A botulinum toxin produced by Clostridium botulinum, anaerobic Gram-positive bacteria, is the most lethal neurotoxin on earth. It is classified into seven types, A, B, C, D, E, F and G, and the property of each type has been elucidated. The types are distinguishable from each other by respective type-specific neutralizing antibodies. Depending on the types, 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 types.
Any botulinum toxin, when produced from Clostridium botulinum, is a complex composed of NTX and relevant nontoxic proteins. A type A botulinum toxin is produced in a molecular form of either 900 kDa (LL toxin), 500 kDa (L toxin), or 300 kDa (M toxin) (FIG. 1). These botulinum toxins are separate to release NTX and NTNH (a nontoxic protein) under alkaline conditions (pH 7.2 or higher). By utilizing this property, it is possible to isolate NTX of 150 kDa (an active center protein that endows a neurotoxin with the activity; also called “S toxin”) alone. These LL, L and M toxins are called a botulinum toxin complex or a progenitor toxin. These botulinum toxins are, upon absorption in the upper small intestine, separate to release nontoxic proteins and a neurotoxin in a lymphatic vessel. The released neurotoxin 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 neuromuscular transmission at the synapse (Non-patent reference 1).
Although a botulinum toxin is a neurotoxin that may lead human to death in botulinum intoxication through blockage of systemic neuromuscular transmission, it may also be utilized as a remedy for treating a disease with a muscle overactivity such as e.g. dystonia by positively making use of its activity and by administering directly into the muscle of a patient suffering from the disease so that a local muscular tension may be relieved (Non-patent reference 2). For instance, a type A botulinum toxin complex (Allergan Inc., BOTOX; registered trademark) has been approved as a medicament for treating blepharospasm, strabismus, hemifacial spasm, and cervical dystonia, and for treating wrinkles at the middle of the forehead by the Food and Drug Administration (FDA). A seritype B botulinum toxin complex (Elan Pharmaceuticals, MYOBLOC; registered trademark) has also been approved as a medicament for treating cervical dystonia by FDA. It is said that a type A botulinum toxin has a higher potency and a longer duration of action as compared to types other than a type A botulinum toxin. An average duration of action of a type A botulinum toxin from its single muscular administration up till amelioration of symptoms is typically about 3 to 4 months.
In recent years, the action of botulinum toxin has been proved at (1) the neuromuscular conjunctions, (2) the ganglions of the autonomic nerves, (3) the terminal of the postganglionic parasympathetic nerve, (4) the terminal of the postganglionic sympathetic nerve, and (5) the pain receptive fibers. For the neuromuscular conjunctions of the skeletal muscles, the terminal of the muscarinic acetylcholinergic nerves is the main active site. Among the ganglions of the autonomic nerves, it is conceived that a direct action to the ganglions of the parasympathetic nerves is related to a clinic action. It is also reported that the action to the peripheral autonomic nerves causes inhibition of release of ATP, VIP (vasoactive intestinal polypeptide) or substance P or inhibition of the action of NO (nitric oxide) synthetase. It also became known that botulinum toxin is useful for alleviating pain. In this action, it is reported that botulinum toxin inhibits release of glutamic acid, substance P and CGRP (calcitonin gene-related peptide) (Non-patent reference 2). As such, botulinum toxin is a useful neuromuscular transmission blocking agent that inhibits release of various neurotransmitters at various nerves.
Currently, a biological potential of a therapeutic preparation of a botulinum toxin such as a type A botulinum toxin is indicated as a mouse LD50 unit. One LD50 is defined as LD50 which is, based on intraperitoneal administration to mice, defined as an amount with which a half number of mice tested dies. Namely, a potential is quantified with a level or an amount of a neurotoxin with which mice die as a consequence of respiratory muscular relaxation. One LD50, i.e. one unit, in mice of commercially available type A botulinum toxin complex (Allergan, Inc., BOTOX; registered trademark; containing 100 units) is about 50 pg.
Therapeutic preparations of botulinum toxin are available from Allergan Inc. (U.S.A.), Ipsen Limited (U.K.) or Elan Pharmaceuticals (Ireland). These commercially available therapeutic preparations of botulinum toxin consist of a purified botulinum toxin complex (LL toxin) alone in a molecular structure bound with relevant non-toxic proteins. In recent years, type A NTX preparations (Merz Pharma, Xeomin (registered trademark), Germany) comprising no non-toxic proteins were sold in 2005, similar other preparations underwent clinical trials in the U.S.A. and development of next-generation preparations has actively been done.
The currently commercially available therapeutic preparations of type A botulinum toxin, i.e. BOTOX (registered trademark) from Allergan Inc. and Dysport (registered trademark) from Ipsen Limited, are consisted of a botulinum toxin complex (LL toxin) comprising as its component Haemagglutinin (HA) protein such as HA17, HA34, and HA70 (HA-positive).
On the other hand, a botulinum toxin isolated from patients suffering from infant botulism in 1990, though belonging to type A, is consisted of M toxin with no HA proteins (HA-negative). Type A Clostridium botulinum that produces M toxin with no HA protein has been first identified in Japan in 1986 from patients suffering from infant botulism (Non-patent reference 3). The clinically isolated strains include Kyoto-F, Chiba-H, Y-8036, 7I03-H, 7I05-H and KZ1828. When compared with the other types A to G of botulinum toxins, a botulinum toxin from Clostridium botulinum that causes infant botulism is a peculiar neurotoxin distinct from any types of these toxin molecules.
From the genetic point of view, a genetic mechanism of Clostridium botulinum as infant botulism pathogen is different from those of the other types of botulinum toxin. Most of the conventional botulinum toxins, typically type A botulinum toxin, has been seen as a botulinum toxin complex having Haemagglutinin (HA) protein as a component thereof. Genes coding for HA proteins such as HA17, HA34 and HA70 are included in neurotoxin genes of types A, B, C, D and G Clostridium botulinum but are completely absent in those of Clostridium botulinum as infant botulism pathogen. Also, genes of Clostridium botulinum as infant botulism pathogen include a regulator gene such as p47 (Non-patent reference 4). Besides, it was shown that a sequence of the NTNH protein of botulinum toxin produced by Clostridium botulinum as infant botulism pathogen is a miscellany, i.e. a mosaic, of non-toxic non-HA protein NTNH genes of type C and type A (Non-patent reference 5).
Furthermore, when botulinum toxin produced by Clostridium botulinum as infant botulism pathogen is compared with the conventional type A botulinum toxin comprising HA proteins, biochemical properties of the purified botulinum toxins are remarkably different from each other. The conventional type A botulinum toxin comprises the NTNH protein and at least three HA proteins (HA17, HA34 and HA70) whereas botulinum toxin produced by Clostridium botulinum as infant botulism pathogen comprises the NTNH protein alone but lacks the HA proteins (Non-patent reference 6). As for neurotoxin molecules per se, a molecular weight is distinct from each other in that a heavy chain of the conventional type A botulinum toxin is 93 kDa whereas botulinum toxin produced by Clostridium botulinum as infant botulism pathogen is 101 kDa. They also show different protease reactivity (Non-patent reference 7). The amino acid sequences of these two isotypes of the botulinum toxins are different by 89.9% as a whole and, in particular, there is great difference in the heavy chain regions, 109 among 847 amino acids (difference of 13%). On the other hand, it is reported that the light chains are different by 95.1% (Non-patent reference 8).
On the other hand, a problem has been presented that repetitive administration of botulinum toxin may diminish its efficacy. This phenomenon is thought to be due to production of antibodies against the toxin. It is pointed out that, as one of the causes, Haemagglutinin (HA) contained in therapeutic preparations has an adjuvant activity for antibody production (Non-patent reference 9).
For a highly purified botulinum toxin, it was formerly reported by Tse C K., et al. (Non-patent reference 10) and also in WO1996/11699 (Patent reference 1) as to a process for purification (p. 6, line 9 to p. 7, line 2) and pharmaceutical compositions (p. 11, Table 2).    Patent reference 1: WO1996/11699    Non-patent reference 1: Jankovic J. et al., Curr. Opin. Neurol., (7): p. 358-366, 1994    Non-patent reference 2: Ryuji Kaji et al., “Dystonia and botulinum therapy”, Shindan-To-Chiryosha, 2005    Non-patent reference 3: Sakaguchi G. et al., Int. J. Food Microbiol., 11: p. 231-242, 1990    Non-patent reference 4: Kubota T. et al., FEMS Microbiology letters, 158: p. 215-221, 1998    Non-patent reference 5: Kubota T. et al., Biochem. Biophys. Res. Commun., 224(3): p. 843-848, 1996    Non-patent reference 6: Sakaguchi G. et al., Int. J. Food Microbiol. 11: p. 231-242, 1990    Non-patent reference 7: Kozaki S. et al., Microbiol. Immunol. 39(10): p. 767-774, 1995    Non-patent reference 8: Cordoba J. et al., System. Appl. Microbiol. 18: p. 13-22, 1995    Non-patent reference 9: Arimitsu H. et al., Infect. Immun., 71(3): p. 1599-1603, 2003    Non-patent reference 10: Tse C K. et al., Eur. J. Biochem., 122(3): p. 493-500, 1982