Unmasking Botulinum Toxin Myths

Image by Chrissy Bolton /MJH Life Sciences using AI

Botulinum toxin treatments are among the most common approaches to address aesthetic concerns worldwide.^1,2 Starting as a potent neurotoxin produced by the bacteria Clostridium botulinum, botulinum toxin is now used globally in clinical practice, providing a highly accessible and minimally invasive solution to undesirable conditions such as facial wrinkles.^3–5 Currently, there are many different botulinum toxin products globally available, with more coming at an accelerated pace.

Nearly all aspects of botulinum toxins have changed dramatically since the first product was approved for therapeutic use in 1989,^6,7 due to factors such as new manufacturing technologies, advances in toxin science, increases in the number of conditions treated and amount of toxin injected, and a shift in the patient demographics receiving these products.^8,9 As these changes have occurred, so have inaccurate marketing messages, misuse of toxin-related scientific testing data, incomplete analyses of published data, and a general unawareness among health care professionals administering the toxins of the scope and scale of toxin usage.

Notably, the aesthetic space is permeated with incorrect or incomplete information regarding toxin science, with misleading information shared via marketing materials and even health care clinicians. Many professionals consider themselves “toxin experts” because they have been injecting botulinum toxin for many years and may rely on their personal experience as an interpretation of science, anecdotal usage, or an incomplete understanding of research studies. Meanwhile, toxin manufacturers have, at times, misrepresented and falsely claimed scientific data in attempts to differentiate toxins and advance market share.^10,11

Brief Description of BoNT-A Manufacturing

The bacteria Clostridium botulinum produces botulinum toxin type A (BoNT-A).¹ Manufacturing BoNT-A for injection involves extracting the BoNT-A from the Clostridium botulinum bacteria, resulting in a BoNT-A neurotoxin core of 150 kDa surrounded by 150 to 750 kDa of complexing proteins. The total molecular weight of the BoNT-A complex can thus range between 300 and 900 kDa but only the 150-kDa core imparts the therapeutic effect and is necessary for efficacy.^12–14 Complexing proteins protect the 150-kDa core against enzymatic degradation in the gastrointestinal tract when ingested, but they are unnecessary when injected into patients (and contribute to an increased protein load).^1,12,15 The BoNT-A molecule with complexing proteins is present in numerous commercially available formulations, including Botox (onabotulinumtoxinA; AbbVie),¹⁶ Dysport (abobotulinumtoxinA; Galderma),¹⁷ Jeuveau (prabotulinumtoxinA; Evolus),¹⁸ and Letybo (letibotulinumtoxinA; Hugel).¹⁹ Various excipients are then added to aid BoNT-A stabilization, help prevent it from adhering to the glass vial, and prevent aggregate formation.¹³

BoNT-A is a 3D protein that unfolds and will refold when extracted from the bacteria. Some of the BoNT-A does not refold properly, which renders it broken, also known as inactive or denatured. These BoNT-A molecules cannot impart activity for an injectable treatment. A few formulations, including Xeomin (incobotulinumtoxinA; Merz)²⁰ and Daxxify (daxibotulinumtoxinA; Revance),²¹ undergo a more thorough purification process in which the complexing proteins are removed from the 150-kDa core, leaving only the necessary part of the BoNT-A molecule for effect.¹²

Measuring BoNT-AContent in Formulations

BoNT-A is measured in terms of both potency and total protein content. Potency is measured by proprietary biological tests for each manufacturer and defined as units (U) specific to each respective formulation. The total protein content depends on the company’s manufacturing process, and includes a 150-kDA core, complexing proteins, and inactive molecules.

Myth: The ELISA Test CanMeasure “Active” Neurotoxin

Enzyme-linked immunosorbent assay (ELISA) is a well-established method of quantifying the amount of protein present in a product. However, ELISA can measure only the amount of a specific protein in a formulation; it cannot measure whether the protein is active or inactive. An analogy is putting good apples and rotten apples on a scale: The scale measures the apples’ weight but cannot distinguish between the good and rotten apples.

A scientific publication sponsored by a toxin manufacturer used ELISA to measure the amount of BoNT-A in various commercially available formulations.²² The authors claimed that this test allowed them to determine the amount of active toxin in an on-label dose for glabellar lines for each product. The amounts calculated were 0.27 ng (50 U) in Dysport, 0.18 ng (20 U) in Botox, and 0.08 ng (20 U) in Xeomin; these numbers have been widely used across other publications and marketing materials in the industry.

Herein is the first myth: Toxin potency can be determined by an ELISA test. ELISA can only determine the total amount of the BoNT-A protein present and cannot measure any biological function or activity of this protein. Although an ELISA is useful in determining the total amount of protein present, using it to make claims on the amount of active toxin is inappropriate, false, and misleading. The Table lists the total BoNT-A protein in various formulations, broken down into the amount of active and inactive neurotoxin.

Measuring the 3 Domains

The botulinum toxin 150-kDa core comprises 3 distinct domains, each performing an action required to achieve clinical effect: (1) the binding domain, (2) the translocation domain, both part of the core toxin in the heavy chain region, and (3) a catalytic domain that cleaves SNAP-25, found in the light chain. Not only are these essential for the entire function and, thus, efficacy of the toxin product, but the actions performed by both the heavy and light chains must occur in the appropriate order. Investigating 1 of these 3 actions will not provide insight into the whole context of the product’s efficacy and clinical effect.

Myth: Measuring Only the Light Chain Component Will Determine the Potency of a Toxin

An example of this occurred in the aforementioned paper. The authors used Endopep–mass spectrometry (Endopep-MS) assay data, which included only the amount of light chain activity of the toxin, to make claims regarding the toxin potency in several BoNT-A products.22 Similarly to the ELISA potency myth, the authors used an assay that has critical limitations when being used in the context of measuring the full activity of the 150-kDa toxin core. Although the Endopep-MS assay is an established method for measuring the activity of the light chain, it is not adequate to measure the entirety of the toxin activity. It is limited to determining only 1 of the 3 steps involved in the toxin action.

Although the light chain does impart toxin activity once within the neuron, the amount of light chain is irrelevant if the heavy chain is not included to allow neuronal binding and internalization of the light chain. The activity, and therefore the potency, of a BoNT-A can be measured only via biological tests that contain all 3 domains, including the gold-standard animal model, the LD50 or median lethal dose, along with cell-based and ex vivo assays. In fact, these methods are what the FDA requires to establish potency.

Marketing messages have used the results of both the ELISA and Endopep-MS assays to claim that higher numbers mean more toxin activity and, therefore, more efficacy and/or longer duration for a given BoNT-A product. These “more is better” claims derived from incomplete ELISA and Endopep-MS data sets are inherently false and thus misleading. Clever marketing campaigns using syringes depicting more or less toxin amounts based on these data were widely picked up by aesthetic clinicians on social media, with the vast majority continuing to misinterpret the data and make claims of increased efficacy and/or duration of 1 BoNT-A product. These claims, however, are not supported by clinical data and serve only to further feed this myth.

Myth: Different Measurements Are a Valid Way to Compare Duration of Efficacy

The duration of effect for neuromodulator treatments has been heavily studied since the first BoNT-A product became available. Botox Cosmetic, the first commercially available BoNT-A in aesthetics, used a rating of none (0) or mild (1) to denote a “responder” on a 4-point severity scale, with a duration of approximately 4 months.¹⁶ Subsequently, the FDA has required a minimum of a 2-point improvement as rated by the treating investigator and the patient, both having to agree on at least the 2-point improvement.^17,20 This measurement is termed a composite score. Based on this measure, duration is considerably shortened compared with a single investigator score. With these different end point measurements used across research articles and communications, it is very easy for inappropriate comparisons to be made between measurements. In some cases, mismatched comparisons could suggest a particular toxin formulation has increased duration of efficacy compared to another. Therefore, it is critical for any discussion regarding clinical end point comparisons to be mindful of the measurements used and to utilize the same clinical end point. Additionally, Botox, Dysport, Xeomin, and Jeuveau, the first 4 toxins approved in the US, have published analyses of their clinical trial data, including 1-point improvement as well as ratings of none or mild, with all showing a duration of 3 to 4 months when using the same outcome measurement.^23–27

Furthermore, data on these 4 toxins demonstrates that as the dose per treatment increases, the duration of efficacy increases as well.^28–30 This result is consistent among all BoNT-A and is a class effect. The data seem to indicate a saturation point specific to each brand’s, at which point the duration of efficacy does not continue to increase. Additionally, the safety of each brand’s dosing must be considered, especially in context of the boxed warnings of the potential spread of neurotoxin to adjoining musculature.

Myth: Excipients or Peptides Increase Toxin Duration of Effect

Botulinum toxin science has long focused on understanding and maximizing the potential of the 150-kDa core neurotoxin molecule. Manufacturers of new toxin products on the market have claimed to have added molecules that aid the core neurotoxin, resulting in increased functionality.³¹ A recently available neurotoxin product has an additional component: a peptide excipient that is derived from a protein found in HIV-1.³² Initially, this viral peptide was included to allow for transdermal delivery of the toxin, as the first iteration of the investigational drug was for a topical application instead of an intramuscular or intradermal injectable.³³ When the phase 3 clinical trials did not achieve their primary end points, the application of the product was changed to be injectable for the treatment of glabellar lines, similar to the other available toxin products. Because of this pivot, the purpose of the peptide component in the injectable formulation has become unclear and potentially dispensable, as not only was its original task to allow for transdermal delivery, but the peptide was specifically added for that purpose.³³

However, there have been some seemingly inappropriate marketing claims regarding the peptide’s purpose, including (1) increasing efficacy, (2) extending duration, (3) increasing speed of onset, and (4) allowing diffusion of the toxin after injection.10,11 Data supporting these claims are either lacking with limited in vitro or ex vivo work or have not been published in peer-reviewed sources.^31,34 Additionally, and most confounding, is the fact that the peptide is listed as an excipient on the package insert, thus rendering it, by definition, inactive and having no clinical effect.²¹

The most meaningful data can be found with the most empirical approaches, with the clinical data from injectable phase 2 and 3 studies. The phase 2 clinical trials provided a great opportunity to compare 3 doses (20 U, 40 U, 60 U) of the neurotoxin including the virally derived peptide to the on-label dose of Botox (20 U), which lacks the peptide.³⁵ If the peptide provided a significant benefit, it would result in an apparent change in efficacy or duration. However, this was not found because there was no significant difference in duration when comparing 20 U of a toxin to the peptide (Daxxify) to 20 U without the peptide (Botox).³⁵ As expected, 40 U had a longer duration than 20 U and is the on-label dose of Daxxify. Surprisingly, the 60 U dose of Daxxify did not have a significant increase in duration compared with 20 U of Botox.³⁵

In an effort to claim that the increased duration of response is not simply due to the doubled dose, the manufacturer of Daxxify used the same misrepresented data previously discussed in this paper and reported that a 40 U of its product contains 0.18 ng of core neurotoxin, the same as 20 U of Botox.^22,32,36 They also claimed a similar myth: Differences in neurotoxin formulation are responsible for increased duration.

The toxin plus peptide formulation of Daxxify contains only the 150-kDa neurotoxin core and is stated to have 0.18 ng of toxin in 40 U, of which all is considered to be active toxin.³² The 0.18 ng of 20 U of Botox consists of both inactive and active neurotoxins; it was measured via ELISA, which is not able to differentiate between active and inactive neurotoxin.^22,37 Xeomin has been reported to have 0.08 to 0.088 ng of active toxin per 20 U.^37,38 Doubling the dose of Xeomin to 40 U would equate to 0.16 to 0.176 ng, similar to the 0.18 ng in Daxxify. Examining the amount of core neurotoxin in Xeomin, which also consists of only the 150-kDa core, clarifies the math that the dose—not the peptide—increases duration.

As previously discussed, further clinical evidence can be found in the phase 2 study that showed no difference in 20 U with the peptide (Daxxify) compared with 20 U of a toxin without the peptide (Botox). Similarly, multiple head-to-head studies have shown equivalent efficacy and duration between same doses of Botox and Xeomin.^39–41

Myth: Human Serum AlbuminIs Potentially Harmful

Historically, all BoNT-A products have utilized human serum albumin (HSA) as a critical excipient in their formulations, HSA acts as an important stabilizer protein for the neurotoxin, preventing protein aggregation and preventing the toxin from adhering to the glass surfaces of the vial.^13,42,43 Recent formulations, however, have switched from HSA to polysorbates as the stabilizing excipient.

There have been marketing claims that some products are superior because they do not use HSA, some even purporting that polysorbates are an improvement over HSA. Although polysorbates have been used in other pharmaceuticals, they are newly used in toxin formulations. They have several distinct characteristics that differentiate them from HSA, with some differences noteworthy enough to rethink any such statements claiming superiority over HSA-containing formulations.⁴²

HSA is the most abundant protein found in blood plasma and has more than 80 years of clinical use as a treatment across several conditions.⁴⁴ It has been utilized in large volumes for severe conditions in intensive care units. (Notably, the amounts of HSA used in toxin products are drastically lower than the large volumes used in environments such as the intensive care unit.) This long history and very high dosing have created a substantial log of safety data, showing that HSA is very safe, nontoxic and no evidence of immunogenicity.⁴² Whereas the FDA guidelines state that there is a theoretical risk of transmission of Creutzfeldt-Jakob disease, no cases have been found in the United States.⁴² Indeed, there is no evidence that these blood-derived products transmit prion-type agents to humans.

HSA is also a natural human protein, so the body understands it and how to break it down. This is supported by the lack of immunogenicity seen with HSA across all uses. Its safety is also evidenced by the fact that HSA is used as a control in allergy testing and the FDA does not require immunogenicity testing for HSA.⁴²

In contrast, polysorbates are synthetic molecules in the ethoxylated nonionic surfactants family. They are used in several industries, including pharmaceuticals, industrial detergents, and surfactants.⁴⁵ Polysorbates generally serve as protein stabilizers, with the FDA classifying polysorbate 20 and polysorbate 80 as inactive ingredients. Polysorbates have been associated with a variety of adverse events, including hypersensitivity, injection-site reactions, angioedema, dyspnea, and even anaphylactic shock.⁴²

The practice of using polysorbates in botulinum toxin products is new and limited to only a few products with modest time in the market. Comparing the safety track record of HSA and polysorbate excipients creates a clear depiction of HSA’s safety both as an excipient in drugs such as toxins and across its variety of indications.⁴² Similar safety data for polysorbates are lacking. The rationale to avoid HSA as the preferred excipient in toxins must be firmly substantiated before switching from a well-established protein.

Myth: Immunogenicity to Botulinum Toxin Does Not Occur From Aesthetics Indications Treatment

The development of neutralizing antibodies (NABs) as a reaction to BoNT-A resulting in the patient not responding to treatment is well established in the literature.^46–48 Studies describing the rate of NAB development for aesthetic use are based on clinical trials in which patients received 2 to 3 sessions of the on-label dose. This, however, does not reflect current real-world use of BoNT-A for aesthetics. Consequently, this may lead aesthetic clinicians to a false sense of security.

Myth: Immunogenicity to BoNT-A Does Not Occur in Aesthetics

Increased risk of developing NABs are related to higher doses, greater overall protein load, and frequency of the immune system’s exposure to neurotoxin, including the duration of toxin treatment and the relative frequency between toxin treatments.⁴⁹ Recent research and surveys have shown considerable changes in patients’ lifetime toxin journeys, including initiation of toxin treatment at a much younger age, receiving much higher doses per session than the on-label indication, and receiving treatments more frequently than in years past.^8,50 For instance, patients presenting for initial toxin treatment used to be in their mid-40s, but data now shows at least 25% of patients presenting for their first toxin treatment are younger than 30 years of age; younger age of initiation thus increases lifetime toxin exposure.^8,51 On-label treatment of upper facial lines (glabellar, lateral canthal, and forehead lines) is commonly 64 U. However, the total cumulative dosage can easily reach or exceed 150 U due to the myriad of on-label and off-label uses (eg, masseteric hypertrophy, platysma band treatment, body indications such as axillary hyperhidrosis and, calf and trapezius slimming).⁸ Additionally, recent research has described that 44% of patients receiving toxin for aesthetic use are also receiving toxin for therapeutic conditions. Thus, toxin exposure is on the rise.⁵⁰

Overall protein load and the correlation to NABs development coupled with growing efficacy resistance among patients, led Allergan in 1997 to improve its initial Botox formulation with a lower overall protein load.^48,52,53 Manufacturers have seemingly acknowledged the dispensability of complexing proteins in injectable BoNT-A administration, and new products have moved toward formulations that contain only the 150-kDa neurotoxin core.^32,54 Commercially available since 2010, Xeomin contains only the 150-kDa neurotoxin core. It was the first product to reduce the overall protein load to 150 kDa and is the only toxin that has not had any patients develop NABs across all clinical trials.^8,20,55

Conclusion

As the number of commercially available BoNT-A products continues to increase globally, manufacturers have engaged in “toxin wars” to gain market share. In doing so, some have misrepresented scientific data and promoted claims without sufficient scientific substantiation. Unfortunately, social media has promulgated some of these marketing myths, and social media–savvy aesthetic practitioners may be inadvertently further cultivated these inaccuracies by interpreting misrepresented or incomplete data.

We encourage practitioners to ask for supporting scientific and/or clinical data to educate both themselves and their patients on BoNT-A safety.

Acknowledgments

The authors would like to thank Diana Romero, PhD, for editing the manuscript.

Andy Curry, PhD, is the associate global medical director of Xeomin at Merz North America, Inc., in Raleigh, North Carolina.

Jennifer Rosenfeld, NP-C, is a nurse practitioner specializing in aesthetics and founder of Jenn Rosenfeld Aesthetics in Newport Beach, California.

Melissa Kanchanapoomi Levin, MD, is a board-certified dermatologist at the Mount Sinai Icahn School of Medicine and founder of Entière Dermatology in New York, New York.

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