data, we can conclude that the newly synthesized Proline Derivative of tocainide, To5, may be considered as a potential therapeutic agent in the treatment of myotonic syndromes. Figure 1 Chemical structure of tocainide and its newly synthesized α-Proline and pyrrolo-imidazolic analogues. Methods Fibre preparation and voltage clamp apparatus Segments of undamaged single muscle fibres (about 1 cm in length) were obtained by microsurgery (plucking procedure) from the ventral branch of the semitendinosus muscle of Rana Esculenta bathed in normal physiological solution at room temperature. The cut-end fibre was then superfused with an internal solution and mounted across three chamber partitions, which delineated the four pools. Three strips of vaseline were applied over the fibre and carefully sealed to the fibre to reduce leakage. The width of the gaps of the central pools (A and B) had been previously set to 70 – 100 μM and 200 μM, respectively. Four KCl/agar bridges electrodes connected the recording chamber to the voltage clamp amplifier based on methods described by Hille & Campbell (1976) and detailed elsewhere (de luca et al., 1995; 1997a). When the solution level was lowered below the vaseline strips, the four pools were physically and electrically independent from each other: this was confirmed by verifying that no leak current was flowing upon increasing the amplifier gain. Then the solution in the pool A was replaced with the external solution and after about 10 min of equilibration the recordings were performed at 10°C. The usual holding potential (h.p.) was −100 mV. Sodium currents were recorded using an amplifier connected via a A/D and D/A Digidata 1200 Interface (Axon Instruments, Foster City, CA., U.S.A.) to a 486 DX2/66 personal computer and stored on the hard disk. The stimulation protocols and data acquisition were driven by the Clampex program (pClamp6 software package; Axon Instruments). The currents flowing in response to depolarizing command voltages were low pass filtered at 10 kHz (Frequency Devices, U.S.A.), visualized on an oscilloscope and sampled at 20 kHz. When necessary, leak and capacities currents were subtracted by P/4 method. The acquired traces were analysed later using Clampfit program (pClamp6 software package; Axon Instruments).
Filomena Corbo - One of the best experts on this subject based on the ideXlab platform.
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searching for novel anti myotonic agents pharmacophore requirement for use dependent block of skeletal muscle sodium channels by n benzylated cyclic Derivatives of tocainide
Neuromuscular Disorders, 2012Co-Authors: Annamaria De Luca, Michela De Bellis, Filomena Corbo, Carlo Franchini, Marilena Muraglia, Alessia Catalano, Alessia Carocci, Diana Conte CamerinoAbstract:Drug screening on sodium currents of native myofibers by means of voltage-clamp recordings is predictive of pre-clinical anti-myotonic activity in vivo and ex vivo. By this approach we identified the N-benzylated beta-Proline Derivative of tocainide (To10) as the most potent use-dependent blocker of Nav1.4 so far. We tested novel analogs with modifications on the pharmacophore groups of To10. The substitution of the Proline cycle with less planar piperidine or piperazine rings disclosed the importance of a two carbon atom distance and/or an additional nitrogen atom for potency. Structural changes on the xylididic group corroborated the role of a proper electronic cloud for hydrophobic interactions with the binding site. The N-benzylated moiety lead to a stereoselective behavior only in the rigid alpha-Proline analog To11 vs. To10 and N-benzylated tocainide (To12). The results confirm the strict structural requirements of Nav1.4 blockers and allow to refine the drug design toward novel anti-myotonic drugs.
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increased rigidity of the chiral centre of tocainide favours stereoselectivity and use dependent block of skeletal muscle na channels enhancing the antimyotonic activity in vivo
British Journal of Pharmacology, 2001Co-Authors: S Talon, Jf Desaphy, Annamaria De Luca, Michela De Bellis, Filomena Corbo, Carlo Franchini, Giovanni Lentini, Antonio Scilimati, Paolo Tortorella, Harald JockuschAbstract:Searching for the structural requirements improving the potency and the stereoselectivity of Na+ channel blockers as antimyotonic agents, new Derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid α-Proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers. Their ability to block Na+ currents, elicited from −100 to −20 mV at 0.3 Hz (tonic block) and 2 – 10 Hz (use-dependent block) frequencies, was investigated in vitro on single fibres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method. The α-Proline Derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2- and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. Contrarily to tocainide, all Proline Derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic Derivatives markedly decreased the drug potency with respect to tocainide. Antimyotonic activity, evaluated as the shortening of the time of righting reflexes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more effective for R-To5 than for R-Tocainide. Thus, constraining the chiral centre of tocainide in α-Proline cycle leads to more potent and stereoselective use-dependent Na+ channel blockers with improved therapeutic potential. Keywords: Na+ channel, native frog skeletal muscle fibres, vaseline-gap voltage-clamp, tocainide Derivatives, use-dependent block, stereoselectivity, antimyotonic activity, adr/adr mice Introduction Tocainide is an orally effective lidocaine Derivative, able to block the voltage-gated Na+ channels in a use-dependent manner, a feature that enhances its potency in situations of prolonged depolarization and/or excessive firing of action potentials with respect to a physiological excitability (Catterall, 1987; de luca et al., 1997b; Conte Camerino et al., 2000). Thus, tocainide is actually among the few drugs clinically used for the symptomatic treatment of hyperexcitability of myotonic syndromes, hereditary disorders of skeletal muscle due to genetic mutations on either sodium or chloride channel genes (Streib, 1986; Kwiecinski et al., 1992, Rudel et al., 1994; Lehmann-Horn & Rudel, 1996; Cannon, 1997; Ptacek, 1998). However, its clinical use presents some disadvantages, due to the high doses required for its antimyotonic effectiveness with the risk of a greater incidence of serious side effects, especially at the cardiac, haematopoietic and central nervous system levels (Roden & Woosley, 1986; Puniani & Bertorini, 1991; Rudel et al., 1994). The use-dependent inhibition of Na+ channels by tocainide and other local anaesthetic (LA) drugs relies on the higher affinity of these drugs for their receptor site on the α-subunit of the channel when this latter is open and/or inactivated, and to a slow recovery from inactivation of the drug-bound channels during membrane repolarization (Catterall, 1987; de luca et al., 1997a). At the resting state, the LA interacts with its presumed receptor through hydrophobic interactions with two aromatic amino acid residues at the D4 – S6 segment (Ragsdale et al., 1994; Wright et al., 1998). Gating movements due to the channel opening may then allow further drug interactions with other residues in the pore that could stabilize the drug-bound channel in an inactivated state (Wang et al., 2000; Yarov-Yarovoy et al., 2001). Furthermore, a site in D1 – S6 segment would also be involved in determining the drug stereoselectivity (Nau et al., 1999). This feature is important because tocainide and mexiletine, another LA-like compound, have a chiral carbon atom that contributes to the potency and the stereoselectivity of the drug (Tricarico et al., 1991; de luca et al., 1997a; 2000; Desaphy et al., 1999). In particular, we found that the introduction of an aromatic lipophilic group on the chiral centre of mexiletine highly increases the drug potency suggesting the establishment of a strong two-points interaction with the two hydrophobic pockets of the receptor in D4 – S6 segment (Wright et al., 1998; de luca et al., 2000). Furthermore, the nature of the group on the stereogenic centre is important for the drug stereoselectivity because the introduction of an unbending lipophilic group such as the isopropyl one enhanced the stereoselectivity, likely due to the occurrence of a three-points interaction of this more rigid structure with the receptor (de luca et al., 1997a; 2000). In agreement with previous data, we confirmed that a higher basicity in the molecule determines a greater use-dependent behaviour (de luca et al., 1997a). To go further in the study of the structure-activity relationship and to better evaluate the role of the chiral centre in the potency and/or stereoselectivity of drug for blocking Na+ channels, the effects of the pure enantiomers of a new Derivative of tocainide, in which the asymmetric carbon atom is constrained in a rigid α-Proline cycle (To5, Figure 1) were evaluated on Na+ currents of native frog skeletal muscle fibres. In addition, by introducing methyl groups on one or both of the nitrogen atoms of the Proline Derivative (as in To6, To7 and To8, Figure 1) or by constraining the chiral centre in a pyrrolo-imidazolic cycle (To3 and To4, Figure 1), we have investigated whether a further decrease in the ability of the molecule to transit through various conformations could modify the drug potency and/or stereoselectivity, and consequently the interaction with the receptor. The results indicate that constraining the stereogenic centre in a α-Proline cycle, as in To5, highly improves potency, stereoselectivity and use-dependent behaviour of Na+ channel blockers. Accordingly, an acute in vivo administration of tocainide and To5 in myotonic adr/adr mice showed that To5 was 3 fold more effective than its parent compound in decreasing the time of righting reflex of myotonic mice. On the basis of these data, we can conclude that the newly synthesized Proline Derivative of tocainide, To5, may be considered as a potential therapeutic agent in the treatment of myotonic syndromes. Figure 1 Chemical structure of tocainide and its newly synthesized α-Proline and pyrrolo-imidazolic analogues. Methods Fibre preparation and voltage clamp apparatus Segments of undamaged single muscle fibres (about 1 cm in length) were obtained by microsurgery (plucking procedure) from the ventral branch of the semitendinosus muscle of Rana Esculenta bathed in normal physiological solution at room temperature. The cut-end fibre was then superfused with an internal solution and mounted across three chamber partitions, which delineated the four pools. Three strips of vaseline were applied over the fibre and carefully sealed to the fibre to reduce leakage. The width of the gaps of the central pools (A and B) had been previously set to 70 – 100 μM and 200 μM, respectively. Four KCl/agar bridges electrodes connected the recording chamber to the voltage clamp amplifier based on methods described by Hille & Campbell (1976) and detailed elsewhere (de luca et al., 1995; 1997a). When the solution level was lowered below the vaseline strips, the four pools were physically and electrically independent from each other: this was confirmed by verifying that no leak current was flowing upon increasing the amplifier gain. Then the solution in the pool A was replaced with the external solution and after about 10 min of equilibration the recordings were performed at 10°C. The usual holding potential (h.p.) was −100 mV. Sodium currents were recorded using an amplifier connected via a A/D and D/A Digidata 1200 Interface (Axon Instruments, Foster City, CA., U.S.A.) to a 486 DX2/66 personal computer and stored on the hard disk. The stimulation protocols and data acquisition were driven by the Clampex program (pClamp6 software package; Axon Instruments). The currents flowing in response to depolarizing command voltages were low pass filtered at 10 kHz (Frequency Devices, U.S.A.), visualized on an oscilloscope and sampled at 20 kHz. When necessary, leak and capacities currents were subtracted by P/4 method. The acquired traces were analysed later using Clampfit program (pClamp6 software package; Axon Instruments).
Carlo Franchini - One of the best experts on this subject based on the ideXlab platform.
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searching for novel anti myotonic agents pharmacophore requirement for use dependent block of skeletal muscle sodium channels by n benzylated cyclic Derivatives of tocainide
Neuromuscular Disorders, 2012Co-Authors: Annamaria De Luca, Michela De Bellis, Filomena Corbo, Carlo Franchini, Marilena Muraglia, Alessia Catalano, Alessia Carocci, Diana Conte CamerinoAbstract:Drug screening on sodium currents of native myofibers by means of voltage-clamp recordings is predictive of pre-clinical anti-myotonic activity in vivo and ex vivo. By this approach we identified the N-benzylated beta-Proline Derivative of tocainide (To10) as the most potent use-dependent blocker of Nav1.4 so far. We tested novel analogs with modifications on the pharmacophore groups of To10. The substitution of the Proline cycle with less planar piperidine or piperazine rings disclosed the importance of a two carbon atom distance and/or an additional nitrogen atom for potency. Structural changes on the xylididic group corroborated the role of a proper electronic cloud for hydrophobic interactions with the binding site. The N-benzylated moiety lead to a stereoselective behavior only in the rigid alpha-Proline analog To11 vs. To10 and N-benzylated tocainide (To12). The results confirm the strict structural requirements of Nav1.4 blockers and allow to refine the drug design toward novel anti-myotonic drugs.
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increased rigidity of the chiral centre of tocainide favours stereoselectivity and use dependent block of skeletal muscle na channels enhancing the antimyotonic activity in vivo
British Journal of Pharmacology, 2001Co-Authors: S Talon, Jf Desaphy, Annamaria De Luca, Michela De Bellis, Filomena Corbo, Carlo Franchini, Giovanni Lentini, Antonio Scilimati, Paolo Tortorella, Harald JockuschAbstract:Searching for the structural requirements improving the potency and the stereoselectivity of Na+ channel blockers as antimyotonic agents, new Derivatives of tocainide, in which the chiral carbon atom is constrained in a rigid α-Proline or pyrrolo-imidazolic cycle, were synthesized as pure enantiomers. Their ability to block Na+ currents, elicited from −100 to −20 mV at 0.3 Hz (tonic block) and 2 – 10 Hz (use-dependent block) frequencies, was investigated in vitro on single fibres of frog semitendinosus muscle using the vaseline-gap voltage-clamp method. The α-Proline Derivative, To5, was 5 and 21 fold more potent than tocainide in producing tonic and 10 Hz-use-dependent block, respectively. Compared to To5, the presence of one methyl group on the aminic (To6) or amidic (To7) nitrogen atom decreased use-dependence by 2- and 6-times, respectively. When methylene moieties were present on both nitrogen atoms (To8), both tonic and use-dependent block were reduced. Contrarily to tocainide, all Proline Derivatives were stereoselective in relation to an increased rigidity. A further increase in the molecular rigidity as in pyrrolo-imidazolic Derivatives markedly decreased the drug potency with respect to tocainide. Antimyotonic activity, evaluated as the shortening of the time of righting reflexes of myotonic adr/adr mice upon acute drug in vivo administration was 3 fold more effective for R-To5 than for R-Tocainide. Thus, constraining the chiral centre of tocainide in α-Proline cycle leads to more potent and stereoselective use-dependent Na+ channel blockers with improved therapeutic potential. Keywords: Na+ channel, native frog skeletal muscle fibres, vaseline-gap voltage-clamp, tocainide Derivatives, use-dependent block, stereoselectivity, antimyotonic activity, adr/adr mice Introduction Tocainide is an orally effective lidocaine Derivative, able to block the voltage-gated Na+ channels in a use-dependent manner, a feature that enhances its potency in situations of prolonged depolarization and/or excessive firing of action potentials with respect to a physiological excitability (Catterall, 1987; de luca et al., 1997b; Conte Camerino et al., 2000). Thus, tocainide is actually among the few drugs clinically used for the symptomatic treatment of hyperexcitability of myotonic syndromes, hereditary disorders of skeletal muscle due to genetic mutations on either sodium or chloride channel genes (Streib, 1986; Kwiecinski et al., 1992, Rudel et al., 1994; Lehmann-Horn & Rudel, 1996; Cannon, 1997; Ptacek, 1998). However, its clinical use presents some disadvantages, due to the high doses required for its antimyotonic effectiveness with the risk of a greater incidence of serious side effects, especially at the cardiac, haematopoietic and central nervous system levels (Roden & Woosley, 1986; Puniani & Bertorini, 1991; Rudel et al., 1994). The use-dependent inhibition of Na+ channels by tocainide and other local anaesthetic (LA) drugs relies on the higher affinity of these drugs for their receptor site on the α-subunit of the channel when this latter is open and/or inactivated, and to a slow recovery from inactivation of the drug-bound channels during membrane repolarization (Catterall, 1987; de luca et al., 1997a). At the resting state, the LA interacts with its presumed receptor through hydrophobic interactions with two aromatic amino acid residues at the D4 – S6 segment (Ragsdale et al., 1994; Wright et al., 1998). Gating movements due to the channel opening may then allow further drug interactions with other residues in the pore that could stabilize the drug-bound channel in an inactivated state (Wang et al., 2000; Yarov-Yarovoy et al., 2001). Furthermore, a site in D1 – S6 segment would also be involved in determining the drug stereoselectivity (Nau et al., 1999). This feature is important because tocainide and mexiletine, another LA-like compound, have a chiral carbon atom that contributes to the potency and the stereoselectivity of the drug (Tricarico et al., 1991; de luca et al., 1997a; 2000; Desaphy et al., 1999). In particular, we found that the introduction of an aromatic lipophilic group on the chiral centre of mexiletine highly increases the drug potency suggesting the establishment of a strong two-points interaction with the two hydrophobic pockets of the receptor in D4 – S6 segment (Wright et al., 1998; de luca et al., 2000). Furthermore, the nature of the group on the stereogenic centre is important for the drug stereoselectivity because the introduction of an unbending lipophilic group such as the isopropyl one enhanced the stereoselectivity, likely due to the occurrence of a three-points interaction of this more rigid structure with the receptor (de luca et al., 1997a; 2000). In agreement with previous data, we confirmed that a higher basicity in the molecule determines a greater use-dependent behaviour (de luca et al., 1997a). To go further in the study of the structure-activity relationship and to better evaluate the role of the chiral centre in the potency and/or stereoselectivity of drug for blocking Na+ channels, the effects of the pure enantiomers of a new Derivative of tocainide, in which the asymmetric carbon atom is constrained in a rigid α-Proline cycle (To5, Figure 1) were evaluated on Na+ currents of native frog skeletal muscle fibres. In addition, by introducing methyl groups on one or both of the nitrogen atoms of the Proline Derivative (as in To6, To7 and To8, Figure 1) or by constraining the chiral centre in a pyrrolo-imidazolic cycle (To3 and To4, Figure 1), we have investigated whether a further decrease in the ability of the molecule to transit through various conformations could modify the drug potency and/or stereoselectivity, and consequently the interaction with the receptor. The results indicate that constraining the stereogenic centre in a α-Proline cycle, as in To5, highly improves potency, stereoselectivity and use-dependent behaviour of Na+ channel blockers. Accordingly, an acute in vivo administration of tocainide and To5 in myotonic adr/adr mice showed that To5 was 3 fold more effective than its parent compound in decreasing the time of righting reflex of myotonic mice. On the basis of these data, we can conclude that the newly synthesized Proline Derivative of tocainide, To5, may be considered as a potential therapeutic agent in the treatment of myotonic syndromes. Figure 1 Chemical structure of tocainide and its newly synthesized α-Proline and pyrrolo-imidazolic analogues. Methods Fibre preparation and voltage clamp apparatus Segments of undamaged single muscle fibres (about 1 cm in length) were obtained by microsurgery (plucking procedure) from the ventral branch of the semitendinosus muscle of Rana Esculenta bathed in normal physiological solution at room temperature. The cut-end fibre was then superfused with an internal solution and mounted across three chamber partitions, which delineated the four pools. Three strips of vaseline were applied over the fibre and carefully sealed to the fibre to reduce leakage. The width of the gaps of the central pools (A and B) had been previously set to 70 – 100 μM and 200 μM, respectively. Four KCl/agar bridges electrodes connected the recording chamber to the voltage clamp amplifier based on methods described by Hille & Campbell (1976) and detailed elsewhere (de luca et al., 1995; 1997a). When the solution level was lowered below the vaseline strips, the four pools were physically and electrically independent from each other: this was confirmed by verifying that no leak current was flowing upon increasing the amplifier gain. Then the solution in the pool A was replaced with the external solution and after about 10 min of equilibration the recordings were performed at 10°C. The usual holding potential (h.p.) was −100 mV. Sodium currents were recorded using an amplifier connected via a A/D and D/A Digidata 1200 Interface (Axon Instruments, Foster City, CA., U.S.A.) to a 486 DX2/66 personal computer and stored on the hard disk. The stimulation protocols and data acquisition were driven by the Clampex program (pClamp6 software package; Axon Instruments). The currents flowing in response to depolarizing command voltages were low pass filtered at 10 kHz (Frequency Devices, U.S.A.), visualized on an oscilloscope and sampled at 20 kHz. When necessary, leak and capacities currents were subtracted by P/4 method. The acquired traces were analysed later using Clampfit program (pClamp6 software package; Axon Instruments).
