Novel recombinant botulinum neurotoxins with increased duration of effect

Abstract

This invention relates to novel recombinant botulinum neurotoxins serotype A exhibiting both (i) an increased duration of effect and (ii) a high specific biological activity. These novel recombinant botulinum neurotoxins comprise at least two additional domains consisting of proline, alanine and an additional amino acid residue and at least one amino acid modification which is located at the alpha-exosite or at the beta-exosite of the light chain of the neurotoxin. The invention further relates to novel recombinant single-chain precursor botulinum neurotoxins and compositions comprising the recombinant botulinum neurotoxin with an increased duration of effect and a high specific biological activity.

1 .- 15 . (canceled) 
     
     
         16 . A method of treating a disease requiring improved chemodenervation, said method comprising the step of administering a recombinant neurotoxin to a patient in need thereof, said recombinant neurotoxin comprising:
 at least two domains wherein each domain comprises an amino acid sequence comprising at least 50 amino acid residues, wherein said amino acid sequence comprises at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, wherein the neurotoxin further comprises at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin,   wherein the said at least one amino acid modification is located at at least one position selected from D102, T109, K340, I348, N353, and K356 of the alpha-exosite, or at at least one position selected from G169, T220, P239, and S254 of the beta-exosite, and   wherein the recombinant neurotoxin causes both (i) an increased duration of effect relative to a wildtype botulinum neurotoxin serotype A and (ii) an increased specific biological activity relative to a botulinum neurotoxin serotype A comprising two domains consisting of proline, alanine and serine residues without said modifications in the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.   
     
     
         17 . The method of  claim 16 , wherein six amino acid modifications are located at the alpha-exosite at positions D102, T109, K340, I348, N353, K356. 
     
     
         18 . The method of  claim 17 , wherein the six amino acids are substituted as follows: D102F, T109R, K340M, I348L, N353M, K356R. 
     
     
         19 . The method of  claim 16 , wherein four amino acid modifications are located at the beta-exosite at positions G169, T220, P239, S254 
     
     
         20 . The method of  claim 19 , wherein the four amino acids are substituted as follows: G169I, T220R, P239M, S254T. 
     
     
         21 . The method of  claim 16 , wherein the recombinant neurotoxin comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 260 amino acid residues. 
     
     
         22 . The method of  claim 16 , wherein the recombinant neurotoxin is administered with a solvent or excipient. 
     
     
         23 . The method of  claim 16 , wherein the recombinant neurotoxin is administered with one or more pharmaceutically acceptable carriers. 
     
     
         24 . The method of  claim 16 , wherein the recombinant neurotoxin is administered through an injection. 
     
     
         25 . The method of  claim 16 , wherein the disease is selected from the group consisting of: cervical dystonia (spasmodic torticollis), blepharospasm, severe primary axillary hyperhidrosis, achalasia, lower back pain, benign prostate hypertrophy, chronic focal painful neuropathies, migraines and other headache disorders. 
     
     
         26 . A method of using a recombinant neurotoxin for cosmetic treatment, said method comprising the step of administering the recombinant neurotoxin to a subject, said recombinant neurotoxin comprising:
 at least two domains wherein each domain comprises an amino acid sequence comprising at least 50 amino acid residues, wherein said amino acid sequence comprises at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, wherein the neurotoxin further comprises at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin,   
       wherein the said at least one amino acid modification is located at at least one position selected from D102, T109, K340, I348, N353, and K356 of the alpha-exosite, or at at least one position selected from G169, T220, P239, and S254 of the beta-exosite, and
 wherein the recombinant neurotoxin causes both (i) an increased duration of effect relative to a wildtype botulinum neurotoxin serotype A and (ii) an increased specific biological activity relative to a botulinum neurotoxin serotype A comprising two domains consisting of proline, alanine and serine residues without said modifications in the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin. 
 
     
     
         27 . The method of  claim 26 , wherein six amino acid modifications are located at the alpha-exosite at positions D102, T109, K340, I348, N353, K356. 
     
     
         28 . The method of  claim 27 , wherein the six amino acids are substituted as follows: D102F, T109R, K340M, I348L, N353M, K356R. 
     
     
         29 . The method of  claim 26 , wherein four amino acid modifications are located at the beta-exosite at positions G169, T220, P239, S254 
     
     
         30 . The method of  claim 29 , wherein the four amino acids are substituted as follows: G169I, T220R, P239M, S254T. 
     
     
         31 . The method of  claim 26 , wherein the recombinant neurotoxin comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 260 amino acid residues. 
     
     
         32 . The method of  claim 26 , wherein the recombinant neurotoxin is administered with a solvent or excipient. 
     
     
         33 . The method of  claim 26 , wherein the recombinant neurotoxin is administered with one or more pharmaceutically acceptable carriers. 
     
     
         34 . The method of  claim 26 , wherein cosmetic treatment is selected from the group consisting of: treatment of wrinkles, treatment of crow's feet, treatment of glabella frown lines, reduction of masseter muscles, reduction of calves, and removing of facial asymmetries.