Nuclei density and nuclei area methods for determining the effects of a botulinum toxin on muscles

Abstract

Methods for determining the effect of a Clostridial neurotoxin on muscle are disclosed. In particular, methods for determining a potency and/or diffusion of a neurotoxin based on a nuclear index and/or an amount of muscle atrophy are disclosed. The methods may be used to distinguish two different Clostridial neurotoxins. In certain embodiments, the neurotoxins are obtained from Clostridium botulinum.

1 . A method for distinguishing between two or more  Clostridial  neurotoxins, comprising the steps of: 
 administering a first Clostridial neurotoxin to a first muscle of a first mammal;    administering a second Clostridial neurotoxin to a second muscle of second mammal, wherein the first and second muscles are the same type of muscle;    comparing a resultant neurotoxic effect that each of said first and second Clostridial neurotoxin has on each of said first and second muscle by determining and comparing a nuclear index determined for each of said first and second muscle; and    determining potency of the first and second Clostridial neurotoxins by constructing a dose response curve.    
   
   
       2 . The method of  claim 1 , wherein the first and second  Clostridial  neurotoxin are botulinum neurotoxins selected from the group consisting of botulinum neurotoxin types A, B, C, D, E, F, G and mixtures thereof.  
   
   
       3 . The method of  claim 1 , wherein the first Clostridial neurotoxin is a first botulinum neurotoxin type and the second Clostridial neurotoxin is a second botulinum neurotoxin type different from the first botulinum neurotoxin type.  
   
   
       4 . The method of  claim 1 , wherein said first and second mammals are selected from the group consisting of humans, rats, rabbits, mice and dogs.  
   
   
       5 . The method of  claim 1 , wherein the first muscle and second muscle are gastrocnemius muscles.  
   
   
       6 . The method of  claim 1 , further comprising the step of comparing the nuclear index for each of said first and second muscle to a muscle of the same type to which no Clostridial neurotoxin was administered.  
   
   
       7 . The method of  claim 1 , wherein administration of the first and second Clostridial neurotoxin is achieved by intramuscular injection, needless injection or placement of a neurotoxin-containing implant.  
   
   
       8 . A method for distinguishing a first Clostridial neurotoxin from a second Clostridial neurotoxin, comprising the steps of: 
 quantifying muscle atrophy of a first muscle in which the first  Clostridial  neurotoxin has been administered to determine potency of the first  Clostridial  neurotoxin;    quantifying muscle atrophy of a second muscle in which the second Clostridial neurotoxin has been administered to determine the potency of the second Clostridial neurotoxin, wherein the second muscle is a same muscle type as the first muscle; and    comparing the potency of the first Clostridial neurotoxin to the potency of the second Clostridial neurotoxin, wherein muscle atrophy is determined by a) staining muscle nuclei present in a first muscle sample and a second muscle sample taken from each of said first and second muscle, respectively, wherein the muscle nuclei have a size; (b) measuring a first nuclei density and/or nuclei area of the muscle sample from the first muscle, and a second nuclei density and/or nuclei area of the muscle sample from the second muscle, by measuring the size of each of one or more stained muscle nuclei, and; (c) comparing the first nuclei density and/or nuclei area with a second nuclei density and/or nuclei area, wherein a difference in potency is effective in distinguishing the first Clostridial neurotoxin from the second Clostridial neurotoxin.    
   
   
       9 . The method of  claim 8 , wherein the first Clostridial neurotoxin and the second Clostridial neurotoxin are the same type of neurotoxin obtained from different strains of  Clostridial  bacteria.  
   
   
       10 . The method of  claim 9 , wherein the first Clostridial neurotoxin and the second Clostridial neurotoxin are both botulinum toxin type A.  
   
   
       11 . The method of  claim 8 , wherein the first Clostridial neurotoxin and the second Clostridial neurotoxin are different types of botulinum toxin.  
   
   
       12 . The method of  claim 11 , wherein the first Clostridial neurotoxin is botulinum toxin type A, and wherein the second Clostridial neurotoxin is a botulinum toxin selected from the group consisting of botulinum toxin types B, C 1 , D, E, F, and G.  
   
   
       13 . The method of  claim 8 , wherein the first muscle and the second muscle are gastrocnemius muscles.  
   
   
       14 . The method of  claim 13 , wherein the first muscle and the second muscle are rodent muscles.  
   
   
       15 . The method of  claim 8 , wherein administration of the first  Clostridial  neurotoxin and the second Clostridial neurotoxin is to a midbelly area or a lateral head of the first or second muscle.  
   
   
       16 . The method of  claim 8 , wherein the comparing step includes determining an ED50 of the first Clostridial neurotoxin and an ED50 of the second Clostridial neurotoxin.  
   
   
       17 . The method of  claim 8 , wherein the first muscle and the second muscle are muscles of different animals.  
   
   
       18 . The method of  claim 8 , further comprising repeating administration of the first Clostridial neurotoxin to a muscle in each of a plurality of animals, and repeating administration of the second Clostridial neurotoxin to a muscle in each of a plurality of different animals.  
   
   
       19 . The method of  claim 8 , wherein administration of the first and second Clostridial neurotoxin is achieved by intramuscular injection, needless injection or placement of a neurotoxin-containing implant.