Evaluation of the Efficacy of a Mouthwash Formulation for Minor Oral Ulcers: A Triple-Blind Randomized Controlled Trial
Phase: NA
Conditions: Recurrent Aphthous Ulcer, Recurrent Aphthous Stomatitis
Sponsor: G. d'Annunzio University

 
 
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CLINICAL STUDY PROTOCOL  
 
 
 
Evaluation of the Efficacy of a Mouthwash Formulation for the 
Treatment of Minor Oral Ulcers: A Triple-blind Randomized Controlled 
Trial  
 
 
Version 1.0 
Date 05/10/2021 
 
Scientific Coordinator: 
Prof. Antonio Scarano 
Department of Innovative Technologies in Medicine and Dentistry 
University of Chieti-Pescara 
Via dei Vestini 31, 66100 Chieti (CH) 
 
Trial Center: 
Department of Innovative Technologies 
In Medicine and Dentistry 
University of Chieti-Pescara 
Via dei Vestini 31, 66100 Chieti (CH) 
 
    
 
 
 
 

 
 
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Table of Contents 
1. INTRODUCTION ................................................................................................................................ 3 
2. PURPOSE OF THE RESEARCH ........................................................................................................ 3 
3. STUDY DESIGN .................................................................................................................................. 4 
3.1 Inclusion Criteria............................................................................................................................. 5 
3.2 Exclusion Criteria ........................................................................................................................... 5 
4. Procedure .............................................................................................................................................. 5 
4.1 Treatment ........................................................................................................................................ 5 
4.2 Compliance of the processing ......................................................................................................... 5 
4.4 Curriculum ...................................................................................................................................... 6 
6 
CLINICAL PROCEDURES .............................................................................................................. 8 
6.1.1 Summary of procedures and follow-up ............................................................................................ 8 
6.1.2 Drop-out ........................................................................................................................................... 8 
6.1 
Evaluation of the primary objectives .......................................................................................... 8 
6.2 
Evaluation of secondary objectives ............................................................................................ 8 
7 
ANALYSIS........................................................................................................................................ 9 
8 
Data management ............................................................................................................................ 12 
 
 
 
 
 

 
 
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1. INTRODUCTION 
Recurrent aphthosis is a disease with a multifactorial and immune-mediated inflammatory etiology. 
Clinically it is often associated with the manifestation of ulcers affecting the soft tissues and oral mucosa 
[1,2].  Recurrent aphthosis is epidemiologically the pathology of the oral district with the highest 
incidence around and occurs most frequently in subjects between 20 and 30 years of age [3,4].. The 
clinical course is often associated with periods of quiescence followed by phases of recrudescence and 
tends to occur less frequently and severely in the late phase of the adult subject [5,6]. In the case of an 
individual predisposition, this pathology can produce the appearance of recurrent lesions in adulthood, 
often also in association with systemic diseases or immunodepression [2,5,7]. The main individual and 
environmental predisposing factors are: 
o Familiarity and heredity  
o Microbiological factors  
o Hypersensitivity  
o Local traumas 
o Hormonal disorders  
o Immunological disorders 
o States of stress and psychological disorders 
o Smoking 
 
 
An association with vitamin deficiencies (zinc, iron, vitamin B1, B2, B6, B12, folic acid) as well as 
gastrointestinal diseases, celiac disease and chronic inflammatory bowel diseases (Crohn's disease) is 
also reported in the literature [4,5,8,9]. In addition, recurrent aphosis has been associated with Behcet's 
syndrome, Reiter's disease and AIDS. In addition, neutropenia and administration of beta-blockers, 
bisphosphonates, NSAIDs, protease inhibitors, sulfonamides are also predisposing factors [4–6,8–11]. 
In the literature, several protocols have been proposed for the treatment of recurrent aphthosis with 
symptomatic therapies and active ingredients such as chlorhexidine 0.2% in mouthwash, benzidamine, 
zinc chloride and polycresulene, where, however, many ulcerative lesions can sometimes recur and be 
refractory to such administrations [5,6,12–15].  Among the possible correlations to this failure is the 
effect of dilution and Wash-out of saliva. Bio-adhesion to oral tissues affected by canker sores is also a 
key factor in determining the effectiveness of the treatment [6,8–11]. 
 
2. PURPOSE OF THE RESEARCH 
 
The aim of the present research is to evaluate the efficacy of a mouthwash formulation for a total 
treatment of 14 days for the treatment of oral ulcers of traumatic or aphthous nature. 
The study groups are as follows: 
 
• Group I (Test): Curasept Canker Sores Rapid Mouthwash: Dicaprylyl Ether, Coco-
Caprylate/Caprate, Hydrolyzed RNA/DNA, Hyaluronic Acid, Cetyl Palmitate, Beta-Glucan. 

 
 
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Versus 
 
• Group II (Control): Placebo mouthwash  
 
 
Null hypothesis (H0): There is no difference at 14 days in the reduction of the mean pain sensation and 
ulcer area score, thermography [16,17] and Ulcer Severity Score (USS) [18] in the two groups, against 
the alternative hypothesis of a difference between them. 
 
3. STUDY DESIGN 
The present study is a 2-week double-arm randomized controlled trial.  
The study will be carried out in the following ways:  
 
- 
Randomization: Using a computer-generated, centralized random number with random codes 
(A, B) placed in sequentially numbered sealed opaque envelopes provided by the study 
consultant. A simple randomization procedure will be used. 
- 
Concealment of assignment: Doctors involved in patient treatment will never know which 
mouthwash will be delivered to patients. Identical and indistinguishable bottles with mouthwash 
will be prepared by a person designated by the sponsor. The mouthwash will be poured into 
identical vials labeled with only one letter (A, B), and only the person designated by the sponsor 
will be of which product matches the letters. The random assignment will be made by opening 
the sequentially numbered sealed envelope just before the surgery.  
- 
Blinded assessment: A single, independent, blinded assessor will perform all clinical 
measurements. Patients will not be aware of the mouthwash they will use. In addition, the 
operator evaluators and statistician will remain blinded until statistical analysis (blinded study) is 
performed. The codices (A, B) will be broken at the time of writing the manuscript. 
 
The principles outlined in the Declaration of Helsinki on clinical research involving human subjects will 
be respected. Written informed consent will be obtained for each patient. 
 
The study will be reported according to the CONSORT statement to improve the quality of reports of 
randomized parallel-group trials (http://www.consort-statement.org/). 
A total of 3 visits will therefore be carried out:  
• A screening/baseline visit will help establish patients' eligibility for inclusion in the study, followed 
by baseline data recording and first supervised mouthwash application 
• A 1-week experimental visit 
• A 2-week experimental visit and end of study 
 
 

 
 
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3. CRITERIA FOR THE SELECTION OF STUDY SUBJECTS 
 
3.1 Inclusion Criteria 
 
Patients must meet all of the following inclusion criteria to be eligible for study enrollment: 
 
1. Patients with at least one well-defined ulcer 
2. Any patient who is at least 18 years old and able to sign an informed consent.  
 
3.2 Exclusion Criteria 
The presence of any of the following will exclude a subject from study enrollment:  
 
1. Intolerance or allergy to the product. 
2. Patients with hematologic deficiency such as anemia, iron, vitamin B12 and/or folic acid deficiency; 
systemic diseases such as ulcerative colitis, Crohn's disease, Behcet's syndrome in which RAS is part of 
their clinical presentation; alcohol consumption and smoking; history of allergy; treatment of ulcers with 
systemic steroids, vitamins, antibiotics, antihistamines, oral retinoids, or immunomodulatory agents 
within three months prior to study entry; and the use of nonsteroidal anti-inflammatory drugs or 
mouthwash for the treatment of ulcer prior to 72 hours of study entry. 
3. Patients who are pregnant or breastfeeding. 
 
 
4. Procedure 
4.1 Treatment 
The local trial manager will open the sequentially numbered sealed pouch corresponding to the patient's 
enrollment number and provide the patient with the randomly assigned coded vial (A, B) instructing the 
patient to rinse the ulcer with mouthwash, twice a day, after oral hygiene. 
 
The treatments assigned are: 
• Curasept Afte Rapid Mouthwash: Dicaprylyl Ether, Coco-Caprylate/Caprate, Hydrolyzed RNA/DNA, 
Hyaluronic Acid, Cetyl Palmitate, Beta-Glucan. 
 
Versus 
 
• Placebo mouthwash 
 
4.2 Treatment compliance 
Compliance will be assessed based on the information recorded at the last visit by the subject and the 
information recorded in the CRF during the treatment visits. 
 
4.3 Product Storage 
Store in a cool place (< 30°C) and keep away from light sources in an unopened original vial 
STUDY PLAN 

 
 
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4.4 Study program  
 
Screening and baseline visit (V1)  
Potentially eligible patients are screened to determine their eligibility for the study. Complete (Patient 
Eligibility CRF) and record the number and reason for patients not included. 
Prior to enrollment, all patients will be asked to sign an informed consent form to document that they 
understand the purpose of the study (including procedures, follow-up assessments, and any potential risks 
involved), have been given the opportunity to ask questions related to this study, and have been informed 
of treatment alternatives. 
At the recruitment visit, the clinician should assess the presence of an oral ulcer, rule out lesions of 
neoplastic, infectious, or allergic origin, and remove, if present, any causative agents (such as sharp edges 
of teeth, fillings, crowns, dentures, or braces). 
 
Primary Results: 
 
• Pain intensity. It is measured using the VAS consisting of a 10 cm line [13], where 0 indicates no pain 
and 10 indicates severe pain. 
 
• Size of each ulcer. A sheet of clear plastic will be applied directly to the ulcer and using a permanent 
waterproof marker the circumference of the ulcer will be traced and then placed on a graph paper and 
the number of mm2 units included within the drawn area will be counted. 
 
Secondary Results: 
 
•Thermography 
• Ulcer Severity Score (USS) 
 
 Treatment will be started on the same day with strict infection control measures. 
The assessor will open the sequentially sealed envelope corresponding to the patient's recruitment 
number and write on the CRF the progressive number of the patient contained in the envelope and to 
which group (A or B) the patient will be assigned. 
 
Patients will receive three anonymous bottles, coded with A, B containing the mouthwash and a scoop. 
Patients will apply mouthwash. This procedure will be performed under the supervision of the outcome 
measure which will provide further explanations to patients if necessary. 
 
Participants will be asked to flush with 10mL of mouthwash twice daily directly onto the ulcer. Patients 
were prevented from eating, drinking, or rinsing their mouths for at least one hour after each 
application. 
 
 
Observation at 1 week (V2) and at 2 weeks (V3):  
 

 
 
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• Pain intensity using the VAS scale. The following outcome measures will be recorded by the 
blinded outcome assessor: 
• Size of each ulcer using a clear plastic sheet 
 
• Thermography/ Ulcer severity score (USS) 
 
In the event of any complication observed during any of the scheduled visits or during an emergency 
visit, protocol deviation and drop out should be filled out CRF. 
 
Unscheduled visits may be made during the study at the discretion of the Investigator. 
Relevant information will be collected in the CRF. 
The study plan and the scheduled items are summarized in the following flowchart: 
 
 
 
Visit 
Screening and 
Baseline (V1) 
1 week (V2)  
 
2 week (V3) 
Day 0 
Day 7 
Day 14 
Evaluation 
 
 
 
Inclusion and Exclusion 
Criteria 

 
 
Informed consent 

 
 
First mouthwash 
application  

 
 
Pain Assessment (VAS) 
 
 

Ulcer measurement 
 
 

Thermography 



Ulcer severity Score 
(USS) 



Complications 
 
 


 
 
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6 CLINICAL PROCEDURES 
 
 6.1.1 Summary of procedures and follow-up 
 
Day 0 Patient Recruitment, Informed Consent. 
        
Pain Intensity (VAS), Lesion Measurement, Thermography, USS and Mouthwash 
Application  
 
 
Day 7 
Pain Intensity (VAS), Lesion Measurement, Thermography, USS, and Recording of 
Complications and Adverse Effects  
Day 14 Pain Intensity (VAS), Lesion Measurement, Thermography, USS and Recording of 
Complications and Adverse Effects 
 
6.1.2 Drop-out 
All drop outs will need to be reported by recording the study group's reason for drop out. No included 
patient can be excluded from the final evaluation, for any reason, by the clinical investigators. 
6.1 Evaluation of the primary objectives  
 
• Pain intensity. It will be measured using the VAS consisting of a 10 cm line [13], where 0 
indicates no pain and 10 indicates severe pain. 
• Size of each ulcer. A sheet of transparent plastic will be applied directly to the ulcer and using a 
permanent waterproof marker the circumference of the ulcer has been traced and then placed on 
a graph paper counting them units of mm2 included within the drawn area. Treatment will begin 
on the same day with strict infection control measures. 
 
6.2 Evaluation of secondary objectives  
a. Thermography [16,17,19] 
b. Ulcer severity Score (USS) [18]: 
 
 
 
Ulcer severity Score (USS) 
 
 
Characteristics of the 
lesion 
Score 
Description 
of 
the 
USS 
Average number of 
injuries 
 
 
Score 
= 
average 
number of ulcers in a 
culture Maximum score 
= 20 
Average lesion size (in 
mm) 
 
 
Score = mean size of 
ulcers in mm Maximum 
score = 20 
Average duration of 
lesions (in weeks) 
 
 
Score = number of 1/2 
weeks 
i.e. Half a week (3 
days) scores 1, one and 

 
 
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Complications and adverse events: Any post-operative complications and adverse events (mucosal 
irritation, allergic reactions, etc.) will be recorded and reported by study group by blinded evaluators in 
the Complications, protocol deviation, and drop out. 
  
 
7 ANALYSIS 
 
7.1 Sample size 
a half weeks (10 days) 
scores 3. 
Maximum score = 10 
Injury-free period (in 
weeks) 
 
 
Score = 10 minus the 
average 
ulcer-free 
period in weeks 
Maximum score = 10 
(never ulcer-free) 
Patient-perceived 
pain (scaled from 0–
10) 
 
 
1 for slight discomfort 
in 
the presence of 
ulcers.10 
for 
excruciating ulcers that 
interfere with eating 
and speaking 
Maximum score = 10 
Site of injury 
Group 1  
Labial mucosa Buccal 
mucosa Buccal sulcus 
Soft palate Belly of the 
tongue Floor of the 
mouth  
Group 2  
Hard palate Adherent 
gum 
Alveolar 
ridge 
Dore of tongue Tonsils 
Tonsil pillars Uvula 
Oropharynx 
 
Score = total of sites 
involved 
1 for each site of group 
1 
(non-keratinized 
mucosa) 
2 for each site of group 
2 
(keratinized, 
specialized) 
Maximum score = 10 
Scarring 
Yes/No 
 
USS Total 

 
 
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A sample size of 29 subjects for each group (power 80%) to detect a significant difference in means of 
0.820 assuming the common standard deviation is 1.80 using a two-group t Student test with a significance 
level for p<0.05. Considering a 3% drop out, a sample of 30 subjects per study group will be enrolled. 
7.2 Population 
 
• Intention-to-Treat population (ITT): All randomized subjects who complete the clinical procedure and 
use mouthwash at least one day and with at least one available assessment of variables after baseline will 
be considered in this population. 
 
• Per-protocol population (PP): All subjects in the ITT population with primary outcome measures 
available and without major protocol violations (e.g., misinclusions, poor compliance). 
 
7.3 Statistical analysis 
 
Descriptive statistics will be provided in summary tables by group based on the type of outcome 
measure summarized. 
General descriptive statistics for continuous outcome measures will include n (number of observed 
values), mean with its 95% CI, standard deviation, median, minimum and maximum values. For 
categorical variables, the number and percentage of subjects with a specific level of the variable will be 
presented. 
This primary efficacy analysis will be performed on ITT, using the null hypothesis of no between-
group difference in overall VAS change from baseline between both groups: 
 
H0: μAfteRapidDNA = μ Placebo 
H1: μAfteRapidDNA μ Placebo 
 
Where μ is the mean change from baseline on Day 7 within the given product. 
This hypothesis will be tested with an Analysis of Variance (ANOVA) with baseline values of VAS as 
a covariate. The groups will be compared to the bilateral significance level of 5%. 
The assumption of normality will be verified by means of QQ graphs and by plotting the studentized 
residuals with respect to the expected values of the variable. If there are strong indications of non-
normality of the data, the Wilcoxon Rank-Sum Test will be used to test the hypothesis of no median 
difference between buccacollutorio and placebo at Day 7 in a nonparametric manner. Summaries for 
each treatment group will include sample size, mean, median, standard deviation, minimum, maximum, 
Wilcoxon Rank-Sum test p-value, Hodges-Lehman point estimates of median treatment difference, and 
its 95% confidence interval. 
From the Day 7 endpoint ANOVA model, a least squares estimate (LSMEANS) of the group effect and 
group difference (both with 95% confidence intervals) will be displayed along with the p-value of the 
difference between the groups. 
Analysis will also be performed for the PP Analysis Set to test the robustness of the results. 
For secondary quantitative efficacy variables, cross-treatment group comparison of change from 
baseline will be done by the same ANOVA model used in the primary efficacy analysis. Categorical 
derived variables (complications/adverse events) will be compared via a chi-square test or Fisher's 
exact test, as appropriate (chi-square test will be used if all groups have sufficient observations, 

 
 
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otherwise Fisher's exact test will be used). All statistical comparisons will be conducted at the 
significance level 0.05. SAS version 9.4 will be used for scanning. 
 
 
 
 
 

 
 
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8 Data management 
An electronic CRF (eCRF) will be completed by the Investigator and/or his/her delegate. 
All patients who sign the informed consent will be archived. A minimum set of information is required 
for patients who are screened but not randomized: date of signed informed consent, demographics, 
assessment of inclusion/exclusion criteria where applicable, primary reason for non-continuation, adverse 
events, and any concomitant medications. 
Front-end change checks will be performed at the time of data collection, and back-end change checks 
will be used by the Data Manager to check for discrepancies and ensure data consistency and 
completeness. 
 
Administrative practices and data management  
Medical record forms (CRFs) must be completed at the time of data collection. The complete CRF 
memory device containing the digitized clinical images should be attached to each patient's study file. 
All data required for this study will be collected using paper-based CRFs. CRFs will be kept in a secure, 
locked location that will only be accessed by the Principal Investigator and the data collector. The data 
will also be accessible to the sponsor for monitoring and evaluation purposes at its discretion. After 
completion of the 1-week follow-up, copies of all CRFs will be sent to the investigator who will check 
the data and store them in a protected computer with a password known only to him. The data will be 
processed by an expert statistician. The patient's identity will be protected and known only to the study 
participants listed in this protocol. 
 
Case Report Forms (CRFs) 
- Patient informed consent form 
- CRF Patient Eligibility 
- CRF Rating 
- Complications, protocol deviation and CRF drop-out 
 
Changes and violations of the Protocol  
Changes to this protocol may be necessary, and protocol violations are likely to occur. Any protocol 
violation should be reported in the "Complications, Protocol Deviation, and CRF Dropout" section. If 
you experience any problems, contact your studio monitor immediately. 
 
Data Ownership  
The data belongs to the sponsor. 
 
 
 
 
 
 
 
 
 
 
Essential bibliography 

 
 
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