Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined... – Nervous system
Patent
1997-09-26
1999-11-16
Weber, Jon P.
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
Nervous system
435353, 530350, 549426, A61K 3530, C12N 506, C07K 100, C07D31500
Patent
active
059853344
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a novel receptor in substantially pure form, to a soluble form of the receptor and it's use as a therapeutic agent, to a method of screening compounds useful in treating disorders by interacting with the receptor, to novel compounds discovered by carrying out the method of screening; to the recombinant receptor and to it's use in such a method of screening; to the preparation of monoclonal and polyclonal antibodies which bind to the receptor; to the use of such antibodies as therapeutic agents and to a method of determining the effectiveness of therapeutic agents which bind to the receptor.
WO 92.backslash.22293 (SmithKline Beecham plc) describes a class of compounds which have been shown by behavioural models to possess certain CNS activities, in particular, in the treatment and/or prevention of epilepsy. An example of such a compound described in the above patent application is trans-(+)-6-Acetyl-4S-(4-fluorobenzolamino)-3,4-dihydro-2, 2-dimethyl-2H-1-benzopyran-3R-ol. (hereinafter referred to as Compound A).
WO.backslash.94.backslash.13656, WO.backslash.94.backslash.13657, WO.backslash.94.backslash.13292, WO.backslash.94.backslash.13297, PCT.backslash.EP.backslash.95.backslash.02076, PCT.backslash.EP.backslash.95.backslash.02249 and PCT.backslash.EP.backslash.95.backslash.02246 all describe other compounds possessing certain CNS activities, in particular PCT.backslash.EP.backslash.95.backslash.02076 describes the `cold` compound of example 4 in the present application i.e. compound B.
The compounds described in the above mentioned patents do not bind at any known receptor and a novel receptor has now been identified to which such compounds bind.
Accordingly the present invention provides a receptor in substantially pure form obtainable from rat forebrain tissue which is characterised in that;
a) compound A binds to it with a Kd of 40 nM for rat forebrain tissue,
b) compound A binds to it with a B.sub.max of 220 pmol/g protein for rat forebrain tissue
c) compound B binds to it with a Kd of 2 nM for rat forebrain tissue
d) compound B binds to it with a Bmax of 220 pmol/g protein for rat forebrain tissue; with the rat forebrain tissue.
Other known anticonvulsant compounds including diazepam, phenyotin, pentobarbitone, valproate, carbamazepine, vigabatrin, lamotrigine, ethosuximide and gabapentin do not bind to the novel receptor.
In addition, the novel receptor can be found in human or rodent neuroblastoma and glioma cell cultures. These can be used without preparation, or can be prepared by the same method as for brain tissue. In human neuroblastoma cell lines e.g. SHSY5Y or IMR 32, compound B binds at the novel receptor with a Kd of 2 nM and a Bmax of 150 pmol/g protein.
The novel receptor is isolated in substantially pure form by conventional techniques. For example, an aliquot (for example containing 1 to 10 mg protein/ml) of the tissue containing the novel receptor (for example that described above) is mixed with a radioactively-labelled (for example .sup.125 I) photoaffinity label compound (for example Compound C--see Example 6). Preferably the final concentration of the photoaffinity label compound in the mixture is 0.1 to 1000 pM. The mixture may be suitably incubated for about 1 hour at ambient temperature. The mixture is then exposed to UV light (for example 366 nm for a 6 W lamp) for about 30 min. The tissue is then washed by centrifugation to remove unbound photoaffinity label compound. The photoaffinity labelled receptor can be initially separated from other proteins by gel permeation chromatography (for example with Superose 6) under non-reducing conditions. Protein fractions containing the receptor can then be precipitated with trichloroacetic acid as described by Bensadoun and Weinstein (Bensadoun A., and Weinstein D. (1976) Anal. Biochem. 70, 241-250). The proteins are then further separated by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) under reducing conditions 6% (w/v) rod gels overlayed with a 5% (w/v) stacking gels, or vertica
Chan Wai Ngor
Herdon Hugh Jonathan
Jerman Jeffrey Clifford
Gimmi Edward R.
Hanley Susan
Hecht Elizabeth J.
King William T.
SmithKline Beecham p.l.c.
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