Abobotulinum toxin A: Science and clinical usage

With more than 6.5 million treatments each year in the United States reported by the American Society of Plastic Surgeons, and perhaps more than 10 million treatments worldwide, botulinum neurotoxin (BoNT) administration is by far the most common aesthetic procedure performed in the world and has truly revolutionized the field of aesthetic medicine. Since the US Food and Drug Administration (FDA) first approved the use for the treatment of glabellar lines, many other aesthetic and therapeutic applications have been tried for aging skin of the face and neck, but none has surpassed the efficacy and durability of BoNT treatments.

Botulinum neurotoxin A (BoNT-A) is the most commonly used serotype of BoNT for both clinical and aesthetic applications. Though all serotypes are synthesized as a continuous 150-kDA protein, biological activity requires posttranslational proteolysis, or nicking, which cuts the BoNT polypeptide into two separate moieties of approximately 100 kDa (the heavy chain [Hc]) and 50 kDa (the light chain [Lc]). The Hc and Lc remain bound together by a single disulfide bridge, although they carry out separate functions at the nerve terminal. One part of the Hc is bound by protein and ganglioside receptors on the presynaptic nerve terminals so that the bound molecule enters the nerve terminal by endocytosis in an endosome. The second part of the Hc then forms a channel in the endosomal membrane as the disulfide bond is reduced and the Lc travels to the cytosol, where it cleaves a portion of the protein receptor (soluble N -ethylmaleimide-sensitive factor attachment protein receptor [SNARE]) complex, thus blocking the release of acetylcholine and hence nerve transmission. The molecular target for all BoNT-A in the presynaptic terminal is the synaptosomal-associated protein, 25 kDa (SNAP-25), regardless of commercial preparation ( Fig. 4.1 ).

Open full size image

The multiple steps in the mechanism of action of botulinum toxin type A: (1) After binding, the 150-kDa core neurotoxin is internalized into the neuron via a process of endocytosis. (2) The endosome is acidified, which facilitates (3) the translocation of the light chain into the neuronal cytosol. (4) Once in the neuronal cytosol the light chain of type A botulinum toxin (yellow) cleaves the membrane-associated SNAP-25 (brown) , one component of the SNARE protein complex. Another neurotoxin serotype, type B, botulinum toxin cleaves a different protein known as synaptobrevin or vesicle-associated membrane protein (VAMP; green) . ACh, Acetylcholine; SNARE, soluble N -ethylmaleimide-sensitive factor attachment protein receptor.

BoNT-A exists in nature as a complex with a collection of surrounding protective proteins. These are present in both onabotulinumtoxinA (OnaBoNT/Vistabel; Allergan, Inc., Irvine, California) and abobotulinumtoxinA (AboBoNT/Azzalure; Ipsen/Galderma) but apparently not in incobotulinumtoxinA (IncoBoNT, Merz Aesthetics). The protective proteins are known as neurotoxin-associated proteins (NAPs). NAPs are composed of four distinct hemagglutinin proteins and a nontoxic, nonhemagglutinin protein (NTNH). The NAPs are also synthesized by the clostridial bacterium simultaneously with the active BoNT molecule. The purpose of these NAPs was originally thought to be to shield the BoNT neurotoxin from potential destruction by gastric acid, the first environment the BoNT encounters when ingested from a contaminated foodstuff. However, structural data on the toxin complex indicate the BoNT molecule is actually isolated from many NAPs and only closely associated with one protein, the NTNH. The structure of the NTNH is remarkably similar to that of the neurotoxin molecule itself. Because the conditions under which the bacteria exist can vary widely in nature, from rich protein sources to sparse minimal environments, there are three sizes of the BoNT-A progenitor complexes that are known to exist: 300, 500, and 900 kDa. Different methods of neurotoxin isolation and purification have shown these different molecular weight complexes, but early data from minimally manipulated, BoNT-rich sources have shown their existence to be correct.

OnaBoNT has been reported as a complex of varying molecular weight. The chromatographic method of purifying AboBoNT also produces a complex. However, any molecular weight differences between the products, if indeed they exist, are irrelevant because all the products are now known to exist as free BoNT in the vial, immediately after reconstitution. Previous theories that certain product BoNT complexes confer a more rapid diffusion in tissue (ostensibly based on their smaller size) have now been dispelled due to the biophysical data. At this time, there is no convincing evidence to support any argument of clinical differences due to physical product differences, and there is scientific evidence to support the similarities in the performance of the two complexed neuroproteins. Both in vivo are base 150 kDa molecules.

Before the BoNT-A neurotoxin can become active, the NAPs must release the active BoNT-A 150-kDa molecule from the progenitor complex. This occurs with a change in environment to a physiologic pH. A more recent study by Merz Pharmaceuticals, summarizing some years of collected experimental data, reported that the naked neurotoxin was released from the associated complex in less than 1 minute with a change to physiologic pH. This occurs with both OnaBoNT and AboBoNT. The investigations demonstrated release of the neurotoxin probably occurs in the vial during reconstitution with sterile saline, well before injection and tissue spread.

These biochemical studies do have implications for clinical use in understanding the efficacy and safety of each product. With the neurotoxin protein of 150 kDa released before injection, the active BoNT-A molecules are stoichiometrically similar and no difference in diffusion should be expected because complex size is now known to be irrelevant. In the authors’ opinion, the difference seen with the products has to do with dosage-unit differences (and potentially volume of reconstitution/volume of injection). Eisele (Merz Pharmaceuticals, the manufacturer of incobotulinumtoxinA, a noncomplexed 150-kDa protein), using standard stability tests, found no difference in any of the three commercially available neurotoxins as to potency loss or shelf life.

The issue of diffusion between BoNT-A products due to complex size has been put to rest. The conflicting studies of hyperhidrosis “halos” on the forehead by different companies are now found to be due to dose-conversion differences. The diffusion differences were the result of differences in dosage and volume injected and not an inherent difference in the molecules. The most important differences between OnaBoNT and AboBoNT are the dosage or activity units defined by the respective manufacturers: OnaBoNT units (often called Botox units) for OnaBoNT and Speywood units (s.U) for AboBoNT. Both use the LD50 potency test on mice to define a potency unit, but there are differences in the experimental designs of the assays, making the units non-equivalent. Indeed, in the original small animal study comparing the assay methodology of each product, Hambleton & Pickett showed that, when tested in the AboBoNT assay, the LD50 of OnaBoNT was significantly higher than the labeled potency, indicating that the AboBoNT assay “recovered” more neurotoxin than the OnaBoNT assay. Conversely, when AboBoNT was assayed using the OnaBoNT method, significantly less neurotoxin was measured, indicating the OnaBoNT assay method actually inactivated the neurotoxin present. The assay difference was identified as the diluent used, which significantly affected product neurotoxin recovery for measurement. This subject of assay methodology has been debated for many years since, with each manufacturer claiming that their method is the most appropriate and scientifically accurate. The subject can only be dealt with conclusively by the adoption of both an international standard unit of neurotoxin and an accompanying assay method, but attempts to introduce these in the early 2000s were not successful. Therefore there is no direct conversion factor between units, and mention of this has been discouraged by both the manufacturers and, most importantly, forbidden by the regulatory authorities worldwide. Thus we do not have a direct conversion factor between the toxin products. Nevertheless, practitioners have sought to define a conversion factor to guide the novice injector when transitioning from one toxin to the other, for a given application.

A number of attempts have been made by the clinical community to define a conversion number. A summary of recent dosage studies places the ratio between 1:2 and 1:2.5 (OnaBoNT to AboBoNT units), as discussed previously. Multiple other studies, both therapeutic and aesthetic, have suggested ratios of 1:2.5, 1:3, and 1:4 for bioequivalence. An earlier review concluded that a 1:4 ratio was too high, and a 1:3 ratio approached bioequivalence, although the included studies suggested that an even lower ratio might be more appropriate. An independently funded, double-blind study of AboBoNT versus OnaBoNT for the treatment of glabellar lines found a longer duration of action as assessed by electromyography studies with AboBoNT when used at a 1:3 ratio. Though the aesthetic clinical trials of AboBoNT for efficacy and safety were performed at a (theoretical) ratio of 1:2.5, other recent studies have suggested a ratio of 1:3 provides AboBoNT with a greater longevity and equivalent safety to OnaBoNT. Thus we can see that dosage is really a determining and defining factor in efficacy. The dosage should be determined by the physiologic response, using the individual product units and manufacturers’ recommendations, rather than by comparing product dosage units between the products.

As we correlate these data to clinical practice, we must realize there are subtle differences in the properties of the BoNT-A products. At this point, exact data on the clinical composition, diffusion properties, and potencies are not completely known. For example, do the excipients play any role in these properties and, if so, why? Until we have a more complete understanding of these differences, the clinician should think and treat each of these products independently and avoid relying on conversion factors. If asked, the authors recommend a conversion factor of 1:2.5, which has become the most commonly quoted unit dose ratio among experienced injectors. The multiple studies that underpinned the FDA-approved dosages for glabellar lines (50 s.U of AboBoNT and 20 units of OnaBoNT) demonstrated good efficacy and safety from the two BoNT-A products, again supporting the 1:2.5 ratio as a starting point for aesthetic applications.

According to prescribing information in the package insert, the AboBoNT vial with 300 s.U of neurotoxin should be reconstituted with 2.5 mL of unpreserved saline. The FDA clinical studies, which used 500 s.U per vial, were reconstituted with 2.5 mL. The equivalency would be 1.5 mL per 300 s.U vial. Other dilutions used are 3.0 mL per 300 s.U vial. Though the package insert recommends nonpreserved saline, most injectors prefer preserved saline, which has been shown to have equal efficacy with less pain.

Dilution (actually, the reconstitution volume) is one important factor in evaluating FoE. Does a greater reconstitution volume (and hence a greater injection volume to deliver the same number of units) create a greater FoE? The data on this subject are ambiguous. One study shows clearly that the same number of units injected in different volumes per injection point have no difference in effect. Other studies show the contrary. The likelihood is that operating within a quite wide range of dilution seems to have no effect but, outside of that range (which has yet to be firmly established), dilution volume/volume of injection per point may well be affected. One author (GM) prefers the 3.0 mL dilution, thus making it easy to calibrate the unit dosage in the 1-mL syringes used. At a 3 mL dilution for a 300 s.U vial, a concentration of one unit per 0.01 mL can be drawn up corresponding to the one-unit gradation on an insulin syringe. This also corresponds to the concentration of OnaBoNT, giving a similar one-unit gradation on the insulin syringe when diluted at 1 mL per 100 OnaBoNT unit vial.