Botulinum neurotoxin-substance P conjugate or fusion protein for treating pain
View Patent ↗Agents for treating pain, methods for producing the agents and methods for treating pain by administration to a patient of a therapeutically effective amount of the agent. The agent can include a clostridial neurotoxin, or a component or fragment or derivative thereof, attached to a targeting moiety, wherein the targeting moiety is selected from a group consisting of transmission compounds which can be released from neurons upon the transmission of pain signals by the neurons, and compounds substantially similar to the transmission compounds.
1. 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.
2. A method of making a modified clostridial neurotoxin, the method comprising:
(a) inserting the plasmid of claim 1 into a suitable host cell,
(b) culturing the host cell under conditions sufficient to express the clostridial neurotoxin, and
(c) isolating the clostridial neurotoxin.
3. A method of obtaining an agent for alleviating pain, the method comprising:
(a) producing the plasmid of claim 1 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.
4. The method of claim 3 , wherein the modified clostridial neurotoxin further comprises at least one spacer component.
5. The method of claim 3 , wherein the clostridial neurotoxin comprises an H N and an L chain.
6. The method of claim 5 , 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.
7. The method of claim 5 , 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.
8. The method of claim 5 , 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 C1, serotype D, seretype E, serotype F, and serotype G.
9. The method of claim 5 , 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 C1, 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 C1, serotype D, serotype E, serotype F, and serotype G.
10. The method of claim 5 , 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.