Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-10-06
2001-06-12
Kunz, Gary L. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C530S300000
Reexamination Certificate
active
06245893
ABSTRACT:
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\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\94\13656, WO\94\13657, WO\94\13292, WO\94\13297, PCT\EP\95\02076, PCT\EP95\02249 and PCT\EP95\02246 all describe other compounds possessing certain CNS activities, in particular PCT\EP95\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
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;
and homologous receptors from other sources sharing at least 85% homology with the rat forebrain tissue.
Other known anticonvulsant compounds including diazepam, phenytoin, 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 AM 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
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 from 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 vertical 10% (w/v), or 4-20% (w/v) gradient, slab gels can be used, based on the method of Laemmli (see Laemmli U.K. (1970) Nature, 227, 680-685). Further purification of the receptor can be achieved by Isoelectric Focussing (IEF) in immobilised pH gradient polyacrylamide gels of material prepared by preparative SDS-PAGE.
The molecular weight of the receptor is in the region of 130 kiloDaltons when analysed by gel electrophoresis (SDS-PAGE).
A second aspect of the invention provides a soluble form of the above receptor.
Soluble forms of said receptor can be prepared according to conventional techniques.
Such soluble forms of said receptor are believed to possess therapeutic utility and therefore the present invention extends to the use of a soluble form of the said receptor as a therapeutic agent.
The present invention also extends to the use of a soluble form of the said receptor in the manufacture of a medicament for treating and/or preventing of anxiety, mania, depression, disorders associated with a subarachnoid haemorrhage or neural shock, the effects associated with withdrawal from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders treatable and/or preventable with anti-convulsive agents, such as epilepsy; Parkinson's disease, psychosis, migraine and/or cerebral ischaemia.
The present invention also extends to a method of treating and/or preventing anxiety, mania, depression, disorders associated with a subarachnoid haemorrhage or neural shock, the effects associated with withdrawal from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders treatable and/or preventable with anti-convulsive agents, such as epilepsy; Parkinson's disease, psychosis, migraine and/or cerebral ischaemia, which comprises administering an effective or prophylactic amount of the soluble receptor to a sufferer in need thereof.
The present invention also extends to a pharmaceutical composition for use in the treatment or prevention of anxiety, mania, depression, disorders associated with a subarachnoid haemorrhage or neural shock, the effects associated with withdrawal from substances of abuse such as cocaine, nicotine, alcohol and benzodiazepines, disorders treatable and/or preventable with anti-convulsive agents, such as epilepsy Parkinson's disease, psychosis, migraine and/or cerebral ischaemia, which comprises the soluble receptor admixed with pharmaceutically acceptable carriers.
Such pharmaceutical compositions and carriers are well known in the art and can be prepared by conventional techniques.
In a third aspect the present invention provides a method of screening compounds having therapeutic activity associated with binding to the said receptor which comprises; contacting a test compound with a substrate in which the novel receptor is present and measuring the degree of binding.
Substrates in which the novel receptor can be found include but are not limited to brain tissue from rats, humans, marmosets, dogs, cats and mice. Such brain tissue can suitably be homogenised in a buffered aqueous medium such as 5 to 50 mM HEPES, pH 7.4 or Tris/HCl buffer. The homogenised tissue can be washed by centrifugation and resuspension. Subcellular fractions prepared from the tissue can also be used.
Contacting the test compound with a substrate in which the novel receptor is present may be carried out by mixing an aliquot (for example containing 1 to 10 mg protein/ml) of the tissue containing the novel receptor (for example, that described above) with a radioactively-labelled (for example with
3
H or
125
I) test compound. Preferably the final concentration of the test compound in the mixture is 0.1 to 1000 nM, more preferably 1 to 50 nM.
The mixture may be suitably incubated for about 1 hour at ambient temperature. Unbound test compound is then separated from bound test compound by filtration. This may be carried out using Whatman glass fibre filters preferably of the type GF/B or GF/C. The filters may suitably be washed with ice cold buffered medium (preferably of the
Chan Wai Ngor
Herdon Hugh Jonathan
Jerman Jeffrey Clifford
Gimmi Edward R.
Hecht Elizabeth J.
Kinzig Charles M.
Kunz Gary L.
Landsman Robert S.
LandOfFree
Receptor that binds anti-convulsant compounds does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Receptor that binds anti-convulsant compounds, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Receptor that binds anti-convulsant compounds will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2492492