Onabotulinum toxin A (BoNT-A) inhibits neural conduction by interfering with the release of acetylcholine. After binding selectively to presynaptic receptors, Onabotulinum toxin A enters the cell and cleaves a membrane protein causing acetylcholine exocytosis [1]. The use of Onabotulinum toxin A for aesthetic purposes was introduced by Carruthers in 1992 for glabellar frown lines [2], and after ten years, in 2002, the Food and Drug Administration (FDA) approved its application for aesthetic intervention.

Although Onabotulinum toxin A it’s a widely used treatment method and a lot of data from published studies support its clinical efficacy, few of them are based on clinical evaluation tools able to exclude the subjective component of measurements [3-15]. Then, an objective and quantitative scale is still needed for the evaluation of lines severity, skin texture improvement and for the monitoring of the effectiveness of Onabotulinum toxin A treatment. Here in this study, we propose a digital evaluation tool able to detect lines improvements with objectivity, evaluating the same areas of the skin at baseline and after treatments with Onabotulinum toxin A.

This is a prospective, single group, not randomized and open label observational study conducted between September 2014 and June 2015 in Milan, Italy. Twenty healthy Caucasian females, with median age of 54 years (range: 35-76) were eligible for the study. Exclusion criteria were age <18 years, treatment of glabellar lines and crow’s feet lines with absorbable fillers in the last 6 months previous the study, botulinum toxin treatment or laser treatment in the upper face in the previous 6 months, blepharoplasty and face lifting in the last 1 year, pregnancy and nursing, and immunological disorders.

Subjects were at least 18 years of age who met severity criteria for both CFL and GL (moderate-to-severe bilaterally symmetrical CFL at maximum smile and moderate-to-severe GL at maximum frown) requiring treatment.

Baseline CFL and GL severity determination was based on investigator ratings using the Facial Wrinkle Scale (FWS; 0=none, 1=mild, 2=moderate, and 3=severe). Each patient provided a written informed consent. This study complied with the ethical rules recommended by 2008 Declaration of Helsinki. Authors ensure adherence to the principles of good clinical practices (GCPs), including adequate human subject protection (HSP).

Onabotulinum toxin A (Vistabex^®, Allergan^®, Inc.) was reconstituted with 1.25 ml of saline solution and each patient received a total of 44 U (20 U and 24 U for the treatment of glabellar lines and crow’s feet lines, respectively). Information on injection procedures are reported in Table 1.

Table 1: Injection procedures for the treatment of glabellar lines and crow’s feet.

All the patients enrolled in the present study were evaluated by a special software, Digital Analysis of the Cutaneous Surface [14-20], performed by Antera 3D^® device (Antera3D^®, Miravex Limited , Dublin. Ireland) as previously reported [14]. The assessment tool is provided by a digital camera (Antera 3D^®) 56 x 56 mm amplitude, placed on the area of the skin where measurements are performed. This method relies on multi-directional illumination and computeraided reconstruction of the skin surface, illuminating the surface from different angles and using the differences between these images to reconstruct the surface in three dimensions. The skin topography and chromophore concentration are derived from the spatial and spectral analysis of the acquired image data, obtained by illuminating the skin with light emitting diodes (LEDs) of different wavelengths shining from different directions (Figure 1).

Figure 1: Scheme of multi-spectral illumination of the skin with the Antera 3D^®.

The reconstruction of the skin surface was achieved using a technique based on Shape from Shading (SFS) [B.K. Horn, The Psychology of Computer Vision, P.H. Winston ed., New York, 1975], extensively modified in order to eliminate skin glare and improve the accuracy of measured data [PCT patent application PCT/ EP2010/001168, Irish Patent No. S85695]. The reconstructed surface is then used for quantitative skin analysis, such as depth and width of wrinkles and skin roughness.

For wrinkles assessment, a filter was applied to the reconstructed 3D skin surface, in order to discriminate between wrinkles-related curves (i.e. sharp, fine skin indentations) and the standardized reference surface, the skin as it would be in the absence of wrinkles. The standardized reference shape was then removed to obtain wrinkles representation only (Figure 2).

Figure 2: (a) Wrinkle cross-section showing the reconstructed skin surface (in black) and the reference surface (in red). A representation of the wrinkles alone can be obtained by removing the normalized reference surface from the reconstructed skin surface; (b) Top view representing a wrinkle width measurment.

Wrinkles were assessed for both depth and width. Wrinkle depth was defined as the average of the maximum depths of each crosssection along the strip selection’s centerline. Wrinkle’s width was defined as the average width of each cross-section along the strip selection’s centerline. The overall size of wrinkles was proportional to the average cross-section area along the strip selection centerline.

For glabellar lines analysis, the most depth and/or more representative wrinkle was selected. For crow’s feet, the most representative wrinkle from the upper area, delimited by the lateral canthal muscle, eyebrow’s tail extremities and the external canthus, was assessed.

Antera 3D^® digital images and photographs were performed at 3 different times, at rest and at maximum muscular activity of glabellar (maximal frown) (Figures 3-5) and periocular areas (maximum smile and contraction of orbicularis oculi muscle) (Figures 6-8). In particular, two images were taken both frontal (at rest and maximum muscular activity) for the glabellar area, right and left profiles for the periocular area, at rest and maximum muscular activity respectively as recommended by the international guidelines [18].

Figure 3: Case (A) Caucasican Female, 36 years old. Antera 3D^® Digital Image of Glabella in maximal frown. Preoperative.

Figure 4: Case (A) Caucasican Female, 36 years old. Antera 3D^® Digital Image of Glabella in maximal frown. Postoperative 1 month later.

Figure 5: Case (A) Caucasican Female, 36 years old. Antera 3D^® Digital Image of Glabella in maximal frown. Postoperative 4 month later.

Figure 6: Case (B) Caucasican Female, 46 years old. Antera 3D^® Digital Image of periocular area in maximal contraction. Preoperative.

Figure 7: Case (B) Caucasican Female, 46 years old. Antera 3D^® Digital Image of periocular area in maximal contraction. Postoperative 1 month later.

Figure 8: Case (B) Caucasican Female, 46 years old. Antera 3D^® Digital Image of periocular area in maximal contraction. Postoperative 4 month later.

Furthermore, a clinical evaluation was also performed using the Facial Wrinkle Scale (FWS), which assess wrinkles severity in 4 degrees : 0 (none), 1 (mild), 2 (moderate), and 3 (severe). All the pictures were captured with predefined reference anatomic points in standardize reproducible method for each measurement in each patient, using the software graduate visual scale of the digital camera. Collection and statistical analysis of data obtained was then processed by both study clinician and a second expert.

At the first visit, all the clinical information about side effects, adverse events and/or any other information were noted on a register at baseline, 4 weeks and 4 months after treatment.

The primary endpoint was glabellar lines and crow’s feet improvements after the treatment with Onabotulinum toxin A. Duration and safety of the treatment represented the secondary endpoints. Patient satisfaction was not an outcome measured in this study.

Wrinkles were assessed as follows

For glabellar area, the deepest and/or most significant line was selected and analyzed for both the global improvement of the wrinkle by the length/width ratio (percentage of improvement/100%) and wrinkle depth improvement (millimeters, mm) measured by Antera 3D^® software (Figures 9-11)

Figure 9: Case (A) Caucasican Female, 36 years old Antera 3D^® Software analyzed image of glabellar lines in maximal frown. Preoperative.

Figure 10: Case (A) Caucasican Female, 36 years old Antera 3D^® Software analyzed image of glabellar lines in maximal frown. Postoperative 1 month later.

Figure 11: Case (A) Caucasican Female, 36 years old Antera 3D^® Software analyzed image of glabellar lines in maximal frown. Postoperative 4 month later.

For crow’s feet, the most representative wrinkle from the upper area, delimited by the lateral canthus, eyebrow’s tail extremities and the external canthus, was assessed (Figures 12-14).

Figure 12: Case (B) Caucasican Female, 46 years old. Antera 3D^® Software analyzed image of periocular lines in maximal frown. Preoperative.

Figure 13: Case (B) Caucasican Female, 46 years old. Antera 3D^® Software analysed image of periocular lines in maximal frown. Postoperative 1 month later.

Figure 14: Case (B) Caucasican Female, 46 years old. Antera 3D^® Software analysed image of periocular lines in maximal frown. Postoperative 4 month later.

These criteria were chosen to exclude wrinkles resulting from the activity of muscles other than the orbital, or from the combination of more than a muscle as happen for zygomatic muscles. We found the method described as the only one suitable for analysis, since any different method/area selected for the comparison between pre- and post-treatment could include the zigomatic muscles action that interfere with the orbicularis contraction and CFL formations. The power of our method is represented by the fact we can be sure that we are analyzing only the contraction of the orbicularis muscle, which is the target of Onabotulinum toxin A treatment (Figures 15-18).

Figure 15: Case (A) Antera 3D^® Software automated chart; overall dimension improvement of glabellar line in Caucasican Female, 36 years old.

Figure 16: Case (A) Antera 3D^® Software automated chart; depth improvement of glabellar line in Caucasican Female, 36 years old.

Figure 17: Case (B) Antera 3D^® Software automated chart; overall dimension improvement of periocular line in Caucasican Female, 46 years old.

Figure 18: Case (B) Antera 3D^® Software automated chart; depth improvement of periocular line in Caucasican Female, 46 years old.

For glabellar area, the improvement from baseline of the deepest wrinkle was, for the 75% of patients, a value ranging from 45% to 88.6% at 4 weeks, and for the 33% of patient, a value ranging from 44.7% to 61.8% at 4 months after treatment with Onabotulinumtoxin A. Wrinkle depth improved from 0.589 mm at baseline, to 0.248 mm after 4 weeks and 0.416 mm after 4 months from treatment injections.

During the assessment of crow’s feet improvements, changes in wrinkles position were considered because of their affection on the treatment of this area with Onabotulinumtoxin A. The improvement of the most representative wrinkle was, for the 85% of patients, a value ranging from 44.5% to 83.3% at 4 weeks and, for the 30% of patients, a value ranging from 31.1% to 61.9% at 4 months after treatment with Onabotulinumtoxin A. Wrinkle depth improved from 0.37 mm before treatment, to 0.15 mm at 4 weeks and 0.3 mm at 4 months after treatment injections.

As for the evaluation of GL on the FWS, 100% of patients had an improvement of at least 2 grades after 4 weeks and 95% of patient still had an improvement of at least 1 grade after 4 months (investigator assessment).

Concerning the analysis of CFL on the FWS, 90% of patients had an improvement of at least 2 grades after 4 weeks at least 1 grade after 4 months (investigator assessment). Two examples are reported in Table 2.

Table 2: Results obtained for two patients with both FWS and Antera 3D^® evaluations.

No serious adverse events were observed during the study treatment. Headache was referred by 3 patients (15%) few hours after being treated with Onabotulinuntoxin A, resolved 12-48 hours from its occurrence and without any treatment need. Two patients (10%) reported bruising at the periocular area, and 1 patient (5%) referred eyelid heaviness which resolved spontaneously 2 weeks after Onabotulinum toxin A injection.

Although Onabotulinum toxin A effectiveness for wrinkles aesthetic treatment of is proved and undisputed, the evaluations scales used to assess wrinkles improvements are still debated. These scales are based on different scoring (from 0 to 5 for glabellar lines, and from 0 to 4 for crow’s feet) [8] and have a subjective component, living space to individual interpretations by clinician and, in double blind studies, by patient.

Previous studies tried to establish objectives methods to quantify wrinkles improvements after treatment with Onabotulinum toxin A. Heckman et al. [11] established objective parameters to measure the efficacy of Onabotulinum toxin A for correction of hyperkinetic facial lines, which were reproducible and based on serial digital photographs used to measure brows mobility and brow to brow distance.

The Antera 3D^® Software (Digital Analysis of the Cutaneous Surface) digital tool usefulness has been recently described to evaluate wrinkles severity and texture analysis [14]. In the study, The Antera 3D^® Software provided an objective, direct, fast, and reproducible method to measure OnabotulimuntoxinA treatment for rhytides. With Antera 3D^® Software the operator is able to select the investigated area, which is automatically recognized by the software during the subsequent acquisitions. An algorithm is then used to analyze skin surface and measure parameter changes from baseline, without any subjective evaluation.

In this paper, we applied the Antera 3D^® Software digital tool for the quantitative assessment of glabellar lines and crow’s feet improvements after Onabotulinumtoxin A treatment.

Onabotulinum toxin A was administered following guidelines recommendations, based on FDA, EMA, AIFA and the summary of product characteristics.

Treatment efficacy was evaluated numerically, therefore objectively, based on depth changes of the most representative wrinkle from both glabellar and crow’s feet areas. The most representative wrinkle has the highest aesthetic impact and is the most difficult to treat, then reflecting properly treatment success after Onabotulinum toxin A injections. Specifically, for the periocular area we selected the upper wrinkle, delimited by the lateral canthus, eyebrow’s tail extremities and the external canthus, which derives from the orbital muscle activity. This method excluded measurements of zygomatic muscles, which are involved at maximum of smile extension and eye wink during photographs acquisition and their activity is subjectively different between patients. Moreover, periocular area dynamics change after treatment, included wrinkle position and the area selected by the investigator during the next assessments. All together, these criteria represent an objective evaluation of wrinkle improvements over a treatment cycle with Onabotulinum toxin A. Wrinkles improvement was assessed by wrinkles depth/length ratio, where depth decreases and length increases as a consequence of treatment efficacy. All data obtained with Antera 3D^® Software indicated a significant improvement of glabellar lines and crow’s feet after treatment with Onabotulinum toxin A, confirming its clinical effectiveness for aesthetic use. Glabellar lines improved up to 88.6% and 73.9% respectively after 4 weeks and 4 months from treatment. For crow’s feet, wrinkles improved up to 83.3% and 61.9% after 4 weeks and 4 months from the first Onabotulinum toxin A injection.

We observed, in the group of patients, a large distribution of the improvement percentages. In this study we had used a standard treatment protocol based on the guidelines recommended in the S.P.C. However, the glabellar and periocular area have a most variable muscular contraction patterns in the population. To obtain better results in these areas, as described by De Almeida et al. [20] for the glabellar lines, the injection points should be adapted to the specific contraction patterns in each patient.

A comparison between FWS evaluation and Antera 3D^® results has been reported in Table 2, which report data for two patients enrolled in the study. It seems that approximatively 1 grade on the FWS correspond to 25 percentage points of Antera 3D^® Software results. The Table 2 clearly shows two important points: first, the “correspondence” FWS/Antera 3D^® Software is not always respected (patient 2, GL improvement after 4 months), underlying the subjective bias of the FWS. Second, FWS is just a 4 grades scale, while Antera 3D^® Software uses a 100 point scale, allowing a more precise evaluation of lines.

The presence of few and transient side effects during the treatment period, confirmed the safety of Onabotulinum toxin A previously described [18].

In this work, no self-assessment was conducted by patients. Although, we believe that this innovative method could be a valid tool to involve patients during the treatment period, as photographs and measurements show the objective improvement of wrinkle at any time of the treatment period. Furthermore, the objectivity of this tool could help on comparing with more precision the treatment efficacy of the different toxin formulation available for aesthetic use.

Images acquired with Antera 3D^® device and analyzed by the special software is a valid and alternative method to assess wrinkles severity as well as their improvement after Onabotulinumtoxin A treatment. Authors find that this fast, objective, reproducible and self-consistent evaluation method could be a very useful tool to be used for both in the clinical practice during dialog with patient and in clinical trials.

Efficacy and safety observed in this study represent a further confirmation of the positive experience of Onabotulinumtoxin A in the aesthetic use.

This research received a financial support from Allergan, Inc. Allerga, Inc. didn't have any active role in this study; only authors provide to organize the study, to execute the treatments, to analyze and interpret data, to write and submit the report [Grant number 4301148828].