Neurotoxins

2
Neurotoxins: The Cosmetic Use of Botulinum Toxin A

Jon D. Perenack^1,2 and Shelly Williamson‐Esnard²

¹ Oral and Maxillofacial Surgery, Louisiana State University, New Orleans, LA, USA

² Williamson Cosmetic Center/Perenack Esthetic Surgery, Baton Rouge, LA, USA

2.1 Botulinum Neurotoxins Introduction

The cosmetic use of botulinum neurotoxin A has been one of the main revolutionary forces in the cosmetic surgery world over the past 30 years. From its initial description for use to smooth mimetic lines of the upper face in the 1980s, it has grown to become the most commonly performed cosmetic procedure in the world. Its success is easy to understand; the technique is relatively simple to learn and perform, results are predictable, and downtime is minimal. Complications are rare and typically self‐limited. Few cosmetic procedures offer so much for the patient and practitioner, with so few negatives.

The effects of botulinum neurotoxins (BoNTs) had been noted as early as the first recognized outbreak of botulism in Wilbad, Germany in 1793. Initially linked to consumption of blood sausage, the illness became known as botulism, based on “botulus,” the Latin word for sausage. In 1897 the clinical illness of botulism was eventually linked to an exotoxin, and not an infection, produced by an obligate anaerobic bacterium, Clostridium botulinum. Within 50 years two distinct strains (A and B) of the Clostridium bacterium were identified and eventually eight distinct toxic serotypes would be described (A, B, C1, C2, D, E, F, and G). The exotoxins released were found to inhibit acetylcholine (ACh) vesicular neurotransmitter release by interacting with the exocytic release mechanism. At the neuromuscular junction, the clinical effect was to reduce muscular contraction. In other tissues that rely on ACh as a neurotransmitter, the clinical effect was related to the function of the target tissue; decreased sweating from sweat glands, or decreased salivary flow from salivary glands [1].

The medical use of BoNT was studied as early as the 1940s. In the late 1970s, the drug Oculinum, botulinum toxin type A, (BoNTA) was used in studies to evaluate treatment of strabismus and blepharospasm. Observations during the treatment of strabismus noted that while the muscular disorder was improved, the muscular blockade also occasionally extended to the lateral aspect of the orbicularis oculi muscle causing the relaxation of the mimetic “crow’s feet” lines. Patients in the study appreciated the softening effect on their “crow’s feet lines” and asked for treatment of the opposite non‐strabismus eye to make the effect symmetric. Thus, the first cosmetic use of botulinum toxin was performed [1–3].

Subsequent studies led to Food and Drug Administration (FDA) approval in the USA for treatment of numerous medical conditions related to BoNT’s ability to locally interfere with the release of acetylcholine. Strabismus and blepharospasm (1989), cervical dystonias (2000), glabellar rhytids (2002), axillary hyperhidrosis (2004), chronic migraine headaches in adults (2010), crow’s feet lines (2013), and overactive bladder (2013). Additionally, BoNT is used “off‐label” for numerous other conditions including temporomandibular dysfunction (TMD), trigeminal neuralgia, and all other cosmetic uses related to mimetic line treatment, and/or cosmetic muscular weakening effects.

The use of BoNT for cosmetic purposes has continued to grow in the USA year over year with a nearly 6500% increase from 1997 to 2015. The American Society for Aesthetic Plastic Surgery estimates that there were over four million cosmetic BoNT injections performed in the USA during 2015 [4].

2.2 Botulinum Toxins Physiology and Characteristics

While there exist medical grade preparations of both BoNTA and BoNTB in the USA, clinically only BoNTA is used for cosmetic purposes. The active particle in BoNTA preparations is a 150 kDa dichain protein linked by a disulfide bond [5, 6]. After injection into a tissue this particle binds to an ACh producing neuronal endplate (typically at a neuromuscular junction) and is internalized. Once inside the neuron, the neurotoxin proteolytically cleaves the SNAP 25 protein of the Snare complex. This complex is responsible for moving acetylcholine containing vesicles to the neuronal membrane for neurotransmitter release. In muscular tissue, the absence of ACh release results in a flaccid paralysis of the associated striated muscle. Clinically this results in muscle weakening noticeable within a few days. This muscle weakening progresses until the maximum result is achieved somewhere between two and three weeks post injection [7]. The delay in this final result appears to be due to ACh vesicles that were already bound to the membrane and available for release prior to the internalization of the BoNT. Once these residual vesicles are depleted the maximum effect is reached. This effect on the neuronal endplate is irreversible, however, due to neuronal production of new axonal sprouts, the tissue is reinnervated within three to six months. Clinically this correlates with the duration of the product’s efficacy [7–9].

There are three main BoNTA products available in the USA today. OnabotulinumtoxinA, produced by Allergan, Inc., Irvine CA, USA, is marketed as Botox Cosmetic. AbobotulinumtoxinA, produced by Ipsen Ltd, Slough, UK, is marketed as Dysport. IncobotulinumtoxinA, produced by Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany, is marketed as Xeomin [10–13] (Figure 2.1).

Image described by caption and surrounding text. — **Figure 2.1** Packaging for the three BoNTA products. Note that a 50 unit vial of Botox Cosmetic and Xeomin are pictured. These half‐dose vials are often given for staff usage only. The dilution volume should be adjusted by half.

While all three BoNTA products block ACh vesicular release, clinical performance of the three show differences related to dose and efficacy. These differences arise from variation of manufacturing processes, formulation, and potency testing methods (Table 2.1). For the clinician attempting to decide which product to use, this creates a confusing decision‐making process that is worsened by conflicting scientific studies on the three BoNTA.

Table 2.1 Comparison of BoNTA products and manufacturing methods.

Manufacturer	Allergan [5, 6, 13, 14]	Ipsen [5, 6, 11, 14]	Merz [5, 6, 10, 14]
Proprietary name	Botox cosmetic	Dysport	Xeomin
Nonproprietary name	OnabotulinumtoxinA	AbobotulinumtoxinA	IncobotulinumtoxinA
Purification Method	Crystallization	Chromatography	Chromatography
Purification product	BoNTA – 900 kDa complex protein	BoNTA complex sizes <500 kDa (exact weights and composition has not been reported by the manufacturer)	150 kDa BoNTA protein only
Excipients	In 100‐unit vial: –900 μg NaCl –500 μg human serum albumin	In 500‐unit vial: −2.5 mg lactose 125 μg human serum albumin	In 100‐unit vial: −4.7 mg sucrose −1 mg human serum albumin
Finishing	Vacuum dried	Freeze dried	Lyophilized

2.3 Manufacturing Process

Unlike chemically synthesized drugs, BoNT is a biologic protein complex harvested and purified from a living bacterium. Neurotoxin serotype and protein composition of the complex is dependent upon the strain of the organism [14, 15]. The manufacturing process to grow the bacterium and isolate the neurotoxin is tightly controlled, as small alterations in technique may lead to large changes in the clinical characteristics of the BoNTA produced. All three commercial bacterium strains produce the 150 kDa neurotoxin particle that is responsible for the BoNT muscle‐relaxing effect. The Xeomin product consists only of the 150 kDa particle, while Botox Cosmetic and Dysport also contain neurotoxin accessory proteins (NAPs) of various sizes (300, 500, 900 kDa) depending upon the strain. While associated NAPs theoretically act to help stabilize and protect the 150 kDa particle, research has been inconclusive of their impact on clinical effect [16–18]. The 150 kDa particle must first dissociate from the NAP within the injected tissue to bind to the neuron, but the exact duration of this process is undetermined. Some studies have suggested that at physiologic pH levels the NAPs remain largely associated with the NT, other studies have suggested that NAP dissociation occurs either shortly before or after injection [16, 17], within less than one minute. Clinical studies have shown that all three products will initiate muscle weakness in the target tissue within three to five days after injection. Conflicting studies exist that directly compare one product to another in terms of onset of action but there appears to be no clear difference [7, 18].

Immunogenicity of the 150 kDa particle and NAPs has also been studied. It was initially theorized that NAP‐containing BoNTA products would have a greater chance of stimulating an immunogenic response that would in turn neutralize the 150 kDa particle. However, subsequent studies have confirmed that although NAPs may stimulate an immunogenic response, it does not appear to harm the activity of the 150 kDa particle and are non‐neutralizing. Preclinical studies have suggested the NAPs may in fact act to protect the 150 kDa particle from stimulating an immune response by physically “hiding” the antigenic portion of the 150 kDa protein from the immune system [19–21]. Clinically, allergic reaction and non‐reactivity due to particle inactivation is exceedingly rare with no differences conclusively shown between the three products. Non‐reaction after therapeutic injection is far more likely due to other factors such as product storage, handling, preparation, and technique of injection. Additionally, patient satisfaction which correlates highly to efficacy and duration of effect may decrease over time with repeated injections due to muscle compensation in the area, repeated frequent injections resulting in overlapping reinnervation of the muscle, and increased expectations.

Dosing of the three products also creates a dilemma for the practitioner looking to achieve similar results between different products on the same patient. As no international standards exist for measuring potency for BoNT, each manufacturer has its own proprietary method for testing potency units and a product‐specific reference standard. Clinically, Botox Cosmetic and Xeomin are supplied in 100 unit vials while Dysport supplies a 300 unit vial. Studies comparing unit dosage equivalency between the three products have yielded conflicting results. In general, 1 unit of Botox Cosmetic is roughly equal to 1 unit of Xeomin, with both equal to 2.5–3 units of Dysport. As a matter of safety the LD50 for Botox Cosmetic in mice is 1 unit. The LD50 for humans is estimated at 2500–3000 units for a 70 kg man, or 40 U kg⁻¹. Assuming a 25 unit treatment for glabellar lines, this represents a 100‐fold margin of safety [6, 10, 11, 13].

Diffusion of BoNTA is also an important consideration when performing an injection. Ideally, the practitioner would be able to target only the intended muscle with no interaction of adjacent musculature. In general, there is little evidence that there exists any difference in diffusion of action between the three products. A number of studies have attempted to quantify diffusion. Injection halo studies use a BoNT injection to block sweating in the underarm area. Starch is applied to the area after maximum effect has been achieved to delineate the area of decreased sweating thereby allowing mapping of diffusion. Difficulties in standardizing dosing and dilution create uncertainty in these studies [22–25]. Clinically, increasing the unit dilution seems to correlate with greater diffusion. BoNTA in standard injection dilution and dosage typically may diffuse in a 1 cm radius from injection delivery point.

Duration of action is physiologically correlated with new axonal sprouting and subsequent reinnervation. However, in clinical practice, the duration of the achieved final result may vary depending upon numerous factors. Practically speaking, most patients do not desire a “frozen” look and wish to maintain some movement in the muscles of facial expression. Treatment thus is aimed at delivering a result somewhere between no movement, “frozen,” and total movement, a “non‐result.” In general, the closer the patient is treated with little to no movement, the longer the perceived result appears to last. Anecdotally, patients who desire a very mild weakening of a mimetic line, seem to recover movement faster. Similarly, patients who report having a suboptimal result at three weeks post injection often relate a story that the “Botox didn’t last” as long as previous treatments. This effect may be due to adjacent untreated muscle recruitment, muscle accommodation or patient perception due to dissatisfaction. On the opposite side of the spectrum, patients requesting the “frozen look” may also occasionally self‐report a sense that the treatment didn’t last as long as desired, even though the practitioner evaluation evidences a normal efficacy response and duration of three to six months. These patients often appear for an appointment at two months post treatment requesting BoNTA reinjection at the first sign of subtle movement. One problematic aspect in this scenario relates to patient expectations and perceptions of what is achievable with BoNTA treatment. Patients need to realize that the BoNTA effect gradually wears off somewhere between three and six months, and that sometimes a complete muscle block is not achievable. The other difficulty arises if the clinician agrees to reinject the patient earlier than three to four months. This sets up an unfortunate cycle where the neuronal axon sprouting is on two different schedules, thus requiring retreatment every two months or sooner. This should be avoided as not only does this tend to increase patient dissatisfaction and cost to the patient, but also repeat exposure increases the risk of antibody formation and inactivation of BoNTA. Ideally, these patients should be encouraged to completely allow muscle function to return, then proceed with an appropriate BoNTA treatment.

In comparison studies of clinical duration of efficacy between the three commercially available products, results have been conflicting. The difficulty in these studies arises from the non‐interchangeability of the products regarding unit dosing and dilution technique. As previously discussed, all three products are produced and tested in very different proprietary ways, thus they are not exactly “apples to apples.” Most clinicians who use multiple BoNTA products in their office tend to adjust their technique and dosing to achieve clinically similar results with similar duration of efficacy [26–29].

Contraindications to treatment with BoNTA are generally the same between the three products. These include a known hypersensitivity to any component of the preparation. All three BoNTA contain human serum albumin, but there is variation in the excipients (NaCl, Lactose, Sucrose) used in each, as well as the presence or absence of NAPs. Patients with systemic neuromuscular diseases must be treated with caution, or not at all, depending upon the severity of their condition to avoid an exaggerated response to BoNTA. Patients being treated with aminoglycoside or spectinomycin antibiotics must also be treated with caution as these medications have been known to potentiate the effects of BoNTA. The most common absolute contraindication to treatment with BoNTA is for women who are pregnant or attempting to become pregnant, and those who are breastfeeding [10, 12, 13].

2.4 Clinical Usage

Patient Consultation for possible cosmetic treatment with BoNTA should be treated in a comprehensive fashion as much as possible. This should include evaluation of skin for actinic changes, vascularity and dyspigmentation, and so on and assessment of facial contour and volume depletion or excess. The face should be assessed for the possible role of cosmetic surgical tightening and repositioning procedures. The patient’s expectations and goals should be explored to achieve a consensus of what is hoped to be achieved. We recommend having a large mirror in the consultation room to aid in showing the patient their distinct features and what might be improved with BoNTA treatment (Figure 2.2).

Photo of a consult room depicting a therapist sitting in front of a seated woman and touching her chin. At the right side of the woman is where a large mirror is located and at the left side is a computer for displaying multiple photos of a woman. — **Figure 2.2** The consult room should have a large mirror available, and a computer for displaying before and after photos.

In addition, it is helpful to have available multiple before and after photos of not only BoNTA patients, but also cosmetic skin and surgical patients. There often exists a knowledge gap with patients as to what BoNTA treatment can achieve. Confusion about the difference between BoNTA and facial filler treatments is common in the novice patient. These issues provide an excellent opportunity to discuss a complete treatment plan that may lead to additional services beyond BoNTA treatment. The discussion also serves to “credential” the practitioner and helps assure the patient that they are being seen by a knowledgeable and caring provider. Even if the patient adamantly wishes to only proceed with BoNTA injection, the consultation provides a platform for proper informed consent and leads to greater patient satisfaction as expectations have been appropriately managed.

Often a patient new to the practice may be seen who has a history of BoNTA treatment and has extensive knowledge of which product and dosing they want. These patients are often resistant to having a comprehensive evaluation and only “want my Botox.” However, not uncommonly these patients also exhibit knowledge gaps in various aspects of the treatment and expectations. Patients with previous BoNTA treatment from other providers may have been seen by an individual with a limited cosmetic and surgical knowledge base, and may have received less than optimal information and treatment. Often patients “shop” doctors, looking for either a lower price or better treatment outcomes. All these issues provide an entry point for discussion by the practitioner to credential themselves and better inform the patient.

Most providers find that the initial consultation appointment and first treatment, if performed on the same day, takes about 45 minutes. Subsequent reinjection appointments typically only take 10–15 minutes.

2.4.1 Age of Patient Treated

With the advent of social media and celebrity figures openly discussing cosmetic procedures, BoNTA and filler treatment have become much more acceptable at a younger age. The rationale for treating patients in their young 20s (or earlier) revolves around the attempt to prevent the formation of resting lines in the epidermis and dermis. Indeed, studies of stroke victims and anecdotal observation of BoNTA patients treated from a young age into their late 30s would appear to substantiate the preventative effect of muscle paralysis on wrinkle formation. Younger patients also sometimes desire to achieve a certain look, or to soften muscular tone to look less angry. (“Resting Angry Face,” RAF) These are in fact often the same goals of older patients. While the treatment of patients under the age of 18 with parental consent is legal, it is certainly still controversial. Treatment of patients over the age of 18 should follow the standard protocols of informed consent that one would offer any other aged patient.

While the most common patient age group for cosmetic BoNTA injections is in the 30s and 40s, there is also a large group in their 50s, 60s, and above, desiring treatment [4]. In this older age cohort the practitioner must be cautious of patients possessing a high degree of brow/forehead laxity and ptosis, where BoNTA treatment may be perceived as ineffective. In these cases, the facial rhytids are primarily caused by skin redundancy and folding, and are not improved with muscle relaxation. Often these patients rely on unconscious chronic frontalis activation to lift the brow out of the superior visual field and blockage of the frontalis will lead to an unpleasant sense of brow heaviness and may restrict the superior visual field. The use of BoNTA to treat forehead rhytids is not indicated and a browlift should be performed instead (Figure 2.3).

Image described by caption. — **Figure 2.3** (a) This patient has significant browptosis and would not be a good candidate for BoNTA treatment. (b) The same patient after browlift, upper and lower blepharoplasty and laser resurfacing, will now see some benefit for BoNTA treatment of the upper face.

Interestingly, a well‐performed browlift may convert these patients back to being an optimal BoNTA recipient.

Who can provide cosmetic BoNTA treatment varies from state to state and is regulated by the respective state medical, dental and nursing boards as well as state legislation. Dermatologists, plastic surgeons, ENT/facial plastic surgeons, oculoplastic surgeons and oral and maxillofacial surgeons are common providers. Most state medical boards allow any MD or DO licensed physician to provide cosmetic BoNTA treatment regardless of training. It is not uncommon for family practitioners/emergency medicine doctors to provide BoNTA treatment in medical spas. Physician assistants are regulated by the state medical board and may also be allowed to perform BoNTA treatments with some degree of physician supervision. Depending on state dental board and state legislation, some states allow general dentists to provide treatment. State nursing boards may allow nurse practitioners or registered nurses (RNs) to provide BoNTA, sometimes with or without direct physician supervision. This extreme variation in experience and training among providers gives the facial cosmetic surgeon cause to discuss with patients any previous experiences they had with BoNTA or other cosmetic services. It is important to always remain complimentary and professional when discussing other providers, but at the same time, any patient misconceptions or untoward experiences should be addressed.

Not uncommonly nurse practitioners or physician assistants may act as physician extenders and provide BoNTA treatment in busy cosmetic practices. For the facial cosmetic surgeon, this has both benefits and disadvantages. While this arrangement frees the surgeon up to have more time for surgery and seeing pre‐ and post‐op patients, it also distances him/her from interacting with the patient on the regular four to six month intervals, as the patient returns for follow‐up BoNTA treatment. It is important for the provider team to have clear guidelines on initial evaluation, care protocols, and when referral to the facial surgeon is mandated. It has been the author’s experience that the use of physician extenders can provide a non‐threatening atmosphere for facial cosmetic surgical procedures to be discussed. Patients who are “not sure” if they want surgery or have a general fear of cosmetic surgery report feeling more comfortable initially discussing the topic with someone they know does not provide the service directly, but is informed. The physician extenders often develop a close relationship with their patients and can add an extra level of credentialing and security when referring them to the facial surgeon.

2.4.2 Storage and Preparation of BoNTA

All three products present from the manufacturer as a scant amount of clear to white powder in the bottom of a small vial. To an untrained eye, the vial may appear empty. Botox Cosmetic comes packaged frozen and is recommended to be kept frozen until reconstitution. In a personal communication with Allergan, representatives suggested that the un‐reconstituted product could in fact be kept at room temperature with little to no effect on the product. Functionally, in our office, Botox Cosmetic is kept refrigerated at 2–8°C both before and after reconstitution. It is recommended that Dysport also be refrigerated at 2–8°C after reconstitution. Xeomin alone does not recommend a need for refrigeration of their product, although exposure to extreme heat seems ill‐advised. All three manufacturers provide their product as a single dose vial meant to be used on one patient. In practice, this rarely occurs. All three manufacturers recommend the product be fully used within hours of reconstitution, with Xeomin being the longest at 24 hours [10, 11, 13]. Numerous studies have in fact reported little to no change in efficacy if the reconstituted product is kept refrigerated for two weeks. Studies have also shown that refrigeration versus freezing of the product after reconstitution does not change its efficacy [30].

Dilution. When planning to dilute the product, volumes from 1 to 10 ml have been used. Our practice prefers the 2.5cc dilution as this yields a reconstitution such that each 0.1cc conveniently contains 4 units of Botox Cosmetic or Xeomin, or 12 units of Dysport (Figure 2.4).

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