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Comparative study of the diffusion of five botulinum toxins type-A in five dosages of use: Are there differences amongst the commercially-available products?

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Comparative study of the diffusion of five botulinum toxins type-A in five dosages of use: Are there differences amongst the commercially-available products?
Adilson Costa MD MSc PhD, Elisangela Samartin Pegas Pereira MD, Margareth de Oliveira Pereira MD, Felipe Borba Calixto dos Santos MD, Raquel Favaro MD, Paula Luz Stocco MD, Lúcia Helena Fávaro de Arruda MD MSc
Dermatology Online Journal 18 (11): 2

Pontifitial Catholic University of Campinas, Campinas/SP, Brazil and KOLderma Clinical Trials Institute Ltd., Campinas/SP, Brazil


BACKGROUND: Diffusion halo profiles of different commercially available type-A botulinum toxins (BTX-A) have been studied. OBJECTIVE: To evaluate and compare the largest diameter of diffusion halos of 5 different doses of 5 different commercially available BTX-A. METHODS: Twenty-five adult female volunteers were included. Products with 100 units (100 UI) and the product with 500 units (500 UI) were reconstituted in a ratio of 1:2.5. Products were applied in five different concentrations (1U/2.5U, 2U/5U, 3U/7.5U, 4U/10U, and 5U/12.5U). After 30 days, a starch-iodine test was conducted to obtain the largest diameter of each halo. RESULTS: For all BTX-As, the higher the number of units used, the larger the diameter of the diffusion halo p<0.05). Statistically significant differences (p<0.05) were observed between the North American and Chinese BTX-As for the three lowest doses, between the Korean and German BTX-As for all doses, between the French and Chinese BTX-As for the four highest doses, between the French and German BTX-As for all doses, between the Chinese and German BTX-As for the four highest doses, and between the North American and German BTX-As for all doses (p<0.01). CONCLUSIONS: Differences were observed between all brands of BTX-As and between the different doses of each brand.


The United States Food and Drug Administration (FDA) has approved the cosmetic use of type-A botulinum toxin (BTX-A) for treatment of wrinkles in the glabella area and for axillary hyperhidrosis. However, there are numerous off-label uses for BTX-A, including correcting lines and wrinkles on the face, neck, and chest [1-7].

Botulinum toxin is a neurotoxin produced by the anaerobic bacteria, Clostridium botulinum, which is found in soil, plants, and water [5]. This bacteria produces seven different antigenic exotoxins, of which type A is the most powerful, followed by types B and F [5]. This toxin acts on the neuromuscular junction, the autonomic ganglion, and on sympathetic and parasympathetic postganglionic nerve endings [1, 6]. The toxin interferes with neural transmission by blocking the release of acetylcholine, which is the primary neurotransmitter in the neuromuscular junction of the presynaptic plaque, thus causing muscle paralysis [3, 5].

Another mechanism by which BTX-A exerts its activity is by inducing muscle weakness through the inhibition of the transmission of alpha-motor neurons in the neuromuscular junction, a fact that has led to the use of this substance for conditions of muscular hyperactivity and dystonia [8]. BTX-A also inhibits acetylcholine release in all sympathetic and parasympathetic postganglionic nerves, which has served as the basis for its use in smooth muscles for conditions such as achalasia and for hyperhidrosis, which is characterized by abnormal glandular activity [9].

Relatively few studies have compared the diffusion halos of different commercially available BTX-As. BTX-A acts on the sympathetic nerves of eccrine sweat glands, where it inhibits acetylcholine release in the neuromuscular junction, which results in decreased sweating [1, 4, 5]. For this reason, studies in animals and humans have used this clinical model as a parameter to compare the diffusion halos of several commercially available BTX-As [10-14].

This study is the first to compare the formation of diffusion halos in 5 commercially available BTX-As (commercialized by North American, Korean, French, Chinese and German companies) at 5 different doses 30 days after their application on the backs of patients diagnosed with trunk hyperhidrosis in order to verify differences in the maximum average of diffusion halo diameters among such products; different clinical doses are compared.

Materials and Methods

This open, single-center, double-blind clinical study (neither the volunteers nor the evaluating physician knew which BTX-A was applied to each site) was approved by a university’s Research Ethics Committee and followed the Declaration of Helsinki and all other international and local rules regarding the performance of clinical trials. This study involved a comparison of the effects of applying 5 different commercially available brands of BTX-A: a North American (Allergan Inc., Irvine, USA), a Korean (Medy-Tox, Inc., Ochang Myeon Cheongwon-gun Chungcheongbuk-do, South Korea), a French (Ipsen Société Anonyme, Boulogne Billancourt, France), a Chinese (Lanzhou Institute of Biological Products, Lanzhou, Gansu, China), and a German (Merz Group Service GmbH, Dessau-Rosslau, Germany) brand.

The applications of BTX-As were made by a non-blinded physician on the left upper back of 25 female volunteers who had been diagnosed with trunk hyperhidrosis, were between the ages of 18 and 45, and had skin phototypes I-IV. All volunteers read, understood, and signed a consent form before being included in the clinical study. Except for the French BTX-A, which contained 500 U per bottle, all others contained 100 U per bottle. Each bottle was reconstituted in 1 mL of 0.9 percent sodium chloride solution (0.9% SCS), except for the French product, which was reconstituted in 2 mL (1:2.5) of 0.9 percent SCS.

Therefore, 5 different doses of each 100 UI BTX-A (1U, 2U, 3U, 4U, and 5U) were applied. These 5 doses corresponded to 2.5U, 5.0U, 7.5U, 10.0U, and 12.5U of the French product, respectively, and were administered in volumes of 0.01 mL, 0.02 mL, 0.03 mL, 0.04 mL, and 0.05 mL, respectively. The applications were made at a depth of 3 mm (Figure 1) using 30 UI syringes (BD®, Franklin Lakes, USA), with a distance of 3 cm in between each individual application (Figure 2).

Figure 1Figure 2

Figure 3

After 30 days, the diffusion halo formed by injecting each dose of each commercially available BTX-A was evaluated by a blinded physician, who measured its clearest and largest diameter using starch-iodine tests. This test involves the application of a 3 percent alcoholic iodine solution and after natural drying, the application of a thin layer of starch. After each volunteer rested for 30 minutes in a temperature-controlled room at 38° Celsius and 50.2 percent constant humidity (Duracraft CZ-60, HoneyWell Inc., South Borought, USA), the largest diameter obtained in each halo of sweat inhibition (BTX-A diffusion halo) was measured and the area was photographed (Figure 3).


Figure 4

The Mann-Whitney U test was used to compare differences between the various toxins used and between different doses of each toxin. The significance level was set to p<0.05 for all comparisons. As shown in Table 1, statistically significant differences were observed in the diffusion halo diameters of each commercially available BTX-A, depending on the dose applied (p<0.05) (Figure 4). However, no significant differences were observed between the halo diameters of doses 3 and 4 of the North American BTX-A. Similarly, no significant differences in halo diameter were observed between doses 3 and 4 or 4 and 5 of the Korean toxin or between doses 2 and 3 of the French toxin (Table 2).

Table 3 contains the results of our comparisons between different BTX-A toxins at each dose. These results revealed statistically significant differences in the halo diameters between the North American and Chinese toxins for doses 1, 2, and 3; the halos for the North American vs. the Chinese toxin were (in cm) 1.7 vs. 1.5 (p=0.007), 2.0 vs. 1.8 (p=0.005), and 2.3 vs. 2.0 (p=0.010), respectively. When compared to the German BTX-A, the North American product showed significant halo size differences at all applied doses (1, 2, 3, 4, and 5). In order of increasing dose, the average halo sizes were 1.7 vs. 1.3 (p<0.001), 2.0 vs. 1.5 (p<0.001), 2.3 vs. 1.7 (p<0.001), 2.4 vs. 1.9 (p<0.001), and 2.6 vs. 2.1 (p<0.001) for the North American vs. the German product. A significant difference between the North American and the French BTX-A was observed only for dose 1, for which the average halo size was 1.7 for the North American BTX-A vs. 1.5 for the French BTX-A (p=0.0024). No significant differences were observed between the North American and Korean BTX-A products at any of the doses studied.

As shown in Table 3, when the Korean BTX-A was compared to the German product, we observed significant differences for all of the doses tested (1, 2, 3, 4, and 5). In order of increasing dose, the observed halo sizes were 1.6 vs. 1.3 (p=0.008), 1.9 vs. 1.5 (p<0.001), 2.2 vs. 1.7 (p<0.001), 2.4 vs. 1.9 (p<0.001), and 2.5 vs. 2.1 (p<0.001) for the Korean vs. the German product. A comparison between the Korean and Chinese BTX-As revealed a statistically significant difference at dose 3, with the Korean product exhibiting a 2.2 cm halo and the Chinese product inducing a 2.0 cm halo (p=0.016). No significant differences were observed between the Korean and French BTX-A for any of the doses examined.

Upon further analysis of Table 3, we can observe that the average halo diameter for the French vs. Chinese toxins for doses 2, 3, 4, and 5 were 2.1 vs. 1.8 (p=0.002), 2.2 vs. 2.0 (p=0.018), 2.4 vs. 2.2 (p=0.037), and 2.6 vs. 2.4 (p= 0.041), respectively. Similarly, we can observe that the average halo diameter for the French vs. the German BTX-A for doses 1, 2, 3, 4, and 5 were 1.5 vs. 1.3 (p=0.014), 2.1 vs. 1.5 (p<0.001), 2.2 vs. 1.7 (p<0.001), 2.4 vs. 1.9 (p<0.001), and 2.6 vs. 2.1 (p<0.001), respectively.

Finally, Table 3 shows that the average halo diameters for the Chinese vs. the German toxin for doses 2, 3, 4, and 5 were 1.8 vs. 1.5 (p= 0.018), 2.0 vs. 1.7 (p=0.003), 2.2 vs. 1.9 (p<0.001), and 2.4 vs. 2.1 (p=0.005), respectively.


BTX-A is currently used in the treatment of a wide variety of medical conditions, including strabismus, focal dystonia, hemifacial spasms, spastic diseases, headaches, drooling, hyperhidrosis, and expression line minimization [5]. In the treatment of focal hyperhidrosis, BTX-A is injected intradermally, where it inhibits the sympathetic nerves that innervate the eccrine glands, thus resulting in the reduction or cessation of sweat [1]. When used for this non-aesthetic purpose, it has been shown to induce a significant improvement in 95 percent of patients after one week and its effects have been shown to last an average of 7 months [1].

The number of aesthetic procedures using BTX-A has increased exponentially [16]. Studies of BTX-A diffusion halos aim to perfect the desired response and to ensure that the applied BTX-A reaches the intended target without leading to local or systemic damage. Among the several factors that could potentially influence BTX-A diffusion halos are the following: 1) the characteristics of each commercial brand, such as the type of protein contained in its formulation, its molecular weight, its structure, and the dose and volume used; 2) the concentration of BTX-A; 3) the number of injections; 4) the application technique; and 5) the area to which BTX-A is applied [16, 17]. Other potentially important factors in the regulation of BTX-A diffusion halos include the muscle and fascia depth where BTX-A is applied, the needle diameter, and the distance from the needle tip to the neuromuscular junction [17].

Evaluating the diffusion halos of BTX-A, which are calculated by measuring the size of the areas characterized by the cessation of sweat, is a clinical tool that can be used to measure BTX-A’s effective range. The diffusion halo of BTX-A can be quickly and objectively evaluated by observing the decreased sweat in the area where it is applied. In the scientific literature, this method has been described as a means to evaluate the diffusion halo of several commercially available forms of BTX-A [10]. However, our study is the first to compare the diffusion halo of 5 different types of BTX-A that are commercially available on the international market.

One of the most undesired side effects of BTX-A occurs when its diffusion extends beyond the intended area for aesthetic purposes and reaches muscle, which can cause unintentional muscle weakness. This weakness may be related to inaccurate targeting or to the diffusion of BTX-A through the intended muscle fibers, which can cause it to reach other undesired muscle groups.

Trindade de Almeida et al [10] compared the North American and French BTX-A products by applying each to an isolated forehead at dose ratios of 1:2.5, 1:3, or 1:4. The authors reported that the diffusion halo obtained in the forehead area was higher for the French BTX-A (in 93% of the cases) for all dilutions tested and no effects on muscle contraction capacity were observed for either product at any dose. In contrast, our results revealed no significant differences (p>0.05) between these drugs, even though the same doses (3 UI of the North-American toxin and 7.5 UI of the French toxin, i.e., a ratio of 1:2.5) were used (Table 3).

The French BTX-A contains a higher quantity of units per bottle (500 UI) than the other forms of BTX-A. According to recent agreements regarding the cosmetic use of BTX-A, it has been observed that the most commonly used ratio of this form of BTX-A compared to those that contain 100 UI is 1:2 [18]. In our study, we wanted to compare a more concentrated form of the French product (2 mL of 0.9% SCS, that is, 2.5 UI for each 1 UI of the other toxins) compared to other traditional trials examining the same drugs [10]. Using this approach, we observed that the French BTX-A is inferior to the North American product only at dose 1 (its average halo diameter of 1.5 cm is less than the 1.7 cm average for the North American product, p=0.024) (Table 1 and Table 3).

In a randomized prospective study with 10 patients, low concentrations and high volumes of BTX-A have been demonstrated to exhibit increased diffusion and to affect larger areas [11]. Paradoxically, we observed a direct connection between halo size and the number of BTX-A units used with 5 different commercially available BTX-As, as shown in Table 1 and observed in Figure 3.

In an experimental study that used histological analyses to measure BTX-A diffusion in the back muscle of rabbits, it has been verified that low doses of BTX-A (1 UI) lead to a diffusion gradient that extends 15-30 mm beyond the muscular segment where the BTX-A was applied. With higher doses (5-10 UI), the diffusion of BTX-A occurs throughout the muscle, without evidence of limit, and BTX-A produces a denervation gradient in the muscle in which both the magnitude and the extent of denervation are dose dependent [19].

In our work, we observed that both the applied dose and the origin of the product can lead to differences in the diffusion halos of commercially available BTX-As (Table 1). For the North American, Korean, and French products, several concentrations of BTX-A showed equal or very similar inhibition halos (Table 1).

It is possible that the lack of randomization in BTX-A placement might have allowed the grid to play a role in the results obtained. However, we do not believe that the lack of randomization significantly affected our results because the volunteers examined in this study exhibited similar patterns of sweating in a large area of their trunks, as indicated by the starch-iodine test.

The North American toxin was observed to exhibit increased diffusion halo sizes compared to the German toxin at all doses examined (Table 3). At the three lowest doses, the North American toxin was superior to the Chinese toxin. The Korean toxin was superior to the German toxin at all doses tested. The French toxin was superior to the Chinese toxin in producing large diffusion halos at the four highest doses examined (2, 3, 4, and 5 UI) and was superior to the German toxin at all doses analyzed. The Chinese toxin, however, was more efficient in promoting diffusion than the German toxin at the 2, 3, 4, and 5 UI doses.

The fact that differences were observed in the extension of diffusion halos between different doses and brands of commercially available BTX-A does not necessarily indicate that any particular commercial product should be used instead of another. Rather, dermatologists are ultimately responsible to know which product is the best product based on his or her personal experience and on the specific anatomical area and condition to be treated.

It should be noted that high-risk areas, such as the palm of the hands and the face, require more conservative dosing and products with a lower diffusion range than other areas [10]. However, the use of products with larger diffusion halos could be useful to treat areas that are at lower risk of complications and/or for clinical indications in which higher product profitability is desired (such as, for instance, the treatment of axillary hyperhidrosis) [1].

It should also be noted that this study did not evaluate differences in the efficacy of these different BTX-As. Rather, this clinical trial focused on comparing the diffusion halos of each of the 5 commercial BTX-As evaluated. To make conclusions regarding the relative efficacies of these products would have required a different measurement methodology (for instance, a gravimetric analysis of sweat in each location).

Finally, additional clinical studies examining the diffusion halos of BTX-A appear to be warranted. For example, it will be of interest to determine a more complete time course of the behavior of halos generated using different commercially available products, not only at the peak of their efficacy, but also in the months following BTX-A application. Once those types of studies have been conducted, it will be possible to make conclusions regarding the overall superiority of one commercial BTX-A in relation to the others.


This study has shown that the application of higher numbers of units of BTX-A leads to larger diffusion halo diameters. In addition, differences in the size of diffusion halos were observed between different brands at various doses. In general, the North American, Korean, French, and Chinese toxins produced similar diffusion halos, although several significant differences were observed between these products. The German toxin consistently produced the smallest diffusion halo diameters of any of the BTX-A products evaluated.

This clinical trial was sponsored by Cristália Produtos Químicos Farmacêuticos Ltda., Itapira/SP, Brazil.


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