FormalPara Key Summary Points

Neurotoxins, such as botulinum toxin type A, are the mainstay treatment for dynamic wrinkles on the upper face. The disparity of the results in clinical practice can be attributed to factors like varying dosages, injection sites, dilution, wrinkle severity, muscle mass, outcome protocols, and follow-up procedures.

The persistence of effective neuromuscular blockade for approximately 4 months allows an aesthetic adjustment of facial mimicry that can persist for months after the pharmacological effect of the drug wears off.

This open-label, longitudinal follow-up, evaluator-blinded study enrolled 28 women with dynamic lines on the periocular, glabella, and forehead regions that were treated with incobotulinumtoxinA following an individualized protocol (ONE21 and glabellar contraction pattern).

Reduction from baseline in static forehead lines was achieved in 100%, 89%, and 46% of participants at days 30, 120, and 180, respectively. For dynamic lines, the rates were 96%, 70%, and 67%. Reduction in static glabellar lines was achieved in 96%, 93%, and 54% of participants at days 30, 120, and 180. For dynamic lines, the rates were 100%, 85%, and 58%. Reduction in static crow’s feet lines was achieved in 96%, 96%, and 58% of participants at days 30, 120, and 180. For dynamic lines, the rates were 100%, 82%, and 42%.

Introduction

Facial aging encompasses a series of anatomical and physiological processes, including deficits in collagen production, reduced cellular turnover, connective tissue laxity, decreased skin barrier function, superficial dermal elastosis, and redistribution of facial fat. Furthermore, repetitive muscle activity in facial mimicry leads to expression lines, which further contributes to the appearance of aging [1,2,3,4,5].

Neurotoxins, such as botulinum toxin type A, are the mainstay treatment for dynamic wrinkles on the upper face. The disparity of the results in clinical practice can be attributed to factors like varying dosages, injection sites, dilution, wrinkle severity, muscle mass, outcome protocols, and follow-up procedures [6,7,8,9,10].

Numerous techniques involving the facial application of botulinum toxin have shown effective improvement in expression lines and the repositioning of facial structures. Generally, type A botulinum toxin rapidly acts on the motor endplate, stopping muscle fiber contraction for an average period of 4 months until synaptic sprouting restores neuromuscular activity. In addition to relaxing facial muscles, it also reduces sweating, depressive symptoms, and headaches [11]. In addition, as acetylcholine receptors are found on the surface of melanocytes, keratinocytes, and a much broader range of cellular targets, including dermal fibroblasts, sebocytes, and vascular endothelial cells, several aesthetic skin benefits can be perceived after facial aesthetic use of botulinum toxin [12].

The persistence of effective neuromuscular blockade for approximately 4 months allows an aesthetic adjustment of facial mimicry that can persist for months after the pharmacological effect of the drug wears off [13, 14]. To date, no systematic study on the maintenance of the benefits of incobotulinumtoxinA (INCO) in the treatment of the upper thirds of the face has been conducted.

This study aimed to assess the effectiveness of INCO in treating the upper face of adult women, focusing on both the duration of the effects and the aesthetic benefits. In this study, an individualized treatment technique was employed.

Methods

This was a quasi-experimental intervention (before vs. after), prospective, open-label, longitudinal follow-up, self-controlled, evaluator-blinded, sequential allocation trial. A total of 28 women with dynamic lines in the periocular, glabella, and forehead regions were enrolled in the study, which was carried out in São Paulo-SP (Brazil) from November 2021 to August 2022.

The inclusion criteria encompassed women aged between 30 and 60 years, exhibiting at least mild dynamic lines in the forehead, glabella, and periocular regions (crow’s feet). Exclusion criteria were applied to women with liver, neurologic, or hematologic diseases, those under anticoagulant treatment, pregnant individuals, those with a history of botulism, or those who had undergone neurotoxin treatments within 1 year before the selection visit.

Eligible and consenting participants were evaluated by the same researcher and received pan facial neuromodulator treatment. In the upper face, the injection plan was based on previously described and published techniques such as ONE21 and the glabellar contraction patterns, and performed by two experienced dermatologists (CP and ATA). For the glabella, injection points and doses were selected according to muscle strength and inner eyebrow movement of depression, elevation, and/or approximation, corresponding to the enrollment of corrugators, frontalis, depressor supercilii, and procerus in related “U”, “V”, “omega”, “converging arrows,” and “inverted omega.” Five, seven, or more injection points were distributed according to muscle recruitment. For frontal treatment, the ONE21 technique suggests 21 possible injection points, after the design of seven vertical lines and three horizontal lines in the forehead. The dose was also selected according to muscle strength and recruitment. These two techniques were used as a basis for the injectors to personalize each subject’s treatment plan, addressing their individual needs, differently than a standardized injection protocol applied in other clinical trials [9, 15,16,17]. The 100-UI vials of INCO (Xeomin, Merz Pharmaceuticals GmbH) were reconstituted in 2 ml of sterile saline solution shortly before treatments.

At visits on days 0, 30, 120, 180, and 240, all participants underwent standardized facial photographs at rest and under maximal contraction of the muscles of the upper and lower thirds of the face. These photographs were taken by the same photographer, in the same environment and lighting conditions, without makeup or accessories [18]. At each follow-up visit, participants were asked about adverse effects and their level of satisfaction, rated on a scale of 1–5, ranging from very unsatisfied to very satisfied.

At the end of the study, the standardized photographs from each visit were randomly presented and consensually evaluated, blinded to the timing, by two experienced raters, to classify the severity of dynamic and static lines according to the Merz Aesthetics Scales (MAS), a 5-point scale in which the scores range from 0 (no lines) to 4 (very severe lines) [19]. These photographs were also arranged side by side with those from day 0 to assess the GAIS (Global Aesthetic Improvement Scale) for overall improvement. GAIS is a 5-point scale used in aesthetic medicine to assess the overall improvement in the appearance of a patient after a specific treatment or procedure and ranges from “worse” to “very much improved” [20].

The main outcomes were any reduction of the MAS score (periocular, glabella, and forehead) and its reduction by 1 point from baseline (day 0), at each visit. Secondary outcomes include patient satisfaction and GAIS for overall improvement.

The 95% confidence intervals (95% CI) were calculated for the percentiles of participants with ≥ 1-point MAS reductions through bootstrapping (5000 resamples) [21].

The change in the MAS assessment before and after treatment was analyzed by generalized mixed regression, and the multiple comparisons were corrected by the Šidák procedure [22,23,24]. The missing values were estimated throughout the mixed model [25]. The correlations between the dose or the points of injections used to treat each region and MAS severity were explored by Spearman’s (rho) rank correlation [26]. Data was analyzed using IBM SPSS v29 software. Significance was set as a (two-tailed) p value ≤ 0.05 [27].

The sample size was determined on the basis of the expectation that at least 35% of the women would maintain photographic scores of muscle contraction at day 240 lower than those at day 0, considering a standard error of 15% and a dropout rate of up to 10% [28].

The study was approved by the Research Ethics Committee of the Universidade São Francisco (#5.045.791). All procedures in studies involving human participants were performed in accordance with the Declaration of Helsinki (as revised in 2013). The study was registered in the Brazilian Clinical Trials Registry (UTN U1111-1307-7897). The participants in this study gave written informed consent to the publication of their case details and images.

Results

A total of 28 women were enrolled in the trial. Two of them (7%) missed the day 180 visit, and 2 (7%) dropped out of the study: one at day 30 and one at day 180. The reasons for these dropouts and noncompliance were not associated with the treatment but were due to challenges in attending the clinic amid the COVID-19 pandemic.

Their ages ranged from 30 to 60 years, with a mean (SD) age of 47 (9) years. In terms of self-declared race, 26 (93%) were white, and 2 (7%) were black. Phototypes were distributed as I–II (39%), III (54%), and VI (7%). Previous aesthetic treatments with neurotoxins (more than 1 year before enrollment) were reported by 10 (36%) women.

The baseline severity of dynamic and static lines according to the Merz Aesthetics Assessment Scales are presented in Table 1. Most (> 90%) of the participants presented moderate or severe dynamic lines at baseline. The treatment protocol was personalized on the basis of individual aesthetic severity following the assessment. Despite this study addressing the upper face, all patients received pan-facial treatment with INCO, and some also received injections in the platysma. The total doses used were distributed as follows: 39.3% (11 patients) received from 45 to 75 UI of INCO; 32.1% (9 patients) received 76–100 UI; and 28.57% (8 patients) received 101–122 UI.

Table 1 Distribution of the severity of dynamic and static lines of the sample (n = 28) according to the MAS (Merz Aesthetics Scales) at baseline (day 0)
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The average (SD) units of INCO for each upper face region were 15 (6) for the forehead, 20 (5) for the glabella, and 19 (5) for the periocular area. The correlation coefficients (rho) between the severity of the dynamic MAS and the dose of INCO utilized were 0.56 (p < 0.01) for the forehead, 0.37 (p = 0.05) for the glabella, and 0.62 (p < 0.01) for the periocular area.

Additionally, the mean (SD) injection points for each region were 18 (4) for the forehead, 7 (1) for the glabella, and 9 (2) for the periocular area. There were no correlations between the severity of the dynamic MAS and the injection points at the forehead and glabella (rho < 0.3); nevertheless, the number of injection points was correlated to MAS severity on the periocular area (rho = 0.39, p = 0.04).

The longitudinal evaluation of durability and efficacy maintenance was assessed through standardized photos as illustrated in Figs. 1 and 2. A prolonged benefit was perceived at day 240 on the upper face dynamic lines.

Fig. 1
figure 1

Sequential visits (previous to 240 days) from a participant treated with incobotulinumtoxinA on the forehead and glabella. Photos under maximum contraction of the forehead and glabella

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Fig. 2
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Sequential visits (previous to 240 days) from a participant treated with incobotulinumtoxinA on the glabella and forehead. Photos under maximum contraction of the crow’s feet

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The consensual blinded assessments of the participants (dynamic and at rest) from each visit are presented in Figs. 3, 4, 5, 6, 7, and 8. Compared to baseline, reductions in the MAS scores for forehead, glabella, and crow’s feet were observed at days 30, 120, 180, and 240 for both dynamic and static lines (p < 0.01).

Fig. 3
figure 3

MAS (Merz Aesthetics Scales) scores of dynamic lines of the forehead at day 0 (D0), D30, D120, D180, and D240 (n = 28)

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Fig. 4
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MAS (Merz Aesthetics Scales) scores of static lines of the forehead at day 0 (D0), D30, D120, D180, and D240 (n = 28)

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Fig. 5
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MAS (Merz Aesthetics Scales) scores of dynamic lines of the glabella at day 0 (D0), D30, D120, D180, and D240 (n = 28)

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Fig. 6
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MAS (Merz Aesthetics Scales) scores of static lines of the glabella at day 0 (D0), D30, D120, D180, and D240 (n = 28)

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Fig. 7
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MAS (Merz Aesthetics Scales) scores of dynamic lines of the periocular area at day 0 (D0), D30, D120, D180, and D240 (n = 28)

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Fig. 8
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MAS (Merz Aesthetics Scales) scores of static lines of the periocular area at day 0 (D0), D30, D120, D180, and D240 (n = 28)

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A reduction from baseline of at least 1 point in MAS for static forehead lines was achieved in 100%, 89%, and 46% of participants at days 30, 120, and 180, respectively. For dynamic lines, the rates were 96%, 70%, and 67%, respectively. The same reduction for static glabellar lines was achieved in 96%, 93%, and 54% of participants at days 30, 120, and 180, respectively. For dynamic lines, the rates were 100%, 85%, and 58%, respectively. Reduction from baseline of at least 1 point in MAS for static crow’s feet lines was achieved in 96%, 96%, and 58% of participants at days 30, 120, and 180, respectively. For dynamic lines, the results were 100%, 82%, and 42%, respectively (Figs. 9, 10, 11).

Fig. 9
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Percentage of participants with any improvement in forehead MAS (Merz Aesthetics Scales) dynamic lines severity, according to the visits (n = 28)

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Fig. 10
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Percentage of participants with any improvement in glabellar MAS (Merz Aesthetics Scales) dynamic lines severity, according to the visits (n = 28)

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Fig. 11
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Percentage of participants with any improvement in crow´s feet MAS (Merz Aesthetics Scales) dynamic lines severity, according to the visits (n = 28)

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At day 240, dynamic MAS scores were lower than baseline for forehead lines in 15.4% (95% CI 0.8‒30.0%) of the participants, for glabellar lines in 38.5% (95% CI 18.8‒58.1%), and for crow’s feet lines in 26.9% (95% CI 9.0‒44.8%) of the participants. These percentages were even higher for the MAS assessment of the static lines on the forehead: 38.5% (95% CI 18.8‒58.1%), glabella 46.2% (95% CI 26.0‒66.3%), and crow’s feet 42.3% (95% CI 22.4‒62.3%).

The blind assessment of GAIS for each visit is presented in Fig. 12. At day180 and 240, aesthetic improvement in comparison to baseline was identified in 63% (CI 95% 39–71%) and 35% (CI 95% 23‒50%) of the participants, respectively. There was a strong correlation between the absolute reduction of dynamic MAS for the three areas and the GAIS rating (rho > 0.63, p < 0.01).

Fig. 12
figure 12

Blind assessment of GAIS (Global Aesthetic Improvement Scale) for each visit, according to the visits (n = 28)

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There were no serious adverse effects following the 28 treatments. The sensation of a heavy forehead/glabella (14%), headache (14%), and pain at the injection points (4%) were transient until 2 weeks after the procedure, and no interventions were necessary.

The degree of overall participant satisfaction with the treatment results during the visits is presented in Table 2. At day 240 up to 62% (CI 95% 42‒81%) of the participants reported some satisfaction with the procedure.

Table 2 Distribution of the rates of satisfaction with the treatment during the visits (n = 28)
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Discussion

In this prospective evaluator-blind trial, the positive effects of upper face aesthetic treatment with INCO persisted beyond the expected muscle paralysis which usually fades after 120 days [29]. This was evidenced by the long-term reduction in both dynamic and static lines and the satisfaction of most participants lasting more than 180 days after the treatment.

The long-term effect of INCO follows a dose range, and the median duration effect for a 50-U injection in the glabella is 185 days, increasing to 215 days if 75 UI was used [30]. In this study, we achieved an 85% efficacy rate at day 120 in the glabella region by employing a personalized injection technique (ONE21 and glabellar contraction patterns), utilizing up to 25 UI of INCO (mean 20.5; SD 3.4) guided by anatomical landmarks [15]. Additionally, when comparing these results with other series, the median duration of efficacy (defined as ≤ 1-point reduction from baseline) for glabellar lines was 150 days for abobotulinumtoxinA (120 U), 168 days for onabotulinumtoxinA (40 U), and 168.7 days for daxibotulinumtoxinA (40 U) [31, 32].

The early assessment at day 30 also confirmed the efficacy in over 95% of the participants. This outcome aligns with findings from previous trials and supports the reliability of the aesthetic results from INCO [30, 33,34,35,36,37]. Furthermore, the individualized approach, guided by the assessment of upper face muscle activity, allowed for the use of lower dosages than standardized recommendations, which prevents a “frozen look” of the face [38].

This study provided the first evaluation of the overall improvement in facial appearance over a long time following the treatment. The sustained efficacy in more than 60% of the participants at day 180 supports the notion that regular treatments with INCO can yield progressive benefits and potentially prevent facial aging [39]. This preventive effect of the sequential treatments was evidenced by the comparison of a pair of twins regarding the differences in the smoothness and texture of their skin [40].

Blinded studies, with homogeneous outcomes, on assessing the long-term effects of botulinum toxin A among brands and different techniques are scarce [7]. In a double-blinded study, 250 women were randomized to receive a single treatment with 20 U of INCO or onabotulinumtoxinA on the glabella and followed for 4 months. Similarly to our results, satisfaction occurred in 93% after 4 months from those treated with INCO, without difference from those who received onabotulinumtoxinA [41]. However, in our study, up to 84% and 63% of the participants rated themselves as “satisfied” and “very satisfied” with the treatment at days 180 and 240, respectively, suggesting that the application protocol utilized may lead to longer satisfaction.

Furthermore, the aesthetic outcomes of the upper face treatment with INCO significantly enhance the patient’s quality of life. A total of 121 female Brazilian patients participated in a phase III open-label, prospective clinical study, and over 4 months, they exhibited an impressive 80% improvement from the baseline quality of life assessment conducted at day 120 [42].

The results of the efficacy and safety of INCO are well established in different populations. In a Brazilian prospective study that included 121 women undergoing treatment of upper facial dynamic wrinkles with INCO, 89% of the patients presented an improvement of at least 1 point in the overall assessment for dynamic wrinkles (forehead, glabella, and crow-feet) at day 30. Moreover, the patient self-perception of aesthetic improvement at day 120 was perceived by more than 50% of the subjects [42]. In addition, in comparative studies, no general outcome nor adverse effect differed in the comparisons of efficacy between INCO and abobotulinuntoxinA or onabotulinumtoxinA [33, 34, 41, 43, 44].

A real-life (dosage-free) case series involved 20 subjects undergoing treatment of the upper facial dynamic lines with INCO in an individualized manner. The forehead dynamic lines presented at least a 1-point improvement in 85% and 70% of participants at days 90 and 120; and for the glabellar lines, the rates were 95% and 80% at days 90 and 120, respectively [13]. In our study, the efficacy for treating dynamic lines on the forehead lasted in 70% and 67% at days 120 and 180, respectively. The efficacy for dynamic glabellar lines was 93% and 58% at days 120 and 180, respectively. Moreover, in comparison to the baseline, the blind assessment of overall improvement was evidenced in 63% and 35% of the participants at days 180 and 240, respectively.

A meta-analysis that pooled the data from four trials, with 523 onabotulinumtoxinA-treated subjects with a standard dosage of glabella, resulted in 50% of the subjects with any reduction of contraction scales after 120 days of treatment [45]. These results are also numerically inferior to ours, which reinforces the importance of tailoring the treatment with INCO according to the patient’s needs and anatomical patterns, as utilized in this study.

A longitudinal evaluation of upper face treatment efficacy with INCO, as perceived by both patients and investigators in 30 German women at day 112, also showed lower performance compared to the outcomes attained in our study for the glabella, forehead, and crow’s feet [46]. One potential explanation for these disparities could be the absence of personalized assessment doses and injection points provided by ONE21 and glabellar patterns of contraction. Differences in muscle contraction patterns necessitate tailored approaches in botulinum toxin treatments, leading to more satisfactory results [16].

The aesthetic treatment of the upper face with INCO induced a comprehensive enhancement in skin and facial appearance. Its long-term benefits are attributed to reduced muscle contraction, resulting in diminished muscle mass and decreased strength in its recovery [47]. Additionally, behavioral conditioning could lead to lower muscle contraction during facial expressions. Positive reinforcement following botulinum toxin A treatment is likely more significant in severe cases, contributing to sustained improvements [48, 49]. The ultrasonographic assessment of the reduction of muscle thickness following aesthetic treatment of the upper face is warranted.

The overall improvement of the appearance of the skin induced by neurotoxins leads to the hypothesis of its effect beyond the neuromuscular junction blockade. Acetylcholine receptors are also found on the surface of keratinocytes, dermal fibroblasts, sebocytes, and vascular endothelial cells. Beyond the reduction of the dynamic lines, an overall improvement of skin surface, redness, and smoothness is frequently perceived following aesthetic treatment with botulinum toxins [50,51,52,53].

Finally, botulinum toxin A stimulates the expression of type 1 collagen, without promoting fibroblast proliferation. This suggests the potential to induce the remodeling of aged and photoaged skin [54]. The preventive effect of sequential treatments with botulinum toxin was also evidenced by the comparison of a pair of twins regarding the differences in smoothness and fine texture of their skin [40].

This study has limitations as a result of being non-controlled. Nevertheless, as aging did not reverse by itself and the dynamic lines of the upper face were not prone to self-smoothing, it did not hinder the assessment of the results. Moreover, the homogeneity of the sample did not allow for the exploration of demographic characteristics associated with the long-term benefits following aesthetic treatment with INCO.

Conclusion

The aesthetic treatment of the upper face with INCO demonstrates enduring clinical benefits, and patient satisfaction lasting up to 180 days in most participants. The length of efficacy and satisfaction rates, which exceeded those reported in the literature, may be attributed to the utilization of techniques based on individualized assessment such as ONE21 and patterns of glabellar contraction.