WO2004005345A1

WO2004005345A1 - Inhibitors of the ephrin/ephb receptor interaction

Info

Publication number: WO2004005345A1
Application number: PCT/GB2003/002859
Authority: WO
Inventors: Stephen Mcmahon; Isabella Gavazzi
Original assignee: King's College London
Priority date: 2002-07-03
Filing date: 2003-07-03
Publication date: 2004-01-15
Also published as: AU2003251149A1; GB0215389D0

Abstract

The invention relates to the use of compounds which disrupt the interaction between EphB receptors and ephrinB ligands in the preparation of analgesic medicaments, and to assays for identifying such compounds.

Description

INHIBITORS OF THE EPHRIN/EPHB RECEPTOR INTERACTION

The present invention relates to compounds for use as analgesics and to methods for the identification thereof.

Analgesics are one of the most commonly used types of medicines, ranging from the occasional use of a mild painkiller, to constant use of a high strength analgesic to combat more serious pain. Chronic pain represents a large economic drain on resources, in the United States of America alone more than two million people are incapacitated by pain at any given time. A range of new analgesics would be beneficial because providing a wider spectrum of treatments is likely to reduce side effects and resistance. Some individuals become resistant after long term use of some analgesics, requiring increasing doses to achieve same level of pain relief.

The Ephrin (Eph) family of receptors, and their cognate ligands has been described by Renping Zhou (The Eph Family Receptors and Ligands, Pharmacol. Ther., Vol 77, No. 3, pp 151-181, 1998). Eph receptors are a family of receptor tyrosine kinases, and with their ephrin ligands are known to be involved in crucial aspects of nervous circuits assembly in development. Eph receptors are divided into 2 subclasses, EphA and EphB. The inventors have concentrated research on EphB receptors, which bind transmembrane ephrinB ligands. Six different EphB receptors and three ephrinB ligands have been described. Both receptors and ligands are expressed, particularly during embryonic development, on a large number of cell types. When the EphB receptor on one cell binds to the ligand on another cell, intracellular signalling pathways can be activated in both cells. All EphB receptors bind to all ephrinB ligands, albeit with different affinity. Recently the EphB subclass of receptors have also been implicated in synaptogenesis and in modification of synaptic currents in hippocampal cultures and slices. The inventors have found that EphB-ephrinB interactions modulate synaptic function and pain proceeding in the dorsal horn of the spinal cord. The inventors have found that EphB receptors are present in neurons in the superficial laminae of the dorsal horn of the spinal cord. These neurons receive inputs from nociceptive afferents in the dorsal root ganglia (DRGs), which constitutively express ephrinB ligands. The synapses (sites of contact) between sensory afferents and dorsal horn neurons are the first relay station along the pain pathway from peripheral tissue to the brain, and one of the commonly used targets for the development and delivery of analgesic drugs. The principal excitatory neurotransmitter released at the synapses is glutaniate; glutamate-mediated excitation of interneurons in the dorsal horn is a fundamental step in the transmission of painful stimuli.

There are no prior publications related to Eph receptors and ephrin in the context of pain processing. There is abundant literature on the biology of these molecules, in relation to their role in development of the nervous system, angiogenesis and tumours, which is not relevant, however, to the invention. There are two publications which suggest a role of EphB2 in modulating synaptic plasticity in a different system (hippocampus) (Grunwald et al, 2001, Neuron 32: 1027-1040, and Henderson et al., 2001, Neuron 32: 1041-1056).

As indicated above, there is a need for new analgesics.

The inventors have identified a new group of compounds for use as analgesics, and have produced a method for identifying such compounds.

The invention provides the use of a compound which disrupts the EphB receptor and ephrinB ligand interaction in the preparation of a medicament for the treatment of pain.

A compound which disrupts the interaction between EphB receptors and ephrinB ligands can be used, according to the invention to produce medicaments to treat pain. The compound may disrupt the interaction by affecting either the receptor or the ligand. Such compounds will provide alternative analgesic medicines to those already available, which will reduce the problems of side effects and resistance. The compound may be an antagonist of the ligand and may bind to, modify or cleave the ligand such that it cannot bind to the receptor, or so that even if the ligand binds to the receptor the receptor is not activated to release its neurotransmitter.

Alternatively the compound may be an antagonist of the receptor. An EphB receptor antagonist is a compound which disrupts the interaction of EphB with its ligands. An antagonist may act competitively, competing with the ligand for the EphB binding site, without causing receptor activation and release of the neurotransmitter. Alternatively the antagonist may be non-competitive, binding to some part of the receptor so as to alter the ligand binding site preventing ligand binding.

Equally the compound may have an effect on the expression, turnover or grouping of EphB receptors or ephrinB ligands, disrupting the interaction between the receptor and ligand, by reducing the overall number of receptors or ligands or by affecting their arrangement on the neurons .

Preferably the compound blocks the interaction between the EphB receptor and its ligand.

The candidate compound may be a protein, including a polypeptide or peptide (i.e. an enzyme, antibody molecule or receptor protein), a carbohydrate or organic or inorganic compounds. Preferred compounds are those that either bind to the EphB receptor at the site of its interaction with the ephrinB ligand, so precluding the two from interacting, or that bind to the ephrinB ligand at the site at which it binds the EphB receptor, again precluding the interaction of the two.

Preferred protein include enzymes that disrupt the function or binding affinity of the ephrinB ligand. A protein may be an antibody molecule or other molecule that exhibits affinity for, and binds to the ephrinB ligand. The term "antibody molecule" refers to polyclonal antibodies, monoclonal antibodies or antigen binding fragments thereof, such as Fv, Fab, F(ab')₂ fragments and single chain Fv fragments. The antibody molecule may be a recombinant antibody molecule, such as a chimeric antibody molecule preferably having human constant regions and mouse variable regions, a humanised CDR-grafted antibody molecule or a fragment thereof. Methods for producing such antibodies are well known to those skilled in the art and are described in published European patent applications EP-A-0120694 and EP-A-0125023.

A carbohydrate compound can be any carbohydrate that disrupts the interaction between the ephrinB ligand and the EphB receptor. Preferably the carbohydrate binds to the ephrinB ligand.

Organic and inorganic compounds may be naturally-occurring or chemically synthesised compounds that disrupt the interaction between the ephrinB ligand and the EphB receptor. There are numerous commercially available libraries of chemical compounds that can be assayed for their activity. Preferably the organic and inorganic chemical inhibitor are small molecules having a molecular weight of less than 1000. Libraries of small molecules are commercially available and can be analysed for suitable compounds using the method of the present invention.

The action of the compound is preferably reversible.

Preferably the compound is selected from the group consisting of antibody molecules to either the EphB receptor or the ephrinB ligand, EphBl-Fc chimeras, and fragments of the EphB receptor or ephrinB ligands.

The medicament is preferably suitable for treating all types of pain, including those caused by hyperalgesia and allodynia. Hyperalgesia is an increased sensation to pain, usually following tissue damage. Allodynia is the sensation of pain in response to normally non-painful stimuli. Preferably the pain is related to tissue injury. Alternatively it may be related to nerve injury. The invention encompasses the use of all known compounds and all compounds which become known that disrupt the EphB receptor and ephrinB ligand interaction.

The invention further provides a method of treating pain comprising the step of administering to a subject an effective amount of a compound which disrupts the EphB receptor and ephrinB ligand interaction.

Also provided is a method of identifying compounds useful for the treatment of pain comprising the step of measuring the ability of a test compound to disrupt the EphB receptor and ephrinB ligand interaction. Preferably that step further comprises the step of determining the binding of the ligand to an EphB receptor, before and after the addition of a test compound.

There are numerous methods of determining if a candidate compound binds at a site on the ephrinB ligand or EphB receptor that prevents binding between the ephrinB ligand and the EphB receptor. In particular, competition assays can be used to determine if a candidate inhibitor prevents binding of an ephrinB ligand to EphB receptors.

By identifying the sites of interaction between the ephrinB ligand and EphB receptor, it can easily be determined whether a candidate compound binds to the site of interaction on the ephrinB ligand or the site of interaction on the EphB receptor and thereby prevents binding between the ephrinB ligand and EphB receptor. Methods for identifying sites of interaction are well known to those skilled in the art and include X-ray crystallography and nuclear magnetic resonance.

In order to identify candidate compounds of the present invention, both the ephrinB ligand and/or EphB receptors can be used to screen libraries of compounds in any of a variety of drug screening techniques. Candidate compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries, or natural product mixtures. These candidate compounds may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al, Current Protocols in Immunology l(2):Chapter 5 (1991).

The EphB receptor or ephrinB ligand that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The adherence of a candidate compound a surface bearing ephrinB ligand or the EphB receptor can be detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labelled competitor. In general, such screening procedures may involve using appropriate cells or cell membranes that express the appropriate protein, that are then contacted with a candidate inhibitor to observe binding, or inhibition of a functional response. The functional response of the cells contacted with the candidate compound is then compared with control cells that were not contacted with the candidate compound. Such compounds will generally be assayed in the presence of a known agonist and the biological effect of by the agonist in the presence of the test compound observed.

In another embodiment, competitive drug screening assays may be used, in which neutralising antibodies or known inhibitors that are capable of binding ephrinB ligand or EphB receptor specifically compete with a candidate inhibitor for binding. In this manner, the antibodies or known inhibitors can be used to detect the presence of any candidate inhibitor that possesses specific binding affinity for ephrinB ligand or EphB receptor.

Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (for example, see International patent application WO84/03564). In this method, large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with ephrinB ligand or EphB receptor and washed. One way of immobilising the target is to use non-neutralising antibodies. Bound target may then be detected using methods that are well known in the art, including using biophysical techniques such as surface plasmon resonance and spectroscopy.

In a preferred embodiment, a competition assay is performed wherein ephrinB ligand is attached to a solid support. Any suitable solid support may be used, such as, for example, sepharose beads.

For example, when ephrinB ligand is attached to a solid support the detection of EphB binding to the ephrinB ligand on the solid support can easily be achieved using a labelled antibody specific for EphB or by using labelled EphB. EphB can be labelled with any suitable label. Suitable labels include: enzymes, such as horseradish peroxidase (HRP) and chloramphenicol acetyl transferase (CAT); digoxygenin (DIG); fluorescein; and radioisotopes such as ^{I 5}I, ³H and ¹⁴C.

In a particularly preferred method a fluorescent label is used, allowing visualisation of the binding of the EphB receptor and ephrinB ligand.

Depending on the label used, the amount of labelled antigen immobilised may be determined using standard methods known to one of skill in the art. For example, if the label is HRP, the degradation of luminol by the enzyme and the associated emission of chemiluminescence can be measured. However, if a radioactive label is used, the presence of the label is measured by detecting the emitted radiation.

Once a compound has been identified using the method of the present invention, it will be desirable to test the compound on a cell expressing EphB in order to determine if the compound has an effect on the binding of ephrinB ligand to EphB in the cell. The invention also provides a kit for identifying compounds useful for the treatment of pain comprising an EphB receptor and an ephrinB ligand and elements for determining the interaction between them.

The invention will now be described by way of example, with reference to the figures, in which:

Figure 1 shows the latencies of rat paw withdrawal from a heat source, (a) shows measurements from control rats, and rats treated intrathecally with CSF, ephrinB2 or EphBl, or MK801 followed after 5 min by CSF or ephrinB2. EphrinB2 caused significant reductions in reaction times (i.e. thermal hyperalgesia) as compared to controls at all times, and (b) shows reductions in latency of response as compared to baseline in rats treated i.t. with CSF or EphBl (5 or 10 microgram/rat) 10 min prior to a carrageenin injection in the tested paw. EphBl at 10 microgram significantly reduced the inflammation-evoked hyperalgesia at 2 h after inj ection. [Bars represent s.e.m.]; and

Figure 2 shows a generic FRET assay format for protein - protein interaction as applied in a high resolution screening platform 1. Injection sample 2. Addition of first fluorescent-labelled protein, 3. Addition of second fluorescent-labelled protein. Affinity complex formation is monitored at 650 nm.

Examples.

It was known from published developmental studies that EphB receptors can interact with one type of glutamate receptors, the NMDA receptors, in the hippocampus. It has also been previously demonstrated that NMDA receptors in the dorsal horn of the spinal cord are involved in the induction and maintenance of phenomena of hypersensitivity, which follow peripheral tissue injury and inflammation. From our observations on the pattern of expression of EphB receptors and ephrinB, together with the knowledge of possible interactions between EphB and NMDA receptors, the inventors have discovered that EphB-ephrinB interactions modulate synaptic function (and therefore pain processing) in the dorsal horn.

The inventors used a well established preclinical animal model for the study of pain states, first described by Hargreaves in 1988 (Hargreaves,K., Dubner,R., Brown,F., Flores,C. & Joris,J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain., 32, 77-88, 1988). The inventors implanted catheters in the subarachnoid space in the lumbar region of adult rats for the delivery of drugs to the spinal cord (equivalent to intrathecal or epidural delivery in humans). Following delivery of drugs or control vehicle solution the inventors examined the response of the rats to radiant heat stimuli by placing a radiant heat source (a bulb) under the rat hindpaw in a plantar test apparatus. Lifting of the paw switched off the bulb and stopped a timer. A significantly reduced latency of response as compared to baseline value is considered a sign of hyperalgesia.

The inventors found that a single intrathecal administration of ephrinB2-Fc chimeric protein, which can activate EphB receptors, induces long-lasting (24 h) behavioural thermal hyperalgesia in adult rats (measured as a reduction in latency of paw withdrawal in a plantar test) within 15 minutes.

Post-synaptically, the inventors demonstrate that intrathecal ephrins lead to activation of the signalling kinase Src and recruitment of NMDA receptors.

The hyperalgesic effect following ephrinB2 treatment in vivo was blocked by co-administration of MK-801, a non-competitive NMDA receptor antagonist, confirming that activated EphB receptors affect responses to thermal stimuli via NMDA receptors activation.

The inventors have in vitro evidence demonstrating that peripheral stimuli, such as inflammation, can cause a redistribution of ephrinB 2 expressed in sensory neurons in vivo, and thus lead to hyperalgesia. Using dorsal root ganglion neurons in culture, the inventors were able to provide immunocytochemical evidence that the nociceptor activation can lead to a redistribution of ephrinB2 receptors. The presynaptic clustering of ephrins is reported to result in post-synaptic Eph activation. The inventors studied the actions of chemical exciters of nociceptors on dissociated adult DRG neurons grown in culture for 2 days. These cells extend neuritic processes and retain many of the features of sensory neurons in vivo. The inventors applied to cultures vehicle or 100 μM of ATP (a ligand for P2X receptors), which selectively activates about 50% of nociceptive sensory neurons. After 5 minutes of stimulation, cultures the were fixed and double stained with anti-ephrinB2 and anti-P2X3 antibodies. Cells which were P2X3-immunopositive (and therefore ATP -responsive) showed higher levels of ephrinB2 immunoreactivity in image analysis (143 +/- 11% vs control; p < 0.05, unpaired t-test ). The number of discrete peaks of ephrinB2 immunofluorescence/unit length was slightly but significantly higher in ATP-treated neurons (119 +/- 3.5 % vs. control, p< 0.01, unpaired t-test), indicating the presence of a higher number of ephrinB2 clusters. These changes in staining intensity and pattern clearly indicate that peripheral stimulation of sensory neurons has the potential to cause redistribution of ephrinB2 in the cell membrane.

The inventors therefore have shown that ephrinB-EphB interactions are important modulators of pain processing at spinal level. This knowledge can be exploited for the development of analgesic drugs for the treatment of chronic pain.

Testing potential analgesic compounds

The inventors have tested our ability to induce analgesic or antihyperalgesic effects by manipulating EphB-ephrinB interactions. The inventors employed a well-characterised model of chronic pain (carrageenin inflammation), which causes persistent hyperalgesia and allodynia within 1-2 hours.

Disruption of ephrinB-EphB interactions by intrathecal injection of EphBl-Fc chimeras prevented the onset of thermal hyperalgesia normally induced by carrageenin-evoked inflammation, thus proving a) that disruption of ephB-ephrinB interaction can prevent hyperalgesia and b) this model can be used to test further compounds (e.g. shorter fragments or peptide sequences of EphB receptors) which interfere with ephrinB-EphB interactions for their ability to act as anti-hyperalgesics in this test.

The inventors can test compounds for their ability to disrupt EphB-ephrinB interactions, prior to their use in vivo, by using an in vitro test. Several adult DRG neurons express EphBl and EphB2 receptors in culture. These receptors bind ephrinB-Fc added to the culture medium, and the binding is visualised by secondary antibodies conjugated with fluorescent markers. Addition of compounds that prevent EphB-ephrinB interactions leads to lack of cellular labelling.

The data have led the inventors to conclude that antagonists of EphB receptors or molecules that disrupt EphrinB - EphB interactions (such as soluble EphB receptors) will act as analgesics. This is a completely novel approach to the development of analgesics that work independently or work by potentiating other analgesic drug effects. The novel mode of action would avoid the side affects associated with other classes of analgesic drugs.

As indicated above, the present invention enables the discovery of new analgesic drugs for the treatment of pain in humans and animals. The novel analgesic drugs would prevent normal interactions between EphB receptors and ephrinB ligands. This might arise because the novel drugs were competitive or non-competitive antagonists of ephB receptors, or because they bound to, modified cleaved ephrin B ligands, or because they prevented the normal expression or turnover or grouping of ephrinBs or EphB receptors, or interfere with or modify EphB receptor or ephrinB ligand expression or function.

The following two practical examples show how candidate drugs can be screened for their ability to disrupt EphB receptor-ephrinB interactions, allowing their identification as novel analgesic drugs. EXAMPLE 1

The identification of potential novel analgesic compounds by testing their ability to disrupt EphB-ephrinB binding in an in vitro assay using cultured sensory neurons.

Principles of assay:

A. Adult primary sensory neurons located in dorsal root ganglia are readily maintained in primary cell cultures. When this is done, some of the neurons express EphBl and EphB2 receptors.

B. Synthetic chimeric molecular conjugates of ephrinB and the Fc portion of human IgG are commercially available. When these chimeric molecules are added to cultures of primary sensory neurons, the ephrinB-Fc molecules bind to the EphB receptors expressed by the cultured neurons.

C. This binding is visualised by adding commercially available secondary antibodies which recognise the Fc portion of the chimeric ephrinB-Fc. The secondary antibodies are themselves conjugated to fluorescent markers such as Cy-3. This fluorescence is observed with standard fluorescence microscopy, and can be readily measured by image analysis.

D. If duplicate culture wells are treated as above, but with the inclusion of novel compounds that interfere with EphB-ephrinB interactions, less secondary antibody will bind to the neurons. After washing, there will be less fluorescence signal in these cultures, which is detected by image analysis.

E. The ability of ephrin-FC chimeric molecules to bind to cells expressing Eph receptors has been extensively documented, as has the ability to use secondary antibodies to visualise ephrin-Fc bound to Eph receptors on cultured cells (see e.g. Krull et al, Current Biology 7, pp. 571-580, 1997; Flenniken et al., Developmental Biology 179, pp. 382-401, 1996; Gale et al., Neuron 17, pp. 9-19, 1996)

Protocol:

1. Adult male Wistar rats are killed by decapitation. 2. Dorsal root ganglia (DRG) are dissected from these animals. The capsules of the ganglia are removed with fine forceps. Nerve roots are trimmed from the ganglia. 3. The ganglia are then chemically dissociated in 0.125% collagenase (Sigma, UK) in modified Bottenstein and Sato medium (Ham's F-12, Life Technologies, UK, with N2 supplement, 10 ml/1, Life Technologies, UK, and 0.3% BSA, Sigma, UK) for 2 hours at 37°C. 4. The ganglia are then mechanically dissociated by trituration with a PI 000 Gilson pipette (the tip of the pipette is cut approximately 0.5 mm from the end to enlarge the bore) in 1 ml modified Bottenstein and Sato's culture medium (as above).

5. The resulting cell suspension is centrifuged at 800 rpm for 5 minutes through a cushion of 15% bovine serum albumin (BSA) (Sigma); this procedure eliminates much of the cellular debris and results in a neuronally enriched pellet of dissociated neurons.

6. The dissociated neurons are resuspended in 100 μl of calcium and magnesium-free Hank's balanced salts solution (Life Technologies), containing 50 μg/ml DNase (Type I; Sigma) and 250μg/ml soybean trypsin inhibitor (Type II; Sigma) and diluted in culture medium (as above) to a final concentration of 2500-3000 cells/ml. The dissociated neurons are plated onto laminin-coated glass coverslips in 4- well plates (Nunc, UK).

7. Glass coverslips are precoated with poly-L-lysine (2 mg/ml; Sigma) for 2 hours at room temperature, washed 3 times in sterile distilled H2O, allowed to dry and stored in a sterile environment. When required, coverslips are incubated for at least 2 hours at 37°C with a 10 μg/ml solution of EHS laminin (Sigma) in PBS in 4-well plates. Wells are aspirated thoroughly before use.

8. Neuronal cultures are incubated for 42-44 h at 37°C in a humidified atmosphere containing 5% CO₂ prior to use

Steps 1 to 8 above are standard laboratory practice to culture these primary sensory neurons. The methods have been extensively reported and reviewed (Lindsay, J. Neurosci. 8: 2394-2405). There are many hundreds of research publications that have used these or very similar methods to culture primary sensory neurons.

Testing compounds for their ability to disrupt EphB-ephrinB interactions: 9. Primary cultures (prepared as in steps 1-8 above) are incubated with ephrinB2-Fc (R&D Systems) at 10 μg/ml in culture medium for 30 min, in the presence of a range of concentrations of the compound to be tested.

10. The coverslips are then washed 3 times in phosphate buffered saline (PBS, pH 7.4) and the cells treated with fixative (4% paraformaldehyde in PBS, ph 7.4) for 10-15 minutes at room temperature.

11. After PBS washing, the cultures are incubated at room temperature in the dark for 2 hours with Cy3 -conjugated donkey anti-human IgG (Jackson Immunochemicals) diluted 1:200 in PBS (pH 7.4) containing 0.1% sodium azide and 1% normal donkey serum.

12. Following 3 further washes in PBS (as for 10, above) the coverslips are mounted in Cytifluor (Cytifluor Ltd, UK).

To assess the degree of binding between ephrinB and EphB receptors:

13. Cultures that are immunostained (as in 9-12 above) are viewed using a standard fluorescent microscope equipped using 20x or 40x objectives, and filters appropriate for the fluorochrome of the secondary antibody.

14. Representative images of the immunostained cultures are photographed with a digital CCD camera fitted to the microscope. Images of all the cultures to be compared are taken under identical lighting conditions and using the same camera settings. The digital images are stored on a computer.

15. The stored images are subsequently analysed with image analysis software. The average brightness of each image is calculated. The control cultures (where ephrinB-Fc is added to the neurons without any putative blocking compounds) will have a certain degree of brightness. Cultures in which ephrinB-EphB receptor interactions have been blocked will have lower levels of brightness.

16. The average brightness of the images treated with different putative blocking compounds are compared by standard statistical tests such as Analysis of Variance analysis (ANOVA). A statistically significant reduction in average grey value in the presence of a given compound indicates significant inhibition of ephrinB2-Fc binding, and therefore analgesic potential. EXAMPLE 2

The identification of potential novel analgesic compounds by testing their ability to disrupt EphB-ephrinB binding in an in vitro assay using cultured mammalian cell lines which express EphB receptors.

Principles of assay:

A. COS or NIH3T3 cells are stably transfected with EphB receptors, as described in Torres et al. (Neuron vol 21, pp. 1453-1463, 1998) and Brambilla et ah, (Molecular and Cellular Neuroscience vol 8, pp 199-209, 1996). These cells can readily be maintained in cell cultures.

B. Synthetic chimeric molecular conjugates of ephrinB and the Fc portion of human IgG are commercially available. When these chimeric molecules are added to cultures of COS/NIH3T3 cells (transfected as in A, above) the ephrinB-Fc molecules bind to the EphB receptors expressed by the cultured cells.

C. This binding is visualised by adding commercially available secondary antibodies which recognise the Fc portion of the chimeric ephrinB-Fc. The secondaiy antibodies are themselves conjugated to fluorescent markers such as Cy-3. This fluorescence is observed with standard fluorescence microscopy, and can be readily measured by image analysis.

D. If duplicate culture wells are treated as above, but with the inclusion of novel compounds that interfere with EphB-ephrinB interactions, less secondary antibody will bind to the neurons. After washing, there will be less fluorescence signal in these cultures which is detected by image analysis. E. The ability of ephrin-Fc chimeric molecules to bind to cells expressing Eph receptors has been extensively documented, as has the ability to use secondary antibodies to visualise ephrin-Fc bound to Eph receptors on cultured cells (see e.g. Krull et al, Current Biology 7, pp. 571-580, 1997; Flenniken et al, Developmental Biology 179, pp. 382-401, 1996; Gale et al., Neuron 17, pp. 9-19, 1996)

Protocol: 1. COS/NIH3T3 cells transfected with EphB receptors are grown on glass coverslips in 4-well plates (Nunc, UK) in DMEM supplemented with 10% foetal bovine serum at 37 °C

2. Cultures are incubated for 42-44 h at 37°C in a humidified atmosphere containing 5% CO₂ prior to use.

Steps 1 to 2 above are standard laboratory practise to culture this cell line. The methods have been extensively reported and reviewed (eg. Giesse, S., et al, Protein Expr. Purif. 8: 271-282, 1996). There are many research publications that have used these or very similar methods to culture COS cells.

Testing compounds for their ability to disrupt EphB-ephrinB interactions:

3. Cultures (prepared as in steps 1-3 above) are incubated with ephrinB2-Fc (R&D Systems) at 10 μg/ml in culture medium for 30 min, in the presence of a range of concentrations of the compound to be tested.

4. The coverslips are then washed 3 times in phosphate buffered saline (PBS, pH 7.4) and the cells treated with fixative (4% paraformaldehyde in PBS, ph 7.4) for 10-15 minutes at room temperature. 5. After PBS washing, the cultures are incubated at room temperature in the dark for 2 hours with Cy3-conjugated donkey anti-human IgG (Jackson Immunochemicals) diluted 1 :200 in PBS (pH 7.4) containing 0.1% sodium azide and 1% normal donkey serum.

6. Following 3 further washes in PBS (as for 10, above) the coverslips are mounted in Cytifluor (Cytifluor Ltd, UK).

To assess the degree of binding between ephrinB ligands and EphB receptors:

7. Cultures that are immunostained (as in 4-7 above) are viewed using a standard fluorescent microscope equipped using 20x or 40x objectives, and filters appropriate for the fluorochrome of the secondary antibody.

8. Representative images of the immunostained cultures are photographed with a digital CCD camera fitted to the microscope. Images of all the cultures to be compared are taken under identical lighting conditions and using the same camera settings. The digital images are stored on a computer.

9. The stored images are subsequently analysed with image analysis software. The average brightness of each image is calculated. The control cultures (where ephrinB-Fc is added to the neurons without any putative blocking compounds) will have a certain degree of brightness. Cultures in which ephrinB-EphB receptor interactions have been blocked will have lower levels of brightness.

10. The average brightness of the images treated with different putative blocking compounds are compared by standard statistical tests such as Analysis of Variance analysis (ANOVA). A statistically significant reduction in average grey value in the presence of a given compound indicates significant inhibition of ephrinB2-Fc binding, and therefore analgesic potential.

EXAMPLE 3 Ligands for Eph receptors can rapidly be detected by monitoring the affinity interaction between the endogenous ephrinB ligands and the EphB receptor, using fluorescence resonance energy transfer (FRET) based continuous-flow bioassays (Figure 2). The on-line coupling between the FRET-based bioassay and High Performance Liquid Chromatography (HPLC), as applied in High Resolution Screening instruments, enables rapid detection of ligands in complex mixtures and moreover provides access to a high level of chemical diversity, unmatched by the currently used synthetic approaches. This would allow for rapid identification of Eph receptor ligands that would then require further characterisation and investigation using the test systems already described.

All documents cited above are incorporated herein by reference.

Claims

1. The use of a compound which disrupts the interaction between an EphB receptor and an ephrinB ligand in the preparation of a medicament for the treatment of pain.

2. The use of a compound according to claim 1 wherein the compound is an EphB receptor antagonist.

3. The use of a compound according to claim 2, wherein the antagonist is a competitive antagonist.

4. The use of a compound according to claim 2, wherein the antagonist is a non-competitive antagonist.

5. The use of a compound according to any of claims 2 to 4, wherein the antagonist is a reversible antagonist.

6. The use of a compound according to claim 1, wherein the compound binds to, modifies or cleaves the ephrinB ligand.

7. The use of a compound according to claim 1, wherein the compound affects the turnover, expression or function of either the EphB receptor, or the ephrinB ligand.

8. The use of a compound according to claim 1, wherein the compound is selected from the group consisting of antibodies to either the EphB receptor or the ephrinB ligand, EphBl-Fc chimeras fragments of EphB receptor or ephrinB ligands.

9. The use of compound according any preceding claim, wherein the medicament is for the treatment of hyperalgesia and/or allodynia.

10. A method of treating pain comprising administering to a subject an effective amount of a compound which disrupts the interaction between an EphB receptor and an ephrinB ligand.

11. A method according to claim 10, wherein the pain is hyperalgesia or allodynia.

12. A method according to claims 10 or 11, wherein the compound is an EphB receptor antagonist.

13. A method according to claim 12, wherein the antagonist is a competitive antagonist.

14. A method according to claim 12, wherein the antagonist is a non-competitive antagonist.

15. A method according to any of claims 12 to 14 wherein the antagonist is a reversible antagonist.

16. A method according to claim 10 wherein the compound is selected from the group consisting of antibodies to either the EphB receptor or the ephrinB ligand, EphBl-Fc chimeras fragments of EphB receptor or ephrinB ligands .

17. A method of identifying a compound as an analgesic comprising the step of measuring the ability of the test compound to disrupt the interaction between an EphB receptor and an ephrinB ligand.

18. A method according to claim 17 further comprising the steps of binding a marker to an ephrinB ligand; and determining the binding of an ephrinB ligand to an EphB receptor before, and after the addition of the test-compound.

19. A method according to claim 17 or 18 for use in vivo.

20. A method according to claim 17 or 18 for use in vitro.

21. A kit for identifying test-compounds as analgesics comprising an EphB receptor, an ephrinB ligand, a means for measuring the binding of the ligand to the receptor.