2.1 Materials
Cinobufacini, which was approved by the State Food and Drug Administration (approval number: Z34020272), was obtained from Anhui Huarun Jinchan Pharmaceutical Co., LTD (batch number: 210317, 211117). Each tablet contained 0.3g of cinobufacini extract.
2.2 Participants and treatment procedures
2.2.1 Participants
Patients with OPMDs were selected from admissions to the Hospital of Stomatology, Air Force Medical University, during the period from February to July 2022. The inclusion criteria consisted of individuals aged 18 to 75 years with pathologically confirmed mild to severe dysplasia. Exclusion criteria included patients with lesion areas smaller than 1cm×1cm before biopsy, those treated for head and neck cancer within the previous six months, individuals using immune agents within the last month, those with uncontrolled systemic diseases, and pregnant or lactating women. The study protocol was approved by the Ethics Committee of Stomatology, Air Force Medical University (clinical trial batch number: IRB-YJ-2022005) and adhered to the principles of the Declaration of Helsinki.
2.2.2 Treatment
The patients were administered Cinobufacini orally at a dosage of 0.6 grams thrice daily for 4 weeks, were constantly monitored throughout the study, and their adverse drug reactions were recorded. After the completion of the 4-week trial, the clinical response was assessed. Participants who experienced a subjective improvement in their predominant symptoms were given the option to independently choose whether to continue the treatment beyond the initial 4-week period.
2.3 Definitions of response
We used the area scoring system and the activity scoring system to assess the efficacy of Cinobufacini in these patients.
2.3.1 Area scoring system
In this study, patients with assessable disease were defined as those with bidimensionally measurable lesions, where the dimensions were obtained by measuring the longest and greatest perpendicular diameters of lesions with well-defined margins.
Clinical response was categorized as follows: complete response, signifying the total disappearance of all lesions; partial response, indicating a reduction of at least 50% in the sum of products of lesion diameters; stable disease, characterized by a reduction of less than 50% or no change in the sum of products of lesion diameters; and progressive disease, denoting an increase of over 25% in the sum of products of lesion diameters[24]. The overall positive response rate was determined as follows: Overall positive response rate = complete responses (N) + partial responses (N)/totality.
2.3.2 Activity scoring system
Due to the limitations of using area change records to capture certain conditions such as ulcer healing or reduction of congestion and erosion, especially when the area remains unchanged, a novel scoring system adapted from the activity scoring method used for erosive oral lichen planus (OLP) was employed[25], with some modifications for improved applicability in evaluating OPMDs. The scoring criteria were defined as follows: Position score (a): 0, indicating the absence of lesions; 1, signifying visible lesions; 2, denoting involvement of >50% of areas like the cheek, dorsum of the tongue, floor of the mouth, hard palate, soft palate, and pharynx. Severity score (b): 0, representing a normal appearance; 1, reflecting only keratosis/texture changes; 2, indicating mild erythema with or without keratosis; 3, representing marked erythema with or without keratosis; 4, indicating the presence of ulceration. The Activity score was calculated as follows: Activity score = a×b. If there were more than one lesion site in the oral cavity, the sum of the activity score was used (Supplement).
2.4 Pain intensity
Numerical Rating Scale (NRS) was used as a supplementary indicator to assess the intensity of pain experienced by each patient. They were requested to select a number ranging from 0 to 10 to indicate their pain level over the preceding two weeks, whereby 0 indicated no pain, 1-3 signified mild pain, 4-6 denoted moderate pain, 7-9 indicated more severe pain, and the maximum score of 10 represented severe unbearable pain (Supplement).
2.5 Pathologic assessment
Eight participants were treated for 12 weeks, and we performed histopathological examinations before and after treatment. The histologic evaluation of all biopsies was performed independently by two pathologists who were blinded to clinical data. In general, the residual and worst-performing areas of each lesion were biopsied after treatment. If the target lesion was not visible after the intervention, a biopsy was performed at the site of the previous lesion biopsy. Biopsy samples were scored according to the extent of dysplasia on a 5-point ordinal scale (0=no dysplasia; 1=mild dysplasia; 2=moderate dysplasia; 3=severe dysplasia; 4=carcinoma in situ) as previously described[26].
2.6 Safety indicators and evaluation criteria
Adverse events (AEs) occurring during the course of the clinical trial were evaluated and documented. Additionally, participants underwent hematological and blood biochemical assessments both before and 4 weeks after the procedure to further evaluate the safety of this trial by assessing parameters such as white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT), hemoglobin (HGB), mean corpuscular volume (MCV), glucose (Glu), cholesterol (ChOL), triglycerides (TG), albumin (ALB), alkaline phosphatase (ALP), aspartate transaminase (AST), alanine aminotransferase (ALT), uric acid (UA), blood urea nitrogen (BUN), and creatinine (SCr).
2.7 Statistical analysis
Data analysis was performed using GraphPad Prism version 5.01 and SPSS version 22.0 software. Participant characteristics are presented using mean ± SD for continuous variables and frequency (%) for categorical variables. Paired differences were assessed to compare values before and after treatment. Statistical significance was defined as P<0.05.