CPD: Botulinum Neurotoxin Formulations

Dr Souphiyeh Samizadeh explores the science and pharmacological differences of botulinum neurotoxin formulations

There are different botulinum toxin A (BT-A) products/formulations
available on the market that are used for both therapeutic and cosmetic purposes. They vary in terms of composition, units, chemical properties, biologic activities and weight; hence, they are not interchangeable.1 Like many other aesthetic and therapeutic treatments, poor understanding of the clinical pharmacology of BT-A may result in poor product choice for a given indication, patient consent, inadequate treatment planning and a lack of understanding of potential complications. In this paper, I will discuss the basic clinical pharmacology of BT-A and detail some of the differences presented in the literature between three popular formulations.


Overview of botulinum neurotoxin
Botulinum toxin (BT) is a neurotoxic protein that consists of botulinum
neurotoxin (BoNT) and non-toxic proteins (also known as complexing
proteins).2 It is produced by anaerobic spore-forming bacteria, bacterium Clostridium botulinum and related species, including some strains of Clostridium butyricum and Clostridium baratii. 3 BoNT can cause botulism and is one of the most potent toxins known to mankind.4 BT serotypes include A, B, C, D, E, F and G, which are produced by different strains of Clostridium botulinum.4,5 BoNT consists of a heavy and a light chain, which are linked together by a disulfide bond.2 The toxin enters peripheral cholinergic nerve terminals and causes temporary and reversible inhibition of neurotransmitter release.2


Clinical uses

The specificity of action of BoNTs has made it a useful, practical and popular therapeutic agent. The popular use of BoNT is because of:6

  • Potency – amount of drug needed to produce a given effect
  • Complete reversibility – does not cause neuronal death
  • Neurospecificity – high specificity for peripheral cholinergic nerve terminals

There are three well-known BT-A formulations that have been approved by the US Food and Drug Administration, which are currently commercially available in the UK. These are onabotulinum toxin A (Botox/Vistabel), abobotulinum toxin A (Dysport/Azzalure) and incobotulinum toxin A (Bocouture/Xeomin).7 These products have cosmetic product names that differ depending on the country (stated in the brackets above) and a nonproprietary name (the chemical or generic name of a drug, distinguished from a brand name or trademark). Despite having similar efficacies, there is an ongoing debate in the literature and among practitioners regarding the comparability of these various preparations.7 Each of these products is formulated differently, has a different manufacturing process and demonstrates unique characteristics. They have all been approved for various and limited indications in different countries and these indications are still evolving. However, they are used for many on-label and off-label purposes.8 Practitioners need to understand clinical issues of potency, conversion ratio, and safety issues such as toxin spread and immunogenicity, prior to prescribing and administering any of these products. All contributing factors, including efficacy, diffusion, spread, properties of the preparation, muscle characteristic and injection technique, should be considered when comparing different toxin formulations.8

Composition
In nature, BoNT-A, is synthesised as a macromolecular protein complex, consisting of non-toxic accessory proteins (NAPs), bonded to the active neurotoxin. The neurotoxin has a molecular weight of 150kD.9 The therapeutic preparations of BT consist of botulinum neurotoxin, complexing proteins and excipients (Figure 1).9 Various BoNT-A products have different NAP compositions.7 Hence, the molecular weight of the BoNT-A progenitor toxins vary and are determined by the composition of NAPs and the manufacturing process.9 The neurotoxin (150kD) dissociates from NAPs prior to exerting its pharmacologic effects.7 The role of NAPs, in potentially increasing the immunogenic risk, has been debated in the literature, however, there is no consensus that they may modify the immunogenicity of the active toxin.7 BoNT consists of a light amino acid chain with a molecular weight of 50kD and a heavy amino acid chain of 100kD, which are bonded together by a disulphide bond (Figure 2). Both chains are important – they have a different function in the action mechanism of the neurotoxin and the disulphide bond, playing a key role in the biological activity of BT.8,9 The molecular weight of the BoNT-A progenitor toxins varies between 300-900kD.9 IncobotulinumtoxinA contains only 150kD neurotoxin with no complexing proteins.9-11 OnabotulinumtoxinA and abobotulinumtoxinA contain the 150kD neurotoxin as part of a complex with other proteins.11 OnabotulinumtoxinA is composed of a 900kD complex,12 and the size of the abobotulinumtoxinA complex is unknown as it is undisclosed by the manufacturer.13 The neurotoxin has been reported to rapidly dissociate from the complexing proteins after dilution, resulting in drying and reconstitution of the product. Therefore, it has been debated that the molecular weight protein complex size) does not affect the biological activity and pharmacological properties of BoNT.10,14


Mode of action on the striated muscle
All botulinum toxin serotypes disturb the neural transmission by blocking the release of acetylcholine (the principal neurotransmitter at the neuromuscular junction).6,15 Inhibition of the release of acetylcholine, from presynaptic motor neurons, results in muscle paralysis and can be achieved by intramuscular administration of botulinum toxins. This can, in addition, result in muscle atrophy.2 This could be desirable, in the case of masseter and/or temporalis muscle hypertrophy, or could alternatively be an unwanted outcome, depending on the treatment aims and objectives.16,17 The inhibition of neuroexocytosis varies depending on the following:6,15

  • Toxin serotype used
  • The dose injected
  • The type of cholinergic nerve terminal affected
  • The animal species

Duration of action
Paresis occurs after two to five days post injection of BT into a striated muscle and lasts two to three months, prior to wearing off gradually.2 The extent of paresis is associated to the amount of BT injected. For optimisation of BT treatment, the dose-effect correlation can be taken into consideration.2,18 It has been reported that when lower doses of BT are used, the duration of its action is correlated to the amount injected. However, the duration of action is thought to saturate at approximately three months, when higher BT doses are used.2 Patients treated with BoNT-A, for aesthetic purposes, can expect their results to last at least three months.19 The results, however, can last four to five months, depending on various factors including the area treated, the dose, and the formulation used.19 It has been also reported that the results may last longer for some patients, especially after repeated treatment. The mechanism for this is not fully understood.9

Potency
Potency of a given drug is the measure of drug activity, i.e. the amount required to produce an effect of given intensity. The biological potency of BoNT formulations is based on the median lethal dose of the neurotoxin, after intraperitoneal injection in female Swiss-Webster mice. It is known as median lethal dose (LD50) assay.10,20,21 The dose of product for treating patients (discussed further below) is determined by each manufacturer’s result of LD50 potency or, nowadays, cell-based assay. This is dependent on multiple variables, is not standardised, and is different for each manufacturer. The unit of measurement is the proprietary of each manufacturer. As such, the direct comparisons of potency between products is invalid.10,20,21 From a clinical point of view for practitioners, this means that, despite the same active molecule, BoNT-A, the potency of different preparations varies.7


Reconstitution
As all three mentioned products are in powder form, they need to be reconstituted prior to injection. The manufacturer’s suggested diluent is normal saline with no preservatives for all three products. However, there is debate in regards to reconstitution with saline with no preservatives, and preserved saline. Although some authors debate whether the preservatives in the saline deactivate the toxin partially, other authors have reported equivalent clinical effectiveness with BoNT-A diluted with preserved saline.22-24 Measures can be taken to avoid reducing the efficacy of the toxins. These were previously thought to include not shaking, bubbling, and storing the vial for a long period of time. However, reduction of efficacy due to shaking, bubbling or storage has been refuted by other studies.25-27 It is paramount to remember that suboptimal reconstitution of BoNT preparations can reduce or diminish their efficacy. Similarly, inaccurate reconstitution could result in inaccuracy of actual units injected and therefore compromise treatment.28,29 One author has reported that improper reconstitution of incobotulinumtoxinA, for example swirling without inversion of the vial of this product following saline injection, can result in substantial loss of units of the neurotoxin.28


Dosage
There are different amounts of the 150kD toxin (and NAPs)/LD50 unit for different formulations of BoNT-A. Therefore, it is important for practitioners to understand the equivalence ratio of the dose. The clinical conversion ratio between onabotulinumtoxinA and incobotulinumtoxinA has been shown by clinical and preclinical analysis to be very close to 1:1.30,31 OnabotulinumtoxinA / incobotulinumtoxinA / abobotulinumtoxinA dose conversion is a topic of debate. The available clinical and preclinical data suggests a conversion ratio of 1:3 or lower, 1:2.5.7,32,33

Toxin spread
BoNT’s effect on areas away from the injection site is known as toxin spread, or diffusion, and/or field of effect.34 However, there is lack of consistency34 and much confusion about the terminology used; clarity is best described below.34

  • Spread – physical
    movement of toxin from
    original injection site
  • Diffusion – kinetic dispersion of toxin beyond original injection site
  • Migration – distal effect

Toxin spread, or diffusion, and/or increased field of effect may increase the risk of adverse effects and complications. For example, treatment of the periorbital area with BT injection can result in eyelid ptosis due to accidental spread of the product to the levator palpebrae superioris muscle and its temporary reduced/diminished activity.22,35 Factors thought to affect the potential for spread of BoNT include: clinical dose, solution, concentration, injection technique, target site, location within the muscle, muscle hyperactivity, depth of injection and post-injection massage.13,36,37 It is thought that incobotulinumtoxinA and onabotulinumtoxinA have comparable spread, however, abobotulinumtoxinA was reported in one study to have significantly greater spread than incobotulinumtoxinA.35 Kerscher, compared the condition anhidrosis, produced by the three products mentioned. They reported that the mean difference in the maximal area of anhidrosis from incobotulinumtoxinA was 135.2mm2 for abobotulinumtoxinA, and therefore suggested an increased risk of side effects due to migration/diffusion of the toxin.35

Storage
Each manufacturer recommends optimal storage conditions for their products. As shown in Figure 1, onabotulinumtoxinA and abobotulinumtoxinA need to be stored at 2-8°C while incobotulinumtoxinA can be stored at room temperature.10 Studies have suggested that there is no alteration in potency of onabotulinumtoxinA after reconstitution, even when it is refrigerated or refrozen for up to two weeks after reconstitution.38 No significant alterations or clinical response were reported by other studies that stored onabotulinumtoxinA two to six weeks after reconstitution and prior to injection, however, the question of sterility was raised.39-41

Conclusion
There are different BT-A products available on the market and they vary in terms of composition, units, chemical properties, biologic activities and weight. As such, they are not interchangeable. The conversion ratio of onabotulinum toxin A and incobotulinum toxin A are comparable and reported to be 1:1 and 1:2.5 for abobotulinumtoxinA. The products’ regional approved indications, dilution and reconstitution, storage and field of effect need to be understood prior to prescription and administration. Such differences may cause practical challenges for the clinicians if not understood. The key factors affecting the clinical response to BoNT injections are toxin preparation, reconstitution, dose injected, storage prior and after reconstitution, individual anatomy, and immunogenicity. An understanding of clinical pharmacology of BT-A therapy will be useful for standardising techniques used, and achieving consistent and optimal therapeutic results.

Dr Souphiyeh Samizadeh is a dental surgeon with a special interest in aesthetic medicine. She is an honorary clinical teacher at King’s College London and the clinical director of the Revivify London clinic. She has presented at both national and international conferences, and is actively involved with research into aesthetic medicine.

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