WO2004108119A2

WO2004108119A2 - Use of atp or derivatives in medicine

Info

Publication number: WO2004108119A2
Application number: PCT/GB2004/002298
Authority: WO
Inventors: Samuel Aparicio; Jonathan Ellery
Original assignee: Paradigm Therapeutics Limited
Priority date: 2003-06-04
Filing date: 2004-06-01
Publication date: 2004-12-16
Also published as: WO2004108119A3; GB0312845D0

Abstract

We disclose that ATP is capable of binding to and activating the BACH-Gprotein coupled receptor (BACH-GPCR). We describe methods of identifying a compound or an agent which is capable of modulating the ability of ATP to activate the BACH-GPCR. We also describe using ATP or derivatives thereof or modulators of the binding between ATP and the BACH-GPCR for treating diseases associated with BACH-GPCR.

Description

USE OF COMPOUNDS IN MEDICINE

Field

The present invention relates to the treatment of disease.

In particular, the present invention relates to the use of ATP or a derivative thereof in the treatment of disease.

Background

A variety of physiological processes are mediated by proteins involved in signal tranduction pathways. These signal tranduction pathways provide suitable targets for therapeutic intervention. Many of the signal transduction pathways involve G-proteins and/or second messengers, for example, cAMP (Lefkowitz, Nature, 1991, 351: 353- 354). These proteins are referred to as proteins participating in pathways with G- proteins. Some examples of these proteins include the GPC receptors, such as those for adrenergic agents and dopamine (Kobilka, B. K., et al., Proc. Natl Acad. Set, USA, 1987, 84: 46-50; Kobilka B. K., et al, Science, 1987, 238: 650-656; Bunzow, J. R., et al., Nature, 1988, 336: 783-787), G-proteins themselves, effector proteins, for example, phospholipase C, adenyl cyclase, and phosphodiesterase, and actuator proteins, for example, protein kinase A and protein kinase C (Simon, M. I., et al, Science, 1991, 252: 802-8).

Understanding the relationship between the ligands which regulate complex functions in cells and internal organs and their specific receptor proteins, in particular G-protein coupled receptor proteins, would elucidate the functional mechanisms of the of cells and internal organs and thus provide a very important means for development of therapeutic agents having close relation to such functional mechanisms.

BACH-GPCR is a G-Protein Coupled Receptor (GPCR), in particular, an orphan purinoceptor type G-protein coupled receptor. It is also known that BACH is structurally related to other proteins of the G-protein coupled receptor family and that the Human BACH gene is found to map to Homo sapiens chromosome 12pl3.3. The cloning of the gene coding for the BACH-GPCR polypeptide was described in WO 02/38607.

Summary

The present inventors have surprisingly found that adenosine di-phosphate (ATP) can bind and activate the BACH G-protein coupled receptor. Thus, the inventors have identified that ATP can act as the natural ligand of the BACH-GPCR. It is therefore envisaged that ATP or derivatives thereof and/or modulators of the interaction between ATP and the BACH-GPCR receptor can be useful in the treatment of any disease associated with BACH-GPCR.

Statements of invention

According to one aspect of the present invention, there is provided use of adenosine di- phosphate (ATP) or derivatives thereof, in the manufacture of a medicament for the treatment of BACH-G-protein coupled receptor (BACH-GPCR) associated disease in an individual.

There is also provided, according to another aspect of the present invention use of ATP or derivatives thereof for treating a disease or a condition which is caused by or associated with increased, decreased or otherwise abnormal expression or activity of BACH-GPCR.

In a further aspect according to the present invention we provide use of ATP or derivatives thereof modified such that it is capable of specifically modulating the activity of BACH-GPCR for the manufacture of a medicament in the treatment of BACH-GPCR associated disease. By way of example, modified ATP or derivatives thereof may comprise substitutions or additions of one or more groups or atoms which are capable of allowing the modified molecule or a derivative thereof to specifically bind and activate the BACH-GPCR. Exemplary substitutions or additions include hydroxy, amino, halo, aromatic, alkoxy, and alkyl.

A disease associated with increased, decreased or otherwise abnormal expression or activity of BACH-GPCR is referred to in this document as a BACH-GPCR-associated disease.

According to a further aspect of the present invention, there is provided use of ATP or a derivative thereof or a modified ATP or a modified derivative thereof for the manufacture of a medicament in the treatment of BACH-GPCR associated disease wherein the individual is also undergoing treatment with at least one other compound selected from steroids, corticosteroids, antibiotics, antiviral therapy, immunosuppresants and anti-inflammatories.

According to another aspect of the present invention, we provide a method of identifying a compound capable of modulating the binding between BACH-GPCR and ATP, the method comprising the steps of providing a BACH-GPCR polypeptide, providing an ATP molecule, and detecting binding between the BACH-GPCR polypeptide and the ATP molecule in the presence and absence of a candidate compound.

According to another aspect of the present invention, we provide a method of identifying a compound capable of modulating the binding between BACH-GPCR and ATP, wherein binding between the BACH-GPCR polypeptide and the ATP molecule is detected by a fluorescent resonance energy transfer (FRET) assay.

According to yet another aspect of the present invention, we provide a method of identifying a compound capable of modulating the interaction between ATP and BACH-GPCR comprising the steps of providing a fluorescent marker labelled B ACH- GPCR polypeptide comprising a donor or an acceptor group, providing a fluorescent marker labelled ATP molecule comprising a complementary acceptor or donor group, allowing binding of the labelled BACH-GPCR polypeptide and the labelled ATP molecule which permits the donor group to come into sufficient proximity to the acceptor group providing for fluorescent resonance energy transfer and/or quenching to take place and measuring the fluorescence in the presence or absence of a candidate compound. Preferably, a difference in fluorescence between presence of a compound and absence of a candidate compound indicates that the compound is capable of modulating the binding between the BACH-GPCR and ATP molecule.

According to yet another aspect of the present invention, we provide a method of identifying a compound capable of modulating the interaction between ATP and BACH-GPCR comprising the steps of providing a BACH-GPCR polypeptide expressing cell, providing an ATP molecule, and detecting binding between the BACH-GPCR polypeptide and the ATP molecule by measuring calcium mobilisation.

According to yet another aspect of the present invention, we provide a method of identifying a compound capable of modulating the interaction between ATP and BACH-GPCR by measuring calcium mobilisation wherein the mobilisation of calcium is measured by a calcium sensitive dye.

According to yet another aspect of the present invention, we provide a method of identifying a compound capable of modulating the interaction between ATP and BACH-GPCR by measuring calcium mobilisation using a calcium sensitive dye wherein the dye is Flou-3.

We provide, according to a further aspect of the present invention a method of identifying a compound capable of modulating the interaction between ATP and BACH-GPCR comprising the steps of providing a cell comprising a reporter the expression of which is indicative of BACH-GPCR activation, providing an ATP molecule, and detecting binding between the BACH-GPCR and the ATP molecule by measuring reporter expression in the presence and absence of a candidate compound. Preferably, a difference in reporter expression between presence of a compound and absence of a candidate compound indicates that the compound is capable of modulating the binding between the BACH-GPCR and ATP molecule. According to a further aspect of the present invention there is provided a method of identifying a candidate compound capable of modulating the interaction between ATP and BACH-GPCR wherein the ATP or the BACH-GPCR are exposed to a library comprising the candidate compound.

According to yet another aspect of the present invention there is provided a method of identifying a candidate compound capable of modulating the interaction between ATP and BACH-GPCR wherein the ATP or the BACH-GPCR are exposed to a library comprising the candidate compound and where the members of the library are labelled with fluorescent label, radioactive label or luminescent label.

According to a further aspect of the present invention there is provided a method of identifying a candidate compound capable of modulating the interaction between ATP and BACH-GPCR using cells from the central nervous system (CNS), peripheral cells, cell lines derived from peripheral cells, transgenic cells derived from transgenic animals, or human cells or cell lines.

According to a further aspect of the present invention there is provided a method of identifying a candidate compound capable of modulating the interaction between ATP and BACH-GPCR using cells selected from the group consisting of: Flp-In-CHO, HeLa, COS-7, Saos2, U2OS, DG75, NIH-3T3, HEK-293, K-562, LM (TK-).

According to a further aspect, the present invention also provides a method of identifying a compound that reduces, ameliorates, or modulates symptoms of a BACH- GPCR associated disease, comprising administering a compound that modulates the interaction between ATP and BACH-GPCR to an animal model and measuring or observing the reduction, amelioration, or modulation of the symptoms.

We also provide, according to yet another aspect of the present invention a compound identified by any screening method as described in this document. According to another aspect, the present invention provides a compound selected from the group consisting of natural or synthetic peptide, a polypeptide, an antibody or antigen-binding fragment thereof, a lipid, a carbohydrate, a nucleic acid, and a small organic molecule.

According to a further aspect there is provide use of a compound identified by a screening method as described here in the manufacture of a medicament for the treatment of disease or condition associated with BACH-GPCR.

According to yet another aspect we provide use of ATP or derivatives thereof or a compound identified by a screening method as described here for treating a disease or condition selected from the group consisting of trigeminal neuralgia, orofacial pain, pain associated with toothache, pain from visceral organs, inflammatory bowel disease related pain, irritable bowel syndrome, chronic pain from renal cholelithiasis, bladder instability, Barrett's oesophagus, glaucoma, pain associated with cancer, diabetic neuropathies, Herpes virus infections, pain associated with Herpes virus associated diseases or conditions, HIV infections, migraine and skin sensitivity associated with migraine, allodynia, toothache, neuroma caused by amputation, neuroma caused by nerve transaction, neuroma caused by trauma, nerve compression caused by tumour, nerve compression caused by entrapment, nerve compression caused by crush, and pain due to damage of spinal cord or brain.

In highly preferred embodiments, the disease or condition comprises dementia, dyslexia, dyskinesias, tremor, Parkinson's, benign essential tremor, chorea, epilepsy or ballismus, for example occurring through stroke, trauma, degeneration and malignancy, chronic pain (peripheral or visceral) and relapsing remitting pain such as migraine, dry eye disorders, cyctic fibrosis, hyperactive bladder, hypercholesterolaemia, dislipdaemias and obesity.

In yet another aspect, there is provided a use of a compound identified by a screening method as described in this document for treating an individual who is also undergoing treatment with at least one other compound selected from ATP or a derivative thereof, a steroid, a corticosteroid, an antibiotic, an antiviral therapy, an immunosuppresant and an anti-inflammatory.

In another aspect the present invention provides a method of treating a patient suffering from a disease or condition associated with decreased activity of BACH- GPCR, wherein the method comprises administering to the patient ATP or a functional derivative thereof.

Another aspect of the present invention provides a method of treating a patient suffering from a disease or condition associated with decreased activity of BACH- GPCR wherein the method comprises administering to the patient a general modulator which increases or agonises the capacity of ATP or derivatives thereof to activate the BACH-GPCR.

Another aspect of the present invention provides a method of treating a patient suffering from a disease or condition associated with decreased activity of BACH- GPCR wherein the method comprises administering to the patient a modulator which increases or agonises the capacity of ATP or derivatives thereof to activate the BACH-GPCR wherein the modulator is a compound or an agent which is identified by a screening method as described here.

Another aspect of the present invention provides a method of treating a patient suffering from a disease or condition associated with increased activity of BACH- GPCR, wherein the method comprises administering to the patient a general modulator which decreases or antagonises the capacity of ATP or derivatives thereof to activate the BACH-GPCR.

Another aspect of the present invention provides a method of treating a patient suffering from a disease or condition associated with increased activity of BACH- GPCR, wherein the method comprises administering to the patient a modulator which decreases or antagonises the capacity of ATP or derivatives thereof to activate the BACH-GPCR, wherein the modulator is a compound or an agent which is identified by a screening method as described here.

In yet another aspect, the present invention provides a pharmaceutical composition comprising ATP or derivatives thereof and/or a general modulator of the interaction between ATP or derivatives thereof and the BACH-GPCR and/or a compound or an agent identified by a screening method as described here, together with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant for treating a BACH-GPCR associated disease.

According to another aspect, the present invention, provides a method of diagnosing a BACH-GPCR associated disease, the method comprising the steps of providing a BACH-GPCR polypeptide expressed from a nucleic acid sequence obtained from a tissue sample from a patient suspected of suffering from a BACH-GPCR associated disease, providing an ATP molecule, detecting binding between the BACH-GPCR polypeptide and the ATP molecule and comparing the binding between BACH-GPCR polypeptide from the patient suspected of suffering from a BACH-GPCR associated disease and the ATP molecule with the binding of an ATP molecule to BACH-GPCR polypeptide expressed from a nucleic acid sequence obtained from a normal patient, wherein a difference in binding relative to the normal is diagnostic of a disease associated with BACH-GPCR.

According to another aspect, the present invention provides a method of diagnosing a BACH-GPCR associated disease, the method comprising the steps of providing a BACH-GPCR polypeptide, providing ATP sample obtained from a patient suspected of suffering from a BACH-GPCR associated disease, detecting binding between BACH-GPCR polypeptide and the ATP sample and comparing the binding of the BACH-GPCR and the ATP from the sample obtained from a patient suspected of suffering from a BACH-GPCR associated disease, with the binding of BACH-GPCR polypeptide to ATP which is obtained from a normal patient, wherein a difference in binding relative to the normal is diagnostic of a disease associated with BACH-GPCR. According to a another aspect, the present invention provides a method of diagnosing a BACH-GPCR associated disease, the method comprising the steps of isolating a cell from a tissue sample from a patient suspected to be suffering from such a disease or condition, incubating the cell with ATP and determining the binding affinity between the ATP and BACH-GPCR, comparing this binding affinity with that of ATP and cells obtained from a normal patient, wherein a difference in binding relative to the normal is diagnostic of a disease associated with BACH-GPCR.

Accordingly, in one embodiment of the invention, ATP or derivatives thereof or a general modulator of the interaction between ATP or derivatives thereof and the

BACH-GPCR or a compound identified by a screening method as described here, may be useful to diagnose or treat, by any means as described in this document, a disease selected from the group consisting of trigeminal neuralgia, orofacial pain, pain associated with toothache, pain from visceral organs, inflammatory bowel disease related pain, irritable bowel syndrome, chronic pain from renal cholelithiasis, bladder instability, Barrett's oesophagus, glaucoma, pain associated with cancer, diabetic neuropathies, Herpes virus infections, pain associated with Herpes virus associated diseases or conditions, HIV infections, migraine and skin sensitivity associated with migraine, allodynia, toothache, neuroma caused by amputation, neuroma caused by nerve transaction, neuroma caused by trauma, nerve compression caused by tumour, nerve compression caused by entrapment, nerve compression caused by crush, and pain due to damage of spinal cord or brain.

In another embodiment, ATP or derivatives thereof or a general modulator of the interaction between ATP or derivatives thereof and the BACH-GPCR or a compound identified by a screening method as described here, may be useful to diagnose or treat, by any means as described in this document, a disease selected from the group consisting of dementia, dyslexia, dyskinesias, tremor, Parkinson's, benign essential tremor, chorea, epilepsy or ballismus, for example occurring through stroke, trauma, degeneration and malignancy. In a further embodiment, ATP or derivatives thereof and a general modulator of the interaction between ATP or derivatives thereof and the BACH-GPCR or a compound identified by a screening method as described here, may be useful to diagnose or treat, by any means as described in this document, a disease selected from chronic pain (peripheral or visceral) and relapsing remitting pain such as migraine.

In yet another embodiment, ATP or derivatives thereof and a general modulator of the interaction between ATP or derivatives thereof and the BACH-GPCR or a compound identified by a screening method as described here, may be useful to diagnose or treat, by any means as described in this document, a disease selected from the group consisting of dry eye disorders, cyctic fibrosis, hyperactive bladder, hypercholesterolaemia, dislipdaemias and obesity.

ATP or derivatives thereof may be modified such that they are capable of specifically modulating the activity of the BACH-GPCR. Thus, according to an embodiment, these agents can be used to prepare a pharmaceutical composition comprising said modified agents for use as described in this document.

In another embodiment, ATP or derivatives thereof can be chemically coupled to isotopes, enzymes, carrier proteins, cytotoxic agents, fluorescent molecules, radioactive nucleotides and other compounds for a variety of applications.

In a further embodiment the screening methods described here can be used to identify compounds or agents that can modulate the capacity of ATP or derivatives thereof to activate the BACH-GPCR. If the compound inhibits or reduces the capacity of ATP to activate the BACH-GPCR then that compound or agent is termed an antagonist.

An antagonist identified by a screening method described in this document is a compound or a group of compounds able to inhibit or reduce the capacity of ATP or derivatives thereof to activate the BACH-GPCR. Preferably, the compound can reduce the capacity of ATP to activate the BACH-GPCR by at least 3%, 10%, 20%, 30%), 40%), 50% or more of that of the amount induced by ATP in the absence of an antagonist. These antagonists can be used for manufacturing pharmaceutical compositions or diagnostic kits to treat or diagnose any diseases associated with BACH-GPCR.

In another embodiment, the screening methods described here can be used to identify compounds or agents that can modulate the ability of ATP to bind to and activate the BACH-GPCR. If the compound potentiates or enhances the ability of ATP to activate the BACH-GPCR then that compound or agent is termed an agonist.

An agonist identified by a screening method described in this document is a compound or a group of compounds able to increase the capacity of ATP or derivatives thereof to activate the BACH-GPCR. Preferably, the compound can increase the capacity of ATP to activate the BACH-GPCR by at least 3%, 10%, 20%, 30%, 40%, 50% or more of that of the amount induced by ATP in the absence of an agonist. These antagonists can be used for manufacturing pharmaceutical compositions or diagnostic kits to treat or diagnose any diseases associated with BACH-GPCR.

Definitions

As used herein, "ligand" refers to a moiety such as ATP that is capable of associating or binding to a receptor such as the BACH-GPCR. As used herein, "ATP" refers to a nucleotide that is produced by phosphorylating ADP to ATP by the addition of a third phosphate (Pj) and has a structure comprising adenine, ribose and three phosphate groups. It is contemplated that derivatives or analogues of ATP will be considered as ATP equivalents. ATP derivatives or analogues according to the present invention include, but are not limited to, 2MeSATP or Ap3 A. An ATP derivative or analogue of the present invention would exhibit the same basic structure as ATP as defined above. An ATP derivative or analogue according to the invention will exhibit binding to BACH-GPCR that is equivalent to ATP. The ATP derivative may exhibit higher affinity or lower affinity for binding to the BACH-GPCR compared to ATP. For example the higher affinity ATP derivative or analogue is capable of binding to the BACH-GPCR with more than 2%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50% or more of that of ATP. While, the lower affinity ATP derivative or analogue is capable of binding to the BACH-GPCR with less than 99%-95%, 95%- 90%, 90%-80%, 80%-70%, 70%-60%, 60%-50%, 50%-40%, 30%-20%₅ 20%-10%, 10%-5%, 5%-2% or less of that of ATP.

As used herein, "receptor activity" or "biological activity" of BACH GPCR refer to the metabolic or physiological function of the BACH receptor, including similar activities or improved activities or these activities with decreased undesirable side effects. As described here, a biologically active BACH-GPCR can bind and be activated by ATP or derivatives thereof.

The term "general modulator" as used here refers to any compound or agent or a group of compounds or a group of agents capable of modulating the binding between ATP or derivatives thereof and the BACH-GPCR. Preferably, a general modulator is any compound or an agent that is capable of modulating the capacity of ATP or derivatives thereof to activate the BACH-GPCR signaling pathway.

The terms "compound" or "agent" refer to a chemical compound (naturally occurring or synthesised), such as a biological macromolecule (e.g., nucleic acid, protein, non- peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, or a transgenic animal, or an inorganic element or molecule.

As used herein the term "modulator" means a compound or an agent that is capable of acting as an inhibitor or a potentiator of ATP or derivatives thereof to bind to and activate the BACH-GPCR. A modulator can be a protein, a nucleic acid, an antibody, a peptide, a fragment thereof, such as an antigen-binding fragment, a protein, a polypeptide, a peptide, a lipid, a carbohydrate, a small inorganic or organic molecule, etc. A candidate modulator can be a natural or synthetic compound, including for example a small molecule, a compound contained in extracts of animals including humans, a compound contained in extracts of transgenic animals, plants, bacteria or fungal cells, as well as contained medium from such cells. The modulator may be identified by any screening method available in the art.

"Contained medium" is a medium which has been used to grow animal, plant, bacterial or fungal cells which medium contains among other metabolic products, minerals, growth factors and other constituents which may be required for growth or have been secreted in the medium as a consequence of the growth.

As used herein the term "small molecule" or "small compound" refers to a compound having a molecular mass of less than 3000 Daltons. For example the "small molecule" less than 2000 or 1500, or even less than 1000, or even less than 600 Daltons, or even less than 200 Daltons, and more than 50 Daltons.

A "small organic molecule" is a small molecule that comprises carbon.

As used herein the terms "change in binding" or "change in activity" refer to an at least 3%, preferably at least 10%o increase or decrease in binding relative to a standard in a given assay.

The terms "standard" or "normal" refer to a reading or a measurement in a given assay which can be used as a reference point to which all other measurements or readings are compared. By way of example, the normal or standard readings may be obtained from sample derived from a patient who is not suffering from a BACH-GPCR associated disease or if the patient is suffering from BACH-GPCR associated disease, the sample may be obtained from an unaffected tissue or organ.

The term "inhibitor" as used herein includes a compound or an agent that is capable of suppressing or reducing or blocking or removing or masking or eliminating or preventing or thwarting or antagonising the binding between ATP and the BACH- GPCR. As used herein, an "antagonist" is a compound or an agent that is capable of inhibiting or reducing the binding between ATP or derivatives thereof and the BACH-GPCR, and thereby inhibit or reduce the intracellular response induced by ATP or derivatives thereof. By way of example, the "antagonist" is capable of reducing the binding between ATP or derivatives thereof and the BACH-GPCR by at least 3%, 10%, 20%, 30%), 40%), 50% or more of that of the binding between ATP or derivatives thereof and the BACH-GPCR, in the absence of an antagonist.

As used herein the teπn "inhibit or reduce the intracellular response" refers to a compound or an agent which is capable of blocking, suppressing, eliminating, preventing, thwarting or generally lowering the ability of ATP or derivatives thereof to bind to and activate the BACH-GPCR mediated intracellular signalling pathway.

Such compounds may be used in the treatment of diseases associated with higher than normal BACH-GPCR activity.

The term "potentiator" as used herein includes agents capable of increasing or augmenting or enhancing or agonising or ensuring an improvement in the capacity of

ATP or derivatives thereof to bind to the BACH-GPCR.

As used herein, an "agonist" refers to a compound or an agent that is capable of increasing the capacity of ATP or derivatives thereof to bind to the BACH-GPCR and thereby increase or enhance the intracellular response induced by ATP or derivatives thereof. By way of example, the "agonist" is capable of increasing or enhance the capacity of ATP or derivatives thereof to bind to the BACH-GPCR by at least 3%, 10%, 20%), 30%), 40%, 50% or more compared to the capacity of ATP or derivatives thereof to bind to the BACH-GPCR in the absence of an agonist.

As used herein the term "increase or enhance the intracellular response" refers to a compound or an agent that is capable of enhancing, improving, or generally increasing the capacity of ATP or derivatives thereof to bind to and activate the BACH-GPCR mediated intracellular signalling pathway. Such compounds may be used in the treatment of diseases associated with reduced BACH-GPCR activity. As used herein the term "sample" refers to a source of molecules being tested for the presence of a compound or an agent that can modulate the capacity of ATP to bind to and activate the BACH-GPCR. A sample can be an environmental sample, a natural extract of animal, a natural extract of a transgenic animal, plant yeast or bacterial cells or tissues, a clinical sample, a synthetic sample, or a conditioned medium from recombinant cells or a fermentation process. The term "tissue sample" refers to a tissue that is tested for the presence of, abundance, quality of an agent or a compound that is capable of modulating the ability of ATP to bind to and activate the BACH-GPCR.

As used herein the term "tissue" is an aggregate of cells that perform a particular function in an organism. The term "tissue" as used herein refers to cellular material from a particular physiological region. The cells in a particular tissue can comprise several different cell types. By way of example, the tissue may be brain tissue that further comprises neurones and glial cells, as well as vascular and lymphatic endothelial cells and blood cells, all contained in a given tissue section or sample. In addition to solid tissue, the term "tissue" is also intended to encompass non-solid tissue, such as blood and lymph.

The cells that can be used in certain screening methods include, but are not limited to central nervous system (CNS) cells, peripheral cells, cell lines derived from peripheral cells, transgenic cells, transgenic cells derived from transgenic animals, and human cells or cell lines.

As used herein the term, "Fluo-3-based assay" refers to an assay for BACH-GPCR activity that measures intracellular calcium mobilisation from intracellar stores induced by activation of the BACH-GPCR wherein the intracellular calcium is measured by the fluorescence of Fluo-3 in the cell. This dye has routinely been used to study the elementary process of calcium signaling within a cell^1"3 and for cell based pharmacological screening⁴. Mobilised calcium is bound by the dye resulting in a >100 fold increase in dye fluorescence. We therefore describe methods of identifying compound or agents that modulate the ability of ATP to specifically bind and activate the BACH-GPCR by measuring changes in Fluo-3 fluorescence.

As used herein the term "binding" refers to the physical association or binding of ATP with the BACH-GPCR. As the term is used herein binding is "specific" if it occurs with an EC50 or a Kd of 100 nM or less, generally in the range of 100 nM to 10 pM. For example binding is specific if the EC50 or the Kd is lOOnM, 10 nM, 1 nM, 950 pM, 900 pM, 850 pM, 800 pM, 750 pM, 700 pM, 650 pM, 600 pM, 550 pM, 500 pM, 450 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 75 pM, 50 pM, 25 pM, or 10 pM or less.

As used herein the term "EC50" refers to the concentration of a compound or an agent at which a given activity, including binding of ATP or derivatives thereof and a functional activity of BACH-GPCR is 50% of the maximum for BACH-GPCR activity measured using the same assay in the absence of a compound or an agent. Defined differently, the "EC50" is the concentration of a compound or an agent that gives 50% activation, when 100% activation is set at the amount of activity that does not increase with the addition of more ATP or derivatives thereof. It is of note that the "EC50 of ATP or derivatives thereof will vary according to the identity of the molecule used.

As used herein the term "decrease in binding" refers to a decrease of at least 3%> in the amount of binding of ATP to BACH-GPCR, detected in a given assay in the presence of a compound or agent relative to binding detected in an assay lacking the compound or agent.

As used herein, references to "fluorescence" or "fluorescent groups" or "fluorophores" include luminescence, luminescent groups and suitable chromophores. The BACH- GPCR, ATP or derivatives thereof and candidate compounds or agents may be labelled with luminescent labels and luminescence resonance energy transfer is indicative of binding between ATP or derivatives thereof and the BACH-GPCR. Suitable luminescent probes include, but are not limited to, the luminescent ions of europium and terbium introduced as lanthium chelates (Heyduk E. and Heyduk T., 1997, Anal Biochem. 248(2): 216-27). The lanthanide ions are also good donors for energy transfer to fluorescent groups (Selvin, P. R., 1995, Methods Enzymol. 246: 300- 34). Luminescent groups containing lanthanide ions can be incorporated into nucleic acids utilising an 'open cage' chelater phosphoramidite.

As used herein the term "pharmaceutically active ingredient" refers to ATP or derivatives thereof and a general modulator or a compound identified by a screening method as described here which is/are capable of being used as therapeutics in the treatment of BACH-GPCR-associated disease or for prophylactics.

Detailed description of the invention.

The invention is based on the demonstration that ATP is a natural ligand for the BACH-GPCR. We therefore describe methods of using the binding of this ligand to the receptor in a drug (a compound or an agent) screening methods. The ligand and its interaction with the receptor BACH-GPCR also provide for the diagnosis of BACH- GPCR associated diseases. We further describe a kit comprising the BACH-GPCR sequence and its corresponding polypeptide and/or recombinant cells expressing the polypeptide and ATP or derivatives thereof which can be used to identify a compound or an agent that is capable of modulating the ability of ATP or derivatives thereof to bind to and activate the BACH-GPCR. Such kits are useful for the diagnosis, prevention and/or treatment of various BACH-GPCR associated diseases, disorders and conditions.

We also describe agonists and antagonists that modulate the ability of ATP or derivatives thereof to bind to and activate the BACH-GPCR.

Described here is also the use of ATP or derivatives thereof and/or a general modulator or a compound identified by a screening method as described here.

The present invention also relates to the use of ATP or derivatives thereof and/or a general modulator or a compound identified by a screening method as described here in the manufacture of medicament for the treatment of BACH-GPCR associated disease.

Screening methods.

A compound or an agent that modulates the interaction between ATP or derivatives thereof and the BACH-GPCR can be identified in a number of ways by taking advantage of the ability of ATP to activate the BACH-GPCR.

For example, the ability to reconstitute the binding between ATP and the BACH- GPCR either in vivo, in vitro or in cultured cells provides a suitable target for the identification of compounds or agents that interfere or disrupt this binding.

Assays based on interference or disruption of binding can be used to identify agents such as small organic molecules, from libraries or collections of such molecules. Alternatively, similar assays can select a compound or an agent from a pool of compounds or agents present in natural sources such as plants, fungal or bacterial extracts or even in human and transgenic animal tissue samples.

The extract can be made from cells expressing a library of variant nucleic acids, peptides or polypeptides. Modulators of the binding between ATP or derivatives thereof and the BACH-GPCR can then be screened using a binding assay or a functional assay that measures downstream intracellular signaling through the receptor, for example calcium mobilisation from intracellular stores.

This functional assay can be performed in isolated cell membrane fractions or on recombinant cells expressing the BACH-GPCR on their surface.

The screening methods will generally have two basic approaches:

1) Ligand binding screens.

These screens may use cells that express the BACH-GPCR, membrane extracts from such cells or immobilised lipid membranes comprising the BACH-GPCR. The BACH-GPCR are then exposed to labeled ATP or a derivative thereof in the presence or absence of a candidate compound. After a predetermined incubation period, the reaction mixture is measured for binding of the labeled ATP or a derivative thereof to the BACH-GPCR. Compounds that interfere with binding or displace labeled ATP or a derivative thereof from the BACH-GPCR can be agonists or antagonists. Subsequent functional analysis can then be performed on a compound or agent identified as positive by the above method to determine whether the compound or agent is an antagonist or an agonist of the binding. Control reactions should be performed using cells, membrane extracts or lipid membranes that do not express or comprise the BACH-GPCR in order to exclude possible non-specific effects of some candidate modulators.

2) Functional analysis in which the ability of the BACH-GPCR to transmit a signal is determined. When testing for agonists, cells expressing BACH-GPCR or membranes prepared from them are incubated with ATP, and the ability of BACH-GPCR to transmit a signal is determined in the presence or absence of the candidate compound. An agonist or partial agonist will have the capacity to increase the ability of ATP or derivatives thereof to activate the BACH-GPCR by at least 3% of that of ATP or derivatives thereof. Control reactions should be performed using cells or membrane extracts from cells that do not express the BACH-GPCR in order to exclude possible non-specific effects of some candidate modulators.

Ligand binding and displacement based screening methods.

The BACH-GPCR polypeptide expressed on a cell, or isolated membranes comprising such a receptor polypeptide can be used along with ATP or derivatives thereof in order to screen for compounds that inhibit the binding of ATP or derivatives thereof to the BACH-GPCR. When identified in a screening method that measures binding or ATP displacement alone, compounds or agents will also have to be subjected to functional analysis to determine whether they are capable of acting as agonists or antagonists. For the purposes of the displacement type experiments, cells expressing the BACH- GPCR polypeptide e.g. Flp-In-CHO cells (generally 25x10³ cells per experiment or 1 to lOOμg of membrane extract) are incubated in binding buffer with labeled ATP or a derivative thereof in the presence or absence of increasing concentrations of the candidate modulator. To ensure validity of the experiment, the assay can be calibrated against a control displacement reaction using the same increasing concentrations of labeled ATP or a derivative thereof. After the incubation, the cells are washed extensively, and bound, labeled ATP or a derivative thereof is measured as appropriate for the given label.

Labels include but are not limited to radioactive labels, fluorescent labels and luminescent labels.

A decrease of at least 3% in the amount of labeled ATP or a derivative bound to the BACH-GPCR in the presence of candidate modulator indicates displacement of binding by the candidate modulator. Candidate modulators are considered to bind specifically in this or other screening methods described herein if they can displace 50% of labeled ATP or derivative thereof at a concentration of 10 nM or less. It is of note that the 50% displacement is with reference to ATP or a derivative thereof used at a level that is below the saturation level.

Surface plasmon resonance (SPR)

Other methods for screening for compounds or agents that modulate the binding or displacement of ATP from the BACH-GPCR include surface plasmon resonance (SPR).

This type of test can be used as a quantitative method of evaluating binding between two entities by the change in mass near an immobilised sensor caused by the binding or loss of ATP or a derivative thereof from the aqueous phase to a BACH-GPCR polypeptide immobilised on a membrane or a sensor chip. This change in mass is measured as resonance units versus time after addition or removal of the ATP or candidate modulator and is measured using a Biacore Biosensor (Biacore AB).

The BACH-GPCR can be immobilised on a sensor chip e.g. research grade CM5 chip, in a thin film lipid membrane according to methods described by Salamon et al, (Salamon et al., 1996, Biophys J. 71: 283-294; Salamon et al., 2001, Biophys. J. 80: 1557-1567; Salamon et al., 1999, Trends Biochem. Sci. 24: 213-219, also see WO97/49989)

SPR can test for modulators of binding in at least two ways. First, ATP or a derivative thereof can be pre-bound to the immobilised BACH-GPCR polypeptide, followed by addition of candidate modulator at a concentration ranging from 0.1 nM to 1 μM. Displacement of the bound ATP or a derivative thereof can be quantitated which allows for detection of modulator binding. Alternatively, the immobilised BACH- GPCR polypeptide can be pre-incubated with candidate modulator and challenged with ATP or a derivative thereof.

A difference in the binding of ATP or a derivative thereof to the BACH-GPCR exposed to a candidate modulator relative to that on a membrane or a sensor chip not pre-exposed to a candidate modulator demonstrates binding or displacement of ATP or a derivative thereof in the presence of modulator.

A difference in the binding of ATP or derivatives thereof to the BACH-GPCR exposed to a candidate modulator relative to that of a membrane or a sensor chip pre- exposed to modulator demonstrates binding or displacement of ATP or derivatives thereof in the presence of modulator.

For SPR assays, a decrease of 10% or more in the amount of ATP or derivatives thereof bound in the presence of candidate modulator, relative to the amount of ATP or derivatives thereof bound in the absence of candidate modulator indicates that the candidate modulator inhibits the interaction between ATP or derivatives thereof and the BACH-GPCR. Fluorescence resonance energy transfer (FRET).

Another method for detecting inhibition of binding between ATP or a derivative thereof and the BACH-GPCR uses fluorescence resonance energy transfer (FRET). FRET is a quantum mechanical phenomenon that occurs between a fluorescence donor (D) and a fluorescence acceptor (A) in close juxtaposition to each other (usually <100nm of separation) if the emission spectrum of D overlaps with the excitation spectrum of A. The molecules to be tested, ATP or a derivative thereof and the BACH-GPCR polypeptide, are labeled with a complementary pair of donor and acceptor fluorescence markers. While bound closely together by the ATP:BACH- GPCR complex, the fluorescence emitted upon excitation of the fluorophore will have a different wavelength than that emitted in response to that excitation wavelength when the ATP or a derivative thereof and the BACH-GPCR are not bound or not in sufficiently close juxtaposition, providing for quantitation or bound versus modified interactions by measurement of emission intensity at each wavelength.

A variation on FRET uses fluorescence quenching to monitor molecular interactions. One entity in the interaction can be labeled with a fluorophore, and the other with a molecule that quenches the fluorescence when brought into close apposition with it. A change in fluorescence upon excitation is indicative of a change in the association of the molecules tagged with the fluorophore: quencher pair. As an example, if the BACH-GPCR polypeptide is labeled with a fluorophore marker then an increase in fluorescence of the labeled BACH-GPCR polypeptide is indicative that the ATP or a derivative thereof bearing the quencher has been displaced. Alternatively, if the ATP or a derivative thereof is labeled with a fluorophore marker then an increase in fluorescence of the labeled ATP or a derivative thereof is indicative that BACH-GPCR bearing the quencher has been displaced.

For quenching assays, a 10%) or greater increase in the intensity of fluorescent emission in sample containing a candidate modulator, relative to samples without the candidate modulator, indicates that the candidate modulator inhibits the binding between ATP or a derivative thereof and the BACH-GPCR. As used herein, the term "donor" refers to a fluorophore which absorbs at a first wavelength and emits at a second, longer wavelength. The term "acceptor" refers to a fluorophore, chromophore or quencher with an absorption spectrum which overlaps the donor's emission spectrum and is able to absorb some or most of the emitted energy from the donor when it is near the donor group (typically between 1-lOOnm). If the acceptor is a fluorophore capable of exhibiting FRET, it then re-emits at a third, still longer wavelength; if it is a chromophore or quencher, then it releases the energy absorbed from the donor without emitting a photon.

The donor and acceptor groups may independently be selected from suitable fluorescent groups, chromophores and quenching groups. Preferred donors and acceptors include:

5-FAM (also called 5-carboxyfluorescein; also called Spiro(isobenzofuran-l(3H),

9'-(9H)xanthene)-5-carboxylic acid,3',6'-dihydroxy-3-oxo-6-carboxyfluorescein);

5-Hexachloro-Fluorescein ([4,7,2',4',5',7'-hexachloro-(3^,,6'- dipivaloylfluoresceinyl)-6-carboxylic acid ]);

6-Hexachloro-Fluorescein ([4,7,2',4^,,5',7'-hexachloro-(3',6'- dipivaloylfluoresceinyl)-5-carboxylic acid ]);

5-Tetrachloro-Fluorescein ([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceinyl)-5- carboxylic acid ]);

6-Tetrachloro-Fluorescein ([4,7,2',7'-tetrachloro-(3',6'-dipivaloylfluoresceinyl)-6- carboxylic acid ]);

5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium, 9-(2,4- dicarboxyphenyl)-3,6- bis(dimethylamino);

6-TAMRA (6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5- dicarboxyphenyl)-3 ,6- bis(dimethylamino) ;

EDANS (5-((2-aminoethyl)amino)naphthalene- 1-sulfonic acid) ;

1,5-IAEDANS (5-((((2-iodoacetyl)amino)ethyl) amino)naphthalene- 1-sulfonic acid);

DABCYL (4-((4-(dimethylamino)phenyl) azo)benzoic acid);

Cy5 (Indodicarbocyanine-5); ^■ Cy3 (Indodicarbocyanine-3); and

^■ BODIPY™ FL (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3- propionic acid) as well as suitable derivatives thereof.

Fluorescence polarization

In addition to the SPR and FRET methods, fluorescence polarization measurement is useful in quantitating binding. The fluorescence polarization value for a fluorescently- tagged molecule depends on the rotational correlation time or tumbling rate. Complexes, such as those formed between the BACH-GPCR and a fluorescently labeled ATP or derivative thereof, have a higher polarization value than uncomplexed labeled ATP or a derivative thereof. The inclusion of a candidate inhibitor of the BACH-GPCR:ATP binding results in a decrease in fluorescence polarization, relative to a mixture without the candidate inhibitor.

Fluorescence polarization is particularly advantageous to the screening methods described here as it capable of identifying small molecules that are capable of disrupting the formation of receptor: ligand complexes.

A decrease of 10 %> or more in fluorescence polarization in samples containing a candidate modulator, relative to fluorescence polarization in a sample lacking the candidate modulator, indicates that the candidate compound or agent acts as an antagonist of the interaction between ATP or a derivative thereof and the BACH- GPCR polypeptide.

Functional assays

Reporter screening assays

The intracellular signals initiated by binding of ATP or derivatives thereof to the BACH-GPCR sets a cascade of intracellular signalling events, the end result of which is a rapid and detectable change in the transcription or translation of one or more genes. The expression of a receptor can therefore be monitored by detecting the expression of the reporter gene which is controlled by promoter sequences which are responsive to BACH-GPCR activation.

As used herein the term "promoter" refers to the transcriptional control sequences required for reporter mediated regulation of gene expression, including not only the basal promoter, but also any enhancers or transcription-factor binding sites necessary for reporter-regulated expression. By selecting promoters that are responsive to the intracellular signals resulting from ATP or derivatives thereof binding to the BACH- GPCR, and operably linking the selected promoters to reporter genes whose transcription, translation or ultimate activity is readily detectable and measurable, the transcription based reporter assay provides a rapid indication of whether a given reporter is activated.

The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

Reporter genes such as luciferase, chloramphenical acetyl transferase (CAT), green fluorescent protein (GFP), β-lactamase or β-galactosidase are well known in the art, as are assays for measuring the products of their activities.

Genes which are considered to be particularly suited the "immediate early" genes, which are rapidly induced, generally within minutes of activation of the receptor. The induction of immediate early genes transcription does not require the synthesis of new regulatory proteins. In addition to rapid responsiveness to ligand binding, characteristics of preferred genes useful for making reporter constructs include: low or undetectable expression of the reporter in cells that have not been received a stimulus; induction that is transient and independent of new protein synthesis; subsequent shut off of transcription requires new-protein synthesis; and mRNA transcribed from these genes have a short half life. It is preferred, but not necessary that a transcription control element have all of these features for it to be useful.

An example of a gene that is responsive to a number of different stimuli is the c-fos proto-oncogene. The c-fos gene is activated in a protein-synthesis-independent manner by growth factors, hormones, differentiation-specific agents, stress, and other known promoters of cell surface proteins. The induction of c-fos expression is extremely fast, often taking place within minutes of receptor activation. This and other characteristic features makes the c-fos regulatory regions very attractive for use in the reporter screening methods described in this document.

The c-fos regulatory elements include (Verma et ah, 1987, Cell 51: 513-514) a TATA box which is known to be required for initiation of transcription, two upstream elements for basal transcription, and an enhancer, which includes an element with dyad symmetry and which is required for induction of by TPA, serum and EGF.

Additional examples of transcription control elements that are responsive to BACH- GPCR include, but are not limited to the elements of the transcription factor CREB (cyclic AMP responsive element binding protein, see US patent No 5,919,649 for reporter constructs responsive to CREB binding activity), the CBER-binding element (CRE, cAMP responsive), the vasoactive intestinal peptide (VIP) gene promoter (cAMP responsive, Fink et al., Proc. Natl. Acad. Sci. 85: 6662-6666), the somatostatin gene promoter (cAMP responsive, Montminy et al., 1996, Proc. Natl. Acad. Sci, 83: 6682-6686), the proekephalin promoter (cAMP responsive, nicotinis agonists, and phorbol esters, Comb et al., 1986, Nature 232:353-356),

Other examples of transcriptional control elements that are responsive to BACH- GPCR include, but are not limited to those responsive to the AP-1 transcription factor and those responsive to NF-κB. The consensus of the AP-1 binding site a palindromic sequence (5-'TGA(C/G)TCA-3')₅ (Lee et al, 1987, Nature 325:368-372; Lee et al, 1987 Cell 49:741-752). The AP-1 binding site is also responsive to tumour promoters such as the phorbol ester 10-O-tetradecanoylphorbol-β-acetate (TPA), and are therefore sometimes also known as TRE which stands for TPA-response elements. AP-1 can activate a numerous genes that are involved in the early response of cells to growth promoting signals. By way of non-limiting examples, AP-1 responsive genes include the genes Fas and June, Fas-related antigens (Fra) 1 and 2, IκBα, ornithine decarboxylase, and annexins I and II.

The NF-κB binding element comprises the consensus sequence 5'-GGGGACTTTCC- 3'. A large number of genes have been identified as NF-κB responsive and their control elements can be linked to a reporter gene for monitoring the BACH-GPCR activity. By way of non-limiting examples the gene which are regulated by the NF- B include those encoding IL-lb (Hiscott et al, 1993, Mol. Cell. Biol. 13: 6231-6240), Fas ligand (Matsui et al, 1998, J Immunol 161: 3469-3473), GM-CSF (Schrech and Baeuerle, 1990 Mol. Cell. Biol. 10: 1281-1286), TNF-a (Shakhov et al, 1990, J Exp. Med. 171: 35-47) and IκBα (Haskill et al, 1991, Cell 65: 1281-1289).

Vectors encoding NF-κB responsive reporter constructs are also known in the art or can be readily constructed by those of skill in the art using for example synthetic NF- B responsive elements and a minimal promoter, or using the NF-KB responsive sequences obtained from gene known to be regulated by the NF-κB transcription factor.

In order to test whether a particular reporter can be used in the reporter screening methods described here, the reporter should be tested by exposing BACH-GPCR- expressing cells, which have been transfected or transduced with the reporter construct, in the presence of ATP or a derivative thereof. An increase of two fold or more in the expression of the reporter gene in response to ATP or a derivative thereof is indicative that the reporter can be used as an indicator of BACH-GPCR activity.

In order to assay the BACH-GPCR activity with an ATP molecule or a derivative thereof responsive transcriptional construct, cells that stably express a BACH-GPCR polypeptide are stably transfected or transduced with the reporter construct. To screen for activators (agonists), the cells are left untreated, exposed to ATP or derivatives thereof, in the presence or absence of candidate modulators, and expression of the reporter gene is measures. The ATP or a derivative thereof treated cultures are used as a standard for the level of transcription induced by the candidate agonist/enhancer. An increase by 5%> or more in reporter expression in the presence of a candidate modulator, relative to the absence of the candidate modulator, indicates that the candidate is capable of acting as an agonist/enhancer of ATP or derivatives thereof to activate the BACH-GPCR.

To screen for antagonists, the cells expressing BACH-GPCR and carrying the reporter construct are exposed to ATP or derivatives thereof in the presence or absence of candidate modulators. A decrease of 5% or more in reporter expression in the presence of candidate modulator, relative to the absence of the candidate modulator, indicates that the candidate acts as an antagonist/inhibitor to the ability of ATP or derivatives thereof to activate the BACH-GPCR.

Controls for reporter based assays include cells not expressing the BACH-GPCR but carrying the reporter construct, as well as cells which incorporate a construct that comprises a reporter gene but has the regulatory sequences deleted. Compounds that are identified as modulators of the ability of ATP or derivatives thereof to activate the BACH-GPCR should also be analysed whether they modulate transcriptional activity of other regulatory sequences and by other reporter constructs, in order to determine the level of specificity and the scope of their activity.

The reporter based assays and most cell-based assays are well suited for screening libraries which may be expression libraries for compounds or agents that modulate the ability of ATP or derivatives thereof to activate the BACH-GPCR. By way of a non- limiting example, the libraries can comprise cDNA from natural sources, e.g. plants, animals, transgenic animals, bacteria, insects, yeast or they can be libraries expressing randomly or synthetically mutated variants or one or more polypeptides. Genomic libraries in viral vectors can also be used to express the mRNA content of one or more cell or tissue. GTPase/GTP binding assay

For GPCRs such as BACH-GPCR, a measure of receptor activity is the binding of GTP by cell membranes which carry receptors. The method of Traynor and Nahorski, 1995 Mol. Pharmac. 47: 848-854, incorporated herein by reference, essentially measures G-protein coupling to membranes by detecting the binding of labeled GTP. For GTP -binding assays, membranes isolated from cells expressing the receptor are incubated in a buffer containing 20 mM HEPES, pH 7.6, 1000 mM NaCl, and 10 mM MgCl₂, 80 pM ³⁵S-GTPγS and 3 μM GTP. The assay mixture is incubated for 1 (one) hour at 30°C, afetr which unbound radioactive GTP is removed using a filter GF/B. Bound, radioactive GTP is measured using a scintillation counter. In order to assay for ATP or a derivative thereof induced BACH-GPCR activity, membranes prepared from cells expressing the receptor are mixed with ATP or a derivative thereof, and the GTP binding assay is performed in the presence and absence of a candidate modulator of the BACH-GPCR activation.

A decrease of 5%> or more in labeled GTP as measured by scintillation counting in an assay of this kind containing a candidate modulator, relative to an assay without the modulator, indicates that the candidate modulator inhibits BACH-GPCR activation by ATP or a derivative thereof.

Controls include assays using membranes isolated from cells not expressing the BACH-GPCR, in order to exclude possible non-specific effects of the candidate compound.

In order to assay for the effect of a candidate modulator on BACH-GPCR regulated GTPase activity, membrane samples are incubated with ATP or a derivative thereof with and without the modulator, followed by the GTPase assay. A change (increase or decrease) of 5%> or more in the level of GTP binding or GTPase activity relative to samples without modulator is indicative of BACH-GPCR modulation by a candidate compound or agent. Library screening /including high throughput screens)

We also describe high-throughput screening methods for identifying compounds that modulate the binding between ATP or derivatives thereof and the BACH-GPCR. Preferably, all the biochemical steps for this assay are performed in a single solution in, for instance, a test tube or microtitre plate, and the test compounds are analysed initially at a single compound concentration.

For the purposes of high throughput screening, the experimental conditions are adjusted to achieve a proportion of candidate compounds identified as "positive" compounds from amongst the total compounds screened. The assay is preferably set to identify compounds with an appreciable affinity towards the target eg. when 0.1%) to 1% of the total test compounds from a large compound library are shown to bind to a given target with a K_tof 10 μM or less (eg. 1 μM, 100 nM, 10 nM, or less).

Another alternative for monitoring the binding between ATP or a derivative thereof and the BACH-GPCR polypeptide uses biosensor assays. ICS biosensors have been described in the art (Australia Membrane Biotechnology Research Institute; http//www.ambri.com.au/; Cornell B, Braach-Maksvytis V, King L., Osman P., Raguse B., Wieczorek L., and Pace R., "A Biosensor that uses ion-channel switches" Nature, 1997, 387: 580). In this technology, the association of BACH-GPCR and its ligand, is coupled to the closing of gramacidin-facilitated ion channels in suspended membrane biolayers and thus to a measurable change in the admittance (similar or impedance) of the biosensor. This approach is linear over six orders of admittance change and is ideally suited for large scale, high throughput screening of small molecule combinatorial libraries.

A 10%> or greater increase or decrease in admittance in a sample containing a candidate modulator, relative to the admittance of a sample lacking the candidate modulator, indicates that the candidate modulator acts as an agonist or antagonist respectively. It is important to state that in assays testing the interaction of ATP or a derivative thereof and the BACH-GPCR polypeptide, it is possible that a modulator need not necessarily interact directly with the domains of the receptor that mediate the interaction with ATP or the derivative thereof. It is therefore, possible that the candidate molecule by binding to a distant location removed from the site of interaction may cause for instance a conformational change in the BACH-GPCR and thereby modulate the binding of ATP.

Modulators that act in this manner are nonetheless of interest as agents to modulate the ability of ATP or derivatives thereof to bind to and activate the BACH-GPCR.

The compound or agent identified or characterised by the methods described in this document can be used in a method of modulating the ability of ATP or derivatives thereof to bind to and activate the BACH-GPCR in a cell, said method comprising the step of delivering to said cell said compound or said agent such that the ability of ATP or a derivative thereof to bind to and activate the BACH-GPCR is modulated.

Downstream pathway activation assays.

Calcium mobilisation - Fluo-3 dye based assay.

The elementary process of calcium signaling within a cell has been described in the art (Bootman, et al, 1997, "Imaging of hierarchical Ca²⁺ signaling system in HeLa cells"

94-

J Phisiol. 499: 307; Cheng et al, 1996, "Calcium sparks and [Ca ]i waves in cardiac myocytes" Am J Cell Physiol, 270: C148; Clapham D. E., 1995, "Calcium signaling" Cell, 80: 259).

Other well known downstream pathway activation assays which can also be used in order to screen for modulators of the binding between ATP or derivatives thereof and the BACH-GPCR include but are not limited to calcium flux assay (also referred to as the Aequorin-based assay) (Stables et al, 1997, Anal. Biochem. 252: 115-126), the adenylate cyclase assay (Kenimer and Nirenberg, 1981, Mol. Pharmacol. 20: 585- 591), cAMP (Horton and Baxendale, 1995, Methods Mol. Biol. 41 : 91-105), phospholipid bread down (Rudolph et al, 1999, J Biol. Chem. 21 A: 11824-11831), protein kinase C (PKC) assay (Kikkawa et al, 1982, J Biol. Chem. 257: 13341), kinase assay (Pinna and Ruzzene, 1996, Biochem. Biophys. Acta 1314: 191-225).

In vivo - BACH-GPCR Knock-Out Mouse.

BACH-GPCR knock-out mouse are less sensitive to external stimuli and pain (see WO 02/38607). Loss of the BACH-GPCR can also result in abnormal bladder motility and mutants of the protein have hypo active bladder urinating less frequently but releasing a larger volume and suffer urinary retention. BACH-GPCR also plays a role in erectile dysfunction and the control of motor fibers in the prostate. A compound or an agent that modulates the ability of ATP or a derivative thereof can be identified or verified by taking advantage of the phenotypic characteristics of the BACH-GPCR knock-out mouse.

Accordingly, a 5%> or greater increase or decrease in the tested phenotypic characteristic of a normal mouse or a BACH-GPCR knock-out mouse containing a candidate modulator indicates that the candidate modulator acts as an agonist or antagonist respectively. Based on their mode of action the candidate compounds can be grouped into three categories: i) those that act in a BACH-GPCR independent manner; ii) compounds that act by modulating the interaction between ATP and the GPCR; and iii) compounds that affect components which are positioned downstream of ATP in the BACH-GPCR signaling pathway.

The candidate compounds of i) and iii) are anticipated to modulate the phenotypic characteristic in the normal mouse as well as the BACH-GPCR knock-out mouse, while the compounds of ii) would only have an effect in the normal mouse. It is intended that candidate compounds belonging to all three categories can be used in the treatment of BACH-GPCR associated diseases. Cells

A cell that is useful according to the invention may be selected from the group consisting of bacterial cells, yeast cells, insect cells, mammalian cells, central nervous system (CNS) cells, peripheral cells, cell lines derived from peripheral cells, transgenic cells, transgenic cells derived from transgenic animals, or human cells or cell lines. The cells useful in cell based screening methods of the present invention preferably include recombinant cells which express the BACH-GPCR and may or may not express a BACH-GPCR responsive reporter construct. Such recombinant cells include but are not limited to Flp-In-CHO, HeLa, COS-7, Saos2, U2OS, DG75, NIH-3T3, HEK-293, K-562, LM (TK-).

Diagnostic assays based on the interaction between ATP and the BACH-GPCR.

The interaction between ATP and the BACH-GPCR can be used as the basis of assays for the diagnosis or monitoring of BACH-GPCR associated diseases, conditions or disorders.

BACH-GPCR associated diseases can be selected from the group consisting of trigeminal neuralgia, orofacial pain, pain associated with toothache, pain from visceral organs, inflammatory bowel disease related pain, irritable bowel syndrome, chronic pain from renal cholelithiasis, bladder instability, Barrett's oesophagus, glaucoma, pain associated with cancer, diabetic neuropathies, Herpes virus infections, pain associated with Herpes virus associated diseases or conditions, HIV infections, migraine and skin sensitivity associated with migraine, allodynia, toothache, neuroma caused by amputation, neuroma caused by nerve transaction, neuroma caused by trauma, nerve compression caused by tumour, nerve compression caused by entrapment, nerve compression caused by crush, and pain due to damage of spinal cord or brain. Other BACH-GPCR associated diseases include dementia, dyslexia, dyskinesias, tremor, Parkinson's, benign essential tremor, chorea, epilepsy or ballismus, for example occurring through stroke, trauma, degeneration and malignancy.

BACH-GPCR associated diseases also include chronic pain (peripheral or visceral) or relapsing remitting pain such as migraine.

BACH-GPCR associated diseases further include dry eye disorders, cyctic fibrosis, hyperactive bladder, hypercholesterolaemia, dislipdaemias and obesity.

Diagnostic assays for BACH-GPCR associated diseases can be used to evaluate the quantity of the ATP or the BACH-GPCR. For example, assays that determine whether an individual expresses a mutant or variant form of the ligand or receptor can be used diagnostically. Also assays that measure the ability of ATP to bind to and activate the BACH-GPCR can be used diagnostically.

Assay that measures the amount of ATP and/or the BACH-GPCR.

The levels of ATP can be measured and compared to standards in order to determine whether an abnormal level is present in a sample, which may be indicative of a possible cause of the BACH-GPCR associated disease.

The levels of ATP can for example be measured by the reporter based assays described above. By way of example, a sample isolated from an individual suspected of suffering from a BACH-GPCR associated disease is contacted with cells expressing the BACH-GPCR polypeptide and the reporter construct and activation of the BACH- GPCR detected is compared to the activation in a sample of similar tissue from a healthy individual, or from a site on the tested individual that is not so affected.

A difference of 5% or more relative to a standard is diagnostic for a BACH-associated disease, disorder or condition. Diagnosis of BACH-GPCR associated disease can also be diagnosed using the calcium mobilisation assay which is performed in cultured recombinant cells as part of an assay for the identification of modulators of the ability of ATP or derivatives thereof to activate the BACH-GPCR. In this assay, BACH-GPCR activation by ATP is determined by the ability of the dye Flou-3 to associate with calcium released from cellular stores following activation of the BACH-GPCR by ATP. The activity detected is compared to that in a standard sample taken from a healthy individual or from an unaffected area of the tested individual.

A differential of 5% or more in the activity measured, relative to the activity of the standard is diagnostic for a BACH-associated disease, disorder or condition.

When ATP or a derivative thereof and/or a modulator of the ability of ATP or derivatives thereof to bind BACH-GPCR is administered to an animal for the treatment of a BACH-GPCR associated disease, the amount administered can be adjusted by one of skill in the art on the basis of the desired outcome. Successful treatment is achieved when one or more measurable aspects of the pathology (e.g. tumour cell growth, accumulation of inflammatory cells) is change by at least 10% relative to the value for that aspect prior to treatment.

Candidate modulators

Candidate modulator compounds or agents can be screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries can be obtained from Maybridge Chemicals Co (Trevillet, Cornwall, UK) and Comgenex (Princeton, NJ, USA). A rare chemical library is available from Aldrcih (Milwaukee, USA). Combinatorial libraries are available and can be prepared.

Alternatively, libraries of natural compound in the form of bacterial, plant, fungal, and animal extracts are available from e.g. Pan Laboratories (Bothell, USA) or are capable of being produced by methods which are well known in the art. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical processes.

Useful compounds or agents.

By way of non-limiting examples useful modulators of the ability of ATP to bind to and activate the BACH-GPCR includes but is not limited to organic compounds, or small organic compounds. Small organic compounds have a molecular mass of more than 50 and less than 3000 daltons, or less than about 800, or less than about 300 daltons.

Examples include heterocycles, peptides, saccharides, steroids and derivatives thereof. The compound and/or ATP or derivatives thereof may be modified to enhance their specificity, efficacy, stability or pharmaceutical compatibility. Indeed a modified ATP or derivatives thereof or a modulator which is/are capable of increasing the specificity, efficacy and selectivity of affecting the BACH-GPCR activity may be used in the manufacture of a medicament for the treatment of BACH-GPCR associated diseases. For example where peptide agents are identified they may be modified in a variety of ways to enhance their stability such as using a non-natural amino group, such as a:

alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p- Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-amino caproic acid , 7-amino heptanoic acid*, L-methionine sulfone^#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline^#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)^#, L- Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (l,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid and L-Phe (4-benzyl)*. The notation * has been utilised for the purpose of the discussion above (relating to homologous or non- homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.

High throughput screening kit.

A high throughput screening kit as described here comprises all the necessary means and media for performing the detection of a modulator compound or agent including for example a agonist, antagonist of the ability of ATP or a derivative thereof to bind to and activate the BACH-GPCR for instance at a concentration in the range of InM to lOμM.

The kit may be used as follows:

Recombinant cells expressing the BACH-GPCR, are grown on a solid support, such as a microtiter plate, e.g. a 96 well plate, according to methods well known in the art such as described in WO 02/38607. Modulator compounds identified according to the screening methods described here, at concentrations between of InM to lOμM or more, are added to the culture medium of defined wells in the presence of an appropriate concentration of ATP or a derivative thereof for example in the range of InM to lOμM. Results are compared to the standard level of BACH-GPCR activity obtained from recombinant cells in the presence of ATP or a derivative thereof but in the absence of added modulator compound. Wells showing at least 2 fold, or 5 fold, or 10 fold, or 50 fold, or 75 fold or 100 fold or more increase or decrease in BACH- GPCR activity as compared to the level of activity in the absence of modulator are selected for further analysis.

Use of ATP or derivatives thereof and/or modulators.

As stated above ATP or derivatives thereof, and/or modulators such as compounds or agents identified by the methods described in this document can be useful in the treatment and/or diagnosis of BACH GPCR associated diseases. BACH-GPCR associated diseases have been described in WO 02/38607. Accordingly, in one embodiment of the invention, ATP or a derivative thereof may be useful to diagnose or treat, by any means as described in this document, a disease selected from a group consisting of trigeminal neuralgia, orofacial pain, pain associated with toothache, pain from visceral organs, inflammatory bowel disease related pain, irritable bowel syndrome, chronic pain from renal cholelithiasis, bladder instability, Barrett's oesophagus, glaucoma, pain associated with cancer, diabetic neuropathies, Herpes virus infections, pain associated with Herpes virus associated diseases or conditions, HIV infections, migraine and skin sensitivity associated with migraine, allodynia, toothache, neuroma caused by amputation, neuroma caused by nerve transaction, neuroma caused by trauma, nerve compression caused by tumour, nerve compression caused by entrapment, nerve compression caused by crush, and pain due to damage of spinal cord or brain.

In another embodiment, ATP or a derivative thereof may be useful to diagnose or treat, by any means as described in this document, a disease selected from the group consisting of dementia, dyslexia, dyskinesias, tremor, Parkinson's, benign essential tremor, chorea, epilepsy or ballismus, for example occurring through stroke, trauma, degeneration and malignancy.

In a further embodiment, ATP or a derivative thereof may b useful to diagnose or treat by any means as described in this document, a disease selected from chronic pain (peripheral or visceral) and relapsing remitting pain such as migraine.

In yet another embodiment, ATP or a derivative thereof may be useful to diagnose or treat, by any means as described in this document, a disease selected from a group consisting of dry eye disorders, cyctic fibrosis, hyperactive bladder, hypercholesterolaemia, dislipdaemias and obesity.

As noted above, ATP or a derivative thereof and/or modulators identified by the methods of the present invention may be useful to diagnose and/or treat any of these specific diseases ("BACH associated diseases") using any of the methods and compositions described here. In particular, we specifically envisage the use of ATP or derivatives thereof and/or compounds or agents for the treatment or diagnosis of the specific diseases listed above.

We have previously reported that BACH-GPCR may also play a role in urogenital functions (see WO 02/38607). Loss of the BACH-GPCR can result in abnormal bladder motility and mutants of the protein have hypoactive bladder urinating less frequently but releasing a larger volume and suffer urinary retention. BACH-GPCR also plays a role in erectile dysfunction and the control of motor fibers in the prostate.

Accordingly, in a further embodiment, ATP or a derivative thereof and/or agents or compounds identified by the screening methods described in this document may be used in the treatment and management of urogenital conditions, erectile dysfunction and prostate malfunction.

Pain and Sensitivity

ATP or a derivative thereof and/or the agents or compounds identified by the serening methods described in this document may be used in the treatment and management of neuropathic, inflammatory and visceral pain. These analgesic type therapeutics may among other conditions be used to treat group consisting of trigeminal neuralgia, orofacial pain, pain associated with toothache, pain from visceral organs, inflammatory bowel disease related pain, irritable bowel syndrome, chronic pain from renal cholelithiasis, bladder instability, Barrett's oesophagus, glaucoma, pain associated with cancer, diabetic neuropathies, Herpes virus infections, pain associated with Herpes virus associated diseases or conditions, HIV infections, migraine and skin sensitivity associated with migraine, allodynia, toothache, neuroma caused by amputation, neuroma caused by nerve transaction, neuroma caused by trauma, nerve compression caused by tumour, nerve compression caused by entrapment, nerve compression caused by crush, and pain due to damage of spinal cord or brain.

Orofacial pain is a consequence of trigeminal neuralgia, in which paroxysmal pain radiates over one, or two divisions of the trigeminal nerve. The opthalmic division is rarely affected. Drug treatment is usually effective but if it fails surgical treatment is used. None of these surgical treatments has proved satisfactory. No specific drug has been developed yet.

Skin sensitivity appears among the majority of migraine sufferers. Burstein et al, published a study showing that 79 percent of 44 migraine patients had extreme skin sensitivity. Burstein describes the extreme effects of migraine sufferers are unable to undertake day to day tasks such as brushing hair, wearrings or eyeglasses, or shaving beards because of the extreme pain.

In migraine series of neuronal clusters —in the sensory ganglions, the brainstem and the thalamus —become sensitised in a kind of domino effect. If the sensitised cluster, a group of nerve cells that acts like the hub of a computer network, happens to be connected to the skin, the result can be skin sensitivity. The problem starts with the release of inflammatory substances from the dura, and from blood vessels and nerve endings in the brain. This oversensitizes the trigeminal ganglion. When oversensitized, the ganglion interprets normal pressure inside the skull as the throbbing pain of migraine. Because the trigeminal ganglion seems to cause the primary pain of migraine, it is the target of current migraine drugs, which block serotonin receptors in sensory neurons connected to the dura. The drugs are often effective, but only if taken immediately after the headache begins.

The oversensitised trigeminal ganglion may, in turn, send signals to the nucleus caudalis, at the top of the spinal cord. Unlike the trigeminal ganglion, this group of nerves is connected to the skin, particularly near the eye, where the most dramatic skin sensitivity is found in migraine sufferers. In rats, once the trigeminal ganglion has activated the nucleus caudalis for an hour, the nucleus caudalis remains overwrought even if the trigeminal ganglion is calmed by drugs ~ as existing migraine treatments often do. The experiment also indicates that hyper-sensitive neurons in the nucleus caudalis interpret soft touches on the skin as pain. Although current migraine drugs often work if taken quickly after the headache's onset, that is impossible for people who don't have drugs handy or get the headaches while asleep. According to Burnstein, this could explain why current anti-migraine therapies, which work on the primary cluster, are only effective if taken during the firsts hour after an attack has begun. An important target therefore comprises secondary neurons.

The Burnstein study therefore shows that skin sensitivity has a clear origin in the hypervigilance of oversensitized nerve cells. (Burstein, R. et al, 2000, Brain, 123:1703-1709; Burstein et al, 2000, Ann Neurol 47(5):614-624).

Motion Related Disorders and Dementia

Furthermore, ATP or a derivative thereof and/or the agents or compounds identified by the methods described here may be used in the treatment and management of dementia related disorders. Such disorders include dementia, dyslexia, dyskinesias, tremor, Parkinson's, benign essential tremor, chorea, epilepsy and ballismus, for example occurring through stroke, trauma, degeneration or malignancy.

Other diseases may include balance, tremor, epilepsy all of which can be caused by defects in genes expressed the cerebellum.

Dyslexia has been shown to have abnormal cerebellar processing (Nicolson et al,

1999, Lancet 353(9165): 1662-7) that causes (among other factors) dyslexic patients to have a cerebellar deficit that adversely affects learning of new skills and the performance of autonomic, overleamed skills. In other areas of the brain purines have been shown to increase dopamine levels and thereby enhance reward behaviour. Purinoceptors are generally excitatory but have been shown to inhibit the release of the main excitatory neurotransmitter in the CNS, glutamate (Mendoza-Fernandez et al,

2000, J Pharmacol Exp Ther. 293(1): 172-9).

Secretion Related Disorders

Therapeutic agents developed to BACH may be used in the treatment and management of dry-eye disorders, cystic fibrosis, hyperactive bladder, hypercholesterolaemia, dislipdaemias and obesity. Dosages/Formulations

The dosage of ATP or a derivative thereof and/or the agents or compounds identified by the methods of the present invention will depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration.

As stated above, the term "ATP or a derivative thereof and/or the agents or compounds identified by the screening methods described here" when used in the context of a therapeutic or a prophylactics application will be referred to as the therapeutically active ingredient of the present invention.

Accordingly, depending upon the half-life of the pharmaceutically active ingredient of the present invention in the particular animal or human, the ingredient can be administered between several times per day to once a week. It is to be understood that the use can be both human and veterinary applications. The uses described in this document also include single as well as multiple administrations, given either simultaneously or over an extended period of time.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. The pharmaceutically active ingredient of the present invention is effective in treating BACH-GPCR associated disease. The present invention includes a method of treating a BACH-GPCR associated disease with an effective amount of pharmaceutically active ingredient of the present invention. The pharmaceutically active ingredient of the present invention can be provided as isolated and substantially purified ingredients in pharmaceutically acceptable compositions using formulation methods known to those of ordinary skill in the art. These compositions can be administered by standard routes. These include but are not limited to: oral, rectal, ophthalmic (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural) transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) routes.

The formulations comprising the pharmaceutically active ingredient may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

In addition, the pharmaceutically active ingredient may be incorporated into biodegradable polymers allowing for sustained release of the ingredient, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the active ingredient as described herein can be slowly released systemically. The biodegradable polymers and their use are described, for example, in detail in Brem et al, 1991 J Neurosurg 74: 441-446. Osmotic minipumps may also be used to provide controlled delivery of high concentrations of the polypeptide of the present invention through cannulae to the site of interest, such as directly into a solid tumour growth or into tissue of an ischemic diesease. In one embodiment, the pharmaceutically active ingredient may be linked to cytotoxic agents. These are then infused in a manner designed to maximize delivery to the desired location. For example, ricin-linked high affinity pharmaceutically active ingredient of the present invention is delivered through a cannula into vessels supplying the target site or directly into the target. Such agents are also delivered in a controlled manner through osmotic pumps coupled to infusion cannulae.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question.

Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.

The pharmaceutically active ingredient may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine.

It is within the scope of the present invention that the pharmaceutically active ingredient may be administered while the patient is undergoing other forms of treatment. Accordingly, it is contemplated that the pharmaceutically active ingredient of the present invention may be used in conjunction with another pharmaceutically beneficial entity. The other entity need not be administered by the same route. That other entity may be a drug such as steroids, corticosteroids, antibiotics, antiviral therapy, immunosuppresants and anti-inflammatories.

Route of administration.

The pharmaceutical compositions may be adapted for administration by any appropriate route. For example, it may be administered by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes. Such a composition may be prepared by any method known in the art of pharmacy, for example by admixing one or more active ingredients with a suitable carrier.

Different drug delivery systems can be used to administer the pharmaceutical compositions, depending upon the desired route of admirtistration. Drug delivery systems are described, for example, by Langer (Science 249:1527 - 1533 (1991)) and by Ilium and Davis (Current Opinions in Biotechnology 2 254 - 259 (1991)).

Different routes of administration for drug delivery will now be considered in greater detail.

The term "administered" includes delivery by viral or non-viral techniques. Viral delivery mechanisms include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectos, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors. Non-viral delivery mechanisms include lipid mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFAs) and combinations thereof.

The therapeutically active ingredient of the present invention may be administered alone but will generally be administered as a pharmaceutical composition - e.g. when the ingredient is in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the ingredient can be administered (e.g. orally or topically) in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The routes for administration (delivery) include, but are not limited to, one or more of: oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, via the pensis, vaginal, epidural, sublingual. It is to be understood that not all of the ingredients need be administered by the same route. Likewise, if the composition comprises more than one active component, then those components may be administered by different routes.

If the therapeutically active ingredient of the present invention is administered parenterally, then examples of such administration include one or more of: intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques.

Oral administration

Pharmaceutical compositions adapted for oral administration may be provided as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids); as edible foams or whips; or as emulsions. Tablets or hard gelatine capsules may comprise lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Soft gelatine capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. Solutions and syrups may comprise water, polyols and sugars. For the preparation of suspensions oils (e.g. vegetable oils) may be used to provide oil-in- water or water-in-oil suspensions. An active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract (e.g. glyceryl monostearate or glyceryl distearate may be used). Thus the sustained release of an active ingredient may be achieved over many hours and, if necessary, the active agent can be protected from being degraded within the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of an active ingredient at a particular gastrointestinal location due to specific pH or enzymatic conditions.

Transdermal administration Pharmaceutical compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis. (Iontophoresis is described in Pharmaceutical Research, 3(6): 318 (1986).)

Topical administration

Pharmaceutical compositions adapted for topical administration may be provided as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eye or other external tissues a topical ointment or cream is preferably used. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. Here the active ingredient can be dissolved or suspended in a suitable carrier, e.g. in an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouthwashes.

Rectal administration

Pharmaceutical compositions adapted for rectal administration may be provided as suppositories or enemas.

Parenteral administration

If the therapeutically active ingredient is administered parenterally, then examples of such administration include one or more of: intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the agent; and/or by using infusion techniques. For parenteral administration, the ingredient is best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Transmuclular

"Transmucosal" refers to delivery of a drug into the blood stream such that the drag passes through the mucosal tissue and enters into the blood stream.

Transurethral or intraurethral

"Transurethral" or "intraurethral" refers to delivery of a drug into the urethra, such that the drug contacts and passes through the wall of the urethra and enters into the blood stream.

Carriers or vehicles

"Carriers" or "vehicles" refers to carrier materials suitable for compound administration and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, sofubilizer, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner.

Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume Blood brain barrier (BBB)

Within the scope of the present invention, pharmaceutical compositions may be designed to pass across the blood brain barrier (BBB). For example, a carrier such as a fatty acid, inositol or cholesterol may be selected that is able to penetrate the BBB. The carrier may be a substance that enters the brain through a specific transport system in brain endothelial cells, such as insulin-like growth factor I or II. The carrier may be coupled to the active agent or may contain/be in admixture with the active agent. Liposomes can be used to cross the BBB. WO 91/04014 describes a liposome delivery system in which an active agent can be encapsulated/embedded and in which molecules that are normally transported across the BBB (e.g. insulin or insulin-like growth factor I or II) are present on the liposome outer surface. Liposome delivery systems are also discussed in US Patent No. 4704355.

The composition may comprise a brain targeting moiety, such as an anti-insulin receptor antibody (Coloma et al, (2000) Pharm Res 17:266-74), anti-transferrin receptor antibodies (Zhang and Pardridge, (2001) Brain res 889:49-56) or activated T- cells (Westland et al., (1999) Brain 122:1283-91). Alternatively, techniques resulting in modification of the vasculature by the use of vasoactive peptides such as bradykinin or other techniques such as osmotic shock (reviewed in Begley, (1996) J Pharm Pharmacol 48:136-46; Neuwelt et al, (1987) Neurosurgery 20:885-95; Kroll et al, (1998) Neurosurgery 43:879-86; Temsamani et al, (2000) Pharm Sci Technol Today 3:155-162) may be employed.

References

1. M Bootman, E Niggli, M Berridge & P Lipp, 1997. Imaging the hierarchical Ca2+ signalling system in HeLa cells. J Physiol 499 307 2. H Cheng, MR Lederer, WJ Lederer & MB Cannell, 1996. Calcium sparks and [Ca2+]i waves in cardiac myocytes. AM J. Physiol Cell Physiol 270 C148

3. DE Clapham, 1995. Calcium Signalling Cell 80 259

4. Calcium Signalling Protocols (Methods in Molecular Biology, Volume 114) D Lambert, Ed., pp. 125-133, Humana Press 1999.

Each of the applications and patents mentioned in this document, and each document cited or referenced in each of the above applications and patents, including during the prosecution of each of the applications and patents ("application cited documents") and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the applications and patents and in any of the application cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text, are hereby incorporated herein by reference.

Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

Claims

1. Use of ATP or derivatives thereof, in the manufacture of a medicament for the treatment of a BACH-GPCR associated disease in an individual.

2. Use according to claim 1 wherein the disease or condition is caused by or associated with increased, decreased or otherwise abnormal expression or activity of BACH-GPCR.

3. Use according to claim 1 or 2, wherein the ATP or a derivative thereof is modified such that it is capable of specifically modulating the activity of BACH-GPCR.

4. Use according to any one of claims 1 to 3, wherein the individual is also undergoing treatment with at least one other compound selected from steroids, corticosteroids, antibiotics, antiviral therapy, immunosuppresants and anti- inflammatories.

5. A method of identifying a compound capable of modulating the interaction between BACH-GPCR and ATP, the method comprising the steps of: a) providing a BACH-GPCR polypeptide; b) providing an ATP molecule; and c) detecting binding between the BACH-GPCR polypeptide and the ATP molecule in the presence and absence of a candidate compound.

6. A method according to claim 5, wherein binding between the BACH-GPCR polypeptide and the ATP molecule is detected by fluorescent resonance energy transfer (FRET).

7. A method according to claim 6 comprising the steps of: a) providing a fluorescent marker labelled BACH-GPCR polypeptide comprising a donor or an acceptor group; b) providing a fluorescent marker labelled ATP molecule comprising a complementary acceptor or donor group; c) allowing binding of the labelled BACH-GPCR polypeptide and the labelled ATP molecule which permits the donor group to come into sufficient proximity to the acceptor group providing for fluorescent resonance energy transfer and/or quenching to take place; and d) measuring the fluorescence in the presence or absence of a candidate compound.

8. A method of identifying a compound capable of modulating the interaction between ATP and BACH-GPCR comprising the steps of: a) providing a BACH-GPCR polypeptide expressed on the surface of a cell; b) providing an ATP molecule; and c) detecting binding between the BACH-GPCR polypeptide and the

ATP molecule by measuring calcium mobilisation from intracellular stores in the presence and absence of a candidate compound.

9. A method according to claim 8 wherein the mobilisation of calcium is measured by a calcium sensitive dye.

10. A method according to claim 9 wherein the calcium sensitive dye is Fluo-3.

11. A method of identifying a compound capable of modulating the interaction between ATP and BACH-GPCR comprising the steps of: a) providing a cell comprising a reporter the expression of which is indicative of BACH-GPCR activation; b) providing an ATP molecule; and c) detecting binding between the BACH-GPCR and the ATP molecule by measuring reporter expression in the presence and absence of a candidate compound.

12. A method according to anyone of claims 5 to 11 wherein the ATP or the BACH- GPCR are exposed to a library comprising the candidate compound.

13. A method according to claim 12 wherein the members of the library are labelled with a fluorescent label, radioactive label or luminescent label.

14. A method according to anyone of claims 8 to 13 wherein said cells are central nervous system (CNS) cells, peripheral cells, cell lines derived from peripheral cells, transgenic cells, transgenic cells derived from transgenic animals, or human cells or cell lines.

15. A method according to claim 14, wherein the cells are selected from the group consisting of: Flp-In-CHO, HeLa, COS-7, Saos2, U2OS, DG75, NIH-3T3, HEK- 293, K-562, LM (TK-).

16. A method of identifying a compound that reduces, ameliorates, or modulates symptoms of a BACH-GPCR associated disease, comprising administering a compound that modulates the interaction between ATP and BACH-GPCR to an animal model and measuring or observing the reduction, amelioration, or modulation of said symptoms.

17. A compound identified by the method according to any one of claims 5 to 16.

18. A compound according to claim 16 wherein the compound is selected from the group consisting of natural or synthetic peptide, a polypeptide, an antibody or antigen-binding fragment thereof, a lipid, a carbohydrate, a nucleic acid, and a small organic molecule.

19. Use of a compound according to any one of claims 17 or 18 in the manufacture of a medicament for the treatment of disease or condition associated with BACH- GPCR.

20. Use of a compound according to anyone of claims 1, 17 or 18 wherein the disease or condition is selected from the group consisting of trigeminal neuralgia, orofacial pain, pain associated with toothache, pain from visceral organs, inflammatory bowel disease related pain, irritable bowel syndrome, chronic pain from renal cholelithiasis, bladder instability, Barrett's oesophagus, glaucoma, pain associated with cancer, diabetic neuropathies, Herpes virus infections, pain associated with Herpes virus associated diseases or conditions, HIV infections, migraine and skin sensitivity associated with migraine, allodynia, toothache, neuroma caused by amputation, neuroma caused by nerve transaction, neuroma caused by trauma, nerve compression caused by tumour, nerve compression caused by entrapment, nerve compression caused by crush, and pain due to damage of spinal cord or brain.

21. Use of a compound according to anyone of claims 1, 17 or 18 wherein the disease or condition is selected from the group consisting of dementia, dyslexia, dyskinesias, tremor, Parkinson's, benign essential tremor, chorea, epilepsy or ballismus, for example occurring through stroke, trauma, degeneration and malignancy.

22. Use of a compound according to anyone of claims 1, 17 or 18 wherein the disease or condition is selected from chronic pain (peripheral or visceral) and relapsing remitting pain such as migraine.

23. Use of a compound according to anyone of claims 1, 17 or 18 wherein the disease or condition is selected from the group consisting of dry eye disorder, cyctic fibrosis, hyperactive bladder, hypercholesterolaemia, dislipdaemias and obesity.

24. Use according to any one of claims 17 to 23 wherein the individual is also undergoing treatment with at least one other compound selected from ATP or a derivative thereof, steroids, corticosteroids, antibiotics, antiviral therapy, immunosuppresants and anti-inflammatories.

25. A method of treating a patient suffering from a disease or condition associated with decreased activity of BACH-GPCR wherein the method comprises administering to the patient ATP or a functional derivative thereof according to any one of claims l to 4.

26. A method of treating a patient suffering from a disease or condition associated with decreased activity of BACH-GPCR wherein the method comprises administering to the patient a general modulator which increases or agonises the capacity of ATP or derivatives thereof to activate the BACH-GPCR.

27. A method according to claim 26, wherein the modulator which increases or agonises the capacity of ATP or derivatives thereof to activate the BACH-GPCR is identified by the method of any one of claims 5 to 16.

28. A method of treating a patient suffering from a disease or condition associated with increased activity of BACH-GPCR, wherein the method comprises administering to the patient a general modulator which decreases or antagonises the capacity of ATP or derivatives thereof to activate the BACH-GPCR.

29. A method according to claim 28, wherein the modulator which decreases or antagonises the capacity of ATP or derivatives thereof to activate the BACH- GPCR is identified by the methods of any one of claims 5 to 16.

30. A pharmaceutical composition comprising ATP or derivatives thereof according to any one of claims 1-4 and/or a modulator according to any one of claims 26 or 28 and/or a compound identified by the method of anyone of claims 5 to 16 together with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

31. A method of diagnosing a BACH-GPCR associated disease the method comprising the steps of: a) providing a BACH-GPCR polypeptide expressed from a nucleic acid sequence obtained form a patient suspected of suffering from a disease associated with BACH-GPCR; b) providing an ATP molecule; c) detecting binding between the BACH-GPCR polypeptide and the ATP molecule; and d) comparing the binding of c) with the binding of ATP and BACH-GPCR polypeptide expressed from a nucleic acid sequence obtained from a normal patient, wherein a difference in binding relative to the normal is diagnostic of a disease associated with BACH-GPCR.

32. A method of diagnosing a BACH-GPCR associated disease the method comprising the steps of: a) providing a BACH-GPCR polypeptide; b) providing ATP from a sample obtained from a patient suffering from or suspected of suffering from a BACH-GPCR associated disease; c) detecting binding between BACH-GPCR polypeptide and the ATP; and d) comparing the binding of c) with the binding of BACH-GPCR polypeptide to ATP obtained from a normal patient wherein a difference in binding relative to the normal is diagnostic of a disease associated with BACH- GPCR.

33. A method of diagnosing a BACH-GPCR associated disease the method comprising the steps of: a) isolating a cell from a patient suspected to be suffering from such a disease or condition and incubating the cell with ATP and determining the binding affinity between the ATP and BACH-GPCR. b) Comparing the binding affinity of a) with that of ATP and cells obtained from a normal patient, wherein a difference in binding relative to the normal is diagnostic of a disease associated with BACH-GPCR.