Claims for Patent: 8,013,113

Summary for Patent: 8,013,113

Title:	FRET protease assays for clostridial toxins
Abstract:	The present invention provides clostridial toxin substrates useful in assaying for the protease activity of any clostridial toxin, including botulinum toxins of all serotypes as well as tetanus toxins. A clostridial toxin substrate of the invention contains a donor fluorophore; an acceptor having an absorbance spectrum overlapping the emission spectrum of the donor fluorophore; and a clostridial toxin recognition sequence that includes a cleavage site, where the cleavage site intervenes between the donor fluorophore and the acceptor and where, under the appropriate conditions, resonance energy transfer is exhibited between the donor fluorophore and the acceptor.
Inventor(s):	Steward; Lance E. (Irvine, CA), Fernandez-Salas; Ester (Fullerton, CA), Aoki; Kei Roger (Coto de Caza, CA)
Assignee:	Allergan, Inc. (Irvine, CA)
Application Number:	12/620,434
Patent Claims:	1. A botulinum toxin substrate, comprising: a) a donor fluorophore; b) an acceptor having an absorbance spectrum overlapping the emission spectrum of said donor fluorophore, wherein the acceptor is an acceptor fluorophore; and c) a botulinum toxin type B recognition sequence comprising a botulinum toxin type B P5-P4-P3-P2-P1-P1'-P2'-P3'-P4'-P5' cleavage site sequence, said botulinum toxin type B P5-P4-P3-P2-P1-P1'-P2'-P3'-P4'-P5' cleavage site sequence intervening between said donor fluorophore and said acceptor; wherein either of said donor fluorophore, said acceptor, or both said donor fluorophore and said acceptor are genetically encoded; and wherein, under the appropriate conditions, resonance energy transfer is exhibited between said donor fluorophore and said acceptor fluorophore. 2. The substrate of claim 1, wherein said donor fluorophore is genetically encoded. 3. The substrate of claim 1, wherein said acceptor is genetically encoded. 4. The substrate of claim 1, wherein said donor fluorophore and said acceptor are genetically encoded. 5. The substrate of claim 1, wherein said donor fluorophore is a blue fluorescent protein, a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein or a red fluorescent protein. 6. The substrate of claim 1, wherein said acceptor is a fluorophore. 7. The substrate of claim 6, wherein said acceptor fluorophore is a blue fluorescent protein, a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein or a red fluorescent protein. 8. The substrate of claim 1, wherein said acceptor is a non fluorescent acceptor. 9. The substrate of claim 8, wherein said non fluorescent acceptor is a heme protein. 10. The substrate of claim 1, wherein said botulinum toxin type B P5-P4-P3-P2-P1-P1'-P2'-P3'-P4'-P5' cleavage site sequence comprises at least six consecutive residues of synaptobrevin, said six consecutive residues comprising Gln-Phe. 11. The substrate of claim 10, wherein said botulinum toxin type B P5-P4-P3-P2-P1-P1'-P2'-P3'-P4'-P5' cleavage site sequence comprises at least six consecutive residues of human synaptobrevin isotype 2, said six consecutive residues comprising Gln76-Phe77. 12. The substrate of claim 10, wherein said botulinum toxin type B P5-P4-P3-P2-P1-P1'-P2'-P3'-P4'-P5' cleavage site sequence comprises SEQ ID NO: 3. 13. The substrate of claim 10, wherein said botulinum toxin type B recognition sequence comprises residues 55 to 94 of SEQ ID NO: 4, residues 60 to 94 of SEQ ID NO: 4, residues 60 to 88 of SEQ ID NO: 4. 14. The substrate of claim 1, wherein said donor fluorophore and said acceptor are separated by at most 6 residues, at most 8 residues, at most 10 residues, at most 20 residues, at most 30 residues, or at most 40 residues. 15. The substrate of claim 1, wherein said substrate can be cleaved with an activity of at least 1 nanomoles/minute/milligram toxin, at least 20 nanomoles/minute/milligram toxin, at least 50 nanomoles/minute/milligram toxin, at least 100 nanomoles/minute/milligram toxin, or at least 150 nanomoles/minute/milligram toxin.

Details for Patent 8,013,113

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Solstice Neurosciences, Llc	MYOBLOC	rimabotulinumtoxinb	Injection	103846	12/08/2000		2021-08-28