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Patrick Emond - One of the best experts on this subject based on the ideXlab platform.
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The Story of the Dopamine Transporter PET Tracer LBT-999: From Conception to Clinical Use
Frontiers in Medicine, 2019Co-Authors: Sylvie Chalon, Johnny Vercouillie, Denis Guilloteau, Pierre Payoux, Jean-bernard Deloye, Cécile Malherbe, Florence Le Jeune, Nicolas Arlicot, Anne-sophie Salabert, Patrick EmondAbstract:The membrane dopamine transporter (DAT) is involved in a number of brain disorders and its exploration by positron emission tomography (PET) imaging is highly relevant for the early and differential diagnosis, follow-up and treatment assessment of these diseases. A number of carbon-11 and fluor-18 labeled tracers are to date available for this aim, the majority of them being derived from the chemical structure of Cocaine. The development of such a tracer, from its conception to its use, is a long process, the expected result being to obtain the best radiopharmaceutical adapted for clinical protocols. In this context, the Cocaine Derivative (E)-N-(4-fluorobut-2-enyl)2β-carbomethoxy-3β-(4'-tolyl)nortropane, or LBT-999, has passed all the required stages of the development that makes it now a highly relevant imaging tool, particularly in the context of Parkinson's disease. This review describes the different steps of the development of LBT-999 which initially came from its non-fluorinated Derivative (E)-N-(3-iodoprop-2-enyl)-2-carbomethoxy-3-(4-methylphenyl) nortropane, or PE2I, because of its high promising properties. [18F]LBT-999 has been extensively characterized in rodent and non-human primate models, in which it demonstrated its capability to explore in vivo the DAT localized at the dopaminergic nerve endings as well as at the mesencephalic cell bodies, in physiological conditions. In lesion-induced rat models of Parkinson's disease, [18F]LBT-999 was able to precisely quantify in vivo the dopaminergic neuron loss, and to assess the beneficial effects of therapeutic approaches such as pharmacological treatment and cell transplantation. Finally recent clinical data demonstrated the efficiency of [18F]LBT-999 in the diagnosis of Parkinson's disease.
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PE2I: a radiopharmaceutical for in vivo exploration of the dopamine transporter.
CNS neuroscience & therapeutics, 2008Co-Authors: Patrick Emond, Denis Guilloteau, Sylvie ChalonAbstract:The membrane dopamine transporter (DAT) has a pivotal role in the regulation of dopamine (DA) neurotransmission involved in a number of physiological functions and brain disorders. Molecular imaging techniques, such as positron emission tomography (PET) and single photon emission computerized tomography (SPECT), are relevant tools to explore the DAT, and we developed the Cocaine Derivative N-(3-iodopro-2E-enyl)-2β-carbomethoxy-3β-(4′-methylphenyl) nortropane (PE2I) that has proved to be a very potent radiopharmaceutical to image the DAT by these techniques. Several methods are available to obtain PE2I labeled with iodine-123 or -125, carbon-11 and tritium. The pharmacological properties of PE2I have demonstrated that it has good affinity for the DAT (4 nM) and is one of the most selective DAT ligands. [125I]PE2I characterized postmortem in human brains has revealed very intense and selective binding in the basal ganglia. Ex vivo autoradiography in rats has shown that high level of [125I]PE2I accumulates in the striatum and also in the substantia nigra and ventral tegmental area. [125I]PE2I accumulation in the rat striatum is rapid, high, and selective, providing a maximum striatum/cerebellum ratio of 10 during the first 30 min post injection. Using SPECT or PET, rapid, high, and selective accumulation of PE2I was found in the caudate nucleus and putamen in monkeys, whereas rapid wash out from the cerebellum was observed. In vivo investigations in healthy humans have demonstrated that PE2I has high striatal uptake, low nonspecific binding, low radiation exposure, and a fairly short scanning time. A number of findings in various animal models of Parkinson's disease in rats and monkeys have demonstrated the high efficacy of PE2I for detection of reduction in the density of DAT, thus showing the potential value of PE2I for early diagnosis and evaluation of treatment of this disease. The excellent properties of PE2I are basis for the development of new DAT tracers for use in future PET explorations using fluor-18.
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Radiosynthesis of [18F]LBT-999, a selective radioligand for the visualization of the dopamine transporter with PET
Journal of Labelled Compounds and Radiopharmaceuticals, 2006Co-Authors: Frédéric Dollé, Patrick Emond, Lucette Garreau, Sylvie Mavel, Françoise Hinnen, Zoia Mincheva, Wadad Saba, Héric Valette, Marie-anne Schöllhorn-peyronneau, Sylvie ChalonAbstract:LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3-beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2-beta-carboxylicacid methyl ester) is a recently developed Cocaine Derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands (KD: 9 nM for the DAT and IC50 > 1000 nM for the serotonin and norepinephrine transporter). Initial fluorine-18-labelling of LBT-999 was based on the robust and reliable two-step radiochemical pathway often reported for such tropane Derivatives, involving first the preparation of (E)-1-[18F]fluoro-4-tosyloxybut-2-ene followed by a N-alkylation reaction with the appropriate nor-tropane moiety. In the present work, a simple one-step fluorine-18-labelling of LBT-999 is reported, based on a chlorine-for-fluorine nucleophilic aliphatic substitution, facilitating as expected both automation and final high-performance liquid chromatography (HPLC) purification. The process involves: (A) reaction of K[18F]F–Kryptofix®222 with the chlorinated precursor (3.5–4.5 mg) at 165°C for 10 min in DMSO (0.6 mL) followed by (B) C-18 PrepSep cartridge pre-purification and finally (C) semi-preparative HPLC purification on a Waters Symmetry® C-18. Typically, 3.70–5.92 GBq of [18F]LBT-999 (> 95% chemically and radiochemically pure) could be obtained with specific radioactivities ranging from 37 to 111 GBq/µmol within 85–90 min (HPLC purification and Sep-Pak-based formulation included), starting from a 37.0 GBq [18F]fluoride batch (overall radiochemical yields: 10–16%, non-decay-corrected). Copyright © 2007 John Wiley & Sons, Ltd.
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Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET.
Bioorganic & medicinal chemistry, 2005Co-Authors: Frédéric Dollé, Patrick Emond, Sylvie Chalon, Sylvie Mavel, Stéphane Demphel, Françoise Hinnen, Zoia Mincheva, Wadad Saba, Héric Valette, Christer HalldinAbstract:Abstract LBT-999 (8-(( E )-4-fluoro-but-2-enyl)-3β- p -tolyl-8-aza-bicyclo[3.2.1]octane-2β-carboxylic acid methyl ester), a Cocaine Derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid Derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from ( R )-Cocaine. [ 3 H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [ 3 H]methyl iodide, allowing its in vitro pharmacological evaluation ( K D : 9 nM for DAT and IC 50 > 1000 nM for SERT and NET). Routine production batches of 4.5–9.0 GBq of iv injectable solutions of [ 11 C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/μmol) were prepared in 25–30 min (HPLC purification and formulation included) using the efficient methylation reagent [ 11 C]methyl triflate. The preliminary in vivo pharmacological evaluation of [ 11 C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.
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Synthesis of tropane and nortropane analogues with phenyl substitutions as serotonin transporter ligands.
Bioorganic & medicinal chemistry, 2001Co-Authors: Patrick Emond, Julie Helfenbein, Sylvie Chalon, Lucette Garreau, Johnny Vercouillie, Y. Frangin, Jean Claude Besnard, Denis GuilloteauAbstract:The effects of structural modifications of 2 beta-carbomethoxy-3 beta-phenyl tropane analogues were evaluated on in vitro affinity to the dopamine (DAT) and serotonin (5-HTT) transporters in rat brain tissue. The introduction of a large alkyl group at the 4'-position of the phenyl ring, affording 2 beta-carbomethoxy-3 beta-(4'-alkylphenyl) tropane, diminished the affinity for the DAT whereas moderate 5-HTT affinity was obtained. The introduction of an iodine at the 3'-position of the 4'-alkylphenyl, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) tropane, and N-demethylation, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) nortropane, improved affinity and specificity for the 5-HTT. It could be assumed from these results that the combination of these three modifications of tropane structure yielded highly selective compounds for the 5-HTT. Of the new compounds synthesized, the most selective Cocaine Derivative, 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-isopropylphenyl) nortropane (8d) labeled with iodine-123 or carbon-11, could be a potential ligand for exploration of the 5-HT transporter by SPET or PET.
Sylvie Chalon - One of the best experts on this subject based on the ideXlab platform.
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The Story of the Dopamine Transporter PET Tracer LBT-999: From Conception to Clinical Use
Frontiers in Medicine, 2019Co-Authors: Sylvie Chalon, Johnny Vercouillie, Denis Guilloteau, Pierre Payoux, Jean-bernard Deloye, Cécile Malherbe, Florence Le Jeune, Nicolas Arlicot, Anne-sophie Salabert, Patrick EmondAbstract:The membrane dopamine transporter (DAT) is involved in a number of brain disorders and its exploration by positron emission tomography (PET) imaging is highly relevant for the early and differential diagnosis, follow-up and treatment assessment of these diseases. A number of carbon-11 and fluor-18 labeled tracers are to date available for this aim, the majority of them being derived from the chemical structure of Cocaine. The development of such a tracer, from its conception to its use, is a long process, the expected result being to obtain the best radiopharmaceutical adapted for clinical protocols. In this context, the Cocaine Derivative (E)-N-(4-fluorobut-2-enyl)2β-carbomethoxy-3β-(4'-tolyl)nortropane, or LBT-999, has passed all the required stages of the development that makes it now a highly relevant imaging tool, particularly in the context of Parkinson's disease. This review describes the different steps of the development of LBT-999 which initially came from its non-fluorinated Derivative (E)-N-(3-iodoprop-2-enyl)-2-carbomethoxy-3-(4-methylphenyl) nortropane, or PE2I, because of its high promising properties. [18F]LBT-999 has been extensively characterized in rodent and non-human primate models, in which it demonstrated its capability to explore in vivo the DAT localized at the dopaminergic nerve endings as well as at the mesencephalic cell bodies, in physiological conditions. In lesion-induced rat models of Parkinson's disease, [18F]LBT-999 was able to precisely quantify in vivo the dopaminergic neuron loss, and to assess the beneficial effects of therapeutic approaches such as pharmacological treatment and cell transplantation. Finally recent clinical data demonstrated the efficiency of [18F]LBT-999 in the diagnosis of Parkinson's disease.
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PE2I: a radiopharmaceutical for in vivo exploration of the dopamine transporter.
CNS neuroscience & therapeutics, 2008Co-Authors: Patrick Emond, Denis Guilloteau, Sylvie ChalonAbstract:The membrane dopamine transporter (DAT) has a pivotal role in the regulation of dopamine (DA) neurotransmission involved in a number of physiological functions and brain disorders. Molecular imaging techniques, such as positron emission tomography (PET) and single photon emission computerized tomography (SPECT), are relevant tools to explore the DAT, and we developed the Cocaine Derivative N-(3-iodopro-2E-enyl)-2β-carbomethoxy-3β-(4′-methylphenyl) nortropane (PE2I) that has proved to be a very potent radiopharmaceutical to image the DAT by these techniques. Several methods are available to obtain PE2I labeled with iodine-123 or -125, carbon-11 and tritium. The pharmacological properties of PE2I have demonstrated that it has good affinity for the DAT (4 nM) and is one of the most selective DAT ligands. [125I]PE2I characterized postmortem in human brains has revealed very intense and selective binding in the basal ganglia. Ex vivo autoradiography in rats has shown that high level of [125I]PE2I accumulates in the striatum and also in the substantia nigra and ventral tegmental area. [125I]PE2I accumulation in the rat striatum is rapid, high, and selective, providing a maximum striatum/cerebellum ratio of 10 during the first 30 min post injection. Using SPECT or PET, rapid, high, and selective accumulation of PE2I was found in the caudate nucleus and putamen in monkeys, whereas rapid wash out from the cerebellum was observed. In vivo investigations in healthy humans have demonstrated that PE2I has high striatal uptake, low nonspecific binding, low radiation exposure, and a fairly short scanning time. A number of findings in various animal models of Parkinson's disease in rats and monkeys have demonstrated the high efficacy of PE2I for detection of reduction in the density of DAT, thus showing the potential value of PE2I for early diagnosis and evaluation of treatment of this disease. The excellent properties of PE2I are basis for the development of new DAT tracers for use in future PET explorations using fluor-18.
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Radiosynthesis of [18F]LBT-999, a selective radioligand for the visualization of the dopamine transporter with PET
Journal of Labelled Compounds and Radiopharmaceuticals, 2006Co-Authors: Frédéric Dollé, Patrick Emond, Lucette Garreau, Sylvie Mavel, Françoise Hinnen, Zoia Mincheva, Wadad Saba, Héric Valette, Marie-anne Schöllhorn-peyronneau, Sylvie ChalonAbstract:LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3-beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2-beta-carboxylicacid methyl ester) is a recently developed Cocaine Derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands (KD: 9 nM for the DAT and IC50 > 1000 nM for the serotonin and norepinephrine transporter). Initial fluorine-18-labelling of LBT-999 was based on the robust and reliable two-step radiochemical pathway often reported for such tropane Derivatives, involving first the preparation of (E)-1-[18F]fluoro-4-tosyloxybut-2-ene followed by a N-alkylation reaction with the appropriate nor-tropane moiety. In the present work, a simple one-step fluorine-18-labelling of LBT-999 is reported, based on a chlorine-for-fluorine nucleophilic aliphatic substitution, facilitating as expected both automation and final high-performance liquid chromatography (HPLC) purification. The process involves: (A) reaction of K[18F]F–Kryptofix®222 with the chlorinated precursor (3.5–4.5 mg) at 165°C for 10 min in DMSO (0.6 mL) followed by (B) C-18 PrepSep cartridge pre-purification and finally (C) semi-preparative HPLC purification on a Waters Symmetry® C-18. Typically, 3.70–5.92 GBq of [18F]LBT-999 (> 95% chemically and radiochemically pure) could be obtained with specific radioactivities ranging from 37 to 111 GBq/µmol within 85–90 min (HPLC purification and Sep-Pak-based formulation included), starting from a 37.0 GBq [18F]fluoride batch (overall radiochemical yields: 10–16%, non-decay-corrected). Copyright © 2007 John Wiley & Sons, Ltd.
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Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET.
Bioorganic & medicinal chemistry, 2005Co-Authors: Frédéric Dollé, Patrick Emond, Sylvie Chalon, Sylvie Mavel, Stéphane Demphel, Françoise Hinnen, Zoia Mincheva, Wadad Saba, Héric Valette, Christer HalldinAbstract:Abstract LBT-999 (8-(( E )-4-fluoro-but-2-enyl)-3β- p -tolyl-8-aza-bicyclo[3.2.1]octane-2β-carboxylic acid methyl ester), a Cocaine Derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid Derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from ( R )-Cocaine. [ 3 H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [ 3 H]methyl iodide, allowing its in vitro pharmacological evaluation ( K D : 9 nM for DAT and IC 50 > 1000 nM for SERT and NET). Routine production batches of 4.5–9.0 GBq of iv injectable solutions of [ 11 C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/μmol) were prepared in 25–30 min (HPLC purification and formulation included) using the efficient methylation reagent [ 11 C]methyl triflate. The preliminary in vivo pharmacological evaluation of [ 11 C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.
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Synthesis of tropane and nortropane analogues with phenyl substitutions as serotonin transporter ligands.
Bioorganic & medicinal chemistry, 2001Co-Authors: Patrick Emond, Julie Helfenbein, Sylvie Chalon, Lucette Garreau, Johnny Vercouillie, Y. Frangin, Jean Claude Besnard, Denis GuilloteauAbstract:The effects of structural modifications of 2 beta-carbomethoxy-3 beta-phenyl tropane analogues were evaluated on in vitro affinity to the dopamine (DAT) and serotonin (5-HTT) transporters in rat brain tissue. The introduction of a large alkyl group at the 4'-position of the phenyl ring, affording 2 beta-carbomethoxy-3 beta-(4'-alkylphenyl) tropane, diminished the affinity for the DAT whereas moderate 5-HTT affinity was obtained. The introduction of an iodine at the 3'-position of the 4'-alkylphenyl, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) tropane, and N-demethylation, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) nortropane, improved affinity and specificity for the 5-HTT. It could be assumed from these results that the combination of these three modifications of tropane structure yielded highly selective compounds for the 5-HTT. Of the new compounds synthesized, the most selective Cocaine Derivative, 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-isopropylphenyl) nortropane (8d) labeled with iodine-123 or carbon-11, could be a potential ligand for exploration of the 5-HT transporter by SPET or PET.
Denis Guilloteau - One of the best experts on this subject based on the ideXlab platform.
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The Story of the Dopamine Transporter PET Tracer LBT-999: From Conception to Clinical Use
Frontiers in Medicine, 2019Co-Authors: Sylvie Chalon, Johnny Vercouillie, Denis Guilloteau, Pierre Payoux, Jean-bernard Deloye, Cécile Malherbe, Florence Le Jeune, Nicolas Arlicot, Anne-sophie Salabert, Patrick EmondAbstract:The membrane dopamine transporter (DAT) is involved in a number of brain disorders and its exploration by positron emission tomography (PET) imaging is highly relevant for the early and differential diagnosis, follow-up and treatment assessment of these diseases. A number of carbon-11 and fluor-18 labeled tracers are to date available for this aim, the majority of them being derived from the chemical structure of Cocaine. The development of such a tracer, from its conception to its use, is a long process, the expected result being to obtain the best radiopharmaceutical adapted for clinical protocols. In this context, the Cocaine Derivative (E)-N-(4-fluorobut-2-enyl)2β-carbomethoxy-3β-(4'-tolyl)nortropane, or LBT-999, has passed all the required stages of the development that makes it now a highly relevant imaging tool, particularly in the context of Parkinson's disease. This review describes the different steps of the development of LBT-999 which initially came from its non-fluorinated Derivative (E)-N-(3-iodoprop-2-enyl)-2-carbomethoxy-3-(4-methylphenyl) nortropane, or PE2I, because of its high promising properties. [18F]LBT-999 has been extensively characterized in rodent and non-human primate models, in which it demonstrated its capability to explore in vivo the DAT localized at the dopaminergic nerve endings as well as at the mesencephalic cell bodies, in physiological conditions. In lesion-induced rat models of Parkinson's disease, [18F]LBT-999 was able to precisely quantify in vivo the dopaminergic neuron loss, and to assess the beneficial effects of therapeutic approaches such as pharmacological treatment and cell transplantation. Finally recent clinical data demonstrated the efficiency of [18F]LBT-999 in the diagnosis of Parkinson's disease.
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PE2I: a radiopharmaceutical for in vivo exploration of the dopamine transporter.
CNS neuroscience & therapeutics, 2008Co-Authors: Patrick Emond, Denis Guilloteau, Sylvie ChalonAbstract:The membrane dopamine transporter (DAT) has a pivotal role in the regulation of dopamine (DA) neurotransmission involved in a number of physiological functions and brain disorders. Molecular imaging techniques, such as positron emission tomography (PET) and single photon emission computerized tomography (SPECT), are relevant tools to explore the DAT, and we developed the Cocaine Derivative N-(3-iodopro-2E-enyl)-2β-carbomethoxy-3β-(4′-methylphenyl) nortropane (PE2I) that has proved to be a very potent radiopharmaceutical to image the DAT by these techniques. Several methods are available to obtain PE2I labeled with iodine-123 or -125, carbon-11 and tritium. The pharmacological properties of PE2I have demonstrated that it has good affinity for the DAT (4 nM) and is one of the most selective DAT ligands. [125I]PE2I characterized postmortem in human brains has revealed very intense and selective binding in the basal ganglia. Ex vivo autoradiography in rats has shown that high level of [125I]PE2I accumulates in the striatum and also in the substantia nigra and ventral tegmental area. [125I]PE2I accumulation in the rat striatum is rapid, high, and selective, providing a maximum striatum/cerebellum ratio of 10 during the first 30 min post injection. Using SPECT or PET, rapid, high, and selective accumulation of PE2I was found in the caudate nucleus and putamen in monkeys, whereas rapid wash out from the cerebellum was observed. In vivo investigations in healthy humans have demonstrated that PE2I has high striatal uptake, low nonspecific binding, low radiation exposure, and a fairly short scanning time. A number of findings in various animal models of Parkinson's disease in rats and monkeys have demonstrated the high efficacy of PE2I for detection of reduction in the density of DAT, thus showing the potential value of PE2I for early diagnosis and evaluation of treatment of this disease. The excellent properties of PE2I are basis for the development of new DAT tracers for use in future PET explorations using fluor-18.
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Synthesis of tropane and nortropane analogues with phenyl substitutions as serotonin transporter ligands.
Bioorganic & medicinal chemistry, 2001Co-Authors: Patrick Emond, Julie Helfenbein, Sylvie Chalon, Lucette Garreau, Johnny Vercouillie, Y. Frangin, Jean Claude Besnard, Denis GuilloteauAbstract:The effects of structural modifications of 2 beta-carbomethoxy-3 beta-phenyl tropane analogues were evaluated on in vitro affinity to the dopamine (DAT) and serotonin (5-HTT) transporters in rat brain tissue. The introduction of a large alkyl group at the 4'-position of the phenyl ring, affording 2 beta-carbomethoxy-3 beta-(4'-alkylphenyl) tropane, diminished the affinity for the DAT whereas moderate 5-HTT affinity was obtained. The introduction of an iodine at the 3'-position of the 4'-alkylphenyl, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) tropane, and N-demethylation, affording 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-alkylphenyl) nortropane, improved affinity and specificity for the 5-HTT. It could be assumed from these results that the combination of these three modifications of tropane structure yielded highly selective compounds for the 5-HTT. Of the new compounds synthesized, the most selective Cocaine Derivative, 2 beta-carbomethoxy-3 beta-(3'-iodo-4'-isopropylphenyl) nortropane (8d) labeled with iodine-123 or carbon-11, could be a potential ligand for exploration of the 5-HT transporter by SPET or PET.
Frieder W. Scheller - One of the best experts on this subject based on the ideXlab platform.
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A Set of Piezoelectric Biosensors Using Cholinesterases
Methods in molecular biology (Clifton N.J.), 2009Co-Authors: Carsten Teller, Jan Halámek, Alexander Makower, Frieder W. SchellerAbstract:Piezoelectric sensors have become a versatile tool in biosensorics to study protein-protein and protein-small molecule interactions. Here we present theoretical background on piezoelectric sensors and instructions, how to modify their surface with various recognition elements for cholinesterases. These recognition elements comprise an organophosphate (paraoxon), a Cocaine Derivative (BZE-DADOO), and a tricyclic, aromatic compound (propidium). Additionally, a guide to the kinetic evaluation of the obtained binding curves is given in this chapter.
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Development of a bifunctional sensor using haptenized acetylcholinesterase and application for the detection of Cocaine and organophosphates
Biosensors & bioelectronics, 2008Co-Authors: Carsten Teller, Jan Halámek, Jiri Žeravík, Walter Stöcklein, Frieder W. SchellerAbstract:We developed a dual piezoelectric/amperometric sensor for the detection of two unrelated analytes in one experiment that uses propidium to anchor acetylcholinesterases (AChE) at the surface. This mass-sensitive sensor does not only allow the examination of the interaction between AChE and the modified surface but also the detection of in situ inhibition of the surface-bound AChE. Here we describe the application of the propidium-based sensor in combination with a modified AChE. For this reason the Cocaine Derivative benzoylecgonine (BZE) was coupled via a 10 A long hydrophilic linker – 1,8-diamino-3,4-dioxaoctane – to carboxylic groups of the AChE after EDC/NHS activation. Thus the modified AChE (BZE–AChE) possesses an additional recognition element besides the inhibitor binding site. After the deposition of BZE–AChE on the sensor surface the binding of an anti-BZE-antibody to the BZE–AChE can be monitored. This makes it possible to determine two analytes – Cocaine and organophosphate – in one experiment by measuring antibody binding and decrease in enzymatic activity, respectively. Furthermore it was also shown that other Cocaine-binding enzymes, e.g., butyrylcholinesterase, can bind to the modified BZE–AChE. The competitive immunoassay allowed the detection of Cocaine with a dynamic range from 10−9 to 10−7 M. The organophosphate chlorpyrifos-oxon could be detected in concentrations from 10−6 down to 10−8 M after 20 min of injection time (equals to 500 L sample volume.
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Piezoelectric affinity sensors for Cocaine and cholinesterase inhibitors
Talanta, 2005Co-Authors: Jan Halámek, Kristina Knösche, Alexander Makower, Petr Skládal, Frieder W. SchellerAbstract:We report here the development of piezoelectric affinity sensors for Cocaine and cholinesterase inhibitors based on the formation of affinity complexes between an immobilized Cocaine Derivative and an anti-Cocaine antibody or cholinesterase. For both binding reactions benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on the surface of the sensor. For immobilization, pre-conjugated BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-te trafluoroborate (TNTU) allowed the formation of a chemisorbed monolayer on the piezosensor surface. The detection of Cocaine was based on a competitive assay. The change of frequency measured after 300 s of the binding reaction was used as the signal. The maximum binding of the antibody resulted in a frequency decrease of 35 Hz (with an imprecision 3%, n = 3) while the presence of 100 pmol l1 Cocaine decreased the binding by 11%. The limit of detection was consequently below 100 pmol l1 for Cocaine. The total time of one analysis was 15 min. This BZE-DADOO-modified sensor was adapted for the detection of organophosphates. BZE-DADOO a competitive inhibitor served as binding element for cholinesterase in a competitive assay.
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Molecular recognition of Cocaine by acetylcholinesterases for affinity purification and bio-sensing
Biosensors & bioelectronics, 2004Co-Authors: Kristina Knösche, Jan Halámek, Alexander Makower, Didier Fournier, Frieder W. SchellerAbstract:Abstract Cholinesterases can be used as sensitive biorecognition elements for widely used agricultural pesticides. This requires highly purified and inhibitor-free enzyme preparations. In the present work the Cocaine Derivative benzoylecgonine was for the first time used as the molecular recognition element for the purification of acetylcholinesterase from Electrophorus electricus by affinity chromatography. The preparation of enriched enzyme without the contamination by an inhibitor, which is traditionally used for eluting the “affinity” bound protein, was achieved. The specific activity was 2.2-fold increased to 3100 U mg−1. The same Cocaine Derivative was immobilized on the surface of a piezoelectric crystal in order to analyze the binding of acetylcholinesterases from two different species, E. electricus and Drosophila melanogaster, to the immobilized inhibitor. Evaluation of the binding curves allowed the analysis of the binding kinetics. These experiments are fundamental for the development of a (competitive) biosensor for inhibitors of cholinesterase.
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The development of a non-competitive immunoenzymometric assay of Cocaine
Analytica Chimica Acta, 1998Co-Authors: Arkadi V. Eremenko, Alexander Makower, Christian G. Bauer, Beata Kanne, Horst Dr. Rer. Nat. Baumgarten, Frieder W. SchellerAbstract:Abstract A non-competitive immunoenzymometric assay (IEMA) of Cocaine has been developed. An excess of alkaline phosphatase (ALP) labelled polyclonal antibody (pAb) is added to Cocaine in solution. After short-time incubation, the free antibody-ALP conjugate is separated from the Cocaine–pAb–ALP complex by passing an affinity column POROS 50 OH derivatized with an immobilized Cocaine Derivative (benzoylecgonine-1,8-diamino-3,4-dioxaoctane or ecgonine-1,8-diamino-3,4-dioxaoctane). The Cocaine–pAb–ALP complex is collected after passing the column. The Cocaine concentration is quantified spectrophotometrically by measuring the ALP activity using p-nitrophenyl phosphate as substrate. The analytical response was proportional to the Cocaine concentration in the sample with a detection limit of 0.5 nM (0.15 ng ml−1) Cocaine. The combined approach developed with an amperometric bienzyme electrode, and the potential for on-line Cocaine monitoring are discussed.
Ursula Scheffel - One of the best experts on this subject based on the ideXlab platform.
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In Vivo Detection of Short- and Long-Term MDMA Neurotoxicity—A Positron Emission Tomography Study in the Living Baboon Brain
2013Co-Authors: Ursula Scheffel, Zsolt Szabo, William B. Mathews, Paige A. Finley, Robert F. Dannals, Hayden T. Ravert, Katalina Szabo, Jie Yuan, George A. RicaurteAbstract:ABSTRACT The present study evaluated short- and long-term effects of MDMA (3,4-methylenedioxymethamphetamine) in the baboon brain using PET and [ 11C](�)McN 5652, a potent 5-HT transporter ligand, as well as [ 11C]RTI-55, a Cocaine Derivative which labels both 5-HT and dopamine transporters. Following baseline PET scans with [ 11C](�)McN5652, [ 11C](�)McN5652 (the inactive enantiomer of the active enantiomer [ 11C](�)McN5652) and [ 11C]RTI-55, a baboon was treated with MDMA (5 mg/kg, s.c., twice daily for four consecutive days). PET studies at 13, 19, and 40 days post-MDMA revealed decreases in mean radioactivity levels in all brain regions when using [ 11C](�)McN 5652, but not with [ 11C](�)McN5652 or [ 11C]RTI-55. Reductions in specific [ 11C](�)McN5652 binding (calculated as the difference in radioactivity concentrations between (�) and (�) [ 11C]McN5652) ranged from 44 % in the pons to 89 % in the occipital cortex. PET studies at 9 and 13 months showed regional differences in the apparent recovery of 5-HT transporters, with increases in some brain regions (e.g., hypothalamus) and persistent decreases in others (e.g., neocortex). Data obtained from PET studies correlated well with regional 5-HT axonal marker concentrations in the CNS measure
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in vivo detection of short and long term mdma neurotoxicity a positron emission tomography study in the living baboon brain
Synapse, 1998Co-Authors: Ursula Scheffel, Zsolt Szabo, William B. Mathews, Robert F. Dannals, Hayden T. Ravert, Katalina Szabo, Jie Yuan, Paige Finley, George A. RicaurteAbstract:The present study evaluated short- and long-term effects of MDMA (3,4-methylenedioxymethamphetamine) in the baboon brain using PET and [11C](+)McN 5652, a potent 5-HT transporter ligand, as well as [11C]RTI-55, a Cocaine Derivative which labels both 5-HT and dopamine transporters. Following baseline PET scans with [11C](+)McN5652, [11C](−)McN5652 (the inactive enantiomer of the active enantiomer [11C](+)McN5652) and [11C]RTI-55, a baboon was treated with MDMA (5 mg/kg, s.c., twice daily for four consecutive days). PET studies at 13, 19, and 40 days post-MDMA revealed decreases in mean radioactivity levels in all brain regions when using [11C](+)McN 5652, but not with [11C](−)McN5652 or [11C]RTI-55. Reductions in specific [11C](+)McN5652 binding (calculated as the difference in radioactivity concentrations between (+) and (−)[11C]McN5652) ranged from 44% in the pons to 89% in the occipital cortex. PET studies at 9 and 13 months showed regional differences in the apparent recovery of 5-HT transporters, with increases in some brain regions (e.g., hypothalamus) and persistent decreases in others (e.g., neocortex). Data obtained from PET studies correlated well with regional 5-HT axonal marker concentrations in the CNS measured after sacrifice of the animal. The results of these studies indicate that PET imaging of the living nonhuman primate brain with [11C](+)McN 5652 can detect changes in regional 5-HT transporter density secondary to MDMA-induced neurotoxicity. Using PET, it should also be feasible to use [11C](+)McN5652 to determine whether human MDMA users are also susceptible to MDMA's neurotoxic effects. Synapse 29:183–192, 1998. © 1998 Wiley-Liss, Inc.