US20140056870A1 - Fusion proteins - Google Patents

Fusion proteins Download PDF

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Publication number
US20140056870A1
US20140056870A1 US13/595,927 US201213595927A US2014056870A1 US 20140056870 A1 US20140056870 A1 US 20140056870A1 US 201213595927 A US201213595927 A US 201213595927A US 2014056870 A1 US2014056870 A1 US 2014056870A1
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Prior art keywords
pain
protease
amino acid
fusion protein
seq
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Abandoned
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US13/595,927
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English (en)
Inventor
Peter James
Keith Foster
John Chaddock
Kei Roger Aoki
Lance Steward
Joseph Francis
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Syntaxin Ltd
Allergan Inc
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Syntaxin Ltd
Allergan Inc
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Priority to US13/595,927 priority Critical patent/US20140056870A1/en
Assigned to ALLERGAN, INC. reassignment ALLERGAN, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FRANCIS, JOSEPH, STEWARD, LANCE E., AOKI, KEI ROGER
Assigned to SYNTAXIN LIMITED reassignment SYNTAXIN LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHADDOCK, JOHN, FOSTER, KEITH, JAMES, PETER
Priority to ES13759562.5T priority patent/ES2632470T3/es
Priority to US14/422,574 priority patent/US20150197739A1/en
Priority to PL13759562T priority patent/PL2888359T3/pl
Priority to EP13759562.5A priority patent/EP2888359B1/en
Priority to UAA201502728A priority patent/UA117349C2/uk
Priority to PT137595625T priority patent/PT2888359T/pt
Priority to JP2015529117A priority patent/JP2015528461A/ja
Priority to AU2013308233A priority patent/AU2013308233B2/en
Priority to HUE13759562A priority patent/HUE035286T2/en
Priority to KR1020157005061A priority patent/KR102283218B1/ko
Priority to EP17165621.8A priority patent/EP3246405B1/en
Priority to RU2015111001A priority patent/RU2652954C2/ru
Priority to DK13759562.5T priority patent/DK2888359T3/en
Priority to CN201380045083.XA priority patent/CN104769108A/zh
Priority to MX2015002588A priority patent/MX369005B/es
Priority to CA2882233A priority patent/CA2882233A1/en
Priority to ES17165621T priority patent/ES2737850T3/es
Priority to PCT/GB2013/052243 priority patent/WO2014033441A1/en
Priority to BR112015003947A priority patent/BR112015003947A8/pt
Publication of US20140056870A1 publication Critical patent/US20140056870A1/en
Priority to HK15111817.1A priority patent/HK1211056A1/xx
Priority to US15/661,433 priority patent/US20170327810A1/en
Priority to HK18105260.2A priority patent/HK1245833B/zh
Priority to JP2018172942A priority patent/JP2019004909A/ja
Priority to US16/416,435 priority patent/US11248219B2/en
Abandoned legal-status Critical Current

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21072—IgA-specific serine endopeptidase (3.4.21.72)
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    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24068—Tentoxilysin (3.4.24.68), i.e. tetanus neurotoxin
    • C—CHEMISTRY; METALLURGY
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    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
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    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24013—IgA-specific metalloendopeptidase (3.4.24.13)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification.
  • the name of the text file containing the sequence listing is 39905_Sequence Listing_FINAL — 2012-08-24_ST25.txt.
  • the text file is 352 KB; was created on Aug. 24, 2012; and is being submitted via EFS-Web with the filing of the specification.
  • This invention relates to non-cytotoxic fusion proteins, and to the therapeutic application thereof as analgesic molecules.
  • Toxins may be generally divided into two groups according to the type of effect that they have on a target cell.
  • the first group of toxins kill their natural target cells, and are therefore known as cytotoxic toxin molecules.
  • This group of 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 have attracted much interest in the design of “magic bullets” (e.g., immunoconjugates, which comprise a cytotoxic toxin component and an antibody that binds to a specific marker on a target cell) for the treatment of cellular disorders and conditions such as cancer. Cytotoxic toxins typically kill their target cells by inhibiting the cellular process of protein synthesis.
  • 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.
  • Non-cytotoxic toxins are produced by a variety of plants, and by a variety of microorganisms such as Clostridium sp. and Neisseria sp.
  • 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 genus Clostridium , most importantly C. tetani and 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, C1, D, E, F and G, and they all share similar structures and modes of action.
  • Clostridial neurotoxins represent a major group of non-cytotoxic toxin molecules, and are synthesized 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 (L-chain), which has a molecular mass of approximately 50 kDa.
  • L-chains possess a protease function (zinc-dependent endopeptidase activity) and exhibit a high substrate specificity for vesicle and/or plasma membrane associated proteins involved in the exocytic process.
  • L-chains from different clostridial species or serotypes may hydrolyze 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.
  • Neisseria sp. most importantly from the species N. gonorrhoeae , produce functionally similar non-cytotoxic proteases.
  • An example of such a protease is IgA protease (see WO99/58571).
  • toxin molecules may be re-targeted to a cell that is not the toxin's natural target cell.
  • the modified toxin 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.
  • 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 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.
  • TM replacement may be effected by conventional chemical conjugation techniques, which are well known to a skilled person.
  • recombinant techniques may be employed, such as those described in WO98/07864. All of the above cited references are incorporated by reference herein.
  • the present invention seeks to address one or more of the above problems by providing unique fusion proteins.
  • the present invention addresses one or more of the above-mentioned problems by providing a single chain, polypeptide fusion protein, comprising:
  • FIGS. 1A and 1B Depict the Purification of a LC/A-Spacer-Galanin-Spacer-H N /A Fusion Protein.
  • LC/A-GS18-galanin-GS20-H N /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 enterokinase to activate the fusion protein and treated with factor Xa to remove the maltose-binding protein (MBP) tag. Activated fusion protein was then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE ( FIG. 1A ) and Western blotting ( FIG. 1B ).
  • Anti-galanin antisera obtained from Abcam
  • Anti-histag antisera obtained from Qiagen
  • the final purified material in the absence and presence of reducing agent is identified in the lanes of FIG. 1A marked [ ⁇ ] and [+] respectively.
  • FIGS. 2A and 2B Depict the Purification of a LC/C-Spacer-Galanin-Spacer-H N /C Fusion Protein.
  • an LC/C-galanin-H N /C 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 enterokinase to activate the fusion protein, then re-applied to a second nickel-charged affinity capture column. Samples from the purification procedure were assessed by SDS-PAGE ( FIG. 2A ) and Western blotting ( FIG. 2B ).
  • Anti-galanin antisera obtained from Abcam
  • Anti-histag antisera obtained from Qiagen
  • the final purified material in the absence and presence of reducing agent in FIG. 2A is identified in the lanes marked [ ⁇ ] and [+] respectively.
  • FIGS. 3A and 3B Depict a Comparison of SNARE Cleavage Efficacy of a LC-Spacer-Galanin-Spacer-H N Fusion Protein and a LC-H N -Galanin Fusion Protein.
  • FIGS. 3A and 3B The ability of galanin fusions to cleave SNAP-25 in a CHO GALR1 SNAP25 cell was assessed.
  • Chinese hamster ovary (CHO) cells were transfected so that they express the GALR1 receptor. Said cells were further transfected to express a SNARE protein (SNAP-25). The transfected cells were exposed to varying concentrations of different galanin fusion proteins 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.
  • FIG. 4 Depicts GALR1 Receptor Activation Studies in the CHO-GALCHO-GALR1 SNAP-25 Cleavage Assay with Galanin Fusion Proteins of the Present Invention Having Different Serotype Backbones.
  • CHO cells Chinese hamster ovary (CHO) cells were transfected so that they express the GALR1 receptor and SNAP-25. Said cells were used to measure cAMP deletion that occurs when the receptor is activated with a galanin ligand, using a FRET-based cAMP kit (LANCE kit from Perkin Elmer). The transfected cells were exposed to varying concentrations of galanin (GA16) fusion proteins having different serotype backbones (i.e., botulinum neurotoxin serotypes A, B, C and D) for 2 hours.
  • GA16 galanin
  • serotype backbones i.e., botulinum neurotoxin serotypes A, B, C and D
  • cAMP levels were then detected by addition of a detection mix containing a fluorescently labeled cAMP tracer (Europium-streptavadi/biotin-cAMP) and fluorescently (Alexa) labeled anti-cAMP antibody and incubating at room temperature for 24 hours. Then samples were excited at 320 nM and emitted light measured at 665 nM to determine cAMP levels.
  • a detection mix containing a fluorescently labeled cAMP tracer (Europium-streptavadi/biotin-cAMP) and fluorescently (Alexa) labeled anti-cAMP antibody
  • FIG. 5 Depicts the Cleavage of SNARE Protein by Galanin (GA16 and GA30) Fusion Proteins in CHO-GALR1 SNAP-25 Cleavage Assay.
  • CHO cells Chinese hamster ovary (CHO) cells were transfected so that they express the GALR1 receptor. Said cells were further transfected to express a SNARE protein (SNAP-25). The transfected cells were exposed to varying concentrations of different galanin fusion proteins 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 data demonstrate that galanin fusion proteins having galanin-16 and galanin-30 ligands cleave SNARE protein. In addition, the data confirm that galanin fusion proteins having GS5, GS10 and GS18 spacers between the non-cytotoxic protease component and the protease cleavage site are functional.
  • FIG. 6 Depicts the Results of In Vivo Paw Guarding Assay Employing Galanin Fusion Proteins.
  • the nociceptive flexion reflex (also known as paw guarding assay) is a rapid withdrawal movement that constitutes a protective mechanism against possible limb damage. It can be quantified by assessment of electromyography (EMG) response in anesthetized rat as a result of low dose capsaicin, electrical stimulation or the capsaicin-sensitized electrical response.
  • EMG electromyography
  • Induction of paw guarding was achieved by 0.006% capsaicin, 10 ⁇ l in PBS (7.5% DMSO), injected in 10 seconds. This produced a robust reflex response from biceps feroris muscle.
  • a reduction/inhibition of the nociceptive flexion reflex indicates that the test substance demonstrates an anti-nociceptive effect.
  • FIG. 7 Depicts Galanin Fusion Protein Efficacy in Capsaicin-Induced Thermal Hyperalgesia Assay.
  • FIG. 8 Depicts Galanin Fusion Protein Efficacy in Capsaicin-Induced Thermal Hyperalgesia Assay.
  • FIGS. 9A Through 9C Depict the Activation of Galanin Fusion Proteins with Single and Double-Spacers.
  • FIGS. 9A and 9B Galanin fusion proteins lacking a first spacer (spacer 1) of the present invention located between the non-cytotoxic protease component and the Targeting Moiety component showed poor activation with protease ( FIGS. 9A and 9B ).
  • FIG. 9C demonstrates the enhanced activation of galanin fusion proteins of the present invention having both first (spacer 1) and second (spacer 2) spacers.
  • the non-cytotoxic protease component of the present invention is a non-cytotoxic protease, which protease is capable of cleaving different but specific peptide bonds in one of three substrate proteins, namely synaptobrevin, syntaxin or SNAP-25, of the exocytic fusion apparatus in a nociceptive sensory afferent. These substrates are important components of the neurosecretory machinery.
  • the non-cytotoxic protease component of the present invention is preferably a neisserial IgA protease or a clostridial neurotoxin L-chain.
  • the term non-cytotoxic protease embraces functionally equivalent fragments and derivatives of said non-cytotoxic protease(s).
  • a particularly preferred non-cytotoxic protease component is a botulinum neurotoxin (BoNT) L-chain.
  • the translocation component of the present invention enables translocation of the non-cytotoxic protease (or fragment thereof) into the target cell such that functional expression of protease activity occurs within the cytosol of the target cell.
  • the translocation component is preferably capable of forming ion-permeable pores 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 component may be obtained from a microbial protein source, in particular from a bacterial or viral protein source.
  • the translocation component is a translocating domain of an enzyme, such as a bacterial toxin or viral protein.
  • the translocation component of the present invention is preferably a clostridial neurotoxin H-chain or a fragment thereof. Most preferably it is the H N domain (or a functional component thereof), wherein H N 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.
  • the galanin TM component of the present invention is responsible for binding the fusion protein of the present invention to a Binding Site on a target cell.
  • the galanin TM component is a ligand through which the fusion proteins of the present invention bind to a selected target cell.
  • the target cell is a nociceptive sensory afferent, preferably a primary nociceptive afferent (e.g., an A-fiber such as an A ⁇ -fiber or a C-fiber).
  • a primary nociceptive afferent e.g., an A-fiber such as an A ⁇ -fiber or a C-fiber.
  • the fusion proteins of the present invention are capable of inhibiting neurotransmitter or neuromodulator (e.g., glutamate, substance P, calcitonin-gene related peptide (CGRP), and/or neuropeptide Y) release from discrete populations of nociceptive sensory afferent neurons.
  • the fusion proteins reduce or prevent the transmission of sensory afferent signals (e.g., neurotransmitters or neuromodulators) from peripheral to central pain fibers, and therefore have application as therapeutic molecules for the treatment of pain, in particular chronic pain.
  • TM binds to a nociceptive sensory afferent.
  • tissue or cells representative of the nociceptive sensory afferent for example DRGs
  • labeled e.g., tritiated
  • 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.
  • the assay may include one or more binding antagonists, and the assay may further comprise 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. Hulme, Oxford University Press.
  • the fusion proteins of the present invention generally demonstrate a reduced binding affinity (in the region of up to 10-fold) for the galanin receptor (e.g., GALR1) when compared with the corresponding ‘free’ TM (e.g., gal16).
  • the fusion proteins of the present invention surprisingly demonstrate good 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.
  • the galanin 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 mediated pharmacological response.
  • the fusion proteins of the present invention may be administered at a dosage that is much lower than would be employed for other types of analgesic molecules such as NSAIDS, morphine, and gabapentin.
  • the latter molecules are typically administered at high microgram to milligram (even up to hundreds of milligram) quantities, whereas the fusion proteins of the present invention may be administered at much lower dosages, typically at least 10-fold lower, and more typically at 100-fold lower.
  • the galanin TM of the invention can also be a molecule that acts as an “agonist” at one or more of the galanin receptors present on a nociceptive sensory afferent, more particularly on a primary nociceptive afferent.
  • 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.
  • the conventional meaning of an agonist 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.
  • an agonist is more specifically defined as a molecule that is capable of stimulating the process of exocytic fusion in a 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.
  • nerve growth factor is an agonist in respect of its ability to promote neuronal differentiation via binding to a TrkA receptor.
  • NGF nerve growth factor
  • NGF is not an agonist when assessed by the above criteria because it is not a principal inducer of exocytic fusion.
  • the process that NGF stimulates i.e., cell differentiation
  • the fusion proteins according to the present invention demonstrate preferential receptor binding and/or internalization properties. This, in turn, may result in more efficient delivery of the protease component to a pain-sensing target cell.
  • an agonist as a TM is self-limiting with respect to side-effects.
  • binding of an agonist TM to a pain-sensing target cell increases exocytic fusion, which may exacerbate the sensation of pain.
  • the exocytic process that is stimulated by agonist binding is subsequently reduced or inhibited by the protease component of the fusion protein.
  • the Targeting Moiety of the present invention comprises or consists of galanin and/or derivatives of galanin.
  • Galanin receptors e.g., GALR1, GALR2 and GALR3 are found pre- and post-synaptically in dorsal root ganglia (DRGs) (Liu and Hokfelt, (2002) Trends Pharm. Sci., 23(10):468-474), and are enhanced in expression during neuropathic pain states.
  • DDGs dorsal root ganglia
  • Xu et al., (2000) Neuropeptides, 34(3-4):137-147 provides further information in relation to galanin. All of the above cited references are incorporated by reference herein.
  • the target for the galanin TM is the GALR1, GALR2 and/or the GALR3 receptor. These receptors are members of the G-protein-coupled class of receptors, and have a seven transmembrane domain structure.
  • the galanin TM is a molecule that binds (preferably that specifically binds) to the GALR1, GALR2 and/or the GALR3 receptor. More preferably, the galanin TM is an “agonist” of the GALR1, GALR2 and/or the GALR3 receptor.
  • the term “agonist” in this context is defined as above.
  • Wild-type human galanin peptide is a 30 amino acid peptide, abbreviated herein as “GA30” (represented by SEQ ID NO: 7).
  • the galanin TM comprises or consists of SEQ ID NO: 7.
  • the invention also encompasses fragments, variants, and derivatives of the galanin TM described above. These fragments, variants, and derivatives substantially retain the properties that are ascribed to said galanin TM (i.e., are functionally equivalent). For example, the fragments, variants, and derivatives may retain the ability to bind to the GALR1, GALR2 and/or GALR3 receptor.
  • the galanin TM of the invention comprises or consists of a 16 amino acid fragment of full-length galanin peptide and is referred to herein as GA16 (represented by SEQ ID NO: 8).
  • the galanin TM comprises or consists of an amino acid sequence having at least 70%, preferably at least 80% (such as at least 82, 84, 85, 86, 88 or 89%), more preferably at least 90% (such as at least 91, 92, 93 or 94%), and most preferably at least 95% (such as at least 96, 97, 98, 99 or 100%) amino acid sequence acid identity to SEQ ID NO: 7 or SEQ ID NO: 8.
  • the galanin TM comprises or consists of an amino acid sequence having at least 70% (such as at least 80, 82, 84, 85, 86, 88 or 89%), more preferably at least 90% (such as at least 91, 92, 93 or 94%), and most preferably at least 95% (such as at least 96, 97, 98, 99 or 100%) amino acid sequence acid identity to full-length amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or a fragment of SEQ ID NO: 7 or SEQ ID NO: 8 comprising or consisting of at least 10 (such as at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) contiguous amino acid residues thereof.
  • the galanin Targeting Moiety comprises or consists of an amino acid sequence according to SEQ ID NO: 7 or a fragment comprising or consisting of at least 16 (such as at least 10, 11, 12, 13, 14 or 15) contiguous amino acid residues thereof, or a variant amino acid sequence of said SEQ ID NO: 7 or said fragment having a maximum of 6 (such as a maximum of 5, 4, 3, 2 or 1) conservative amino acid substitutions.
  • the protease cleavage site of the present invention allows cleavage (preferably controlled cleavage) of the fusion protein at a position between the non-cytotoxic protease component and the TM component. It is this cleavage reaction that converts the fusion protein from a single chain polypeptide into a disulphide-linked, di-chain polypeptide.
  • the galanin TM binds via a domain or amino acid sequence that is located away from the C-terminus of the galanin TM.
  • the relevant binding domain may include an intra domain or an amino acid sequence located towards the middle (i.e., of the linear peptide sequence) of the TM.
  • the relevant binding domain is located towards the N-terminus of the galanin TM, more preferably at or near to the N-terminus.
  • the single chain polypeptide fusion 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 embodiment, it is preferred that the single chain polypeptide fusion has a single protease cleavage site.
  • 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 (e.g., the MapDraw program by DNASTAR, Inc.).
  • protease cleavage site Whilst any protease cleavage site may be employed, the following are preferred:
  • Enterokinase SEQ ID NO: 60
  • DDDDK ⁇ Factor Xa
  • SEQ ID NO: 61/SEQ ID NO: 62 IEGR ⁇ /IDGR ⁇
  • TEV(Tobacco Etch virus) SEQ ID NO: 63
  • ENLYFQ ⁇ G TEV(Tobacco Etch virus)
  • Thrombin SEQ ID NO: 64
  • LVPR ⁇ GS PreScission
  • SEQ ID NO: 65 LEVLFQ ⁇ GP
  • the protease cleavage site is an enterokinase cleavage site (DDDDK ⁇ ).
  • enterokinase protease is used to cleave the enterokinase cleavage site and activate the fusion protein.
  • 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.
  • the protease cleavage site is cleaved and the N-terminal region (preferably the N-terminus) of the TM becomes exposed.
  • the resulting polypeptide has a TM with an N-terminal domain or an intra domain that is substantially free from the remainder of the fusion protein. This arrangement ensures that the N-terminal component (or intra domain) of the TM may interact directly with a Binding Site on a target cell.
  • the TM and the protease cleavage site are distanced apart in the fusion protein by at most 10 amino acid residues, more preferably by at most 5 amino acid residues, and most preferably by zero amino acid residues. In one embodiment, the TM and the protease cleavage site are distanced apart in the fusion protein by 0-10 (such as 0-9,0-8, 0-7,0-6, 0-5,0-4, 0-3, 0-2) and preferably 0-1 amino acid residues
  • a fusion is provided with a TM that has an N-terminal domain that is substantially free from the remainder of the fusion. This arrangement ensures that the N-terminal component of the Targeting Moiety may interact directly with a Binding Site on a target cell.
  • One advantage associated with the above-mentioned activation step is that the TM only becomes susceptible to N-terminal degradation once proteolytic cleavage of the fusion protein has occurred.
  • the selection of a specific protease cleavage site permits selective activation of the polypeptide fusion into a di-chain conformation.
  • Construction of the single-chain polypeptide fusion of the present invention places the protease cleavage site between the TM and the non-cytotoxic protease component.
  • the TM is located between the protease cleavage site and the translocation component. This ensures that the TM is attached to the translocation domain (i.e., as occurs with native clostridial holotoxin), though in the case of the present invention the order of the two components is reversed vis-à-vis native holotoxin.
  • a further advantage with this arrangement is that the TM is located in an exposed loop region of the fusion protein, which has minimal structural effects on the conformation of the fusion protein.
  • said loop is variously referred to as the linker, the activation loop, the inter-domain linker, or just the surface exposed loop (Schiavo et al., (2000) Phys. Rev. 80:717-766; Turton et al., (2002) Trends Biochem. Sci. 27:552-558).
  • the single chain fusion protein of the present invention comprises a first spacer located between the non-cytotoxic protease and the protease cleavage site, wherein said first spacer comprises (or consists of) an amino acid sequence of from 4 to 25 (such as from 6 to 25, 8 to 25, 10 to 25, 15 to 25 or from 4 to 21, 4 to 20, 4 to 18, 4 to 15, 4 to 12 or 4 to 10) amino acid residues.
  • the first spacer comprises (or consists of) an amino acid sequence of at least 4 (such as at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acid residues.
  • the first spacer comprises (or consists of) an amino acid sequence of at most 25 (such as at most 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10) amino acid residues. Said first spacer enables cleavage of the fusion protein at the protease cleavage site.
  • protease cleavage and activation of the fusion protein is markedly poor.
  • the galanin Targeting Moiety may sterically block or interact with the protease cleavage site resulting in poor activation of fusion proteins lacking a first spacer of the present invention.
  • the present inventors believe that it is the flexibility afforded by the first spacer which provides for the enhanced/improved activation properties of the presently claimed fusion proteins. Rigid linkers such as alpha-helical linkers do not afford the necessary flexibility.
  • Particularly preferred amino acid residues for use in the first spacer include glycine, threonine, arginine, serine, alanine, asparagine, glutamine, aspartic acid, proline, glutamic acid and/or lysine.
  • the aforementioned amino acids are considered to be the most flexible amino acids—see Smith et al., (2003) Protein Sci. 12:1060-1072.
  • the amino acid residues of the first spacer are selected from the group consisting of glycine, threonine, arginine, serine, asparagine, glutamine, alanine, aspartic acid, proline, glutamic acid, lysine, leucine and/or valine. In one embodiment, the amino acid residues of the first spacer are selected from the group consisting of glycine, serine, alanine, leucine and/or valine. In one embodiment, the amino acid residues of the first spacer are selected from the group consisting of glycine, serine and/or alanine Glycine and serine are particularly preferred.
  • the first spacer comprises or consists of one or more pentapeptides having glycine, serine, and or threonine residues.
  • One way of assessing whether the first spacer possesses the requisite flexibility in the presently claimed fusion proteins is by performing a simple protease cleavage assay. It would be routine for a person skilled in the art to assess cleavage/activation of a fusion protein—standard methodology is described, for example, in Example 1.
  • the first spacer may be selected from a GS5, GS10, GS15, GS18, GS20, FL3 and/or FL4 spacers.
  • the sequence of said spacers is provided in Table 1, below.
  • the first spacer enables at least 45% (such as at least 50, 55, 60, 65, 70, 75, 80, 90, 95, 98, 99 or 100%) activation of the fusion protein by protease cleavage. In one embodiment, the first spacer enables at least 70% activation of the fusion protein by protease cleavage.
  • the first spacer is not a naturally-occurring spacer sequence. In one embodiment, the first spacer does not comprise or consist of an amino acid sequence native to the natural (i.e., wild-type) clostridial neurotoxin, such as botulinum neurotoxin. In other words, the first spacer may be a non-clostridial sequence (i.e., not found in the native clostridial neurotoxin).
  • the fusion protein does not comprise or consist of the amino acid sequence GIITSK (BoNT/A) (SEQ ID NO:74); VK (BoNT B); AIDGR (BoNT/C) (SEQ ID NO:75); LTK (BoNT/D); IVSVK (BoNT/E) (SEQ ID NO:76); VIPR (BoNT/F) (SEQ ID NO:77); VMYK (BoNT/G) (SEQ ID NO:78) and/or IIPPTNIREN (TeNT) (SEQ ID NO:79) as the first spacer.
  • GIITSK BoNT/A
  • VK BoNT B
  • AIDGR BoNT/C
  • LTK BoNT/D
  • IVSVK BoNT/E
  • VIPR BoNT/F
  • VMYK BoNT/G
  • IIPPTNIREN TeNT
  • the first spacer begins on the third amino acid residue following the conserved cysteine residue in the clostridial neurotoxin L-chain (see Table 3 below). In one embodiment, the first spacer begins after the VD amino acid residues of a non-cytotoxic protease clostridial L-chain engineered with a SalI site following the conserved cysteine residue. In one embodiment, the first spacer ends with the amino acid residue marking the beginning of the protease cleavage sites mentioned above.
  • the single chain fusion protein comprises a second spacer, which is located between the galanin Targeting Moiety and the translocation domain.
  • Said second spacer may comprise (or consist of) an amino acid sequence of from 4 to 35 (such as from 6 to 35, 10 to 35, 15 to 35, 20 to 35 or from 4 to 28, 4 to 25, 4 to 20 or 4 to 10) amino acid residues.
  • the present inventors have unexpectedly found that the fusion proteins of the present invention may demonstrate an improved binding activity when the size of the second spacer is selected so that (in use) the C-terminus of the TM and the N-terminus of the translocation component are separated from one another by 40-105 angstroms, preferably by 50-100 angstroms, and more preferably by 50-90 angstroms.
  • Suitable second spacers may be routinely identified and obtained according to Crasto and Feng, (2000) Protein Eng. 13(5):309-312.
  • the second spacer is selected from a GS5, GS10, GS15, GS18, GS20 or HX27 spacer.
  • the sequence of said spacers is provided in Table 2, below.
  • the presently claimed fusion proteins having said first and second spacer features display enhanced activation properties and increased yield during recombinant expression.
  • the presently claimed fusion proteins display enhanced potency compared to fusion proteins wherein the galanin TM is C-terminal of the translocation domain component.
  • the invention provides a single-chain polypeptide fusion protein comprising (or consisting of) an amino acid sequence having at least 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the amino acid sequence of SEQ ID NOs: 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 56 and/or 59.
  • the invention provides a single-chain polypeptide fusion protein comprising (or consisting of) an amino acid sequence having at least 80% (such as at least 85, 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the full-length amino acid sequence of SEQ ID NOs: 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 53, 56 and/or 59.
  • the non-cytotoxic protease component and the translocation component are linked together by a disulphide bond.
  • the polypeptide assumes a di-chain conformation, wherein the protease and translocation components remain linked together by the disulphide bond.
  • the protease and translocation components are distanced apart from one another in the single chain fusion protein by a maximum of 100 amino acid residues, more preferably a maximum of 80 amino acid residues, particularly preferably by a maximum of 60 amino acid residues, and most preferably by a maximum of 50 amino acid residues.
  • the non-cytotoxic protease component forms a disulphide bond with the translocation component of the fusion protein.
  • the amino acid residue of the protease component that forms the disulphide bond is located within the last 20, preferably within the last 10 C-terminal amino acid residues of the protease component.
  • the amino acid residue within the translocation component that forms the second part of the disulphide bond may be located within the first 20, preferably within the first 10 N-terminal amino acid residues of the translocation component.
  • the non-cytotoxic protease component and the TM may be linked together by a disulphide bond.
  • the amino acid residue of the TM that forms the disulphide bond is preferably located away from the N-terminus of the TM, more preferably towards the C-terminus of the TM.
  • the non-cytotoxic protease component forms a disulphide bond with the TM component of the fusion protein.
  • the amino acid residue of the protease component that forms the disulphide bond is preferably located within the last 20, more preferably within the last 10 C-terminal amino acid residues of the protease component.
  • the amino acid residue within the TM component that forms the second part of the disulphide bond is preferably located within the last 20, more preferably within the last 10 C-terminal amino acid residues of the TM.
  • the above disulphide bond arrangements have the advantage that the protease and translocation components are arranged in a manner similar to that for native clostridial neurotoxin.
  • the respective cysteine amino acid residues are distanced apart by between 8 and 27 amino acid residues—taken from Popoff and Marvaud, 1999, Structural and genomic features of clostridial neurotoxins, Chapter 9, in The Comprehensive Sourcebook of Bacterial Protein Toxins. eds. Alouf and Freer, Elsevier:
  • the fusion protein may comprise one or more purification tags, which are located N-terminal to the protease component and/or C-terminal to the translocation component.
  • His-tag e.g., 6 ⁇ histidine
  • MBP-tag maltose binding protein
  • glutthione-S-transferase a C-terminal tag binding protein
  • His-MBP-tag glutathione-S-transferase
  • His-MBP-tag preferably as an N-terminal tag His-MBP-tag
  • Thioredoxin-tag preferably as an N-terminal tag CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.
  • one or more additional peptide spacer molecules may be included in the fusion protein.
  • a peptide spacer may be employed between a purification tag and the rest of the fusion protein molecule (e.g., between an N-terminal purification tag and a protease component of the present invention; and/or between a C-terminal purification tag and a translocation component of the present invention.
  • a DNA sequence that encodes the above-mentioned single chain polypeptide is prepared as part of a DNA vector, wherein the vector comprises a promoter and terminator.
  • the vector has a promoter selected from:
  • the DNA construct of the present invention is preferably designed in silico, and then synthesized by conventional DNA synthesis techniques.
  • the above-mentioned DNA sequence information is optionally modified for codon-biasing according to the ultimate host cell (e.g. E. coli ) expression system that is to be employed.
  • the ultimate host cell e.g. E. coli
  • the DNA backbone is preferably screened for any inherent nucleic acid sequence, which when transcribed and translated would produce an amino acid sequence corresponding to the protease cleave site encoded by the second peptide-coding sequence. This screening may be performed manually or with the assistance of computer software (e.g., the MapDraw program by DNASTAR, Inc.).
  • a method of preparing a non-cytotoxic agent comprising:
  • di-chain polypeptide which generally mimics the structure of clostridial holotoxin.
  • the resulting di-chain polypeptide typically has a structure wherein:
  • the single chain or di-chain polypeptide of the invention treat, prevent or ameliorate pain.
  • a therapeutically effective amount of a single chain or di-chain polypeptide of the invention is administered to a patient.
  • a single chain or di-chain polypeptide of the invention for the manufacture of a medicament for treating, preventing or ameliorating pain.
  • a method of treating, preventing or ameliorating pain in a subject comprising administering to said patient a therapeutically effective amount of a single chain or di-chain polypeptide of the invention.
  • the compounds described here may be used to treat a patient suffering from one or more types of chronic pain including neuropathic pain, inflammatory pain, headache pain, somatic pain, visceral pain, and referred pain.
  • treat means to deal with medically. It includes, for example, administering a compound of the invention to prevent pain or to lessen its severity.
  • Pain 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 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 includes neuropathic pain, inflammatory pain, headache pain, somatic pain visceral pain and referred pain.
  • Neuroopathic pain means abnormal sensory input, resulting in discomfort, from the peripheral nervous system, central nervous systems, or both.
  • 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 prolonged severe pain).
  • 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
  • hyperpathia where a short discomfort becomes a prolonged severe pain
  • Neuropathic pain may be caused by any of the following.
  • a traumatic insult such as, for example, a nerve compression injury (e.g., a nerve 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.
  • a nerve compression injury e.g., a nerve 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 e.g., a contusion
  • an inflammation e.g., an inflammation of the spinal cord
  • surgical procedure e.g., a surgical procedure.
  • An ischemic event including, for example, a stroke and heart attack.
  • 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.
  • 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.
  • a disease including, for example, an inflammatory disorder, a neoplastic tumor, an acquired immune deficiency syndrome (AIDS), Lyme disease, a leprosy, a metabolic disease, a peripheral nerve disorder, like neuroma, a mononeuropathy or a polyneuropathy.
  • AIDS acquired immune deficiency syndrome
  • Lyme disease a leprosy
  • a metabolic disease a peripheral nerve disorder, like neuroma, a mononeuropathy or a polyneuropathy.
  • a neuralgia is a pain that radiates along the course of one or more specific nerves 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 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 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 “stabbing,” “sharp,” “like lightning,” “burning,” and even “itchy”.
  • 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.
  • 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, 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 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.
  • 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 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 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 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 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 occurs at the location of the shingles rash. Depression is not uncommon due to the pain and social isolation during the illness.
  • 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.
  • 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 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.
  • neuralgias 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.
  • Deafferentation indicates a loss of the sensory input from a portion of the body, and can be caused by interruption of either peripheral sensory fibers 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 paraplegia, a brachial plexus avulsion injuries, lumbar radiculopathies.
  • CRPS is a chronic pain syndrome resulting from sympathetically-maintained pain, and presents in two forms.
  • CRPS 1 currently replaces the term “reflex sympathetic 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 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).
  • 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 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 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.
  • 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 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, 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 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 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 nerve or nerve group (mononeuropathy) or a function or pathological change affecting multiple nerves (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, postmastectomy syndrome, diabetic neuropathy, alcohol neuropathy, amyloid, toxins, AIDS, hypothyroidism, uremia, vitamin deficiencies, chemotherapy-induced pain, 2′,3′-didexoycytidine (ddC) treatment, Guillain-Barré syndrome or Fabry's disease.
  • ddC 2′,3′-didexoycytidine
  • 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 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 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 III (compression type), a cranial mononeuropathy III (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
  • peripheral neuropathies 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:
  • Distal axonopathies are the result of some metabolic or toxic 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 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.
  • PNS peripheral nervous system
  • the most common cause of distal axonopathy is diabetes, and the most common distal axonopathy is diabetic neuropathy.
  • 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-Barré syndrome), though other causes include chronic inflammatory demyelinating syndrome (CIDP), genetic metabolic disorders (e.g., leukodystrophy), or toxins.
  • CIDP chronic inflammatory demyelinating syndrome
  • Myelinopathy is due to primary 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 electrical impulses through the nerve.
  • ADP acute inflammatory demyelinating polyneuropathy
  • CIDP chronic inflammatory demyelinating polyneuropathy
  • genetic metabolic disorders e.g., leukodystrophy or Charcot-Marie-Tooth disease
  • Neuronopathies are the result of destruction of peripheral nervous system (PNS) neurons. They may be caused by motor neuron diseases, sensory neuronopathies (e.g., Herpes zoster), toxins or autonomic dysfunction. Neurotoxins may cause neuronopathies, such as the chemotherapy agent vincristine. 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 neuron 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 nerve cell that is most affected.
  • PNS peripheral nervous system
  • a person with neuronopathy may present in different ways, depending on the cause, the way it affects the nerve cells, and the type of nerve cell that is most affected.
  • Focal entrapment neuropathies e.g., carpal tunnel syndrome
  • the compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following inflammatory conditions
  • Arthritic disorders include, for example, a rheumatoid arthritis; a juvenile rheumatoid arthritis; a systemic lupus erythematosus (SLE); a gouty arthritis; a scleroderma; an 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 blastomycosis
  • Autoimmune diseases include, for example, a Guillain-Barré 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 syndrome, a lupus erythematosus, a multiple sclerosis, a myasthenia gravis, a Reiter's syndrome and a Grave's disease.
  • Connective tissue disorders include, for example, a spondyloarthritis a dermatomyositis, and a fibromyalgia.
  • Inflammation caused by injury including, for example, a crush, puncture, stretch of a tissue or joint, may cause chronic inflammatory pain.
  • Inflammation caused by infection including, for example, a tuberculosis or an interstitial keratitis may cause chronic inflammatory pain.
  • Neuritis is an inflammatory process affecting a nerve or group of nerves. Symptoms depend on the nerves involved, but may include pain, paresthesias, paresis, or hypesthesia (numbness).
  • Inflammation of the joint such as that caused by bursitis or tendonitis, for example, may cause chronic inflammatory pain.
  • 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.
  • 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.
  • 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 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 tension headache.
  • 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 pain, and headaches resulting from high blood pressure
  • 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.
  • 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’ 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 non-infective form of chronic sinusitis.
  • Ictal headaches are headaches associated with seizure activity.
  • 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; examples include sprains and broken bones. Additional examples include the following.
  • Excessive muscle tension can be caused, for example, by a sprain or a strain.
  • Repetitive motion disorders can result from overuse of the hands, wrists, elbows, shoulders, neck, back, hips, knees, feet, legs, or ankles.
  • Muscle disorders causing somatic pain include, for example, a polymyositis, a dermatomyositis, a lupus, a fibromyalgia, a polymyalgia rheumatica, and a rhabdomyolysis.
  • 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 deficiencies or chronic fatigue syndrome.
  • Infection can cause somatic pain.
  • infections include, for example, an abscess in the muscle, a trichinosis, an influenza, a Lyme disease, a malaria, a 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.
  • 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)
  • 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 pain.
  • Visceral pain is extremely difficult to localize, and several injuries to visceral tissue exhibit “referred” pain, where the sensation is localized to an area completely unrelated to the site of injury. Examples of visceral pain include the following:
  • Functional visceral pain includes, for example, an irritable bowel syndrome and a chronic functional abdominal pain (CFAP), a functional constipation and a functional dyspepsia, a non-cardiac chest pain (NCCP) and a chronic abdominal pain.
  • CFAP chronic functional abdominal pain
  • NCCP non-cardiac chest pain
  • Chronic gastrointestinal inflammation includes, for example, a gastritis, an inflammatory bowel disease, like, e.g., a Crohn's disease, an ulcerative colitis, a 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.
  • a gastritis an inflammatory bowel disease, like, e.g., a Crohn's disease, an ulcerative colitis, a microscopic colitis, a diverticulitis and a gastroenteritis
  • an interstitial cystitis an intestinal ischemia
  • a cholecystitis cholecystitis
  • an appendicitis a gastroesophageal reflux
  • Autoimmune pain includes, for example, a sarcoidosis and a vasculitis.
  • 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 sensory innervation.
  • Treatment-induced visceral pain includes, for example, a pain attendant to chemotherapy therapy or a pain attendant to radiation therapy.
  • the compounds of the invention may be used to treat pain caused by or otherwise associated with any of the following referred pain conditions.
  • 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 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 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 left shoulder, arm or even hand.
  • the present invention addresses a wide range of pain conditions, in particular chronic pain conditions.
  • Preferred conditions include cancerous and non-cancerous pain, inflammatory pain and neuropathic pain.
  • the opioid-fusions of the present application are particularly suited to addressing inflammatory pain, though may be less suited to addressing neuropathic pain.
  • the galanin-fusions are more suited to addressing neuropathic pain.
  • polypeptides of the present invention are typically employed in the form of a pharmaceutical composition in association with a pharmaceutical carrier, diluent and/or excipient, although the exact form of the composition may be tailored to the mode of administration. Administration is preferably to a mammal, more preferably to a human.
  • polypeptides may, for example, be employed in the form of a sterile solution for intra-articular administration or intra-cranial administration.
  • Spinal injection e.g., epidural or intrathecal
  • epidural or intrathecal is preferred.
  • the dosage ranges for administration of the polypeptides of the present invention are 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 judgment of the attending physician.
  • Suitable daily dosages are in the range 0.0001 to 1 mg/kg, preferably 0.0001 to 0.5 mg/kg, more preferably 0.002 to 0.5 mg/kg, and particularly preferably 0.004 to 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 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 as the 1 ⁇ dose.
  • preferred dosages are in the range 1 ⁇ -100 ⁇ (i.e., 2.5 to 250 ng). This dosage range is significantly lower (i.e., at least 10-fold, typically 100-fold lower) than would be employed with other types of analgesic molecules such as NSAIDS, morphine, and gabapentin.
  • 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 molecular weight than do conventional ‘small’ molecule therapeutics.
  • 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.
  • Fluid unit dosage forms are typically prepared utilizing a pyrogen-free sterile vehicle.
  • the active ingredients depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle.
  • the polypeptides can be dissolved in a vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilized by filtration through a sterile filter using aseptic 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 sterilized by autoclaving.
  • compositions such as buffering, solubilizing, stabilizing, preservative or bactericidal, suspending or emulsifying 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-sterilized drug substance and other ingredients into a sterile container using aseptic technique in a sterile area.
  • polypeptides and other ingredients may be dissolved in an aqueous 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.
  • 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 sterilization cannot be accomplished by filtration.
  • the components may be isolated in a sterile state or alternatively it may be sterilized after isolation, e.g., by gamma irradiation.
  • a suspending agent for example polyvinylpyrrolidone is included in the composition/s to facilitate uniform distribution of the components.
  • Targeting Moiety means any chemical structure associated with an agent that functionally interacts with a Binding Site to cause a physical association between the agent and the surface of a target cell.
  • the target cell is a nociceptive sensory afferent.
  • 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 Binding Site on the target cell, which Binding Site is capable of internalization (e.g., endosome formation)—also referred to as receptor-mediated endocytosis.
  • the TM may possess an endosomal membrane translocation function, in which case separate TM and Translocation Domain components need not be present in an agent of the present invention.
  • the TM of the present invention binds (preferably specifically binds) to a nociceptive sensory afferent (e.g., a primary nociceptive afferent).
  • a nociceptive sensory afferent e.g., a primary nociceptive afferent
  • specifically binds means that the TM binds to a nociceptive sensory afferent (e.g., a primary nociceptive afferent) with a greater affinity than it binds to other neurons such as non-nociceptive afferents, and/or to motor neurons (i.e., the natural target for clostridial neurotoxin holotoxin).
  • TM binds to a given receptor, for example galanin receptors, such as GALR1, GALR2 and/or GALR3 receptors, with a binding affinity (Ka) of 10 6 M ⁇ 1 or greater, preferably 10 7 M ⁇ 1 or greater, more preferably 10 8 M ⁇ 1 or greater, and most preferably, 10 9 M ⁇ 1 or greater.
  • Ka binding affinity
  • an agonist is defined as a molecule that is capable of stimulating the process of exocytic fusion in a target cell, which process is susceptible to inhibition by a protease capable of cleaving a protein of the exocytic fusion apparatus in said target cell.
  • the particular agonist definition of the present invention would exclude many molecules that would be conventionally considered as agonists.
  • nerve growth factor is an agonist in respect of its ability to promote neuronal differentiation via binding to a TrkA receptor.
  • NGF nerve growth factor
  • the process that NGF stimulates i.e., cell differentiation
  • fragment when used in relation to a protein, means a peptide having at least thirty-five, preferably at least twenty-five, more preferably at least twenty, and most preferably at least 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues of the protein in question.
  • variant when used in relation to a protein, means a peptide or peptide fragment of the protein that contains one or more analogues of an amino acid (e.g., an unnatural amino acid), or a substituted linkage.
  • derivative when used in relation to a protein, means a protein that comprises the protein in question, and a further peptide sequence.
  • the further peptide sequence should preferably not interfere with the basic folding and thus conformational structure of the original protein.
  • Two or more peptides (or fragments, or variants) may be joined together to form a derivative.
  • a peptide (or 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 polypeptide components may be included.
  • non-cytotoxic means that the protease molecule in question does not kill the target cell to which it has been re-targeted.
  • 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 apparatus in eukaryotic cells.
  • the non-cytotoxic protease of the present invention is preferably a bacterial protease.
  • the non-cytotoxic protease is selected from the genera Clostridium or Neisseria (e.g., a clostridial L-chain, or a neisserial IgA protease preferably from N. gonorrhoeae ).
  • the term protease embraces functionally equivalent fragments and molecules thereof.
  • the present invention also embraces modified non-cytotoxic proteases, which include amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified proteases still demonstrate the above-mentioned protease activity.
  • 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, synaptobrevin/VAMP, or syntaxin).
  • protease domains of neurotoxins for example the protease domains of bacterial neurotoxins.
  • the present invention embraces the use of neurotoxin domains, which occur in nature, as well as recombinantly prepared versions of said naturally-occurring neurotoxins.
  • 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 through G, as well as the closely related BoNT-like neurotoxins produced by C. baratii and C. butyricum .
  • TeNT C. tetani
  • BoNT botulinum
  • BoNT/A denotes the source of neurotoxin as BoNT (serotype A).
  • Corresponding nomenclature applies to other BoNT serotypes.
  • L-chain fragment means a component of the L-chain of a neurotoxin, which fragment demonstrates a metalloprotease activity and is capable of proteolytically 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 target cell such that a functional expression of 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.
  • Shone (1987) Eur. J. Biochem 167(1):175-180, describes an in vitro assay employing liposomes, which are challenged with a test molecule. Presence of the requisite translocation function is confirmed by release from the liposomes of K + and/or labeled NAD, which may be readily monitored.
  • the Translocation Domain is preferably capable of formation of ion-permeable pores 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 particular from a bacterial or viral protein source.
  • the Translocation Domain is a translocation domain of an enzyme, such as a bacterial toxin or viral protein.
  • the Translocation Domain may be of a clostridial origin, namely the H N domain (or a functional component thereof).
  • H N 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.
  • the H-chain substantially lacks the natural binding function of the H C component of the H-chain.
  • the H C function may be removed by deletion of the H C amino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment).
  • the H e function may be inactivated by chemical or biological treatment.
  • the H-chain is preferably incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e., holotoxin) binds.
  • the translocation domain is a H N domain (or a fragment thereof) of a clostridial neurotoxin.
  • suitable clostridial Translocation Domains include:
  • Botulinum type B neurotoxin amino acid residues (441-858)
  • 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)
  • H N embraces naturally-occurring neurotoxin H N portions, and modified H N portions having amino acid sequences that do not occur in nature and/or synthetic amino acid residues, so long as the modified H N portions still demonstrate the above-mentioned translocation function.
  • the Translocation Domain may be of a non-clostridial origin (see Table 4).
  • non-clostridial Translocation Domain origins include, but not be restricted to, the translocation domain of diphtheria toxin (O'Keefe et al., (1992) Proc. Natl. Acad. Sci. USA 89:6202-6206; Silverman et al., (1993) J. Biol. Chem. 269:22524-22532; and London, (1992) Biochem. Biophys. Acta.
  • the Translocation Domain may mirror the Translocation Domain present in a naturally-occurring protein, or may include amino acid variations so long as the variations do not destroy the translocating ability of the Translocation Domain.
  • viral Translocation Domains suitable for use in the present invention include certain translocating domains of virally expressed membrane fusion proteins.
  • translocation i.e., membrane fusion and vesiculation
  • the translocation i.e., membrane fusion and vesiculation
  • virally expressed membrane fusion proteins 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.
  • SFV Semliki Forest Virus
  • VSV vesicular stomatitis virus
  • SER virus F protein a translocating domain of Foamy virus envelope glycoprotein.
  • Virally encoded Aspike proteins have particular application in the context of the present invention, for example, the E1 protein of SFV and the G protein of the G protein of VSV.
  • Translocation Domains listed in Table 4 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 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.
  • the following procedure creates the LC and H N fragments for use as the component backbone for multidomain fusion expression.
  • This example is based on preparation of a serotype A based clone (SEQ ID NO: 1 and SEQ ID NO: 2), though the procedures and methods are equally applicable to the other serotypes (i.e., A, B, C, D and E serotypes) as illustrated by the sequence listing for serotype B (SEQ ID NO: 3 and SEQ ID NO: 4) and serotype C (SEQ ID NO: 5 and SEQ ID NO: 6).
  • 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 (BamHI-SalI-PstI-HindIII). A fragment of the expression vector has been removed to create a non-mobilizable plasmid and a variety of different fusion tags have been inserted to increase purification options.
  • the LC/A (SEQ ID NO: 1) 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 (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 v 2.0 (Entelechon)). BamHI/SalI 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 restriction enzyme cleavage sequences incorporated during the back translation.
  • GenBank accession number P10845
  • Swissprot accession locus BXA1_CLOBO
  • BamHI/SalI 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.)
  • 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 Sep. 2004).
  • This optimized 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.
  • the alternative method is to use PCR amplification from an existing DNA sequence with BamHI and SalI restriction enzyme sequences incorporated into the 5′ and 3′ PCR primers respectively.
  • Complementary oligonucleotide primers are chemically synthesized 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.
  • 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 machine’) that can change the incubation temperature of the reaction tube automatically, cycling between approximately 94° C. (for denaturation), 55° C. (for oligonucleotide annealing), and 72° C. (for synthesis).
  • 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 amounts (50 to 200 ⁇ M) and a buffer appropriate for the enzyme optimized for Mg 2+ concentration (0.5 to 5 mM).
  • a DNA polymerase such as Taq or Pfu polymerase
  • each of the four nucleotide dNTP building blocks of DNA in equimolar amounts (50 to 200 ⁇ M)
  • a buffer appropriate for the enzyme optimized for Mg 2+ concentration 0.5 to 5 mM.
  • 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 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.)).
  • the H N /A (SEQ ID NO: 2) is created by one of two ways:
  • the DNA sequence is designed by back translation of the H N /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. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v 2.0 (Entelechon)).
  • a PstI restriction sequence added to the N-terminus and XbaI-stop codon-HindIII 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.
  • 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 Sep. 2004).
  • This optimized DNA sequence 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 PstI and XbaI-stop codon-HindIII 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 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.)).
  • an A serotype linker with the addition of an Enterokinase (EN) site for activation, arranged as BamHI-SalI-GS18-protease site-GS20-PstI-XbaI-stop codon-HindIII is synthesized.
  • the pCR 4 vector encoding the linker is cleaved with BamHI+SalI restriction enzymes. This cleaved vector then serves as the recipient for insertion and ligation of the LC/A DNA (SEQ ID NO: 1) also cleaved with BamHI+SalI.
  • This construct is then cleaved with BamHI+HindIII and inserted into an expression vector such as the pMAL plasmid (NEB) or pET based plasmid (Novagen).
  • the resulting plasmid DNA is then cleaved with PstI+XbaI restriction enzymes and the H N /A DNA (SEQ ID NO 2 ) is then cleaved with PstI+XbaI restriction enzymes and inserted into the a similarly cleaved pMAL vector to create pMAL-LC/A-GS18-EN-CPGA16-GS20-H N /A-XbaI-His-tag-stop codon-HindIII.
  • the final construct contains the GS18-EN-CPGA16-GS20 spacer ORF for expression as a protein of the sequence illustrated in SEQ ID NO: 10.
  • NuPAGE 4-12% Bis-Tris gels (10, 12 and 15 well pre-poured gels) were used to analyze activation of fusion proteins after treatment with protease.
  • Protein samples were prepared with NuPAGE 4 ⁇ LDS sample buffer, typically to a final volume of 100 ⁇ l Samples were either diluted or made up neat (75 ⁇ l of sample, 25 ⁇ l of sample buffer) depending on protein concentration. The samples were mixed and then heated in a heat block at 95° C. for 5 min before loading onto the gel. 5 to 20 ⁇ l of sample was loaded along with 5 ⁇ l of the protein marker (BenchmarkTM protein marker from Invitrogen). The gels were typically run for 50 min at 200 V. The gel was immersed in dH 2 O and microwaved for 2 min on full power.
  • BenchmarkTM protein marker from Invitrogen
  • the gel was rinsed and the microwave step was repeated.
  • the gel was transferred to a staining box and immersed in Simply Blue SafeStain (Invitrogen). It was microwaved for 1 minute on full power and left for 0.5 to 2 h to stain.
  • the gel was then destained by pouring off the Safestain and rinsing the gel with dH 2 O.
  • the gels were left in dH 2 O to destain overnight and an image was taken on a GeneGnome (Syngene) imager.
  • Total activated protein was calculated by comparing the density of the band that corresponded to full-length fusion protein (after protease treatment) in non-reduced and reduced conditions.
  • DNA linkers were prepared that encoded galanin16 and variable spacer content.
  • reverse translation software tools for example EditSeq best E. coli reverse translation (DNASTAR Inc.), or Backtranslation tool v 2.0 (Entelechon)
  • the DNA sequence encoding the Spacer 1-Protease site-ligand-spacer 2 region is determined. Restriction sites are then incorporated into the DNA sequence and can be arranged as BamHI-SalI-Spacer 1-protease site-CPGA16-NheI-spacer 2-SpeI-PstI-XbaI-stop codon-HindIII.
  • 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 Sep. 2004). This optimized DNA sequence is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
  • the linker is cleaved with BamHI+SalI restriction enzymes.
  • This cleaved vector then serves as the recipient vector for insertion and ligation of the LC/A DNA (SEQ ID NO: 1) also cleaved with BamHI+SalI.
  • the resulting plasmid DNA is then cleaved with BamHI+HindIII restriction enzymes and the LC/A-linker fragment inserted into a similarly cleaved vector containing a unique multiple cloning site for BamHI, SalI, PstI, and HindIII such as the pMAL vector (NEB) or the pET vector (Novagen).
  • the H N /A DNA (SEQ ID NO: 2) is then cleaved with PstI+HindIII restriction enzymes and inserted into the similarly cleaved pMAL-LC/A-linker construct.
  • the final construct contains the LC/A-GS5-EN-CPGA16-GS20-H N /A ORF for expression as a protein of the sequence illustrated in SEQ ID NO: 13.
  • a falcon tube containing 25 ml 50 mM HEPES pH 7.2, 200 mM NaCl and approximately 10 g of E. coli BL21 cell paste was defrosted.
  • the thawed cell paste was made up to 80 ml with 50 mM HEPES pH 7.2, 200 mM NaCl and sonicated on ice for 30 seconds on, 30 seconds off for 10 cycles at a power of 22 microns ensuring that the sample remained cool.
  • the lysed cells were centrifuged at 18,000 rpm, 4° C. for 30 minutes.
  • the supernatant was loaded onto a 0.1 M NiSO 4 charged Chelating column (a 20-30 ml column was sufficient) equilibrated with 50 mM HEPES pH 7.2, 200 mM NaCl.
  • a step gradient of 10 and 40 mM imidazole was used, the non-specific bound protein was washed and the fusion protein with eluted with 100 mM imidazole.
  • the eluted fusion protein was dialyzed against 5 L of 50 mM HEPES pH 7.2, 200 mM NaCl at 4° C. overnight and the OD of the dialysed fusion protein was measured.
  • enterokinase 1 mg/ml was added per 100 ⁇ g of purified fusion protein along with 10 ⁇ l of factor Xa per mg of purified fusion protein if the fusion protein contained a maltose binding protein. Incubation was at 25° C. static overnight. Dialysate was loaded 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. The column was washed to baseline with 50 mM HEPES pH 7.2, 200 mM NaCl.
  • the non-specific bound protein was washed away and the fusion protein was eluted with 100 mM imidazole.
  • the eluted fusion protein was dialyzed against 5 L of 50 mM
  • H N /C (SEQ ID NO: 6) are created and inserted into the A serotype linker arranged as BamHI-SalI-Spacer 1-protease site-GA16-NheI-spacer 2-SpeI-PstI-XbaI-stop codon-HindIII.
  • the final construct contains the LC-spacer 1-GA16-spacer 2-H N ORF for expression as a protein of the sequence illustrated in SEQ ID NO: 25.
  • 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 (Pohlner et al., (1987) Nature 325(6103):458-462). Using Backtranslation tool v 2.0 (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 SalI 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 were found to 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 optimized DNA sequence (SEQ ID NO: 51) containing the IgA open reading frame (ORF) was then commercially synthesized.
  • the IgA (SEQ ID NO: 51) was inserted into the LC-GS5-GA16-GS18-H N ORF using BamHI and SalI restriction enzymes to replace the LC with the IgA protease DNA.
  • the final construct contains the IgA-GS5-GA16-GS18-H N ORF for expression as a protein of the sequence illustrated in SEQ ID NO: 53.
  • 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 v 2.0 (Entelechon)). BamHI/SalI recognition sequences are incorporated at the 5′ and 3′ ends respectively of the sequence maintaining the correct reading frame (SEQ ID NO: 57). The DNA sequence is screened (using software such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences incorporated during the back translation.
  • 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 Sep. 2004).
  • This optimized DNA sequence containing the tetanus toxin LC open reading frame (ORF) is then commercially synthesized (for example by Entelechon, Geneart or Sigma-Genosys) and is provided in the pCR4 vector (Invitrogen).
  • the pCR4 vector encoding the TeNT LC is cleaved with BamHI and SalI.
  • the BamHI-SalI fragment is then inserted into the LC/A-GA16-H N /A vector that has also been cleaved by BamHI and SalI.
  • the final construct contains the TeNT LC-GS5-GA16-GS20-H N ORF sequences for expression as a protein of the sequence illustrated in SEQ ID NO: 58.
  • CHO-K1 cells stably expressing either the human galanin 1 receptor (CHO-K1-Gal-1R; product number ES-510-C) or human galanin 2 receptor (CHO-K1-Gal-2R; product number ES-511-C) were purchased from Perkin-Elmer (Bucks, UK).
  • cells were transfected with SNAP-25 DNA using LipofectamineTM 2000 and incubated for 4 hours before media replacement. After 24 hours, cells were transferred to a T175 flask. 100 ⁇ g/ml Zeocin was added after a further 24 hours to begin selection of SNAP-25 expressing cells, and 5 ⁇ g/ml 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 to maintain 30 to 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 wells containing single colonies were marked. Media in these wells was changed weekly.
  • the GALR1 receptor activation assay measures the potency and intrinsic efficacy of ligands at the GALR1 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).
  • FRET-based cAMP Perkin Elmer LANCE cAMP kit.
  • a fluorescently labeled cAMP tracer Europium-streptavadin/biotin-cAMP
  • fluorescently (Alexa) labeled anti-cAMP antibody are added to the cells in a lysis buffer.
  • cAMP from the cells competes with the cAMP tracer for antibody binding sites.
  • 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-labeled 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 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 increase in signal at 665 nm will be observed compared with samples treated with forskolin alone. The signal at 665 nm signal is converted to cAMP concentration by interpolation to a cAMP standard curve which is included in each experiment.
  • test materials and standards were diluted to the appropriate concentrations in the wells of the first two columns of an Eppendorf 500 ⁇ l deep-well lo-bind plate, in assay buffer containing 10 ⁇ M forskolin. The chosen concentrations in columns one and two were half a log unit 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 buffer only was added as a ‘basal’. Using a 12 channel digital pipette, 10 ⁇ l of sample from the lo-bind plate was transferred to the OptiplateTM 96 well microplate.
  • the GALR2 receptor activation assay measures the potency and intrinsic efficacy of ligands at GALR2 receptor in transfected CHO-K1 cells by measuring the calcium mobilization that occurs when the receptor is activated.
  • the transfected cells are pre-loaded with a calcium sensitive dye (FLIPR) before treatment.
  • FLIPR calcium sensitive dye
  • a light pulse at 485 nm excites the fluorescent dye and causes an emission at 525 nm. This provides real-time fluorescence data from changes in intracellular calcium.
  • agonist treated cells there will be activation of the receptor, leading to an increase in calcium mobilization. This will be measured as an increase in the relative fluorescence units (RFU) at 525 nM.
  • 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 ⁇ g for 3 min and the supernatant removed. Cells were gently re-suspended in 10 ml media (if freezing cells) or assay buffer (if using ‘fresh’ cells), 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 ⁇ g; 3 min), the supernatant removed and cells re-suspended in Synth-a-freeze at 4° C. to 3 ⁇ 10 6 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° C. freezer. The following day vials were transferred to the vapor phase of a liquid nitrogen storage tank.
  • FIG. 4 demonstrates that galanin fusion proteins of the present invention having different galanin ligands (i.e., galanin-16 and galanin-30) and different serotype backbones (i.e., LC/A-H N /A, LC/B-H N /B, LC/C—H N /C and LC/D-H N /D) activate GALR1 receptors.
  • galanin ligands i.e., galanin-16 and galanin-30
  • different serotype backbones i.e., LC/A-H N /A, LC/B-H N /B, LC/C—H N /C and LC/D-H N /D
  • Cells at 2 ⁇ 10 5 cells/ml were prepared and seeded at 125 ⁇ l per well of a 96 well plate using 500 ml Gibco Ham F12 with Glutamax, 50 ml FBS, 5 ⁇ g/ml Blasticidin (Calbiochem, 10 ml at 10 mg/ml), 100 ⁇ g/ml Zeocin. Cells were allowed to grow for 24 hrs (37° C., 5% CO 2 , humidified atmosphere).
  • test protein Dilutions of test protein were prepared for a dose range of each test proteins.
  • CHO GALR1 feeding medium was filter sterilized (20 ml syringe, 0.2 ⁇ m syringe filter) to make the dilutions. Filtered medium was added into 5 labeled Bijoux's (7 ml tubes), 0.9 ml each using a Gilson pipette or multi-stepper.
  • the stock test protein was diluted to 2000 nM (working stock solution 1) and 600 nM (working stock solution 2).
  • Using a Gilson pipette a 10-fold serial dilutions of each working stock was prepared, by adding 100 ⁇ l to the next concentration in the series. Each dilution was pipetted up and down to mix thoroughly.
  • Test sample (125 ⁇ l, double concentration) was applied per well. Each test sample was applied to triplicate wells and each dose range tested included a 0 nM control. Samples were incubated for 24 hrs (37° C., 5% CO 2 , humidified atmosphere).
  • Fresh lysis buffer (20 mls per plate) was prepared with 25% (4 ⁇ ) NuPAGE LDS sample buffer, 65% dH 2 0 and 10% 1 M DTT.
  • Medium was removed from the CHO GALR1 plate by inverting over a waste receptacle. The remaining media was drained from each well using a fine-tipped pipette. The cells were lysed by adding 125 ⁇ l of lysis buffer per well using a multi-stepper pipette. After a minimum of 20 min, the buffer was removed from each well to a 1.5 ml microcentrifuge tube. Tubes were numbered to allow tracking of the CHO GALR1 treatments throughout the blotting procedure.
  • Each sample was mixed by vortex and heated at 90° C. for 5 to 10 min in a pre-warmed heat block. The samples were either stored at ⁇ 20° C. or separated on the same day on an SDS gel.
  • Nitrocellulose membranes were put in individual small trays and incubated 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.
  • Primary antibody Anti-SNAP-25 1:1000 dilution
  • Primary antibody was applied and the membranes were incubated with primary antibody (diluted in blocking buffer) for 1 hour on a rocker at room temperature.
  • Membranes were washed by rinsing 3 times with PBS/Tween (0.1%).
  • the secondary (Anti-Rabbit-HRP conjugate diluted 1:1000) was then applied and the membranes were incubated with secondary antibody (diluted in blocking buffer) at room temperature on a rocker for 1 hour. Membranes were again washed by rinsing 3 times with PBS/Tween (0.1%) and the membranes were left for a minimum of 20 min for the last wash. Bound antibody was detected using Syngene: Blots were drained of PBS/Tween, and WestDura reagents were mixed 1:1 and added to completely cover the blots for 5 minutes. The membranes were placed in a Syngene tray, and the Syngene software was set for a 5 min expose time.
  • FIGS. 3 and 5 demonstrate that galanin fusion proteins of the invention effectively cleave SNAP-25.
  • the nociceptive flexion reflex (also known as paw guarding assay) is a rapid withdrawal movement that constitutes a protective mechanism against possible limb damage. It can be quantified by assessment of electromyography (EMG) response in anesthetized rat as a result of low dose capsaicin, electrical stimulation or the capsaicin-sensitized electrical response.
  • EMG electromyography
  • a reduction/inhibition of the nociceptive flexion reflex indicates that the test substance demonstrates an anti-nociceptive effect.
  • FIGS. 7 and 8 The ability of different galanin fusion proteins of the invention to inhibit capsaicin-induced thermal hyperalgesia was evaluated ( FIGS. 7 and 8 ). Intraplantar pretreatment of fusion proteins into Sprague-Dawley rats and 24 hours later 0.3% capsaicin was injected and rats were put on 25° C. glass plate (rats contained in acrylic boxes, on 25° C. glass plate). The light beam (adjustable light Intensity) was focused on the hind paw and sensors detected movement of a paw, stopping the timer. Paw Withdrawal Latency is time to remove paw from heat source (Cut-off of 20.48 seconds). A reduction/inhibition of the paw withdrawal latency indicates that the test substance demonstrates an anti-nociceptive effect. The data demonstrated the enhanced anti-nociceptive effect of the galanin fusion proteins of the present invention compared to fusion proteins with a C-terminally presented ligand.
  • Substance P EIA was obtained from R&D Systems, UK.
  • the amount of Substance P released by the neuronal cells in the presence of the TM of interest was compared to the release obtained in the presence and absence of 100 mM KCl. Stimulation of Substance P release by the TM of interest above the basal release, establishes that the TM of interest was an “agonist ligand” as defined in this specification. If desired the stimulation of Substance P released by the TM of interest could be compared to a standard Substance P release-curve produced using the natural ORL-1 receptor ligand, nociceptin (Tocris).
  • 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 malignant disease, chronic pain not caused by malignant disease (peripheral neuropathies).
  • a 73 year old woman suffering from severe pain caused by [postherpatic?] neuralgia is treated by a peripheral injection with fusion protein to reduce neurotransmitter release at the synapse of nerve terminals to reduce the pain.
  • 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.
  • 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.
  • 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.

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US13/595,927 US20140056870A1 (en) 2012-08-27 2012-08-27 Fusion proteins
PCT/GB2013/052243 WO2014033441A1 (en) 2012-08-27 2013-08-27 Fusion proteins and methods for treating, preventing or ameliorating pain
BR112015003947A BR112015003947A8 (pt) 2012-08-27 2013-08-27 Proteínas de fusão e métodos para tratar, prevenir ou melhorar dor
EP17165621.8A EP3246405B1 (en) 2012-08-27 2013-08-27 Fusion proteins and methods for treating, preventing or ameliorating pain
CN201380045083.XA CN104769108A (zh) 2012-08-27 2013-08-27 用于治疗、预防或缓解疼痛的融合蛋白和方法
PL13759562T PL2888359T3 (pl) 2012-08-27 2013-08-27 Białka fuzyjne i sposoby leczenia, zapobiegania lub łagodzenia bólu
EP13759562.5A EP2888359B1 (en) 2012-08-27 2013-08-27 Fusion proteins and methods for treating, preventing or ameliorating pain
UAA201502728A UA117349C2 (uk) 2012-08-27 2013-08-27 Одноланцюговий гібридний білок та спосіб лікування, попередження або усунення болю
PT137595625T PT2888359T (pt) 2012-08-27 2013-08-27 Proteínas de fusão e métodos para o tratamento, prevenção ou amenização da dor
JP2015529117A JP2015528461A (ja) 2012-08-27 2013-08-27 融合タンパク質および疼痛を治療、予防または緩和するための方法
AU2013308233A AU2013308233B2 (en) 2012-08-27 2013-08-27 Fusion proteins and methods for treating, preventing or ameliorating pain
HUE13759562A HUE035286T2 (en) 2012-08-27 2013-08-27 Fusion proteins and methods for treating, preventing or alleviating pain
KR1020157005061A KR102283218B1 (ko) 2012-08-27 2013-08-27 통증을 치료, 예방 또는 개선하는 융합단백질 및 그의 제조방법
ES13759562.5T ES2632470T3 (es) 2012-08-27 2013-08-27 Proteínas de fusión y métodos para tratar, prevenir o mejorar el dolor
RU2015111001A RU2652954C2 (ru) 2012-08-27 2013-08-27 Слитые белки и способы лечения, профилактики или облегчения боли
DK13759562.5T DK2888359T3 (en) 2012-08-27 2013-08-27 FUSION PROTEINS AND PROCEDURES FOR TREATMENT, PREVENTION, OR RELIEFING Pain
US14/422,574 US20150197739A1 (en) 2012-08-27 2013-08-27 Fusion proteins and methods for treating, preventing or ameliorationg pain
MX2015002588A MX369005B (es) 2012-08-27 2013-08-27 Proteinas de fusión y usos de las mismas para tratar, prevenir o aliviar el dolor.
CA2882233A CA2882233A1 (en) 2012-08-27 2013-08-27 Fusion proteins and methods for treating, preventing or ameliorating pain
ES17165621T ES2737850T3 (es) 2012-08-27 2013-08-27 Proteínas de fusión y métodos para tratar, prevenir o mejorar el dolor
HK15111817.1A HK1211056A1 (en) 2012-08-27 2015-12-01 Fusion proteins and methods for treating, preventing or ameliorating pain
US15/661,433 US20170327810A1 (en) 2012-08-27 2017-07-27 Fusion proteins and methods for treating, preventing or ameliorating pain
HK18105260.2A HK1245833B (zh) 2012-08-27 2018-04-23 用於治療、預防或緩解疼痛的融合蛋白和方法
JP2018172942A JP2019004909A (ja) 2012-08-27 2018-09-14 融合タンパク質および疼痛を治療、予防または緩和するための方法
US16/416,435 US11248219B2 (en) 2012-08-27 2019-05-20 Fusion proteins comprising a non-cytotoxic protease, a translocation domain, and a targeting moiety that binds a galanin receptor and methods for treating, preventing or ameliorating pain using such fusion proteins

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US11248219B2 (en) 2022-02-15
KR20150068353A (ko) 2015-06-19
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ES2632470T3 (es) 2017-09-13
EP2888359B1 (en) 2017-04-12
AU2013308233A1 (en) 2015-02-26
EP3246405B1 (en) 2019-04-24
UA117349C2 (uk) 2018-07-25
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