This study was a prospective, parallel-arm, 4-arm, multicenter, triple-blind randomized, placebo-controlled clinical trial performed from 2020 to 2022 on 729 participants in a private clinic and a hospital in Tehran, Iran with a treatment/placebo allocation ratio of about 1:2. All participants were briefed completely before the study and signed written informed consent. The ethics of this study was approved by two independent bodies: the research committee of the Islamic Azad University (ethics code: IR.IAU.DENTAL.REC.1399.301, date: 07/03/2021) as well as an international organization responsible for examining, approving, and registering randomized clinical trials (IRCT.ir) following the Helsinki declaration (RCT code: IRCT20210401050804N1, date: 26/04/2021). After the registration of the study, the sample size was augmented to 729 from an original size of 96 individuals, per more conservative criteria to ensure high power, reliability, and generalizability (given that the treatment was harmless; detailed below). The experiments began on 25/04/2021 and continued until May 2022.
Eligibility criteria and the sample
Included were all patients who had indications for BTX-A injection in the forehead area and had been referred to BouAli Hospital, Tehran, and a private clinic at the time of the examination and who did not have a previous history of an unpleasant postinjection issue. Each person needed to have symmetric mimetic forehead muscle function. Patients with any history of allergy to botulinum toxin type A or lidocaine as well as any history of a neuromuscular disorder would be excluded. Initially, after filling out the file for patients and taking a medical history, all patients with a previous unpleasant history (including allergies) and all patients with asymmetry in injection sites, eyelid ptosis, and diplopia were excluded.
Diagnosis of diplopia was made according to the patient’s statements; asymmetry and ptosis were diagnosed via observation by a specialist physician. Only patients with full data were included; in other words, if a patient would not attend the follow-up session 2 weeks after the injection, the recorded pain score would be discarded as well. The patients lost to follow-up would be replaced by new patients in order to reach the desired sample size.
Interventions
Based on the dilution materials and the Botulinum toxin brands, there were 4 subgroups:
A: Each participant received 100 units of BTX-A (Dysport, Ipsen, France) at 20 injection points, combined with 1 ml of normal saline without preservatives.
B: Each person received 100 units of BTX-A (Dysport) combined with 1 ml of lidocaine 2% without epinephrine at 20 injection points.
C: Each individual received 50 units of BTX-A (Xeomin, Merz, Germany) combined with 1 ml of normal saline without preservatives at 20 injection points.
D: Each patient received 50 units of BTX-A (Xeomin) attenuated with 1 ml of lidocaine 2% without epinephrine.
The allocation ratio of experimental-to-placebo groups was about 1:2. The allocation ratio of the botulinum toxin brands was determined according to their market availability, i.e. subgroups utilizing the Dysport brand were larger than Xeomin subgroups because Dysport was more available than Xeomin. The amount of botulinum toxin to be used for each patient was instructed by the manufacturer.
Injection protocol
The dilution of botulinum toxin was performed by an experienced technician. Injections were performed in an upright position by an experienced surgeon. This was considered safe because for the dilution with normal saline, we followed the exact instructions of the manufacturers (available in product packages), and for the dilution with lidocaine, previous numerous studies on lidocaine in combination with different materials or alone have found it safe22. A 29-gauge needle was used in all subgroups (Terumo K-Pack®II, Japan). In the placebo group, in which botulinum toxin was diluted with normal saline, the normal saline was a 9% injectable sodium chloride solution without any preservatives (Injectable and Pharmaceutical Products, Tehran, Iran). In the experimental group, in which botulinum toxin was diluted with lidocaine, the lidocaine in use was 2% with a preservative (Pasteur Institute, Tehran, Iran). The injection was performed carefully by an experienced surgeon for each person at 20 points (for Dysport: 5 units each, 100 units in total; for Xeomin: 2.5 units each, 50 units in total, Fig. 1). These were standard points recommended in textbooks, and hence effective and safe23. The amount of injected toxin from each brand was in accordance with its manufacturer’s instructions.
Injection sites: 1. Edge of the corrugator muscle; 2. The distance between the previous 2 points on the nose; 3. About 1 cm above the supraorbital notch; 4. About 1 cm above point #3 on each side, two new points were selected on the frontal muscle with equal distances; 5. About 1 cm above points #4 and between these points, three new points with equal distances were selected on the frontal muscle; 6. The tail of the eyebrow; 7. The edge of the temporal line; 8. Around the eye at the location of the orbicularis oculi muscle on the lateral rim of the eye, two points were selected.
Sample size
Since there was no comparative study on the pain intensity after the injection of BTX-A at the time of designing this research, no effect size was available from the literature. Therefore, the authors assumed a conservative effect size (0.25) as well as a high power (90%), resulting in a sample size of 676 participants. The sample size was augmented to 730 patients to offset additional variables besides pain (i.e. 2-week complications). Since there was no limit on the time frame of the study and also since there was a large number of individuals agreeing to participate, we did not anticipate any particular issues in curating the needed data.
Randomization and blinding
The study was triple-blind, meaning that the surgeon, the participant, and the observer were unaware of the materials and grouping: The surgeon administering the botulinum toxin was blinded to the experimental/placebo assignments and the botulinum brands. Although the numbers of units used from the brands differed, their volumes after dilution would be similar and undetectable from each other. The observers were blinded to the treatment/control assignments and to the botulinum brands as well. The patients were blinded to the grouping as well.
All documents and questionnaires were coded. The coded documents were kept in sealed envelopes. The person who did the randomization also performed the concealment. She was not involved in any of the examinations.
The participants were randomized using a computer program at two levels (Excel, Microsoft, Redmond, Washington, USA). First, they were randomized into one of the two groups (experimental versus placebo) with a ratio of about 1:2. Since the study was large and multicentered, and since many people could be excluded or lost to follow-up, an exact ratio of 1:2 was not necessitated. After the first randomization, the individuals were randomized into either of the two brands of BTX-A with a ratio similar to the market share and availability of these brands. The syringes were coded and given to the surgeon who injected the botulinum toxin. The surgeon, who was the same person in all groups, performed the injection and completed the form without knowing the botulinum toxin brand and the dilution material.
Outcomes
Primary outcome
The post-injection pain was assessed using an 11-level numerical rating scale questionnaire (0 representing no pain and 10 representing maximum and intolerable pain needing emergency attention).
Secondary outcomes
Two weeks after injection, ptosis (assessed by observation), asymmetry (through observation), diplopia (assessed by asking the participant), and any need for a botulinum toxin retouch (expressed by the patient) were recorded. Ptosis of the eyelid is one of the most common ocular complications associated with botulinum toxin injection that appears in the first 2 weeks after botulinum injection24. Ptosis refers to drooping of the upper eyelid and can affect one or both eyelids. In the mild form, drooping eyelids are above the pupil of the eye, but in more severe cases drooping eyelids cover a part of the pupil so that it reduces the upper visual amplitude24. Diplopia is another ocular complication associated with botulinum toxin injection. Diplopia is secondary to extraocular muscle dysfunction. The patient complains of diplopia resulting from the injection, usually in the follow-up 1 week after the injection, which occurs following the paralysis of the lateral rectus muscle24. Eyebrow asymmetry is sometimes seen in the upper face third at the tail of the eyebrows, following the injection into the frontalis muscle to treat hyperkinetic forehead lines24.
Statistical analysis
Descriptive statistics and 95% confidence intervals (CIs) were calculated for the pain levels in different groups and subgroups. An independent-samples t-test was used to compare the main groups with each other in terms of their pain and age. Furthermore, a 3-way analysis of covariance (ANCOVA) was used to assess the simultaneous effects of the independent variables (lidocaine incorporation, the brand of botulinum toxin, and participants’ age and sex) on postinjection pain. Age was a continuous variable recorded as an integer number; it was without any grouping and with a unit of change of 1 year. A Pearson correlation coefficient and a partial correlation coefficient were used to examine the correlation between patients’ age (continuous) and their pain levels. Since the correlations were significant, descriptive statistics and 95% CIs were calculated for patients’ pain in different decades of life (as discrete age groups). As well, a histogram was drawn to show the distribution of patients’ ages. For the assessment of the effects of the same 4 independent variables on each of the 2-week post-injection complications, a point-biserial correlation coefficient, a Fisher, and a multiple binary logistic regression were used. Again, the variable “age” was an integer continuous variable without grouping. However, since its role became significant in some complications, the incidences of complications in different life decades (as discrete groups) were summarized as well. The level of significance was set at 0.05.
Ethics approval and consent to participate
The participants were briefed completely before the study and signed written informed consents. The ethics of this study was approved by two independent bodies: the research committee of the Islamic Azad University (ethics code: IR.IAU.DENTAL.REC.1399.301, date: 07/03/2021) as well as an international organization responsible for examining, approving, and registering randomized clinical trials (IRCT.ir) in accordance with the Helsinki declaration (RCT code: IRCT20210401050804N1, date: 26/04/2021).