Purity

Botulinum toxin is produced by anaerobic fermentation of the bacterium Clostridium botulinum type A (Hall strain). During the manufacture of incobotulinumtoxinA, following fermentation and extraction of the toxin, the complexing proteins are removed by chromatography. Thus incobotulinumtoxinA contains markedly less clostridial protein than do onabotulinumtoxinA and abobotulinumtoxinA (Fig. 6.1). In addition, incobotulinumtoxinA has the highest specific biological activity of all three products. A high-sensitivity sandwich ELISA was used in a study by Frevert to measure the amount of botulinum toxin type A per 100 U of onabotulinumtoxinA, incobotulinumtoxinA and abobotulinumtoxinA. The results were 0.73 ng, 0.44 ng and 0.65 ng, respectively, giving incobotulinumtoxinA the highest specific biological activity (U/ng neurotoxin) at 227 U/ng compared with 137 U/ng for onabotulinumtoxinA and 154 U/ng for abobotulinumtoxinA (but it must be noted that the units of abobotulinumtoxinA are different from those of onabotulinumtoxinA and incobotulinumtoxinA). This suggests that, in addition to containing complexing proteins, onabotulinumtoxinA may also contain denatured / inactivated neurotoxin, unlike incobotulinumtoxinA (see Fig. 6.1). Like the other botulinum toxin type A products, incobotulinumtoxinA contains human serum albumin as an excipient but, whereas incobotulinumtoxinA contains sucrose, abobotulinumtoxinA contains lactose, and onabotulinumtoxinA contains neither of these sugars (Table 6.1).

Figure 6.1 The amount of clostridial protein and neurotoxin protein contained in incobotulinumtoxinA, onabotulinumtoxinA and abobotulinumtoxinA per dose approved for the treatment of glabellar frown lines.

Stability

It was assumed that complexing proteins are required to stabilize the neurotoxin, but incobotulinumtoxinA can be stored at room temperature and without refrigeration for 4 years. In addition to the practical advantage of not requiring cold storage, incobotulinumtoxinA did not lose activity in a temperature stress stability study by Grein & Fink. Furthermore, its quality did not decrease following storage at 60°C for 1 month. In accordance with the prescribing information, once reconstituted, incobotulinumtoxinA should be stored at 2–8°C and used within 24 hours to limit the risk of bacterial growth.

Diffusion

It is important to understand the difference between diffusion and spread of the neurotoxin. Diffusion is the passive movement of the toxin down a concentration gradient within the medium. In contrast, spread occurs when the injected molecule travels from the site of injection. The latter is usually a result of the injection technique, skills of the injector, injected volume, and needle size and the consistency of the treated tissue and can cause undesirable effects in adjacent muscles.

Under acidic conditions the complexing proteins form a high-molecular-weight complex (900 kDa) with the neurotoxin. Accordingly, it was speculated that complexing proteins limit the diffusion of the active neurotoxin within the target muscle. However, experiments in animals and humans suggest otherwise. The diffusion of onabotulinumtoxinA, abobotulinumtoxinA, and incobotulinumtoxinA was investigated by Carli and colleagues in a mouse model using staining for neural cell adhesion molecule (N-CAM) as a marker. N-CAM is expressed only by denervated muscle cells and so is suitable for testing for muscles affected by botulinum toxin type A. Results indicated that, following injection into the tibialis anterior (TA) muscle of the mouse leg, there was limited diffusion into tissue that was adjacent to the site of injection and no difference in diffusion was observed between incobotulinumtoxinA, onabotulinumtoxinA, and abobotulinumtoxinA using a 1 : 1 : 4 dose conversion ratio.

A recent clinical study by Kerscher and colleagues showed no difference in the size of the anhidrotic area produced following injection of 5 U of incobotulinumtoxinA (free from complexing proteins) compared with 5 U of onabotulinumtoxinA (containing complexing proteins) on either side of the forehead after 6 weeks and 6 months.

These results suggest that complexing proteins have no effect on diffusion. The reason for this was revealed by a recent study by Eisele and co-workers on the stability of the complexes and the kinetics of their dissociation. This report showed that the high molecular weight complexes presumably present in onabotulinumtoxinA and abobotulinumtoxinA are dissociated by reconstitution and physiological pH. Reconstitution of the commercially available complexing-protein-containing products, onabotulinumtoxinA and abobotulinumtoxinA, with saline (as in the clinic) led to a complete dissociation of the 900 kDa complexes and resulted in at least 85% of the neurotoxin being in its free form. In addition, it was shown that more than 80% of the 150 kDa neurotoxin was released from complexes in under a minute at physiological pH. Therefore, following injection into target tissue, where the pH is usually close to neutral, any remaining complexes dissociate immediately, making the presence or absence of complexing proteins in the original preparation irrelevant in terms of diffusion. Complexing proteins do not contribute to the therapeutic effect and are therefore only clostridial impurities.

Immunogenicity

Botulinum toxin is a foreign protein and is therefore antigenic. Immunogenicity remains a controversial subject in the field of botulinum toxin type A treatment in aesthetics. While the generation of neutralizing antibodies is more common in therapeutic indications, where dosages tend to be much larger, cases of secondary non-responsiveness have been reported in aesthetics. In a study by Blümel and colleagues investigating the generation of neutralizing antibodies following repeated injections of onabotulinumtoxinA, abobotulinumtoxinA or incobotulinumtoxinA into New Zealand white rabbits, neutralizing antibodies were detected after both abobotulinumtoxinA and onabotulinumtoxinA treatment. In contrast, incobotulinumtoxinA treatment did not induce the production of neutralizing antibodies. This suggests that the presence of complexing proteins might be clinically relevant.

Introduction

IncobotulinumtoxinA has been assessed as a neurological and aesthetic treatment in a robust preclinical and clinical development program. In an open label, intraindividual, controlled study by Jost and colleagues in healthy male volunteers, incobotulinumtoxinA was compared with onabotulinumtoxinA in terms of its ability to reduce the compound muscle action potential in the extensor digitorum brevis (EDB) muscle of the foot at various timepoints post-injection. There were no significant differences in the degree of paralysis at a 1 : 1 dose ratio. In a similar study by Wohlfarth and colleagues in healthy volunteers injected in the EDB muscle with a range of doses of incobotulinumtoxinA compared with equal doses of onabotulinumtoxinA, there was no difference in the efficacy of these products and neither treatment diffused into adjacent muscles.

Neurological indications

IncobotulinumtoxinA is indicated for the treatment of three neurological conditions: the symptomatic treatment of blepharospasm, cervical dystonia of a predominantly rotational form (spasmodic torticollis), and post-stroke spasticity of the upper limb presenting with flexed wrist and clenched fist in adults.

A large, prospective, randomized, double-blind trial by Benecke and colleagues demonstrated that incobotulinumtoxinA was not inferior to onabotulinumtoxinA for the treatment of cervical dystonia. In this study, 463 patients received intramuscular injections of 70–300 U of incobotulinumtoxinA or onabotulinumtoxinA and, in both groups, the severity score on the Toronto Western Spasmodic Torticollis Scale (TWSTRS) improved by 11 points by day 28. For the treatment of blepharospasm, in a large, multi-center double-blind Phase III study by Roggenkämper and co-workers, 256 patients received a similar mean total dose of either incobotulinumtoxinA or onabotulinumtoxinA (39.6 U and 40.8 U, respectively). Three weeks after injection, patients in both groups showed a significant improvement in the symptoms of blepharospasm, indicating that both products were effective treatments and the non-inferiority of incobotulinumtoxinA to onabotulinumtoxinA was confirmed.

In a randomized, double-blind, placebo-controlled, Phase III study by Kanovsky and colleagues, 148 patients with an Ashworth Scale score of at least two for wrist and finger flexors following stroke were randomly assigned to receive incobotulinumtoxinA or placebo. The median dose of incobotulinumtoxinA administered was 320 U. Four weeks after treatment, a significantly larger proportion of incobotulinumtoxinA-treated patients had shown an improvement of at least one point on the five-point Ashworth Scale in wrist flexors compared with placebo-treated patients. IncobotulinumtoxinA treatment led to statistically significant improvements in muscle tone and disability in patients with post-stroke upper limb spasticity compared with placebo, confirming incobotulinumtoxinA as an effective treatment.

Safety summary in neurological conditions

In all three Phase III clinical trials in neurological indications, incobotulinumtoxinA was well tolerated and most adverse events were of mild or moderate intensity. When compared with onabotulinumtoxinA using a 1 : 1 dose ratio, the safety and tolerability profiles of both products were similar.

Aesthetic indications

IncobotulinumtoxinA has been studied by Sattler and colleagues in a large Phase III prospective, multicenter, randomized, rater- and patient-blind clinical trial designed to investigate the non-inferiority of incobotulinumtoxinA compared with onabotulinumtoxinA for the treatment of glabellar frown lines. A total of 381 women with moderate to severe glabellar frown lines at maximum frown (severity score of 2 or 3 on the four-point Facial Wrinkle Scale [FWS]) were randomized in a 3 : 1 ratio (incobotulinumtoxinA : onabotulinumtoxinA) to receive 24 U of either product. Four weeks after injection, response rates at maximum frown were assessed by independent raters and were found to be 96.4% in the incobotulinumtoxinA group and 95.7% in the onabotulinumtoxinA group. Twelve weeks after injection, the response rates at maximum frown remained high at 80.1% and 78.5% for incobotulinumtoxinA and onabotulinumtoxinA respectively. A responder was defined as a patient with at least a one-point improvement on the FWS. Statistical analysis confirmed that incobotulinumtoxinA was not inferior to onabotulinumtoxinA for the treatment of glabellar frown lines.

In another prospective, open-label, multicenter Phase III clinical trial by Imhof & Kuhne, 105 patients received one cycle of 20 U of incobotulinumtoxinA for the treatment of moderate to severe glabellar frown lines. A responder was defined as a patient with an improvement of at least one point on the FWS and the response rate at maximum frown was 98.1% at day 28 and remained high at 80.0% on day 84, as assessed by the investigator demonstrating the efficacy of incobotulinumtoxinA in the treatment of glabellar frown lines.

Two recent large, randomized, double-blind, placebo-controlled Phase III trials of incobotulinumtoxinA in the treatment of glabellar frown lines confirmed the efficacy and tolerability previously demonstrated in this indication (Merz Pharmaceuticals GmbH, data on file). In addition, these trials used new, more stringent definitions of responders, mandated by the FDA. A responder was defined as a patient with a greater than two-point improvement in glabellar frown lines on a four-point scale, as assessed by both investigator and patient at day 30 at maximum frown. In both studies, the responder rate with incobotulinumtoxinA was superior to that with placebo (p < 0.0001). In addition, when a responder was defined as a patient with a score of 0 (none) or 1 (mild wrinkles) at day 30 post-injection on the FWS, as assessed by the investigator, 79.9% and 76.4% of patients respectively were responders at maximum frown (p < 0.0001 compared with placebo in both trials) and responder rates were also superior to placebo (p < 0.0001) at rest (66.8% and 86.3% respectively; Merz Pharmaceuticals GmbH, data on file).

In a further recent clinical trial of incobotulinumtoxinA for the treatment of glabellar frown lines, the efficacy and safety of repeated dosing were studied. Responder rates remained high, in the range of 79–90% at maximum frown, as assessed by the investigator 30 days after treatment in each cycle over a maximum 2-year period at maximum frown, where responders were defined as patients with a score of 0 or 1 on the FWS (Merz Pharmaceuticals GmbH).

IncobotulinumtoxinA has also demonstrated efficacy in the off-label treatment of periorbital lines. In a double-blind, randomized, proof-of-concept study by Prager and colleagues, the clinical efficacy of incobotulinumtoxinA was compared with onabotulinumtoxinA in a 1 : 1 dose ratio. Twelve units of each product were compared in an intraindividual, split-face study. One month post-treatment, response rates were 95% for incobotulinumtoxinA compared with 90% for onabotulinumtoxinA, although this difference was not statistically significant. The response rates after 3 and 4 months were 89% and 84% respectively, for both products. These results demonstrate that there was no difference in the efficacy of incobotulinumtoxinA and onabotulinumtoxinA for the treatment of periorbital lines.

Another double-blind, randomized pilot study by the same team compared the efficacy of incobotulinumtoxinA with abobotulinumtoxinA in an intraindividual, split-face design for the treatment of periorbital lines over a period of 4 months. The results showed no significant difference in efficacy between incobotulinumtoxinA and abobotulinumtoxinA at the dose ratio of 1 : 3 used in this study.

Safety in aesthetics

In the head-to-head study by Sattler et al comparing incobotulinumtoxinA with onabotulinumtoxinA for the treatment of glabellar frown lines, the incidence of adverse events (AEs) was low. Treatment-emergent AEs that were considered to be related to study treatment occurred in 3.2% of patients in the incobotulinumtoxinA group and 5.2% of patients in the onabotulinumtoxinA group. The most commonly reported AE was headache. In Imhof & Kuhne’s Phase III trial of incobotulinumtoxinAs for glabellar frown lines, four patients (3.8%) experienced TEAEs that were deemed related to study treatment: three headaches and one case of worsening of pre-existing carotid artery stenosis. In the study of crow’s feet by Prager and co-workers, the majority of patients (>68%) rated tolerability as very good over the whole study period, according to a subjective five-point scale.

Summary

This is reflected by the fact that, in clinical practice, incobotulinumtoxinA and onabotulinumtoxinA are treated as equivalent in terms of dosage and the approved dose for glabellar frown lines is 20 U for both products.

The total face approach with incobotulinumtoxinA

To obtain the most effective improvement in facial wrinkles, treatment of the whole face, as described below, is recommended. The glabellar region can display single and double vertical lines and can be treated with one or two injections into the superior region of the procerus along the median line (total dose of 4–6 U), along with further medial injections into the central part of the muscle (total dose of 2–4 U). In addition, treatment should include one medial injection (1–2 U) in the direction of the fibers of the corrugator supercilii and one superficial injection into the medial aspect of the orbicularis oculi (2–4 U; Fig. 6.3A). Treatment of the eyebrow includes four injections in the part of the eyebrow above the orbital rim: superficial injections in fibers of the orbicularis and deep injections in caudal fibers of the frontalis (Fig. 6.3B). The mephisto appearance should be treated at the point of maximum contraction, with 1 U incobotulinumtoxinA injected into the lateral part of the frontalis (Fig. 6.3C). For lateral canthal lines, lateral intracutaneous injections and deep subcutaneous injections above the lateral orbital rim (1–2 U per injection) can be performed into the orbicularis oculi (Fig. 6.3D). There may be no need to treat the center of the frontalis as this can enhance brow ptosis and a ‘tired’ look. Horizontal forehead lines form part of normal expressions of surprise and interest and hence do not necessarily have negative connotations. It is therefore advisable to delay treatment of the frontalis until approximately 4 weeks after treatment of the glabellar region if it is still deemed necessary. The forehead can be treated by five deep muscle injections of 1–2 U each administered in a V-shape configuration in the medial area of the frontalis and multiple superficial injections of (0.5–1 U lateral to the midline (Fig. 6.3E)).

Figure 6.3 The total face approach: injection sites to treat the glabellar region (A), eyebrows (B), mephisto (C), lateral canthal lines (D), forehead (E), lower eyelid (F), open eye (G), bunny lines (H), gummy smile (I), upper and lower lip (J), marionette lines (K), mentalis (L), and platysma (M). Blue dots, intramuscular injections of 1–2 U depending on the clinical situation and gender. Light blue circles, intradermal injections of 0.5–1 U.

Reproduced from Sattler G, et al 2009 Bildatlas der ästhetischen Botulinumtoxin-Therapie. KVM Verlag with permission from KVM Verlag.

In cases of fine wrinkles in the area of the lower eyelid four intradermal injections of 0.5 U incobotulinumtoxinA, in a volume of approximately 0.04 mL (i.e. a more diluted preparation of incobotulinumtoxinA), can be used treat each lower eyelid. In order to open the eye, a single superficial pretarsal injection of 1–2 U in the orbicularis oculi along the mid-pupillary line can also be administered (Fig. 6.3F, G). Treatment of bunny lines can be achieved by one or two superficial injections of 1–2 U in the medial, superior part of the nasal wing (Fig. 6.3H). A single deep injection of 1 U in part of the levator labii superioris at the base of the nasolabial fold can treat a ‘gummy’ smile (Fig. 6.3I). An injection of 0.5 U between the vermilion border and the white portion of the lip can improve the appearance of the upper and lower lips (Fig. 6.3J), and a deep injection (1–2 U) lateral to the marionette line will cover the superior parts of the platysma muscle and the depressor anguli oris to treat marionette lines. Care should be taken to ensure that the distance to the corner of the mouth is at least 1 cm in the inferior direction to prevent diffusion into the orbicularis oris (Fig. 6.3K). To treat the chin, both the left and right mentalis muscles should be injected. This should preferably be done at the top of the corner of the chin in the direction of the neck to prevent treatment of the orbicularis oris (Fig. 6.3L). Finally the platysma muscle can be treated by multiple injections of 1–2 U along the hypertrophic bands, 2–3 cm apart (Fig. 6.3M).