Sociedade Brasileira de Dermatolodia Surgical & Cosmetic Dermatology


ISSN-e 1984-8773

Volume 3 Number 4

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Original Article

Correction of dynamic wrinkles with incobotulinumtoxinA

Resultados da aplicação de incobotulinumtoxinA em pacientes para correção de rugas dinâmicas

Natalia Cymrot Cymbalista1

Masters in Dermatology, Faculdade de Medicina da Universidade de São Paulo (USP) – São Paulo (SP), Brazil.1

Received on: 15 October 2011
Approved on: 1 December 2011

This study was carried out at the author’s private practice – São Paulo (SP), Brazil.

Conflicts of interest: None
Financial support: None



Introduction: Patients (n = 56) received incobotulinumtoxinA for the treatment of dynamic facial wrinkles.
Objective: To test the efficacy of incobotulinumtoxinA in correcting facial wrinkles.
Methods: The toxin was applied in in the usual doses and regions.
Results: Only 38 patients returned for evaluation after treatment. Of those, 19 complained about the low efficacy, short duration or absence of effects of the toxin. The total dose applied in patients who expressed satisfaction with the outcome (19 patients) was 872 units, while in the dissatisfied group (19 patients) it was 748 units – which was later increased by an additional 30.62% (229 units) of the initial dose.
Conclusion: Although some clinical studies demonstrate that this botulinum toxin has an efficacy similar to others available, this study has not succeeded in reproducing that fact.



Many preparations of botulinum toxin type A are curren- tly available for use, with diverse chemical denominations, such as onabotulinumtoxinA (Botox®, Allergan, Inc., Irvine, CA, USA), abobotulinumtoxinA (Dysport ®, Ipsen Ltd., Berkshire, UK), and incobotulinumtoxinA (Xeomin® Merz Pharma, Frankfurt, Germany). Some studies in the literature show a simi- lar efficacy between incobotulinumtoxin the onabotulinumtoxinA (using an equivalent dose of 1:1) and bet- ween incobotulinumtoxinA and abobotulinumtoxinA (with an equivalent dose of 1:3). 1-4 This study sought to verify the effi- cacy of incobotulinumtoxin in the treatment of dynamic facial wrinkles.


Fifty-six patients (53 women and 3 men), aged 19-78, were treated with incobotulinumtoxinA (Xeomin® Merz Pharma, Frankfurt) in this short, prospective, monocentric study. According to the manufacturer, a vial contains 100 DL50 units of neurotoxin, without complex proteins, in addition to human albumin and sucrose. The study was carried out at a private practice, according to good clinical practice guidelines.

The areas of application and doses applied (in units) by region, are described in table 2. The product was stored at room temperature (15-30º C) before reconstitution and refrigerated (2-8ºC) after dilution. The product was used within 24 hours after reconstitution. The dilution of all vials was carried out by dermatologist physicians with experience in the technique, using 1.07 ml of 0.9% sodium chloride, without preservatives, in sterile vials (the excess 0.7 ml of saline solution was used to compensate the loss of liquid in the syringe).

The saline solution was injected into the vial randomly, i.e., the needle sometimes pointed towards the vial''''''''s wall and sometimes did not. All vials contained vacuum. Asepsis of the patients'''''''' skin and of the vials'''''''' rubber stoppers were carried out with 0.5% chlorhexidine digluconate solution. The patients'''''''' skin was dried with sterile gauze before the puncture was carried out. The injection sites did not receive massage or any substan- ce after the application of the toxin. The evaluation of the toxin''''''''s efficacy was clinical and by comparing standardized pictures of each treated region, before and 15 days after the procedure. The patients were clinically re-evaluated after four months.


Eighteen of the 56 patients did not return for the 15-day follow-up, which prevented the collection of important data to evaluate efficacy and patient satisfaction. Of the 38 patients who returned for re-evaluation 15 days after the application, 19 (50%) did not present complaints; 19 patients (50%) reported low efficacy, short duration or absence of effects of the toxin.

Patient 1 received 15 toxin units, distributed in the glabel- la, forehead and periocular region, and complained about the short duration of the toxin''''''''s effect (one month).

Patients 2, 6 and 18 (Figures 1, A, B and C) presented no effects.

Patients 6 and 18 received a new application of the toxin in doses similar to the initial application, again without any effect. Patient 2 declined to repeat the treatment.

Patient 3 presented an unsatisfactory response in the fore- head with an 11-unit dose. He had previously received another type of botulinum toxin (abobotulinumtoxinA) in the equiva- lent dose of 1:3 with a positive response and did not want to repeat the treatment in the same site. In the additional sites where it was applied, the incobotulinumtoxinA''''''''s response was satisfactory.

Patients 4, 19 and 15 received 15, 15 and 24 units, respec- tively, in the glabella only, and required 8, 8 and 9 units as com- plementary doses, respectively.

Despite having received, respectively, 20, 18, 18 and 23 units in the orbicularis muscles of the eyes, patients 5, 9, 13 and 17 needed 6, 4, 6 and 6 complementary units in those points, respectively. Patient 17 remained unaffected, and was later trea- ted with 36 abobotulinumtoxinA units (18 units per side), in order to obtain a complete effect.

Despite having used 24 units in the glabella and 15 in the forehead, patient 7 needed complementary doses of 11 and 4 units, respectively.

Patients 8 (Figures 2 A, B and C) and 10 respectively used 16 and 20 units in the glabella, 18 and 12 units in the orbicular muscle of the eyes and 11 and 8 in the forehead. They required re-applications of 10 and 5, 8 and 4, 6 and 4 units in those regions, respectively. Patient 10 did not demonstrate a response even after the complementary dose; a third application (with an additional 4, 4 and 3 units in those areas, respectively) was necessary.

Patient 11 received 30, 20 and 15 units, in the glabella, in the periorbicular regions and in the forehead, respectively, and needed 6 complementary units in each region. As no satisfacto- ry effect was obtained, injections of a different toxin trademark, of 8 and 4 units (in the glabella and forehead, respectively) pro- duced a satisfactory response.

Patient 12 received 19 units in the glabella, 14 in the orbi- cular muscle of the eyes, 11 in the forehead and 2 in the bunny lines. He/she needed 1 additional unit in the orbicularis muscle, 5 in the forehead and 1 in the bunny lines.

Patient 14 received 10 units in the glabella and 8 in the forehead, but needed 2 additional units in the glabella and 4 in the forehead. The doses in this case were considered low, howe- ver the patient was elderly, with atrophic musculature, and had previously received similar doses of a different toxin trademark without the need for complementary doses.

Despite having received 26 initial units in the glabella, patient 16 needed 7 more complementary units. In the malar region, this patient initially received 6 units (distributed in 6 one-unit points, applied intradermally to correct superficial wrinkles) and required 1 additional unit at one of the sites.

In some cases, the complementary dose was applied on the first follow-up visit (15 days after the initial application), but rat- her on variable subsequent days, at the patient''''''''s request (See Table 1). Four months after the application, all patients (inclu- ding those who expressed satisfaction and those who expressed dissatisfaction at the first follow-up visit) described the effect of the toxin as lasting only two months in all areas of the body where it had been applied.


The data from the 38 patients who returned for the fol- low-up visits underwent statistical analysis. Non-parametric Mann-Whitney tests were carried out in order to evaluate whether the average doses used in the application sites in the groups with satisfactory results (S) were similar to those in the groups with unsatisfactory results (NS). It was not possible to use the parametric t-test due to the reduced size of the groups and the absence of supposition of normal distribution of the values. 5

A hypothesis test''''''''s descriptive level of significance is the pro- bability of obtaining more unfavorable estimates than those provi- ded by the sample, in light of the alternative hypothesis. 6 All hypot- hesis tests were carried out with a 5% significance level; their res- pective descriptive levels (p-value) were also calculated. In this man- ner, only the hypotheses with p-values less than 0.05 were rejected.

The tests were conducted in order, to prove that there was no difference in the two groups regarding the variables age or doses applied. The tests were carried out using a 5% significan- ce level, with no statistically significant differences in the ave- rage doses applied in the glabella (p = 0.6456), the orbicular muscle of the eyes (p = 0.0823) or the forehead (p = 0.3760) between the two groups. The test was not carried out for the remaining application sites, due to the small number of appli- cations in both groups. There was also no difference in the patients'''''''' average age between the two groups (p = 0.2607). Table 2 presents some descriptive statistics for the number of units applied per dose in the first application, calculated for the area of the body and group, with satisfactory (S) and unsatisfac- tory (NS) outcomes.

Next, the 95% confidence interval for the proportion of patients who reported satisfaction in the follow-up visit was cal- culated ([34.10%; 65.90%]). The percentage values for the ratios second dose/first dose and total complementary dose (2nd + 3rd doses)/ first dose, were also computed. The latter are depic- ted in Graph 1.

Of the 19 patients who were not satisfied, 4 (21.05%) declined to receive complementary injections. A total of 30 complementary doses (2nd doses) were applied in various sites in the 15 remaining patients. The average application was 47.14% of the initial dose (minimum 7.14%, maximum 175%). The 95% confidence interval for the average percentage of the 2nd dose compared to the initial dose was [34.93%, 59.35%].

The total number of initial doses applied in the unsatisfied group was 748 units, and the total number of second doses applied in the unsatisfied group was 194 units – which corres- ponds to 25.94% of the first. It was also verified that the num- ber of 3rd doses applied in the unsatisfied group was 35 units, corresponding to more than 4.68% of the value of the previous application.

Nine 3rd doses were applied in diverse sites in 3 patients only. Taking into account the complementary total dose (2nd + 3rd doses), the average application corresponded to 53.69% of the initial dose (minimum 7.14%, maximum 175%). The 95% confidence interval for the average of the percentage total com- plementary dose/initial dose was [41.45%, 65.93%].


Of the 56 patients treated, 38 returned to be re-evaluated after the botulinum toxin application. Of the latter, 25 had pre- viously received previous applications of onabotulinumtoxinA (in the equivalent dose of 1:1) or abobotulinumtoxinA (in the equivalent dose of 1:3). The therapeutic efficacy was satisfacto- ry with the previously applied toxins in those patients. Of the 19 patients dissatisfied with the outcome, 15 had earlier recei- ved one of the two other botulinum toxin type A trademarks, with the same dermatologist physicians, in the above mentioned dose proportions, with satisfactory effects and duration, and wit- hout the need for complementary doses. There are no data about the other three patients'''''''' previous applications. It is only known that one patient had never used botulinum toxin befo- re. All patients reported the effect of the toxin as lasting only two months in all application sites.

Total or partial absence of efficacy of the incobotulinumtoxinA was verified in 50% of the patients who returned for re-evaluation. Statistical analysis demonstrated that the 95% confidence interval for the patients who were unsatis- fied in the follow-up visits was [34.10%, 65.90%]. That result suggests an excessively high degree of dissatisfaction that is not usually observed in clinical daily practice with other botulinum toxin trademarks routinely used (onabotulinumtoxinA at 1:1 dose equivalence and abobotulinumtoxinA at 1:3 dose equiva- lence). Usually, there is no need for complementary applica- tions. The conservation, dilution (carried out by three physician dermatologists in all cases) and application of incobotulinumtoxinA complied with the theoretical and practi- cal instructions provided by the manufacturer at the time of the launching of the product in Brazil – when no emphasis was given to the need to point the needle towards the vial''''''''s wall when injecting the saline solution for dilution.

The manufacturer (Merz Pharma, Frankfurt, Germany) tested the batch used, confirming it was in good condition. IncobotulinumtoxinA is the most purified form of botulinum toxin, lacking complexing proteins. According to the manufac- turer, complexing proteins do not seem to affect the diffusion and therapeutic efficacy of the neurotoxin, or to improve the product''''''''s stability during storage. However, they can be respon- sible for forming neutralizing antibodies of botulinum toxin type A 7 that can result in therapeutic failure.

One study using incobotulinumtoxinA suggests that it can remain stable for three years at room temperature – unlike onabotulinumtoxinA and abobotulinumtoxinA, which need refrigerated storage. 8 In addition, Frevert verified that incobotulinumtoxinA is the type A botulinum toxin that con- tains the highest specific activity of neurotoxin, when compared to onabotulinumtoxinA and abobotulinumtoxinA. This finding suggests that incobotulinumtoxinA contains active neurotoxin only, in contrast with the other trademarks, which also contain denatured or inactive neurotoxin. 9

In animal and human models, incobotulinumtoxinA was not correlated to the development of neutralizing antibodies – even after the administration of high doses in short time intervals. 10

In addition, despite the existence of some reports in the literature of the partial or total loss of efficacy of the botulinum toxin due to the presence of specific neutralizing antibodies, such an occurrence, which has been described with abobotulinumtoxinA and onabotulinumtoxinA, is extremely rare. 11-17 It therefore seems unlikely that all patients in this study who did not demonstrate an adequate response with incobotulinumtoxinA presented antibodies secondary to pre- vious applications of onabotulinumtoxinA or abobotulinumtoxinA. It is assumed that high doses per applica- tion, short intervals between applications (less than 12 weeks), a high number of applications, chemical characteristics of the toxin used, and the patient''''''''s predisposition to develop an immu- nological reaction are risk factors for developing anti-botulinum toxin neutralizing antibodies. 18,19 Dissatisfied patients, who had received few previous applications, were treated with average doses and usual intervals. Nevertheless, the presence of neutrali- zing antibodies was not laboratorially evaluated.

On the other hand, it is possible that the dose proportions described in some studies (1:1 incobotulinu - mtoxinA:onabotulinumtoxinA20-22 and 1:3 incobotulinum - toxinA: abobotulinumtoxinA) is incorrect. Comparing incobotulinumtoxinA with onabotulinumtoxinA, Beylot veri- fied that the former has a slightly lower efficacy in the same dose proportion. 23 In 2006, Hunt and Clarke tested three different and valid batches of incobotulinumtoxinA at a qualified labora- tory, using the same trial method that Allergan uses to test the potency of onabotulinumtoxinA. That method is approved by regulatory agencies to quantify onacobo tulinumtoxinA''''''''s biolo- gical activity for commercial use. Four different tests with six different incobotulinumtoxinA dilutions were carried out. The results suggested a considerably lower potency than that indica- ted on the packaging (i.e., 100 U/vial) compared to the poten- cy of onabotulinumtoxinA. The average potency of the three incobotulinumtoxinA batches studied was 69-78 U/vial at the beginning of the study and 64-67 U/vial one year later. The authors proposed two explanations for this finding. The first hypothesis suggested that the different methods used by the two manufacturers (Merz Pharma and Allergan) to determine the potency affected the biological activity measurements. 24-25 The second hypothesis was that incobotulinumtoxinA loses its potency over time when stored at room temperature, perhaps due to the lack of complexing proteins that protect against the degradation of the neurotoxin, which in turn has greater expo- sure and reduced molecular stability.26-28 Moreover, it is also possible that both toxin trademarks have similar potencies if incobotulinumtoxinA is evaluated immediately after its manu- facturing. In practice, however, the product is not used imme- diately after leaving the production line, but rather after variable periods of time.

A study testing incobotulinumtoxinA''''''''s efficacy in the treatment of neurological disorders (dystonia, spasticity, synkine- tic reinnervation, hypersalivation and hyperhidrosis) combined with onabotulinumtoxinA in the dose proportion of 1:1 in 263 patients did not find differences regarding the appearance of the effect, duration, efficacy or side effects at doses of up to 840 U during a three-year follow-up period.4 Since the doses to treat these conditions are larger than those used in botulinum toxin cosmetic procedures, the results might be skewed in favor of incobotulinumtoxinA.

A further hypothesis that could explain the high degree of dissatisfaction verified in the present study is the requirement of special procedures in the storage, dilution and application of this new toxin. One study demonstrated that 2.5 ml and 4 ml sodium chloride solution dilutions in the preparation of the toxin for the treatment of glabellar wrinkles do not affect the aesthetic result. The patients who responded to the treatment demonstrated 100% and 89.5% improvement, respectively, two weeks after application. Three months after the procedure, those rates rose to 84.2% and 64.7%, and increased four months after to 53.3% and 61.5%, respectively. Although the study concluded that there was no apparent difference in the efficacy of the two dilutions, the data suggested a slightly higher efficacy at lower dilutions. A dilution of 1.07 ml was used in the present study, which suggests that the low efficacy was not correlated to the level of dilution. 29

Although it does not offer an explanation for the guideli- ne, incobotulinumtoxinA''''''''s package insert specifies that the dilution should be carried out with the needle pointing towards the vial''''''''s wall – not towards the bottom – when injecting the saline solution. Previous studies have demonstrated when dilu- ting onabotulinumtoxinA it is not necessary to point the needle in a specific direction. Furthermore, it was shown that conti- nuous and vigorous agitation of the vial (even to an intensity that may cause bubbles to form) and storage for up to six weeks after reconstitution do not affect the toxin''''''''s efficacy. 30-32 Therefore, it is unclear why there is advice to direct the needle towards the vial''''''''s wall. One hypothesis could be that the toxin is more fragile since it lacks complexing proteins. However, this issue not been clarified by the manufacturer.

Given these results, more studies are necessary to establish the correct equivalent doses and test the diverse storage, dilution and application conditions. Future studies should be conducted by independent investigators who have no connections to botu- linum toxin manufacturers.


Although some studies demonstrate that incobotulinumtoxinA''''''''s efficacy is similar to that of other botu- linum toxin trademarks, it was not possible to reproduce the duration of the effect and the efficacy of the blocking of the muscular contraction in this study. Therefore, it remains in doubt whether incobotulinumtoxinA requires any special care in its handling or whether its effects are less evident than other botu- linum toxin trademarks. If so, the equivalence rate between incobotulinumtoxinA and onabotulinumtoxinA Botox® should perhaps be greater than 1:1.


We would like to thank Raquel Cymrot for the statistical analysis.


1 . Noninferiority of incobotulinumtoxin A, free from complexing proteins, compared with another botulinum toxin type A in the treatment of glabellar frown lines. Dermatol Surg. 2010;36 (Suppl 4): 2146-54.

2 . Treatment of crow''s feet with two different botulinum toxin type: A preparations in split-face technique. Hautarzt. 2011; 62(5): 375-9.

3 . Comparing Botox and Xeomin for axillar hyperhidrosis. J Neural Transm. 2010;117(3):317-9.

4 . Routine use of Xeomin in patients previously treated with Botox: long term results. Eur J Neurol. 2009;16(Suppl 2):2-5.

5 . Estatística não-paramétrica para ciências do comportamento. Métodos de Pesquisa. 2 ed. Porto Alegre: Bookman, 2008.

6 . Noções de Probabilidade e Estatística. 7 ed. São Paulo: Edusp, 2010.

7 . Complexing proteins in botulinum toxin type A drugs: a help or a hindrance? Biologics. 2010; 4:325-32.

8 . Xeomin is free from complexing proteins. Toxicon. 2009;54(5):697-701

9 . Content of botulinum neurotoxin in Botox®/ Vistabel ®, Dysport®/ Azzalure ®, and Xeomin ®/ Bocouture .®. Drugs RD. 2010;10(2):67-73.

10 . Botulinum neurotoxin type A free of complexing proteins (Xeomin®) in focal dystonia. Drugs. 2007a;67(5):669-83.

11 . Antibody – Induced Failure of Botulinum Toxin A Therapy in Cosmetic Indications. Dermatol Surg 2010:36(Suppl 4): 2182-7.

12 . Immunologic resistance after repeated botulinum toxin type A injections for facial rhytides. Ophthal Plast Reconstr Surg. 2006;22(3):239-40.

13 . Antibody-induced failure of botulinum toxin type A therapy in a patient with masseteric hypertrophy. Dermatol Surg. 2007;33 (suppl 2):S105-10.

14 . New formation of BOTOX®: complete antibody-induced therapy failure in hemifacial spasm. J Neurol. 2004;25(1):360.

15 . New Formulation of BOTOX®: complete antibody –induced therapy, failure in cervical in hemifacial spasm. J Neurol. 2004;251(3):360.

16 . Complete secondary botulinum toxin therapy failure in blepharospasm. J neurol 2000;247(10): 809-10.

17 . Clinical features of secondary failure of botulinum toxin therapy. Eur Neurol 2002;48(1):26-9.

18 . Botulinum toxin therapy:risk factors for therapy failure. Mov Disord. 2000;15 (suppl 2): 51.

19 . Pharmacology of therapeutic botulinum toxin preparations. Disabil Rehabil. 2007;29(23):1761-8.

20 . Comparison of two botulinum toxin type A preparations for treating crow''s feet: A split-face, double-blind, proof-of-concept study. Dermatol Surg. 2010;36 (Suppl 4):2155-60.

21 . . Efficacy and safety of a new botulinum toxin type Afree of complexing proteins in the treatment of blepharospasm. J Neural transm. 2006;113(3):303-12.

22 . A new botulinum toxin type A free of complexing proteins for treatment of cervical dystonia. Neurology. 2005;64(11):1949-51.

23 . Different botulinum toxins and their specifications. Ann Dermatol Venereol. 2009;136( Suppl 4):S77-85.

24 . Therapeutic botulinum type A toxin: factors affecting potency. Toxicon. 1996;34(9):975-85.

25 . Significance of the LD50-test for the toxicological evaluation of chemical substances. Arch Toxicol. 1981;47(2):77-99

26 . Potency evaluation of a formulated drug product containing 150-kd botulinum neurotoxin type A. Clin Neuropharmacol. 2009;32(1):28-31.

27 . Biophysical characterization of the stability of the 150-kilodalton botulinum toxin, the nontoxic component, and the 900-kilodalton botulinum toxin complex species. Infect Immun. 1998;66(6):2420-5.

28 . Hemagglutinin binding mediated protection of botulinum neurotoxin from proteolysis. J Nat Toxins; 1998;7(3):239-53.

29 . Does dilution have an impact on cosmetic results with BoNT/A? Complex-protein- free BoNT/A for treatment of glabella lines. Hautarzt. 2009;60(10):815-20.

30 . Botulinum toxin A: Is it really that fragile molecule?. Dermatol Surg. 2010;36(Suppl 4):2106-10.

31 . Foam during reconstitution does not affect the potency of botulinum toxin type A. Dermatol Surg. 2003;29(5):530-2.

32 . Botox (OnabotulinumtoxinA): shaken, not stirred. Ophthal Plast Reconstr Surg. 2008;24:10-2.

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