Methods for the identification of agents for the treatment of seizures, neurological diseases, endocrinopathies and hormonal diseases

a technology for neurodegenerative disorders and agents, applied in the field of drug discovery in neurological disorders, endocrinopathies and hormonal diseases, can solve the problems of disease condition, adverse effects of synaptic vesicle function on neurotransmission in general, and control of neurotransmitter releas

Inactive Publication Date: 2007-06-14
UCB SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0034] In another preferred embodiment, the invention includes a method of discovering or modeling an interaction between an SV2 protein and a compound or agent selected from the group consisting of: levetiracetam, an analog or derivative of levetiracetam, or a compound or agent which competes with levetiracetam or an analog or derivative thereof for binding to the levetiracetam binding site. The method comprises determining a biochemical, pharmacological, organismal, cellular or molecular effect of a potential CNS active molecule in a genetically wild-type animal or in molecules, cells or tissues derived from such animals and comparing the measured effect of that compound in an equivalent study in a system with an SV2 protein knocked out or knocked down.

Problems solved by technology

Neurological disorders afflict a substantial number of individuals and present an increasing economic challenge to health care systems since little is known regarding their causes, their diagnosis is often subjective, and many lack effective treatment.
Likewise, in several psychiatric diseases, movement disorders and neurodegenerative diseases the conduction currents become aberrant, disorganized or reduced, thereby causing the disease condition.
Accordingly, defects in synaptic vesicle functions will have an adverse effect upon neurotransmission in general and control of neurotransmitter release in particular.
Obviously, endocrinopathies involving either hyper- or hyposecretion of hormones have pathological consequences.

Method used

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  • Methods for the identification of agents for the treatment of seizures, neurological diseases, endocrinopathies and hormonal diseases
  • Methods for the identification of agents for the treatment of seizures, neurological diseases, endocrinopathies and hormonal diseases
  • Methods for the identification of agents for the treatment of seizures, neurological diseases, endocrinopathies and hormonal diseases

Examples

Experimental program
Comparison scheme
Effect test

example 1

Development of a Levetiracetam Analog for Binding Studies

[0181] LEV has been shown to bind to a specific binding site located preferentially in the brain (levetiracetam binding site or LBS : Noyer et al., Euro. J. Pharmacol. 286:137-146. (1995); Gillard et al. 2003)). However, [3H]LEV displayed only micromolar affinity for this site, making it unsuitable for in depth characterization. This example describes the binding properties of [3H]ucb 30889, (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide, an analogue of levetiracetam. Binding experiments were conducted on crude rat brain membranes at 4° C. as described in Noyer et al. (Euro. J. Pharmacol. 286:137-146 (1995)). Incubation time for equilibrium studies was 120 min. For kinetic and competition studies, [3H]ucb 30889 (30 Ci / mmol) was used at a concentration of 1.3 nM in 0.5 ml of a Tris-HCl (pH 7.4) buffer containing 2 mM Mg2+. Localization of the LBS in brain substructures was assessed by autoradiography on 25 μm thick ...

example 2

Cellular and Subcellular Distribution of the LBS

[0188] To identify and characterize the LBS in situ, [3H]ucb 30889 was used to map the LBS within the brain and to study both its cellular and subcellular distribution. For rat brain autoradiography, 25 μm slices were incubated with 1.3 nM [3H]ucb 30889 for 120 min at 4° C. in 50 mM Tris-HCl buffer (pH 7.4). Binding assays with rat brain membranes and various neuronal cell lines were performed under similar conditions. Non-specific binding was determined by the inclusion of 1 mM levetiracetam in the assay. For photolabeling, membranes were incubated with 40 nM [3H]ucb 30889 for 120 min at 4° C. in the same buffer, followed by irradiation with UV-light for 30 min (Fuks et al., Eur. J. Pharmacol. 478:11-19 (2003)).

[0189] For rat brain autoradiography, 25 μm slices were incubated with 1.3 nM [3H]ucb 30889 for 120 min at 4° C. in 50 mM Tris-HCl buffer (pH 7.4). FIG. 7 shows that ucb 30889 binding sites are heterogeneously distributed in ...

example 3

The LBS is on SV2A

[0195] In this example, the biochemical characterization of LBS in rat brain led to studies to identify potential candidate LBS proteins for cloning and binding characterization. Based on the integral membrane nature of the protein, brain specific expression, apparent size, and synaptic vesicle localization, the SV2 protein family was analyzed as a candidate for localization of the LBS. Accordingly, SV2 proteins were cloned and assayed for binding of LBS ligands.

[0196] Materials: Levetiracetam and derivatives were synthesized at UCB Pharma (Braine-l'Alleud, Belgium). [3H]ucb 30889, (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide (32 Ci / mmol), was custom labelled by Amersham Biosciences (Roosendaal, The Netherlands). The monoclonal antibody against SV2 proteins developed by Buckley and Kelly (Buckley et al., J. Cell. Biol., 100, 1284-94 (1985)) was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and mainta...

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Abstract

The present invention is drawn to methods of characterization of the properties and functions of SV2 proteins. The invention further includes methods of identifying compounds or agents which modulate the activity of SV2 proteins. Included in these methods is the identification of compounds or agents which modulate the binding of levetiracetam to SV2 proteins, including SV2A. Additionally, the present invention provides biotinylated ligands as a tool to screen chemical libraries and characterize the SV2 proteins. Further, the present invention provides a method of solubilizing and purifying functionally active membrane associated proteins, such as SV2.

Description

RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application 60 / 506,764, filed Sep. 30, 2003, and U.S. Provisional Application 60 / 430,372, filed Dec. 3, 2002, which are herein incorporated by reference in their entirety.FIELD OF THE INVENTION [0002] The present invention is generally drawn to the field of drug discovery in neurological disorders, endocrinopathies and hormonal diseases. BACKGROUND OF THE INVENTION [0003] Neurological disorders afflict a substantial number of individuals and present an increasing economic challenge to health care systems since little is known regarding their causes, their diagnosis is often subjective, and many lack effective treatment. In general, brain activity is ultimately determined by the capacity of neurons to communicate at synapses. Specific neurotransmitter chemicals are packaged in presynaptic neurons into synaptic vesicles which fuse with the presynaptic membrane to release quanta of the neurotransmitter ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/4015A61K31/445A61K31/55A61K31/655A61K45/00C07K1/14C07K14/435C07K14/47C12N15/12C12Q1/68G01NG01N23/207G01N33/53G01N33/68G06F19/00
CPCA61K31/4015G01N33/6896G01N33/9473G01N2333/705G01N2500/00G01N2800/28A61P5/00A61P25/00A61P25/04A61P25/06A61P25/08A61P25/16A61P25/28A61P43/00Y02A90/10
Inventor LYNCH, BERKLEYNOCKA, KARLFUKS, BRUNO
Owner UCB SA
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