Structure and mechanism of action

Botulinum toxin is produced by various species of gram-positive, spore-forming bacilli of the genus, Clostridium , but chiefly from strains of C botulinum . Seven serotypes of BTX have been identified to date, which are labeled alphabetically, A to G. Many of these possess additional subtypes; for example, there are 4 described distinct subtypes of serotype A toxins. All of the serotypes have a similar chemical structure and, except subtype C ₂ , are neurotoxins. Each botulinum toxin is initially synthesized as a continuous 150-kDa gene product. Biologic activity requires posttranslational proteolysis, or nicking, which clips the BTX polypeptide into 2 separate moieties of 100 kDa and 50 kDa in size. This process results in a heavy chain and light chain that remain covalently bound by a single disulfide bridge. In human tissue, the heavy chain is recognized by receptors on presynaptic nerve terminals and the active di-chain neurotoxin is endocytosed. Upon acidification of the endosome, the heavy chain forms a channel in the endosomal membrane and the disulfide bond between the chains is reduced. The liberated light chain translocates to the cytosol where its zinc-dependent protease domain cleaves a member of the soluble NSF attachment protein receptor (SNARE) complex. The loss of any of these proteins abrogates exocytosis of presynaptic acetylcholine-rich vesicles, thereby eliminating signal conduction in afflicted cholinergic neurons. The various serotypes have specific molecular targets: SNAP-25 for toxins A and E; VAMP/synaptobrevin for B, D, F, G; and both SNAP-25 and syntaxin for C.

Botulinum toxin serotype A (BTX-A), the serotype in both onabotulinumtoxinA (Botox/Vistabel; Allergan, Inc) and abobotulinumtoxinA (Dysport), naturally exists as a complex with a surrounding coat of catalytically inactive, protective proteins, known collectively as neurotoxin- associated proteins (NAPs). NAPs, including 4 distinct hemagglutinin proteins and a nontoxic nonhemagglutinin protein, are synthesized by the clostridial bacterium and shield the neurotoxin from potential destruction by gastric acidity. Clostridial cultures yield 3 sizes of progenitor complexes: 300 kDa, 500 kDa, and 900 kDa. Allergan asserts their proprietary purification method for onabotulinumtoxinA (OnaA) isolates the 900-kDa complex exclusively. It has been suggested that the column chromatography purification method used to isolate abobotulinumtoxinA (AboA) results in a heterogeneous mixture of the 3 progenitor complexes, with the smaller complexes conferring more rapid diffusion in tissues. These claims have been disputed by Ipsen and admittedly the authors were unable to identify any convincing studies that establish a direct correlation between tissue diffusion properties and complex size.

Pharmacologic activity of BTX-A requires dissociation of the progenitor complex and release of the active BTX-A 150-kDa monomer. This process does occur at physiologic pH but the kinetics of the dissociation are not fully clarified. Some have suggested that dissociation is nearly immediate. A recent study conducted by Merz Pharmaceuticals (manufacturers of one of the new naked neurotoxin agents discussed later) reported that the 150-kDa toxin was released from both OnaA and AboA in less than 1 minute at neutral pH. The investigators suggested that dissociation may occur in the vial even before injection. The relative kinetics of dissociation versus diffusion have implications for the safety profiles of the various formulations of BTX-A in current or future clinical use and, therefore, these remain contentious issues for their respective manufacturers.

In addition to the well-characterized effects previously detailed, a growing body of evidence indicates that BTX-A targets some noncholinergic neurons. Inhibition of neurotransmitters, such as substance P, glutamate, and calcitonin gene-related peptide, has been demonstrated, supplying the mechanistic underpinning for the use of BTX-A in the treatment of chronic pain, one of its most exciting new nonaesthetic applications. It is hoped that future research in this area will establish novel therapeutic indications for BTX-A.