Methods and compounds for the treatment of mucus hypersecretion
A method of treating mucus hypersecretion, the causative factor in chronic obstructive pulmonary disease (COPD), asthma and other clinical conditions involving COPD, comprises administering a compound that inhibits exocytosis in mucus secreting cells or neurones that control or direct mucus secretion. Also described is a compound, for use in the treatment of hypersecretion of mucus, which inhibits mucus secretion by inhibiting mucus secretion by mucus secreting cells, and/or inhibiting neurotransmitter release from neuronal cells controlling or directing mucus secretion.
1 - 54 . (canceled)
55 . An agent for treating pain comprising a modified botulinum neurotoxin comprising a botulinum neurotoxin H N , a botulinum neurotoxin L chain, and no functional H C domain, the modified botulinum neurotoxin being covalently coupled to substance P so that the modified botulinum neurotoxin no longer binds to botulinum neurotoxin receptors at a neuromuscular junction with the same affinity as the native botulinum neurotoxin.
56 . The agent of claim 55 , wherein the botulinum neurotoxin is a botulinum toxin selected from the group consisting of serotype A, serotype B, serotype C 1 , serotype D, serotype E, serotype F and serotype G.
57 . The agent of claim 55 , wherein the botulinum neurotoxin is botulinum toxin serotype A.
58 . The agent of claim 55 , wherein the agent is a fusion protein.
59 . The agent of claim 55 , wherein the L chain is obtained from a botulinum toxin selected from the group consisting of botulinum toxin serotype A, serotype B, serotype C 1 , serotype D, serotype E, serotype F and serotype G.
60 . The agent of claim 55 , wherein the H N is obtained from a botulinum toxin selected from the group consisting of botulinum toxin serotype A, serotype B, serotype C 1 , serotype D, serotype E, serotype F and serotype G.
61 . A plasmid encoding a modified clostridial neurotoxin, comprising: (a) a first nucleotide sequence comprising; (i) a first nucleotide segment encoding an amino acid sequence comprising substance P as the targeting moiety; and (ii) a second nucleotide segment encoding an amino acid sequence comprising a translocation domain (H N ) of a clostridial neurotoxin or a fragment thereof, which fragment translocates a light chain (L-chain) or L-chain fragment of a clostridial neurotoxin across an endosome membrane, wherein the first and second nucleotide segments encode an amino acid sequence comprising a fusion protein of a translocation domain and substance P, and wherein the H C has been removed from said clostridial neurotoxin or has been modified so as to reduce the ability of the clostridial neurotoxin to bind to a receptor for the H C at a neuromuscular junction; (b) a second nucleotide sequence encoding an amino acid sequence comprising the L-chain or an L-chain fragment of a clostridial neurotoxin, which L-chain fragment comprises the active protease domain of L-chain and (c) an element for plasmid replication by a host cell.
62 . A method of making a modified clostridial neurotoxin, the method comprising: (a) inserting the plasmid of claim 61 into a suitable host cell, (b) culturing the host cell under conditions sufficient to express the clostridial neurotoxin, and (c) isolating the clostridial neurotoxin.
63 . A method of obtaining an agent for alleviating pain, the method comprising: (a) producing the plasmid of claim 61 which encodes a modified clostridial neurotoxin; (b) inserting the plasmid into a suitable host cell; (c) culturing the host cell under conditions sufficient to express the clostridial neurotoxin; and isolating the clostridial neurotoxin as the agent for alleviating pain.
64 . The method of claim 63 , wherein the modified clostridial neurotoxin further comprises at least one spacer component.
65 . The method of claim 63 , wherein the clostridial neurotoxin comprises an H N and an L chain.
66 . The method of claim 65 , wherein the H N has an amino acid sequence identical to the translocation domain of a clostridial neurotoxin from an organism selected from the group consisting of Clostridial beratti, Clostridial butyricum, Clostridial botulinum , and Clostridial tetani.
67 . The method of claim 65 , wherein the L-chain has an amino acid sequence identical to the light chain of a clostridial neurotoxin from an organism selected from the group consisting of Clostridial beratti, Clostridial butyricum, Clostridial botulinum , and Clostridial tetani.
68 . The method of claim 65 , wherein the H N has an amino acid sequence identical to the translocation domain of a botulinum toxin selected from the group consisting of botulinum toxin serotype A, serotype B, serotype C 1 , serotype D, seretype E, serotype F, and serotype G.
69 . The method of claim 65 , wherein the H N has an amino acid sequence identical to the translocation domain of a botulinum toxin selected from the group consisting of botulinum toxin serotype A, serotype B, serotype C 1 , serotype D, serotype E, serotype F, and serotype G, and wherein the L-chain has an amino acid sequence identical to the light chain of a botulinum toxin selected from the group consisting of botulinum toxin serotype A, serotype B, serotype C 1 , serotype D, serotype E, serotype F, and serotype G.
70 . The method of claim 65 , wherein the H N has an amino acid sequence identical to the translocation domain of botulinum toxin serotype A, and wherein L-chain has an amino acid sequence identical to the light chain of botulinum toxin serotype A.
71 . A compound comprising a modified botulinum neurotoxin comprising a botulinum neurotoxin H N , a botulinum neurotoxin L chain, wherein said modified neurotoxin is modified so as to disable or remove its H C portion, the modified botulinum neurotoxin being covalently linked to a Targeting Moiety (TM) so that the modified botulinum neurotoxin no longer binds to botulinum neurotoxin receptors at a neuromuscular junction with the same affinity as the native botulinum neurotoxin.
72 . The compound of claim 71 , wherein the TM is substance P.
73 . A plasmid encoding a modified clostridial neurotoxin, comprising: (a) a first nucleotide sequence comprising; (i) a first nucleotide segment encoding an amino acid sequence as the targeting moiety (TM); and (ii) a second nucleotide segment encoding an amino acid sequence comprising a translocation domain (H N ) of a clostridial neurotoxin or a fragment thereof, which fragment translocates a light chain (L-chain) or L-chain fragment of a clostridial neurotoxin across an endosome membrane, wherein the first and second nucleotide segments encode an amino acid sequence comprising a fusion protein of a translocation domain and a TM, and wherein the H C has been disabled or removed from said clostridial neurotoxin so as to reduce the ability of the clostridial neurotoxin to bind to a receptor for the H C at a neuromuscular junction; (b) a second nucleotide sequence encoding an amino acid sequence comprising the L-chain or an L-chain fragment of a clostridial neurotoxin, which L-chain fragment comprises the active protease domain of L-chain and (c) an element for plasmid replication by a host cell.
74 . The plasmid of claim 73 , wherein the TM is substance P.
75 . A method of making a modified clostridial neurotoxin, the method comprising: (a) inserting the plasmid of claim 73 into a suitable host cell, (b) culturing the host cell under conditions sufficient to express the clostridial neurotoxin, and (c) isolating the clostridial neurotoxin.
76 . A method of obtaining a compound, the method comprising: (a) producing the plasmid of claim 73 which encodes a modified clostridial neurotoxin; (b) inserting the r plasmid into a suitable host cell; (c) culturing the host cell under conditions sufficient to express the clostridial neurotoxin; and isolating the clostridial neurotoxin.