AU2011202225B2 - Non-cytotoxic protein conjugates - Google Patents

Abstract

Non-cytotoxic Protein Conjugates 5 A non-cytotoxic protein conjugate for inhibition or reduction of exocytic fusion in a nociceptive sensory afferent cell, comprising: (i) a dynorphin Targeting Moiety (TM), wherein said TM is an agonist of a receptor present on said nociceptive sensory afferent cell, and wherein said receptor undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell; (ii) a non-cytotoxic 1o protease or a fragment thereof, wherein the protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of said nociceptive sensory afferent cell; and (iii) a Translocation Domain, wherein the Translocation Domain translocates the protease or protease fragment from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory is afferent cell. Nucleic acid sequences encoding the protein conjugates, methods of preparing same and uses thereof are also described.

Description

AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant(s): ALLERGAN, INC. and HEALTH PROTECTION AGENCY Invention Title: NON-CYTOTOXIC PROTEIN CONJUGATES The following statement is a full description of this invention, including the best method for performing it known to me/us: 11 Non-cytotoxic Protein Conjugates This invention relates to a non-cytotoxic protein conjugate, and to the use of said 5 conjugate for treating pain. Toxins may be generally divided into two groups according to the type of effect that they have on a target cell. In more detail, the first group of toxins kill their natural target cells, and are therefore known as cytotoxic toxin molecules. This group of 1o toxins is exemplified inter alia by plant toxins such as ricin, and abrin, and by bacterial toxins such as diphtheria toxin, and Pseudomonas exotoxin A. Cytotoxic toxins typically kill their target cells by inhibiting the cellular process of protein synthesis. 15 In contrast, the second group of toxins, which are known as non-cytotoxic toxins, do not (as their name confirms) kill their natural target cells. Non-cytotoxic toxins have attracted much less commercial interest than have their cytotoxic counterparts, and exert their effects on a target cell by inhibiting cellular processes other than protein synthesis. As with their cytotoxic counterparts, non-cytotoxic toxins are produced 20 from a variety of sources such as plants, and bacteria. Bacterial non-cytotoxic toxins are now described in more detail. Clostridial neurotoxins are proteins that typically have a molecular mass of the order of 150 kDa. They are produced by various species of bacteria, especially of the 25 genus Clostridium, most importantly C. tetaniand several strains of C. botulinum, C. butyricum and C. argentinense. There are at present eight different classes of the clostridial neurotoxin, namely: tetanus toxin, and botulinum neurotoxin in its serotypes A, B, C 1 , D, E, F and G, and they all share similar structures and modes of action. 30 Clostridial neurotoxins represent a major group of non-cytotoxic toxin molecules, and 2 are synthesised by the host bacterium as single polypeptides that are modified post translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. The two chains are termed the heavy chain (H chain), which has a molecular mass of approximately 100 kDa, and the light chain 5 (L-chain), which has a molecular mass of approximately 50 kDa. L-chains possess a protease function (zinc-dependent endopeptidase activity) and exhibit high substrate specificity for vesicle and/or plasma membrane associated proteins involved in the exocytic process. L-chains from different clostridial species 1o or serotypes may hydrolyse different but specific peptide bonds in one of three substrate proteins, namely synaptobrevin, syntaxin or SNAP-25. These substrates are important components of the neurosecretory machinery. Non-cytotoxic toxins are also produced by other bacteria, such as from the genus 15 Neisseria, most importantly from the species N. gonorrhoeae. For example, Neisseria sp. produces the non-cytotoxic toxin IgA protease (see W099/58571). It has been well documented in the art that toxin molecules may be re-targeted to a cell that is not the toxin's natural target cell. When so re-targeted, the modified toxin 20 is capable of binding to a desired target cell and, following subsequent translocation into the cytosol, is capable of exerting its effect on the target cell. Said re-targeting is achieved by replacing the natural Targeting Moiety (TM) of the toxin with a different TM. In this regard, the TM is selected so that it will bind to a desired target cell, and allow subsequent passage of the modified toxin into an endosome within 25 the target cell. The modified toxin also comprises a translocation domain to enable entry of the non-cytotoxic protease into the cell cytosol. The translocation domain can be the natural translocation domain of the toxin or it can be a different translocation domain obtained from a microbial protein with translocation activity. 30 For example, in the context of non-cytotoxic toxin molecules, it has been well documented that a clostridial neurotoxin may be re-targeted by incorporation of a Targeting Moiety (TM), which is not the natural TM of a clostridial neurotoxin. The 3 described chemical conjugation and recombinant methodologies are now regarded as conventional, and reference is made to Hermanson, G.T. (1996), Bioconjugate techniques, Academic Press, and to Wong, S.S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press. 5 For example, W094/21300 describes modified clostridial neurotoxin molecules that are capable of regulating Integral Membrane Protein (IMP) density present at the cell surface of the target cell. The modified neurotoxin molecules are thus capable of controlling cell activity (e.g. glucose uptake) of the target cell. W096/33273 and 1o W099/17806 describe modified clostridial neurotoxin molecules that target peripheral sensory afferents. The modified neurotoxin molecules are thus capable of demonstrating an analgesic effect. WOOO/10598 describes the preparation of modified clostridial neurotoxin molecules that target mucus hypersecreting cells (or neuronal cells controlling said mucus hypersecreting cells), which modified 15 neurotoxins are capable of inhibiting hypersecretion from said cells. WOO1/21213 describes modified clostridial neurotoxin molecules that target a wide range of different types of non-neuronal target cells. The modified molecules are thus capable of preventing secretion from the target cells. Additional publications in the technical field of re-targeted toxin molecules include: WOOO/62814; WOOO/04926; 20 US5,773,586; W093/15766; WOOO/61192; and W099/58571. Thus, from the above-described publications, it will be appreciated that the basic concept of re-targeting a non-cytotoxic protease to a desired target cell, by selecting a TM that has a corresponding receptor present on the target cell, has been well 25 documented. However, different receptors present on a target cell of interest demonstrate different binding affinities for different TMs. This may be a particular problem with pain sensing cells, which possess a wide range of receptor types having different binding 30 affinities for different TMs. Thus, a re-targeted conjugate comprising a particular TM (that binds to a receptor on a pain-sensing cell) may demonstrate a low binding affinity for a pain-sensing target cell, which is undesirable.

4 There is therefore a need to develop modified non-cytotoxic conjugates that address one or more of the above problems. Of particular interest is the development of an improved conjugate for use in treating pain. 5 The present invention seeks to address one or more of the above problems by using as the conjugate's Targeting Moiety (TM) an "agonist" of a receptor that is present on the pain-sensing target cell of interest. In preferred embodiments, the pain sensing target cell is a nociceptive sensory afferent, more preferably a primary nociceptive sensory afferent. In particularly preferred embodiments, the TM is an 10 agonist of the opioid-like receptor-1 (ORL 1 ) receptor. Accordingly, in a first aspect, the present invention provides a non-cytotoxic conjugate for inhibition or reduction of exocytic fusion in a nociceptive sensory afferent cell, comprising: 15 (i) a Targeting Moiety (TM), wherein said TM is an agonist of a receptor present on said nociceptive sensory afferent cell, and wherein said receptor 20 undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell (ii) a non-cytotoxic protease or a fragment thereof, 25 wherein the protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of said nociceptive sensory afferent cell; and (iii) a Translocation Domain, 30 wherein the Translocation Domain translocates the protease or protease 6615129_2 (GHMatters) P73912.AU.1 JESSIEL 4a fragment from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory afferent cell. The present invention as claimed herein is described in the following items 1 to 9: 5 1. A non-cytotoxic protein conjugate for inhibition or reduction of exocytic fusion in a nociceptive sensory afferent cell, comprising: (i) a dynorphin Targeting Moiety (TM), 10 wherein said TM is an agonist of a receptor present on said nociceptive sensory afferent cell, and wherein said receptor undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell 15 (i) a non-cytotoxic protease or a fragment thereof, wherein the protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of said 20 nociceptive sensory afferent cell; and (ii) a Translocation Domain, wherein the Translocation Domain translocates the protease or 25 protease fragment from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory afferent cell, wherein said conjugate comprises an amino acid sequence selected from 30 the group consisting of SEQ ID NOs 103, 104, 105, 106, 107, 108 and 109. 6615129_2 (GHMatters) P73912.AU.1 JESSIEL 5 2. A pharmaceutical composition, comprising a conjugate according to item 1 and a pharmaceutically acceptable carrier. 5 3. A DNA construct encoding the conjugate of item 1. 4. A DNA construct according to item 3, wherein the construct comprises a DNA sequence selected from SEQ ID NOs 102. 10 5. A method of preparing the conjugate of item 1, comprising expressing the DNA construct of Claim 3 in a host cell. 6. A method for treating, preventing or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of a conjugate 15 according to item 1 or a composition according to item 2. 7. A method according to item 6, wherein the pain is chronic pain selected from neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain and referred pain. 20 8. Use of a conjugate according to item 1 or a composition according to item 2, for the manufacture of a medicament for treating, preventing or ameliorating pain. 25 9. Use according to item 8, wherein the pain is chronic pain selected from neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain and referred pain. The use of an "agonist", which would normally stimulate a biological process, 30 particularly exocytosis (for example, an increase in cellular secretion, or an up 6615129_2 (GHMatters) P73912.AU.1 JESSIEL 5a regulation in membrane protein expression), is an exciting development in the technical field of re-targeted toxins. Furthermore, it is particularly surprising that an agonist may be employed in a therapeutic composition to achieve a reduction or inhibition of a biological process that the agonist would normally stimulate. 5 The agonist-containing conjugates of the present invention represent a distinct sub set of toxin conjugates. In more detail, the conjugates of the present invention comprise TMs that have been selected on the basis of specific agonist properties rather than on the simple basis that they have a corresponding receptor on a pain 10 sensing target cell of interest. Conventionally, an agonist has been considered any molecule that can either increase or decrease activities within a cell, namely any molecule that simply causes an alteration of cell activity. For example, the conventional meaning of an agonist 15 would include: a chemical substance capable of combining with a receptor on a cell and initiating a reaction or activity, or a drug that induces an active response by activating receptors, whether the response is an increase or decrease in cellular activity. 20 However, for the purposes of this invention, an agonist is more specifically defined as a molecule that is capable of stimulating the process of exocytic fusion in a pain sensing target cell, which process is susceptible to inhibition by a protease (or fragment thereof) capable of cleaving a protein of the exocytic fusion apparatus in said target cell. 25 Accordingly, the particular agonist definition of the present invention would exclude 6615129_2 (GHMatters) P73912.AU.1 JESSIEL 6 many molecules that would be conventionally considered as agonists. For example, nerve growth factor (NGF) is an agonist in respect of its ability to promote neuronal differentiation via binding to a TrkA receptor. However, NGF is not an agonist when assessed by the above criteria because it is not a principal inducer of exocytic 5 fusion. In addition, the process that NGF stimulates (i.e. cell differentiation) is not susceptible to inhibition by the protease activity of a non-cytotoxic toxin molecule. In use, an agonist-containing conjugate of the present invention does not deactivate an agonist receptor on a pain-sensing target cell, but rather the protease activity of 1o the conjugate serves to negate the agonist-mediated response. Furthermore, once delivered to the cytosol of the pain-sensing target cell, the protease component of a conjugate of the present invention inhibits or blocks the action of all subsequent agonists capable of causing the same effect (i.e. increased 15 exocytic fusion) in the same target cell. This is advantageous and means that the conjugates of the present invention have application in situations where multiple agonists may be responsible for causing the sensation of pain. Thus, when designing a conjugate of the present invention, the TM that is selected for delivery need not necessarily be the principal agonist involved in causing the sensation of 20 pain. Agonist-mediated delivery according to the present invention provides the following significant advantage over previous non-cytotoxic protease-containing therapeutics: use of an agonist may confer preferential binding and/or internalisation properties on 25 the conjugate. This, in turn, may result in more efficient delivery of the protease component to a pain-sensing target cell. In addition, use of an agonist as a TM is self-limiting with respect to side-effects. In more detail, binding of an agonist to a pain-sensing target cell increases exocytic 30 fusion, which may exacerbate the sensation of pain. However, the exocytic process that is stimulated by agonist binding is subsequently reduced or inhibited by the protease component of the conjugate.

7 In preferred embodiments of the invention, the TM is an agonist of the ORL 1 receptor. The ORL 1 receptor is present on pain-sensing cells in the body. 5 The ORL 1 receptor is a member of the G-protein-coupled class of receptors, and has a seven transmembrane domain structure. The properties of the ORL 1 receptor are discussed in detail in Mogil & Pasternak (2001), Pharmacological Reviews, Vol. 53, No. 3, pages 381-415. io Throughout this specification, reference to the "ORL 1 receptor" embraces all members of the ORL, receptor family. Members of the ORL 1 receptor family typically have a seven transmembrane domain structure, and are coupled to G-proteins of the Gi and Go families. A method for determining the G-protein-stimulating activity of ligands of the ORL, receptor is given in Example 17. A method for measuring 15 reduction in cellular cAMP levels following ORL, activation is given in Example 16. A further characteristic of members of the ORL 1 receptor family is that they are typically able to bind nociceptin (the natural ligand of ORL 1 ). As an example, all alternative splice variants of the ORL, receptor, are members of the ORL, receptor family. 20 The conjugates of the present invention generally demonstrate a reduced binding affinity (in the region of up to 100-fold) for nociceptive sensory afferent target cells when compared with the corresponding 'free' TM. However, despite this observation, the conjugates of the present invention surprisingly demonstrate good 25 efficacy. This can be attributed to two principal features. First, the non-cytotoxic protease component is catalytic - thus, the therapeutic effect of a few such molecules is rapidly amplified. Secondly, the receptors present on the nociceptive sensory afferents need only act as a gateway for entry of the therapeutic, and need not necessarily be stimulated to a level required in order to achieve a ligand-receptor 30 mediated pharmacological response. Accordingly, the conjugates of the present invention may be administered at a dosage that is much lower that would be employed for other types of analgesic molecules such as NSAIDS, morphine, and 8 gabapentin. The latter molecules are typically administered at high microgram to milligram (even up to hundreds of milligram) quantities, whereas the conjugates of the present invention may be administered at much lower dosages, typically at least 10-fold lower, and more typically at 100-fold lower. 5 In a particularly preferred embodiment of the invention, the TM of the conjugate is nociceptin - the natural ligand for the ORL 1 receptor. Nociceptin targets the ORL 1 receptor with high affinity. 1o Examples of other preferred TMs include: Code Sequence Ref. SEQ ID No. Nociceptin 1-17 FGGFTGARKSARKLANQ [1] 1,2 Nociceptin 1-11 FGGFTGARKSA [1] 3,4 Nociceptin [Y10]1-11 FGGFTGARKYA [1] 5,6 Nociceptin [Y11]1-11 FGGFTGARKSY [1] 7,8 Nociceptin [Y14]1-17 FGGFTGARKSARKYANQ [1] 9,10 Nociceptin 1-13 FGGFTGARKSARK [2] 11,12 Nociceptin [R14K15] FGGFTGARKSARKRKNQ [3,4] 13,14 1-17 (also known as "variant" nociceptin) Nociceptin 1-13-NH 2

FGGFTGARKSARK-NH

2 (5] Nociceptin (pNO 2 )FGGFTGARKSARKLANQ [5] Phe (p-NO 2 ) 1-17 Lofentanil Non-peptide agonists [5] - 9 Code Sequence Ref. SEQ ID No. Etorphine Non-peptide agonists [5] Peptide agonist Peptide agonists from combinatorial [6] library approach [1] Mogil & Pasternak, 2001, Pharmacol. Rev., 53, 381-415 [2] Maile et al., 2003, Neurosci. Lett., 350, 190-192 [3] Rizzi et al., 2002, J. Pharmacol. Exp. Therap., 300, 57-63 5 [4] Okada et al., 2000, Biochem. Biophys. Res. Commun., 278, 493-498 [5] Zaveri, 2003, Life Sci., 73, 663-678. [6] Dooley et al., 1997, J Pharmacol Exp Ther. 283(2), 735-41. The TM preferably comprises a maximum of 50 amino acid residues, more 10 preferably a maximum of 40 amino acid residues, particularly preferably a maximum of 30 amino acid residues, and most preferably a maximum of 20 amino acid residues. For example, nociceptin is a 17 amino acid residue peptide. The above-identified "variant" TM demonstrates particularly good binding affinity 15 (when compared with natural nociceptin) for nociceptive sensory afferents. Generally speaking, a TM-containing conjugate will demonstrate an approximate 100-fold reduction in binding ability vis-e-vis the TM per se. The above-mentioned "variant" TM per se demonstrates an approximate 3- to 10-fold increase in binding ability for a nociceptive sensory afferent vis-e-vis natural nociceptin. Thus, a "variant" TM 20 containing fusion might be expected to demonstrate an approximate 10-fold reduction in binding ability for a nociceptive sensory afferent vis-e-vis 'free' nociceptin. However, the present inventors have demonstrated that conjugates comprising said "variant" TM demonstrate a binding ability that (most surprisingly) closely mirrors that of 'free' nociceptin - see Figure 17. 25 In the context of the present invention, the term agonist of the ORL 1 receptor (such as nociceptin, or any one of the peptides listed in the table above) embraces 10 molecules having at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology with said agonist. The agonist homologues retain the agonist properties of nociceptin at the ORL 1 receptor, which may be tested using the methods provided in Example 10. 5 The invention also encompasses fragments, variants, and derivatives of any one of the TMs described above. These fragments, variants, and derivatives will substantially retain the properties that are ascribed to said TMs. io In addition to the above-mentioned opioid and non-opioid classes of TMs, a variety of other polypeptides are suitable for targeting the conjugates of the present invention to nociceptive sensory afferents (e.g. to nociceptors). In this regard, particular reference is made to galanin and derivatives of galanin. Galanin receptors are found pre- and post-synaptically in DRGs (Liu & Hokfelt, (2002), Trends Pharm. 15 Sci., 23(10), 468-74), and are enhanced in expression during neuropathic pain states. Proteinase-activated receptors (PARs) are also a preferred group of TMs of the present invention, most particularly PAR-2. It is known that agonists of PAR-2 induce/ elicit acute inflammation, in part via a neurogenic mechanism. PAR2 is expressed by primary spinal afferent neurons, and PAR2 agonists stimulate release 20 of substance P (SP) and calcitonin gene-related peptide (CGRP) in peripheral tissues. A particularly preferred set of TMs of the present invention includes: Ligand Reference Nociceptin Guerrini, et al., (1997) J. Med. Chem., 40, pp. 1789-1793 p-endorphin Blanc, et al., (1983) J. Biol. Chem., 258(13), pp. 8277-8284 Endomorphin-1; Zadina, et al., (1997). Nature, 386, pp. Endomorphin-2 499-502 11 Ligand Reference Dynorphin Fields & Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds. Met-enkephalin Fields & Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds. Leu-enkephalin Fields & Basbaum (2002) Chapter 11, In The Textbook of Pain, Wall & Melzack eds. Galanin Xu et al., (2000) Neuropeptides, 34 (3&4), 137-147 PAR-2 peptide Vergnolle et al., (2001) Nat. Med., 7(7), 821-826 The agonist properties of a TM can be confirmed using the methods described in Example 1. These methods are based on previous experiments (see Inoue et al. 5 (1998) Proc. Natl. Acad. Sci., 95, 10949-10953), which confirm that the natural agonist of the ORL 1 receptor, nociceptin, causes the induction of substance P release from nociceptive primary afferent neurons. This is supported by the facts that: 10 > the nociceptin-induced responses are abolished by specific NK1 receptor (the substance P receptor) antagonists; and > pre-treatment of the cells with capsaicin (which depletes substance P from small diameter primary afferent neurons) attenuates the 15 nociceptin-induced responses. Similarly, Inoue et al. confirm that an intraplantar injection of botulinum neurotoxin type A abolishes the nociceptin-induced responses. Since it is known that BoNT 12 inhibits the release of substance P from primary afferent neurons (Welch et al., (2000), Toxicon, 38, 245-258), this confirms the link between nociceptin-ORL 1 interaction and subsequent release of substance P. 5 Thus, a TM can be said to have agonist activity at the ORL 1 receptor if the TM causes an induction in the release of substance P from a nociceptive sensory afferent neuron (see Example 1). In another embodiment, opioids represent a preferred group of TMs of the present 10 invention. Within this family of peptides is included enkephalins (met and leu), endomorphins 1 and 2, p-endorphin and dynorphin. Opioid peptides are frequently used in the clinic to modify the activity to nociceptors, and other cells involved in the pain response. As exemplified by the three-step World Health Organisation Analgesic Ladder, opioids have entry points into the pharmacological treatment of 15 chronic cancer and non-cancer pain at all three stages, underlining their importance to the treatment of pain. Reference to opioids embraces fragments, variants and derivatives thereof, which retain the ability to bind to nociceptive sensory afferents. The protease of the present invention embraces all naturally-occurring non-cytotoxic proteases that are capable of cleaving one or more proteins of the exocytic fusion 20 apparatus in eukaryotic cells. The protease of the present invention is preferably a bacterial protease. More preferably, the bacterial protease is selected from the genera Clostridium or 25 Neisseria (e.g. a clostridial L-chain, or a neisserial IgA protease preferably from N. gonorrhoeae). The present invention also embraces modified non-cytotoxic proteases, which include amino acid sequences that do not occur in nature and/or synthetic amino 30 acid residues, so long as the modified proteases still demonstrate the above mentioned protease activity.

13 The protease of the present invention preferably demonstrates a serine or metalloprotease activity (e.g. endopeptidase activity). The protease is preferably specific for a SNARE protein (e.g. SNAP-25, synaptobrevinNAMP, or syntaxin). 5 Particular mention is made to the protease domains of neurotoxins, for example the protease domains of bacterial neurotoxins. Thus, the present invention embraces the use of neurotoxin domains, which occur in nature, as well as recombinantly prepared versions of said naturally-occurring neurotoxins. 10 Exemplary neurotoxins are produced by clostridia, and the term clostridial neurotoxin embraces neurotoxins produced by C. tetani (TeNT), and by C. botulinum (BoNT) serotypes A-G, as well as the closely related BoNT-like neurotoxins produced by C. barati and C. butyricum. The above-mentioned abbreviations are used throughout the present specification. For example, the nomenclature BoNT/A denotes the 15 source of neurotoxin as BoNT (serotype A). Corresponding nomenclature applies to other BoNT serotypes. The term L-chain fragment means a component of the L-chain of a neurotoxin, which fragment demonstrates a metalloprotease activity and is capable of proteolytically 20 cleaving a vesicle and/or plasma membrane associated protein involved in cellular exocytosis. A Translocation Domain is a molecule that enables translocation of a protease (or fragment thereof) into a pain-sensing target cell such that a functional expression of 25 protease activity occurs within the cytosol of the target cell. Whether any molecule (e.g. a protein or peptide) possesses the requisite translocation function of the present invention may be confirmed by any one of a number of conventional assays. For example, Shone C. (1987) describes an in vitro assay employing liposomes, 30 which are challenged with a test molecule. Presence of the requisite translocation function is confirmed by release from the liposomes of K+ and/or labelled NAD, which may be readily monitored (see Shone C. (1987) Eur. J. Biochem; vol. 167(1): 14 pp. 175-180). A further example is provided by Blaustein R. (1987), which describes a simple in vitro assay employing planar phospholipid bilayer membranes. The membranes are 5 challenged with a test molecule and the requisite translocation function is confirmed by an increase in conductance across said membranes (see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp. 115-120). Additional methodology to enable assessment of membrane fusion and thus 1o identification of Translocation Domains suitable for use in the present invention are provided by Methods in Enzymology, Vols. 220 and 221, Membrane Fusion Techniques, Parts A and B, Academic Press 1993. The Translocation Domain is preferably capable of formation of ion-permeable pores 15 in lipid membranes under conditions of low pH. Preferably, it has been found to use only those portions of the protein molecule capable of pore-formation within the endosomal membrane. The Translocation Domain may be obtained from a microbial protein source, in 20 particular from a bacterial or viral protein source. Hence, in one embodiment, the Translocation Domain is a translocating domain of an enzyme, such as a bacterial toxin or viral protein. It is well documented that certain domains of bacterial toxin molecules are capable 25 of forming such pores. It is also known that certain translocation domains of virally expressed membrane fusion proteins are capable of forming such pores. Such domains may be employed in the present invention. The Translocation Domain may be of a clostridial origin, namely the HN domain (or a 30 functional component thereof). HN means a portion or fragment of the H-chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H chain, or the domain corresponding to that fragment in the intact H-chain. Examples 15 of suitable clostridial Translocation Domains include: Botulinum type A neurotoxin - amino acid residues (449-871) Botulinum type B neurotoxin - amino acid residues (441-858) 5 Botulinum type C neurotoxin - amino acid residues (442-866) Botulinum type D neurotoxin - amino acid residues (446-862) Botulinum type E neurotoxin - amino acid residues (423-845) Botulinum type F neurotoxin - amino acid residues (440-864) Botulinum type G neurotoxin - amino acid residues (442-863) 10 Tetanus neurotoxin - amino acid residues (458-879) For further details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, we refer to Henderson et al. (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press. 15 The term HN embraces naturally-occurring neurotoxin HN portions, and modified HN portions having amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified HN portions still demonstrate the above-mentioned translocation function. 20 Alternatively, the Translocation Domain may be of a non-clostridial origin (see table below). Examples of non-clostridial Translocation Domain origins include, but are not restricted to, the translocation domain of diphtheria toxin [O'Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89, 6202-6206; Silverman et al., J. Biol. Chem. 25 (1993) 269, 22524-22532; and London, E. (1992) Biochem. Biophys. Acta., 1112, pp.25-51], the translocation domain of Pseudomonas exotoxin type A [Prior et al. Biochemistry (1992) 31, 3555-3559], the translocation domains of anthrax toxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], a variety of fusogenic or hydrophobic peptides of translocating function [Plank et al. J. Biol. 30 Chem. (1994) 269, 12918-12924; and Wagner et al (1992) PNAS, 89, pp.7934 7938], and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp. 1986-1992]. The Translocation Domain may mirror the Translocation Domain present in a 16 naturally-occurring protein, or may include amino acid variations so long as the variations do not destroy the translocating ability of the Translocation Domain. Particular examples of viral Translocation Domains suitable for use in the present 5 invention include certain translocating domains of virally expressed membrane fusion proteins. For example, Wagner et al. (1992) and Murata et al. (1992) describe the translocation (i.e. membrane fusion and vesiculation) function of a number of fusogenic and amphiphilic peptides derived from the N-terminal region of influenza virus haemagglutinin. Other virally expressed membrane fusion proteins io known to have the desired translocating activity are a translocating domain of a fusogenic peptide of Semliki Forest Virus (SFV), a translocating domain of vesicular stomatitis virus (VSV) glycoprotein G, a translocating domain of SER virus F protein and a translocating domain of Foamy virus envelope glycoprotein. Virally encoded "spike proteins" have particular application in the context of the present invention, 15 for example, the El protein of SFV and the G protein of VSV. Use of the Translocation Domains (listed below) includes use of sequence variants thereof. A variant may comprise one or more conservative nucleic acid substitutions and/or nucleic acid deletions or insertions, with the proviso that the variant 20 possesses the requisite translocating function. A variant may also comprise one or more amino acid substitutions and/or amino acid deletions or insertions, so long as the variant possesses the requisite translocating function. Translocation Amino acid References Domain source residues Diphtheria toxin 194-380 Silverman et al., 1994, J. Biol. Chem. 269, 22524-22532 London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 17 Translocation Amino acid References Domain source residues Domain 11 of 405-613 Prior et al., 1992, Biochemistry pseudomonas 31, 3555-3559 exotoxin Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virus GLFGAIAGFIENGWE Plank et al., 1994, J. Biol. Chem. haemagglutinin GMIDGWYG, and 269,12918-12924 Variants thereof Wagner et al., 1992, PNAS, 89, 7934-7938 Murata et al., 1992, Biochemistry 31, 1986-1992 Semliki Forest virus Translocation domain Kielian et al., 1996, J Cell Biol. fusogenic protein 134(4), 863-872 Vesicular Stomatitis 118-139 Yao et al., 2003, Virology 310(2), virus glycoprotein G 319-332 SER virus F protein Translocation domain Seth et al., 2003, J Virol 77(11) 6520-6527 Foamy virus Translocation domain Picard-Maureau et al., 2003, J envelope Virol. 77(8), 4722-4730 glycoprotein Once a potential receptor agonist (e.g. an ORL1 agonist) has been identified, one or more of the following optional steps may be carried out: 5 (A) confirming that the putative agonist molecule or agonist is capable of being combined with a non-cytotoxic protease (or a fragment thereof) and optionally a Translocation Domain to form a conjugate of the present invention; and/or 10 (B) confirming that said putative agonist molecule or agonist binds to the receptor on the pain-sensing target cell, which 18 receptor is susceptible to receptor-mediated endocytosis; and/or (C) confirming that said putative agonist molecule or agonist is 5 able to deliver a non-cytotoxic protease (or fragment thereof) into the cytosol of a pain-sensing target cell. The above steps (A)-(C) may be confirmed by routine tests that would be readily available to a skilled person. 1o For example, step (A) may be performed by a simple chemical conjugation experiment using conventional conjugation reagents and/or linker molecules, followed by native polyacrylamide gel electrophoresis to confirm that a conjugate of the present invention is formed that has the anticipated molecular weight. The conjugate components are typically linked together (optionally via linker molecules) is by covalent bonds. For example, step (B) may be performed by any one of a range of methodologies for assessment of binding of a ligand. Standard text, for example "Receptor-Ligand Interactions. A Practical Approach. Ed. E. C. Hulme, IRL Press, 1992" are available 20 that describe such approaches in detail. In brief, the agonist or putative agonist molecule is labelled (for example, with 125-iodine) and applied to a cell preparation in vitro in the presence of an excess of unlabelled agonist. The purpose of the unlabelled material is to saturate any non-specific binding sites. The agonist is incubated with the cell preparation for sufficient time to achieve equilibrium, and the 25 amount of label bound to the cells assessed by measuring cell associated radioactivity, for example by scintillation or gamma counting. A further example involves gold-labelling of the agonist (or putative agonist), followed by the use of electron microscopy to monitor the cellular transport progress 30 of the labelled agonist [see the basic methodology described by Rabinowitz S. (1992); J. Cell. Biol. 116(1): pp. 95-112; and that described by van Deurs (1986); J. Cell. Biol. 102: pp. 37-47].

19 For example, step (C) may be performed by contacting the conjugate prepared in step (A) with a suitable target cell and assessing cleavage of the substrate. This is performed by extraction of the SNARE proteins, followed by Western blotting of 5 SDS-PAGE-separated samples. Cleavage of substrate is indicative of delivery of the protease into the target cell. In this regard, cleavage may be monitored by disappearance of substrate and/or appearance of cleavage product. A particularly useful antibody that selectively binds to the cleaved substrate product is described in W095/33850. 10 Preparation of a conjugate according to the present invention is now discussed. It is known in the art that the Hc portion of a neurotoxin molecule can be removed from the other portion of the H-chain, known as HN, such that the HN fragment 15 remains disulphide linked to the L-chain of the neurotoxin providing a fragment known as LHN. Thus, in one embodiment of the present invention the LHN fragment of a neurotoxin is covalently linked, using linkages which may include one or more spacer regions, to a TM. 20 In another embodiment of the invention, the Hc domain of a neurotoxin is mutated, blocked or modified, e.g. by chemical modification, to reduce or preferably incapacitate its ability to bind the neurotoxin to receptors at the neuromuscular junction. This modified neurotoxin is then covalently linked, using linkages which may include one or more spacer regions, to a TM. 25 In another embodiment of the invention, the H-chain of a neurotoxin, in which the Hc domain is mutated, blocked or modified, e.g. by chemical modification, to reduce or preferably incapacitate its native binding ability, is combined with the L-chain of a different neurotoxin, or another protease capable of cleaving a protein of the 30 exocytic fusion apparatus (e.g. IgA protease of N. gonorrhoeae). This hybrid, modified neurotoxin is then covalently linked, using linkages which may include one or more spacer regions, to a TM.

20 In another embodiment of the invention, the HN domain of a neurotoxin is combined with the L-chain of a different neurotoxin, or another protease capable of cleaving a protein of the exocytic fusion apparatus (e.g. IgA protease of N. gonorrhoeae). This 5 hybrid is then covalently linked, using linkages which may include one or more spacer regions, to a TM. In another embodiment of the invention, the protease (for example the L-chain component of a neurotoxin) is covalently linked, using linkages that may include one 10 or more spacer regions, to a TM that can also effect the internalisation of the protease into the cytoplasm of the relevant target cell(s). In another embodiment of the invention, the protease (for example the L-chain component of a neurotoxin) is covalently linked, using linkages which may include 15 one or more spacer regions, to a translocation domain to effect transport of the protease fragment into the cytosol. In use, the domains of a conjugate according to the present invention are associated with each other. In one embodiment, two or more of the domains may be joined 20 together either directly (e.g. by a covalent linkage), or via a linker molecule. A variety of different linker/ spacer molecules may be employed in any of the fusion proteins of the present invention. Examples of such spacer molecules include those illustrated in Figures 31 and 32. Particular mention here is made to GS15, GS20, 25 GS25, and Hx27 - see Figures 31 and 32. The present inventors have unexpectedly found that non-cytotoxic protease-TM conjugates (eg. CPNv/A) may demonstrate an improved binding activity for nociceptive sensory afferents when the size of the spacer is selected so that (in use) 30 the TM (preferably the C-terminus thereof) and the translocation domain (preferably the N-terminus thereof) are separated from one another by 40-105 angstroms, preferably by 50-100 angstroms, and more preferably by 50-90 angstroms. In 21 another embodiment, the preferred spacers have an amino acid sequence of 11-29 amino acid residues, preferably 15-27 amino acid residues, and more preferably 20 27 amino acid residues. Suitable spacers may be routinely identified and obtained according to Crasto, C.J. and Feng, J.A. (2000) May, 13(5), pp. 309-312 - see also 5 http://www.fccc.ledu/research/labs/fenillimker.html. Conjugation techniques suitable for use in the present invention have been well documented and are routine for a person skilled in the art. io The methodology involved in coupling two protein molecules (A and B) together is simple, and is achieved through the use of a cross-linking agent (also known as a chemical coupling agent). For example, molecules A and B are separately contacted with a cross-linking agent, which chemically modifies a specific surface group on each of molecules A and B thereby forming derivatised molecules A' and 15 B'. The modified surface group on molecule A' is capable of covalently bonding with the modified surface group on molecule B'. Thus, the coupling reaction is completed by mixing together the two protein molecules A' and B'. Chemical conjugation is illustrated by reference to the following embodiments, where 20 P = non-cytotoxic protease component, T = translocation component, and TM = targeting moiety. In one embodiment, a single chain P - T is prepared, which is then conjugated to a TM. In another embodiment, a single chain TM - T (or T - TM) is prepared, which is 25 then conjugated to a P. In a further embodiment, a single chain P - TM (or TM - P) is prepared, which is then conjugated to a T. Another particularly preferred conjugate has the structure P - TM - T (with an optional protease cleavage site between P and TM). 30 Where the T and P components are prepared as a single chain polypeptide, a protease cleavage site is typically included between said components. Any protease cleavage site may be employed in this regard.

22 In an alternative embodiment, the three components may be simultaneously or sequentially conjugated together. Thus, the conjugation may be a one- or two-step process, and may include one or more different coupling agents. 5 Chemical coupling agents and cross-linking agents have been commercially available for many years. Example 5 of the present invention describes in detail the use of one such coupling 10 agent, namely SPDP, to chemically couple two protein molecules (nociceptin, and the LHN of botulinum neurotoxin). The two molecules are separately contacted with SPDP, and then mixed together to allow covalent conjugation. The conjugate described in Example 6 confirms that another coupling agent, 15 PDPH/EDAC, or Traut's reagent, may be employed as an alternative coupling agent to SPDP. SPDP and Traut's reagent are popular and well-documented coupling agents in the technical field of protein conjugation chemistry and are presented here simply as two 20 examples of a well known class of compounds that may be employed to covalently link together the Targeting Moiety component and the clostridial neurotoxin component of the conjugate of the present invention. Other suitable agents include SMPB, SMCC (succinimidyl 4-(N-maleimidomethyl) cyclohexan-1-carboxylate), and LC-SPDP. 25 In more detail, commercially available members of the well-known coupling agents may be used for conjugation purposes to produce a conjugate of the invention. Details of such agents can be found in the following publications: 30 Hermanson, G.T. (1996), Bioconjugate techniques, Academic Press; Wong, S.S. (1991), Chemistry of protein conjugation and cross-linking, 23 CRC Press; Thorpe et al (1987), Cancer Res, 1987, 47, 5924-31. This paper describes the use of SMBT (sodium S-4-succinimidyloxycarbonyl 5 alpha-methyl benzyl thiosulfate) and SMPT (4 succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene); and Peeters et al (1989), J Immunol Methods. 1989, 120, 133-43. This 10 paper describes the use of 4 coupling reagents, MHS (succinimidyl 6 (N-maleimido)-n-hexanoate), SMCC (succinimidyl 4-(N maleimidomethyl)-cyclohexane-1 -carboxylate), MBS (succinimidyl m maleimidobenzoate), and SPDP. 15 The conjugates according to the present invention may also be prepared recombinantly, as detailed in Examples 9 to 12. In one embodiment, the preparation of a recombinant conjugate involves arrangement of the coding sequences of a selected TM, a selected non-cytotoxic 20 protease component, and a translocation component (in any order) in a single genetic construct. These coding sequences may be arranged in-frame so that subsequent transcription and translation is continuous through both coding sequences and results in a fusion protein. All constructs would have a 5' ATG codon to encode an N-terminal methionine, and a C-terminal translational stop codon. 25 Thus, the recombinant preparation method results in the generation of a single chain polypeptide. In order to activate this polypeptide, a protease cleavage site is present between the non-cytotoxic protease component and the translocation component. Cleavage of this site generates a di-chain polypeptide in which the protease and 30 translocation domains are linked together by way of a covalent bond, preferably a disulphide bond. In this regard, any protease cleavage site may be employed.

24 In the single polypeptide aspect of the present invention, the TM is preferably either N- or C-terminally located with respect to the fusion protein. In other words, it is preferred that the TM is not located between the P and T components of the single polypeptide fusion protein. In a particularly preferred embodiment, the TM is N 5 terminally located with respect to the fusion protein. In one embodiment, an L-chain of a clostridial neurotoxin or another protease capable of cleaving a protein of the exocytic fusion apparatus (e.g. an IgA protease), or a fragment/variant thereof, may be expressed recombinantly as a fusion protein 10 with a TM, which TM can also effect the internalisation of the L-chain component into the cytoplasm of the relevant target cell(s) responsible for secretion. Alternatively, the fusion protein may further comprise a Translocation Domain. The expressed fusion protein may include one or more spacer regions. 15 By way of example, the following information is required to produce, recombinantly, an agent of the present invention: (1) DNA sequence data relating to a selected TM; (II) DNA sequence data relating to the protease component; 20 (111) DNA sequence data relating to the translocation domain; and (IV) a protocol to permit construction and expression of the construct comprising (1), (11) and (111). All of the above basic information (I)-(IV) are either readily available, or are readily 25 determinable by conventional methods. For example, both W098/07864 and W099/17806 exemplify recombinant technology suitable for use in the present application. In addition, methods for the construction and expression of the constructs of the 30 present invention may employ information from the following references and others: Lorberboum-Galski, H., FitzGerald, D., Chaudhary, V., Adhya, S., 25 Pastan, I. (1988), Cytotoxic activity of an interleukin 2-Pseudomonas exotoxin chimeric protein produced in Escherichia coli. Proc.Natl. Acad. Sci. USA, 85(6):1922-6; 5 Murphy, J.R. (1988), Diphtheria-related peptide hormone gene fusions: a molecular genetic approach to chimeric toxin development. Cancer Treat. Res.; 37:123-40; Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., to Genbauffe, F., Strom, T.B., Murphy, J.R. (1987), Diphtheria toxin receptor binding domain substitution with interleukin-2: genetic construction and properties of a diphtheria toxin-related interleukin-2 fusion protein. Protein Eng;1(6):493-8; 15 Arora, N., Williamson, L.C., Leppla, S.H., Halpern, J.L. (1994), Cytotoxic effects of a chimeric protein consisting of tetanus toxin light chain and anthrax toxin lethal factor in non-neuronal cells J. Biol. Chem., 269(42):26165-71; 20 Brinkmann, U., Reiter, Y., Jung, S.H., Lee, B., Pastan, I. (1993), A recombinant immunotoxin containing a disulphide-stabilized Fv fragment. Proc. Natl. Acad. Sci. USA, 90(16):7538-42; and O'Hare, M., Brown, A.N., Hussain, K., Gebhardt, A., Watson, G., 25 Roberts, L.M., Vitetta, E.S., Thorpe, P.E., Lord, J.M. (1990), Cytotoxicity of a recombinant ricin-A-chain fusion protein containing a proteolytically-cleavable spacer sequence. FEBS Lett Oct 29;273(1 2):200-4. 30 Suitable clostridial neurotoxin sequence information relating to L- and LHN-chains may be obtained from, for example, Kurazono, H. (1992) J. Biol. Chem., vol. 267, No. 21, pp.14721-14729; and Popoff, M.R., and Marvaud, J.-C. (1999) The 26 Comprehensive Sourcebook of Bacterial Protein Toxins, 2nd edition (ed. Alouf, J.E., and Freer, J.H.), Academic Press, pp.174-201. All of the aforementioned publications are hereby incorporated into the present 5 specification by reference thereto. Similarly, suitable TM sequence data are widely available in the art. Alternatively, any necessary sequence data may be obtained by techniques which are well-known to the skilled person. 10 For example, DNA encoding the TM component may be cloned from a source organism by screening a cDNA library for the correct coding region (for example by using specific oligonucleotides based on the known sequence information to probe the library), isolating the TM DNA, sequencing this DNA for confirmation purposes, 15 and then placing the isolated DNA in an appropriate expression vector for expression in the chosen host. As an alternative to isolation of the sequence from a library, the available sequence information may be employed to prepare specific primers for use in PCR, whereby 20 the coding sequence is then amplified directly from the source material and, by suitable use of primers, may be cloned directly into an expression vector. Another alternative method for isolation of the coding sequence is to use the existing sequence information and synthesise a copy, possibly incorporating alterations, 25 using DNA synthesis technology. For example, DNA sequence data may be generated from existing protein and/or RNA sequence information. Using DNA synthesis technology to do this (and the alternative described above) enables the codon bias of the coding sequence to be modified to be optimal for the chosen expression host. This may give rise to superior expression levels of the fusion 30 protein. Optimisation of the codon bias for the expression host may be applied to the DNA 27 sequences encoding the TM and clostridial components of the construct. Optimisation of the codon bias is possible by application of the protein sequence into freely available DNA/protein database software, e.g. programs available from Genetics Computer Group, Inc. 5 Having prepared a conjugate of the invention, it is a matter of routine to confirm that the various domains have retained their specified function. Protease function after conjugation may be tested by using, for example, any one of o the following routine tests: SNAP-25 (or synaptobrevin, or syntaxin) may be challenged with a conjugate to be tested, and then analysed by SDS-PAGE peptide separation techniques. Subsequent detection of peptides (e.g. by silver staining) having molecular weights 15 corresponding to the cleaved products of SNAP-25 (or other component of the neurosecretory machinery) would confirm the presence of a functional L-chain. As a further alternative, the conjugate may be tested by assaying for SNAP-25 (or synaptobrevin, or syntaxin) cleavage products via antibody-specific binding (see 20 W095/33850). In more detail, a specific antibody is employed for detecting cleavage of SNAP-25. Since the antibody recognises cleaved SNAP-25, but not uncleaved SNAP-25, identification of the cleaved product by the antibody confirms the presence of L-chain proteolytic function. By way of exemplification, such a method is described in Examples 2 and 3 of W096/33273. 25 Translocation component function after conjugation may be tested using, for example, any one of the following routine tests: Suitable methods are, for example, described by Shone et al. (1987) Eur. J. 30 Biochem. 167, pp.175-180; and by Blaustein et al. (1987) FEBS 226 (1), pp.115 120.

28 The Shone et al. method employs artificial liposomes loaded with potassium phosphate buffer (pH 7.2) and radiolabelled NAD. Release of K+ and NAD from the liposomes correlates with a positive result for channel forming activity and hence translocation activity. In this regard, K+ release from liposomes may be measured 5 using an electrode and NAD release calculated by measuring the radioactivity in the supernatant (see page 176, column 1, line 33 - column 2, line 17). The Blaustein et al. method employs planar phospholipid bilayer membranes, which are used to test for channel forming activity. In more detail, salt solutions on either 10 side of the membrane are buffered at a different pH - on the cis side, pH 4.7 or 5.5 and on the trans side, pH 7.4. The "conjugate" to be tested is added to the cis side of the membrane and electrical measurements are made under voltage clamp conditions, in order to monitor the flow of current across the membrane (see paragraph 2.2, pages 116-118). The presence of an active translocation function is 15 confirmed by a steady rate of channel turn-on (i.e. a positive result for channel formation) -see paragraph 3, page 118. Targeting Moiety (TM) function after conjugation may be tested by assaying for the agonist function inherent to the TM. Suitable methods include those described in 20 Example 1. The ability of the conjugate of the invention to inhibit substance P release from nociceptive afferent cells can be assessed using the methods described in Example 15. 25 In Example 15, a nociceptin-LHN/A conjugate according to the first aspect of the invention is assessed for its ability to inhibit the release of substance P from primary nociceptive sensory afferent neurons. As can be seen from Table 1, incubation of the conjugate with cultures of nociceptive afferent neurons results in a significant 30 inhibition of release of substance P (when compared to incubation of the cells with LHN/A alone). The experiment therefore confirms that the conjugate is inhibiting substance P release from these cells.

29 In use of the present invention, a pain-sensing target cell is selected in which it is desired to reduce or inhibit the process of exocytic fusion, which exocytic process contributes to the symptoms associated with the sensation of pain. For example, the 5 target cell in question may demonstrate an undesirable phenotype (e.g. an undesirable secretion, or the expression of an undesirable concentration of membrane receptor, transporter or membrane channel), which contributes to the symptoms associated with pain. Alternatively, a target cell may be selected in which the process of exocytic fusion contributes to the sensation of pain. 10 In preferred embodiments of the invention, the target cell is a nociceptive sensory afferent cell, preferably a primary nociceptive afferent cell (e.g. an A-fibre such as an A6-fibre or a C-fibre). Thus, the conjugates of the present invention are capable of inhibiting neurotransmitter or neuromodulator (e.g. glutamate, substance P, 15 calcitonin-gene related peptide (CGRP), and/or neuropeptide Y) release from discrete populations of nociceptive sensory afferent neurons. In use, the conjugates reduce or prevent the transmission of sensory afferent signals (e.g. neurotransmitters or neuromodulators) from peripheral to central pain fibres, and therefore have application as therapeutic molecules for the treatment of pain, in 20 particular chronic pain. It is routine to confirm that a TM binds to a nociceptive sensory afferent. For example, a simple radioactive displacement experiment may be employed in which tissue or cells representative of the nociceptive sensory afferent (for example 25 DRGs) are exposed to labelled (e.g. tritiated) ligand in the presence of an excess of unlabelled ligand. In such an experiment, the relative proportions of non-specific and specific binding may be assessed, thereby allowing confirmation that the ligand binds to the nociceptive sensory afferent target cell. Optionally, the assay may include one or more binding antagonists, and the assay may further comprise 30 observing a loss of ligand binding. Examples of this type of experiment can be found in Hulme, E.C. (1990), Receptor-binding studies, a brief outline, pp 303-311, in Receptor biochemistry, A Practical Approach, Ed. E.C. Hulme, Oxford University 30 Press. According to a second aspect, the present invention provides a non-cytotoxic conjugate for inhibition or reduction of exocytotic fusion in a nociceptive sensory 5 afferent cell, comprising: (i) a Targeting Moiety (TM), wherein said TM is an agonist of a receptor that is present on 10 said nociceptive sensory afferent cell, and wherein said receptor undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell; (ii) a DNA sequence encoding a non-cytotoxic protease or a is fragment thereof, wherein the DNA sequence is expressible in the nociceptive sensory afferent cell and when so expressed provides a protease or protease fragment capable of cleaving a protein of 20 the exocytic fusion apparatus of said nociceptive sensory afferent cell; and (iii) a Translocation Domain, 25 wherein the Translocation Domain translocates the DNA sequence encoding the protease or protease fragment from within the endosome, across the endosomal membrane, and into the nociceptive sensory afferent cell. 30 In a preferred embodiment, the receptor is an ORL 1 receptor. DNA encoding a protein of interest can be transfected into eukaryotic cells through 31 receptor-mediated endocytosis of a protein-DNA conjugate, as confirmed by Cotton et al. (Cotton, M., Wagner, E. and Birnstiel, L. (1993) Receptor-mediated transport of DNA into eukaryotic cells. Methods in Enzymol. 217, 619-645). Several methods exist for condensing DNA to a suitable size using polycationic ligands. These 5 include: polylysine, various cationic peptides and cationic liposomes. Of these, polylysine was used in the present study because of its successfully reported use in receptor-mediated transfection studies (Cotton et al., 1993). The DNA sequence encoding the non-cytotoxic protease component may be io expressed under the control of an operably linked promoter present as part of the agent (e.g. as part of the protease DNA sequence upstream of the coding region). Alternatively, expression of the protease component in the target cell may rely on a promoter present in the target cell. 15 The DNA sequence encoding the protease component may integrate into a DNA sequence of the target cell. One or more integration site(s) may be provided as part of the conjugate (e.g. as part of the protease DNA sequence). The TM, Translocation Domain and protease components of this second aspect of 20 the invention are as defined for the first aspect of the invention. Examples 13 and 14 describe the preparation of conjugates according to the second aspect of the invention. According to a third aspect, the present invention provides a pharmaceutical 25 composition comprising a conjugate according to the first and/or second aspect of the present invention. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, and/or a suitable diluent and/or excipient, although the exact form 30 of the composition may be tailored to the mode of administration. Administration is preferably to a mammal, more preferably to a human.

32 The components of the composition may, for example, be employed in the form of an aerosol or nebulisable solution for inhalation or a sterile solution for parenteral administration, intra-articular administration or intra-cranial administration. 5 The composition may also be administered by i.v. injection, which includes the use of pump systems. Spinal injection (e.g. epidural or intrathecal) or indwelling pumps may also be used. The dosage ranges for administration of the components of the present invention are io those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the components, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. 15 Suitable daily dosages (for each component) are in the range 0.0001-1 mg/kg, preferably 0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and particularly preferably 0.004-0.5 mg/kg. The unit dosage can vary from less that 1 microgram to 30 mg, but typically will be in the region of 0.01 to 1 mg per dose, which may be 20 administered daily or preferably less frequently, such as weekly or six monthly. A particularly preferred dosing regimen is based on 2.5 ng of fusion protein (e.g. CPNv/A) as the 1X dose. In this regard, preferred dosages are in the range 1X 10OX (i.e. 2.5-250 ng). This dosage range is significantly lower (i.e. at least 10-fold, 25 typically 100-fold lower) than would be employed with other types of analgesic molecules such as NSAIDS, morphine, and gabapentin. Moreover, the above mentioned difference is considerably magnified when the same comparison is made on a molar basis - this is because the fusion proteins of the present invention have a considerably greater Mw than do conventional 'small' molecule therapeutics. 30 Wide variations in the required dosage, however, are to be expected depending on the precise nature of the components, and the differing efficiencies of various routes 33 of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines 5 for optimisation, as is well understood in the art. Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use. 10 Fluid unit dosage forms are typically prepared utilising a pyrogen-free sterile vehicle. The active ingredients, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. 15 Solutions may be used for all forms of parenteral administration, and are particularly used for intravenous injection. In preparing solutions the components can be dissolved in the vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic 20 techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Advantageously additives such as buffering, solubilising, stabilising, preservative or 25 bactericidal, suspending or emulsifying agents and/or local anaesthetic agents may be dissolved in the vehicle. Dry powders which are dissolved or suspended in a suitable vehicle prior to use may be prepared by filling pre-sterilised drug substance and other ingredients into a 30 sterile container using aseptic technique in a sterile area. Alternatively the components of the composition may be dissolved in an aqueous 34 vehicle, the solution is sterilized by filtration and distributed into suitable containers using aseptic technique in a sterile area. The product is then freeze-dried and the containers are sealed aseptically. 5 Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma io irradiation. Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition(s) to facilitate uniform distribution of the components. 15 Compositions suitable for administration via the respiratory tract include aerosols, nebulisable solutions or microfine powders for insufflation. In the latter case, particle size of less than 50 microns, especially less than 10 microns, is preferred. Such compositions may be made up in a conventional manner and employed in conjunction with conventional administration devices. 20 The compositions described in this invention can be used in vivo, either directly or as a pharmaceutically acceptable salt, for the treatment of conditions involving exocytosis (for example secretion, or the delivery of proteins such as receptors, transporters, and membrane channels to the plasma membrane of a cell). 25 According to a fourth aspect, the present invention provides a DNA construct that encodes a conjugate according to the first or second aspects of the invention. By expressing the construct in a host cell, conjugates of the invention may be 30 prepared. According to a fifth aspect, the present invention provides a method of treatment of 35 pain by administration to a patient of a conjugate, composition, or construct according to the first to fourth aspects of the invention, or any combination thereof. In a preferred embodiment, the invention provides a method of treating chronic pain. 5 According to a sixth aspect, the present invention provides for the use of a conjugate, composition or construct according to the first to fourth aspects of the invention, for the manufacture of a medicament for treating pain, preferably chronic pain. io According to a further aspect of the present invention, there is provided use of a conjugate of the invention, for the manufacture of a medicament for treating, preventing or ameliorating pain. According to a related aspect, there is provided a method of treating, preventing or 15 ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of a conjugate or composition of the invention. The conjugates and compositions described here may be used to treat a patient suffering from one or more types of chronic pain including neuropathic pain, 20 inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain. To "treat," as used here, means to deal with medically. It includes, for example, administering a compound of the invention to prevent pain or to lessen its severity. 25 The term "pain," as used here, means any unpleasant sensory experience, usually associated with a physical disorder. The physical disorder may or may not be apparent to a clinician. Pain is of two types: chronic and acute. An "acute pain" is a pain of short duration having a sudden onset. One type of acute pain, for example, is cutaneous pain felt on injury to the skin or other superficial tissues, such as 30 caused by a cut or a burn. Cutaneous nociceptors terminate just below the skin, and due to the high concentration of nerve endings, produce a well-defined, localized pain of short duration. "Chronic pain" is a pain other than an acute pain. Chronic 36 pain includes neuropathic pain, inflammatory pain, headache pain, somatic pain visceral pain and referred pain. I. Neuropathic Pain 5 The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following neuropathic pain conditions. "Neuropathic pain" means abnormal sensory input, resulting in discomfort, from the peripheral nervous system, central nervous systems, or both. 10 A. Symptoms of neuropathic pain Symptoms of neuropathic pain can involve persistent, spontaneous pain, as well as allodynia (a painful response to a stimulus that normally is not painful), hyperalgesia (an accentuated response to a painful stimulus that usually causes only a mild discomfort, such as a pin prick), or hyperpathia (where a short discomfort becomes a 15 prolonged severe pain). B. Causes of neuropathic pain Neuropathic pain may be caused by any of the following. 1. A traumatic insult, such as, for example, a nerve compression injury (e.g., a nerve 20 crush, a nerve stretch, a nerve entrapment or an incomplete nerve transsection); a spinal cord injury (e.g., a hemisection of the spinal cord); a limb amputation; a contusion; an inflammation (e.g., an inflammation of the spinal cord); or a surgical procedure. 2. An ischemic event, including, for example, a stroke and heart attack. 25 3. An infectious agent 4. Exposure to a toxic agent, including, for example, a drug, an alcohol, a heavy metal (e.g., lead, arsenic, mercury), an industrial agent (e.g., a solvent, fumes from a glue) or nitrous oxide. 5. A disease, including, for example, an inflammatory disorder, a neoplastic tumor, 30 an acquired immune deficiency syndrome (AIDS), Lymes disease, a leprosy, a metabolic disease, a peripheral nerve disorder, like neuroma, a mononeuropathy or a polyneuropathy.

37 C. Types of neuropathic pain 1. Neuralgia. A neuralgia is a pain that radiates along the course of one or more specific nerves 5 usually without any demonstrable pathological change in the nerve structure. The causes of neuralgia are varied. Chemical irritation, inflammation, trauma (including surgery), compression by nearby structures (for instance, tumors), and infections may all lead to neuralgia. In many cases, however, the cause is unknown or unidentifiable. Neuralgia is most common in elderly persons, but it may occur at any 10 age. A neuralgia, includes, without limitation, a trigeminal neuralgia, a post-herpetic neuralgia, a postherpetic neuralgia, a glossopharyngeal neuralgia, a sciatica and an atypical facial pain. Neuralgia is pain in the distribution of a nerve or nerves. Examples are trigeminal 15 neuralgia, atypical facial pain, and postherpetic neuralgia (caused by shingles or herpes). The affected nerves are responsible for sensing touch, temperature and pressure in the facial area from the jaw to the forehead. The disorder generally causes short episodes of excruciating pain, usually for less than two minutes and on only one side of the face. The pain can be described in a variety of ways such as 20 "stabbing," "sharp," "like lightning," "burning," and even "itchy". In the atypical form of TN, the pain can also present as severe or merely aching and last for extended periods. The pain associated with TN is recognized as one the most excruciating pains that can be experienced. 25 Simple stimuli such as eating, talking, washing the face, or any light touch or sensation can trigger an attack (even the sensation of a gentle breeze). The attacks can occur in clusters or as an isolated attack. Symptoms include sharp, stabbing pain or constant, burning pain located anywhere, 30 usually on or near the surface of the body, in the same location for each episode; pain along the path of a specific nerve; impaired function of affected body part due to pain, or muscle weakness due to concomitant motor nerve damage; increased 38 sensitivity of the skin or numbness of the affected skin area (feeling similar to a local anesthetic such as a Novacaine shot); and any touch or pressure is interpreted as pain. Movement may also be painful. 5 Trigeminal neuralgia is the most common form of neuralgia. It affects the main sensory nerve of the face, the trigeminal nerve ("trigeminal" literally means "three origins", referring to the division of the nerve into 3 branches). This condition involves sudden and short attacks of severe pain on the side of the face, along the area supplied by the trigeminal nerve on that side. The pain attacks may be severe io enough to cause a facial grimace, which is classically referred to as a painful tic (tic douloureux). Sometimes, the cause of trigeminal neuralgia is a blood vessel or small tumor pressing on the nerve. Disorders such as multiple sclerosis (an inflammatory disease affecting the brain and spinal cord), certain forms of arthritis, and diabetes (high blood sugar) may also cause trigeminal neuralgia, but a cause is is not always identified. In this condition, certain movements such as chewing, talking, swallowing, or touching an area of the face may trigger a spasm of excruciating pain. A related but rather uncommon neuralgia affects the glosso-pharyngeal nerve, which provides sensation to the throat. Symptoms of this neuralgia are short, shock-like 20 episodes of pain located in the throat. Neuralgia may occur after infections such as shingles, which is caused by the varicella-zoster virus, a type of herpesvirus. This neuralgia produces a constant burning pain after the shingles rash has healed. The pain is worsened by movement 25 of or contact with the affected area. Not all of those diagnosed with shingles go on to experience postherpetic neuralgia, which can be more painful than shingles. The pain and sensitivity can last for months or even years. The pain is usually in the form of an intolerable sensitivity to any touch but especially light touch. Postherpetic neuralgia is not restricted to the face; it can occur anywhere on the body but usually 30 occurs at the location of the shingles rash. Depression is not uncommon due to the pain and social isolation during the illness.

39 Postherpetic neuralgia may be debilitating long after signs of the original herpes infection have disappeared. Other infectious diseases that may cause neuralgia are syphilis and Lyme disease. 5 Diabetes is another common cause of neuralgia. This very common medical problem affects almost 1 out of every 20 Americans during adulthood. Diabetes damages the tiny arteries that supply circulation to the nerves, resulting in nerve fiber malfunction and sometimes nerve loss. Diabetes can produce almost any neuralgia, including trigeminal neuralgia, carpal tunnel syndrome (pain and numbness of the hand and 1 o wrist), and meralgia paresthetica (numbness and pain in the thigh due to damage to the lateral femoral cutaneous nerve). Strict control of blood sugar may prevent diabetic nerve damage and may accelerate recovery in patients who do develop neuralgia. 15 Other medical conditions that may be associated with neuralgias are chronic renal insufficiency and porphyria -- a hereditary disease in which the body cannot rid itself of certain substances produced after the normal breakdown of blood in the body. Certain drugs may also cause this problem. 20 2. Deafferentation. Deafferentation indicates a loss of the sensory input from a portion of the body, and can be caused by interruption of either peripheral sensory fibres or nerves from the central nervous system. A deafferentation pain syndrome, includes, without limitation, an injury to the brain or spinal cord, a post-stroke pain, a phantom pain, a 25 paraplegia, a brachial plexus avulsion injuries, lumbar radiculopathies. 3. Complex regional pain syndromes (CRPSs) CRPS is a chronic pain syndrome resulting from sympathetically-maintained pain, and presents in two forms. CRPS 1 currently replaces the term "reflex sympathetic 30 dystrophy syndrome". It is a chronic nerve disorder that occurs most often in the arms or legs after a minor or major injury. CRPS 1 is associated with severe pain; changes in the nails, bone, and skin; and an increased sensitivity to touch in the 40 affected limb. CRPS 2 replaces the term causalgia, and results from an identified injury to the nerve. A CRPS, includes, without limitation, a CRPS Type I (reflex sympathetic dystrophy) and a CRPS Type II (causalgia). 5 4. Neuropathy. A neuropathy is a functional or pathological change in a nerve and is characterized clinically by sensory or motor neuron abnormalities. Central neuropathy is a functional or pathological change in the central nervous io system. Peripheral neuropathy is a functional or pathological change in one or more peripheral nerves. The peripheral nerves relay information from your central nervous system (brain and spinal cord) to muscles and other organs and from your 15 skin, joints, and other organs back to your brain. Peripheral neuropathy occurs when these nerves fail to carry information to and from the brain and spinal cord, resulting in pain, loss of sensation, or inability to control muscles. In some cases, the failure of nerves that control blood vessels, intestines, and other organs results in abnormal blood pressure, digestion problems, and loss of other basic body processes. Risk 20 factors for neuropathy include diabetes, heavy alcohol use, and exposure to certain chemicals and drugs. Some people have a hereditary predisposition for neuropathy. Prolonged pressure on a nerve is another risk for developing a nerve injury. Pressure injury may be caused by prolonged immobility (such as a long surgical procedure or lengthy illness) or compression of a nerve by casts, splints, braces, 25 crutches, or other devices. Polyneuropathy implies a widespread process that usually affects both sides of the body equally. The symptoms depend on which type of nerve is affected. The three main types of nerves are sensory, motor, and autonomic. Neuropathy can affect any one or a combination of all three types of nerves. Symptoms also depend on whether the condition affects the whole body or 30 just one nerve (as from an injury). The cause of chronic inflammatory polyneuropathy is an abnormal immune response. The specific antigens, immune processes, and triggering factors are variable and in many cases are unknown. It 41 may occur in association with other conditions such as HIV, inflammatory bowel disease, lupus erythematosis, chronic active hepatitis, and blood cell abnormalities. Peripheral neuropathy may involve a function or pathological change to a single 5 nerve or nerve group (monneuropathy) or a function or pathological change affecting multiple nerves (polyneuropathy). Peripheral neuropathies Hereditary disorders 10 Charcot-Marie-Tooth disease Friedreich's ataxia Systemic or metabolic disorders Diabetes (diabetic neuropathy) Dietary deficiencies (especially vitamin B-12) 15 Excessive alcohol use (alcoholic neuropathy) Uremia (from kidney failure) Cancer Infectious or inflammatory conditions AIDS 20 Hepatitis Colorado tick fever diphtheria Guillain-Barre syndrome HIV infection without development of AIDS 25 leprosy Lyme polyarteritis nodosa rheumatoid arthritis sarcoidosis 30 Sjogren syndrome syphilis systemic lupus erythematosus 42 amyloid Exposure to toxic compounds sniffing glue or other toxic compounds nitrous oxide 5 industrial agents -- especially solvents heavy metals (lead, arsenic, mercury, etc.) Neuropathy secondary to drugs like analgesic nephropathy Miscellaneous causes ischemia (decreased oxygen/decreased blood flow) 10 prolonged exposure to cold temperature a. Polyneuropathy Polyneuropathy is a peripheral neuropathy involving the loss of movement or sensation to an area caused by damage or destruction to multiple peripheral nerves. Polyneuropathic pain, includes, without limitation, post-polio syndrome, 15 postmastectomy syndrome, diabetic neuropathy, alcohol neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia, vitamin deficiencies, chemotherapy-induced pain, 2',3'-didexoycytidine (ddC) treatment, Guillain-Barre syndrome or Fabry's disease. b. Mononeuropathy 20 Mononeuropathy is a peripheral neuropathy involving loss of movement or sensation to an area caused by damage or destruction to a single peripheral nerve or nerve group. Mononeuropathy is most often caused by damage to a local area resulting from injury or trauma, although occasionally systemic disorders may cause isolated nerve damage (as with mononeuritis multiplex). The usual causes are direct 25 trauma, prolonged pressure on the nerve, and compression of the nerve by swelling or injury to nearby body structures. The damage includes destruction of the myelin sheath (covering) of the nerve or of part of the nerve cell (the axon). This damage slows or prevents conduction of impulses through the nerve. Mononeuropathy may involve any part of the body. Mononeuropathic pain, includes, without limitation, a 30 sciatic nerve dysfunction, a common peroneal nerve dysfunction. a radial nerve dysfunction, an ulnar nerve dysfunction, a cranial mononeuropathy VI, a cranial mononeuropathy VII, a cranial mononeuropathy Ill (compression type), a cranial 43 mononeuropathy Ill (diabetic type), an axillary nerve dysfunction, a carpal tunnel syndrome, a femoral nerve dysfunction, a tibial nerve dysfunction, a Bell's palsy, a thoracic outlet syndrome, a carpal tunnel syndrome and a sixth (abducent) nerve palsy 5 c. Generalized peripheral neuropathies Generalized peripheral neuropathis are symmetrical, and usually due to various systematic illnesses and disease processes that affect the peripheral nervous system in its entirety. They are further subdivided into several categories: i. Distal axonopathies are the result of some metabolic or toxic io derangement of neurons. They may be caused by metabolic diseases such as diabetes, renal failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. Distal axonopathy (aka dying back neuropathy) is a type of peripheral neuropathy that results from some metabolic or toxic derangement of peripheral nervous system (PNS) neurons. It is the most common response of is nerves to metabolic or toxic disturbances, and as such may be caused by metabolic diseases such as diabetes, renal failure, deficiency syndromes such as malnutrition and alcoholism, or the effects of toxins or drugs. The most common cause of distal axonopathy is diabetes, and the most common distal axonopathy is diabetic neuropathy. 20 ii. Myelinopathies are due to a primary attack on myelin causing an acute failure of impulse conduction. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP; aka Guillain-Barre syndrome), though other causes include chronic inflammatory demyelinating syndrome (CIDP), genetic metabolic disorders (e.g., leukodystrophy), or toxins. Myelinopathy is due to primary 25 destruction of myelin or the myelinating Schwann cells, which leaves the axon intact, but causes an acute failure of impulse conduction. This demyelination slows down or completely blocks the conduction of electical impulses through the nerve. The most common cause is acute inflammatory demyelinating polyneuropathy (AIDP, better known as Guillain-Barre syndrome), though other causes include chronic 30 inflammatory demyelinating polyneuropathy (CIDP), genetic metabolic disorders (e.g., leukodystrophy or Charcot-Marie-Tooth disease), or toxins.

44 iii. Neuronopathies are the result of destruction of peripheral nervous system (PNS) neurons. They may be caused by motor neurone diseases, sensory neuronopathies (e.g., Herpes zoster), toxins or autonomic dysfunction. Neurotoxins may cause neuronopathies, such as the chemotherapy agent vincristine. 5 Neuronopathy is dysfunction due to damage to neurons of the peripheral nervous system (PNS), resulting in a peripheral neuropathy. It may be caused by motor neurone diseases, sensory neuronopathies (e.g., Herpes zoster), toxic substances or autonomic dysfunction. A person with neuronopathy may present in different ways, depending on the cause, the way it affects the nerve cells, and the type of 1o nerve cell that is most affected. iv. Focal entrapment neuropathies (e.g., carpal tunnel syndrome). II. Inflammatory pain The compounds of the invention may be used to treat pain caused by or otherwise 15 associated with any of the following inflammatory conditions A. Arthritic disorder Arthritic disorders include, for example, a rheumatoid arthritis; a juvenile rheumatoid arthritis; a systemic lupus erythematosus (SLE); a gouty arthritis; a scleroderma; an 20 osteoarthritis; a psoriatic arthritis; an ankylosing spondylitis; a Reiter's syndrome (reactive arthritis); an adult Still's disease; an arthritis from a viral infection; an arthritis from a bacterial infection, such as, e.g., a gonococcal arthritis and a non gonococcal bacterial arthritis (septic arthritis); a Tertiary Lyme disease; a tuberculous arthritis; and an arthritis from a fungal infection, such as, e,g. a 25 blastomycosis B. Autoimmune diseases Autoimmune diseases include, for example, a Guillain-Barre syndrome, a Hashimoto's thyroiditis, a pernicious anemia, an Addison's disease, a type I diabetes, a systemic lupus erythematosus, a dermatomyositis, a Sjogren's 30 syndrome, a lupus erythematosus, a multiple sclerosis, a myasthenia gravis, a Reiter's syndrome and a Grave's disease.

45 C. Connective tissue disorder Connective tissue disorders include, for example, a spondyloarthritis a dermatomyositis, and a fibromyalgia. 5 D. Injury Inflammation caused by injury, including, for example, a crush, puncture, stretch of a tissue or joint, may cause chronic inflammatory pain. E. Infection io Inflammation caused by infection, including, for example, a tuberculosis or an interstitial keratitis may cause chronic inflammatory pain. F. Neuritis Neuritis is an inflammatory process affecting a nerve or group of nerves. Symptoms 15 depend on the nerves involved, but may include pain, paresthesias, paresis, or hypesthesia (numbness). Examples include: a. Brachial neuritis b. Retrobulbar neuropathy, an inflammatory process affecting the part of the 20 optic nerve lying immediately behind the eyeball. c. Optic neuropathy, an inflammatory process affecting the optic nerve causing sudden, reduced vision in the affected eye. The cause of optic neuritis is unknown. The sudden inflammation of the optic nerve (the nerve connecting the eye and the brain) leads to swelling and destruction of the myelin sheath. The 25 inflammation may occasionally be the result of a viral infection, or it may be caused by autoimmune diseases such as multiple sclerosis. Risk factors are related to the possible causes. d. Vestibular neuritis, a viral infection causing an inflammatory process affecting the vestibular nerve. 30 G. Joint inflammation 46 Inflammation of the joint, such as that caused by bursitis or tendonitis, for example, may cause chronic inflammatory pain. III. Headache Pain 5 The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following headache conditions. A headache (medically known as cephalgia) is a condition of mild to severe pain in the head; sometimes neck or upper back pain may also be interpreted as a headache. It may indicate an underlying local or systemic disease or be a disorder in itself. 10 A. Muscular/myogenic headache Muscular/myogenic headaches appear to involve the tightening or tensing of facial and neck muscles; they may radiate to the forehead. Tension headache is the most common form of myogenic headache. 15 A tension headache is a condition involving pain or discomfort in the head, scalp, or neck, usually associated with muscle tightness in these areas. Tension headaches result from the contraction of neck and scalp muscles. One cause of this muscle contraction is a response to stress, depression or anxiety. Any activity that causes 20 the head to be held in one position for a long time without moving can cause a headache. Such activities include typing or use of computers, fine work with the hands, and use of a microscope. Sleeping in a cold room or sleeping with the neck in an abnormal position may also trigger this type of headache. A tension-type headache, includes, without limitation, an episodic tension headache and a chronic 25 tension headache. B. Vascular headache The most common type of vascular headache is migraine. Other kinds of vascular headaches include cluster headaches, which cause repeated episodes of intense 30 pain, and headaches resulting from high blood pressure 1. Migraine 47 A migraine is a heterogeneous disorder that generally involves recurring headaches. Migraines are different from other headaches because they occur with other symptoms, such as, e.g., nausea, vomiting, or sensitivity to light. In most people, a throbbing pain is felt only on one side of the head. Clinical features such 5 as type of aura symptoms, presence of prodromes, or associated symptoms such as vertigo, may be seen in subgroups of patients with different underlying pathophysiological and genetic mechanisms. A migraine headache, includes, without limitation, a migraine without aura (common migraine), a migraine with aura (classic migraine), a menstrual migraine, a migraine equivalent (acephalic io headache), a complicated migraine, an abdominal migraine and a mixed tension migraine. 2. Cluster headache Cluster headaches affect one side of the head (unilateral) and may be associated with tearing of the eyes and nasal congestion. They occurs in clusters, 15 happening repeatedly every day at the same time for several weeks and then remitting. D. High blood pressure headache 20 E. Traction and inflammatory headache Traction and inflammatory headaches are usually symptoms of other disorders, ranging from stroke to sinus infection. F. Hormone headache 25 G. Rebound headache Rebound headaches, also known as medication overuse headaches, occur when medication is taken too frequently to relieve headache. Rebound headaches frequently occur daily and can be very painful. 30 H. Chronic sinusitis headache 48 Sinusitis is inflammation, either bacterial, fungal, viral, allergic or autoimmune, of the paranasal sinuses. Chronic sinusitis is one of the most common complications of the common cold. Symptoms include: Nasal congestion; facial pain; headache; fever; general malaise; thick green or yellow discharge; feeling of facial 'fullness' 5 worsening on bending over. In a small number of cases, chronic maxillary sinusitis can also be brought on by the spreading of bacteria from a dental infection. Chronic hyperplastic eosinophilic sinusitis is a noninfective form of chronic sinusitis. 1. An organic headache 10 J. Ictal headaches Ital headaches are headaches associated with seizure activity. IV. Somatic pain 15 The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following somatic pain conditions. Somatic pain originates from ligaments, tendons, bones, blood vessels, and even nerves themselves. It is detected with somatic nociceptors. The scarcity of pain receptors in these areas produces a dull, poorly-localized pain of longer duration than cutaneous pain; 20 examples include sprains and broken bones. Additional examples include the following. A. Excessive muscle tension Excessive muclse tension can be caused, for example, by a sprain or a strain. 25 B. Repetitive motion disorders Repetitive motion disorders can result from overuse of the hands, wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, or ankles. 30 C. Muscle disorders 49 Muscle disorders causing somatic pain include, for example, a polymyositis, a dermatomyositis, a lupus, a fibromyalgia, a polymyalgia rheumatica, and a rhabdomyolysis. 5 D. Myalgia Myalgia is muscle pain and is a symptom of many diseases and disorders. The most common cause for myalgia is either overuse or over-stretching of a muscle or group of muscles. Myalgia without a traumatic history is often due to viral infections. Longer-term myalgias may be indicative of a metabolic myopathy, some nutritional io deficiencies or chronic fatigue syndrome. E. Infection Infection can cause somatic pain. Examples of such infection include, for example, an abscess in the muscle, a trichinosis, an influenza, a Lyme disease, a malaria, a 15 Rocky Mountain spotted fever, Avian influenza, the common cold, community acquired pneumonia, meningitis, monkeypox, Severe Acute Respiratory Syndrome, toxic shock syndrome, trichinosis, typhoid fever, and upper respiratory tract infection. F. Drugs 20 Drugs can cause somatic pain. Such drugs include, for example, cocaine, a statin for lowering cholesterol (such as atorvastatin, simvastatin, and lovastatin), and an ACE inhibitor for lowering blood pressure (such as enalapril and captopril) V. Visceral pain 25 The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following visceral pain conditions. Visceral pain originates from body's viscera, or organs. Visceral nociceptors are located within body organs and internal cavities. The even greater scarcity of nociceptors in these areas produces pain that is usually more aching and of a longer duration than somatic 30 pain. Visceral pain is extremely difficult to localise, and several injuries to visceral tissue exhibit "referred" pain, where the sensation is localised to an area completely unrelated to the site of injury. Examples of visceral pain include the following.

50 A. Functional visceral pain Functional visceral pain includes, for example, an irritable bowel syndrome and a chronic functional abdominal pain (CFAP), a functional constipation and a functional 5 dyspepsia, a non-cardiac chest pain (NCCP) and a chronic abdominal pain. B. Chronic gastrointestinal inflammation Chronic gastrointestinal inflammation includes, for example, a gastritis, an inflammatory bowel disease, like, e.g., a Crohn's disease, an ulcerative colitis, a to microscopic colitis, a diverticulitis and a gastroenteritis; an interstitial cystitis; an intestinal ischemia; a cholecystitis; an appendicitis; a gastroesophageal reflux; an ulcer, a nephrolithiasis, an urinary tract infection, a pancreatitis and a hernia. C. Autoimmune pain 15 Autoimmune pain includes, for example, a sarcoidosis and a vasculitis. D. Orangic visceral pain Organic visceral pain includes, for example, pain resulting from a traumatic, inflammatory or degenerative lesion of the gut or produced by a tumor impinging on 20 sensory innervation. E. Treatment-induced visceral pain Treatment-induced visceral pain includes, for example, a pain attendant to chemotherapy therapy or a pain attendant to radiation therapy. 25 VI. Referred pain The compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following referred pain conditions. 30 Referred pain arises from pain localized to an area separate from the site of pain stimulation. Often, referred pain arises when a nerve is compressed or damaged at or near its origin. In this circumstance, the sensation of pain will generally be felt in 51 the territory that the nerve serves, even though the damage originates elsewhere. A common example occurs in intervertebral disc herniation, in which a nerve root arising from the spinal cord is compressed by adjacent disc material. Although pain may arise from the damaged disc itself, pain will also be felt in the region served by 5 the compressed nerve (for example, the thigh, knee, or foot). Relieving the pressure on the nerve root may ameliorate the referred pain, provided that permanent nerve damage has not occurred. Myocardial ischaemia (the loss of blood flow to a part of the heart muscle tissue) is possibly the best known example of referred pain; the sensation can occur in the upper chest as a restricted feeling, or as an ache in the 1o left shoulder, arm or even hand. Definitions Section Exocytic fusion is a process by which intracellular molecules are transported from 15 the cytosol of a pain-sensing target cell to the plasma (i.e. cell) membrane thereof. Thereafter, the intracellular molecules may become displayed on the outer surface of the plasma membrane, or may be secreted into the extracellular environment. In a healthy individual, the rate of exocytic fusion is carefully regulated and allows 20 control of the transport of molecules between the cytosol and the plasma membrane of a pain-sensing cell. For example, regulation of the exocytic cycle allows control of the density of receptors, transporters, or membrane channels present at the cell's surface, and/or allows control of the secretion rate of intracellular components (e.g. neurotransmitters) from the cytosol of the cell. 25 However, in an unhealthy individual, the regulation of exocytic fusion may be modified. For example, exocytic fusion may cause affected pain-sensing cells to enter a state of hypersecretion. Alternatively, exocytic fusion may result in the display of an increased concentration of receptors, transporters, or membrane 30 channels present on the surface of the pain-sensing, which may expose the cell to undesirable external stimuli. Thus, the process of exocytic fusion may contribute to the progression and/or severity of pain, and therefore provides a target for 52 therapeutic intervention. It should also be appreciated that otherwise normal rates of cellular exocytic fusion may contribute to the progression and severity of pain in compromised patients. 5 Thus, by targeting exocytic fusion in accordance with the present invention, it is also possible to provide therapy in such patients Targeting Moiety (TM) means any chemical structure associated with a conjugate that functionally interacts with a receptor, e.g. an ORL 1 receptor, to cause a physical io association between the conjugate and the surface of a pain-sensing target cell. The term TM embraces any molecule (i.e. a naturally occurring molecule, or a chemically/physically modified variant thereof) that is capable of binding to a receptor on the target cell, which receptor is capable of internalisation (e.g. endosome formation) - also referred to as receptor-mediated endocytosis. The TM is may possess an endosomal membrane translocation domain, in which case separate TM and Translocation Domain components need not be present in an agent of the present invention. The term "fragment" means a peptide having at least thirty-five, preferably at least 20 twenty-five, more preferably at least fifteen, and most preferably at least ten amino acid residues of the TM in question. In one embodiment, the first amino acid residue of the fragment is the N-terminal amino acid residue of the TM from which the fragment has been derived. 25 An example of a "variant" is a peptide or peptide fragment of a TM that contains one or more analogues of an amino acid (e.g. an unnatural amino acid), or a substituted linkage. A "derivative" comprises the TM in question, and a further peptide sequence. The 30 further peptide sequence should preferably not interfere with the basic folding and thus conformational structure of the TM. Two or more peptides (or fragments, or variants) may be joined together to form a derivative. Alternatively, a peptide (or 53 fragment, or variant) may be joined to an unrelated molecule (e.g. a second, unrelated peptide). Derivatives may be chemically synthesized, but will be typically prepared by recombinant nucleic acid methods. Additional components such as lipid, and/or polysaccharide, and/or polyketide components may be included. 5 The term non-cytotoxic means that the protease molecule in question does not kill the pain-sensing target cell to which it has been re-targeted. The "protease cleavage site" of the present invention allows cleavage (preferably io controlled cleavage) of the conjugate at a position between the non-cytotoxic protease component and the TM component. In one embodiment, the conjugate may include more than one proteolytic cleavage site. However, where two or more such sites exist, they are different, thereby substantially preventing the occurrence of multiple cleavage events in the presence of a single protease. In another 15 embodiment, it is preferred that the conjugate has a single protease cleavage site. The protease cleavage sequence(s) may be introduced (and/or any inherent cleavage sequence removed) at the DNA level by conventional means, such as by site-directed mutagenesis. Screening to confirm the presence of cleavage sequences may be performed manually or with the assistance of computer software 20 (e.g. the MapDraw program by DNASTAR, Inc.). Whilst any protease cleavage site may be employed, the following are preferred: Enterokinase (DDDDKJ) 25 Factor Xa (IEGRJ / IDGRJ) TEV(Tobacco Etch virus) (ENLYFQ4G) Thrombin (LVPRIGS) PreScission (LEVLFQIGP). 30 Also embraced by the term protease cleavage site is an intein, which is a self cleaving sequence. The self-splicing reaction is controllable, for example by varying the concentration of reducing agent present.

54 The present invention is now described by reference to the following Examples and Figures, without intended limitation thereto. 5 Figures Figure 1 Expression and purification of recLHN/B fusion protein Figure 2 Expression and purification of LHN/C fusion protein Figure 3 Expression and purification of N[1 -1 7]-LHN/A fusion protein io Figure 4 Purification of a LC/A-nociceptin-HN/A fusion protein Figure 5 Purification of a nociceptin-LC/A-HN/A fusion protein Figure 6 Purification of a LC/C-nociceptin-HN/C fusion protein Figure 7 Purification of a LC/A-met enkephalin-HN/A fusion protein Figure 8 Comparison of binding efficacy of a LC/A-nociceptin-HN/A fusion 15 protein and a nociceptin-LC/A-HN/A fusion protein Figure 9 In vitro catalytic activity of a LC/A-nociceptin-HN/A fusion protein Figure 10 Purification of a LC/A-nociceptin variant-HN/A fusion protein Figure 11 Comparison of binding efficacy of a LC/A-nociceptin-HN/A fusion protein and a LC/A-nociceptin variant-HN/A fusion protein 20 Figure 12 Expressed / purified LC/A-nociceptin-HN/A fusion protein family with variable spacer length product(s) Figure 13 Inhibition of SP release and cleavage of SNAP-25 by CPN-A Figure 14 Inhibition of SP release and cleavage of SNAP-25 over extended time periods after exposure of DRG to CPN-A 25 Figure 15 Cleavage of SNAP-25 by CPNv-A Figure 16 Cleavage of SNAP-25 over extended time periods after exposure of DRG to CPNv-A Figure 17 CPNv-A fusion-mediated displacement of [3H]-nociceptin binding Figure 18 Expressed I purified CPNv(Ek)-A product 30 Figure 19 Cleavage of SNAP-25 by CPNv(Ek)-A Figure 20 Expressed / purified CPNv-C product Figure 21 Cleavage of syntaxin by CPNv-C 55 Figure 22 CPN-A efficacy in the Acute Capsaicin-Induced Mechanical Allodynia model Figure 23 CPN-A efficacy in the Streptozotocin (STZ)-Induced Peripheral Diabetic Neuropathy (Neuropathic Pain) model 5 Figure 24 CPNv-A efficacy in the Acute Capsaicin-Induced Mechanical Allodynia model Figure 25 Expressed / purified LC/A-CPLE-HN/A product Figure 26 Expressed / purified LC/A-CPBE-HN/A product Figure 27 Expressed / purified CPOP-A product io Figure 28 Expressed / purified CPOPv-A product Figure 29 In vitro SNAP-25 cleavage in a DRG cell model Figure 30 Expressed / purified CPNv-A-FXa-HT (removable his-tag) Figure 31 In vitro efficacy of LC/A-nociceptin-HN/A fusion proteins with variable spacer length, as assessed by ligand competition assay 15 Figure 32 In vitro efficacy of LC/A-nociceptin-HN/A fusion proteins with variable spacer length, as assessed by in vitro SNAP-25 cleavage Figure 33 Cleavage of SNARE protein by dynorphin conjugates in embryonic spinal cord neurons (eSCNs) Figure 34 Cleavage of SNARE protein by dynorphin conjugates in Chinese 20 hamster ovary cells (CHO-K1 cells) transfected with OP2 receptor and SNAP-25 Figure 35 Cleavage of SNARE protein by dynorphin conjugates in embryonic spinal cord neurons (eSCNs) Figure 36 Kappa receptor activation studies with a range of dynorphin 25 conjugates Figure 37 Kappa receptor activation studies with a range of dynorphin conjugates The Figures are now described in more detail. 30 Figure 1 - Expression and purification of recLHN/B fusion protein 56 SDS-PAGE analysis of expression and purification of recLHN/B from E. coli. In Figure, recLHN/B is purified from cell paste using a three column strategy as described in Example 3. Protein samples are separated by SDS-PAGE and visualised by staining with simplyblue safestain coomassie reagent. Crude, soluble 5 MBP-LHN/B fusion protein contained within the clarified extract (lane 2) is loaded onto Q-Sepharose FF anion-exchange resin. Lane 3 represents recombinant MBP LHN/B fusion eluted from column at 150-200 mM salt. This sample is treated with factor Xa protease to remove MBP affinity tag (lane 4), and cleaved mixture diluted to lower salt concentration prior to loading onto a Q-Sepharose FF anion-exchange io column. Material eluted between 120-170 mM salt was rich in LHN/B (lane 5). Protein in lanes 6 and 8 represents LHN/B harvested after treatment with enterokinase and final purification using Benzamidine Sepharose, under non reducing and reducing conditions respectively. Lanes 1 and 7 represent molecular mass markers [Mark 12 (Invitrogen)]. 15 Figure 2 - Expression and purification of LHN/C fusion protein SDS-PAGE analysis of expression and purification of LHN/C from E. coli. In Figure 2, recLHN/C is purified from E. coli cell paste using a two-step strategy described in 20 Example 4. Protein samples are separated by SDS-PAGE and visualised by staining with coomassie blue. Clarified Crude cell lysate (lane 2) is loaded onto Q Sepharose FF anion-exchange resin. Fusion protein, MBP-LHN/C is eluted with 0.1 M NaCl (lane 3). Eluted material incubated at 22*C for 16 h with factor Xa protease (New England Biolabs) to cleave fusion tag MBP and nick recLHN/C at the linker site. 25 The protein of interest is further purified from cleaved fusion products (lane 4) using Q-Sepharose FF. Lanes 5 and 7 show purified recLHN/C under non-reducing conditions and reduced with 10 mM DTT respectively, to illustrate disulphide bonding at the linker region between LC and HN domains after nicking with factor Xa. Lanes 1 and 6 represent molecular mass markers (shown in KDa); Mark 12 (Invitrogen). 30 Figure 3 - Expression and purification of N[1-17]-LHNIA fusion protein 57 SDS-PAGE analysis of expression and purification of N[l-1 7]-LHN/A from E. coli. In Figure 3, N[1-17]-LHN/A is purified from E. coli BL21 cell paste using the methodology outlined in Example 9. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound 5 proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (Panel A) and Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were used as the primary antibody io for Western blotting. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. Figure 4 - Purification of a LC/A-nociceptin-HNiA fusion protein 15 Using the methodology outlined in Example 26, a LC/A-nociceptin-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then 20 re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (Panel A) and Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were used as the primary antibody for Western blotting. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. 25 Figure 5 - Purification of a nociceptin-LCA-HN/A fusion protein Using the methodology outlined in Example 26, a nociceptin-LC/A-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained 30 following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then 58 re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE (Panel A) and Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were used as the primary antibody for Western blotting. The final purified material in the absence and 5 presence of reducing agent is identified in the lanes marked [-] and [+] respectively. Figure 6 - Purification of a LC/C-nociceptin-HN/C fusion protein Using the methodology outlined in Example 26, an LC/C-nociceptin-HN/C fusion 1o protein was purified from E. coi BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from the 15 purification procedure were assessed by SDS-PAGE (Panel A) and Western blotting (Panel B). Anti-nociceptin antisera (obtained from Abcam) were used as the primary antibody for Western blotting. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. 20 Figure 7 - Purification of a LC/A-met enkephalin-HN/A fusion protein 25 Using the methodology outlined in Example 26, an LC/A-met enkephalin-HN/A fusion protein was purified from E. coi BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then 30 re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE. The final purified material in 59 the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. Figure 8 - Comparison of binding efficacy of a LCIA-nociceptin-HN/A fusion 5 protein and a nociceptin-LC/A-HN/A fusion protein The ability of nociceptin fusions to bind to the ORL 1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of test material in the presence of 1 nM [3H] io nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). It is clear that the LC/A-nociceptin-HN/A fusion is far superior to the nociceptin-LC/A-HN/A fusion at interacting with the ORL 1 receptor. 15 Figure 9 - In vitro catalytic activity of a LC/A-nociceptin-HN/A fusion protein The in vitro endopeptidase activity of the purified LC/A-nociceptin-HN/A fusion protein was determined essentially as described in Chaddock et al 2002, Prot. 20 Express Purif. 25, 219-228. Briefly, SNAP-25 peptide immobilised to an ELISA plate was exposed to varying concentrations of fusion protein for 1 hour at 37 0 C. Following a series of washes, the amount of cleaved SNAP-25 peptide was quantified by reactivity with a specific antisera. 25 Figure 10 - Purification of a LC/A-nociceptin variant-HN/A fusion protein Using the methodology outlined in Example 26, an LC/A-nociceptin variant-HN/A fusion protein was purified from E. coli BL21 cells. Briefly, the soluble products obtained following cell disruption were applied to a nickel-charged affinity capture 30 column. Bound proteins were eluted with 100 mM imidazole, treated with Factor Xa to activate the fusion protein and remove the maltose-binding protein (MBP) tag, then re-applied to a second nickel-charged affinity capture column. Samples from 60 the purification procedure were assessed by SDS-PAGE. The final purified material in the absence and presence of reducing agent is identified in the lanes marked [-] and [+] respectively. 5 Figure 11 - Comparison of binding efficacy of a LC/A-nociceptin-HN/A fusion protein and a LC/A-nociceptin variant-HN/A fusion protein The ability of nociceptin fusions to bind to the ORL 1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were io exposed to varying concentrations of test material in the presence of 1nM [3H] nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). It is clear that the LC/A-nociceptin variant-HN/A fusion (CPNv-LHA) is superior to the LC/A-nociceptin variant-HN/A fusion (CPN 15 LHA) at interacting with the ORL 1 receptor. Figure 12 - Expressed / purified LC/A-nociceptin-HN/A fusion protein family with variable spacer length product(s) 20 Using the methodology outlined in Example 26, variants of the LC/A-CPN-HN/A fusion consisting of GS10, GS30 and HX27 are purified from E. coli cell paste. Samples from the purification of LC/A-CPN(GS10)-HN/A, LC/A-CPN(GS15)-HN/A, LC/A-CPN(GS25)-HN/A, LC/A-CPN(GS30)-HN/A and LC/A-CPN(HX27)-HN/A were assessed by SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis 25 profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPBE-A. Top panel: M = benchmark molecular mass markers; S = total E. co/i protein soluble fraction; FT = proteins that did not bind to the Ni 2 +_ charged Sepharose column; FUSION = fusion protein eluted by the addition of imidazole. Bottom panel: Lane 1 = benchmark molecular mass markers; Lane 2 = 30 total E. coli protein soluble fraction; Lane 3 = purified material following initial capture on Ni 2 +-charged Sepharose; Lane 4 = Factor Xa treated material prior to final capture on Ni 2 +-charged Sepharose; Lane 5 = purified final material post activation 61 with Factor Xa (5 pl); Lane 6 = purified final material post activation with Factor Xa (10 pl); Lane 7 = purified final material post activation with Factor Xa (20 pl); Lane 8 = purified final material post activation with Factor Xa + DTT (5 pl); Lane 9 = purified final material post activation with Factor Xa + DTT (10 pl); Lane 10 = purified final 5 material post activation with Factor Xa + DTT (20 pl). Figure 13 - Inhibition of SP release and cleavage of SNAP-25 by CPN-A Briefly, primary cultures of dorsal root ganglia (DRG) were exposed to varying io concentrations of CPN-A for 24 hours. Cellular proteins were separated by SDS PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis and plotted against fusion concentration (dashed line). Material was also recovered for an analysis of substance P content using a specific is EIA kit. Inhibition of substance P release is illustrated by the solid line. The fusion concentration required to achieve 50% maximal SNAP-25 cleavage is estimated to be 6.30±2.48 nM. Figure 14 - Inhibition of SP release and cleavage of SNAP-25 over extended 20 time periods after exposure of DRG to CPN-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPN-A for 24 hours. Botulinum neurotoxin (BoNT/A) was used as a control. After this initial exposure, extracellular material was removed by washing, 25 and the cells incubated at 37*C for varying periods of time. At specific time points, cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis and illustrated by the dotted lines. Material was also recovered for an analysis of substance P content 30 using a specific EIA kit. Inhibition of substance P release is illustrated by the solid lines.

62 Figure 15 - Cleavage of SNAP-25 by CPNv-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPNv-A for 24 hours. Cellular proteins were separated by SDS 5 PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. The fusion concentration required to achieve 50% maximal SNAP-25 cleavage is estimated to be 1.38±0.36 nM. 10 Figure 16 - Cleavage of SNAP-25 over extended time periods after exposure of DRG to CPNv-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPNv-A for 24 hours. CPN-A was used as a control. After this 15 initial exposure, extracellular material was removed by washing, and the cells incubated at 37*C for varying periods of time. At specific time points, cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. 20 Figure 17 - CPNv-A fusion-mediated displacement of [3H]-nociceptin binding The ability of nociceptin fusions to bind to the ORL 1 receptor was assessed using a 25 simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of test material in the presence of 1 nM [3H] nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). It is clear that the LC/A-nociceptin variant-HN/A 30 fusion (labelled as CPNv-LHnA) is superior to the LC/A-nociceptin-HN/A fusion (labelled as CPN-LHnA) at interacting with the ORL 1 receptor.

63 Figure 18 - Expressed / purified CPNv(Ek)-A product Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species 5 of the expected molecular mass of CPNv(Ek)-A. Lane 1 = benchmark molecular mass markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = purified material following initial capture on Ni 2 +-charged Sepharose; Lane 4 = purified final material post activation with enterokinase (5 pl); Lane 5 = purified final material post activation with enterokinase (10 pl); Lane 6 = purified final material post activation 1o with enterokinase (20 pl); Lane 7 = purified final material post activation with enterokinase + DTT (5 pl); Lane 8 = purified final material post activation with enterokinase + DTT (10 pl); Lane 9 = purified final material post activation with enterokinase + DTT (20 pl). 15 Figure 19 - Cleavage of SNAP-25 by CPNv(Ek)-A Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPNv(Ek)-A for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an 20 assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. CPNv-A as prepared in Example 26 was used for comparison purposes. The percentage cleavage of SNAP-25 by CPNv(Ek)-A (labelled as En activated) and CPNv-A (labelled as Xa activated) are illustrated. 25 Figure 20 - Expressed / purified CPNv-C product Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPNv-C. Lane 1 = benchmark molecular mass 30 markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = purified material following initial capture on Ni 2 +-charged Sepharose; Lane 4 = Factor Xa treated material prior to final capture on Ni 2 +-charged Sepharose; Lane 5 = purified material 64 following second capture on Ni 2 +-charged Sepharose; Lane 6 = final purified material; Lane 7 = final purified material + DTT; Lane 8 = benchmark molecular mass markers. 5 Figure 21 - Cleavage of syntaxin by CPNv-C Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPNv-C for 24 hours. Cellular proteins were separated by SDS PAGE, Western blotted, and probed with anti-syntaxin to facilitate an assessment of 10 syntaxin cleavage. The percentage of cleaved syntaxin was calculated by densitometric analysis. The fusion concentration required to achieve 50% maximal syntaxin cleavage is estimated to be 3.13±1.96 nM. Figure 22 - CPN-A efficacy in the Acute Capsaicin-Induced Mechanical is Allodynia model The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibit capsaicin-induced mechanical allodynia was evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals were evaluated for paw withdrawal frequency 20 (PWF%) in response to a 10 g Von Frey filament stimulus series (10 stimuli x 3 trials) prior to recruitment into the study (Pre-Treat); after subcutaneous intraplantar treatment with CPN/A but before capsaicin (Pre-CAP); and following capsaicin challenge post-injection of CPN/A (average of responses at 15' and 30'; CAP). Capsaicin challenge was achieved by injection of 10 pL of a 0.3% solution. Sample 25 dilutions were prepared in 0.5% BSA/saline. Figure 23 - CPN-A efficacy in the Streptozotocin (STZ)-Induced Peripheral Diabetic Neuropathy (Neuropathic Pain) model 30 Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ in citrate buffer (l.V.) and blood glucose and lipid are measured weekly to define the readiness of the model. Paw Withdrawal Threshold (PWT) is measured in response 65 to a Von Frey filament stimulus series over a period of time. Allodynia is said to be established when the PWT on two consecutive test dates (separated by 1 week) measures below 6 g on the scale. At this point, rats are randomized to either a saline group (negative efficacy control), gabapentin group (positive efficacy control) or a 5 test group (CPN/A). Test materials (20-25 pl) are injected subcutaneously as a single injection (except gabapentin) and the PWT is measured at 1 day post treatment and periodically thereafter over a 2 week period. Gabapentin (30 mg/kg i.p. @ 3 ml/kg injection volume) is injected daily, 2 hours prior to the start of PWT testing. 10 Figure 24 - CPNv-A efficacy in the Acute Capsaicin-Induced Mechanical Allodynia model The ability of an LC/A-nociceptin variant-HN/A fusion (CPNv/A) to inhibit capsaicin 15 induced mechanical allodynia was evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals were evaluated for paw withdrawal frequency (PWF%) in response to a 10 g Von Frey filament stimulus series (10 stimuli x 3 trials) prior to recruitment into the study (Pre-Treat), after subcutaneous intraplantar treatment with CPNv/A but before capsaicin (Pre-CAP), and following 20 capsaicin challenge post-injection of CPNv/A (average of responses at 15' and 30'; CAP). Capsaicin challenge was achieved by injection of 10 pL of a 0.3% solution. Sample dilutions were prepared in 0.5% BSA/saline. These data are expressed as a normalized paw withdrawal frequency differential, in which the difference between the peak response (post-capsaicin) and the baseline response (pre-capsaicin) is 25 expressed as a percentage. With this analysis, it can be seen that CPNv/A is more potent than CPN/A since a lower dose of CPNv/A is required to achieve similar analgesic effect to that seen with CPN/A. Figure 25 - Expressed / purified LC/A-CPLE-HN/A product 30 Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species 66 of the expected molecular mass of CPLE-A. Lane 1 = benchmark molecular mass markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = purified material following initial capture on Ni 2 +-charged Sepharose; Lane 4 = Factor Xa treated material prior to final capture on Ni 2 +-charged Sepharose; Lane 5 = purified material 5 following second capture on Ni 2 +-charged Sepharose; Lane 6 = final purified material; Lane 7 = final purified material + DTT. Figure 26 - Expressed / purified LC/A-CPBE-HN/A product io Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPBE-A. Lane 1 = total E. coli protein soluble fraction; Lane 2 = purified material following initial capture on Ni 2 +-charged Sepharose; Lane 3 = Factor Xa treated material prior to final capture on Ni2+_ 15 charged Sepharose; Lane 4 = purified final material post activation with Factor Xa (5 pl); Lane 5 = purified final material post activation with Factor Xa (10 pl); Lane 6 = purified final material post activation with Factor Xa (20 pl); Lane 7 = purified final material post activation with Factor Xa + DTT (5 pl); Lane 8 = purified final material post activation with Factor Xa + DTT (10 pl); Lane 9 = purified final material post 20 activation with Factor Xa + DTT (20 pl); Lane 10 = benchmark molecular mass markers. Figure 27 - Expressed / purified CPOP-A product 25 Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPOP-A. Lane 1 = benchmark molecular mass markers; Lane 2 = purified material following initial capture on Ni 2 +-charged Sepharose; Lane 3 = Factor Xa treated material prior to final capture on Ni 2 +_ 30 charged Sepharose; Lane 4 = purified material following second capture on Ni2+_ charged Sepharose; Lane 5 = purified final material post activation with Factor Xa (5 pl); Lane 6 = purified final material post activation with Factor Xa (10 pl); Lane 7 = 67 purified final material post activation with Factor Xa (20 pl); Lane 8 = purified final material post activation with Factor Xa + DTT (5 pl); Lane 9 = purified final material post activation with Factor Xa + DTT (10 pl); Lane 10 = purified final material post activation with Factor Xa + DTT (20 pl). 5 Figure 28 - Expressed / purified CPOPv-A product Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species 10 of the expected molecular mass of CPOPv-A. Lane 1 = benchmark molecular mass markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = purified material following initial capture on Ni 2 +-charged Sepharose; Lane 4 = Factor Xa treated material prior to final capture on Ni 2 +-charged Sepharose; Lane 5 = purified final material post activation with Factor Xa (5 pl); Lane 6 = purified final material post 15 activation with Factor Xa (10 pl); Lane 7 = purified final material post activation with Factor Xa (20 pl); Lane 8 = purified final material post activation with Factor Xa + DTT (5 pl); Lane 9 = purified final material post activation with Factor Xa + DTT (10 pl); Lane 10 = purified final material post activation with Factor Xa + DTT (20 pl). 20 Figure 29 - In vitro SNAP-25 cleavage in a DRG cell model Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of CPOPv-A for 24 hours. Cellular proteins were separated by SDS PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment 25 of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. Figure 30 - Expressed / purified CPNv-A-FXa-HT (removable his-tag) 30 Proteins were subjected to SDS-PAGE prior to staining with Coomassie Blue. The electrophoresis profile indicates purification of a disulphide-bonded di-chain species of the expected molecular mass of CPNv-A-FXa-HT. Lane 1 = benchmark 68 molecular mass markers; Lane 2 = total E. coli protein soluble fraction; Lane 3 = Factor Xa treated material prior to final capture on Ni 2 '-charged Sepharose; Lane 4 = purified final material post activation with Factor Xa; Lane 5 = purified final material post activation with Factor Xa + DTT. 5 Figure 31 - In vitro efficacy of LC/A-nociceptin-HN/A fusion proteins with variable spacer length, as assessed by ligand competition assay The ability of LC/A-nociceptin-HN/A fusions of variable spacer length to bind to the 10 ORL 1 receptor was assessed using a simple competition-based assay. Primary cultures of dorsal root ganglia (DRG) were exposed to varying concentrations of test material in the presence of 1 nM [3H]-nociceptin. The reduction in specific binding of the radiolabelled ligand was assessed by scintillation counting, and plotted in comparison to the efficacy of unlabelled ligand (Tocris nociceptin). The upper panel 15 illustrates the displacement characteristics of the GSO, GS20, GS30 and Hx27 spacers, whilst the lower panel illustrates the displacement achieved by the GS10, GS15 and GS25 spaced fusion proteins. It is concluded that the GSO and GS30 spacers are ineffective, and the GS10 is poorly effective, at displacing nociceptin from the ORL1 receptor. 20 Figure 32 - In vitro efficacy of LC/A-nociceptin-HN/A fusion proteins with variable spacer length, as assessed by in vitro SNAP-25 cleavage Primary cultures of dorsal root ganglia (DRG) were exposed to varying 25 concentrations of CPN-A (of variable spacer length) for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP 25 was calculated by densitometric analysis. The poorly effective binding characteristics of the GS10 spaced fusion protein (see Figure 28) are reflected in the 30 higher concentrations of fusion required to achieve cleavage of intracellular SNAP 25. GSO and GS30 spaced fusion proteins were completely ineffective (date not shown). GS15, 20 and 25 spaced fusion proteins were similarly effective.

69 Figure 33 - Cleavage of SNARE protein by dynorphin conjugates in embryonic spinal cord neurons (eSCNs) Embryonic spinal cord neurons were exposed to varying concentrations of dynorphin 5 conjugates of the present invention for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. It is clear that LC/A-dynorphin-HN/A fusion is more potent than an unliganded LC/A-HN/A control molecule. The concentration of io LC/A-dynorphin-HN/A fusion required to achieve 50% maximal SNAP-25 cleavage is estimated to be 35.3 nM and the concentration for the LC/A-HN/A control required to achieve 50 % maximal SNAP-25 cleavage could not be determined due to it's low potency. 15 Figure 34 - Cleavage of SNARE protein by dynorphin conjugates in Chinese hamster ovary cells (CHO-KI cells) transfected with OP2 receptor and SNAP 25 Chinese hamster ovary (CHO) cells were transfected so that they express the OP2 receptor. Said cells were further transfected to express a SNARE protein (SNAP-25). 20 The transfected cells were exposed to varying concentrations of different dynorphin conjugates for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was calculated by densitometric analysis. It is clear that LC/A-CPDY-HN/A conjugates are more potent than the 25 unliganded LC/A-HN/A control molecule (labelled as LC/A-HN/A). Figure 35 - Cleavage of SNARE protein by dynorphin conjugates in embryonic spinal cord neurons (eSCNs) Embryonic spinal cord neurons were exposed to varying concentrations of dynorphin 30 conjugates of the present invention for 24 hours. Cellular proteins were separated by SDS-PAGE, Western blotted, and probed with anti-SNAP-25 to facilitate an assessment of SNAP-25 cleavage. The percentage of cleaved SNAP-25 was 70 calculated by densitometric analysis. It is clear that LC/A-CPDY-HN/A conjugates are more potent than the unliganded LC/A-HN/A control molecule (labelled as LC/A HN/A). 5 Figure 36 - Kappa receptor activation studies with a range of dynorphin conjugates Chinese hamster ovary (CHO) cells were transfected so that they express the OP2 receptor and SNAP-25. Said cells were used to measure cAMP deletion that occurs when the receptor is activated with a dynorphin ligand, using a FRET-based cAMP 1o kit (LANCE kit from Perkin Elmer). The transfected cells were exposed to varying concentrations of dynorphin conjugates of the present invention for 2 hours. cAMP levels were then detected by addition of a detection mix containing a fluorescently labelled cAMP tracer (Europium-streptavadi/biotin-cAMP) and fluorescently (Alexa) labelled anti-cAMP antibody and incubating at room temperature for 24 hours. Then is samples are excited at 320 nM and emitted light measured at 665 nM to determine cAMP levels. It is clear that LC/A-CPDY-HN/A conjugates are more potent than the unliganded LC/A-HN/A control molecule (labelled as LC/A-HN/A). Figure 37 - Kappa receptor activation studies with a range of dynorphin 20 conjugates Chinese hamster ovary (CHO) cells were transfected so that they express the OP2 receptor (purchased from Perkin Elmer). Said cells were transfected so they express SNAP-25 and used to measure cAMP deletion that occurs when the receptor is activated with a dynorphin ligand, using a FRET-based cAMP kit (LANCE kit from 25 Perkin Elmer). The transfected cells were exposed to varying concentrations of dynorphin conjugates of the present invention for 2 hours. cAMP levels were then detected by addition of a detection mix containing a fluorescently labelled cAMP tracer (Europium-streptavadi/biotin-cAMP) and fluorescently (Alexa) labelled anti cAMP antibody and incubating at room temperature for 24 hours. Then samples are 30 excited at 320 nM and emitted light measured at 665 nM to determine cAMP levels. It is clear from the figure by the reduction in maximum cAMP that the OP2 receptor is activated by LC/A-CPDY-HN/A (labelled as CPDY/A), LC/B-CPDY-HN/B (labelled 71 as CPDY/B), LC/C-CPDY-HN/C (labelled as CPDY/C), and LC/D-CPDY-HN/D (labelled as CPDY/D). The concentration required to achieve 50 % reduction in cAMP with LC/A-CPDY-HN/A, LC/B-CPDY-HN/B, LC/C-CPDY-HN/C (labelled as CPDY/, and LC/D-CPDY-HN/D is 10.47 nM, 14.79 nM, 14.79 nM and 23.99 nM, 5 respectively. Dynorphin peptide containing amino acids 1-17 of dynorphin A (labelled as dynorphin (1-17) was more potent than the fusions; 0.15 nm concentration required to achieve 50 % reduction of cAMP. SEQ ID Nos io Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/ codon is optional. SEQ ID1 DNA sequence of N[1-17] SEQ ID2 Protein Sequence of N[1-17] 15 SEQ ID3 DNA sequence of N[1-11] SEQ ID4 Protein sequence of N[1-11] SEQ ID5 DNA sequence of N[[Y10]1-11] SEQ ID6 Protein sequence of N[[Y10]1-11] SEQ ID7 DNA sequence of N[[Y11]1-11] 20 SEQ ID8 Protein sequence of N[[Y1 1]1-1 1] SEQ ID9 DNA sequence of N[[Y14]1-17] SEQ ID10 Protein sequence of N[[Y14]1-17] SEQ ID11 DNA sequence of N[1-13] SEQ ID12 Protein sequence of N[1-13] 25 SEQ ID13 DNA sequence of Nv (also known as N[[R14K15]1-17]) SEQ ID14 Protein sequence of Nv (also known as N[[R14K15]1-17]) SEQ ID15 DNA sequence of N[1-17]-LHN/A fusion protein SEQ ID16 Protein sequence of N[1-17]-LHN/A fusion protein SEQ ID17 DNA sequence of N[[Y1 1]1-11]-LHN/A fusion protein 30 SEQ ID18 Protein sequence of N[[Y1 1]1-11]-LHN/A fusion protein SEQ ID19 DNA sequence of N[1-13]-LHN/A fusion protein SEQ ID20 Protein sequence of N[1-13]-LHN/A fusion protein 72 SEQ ID21 DNA sequence of LHN/A-N[1-17] fusion protein SEQ ID22 Protein sequence of LHN/A-N[1-17] fusion protein SEQ ID23 DNA sequence of LHN/C-N[1-11] fusion protein SEQ ID24 Protein sequence of LHN/C-N[1-11] fusion protein 5 SEQ ID25 DNA sequence of N[[Y14]1-17]-LHN/C fusion protein SEQ ID26 Protein sequence of N[[Y14]1-17]-LHN/C fusion protein SEQ ID27 DNA sequence of the LC/A SEQ ID28 DNA sequence of the HN/A SEQ ID29 DNA sequence of the LC/B 10 SEQ ID30 DNA sequence of the HN/B SEQ ID31 DNA sequence of the LC/C SEQ ID32 DNA sequence of the HN/C SEQ ID33 DNA sequence of the CPN-A linker SEQ ID34 DNA sequence of the A linker 15 SEQ ID35 DNA sequence of the N-terminal presentation nociceptin insert SEQ ID36 DNA sequence of the CPN-C linker SEQ ID37 DNA sequence of the CPBE-A linker SEQ ID38 DNA sequence of the CPNvar-A linker SEQ ID39 DNA sequence of the LC/A-CPN-HN/A fusion 20 SEQ ID40 Protein sequence of the LC/A-CPN-HN/A fusion SEQ ID41 DNA sequence of the N-LC/A-HN/A fusion SEQ ID42 Protein sequence of the N-LC/A-HN/A fusion SEQ ID43 DNA sequence of the LC/C-CPN-HN/C fusion SEQ ID44 Protein sequence of the LC/C-CPN-HN/C fusion 25 SEQ ID45 DNA sequence of the LC/C-CPN-HN/C (A-linker) fusion SEQ ID46 Protein sequence of the LC/C-CPN-HN/C (A-linker) fusion SEQ ID47 DNA sequence of the LC/A-CPME-HN/A fusion SEQ ID48 Protein sequence of the LC/A-CPME-HN/A fusion SEQ ID49 DNA sequence of the LC/A-CPBE-HN/A fusion 30 SEQ ID50 Protein sequence of the LC/A-CPBE-HN/A fusion SEQ ID51 DNA sequence of the LC/A-CPNv-HN/A fusion SEQ ID52 Protein sequence of the LC/A-CPNv-HN/A fusion 73 SEQ ID53 DNA sequence of the LC/A-CPN[1-11]-HN/A fusion SEQ ID54 Protein sequence of the LC/A-CPN[1-11]-HN/A fusion SEQ ID55 DNA sequence of the LC/A-CPN[[Y10]1-11]-HN/A fusion SEQ ID56 Protein sequence of the LC/A-CPN[[Y1O]1-11]-HN/A fusion 5 SEQ ID57 DNA sequence of the LC/A-CPN[[Y11}1-11]-HN/A fusion SEQ ID58 Protein sequence of the LC/A-CPN[[Y 11]1-1 1]-HN/A fusion SEQ ID59 DNA sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusion SEQ ID60 Protein sequence of the LC/A-CPN[[Y14]1-17]-HN/A fusion SEQ ID61 DNA sequence of the LC/A-CPN[1-13]-HN/A fusion io SEQ ID62 Protein sequence of the LC/A- CPN[1-13]-HN/A fusion SEQ ID63 DNA sequence of the nociceptin-spacer-LC/A-HN/A fusion SEQ ID64 Protein sequence of the nociceptin-spacer-LC/A-HN/A fusion SEQ ID65 DNA sequence of the CPN-A GS10 linker SEQ ID66 DNA sequence of the CPN-A GS15 linker i5 SEQ ID67 DNA sequence of the CPN-A GS25 linker SEQ ID68 DNA sequence of the CPN-A GS30 linker SEQ ID69 DNA sequence of the CPN-A HX27 linker SEQ ID70 DNA sequence of the LC/A-CPN(GS1 5)-HN/A fusion SEQ ID71 Protein sequence of the LC/A-CPN(GS15)-HN/A fusion 20 SEQ 1072 DNA sequence of the LC/A-CPN(GS25)-HN/A fusion SEQ ID73 Protein sequence of the LC/A-CPN(GS25)-HN/A fusion SEQ ID74 DNA sequence of the CPNvar-A Enterokinase activatable linker SEQ ID75 DNA sequence of the LC/A-CPNv(Ek)-HN/A fusion SEQ ID76 Protein sequence of the LC/A-CPNv(Ek)-HN/A fusion 25 SEQ ID77 DNA sequence of the CPNvar-A linker SEQ ID78 DNA sequence of the LC/C-CPNv-HN/C fusion (act. A) SEQ ID79 Protein sequence of the LC/C-CPNv-HN/C fusion (act. A) SEQ ID80 DNA sequence of the LC/A-CPLE-HN/A fusion SEQ ID81 Protein sequence of the LC/A-CPLE-HN/A fusion 30 SEQ ID82 DNA sequence of the LC/A-CPOP-HN/A fusion SEQ ID83 Protein sequence of the LC/A-CPOP-HN/A fusion SEQ ID84 DNA sequence of the LC/A-CPOPv-HN/A fusion 74 SEQ ID85 Protein sequence of the LC/A-CPOPv-HN/A fusion SEQ ID86 DNA sequence of the IgA protease SEQ ID87 DNA sequence of the IgA-CPNv-HN/A fusion SEQ ID88 Protein sequence of the IgA-CPNv-HN/A fusion 5 SEQ ID89 DNA sequence of the FXa-HT SEQ ID90 DNA sequence of the CPNv-A-FXa-HT SEQ ID91 Protein sequence of the CPNv-A-FXa-HT fusion SEQ ID92 DNA sequence of the DT translocation domain SEQ ID93 DNA sequence of the CPLE-DT-A 1o SEQ ID94 Protein sequence of the CPLE-DT-A fusion SEQ ID95 DNA sequence of the TeNT LC SEQ ID96 DNA sequence of the CPNv-TENT LC SEQ ID97 Protein sequence of the CPNV-TeNT LC fusion SEQ ID98 DNA sequence of the CPNvar-C linker 15 SEQ ID99 DNA sequence of the LC/C-CPNv-HN/C fusion (act. C) SEQ ID100 Protein sequence of the LC/C-CPNv-HN/C fusion (act. C) SEQ ID101 Protein sequence of dynorphin SEQ ID102 DNA sequence of LC/A-CPDY-HN/A fusion SEQ ID103 Protein sequence of LC/A-CPDY-HN/A fusion 20 SEQ ID104 Protein sequence of LC/A-CPDY(GS10)-HN/A fusion SEQ ID105 Protein sequence of LC/A-CPDY(GS15)-HN/A fusion SEQ ID106 Protein sequence of LC/A-CPDY(GS25)-HN/A fusion SEQ ID107 Protein sequence of LC/C-CPDY-HN/C fusion SEQ ID108 Protein sequence of IgA-CPDY-HN/A fusion 25 SEQ ID109 Protein sequence of CPDY-TeNT LC fusion Examples 30 Example 1 - Confirmation of TM Agonist Activity by measuring release of substance P from neuronal cell cultures 75 Materials Substance P EIA is obtained from R&D Systems, UK. Methods 5 Primary neuronal cultures of eDRG are established as described previously (Duggan et al., 2002). Substance P release from the cultures is assessed by EIA, essentially as described previously (Duggan et al., 2002). The TM of interest is added to the neuronal cultures (established for at least 2 weeks prior to treatment); control cultures are performed in parallel by addition of vehicle in place of TM. Stimulated 1o (100 mM KCI) and basal release, together with total cell lysate content, of substance P are obtained for both control and TM treated cultures. Substance P immunoreactivity is measured using Substance P Enzyme Immunoassay Kits (Cayman Chemical Company, USA or R&D Systems, UK) according to manufacturers' instructions. 15 The amount of Substance P released by the neuronal cells in the presence of the TM of interest is compared to the release obtained in the presence and absence of 100 mM KCI. Stimulation of Substance P release by the TM of interest above the basal release, establishes that the TM of interest is an "agonist ligand" as defined in 20 this specification. If desired the stimulation of Substance P release by the TM of interest can be compared to a standard Substance P release-curve produced using the natural ORL-1 receptor ligand, nociceptin (Tocris). Example 2 - Expression and purification of catalytically active LHN/A 25 Materials Synthetic DNA obtained from Sigma Genosys. Restriction enzymes obtained from New England Biolabs. 30 Methods The expression and purification of catalytically active LHN/A was carried out essentially as described in Sutton et al., (2005), Prot. Express. Purif., 40, pp 31-41.

76 Briefly, DNA encoding the light chain plus 423 amino acids from the N-terminal of the heavy chain of BoNT/A was synthesised by Sigma-Genosys to produce a synthetic LHN/A gene with an E. coi codon bias. The linker region between the light chain and 5 HN domain was engineered to contain a Factor Xa cleavage site by splice-overlap extension PCR. Two PCR products were generated using primer pairs consisting of a long, mutagenic primer and a shorter, non-mutagenic primer: (5'-tccaaaactaaatctctgATAGAAGGTAGAaacaaagcgctgaacgac) with 10 (5'-CTTGATGTACTCTGTGAACGTGCTC); and (5'-gtcgttcagcgctttgttTCTACCTTCTATcagagatttagttttgga) with (5'-ATGGAGTTCGTTAACAAACAGTTC). 15 The products from these two reactions were used as templates for the splice-overlap extension PCR. A further PCR reaction was set up to add BamHl and Sall sites at either end of the activatable recLHN/A gene and these sites were used for insertion into an Invitrogen gateway entry vector. The entry vector was then used, along with a gateway recombination site adapted pMAL c2x, in a LR clonase reaction to form 20 pMAL c2x recLHN/A. The pMAL c2x recLHN/A was modified to incorporate a 6'HIS tag at the N-terminus of the MBP. This was achieved by the insertion of annealed oligonucleotides encoding the HIS tag into the Ndel site of pMAL. The expression vector expressing LHN/A was transformed into E. coli HMS174 or 25 AD494(DE3) (Novagen). Cultures were grown in Terrific broth complex medium supplemented with ZnCl 2 (1 pM), ampicillin (100 pg/ml), 0.2% (w/v) glucose. Parameters for expression of all the constructs were initially determined in shake flask cultures before transferring into 8 L fermentor systems. Starter cultures were grown for 16 hours at 37*C, 220 rpm and used to inoculate 1 L in which growth was 30 continued at 37 0 C, 250 rpm. At an OD600 nm of 0.6 the temperature was reduced to 250C for 30 minutes before induction with 1 mM IPTG. Induction was continued for 4 hours before the cells were harvested and stored at -70 0

C.

77 Typically 16 g of cell paste was suspended in 160 ml PBS and lysed by sonication (MSE Soniprep 150). The resulting lysate was clarified by centrifugation prior loading onto a 25 ml amylose column and eluted with 10 mM maltose in PBS. The 5 eluant contained approx. 50% pure fusion protein and was treated with Factor Xa (1 unit Factor Xa /100 pg fusion protein; 20 hours; 26'C) to remove the HISMBP and cleave the LC-HN junction to activate the protein. After incubation the sample was filtered (0.45 mm) and diluted two fold with water to give a 0.5 x PBS buffer composition. The cleaved, filtered and diluted recLHN/A was processed through a Q io Sepharose FF column (10 ml) and eluted with a step gradient of 80 mM NaCl containing HISMBP and 120 mM NaCI containing approx. 75% pure recLHN/A. The addition of His tag to MBP overcame previous co-elution problems with LHN/A and MBP. As a final polishing step to ensure complete removal of the HISMBP, the 120 mM NaCI elution from the Q Sepharose column was passed through a Nickel 15 charged 5 ml HisTrap column (Amersham). The flow through from the HisTrap column contained approx. 95% pure recLHN/A (see the Figures in Sutton et al., (2005), Prot. Express. Purif., 40, pp 31-41 for an illustration of the purification scheme for LHN/A). 20 Example 3 - Expression and purification of catalytically active recombinant LHN/B The methodology described below will purify catalytically active LHN/B protease from E. coli transformed with the appropriate plasmid encoding the LHN/B polypeptide. It 25 should be noted that various sequences of suitable LHN/B polypeptides have been described in PCT/GB97/02273, granted US 6 461617 and US patent application 10/241596, incorporated herein by reference. Methods The coding region for LHN/B is inserted in-frame to the 3' of the gene encoding maltose binding protein (MBP) in the expression vector pMAL (New England Biolabs) to create pMAL- c2x-LHN/B. In this construct, the expressed MBP and 78 LHN/B polypeptides are separated by a Factor Xa cleavage site, and the LC and HN domains are separated by a peptide that is susceptible to cleavage with enterokinase. The expression clone is termed pMAL-c2X-synLHN/B. pMAL-c2X-synLHN/B is transformed into E. coli HMS174 and cultured in Terrific broth complex medium in 8 L fermentor systems. Pre-induction bacterial growth is maintained at 37 0 C to an OD600 nm of 5.0, at which stage expression of recMBP LHN/B is induced by addition of IPTG to 0.5 mM and a reduction in temperature to 30 0 C. After four hours at 30 0 C the bacteria are harvested by centrifugation and the resulting paste stored at -70 0 C. The cell paste is resuspended in 20 mM Hepes pH 7.2, 125 mM NaCl, 1 pM ZnCl 2 and cell disruption achieved using an APV-Gaulin lab model 1000 homogeniser or a MSE Soniprep 150 sonicator. The resulting suspension is clarified by centrifugation prior to purification. Following cell disruption, the MBP-fusion protein is captured either on an amylose affinity resin in 20 mM Hepes pH 7.2, 125 mM NaCl, 1 pM ZnCl 2 , or on a Q Sepharose FF anion-exchange resin in 50 mM Hepes pH 7.2, 1 pM ZnCl 2 with no salt. A single peak is eluted from the amylose resin in the same buffer plus 10 mM maltose and from the Q-Sepharose in 150-200 mM salt. Cleavage of the MBP LHN/B junction is completed in an 18 hours incubation step at 22*C with Factor Xa (NEB) at 1 U/50 pg fusion protein. A substrate (MBP-LHN/B) concentration of at least 4 mg/ml is desirable for efficient cleavage to take place. The cleaved protein is diluted with 20 mM Hepes to a buffer composition of 20 mM Hepes, 25 mM NaCl, 1 pM ZnCl 2 , pH 7.2 and processed through a Q Sepharose column to separate the MBP from LHN/B. The LHN/B is eluted from the Q Sepharose column with 120-170 mM salt. The linker between the light chain and HN domain is then nicked by incubation with enterokinase at 1 U/100 pg of LHN/B at 22 0 C for 16 hours. Finally, the enterokinase is separated from the nicked LHN/B and other contaminating proteins on a Benzamidine Sepharose column, the enzyme 79 preferentially binding to the resin over an incubation of 30 minutes at 4*C. Purified LHN/B is stored at -200C until required. See Figure 1 for an illustration of the purification scheme for recLHN/B. Example 4 - Expression and purification of catalytically active recombinant LHNIC The coding region for LHN/C is inserted in-frame to the 3' of the gene encoding maltose binding protein (MBP) in the expression vector pMAL (New England Biolabs) to create pMAL- c2x-LHN/C. In this construct the expressed MBP and LHN/C polypeptides are separated by a Factor Xa cleavage site. pMAL-c2x-LHN/C is transformed into E. coli AD494 (DE3, IRL) and cultured in Terrific broth complex medium in 8 L fermentor systems. Pre-induction bacterial growth are maintained at 30*C to an OD600 nm of 8.0, at which stage expression of recMBP-c2x-LHN/C is induced by addition of IPTG to 0.5 mM and a reduction in temperature of culture to 25*C. After 4 hours at 250C the bacteria are harvested by centrifugation and the resulting paste stored at -70*C. The cell paste is resuspended in 50 mM Hepes pH 7.2, 1 pM ZnCl 2 at 1:6 (w/v) and cell disruption is achieved using an APV-Gaulin lab model 1000 homogeniser or a MSE Soniprep 150 sonicator. The resulting suspension is clarified by centrifugation prior to purification. 5 Following cell disruption and clarification, the MBP-fusion protein is separated on a Q-Sepharose Fast Flow anion-exchange resin in 50 mM Hepes pH 7.2, 1 pM ZnC1 2 and eluted with the same buffer plus 100 mM NaCl. A double point cleavage is performed at the MBP-LHN/C junction and the HN-LC linker in a single incubation io step with Factor Xa. The reaction is completed in a 16-hour incubation step at 220C with Factor Xa (NEB) at 1 U/100 ig fusion protein. The cleaved protein is diluted with 20 mM Hepes to a buffer composition of 20 mM Hepes, 25 mM NaCl, pH 7.2 and processed through a second Q-Sepharose column to separate the MBP from 80 LHN/C. Activated (disulphide-bonded cleaved linker) LHN/C is eluted from the Q Sepharose column by a salt gradient (20 mM Hepes, 500 mM NaCl, 1 pM ZnC1 2 , pH 7.2) in 120-170 mM salt. See Figure 2 for an illustration of the purification of LHN/C. 5 Example 5 - Production of a chemical conjugate of nociceptin and LHN/A Materials C-terminally extended nociceptin peptide obtained from Sigma Genosys. io Conjugation chemicals obtained from Pierce. Methods In order to couple the nociceptin peptide via a C-terminal Cys, the peptide was first synthesised (by standard procedures, commercially obtainable) to include a Cys as 15 the final C-terminal amino acid. This peptide was then used as the second component in a sulphydryl based coupling reaction as described below (see also previous publications WO 99/17806 and WO 96/33273 and Duggan et al., (2002), J. Biol. Chem. 277, 24846-34852 and 20 Chaddock et al., (2000), Infect Immun., 68, 2587-2593). Sulphydryl based coupling reaction Briefly, approximately two reactive leaving groups were introduced into LHN/A (5 mg/mI in phosphate-buffered saline) by reaction with N-succinimidyl-3-(2 25 pyridyldithio)propionate (SPDP). Derivatised material was isolated from excess SPDP by size exclusion chromatography. Reconstituted cysteine-tagged nociceptin ligand was mixed with the derivatised LHN/A in a 4:1 molar ratio, and incubated at room temperature for 30 1 hour with gentle agitation in order to create a chemical conjugate through a reducible covalent disulphide bond. Initial fractionation of the conjugate mixture to remove unconjugated peptide was performed by size exclusion chromatography 81 (Superose-12, or Superdex G-200 depending on scale of conjugation). 5 Example 6 - Production of a chemical conjugate of nociceptin and LHN/B Materials C-terminally extended nociceptin peptide obtained from Sigma Genosys. Conjugation chemicals obtained from Pierce. 10 Methods Lyophilised nociceptin was dissolved by the addition of water and dialysed into MES buffer (0.1 M MES, 0.1 M NaCl, pH 5.0). To this solution (at a concentration of about 0.3 mg/ml) was added PDPH (100 mg/mI in DMF) to a final concentration of 15 1 mg/ml. After mixing, solid EDAC was added to produce a final concentration of about 0.2 mg/ml. The reaction was allowed to proceed for at least 30 minutes at room temperature. Excess PDPH was then removed by desalting over a PD-10 column (Pharmacia) previously equilibrated with MES buffer. 20 An amount of LHN/B equivalent to half the weight of nociceptin used dissolved in triethanolamine buffer (0.02 M triethanolamine/HCI, 0.1 M sodium chloride, pH 7.8) at a concentration of about 1 mg/ml, was reacted with Traut's reagent (100 mM stock solution in 1 M triethanolamine/HCI, pH 8.0) at a final concentration of 2 mM. After 1 hour, the LHN/B was desalted into PBSE (phosphate buffered saline with 1 mM 25 EDTA) using a PD-10 column (Pharmacia). The protein peak from the column eluate was concentrated using a Microcon 50 (Amicon) to a concentration of about 2 mg/ml. The derivatised nociceptin was subjected to a final concentration step resulting in a 30 reduction in volume to less than 10% of the starting volume and then mixed with the derivatised LHN/B overnight at room temperature. The products of the reaction were analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl- 82 sulphate (SDS-PAGE). The conjugate resulting from the above reaction was partially purified by size exclusion chromatography over Bio-Gel P-100 (BioRad). The elution profile was 5 followed by measuring the optical density at 280 nm and SDS-PAGE analysis of the fractions. This allowed the separation of conjugate from free nociceptin and by products of the reaction. Example 7 - Production of a chemical conjugate of nociceptin 1-11 and LHN/B 10 Materials C-terminally extended nociceptin 1-11 peptide obtained from Sigma Genosys. Conjugation chemicals obtained from Pierce. 15 Methods In order to couple the nociceptin 1-11 peptide via a C-terminal Cys, the peptide was first synthesised (by standard procedures, commercially obtainable) to include a Cys as the final C-terminal amino acid. 20 This peptide was then used as the second component in a sulphydryl based coupling reaction as described in Example 5. Example 8 - Production of a chemical conjugate of nociceptin N[[Y14]1-17] and LHN/C 25 Materials C-terminally extended nociceptin N[[Y14]1-17] peptide obtained from Sigma Genosys. Conjugation chemicals obtained from Pierce. 30 Methods In order to couple the peptide via a C-terminal Cys, the peptide was first synthesised 83 (by standard procedures, commercially obtainable) to include a Cys as the final C terminal amino acid. This peptide was then used as the second component in a sulphydryl based 5 coupling reaction as described in Example 5. Example 9 - Recombinant production of a single polypeptide fusion of nociceptin-LHN/A (SEQ ID15 and SEQ ID16) 10 The DNA sequence for the nociceptin-LHN/A was designed by back translation of the LC/A, HN/A, and nociceptin amino acid sequences. The complete ORF containing the nociceptin-LC/A-activation loop-HN/A sequence was assembled within standard DNA sequence manipulation software (EditSeq). The activation loop between the LC/A cysteine and the HN/A cysteine (CVRGIITSKTKSLDKGYNKALNDLC) was 15 modified to incorporate a Factor Xa protease recognition site. Restriction sites appropriate to facilitate cloning into the required expression vector (for example BamHI/Sall) were incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame. The DNA sequence was screened 20 (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that were found to be common to those required by the cloning system were removed manually from the proposed coding sequence ensuring common E. coi codon usage was maintained. E. coli codon usage was assessed by reference to software 25 programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence containing the nociceptin-LC/A-activation loop-HN/A 30 open reading frame (ORF) was then commercially synthesized and provided in the pCR 4 vector.

84 The DNA encoding the nociceptin-LHN/A fusion was isolated from pCR 4 and transferred into pMAL vector backbone to facilitate protein expression. The resultant pMAL NO-LHN/A vector was transformed into competent E. coli BL21 and correct transformants selected. A single colony of pMAL NO-LHN/A was grown in Terrific 5 broth complex medium supplemented with ZnCl 2 (1 mM), ampicillin (100 pg/ml), 0.2% (w/v) glucose. Expression of the insert was induced by the addition of IPTG (0.1 mM) and the culture maintained at 160C for 16 hours. After this period of expression the bacteria were isolated by centrifugation and the cell pellet stored at -20 0 C until use. 10 10 g of E. coli BL21 cell paste was defrosted in a falcon tube containing 25 ml 50 mM HEPES, pH 7.2, 200 mM NaCl. The thawed cell paste was made up to 80 ml with 50 mM HEPES, pH 7.2, 200 mM NaCl and sonicated on ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring the sample remained 15 cool. The lysed cells were centrifuged at 18 000 rpm, 40C for 30 minutes. The supernatant was loaded onto a 0.1 M NiSO 4 charged chelating column (20-30 ml column is sufficient) and equilibrated with 50 mM HEPES, pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, the non-specific bound protein was 20 washed away and the fusion protein eluted with 100 mM imidazol. The eluted fusion protein was dialysed against 5 L of 50 mM HEPES, pH 7.2, 200 mM NaCl at 40C overnight and the OD of the dialysed fusion protein measured. 1 unit of Factor Xa was added per 100 pg fusion protein and incubated at 250C static overnight. The cleavage mixture was loaded onto a 0.1 M NiSO 4 charged Chelating column (20 25 30 ml column is sufficient) and equilibrated with 50 mM HEPES, pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, the non-specific bound protein was washed away and the fusion protein eluted with 100 mM imidazol. The eluted fusion 30 protein was dialysed against 5 L of 50 mM HEPES, pH 7.2, 200 mM NaCl at 40C overnight and the fusion concentrated to about 2 mg/ml, aliquoted and stored at -20*C.

85 Figure 3 shows the SDS-PAGE analysis of expression and purification of N[1-17] LHN/A. 5 Example 10 - Recombinant production of a single polypeptide fusion of (nociceptin 1-11)-LHN/B The DNA sequence for the (nociceptin 1-11)-LHN/B was designed by back translation of the LC/B, HN/B, and nociceptin 1-11 amino acid sequences. The io complete ORF containing the (nociceptin1-1 1)-LC/B-activation loop-HN/B sequence was assembled within standard DNA sequence manipulation software (EditSeq). The activation loop between the LC/B cysteine and the HN/B cysteine was modified to incorporate a Factor Xa protease recognition site. 15 The recombinant fusion protein was then produced essentially as described in Example 9. Example 11 - Recombinant production of a single polypeptide fusion of (nociceptin N[[Y14]1-17]) - LHN/C (SEQ ID25 and SEQ ID26) 20 The DNA sequence for the nociceptin N[[Y1 4]1-17] was designed by back translation of the LC/C, HN/C, and nociceptin N[[Y14]1-17] amino acid sequences. The complete ORF containing the (nociceptin N[[Y14]1-171)-LC/C-activation loop-HN/C sequence was assembled within standard DNA sequence manipulation software 25 (EditSeq). The activation loop between the LC/C cysteine and the HN/C cysteine was modified to incorporate a Factor Xa protease recognition site. The recombinant fusion protein was then produced essentially as described in Example 9. 30 Example 12 - Recombinant production of a single polypeptide fusion of LHN/C- 86 (nociceptin 1-11) (SEQ ID23 and SEQ ID24) The DNA sequence for the LHN/C-(nociceptin 1-11) was designed by back translation of the LC/C, HN/C and nociceptin 1-11 amino acid sequences. The 5 complete ORF (SEQ ID23) containing the LC/C-activation loop-HN/C-flexible spacer (nociceptin 1-11) was assembled within standard DNA sequence manipulation software (EditSeq). The recombinant fusion protein (SEQ ID24) was then produced essentially as 10 described in Example 9. Example 13 - Production of a conjugate for delivery of DNA encoding LC/C into a cell 15 The construction of a nociceptin-HN-[LC/C] conjugate is described below, where [LC/C] represents the polylysine condensed DNA encoding the light chain of botulinum neurotoxin type C. Materials 20 SPDP is from Pierce Chemical Co. Additional reagents are obtained from Sigma Ltd. Methods Using a plasmid containing the gene encoding LC/C under the control of a CMV 25 (immediate early) promoter, condensation of DNA was achieved using SPDP derivatised polylysine to a ratio of 2 DNA to 1 polylysine. Conjugates were then prepared by mixing condensed DNA (0.4 mg/ml) with HN-nociceptin (100 pg/ml) for 16 h at 25 0 C. The SPDP-derivatised polylysine and the free -SH group present on the HN domain combine to facilitate covalent attachment of the DNA and protein. 30 Example 14 - Production of a conjugate for delivery of DNA encoding LC/B into a cell 87 The construction of a (nociceptin 1-11)-HN-[LC/B] conjugate is described below, where [LC/B] represents the polylysine condensed DNA encoding the light chain of botulinum neurotoxin type B. 5 Materials SPDP is from Pierce Chemical Co. Additional reagents are obtained from Sigma Ltd. io Methods Using a plasmid containing the gene encoding LC/B under the control of a CMV (immediate early) promoter, condensation of DNA was achieved using SPDP derivatised polylysine to a ratio of 2 DNA to 1 polylysine. Conjugates were then prepared by mixing condensed DNA (0.4 mg/ml) with HN-(nociceptin 1-11) 15 (100 pg/ml) for 16 h at 25 0 C. The SPDP-derivatised polylysine and the free -SH group present on the HN domain combine to facilitate covalent attachment of the DNA and protein. Example 15 - Assessment of the activity of nociceptin-LHN/A in substance P 20 releasing neuronal cells Using methodology described in Duggan et al., (2002, J. Biol. Chem., 277, 34846 34852), the activity of nociceptin-LHN/A in substance P releasing neuronal cells was assessed. 25 Nociceptin-LHN/A fusion protein was applied to 2-week old dorsal root ganglia neuronal cultures, and incubated at 37 0 C for 16 hours. Following the incubation, the media was removed and the ability of the cells to undergo stimulated release of substance P (SP) was assessed. 30 The release of SP from the neuronal cells incubated with the nociceptin-LHN/A fusion protein was assayed in comparison to (i) LHN/A-only treated cells and (ii) cells 88 treated with media alone. This allowed the % inhibition of substance P from the eDRG to be calculated. The ability of the nociceptin-LHN/A fusion protein to inhibit SP release (relative to cells treated with media alone) was reported in Table 1. The data represent the mean of 3 determinations: 5 Table 1 Test Material (pM) nociceptin-LHN/A fusion protein LHN/A-only % Inhibition % Inhibition 1.0 47.3 25.6 0.1 13.8 -11.5 Example 16 - Confirmation of ORL 1 receptor activation by measuring io forskolin-stimulated cAMP production Confirmation that a given TM is acting via the ORL 1 receptor is provided by the following test, in which the TMs ability to inhibit forskolin-stimulated cAMP production is assessed. 15 Materials

[

3 H]adenine and [1 4 C]cAMP are obtained from GE Healthcare Methods 20 The test is conducted essentially as described previously by Meunier etal. [Isolation and structure of the endogenous agonist of opioid receptor-like ORL, receptor. Nature 377: 532-535, 1995] in intact transfected-CHO cells plated on 24-well plastic plates. 25 To the cells is added [3H]adenine (1.0 pCi) in 0.4 ml of culture medium. The cells remain at 37 0 C for 2 h to allow the adenine to incorporate into the intracellular ATP pool. After 2 h, the cells are washed once with incubation buffer containing: 130 mM 89 NaCl, 4.8 mM KCI, 1.2 mM KH 2

PO

4 , 1.3 mM CaCl 2 , 1.2 mM MgSO 4 , 10 mM glucose, 1 mg/ml bovine serum albumin and 25 mM HEPES, pH 7.4, and replaced with buffer containing forskolin (10 pM) and isobutylmethylxanthine (50 pM) with or without the TM of interest. After 10 min., the medium is aspirated and replaced with 0.5 ml, 5 0.2 M HCI. Approximately 1000 cpm of [ 1 4 C]cAMP is added to each well and used as an internal standard. The contents of the wells are then transferred to columns of 0.65 g dry alumina powder. The columns are eluted with 4 ml of 5 mM HCI, 0.5 ml of 0.1 M ammonium acetate, then two additional millilitres of ammonium acetate. The final eluate is collected into scintillation vials and counted for 1 4 C and tritium. 10 Amounts collected are corrected for recovery of [ 14 C]cAMP. TMs that are agonists at the ORL 1 receptor cause a reduction in the level of cAMP produced in response to forskolin. Example 17 - Confirmation of ORL 1 receptor activation using a GTPyS 15 binding functional assay Confirmation that a given TM is acting via the ORL, receptor is also provided by the following test, a GTPyS binding functional assay. 20 Materials

[

35 S]GTPyS is obtained from GE Healthcare Wheatgerm agglutinin-coated (SPA) beads are obtained from GE Healthcare Methods 25 This assay is carried out essentially as described by Traynor and Nahorski [Modulation by p-opioid agonists of guanosine-5 -O-(3-[ 35 S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Mol. Pharmacol. 47: 848 854, 1995]. 30 Cells are scraped from tissue culture dishes into 20 mM HEPES, 1 mM ethylenediaminetetraacetic acid, then centrifuged at 500 x g for 10 min. Cells are resuspended in this buffer and homogenized with a Polytron Homogenizer.

90 The homogenate is centrifuged at 27,000 x g for 15 min., and the pellet resuspended in buffer A, containing: 20 mM HEPES, 10 mM MgC 2 , 100 mM NaCl, pH 7.4. The suspension is recentrifuged at 20,000 x g and suspended once more in 5 buffer A. For the binding assay, membranes (8-15 pg protein) are incubated with ["S]GTP S (50 pM), GDP (10 pM), with and without the TM of interest, in a total volume of 1.0 ml, for 60 min. at 25*C. Samples are filtered over glass fibre filters and counted as described for the binding assays. 10 Example 18 - Preparation of a LC/A and HNIA backbone clones The following procedure creates the LC and HN fragments for use as the component backbone for multidomain fusion expression. This example is based on preparation of a serotype A based clone (SEQ ID27 and SEQ ID28), though the procedures and 15 methods are equally applicable to the other serotypes [illustrated by the sequence listing for serotype B (SEQ ID29 and SEQ ID30) and serotype C (SEQ ID31 and SEQ ID32)]. Preparation of cloning and expression vectors 20 pCR 4 (Invitrogen) is the chosen standard cloning vector, selected due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for easy construct confirmation. The expression vector is based on the pMAL (NEB) expression vector, which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (BamHl-Sall-Pst-Hindlll). 25 A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase purification options. Preparation of protease (e.g. LC/A) insert 30 The LC/A (SEQ ID27) is created by one of two ways: The DNA sequence is designed by back translation of the LC/A amino acid sequence [obtained from freely available database sources such as GenBank 91 (accession number P10845) or Swissprot (accession locus BXA1_CLOBO) using one of a variety of reverse translation software tools (for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. BamHI/Sal recognition sequences are incorporated at the 5' and 3' ends 5 respectively of the sequence, maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring 1o common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence containing the LC/A open reading frame 15 (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. The alternative method is to use PCR amplification from an existing DNA sequence with BamHI and Sall restriction enzyme sequences incorporated into the 5' and 3' 20 PCR primers respectively. Complementary oligonucleotide primers are chemically synthesised by a supplier (for example MWG or Sigma-Genosys), so that each pair has the ability to hybridize to the opposite strands (3' ends pointing "towards" each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands. To generate a PCR product the pair of short oligonucleotide 25 primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the 'PCR machine') that can change the incubation temperature of the reaction tube automatically, cycling between approximately 940C (for denaturation), 550C (for oligonucleotide annealing), and 72*C (for synthesis). Other reagents required for 30 amplification of a PCR product include a DNA polymerase (such as Taq or Pfu polymerase), each of the four nucleotide dNTP building blocks of DNA in equimolar 92 amounts (50-200 pM) and a buffer appropriate for the enzyme optimised for Mg 2 + concentration (0.5-5 mM). The amplification product is cloned into pCR 4 using either, TOPO TA cloning for 5 Taq PCR products or Zero Blunt TOPO cloning for Pfu PCR products (both kits commercially available from Invitrogen). The resultant clone is checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis [for example, using Quickchange (Stratagene Inc.)]. 10 Preparation of translocation (e.g. HN) insert The HN/A (SEQ ID28) is created by one of two ways: The DNA sequence is designed by back translation of the HN/A amino acid sequence [obtained from freely available database sources such as GenBank 15 (accession number P10845) or Swissprot (accession locus BXA1_CLOBO)] using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)}. A Pstl restriction sequence added to the N-terminus and Xbal-stop codon-HindIll to the C-terminus ensuring the correct reading frame is maintained. The DNA 20 sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by 25 reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 30 vector.

93 The alternative method is to use PCR amplification from an existing DNA sequence with Pstl and Xbal-stop codon-HindIll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as described above. The PCR product is inserted into pCR 4 vector and checked by 5 sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis [for example using Quickchange (Stratagene Inc.)]. Example 19 - Preparation of a LC/A-nociceptin-HN/A fusion protein (nociceptin 10 is N-terminal of the HN-chain) Preparation of linker-nociceptin-spacer insert The LC-HN linker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype A linker (in this 15 case defined as the inter-domain polypeptide region that exists between the cysteines of the disulphide bridge between LC and HN) is 23 amino acids long and has the sequence VRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood that proteolytic activation in nature leads to an HN domain that has an N terminus of the sequence ALNDL. This sequence information is freely available from 20 available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO). Into this linker a Factor Xa site, nociceptin and spacer are incorporated; and using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence 25 encoding the linker-ligand-spacer region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as BamHI-Sall-linker protease site-nociceptin-Nhel-spacer-Spel-Pstl-Xbal-stop codon-HindIll (SEQ ID33). It is important to ensure the correct reading frame is maintained for the spacer, nociceptin and restriction sequences and that the Xbal sequence is not preceded by 30 the bases, TC, which would result on DAM methylation. The DNA sequence is screened for restriction sequence incorporation, and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon 94 usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example, GenBank Release 143, 13 September 2004). This optimised 5 DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. Preparation of the LC/A-nociceptin-HvA fusion In order to create the LC-linker-nociceptin-spacer-HN construct (SEQ ID39), the pCR io 4 vector encoding the linker (SEQ ID33) is cleaved with BamHl + Sall restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID27) cleaved with BamHI + Sall. The resulting plasmid DNA is then cleaved with Pst + Xbal restriction enzymes and serves as the recipient vector for the insertion and ligation of the HN/A DNA (SEQ ID28) cleaved 15 with PstI + Xbal. The final construct contains the LC-linker-nociceptin-spacer-HN ORF (SEQ ID39) for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID40. Example 20 - Preparation of a nociceptin-LCA-HN/A fusion protein (nociceptin 20 is N-terminal of the LC-chain) The LC/A-HN/A backbone is constructed as described in Example 19 using the synthesised A serotype linker with the addition of a Factor Xa site for activation, arranged as BamHI-Sall-linker-protease site-linker-Pstl-Xbal-stop codon-Hindlll 25 (SEQ ID34). The LC/A-HN/A backbone and the synthesised N-terminal presentation nociceptin insert (SEQ ID35) are cleaved with BamHl + Hindlll restriction enzymes, gel purified and ligated together to create a nociceptin-spacer-LC-linker-HN. The ORF (SEQ ID41) is then cut out using restriction enzymes Aval + Xbal for transfer into expression vectors for expression to result in a fusion protein of the sequence 30 illustrated in SEQ ID42. Example 21 - Preparation of a LC/C-nociceptin-HN/C fusion protein 95 Following the methods used in Examples 1 and 2, the LC/C (SEQ ID31) and HN/C (SEQ ID32) are created and inserted into the C serotype linker arranged as BamHl Sal-linker-protease site-nociceptin-Nhel-spacer-Spel-Psti-Xbal-stop codon-HindIll 5 (SEQ ID36). The final construct contains the LC-linker-nociceptin-spacer-HN ORF (SEQ ID43) for expression as a protein of the sequence illustrated in SEQ ID44. Example 22 - Preparation of a LC/C-nociceptin-HN/C fusion protein with a serotype A activation sequence 10 Following the methods used in Examples 1 and 2, the LC/C (SEQ ID31) and HN/C (SEQ ID32) are created and inserted into the A serotype linker arranged as BamHl Sal-linker-protease site-nociceptin-Nhel-spacer-Spel-Pstl-Xbal-stop codon-Hindll (SEQ ID33). The final construct contains the LC-linker-nociceptin-spacer-HN ORF 15 (SEQ ID45) for expression as a protein of the sequence illustrated in SEQ ID46. Example 23 - Preparation of a LC/A-met enkephalin-HN/A fusion protein Due to the small, five-amino acid, size of the met-enkephalin ligand the LC/A-met 20 enkephalin-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin-HN/A fusion (SEQ ID39) as a template. Oligonucleotides are designed encoding the YGGFM met-enkephalin peptide, ensuring standard E.coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker 25 region of the LC/A-nociceptin-HN/A fusion (SEQ ID39) either side on the nociceptin section. The SDM product is checked by sequencing and the final construct containing the LC-linker-met enkephalin-spacer-HN ORF (SEQ ID47) for expression as a protein of the sequence illustrated in SEQ ID48. 30 Example 24 - Preparation of a LC/A-p endorphin-HN/A fusion protein 96 Following the methods used in Examples 1 and 2, the LC/A (SEQ ID27) and HN/A (SEQ ID28) are created and inserted into the A serotype P endorphin linker arranged as BamHI-Sall-linker-protease site-p endorphin-Nhel-spacer-Spel-Pstl-Xbal-stop codon-HindIll (SEQ ID37). The final construct contains the LC-linker-p endorphin 5 spacer-HN ORF (SEQ ID49) for expression as a protein of the sequence illustrated in SEQ ID50. Example 25 - Preparation of a LC/A-nociceptin variant-HN/A fusion protein io Following the methods used in Examples 1 and 2, the LC/A (SEQ ID27) and HN/A (SEQ ID28) are created and inserted into the A serotype nociceptin variant linker arranged as BamHI-Sal-linker-protease site-nociceptin variant-Nhel-spacer-Spel Pstl-Xbal-stop codon-Hindlli (SEQ ID38). The final construct contains the LC linker-nociceptin variant-spacer-HN ORF (SEQ ID51) for expression as a protein of is the sequence illustrated in SEQ ID52. Example 26 - Purification method for LC/A-nociceptin-HN/A fusion protein Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl and 20 approximately 10 g of E. coli BL21 cell paste. Make the thawed cell paste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate on ice 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4 0 C for 30 minutes. Load the supernatant onto a 0.1 M NiSO 4 charged Chelating column (20-30 ml column is sufficient) 25 equilibrated with 50 mM HEPES pH 7.2, 200mM NaCl. Using a step gradient of 10 and 40 mM imidazol, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 40C overnight and measure the OD of the dialysed fusion protein. Add 1 unit of factor Xa per 100 tg fusion protein and 30 Incubate at 250C static overnight. Load onto a 0.1 M NiSO 4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 97 mM NaCl. Wash column to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4 0 C overnight and 5 concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at -20 0 C. Test purified protein using OD, BCA, purity analysis and SNAP-25 assessments. Example 27 - Preparation of a LC/A-nociceptin-HN/A fusion protein (nociceptin is N-terminal of the HN-chain) 10 The linker-nociceptin-spacer insert is prepared as described in Example 19. Preparation of the LC/A-nociceptin-HN/A fusion In order to create the LC-linker-nociceptin-spacer-HN construct (SEQ ID39), the pCR 15 4 vector encoding the linker (SEQ ID33) is cleaved with BamHI + Sall restriction enzymes. This cleaved vector then serves as the recipient for insertion and ligation of the LC/A DNA (SEQ ID27) also cleaved with BamHI + Sall. The resulting plasmid DNA is then cleaved with BamHI + Hindlll restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning 20 site for BamHl, Sall, Pstl, and Hindlll such as the pMAL vector (NEB). The HN/A DNA (SEQ ID28) is then cleaved with Pstl + Hindill restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC-linker-nociceptin-spacer-HN RF (SEQ ID39) for expression as a protein of the sequence illustrated in SEQ ID40. 25 Example 28 - Preparation of a nociceptin-LC/A-HNIA fusion protein (nociceptin is N-terminal of the LC-chain) In order to create the nociceptin-spacer-LC/A-HN/A construct, an A serotype linker 30 with the addition of a Factor Xa site for activation, arranged as BamHI-Sal-linker protease site-linker-Pstl-Xbal-stop codon-HindIll (SEQ ID34) is synthesised as 98 described in Example 27. The pCR 4 vector encoding the linker is cleaved with BamHI + Sall restriction enzymes. This cleaved vector then serves as the recipient for insertion and ligation of the LC/A DNA (SEQ ID27) also cleaved with BamHl + Sall. The resulting plasmid DNA is then cleaved with BamHI + Hindlll restriction 5 enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing the synthesised N-terminal presentation nociceptin insert (SEQ ID35). This construct is then cleaved with Aval + Hindlll and inserted into an expression vector such as the pMAL plasmid (NEB). The HN/A DNA (SEQ ID28) is then cleaved with Pstl + Hindlll restriction enzymes and inserted into the similarly cleaved pMAL 10 nociceptin-LC/A-linker construct. The final construct contains the nociceptin-spacer LC/A-HN/A ORF (SEQ ID63) for expression as a protein of the sequence illustrated in SEQ ID64. Example 29 - Preparation and purification of an LC/A-nociceptin-HN/A fusion 15 protein family with variable spacer length Using the same strategy as employed in Example 19, a range of DNA linkers were prepared that encoded nociceptin and variable spacer content. Using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse 20 translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the linker-ligand-spacer region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as BamHI-Sall linker-protease site-nociceptin-Nhel-spacer-Spel-Pst-Xbal-stop codon-HindIll (SEQ ID65 to SEQ ID69). It is important to ensure the correct reading frame is maintained 25 for the spacer, nociceptin and restriction sequences and that the Xbal sequence is not preceded by the bases, TC which would result on DAM methylation. The DNA sequence is screened for restriction sequence incorporation and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to 30 software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This 99 optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. The spacers that were created included: 5 Table 2 Code Protein sequence of the linker SEQ ID of the linker DNA GS10 ALAGGGGSALVLQ 53 GS15 ALAGGGGSGGGGSALVLQ 54 GS25 ALAGGGGSGGGGSGGGGSGGGGSALVLQ 55 GS30 ALAGGGGSGGGGSGGGGSGGGGSGGGGSALVLQ 56 HX27 ALAAEAAAKEAAAKEAAAKAGGGGSALVLQ 57 By way of example, in order to create the LC/A-CPN(GS1 5)-HN/A fusion construct (SEQ ID70), the pCR 4 vector encoding the linker (SEQ ID66) is cleaved with BamHl io + Sall restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID27) also cleaved with BamHl + Sal. The resulting plasmid DNA is then cleaved with BamHl + HindIll restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHl, Sall, Pstl, and Hindlll such as is the pMAL vector (NEB). The HN/A DNA (SEQ ID28) is then cleaved with Pstl + Hindlll restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC/A-CPN(GS1 5)-HN/A ORF (SEQ ID70) for expression as a protein of the sequence illustrated in SEQ ID71. 20 As a further example, to create the LC/A-CPN(GS25)-HN/A fusion construct (SEQ ID72), the pCR 4 vector encoding the linker (SEQ ID67) is cleaved with BamHI + Sall restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID27) cleaved with BamHI + Sall. The resulting plasmid DNA is then cleaved with BamHl + Hindlll restriction enzymes and 100 the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHl, Sal, Pstl, and Hindlll such as the pMAL vector (NEB). The HN/A DNA (SEQ ID28) is then cleaved with Pstl + Hindlll restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The 5 final construct contains the LC/A-CPN(GS25)-HN/A ORF (SEQ ID72) for expression as a protein of the sequence illustrated in SEQ ID73. Variants of the LC/A-CPN-HN/A fusion consisting of GS10, GS30 and HX27 are similarly created. Using the purification methodology described in Example 26, 1o fusion protein is purified from E. coli cell paste. Figure 12 illustrates the purified product obtained in the case of LC/A-CPN(GS10)-HN/A, LC/A-CPN(GS15)-HN/A, LC/A-CPN(GS25)-HN/A, LC/A-CPN(GS30)-HN/A and LC/A-CPN(HX27)-HN/A. Example 30 - Assessment of in vitro efficacy of an LC/A-nociceptin-HN/A 15 fusion Fusion protein prepared according to Examples 2 and 9 was assessed in the eDRG neuronal cell model. 20 Assays for the inhibition of substance P release and cleavage of SNAP-25 have been previously reported (Duggan et al., 2002, J. Biol. Chem., 277, 34846-34852). Briefly, dorsal root ganglia neurons are harvested from 15-day-old fetal Sprague Dawley rats and dissociated cells plated onto 24-well plates coated with Matrigel at a density of 1 x 106 cells/well. One day post-plating the cells are treated with 10 pM 25 cytosine p-D-arabinofuranoside for 48 h. Cells are maintained in Dulbecco's minimal essential medium supplemented with 5% heat-inactivated fetal bovine serum, 5 mM L-glutamine, 0.6% D-glucose, 2% B27 supplement, and 100 ng/ml 2.5S mouse nerve growth factor. Cultures are maintained for 2 weeks at 37 0 C in 95% air/5% C02 before addition of test materials. 30 Release of substance P from eDRG is assessed by enzyme-linked immunosorbent 101 assay. Briefly, eDRG cells are washed twice with low potassium-balanced salt solution (BSS: 5 mM KCI, 137 mM NaCl, 1.2 mM MgC 2 , 5 mM glucose, 0.44 mM

KH

2

PO

4 , 20 mM HEPES, pH 7.4, 2 mM CaCl 2 ). Basal samples are obtained by incubating each well for 5 min. with 1 ml of low potassium BSS. After removal of this 5 buffer, the cells are stimulated to release by incubation with 1 ml of high potassium buffer (BSS as above with modification to include 100 mM KCI isotonically balanced with NaCl) for 5 min. All samples are removed to tubes on ice prior to assay of substance P. Total cell lysates are prepared by addition of 250 pl of 2 M acetic acid/0.1% trifluoroacetic acid to lyse the cells, centrifugal evaporation, and 1o resuspension in 500 pl of assay buffer. Diluted samples are assessed for substance P content. Substance P immunoreactivity is measured using Substance P Enzyme Immunoassay Kits (Cayman Chemical Company or R&D Systems) according to manufacturers' instructions. Substance P is expressed in pg/ml relative to a standard substance P curve run in parallel. 15 SDS-PAGE and Western blot analysis were performed using standard protocols (Novex). SNAP-25 proteins were resolved on a 12% Tris/glycine polyacrylamide gel (Novex) and subsequently transferred to nitrocellulose membrane. The membranes were probed with a monoclonal antibody (SMI-81) that recognises cleaved and intact 20 SNAP-25. Specific binding was visualised using peroxidase-conjugated secondary antibodies and a chemiluminescent detection system. Cleavage of SNAP-25 was quantified by scanning densitometry (Molecular Dynamics Personal SI, ImageQuant data analysis software). Percent SNAP-25 cleavage was calculated according to the formula: (Cleaved SNAP-25/(Cleaved+lntact SNAP-25))x100. 25 Following exposure of eDRG neurons to an LC/A-nociceptin-HN/A fusion (termed CPN-A), both inhibition of substance P release and cleavage of SNAP-25 are observed (Figure 13). After 24 h exposure to the fusion, 50% of maximal SNAP-25 cleavage is achieved by a fusion concentration of 6.3±2.5 nM. 30 The effect of the fusion is also assessed at defined time points following a 16 h exposure of eDRG to CPN-A. Figure 14 illustrates the prolonged duration of action 102 of the CPN-A fusion protein, with measurable activity still being observed at 28 days post exposure. 5 Example 31 - Assessment of in vitro efficacy of an LC/A-nociceptin variant HN/A fusion Fusion protein prepared according to Examples 8 and 9 was assessed in the eDRG neuronal cell mode using the method described in Example 30. 10 Following exposure of eDRG neurons to an LC/A-nociceptin variant-HN/A fusion (termed CPNv-A), both inhibition of substance P release and cleavage of SNAP-25 are observed. After 24 h exposure to the fusion, 50% of maximal SNAP-25 cleavage is achieved by a fusion concentration of 1.4±0.4 nM (Figure 15). 15 The effect of the fusion is also assessed at defined time points following a 16 h exposure of eDRG to CPN-A. Figure 16 illustrates the prolonged duration of action of the CPN-A fusion protein, with measurable activity still being observed at 24 days post exposure. 20 The binding capability of the CPNv-A fusion protein is also assessed in comparison to the CPN-A fusion. Figure 17 illustrates the results of a competition experiment to determine binding efficacy at the ORL-1 receptor. CPNv-A is demonstrated to displace [3H]-nociceptin, thereby confirming that access to the receptor is possible 25 with the ligand in the central presentation format. Example 32 - Preparation of an LC/A-nociceptin variant-HN/A fusion protein that is activated by treatment with Enterokinase 30 Following the methods used in Examples 1 and 2, the LC/A (SEQ ID27) and HN/A (SEQ ID28) are created and inserted into the A serotype nociceptin variant linker arranged as BamHI-Sal-linker-enterokinase protease site-nociceptin variant-Nhel- 103 spacer-Spel-Pstl-Xbal-stop codon-Hind IllI (SEQ ID74). The final construct contains the LC-linker-nociceptin variant-spacer-HN ORF sequences (SEQ ID75) for expression as a protein of the sequence illustrated in SEQ ID76. The fusion protein is termed CPNv(Ek)-A. Figure 18 illustrates the purification of CPNv(Ek)-A from E. 5 coli following the methods used in Example 26 but using Enterokinase for activation at 0.00064 pg per 100 pg of fusion protein. Example 33 - Assessment of in vitro efficacy of an LC/A-nociceptin variant HN/A fusion that has been activated by treatment with enterokinase l0 The CPNv(Ek)-A prepared in Example 32 is obtained in a purified form and applied to the eDRG cell model to assess cleavage of SNAP-25 (using methodology from Example 30). Figure 19 illustrates the cleavage of SNAP-25 following 24 h exposure of eDRG to CPNv(Ek)-A. The efficiency of cleavage is observed to be similar to that 15 achieved with the Factor Xa-cleaved material, as recorded in Example 31. Example 34 - Preparation of an LC/C-nociceptin variant-HN/C fusion protein with a Factor Xa activation linker derived from serotype A 20 Following the methods used in Example 21, the LC/C (SEQ ID31) and HN/C (SEQ ID32) are created and inserted into the A serotype nociceptin variant linker arranged as BamHI-Sanl-linker-nociceptin variant-Nhel-spacer-Spel-Pstl-Xbal-stop codon HindIll (SEQ ID77). The final construct contains the LC-linker-nociceptin variant spacer-HN ORF sequences (SEQ ID78) for expression as a protein of the sequence 25 illustrated in SEQ ID79. The fusion protein is termed CPNv-C (act. A). Figure 20 illustrates the purification of CPNv-C (act. A) from E. coli following the methods used in Example 26. Example 35 - Assessment of in vitro efficacy of an LC/C-nociceptin variant 30 HN/C fusion protein Following the methods used in Example 26, the CPNv-C (act. A) prepared in 104 Example 34 is obtained in a purified form and applied to the eDRG cell model to assess cleavage of SNAP-25 (using methodology from Example 30). After 24 h exposure to the fusion, 50% of maximal syntaxin cleavage is achieved by a fusion concentration of 3.1±2.0 nM. Figure 21 illustrates the cleavage of syntaxin following 5 24 h exposure of eDRG to CPNv-C (act. A). Example 36 - Assessment of in vivo efficacy of an LC/A-nociceptin-HN/A fusion io The ability of an LC/A-nociceptin- HN/A fusion (CPN/A) to inhibit acute capsaicin induced mechanical allodynia is evaluated following subcutaneous intraplantar injection in the rat hind paw. Test animals are evaluated for paw withdrawal frequency (PWF%) in response to a 10 g Von Frey filament stimulus series (10 stimuli x 3 trials) prior to recruitment into the study, after subcutaneous treatment 15 with CPN/A but before capsaicin, and following capsaicin challenge post-injection of CPN/A (average of responses at 15' and 30'). Capsaicin challenge is achieved by injection of 10 pL of a 0.3% solution. Sample dilutions are prepared in 0.5% BSA/saline. Figure 22 illustrates the reversal of mechanical allodynia that is achieved by pre-treatment of the animals with a range of concentrations of LC/A 20 nociceptin-HN/A fusion. The ability of an LC/A-nociceptin-HN/A fusion (CPN/A) to inhibit streptozotocin (STZ)-induced mechanical (tactile) allodynia in rats is evaluated. STZ-induced mechanical allodynia in rats is achieved by injection of streptozotocin (i.p. or i.v.) 25 which yields destruction of pancreatic p-cells leading to loss of insulin production, with concomitant metabolic stress (hyperglycemia and hyperlipidemia). As such, STZ induces Type I diabetes. In addition, STZ treatment leads to progressive development of neuropathy, which serves as a model of chronic pain with hyperalgesia and allodynia that may reflect signs observed in diabetic humans 30 (peripheral diabetic neuropathy). Male Sprague-Dawley rats (250-300 g) are treated with 65 mg/kg STZ in citrate 105 buffer (l.V.) and blood glucose and lipid are measured weekly to define the readiness of the model. Paw Withdrawal Threshold (PWT) is measured in response to a Von Frey filament stimulus series over a period of time. Allodynia is said to be established when the PWT on two consecutive test dates (separated by 1 week) 5 measures below 6 g on the scale. At this point, rats are randomized to either a saline group (negative efficacy control), gabapentin group (positive efficacy control) or a test group (CPN/A). Test materials (20-25 pl) are injected subcutaneously as a single injection (except gabapentin) and the PWT is measured at 1 day post treatment and periodically thereafter over a 2-week period. Gabapentin (30 mg/kg io i.p. @ 3 ml/kg injection volume) is injected daily, 2 hours prior to the start of PWT testing. Figure 23 illustrates the reversal of allodynia achieved by pre-treatment of the animals with 750 ng of CPN/A. Data were obtained over a 2-week period after a single injection of CPN/A 15 Example 37 - Assessment of in vivo efficacy of an LC/A-nociceptin variant HN/A fusion The ability of an LC/A-nociceptin variant-HN/A fusion (CPNv/A) to inhibit capsaicin induced mechanical allodynia is evaluated following subcutaneous intraplantar 20 injection in the rat hind paw. Test animals are evaluated for paw withdrawal frequency (PWF%) in response to a 10 g Von Frey filament stimulus series (10 stimuli x 3 trials) prior to recruitment into the study (Pre-Treat); after subcutaneous intraplantar treatment with CPNv/A but before capsaicin (Pre-CAP); and following capsaicin challenge post-injection of CPNv/A (average of responses at 15' and 30'; 25 CAP). Capsaicin challenge is achieved by injection of 10 pL of a 0.3% solution. Sample dilutions are prepared in 0.5% BSA/saline. Figure 24 illustrates the reversal of allodynia that is achieved by pre-treatment of the animals with a range of concentrations of LC/A-nociceptin variant-HN/A fusion in 30 comparison to the reversal achieved with the addition of LC/A-nociceptin-HN/A fusion. These data are expressed as a normalized paw withdrawal frequency differential, in which the difference between the peak response (post-capsaicin) and 106 the baseline response (pre-capsaicin) is expressed as a percentage. With this analysis, it can be seen that CPNv/A is more potent than CPN/A since a lower dose of CPNv/A is required to achieve similar analgesic effect to that seen with CPN/A. 5 Example 38 - Preparation of an LC/A-leu enkephalin-HN/A fusion protein Due to the small, five-amino acid, size of the leu-enkephalin ligand the LC/A-leu enkephalin-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin-HN/A fusion (SEQ ID39) io as a template. Oligonucleotides are designed encoding the YGGFL leu-enkephalin peptide, ensuring standard E. coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin-HN/A fusion (SEQ ID39) either side on the nociceptin section. The SDM product is checked by sequencing and the final construct 15 containing the LC-linker-leu enkephalin-spacer-HN ORF (SEQ ID80) for expression as a protein of the sequence illustrated in SEQ ID81. The fusion protein is termed CPLE-A. Figure 25 illustrates the purification of CPLE-A from E. coli following the methods used in Example 26. 20 Example 39 - Expression and purification of an LC/A-beta-endorphin-HN/A fusion protein Following the methods used in Example 26, and with the LC/A-beta-endorphin-HN/A fusion protein (termed CPBE-A) created in Example 24, the CPBE-A is purified from 25 E. coli. Figure 26 illustrates the purified protein as analysed by SDS-PAGE. Example 40 - Preparation of an LCiA-nociceptin mutant-HN/A fusion protein Due to the single amino acid modification necessary to mutate the nociceptin 30 sequence at position 1 from a Phe to a Tyr, the LC/A-nociceptin mutant-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin-HN/A fusion (SEQ ID39) as a template.

107 Oligonucleotides are designed encoding tyrosine at position 1 of the nociceptin sequence, ensuring standard E. coli codon usage is maintained and no additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin-HN/A fusion (SEQ ID39) either side on the nociceptin 5 section. The SDM product is checked by sequencing and the final construct containing the LC/A-nociceptin mutant-spacer-HN/A fusion ORF (SEQ ID82) for expression as a protein of the sequence illustrated in SEQ ID83. The fusion protein is termed CPOP-A. Figure 27 illustrates the purification of CPOP-A from E. coli following the methods used in Example 26. 10 Example 41 - Preparation and assessment of an LC/A-nociceptin variant mutant-HN/A fusion protein Due to the single amino acid modification necessary to mutate the nociceptin 15 sequence at position 1 from a Phe to a Tyr, the LC/A-nociceptin variant mutant-HN/A fusion is created by site directed mutagenesis [for example using Quickchange (Stratagene Inc.)] using the LC/A-nociceptin variant-HN/A fusion (SEQ ID51) as a template. Oligonucleotides are designed encoding tyrosine at position 1 of the nociceptin sequence, ensuring standard E. coi codon usage is maintained and no 20 additional restriction sites are incorporated, flanked by sequences complimentary to the linker region of the LC/A-nociceptin variant-HN/A fusion (SEQ ID51) either side on the nociceptin section. The SDM product is checked by sequencing and the final construct containing the LC/A-nociceptin mutant-spacer-HN/A fusion ORF (SEQ ID84) for expression as a protein of the sequence illustrated in SEQ ID85. The 25 fusion protein is termed CPOPv-A. Figure 28 illustrates the purification of CPOPv-A from E. coli following the methods used in Example 26. Using methodology described in Example 30, CPOPv-A is assessed for its ability to cleave SNAP-25 in the eDRG cell model. Figure 29 illustrates that CPOPv-A is able 30 to cleave SNAP-25 in the eDRG model, achieving cleavage of 50% of the maximal SNAP-25 after exposure of the cells to approximately 5.9 nM fusion for 24 h.

108 Example 42 - Preparation of an IgA protease-nociceptin variant-HN/A fusion protein The IgA protease amino acid sequence was obtained from freely available database 5 sources such as GenBank (accession number P09790). Information regarding the structure of the N. Gonorrhoeae IgA protease gene is available in the literature (Pohiner et al., Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease, Nature, 1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon), the DNA sequence encoding the IgA protease modified for E. coli 1o expression was determined. A BamHI recognition sequence was incorporated at the 5' end and a codon encoding a cysteine amino acid and Sall recognition sequence were incorporated at the 3' end of the IgA DNA. The DNA sequence was screened using MapDraw, (DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to 15 be common to those required for cloning were removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage was assessed Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables. This optimised DNA sequence (SEQ ID86) containing the IgA open 20 reading frame (ORF) is then commercially synthesized. The IgA (SEQ ID86) is inserted into the LC-linker-nociceptin variant-spacer-HN ORF (SEQ ID51) using BamHl and Sall restriction enzymes to replace the LC with the IgA protease DNA. The final construct contains the IgA-linker-nociceptin variant-spacer 25 HN ORF (SEQ ID87) for expression as a protein of the sequence illustrated in SEQ ID88. Example 43 - Preparation and assessment of a nociceptin targeted endopeptidase fusion protein with a removable histidine purification tag. 30 DNA was prepared that encoded a Factor Xa removable his-tag (his6), although it is clear that alternative proteases site such as Enterokinase and alternative purification 109 tags such as longer histidine tags are also possible. Using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the Factor Xa removable his-tag region is determined. 5 Restriction sites are then incorporated into the DNA sequence and can be arranged as Nhel-linker-Spel-Pstl-HN/A-Xbal-LEIEGRSGHHHHHHStop codon-Hindlll (SEQ ID89). The DNA sequence is screened for restriction sequence incorporated and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is 1o assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 15 4 vector. In order to create CPNv-A-FXa-HT (SEQ ID90, removable his-tag construct) the pCR 4 vector encoding the removable his-tag is cleaved with Nhel and Hindlll. The Nhel - Hindlll fragment is then inserted into the LC/A-CPNv-HN/A vector (SEQ ID51) that has also been cleaved by Nhel and Hindlll. The final construct contains the LC/A-linker-nociceptin variant-spacer-HN-FXa-Histag-Hindlll ORF 20 sequences (SEQ ID90) for expression as a protein of the sequence illustrated in SEQ ID91. Figure 30 illustrates the purification of CPNv-A-FXa-HT from E. coli following the methods used in Example 26. Example 44 - Preparation of a leu-enkephalin targeted endopeptidase fusion 25 protein containing a translocation domain derived from diphtheria toxin The DNA sequence is designed by back translation of the amino acid sequence of the translocation domain of the diphtheria toxin (obtained from freely available database sources such as GenBank (accession number 1XDTT) using one of a 30 variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. Restriction sites are then incorporated into the DNA sequence and can be arranged as Nhel- 110 Linker-Spel-Pstl- diphtheria translocation domain-Xbal-stop codon-HindIll (SEQ ID92). PstV/Xbal recognition sequences are incorporated at the 5' and 3' ends of the translocation domain respectively of the sequence maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, 5 DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. co/i codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage io Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence containing the diphtheria translocation domain is then commercially synthesized as Nhel-Linker-Spel-Pstl diphtheria translocation domain-Xbal-stop codon-Hindll (for example by Entelechon, 15 Geneart or Sigma-Genosys) and is provided in the pCR 4 vector (Invitrogen). The pCR 4 vector encoding the diphtheria translocation domain is cleaved with Nhel and Xbal. The Nhel - Xbal fragment is then inserted into the LC/A-CPLE-HN/A vector (SEQ ID80) that has also been cleaved by Nhel and Xbal. The final construct contains the LC/A-leu-enkephalin-spacer-diphtheria translocation domain ORF 20 sequences (SEQ ID93) for expression as a protein of the sequence illustrated in SEQ ID94. Example 45 - Preparation of a nociceptin variant targeted endopeptidase fusion protein containing a LC domain derived from tetanus toxin. 25 The DNA sequence is designed by back translation of the tetanus toxin LC amino acid sequence (obtained from freely available database sources such as GenBank (accession number X04436) using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or 30 Backtranslation tool v2.0 (Entelechon)]. BamHlI/Sall recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame (SEQ ID95). The DNA sequence is screened (using software 111 such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. 5 coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence containing the tetanus toxin LC open reading frame (ORF) is then commercially synthesized (for 10 example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector (invitrogen). The pCR 4 vector encoding the TeNT LC is cleaved with BamHI and Sall. The BamHI - Sall fragment is then inserted into the LC/A-CPNv-HN/A vector (SEQ ID51) that has also been cleaved by BamHl and Sal. The final construct contains the TeNT LC-linker-nociceptin variant-spacer-HN ORF sequences 15 (SEQ ID96) for expression as a protein of the sequence illustrated in SEQ ID97. Example 46 - Preparation of an LC/C-nociceptin variant-HN/C fusion protein with a native serotype C linker that is susceptible to Factor Xa cleavage 20 Following the methods used in Example 21, the LC/C (SEQ ID31) and HN/C (SEQ ID32) are created and inserted into the C serotype nociceptin variant linker arranged as BamHl-Sail-linker-nociceptin variant-Nhel-spacer-Spel-Pstl-Xbal-stop codon Hindlll (SEQ ID98). The final construct contains the LC-linker-nociceptin variant spacer-HN ORF sequences (SEQ ID99) for expression as a protein of the sequence 25 illustrated in SEQ ID100. The fusion protein is termed CPNv-C (act. C). Example 47 - Construction of CHO-K1 OP2 receptor activation assay and SNAP-25 cleavage assay Cell-line creation 30 CHO OP2 cell line was purchased from Perkin Elmer (ES-541-C, lot 451-719-A). Cells were transfected with SNAP-25 DNA using Lipofectamine T M 2000 and incubated for 4 hours before media replacement. After 24 hours, cells were 112 transferred to a T1 75 flask. 1 00ug/ml Zeocin was added after a further 24 hours to begin selection of SNAP-25 expressing cells, and Sug/mi Blasticidin added to maintain selective pressure for the receptor. Cells were maintained in media containing selection agents for two weeks, passaging cells every two to three days 5 to maintain 30-70% confluence. Cells were then diluted in selective media to achieve 0.5 cell per well in a 96 well microplate. After a few days, the plates were examined under a microscope, and those containing single colonies were marked. Media in these wells was changed weekly. As cells became confluent in the wells, they were transferred to T25 flasks. When they had expanded sufficiently each clone was io seeded to 24 wells of a 96 well plate, plus a frozen stock vial created. LC/A-CPDY HNA fusion and LC/A-HNA were applied to the cells for 24 hours, and then western blots performed to detect SNAP-25 cleavage. Clones from which SNAP-25 bands were strong and cleavage levels were high with fusion were maintained for further investigation. Full dose curves were run on these, and the clone (D30) with the 15 highest differential between LC/A-CPDY-HNA fusion and LC/A-HNA cleavage levels was selected. OP2 receptor activation assay The OP2 receptor activation measures the potency and intrinsic efficacy of ligands at 20 OP2 receptor in transfected CHO-K1 cells by quantifying the reduction of forskolin stimulated intracellular cAMP using a FRET-based cAMP (Perkin Elmer LANCE cAMP kit). After stimulation, a fluorescently labelled cAMP tracer (Europium streptavadin/biotin-cAMP) and fluorescently (Alexa) labelled anti-cAMP antibody are added to the cells in a lysis buffer. cAMP from the cells competes with the cAMP 25 tracer for antibody binding sites. When read, a light pulse at 320 nm excites the fluorescent portion (Europium) of the cAMP tracer. The energy emitted from the europium is transferred to the Alexa fluor-labelled antibodies bound to the tracer, generating a TR-FRET signal at 665 nm (Time-resolved fluorescence resonance energy transfer is based on the proximity of the donor label, europium, and the 30 acceptor label, Alexa fluor, which have been brought together by a specific binding reaction). Residual energy from the europium produces light at 615 nm. In agonist treated cells there will be less cAMP to compete with the tracer so a dose dependant 113 increase in signal at 665nm will be observed compared with samples treated with forskolin alone. The signal at 665nm signal is converted to cAMP concentration by interpolation to a cAMP standard curve which is included in each experiment. 5 Culture of cells for receptor activation assay: Cells were seeded and cultured in T175 flasks containing Ham F12 with Glutamax, 10% Foetal bovine serum, 5pg ml-1 Blasticidin and 1OOpg ml-1 Zeocin. The flasks were incubated at 370C in a humidified environment containing 5% CO2 until 60-80% confluent. On the day of harvest the media was removed and the cells washed twice 10 with 25 ml PBS. The cells were removed from the flask by addition of 1Oml of Tryple Express, and incubation at 37 *C for 10 min followed by gentle tapping of the flask. The dislodged cells were transferred to a 50 ml centrifuge tube and the flask washed twice with 10 ml media which was added to the cell suspension. The tube was centrifuged at 1300 x g for 3 min and the supernatant removed. Cells were gently re is suspended in 10 ml media (if freezing cells) or assay buffer (if using 'fresh' cells in assay), and a sample was removed for counting using a nucleocounter (ChemoMetec). Cells for use 'fresh' in an assay were diluted further in assay buffer to the appropriate concentration. Cells harvested for freezing were re-centrifuged (1300 x g; 3 min), the supernatant removed and cells re-suspended in Synth-a 20 freeze at 4 *C to 3x 106 cells/ml. Cryovials containing 1 ml suspension each were placed in a chilled Nalgene Mr Frosty freezing container (-1*C/minute cooling rate), and left overnight in a -80 0C freezer. The following day vials were transferred to the vapour phase of a liquid nitrogen storage tank. 25 Dilution of test materials and Cell assay Using Gilson pipettes and Sigmacoted or lo-bind tips, test materials and standards were diluted to the appropriate concentrations in the wells of the first two columns of an eppendorf 500pl deep-well lo-bind plate, in assay buffer containing 10pM forskolin. The chosen concentrations in columns one and two were half a log unit 30 apart. From these, serial 1:10 dilutions were made across the plate (using an electronic eight channel pipette with sigmacote or lo-bind tips) until eleven concentrations at half log intervals had been created. In the twelfth column, assay 114 buffer only was added as a 'basal'. Using a 12 channel digital pipette, 1 Opl of sample from the lo-bind plate was transferred to the optiplate 96 well microplate. To wells containing the standard curve, 10ul of assay buffer was added using a 5 multichannel digital pipette. To wells containing the test materials, 10ul of cells in assay buffer at the appropriate concentration were added. Plates were sealed and incubated for 120 min at room temperature, for the first hour on an IKA MTS 2/4 orbital shaker set to maximum speed. 10 Detection LANCE Eu-W8044 labelled streptavidin (Eu-SA) and Biotin-cAMP (b-cAMP) were diluted in cAMP Detection Buffer (both from Perkin Elmer LANCE cAMP kit) to create sub-stocks, at dilution ratios of 1:17 and 1:5, respectively. The final detection mix was prepared by diluting from the two sub stocks into detection buffer at a ratio 15 of 1:125. The mixture was incubated for 15-30 min at room temperature before addition of 1:200 Alexa Fluor@ 647-anti cAMP Antibody (Alexa-Fluor Ab). After briefly vortex mixing, 20 pl was immediately added to each well using a digital multichannel pipette. Microplate sealers were applied and plates incubated for 24h at room temperature (for the first hour on an IKA MTS 2/4 orbital shaker set to 20 maximum speed). Plate sealers were removed prior to reading on the Envision. Figures 36 and 37 show that dynorphin conjugates with LC/A-HN/A, LC/B-HN/B, LC/C-HN/C and LC/D-HN/D backbones active the OP2 receptor. 25 CHO-K1 OP2 SNAP-25 cleavage assay Cultures of cells were exposed to varying concentrations of fusion protein for 24 hours. Cellular proteins were separated by SDS-PAGE and western blotted with anti SNAP-25 antibody to facilitate assessment of SNAP-25 cleavage. SNAP-25 cleavage calculated by densitometric analysis (Syngene). 30 Plating cells 115 Prepare cells at 2x10e5 cells/ml and seed 125 pl per well of 96 well plate. Use the following media: 500 ml Gibco Ham F1 2 with Glutamax (product code 31765068), 50 ml FBS, Sug/mi Blasticidin (250 pl aliquot from box in freezer, G13) (Calbiochem #203351, 10 ml at 10 mg/ml), 100 ug/ml Zeocin (500 pl from box in freezer, G35). 5 (Invitrogen from Fisher, 1 g in 8x 1.25 ml tubes at 100 mg/ml product code VXR25001). Allow cells to grow for 24hrs (37 0 C, 5 % C02, humidified atmosphere). Cell treatment Prepare dilutions of test protein for a dose range of each test proteins ( make up t o double (2x) the desired final concentrations because 125 pl will be applied directly onto 125 pl of media already in each well). Filter sterilize CHO KOR D30 feeding medium (20 ml syringe, 0.2 pm syringe filter) to make the dilutions. Add the filtered medium into 5 labelled bijoux's (7 ml tubes), 0.9 ml each using a Gilson pipette or multi-stepper. Dilute the stock test protein to 2000 nM (working stock solution 1) and 15 600 nM (working stock solution 2). Using a Gilson pipette prepare 10-fold serial dilutions of each working stock, by adding 100 pl to the next concentration in the series. Pipette up and down to mix thoroughly. Repeat to obtain 4 serial dilutions for solution 1, and 3 serial dilutions for solution 2. A OnM control (filtered feeding medium only) should also be prepared as a negative control for each plate. Repeat 20 the above for each test protein. In each experiment a 'standard' batch of material must be included as control/reference material , this is unliganded LC/A-HN/A. Apply diluted Sample to CHO KOR D30 Plates Apply 125 pl of test sample (double concentration) per well. Each test sample should 25 be applied to triplicate wells and each dose range should include a 0 nM control. Incubate for 24hrs (37 *C, 5 % C02, humidified atmosphere). Cell Lysis Prepare fresh lysis buffer (20 mls per plate) with 25 % (4x) NuPAGE LDS sample 30 buffer, 65 % dH 2 0 and 10 % 1 M DTT. Remove medium from the CHO KOR D30 plate by inverting over a waste receptacle. Drain the remaining media from each well using a fine-tipped pipette. Lyse the cells by adding 125 pl of lysis buffer per well 116 using a multi-stepper pipette. After a minimum of 20 mins, remove the buffer from each well to a 1.5 ml microcentrifuge tube. Tubes must be numbered to allowing tracking of the CHO KOR treatments throughout the blotting procedure. A1-A3 down to H1-H3 numbered 1-24, A4-A6 down to H4-H6 numbered 25-48, A7-A9 down to 5 H7-H93 numbered 49-72, Al 0-A12 down to H1 0-H12 numbered 73-96. Vortex each sample and heat at 90*C for 5-10 mins in a prewarmed heat block. Store at -20*C or use on the same day on an SDS gel. Gel electrophoresis 10 If the sample has been stored o/n or longer, put in a heat block prewarmed to 90*C for 5-10 mins. Set up SDS page gels, use 1 gel per 12 samples, prepare running buffer (1x, Invitrogen NuPAGE MOPS SDS Running Buffer (20x) (NP0001)) ~ 800 ml / gel tank. Add 500pl of NuPAGE antioxidant to the upper buffer chamber. Load 15ul samples onto gel lanes from left to right as and load 2.5ul of Invitrogen Magic 15 Marker XP and 5ul Invitrogen See Blue Plus 2 pre-stained standard and 15ul of non treated control. It is important to maximize the resolution of separation during SDSPAGE. This can be achieved by running 12 % bis-tris gels at 200 V for 1 hour and 25 minutes (until the pink (17 kDa) marker reaches the bottom of the tank). 20 Western blotting Complete a Semi-dry transfer: using an Invitrogen iBlot (use iBlot Programme 3 for 6 minutes). Put the nitrocellulose membranes in individual small trays. Incubate the membranes with blocking buffer solution (5 g Marvel milk powder per 100 ml 0.1 %PBS/Tween) at room temperature, on a rocker, for 1 hour. Apply primary antibody 25 (Anti-SNAP-25 1:1000 dilution) and incubate the membranes with primary antibody (diluted in blocking buffer) for 1 hour on a rocker at room temperature. Wash the membranes by rinsing 3 times with PBS/Tween (0.1 %). Then apply the secondary (Anti-Rabbit-HRP conjugate diluted 1:1000) and incubate the membranes with secondary antibody (diluted in blocking buffer) at room temperature, on a rocker, for 30 1 hour. Wash the membranes by rinsing 3 times with PBS/Tween (0.1%), leave membrane a minimum of 20 mins for the last wash. Detect the bound antibody using Syngene: Drain blots of PBS/Tween, mix WestDura reagents 1:1 and add to blots for 117 5 minutes. Ensure enough solution is added to the membranes to completely cover them. Place membrane in Syngene tray, set up Syngene software for 5min expose time. 5 Figure 34 clearly shows that LC/A-CPDY-HN/A conjugates effectively cleave SNAP 25. Example 48 - Construction and activation of dynorphin conjugates Preparation of a LC/A and HN/A backbone clones i o The following procedure creates the LC and HN fragments for use as the component backbone for multidomain fusion expression. This example is based on preparation of a serotype A based clone (SEQ ID27 and SEQ ID28), though the procedures and methods are equally applicable to the other serotypes [illustrated by the sequence listing for serotype B (SEQ ID29 and SEQ ID30) and serotype C (SEQ ID31 and 15 SEQ ID32)]. Preparation of cloning and expression vectors pCR 4 (Invitrogen) is the chosen standard cloning vector, selected due to the lack of restriction sequences within the vector and adjacent sequencing primer sites for 20 easy construct confirmation. The expression vector is based on the pMAL (NEB) expression vector, which has the desired restriction sequences within the multiple cloning site in the correct orientation for construct insertion (BamHl-Sall-Pst-Hindlll). A fragment of the expression vector has been removed to create a non-mobilisable plasmid and a variety of different fusion tags have been inserted to increase 25 purification options. Preparation of protease (e.g. LC/A) insert The LC/A (SEQ ID27) is created by one of two ways: The DNA sequence is designed by back translation of the LC/A amino acid 30 sequence [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO) using one of a variety of reverse translation software tools (for example EditSeq best E.

118 coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. BamHI/Sal recognition sequences are incorporated at the 5' and 3' ends respectively of the sequence, maintaining the correct reading frame. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for 5 restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. co/i codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser 10 (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence containing the LC/A open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. 15 The alternative method is to use PCR amplification from an existing DNA sequence with BamHI and Sall restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. Complementary oligonucleotide primers are chemically synthesised by a supplier (for example MWG or Sigma-Genosys), so that each pair 20 has the ability to hybridize to the opposite strands (3' ends pointing "towards" each other) flanking the stretch of Clostridium target DNA, one oligonucleotide for each of the two DNA strands. To generate a PCR product the pair of short oligonucleotide primers specific for the Clostridium DNA sequence are mixed with the Clostridium DNA template and other reaction components and placed in a machine (the 'PCR 25 machine') that can change the incubation temperature of the reaction tube automatically, cycling between approximately 94 0 C (for denaturation), 550C (for oligonucleotide annealing), and 72 0 C (for synthesis). Other reagents required for amplification of a PCR product include a DNA polymerase (such as Taq or Pfu polymerase), each of the four nucleotide dNTP building blocks of DNA in equimolar 30 amounts (50-200 pM) and a buffer appropriate for the enzyme optimised for Mg 2 + concentration (0.5-5 mM).

119 The amplification product is cloned into pCR 4 using either, TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for Pfu PCR products (both kits commercially available from Invitrogen). The resultant clone is checked by 5 sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis [for example, using Quickchange (Stratagene Inc.)]. Preparation of translocation (e.g. HN) insert io The HN/A (SEQ ID28) is created by one of two ways: The DNA sequence is designed by back translation of the HN/A amino acid sequence [obtained from freely available database sources such as GenBank (accession number P10845) or Swissprot (accession locus BXA1_CLOBO)] using one of a variety of reverse translation software tools [for example EditSeq best E. 15 coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. A Pstl restriction sequence added to the N-terminus and Xbal-stop codon-Hindll to the C-terminus ensuring the correct reading frame is maintained. The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. 20 Any sequences that are found to be common to those required by the cloning system are removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coi codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to 25 published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector. 30 The alternative method is to use PCR amplification from an existing DNA sequence with Pstl and Xbal-stop codon-HindIll restriction enzyme sequences incorporated into the 5' and 3' PCR primers respectively. The PCR amplification is performed as 120 described above. The PCR product is inserted into pCR 4 vector and checked by sequencing. Any additional restriction sequences which are not compatible with the cloning system are then removed using site directed mutagenesis [for example using Quickchange (Stratagene Inc.)]. 5 Preparation of linker-dynorphin-spacer insert The LC-HN tinker can be designed from first principle, using the existing sequence information for the linker as the template. For example, the serotype A linker (in this case defined as the inter-domain polypeptide region that exists between the 1o cysteines of the disulphide bridge between LC and HN) is 23 amino acids long and has the sequence VRGIITSKTKSLDKGYNKALNDL. Within this sequence, it is understood that proteolytic activation in nature leads to an HN domain that has an N terminus of the sequence ALNDL. This sequence information is freely available from available database sources such as GenBank (accession number P10845) or 15 Swissprot (accession locus BXA1_CLOBO). Into this linker an enterokinase site, dynorphin and spacer are incorporated; and using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the linker-ligand-spacer region is determined. Restriction sites are then 20 incorporated into the DNA sequence and can be arranged as BamHI-Sall-linker protease site-dynorphin-Nhel-spacer-Spel-Pstl-Xbal-stop codon-Hindlli. It is important to ensure the correct reading frame is maintained for the spacer, dynorphin and restriction sequences and that the Xbal sequence is not preceded by the bases, TC, which would result on DAM methylation. The DNA sequence is 25 screened for restriction sequence incorporation, and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage 30 tables (for example, GenBank Release 143, 13 September 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.

121 Preparation of the LC/A-dynorphin-HN/A fusion In order to create the LC-linker-dynorphin-spacer-HN construct (SEQ ID102), the pCR 4 vector encoding the linker is cleaved with BamHI + Sall restriction enzymes. 5 This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID27) cleaved with BamHI + Sall. The resulting plasmid DNA is then cleaved with Pstl + Xbal restriction enzymes and serves as the recipient vector for the insertion and ligation of the HN/A DNA (SEQ ID28) cleaved with Pstl + Xbal. The final construct contains the LC-linker-dynorphin-spacer-HN ORF (SEQ ID102) io for transfer into expression vectors for expression to result in a fusion protein of the sequence illustrated in SEQ ID103. Examples 49 - Preparation and purification of an LC/A-dynorphin-HN/A fusion protein family with variable spacer length 15 Using the same strategy as employed in Example 48, a range of DNA linkers were prepared that encoded dynorphin and variable spacer content. Using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)], the DNA sequence encoding the linker-ligand-spacer region is determined. Restriction sites 20 are then incorporated into the DNA sequence and can be arranged as BamHI-Sall linker-protease site-dynorphin-Nhel-spacer-Spel-Pstl-Xbal-stop codon-Hindlll. It is important to ensure the correct reading frame is maintained for the spacer, dynorphin and restriction sequences and that the Xbal sequence is not preceded by the bases, TC which would result on DAM methylation. The DNA sequence is 25 screened for restriction sequence incorporation and any additional sequences are removed manually from the remaining sequence ensuring common E. coli codon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage 30 tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.

122 The spacers that were created included: Code Protein sequence of the linker SEQ ID of the linker DNA GS10 ALAGGGGSALVLQ 104 GS1 5 ALAGGGGSGGGGSALVLQ 105 GS25 ALAGGGGSGGGGSGGGGSGGGGSALVLQ 106 (1) By way of example, in order to create the LC/A-CPDY(GS25)-HN/A fusion construct 5 (SEQ ID106), the pCR 4 vector encoding the linker is cleaved with BamHl + Sall restriction enzymes. This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID27) also cleaved with BamHI + Sall. The resulting plasmid DNA is then cleaved with BamHl + Hindlll restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a io unique multiple cloning site for BamHl, Sall, Pstl, and Hindlll such as the pMAL vector (NEB). The HN/A DNA (SEQ ID28) is then cleaved with Pstl + Hindlll restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct. The final construct contains the LC/A-CPDY(GS25)-HN/A ORF for expression as a protein of the sequence illustrated in SEQ ID106. 15 Example 50 - Purification method for LC/A-Dynorphin-HN/A fusion protein Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl and approximately 10 g of E. coli BL21 cell paste. Make the thawed cell paste up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicate on ice 30 seconds on, 30 20 seconds off for 10 cycles at a power of 22 microns ensuring the sample remains cool. Spin the lysed cells at 18 000 rpm, 4 0 C for 30 minutes. Load the supernatant onto a 0.1 M NiSO 4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl. Using a step gradient of 10 and 40 mM imidazol, wash away the non-specific bound protein and elute the fusion 25 protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 40C overnight and measure the OD of the 123 dialysed fusion protein. Add 3.2 pl of enterokinase (2pg/ml) per 1mg fusion protein and Incubate at 250C static overnight. Load onto a 0.1 M NiSO 4 charged Chelating column (20-30 ml column is sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCI. Wash column to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl. 5 Using a step gradient of 10 and 40 mM imidazol, wash away the non-specific bound protein and elute the fusion protein with 100 mM imidazol. Dialyse the eluted fusion protein against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCI at 4 0 C overnight and concentrate the fusion to about 2 mg/ml, aliquot sample and freeze at -200C. Test purified protein using OD, BCA, purity analysis and SNAP-25 assessments. 10 Example 51 - Preparation of a LC/C-dynorphin-HN/C fusion protein with a serotype A activation sequence Following the methods used in Examples 18 and 19, the LC/C (SEQ ID31) and HN/C (SEQ ID32) are created and inserted into the A serotype linker arranged as BamHl 15 Sal-linker-protease site-dynorphin-Nhel-spacer-Spel-Pstl-Xbal-stop codon-HindIll. The final construct contains the LC-linker-dynorphin-spacer-HN ORF for expression as a protein of the sequence illustrated in SEQ ID107. Example 52 - Preparation of an IgA protease-dynorphin variant-HN/A fusion 20 protein The IgA protease amino acid sequence was obtained from freely available database sources such as GenBank (accession number P09790). Information regarding the structure of the N. Gonorrhoeae IgA protease gene is available in the literature (PohIner et al., Gene structure and extracellular secretion of Neisseria gononThoeae 25 IgA protease, Nature, 1987, 325(6103), 458-62). Using Backtranslation tool v2.0 (Entelechon), the DNA sequence encoding the IgA protease modified for E. coli expression was determined. A BamHI recognition sequence was incorporated at the 5' end and a codon encoding a cysteine amino acid and Sail recognition sequence were incorporated at the 3' end of the IgA DNA. The DNA sequence was screened 30 using MapDraw, (DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to 124 be common to those required for cloning were removed manually from the proposed coding sequence ensuring common E. coli codon usage is maintained. E. coli codon usage was assessed Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon 5 usage tables. This optimised DNA sequence (SEQ ID86) containing the IgA open reading frame (ORF) is then commercially synthesized. The IgA (SEQ ID86) is inserted into the LC-linker-dynorphin -spacer-HN ORF (SEQ ID102) using BamHI and Sall restriction enzymes to replace the LC with the IgA 1o protease DNA. The final construct contains the IgA-linker-dynorphin-spacer-HN ORF for expression as a protein of the sequence illustrated in SEQ ID108. Example 53 - Preparation of a dynorphin targeted endopeptidase fusion protein containing a LC domain derived from tetanus toxin. 15 The DNA sequence is designed by back translation of the tetanus toxin LC amino acid sequence (obtained from freely available database sources such as GenBank (accession number X04436) using one of a variety of reverse translation software tools [for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)]. BamHl/Sall recognition sequences are 20 incorporated at the 5' and 3' ends respectively of the sequence maintaining the correct reading frame (SEQ ID95). The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation. Any cleavage sequences that are found to be common to those required by the cloning system are removed manually from the 25 proposed coding sequence ensuring common E. colicodon usage is maintained. E. coli codon usage is assessed by reference to software programs such as Graphical Codon Usage Analyser (Geneart), and the %GC content and codon usage ratio assessed by reference to published codon usage tables (for example GenBank Release 143, 13 September 2004). This optimised DNA sequence containing the 30 tetanus toxin LC open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector (invitrogen). The pCR 4 vector encoding the TeNT LC is cleaved with BamHI 125 and Sall. The BamHl - Sall fragment is then inserted into the LC/A-dynorphin-HN/A vector (SEQ ID102) that has also been cleaved by BamHl and Sall. The final construct contains the TeNT LC-linker-dynorphin-spacer-HN ORF sequences for expression as a protein of the sequence illustrated in SEQ ID109. 5 Example 54 A method of treating, preventing or ameliorating pain in a subject, comprising administration to said patient a therapeutic effective amount of fusion protein, wherein said pain is selected from the group consisting of: chronic pain arising from io malignant disease, chronic pain not caused by malignant disease (peripheral neuropathies). Patient A A 73 year old woman suffering from severe pain caused by posthepatic neuralgia is 15 treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain. The patient experiences good analgesic effect within 2 hours of said injection. Patient B 20 A 32 year old male suffering from phantom limb pain after having his left arm amputated following a car accident is treated by peripheral injection with fusion protein to reduce the pain. The patient experiences good analgesic effect within 1 hour of said injection. 25 Patient C A 55 year male suffering from diabetic neuropathy is treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain. The patient experiences good analgesic effect within 4 hours of said injection. 30 Patient D 126 A 63 year old woman suffering from cancer pain is treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain. The patient experiences good analgesic effect within 4 hours of said injection. 5 All documents, books, manuals, papers, patents, published patent applications, guides, abstracts and other reference materials cited herein are incorporated by reference in their entirety. While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be 10 appreciated by one skilled in the art from reading this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. 15 In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in 20 various embodiments of the invention. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. 25 66151292 (GHMatters) P73912.AU.1 JESSIEL

Claims (10)

1. A non-cytotoxic protein conjugate for inhibition or reduction of exocytic fusion in a nociceptive sensory afferent cell, comprising: 5 (i) a dynorphin Targeting Moiety (TM), wherein said TM is an agonist of a receptor present on said nociceptive sensory afferent cell, and wherein said receptor 10 undergoes endocytosis to be incorporated into an endosome within the nociceptive sensory afferent cell (ii) a non-cytotoxic protease or a fragment thereof, 15 wherein the protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of said nociceptive sensory afferent cell; and (iii) a Translocation Domain, 20 wherein the Translocation Domain translocates the protease or protease fragment from within the endosome, across the endosomal membrane, and into the cytosol of the nociceptive sensory afferent cell, 25 wherein said conjugate comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 103, 104, 105, 106, 107, 108 and 109. 30

2. A pharmaceutical composition, comprising a conjugate according to Claim 1 and a pharmaceutically acceptable carrier. 6615129_2 (GHMatters) P73912.AU.1 JESSIEL 128

3. A DNA construct encoding the conjugate of Claim 1.

4. A DNA construct according to Claim 3, wherein the construct comprises a 5 DNA sequence selected from SEQ ID NO 102.

5. A method of preparing the conjugate of Claim 1, comprising expressing the DNA construct of Claim 3 in a host cell. 10

6. A method for treating, preventing or ameliorating pain in a subject, comprising administering to said patient a therapeutically effective amount of a conjugate according to Claim 1 or a composition according to Claim 2.

7. A method according to Claim 6, wherein the pain is chronic pain selected 15 from neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain and referred pain.

8. Use of a conjugate according to Claim 1 or a composition according to Claim 2, for the manufacture of a medicament for treating, preventing or 20 ameliorating pain.

9. Use according to Claim 8, wherein the pain is chronic pain selected from neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain and referred pain. 25

10. The non-cytotoxic protein conjugate according to claim 1, the pharmaceutical composition according to claim 2, the DNA construct according to claim 3, the method according to claim 5 or 6, or the use according to claim 8, substantially as hereinbefore described with reference to any one of the 30 Examples. 6615129_2 (GHMatters) P73912.AU.1 JESSIEL