Method for quantification of neurotoxin

#The present invention relates to a method for quantitatively measuring the efficacy of a neurotoxin. Specifically, the present invention relates to a method for quantifying the effect of a neurotoxin with an index of a muscular relaxation in mammals. In some embodiment, the present invention provides a method for quantifying and defining “a potential” of a neurotoxin and/or “a diffusion reaction” from an administration site of a neurotoxin.

#There is known a neurotoxin including one produced by bacteria of Clostridium or by several fish and shellfish, typically a swellfish poison tetrodotoxin, a snake toxin alpha-bungarotoxin, and the like. These toxins, though different in their point of action, commonly block neurotransmission at the nerve end to thereby exhibit a muscular relaxation in mammals to which the toxins are inoculated. Among these, Clostridium toxin is a neurotoxin produced by bacteria of Clostridium which is divided into more than a hundred groups based on the form and function. For bacteria of Clostridium, Clostridium baratii, Clostridium butyricum, Clostridium botulinum, Clostridium tetani and the like are known. A botulinum toxin produced by Clostridium botulinum, aerobic Gram-positive bacteria, is the most lethal neurotoxin on earth. It is classified into seven serotypes, A, B, C, D, E, F and G, and the property of each serotype has been elucidated. The serotypes are distinguishable from each other by respective serotype-specific neutralizing antibodies. Depending on the serotypes, a botulinum toxin may vary in animal species it may affect, severity of paralysis it induces, duration of time of its action, and the like.

#An active center protein of a botulinum toxin has a molecular weight of about 150 kDa (NTX) as common in all the known seven serotypes. Any botulinum toxin, when produced from Clostridium botulinum, is a complex composed of NTX and a relevant nontoxic protein. A serotype A botulinum toxin is produced in a molecular form of either 900 kDa (LL toxin), 500 kDa (L toxin), or 300 kDa (M toxin) These LL, L and M toxins are called a botulinum toxin complex. These botulinum toxins are, upon absorption in the upper small intestine, degraded to release a nontoxic protein and an active center protein, NTX, under alkaline conditions (in a lymphatic vessel). The released NTX is then bound to a receptor at the nerve end at its C-terminus of a heavy chain and taken into neurons via the receptor. Then, it specifically cleaves a protein in the presynaptic membrane through a light chain zinc methaloendopeptidase activity and inhibits a calcium-dependant release of acetylcholine to thereby block neurotransmission at the synapse (Non-patent reference 1).

#FIG. 1 shows an administration site and sites for measurement of a compound muscle action potential (CMAP) in a non-human mammal (rat) to which a neurotoxin is to be administered in the quantification method of the present invention.

#(a): stimulating electrode (+);

#(b): stimulating electrode (−);

#The term “efficacy” as used herein refers to an index of an amount of a certain chemical drug, e.g. a neurotoxin, necessary for inducing an extent of a physiological or chemical activity. For instance, an efficacy of a botulinum toxin means an extent or duration of term of inhibiting release of acetylcholine from a target tissue. Alternatively, an efficacy of a botulinum toxin means an extent of change in contraction of a certain muscle caused by a certain dose of a neurotoxin.

#The term “potential” as used herein means a desirable efficacy of interest that is expressed numerically.

#The term “diffusion reaction” as used herein means spreading of a neurotoxin to the muscle other than the affected site in clinics that is expressed numerically.