Basic science: Abobotulinum toxin A

5 Basic science

Abobotulinum toxin A

With more than 11 million injections since 2001, botulinum toxin (BoNT) administration is by far the most common cosmetic procedure performed in the United States and has truly revolutionized the field of cosmetic medicine. Since the FDA first approved its use for the treatment of glabellar lines, many other aesthetic applications have been tried for aging skin of the face and neck.

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 post-translational proteolysis, or nicking, which snips the BoNT polypeptide into two separate moieties of approximately 100 kDA and 50 kDA. The heavy chain and light chain remain bound together by a single disulfide bridge although they carry out separate functions at the nerve terminal. One part of the heavy chain is bound by receptors on the presynaptic nerve terminals so that the bonded molecule enters the nerve terminal by endocytosis. The second part of the heavy chain then forms a channel in the endosomal membrane as the disulfide bond is reduced and the light chain travels to the cytosol where it cleaves a portion of the protein receptor (SNARE) complex thus blocking the release of acetylcholine and hence nerve transmission. The molecular target for all BoNT-A is SNAP-25, regardless of commercial preparation.

BoNT-A exists in nature as a complex with a shell of surrounding protective proteins. These are present in both onabotulinumtoxinA (onabotA) (Botox^® / Vistabel^®; Allergan Inc., Irvine, CA) and abobotulinumtoxinA (abobotA) (Dysport^® / Azzalure^®; Ipsen / Galderma). These are known as neurotoxin-associated proteins (NAPS). NAPS are composed of four distinct hemaglutinin proteins and a non-toxic non-hemaglutinin protein. NAPS are also synthesized by the clostridial bacterium. The purpose of this protective protein coat is to shield the neurotoxin from potential destruction by gastric acid, the first environment the BoNT encounters when ingested from a contaminated foodstuff. As clostridial culture conditions can vary, there are three sizes of the progenitor complexes: 300 kDa, 500 kDa, and 900 kDa. Different methods of neurotoxin isolation and purification have produced these different molecular weight complexes.

Lietzow et al reported onabotA to be a 925 kDa complex. The chromatography method of purifying abobotA also produces a complex. Any molecular weight differences are irrelevant since all the products are now known to exist as free BoNT in the vial, before even reconstitution. Whether any clinical differences occur due to differences in the products is a point of debate between the manufacturing companies. Previous theories that certain product BoNT complexes confer a more rapid diffusion in tissue have now been dispelled as a result of recent biophysical data. At this point, there is no convincing evidence to support any argument of clinical differences due to product differences and in fact there is scientific evidence to support the similarities in the performance of the two complexed neuroproteins.

Before the BoNT-A neuroprotein can become active, the NAPS must release the active BoNT-A 150 kDa neuroprotein from the progenitor complex. This occurs with a change in environment to a physiological pH. The issue arises as to when this occurs. Earlier pilot in vitro studies by Eisele & Taylor showed the timescale of release varied from immediate with pH change to a delay from minutes to hours, depending on the product serotype with BoNT type B taking longest. A recent study by Merz Pharmaceuticals, reported by Eisele et al in 2011, found that the naked neuroprotein was released from its associated complex in less than 1 minute with a change to physiological pH; this occurs with both onabotA and abobotA. The investigation implied that release of the naked neuroprotein probably occurs in the vial during reconstitution, well before injection and tissue spread. However, definitive studies on complex dissociation by the same group were also published in 2011 and clearly demonstrated that all the BoNT type A products existed as free neurotoxin within the vials even before reconstitution. In other words, the manufacturing processes for the products all released the free BoNT neurotoxin during manufacture.

These biochemical studies do have implications for clinical usage in understanding efficacy and safety of each product. With the neurotoxin protein of 150 kDa released before injection, the active toxins are stoichiometrically similar and one would not expect a difference in diffusion since complex size is now shown to be irrelevant. It is the authors’ opinion that the difference seen with the products has to do with dosage-unit differences and volume reconstitution. The relative kinetics of dissociation versus diffusion have implications for the safety profiles of the various formulations of BoNT-A in current or future clinical use, and therefore these remain contentious issues for their respective manufacturers, as reported by Pickett in 2009.

An additional issue emerged concerning the stability of the neuroprotein as related to complex size. In 2009 Dr Eisele (of Merz Pharmaceuticals, the manufacturer of incobotulinumtoxinA (incobotA), a non-complexed 150 kDa protein) using standard stability tests had found no difference in any of the three commercially available neurotoxins with respect to potency loss or shelf life.

Another myth surrounding botulinum neurotoxin type-A formulations is that diffusion is product specific, and that some products have a greater area of diffusion throughout the target muscles than others. That one botulinum neurotoxin type-A product may diffuse to a greater extent than another would again have safety implications. A 2007 study by Trindade de Almeida and colleagues compared the diffusion of 3 U per injection of Botox^® (total of four injections) with Dysport^® given in dose ratios of 1 : 2.5, 1 : 3, and 1 : 4 to randomized patients with forehead hyperhidrosis. Dysport^® was found to diffuse further from the injection site than Botox^® in a dose-dependent manner. The so-called ‘diffusion differences’ seen between the products were, in fact, due simply to dose differences. These studies on dose, not diffusion, differences are supported by others including Wohlfarth et al and Hexsel et al. Wohlfarth and co-workers reported that diffusion of BoNT type-A beyond the target muscles occurs with both Botox^® and Dysport^® when injected into the feet, and that diffusion was associated with injection volume – the greater the volume, the greater the diffusion – and diffusion beyond the injection site is not an inherent characteristic of botulinum neurotoxin type-A agents. Likewise, the 2008 comparison of Botox^® and Dysport^® by Hexsel and colleagues reported comparable action halos for both products when used for the treatment of forehead wrinkles. This study utilized a ration of 1 : 2.5 U injected with the same volume and at the same depth, and both products were found to be safe and predictable.

The most important differences between onabotA and abobotA are the dosage or activity units defined by the respective manufacturers: Botox units (BU) for onabotA and Speywood units (s.U) for abobotA. Both use the LD₅₀ test on mice to define a unit, but there are differences in the experimental designs of the assays making the units non-equivalent. The s.U (Speywood or Dysport unit assay) has a greater sensitivity indicating less toxin needed to kill a mouse. Indeed, in a small study by Hambleton & Pickett, it was shown that, when tested in the Dysport^® assay, the LD₅₀ of Botox^® is achieved with 0.32 B.U (68% less product than that required for LD₅₀ in the Botox^® assay). Thus a Speywood unit corresponds to a smaller quantity of active toxin than a Botox unit. There is no direct conversion factor between units and this has been discouraged by the manufacturers. Thus we do not have a direct conversion factor between the two toxins. Nevertheless, practitioners have sought to define a conversion factor to guide the novice injector when changing from one toxin to the other for a given application.

A number of attempts have been made to define a conversion number. A summary of recent dosage studies places the ratio between 1 : 2 and 1 : 2.5. Multiple other studies – both therapeutic and cosmetic – have suggested ratios of 1 : 2.5, 1 : 3, and 1 : 4 for bioequivalence. An earlier review by Sampaio and colleagues 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 by Karsai and co-workers of Dysport^® versus Botox^® for the treatment of glabellar lines found a longer duration of action as assessed by electromyographic studies with Dysport^® utilized at a 1 : 3 ratio. This led the authors to conclude the bioequivalence ratio was less than 1 : 3. Though these clinical trials for efficacy and safety were performed at a ratio of 1 : 2.5, other recent studies by Nettar et al and Kerscher et al have suggested a ratio of 1 : 3 provides abobotA with a greater longevity and equivalent safety to Ona-A. At a lower dosage (1 : 25) the study by Lowe and colleagues found a greater longevity to onabotA for glabellar lines. Thus we can see that dosage is really a determining factor in efficacy. The dosage should be determined by physiological response using the individual units rather than by comparing product dosage units.

If we re-examine the safety studies previously presented one can see that the halos of diffusion (called the fields of effect) found by both Almeida and Hexsel are related more to the dosage equivalents than to intrinsic differences in the products.

As we correlate this data with clinical practice, we must realize there are subtle differences in the properties of the BoNT-A products. At this point, exact data on clinical composition, diffusion properties. and potencies are not fully known. 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 conversions factors. 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 Dysport^® and 20 B.U of Botox^®) demonstrated comparable efficacy with the two BoNT-A products, further supporting the 1 : 2.5 ratio as a starting point for aesthetic applications.

According to prescribing information in the package insert, the abobotulinumtoxin vial with 300 s.U of neurotoxin should be reconstituted with 2.5 mL of unpreserved saline. The FDA clinical studies, which had 500 s.U per vial, were reconstituted with 2.5 mL s.U, and 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 non-preserved saline, most injectors prefer preserved saline, which has been shown by both Alam et al and Allen et al to have equal efficacy with less pain.

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