WO2011148200A1

WO2011148200A1 - Treatment of pain

Info

Publication number: WO2011148200A1
Application number: PCT/GB2011/051018
Authority: WO
Inventors: Lucy Donaldson; Richard Hulse
Original assignee: The University Of Bristol
Priority date: 2010-05-28
Filing date: 2011-05-27
Publication date: 2011-12-01
Also published as: GB201009173D0

Abstract

The invention provides an active agent comprising one or more VEGFR2 agonist for use in treating or preventing VEGFR2-mediated non-inflammatory pain. The VEGFR2 agonist may be selected from the VEGF_xxxb family of proteins, particularly VEGF_165b, soluble VEGF receptors having activity as inhibitors of VEGFR2 antagonists and VEGF_xxxisoforms that have the C-terminal hexapeptide corresponding to expression immediately after exon 7, or VEGF_xxx isoforms that lack neuropilin binding mediating portions of the amino acid sequence.

Description

TREATMENT OF PAIN

Field of the Invention The present invention relates to novel pain treatments.

Background of the Invention

Vascular endothelial growth factor (VEGF), acting through its receptor (VEGF Receptor 2 or VEGFR2), is the principal agent responsible for blood vessel growth in physiological and pathological angiogenesis (Carmeliet P., Nat Med. 2003 Jun;9(6):653-60. Review). It is required for the recovery of tissues after wounding as part of the healing response (Bates DO, Int J Low Extreme Wounds. 2003 Jun; 2(2): 107-20), and also for the development of many pathologies, including cancer (Ferrara N. Nat Rev Cancer. 2002 Oct;2(10):795-803), diabetes (Chou E Circulation 2002 Jan 22; 105(3):373-9) and atherosclerosis (Celletti FL Nat Med. 2001 Apr;7(4):425-9). It has become clear recently that the action of VEGF is not restricted to the vascular system, but that VEGF is a key component of neuronal protective mechanisms (Jin KL et al Neuroscience. 2000;99(3):577-85) (Jin KL et al Proc Natl Acad Sci U S A. 2000 Aug 29;97(18): 10242-7) (Oosthuyse et al Nat Genet., 2001 Jun;28(2):131 - 8). This was first identified in vivo through the role of VEGF in preventing progression of the motor neuron disease, amyotrophic lateral sclerosis, by preventing motor neuron cell death (Oosthuyse et al, Nat Genet., 2001 Jun;28(2): 131 -8). There is also a substantial body of evidence to suggest that VEGF may be involved in sensory neuropathy. VEGF is expressed at low levels in adult dorsal root ganglion (DRG) under normal conditions, yet is detectable in up to 35% of neurons, mainly in small (<30μηι diameter) DRG neurons, although there are no reports on the modality of sensory neuron this population represents. VEGFR2 is also normally expressed in the DRG (<10% of DRG neurons). It is unclear which specific subsets of neurons express VEGFR2 but it is thought that the receptor is not co-expressed with VEGF (Sondell M. Neuroreport. 2001 Jan 22; 12(l): 105-8).

In WO-A-2009/106855, the disclosure of which is incorporated herein by reference, we describe and claim the use of a family of anti-angiogenic alternative splice variants of VEGF in the treatment and prevention of neuropathic and neurodegenerative disorders, for example pain such as neuropathic pain. These variants ((WO-A-03/012105; Bates et al, Cancer Res. 62, 4123-4131 (2002); Bates et al, Clinical Science 1 10, 575-585 (2006)) are formed by splicing from exon 7 into the previously assumed 3' UTR of the VEGF mRNA, forming proteins of the same length as other forms, but with a different C terminal amino acid sequence. The family of splice variants has been termed

where "xxx" refers to the number of amino acids in the isoform, e.g. 165 in the case of VEGFi₆sb. We have identified VEGF₁₆₅b, VEGF₁₈₉b, VEGF₁₄₅b, VEGF₁₈₃b and VEGF,₂,b proteins (O. Konopatskaya, Molecular Vision. 2006; 12:626-632). In WO-A-2008/1 10777, the disclosure of which is incorporated herein by reference, we describe and claim agents for selectively promoting the expression of VEGF_xxxb in preference to VEGF_XXX in cells of a subject or in vitro.

Interestingly, the binding domains for VEGFR2 and its co-receptor NP-1 (Neuropilin-1 ) and the dimerisation domains are intact in these newly identified isoforms, although the new C-tenninal sequence of VEGF_xxxb proteins abuts the NP-1 binding site and appears to interfere with NP-1 binding (Cebe Suarez 2006; Cell Mol Life Sci. 2006 Sep; 63(17):2067- 77). Non-natural mutants of the VEGF_xxxb proteins have been developed. One example is the protein NRC L-11885 of the National Research Council of Canada, which differs from VEGF₁₆₅b by two amino acids and has been reported to be more potent than the natural material VEGF^sb (see http://www.nrc-cnrc.gc.ca/eng/licensing/bri/vegf-antagonists.html). Such proteins are encompassed by the term "VEGF^b" used herein.

The present invention is based on our unanticipated finding that VEGFR2 agonists, including for example and without limitation VEGF_xxxb proteins and their functional analogues, fragments and mutants, have an analgesic effect on VEGFR2-mediated noninflammatory pain in mammals, including humans. This is surprising, in view of the prior reports of an analgesic effect of VEGFR2 antagonists against acute inflammatory pain (Grosios et al, Inflamm. Res. 2004; 53(4): 133-42). Brief Description of the Invention

According to a first aspect of the present invention, there is provided one or more VEGFR2 agonist for use in treating or preventing VEGFR2~mediated non-inflammatory pain.

According to a second aspect of the present invention, there is provided a method of treating or preventing non- inflammatory pain which comprises administering to a subject suffering from or susceptible to VEGFR2-mediated non-inflammatory pain an effective amount of one or more VEGFR2 agonist.

According to a third aspect of the present invention, there is provided the use of one or more VEGFR2 agonist in the manufacture of a composition (e.g. a pharmaceutical composition, foodstuff, food supplement, beverage or beverage supplement) for treating or preventing VEGFR2-mediated non-inflammatory pain.

The term "VEGFR2 agonist" used herein includes, for example, (1) agonists of the VEGFR2 receptor, preferably partial agonists thereof, (2) inhibitors of antagonists of the VEGFR2 receptor, preferably such as to promote or obtain partial agonism thereof by endogenous partial agonists thereof, (3) inhibitors of endogenous VEGFR2 full agonists to promote or obtain partial agonism of the VEGFR2 receptor by endogenous partial agonists thereof, (4) promoters of phosphorylation of the VEGFR2 receptor to promote or obtain partial agonism thereof by endogenous partial agonists thereof, or (5) any mixture or combination thereof. The term "VEGFR2 agonist" used herein also includes (6) agents which promote the endogenous expression of one or more VEGFR2 agonist of categories (1 ) to (5) above in cells of a subject. The endogenous expression may, for example, be preferential expression as compared with one or more other proteins. The VEGFR2 agonist, used as active agent in the present invention, if naturally occurring, may be used in isolated form, that is, separated from its natural environment.

The VEGFR2 agonist may optionally be used alone or in the presence of other active or inactive components, as described in more detail below. The analgesic effect against VEGFR2-mediated non-inflammatory pain is obtained in a human or animal subject, for example a human or other mammalian subject, most preferably a human subject. The term "subject" used herein will be understood in this way throughout.

The activity of the agents according to the present invention against pain in a subject appears to depend on whether the neuronal state of the subject is normal or abnormal. This in turn indicates that the functional state of the VEGFR2 receptor, which appears to have a different functional state in neuronally abnormal (e.g. nerve-injured) subjects, is important in detennining the extent to which the agents according to the present invention will alleviate pain of different types and origins.

Without wishing to be bound by theory, we believe that the agents according to the present invention so control the functional state of the VEGFR2 receptor as to establish a pain- insensitive state, adjusted from the untreated pain-sensitive state(s) in the (neuronally normal or neuronally abnormal) subject, and different from the VEGFR2 -blockaded state, thereby alleviating the pain sensation for the subject.

Thus, in one aspect of the present invention, the agents may be used to alleviate pain in a subject experiencing pain as a result of nerve injury or other neuronal abnormality.

In another aspect of the present invention, the agents may be used to alleviate pain in a subject experiencing pain without nerve injury or other neuronal abnormality. As discussed below, the pain can include non-inflammatory allodynia and pain, the agents having a generally antiallodynic and analgesic activity, particularly against VEGFR2- mediated non-inflammatory pain.

The finding of such an effect of VEGFR2 agonists is surprising and provides the basis for an effective treatment of pain states of the types described. Hitherto there was no appreciation that control of agonism at the VEGFR2 receptor could provide the basis for alleviation of pain.

Furthermore, the work underlying the present invention makes available for the first time the possibility to counteract excessively analgesic properties of other pain-reducing agents, for example by adjusting the functional state of the VEGFR2 receptor towards a pain- sensitive state. In a further aspect of the present invention, therefore, the agents may be used in association with one or more different analgesic or antiallodynic agent for the purpose of enhancing or reducing the sensitivity of the subject to pain (which expression includes without limitation allodynia and hyperalgesia, as well as all conditions recognised as pain by the subject).

Detailed Description of the Invention Agonists of the VEGFR2 receptor

The expression "agonists of the VEGFR2 receptor" refers to all materials that produce an agonistic effect at the VEGFR2 receptor. The expression includes agonist compounds which actually bind to the VEGFR2 receptor and compounds that produce an agonistic effect without actual binding to the receptor. The agonist is preferably a partial agonist. A VEGFR2 agonist may, for example, be selective (specific) to the VEGFR2 receptor, or it may be agonistic or antagonistic at one or more receptors in addition to VEGFR2 (nonselective or non-specific). The agonistic effects of the active agent are generally exhibited at neuronal cell receptors.

It is preferred that the one or more VEGFR2 agonist comprises, and more preferably consists of, one or more partial VEGFR2 agonists.

Examples of partial agonists of the VEGFR2 receptor for use in the present invention include, for example, one or more VEGF_xxxb protein agents and functional analogues and fragments thereof, one or more peptoid agents of general formula I as defined below, one or more VEGF_XXX isoforms (VEGF protein agents) that lack neuropilin binding mediating portions of the amino acid sequence, particularly in exons 7 and 8, and all combinations and mixtures thereof. Such VEGF_xxX isoforms (VEGF protein agents) may be non-angiogenic, at least at physiologically compatible concentrations, and so may be useful in the present invention where angiogenesis would be detrimental to the subject. Examples of such VEGFR2 agonists include, for example,

VEGF,₄₅ and VEGF_]48 protein agents and functional analogues and fragments thereof. The term "VEGF^b" used herein includes endogenous proteins and functional fragments and mutants thereof, including non-natural mutants (that is: mutants which are not known to be expressed in any wild-type organism). For avoidance of doubt, a "non-natural" mutant may be expressible in a recombinant organism, and so may be obtained in this way, or it may be preparable by synthesis. VEGF_xxxb and agents which promote the endogenous expression of one or more VEGF^b in preference to one or more VEGF_XXX in cells of a subject are encompassed by the term

active agent" used herein. The VEGF_xxxb may for example, comprise one or more of VEGF₁₆₅b, VEGF_{l g9}b, VEGFi₄₅b, VEGF_{] 83}b and VEGF₁₂ib. The VEGF_xxxb suitably comprises recombinant VEGF_xxxb, preferably recombinant human VEGF_xxxb (rhVEGF_xxxb). The VEGF^b preferably comprises VEGF₁₆₅b, e.g. recombinant VEGFi₆₅b, such as recombinant human VEGF₁₆₅b (rhVEGFi₆₅b). The VEGF_xxxb may, for example, consist essentially of VEGF₁₆₅b, e.g. recombinant VEGF^sb, such as recombinant human VEGFi₆₅b (rhVEGF₁₆₅b). The VEGF_xxxb may, for example, consist of VEGFi₆₅b, e.g. recombinant VEGF^sb, such as recombinant human VEGF^sb (rhVEGFi₆₅b).

Where the VEGFR2 agonist comprises, or promotes expression of, VEGF protein agents or VEGF_xxxb protein agents, such protein agents may suitably contain at least one, for example all, of the sequence of six C-terminal amino acids of VEGF₁₆₅ (namely CysAspLysProArgArg - SEQ ID NO: 1) or, respectively, VEGF₁₆₅b (namely SerLeuThrArgLysAsp - SEQ ID NO: 2), at the amino acid positions corresponding to C- terminal expression immediately after exon 7 (see WO-A-03/012105; Bates et al, Cancer Res. 62, 4123-4131 (2002); Bates et al, Clinical Science 110, 575-585 (2006)). Experimental work in the Examples below (see Figure 10) shows that protein agents which lack all of the C-terminal hexapeptide (e.g. VEGF₁₅₉) do not show an effect on nociception.

The term "peptoid" molecules referred to herein are particularly those reported in Udugamasooriya, D G et al, J. Am. Chem. Soc. (2008), 130, 5744-52. Although putatively characterised as VEGFR2 antagonists, it seems that the peptoids reported in the Udugamasooriya et al reference, for example GU40C and GU40E whether in monomer or dimer fonn, or the dimer GU40C4 (see the reference for structural formulae of the peptoids) appear likely to have partial agonist characteristics in relation to to the VEGFR2 receptor, which make them useful in the present invention. These peptoid molecules are characterised generally by structural formulae I

wherein R is selected from the following species and more than one different R may be present in the repeating units of the left hand end of the molecule as drawn in Formula I,

and the dimer form II

wherein the terms "6mer - diversified region", "Nleu", "Nlys2" and "Nlysl" refer to the monomer forms shown in Formula I.

For further information about the structures and preparation of the peptoids, please refer to the Udugamasooriya et al reference. Inhibitors of antagonists of the VEGFR2 receptor

The expression "inhibitors of antagonists of the VEGFR2 receptor" refers to all materials that produce an inhibition of an antagonistic effect at the VEGFR2 receptor, particularly inhibition of endogenous antagonism of the VEGFR2 receptor. The expression includes inhibitor compounds which actually bind to the antagonists of the VEGFR2 receptor and compounds that produce an inhibitory effect without actual binding to the antagonists of the VEGFR2 receptor. An inhibitor of antagonists of the VEGFR2 agonist may, for example, be selective (specific) to antagonists of the VEGFR2 receptor, or it may be inhibitory of other biochemical activities in addition to antagonists of the VEGFR2 receptor (nonselective or non-specific).

It is preferred that the one or more inhibitors of antagonists of the VEGFR2 receptor comprises, and more preferably consists of, one or more inhibitors that are such as to promote or obtain partial agonism of the VEGFR2 receptor by endogenous partial agonists thereof.

Examples of inhibitors of antagonists of the VEGFR2 receptor for use in the present invention include, for example, one or more inhibitors of endogenous soluble VEGF receptors (these may compete with the VEGFR2 receptor for binding with endogenous VEGFR2 agonists and so inhibition of the soluble VEGF receptors can promote or obtain partial agonism of the VEGFR2 receptor), one or more inhibitors of NP-1 binding (inhibition may prevent antagonistic binding at the VEGFR2 receptor), one or more inhibitor of other VEGFR2 blocking agents such as molecules (e.g. PTK787 and ZM323881) which inhibit tyrosine phosphorylation of the VEGFR2 receptor, and all combinations and mixtures thereof.

Inhibitors of antagonists of the VEGFR2 receptor may include, for example, specific or non-specific antibodies directed to the antagonists or to the binding domain of NP-1 , or specific or non-specific inlribitory small molecule agents directed to the antagonists or to the binding domain of NP-1. The term "antibodies" used herein includes complete antibodies and functional fragments thereof, for example Fab, Fab', F(ab')2, Fd, Fd\ Fv, dAb, isolated CDR region, single chain antibody, diabodies and linear antibodies. Antibodies may be monoclonal or polyclonal. Inhibitors of endogenous VEGFR2 full agonists

The expression "inhibitors of endogenous VEGFR2 full agonists" refers to all materials that produce an inhibition of a full endogenous agonistic effect at the VEGFR2 receptor. The expression includes inhibitor compounds which actually bind to the full agonists of the VEGFR2 receptor and compounds that produce an inhibitory effect without actual binding to the full agonists of the VEGFR2 receptor. An inhibitor of endogenous full agonists of the VEGFR2 receptor may, for example, be selective (specific) to full agonists of the VEGFR2 receptor, or it may be inhibitory of other biochemical molecules in addition to full agonists of the VEGFR2 receptor (non-selective or non-specific).

It is preferred that the one or more inhibitors of endogenous full agonists of the VEGFR2 receptor comprises, and more preferably consists of, one or more inhibitors that are such as to promote or obtain partial agonism of the VEGFR2 receptor.

Examples of inhibitors of endogenous full agonists of the VEGFR2 receptor for use in the present invention include, for example, one or more inhibitors of endogenous VEGF proteins. The one or more inhibitors of endogenous VEGF proteins may preferably be selective (specific) inhibitors of endogenous VEGF proteins, at least to the extent of not being inhibitory of endogenous VEGF_xxXb protein agents, which as described above are useful as active agents in the present invention.

Inhibitors of endogenous full agonists of the VEGFR2 receptor may include, for example, specific or non-specific antibodies directed to the full agonists, or specific or non-specific inhibitory small molecule agents directed to the full agonists. The term "antibodies" used herein includes complete antibodies and functional fragments thereof, for example Fab, Fab', F(ab')₂, Fd, Fd', Fv, dAb, isolated CDR region, single chain antibody, diabodies and linear antibodies. Antibodies may be monoclonal or polyclonal. Certain anti-VEGF antibodies are commercially available, for example avastin and ranibizumab.

Inhibitors of endogenous full agonists of the VEGFR2 receptor may also include, for example, specific or non-specific inhibitory aptamers directed to the full agonists. Certain anti-VEGF aptamers are commercially available, for example pegatanib.

Promoters of phosphorylation of the VEGFR2 receptor The expression "promoters of phosphorylation of the VEGFR2 receptor" refers to all materials that produce an enhanced extent of phosphorylation of the VEGFR2 receptor, A promoters of phosphorylation of the VEGFR2 receptor may, for example, be selective (specific) to the VEGFR2 receptor, or it may have other biochemical activities in addition to being a promoter of phosphorylation of the VEGFR2 receptor (non-selective or nonspecific).

It is preferred that the one or more promoters of phosphorylation of the VEGFR2 receptor are such as to promote or obtain partial agonism of the VEGFR2 receptor by endogenous partial agonists thereof.

Examples of promoters of phosphorylation of the VEGFR2 receptor include kinases and functional analogues and fragments thereof.

Agents which promote the endogenous expression of one or more VEGFR2 agonist

The expression "agents which promote the endogenous expression of one or more VEGFR2 agonist" used herein includes expression vector systems for transforming host cells and organisms in this way. Such an expression vector system suitably comprises a promoter nucleotide sequence operably associated with one or more nucleotide sequence coding for the one or more VEGFR2 agonist and capable of promoting expression of the same in vivo, whereby the agent promoting expression of the one or more VEGFR2 agonist can be expressed in a host organism, suitably the subject to be treated, under suitable conditions of transfection and incubation. Further details are provided below in the section headed "Gene Therapy".

In one embodiment, the agent which promote the endogenous expression of one or more VEGFR2 agonist may include an agent, such as those described and claimed in WO-A- 2008/110777, which selectively promotes the expression of one or more VEGF_xxxb in preference to one or more VEGF_XXX in cells of a subject or in vitro. In particular, there may be mentioned agents that favour distal splice site (DSS) splicing during processing of VEGF pre-mRNA transcribed from the C terminal exon 8 of the VEGF gene. Such agents may, if desired be used in association with one or more controlling agents for the splicing which suppresses or inhibits proximal splice site (PSS) splicing during processing of VEGF pre-mR A transcribed from the C terminal exon 8 of the VEGF gene (see WO-A- 2008/110777). These agents are discussed further below.

Protein agents, functional analogues and functional fragments

The expression "protein agents" used herein includes polypeptides in which the stated protein forms a part of a longer amino acid sequence, for example immature protein sequences carrying signal secretory or other additional amino acid sequences, or synthetic polypeptides in winch the functional epitope(s) form a part of the sequence.

The expression "functional analogues" used herein in relation to an agent includes polypeptides or peptide sequences which have functional efficacy for the purposes of the present invention which generally corresponds to that of stated agent, for example non- natural mutants thereof (that is: mutants which are not known to be expressed in any wild- type organism). For avoidance of doubt, a "non-natural" mutant may be expressible in a recombinant organism, and so may be obtained in this way, or it may be preparable by synthesis.

The expression "functional fragments" used herein in relation to protein agents and functional analogues includes polypeptides or peptide sequences which have a sequence length shorter than the stated protein agents and functional efficacy for the purposes of the present invention which generally corresponds to that of the stated protein agents.

The functional analogues referred to above may include, for example, a polypeptide in which the amino acid sequence of a stated protein agent has been modified by insertion, substitution or deletion of one or more amino acid or has been modified by any combination of such insertion, substitution or deletion without substantial detriment to the functional efficacy of the stated protein agent for the purposes of the present invention. For example, a polypeptide the amino acid sequence of which shows at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, but less than 100%, identity with the stated protein agent or the analogous sequence of the stated protein agent may be considered as a functional analogue of the stated protein agent. Functional analogues of a stated protein agent may, independently of any insertion, substitution or deletion of one or more amino acids, comprise modifications such as, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or a nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation or glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination (see, for instance, PROTEINS -STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed. , T. E. Creighton, W. H. Freeman and Company, New York (1 93) and Wold, F. , Posttranslational Protein Modifications: Perspectives and Prospects, pgs 1 -12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson. Ed. , Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182: 626-646 (1990) and Rattan et al, Protein Synthesis: Posttranslational Modifications and Aging, Ann. N. Y. Acad. Sci. 663: 4862 (1992). Polypeptides can be branched, or cyclic, with or without branching. Cyclic, branched and non-branched polypeptides can result from posttranslational natural processes and can be made by entirely synthetic methods as well.

Agents which selectively promote the expression of VEGF^b in preference to VEGF^ in cells of a subject or in vitro

Such agents are described in the passage from page 6, line 22 to page 8, line 9 of WO-A- 2008/1 10777, and elaborated in the remainder of WO-A-2008/110777 to the extent that favouring of DSS splicing over PSS splicing is concerned. Please refer to these passages of WO-A-2008/1 10777 for the discussion.

In particular, there may be mentioned Transforming Growth Factor (TGF)-bl , TGF-b Rl , SRPK1 specific inhibitors (for example, SRPIN 340), T-cell intercellular antigen-1 (TIA-1), MKK3 MKK6-activatable MAP kinases (for example, p38 MAPK), Cdc20-like (Clk) family kinases Clkl/sty, Clk2, Clk3 and Clk4, the SR splicing factor SRp55, their in vivo activators, upregulators and potentiators, functionally active analogues and functionally active fragments of any of the foregoing, modified forms of any of the foregoing having a secondary functionality useful for control of their primary activity or the effects thereof, expression vector systems for expressing any of the foregoing agents in vivo, transcription/translation blocking agents which bind to the PSS of exon 8a of the pre- mRNA and/or at the region of the pre-mRNA to which a splicing regulatory protein binds, to inhibit proximal splicing (for example, morpholinos or other synthetic blocking agents, peptide conjugates, RNA binding proteins, RNA interference (RNAi) poly- and oligonucleotide blocking agents (for example dsRNA, microRNA (miRNA), siRNA), peptide nucleic acid (PNA), protein kinase C (PKC) inhibitors (for example, bisindolyl maleimide (BHVI) and other mechanistically analogous PKC inhibitors, particularly inhibitors which bind at the PKC catalytic domain), or any combination thereof.

Such an expression vector system suitably comprises a promoter nucleotide sequence operably associated a nucleotide sequence coding for the agent promoting expression of VEGF_xxxb in preference to VEGF_XXX, whereby the agent promoting expression of VEGF_xxxb in preference to

can be expressed in a host organism, suitably the subject to be treated, under suitable conditions of transfection and incubation. Further details are provided below in the section headed "Gene Therapy".

Conditions and Disorders to be Treated VEGFR2-mediated non-inflammatory pain to be treated or prevented according to the present invention includes non-inflammatory neuropathic and nociceptive pain where the VEGFR2 receptor is involved in the cause or transmission of the pain. For example, the agents according to the present invention have activity against non-inflammatory allodynia and pain (antiallodynic and analgesic activity). Pain states of this type include chronic pain, whether of the intermittent or constant form. Such pain states may include, for example, low back pain, neuralgia, atypical pains such as atypical facial pain, pain exhibited post- surgery, post-injury (for example, after surgery or injury causing nerve damage) or in association with cancer or with cancer therapy such as cytotoxic or radiation therapy, or neuropathy associated with diabetes (diabetic neuropathy, insulin neuritis) or other systemic or autoimmune disease or pathology, or the treatment thereof, alcoholism or HIV infection, ageing associated neuropathy, or neuropathy of unknown origin.

The activities of the proteins of the VEGFR2 agonists, for example the VEGF^b family, are predicted to both actively prevent and actively reverse VEGFR2-mediated non- inflammatory pain. However, in view of the anti-angiogenic activity of the proteins of the VEGF_xxxb family, use of VEGFxxxb active agents will be restricted to pain in contexts where possible inhibition of angiogenesis would not be detrimental to the patient. In view of the likely pro-angiogenic activity of full VEGFR2 agonists, use of full VEGFR2 agonists will be restricted to pain in contexts where possible stimulation of angiogenesis would not be detrimental to the patient.

The one or more VEGFR2 agonist may, if desired, be co-administered with one or more additional active agent, for example one or more agent selected from, but not limited to, cholinesterase inhibitors, dopamine agonists (e.g. L-dopa), COMT inhibitors, MAO-B inhibitors, anti-cholinergics, acetylcholine agonists, serotonin agonists, AMPA receptor agonists, GABA receptor agonists, NMDA receptor agonists, β-adrenoceptor agonists, digoxin, dobutamine, anti-inflammatories, neurotrophic factors, statins, adenosine A2a receptor antagonists, aldose reductase inhibitors, immunomodulators, cannabinoid agonists, interferon or tricyclic anti-depressants. The term "active agent" used herein encompasses all permutations of the VEGFxx_Xb family proteins whether singly or in any combination, with or without any one or more co-adminstered active agent of another type. As mentioned above, the VEGFR2 agonist used in the present invention may be employed in association with one or more different pain treatment agent for the purpose of normalising the sensitivity towards pain of the subject treated (or being co-treated) with the said one or more different pain treatment agent. The term "normalising" means moving the subject's pain sensitivity towards normal levels, and may include enhancement of the sensitivity if the one or more different pain treatment agent causes an excessive reduction in feeling or in sensitivity towards pain. The one or more different pain treatment agent may be selected from pain treatment agents currently known or yet to be devised. Such selection will be well within the skill of the person of ordinary skill in this art. Such combination treatments can enable fine control of pain sensitivity in subjects and minimisation of overall side effects according to the particular condition and needs of the subject.

Compositions and Administration

The active agent may be administered in the form of a composition comprising the active agent and any suitable additional component. The composition may, for example, be a pharmaceutical composition (medicament), a foodstuff, food supplement, beverage or beverage supplement.

According to a further aspect of the present invention, there is provided a composition comprising an effective amount of VEGF_xxxb active agent for use in treating or preventing, VEGFR2-mediated non-inflammatory pain.

The active agent according to the present invention may be administered in the form of a composition comprising the active agent and any suitable additional component. The composition may, for example, be a pharmaceutical composition (medicament), suitably for parenteral administration (e.g. injection, implantation or infusion). The composition may alternatively, for example, be a foodstuff, food supplement, beverage or beverage supplement. The term "pharmaceutical composition" or "medicament" in the context of this invention means a composition comprising an active agent and comprising additionally one or more pharmaceutically acceptable carriers. The composition may further contain ingredients selected from, for example, diluents, adjuvants, excipients, vehicles, preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and dosage forms. The compositions may take the form, for example, of tablets, dragees, powders, elixirs, syrups, liquid preparations including suspensions, sprays, inhalants, tablets, lozenges, emulsions, solutions, cachets, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations. Techniques and formulations generally may be found in Remington, The Science and Practice of Pharmacy, Mack Publishing Co., Easton, PA, latest edition.

Liquid form preparations include solutions, suspensions, and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injection. Liquid preparations can also be formulated in solution in aqueous polyethylene glycol solution.

Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions. These particular solid form preparations are most conveniently provided in unit dose form and as such are used to provide a single liquid dosage unit. Alternately, sufficient solid may be provided so that after conversion to liquid form, multiple individual liquid doses may be obtained by measuring predetermined volumes of the liquid form preparation as with a syringe, teaspoon, or other volumetric container or apparatus. The solid form preparations intended to be converted to liquid form may contain, in addition to the active material, flavourings, colourants, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like. The liquid utilized for preparing the liquid form preparation may be water, isotonic water, ethanol, glycerine, propylene glycol, and the like as well as mixtures thereof. Naturally, the liquid utilized will be chosen with regard to the route of administration, for example, liquid preparations containing large amounts of ethanol are not suitable for parenteral use. The terms "foodstuff, "food supplement", "beverage" and "beverage supplement" used herein have the normal meanings for those terms, and are not restricted to pharmaceutical preparations. Other composition forms are also included within the present invention. These may, for example, include pure or substantially pure compound as such, a foodstuff precursor such as a rehydratable powder or a beverage precursor such as a powder dispersible in water, milk or other liquid.

The dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with the smaller dosages which are less than the optimum dose of the compound. Thereafter the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

Gene Therapy

The present invention may alternatively be practiced using gene therapy. Gene therapy techniques are generally known in this art, and the present invention may suitably be put into practice in these generally known ways. The following discussion provides further outline explanation. The gene therapies are broadly classified into two categories, i.e., in vivo and in vitro therapies. The in vivo gene therapy comprises introducing a therapeutic gene directly into the body, and the in vitro gene therapy comprises culturing a target cell in vitro, introducing a gene into the cell, and then, introducing the genetically modified cell into the body.

The gene transfer technologies are broadly divided into a viral vector-based transfer method using virus as a carrier, a non-viral delivery method using synthetic phospholipid or synthetic cationic polymer, and a physical method, such as electroporation or introducing a gene by applying temporary electrical stimulation to a cell membrane.

Among the gene transfer technologies, the viral vector-based transfer method is considered to be preferable for the gene therapy because the transfer of a genetic factor can be efficiently made with a vector with the loss of a portion or whole of replicative ability, which has a gene substituted a therapeutic gene. Examples of virus used as the virus carrier or vector include RNA virus vectors (retrovirus vectors, lentivirus vector, etc.), and DNA virus vectors (adenovirus vectors, adeno-associated virus vectors, etc.). In addition, its examples include herpes simplex viral vectors, alpha viral vectors, etc. Among them, retrovirus and adenovirus vectors are particularly actively studied.

The characteristics of retrovirus acting to integrate into the genome of host cells are that it is harmless to the human body, but can inhibit the function of normal cells upon integration. Also, it infects various cells, proliferates fast, can receive about 1 -7 kb of foreign genes, and is capable of producing replication-deficient virus. However, it has disadvantages in that it is hard to infect cells after mitosis, it is difficult to transfer a gene in vivo, and the somatic cell tissue is needed to proliferate always in vitro. In addition, since it can be integrated into a proto-oncogene, it has the risk of mutation and can cause cell necrosis.

Meanwhile, adenovirus has various advantages for use as a cloning vector; it has moderate size, can be replicated within a cell nucleus, and is clinically nontoxic. Also, it is stable even when inserted with a foreign gene, and does not cause the rearrangement or loss of genes, can transform eucaryotes, and is stably expressed at a high level even when it is integrated into the chromosome of host cells. Good host cells for adenovirus are cells of causing human hematosis, lymphoma and myeloma. However, these cells are difficult to proliferate because they are linear DNAs. Also, it is not easy infected virus to be recovered, and they have low virus infection rate. Also, the expression of a transferred gene is the highest after 1-2 weeks, and in some cells, the expression is kept only for about 3-4 weeks. In addition, these have the problem of high immune antigenicity. Adeno-associated virus (AAV) can overcome the above-described problems and at the same time, has many advantages for use as a gene therapeutic agent and thus is recently considered to be preferable. AAV, which is single-strand provirus, requires an assistant virus for replication, and the AAV genome is 4,680 bp in size and can be inserted into any site of chromosome 19 of infected cells. A trans-gene is inserted into plasmid DNA linked with 145 bp of each of two inverted terminal repeat sequence (ITR) and a signal sequence. This gene is transfected with another plasmid DNA expressing AAV rep and cap genes, and adenovirus is added as an assistant virus. AAV has advantages in that the range of its host cells to be transferred with a gene is wide, immune side effects due to repeated administration are little, and the gene expression time is long. Furthermore, it is stable even when the AAV genome is integrated into the chromosome of a host cell, and it does not cause the modification or rearrangement of gene expression in host cells. Since an AAV vector containing a CFTR gene was approved by ΝΓΗ for the treatment of cystic fibrosis in 1994, it has been used for the clinical treatment of various diseases. An AAV vector containing a factor IX gene, which is a blood coagulation factor, is used for the treatment of hemophilia B, and the development of a therapeutic agent for hemophilia A with the AAV vector is currently being conducted. Also, AAV vectors containing various kinds of anticancer genes were certified for use as tumor vaccines.

Gene therapy, which is a method of treating diseases by gene transfer and expression, is used to adjust a certain gene, unlike the drug therapy. The ultimate purpose of the gene therapy is to obtain useful therapeutic effects by genetically modifying a living gene. The gene therapy has various advantages, such as the accurate transfer of a genetic factor into a disease site, the complete decomposition of the genetic factor in vivo, the absence of toxicity and immune antigenicity, and the long-term stable expression of the genetic factor and thus is spotlighted in connection with the present invention as a potentially suitable route of treatment.

In general, reference herein to the presence of one of a specified group of compounds, for example VEGF^b, soluble VEGF receptors having activity as inhibitors of VEGFR2 antagonists and VEGF^ isoforms that lack neuropilin binding mediating portions of the amino acid sequence, includes within its scope the presence of a mixture of two or more of such compounds.

"Treating or preventing"

The expression "treating or preventing" and analogous terms used herein refers to all forms of healthcare intended to remove or avoid VEGFR2-mediated non-inflammatory pain or to relieve its symptoms, including preventive, curative and palliative care, as judged according to any of the tests available according to the prevailing medical practice. An intervention that aims with reasonable expectation to achieve a particular result but does not always do so is included within the expression "treating or preventing". An intervention that succeeds in slowing or halting progression of a disorder is included within the expression "treating or preventing". "Susceptible to "

The expression "susceptible to" and analogous terms used herein refers particularly to individuals at a higher than normal risk of developing VEGFR2-mediated noninflammatory pain, as assessed using the known risk factors for the individual or disorder.

Mammals

Besides being useful for human treatment, the present invention is also useful in a range of mammals, which can also be affected by VEGFR2 -mediated non-inflammatory pain. Such mammals include non-human primates (e.g. apes, monkeys and lemurs), for example in zoos, companion animals such as cats or dogs, working and sporting animals such as dogs, horses and ponies, farm animals, for example pigs, sheep, goats, deer, oxen and cattle, and laboratory animals such as rodents (e.g. rabbits, rats, mice, hamsters, gerbils or guinea pigs)-

Where the VEGFR2-mediated non-inflammatory pain is exhibited in the context of a disorder or function that is exclusive to humans, then it will be understood that the mammal to be treated is a human. The same applies respectively to any other mammalian species if the disorder or function to be treated is exclusive to that species. Brief Description of the Drawings

In order to illustrate the invention further by way of non-limiting example, reference will now be made to the accompanying drawings and to the Examples which follow.

In the drawings:

Figure 1 (a) and (b) illustrates that inhibition of the VEGFR2 receptor causes a pro- nociceptive response;

Figure 2 illustrates that VEGF] 6₅b has an anti-allodynic effect;

Figure 3(a) and (b) illustrates that VEGF^sb has an analgesic effect; Figure 4(a) and (b) illustrates that inhibition of the VEGFR2 receptor reduces the mechanical activation thresholds in single nociceptive afferent fibres;

Figure 5 illustrates that inhibition of the VEGFR2 receptor does not affect local blood flow; Figure 6 illustrates that inhibition of the VEGFR2 receptor results in hyperalgesia and allodynia;

Figure 7 illustrates that inhibition of the VEGFR2 receptor exacerbates nerve-injury induced allodynia and induces allodynia on the contralateral side to the injury;

Figure 8 illustrates that a full agonist at VEGFR2 (VEGF_{l fl5}) induces mechanical allodynia in normal animals, whereas a partial agonist (VEGF_]65b) has no effect;

Figure 9 illustrates that it is full activation of the VEGFR2 rather than its co-receptor neuropilin that results in enhanced nociception. VEGF₁₂i has the same C terminal as VEGF_{] 65} but does not recruit neuropilin;

Figure 10 illustrates that VEGFi₅₉ (which lacks the C terminal amino acids found in VEGF] 65_/i_2] and VEGF₁₆₅b) does not alter nociceptive behaviours, indicating that it is the C terminal of the partial agonist VEGF_{] 65}b that is responsible for the antinociceptive effects; Figure 1 1 illustrates that full and partial agonists at VEGFR2 exert different effects on primary afferent nociceptors in vivo; Figure 12 illustrates that activation of VEGFR2 on cutaneous nociceptors results in sensitisation;

Figure 13 illustrates that VEGFR2 is expressed on DRG sensory neurones and is upregulated in nociceptive neurones ipsilateral to a nerve injury; and

Figure 14 illustrates that the pronociceptive effects of VEGFR2 activation by a full agonist are mediated through the TRPV1 receptor.

The following legends are applicable to each of the Figures:

Figure 1 (a):

Endogenous VEGFR2 blockade is pro-nociceptive Adult male Wistar rats were injected siibcutaneously with 30 μΐ of vehicle or ZM323881 , a VEGFR2 inhibitor, and mechanical withdrawal thresholds determined by stimulating the plantar surface of the paw with von Frey fibres of increasing force and recording behavioural withdrawal of the paw. VEGFR2- specific blockade by ZM323881 (lOnM, lOOnM) resulted in significant reductions in threshold, indicative of a pro-nociceptive effect of the drug (**p<0.01 vs vehicle, Bonferroni test). VEGFR2 is expressed in dorsal root ganglion neurones and their fibres, so these data suggest that activation of VEGFR2 by endogenous VEGFs exerts an antinociceptive effect on primary afferent neurones.

Figure 1(b): Endogenous VEGF receptor blockade is pro-nociceptive. Adult male Wistar rats were injected siibcutaneously with 30 μΐ of vehicle or PTK787, a VEGFR inhibitor, and mechanical withdrawal thresholds determined by stimulating the plantar surface of the paw with von Frey fibres of increasing force and recording behavioural withdrawal of the paw. VEGFR blockade by PT 787 (200nM) resulted in a significant reduction in threshold, indicative of a pro-nociceptive effect of the drug (**p<0.01 vs vehicle, Bonferroni test). These data suggest that activation of VEGFR2 by endogenous VEGFs exerts an antinociceptive effect on primary afferent neurones.

Figure 2:

Systemic VEGFi₆₅b is anti-allodynic in nerve injured mice. Male C57bl6 mice were subjected to either a partial nerve injury (Partial Saphenous Nerve Injury, PSNI) or sham surgery. Mechanical stimulus withdrawal thresholds on the plantar surface of the ipsilateral foot were measured using von Frey filaments. PSNI surgery has been shown to reduce mechanical stimulus withdrawal thresholds in mice persisting for at least 14 days, providing a model of allodynia. Biweekly i.p. injection of 500ng rhVEGF₁₆₅b in nerve injured mice significantly increased the mechanical thresholds compared to the PSNI control group (biweekly vehicle injection). In this model of nerve injury VEGF^b has an anti-allodynic effect. 2-way ANOVA + Bonferroni post-test. *p<0.05, **p<0.001 , ***p<0.001 Sham vs PSNI + vehicle, ⁺p<0.05 PSNI + vehicle vs PSNI + VEGF]₆₅b. Arrows signify injection time points.

Figure 3(a) and (b): Systemic VEGF₁₆₅b is analgesic in nerve injured mice. Male C57bl6 mice were subjected to a partial nerve injury (Partial Saphenous Nerve Injury, PSNI). Thermal withdrawal latencies on the plantar surface of the ipsilateral and contralateral feet were measured, PSNI surgery does not normally affect thermal withdrawal latencies in mice. Biweekly i.p. injection of 500ng rhVEGF^b in nerve injured mice significantly increased the withdrawal latencies on both sides compared to the PSNI control group (biweekly vehicle injection). In this model of nerve injury VEGF^b has an analgesic effect.

Figure 4(a) and (b): Endogenous VEGFR blockade reduces mechanical activation thresholds in single nociceptive afferent fibres. Electrophysiological recording of single nociceptive afferent fibres in rats before (time point "b") and after subcutaneous injection of vehicle (time point "v") followed by 200nM PTK787 into the receptive field. PTK787 injection resulted in a reduction in mechanical activation threshold (**p<0.01 , one way ANOVA with post Bonferroni n=4, p<0.05 = vehicle (v) vs 15 and 30 mins, ns = baseline (b) vs vehicle). Figure 5 :

Endogenous VEGF receptor 2 blockade does not exert its effects on primary afferent neurones through an alteration in local blood flow. Acute ischaemia or hypoxia can result in sensitisation of primary afferent nociceptors to stimulation. To exclude a possible hypoxic effect as an explanation for the pro-nociceptive effects of ZM323881 , the VEGFR2 inhibitor, adult male Wistar rats were injected subcutaneously with 30 μΐ of adrenaline (vasoconstrictor) or ZM323881 , and local cutaneous blood flow measured using laser Doppler flowmetry. Adrenaline significantly reduced cutaneous blood flow, whereas ZM323881 (Ι ΟΟηΜ) had no effect on blood flow. These data show that the pro-nociceptive effect of VEGFR2 specific blockade by ZM323881 cannot be attributed to local hypoxia or ischaemia. Figure 6:

Local endogenous VEGF receptor 2 blockade with 200nM PTK787 in normal rats resulted in a significant tactile allodynia (a), that is rats flinched in response to brush of the hindpaw with a soft paintbrush. This stimulus does not usually produce a response in rats, but is thought to mimic the allodynia induced by e.g. the touch of clothes as perceived by patients with painful neuropathy, (b) PTK787 also caused cold allodynia (withdrawal response to a drop of acetone placed on the paw), (c) mechanical allodynia (reduction in von Frey withdrawal threshold, and (d) mechanical hyperalgesia (increased withdrawal to a pin prick stimulus.). These findings suggest that VEGFR2 blockade not only results in hyperalgesia, but also the types of allodynia seen in neuropathy that is very difficult to treat (tactile and cold).

Figure 7. (a) In nerve injured rats, slight tactile allodynia developed 3 days after nerve injury (1^st arrow) and this was significantly enhanced following a single intraperitoneal injection of 30mg/kg PTK787 on day 7 compared to vehicle (second arrow), (b) no tactile allodynia developed in the paw contralateral to the nerve injury, but systemic administration of PTK787 just before testing on day 7 resulted in the induction of a contralateral tactile allodynia. (c) Unilateral nerve injury (ipsi) resulted in a mechanical allodynia on day 3 after injury, only ipsilateral to the injury - note the thresholds for the ipsi groups are lower than the contra groups on day 3. PTK787 given just before testing on day 7 resulted in a contralateral mechanical allodynia whereas vehicle had no effect. There was no exacerbation of the previously existing ipsilateral mechanical allodynia.

Figure 8. (a) In normal mice, intraperitoneal injection of the full VEGFR agonist VEGF^ (200ng) at 3 day intervals (arrowheads) induced a reduction in mechanical withdrawal threshold (mechanical allodynia), with no effect on (b) thermal hyperalgesia. In contrast the partial VEGFR agonist VEGF^b had no effect on either mechanical or thermal nociception in normal mice. * p<0.05, ***p<0.001 before vs after, 2 way ANOVA post-hoc Bonferrino.

Figure 9. (a) After 8ng/g VEGF_]2i (i.p.) adult male Wistar rats (n=7), responses to (a) brush (tactile allodynia), (b) pin prick (mechanical hyperalgesia), (c) cooling (cold allodynia), and (e) von Frey hairs (mechanical allodynia) were significantly enhanced compared to vehicle (saline, n=7). (*p<0.05, **p<0.01 , ***p<0.001 two way ANOVA with Bonferroni test). VEGF₁₂1 increased the latency of withdrawal response to (d) radiant heat (thermal hypoalgesia, exerting a significant antinociceptive effect on this modality of stimulation.

Figure 10. After 8ng/g VEGF15₉ (namely VEGF₁₆₅ truncated by absence of the 6 C-terminal acids CysAspLysProArgArg) (i.p.) adult male Wistar rats (n=7), responses to (a) brush (tactile allodynia), (b) pin prick (mechanical hyperalgesia), (c) cooling (cold allodynia), radiant heat (thermal hyperalgesia) and (e) von Frey hairs (mechanical allodynia) were all unaffected compared to vehicle (saline, n=7).

Figure 1 1. Digitised trace of raw data showing the effect of VEGF isoforms on the neuronal response to mechanical stimulation (vF stimulus), after discharge and ongoing firing in cutaneous afferents. Each vertical line in the three traces represents an action potential. VEGF_{) 6}5 enhanced mechanically evoked activity, decreased mechanical activation threshold, and resulted in ongoing activity. These effects were greater than those evoked by local vehicle injection. In contrast, VEGF^sb had no effect on primaiy afferent properties in vivo.

Figure 12. Mean data showing that VEGFig₅ (a) enhanced mechanically evoked activity, (b) reduced mechanical activation threshold, (c) induced ongoing firing, and (d) enhanced after discharge firing in cutaneous mechano-nociceptors. VEGFi₆₅b had no effect on these properties of afferent nociceptors. Figure 13. DRG were removed from nerve injured animals, sectioned and stained for VEGFR2 and the nociceptive neuronal marker TrkA as noted on the images. The numbers of VEGFR2-expressing neurones in the nociceptive population after nerve injury (right side) were quantified, and compared to uninjured animals (left side). VEGFR2 has been previously shown to be expressed in sections of DRG and increased after axotomy (Sondell et al, Eur J Neurosci 2000, 12, 4243-4254).

Figure 14. In normal mice, the mechanical allodynia induced by biweekly intraperitoneal injection of VEGF, ₆5 was blocked by co-administration of 500μg/kg of a TRPV1 antagonist (SB232881), indicating that the pronociceptive action of the VEGFR2 agonist is meditated through TRPV1 activation.

Detailed Description of the Drawings and Examples

Abbreviations min = minutes; hr = hour; ipsi = ipsilateral; contra = contralateral; % are by volume in the case of gases in gases or liquids in liquids, and otherwise by weight; i.p. = intraperitoneal; i.v. = intravenous; ~ = approximately; PC = personal computer; DRG = dorsal root ganglion.

Methods Behavioural nociceptive testing in rats and mice

The day prior to behavioural testing rats or mice were placed in transparent Perspex enclosures with wire (mechanical) or Perspex (Hargreaves) floors and habituated to the testing environment. This was carried out for a minimum of 15 minutes (until they settled). Habituation occurred prior to all behavioural testing sessions.

Mechanical withdrawal thresholds were recorded from both ipsilateral and contralateral hind paws. Mechanical withdrawal thresholds were determined using a series of calibrated von Frey filaments (Linton Instruments, UK) ranging from 0.16g to 2g or until 0-100% withdrawal was achieved from the 5 applications. Each filament was applied to the medial plantar surface of each hindpaw 5 times for a maximum period of 5 seconds. Mechanical withdrawal thresholds, as the force at which the animal withdrew each paw 50% of the time, were then calculated from the full stimulus/response curves generated. Heat withdrawal latencies were determined using a standard Hargreaves apparatus (Linton Instruments, UK). Radiant heat was applied to each hindpaw through the floor and the latency to withdrawal recorded automatically. Each hindpaw was stimulated three times, with an interval of at least 5 min between measurements, and withdrawal latency calculated from the mean of the three measurements. Tactile allodynia was determined by the number of withdrawals to 5 stimuli of the hindpaw plantar surface with a camel hair paintbrush. Cold stimulation was done with a drop (~50μ1) acetone applied to the plantar surface of the hindpaw and the degree of behaviour (flinches, shakes, withdrawals) scored. Mechanical hyperalgesia was scored as the total number of withdrawals to 3 stimulations with a sharp pin. Behavioural testing was done before and after drug administration. Drug administration was either systemic by intraperitoneal injection, or local by subcutaneous injection into the hindpaw under a brief isofluorane anaesthesia (3% in oxygen). Animals recovered completely from the anaesthesia before re-testing - usually 1-2 hours after anaesthetic recovery.

Nerve injury model

Partial saphenous nerve ligation injury was performed as previously described (Hulse et al Neuroreport 2008 May 28; 19(8):825-9) (Walczak et al Neuroscience. 2005; 132(4): 1093- 102). Anaesthesia was induced and maintained with either isoflurane or halothane in O2 (3% induction, 2% maintenance). An incision was made in the inguinal fossa of the right hind leg exposing the superficial saphenous nerve. The saphenous nerve was isolated proximal to any branch points using blunt dissection with fine forceps from the surrounding tissues. A size 4.0 sterile silk suture in rat (size 7.0 in mice) was used to ligate -50% of the total nerve, with the lateral half of the nerve being ligated for consistency. The skin was sutured with a 4.0 sterile silk suture. Animals were monitored and allowed to recover thereafter for the duration of the experiments.

Electrophysioloey

Animals were anaesthetised using sodium pentobarbital (60mg/kg i.p. rats, 150μ1 i.p. l Omg/ml (1.5mg) mice, Sigma- Aldrich, UK) and maintained deeply anaesthetised and areflexive (~20mg/kg/hr i.v. rat, ~2mg/hr i.p. mice). Rats had anaesthetic administered i.v. via an external jugular cannula and also underwent a tracheotomy. Body temperature was maintained within physiological limits (heat provided up to 37.5°C rat, ~36^DC mice) by means of a feedback controlled heater and rectal thermister. At the end of all experiments, rats and mice were killed by an overdose of sodium pentobarbital.

An incision was made along the right inguinal fossa to expose the saphenous nerve. Using blunt dissection, skin was freed from surrounding tissue and this was used to create a pool. To keep the tissue hydrated the pool was filled with warmed mineral oil. In the mouse, the pool was lined with Xantopren XL, which set once mixed with Activator Universal (Heraeus Kulzer, Germany). This prevented mineral oil leakage, due to the porosity of mouse skin. The saphenous nerve was exposed and the epineurium was removed to allow fine filaments to be dissected from the main trunk of the nerve. Filaments were placed on bipolar platinum recording electrodes for differential recordings of neuronal activity to identify individual afferent fibres. This recorded activity was amplified, filtered and captured for off-line analysis by a microl 401 (Cambridge Electronic Design, U.K) and P.C interface (Viglen) using Spike 2 software (C.E.D. Cambridge, UK).

A search protocol was used to identify afferents that innervated the hindpaw (no higher than the ankle) and that had identifiable receptive fields. Initial characterisation of afferents involved mechanical search stimulus (brush/glass rod/probe/pinch with blunt forceps) and monopolar search electrical stimulation. The mechanical search stimulus was used to apply pressure to discrete areas of the foot to stimulate robust responses from mechanically sensitive afferents. Electrical stimulation involved the use of a monopolar (cathode) electrode. The monopolar electrode was used to determine conduction velocity (CV) for fibres in m/s. This was applied to the receptive field on the hindpaw (0.5ms duration, at max. 100V intensity, rate 0.3Hz). The CV for afferents were defined as C fibres <lm/s, and A fibres as >lm/s (Dunham et al Eur J Neurosci 2008: 27:3151-3160 ) (Cain et al J Neurophysiol 2001 : 85: 1561 -1574) These stimuli allowed the identification and characterisation of individual mechanosensitive afferents.

Once a single primary afferent receptive field had been identified, it was further characterised. Brush, pinch with blunt forceps and the use of direct calibrated force with von Frey hairs (vF hair, Linton instruments, UK) were all applied to the receptive field to determine mechanical response properties. Mechanical thresholds were identified by applying vF hairs to the receptive field for 5 seconds as previously described (Dunham et al Eur J Neurosci 2008: 27:3151-3160 ). The lowest force that reproducibly elicited robust firing (firing > 3 action potentials) of the identified afferent fibre was determined to be the mechanical threshold (Cain et al J Neurophysiol 2001 : 85: 1561 -1574). Nociceptive (or high threshold mechanosensitive (HTM)) afferents responded to pinch when applied to the receptive field but not brush, typically von Frey activation thresholds were above l g (Dunham et al, Eur J Neurosci 2008: 27: 3151 -3160 ) (Hulse et al, Neuroreport 2008 May 28; 19(8): 825-829). Mechanically evoked activity was also recorded by applying a range of von Frey hairs (rat; 0.4g-180g: mouse; 0.16-2g) to the receptive field for 5 seconds, each hair being applied three times. Following this mechanical characterisation of the afferent, these responses were also recorded after vehicle and drug administration by local subcutaneous injection just outside the receptive field.

Cutaneous blood flow

Male Wistar rats were anaesthetised using sodium pentobarbital (60mg/kg i.p.) and maintained areflexive for the duration of the experiment via a jugular vein i.v. cannula (i.v. ~20mg/kg/hr sodium pentobarbital). Rat body temperature was maintained at ~37°C through a feedback controller via a rectal thermistor. The right hindpaw was supported and stabilised on a perspex platform. Subcutaneous skin temperature was monitored using a T type thermocouple. A laser doppler flow meter (Moors instruments, UK) was used to record blood flow from the cutanoeus tissues of the hindpaw. An optical probe (type P3, 30mm long) was positioned onto the dorsal surface of the hindpaw. Blood flow readouts were monitored (Moor type MBF3D monitor) until these were stable. Once this had been achieved three recordings were made of both skin temperature and blood flow, with these one minute apart. Following this, a 30 μΐ injection of vehicle (either 1 % PEG300 in saline or 0.001 % DMSO in saline) was administered adjacent to the recording site. This was performed so not to be move the skin/foot position. After five minutes three further readings were taken. Finally 200nM PTK 787, lOOnM ZM 323881 or 63μΜ adrenaline were administered as described with three subsequent recordings taken.

Withdrawal thresholds - electromyography recording (Leith et al, J Neurosci (2007); 27(42); 11296-11305) Anesthesia was induced using 4% halothane in 0₂, and a branch of the external jugular vein was cannulated for anesthetic maintenance using constant intravenous propofol (-30 mg/kg/h; Rapinovet; Schering Plough Animal Health South Harefiled UK) or alphadolone/alphaxalone (-20 mg/kg/h; Saffan; Schering Plough Animal Health) infusion, A branch of the carotid artery was exposed and cannulated for recording of blood pressure, and the trachea was cannulated to allow artificial ventilation of the animal if required. Body temperature was maintained within physiological limits by means of a feedback- controlled heating blanket and rectal probe. Anaesthesia was reduced to a level at which animals were moderately responsive to firm pinch of the contralateral forepaw and brushing of the cornea using a cotton swab. Animals were allowed to stabilize at these levels for a minimum of 30 min before recording EMG activity.

An intramuscular bipolar electrode, custom-made from two short lengths of Teflon-coated 0.075-mm-diameter stainless steel wire, was inserted into the biceps femoris of the left hind leg to record EMG activity during the withdrawal reflex. The EMG signal was amplified (5000%) and filtered (50 Hz to 5 kHz; Neurolog system), before being captured for subsequent analysis via a 1401plus (Cambridge Electronic Design) onto a PC running Spike2 version 5 software (Cambridge Electronic Design). The threshold to mechanical or thermal hindpaw stimulation was determined from the onset of the EMG indicating paw withdrawal.

Immumofliiorescence VEGFR2 and TrkA protein were localized in frozen DRG tissue sections using standard immunohistochemical/immunofluorescent techniques (Bevan et al, Nephron Physiol, 1 10, pages 57-67 (2008)).

Results

See the Figures and the Figure Legends set out above for details of the results. Discussion

The data in Figures 1, 4 and 6 show that VEGFR2 in nonnal animals is under some form of tonic activation and when that is inhibited the nociception is enhanced. This is dramatic in that it affects responses to relatively innocuous stimulation such as gentle brushing or mild cold, in addition to more noxious stimulation.

The data in Figure 5 show that these effects are not mediated through alteration in tissue blood perfusion.

The data in Figures 2 and 3 show that partial activation (agonism) of the VEGFR2 receptor, here with VEGFi₆₅b, is antiallodynic and analgesic in nerve injured animals.

The data in Figure 7 show that blockade of VEGFR2 in nerve injured animals increases allodynia in the whole animal.

The data in Figure 8 show that full agonists at VEGFR2 (VEGF₁₆₅) result in allodynia, whereas partial agonists (VEGF₁₆₅b) have no effect, in the whole animal. The data in Figure 9 show that VEGFR2 agonists that do not effectively recruit the co- receptor neuropilin (VEGF_]2i) increased mechanical and cold allodynia and hyperalgesia in the whole animal.

The data in Figure 10 show that VEGF forms lacking C terminal 6 amino acids (VEGF_]59) do not result in changes in nociceptive behaviour.

The data in Figures 1 1 and 12 show that the full VEGFR2 agonist VEGF_]65 has direct pro- nociceptive actions on primary afferent nociceptors, whereas the partial agonist VEGF^sb has no effect.

The data in Figure 13 show that VEGFR2 is expressed and upregulated in nociceptive neurones 7 days after nerve injury and is therefore a target for agonists in these neurons. The data in Figure 14 show that the allodynic actions of VEGFi₆₅ are mediated through the TRPV1 receptor, which, in the peripheral nervous system, is expressed exclusively on nociceptive afferents. From these data it is seen that agonists at the VEGFR2 receptor are expected to be antiallodynic and analgesic in non-inflammatory pain.

Industrial Applicability The present invention provides a new family of active agents for use against VEGFR2- mediated non-inflammatory pain.

The activity of the VEGFR2 agonists, for example the

family of proteins, particularly VEGFi₆₅b, soluble VEGF receptors having activity as inhibitors of VEGFR2 antagonists and VEGF_XXX isoforms that have the C-terminal hexapeptide corresponding to expression immediately after exon 7, and in some cases also those that lack neuropilin binding mediating portions of the amino acid sequence, to treat or prevent pain such as VEGFR2 -mediated non-inflammatory pain is unexpected in view of the known properties of the proteins.

This finding opens up many new therapeutic and other treatments of human and animal subjects suffering from or susceptible to a range of pain states, including pain states caused by or associated with other disorders of therapies thereof.

Claims

1. An active agent comprising one or more VEGFR2 agonist for use in treating or preventing VEGFR2 -mediated non-inflammatory pain.

2. A method of treating or preventing VEGFR2-mediated non-inflammatory pain which comprises administering to a subject suffering therefrom or susceptible thereto an effective amount of one or more VEGFR2 agonist.

3. Use of one or more VEGFR2 agonist in the manufacture of a composition for treating or preventing VEGFR2-mediated non-inflammatory pain.

4. An agent, method or use according to any one of claims 1 to 3, wherein the VEGFR2 agonist comprises (1) one or more agonist of the VEGFR2 receptor, (2) one or more inhibitor of antagonists of the VEGFR2 receptor, (3) one or more inhibitor of endogenous VEGFR2 full agonists to promote or obtain partial agonism of the VEGFR2 receptor by endogenous partial agonists thereof, (4) one or more promoter of phosphorylation of the VEGFR2 receptor to promote or obtain partial agonism thereof by endogenous partial agonists thereof, (5) any mixture or combination thereof, or (6) one or more agent which promotes the endogenous expression of one or more VEGFR2 agonist of categories (1) to (5) above in cells of a subject.

5. An agent, method or use according to claim 4, wherein the VEGFR2 agonist is a VEGF_xxxb active agent which selectively promotes the expression of VEGF_xxxb in preference to VEGF_XXX in cells of a subject.

6. An agent, method or use according to any one of the preceding claims, wherein the VEGFR2 agonist comprises a soluble VEGF receptor having activity as inhibitor of VEGFR2 antagonists.

7. An agent, method or use according to any one of the preceding claims, wherein the VEGFR2 agonist comprises a VEGF^ isoform that has the C-terminal hexapeptide corresponding to expression immediately after exon 7, or a VEGF_xxX isoform that lacks neuropilin binding mediating portions of the amino acid sequence.

8. An expression vector system which causes the expression of a VEGFR2 agonist in a host organism, for use in treating or preventing VEGFR2-mediated non-inflammatory pain.

9. A method of treating or preventing VEGFR2-mediated non-inflammatory pain in a subject, which comprises administering to the subject an effective amount of an expression vector system which causes the expression of a VEGFR2 agonist in a host organism.

10. Use of an expression vector system which causes the expression of a VEGFR2 agonist in a host organism, in the manufacture of a composition for treating or preventing VEGFR2-mediated non-inflammatory pain.

11. An agent, method or use according to any one of the preceding claims, wherein the VEGFR2 agonist comprises one or more VEGF_xxxb selected from one or more of

VEGF,₆₅b, VEGF_{) g9}b, VEGF,₄₅b, VEGF_{] 83}b and VEGF,_2]b, preferably VEGF_{l 65}b.

12. An agent, method to use according to any one of the preceding claims, wherein the treatment of pain comprises normalising, using one or more VEGFR2 agonist, the sensitivity towards pain of a subject treated or being co-treated with one or more different pain treatment agent.

13. A VEGFR2 agonist for use in treating or preventing VEGFR2-mediated noninflammatory pain, substantially as herein described with reference to the Examples.