Pharmacology of BOTOX® cosmetic

Summary and key features

In 2002 BOTOX® Cosmetic (onabotulinumtoxinA) became the first botulinum neurotoxin FDA approved for the management of facial lines, introducing a new chapter in aesthetic dermatology.
Following local injection into muscles, onabotulinumtoxinA inhibits the release of acetylcholine from motor nerve terminals and reduces muscular contractions.
Botulinum neurotoxins are biological products that are formulated into distinct therapeutics, each with its own basic properties, clinical profile, and unit dosing requirements.
Doses of botulinum neurotoxins are not interchangeable or convertible among different products.
Following injection of onabotulinumtoxinA into facial muscles, clinical effects have been observed within 24 hours, and benefits generally last at least 3 to 4 months; higher doses lead to a longer duration of effect.
As injectable proteins, all botulinum neurotoxin products have the potential to stimulate the immune system, although loss of clinical benefit from onabotulinumtoxinA due to neutralizing antibodies is infrequent.
OnabotulinumtoxinA has an established safety and efficacy profile and has been used by skilled clinicians to manage facial lines for several decades.

Introduction

In 2002 BOTOX® Cosmetic became the first botulinum neurotoxin approved for the management of facial lines in the United States. Its well-documented efficacy and safety profile, along with its accessibility as a locally injected therapeutic, transformed aesthetic medicine.

Following the success of BOTOX® Cosmetic, also known by its non-proprietary name onabotulinumtoxinA, other botulinum neurotoxin products entered the aesthetic market. These products are not interchangeable with BOTOX® Cosmetic or with one another because of their biological nature. That is, botulinum neurotoxins are proteins synthesized by bacteria that are formulated into therapeutics using a series of complex, manufacturing processes that differ among companies. Consequently, the unit doses and biological properties of different botulinum neurotoxin products vary, making the products non-interchangeable. This chapter specifically addresses the pharmacology of BOTOX® Cosmetic. Due to limitations on the number of references in this book chapter, readers are referred to the articles listed for further reading at the end of the chapter.

Serotypes and structure

Botulinum neurotoxins have historically been grouped into seven serotypes, A through G, based on the inability of antisera generated against one type to neutralize the activity of the others. OnabotulinumtoxinA and most other botulinum neurotoxin products currently available are based on serotype A. More recently, numerous subtypes of the serotypes have been identified, as have mosaic toxins that are hybrids of two serotypes. One such mosaic is known as BoNT/FA because its amino acid sequence is largely identical to serotypes A and F; some have characterized this as a separate serotype (serotype H) due to a lack of cross-neutralization, whereas others have found that antisera against type A neutralize its activity.

Botulinum neurotoxins are produced by the bacterium Clostridium botulinum as protein complexes containing a core neurotoxin protein of ∼150 kDa and one or more associated proteins known as neurotoxin associated proteins (NAPs). Different bacterial strains synthesize neurotoxin complexes of different sizes, with serotype A strains producing complex sizes of ∼300, ∼500, or ∼900 kDa. OnabotulinumtoxinA contains the ∼900 kDa neurotoxin complex size synthesized by the type A Hall Allergan strain.

In nature, botulinum neurotoxins are always synthesized in complexes with one or more NAPs, suggesting that the complex conveys a biological advantage. The non-toxin, non-hemagglutinin (NTNH) protein interacts directly with the core neurotoxin to form an interlocked complex that protects the neurotoxin from degradation by proteases found in the gastrointestinal tract. The complex also contains amino acids that respond to pH levels, promoting complex association in acidic environments and dissociation in basic environments.

Few studies have examined the role of the NAPs under conditions relevant to the aesthetic or therapeutic use of botulinum neurotoxins. However, it is likely that NAPs remain at least partially associated with the core neurotoxin for some time following intramuscular injection. Dissociation of NAPs from the 150 kDa neurotoxin is more likely at basic pH values and increasing ionic strength. Laboratory studies have documented a partial association of the neurotoxin and NAPs at pH values of 7.5 and 7.6. This is higher than the pH of skeletal muscle, which is approximately 7.4 at rest and lower during exercise. These findings are supported by in vivo results in mice that found clear physiological differences in the ∼150 and ∼900 kDa molecules when administered intraperitoneally or intravenously. Additionally, muscle tissue contains extracellular enzymes that degrade proteins, and even partial association of the neurotoxin complex may transiently protect the neurotoxin from degradation.

The presence and composition of NAPs is one difference between botulinum neurotoxin products. OnabotulinumtoxinA and several other products contain the ∼900 kDa complex, but incobotulinumtoxinA contains only the ∼150 kDa neurotoxin, and abobotulinumtoxinA contains complexes of approximately 300 to 500 kDa. The properties imparted by these differences have yet to be fully evaluated.

Manufacturing and formulation

Synthesis, isolation, and purification

As biological products, the manufacture of botulinum neurotoxins is highly technical and requires specific expertise and equipment. Each step in the production process must be validated and approved by regulatory agencies. For onabotulinumtoxinA, production begins with fermentation of the Hall Allergan type A clostridial strain of bacteria in an anaerobic environment, leading to synthesis and release of the neurotoxin complex into the bacterial broth. This strain separates or nicks the 150 kDa polypeptide chain to form a 50 kDa light chain and a 100 kDa heavy chain that remain linked by a disulfide bridge. The nicking step is necessary for biological activity.

Next, the neurotoxin complex is isolated and purified from the bacterial broth. Allergan generally follows the methodology of Schantz, consisting of a series of acid precipitation steps to isolate a highly purified crystalline neurotoxin with a homogenous molecular mass of ∼900 kDa. Other manufacturers have their own proprietary methods for isolating and purifying the neurotoxin from the bacterial broth, resulting in unique products that retain all, some, or none of the NAPs.

Unit testing

As biological products, potency and doses of botulinum neurotoxins are expressed in units of biological activity instead of nanograms or milligrams, as is the case with synthesized small molecules. Thus, manufacturers cannot merely weigh these biological products but instead must verify their biological activity prior to release.

There is no single approved test for measuring the biological activity of botulinum neurotoxins. Historically, mouse LD50 assays have been used for this purpose, but reducing animal use in research and testing is an important goal. Allergan pioneered the development of a cell-based potency assay that was approved by the US Food and Drug Administration in 2011. This highly sensitive method is specific to onabotulinumtoxinA, including use of Allergan’s/AbbVie’s own master cell bank as the source of cells for the assay, and has been extensively cross validated against the mouse LD50 assay.

Regardless of the type of assay used for measuring biological activity, botulinum neurotoxins must be tested against a reference standard that is defined to contain a given number of units. There is no international LD50 unit reference standard for all botulinum neurotoxin products. Instead, each manufacturer uses its own proprietary, product-specific reference standard for testing unit activity. Consequently, units of biological activity are specific to each botulinum neurotoxin product and are not interchangeable with those of other products.

Non-interchangeability of unit doses

The differences among botulinum neurotoxins, and especially the different testing methods and conditions used to define units, preclude direct dose comparisons. That is, 100 units of one product will not necessarily test at 100 units when evaluated in another manufacturer’s assay, even if the labeled doses for different products are the same. For some botulinum neurotoxin products, unit doses recommended for a given indication are substantially different, as is the case with onabotulinumtoxinA and abobotulinumtoxinA. Differences in units have led regulatory agencies in the US and some other countries to require manufacturers to state in their product labels that unit doses are not interchangeable or convertible among different botulinum neurotoxin products.

An example of the non-interchangeability of units can be observed in studies that have directly compared different botulinum toxin type A products. When incobotulinumtoxinA was tested in Allergan’s LD50 assay—the test that has been used to define units of onabotulinumtoxinA—vials of incobotulinumtoxinA labeled as 100 U contained only 69 to 78 units. Subsequent studies have examined the biological activity of incobotulinumtoxinA in several other in vitro and in vivo assays: the light-chain activity high-performance liquid chromatography assay, the cell-based potency assay, the rat compound muscle action potential assay, and the mouse digit abduction score assay. In all of these assays, the biological activity of incobotulinumtoxinA was significantly lower than that of onabotulinumtoxinA; for instance, in the light chain activity assay, incobotulinumtoxinA units displayed approximately 54% of the protease activity of label-stated onabotulinumtoxinA units. In another study, the potency of prabotulinumtoxinA labeled at 100 U was only 75% of that obtained with onabotulinumtoxinA labeled at 100 U in the cell based potency assay. These results indicate that units of different botulinum neurotoxin products are not equivalent or interchangeable.

Excipients and stabilization

All botulinum neurotoxin products contain ingredients or excipients added to the vials that enhance product stability. In addition to the 900 kD type A neurotoxin complex, onabotulinumtoxinA contains sodium chloride and human serum albumin. Human serum albumin helps to stabilize the protein during dilution and drying and enhances the recovery of the neurotoxin from the glass vials. Albumin may also act as a cryoprotectant and serve to prevent protein aggregation. Botulinum neurotoxin products contain different excipients, although most contain human serum albumin in varying amounts.

Following the addition of excipients, botulinum neurotoxin products must be stabilized for therapeutic use. OnabotulinumtoxinA is stabilized by vacuum drying, resulting in a powder for reconstitution. Other botulinum toxins are stabilized by freeze drying for reconstitution or are presented in solution.

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