EP4436593A1 - Behandlung von eingeweideschmerzen - Google Patents

Behandlung von eingeweideschmerzen

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Publication number
EP4436593A1
EP4436593A1 EP22817306.8A EP22817306A EP4436593A1 EP 4436593 A1 EP4436593 A1 EP 4436593A1 EP 22817306 A EP22817306 A EP 22817306A EP 4436593 A1 EP4436593 A1 EP 4436593A1
Authority
EP
European Patent Office
Prior art keywords
dermatome
nerve
clostridial neurotoxin
maps
visceral pain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22817306.8A
Other languages
English (en)
French (fr)
Inventor
Jaqueline Caroline MAIGNEL
Stephane Lezmi
Vincent Martin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ipsen Biopharm Ltd
Original Assignee
Ipsen Biopharm Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB2116795.2A external-priority patent/GB202116795D0/en
Application filed by Ipsen Biopharm Ltd filed Critical Ipsen Biopharm Ltd
Publication of EP4436593A1 publication Critical patent/EP4436593A1/de
Pending legal-status Critical Current

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Classifications

    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to methods of treating visceral pain in a patient, more particularly by administration of a clostridial neurotoxin.
  • BACKGROUND Visceral pain is pain that emerges from one’s internal organs, such as the stomach, bladder, uterus, or rectum, usually because of the presence of an injury or insult to the organ caused by an underlying disease.
  • visceral pain a type of nociceptive pain
  • Pelvic pain caused by a bladder infection and abdominal pain caused by irritable bowel syndrome are types of visceral pain.
  • visceral pain is common is because of the wide variety of different conditions/ diseases that can cause such pain, which include Crohn’s disease, ulcerative colitis, irritable bowel syndrome, coeliac disease, endometriosis, gastric ulcers, kidney and bladder stones, gallbladder stones, pancreatitis and peritonitis. Very few treatments are tailored specifically for treating pain that emanates/ emerges from the viscera, and as an alternative a remedy for the underlying cause of the pain is often sought.
  • the patient may be treated with an aim to remove the cause of inflammation e.g. via antibiotic administration.
  • an aim to remove the cause of inflammation e.g. via antibiotic administration.
  • a significant amount of time may be required to remove such underlying insult, such that there remains a need to suppress visceral pain, which may otherwise lead to a significant decrease to a patient’s quality of life.
  • This is made difficult because the viscera are by their very nature ‘internal’ to the subject, such that targeting the viscera (organs) typically requires invasive, and significantly unpleasant, administration routes.
  • BoNT/A clostridial neurotoxin treatment
  • a clostridial neurotoxin to a dermatome nerve, that maps to a thoracic, pelvic, or abdominal organ/ viscera contributing to visceral pain, provides for visceral pain suppression.
  • the inventors have provided data to evidence that, upon administration of clostridial neurotoxin to a dermatome/ dermatome nerve of a patient, retrograde axonal transport of the neurotoxin (more particularly, via a spinal nerve also referred to as a dermatome nerve herein) allows for delivery of the neurotoxin to the spinal cord to act at (e.g. cause SNARE protein cleavage) or target the same area in the spinal cord dorsal horns in which visceral pain is integrated.
  • SNAP-25 cleavage (used as a readout of clostridial neurotoxin transport and activity) was detected in the spinal cords of both animal models tested (rodent and pig) following intradermal administration of clostridial neurotoxin to a dermatome, with the spinal cord area having cleaved SNAP-25 being consistent with the dermatome to which the clostridial neurotoxin was injected, particularly in the ipsilateral dorsal horn.
  • the T8, T9 and T10 dermatomes converge with visceral afferent fibers from the stomach at the same level of the spinal cord.
  • the neurotoxin can be delivered (retrograde transport via dermatome/ spinal nerve(s)) to the same level of the spinal cord that visceral afferent fibers from the stomach can be found.
  • the clostridial neurotoxin can inhibit the activity of this visceral afferent fiber in the spinal cord, and thus suppress the integration of stomach pain. A suppression of perceived pain thus follows.
  • Various particular dermatome-viscera interactions have previously been mapped out (e.g.
  • dermatome maps are used clinically to demonstrate the site(s) of pathology of a patient with a suspected spinal-nerve lesion.
  • a common dermatome map to be used clinically is the American Spinal Injury Association’s (ASIA) worksheet produced as the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI).
  • ASIA American Spinal Injury Association
  • ISNCSCI International Standards for Neurological Classification of Spinal Cord Injury
  • Clostridia produce highly potent and specific protein toxins, which can (temporarily) impair function of neurons and other cells to which they are delivered.
  • Examples of such clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, and X (see WO 2018/009903 A2), as well as those produced by C. baratii and C. butyricum.
  • tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters.
  • clostridial neurotoxins are synthesised as a single-chain polypeptide that is modified post-translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site that is located between the cysteine residues that provide the inter-chain disulphide bond. It is this di-chain form that is the active form of the toxin.
  • 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.
  • the H-chain comprises an N-terminal translocation component (H N domain) and a C-terminal targeting component (HC domain).
  • the cleavage site is located between the L-chain and the translocation domain components.
  • Clostridial neurotoxins provide non-cytotoxic protease activity.
  • Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin).
  • SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor.
  • SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a cell.
  • the protease function is a zinc-dependent endopeptidase activity and exhibits a high substrate specificity for SNARE proteins.
  • the non-cytotoxic protease is capable of inhibiting cellular secretion from the target cell.
  • the L-chain proteases of clostridial neurotoxins are non- cytotoxic proteases that cleave SNARE proteins.
  • clostridial neurotoxins such as botulinum toxin have been successfully employed in a wide range of therapies.
  • the inventors have demonstrated a surprising utility of clostridial neurotoxins in suppressing visceral pain, for example by suppressing visceral pain integration at the spinal cord.
  • a clostridial neurotoxin for use in a method of suppressing visceral pain
  • a method of suppressing visceral pain in a patient the method comprising administering a clostridial neurotoxin to the patient
  • a use of a clostridial neurotoxin in the manufacture of a medicament to suppress visceral pain in a patient Visceral pain is pain that results from the activation of nociceptors of the thoracic, pelvic, or abdominal viscera (organs) and glands.
  • visceral pain may mean pain that is contributed by a thoracic, pelvic, or abdominal viscera and/or gland.
  • examples of such viscera include: urinary bladder, uterus, stomach, liver, gall bladder, pancreas, small intestine, colon, kidney, ureter, ovary, fallopian tube(s), cervix, testes, and/or epididymis.
  • a method of the invention may comprise identifying an organ in the patient that is causing said visceral pain, preferably wherein said organ is selected from the urinary bladder, uterus, stomach, liver, gall bladder, pancreas, small intestine, colon, kidney, ureter, ovary, fallopian tube(s), cervix, testes, and epididymis.
  • a method of the invention may comprise mapping said organ to a specific dermatome nerve; for example, according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): i. urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome nerve(s); ii.
  • uterus maps to the T12 and/ or L1 dermatome nerve(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome nerve(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); vi. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome nerve(s); vii. small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); ix.
  • kidney maps to the T10, T11, T12 and/ or L1 dermatome nerve(s); x. ureter maps to the T11, T12 and/ or L1 dermatome nerve(s); xi. ovary maps to the T11 dermatome nerve(s); xii. fallopian tube(s) maps to the T11 dermatome nerve(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome nerve(s); xiv. testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); xv.
  • the clostridial neurotoxin may be administered to (or proximal to) a “dermatome” toward administering the neurotoxin to a “dermatome nerve”; in which case, the method may comprise mapping said organ to a specific dermatome according to instructions outlined in the paragraph above, thereby matching said organ to one or more specifically designated dermatome(s).
  • a method of the invention may comprise administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s).
  • a method of the invention may comprise administering a therapeutically effective amount of a clostridial neurotoxin to said one or more specifically designated dermatome nerve(s).
  • a method of the invention may comprise administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome(s).
  • a method of the invention may comprise administering a therapeutically effective amount of a clostridial neurotoxin to said one or more specifically designated dermatome(s).
  • the clostridial neurotoxin may suppress visceral pain following administration.
  • the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g. the dorsal horn(s)) where it binds sensory afferent nerve cells, enters said cells (e.g. by receptor-mediated endocytosis) and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • the spinal cord e.g. the dorsal horn(s)
  • a more particular aspect provides a clostridial neurotoxin for use in a method of suppressing visceral pain in a patient, the method comprising: a. identifying an organ in the patient that is causing said visceral pain; b.
  • mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): i. urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome nerve(s); ii. uterus maps to the T12 and/ or L1 dermatome nerve(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome nerve(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); vi.
  • pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome nerve(s); vii. small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome nerve(s); x. ureter maps to the T11, T12 and/ or L1 dermatome nerve(s); xi. ovary maps to the T11 dermatome nerve(s); xii. fallopian tube(s) maps to the T11 dermatome nerve(s); xiii.
  • cervix maps to the S2, S3 and/ or S4 dermatome nerve(s); xiv. testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); and c.
  • a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g. the dorsal horn(s)) where it (the clostridial neurotoxin) binds sensory afferent nerve cells, enters said cells (e.g. by receptor-mediated endocytosis) and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • the spinal cord e.g. the dorsal horn(s)
  • the clostridial neurotoxin binds sensory afferent nerve cells, enters said cells (e.g. by receptor-mediated endocytosis) and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • One aspect provides a method of suppressing visceral pain in a patient, the method comprising: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): i. urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome nerve(s); ii. uterus maps to the T12 and/ or L1 dermatome nerve(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome nerve(s); iv.
  • liver maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); vi. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome nerve(s); vii. small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome nerve(s); x.
  • ureter maps to the T11, T12 and/ or L1 dermatome nerve(s); xi. ovary maps to the T11 dermatome nerve(s); xii. fallopian tube(s) maps to the T11 dermatome nerve(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome nerve(s); xiv. testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); and c.
  • a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g. the dorsal horn(s)) where it (the clostridial neurotoxin) binds sensory afferent nerve cells, enters said cells (e.g. by receptor-mediated endocytosis) and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • the spinal cord e.g. the dorsal horn(s)
  • the clostridial neurotoxin binds sensory afferent nerve cells, enters said cells (e.g. by receptor-mediated endocytosis) and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • suppressing visceral pain comprises: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): i. urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome nerve(s); ii. fallopian tube(s) maps to the T11 dermatome nerve(s); iii. uterus maps to the T12 and/ or L1 dermatome nerve(s); iv.
  • cervix maps to the S2, S3 and/ or S4 dermatome nerve(s); v. stomach maps to the T8, T9 and/ or T10 dermatome nerve(s); vi. liver maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); vii. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); viii. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome nerve(s); ix. small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); x.
  • a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • the term “sensory afferent nerve cells” may be used synonymously with the term “visceral afferent nerve cells”.
  • the visceral afferent nerve cells also known as visceral afferent fibres, or general visceral afferent (GVA) fibers
  • GVA general visceral afferent
  • One aspect provides a clostridial neurotoxin for use in a method of suppressing visceral pain in a patient, the method comprising: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): i.
  • urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome nerve(s); ii. uterus maps to the T12 and/ or L1 dermatome nerve(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome nerve(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); vi. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome nerve(s); vii.
  • small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome nerve(s); x. ureter maps to the T11, T12 and/ or L1 dermatome nerve(s); xi. ovary maps to the T11 dermatome nerve(s); xii. fallopian tube(s) maps to the T11 dermatome nerve(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome nerve(s); xiv.
  • testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); and c. administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g.
  • One aspect provides a method of suppressing visceral pain in a patient, the method comprising: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): i.
  • urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome nerve(s); ii. uterus maps to the T12 and/ or L1 dermatome nerve(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome nerve(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); vi. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome nerve(s); vii.
  • small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome nerve(s); x. ureter maps to the T11, T12 and/ or L1 dermatome nerve(s); xi. ovary maps to the T11 dermatome nerve(s); xii. fallopian tube(s) maps to the T11 dermatome nerve(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome nerve(s); xiv.
  • testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); and c. administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g.
  • a clostridial neurotoxin in the manufacture of a medicament for suppressing visceral pain in a patient, wherein suppressing visceral pain comprises: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): i.
  • urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome nerve(s); ii. fallopian tube(s) maps to the T11 dermatome nerve(s); iii. uterus maps to the T12 and/ or L1 dermatome nerve(s); iv. cervix maps to the S2, S3 and/ or S4 dermatome nerve(s); v. stomach maps to the T8, T9 and/ or T10 dermatome nerve(s); vi. liver maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); vii. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); viii.
  • pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome nerve(s); ix. small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); x. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); xi. kidney maps to the T10, T11, T12 and/ or L1 dermatome nerve(s); xii. ureter maps to the T11, T12 and/ or L1 dermatome nerve(s); xiii. ovary maps to the T11 dermatome nerve(s); xiv.
  • testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome nerve(s); and c.
  • a clostridial neurotoxin for use in a method of suppressing visceral pain in a patient, the method comprising: a.
  • identifying an organ in the patient that is causing said visceral pain b. mapping said organ to a specific dermatome according to the following instructions, thereby matching said organ to one or more specifically designated dermatome(s): i. urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome(s); ii. uterus maps to the T12 and/ or L1 dermatome(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s); vi.
  • pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); vii. small intestine maps to the T10, T11 and/ or T12 dermatome(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome(s); x. ureter maps to the T11, T12 and/ or L1 dermatome(s); xi. ovary maps to the T11 dermatome(s); xii. fallopian tube(s) maps to the T11 dermatome(s); xiii.
  • cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv. testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); and c. administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g.
  • One aspect provides a method of suppressing visceral pain in a patient, the method comprising: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome according to the following instructions, thereby matching said organ to one or more specifically designated dermatome(s): i.
  • urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome(s); ii. uterus maps to the T12 and/ or L1 dermatome(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s); vi. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); vii.
  • small intestine maps to the T10, T11 and/ or T12 dermatome(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome(s); x. ureter maps to the T11, T12 and/ or L1 dermatome(s); xi. ovary maps to the T11 dermatome(s); xii. fallopian tube(s) maps to the T11 dermatome(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv.
  • testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); and c. administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g.
  • a clostridial neurotoxin in the manufacture of a medicament for suppressing visceral pain in a patient, wherein suppressing visceral pain comprises: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome according to the following instructions, thereby matching said organ to one or more specifically designated dermatome(s): i.
  • urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome(s); ii. uterus maps to the T12 and/ or L1 dermatome(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s); vi. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); vii.
  • small intestine maps to the T10, T11 and/ or T12 dermatome(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome(s); x. ureter maps to the T11, T12 and/ or L1 dermatome(s); xi. ovary maps to the T11 dermatome(s); xii. fallopian tube(s) maps to the T11 dermatome(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv.
  • testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); and c. administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g.
  • a clostridial neurotoxin for use in a method of suppressing visceral pain in a patient, the method comprising: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome according to the following instructions, thereby matching said organ to one or more specifically designated dermatome(s): i.
  • urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome(s); ii. uterus maps to the T12 and/ or L1 dermatome(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s); vi. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); vii.
  • small intestine maps to the T10, T11 and/ or T12 dermatome(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome(s); x. ureter maps to the T11, T12 and/ or L1 dermatome(s); xi. ovary maps to the T11 dermatome(s); xii. fallopian tube(s) maps to the T11 dermatome(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv.
  • testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); and c. administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g.
  • One aspect provides a method of suppressing visceral pain in a patient, the method comprising: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome according to the following instructions, thereby matching said organ to one or more specifically designated dermatome(s): i.
  • urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome(s); ii. uterus maps to the T12 and/ or L1 dermatome(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s); vi. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); vii.
  • small intestine maps to the T10, T11 and/ or T12 dermatome(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome(s); x. ureter maps to the T11, T12 and/ or L1 dermatome(s); xi. ovary maps to the T11 dermatome(s); xii. fallopian tube(s) maps to the T11 dermatome(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv.
  • testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); and c. administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g.
  • a clostridial neurotoxin in the manufacture of a medicament for suppressing visceral pain in a patient, wherein suppressing visceral pain comprises: a. identifying an organ in the patient that is causing said visceral pain; b. mapping said organ to a specific dermatome according to the following instructions, thereby matching said organ to one or more specifically designated dermatome(s): i.
  • urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome(s); ii. uterus maps to the T12 and/ or L1 dermatome(s); iii. stomach maps to the T8, T9 and/ or T10 dermatome(s); iv. liver maps to the T8, T9, T10 and/ or T11 dermatome(s); v. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s); vi. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); vii.
  • small intestine maps to the T10, T11 and/ or T12 dermatome(s); viii. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome(s); ix. kidney maps to the T10, T11, T12 and/ or L1 dermatome(s); x. ureter maps to the T11, T12 and/ or L1 dermatome(s); xi. ovary maps to the T11 dermatome(s); xii. fallopian tube(s) maps to the T11 dermatome(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv.
  • testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s); and c. administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g.
  • a method of the invention may comprise identifying an organ in the patient that is “contributing to” (aka “causing”) said visceral pain.
  • a method of the invention may comprise mapping said organ to a specific dermatome nerve; for example, selected from the following combinations: i. stomach maps to the T8, T9 and/ or T10 dermatome(s); ii. liver maps to the T8, T9, T10 and/ or T11 dermatome(s) (preferably the T8, T9 and/ or T10 dermatome(s)); iii. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s) (preferably the T8, T9 and/ or T10 dermatome(s)); iv.
  • pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); v. small intestine maps to the T10, T11 and/ or T12 dermatome(s); vi. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome(s) (preferably the S1, S2, S3 and/ or S4 dermatome(s)); vii. kidney maps to the T10, T11, T12 and/ or L1 dermatome(s) (preferably the T11, T12 and/ or L1 dermatome(s)); viii. ureter maps to the T11, T12 and/ or L1 dermatome(s); ix.
  • urinary bladder maps to the T12, L1, S2, S3 and/ or S4 dermatome(s); x. ovary maps to the T11 dermatome(s); xi. fallopian tube(s) maps to the T11 dermatome(s); xii. uterus maps to the T12 and/ or L1 dermatome(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv. testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s) (preferably the T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s)); xv.
  • a method of the invention may comprise administering a clostridial neurotoxin at a site proximal to the same one or more dermatome nerve that map(s) to the organ identified as contributing to the visceral pain.
  • the clostridial neurotoxin may suppress visceral pain following administration.
  • the neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g. the dorsal horn(s)) and suppresses the underlying visceral pain integration.
  • a more particular aspect provides a clostridial neurotoxin for use in a method of suppressing visceral pain in a patient, the method comprising: a. identifying an organ in the patient that is contributing to said visceral pain; b. mapping said organ to a specific dermatome nerve selected from the following combinations: i. stomach maps to the T8, T9 and/ or T10 dermatome(s); ii.
  • liver maps to the T8, T9, T10 and/ or T11 dermatome(s) (preferably the T8, T9 and/ or T10 dermatome(s)); iii. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s) (preferably the T8, T9 and/ or T10 dermatome(s)); iv. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); v. small intestine maps to the T10, T11 and/ or T12 dermatome(s); vi.
  • uterus maps to the T12 and/ or L1 dermatome(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv. testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s) (preferably the T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s)); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s) (preferably the T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s)); and c.
  • one aspect provides a method of suppressing visceral pain in a patient in need thereof, the method comprising: a. identifying an organ in the patient that is contributing to said visceral pain; b.
  • mapping said organ to a specific dermatome nerve selected from the following combinations: i. stomach maps to the T8, T9 and/ or T10 dermatome(s); ii. liver maps to the T8, T9, T10 and/ or T11 dermatome(s) (preferably the T8, T9 and/ or T10 dermatome(s)); iii. gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s) (preferably the T8, T9 and/ or T10 dermatome(s)); iv. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); v.
  • small intestine maps to the T10, T11 and/ or T12 dermatome(s); vi. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome(s) (preferably the S1, S2, S3 and/ or S4 dermatome(s)); vii. kidney maps to the T10, T11, T12 and/ or L1 dermatome(s) (preferably the T11, T12 and/ or L1 dermatome(s)); viii. ureter maps to the T11, T12 and/ or L1 dermatome(s); ix. urinary bladder maps to the T12, L1, S2, S3 and/ or S4 dermatome(s); x. ovary maps to the T11 dermatome(s); xi.
  • fallopian tube(s) maps to the T11 dermatome(s); xii. uterus maps to the T12 and/ or L1 dermatome(s); xiii. cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv. testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s) (preferably the T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s)); xv.
  • epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s) (preferably the T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s)); and c. administering a clostridial neurotoxin at a site proximal to the same one or more dermatome nerve that map(s) to the organ identified as contributing to the visceral pain, and wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g. the dorsal horn(s)) and suppresses the underlying visceral pain integration.
  • the spinal cord e.g. the dorsal horn(s)
  • One aspect provides use of a clostridial neurotoxin in the manufacture of a medicament for suppressing visceral pain in a patient, wherein suppressing visceral pain comprises: a. identifying an organ in the patient that is contributing to said visceral pain; b. mapping said organ to a specific dermatome nerve selected from the following combinations: i. stomach maps to the T8, T9 and/ or T10 dermatome(s); ii. liver maps to the T8, T9, T10 and/ or T11 dermatome(s) (preferably the T8, T9 and/ or T10 dermatome(s)); iii.
  • gall bladder maps to the T8, T9, T10 and/ or T11 dermatome(s) (preferably the T8, T9 and/ or T10 dermatome(s)); iv. pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome(s); v. small intestine maps to the T10, T11 and/ or T12 dermatome(s); vi. colon maps to the T11, S1, S2, S3 and/ or S4 dermatome(s) (preferably the S1, S2, S3 and/ or S4 dermatome(s)); vii.
  • kidney maps to the T10, T11, T12 and/ or L1 dermatome(s) (preferably the T11, T12 and/ or L1 dermatome(s)); viii. ureter maps to the T11, T12 and/ or L1 dermatome(s); ix. urinary bladder maps to the T12, L1, S2, S3 and/ or S4 dermatome(s); x. ovary maps to the T11 dermatome(s); xi. fallopian tube(s) maps to the T11 dermatome(s); xii. uterus maps to the T12 and/ or L1 dermatome(s); xiii.
  • cervix maps to the S2, S3 and/ or S4 dermatome(s); xiv. testes maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s) (preferably the T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s)); xv. epididymis maps to the T10, T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s) (preferably the T11, L1, L2, S1, S2, S3 and/ or S4 dermatome(s)); and c.
  • a clostridial neurotoxin at a site proximal to the same one or more dermatome nerve that map(s) to the organ identified as contributing to the visceral pain, and wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord (e.g. the dorsal horn(s)) and suppresses the underlying visceral pain integration.
  • the spinal cord e.g. the dorsal horn(s)
  • retrograde axonal transport of the neurotoxin allows for delivery of the neurotoxin to the spinal cord to act at or target the same area in the spinal cord in which visceral pain is integrated.
  • delivery of clostridial neurotoxin to the spinal cord can cause SNARE protein (e.g. SNAP-25) cleavage.
  • the clostridial neurotoxin may bind sensory afferent nerve cells, enter said cells (e.g. by receptor-mediated endocytosis) and suppress neurotransmitter release therefrom e.g. by cleaving a SNARE protein, thereby suppressing said visceral pain.
  • the clostridial neurotoxin may cleave a SNARE protein (thus suppress neurotransmitter release, thereby suppressing said visceral pain).
  • SNARE proteins include synaptobrevin (otherwise known as vesicle-associated membrane protein (VAMP)), SNAP-25 and syntaxin.
  • VAMP vesicle-associated membrane protein
  • BoNT/A, BoNT/C, and BoNT/E cleave the synaptosomal-associated protein of 25 kDa (“SNAP-25”);
  • BoNT/C cleaves syntaxin.
  • SNARE proteins are associated either with the membrane of a secretory vesicle or with a cell membrane and facilitate exocytosis of molecules by mediating the fusion of the secretory vesicle with the cell membrane, thus allowing for the contents of the vesicle to be expelled outside the cell.
  • the cleavage of such SNARE proteins inhibits such exocytosis and thus inhibits the release of neurotransmitter from such neurons.
  • integration of visceral pain into the central nervous system can be suppressed; in other words, visceral pain may be suppressed.
  • “Retrograde axonal transport” is a form of axonal transport (aka.
  • axoplasmic transport or axoplasmic flow a cellular process normally responsible for movement of mitochondria, lipids, synaptic vesicles, proteins, and other organelles to and from a neuron's cell body, through the cytoplasm of its axon called the axoplasm.
  • Axons are on the order of meters long, such that neurons cannot rely on diffusion to carry products of the nucleus and organelles to the end of their axons, hence the use of axonal transport.
  • Axonal transport is also responsible for moving molecules destined for degradation from the axon back to the cell body, where they are broken down by lysosomes.
  • the present invention takes advantage of vesicular retrograde transport to move a clostridial neurotoxin to the spinal cord to exert a central effect.
  • the term “retrograde axonal transport to the spinal cord” may be defined as axonal transport of the clostridial neurotoxin toward the nerve cell body that is positioned in the proximity of the spinal cord.
  • the term “dermatome nerve” may be used synonymously with the term “spinal nerve” and/or “spinal nerve afferent”. Spinal nerves innervate particular dermatomes (e.g.
  • a “dermatome” may be defined as an area of skin that is supplied by any given spinal nerve.
  • the nerve(s) targeted by the present invention may be referred to as “dermatome nerve(s)”, to illustrate the connection between the targeted nerve(s) and the dermatome(s). That being said, it should be noted that throughout this disclosure, a “dermatome nerve” may also (e.g. alternatively) be referred to as a “spinal nerve” and/or “spinal nerve afferent”.
  • the T10 dermatome nerve may be referred to as “the T10 spinal nerve”, and so on for any dermatome nerve described herein.
  • the clostridial neurotoxin may be administered by any route that allows for administration of the clostridial neurotoxin to a dermatome nerve (e.g. spinal nerve) and subsequent retrograde axonal transport of the clostridial neurotoxin to the spinal cord. Examples of suitable administration routes include intradermal and intrathecal administration. In a preferable embodiment, the clostridial neurotoxin is administered by intradermal administration (e.g. to a dermatome).
  • a clostridial neurotoxin may be administered in a minimally invasive manner while targeting pain caused by an insult at an internal organ, which would otherwise require access to internal regions of a patient’s body.
  • intradermal administration provides an enhanced effect when compared with alternative administration routes (e.g. intramuscular or subcutaneous administration).
  • the clostridial neurotoxin is administered by intradermal administration to a dermatome.
  • the clostridial neurotoxin is administered by intradermal administration to a dermatome having a dermatome nerve that maps to the viscera contributing to visceral pain.
  • the clostridial neurotoxin may be administered (e.g., by intradermal injection) at up to 20 or 15 injection sites per treatment session.
  • the clostridial neurotoxin may be administered (e.g., by intradermal injection) at up to 10 injection sites per treatment session, for example up to 9, 8, 7, 6, 5, 4, 3 or 2 sites per treatment session.
  • a clostridial neurotoxin may be administered at 1-10, 3-10, 5-10 or 7-10 injection sites per treatment session.
  • a “dermatome” (aka “peripheral nerve field”) may be defined as an area of skin that is mainly supplied by afferent nerve fibres from the dorsal root of any given spinal nerve.
  • the dermatomes follow the pattern of spinal nerve innervation of the skin.
  • the dermatomes of the thorax and abdomen present as a stack of discs forming an individual. Along the arms and legs, the dermatomes run longitudinally.
  • the general pattern of the dermatomes is shared amongst all individuals. Because each segment (nerve) of the spinal cord innervates a different region of the body, dermatomes can be precisely mapped on the body surface.
  • a typical map of the dermatomes of a human subject that is used clinically is outlined in the American Spinal Injury Association’s (ASIA) worksheet produced as the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI).
  • a “dermatome” may be defined as an area of skin that is supplied by any given spinal nerve which, as mentioned above, is also referred to as a dermatome nerve herein. More particularly, as mentioned above, a “dermatome” may refer to an area of skin that is supplied by afferent nerve fibres from the dorsal root of any given spinal nerve. In humans, there are 30 dermatomes (one less than the number of spinal nerves because the C1 spinal nerve typically doesn’t have a sensory root; as a result, dermatomes begin with spinal nerve C2). Throughout this disclosure, the name given to a dermatome conveniently corresponds to the name of the particular spinal nerve (also referred to a dermatome nerve herein) that innervates said dermatome.
  • a dermatome innervated by the C3 spinal nerve is conveniently referred to as the “C3 dermatome”.
  • a dermatome innervated by the T1 spinal nerve is conveniently referred to as the “T1 dermatome”.
  • the viscera typically transmit visceral nociceptive information to the central nervous system (e.g. the spinal cord) via visceral afferent fibres following stimulation of nociceptive receptors in the viscera.
  • Visceral afferent nerve innervation of the spinal cord is distributed from the cervical to the sacral spinal segments.
  • Second-order spinal neurons receive visceral afferent input to integrate the visceral pain.
  • Said second-order neurons in the spinal cord that receive visceral afferent input are located principally in the superficial spinal laminae, deeper in lamina V (including the intermediolateral cell column in the TL spinal cord and the sacral parasympathetic nucleus), and in the medial LS spinal cord dorsal to the central canal (lamina X).
  • Each viscera comprises a corresponding dermatome to which visceral pain (contributed to by said viscera) may be referred. This is because visceral afferent input (e.g. sensory input) to the spinal cord is also characterized by convergence.
  • the second-order spinal neurons that receive visceral input also receive convergent somatic (or cutaneous) input from skin/ a dermatome, which provides an explanation for referral of visceral sensations to somatic sites (e.g., deep retrosternal pain that radiates to the neck, shoulder, or jaw with angina).
  • somatic sites e.g., deep retrosternal pain that radiates to the neck, shoulder, or jaw with angina.
  • the distribution of referred pain from the viscera can be predicted by the dermatomal distribution of the somatic afferents that enter the spinal cord at the same level as the visceral afferents.
  • the convergence of somatic (e.g. dermatome nerve) and visceral nerves at the spinal cord may be referred to as “somatovisceral convergence”.
  • visceral pain suppression achieved by the inventors herein takes advantage of such somatovisceral convergence onto second- order spinal neurons, by employing dermatome nerves (e.g. spinal nerves/ afferents) to deliver a clostridial neurotoxin (by retrograde transport) to a region of the spinal cord where said dermatome nerve converges with a visceral afferent.
  • dermatome nerves e.g. spinal nerves/ afferents
  • the clostridial neurotoxin can exert an effect in the spinal cord (as evidenced by SNAP-25 cleavage identified in the dorsal horns) to inhibit signal transmission (e.g.
  • visceral afferent fibers have their cell bodies in dorsal root ganglia (DRG) and terminate in the spinal dorsal horn, from which visceral sensory information is transmitted rostrally in the contralateral spinothalamic tract (STT) or ipsilateral dorsal column (DC) to supraspinal sites (the brain). In other words, there is ‘ascending transmission’.
  • DDG dorsal root ganglia
  • STT contralateral spinothalamic tract
  • DC ipsilateral dorsal column
  • rostral (or ‘ascending’) transmission it is believed that suppression of visceral pain at any given segment of the spinal cord may also suppress visceral pain integration at more rostral segments.
  • suppression of visceral pain integration at the T12 segment may also be provided.
  • the neurons that receive visceral afferent input may be referred to as “visceroceptive spinal neurons”.
  • dermatome nerve(s) may refer to a dermatome nerve (e.g.
  • the clostridial neurotoxin may be administered to a “dermatome” toward administering the neurotoxin to a “dermatome nerve”; in which case, the method may comprise mapping said organ to a specific dermatome according to instructions outlined in the claims, thereby matching said organ to one or more specifically designated dermatome(s).
  • one or more specifically designated dermatome(s) may refer to a dermatome innervated by a spinal nerve that converges, in the spinal cord, with a visceral afferent from the organ/ viscera identified as contributing to the visceral pain.
  • convergence of afferent input is characteristic of visceroceptive spinal neurons. It is believed that typically all visceroceptive spinal neurons receive convergent somatic input.
  • the dermatome(s)/ dermatome nerve(s) corresponding to (e.g. mapping to) any given viscera have been mapped out, for example via studies into the phenomenon of “referred pain”.
  • the distribution of referred pain from the viscera can be predicted by the dermatomal distribution of the somatic afferents that enter the spinal cord at the same level as the visceral afferents.
  • the present inventors have thus taken advantage of such dermatomal mapping to target and suppress visceral pain integration via administration of a clostridial neurotoxin to an appropriate dermatome/ dermatome nerve.
  • the term “one or more dermatome nerve that map(s) to the organ identified as contributing to the visceral pain” may refer to a dermatome nerve (e.g. spinal nerve afferent) that converges, in the spinal cord, with a visceral afferent from the organ/ viscera identified as contributing to (e.g.
  • the term “one or more dermatome that map(s) to the organ identified as contributing to the visceral pain” may refer to a dermatome innervated by a spinal nerve that converges, in the spinal cord, with a visceral afferent from the organ/ viscera identified as contributing to the visceral pain.
  • convergence of afferent input is characteristic of visceroceptive spinal neurons. It is believed that typically all visceroceptive spinal neurons receive convergent somatic input.
  • the dermatome(s) corresponding to any given viscera have been mapped out via studies into the phenomenon of “referred pain”.
  • the distribution of referred pain from the viscera can be predicted by the dermatomal distribution of the somatic afferents that enter the spinal cord at the same level as the visceral afferents.
  • the present inventors have thus taken advantage of such dermatomal mapping to target and suppress visceral pain integration via administration of a clostridial neurotoxin to an appropriate dermatome.
  • the term “underlying visceral pain integration” may refer to the activation of second-order spinal neurons (aka. visceroceptive neurons) that receive visceral afferent input.
  • a nociceptive stimulus is detected at the viscera via nociceptive receptors, and nociceptive information is transmitted to the central nervous system which processes the information into an unpleasant sensation perceived as pain.
  • second-order spinal neurons are activated by visceral afferent input to integrate the visceral pain. It is believed that the pain suppression demonstrated by the inventors (see the Examples) is achieved by suppressing signal transfer from a visceral afferent to a visceroceptive neuron in the spinal cord, for example by inhibiting neurotransmitter secretion from the visceral afferent that would otherwise cross the synapse between said visceral afferent and visceroceptive neuron to bind a corresponding receptor on the visceroceptive neuron.
  • SNARE protein (more particularly, SNAP-25) cleavage has been detected in the spinal cord following administration of clostridial neurotoxin to a dermatome, said cleavage correlating with visceral pain suppression.
  • spinal nerves also referred to as dermatome nerves throughout this disclosure.
  • Spinal nerves are part of the peripheral nervous system (PNS) that connects the CNS with the rest of the body. In humans, there are 31 pairs of spinal nerves. They form from nerve roots that branch from the spinal cord. Spinal nerves are named and grouped by the region of the spine that they are associated with. There are five groups of spinal nerves, namely: - Cervical nerves.
  • cervical nerves There are eight pairs of these cervical nerves, numbered C1-C8. They originate from the neck (e.g. spine at the neck); - Thoracic nerves. There are 12 pairs of thoracic nerves, numbered T1-T12. They originate in the part of the spine that makes up the torso; - Lumbar nerves. There are five pairs of lumbar spinal nerves, numbered L1-L5. They come from the part of the spine that makes up your lower back; - Sacral nerves. There are five pairs of sacral spinal nerves, numbered S1-S5. They are associated with the sacrum, one of the bones found in the pelvis; - Coccygeal nerves. There is a single pair of coccygeal spinal nerves.
  • This pair of nerves originates from the area of the coccyx, or tailbone. Each of these nerves relays sensation (including pain) from a particular region of skin (i.e. a dermatome) to the brain.
  • a dermatome a region of skin
  • the dermatomes are like a stack of discs forming a human, each supplied by a different spinal nerve.
  • the pattern is different: the dermatomes run longitudinally along the limbs.
  • the general pattern is similar in all people.
  • the term “at a site proximal to said one or more specifically designated dermatome nerve(s)” may mean a site that is located within (and including) 5mm, 4mm, 3mm, 2mm or 1mm of said specifically designated dermatome nerve(s).
  • “at a site proximal to said one or more specifically designated dermatome nerve(s)” means the clostridial neurotoxin is administered to said specifically designated dermatome nerve.
  • the term “at a site proximal to said one or more specifically designated dermatome nerve(s)” may mean a site that is located within (and including) 5mm, 4mm, 3mm, 2mm or 1mm of the dermatome of said one or more specifically designated dermatome nerve(s).
  • “at a site proximal to said one or more specifically designated dermatome nerve(s)” means the clostridial neurotoxin is administered to the dermatome of said specifically designated dermatome nerve.
  • a site proximal to said one or more specifically designated dermatome(s) may mean a site that is located within (and including) 5mm, 4mm, 3mm, 2mm or 1mm of said one or more specifically designated dermatome(s).
  • “at a site proximal to said one or more specifically designated dermatome nerve(s)” means the clostridial neurotoxin is administered to said one or more specifically designated dermatome nerve(s).
  • spinal nerves also known as dermatome nerves herein
  • a point at least 3 cm behind the ear C3 – In the supraclavicular fossa, at the midclavicular line. C4 – Over the acromioclavicular joint. C5 – On the lateral (radial) side of the antecubital fossa, just proximally to the elbow. C6 – On the dorsal surface of the proximal phalanx of the thumb. C7 – On the dorsal surface of the proximal phalanx of the middle finger. C8 – On the dorsal surface of the proximal phalanx of the little finger.
  • T6 Intersection of the midclavicular line and the horizontal level of the xiphoid process.
  • T7 Intersection of the midclavicular line and the horizontal level at one quarter the distance between the level of the xiphoid process and the level of the umbilicus.
  • T8 – Intersection of the midclavicular line and the horizontal level at one half the distance between the level of the xiphoid process and the level of the umbilicus.
  • T9 Intersection of the midclavicular line and the horizontal level at three quarters of the distance between the level of the xiphoid process and the level of the umbilicus.
  • T10 Intersection of the midclavicular line, at the horizontal level of the umbilicus.
  • the clostridial neurotoxin may be administered in the proximity of at least one cm lateral to the occipital protuberance at the base of the skull (additionally or alternatively, in the proximity of a point at least 3 cm behind the ear); ii.
  • the clostridial neurotoxin may be administered in the proximity of the supraclavicular fossa, at the midclavicular line; iii. C4 dermatome, the clostridial neurotoxin may be administered in the proximity of over the acromioclavicular joint; iv. C5 dermatome, the clostridial neurotoxin may be administered in the proximity of the lateral (radial) side of the antecubital fossa, just proximally to the elbow; v. C6 dermatome, the clostridial neurotoxin may be administered in the proximity of the dorsal surface of the proximal phalanx of the thumb; vi.
  • the clostridial neurotoxin may be administered in the proximity of the dorsal surface of the proximal phalanx of the middle finger; vii. C8 dermatome, the clostridial neurotoxin may be administered in the proximity of the dorsal surface of the proximal phalanx of the little finger; viii. T1 dermatome, the clostridial neurotoxin may be administered in the proximity of the medial (ulnar) side of the antecubital fossa, just proximally to the medial epicondyle of the humerus; ix.
  • the clostridial neurotoxin may be administered in the proximity of the apex of the axilla; x. T3 dermatome, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the third intercostal space; xi. T4 dermatome, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the fourth intercostal space, located at the level of the nipples; xii.
  • the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the fifth intercostal space, horizontally located midway between the level of the nipples and the level of the xiphoid process; xiii. T6 dermatome, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the horizontal level of the xiphoid process; xiv. T7 dermatome, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the horizontal level at one quarter the distance between the level of the xiphoid process and the level of the umbilicus; xv.
  • the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the horizontal level at one half the distance between the level of the xiphoid process and the level of the umbilicus; xvi. T9 dermatome, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the horizontal level at three quarters of the distance between the level of the xiphoid process and the level of the umbilicus; xvii. T10 dermatome, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line, at the horizontal level of the umbilicus; xviii.
  • the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line, at the horizontal level midway between the level of the umbilicus and the inguinal ligament; xix.
  • T12 dermatome the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the midpoint of the inguinal ligament;
  • xx. L1 dermatome the clostridial neurotoxin may be administered in the proximity of the midway between the key sensory points for T12 and L2; xxi.
  • the clostridial neurotoxin may be administered in the proximity of the anterior medial thigh, at the midpoint of a line connecting the midpoint of the inguinal ligament and the medial epicondyle of the femur; xxii. L3 dermatome, the clostridial neurotoxin may be administered in the proximity of the medial epicondyle of the femur; xxiii. L4 dermatome, the clostridial neurotoxin may be administered in the proximity of over the medial malleolus; xxiv.
  • the clostridial neurotoxin may be administered in the proximity of the dorsum of the foot at the third metatarsophalangeal joint; xxv. S1 dermatome, the clostridial neurotoxin may be administered in the proximity of the lateral aspect of the calcaneus; xxvi. S2 dermatome, the clostridial neurotoxin may be administered in the proximity of the midpoint of the popliteal fossa; xxvii. S3 dermatome, the clostridial neurotoxin may be administered in the proximity of over the tuberosity of the ischium or infragluteal fold; xxviii.
  • the clostridial neurotoxin may be administered in the proximity of the perianal area, less than one cm lateral to the mucocutaneous zone.
  • the clostridial neurotoxin may be administered in the proximity of at least one cm lateral to the occipital protuberance at the base of the skull (additionally or alternatively, in the proximity of a point at least 3 cm behind the ear);
  • the clostridial neurotoxin may be administered in the proximity of the supraclavicular fossa, at the midclavicular line; iii.
  • the clostridial neurotoxin may be administered in the proximity of over the acromioclavicular joint; iv. C5 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the lateral (radial) side of the antecubital fossa, just proximally to the elbow; v. C6 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the dorsal surface of the proximal phalanx of the thumb; vi. C7 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the dorsal surface of the proximal phalanx of the middle finger; vii.
  • the clostridial neurotoxin may be administered in the proximity of the dorsal surface of the proximal phalanx of the little finger; viii. T1 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the medial (ulnar) side of the antecubital fossa, just proximally to the medial epicondyle of the humerus; ix. T2 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the apex of the axilla; x.
  • the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the third intercostal space; xi. T4 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the fourth intercostal space, located at the level of the nipples; xii. T5 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the fifth intercostal space, horizontally located midway between the level of the nipples and the level of the xiphoid process; xiii.
  • the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the horizontal level of the xiphoid process;
  • xiv. T7 dermatome nerve the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the horizontal level at one quarter the distance between the level of the xiphoid process and the level of the umbilicus;
  • xv. T8 dermatome nerve the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the horizontal level at one half the distance between the level of the xiphoid process and the level of the umbilicus;
  • the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the horizontal level at three quarters of the distance between the level of the xiphoid process and the level of the umbilicus; xvii. T10 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line, at the horizontal level of the umbilicus; xviii. T11 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line, at the horizontal level midway between the level of the umbilicus and the inguinal ligament; xix.
  • the clostridial neurotoxin may be administered in the proximity of the intersection of the midclavicular line and the midpoint of the inguinal ligament; xx. L1 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the midway between the key sensory points for T12 and L2; xxi. L2 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the anterior medial thigh, at the midpoint of a line connecting the midpoint of the inguinal ligament and the medial epicondyle of the femur; xxii.
  • the clostridial neurotoxin may be administered in the proximity of the medial epicondyle of the femur; xxiii. L4 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of over the medial malleolus; xxiv. L5 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the dorsum of the foot at the third metatarsophalangeal joint; xxv. S1 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the lateral aspect of the calcaneus; xxvi.
  • the clostridial neurotoxin may be administered in the proximity of the midpoint of the popliteal fossa; xxvii. S3 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of over the tuberosity of the ischium or infragluteal fold; xxviii. S4 and/or S5 dermatome nerve, the clostridial neurotoxin may be administered in the proximity of the perianal area, less than one cm lateral to the mucocutaneous zone.
  • the term “at a site proximal to said one or more specifically designated dermatome nerve(s)” may mean a site that is located within (and including) 5mm, 4mm, 3mm, 2mm or 1mm of said location on the body belonging to the dermatome of said one or more specifically designated dermatome nerve.
  • the term “at a site proximal to said one or more specifically designated dermatome(s)” may mean a site that is located within (and including) 5mm, 4mm, 3mm, 2mm or 1mm of said location on the body belonging to said one or more specifically designated dermatome.
  • sensory nerve fibers from dermatomes may come together at the same spinal cord level as the general visceral afferent fibers, such as that from the heart.
  • the general visceral sensory fiber When the general visceral sensory fiber is simulated, the central nervous system does not clearly discern whether the pain is coming from the body wall or from the viscera, so it perceives the pain as coming from somewhere on the body wall, e.g. left arm/hand pain, jaw pain. So the pain is "referred to" the related dermatomes of the same spinal segment.
  • administration (of clostridial neurotoxin) to: - one or more of the T8, T9, or T10 dermatome(s) may suppress visceral pain, wherein the stomach is contributing to the visceral pain; - one or more of the T8, T9, T10 and/or T11 dermatome(s) (preferably the T8, T9, and/or T10 dermatome(s)) may suppress visceral pain, wherein the liver is contributing to the visceral pain; - one or more of the T8, T9, T10 and/or T11 dermatome(s) (preferably the T8, T9, and/or T10 dermatome(s)) may suppress visceral pain, wherein the gall bladder is contributing to the visceral pain; - one or more of the T7, T8, T9, T10, T11 and/or T12 dermatome(s) may suppress visceral pain, wherein the pancrea
  • administration (of clostridial neurotoxin) to: - one or more of the T8, T9, or T10 dermatome nerve(s) may suppress visceral pain, wherein the stomach is contributing to the visceral pain; - one or more of the T8, T9, T10 and/or T11 dermatome nerve(s) (preferably the T8, T9, and/or T10 dermatome nerve(s)) may suppress visceral pain, wherein the liver is contributing to the visceral pain; - one or more of the T8, T9, T10 and/or T11 dermatome nerve(s) (preferably the T8, T9, and/or T10 dermatome nerve(s)) may suppress visceral pain, wherein the gall bladder is contributing to the visceral pain; - one or more of the T7, T8, T9, T10, T11 and/or T12 dermatome nerve(s) may suppress visceral pain, wherein the pancreas is contributing to the visceral pain; - one or more
  • a number of dermatomes/ dermatome nerves may map to any given viscera, and there may be overlap in the dermatomes which map to different viscera.
  • spinal innervation of the viscera e.g. the visceral afferents
  • spinal visceral afferent input has been documented to spread to several segments rostral and caudal from the spinal segment of entry.
  • visceral afferents from the stomach which relay nociceptive signals from the stomach to the spinal cord
  • a dermatome that maps (via a nerve fiber, more particularly a spinal nerve afferent) to any one of the T8, T9 and T10 segments may be employed in the present invention for administering a clostridial neurotoxin to suppress integration of pain from the stomach’s visceral afferents into the central nervous system (more particularly the spinal cord). Visceral afferent fibers from the liver also innervate the T8, T9 and T10 segments, hence dermatomes that map to one viscera may also map to another.
  • the clostridial neurotoxin is administered to (or proximal to) the T8, T9 and/ or T10 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T8 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T9 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T10 dermatome (or dermatome nerve). The clostridial neurotoxin may be administered to (or proximal to) at least two dermatomes (or dermatome nerves) selected from the T8, T9 and T10 dermatome.
  • the clostridial neurotoxin may be administered to (or proximal to) each of the T8, T9 and T10 dermatome (or dermatome nerve). Said dermatome(s) map to the stomach.
  • the clostridial neurotoxin is administered to (or proximal to) the T8, T9, T10 and/ or T11 (e.g. T8, T9, and/or T10) dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T8 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T9 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T10 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T11 dermatome (or dermatome nerve). The clostridial neurotoxin may be administered to (or proximal to) at least two or three dermatomes (or dermatome nerves) selected from the T8, T9, T10 and T11 (e.g.
  • the clostridial neurotoxin may be administered to (or proximal to) each of the T8, T9, T10 and T11 (e.g. T8, T9, and/or T10) dermatome (or dermatome nerve). Said dermatome(s) (or dermatome nerve(s)) map to the liver. Additionally or alternatively, said dermatome(s) map to the gall bladder. In one embodiment, the clostridial neurotoxin is administered to (or proximal to) the T7, T8, T9, T10, T11 and/ or T12 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T7 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T8 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T9 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T10 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T11 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T12 dermatome (or dermatome nerve). The clostridial neurotoxin may be administered to (or proximal to) at least two dermatomes selected from the T7, T8, T9, T10, T11 and T12 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) each of the T7, T8, T9, T10, T11 and T12 dermatomes (or dermatome nerves). Said dermatome(s) (or dermatome nerve(s)) map to the pancreas.
  • the clostridial neurotoxin is administered to (or proximal to) the T10, T11 and/ or T12 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T10 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T11 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T12 dermatome (or dermatome nerve). The clostridial neurotoxin may be administered to (or proximal to) at least two dermatomes (or dermatome nerves) selected from the T10, T11 and T12 dermatome (or dermatome nerve). For example, the clostridial neurotoxin may be administered to (or proximal to) each of the T10, T11 and T12 dermatomes (or dermatome nerves).
  • the clostridial neurotoxin is administered to (or proximal to) the T11, S1, S2, S3 and/ or S4 dermatome (or dermatome nerve) (preferably the S1, S2, S3 and/ or S4 dermatome (or dermatome nerve)).
  • the clostridial neurotoxin may be administered to (or proximal to) the T11 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the S1 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the S2 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S3 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S4 dermatome (or dermatome nerve). The clostridial neurotoxin may be administered to (or proximal to) at least two, three or four dermatomes (or dermatome nerves) selected from the T11, S1, S2, S3 and S4 (e.g.
  • the clostridial neurotoxin may be administered to (or proximal to) each of the T11, S1, S2, S3 and S4 (e.g. S1, S2, S3 and S4) dermatomes (or dermatome nerves). Said dermatome(s) (or dermatome nerves) map to the colon.
  • the clostridial neurotoxin is administered to (or proximal to) the T10, T11, T12 and/ or L1 (e.g. T11, T12 and/ or L1) dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T10 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T11 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the T12 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the L1 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) at least two or three dermatomes selected from the T10, T11, T12 and L1 (e.g. T11, T12 and/ or L1) dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) each of the T10, T11, T12 and L1 (e.g. T11, T12 and L1) dermatomes (or dermatome nerves).
  • Said dermatome(s) (or dermatome nerve(s)) map to the kidney(s).
  • the clostridial neurotoxin is administered to (or proximal to) the T11, T12, L1, S1, S2, S3 and/ or S4 (e.g. T12, L1, S1, S2, S3 and/ or S4) dermatome (or dermatome nerves).
  • the clostridial neurotoxin may be administered to (or proximal to) the T11 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T12 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the L1 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S1 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S2 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S3 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the S4 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) at least two, three or four or five dermatomes (or dermatome nerves) selected from the T11, T12, L1, S1, S2, S3 and S4 (e.g. T12, L1, S1, S2, S3 and/ or S4) dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) each of the T11, T12, L1, S1, S2, S3 and S4 (e.g.
  • T12, L1, S1, S2, S3 and S4) dermatomes (or dermatome nerves).
  • Said dermatome(s) (or dermatome nerve(s)) map to the bladder (urinary bladder).
  • the clostridial neurotoxin is administered to (or proximal to) the T11 dermatome (or dermatome nerve).
  • Said dermatome (or dermatome nerve) maps to the ovary/ ovaries. Additionally or alternatively, said dermatome (or dermatome nerve) maps to the fallopian tube(s).
  • the clostridial neurotoxin is administered to (or proximal to) the T12, and/or L1 dermatome(s) (or dermatome nerve(s)).
  • the clostridial neurotoxin may be administered to (or proximal to) the T12 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the L1 dermatome (or dermatome nerve). The clostridial neurotoxin may be administered to (or proximal to) each of the T12, and L1 dermatomes (or dermatome nerves). Said dermatome(s) (or dermatome nerve(s)) map to the uterus. In one embodiment, the clostridial neurotoxin is administered to (or proximal to) the S2, S3 and/ or S4 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the S2 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S3 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S4 dermatome (or dermatome nerve). The clostridial neurotoxin may be administered to (or proximal to) at least two dermatomes (or dermatome nerves) selected from the S2, S3 and S4 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) each of the S2, S3 and S4 dermatomes (or dermatome nerves). Said dermatome(s) (or dermatome nerves) map to the cervix.
  • the clostridial neurotoxin is administered to (or proximal to) the T10, T11, L1, L2, S1, S2, S3 and/ or S4 (e.g. T11, L1, L2, S1, S2, S3 and/ or S4) dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T10 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the T11 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the L1 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the L2 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S1 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) the S2 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S3 dermatome (or dermatome nerve). Additionally or alternatively, the clostridial neurotoxin may be administered to (or proximal to) the S4 dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) at least two, three, four, five, six or seven dermatomes (or dermatome nerves) selected from the T10, T11, L1, L2, S1, S2, S3 and S4 (e.g. T11, L1, L2, S1, S2, S3 and/ or S4) dermatome (or dermatome nerve).
  • the clostridial neurotoxin may be administered to (or proximal to) each of the T10, T11, L1, L2, S1, S2, S3 and S4 (e.g. the T11, L1, L2, S1, S2, S3 and S4) dermatomes (or dermatome nerves).
  • Said dermatome(s) (or dermatome nerve(s)) map to the testes. Additionally or alternatively, said dermatomes(s) (or dermatome nerve(s)) map to the epididymis.
  • visceral pain may be suppressed in a number of organs identified as contributing to the visceral pain.
  • a number of diseases/ conditions cause injury or insult to internal organs, leading to the unpleasant sensation of visceral pain that can often be chronic and important to manage (e.g. as the disease itself may take time to heal).
  • parts of the digestive system such as the stomach and intestine become inflamed, leading to the emergence of pain from said organs.
  • visceral pain may be bladder pain syndrome (interstitial cystitis), chronic functional abdominal pain (CFAP), functional constipation, functional dyspepsia, a non- cardiac chest pain (NCCP), a chronic abdominal pain, 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, a urinary tract infection, a pancreatitis, a hernia, autoimmune pain including, for example, a sarcoidosis and a vasculitis; organic viscer pain syndrome (interstitial cystitis), chronic functional abdominal pain (CFAP), functional constipation, functional dyspepsia, a non-
  • visceral pain is caused by a condition selected from bladder pain syndrome (interstitial cystitis), cirrhosis, Crohn’s disease, ulcerative colitis, irritable bowel syndrome, coeliac disease, endometriosis, gastric ulcers, kidney stones, bladder stones, gallbladder stones, pancreatitis and peritonitis.
  • the visceral pain may be interstitial cystitis pain, cirrhosis pain, Crohn’s disease pain, ulcerative colitis pain, irritable bowel syndrome pain, coeliac disease pain, endometriosis pain, gastric ulcer pain, kidney stone pain, bladder stone pain, gallbladder stone pain, pancreatitis pain and/or peritonitis pain.
  • the visceral pain may be caused by or associated with a vascular disorder.
  • vascular disorder examples 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, a urinary tract infection, a pancreatitis and a hernia.
  • C. 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 tumour 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 visceral pain may be selected from: endometriosis pain, pancreatitis pain, gastrointestinal pain, and visceral pain caused by or associated with a vascular disorder (more preferably endometriosis pain).
  • the visceral pain may be caused by bladder pain syndrome (interstitial cystitis).
  • the clostridial neurotoxin may be administered to the T12, L1, S2, S3 and/ or S4 dermatome (or dermatome nerve).
  • a method of the invention may comprise (a) identifying the bladder as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): urinary bladder maps to the T11, T12, L1, S1, S2, S3 and/ or S4 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the
  • the visceral pain may be caused by cirrhosis (liver cirrhosis).
  • the clostridial neurotoxin may be administered to the T8, T9, T10 and/or T11 dermatome nerve(s).
  • a method of the invention may comprise (a) identifying the liver as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): liver maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • the visceral pain may be caused by gall bladder stones.
  • the clostridial neurotoxin may be administered to the T8, T9, T10 and/or T11 dermatome nerve(s).
  • a method of the invention may comprise (a) identifying the liver as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): gall bladder maps to the T8, T9, T10 and/ or T11 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal
  • the visceral pain may be caused by Crohn’s disease.
  • the clostridial neurotoxin may be administered to the T8, T9, T10, T11, T12, S1, S2, S3 and/or S4 dermatome.
  • a method of the invention may comprise (a) identifying the stomach, small intestine and/or colon as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): stomach maps to the T8, T9 and/ or T10 dermatome nerve(s), small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s), colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord
  • Crohn’s disease five main types exist, with each typically being defined by the location of inflammation in the gastrointestinal tract.
  • the types of Crohn’s disease include ileocolitis (e.g. inflames the end of the small intestine/ ileum and a portion of the large intestine/ colon), ileitis (e.g. inflames the last section of the small intestine/ ileum); gastroduodenal Crohn’s (e.g. inflames the stomach and the start of the small intestine/ duodenum), jejunoileitis (e.g. inflames the middle part of the small intestine/ jejunum) and Crohn's (granulomatous) colitis (e.g.
  • a method of the invention may comprise (a) identifying the small intestine and/or colon as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s), colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells
  • a method of the invention may comprise (a) identifying the small intestine as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • a method of the invention may comprise (a) identifying the stomach and/or small intestine as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): stomach maps to the T8, T9 and/ or T10 dermatome nerve(s), small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and
  • a method of the invention may comprise (a) identifying the small intestine as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • a method of the invention may comprise (a) identifying the colon as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • the visceral pain may be caused by ulcerative colitis.
  • the clostridial neurotoxin may be administered to the S1, S2, S3 and/ or S4 dermatome.
  • a method of the invention may comprise (a) identifying the colon and/or rectum as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): colon maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s), rectum maps to the T11, S1, S2, S3 and/or S4 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neuro
  • the visceral pain may be caused by irritable bowel syndrome.
  • the clostridial neurotoxin may be administered to the S1, S2, S3 and/or S4 dermatome.
  • a method of the invention may comprise (a) identifying the large intestine as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): large intestine maps to the T11, S1, S2, S3 and/ or S4 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory
  • the visceral pain may be caused by coeliac disease.
  • the clostridial neurotoxin may be administered to the T10, T11, and/or T12 dermatome.
  • a method of the invention may comprise (a) identifying the small intestine as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): small intestine maps to the T10, T11 and/ or T12 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord
  • the visceral pain may be caused by gastric ulcers.
  • the clostridial neurotoxin may be administered to the T8, T9 and/or T10 dermatome.
  • a method of the invention may comprise (a) identifying the stomach as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): stomach maps to the T8, T9 and/ or T10 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory affer
  • the visceral pain may be caused by pancreatitis.
  • the clostridial neurotoxin may be administered to the T7, T8, T9, T10, T11 and/or T12 dermatome.
  • a method of the invention may comprise (a) identifying the pancreas as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): pancreas maps to the T7, T8, T9, T10, T11 and/ or T12 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neuro
  • the visceral pain may be caused by peritonitis (e.g. peritonitis of visceral peritoneum).
  • Peritonitis is inflammation of the peritoneum, a membrane that lines the inner abdominal wall and abdominal organs (viscera). The peritoneum and the organ (viscera) it lines share the same sensory ganglion and spinal cord segment.
  • the appropriate dermatome to target for suppressing visceral pain that is caused by peritonitis depends on the organ that the (inflamed) peritoneum lines.
  • a method of the invention may comprise (a) identifying the stomach as an organ in the patient that is contributing to said visceral pain (e.g. due to inflammation of the peritoneum); (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): stomach maps to the T8, T9 and/ or T10 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and suppresses neurotransmitter release therefrom, thereby suppressing said
  • the visceral pain is caused by endometriosis.
  • endometriosis that is associated with a symptom selected from dysmenorrhea, chronic pelvic pain (CPP), dyspareunia, constipation, dyschezia, and chronic back pain.
  • CPP chronic pelvic pain
  • Endometriosis is a chronic and debilitating condition characterized by chronic pelvic pain and infertility.
  • Endometriosis has a large clinical burden, affecting approximately 1 in 10 women globally. Endometriosis is associated with significant societal and economic burden that costs the US economy $22 billion annually in lost productivity and direct healthcare costs.
  • Endometriosis may be defined as the presence of endometrial glands and stroma in an abnormal or ectopic location outside the uterine cavity, for example ectopic positions such as the ovaries and fallopian tubes. Endometriosis occurs in 20–50% in women with infertility and 71–87% in women with chronic pelvic pain. Endometriosis is a disease of the female reproductive system in which cells similar to those in the endometrium, the layer of tissue that normally covers the inside of the uterus, grow outside the uterus. Most often this is on the ovaries, fallopian tubes, and tissue around the uterus and ovaries; however, it may also occur in other parts of the body.
  • endometriosis is thus a pathology where various dermatomes could be of interest depending on the site of endometriotic growth.
  • the most common symptoms of endometriosis are dysmenorrhea, chronic pelvic pain (CPP), dyspareunia, constipation, dyschezia, and chronic back pain.
  • clostridial neurotoxin treatment has previously been tested for alleviating pain (e.g.
  • the present invention represents a particularly advantageous pain relief strategy for alleviating visceral pain that would otherwise require invasive administration of medicament (e.g. as in the case of visceral pain caused by endometriosis).
  • endometriosis pain can effect a number of different organs, and thus represents a pathology where various dermatomes can advantageously be utilised as an administration route depending on the site of endometriotic growth, thus the organ contributing to the visceral pain.
  • the bladder may be identified as the organ contributing to visceral pain, and the clostridial neurotoxin may thus be administered to a specific dermatome nerve that maps to the bladder.
  • the clostridial neurotoxin may be administered to the T11, T12, L1, S2, S3 and/or S4 dermatome.
  • a method of the invention may comprise (a) identifying an ovary, fallopian tube, uterus and/or cervix as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): ovary maps to the T11 dermatome nerve(s), fallopian tube(s) maps to the T11 dermatome nerve(s), uterus maps to the T12 and/ or L1 dermatome nerve(s), cervix maps to the S2, S3 and/ or S4 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial
  • a method of the invention may comprise (a) identifying an ovary as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): ovary maps to the T11 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to
  • a method of the invention may comprise (a) identifying a fallopian tube as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): fallopian tube(s) maps to the T11 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • a method of the invention may comprise (a) identifying the uterus as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): uterus maps to the T12 and/ or L1 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • a method of the invention may comprise (a) identifying the cervix as an organ in the patient that is contributing to said visceral pain; (b) mapping said organ to a specific dermatome nerve according to the following instructions, thereby matching said organ to one or more specifically designated dermatome nerve(s): cervix maps to the S2, S3 and/ or S4 dermatome nerve(s); and (c) administering a therapeutically effective amount of a clostridial neurotoxin at a site proximal to said one or more specifically designated dermatome nerve(s), wherein following administration of said clostridial neurotoxin to the patient, the clostridial neurotoxin is transported via retrograde axonal transport to the spinal cord where it binds sensory afferent nerve cells, enters said cells by receptor-mediated endocytosis and suppresses neurotransmitter release therefrom, thereby suppressing said visceral pain.
  • the visceral pain may comprise or consist of allodynia.
  • the visceral pain may be allodynia.
  • Allodynia means “other pain”. It is a pain that results from a stimulus that is not normally painful.
  • a subject suffering from bad sunburn may experience intense pain even to light touch.
  • sun exposure can overly sensitize the skin such that wearing a shirt or taking a shower can be very painful.
  • a sufferer of tactile allodynia (aka static tactile allodynia or mechanical allodynia) may experience pain to touch, such as with resting one’s head on a pillow, or with wearing a hat, earrings, or necklace.
  • Allodynia is a condition that is distinct from hyperalgesia, which is a pain stimulus that is more painful than usual. Indeed, as mentioned above, allodynia is by its very definition “pain due to a stimulus that does not usually provoke pain”, as opposed to hyperalgesia (increased pain from a stimulus that does usually provoke pain). Allodynia may also be referred to as “hypersensitivity” herein.
  • Visceral pain With regard to visceral pain, allodynia often occurs in particular as a result of gastrointestinal disorder, such as oesophagitis, gastro-oesophageal reflux disease, non-ulcer dyspepsia, gastroparesis, and irritable bowel syndrome (IBS). Visceral allodynia/ hypersensitivity may also occur in non-gastrointestinal disorders such as interstitial cystitis and ureteric colic (e.g. urinary stone). Visceral allodynia/ hypersensitivity may be defined as an increased intensity of sensations and lowered thresholds for visceral pain in a patient. The present inventors have demonstrated that the present invention may be used to suppress allodynia in a distinct manner from suppressing hyperalgesia.
  • gastrointestinal disorder such as oesophagitis, gastro-oesophageal reflux disease, non-ulcer dyspepsia, gastroparesis, and irritable bowel syndrome (IBS).
  • the visceral pain may be suppressed for at least 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, or 60 days following administration of the clostridial neurotoxin.
  • said visceral pain may be suppressed for at least 60, 64, 68, 72, 76, 80, 84, or 88 days following administration of the clostridial neurotoxin.
  • the visceral pain may be suppressed for at least 1, 2, 3, 4, 5, 7, 8, or 9 months following administration of the clostridial neurotoxin.
  • said visceral pain may be suppressed for at up to an including 9 months following administration of the clostridial neurotoxin.
  • Said visceral pain may be reduced for at least 10 days following administration of the clostridial neurotoxin.
  • visceral pain may be reduced for at least 30 days following administration of the clostridial neurotoxin. More preferably, said visceral pain may be reduced for at least 50 days following administration of the clostridial neurotoxin.
  • Visceral pain may be suppressed within at least 1 day following administration of the clostridial neurotoxin; for example within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days following administration of the clostridial neurotoxin.
  • visceral pain may be suppressed within at least 7 days following administration of the clostridial neurotoxin.
  • visceral pain may be suppressed within at least 10 days following administration of the clostridial neurotoxin.
  • a “patient” (which may be used synonymously with the term “subject”) as used herein may be a mammal, such as a human or other mammal.
  • patient means a human subject.
  • the patient may be a patient having a lower than average (basal) neuroimmune response, e.g. a patient receiving an alternative therapy, such as chemotherapy, which may suppress the immune system.
  • disorder as used herein also encompasses a “disease”. In one embodiment the disorder is a disease.
  • the present invention embraces a method for treating visceral pain in a patient, said method comprising administration of a therapeutically effective amount of a clostridial to a patient suffering from visceral pain.
  • the present invention also embraces a corresponding therapeutic use, namely a clostridial neurotoxin for use in treating visceral pain in a patient.
  • the term “suppress” or “suppressing”, or “treat” or “treating” as used herein encompasses prophylactic suppression and treatment (e.g. to prevent onset of visceral pain) as well as corrective suppression and treatment (suppression and treatment of a subject already suffering from visceral pain).
  • the term “suppress” or “suppressing” as used herein means corrective treatment.
  • the term “treat” or “treating” as used herein means corrective treatment.
  • the term “suppress” or “suppressing”, or “treat” or “treating” encompasses suppressing and treating both the visceral pain and a symptom thereof. In some embodiments the term “suppress” or “suppressing”, or “treat” or “treating” refers to a symptom of visceral pain.
  • a clostridial neurotoxin of the invention suppresses a visceral pain by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or by 100% greater that an otherwise identical administration lacking a clostridial neurotoxin (e.g. a vehicle-only administration).
  • a clostridial neurotoxin of the invention suppresses a visceral pain by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% or by 100% compared to visceral pain perceived by the patient pre-administration of the clostridial neurotoxin.
  • Suppressing (or treating) pain may be used synonymously with “reducing pain”.
  • administration of a clostridial neurotoxin of the invention may reduce visceral pain in a subject.
  • reference to “reduced” or “reducing” preferably means a lower level of visceral pain is perceived by the subject after administration with a clostridial neurotoxin of the invention (post-administration) when compared with a level of visceral pain perceived by the subject prior to administration (pre-administration).
  • the level of visceral pain perceived may be reduced by at least 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85% or 95% post-administration relative to pre-administration.
  • the level of visceral pain perceived may be reduced by at least 75%; preferably at least 85%; more preferably at least 95% post-administration.
  • a variety of means for assessing pain perception are known to those skilled in the art. For example, evaluation of mechanical allodynia (either static or dynamic) is routinely used in human pain studies as described in Pogatzki-Zahn et. al. (Pain Rep.2017 Mar; 2(2): e588), incorporated herein by reference.
  • a suitable (albeit non-limiting) method for assessing pain perception in a subject includes the following: Numerical Rating Scale (NRS) score; although the skilled person is aware of other methods which may be used additionally or alternatively such as sensory threshold, pain perception threshold, static mechanical allodynia, dynamic mechanical allodynia, temporal summation, pressure pain threshold, conditioned pain modulation, and temperature threshold.
  • NRS Numerical Rating Scale
  • pain perception measures include: change from baseline in SF-36 scores at each scheduled time point; amount of rescue medication taken during the study and time to first intake of rescue medication. These may be considered “exploratory” endpoints or pain perception assessment measures.
  • pain perception may be assessed by one or more of: (a) a Numerical Rating Scale (NRS); (b) a stimulus-evoked NRS; (c) temperature of the painful area; (d) size of the painful area; (e) time to onset of analgesic effect; (f) peak analgesic effect; (g) time to peak analgesic effect; (h) duration of analgesic effect; and (i) an SF-36 quality of life assessment.
  • NRS Numerical Rating Scale
  • Numerical Rating Scale Typically pain perception according to the present invention uses the Numerical Rating Scale (NRS).
  • the NRS is an 11-point scale to assess subject pain perception. Subjects are asked to give a number between 0 and 10 that fits best to their pain intensity. Zero represents ‘no pain at all’ whereas the upper limit, 10, represents ‘the worst pain possible’.
  • the NRS can be used to assess numerous facets of pain, including spontaneous average pain, spontaneous worst pain, and spontaneous current pain.
  • Spontaneous average pain is assessed by asking a subject to select a number that best describes the subject’s average pain (e.g. perceived pain) over a period of time, for example at least 6 hours, 12 hours, 24 hours, or at least 48 hours.
  • Spontaneous worst pain is assessed by asking a subject to select a number that best describes the subject’s pain at its worst during a specified period, e.g. at least the previous 6 hours, 12 hours, 24 hours or previous 48 hours.
  • Spontaneous current pain is assessed by asking a subject to select a number that best describes how much pain the subject is in at the time of assessment.
  • the NRS can also be used to assess a subject’s pain perception in response to a variety of different stimuli.
  • the subject will be submitted to stimuli of various nature applied to the painful area.
  • stimuli include: (i) light touch (which can be assessed by measuring pain on the surface of the painful area on radial spokes following application of a von Frey filament as described herein); (ii) pressure (pressure pain threshold), which can be assessed by asking the subject to give a NRS score as increasing pressure is applied using a pressure algometer; and (iii) temperature (which can be assessed by asking the subject for an NRS score for warm, cold and hot stimulation using a thermode applied to the painful area).
  • a clostridial neurotoxin of the invention reduces the subject’s NRS score post-administration (e.g. from a rating of ⁇ 7 to a rating of ⁇ 6) when compared with the subject’s NRS score pre-administration.
  • Quality of Life questionnaire Short Form-36 (SF-36): The SF-36 quality of life questionnaire may be used to assess a subject’s pain perception.
  • the SF-36 is a 36-item, subject-reported survey of subject health.
  • the SF-36 consists of eight scaled scores (vitality, physical functioning, bodily pain, general health perceptions, physical role functioning, emotional role functioning, social role functioning and mental health). Each scale is directly transformed into a 0-100 scale on the assumption that each question carries equal weight.
  • SF-36 The higher the score recorded in the SF-36, the less disability.
  • Relevant parameters commonly tested in clinical trials for the treatment of pain are known in the art and could be readily selected by one of ordinary skill in the art. Examples of such parameters include, but are not limited to NRS; stimulus-evoked NRS; temperature of the painful area; size of the painful area; time to onset of analgesic effect; peak analgesic effect; time to peak analgesic effect; duration of analgesic effect; and/or SF-36 quality of life as described herein. Methods for assessing these parameters are also known in the art and can be carried out by one of ordinary skill using routine methods and procedures.
  • administration of a clostridial neurotoxin of the invention increases the subject’s SF-36 score post-administration (e.g. from a score of ⁇ 50 to a score of ⁇ 50) when compared with the subject’s SF-36 score pre-administration.
  • a clostridial neurotoxin may be administered to a subject in a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” is any amount of the clostridial neurotoxin, which when administered alone or in combination to a subject for suppressing / treating visceral pain (or a symptom thereof) is sufficient to effect such suppression / treatment of the visceral pain, or symptom thereof.
  • a “prophylactically effective amount” is any amount of the clostridial neurotoxin that, when administered alone or in combination to a subject inhibits or delays the onset or reoccurrence of visceral pain (or a symptom thereof). In some embodiments, the prophylactically effective amount prevents the onset or reoccurrence of visceral pain entirely. “Inhibiting” the onset means either lessening the likelihood of visceral pain onset (or symptom thereof), or preventing the onset entirely. CLOSTRIDIAL NEUROTOXINS Clostridial neurotoxins of the invention will now be described in more detail, together with details of how they may be formulated e.g. into pharmaceutical compositions.
  • the clostridial neurotoxins of the invention may be formulated in any suitable manner for administration to a subject, for example as part of a pharmaceutical composition.
  • the invention provides a pharmaceutical composition for use in any method of treatment described herein, the pharmaceutical composition comprising a clostridial neurotoxin of the invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.
  • the clostridial neurotoxin of the invention may be in a single-chain form, while in other embodiments the clostridial neurotoxin may be in a di-chain form, e.g. where the two chains are linked by a di-sulphide bridge.
  • the clostridial neurotoxin is in a di-chain form.
  • the clostridial neurotoxins of the present invention may preferably be formulated for injection to the subject, for example via intradermal injection.
  • 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.
  • the dosage ranges for administration of the clostridial neurotoxins of the present invention are those to produce the desired therapeutic and/or prophylactic effect.
  • a dosage of the clostridial neurotoxin is a flat dose.
  • a flat dose may be in the range of 50 pg to 250 ⁇ g, preferably 100 pg to 100 ⁇ g.
  • a flat dose may be at least 50 pg, 100 pg, 500 pg, 1 ng, 50 ng, 100 ng, 500 ng, 1 ⁇ g or 50 ⁇ g.
  • Said dose may be a single flat dose.
  • a clostridial neurotoxin may be administered by way of a unit dose.
  • Said unit dose may be administered at a single site or, alternatively, less than a unit dose may be administered at an administration site (e.g. where there are two or more administration sites and the dose is divided (equally or unequally) between said sites).
  • a single unit dose may be administered per dermatome injected when carrying out the present invention.
  • a suitable unit dose may be 5 pg to 17,000 pg of the clostridial neurotoxin.
  • An upper limit of the unit dose range may be 16,500, 15,500, 14,500, 13,500, 12,500, 11,500, 10,500, 9,500, 8,500, 7,500, 6,500, 5,500, 4,500, 3,500, 2,500, 1,500 or 500 pg of clostridial neurotoxin, preferably the upper limit is 16,000 pg.
  • a lower limit of the unit dose range may be 10, 20, 30, 50, 100, 200, 250, 350, 450, 550, 650, 750, 850, 950, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500 or 5,000 pg of clostridial neurotoxin, preferably the lower limit is 1,000 pg.
  • the unit dose is 750 pg to 17,000 pg of clostridial neurotoxin. More preferably, the unit dose of clostridial neurotoxin is 1,000 pg to 16,000 pg of (e.g. modified) BoNT/A, e.g.4,000 pg to 6,000 pg.
  • a total dose administered per treatment session may be up to 255,000 pg of the clostridial neurotoxin. This may correspond to 15x the unit dose. In other words, the total amount of clostridial neurotoxin administered at a given treatment session may be up to 255,000 pg.
  • the total dose may be up to 240,000, 220,000, 200,000, 180,000, 160,000, 140,000,110,000, 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 20,000, 10,000 or 5,000 pg.
  • the total dose may be up to 240,000 pg of clostridial neurotoxin.
  • the total dose may be at least 900, 1,000, 2,000, 3,000, 4,000, 5,000, 7,500, 10,000, 12,500, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 120,000, 150,000, 175,000, 200,000 or 220,000 pg.
  • the total dose may be at least 1,500 pg, more preferably at least 2,000 pg of clostridial neurotoxin, e.g. at least 12,000 pg.
  • the total dose may be 2,000-240,000 pg, preferably 128,000-240,000 pg. More preferably, the total dose administered is 15,000-240,000 pg.
  • the total number of unit doses administered in a given treatment may be up to 15x the unit dose.
  • the total number of unit doses administered may be up to 14x, 13x, 12x, 11x, 10x, 9x, 8x or 7x.
  • the total number of unit doses administered may be at least 2x, 3x, 4x, 5x, 6x, 7x the unit dose, preferably at least 2x.
  • the total number of unit doses administered may be 2x to 15x, 7x to 15x or 10x to 14x.
  • the number of unit doses administered is 15x.
  • the term “up to” when used in reference to a value e.g. up to 255,000 pg) means up to and including the value recited.
  • reference to administering “up to 255,000 pg” of clostridial neurotoxin encompasses administration of 255,000 pg of (e.g. modified) BoNT/A as well as administration of less than 255,000 pg of clostridial neurotoxin.
  • the clostridial neurotoxin of the invention may be administered iteratively (e.g. up to 5, 10, 15 or 20 times) as part of a treatment regimen (such as on different days, e.g. with at least 1 day between successive treatments). Iterative administration means administration at least two times, e.g. at least 5, 10, 15 or 20 times.
  • a clostridial neurotoxin of the invention may be administered two or more times to suppress/ treat the visceral pain of a subject. This is particularly pertinent for the treatment of chronic conditions, such as chronic pain, where ongoing treatment is typically necessary.
  • a clostridial neurotoxin of the invention may be administered weekly, twice monthly, monthly, every two months, every six months or annually, preferably at least twice annually or annually.
  • a clostridial neurotoxin of the invention is administered two or more times in a period of 10 years, 5 years, 2 years or 1 year.
  • a clostridial neurotoxin of the invention is administered two or more times in a period of 1 year.
  • Treatment may continue for at least 6 months, 1 year, 2 years, 3 years, 5 years, 10 years, 15 years, 20 years, 25 years or 30 years. It is preferred that the clostridial neurotoxin is not administered together with a further therapeutic or diagnostic agent (e.g. a nucleic acid, protein, peptide or small molecule therapeutic or diagnostic agent) additional to the light-chain and heavy-chain.
  • a further therapeutic or diagnostic agent e.g. a nucleic acid, protein, peptide or small molecule therapeutic or diagnostic agent
  • the clostridial neurotoxin is not administered with a further analgesic.
  • a clostridial neurotoxin of the invention is not administered together with a covalently associated therapeutic agent.
  • a clostridial neurotoxin of the invention is not administered together with a non-covalently associated therapeutic agent.
  • Fluid dosage forms are typically prepared utilising the clostridial neurotoxin and a pyrogen- free sterile vehicle.
  • the clostridial neurotoxin depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle.
  • the clostridial neurotoxin can be dissolved in the vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing.
  • the solution in its sealed containers may be sterilised by autoclaving.
  • Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.
  • Dry powders which are dissolved or suspended in a suitable vehicle prior to use, may be prepared by filling pre-sterilised ingredients into a sterile container using aseptic technique in a sterile area. Alternatively the ingredients may be dissolved 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 an administration route described herein, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration.
  • the components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.
  • a suspending agent for example polyvinylpyrrolidone is included in the composition(s) to facilitate uniform distribution of the components.
  • the clostridial neurotoxin may be administered prior to, simultaneous or subsequent to the onset of visceral pain.
  • the clostridial neurotoxin may be administered prior to, simultaneous or subsequent to the onset of a condition/ disease that cause visceral pain.
  • the clostridial neurotoxin may be administered subsequent to detection of the infection to suppress visceral pain.
  • the term “clostridial neurotoxin” embraces a clostridial neurotoxin fragment thereof. Further information on clostridial neurotoxins, and suitable neurotoxins for use in the invention, is now provided below.
  • the term “clostridial neurotoxin” embraces toxins produced by C.
  • Botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F), as well as modified clostridial neurotoxins or derivatives derived from any of the foregoing.
  • Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex, consisting of BoNT itself complexed to a number of accessory proteins.
  • botulinum neurotoxin serotypes A, B, C1, D, E, F, G, and X all of which share similar structures and modes of action.
  • BoNT serotypes can be distinguished based on inactivation by specific neutralising anti-sera, with such classification by serotype correlating with percentage sequence identity at the amino acid level.
  • BoNT proteins of a given serotype are further divided into different subtypes on the basis of amino acid percentage sequence identity. BoNTs are absorbed in the gastrointestinal tract, and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve terminals and prevent the release of their neurotransmitter acetylcholine.
  • BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave synaptobrevin/vesicle-associated membrane protein (VAMP);
  • BoNT/C1, BoNT/A and BoNT/E cleave the synaptosomal-associated protein of 25 kDa (SNAP-25);
  • BoNT/C1 cleaves syntaxin.
  • BoNT/X has been found to cleave SNAP-25, VAMP1, VAMP2, VAMP3, VAMP4, VAMP5, Ykt6, and syntaxin 1.
  • Tetanus toxin is produced in a single serotype by C. tetani.
  • C. butyricum produces BoNT/E
  • C. baratii produces BoNT/F.
  • a clostridial neurotoxin may be selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, and TeNT (tetanus neurotoxin).
  • a clostridial neurotoxin is a botulinum neurotoxin, such as a botulinum neurotoxin selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, and BoNT/X.
  • the clostridial neurotoxin may be BoNT/A.
  • a reference BoNT/A sequence is shown as SEQ ID NO: 51 (additionally or alternatively, reference BoNT/A sequence may be that shown as SEQ ID NO: 62).
  • the clostridial neurotoxin may be BoNT/B.
  • a reference BoNT/B sequence is shown as SEQ ID NO: 52.
  • the clostridial neurotoxin may be BoNT/C.
  • a reference BoNT/C sequence is shown as SEQ ID NO: 53.
  • the clostridial neurotoxin may be BoNT/D.
  • a reference BoNT/D sequence is shown as SEQ ID NO: 54.
  • the clostridial neurotoxin may be BoNT/E.
  • a reference BoNT/E sequence is shown as SEQ ID NO: 55.
  • the clostridial neurotoxin may be BoNT/F.
  • a reference BoNT/F sequence is shown as SEQ ID NO: 56.
  • the clostridial neurotoxin may be BoNT/G.
  • a reference BoNT/G sequence is shown as SEQ ID NO: 57.
  • the clostridial neurotoxin may be TeNT.
  • a reference TeNT sequence is shown as SEQ ID NO: 58.
  • the clostridial neurotoxin may be BoNT/X.
  • a reference BoNT/X sequence is shown as SEQ ID NO: 59.
  • clostridial neurotoxin is intended to encompass both full length neurotoxins (comprising a clostridial neurotoxin L-chain and a clostridial neurotoxin H-chain), as well as fragments thereof.
  • the “clostridial neurotoxin” may refer to a polypeptide that comprises or consists of a clostridial neurotoxin L-chain, a clostridial neurotoxin translocation domain (H N ) and/or a clostridial neurotoxin receptor binding domain (H C ) domain.
  • the “clostridial neurotoxin” may refer to a polypeptide that comprises or consists of a clostridial neurotoxin L-chain, a clostridial neurotoxin translocation domain (H N ) and/or a clostridial neurotoxin receptor binding domain (H C ) domain, wherein when the polypeptide comprises a clostridial neurotoxin L-chain, the L-chain is a catalytically inactive.
  • the present invention may encompass the use of full-length clostridial neurotoxins comprising a clostridial neurotoxin L-chain and a clostridial neurotoxin H-chain, optionally with the proviso that said clostridial neurotoxin L-chain is catalytically inactive.
  • the use of a clostridial neurotoxin fragment allows for the use of non-toxic (or substantially non-toxic) fragments of clostridial neurotoxins, which given the smaller size (compared to the full-length H-chain or full-length clostridial neurotoxin), are less likely to provoke an adverse immune response (against the fragment) in a subject administered said fragments.
  • the non-toxic (or substantially non-toxic) fragments are less expensive and/or less complex to manufacture than full-length clostridial neurotoxins. Additionally, the non-toxic (or substantially non-toxic) fragments constitute a more well-defined therapeutic than the full-length clostridial toxins, and given the shorter length of the polypeptides there is a reduced probability of, for example, cysteine shuffling between domains.
  • the molecule may conveniently be referred to as a “polypeptide”. That is, the term “clostridial neurotoxin” may be substituted for the term “polypeptide”.
  • the clostridial neurotoxin referred to herein may be a polypeptide that comprises (or consists of) a clostridial neurotoxin light-chain (L-chain), a clostridial neurotoxin translocation domain (HN domain) and/or a clostridial neurotoxin receptor binding domain (HC domain).
  • L-chain clostridial neurotoxin light-chain
  • HN domain clostridial neurotoxin translocation domain
  • HC domain clostridial neurotoxin receptor binding domain
  • the clostridial neurotoxin referred to herein may be a polypeptide that comprises (or consists of) a clostridial neurotoxin light-chain (L-chain), a clostridial neurotoxin translocation domain (HN domain) and/or a clostridial neurotoxin receptor binding domain (HC domain), wherein when the polypeptide comprises a clostridial neurotoxin L-chain, the L-chain is catalytically inactive.
  • L-chain clostridial neurotoxin light-chain
  • HN domain clostridial neurotoxin translocation domain
  • HC domain clostridial neurotoxin receptor binding domain
  • aspects of the invention provide any of the following: - a polypeptide for use in a method of suppressing visceral pain; - a method of suppressing visceral pain in a patient, the method comprising administering a polypeptide to the patient; - use of a polypeptide in the manufacture of a medicament to suppress visceral pain in a patient; wherein said peptide comprises (or consists of) a clostridial neurotoxin light-chain (L- chain), a clostridial neurotoxin translocation domain (H N domain) and/or a clostridial neurotoxin receptor binding domain (H C domain); optionally wherein when the polypeptide comprises a clostridial neurotoxin L-chain, the L-chain is catalytically inactive.
  • L- chain clostridial neurotoxin light-chain
  • H N domain clostridial neurotoxin translocation domain
  • H C domain clostridial neurotoxin receptor binding domain
  • a clostridial neurotoxin of the invention may be encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 60.
  • a clostridial neurotoxin of the invention may be encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 60.
  • a clostridial neurotoxin of the invention may be encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 60.
  • a clostridial neurotoxin of the invention may comprise a clostridial neurotoxin sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.
  • a clostridial neurotoxin of the invention may comprise a clostridial neurotoxin sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.
  • a clostridial neurotoxin of the invention may comprise a clostridial neurotoxin sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.
  • a clostridial neurotoxin of the invention may comprise a clostridial neurotoxin sequence having at least 70% sequence identity to any one of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, or 59 (preferably SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, or 59).
  • a clostridial neurotoxin of the invention may comprise a clostridial neurotoxin sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, or 59 (preferably SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, or 59).
  • a clostridial neurotoxin of the invention may comprise a clostridial neurotoxin sequence of any one of SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, or 59 (preferably SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, 58, or 59 (preferably SEQ ID NOs: 51, 52, 53, 54, 55, 56, 57, or 59).
  • a clostridial neurotoxin of the invention may comprise a clostridial neurotoxin sequence having at least 70% sequence identity to SEQ ID NO: 51.
  • a clostridial neurotoxin of the invention may comprise a clostridial neurotoxin sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 51.
  • a clostridial neurotoxin of the invention may comprise a clostridial neurotoxin sequence of SEQ ID NO: 51.
  • the term “clostridial neurotoxin” is also intended to embrace modified clostridial neurotoxins and derivatives thereof, including but not limited to those described below.
  • a modified clostridial neurotoxin or derivative may contain one or more amino acids that has been modified as compared to the native (unmodified) form of the clostridial neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial neurotoxin.
  • a modified clostridial neurotoxin may have modified amino acid sequences in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may modify functional aspects of the toxin, for example biological activity or persistence.
  • the clostridial neurotoxin of the invention is a modified clostridial neurotoxin, or a modified clostridial neurotoxin derivative, or a clostridial neurotoxin derivative.
  • a modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified HC domain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) clostridial neurotoxin.
  • modifications in the HC domain can include modifying residues in the ganglioside binding site of the HC domain or in the protein (SV2 or synaptotagmin) binding site that alter binding to the ganglioside receptor and/or the protein receptor of the target nerve cell. Examples of such modified clostridial neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are hereby incorporated by reference in their entirety.
  • a modified clostridial neurotoxin may have one or more modifications in the amino acid sequence of the light chain, for example modifications in the substrate binding or catalytic domain which may alter or modify the SNARE protein specificity of the modified L-chain. Examples of such modified clostridial neurotoxins are described in WO 2010/120766 and US 2011/0318385, both of which are hereby incorporated by reference in their entirety.
  • a modified clostridial neurotoxin may comprise one or more modifications that increases or decreases the biological activity and/or the biological persistence of the modified clostridial neurotoxin.
  • a modified clostridial neurotoxin may comprise a leucine- or tyrosine-based motif, wherein said motif increases or decreases the biological activity and/or the biological persistence of the modified clostridial neurotoxin.
  • Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL, and xExxxLM (wherein x is any amino acid) – e.g. SEQ ID Nos: 66-69.
  • Suitable tyrosine-based motifs include Y-x-x-Hy (wherein Hy is a hydrophobic amino acid) – e.g. SEQ ID NO: 70.
  • modified clostridial neurotoxins comprising leucine- and tyrosine-based motifs are described in WO 2002/08268, which is hereby incorporated by reference in its entirety.
  • a modified clostridial neurotoxin (or clostridial neurotoxin fragment) may be one that comprises one or more modifications that increases the isoelectric point of the clostridial neurotoxin when compared to an equivalent unmodified clostridial neurotoxin lacking said one or more modifications.
  • Suitable modified clostridial neurotoxins are described above and in WO 2015/004461 A1 and WO 2016/110662 A1, which are incorporated herein by reference. Exemplary sequences include SEQ ID NOs: 61 and 42 described herein.
  • a modified clostridial neurotoxin may preferably comprise or consist of a sequence of SEQ ID No: 61.
  • the term “clostridial neurotoxin” is intended to embrace hybrid and chimeric clostridial neurotoxins.
  • a hybrid clostridial neurotoxin comprises at least a portion of a light chain from one clostridial neurotoxin or subtype thereof, and at least a portion of a heavy chain from another clostridial neurotoxin or clostridial neurotoxin subtype.
  • the hybrid clostridial neurotoxin may contain the entire light chain of a light chain from one clostridial neurotoxin subtype and the heavy chain from another clostridial neurotoxin subtype.
  • a chimeric clostridial neurotoxin may contain a portion (e.g. the binding domain) of the heavy chain of one clostridial neurotoxin subtype, with another portion of the heavy chain being from another clostridial neurotoxin subtype.
  • the therapeutic element may comprise light chain portions from different clostridial neurotoxins.
  • hybrid or chimeric clostridial neurotoxins are useful, for example, as a means of delivering the therapeutic benefits of such clostridial neurotoxins to subjects who are immunologically resistant to a given clostridial neurotoxin subtype, to subjects who may have a lower than average concentration of receptors to a given clostridial neurotoxin heavy chain binding domain, or to subjects who may have a protease-resistant variant of the membrane or vesicle toxin substrate (e.g., SNAP-25, VAMP and syntaxin).
  • Hybrid and chimeric clostridial neurotoxins are described in US 8,071,110, which publication is hereby incorporated by reference in its entirety.
  • the clostridial neurotoxin (or fragment thereof) of the invention is a hybrid clostridial neurotoxin, or a chimeric clostridial neurotoxin.
  • a polypeptide of the invention may be a chimeric clostridial neurotoxin comprising (preferably consisting of) a BoNT/A light-chain and translocation domain, and a BoNT/B receptor binding domain (H C domain) or a portion thereof (e.g. BoNT/A LH N -BoNT/B H C ).
  • a suitable chimeric and/or hybrid clostridial neurotoxin may be one taught in WO 2017/191315 A1, which is incorporated herein by reference.
  • a chimeric clostridial neurotoxin may comprise (preferably consist of) a sequence of SEQ ID NO: 64 (which may be said to comprise a catalytically inactive L-chain).
  • a chimeric clostridial neurotoxin may comprise (preferably consist of) a sequence of SEQ ID NO: 63.
  • the BoNT/A LH N domain may be covalently linked to the BoNT/B HC domain. Said chimeric BoNT/A is also referred to herein as “BoNT/AB” or a “BoNT/AB chimera”.
  • the C-terminal amino acid residue of the LH N domain may correspond to the first amino acid residue of the 310 helix separating the LH N and HC domains of BoNT/A
  • the N-terminal amino acid residue of the HC domain may correspond to the second amino acid residue of the 310 helix separating the LH N and HC domains in BoNT/B.
  • Reference herein to the “first amino acid residue of the 310 helix separating the LH N and HC domains of BoNT/A” means the N-terminal residue of the 310 helix separating the LH N and HC domains.
  • references herein to the “second amino acid residue of the 310 helix separating the LH N and H C domains of BoNT/B” means the amino acid residue following the N-terminal residue of the 3 10 helix separating the LH N and H C domains.
  • a “3 10 helix” is a type of secondary structure found in proteins and polypeptides, along with ⁇ - helices, ⁇ -sheets and reverse turns. The amino acids in a 3 10 helix are arranged in a right- handed helical structure where each full turn is completed by three residues and ten atoms that separate the intramolecular hydrogen bond between them.
  • a 3 10 helix is a standard concept in structural biology with which the skilled person is familiar. This 3 10 helix corresponds to four residues which form the actual helix and two cap (or transitional) residues, one at each end of these four residues.
  • 3 10 helix separating the LH N and HC domains consists of those 6 residues.
  • a 310 helix separating the LH N and HC domains was identified. This 310 helix is surrounded by an ⁇ -helix at its N- terminus (i.e. at the C-terminal part of the LH N domain) and by a ⁇ -strand at its C-terminus (i.e. at the N-terminal part of the HC domain).
  • the first (N-terminal) residue (cap or transitional residue) of the 310 helix also corresponds to the C-terminal residue of this ⁇ -helix.
  • In silico modelling and alignment tools which are publicly available can also be used to determine the location of the 310 helix separating the LH N and HC domains in other neurotoxins, for example the homology modelling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http://loopp.org), PHYRE (Protein Homology/analogY Recognition Engine, http://www.sbg.bio.ic.ac.uk/phyre2/) and Rosetta (https://www.rosettacommons.org/), the protein superposition server SuperPose (http://wishart.biology.ualberta.ca/superpose/), the alignment program Clustal Omega (http://www.clustal.org/omega/), and a number of other tools/services listed at the Internet Resources for Molecular and Cell Biologists (http://molbiol-tools.ca/).
  • LOOPP Learning, Observing and Outputting Protein Patterns
  • PHYRE Protein Homology/analogY Recognition Engine,
  • the region around the “H N /H CN ” junction is structurally highly conserved which renders it an ideal region to superimpose different serotypes.
  • the following methodology may be used to determine the sequence of this 3 10 helix in other neurotoxins: 1.
  • the structural homology modelling tool LOOP http://loopp.org
  • the structural (pdb) files thus obtained were edited to include only the N-terminal end of the H CN domain and about 80 residues before it (which are part of the H N domain), thereby retaining the “H N /H CN ” region which is structurally highly conserved; 3.
  • the protein superposition server SuperPose http://wishart.biology.ualberta.ca/superpose/ was used to superpose each serotype onto the 3BTA.pdb structure; 4.
  • the superposed pdb files were inspected to locate the 310 helix at the start of the HC domain of BoNT/A1, and corresponding residues in the other serotype were then identified; 5.
  • the other BoNT serotype sequences were aligned with Clustal Omega in order to check that corresponding residues were correct.
  • LH N , HC and 310 helix domains determined by this method are presented below: Using structural analysis and sequence alignments, it was found that the ⁇ -strand following the 3 10 helix separating the LH N and HC domains is a conserved structure in all botulinum and tetanus neurotoxins and starts at the 8 th residue when starting from the first residue of the 310 helix separating the LH N and HC domains (e.g., at residue 879 for BoNT/A1).
  • a BoNT/AB chimera may comprise an LH N domain from BoNT/A covalently linked to a HC domain from BoNT/B, - wherein the C-terminal amino acid residue of the LH N domain corresponds to the eighth amino acid residue N-terminally to the ⁇ -strand located at the beginning (N- term) of the H C domain of BoNT/A, and - wherein the N-terminal amino acid residue of the HC domain corresponds to the seventh amino acid residue N-terminally to the ⁇ -strand located at the beginning (N- term) of the HC domain of BoNT/B.
  • a BoNT/AB chimera may comprise an LH N domain from BoNT/A covalently linked to a HC domain from BoNT/B, - wherein the C-terminal amino acid residue of the LH N domain corresponds to the C- terminal amino acid residue of the ⁇ -helix located at the end (C-term) of LH N domain of BoNT/A, and - wherein the N-terminal amino acid residue of the H C domain corresponds to the amino acid residue immediately C-terminal to the C-terminal amino acid residue of the ⁇ -helix located at the end (C-term) of LH N domain of BoNT/B.
  • the rationale of the design process of the BoNT/AB chimera was to try to ensure that the secondary structure was not compromised and thereby minimise any changes to the tertiary structure and to the function of each domain. Without wishing to be bound by theory, it is hypothesized that by not disrupting the four central amino acid residues of the 310 helix in the BoNT/AB chimera ensures an optimal conformation for the chimeric neurotoxin, thereby allowing for the chimeric neurotoxin to exert its functions to their full capacity.
  • the LH N domain from BoNT/A may correspond to amino acid residues 1 to 872 of SEQ ID NO: 62, or a polypeptide sequence having at least 70% sequence identity thereto.
  • the LH N domain from BoNT/A may correspond to amino acid residues 1 to 872 of SEQ ID NO: 62, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto.
  • the LH N domain from BoNT/A corresponds to amino acid residues 1 to 872 of SEQ ID NO: 62.
  • the HC domain from BoNT/B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52, or a polypeptide sequence having at least 70% sequence identity thereto.
  • the HC domain from BoNT/B may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 52, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto.
  • the HC domain from BoNT/B corresponds to amino acid residues 860 to 1291 of SEQ ID NO: 52.
  • the BoNT/AB chimera comprises a BoNT/A LH N domain and a BoNT/B HC domain. More preferably, the LH N domain corresponds to amino acid residues 1 to 872 of BoNT/A (SEQ ID NO: 62) and the HC domain corresponds to amino acid residues 860 to 1291 of BoNT/B (SEQ ID NO: 52).
  • a BoNT/B HC domain further comprises at least one amino acid residue substitution, addition or deletion in the HCC subdomain which has the effect of increasing the binding affinity of BoNT/B neurotoxin for human Syt II as compared to the natural BoNT/B sequence.
  • Suitable amino acid residue substitution, addition or deletion in the BoNT/B HCC subdomain have been disclosed in WO 2013/180799 and in WO 2016/154534 (both herein incorporated by reference).
  • Suitable amino acid residue substitution, addition or deletion in the BoNT/B H CC subdomain include substitution mutations selected from the group consisting of: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.
  • Suitable amino acid residue substitution, addition or deletion in the BoNT/B H CC subdomain further include combinations of two substitution mutations selected from the group consisting of: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.
  • Suitable amino acid residue substitution, addition or deletion in the BoNT/B H CC subdomain also include a combination of three substitution mutations which are E1191M, S1199W and W1178Q.
  • the suitable amino acid residue substitution, addition or deletion in the BoNT/B HCC subdomain includes a combination of two substitution mutations which are E1191M and S1199Y.
  • the modification may be a modification when compared to unmodified BoNT/B shown as SEQ ID NO: 52, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 52. As the presence of a methionine residue at position 1 of SEQ ID NO: 52 is optional, the skilled person will take the presence/absence of the methionine residue into account when determining amino acid residue numbering.
  • SEQ ID NO: 52 includes a methionine
  • the position numbering will be as defined above (e.g. E1191 will be E1191 of SEQ ID NO: 52).
  • the methionine is absent from SEQ ID NO: 52 the amino acid residue numbering should be modified by -1 (e.g. E1191 will be E1190 of SEQ ID NO: 52).
  • E1191 will be E1190 of SEQ ID NO: 52.
  • the invention provides a clostridial neurotoxin for use in treating a brain disorder by promoting a neuroimmune response in a subject, wherein the clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64 (preferably wherein the clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63).
  • a method for suppressing visceral pain in a subject comprising administering a clostridial neurotoxin to the subject, wherein the clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64 (preferably wherein the clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63).
  • a method for suppressing visceral pain in a subject comprising administering a clostridial neurotoxin to the subject, wherein the clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 or 64 (preferably wherein the clostridial neurotoxin comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63).
  • a clostridial neurotoxin for use according to the invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 63 or 64.
  • a clostridial neurotoxin for use according to the invention comprises (more preferably consists of) a polypeptide sequence shown as SEQ ID NO: 63 or 64.
  • a clostridial neurotoxin for use according to the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 64.
  • a clostridial neurotoxin may comprises (more preferably consist of) a polypeptide sequence shown as SEQ ID NO: 64.
  • a clostridial neurotoxin for use according to the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 64.
  • a particularly suitable clostridial neurotoxin may comprises (more preferably consist of) a polypeptide sequence shown as SEQ ID NO: 63.
  • the clostridial neurotoxin comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 63 comprises a catalytically-inactive L-chain, such as SEQ ID NO: 64.
  • a chimeric and/or hybrid clostridial neurotoxin for use in the present invention may comprise a portion of a BoNT/A polypeptide and a portion of a BoNT/B polypeptide, an example of which includes the polypeptide described herein as SEQ ID NO: 44.
  • Suitable chimeric clostridial neurotoxins may include BoNT/FA.
  • a clostridial neurotoxin (e.g. chimeric clostridial neurotoxin) of the invention may comprise BoNT/FA or a fragment thereof.
  • Catalytically inactive forms of BoNT/FA are described herein as SEQ ID NOs: 26 and 34.
  • Suitable fragments of BoNT/FA are also described herein as SEQ ID NOs: 28, 30, and 32.
  • clostridial neurotoxin may also embrace newly discovered botulinum neurotoxin protein family members expressed by non-clostridial microorganisms, such as the Enterococcus encoded toxin which has closest sequence identity to BoNT/X, the Weissella oryzae encoded toxin called BoNT/Wo (NCBI Ref Seq: WP_027699549.1), which cleaves VAMP2 at W89-W90, the Enterococcus faecium encoded toxin (GenBank: OTO22244.1), which cleaves VAMP2 and SNAP25, and the Chryseobacterium pipero encoded toxin (NCBI Ref.Seq: WP_034687872.1).
  • non-clostridial microorganisms such as the Enterococcus encoded toxin which has closest sequence identity to BoNT/X, the Weissella oryzae encoded toxin called BoNT/Wo (NCBI Ref Se
  • the clostridial neurotoxin of the present invention may lack a functional HC domain of a clostridial neurotoxin and also lack any functionally equivalent exogenous ligand Targeting Moiety (TM).
  • the clostridial neurotoxin of the invention may be a re-targeted clostridial neurotoxin.
  • a clostridial neurotoxin of the invention is not a re-targeted clostridial neurotoxin.
  • the clostridial neurotoxin is modified to include an exogenous ligand known as a Targeting Moiety (TM).
  • the TM is selected to provide binding specificity for a desired target cell, and as part of the re-targeting process the native binding portion of the clostridial neurotoxin (e.g. the HC domain, or the HCC domain) may be removed.
  • Re-targeting technology is described, for example, in: EP-B-0689459; WO 1994/021300; EP-B-0939818; US 6,461,617; US 7,192,596; WO 1998/007864; EP-B- 0826051; US 5,989,545; US 6,395,513; US 6,962,703; WO 1996/033273; EP-B-0996468; US 7,052,702; WO 1999/017806; EP-B-1107794; US 6,632,440; WO 2000/010598; WO 2001/21213; WO 2006/059093; WO 2000/62814; WO 2000/04926; WO 1993/15766; WO 2000/61192; and
  • clostridial neurotoxins are formed from two polypeptide chains, 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.
  • the H- chain comprises a C-terminal targeting component (receptor binding domain or H C domain) and an N-terminal translocation component (H N domain).
  • a clostridial neurotoxin of the invention comprises a fragment of a BoNT/A or a fragment of a BoNT/F.
  • the clostridial neurotoxin of the invention comprises a catalytically inactive L-chain of BoNT/A or BoNT/F.
  • the clostridial neurotoxin of the invention may comprise a catalytically inactive L-chain of BoNT/A.
  • a clostridial neurotoxin described herein has a tag for purification (e.g. a His-tag) and/or a linker, said tag and/or linker are optional. Suitable full-length clostridial neurotoxins are described herein.
  • a clostridial neurotoxin of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65, preferably with the proviso that a clostridial neurotoxin L-chain of said clostridial neurotoxin is catalytically inactive.
  • a clostridial neurotoxin of the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65, preferably with the proviso that a clostridial neurotoxin L-chain of said clostridial neurotoxin is catalytically inactive.
  • a clostridial neurotoxin of the invention may comprise a polypeptide sequence comprising any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65, preferably with the proviso that a clostridial neurotoxin L-chain of said clostridial neurotoxin is catalytically inactive.
  • a clostridial neurotoxin of the invention may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, 33, or 60, preferably with the proviso that the clostridial neurotoxin L-chain of said clostridial neurotoxin is catalytically inactive.
  • a clostridial neurotoxin of the invention is one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, 33, or 60, preferably with the proviso that the clostridial neurotoxin L-chain of said clostridial neurotoxin is catalytically inactive.
  • a clostridial neurotoxin of the invention is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 1, 9, 11, 13, 15, 17, 25, 33, or 60, preferably with the proviso that the clostridial neurotoxin L-chain of said clostridial neurotoxin is catalytically inactive.
  • a clostridial neurotoxin of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65, preferably with the proviso that the clostridial neurotoxin L-chain of said clostridial neurotoxin is catalytically inactive.
  • a clostridial neurotoxin of the invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65, preferably with the proviso that the clostridial neurotoxin L-chain of said clostridial neurotoxin is catalytically inactive.
  • a clostridial neurotoxin of the invention comprises any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 64 or 65, preferably with the proviso that the clostridial neurotoxin L-chain of said clostridial neurotoxin is catalytically inactive.
  • a clostridial neurotoxin of the invention is a full-length clostridial neurotoxin selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, and TeNT.
  • a clostridial neurotoxin of the invention is a full-length clostridial neurotoxin selected from BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X, and TeNT.
  • a clostridial neurotoxin of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 51-59, 61 or 63. In one embodiment a clostridial neurotoxin of the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 51-59, 61 or 63. In one embodiment a clostridial neurotoxin of the invention may comprise a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 51-59, 61 or 63.
  • a clostridial neurotoxin of the invention may comprise (more preferably consist of) a polypeptide sequence comprising any one of SEQ ID NOs: 51-59, 61 or 63.
  • a clostridial neurotoxin of the invention may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 52-59, 61 or 63.
  • a clostridial neurotoxin of the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 52-59, 61 or 63.
  • a clostridial neurotoxin of the invention may comprise a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 52-59, 61 or 63.
  • a clostridial neurotoxin of the invention may comprise (more preferably consist of) a polypeptide sequence comprising any one of SEQ ID NOs: 52-59, 61 or 63.
  • a clostridial neurotoxin of the invention is not a full-length catalytically active clostridial neurotoxin, e.g. is not full-length catalytically active BoNT/A.
  • the clostridial neurotoxin of the present invention may comprise (or consist of) a fragment of a clostridial neurotoxin, e.g. a fragment of any full-length clostridial neurotoxin described herein.
  • a clostridial neurotoxin of the invention may comprise a fragment of a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.
  • a clostridial neurotoxin of the invention may comprise a fragment of a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.
  • a clostridial neurotoxin of the invention may comprise a fragment of a polypeptide sequence comprising any one of SEQ ID NOs: 2, 10, 12, 14, 16, 18, 26, 34, 51, 52, 53, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64 or 65.
  • a clostridial neurotoxin of the invention comprises (or consists of) a clostridial neurotoxin L-chain or fragment thereof.
  • a fragment of a clostridial neurotoxin L- chain may have ⁇ 400, ⁇ 350, ⁇ 300, ⁇ 250, ⁇ 200, ⁇ 150, ⁇ 100 or ⁇ 50 amino acid residues of a clostridial neurotoxin L-chain.
  • a fragment of a clostridial neurotoxin L- chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of a clostridial neurotoxin L-chain.
  • a fragment of a clostridial neurotoxin L-chain may have 20-400, 50-300 or 100-200 amino acid residues of a clostridial neurotoxin L-chain.
  • L-chain reference sequences include: Botulinum type A neurotoxin: amino acid residues 1-448 Botulinum type B neurotoxin: amino acid residues 1-440 Botulinum type C1 neurotoxin: amino acid residues 1-441 Botulinum type D neurotoxin: amino acid residues 1-445 Botulinum type E neurotoxin: amino acid residues 1-422 Botulinum type F neurotoxin: amino acid residues 1-439 Botulinum type G neurotoxin: amino acid residues 1-441 Tetanus neurotoxin: amino acid residues 1-457 For recently-identified BoNT/X, the L-chain has been reported as corresponding to amino acids 1-439 thereof, with the L-chain boundary potentially varying by approximately 25 amino acids (e.g.1-414
  • Botulinum type A neurotoxin amino acid residues M1-K448
  • Botulinum type B neurotoxin amino acid residues M1-K441
  • Botulinum type C1 neurotoxin amino acid residues M1-K449
  • Botulinum type D neurotoxin amino acid residues M1-R445
  • Botulinum type E neurotoxin amino acid residues M1-R422
  • Botulinum type F neurotoxin amino acid residues M1-K439
  • Botulinum type G neurotoxin amino acid residues M1-K446 Tetanus neurotoxin: amino acid residues M1-A457 Suitable clostridial neurotoxin L-chains are described herein.
  • a clostridial neurotoxin L-chain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 6, 24, 32 or 40 or a fragment thereof.
  • a clostridial neurotoxin L-chain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 6, 24, 32 or 40 or a fragment thereof.
  • a clostridial neurotoxin L-chain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 6, 24, 32 or 40 or a fragment thereof.
  • a clostridial neurotoxin L-chain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 5, 23, 31 or 39 or a fragment thereof.
  • a clostridial neurotoxin L-chain is one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 5, 23, 31 or 39 or a fragment thereof.
  • a clostridial neurotoxin L-chain is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 5, 23, 31 or 39 or a fragment thereof.
  • a clostridial neurotoxin of the invention comprises (or consists of) a fragment of a clostridial neurotoxin H-chain.
  • a fragment of a clostridial neurotoxin H-chain may have ⁇ 800, ⁇ 700, ⁇ 600, ⁇ 500, ⁇ 400, ⁇ 350, ⁇ 300, ⁇ 250, ⁇ 200, ⁇ 150, ⁇ 100 or ⁇ 50 amino acid residues of a clostridial neurotoxin H-chain.
  • a fragment of a clostridial neurotoxin H-chain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of a clostridial neurotoxin H-chain.
  • a fragment of a clostridial neurotoxin H-chain may have 20-800, 30-600, 40-400, 50-300 or 100-200 amino acid residues of a clostridial neurotoxin H-chain.
  • a clostridial neurotoxin H-chain comprises two structural/functional domains: the translocation domain (HN) and receptor binding domain (HC).
  • a clostridial neurotoxin of the invention comprises (or consists of) a clostridial neurotoxin translocation domain or a fragment thereof.
  • a fragment of a clostridial neurotoxin translocation domain may have ⁇ 400, ⁇ 350, ⁇ 300, ⁇ 250, ⁇ 200, ⁇ 150, ⁇ 100 or ⁇ 50 amino acid residues of a clostridial neurotoxin translocation domain.
  • a fragment of a clostridial neurotoxin translocation domain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of a clostridial neurotoxin translocation domain.
  • a fragment of a clostridial neurotoxin translocation domain may have 20-400, 50-300 or 100-200 amino acid residues of a clostridial neurotoxin translocation domain.
  • the translocation domain is a 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 HC function of the H-chain may be removed by deletion of the HC amino acid sequence (either at the DNA synthesis level, or at the post-synthesis level by nuclease or protease treatment). Alternatively, the HC function may be inactivated by chemical or biological treatment.
  • the H-chain may be incapable of binding to the Binding Site on a target cell to which native clostridial neurotoxin (i.e. holotoxin) binds.
  • suitable (reference) Translocation Domains include: Botulinum type A neurotoxin - amino acid residues (449-871) 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) Tetanus neurotoxin - amino acid residues (458-879)
  • the above-identified reference sequence should be considered a guide as slight variations may occur according to sub-ser
  • these active fragments can facilitate the release of a non-cytotoxic protease (e.g. a clostridial L-chain) from intracellular vesicles into the cytoplasm of the target cell and thus participate in executing the overall cellular mechanism whereby a clostridial neurotoxin proteolytically cleaves a substrate.
  • a non-cytotoxic protease e.g. a clostridial L-chain
  • the HN regions from the heavy chains of clostridial neurotoxins are approximately 410-430 amino acids in length and comprise a translocation domain. Research has shown that the entire length of a HN region from a clostridial neurotoxin heavy chain is not necessary for the translocating activity of the translocation domain.
  • aspects of this embodiment can include clostridial neurotoxin HN regions comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids and at least 425 amino acids.
  • Other aspects of this embodiment can include clostridial neurotoxin H N regions comprising a translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids and at most 425 amino acids.
  • 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. In one embodiment said modified H N portions still demonstrate the above- mentioned translocation function.
  • a clostridial neurotoxin of the invention comprises (or consists of) a clostridial neurotoxin receptor binding domain (H C ) or a fragment thereof.
  • a fragment of a clostridial neurotoxin receptor binding domain (H C ) may have ⁇ 350, ⁇ 300, ⁇ 250, ⁇ 200, ⁇ 150, ⁇ 100 or ⁇ 50 amino acid residues of a clostridial neurotoxin receptor binding domain (HC).
  • a fragment of a clostridial neurotoxin receptor binding domain has at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or 200 amino acid residues of a clostridial neurotoxin receptor binding domain (HC).
  • a fragment of a clostridial neurotoxin receptor binding domain may have 20-350, 50-300 or 100-200 amino acid residues of a clostridial neurotoxin receptor binding domain (HC).
  • HC clostridial neurotoxin receptor binding domain
  • BoNT/X the HC domain has been reported as corresponding to amino acids 893-1306 thereof, with the domain boundary potentially varying by approximately 25 amino acids (e.g.868-1306 or 918-1306).
  • a clostridial neurotoxin H-chain may further comprise a translocation facilitating domain.
  • Said domain facilitates delivery of the L-chain into the cytosol of the target cell and are described, for example, in WO 08/008803 and WO 08/008805, each of which is herein incorporated by reference thereto.
  • a translocation facilitating domain may comprise a clostridial neurotoxin H CN domain or a fragment or variant thereof.
  • a clostridial neurotoxin H CN translocation facilitating domain may have a length of at least 200 amino acids, at least 225 amino acids, at least 250 amino acids, at least 275 amino acids.
  • a clostridial neurotoxin H CN translocation facilitating domain preferably has a length of at most 200 amino acids, at most 225 amino acids, at most 250 amino acids, or at most 275 amino acids.
  • Specific (reference) examples include: Botulinum type A neurotoxin - amino acid residues (872-1110) Botulinum type B neurotoxin - amino acid residues (859-1097) Botulinum type C neurotoxin - amino acid residues (867-1111) Botulinum type D neurotoxin - amino acid residues (863-1098) Botulinum type E neurotoxin - amino acid residues (846-1085) Botulinum type F neurotoxin - amino acid residues (865-1105) Botulinum type G neurotoxin - amino acid residues (864-1105) Tetanus neurotoxin - amino acid residues (880-1127) The above sequence positions may vary a little according to serotype/ sub-type, and further examples of suitable (
  • a clostridial neurotoxin H C domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48 or 50 or a fragment thereof.
  • a clostridial neurotoxin H C domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48 or 50 or a fragment thereof.
  • a clostridial neurotoxin H C domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 8, 22, 30, 38, 42, 44, 46, 48 or 50 or a fragment thereof.
  • a clostridial neurotoxin H C domain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47 or 49 or a fragment thereof.
  • a clostridial neurotoxin H C domain is one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47 or 49 or a fragment thereof.
  • a clostridial neurotoxin H C domain is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 7, 21, 29, 37, 41, 43, 45, 47 or 49 or a fragment thereof.
  • a clostridial neurotoxin HC domain for use in the invention is a variant BoNT/A HC domain.
  • Said variant BoNT/A HC domain may comprise a modification of one or more amino acids residues selected from Y1117, F1252, H1253, and L1278.
  • a variant BoNT/A HC domain may comprise one or more (preferably two or more) of the following modifications Y1117V, F1252Y, H1253K, and L1278F or L1278H.
  • a variant BoNT/A HC domain comprises the following modifications: Y1117V and H1253K; or Y1117V, F1252Y, H1253K, and L1278F; or Y1117V, F1252Y, H1253K, and L1278H.
  • a variant BoNT/A HC domain comprises the following modifications: Y1117V and H1253K; or Y1117V, F1252Y, H1253K, and L1278H.
  • the modification may be a modification when compared to unmodified BoNT/A shown as SEQ ID NO: 62, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 62.
  • SEQ ID NO: 62 includes a methionine
  • the position numbering will be as defined above (e.g. Y1117 will align against Y1117 of SEQ ID NO: 62).
  • the amino acid residue numbering should be modified by -1 (e.g. Y1117 will align against Y1116 of SEQ ID NO: 52).
  • a variant BoNT/A H C domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 46, 48 or 50 or a fragment thereof with the proviso that the variant BoNT/A H C domain comprises a modification as described above.
  • a variant BoNT/A H C domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 46, 48 or 50 or a fragment thereof with the proviso that the variant BoNT/A H C domain comprises a modification as described above.
  • a variant BoNT/A H C domain comprises a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 46, 48 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above.
  • a variant BoNT/A HC domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 46, 48 or 50 or a fragment thereof.
  • a variant BoNT/A HC domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 46 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above.
  • a variant BoNT/A HC domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 46 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above.
  • a variant BoNT/A HC domain comprises a polypeptide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 46 or 50 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above.
  • a variant BoNT/A HC domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 46 or 50 or a fragment thereof.
  • a variant BoNT/A HC domain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 45, 47 or 49 or a fragment thereof with the proviso that the variant BoNT/A H C domain comprises a modification as described above.
  • a variant BoNT/A H C domain be one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 45, 47 or 49 or a fragment thereof with the proviso that the variant BoNT/A H C domain comprises a modification as described above.
  • a variant BoNT/A H C domain be one encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 45, 47 or 49 or a fragment thereof with the proviso that the variant BoNT/A H C domain comprises a modification as described above.
  • a variant BoNT/A H C domain be one encoded by any one of SEQ ID NOs: 45, 47 or 49 or a fragment thereof.
  • a variant BoNT/A H C domain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 45 or 49 or a fragment thereof with the proviso that the variant BoNT/A H C domain comprises a modification as described above.
  • a variant BoNT/A H C domain be one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 45 or 49 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above.
  • a variant BoNT/A HC domain be one encoded by a nucleotide sequence having at least 99% or 99.9% sequence identity to any one of SEQ ID NOs: 45 or 49 or a fragment thereof with the proviso that the variant BoNT/A HC domain comprises a modification as described above.
  • a variant BoNT/A HC domain be one encoded by any one of SEQ ID NOs: 45 or 49 or a fragment thereof.
  • Any of the above-described facilitating domains may be combined with any of the previously described translocation domain peptides that are suitable for use in the present invention.
  • a non-clostridial facilitating domain may be combined with non- clostridial translocation domain peptide or with clostridial translocation domain peptide.
  • a clostridial neurotoxin HCN translocation facilitating domain may be combined with a non-clostridial translocation domain peptide.
  • a clostridial neurotoxin HCN facilitating domain may be combined with a clostridial translocation domain peptide, examples of which include: Botulinum type A neurotoxin - amino acid residues (449-1110) Botulinum type B neurotoxin - amino acid residues (442-1097) Botulinum type C neurotoxin - amino acid residues (450-1111) Botulinum type D neurotoxin - amino acid residues (446-1098) Botulinum type E neurotoxin - amino acid residues (423-1085) Botulinum type F neurotoxin - amino acid residues (440-1105) Botulinum type G neurotoxin - amino acid residues (447-1105) Tetanus neurotoxin - amino acid residues (458-1127) In some embodiments the clostridial neurotoxins of the present invention may lack a functional H C domain of a clostridial neurotoxin.
  • the clostridial neurotoxins preferably lack the last 50 C-terminal amino acids of a clostridial neurotoxin holotoxin. In another embodiment, the clostridial neurotoxins preferably lack the last 100, preferably the last 150, more preferably the last 200, particularly preferably the last 250, and most preferably the last 300 C-terminal amino acid residues of a clostridial neurotoxin holotoxin.
  • the H C binding activity may be negated/ reduced by mutagenesis – by way of example, referring to BoNT/A for convenience, modification of one or two amino acid residue mutations (W1266 to L and Y1267 to F) in the ganglioside binding pocket causes the H C region to lose its receptor binding function.
  • Analogous mutations may be made to non-serotype A clostridial peptide components, e.g. a construct based on botulinum B with mutations (W1262 to L and Y1263 to F) or botulinum E (W1224 to L and Y1225 to F).
  • Other mutations to the active site achieve the same ablation of HC receptor binding activity, e.g.
  • the HC peptide of a native clostridial neurotoxin comprises approximately 400-440 amino acid residues, and consists of two functionally distinct domains of approximately 25kDa each, namely the N-terminal region (commonly referred to as the HCN peptide or domain) and the C-terminal region (commonly referred to as the HCC peptide or domain).
  • HCC C-terminal region
  • the H CC peptide region may be either partially or wholly deleted, or otherwise modified (e.g. through conventional chemical or proteolytic treatment) to reduce its native binding ability for nerve terminals at the neuromuscular junction.
  • a clostridial neurotoxin H N peptide of the present invention lacks part of a C-terminal peptide portion (H CC ) of a clostridial neurotoxin and thus lacks the H C binding function of native clostridial neurotoxin.
  • the C-terminally extended clostridial H N peptide lacks the C-terminal 40 amino acid residues, or the C-terminal 60 amino acid residues, or the C-terminal 80 amino acid residues, or the C- terminal 100 amino acid residues, or the C-terminal 120 amino acid residues, or the C- terminal 140 amino acid residues, or the C-terminal 150 amino acid residues, or the C- terminal 160 amino acid residues of a clostridial neurotoxin heavy-chain.
  • the clostridial HN peptide of the present invention lacks the entire C-terminal peptide portion (HCC) of a clostridial neurotoxin and thus lacks the HC binding function of native clostridial neurotoxin.
  • HCC C-terminal peptide portion
  • the clostridial HN peptide lacks the C-terminal 165 amino acid residues, or the C-terminal 170 amino acid residues, or the C-terminal 175 amino acid residues, or the C-terminal 180 amino acid residues, or the C-terminal 185 amino acid residues, or the C-terminal 190 amino acid residues, or the C-terminal 195 amino acid residues of a clostridial neurotoxin heavy-chain.
  • the clostridial HN peptide of the present invention lacks a clostridial HCC reference sequence selected from the group consisting of: Botulinum type A neurotoxin - amino acid residues (Y1111-L1296) Botulinum type B neurotoxin - amino acid residues (Y1098-E1291) Botulinum type C neurotoxin - amino acid residues (Y1112-E1291) Botulinum type D neurotoxin - amino acid residues (Y1099-E1276) Botulinum type E neurotoxin - amino acid residues (Y1086-K1252) Botulinum type F neurotoxin - amino acid residues (Y1106-E1274) Botulinum type G neurotoxin - amino acid residues (Y1106-E1297) Tetanus neurotoxin - amino acid residues (Y1128-D1315).
  • Botulinum type A neurotoxin - amino acid residues Y1111-L1296
  • a clostridial neurotoxin of the invention comprises (or consists of) a clostridial neurotoxin L-chain or fragment thereof and a fragment of a clostridial neurotoxin H-chain.
  • a clostridial neurotoxin may comprise (or consist of) a clostridial neurotoxin L-chain or fragment thereof and a clostridial neurotoxin translocation domain (H N ).
  • the clostridial neurotoxin does not further comprise a clostridial neurotoxin receptor binding domain (H C ) or at least the C-terminal portion of a clostridial neurotoxin receptor binding domain (H CC ).
  • H C clostridial neurotoxin receptor binding domain
  • H CC clostridial neurotoxin receptor binding domain
  • such clostridial neurotoxins lack the endogenous clostridial neurotoxin receptor binding capabilities and thus exhibit fewer off-target effects in a subject administered said clostridial neurotoxin.
  • a clostridial neurotoxin of the invention consists essentially of a clostridial neurotoxin L-chain or fragment thereof and/or a fragment of a clostridial neurotoxin H-chain.
  • the term “consists essentially of” as used in this context means that the clostridial neurotoxin does not further comprise one or more amino acid residues that confer additional functionality to the clostridial neurotoxin, e.g. when administered to a subject.
  • a clostridial neurotoxin that “consists essentially of” a clostridial neurotoxin L-chain or fragment thereof and/or a fragment of a clostridial neurotoxin H-chain may further comprise one or more amino acid residues (to those of the clostridial neurotoxin L-chain or fragment thereof and/or fragment of a clostridial neurotoxin H-chain) but said one or more further amino acid residues do not confer additional functionality to the clostridial neurotoxin, e.g. when administered to a subject. Additional functionality may include enzymatic activity, binding activity and/or any physiological activity whatsoever.
  • a clostridial neurotoxin may comprise non-clostridial neurotoxin sequences in addition to any clostridial neurotoxin sequences.
  • the non-clostridial neurotoxin sequences preferably do not disrupt the ability of a clostridial neurotoxin of the invention to promote a neuroimmune response.
  • the non-clostridial neurotoxin sequence is not one having catalytic activity, e.g. enzymatic activity.
  • the non-clostridial sequence is not one that binds to a cellular receptor. In other words, it is most preferred that the non-clostridial sequence is not a ligand for a cellular receptor.
  • a cellular receptor may be a proteinaceous cellular receptor, such as an integral membrane protein. Examples of cellular receptors can be found in the IUPHAR Guide to Pharmacology Database, version 2019.4, available at https://www.guidetopharmacology.org/download.jsp#db_reports.
  • Non- clostridial neurotoxin sequences may include tags to aid in purification, such as His-tags. It is preferred that any clostridial neurotoxin sequences comprised in said clostridial neurotoxin consist of a clostridial neurotoxin L-chain or fragment thereof and/or a fragment of a clostridial neurotoxin H-chain.
  • the clostridial neurotoxin sequence comprised in said clostridial neurotoxin may consist of a clostridial neurotoxin L-chain. In one embodiment, the clostridial neurotoxin sequence comprised in said clostridial neurotoxin may consist of a clostridial neurotoxin translocation domain. In one embodiment, the clostridial neurotoxin sequence comprised in said clostridial neurotoxin may consist of a clostridial neurotoxin receptor binding domain.
  • the clostridial neurotoxin sequence comprised in said clostridial neurotoxin may consist of a clostridial neurotoxin L- chain and a clostridial neurotoxin translocation domain.
  • Suitable clostridial neurotoxins comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain are described herein.
  • a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain may comprise a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4, 20, 28 or 36 or a fragment thereof.
  • a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 4, 20, 28 or 36 or a fragment thereof.
  • a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain comprises (more preferably consists of) a polypeptide sequence comprising any one of SEQ ID NOs: 4, 20, 28 or 36 or a fragment thereof.
  • a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain may be one encoded by a nucleotide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 3, 19, 27 or 35 or a fragment thereof.
  • a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L- chain and translocation domain is one encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to any one of SEQ ID NOs: 3, 19, 27 or 35 or a fragment thereof.
  • a clostridial neurotoxin comprising (or consisting of) a clostridial neurotoxin L-chain and translocation domain is one encoded by a nucleotide sequence comprising any one of SEQ ID NOs: 3, 19, 27 or 35 or a fragment thereof.
  • the clostridial neurotoxin of the present invention may be free from the complexing proteins that are present in a naturally occurring clostridial neurotoxin complex.
  • the clostridial neurotoxins of the present invention can be produced using recombinant nucleic acid technologies.
  • a clostridial neurotoxin (as described above) is a recombinant clostridial neurotoxin.
  • a clostridial neurotoxin of the invention comprises a clostridial neurotoxin L-chain. It is preferred that the L-chain is catalytically inactive. Active clostridial neurotoxin L-chain has non-cytotoxic protease activity. Specifically, active clostridial neurotoxin L-chain has endopeptidase activity and is capable of cleaving a protein of the exocytic fusion apparatus in a target cell.
  • a protein of the exocytic fusion apparatus is preferably a SNARE protein, such as SNAP-25, synaptobrevin/VAMP, or syntaxin.
  • catalytically inactive as used herein in respect of a clostridial neurotoxin L-chain means that said L-chain exhibits substantially no non-cytotoxic protease activity, preferably the term “catalytically inactive” as used herein in respect of a clostridial neurotoxin L-chain means that said L-chain exhibits no non-cytotoxic protease activity.
  • a catalytically inactive clostridial neurotoxin L-chain is one that does not cleave a protein of the exocytic fusion apparatus in a target cell.
  • substantially no non-cytotoxic protease activity means that the clostridial neurotoxin L-chain has less than 5% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L-chain (preferably an L-chain of native BoNT/A shown as SEQ ID NO: 6), for example less than 2%, 1% or preferably less than 0.1% of the non-cytotoxic protease activity of a catalytically active clostridial neurotoxin L-chain.
  • Non-cytotoxic protease activity can be determined in vitro by incubating a test clostridial neurotoxin L-chain with a SNARE protein and comparing the amount of SNARE protein cleaved by the test clostridial neurotoxin L-chain when compared to the amount of SNARE protein cleaved by a catalytically active clostridial neurotoxin L-chain under the same conditions. Routine techniques, such as SDS-PAGE and Western blotting can be used to quantify the amount of SNARE protein cleaved. Suitable in vitro assays are described in WO 2019/145577 A1, which is incorporated herein by reference.
  • Cell-based and in vivo assays may also be used to determine if a clostridial neurotoxin comprising an L-chain and a functional cell binding and translocation domain has non- cytotoxic protease activity.
  • Assays such as the Digit Abduction Score (DAS), the dorsal root ganglia (DRG) assay, spinal cord neuron (SCN) assay, and mouse phrenic nerve hemidiaphragm (PNHD) assay are routine in the art.
  • DAS Digit Abduction Score
  • DRG dorsal root ganglia
  • SCN spinal cord neuron
  • PNHD mouse phrenic nerve hemidiaphragm
  • a suitable assay for determining non- cytotoxic protease activity may be one described in Donald et al (2016), Pharmacol Res Perspect, e00446, 1-14, which is incorporated herein by reference.
  • a catalytically inactive L-chain may have one or more mutations that inactivate said catalytic activity.
  • a catalytically inactive BoNT/A L-chain may comprise a mutation of an active site residue, such as His223, Glu224, His227, Glu262, and/or Tyr366.
  • the position numbering corresponds to the amino acid positions of SEQ ID NO: 62 and can be determined by aligning a polypeptide with SEQ ID NO: 62.
  • the skilled person will take the presence/absence of the methionine residue into account when determining amino acid residue numbering.
  • SEQ ID NO: 62 includes a methionine
  • the position numbering will be as defined above (e.g. His223 will be His223 of SEQ ID NO: 62).
  • the amino acid residue numbering should be modified by -1 (e.g. His223 will be His222 of SEQ ID NO: 62). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present/absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art.
  • a clostridial neurotoxin of the invention may comprise a modified BoNT/A or fragment thereof (preferably a BoNT/A HC domain or fragment thereof).
  • the modified BoNT/A or fragment thereof may be one that comprises a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277.
  • Such a modified BoNT/A or fragment thereof may demonstrate a reduction in, or absence of, side effects compared to the use of known BoNT/A.
  • the increased tissue retention properties of the modified BoNT/A of the invention may also provide increased potency and/or duration of action and can allow for reduced dosages to be used compared to known clostridial toxin therapeutics (or increased dosages without any additional adverse effects), thus providing further advantages.
  • the modification may be a modification when compared to unmodified BoNT/A shown as SEQ ID NO: 62, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO: 62.
  • SEQ ID NO: 62 As the presence of a methionine residue at position 1 of SEQ ID NO: 62 (as well as the SEQ ID NOs corresponding to modified BoNT/A polypeptides or fragments thereof described herein) is optional, the skilled person will take the presence/absence of the methionine residue into account when determining amino acid residue numbering.
  • SEQ ID NO: 62 includes a methionine
  • the position numbering will be as defined above (e.g. ASN 886 will be ASN 886 of SEQ ID NO: 62).
  • the amino acid residue numbering should be modified by -1 (e.g. ASN 886 will be ASN 885 of SEQ ID NO: 62).
  • a modified BoNT/A or fragment thereof may comprise a modification at one or more amino acid residue(s) selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 and THR 1277.
  • one or more amino acid residue(s) when used in the context of modified BoNT/A or fragment thereof preferably means at least 2, 3, 4, 5, 6 or 7 of the indicated amino acid residue(s).
  • a modified BoNT/A may comprise at least 2, 3, 4, 5, 6 or 7 (preferably 7) modifications at the indicated amino acid residue(s).
  • a modified BoNT/A or fragment thereof may comprise 1-30, 3-20, or 5-10 amino acid modifications. More preferably, the term “one or more amino acid residue(s)” when used in the context of modified BoNT/A or fragment thereof means all of the indicated amino acid residue(s).
  • the modified BoNT/A or fragment thereof does not contain any further amino acid modifications when compared to SEQ ID NO: 62.
  • the modification may be selected from: i. substitution of an acidic surface exposed amino acid residue with a basic amino acid residue; ii. substitution of an acidic surface exposed amino acid residue with an uncharged amino acid residue; iii. substitution of an uncharged surface exposed amino acid residue with a basic amino acid residue; iv. insertion of a basic amino acid residue; and v. deletion of an acidic surface exposed amino acid residue.
  • a modification as indicated above results in a modified BoNT/A or fragment thereof that has an increased positive surface charge and increased isoelectric point when compared to the corresponding unmodified BoNT/A or fragment thereof.
  • the isoelectric point (pI) is a specific property of a given protein.
  • proteins are made from a specific sequence of amino acids (also referred to when in a protein as amino acid residues). Each amino acid of the standard set of twenty has a different side chain (or R group), meaning that each amino acid residue in a protein displays different chemical properties such as charge and hydrophobicity. These properties may be influenced by the surrounding chemical environment, such as the temperature and pH. The overall chemical characteristics of a protein will depend on the sum of these various factors.
  • Certain amino acid residues possess ionisable side chains that may display an electric charge depending on the surrounding pH. Whether such a side chain is charged or not at a given pH depends on the pKa of the relevant ionisable moiety, wherein pKa is the negative logarithm of the acid dissociation constant (Ka) for a specified proton from a conjugate base.
  • pKa is the negative logarithm of the acid dissociation constant (Ka) for a specified proton from a conjugate base.
  • acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values of approximately 4.1 (precise pKa values may depend on temperature, ionic strength and the microenvironment of the ionisable group).
  • these side chains exhibit a negative charge at a pH of 7.4 (often referred to as “physiological pH”). At low pH values, these side chains will become protonated and lose their charge. Conversely, basic residues such as lysine and arginine have nitrogen-containing side chain groups with pKa values of approximately 10-12. These side chains therefore exhibit a positive charge at a pH of 7.4. These side chains will become de-protonated and lose their charge at high pH values.
  • the overall (net) charge of a protein molecule therefore depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) and on the surrounding pH. Changing the surrounding pH changes the overall charge on the protein.
  • the isoelectric point is a standard concept in protein biochemistry with which the skilled person would be familiar.
  • the isoelectric point (pI) is therefore defined as the pH value at which a protein displays a net charge of zero.
  • An increase in pI means that a higher pH value is required for the protein to display a net charge of zero.
  • an increase in pI represents an increase in the net positive charge of a protein at a given pH.
  • a decrease in pI means that a lower pH value is required for the protein to display a net charge of zero.
  • a decrease in pI represents a decrease in the net positive charge of a protein at a given pH.
  • Methods of determining the pI of a protein are known in the art and would be familiar to a skilled person.
  • the pI of a protein can be calculated from the average pKa values of each amino acid present in the protein (“calculated pI”). Such calculations can be performed using computer programs known in the art, such as the Compute pI/MW Tool from ExPASy (https://web.expasy.org/compute_pi/), which is the preferred method for calculating pI in accordance with the present invention. Comparisons of pI values between different molecules should be made using the same calculation technique/program.
  • the calculated pI of a protein can be confirmed experimentally using the technique of isoelectric focusing (“observed pI”).
  • This technique uses electrophoresis to separate proteins according to their pI.
  • Isoelectric focusing is typically performed using a gel that has an immobilised pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches the pH at which it has zero net charge, this point being the pI of the protein.
  • Results provided by isoelectric focusing are typically relatively low-resolution in nature, and thus the present inventors believe that results provided by calculated pI (as described above) are more appropriate to use.
  • “pI” means “calculated pI” unless otherwise stated.
  • the pI of a protein may be increased or decreased by altering the number of basic and/or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pI may be provided by reducing the number of acidic residues, or by increasing the number of basic residues.
  • a modified BoNT/A or fragment thereof of the invention may have a pI value that is at least 0.2, 0.4, 0.5 or 1 pI units higher than that of an unmodified BoNT/A (e.g. SEQ ID NO: 62) or fragment thereof.
  • a modified BoNT/A or fragment thereof may have a pI of at least 6.6, e.g. at least 6.8.
  • the properties of the 20 standard amino acids are indicated in the table below:
  • the following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).
  • Aspartic acid and glutamic acid are referred to as acidic amino acid residues.
  • Arginine and lysine are referred to as basic amino acid residues.
  • amino acids are considered uncharged, polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.
  • amino acids are considered uncharged, hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.
  • an additional amino acid residue one that is not normally present is incorporated into the BoNT/A polypeptide sequence or fragment thereof, thus increasing the total number of amino acid residues in said sequence.
  • an amino acid residue is removed from the clostridial toxin amino acid sequence, thus reducing the total number of amino acid residues in said sequence.
  • the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT/A or fragment thereof.
  • an amino acid residue that forms part of the BoNT/A polypeptide sequence or fragment thereof is replaced with a different amino acid residue.
  • the replacement amino acid residue may be one of the 20 standard amino acids, as described above.
  • the replacement amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the standard set of 20 described above).
  • the replacement amino acid may be a basic non-standard amino acid, e.g. L-Ornithine, L-2- amino-3-guanidinopropionic acid, or D-isomers of Lysine, Arginine and Ornithine).
  • Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using E. coli auxotrophic expression hosts.
  • the substitution is selected from: substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue.
  • the substitution is a substitution of an acidic amino acid residue with an uncharged amino acid residue
  • the acidic amino acid residue is replaced with its corresponding uncharged amide amino acid residue (i.e. aspartic acid is replaced with asparagine, and glutamic acid is replaced with glutamine).
  • the basic amino acid residue is a lysine residue or an arginine residue.
  • the substitution is substitution with lysine or arginine.
  • the modification is substitution with lysine.
  • a modified BoNT/A or fragment thereof for use in the invention comprises between 4 and 40 amino acid modifications located in the clostridial toxin H CN domain.
  • Said modified BoNT/A or fragment thereof preferably also has pI of at least 6.6.
  • Said modified BoNT/A preferably comprises modifications of at least 4 amino acids selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, and ASN 1052, wherein said modification comprises substitution of the amino acids with a lysine residue or an arginine residue.
  • said modified BoNT/A or fragment thereof may comprise modifications of at least 5 amino acids selected from: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, and GLN 1229, wherein said modification comprises substitution of the amino acids with a lysine residue or an arginine residue.
  • Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art.
  • amino acid modifications may be introduced by modification of a DNA sequence encoding a polypeptide (e.g. encoding unmodified BoNT/A or a fragment thereof).
  • a clostridial neurotoxin for use according to the invention comprises a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 42.
  • a clostridial neurotoxin for use according to the invention comprises a polypeptide sequence shown as SEQ ID NO: 42.
  • a clostridial neurotoxin for use according to the invention comprises a polypeptide sequence that is encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 41.
  • a clostridial neurotoxin for use according to the invention comprises a polypeptide sequence that is encoded by a nucleotide sequence shown as SEQ ID NO: 41.
  • a clostridial neurotoxin for use according to the invention e.g.
  • a clostridial neurotoxin for use according to the invention may comprise a polypeptide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 61 or 65.
  • a clostridial neurotoxin for use according to the invention may comprise (more preferably consist of) SEQ ID NO: 61 or 65.
  • the clostridial neurotoxin comprises a catalytically-inactive L-chain (e.g. as per SEQ ID NO: 65).
  • a clostridial neurotoxin for use according to the invention may be encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 60.
  • a clostridial neurotoxin for use according to the invention may be encoded by a nucleotide sequence having at least 80%, 90%, 95% or 98% sequence identity to SEQ ID NO: 60.
  • a clostridial neurotoxin for use according to the invention may be encoded by a nucleotide sequence comprising (more preferably consisting of) SEQ ID NO: 60.
  • the clostridial neurotoxin comprises a catalytically-inactive L-chain.
  • SEQ ID NO: 42 is an example of a modified BoNT/A fragment and SEQ ID NOs: 61 and 65 are examples of modified BoNT/A polypeptides that are catalytically active and inactive, respectively. Such modified BoNT/A polypeptides and fragments are particularly preferred for use in the present invention.
  • the polypeptides shown as SEQ ID NO: 42, 61 and 65 have a number of amino acid modifications (e.g. substitutions) when compared to wild-type BoNT/A, which increase the isoelectric point of the polypeptide.
  • undesired effects of a clostridial neurotoxin can be assessed experimentally by measuring percentage bodyweight loss in a relevant animal model (e.g. a mouse, where loss of bodyweight is detected within seven days of administration).
  • desired on-target effects of a clostridial neurotoxin can be assessed experimentally by Digital Abduction Score (DAS) assay, a measurement of muscle paralysis.
  • DAS Digital Abduction Score
  • the DAS assay may be performed by injection of 20 ⁇ l of clostridial neurotoxin, formulated in Gelatin Phosphate Buffer, into the mouse gastrocnemius/soleus complex, followed by assessment of Digital Abduction Score using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001).
  • mice are suspended briefly by the tail in order to elicit a characteristic startle response in which the mouse extends its hind limbs and abducts its hind digits.
  • the Safety Ratio of a clostridial neurotoxin may then be expressed as the ratio between the amount of toxin required for a 10% drop in a bodyweight (measured at peak effect within the first seven days after dosing in a mouse) and the amount of toxin required for a DAS score of 2. High Safety Ratio scores are therefore desired and indicate a toxin that is able to effectively paralyse a target muscle with little undesired off-target effects.
  • a catalytically active modified BoNT/A of the present invention may have a Safety Ratio that is higher than the Safety Ratio of an equivalent unmodified (native) botulinum toxin (e.g. SEQ ID NO: 62).
  • a catalytically active modified BoNT/A of the present invention has a Safety Ratio of at least 10.
  • a modified BoNT/A or fragment thereof of the present invention has a Safety Ratio of at least 15.
  • Clostridial neurotoxins comprising at least 70% sequence identity to SEQ ID NO: 61 are described in WO 2015/004461 A1, which is incorporated herein by reference in its entirety.
  • a clostridial neurotoxin comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42, 61 or 65 and/or comprising a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41 or 60 comprises a substitution at one or more (preferably two or more, three or more, four or more, five or more or six or more, more preferably at all) of positions 930, 955, 991, 1026, 1052, 1229, and 886.
  • the position numbering corresponds to the positions of SEQ ID NO: 62 and can be determined by aligning the polypeptide sequence with SEQ ID NO: 62 (unmodified/wild-type BoNT/A).
  • SEQ ID NO: 62 includes a methionine
  • the position numbering will be as defined above (e.g. position 886 will be ASN 886 of SEQ ID NO: 62).
  • the amino acid residue numbering should be modified by -1 (e.g.
  • position 886 will be ASN 885 of SEQ ID NO: 62). Similar considerations apply when the methionine at position 1 of the other polypeptide sequences described herein is present/absent, and the skilled person will readily determine the correct amino acid residue numbering using techniques routine in the art.
  • the clostridial neurotoxin comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 42, 61 or 65 and/or comprising a polypeptide sequence that is encoded by a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 41 or 60 comprises lysine or arginine (more preferably lysine) at one or more of positions 930, 955, 991, 1026, 1052, 1229, and 886.
  • the clostridial neurotoxin comprises lysine or arginine (more preferably lysine) at least two, three, four, five, six or all of positions 930, 955, 991, 1026, 1052, 1229, and 886. Most preferably, the clostridial neurotoxin comprises lysine or arginine (more preferably lysine) at all of positions 930, 955, 991, 1026, 1052, 1229, and 886.
  • Embodiments related to the various therapeutic uses of the invention are intended to be applied equally to methods of treatment, polypeptides of the invention, and vice versa.
  • SEQUENCE HOMOLOGY Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D.
  • Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992); Gibbs sampling, see, e.g., C. E.
  • % identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues.
  • the polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4- methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo- threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro- glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3- azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine.
  • Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins.
  • an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs.
  • Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
  • a natural amino acid that is to be replaced e.g., phenylalanine
  • the desired non-naturally occurring amino acid(s) e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine.
  • the non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification.
  • Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci.2:395-403, 1993).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989).
  • Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992.
  • the identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.
  • any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • the headings provided herein are not limitations of the various aspects or embodiments of this disclosure.
  • Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.
  • the term “protein”, as used herein, includes proteins, polypeptides, and peptides.
  • amino acid sequence is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”.
  • amino acid sequence is synonymous with the term “enzyme”.
  • protein and “polypeptide” are used interchangeably herein.
  • the conventional one-letter and three-letter codes for amino acid residues may be used.
  • the 3- letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code. Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary.
  • a clostridial neurotoxin includes a plurality of such neurotoxin and reference to “the clostridial neurotoxin” includes reference to one or more clostridial neurotoxin(s) and equivalents thereof known to those skilled in the art, and so forth.
  • the publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. None herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. BRIEF DESCRIPTION OF THE DRAWINGS Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.
  • Figure 1 An illustration of the dermatomes (adapted Beilin, Daniel.
  • Dysport 25 U/kg, i.d. diminished CYP-induced chronic allodynia.
  • Dysport 25 U/kg, i.d.) reduced CYP-induced chronic visceral pain (nociceptive scores).
  • SEQ ID NO: 1 Nucleotide Sequence of Recombinant Catalytically Inactive BoNT/A (rBoNT/A(0)) SEQ ID NO: 2 - Polypeptide Sequence of rBoNT/A(0) SEQ ID NO: 3 - Nucleotide Sequence of rLH N /A (light-chain plus translocation domain only).
  • SEQ ID NO: 4 Polypeptide Sequence of rLH N /A SEQ ID NO: 5 - Nucleotide Sequence of rL/A (light-chain only) SEQ ID NO: 6 - Polypeptide Sequence of rL/A SEQ ID NO: 7 - Nucleotide Sequence of rHC/A SEQ ID NO: 8 - Polypeptide Sequence of rHC/A SEQ ID NO: 9 - Nucleotide Sequence of rBoNT/B(0) SEQ ID NO: 10 - Polypeptide Sequence of rBoNT/B(0) SEQ ID NO: 11 - Nucleotide Sequence of rBoNT/C(0) SEQ ID NO: 12 - Polypeptide Sequence of rBoNT/C(0) SEQ ID NO: 13 - Nucleotide Sequence of rBoNT/E(0) SEQ ID NO: 14 - Polypeptide Sequence of rBoNT/E(0) SEQ ID NO: 15 - Nucle
  • EXAMPLE 1 Suppression of visceral pain following administration of clostridial neurotoxin to a dermatome Introduction to animal model
  • dermatome administration e.g. to a T12, L1, S2, S3 or S4 dermatome in the case of targeting pain in the bladder
  • appropriate dermatome administration e.g. to a T12, L1, S2, S3 or S4 dermatome in the case of targeting pain in the bladder
  • IC/BPS interstitial cystitis/bladder pain syndrome
  • clostridial neurotoxin to the T11 dermatome can be used to suppress visceral pain of the ovary or fallopian tube(s), because visceral pain from the ovary/ fallopian tubes in integrated at the same area from which the T11 spinal nerve emerges.
  • This demonstrates utility in treating visceral pain cause by conditions such as endometriosis.
  • Interstitial cystitis/bladder pain syndrome IC/BPS
  • IC/BPS Interstitial cystitis/bladder pain syndrome
  • Cyclophosphamide (CYP) induced inflammatory visceral pain is commonly used as an experimental model for IC/BPS in rodents.
  • CYP treatment leads to accumulation of its toxic metabolite acrolein in the urinary bladder mediating urinary bladder inflammation and visceral pain.
  • This model is mostly based on single intraperitoneal (i.p.) injection of CYP to induce acute IC/BPS (useful for investigating the acute condition).
  • IC/BPS acute IC/BPS
  • This model shows long-lasting visceral pain which is characterized by both allodynia (painful response to a normally innocuous stimulus) and hyperalgesia (increased response to a noxious stimulus).
  • the aim of the present study was to evaluate the effects of a single intradermal (i.d.) administration of Dysport (25 U/kg) on visceral pain in the CYP-induced chronic cystitis model in female Sprague-Dawley rats.
  • Study Design Protocol design • At D-10, pharmacological treatment was performed as described below (under “pharmacological treatment”). • At D-1, in order to decrease the level of stress due to the new environment, rats were acclimatized to the individual Plexiglas box (von Frey set up) for a minimum of 30 min and to the application of the von Frey filaments. Acclimatization was performed under the same experimental conditions as for pain evaluation described below under “Assessment of visceral pain using von Frey filaments”.
  • Test substance was Dysport, supplied in white lyophilisate from IPSEN Innovation, and was stored as -20 o C and protected from light.
  • Preparation of formulation formulation was made fresh on the day of administration.
  • One vial containing 519 U of Dysport was solubilized in 1.038 mL of saline to obtain a final concentration of 500 U/mL.
  • Working formulation was prepared according to the mean of rat’s body weights from the Dysport group (i.e. 235.7 g at D-11 which is corresponding to a concentration of 294.625 U/mL).
  • Saline was used as vehicle.
  • Saline was purchased from B-Braun via Centravet (Lapalisse, France).
  • Isoflurane (Aerrane®) was purchased from Baxter Laboratories (Maurepas, France).
  • Dolethal® was purchased from Vetoquinol via Centravet (Lapalisse, France).
  • CYP was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France; batch n° MKCG5464).
  • Formaldehyde 4% was purchased from VWR (Fontenay sous bois, France).
  • Phosphate buffered saline (PBS) was purchased from Sigma-Aldrich.
  • Sedated rats were then euthanatized by a second lethal injection of Dolethal® (0.25 mL/rat). Both i.p. injections were made outside the area of skin collection. The skin including the injection site with the underlying abdominal muscles was first collected. The piece of around 2-3 of length was stapled on a plastic sheet and placed into a 500 mL jar containing 4% buffered formaldehyde. Bladder was then excised and lipoid tissue surrounding the bladder was cut away. Bladder was weighed and placed into a tissue processing embedding cassette. Cassette was immerged into 4% buffered formaldehyde.
  • the brain, and the lumbosacral, thoracic and cervical vertebrae were collected.
  • the 3 segments and the brain were placed together in the jar with the skin.
  • the ventral part faced up.
  • the tissue above the thoraco-lumbar area was scratched with a scalpel.
  • the cervical part was separated from the thoracic part by cutting with a scalpel between the third and fourth last thoracic intervertebral discs/ ribs.
  • the third/fourth ribs remained attached to the lumbar part.
  • Bladder and all others collected tissues were fixed at room temperature for around 24h and 48h, respectively. Then samples were transferred into 4% buffered formaldehyde (1/4) / PBS (3/4) solution and stored at +4oC.
  • Immunohistochemistry (IHC) for cleaved SNAP25 (c-SNAP25A) was performed with antibody EF14007, 1/6000 dilution, incubated with sample overnight – same for any related IHC described herein.
  • Laboratory equipment - Mechanical stimulation was performed using von Frey filaments of different forces (reference: Bio-VFM; BioSeb ID Tech, Vitrolles, France). Filaments were purchased pre- calibrated.
  • Bladder weight results of bladder weight are expressed as follows: Bladder weight in g; Bladder weight as a ratio of body weight in g/kg.
  • Statistical analysis Statistical analysis and graphs were performed using GraphPad Prism® (GraphPad Software Inc., La Jolla, CA, USA). A P value ⁇ 0.05 was accepted for statistical significance. Except when two-way analysis was applied, before carrying out any statistical test, the data were tested for normal distribution (Shapiro-Wilk normality test) and their variance evaluated (F test). The appropriate statistical test was consequently applied.
  • D10 a decrease in nociceptive scores was observed in Dysport-treated rats at both time points ( Figures 6B and C).
  • c-SNAP 25 immunohistochemistry results in thoracic spinal cord Following intradermal (I.D.) injection of Dysport to a dermatome nerve, c-SNAP25 staining was seen in the thoracic spinal cord, at both ventral and dorsal horns. Interestingly, such c- SNAP25 staining was also seen following or intradetrusor (I.DT) injection (injection into the detrusor muscle of the bladder), for example similar localization, distribution and severity of c-SNAP25 staining following I.D and I.DT administrations were seen in the thoracic spinal cord. This suggests that I.D Dysport administration at the abdomen level resulted in the same retrograde transport (i.e.
  • I.D Dysport administration at the abdomen level resulted in the same retrograde transport (i.e. to the same area of spinal cord) as I.DT administration, the latter believe to be due to retrograde axonal transport via visceral afferents. See Figure 9.
  • Dysport or saline were injected intradermally using 30G needles attached to 1 ml syringes and into 10 sites spread around an incision on the left leg of the pig (20 U Dysport injected into each site) – 6 animals (8-10 weeks old) were used. Immunohistochemistry was performed on tissue/ skin samples at the site of Dysport injection (left leg of the pig) and at different regions of the spinal cord (see Figure 10). Cleaved SNAP- 25 was not detected in the nerves of skin samples on samples collected 5-7 days after the injection of Dysport. Cleaved SNAP-25 was visualised in the ipsilateral dorsal horn of the spinal cord, e.g.
  • L5-L6 lumbar regions L5-L6 (see Figure 10) of Dysport injected pigs (but not saline injected pigs).
  • the spinal nerves (L5-L6) of these lumbar regions map to and innervate dermatomes on the left leg (e.g. where the Dysport was injection), and are indicative of BoNT/A activity in the spinal cord and that BoNT/A is transported to this region of the spinal cord after the intradermal injection to the dermatome.
  • cleaved SNAP-25 staining in the ipsilateral dorsal horn was different when compared to cleaved SNAP-25 staining in pigs where Dysport was injected (intradermally) to their left flank, indicating that injecting into different dermatomes allows for BoNT/A activity in different parts of the spinal cord.
  • expression levels of a marker of astrocyte activation, GFAP were decreased in the spinal cord, particularly in the dorsal horns, of pigs (those treated with Dysport when compared to untreated) in the same areas that an increase in SNAP-25 cleavage was seen.
  • the aim of the present study was to evaluate the effects of intradermal administration of botulinum toxin (Dysport) at 25 U/kg on visceral pain in the chronic CYP-induced cystitis model in female Sprague-Dawley rats. Consistent with previous and published data, 3 injections of CYP (40 mg/kg, i.p.) induced chronic visceral pain (up to 10 days). Effects of CYP were characterized by allodynia (i.e. decreased nociceptive threshold and increased AUC 1-6 g) and hyperalgesia (i.e. increased AUC 6-26 g). This study showed that single injection of clostridial neurotoxin (e.g.
  • BoNT/A aka Dysport
  • BoNT/A to a dermatome nerve, particularly via intradermal injection, provided anti-nociceptive properties on CYP-induced visceral pain. Allodynia and hyperalgesia induced by CYP were lowered by clostridial neurotoxin/ Dysport (intradermal) injection, with allodynia being a particular target for suppression. Dysport effect was long lasting as clostridial neurotoxin/ Dysport was still efficient 20 days after its administration. EXAMPLE 2 – Suppression of pain caused by bladder pain syndrome A patient presents with severe pelvic pain perceived as visceral pain, and abnormal urination frequency.
  • the patient is diagnosed with interstitial cystitis/bladder pain syndrome (IC/BPS), and the bladder is identified as the organ contributing to the visceral pain.
  • IC/BPS interstitial cystitis/bladder pain syndrome
  • ASIA American Spinal Injury Association
  • ISNCSCI International Standards for Neurological Classification of Spinal Cord Injury
  • T12, L1, S2, S3 and S4 dermatomes map to the urinary bladder.
  • clostridial neurotoxin (5,000 pg Dysport) is administered to the T12 dermatome by intradermal administration to the skin in the proximity of the intersection of the midclavicular line and the midpoint of the inguinal ligament.
  • levels of visceral (bladder) pain are assessed, and found to be significantly reduced. Suppression of visceral (bladder) pain is also noted at 30 days post-administration of the clostridial neurotoxin and at 90 days post- administration.
  • EXAMPLE 3 Suppression of pain caused by endometriosis
  • a female patient presents with severe pelvic pain, perceived as visceral pain, worsening during menstruation.
  • the patient is diagnosed by her GP with endometriosis, and the ovaries are identified as the organ contributing to the visceral pain.
  • a map of the dermatomes and their corresponding organs/ viscera is consulted, such as that outlined in the American Spinal Injury Association’s (ASIA) worksheet produced as the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI).
  • ASIA American Spinal Injury Association
  • clostridial neurotoxin (5,000 pg Dysport) is administered to the T11 dermatome by intradermal administration to the skin in the proximity of the intersection of the midclavicular line, at the horizontal level midway between the level of the umbilicus and the inguinal ligament. After 10 days, levels of visceral (ovary) pain are assessed, and found to be significantly reduced. Suppression of visceral (ovary) pain is also noted at 30 days post- administration of the clostridial neurotoxin and at 90 days post-administration. EXAMPLE 4 – Suppression of pain caused by pancreatitis A patient presents with severe abdominal pain, perceived as visceral pain.
  • pancreas Inflammation of the pancreas is noted, the patient is diagnosed by his GP with pancreatitis, and the pancreas is identified as the organ contributing to the visceral pain.
  • ASIA American Spinal Injury Association
  • INCSCI International Standards for Neurological Classification of Spinal Cord Injury
  • clostridial neurotoxin (5,000 pg Dysport) is administered to the T10 dermatome by intradermal administration to the skin in the proximity of the intersection of the midclavicular line, at the horizontal level of the umbilicus. After 10 days, levels of visceral (pancreas) pain are assessed, and found to be significantly reduced. Suppression of visceral (pancreas) pain is also noted at 30 days post-administration of the clostridial neurotoxin and up to 90 days post-administration. EXAMPLE 5 – Suppression of pain caused by gastric ulcers A patient presents with severe abdominal pain, perceived as visceral pain.
  • the patient is diagnosed by his GP with gastric ulcers, and the stomach is identified as the organ contributing to the visceral pain.
  • ASIA American Spinal Injury Association
  • INCSCI International Standards for Neurological Classification of Spinal Cord Injury
  • FIG. 1B illustrates the segmental level of visceral innervation and the corresponding dermatomes that various viscera e.g. the dermatomes that ‘map’ to the viscera. It is noted that the T8-T10 dermatomes map to the pancreas.
  • clostridial neurotoxin (5,000 pg Dysport) is administered to the T8 dermatome by intradermal administration to the skin in the proximity of the intersection of the midclavicular line and the horizontal level at one half the distance between the level of the xiphoid process and the level of the umbilicus.
  • levels of visceral (stomach) pain are assessed, and found to be significantly reduced. Suppression of visceral (stomach) pain is also noted at 30 days post-administration of the clostridial neurotoxin and at 90 days post- administration.

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EP22817306.8A 2021-11-22 2022-11-21 Behandlung von eingeweideschmerzen Pending EP4436593A1 (de)

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GBGB2116795.2A GB202116795D0 (en) 2021-11-22 2021-11-22 Treatment of visceral pain
PCT/GB2022/052947 WO2023089338A1 (en) 2021-11-22 2022-11-21 Treatment of visceral pain

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CN119791059A (zh) * 2025-02-08 2025-04-11 合肥综合性国家科学中心大健康研究院 一种用于检测内源性疼痛调节功能的小鼠模型及其构建方法和应用

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WO2007143294A1 (en) * 2006-05-02 2007-12-13 Allergan, Inc. Use of botulinum toxin for alleviating testicular pain
WO2014053651A1 (en) * 2012-10-04 2014-04-10 Dublin City University Biotherapy for pain
WO2023287729A1 (en) * 2021-07-12 2023-01-19 Penland Foundation Treatment of acute and chronic kidney disease

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WO2014053651A1 (en) * 2012-10-04 2014-04-10 Dublin City University Biotherapy for pain
WO2023287729A1 (en) * 2021-07-12 2023-01-19 Penland Foundation Treatment of acute and chronic kidney disease

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JP2024540580A (ja) 2024-10-31
US20250082738A1 (en) 2025-03-13

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