Abstract
The aim of this study was to investigate the effect of benzydamine hydrochloride bioadhesive gel on healing of oral mucosal ulceration in an animal model. For in vivo determination of the effects of the bioadhesive gel, 36 rabbits were separated into three groups: the first group was treated with the gel formulation without active agent, the second group with the gel formulation containing benzydamine, and the third group received no treatment. Clinical healing was established by measuring the area of the ulcer in each test group on days 3, 6, 9 and 12. Histological healing was determined on the same days. Benzydamine containing gel applications resulted in a decrease in the ulcer area in 12 days ( p = 0.000). Histological evaluation showed that the benzydamine group had a higher mean histological score than the base and the control groups during the whole test period, and the difference between the benzydamine group and the control group was significant ( p = 0.04). The bioadhesive gel formulation of benzydamine hydrochloride showed a statistically significant increased rate of mucosal repair in this experimental standard mucosal wound animal study. It is a candidate for the topical treatment of oral mucosal ulcerative lesions.
Oral ulcers, an end-result of epithelial damage, may occur due to a number of causes including trauma , immune-mediated conditions such as aphthous stomatitis and lichen planus and lichenoid reactions , cytotoxic therapy (such as radiation and chemotherapy) , drug-induced hypersensitivity reactions of the mucosa and malignant changes .
General treatment modalities for oral ulcers include application of topical corticosteroids, topical tetracycline or tetracycline plus nicotinamide , systemic immunomodulators , topical analgesics , antiseptic mouth washes , amlexanox and benzydamine hydrochloride . Benzydamine hydrochloride (Bnz HCl) is available in a number of countries and is recommended for the relief of inflammatory conditions of the oral cavity, soft tissues and skin . It is a nonsteroidal drug that possesses analgesic, anaesthetic, anti-inflamatory, antipyretic and antimicrobial properties . Studies have shown anti-TNF alpha effects that may have utility in the management of mucosal ulcerations . Other theories about its mechanism of action include membrane stabilization, and anti-inflammatory effects. Even though it has been administered as tablets for systemic use, it is commonly used as a mouth wash or mouth spray in a concentration of 0.15% for the relief of inflammatory conditions of the mouth and throat, and in some countries gel preparations are applied to the skin to treat inflammatory conditions .
In their thorough review, Q uane et al. reported that the recommended dose of benzydamine as a mouthwash is 15 ml of a 4-mmol/l solution of the hydrochloride salt in water as stated by B aldock et al. Following mouthwash administration of benzydamine to rats (1 mg/kg), tissue concentrations in the oral tissues are reported to be as high as 100 μmol/l . The depth of diffusion of the drug into oral tissues is not known but it is probable that surface concentrations are higher than 100 kmol/l. Commercially available benzydamine mouthwash has a pH of 4.5–5.0 but is unbuffered, so should rise quickly to salivary pH (about 7) in the presence of saliva. During a 30 s mouth rinse, only a limited amount is absorbed into the buccal tissue . The small amount of absorption into buccal tissue is confirmed by the poor systemic availability (5%). Peak plasma concentrations are obtained at 3 h and reach 0.5 μmol/l 2 . The excretion of unchanged benzydamine has been reported to vary from 5 to 50–60% in the urine . Bnz HCl has been studied in a multicenter phase III clinical trial as prophylaxis for radiation-induced mucositis and mucosal pain in cancer patients, and statistically significant benefits of benzydamine rinse were shown compared with placebo .
Topical application of appropriate medications for the treatment of ulcerative and inflammatory mucosal conditions is commonly chosen by most dentists and physicians . A recent drug delivery method that can be applied and removed by the patient is a mucoadhesive system, which is especially appropriate for hydrogel-forming polymers, such as cellulose derivatives, natural gums, polyoxyethylenes, polyacrylates and sodium alginate . The application of ointments, creams and gels with bioadhesive properties can enhance the retention time of the formulation at the site of action . Gel formulations are preferred for topical applications due to their ability to release the drug molecules from the systems and are more easily applied to mucosal surfaces than creams and ointments . Hydroxypropylmethylcellulose (HPMC) is suitable for use in buccoadhesive preparations because it can adhere to oral mucosa when hydrated with water and withstand salivation, tongue movements and swallowing for a significant period of time . Even though the advantages of Bnz HCl and buccoadhesive formulations are known, there is no intraoral buccoadhesive Bnz HCl formulation available for oral pathologic conditions.
The aim of this study was to evaluate the effect of a new mucoadhesive gel formulation containing Bnz HCl that was developed by the authors for the clinical and histological healing of oral mucosal ulcers.
Materials and methods
Bnz HCl (Abdi İbrahim Pharmaceutical Company, İstanbul, Turkey) and HPMC K 100-M (Colorcon Ltd., İstanbul, Turkey) were compounded with acetic acid, methyl paraben and pentobarbital (Sigma–Aldrich Chemical Co, St. Louis, MO, USA). The study consisted of three sequential steps: preparation of bioadhesive gels; preliminary investigations for in vivo examination; and evaluation of in vivo performance of the gels in the animal study.
Preparations of gels
The gels for use in the in vivo studies were prepared as described . Briefly, HPMC E 5, E 15 and E 50 were used at the percentages of 5–10–15 and HPMC K 100-M was used at the percentages 1–1.5–2–2.5. HPMC was placed in a 100-ml beaker and wetted with water for 24 h. Bnz HCl and methyl paraben were dissolved in appropriate amounts of water and this solution was added in small portions to the wetted gel base and mixed thoroughly with the aid of vortex (IKA-Labortechnik RW 20 DZM, Germany) . After preparation of bioadhesive gels, rheological characterization, mechanical properties, muco-adhesion studies, drug diffusion studies and stability test of formulation were carried out . These studies guided the most suitable bioadhesive gel formulation for this trial, which was compounded with HPMC K 100-M 25% (80–120 cps), benzydamine 0.15, methyl paraben 0.15, distilled water q.s. 100 (w/w%) .
Preliminary investigations for in vivo studies
The housing care and experimental protocol of the study were approved by the Animal Ethics Committee. Adequate measures were taken to minimize the pain or discomfort of the animals. Experiments were carried out in accordance with the Guidelines laid down by the National Institute of Health (NIH) in the USA regarding the care and use of animals for experimental procedures and in accordance with local laws and regulations.
Complete wound healing of oral ulcerations occurs in 17 days . In order to establish the observation intervals and to determine the days of histological sample collection, an oral ulcer model was established for this study: oral ulcers were created on the oral mucosa of 17 young adult male New Zealand rabbits, weighing 2.5–3.0 kg. The animals were kept in a room with environmental conditions of 22 ± 2 °C, 50 ± 10% relative humidity and were fed a standard laboratory diet and water ad libitum. Each day, one animal was randomly selected and killed. The wound healing process on each day was evaluated histologically by an expert pathologist (A.V.). Thus, the observation intervals and the days of histological sample collection were set, and the length of the observation period for the oral ulcer model was determined.
Prior to the creation of the ulcers, all animals were anaesthetized with administration of pentobarbital (50 mg/kg). A round filter paper (Whatman, Madison, UK), 5 mm in diameter was soaked in 15 μl of 50% acetic acid and was used to cause aseptic tissue necrosis. In order to create round ulcers, the acid-soaked paper was pressed onto the labial gingival tissue of the rabbits for 60 s ( Fig. 1 ). On each day, one rabbit was killed with an excess dose of pentobarbital. The histological characteristics of the ulcer observed on day 1 were accepted as the baseline histological features. During 17 days, one animal was killed each day, and the histological wound healing process was established daily by the same pathologist. These characteristics were used to create a ‘custom-made scale’ to evaluate healing progression ( Table 1 ).
| Scores | Histological level of healing |
|---|---|
| 1 | Presence of epithelial necrosis, but no signs of inflammation |
| 2 | The inflammatory reaction has started, with no new capillary proliferation |
| 3 | The inflammatory reaction is prominent with few capillary proliferations on the basis of the ulcer, but no epithelization at the surface |
| 4 | The inflammatory reaction is decreased, new capillary proliferation has reached the surface and epithelization has started at the surface |
| 5 | The epithelization is complete |
Complete wound healing was observed in the specimens obtained on day 12. Therefore, the total observation period for the present study was set as 12 days. The healing process presented histological differences on days 3, 6, 9 and 12, which can be defined as the steps of wound healing for this investigation and determined the schedule of tissue biopsy for the study.
Evaluation of bioadhesive gels
The animals were separated into three groups: the first group (base) comprised animals treated twice a day with the gel formulation (without active agent); the second group (Bnz HCl) was treated with the Bnz HCl gel formulation twice a day; the third group (control) received no treatment and their measurements served as the control.
The clinical healing of each oral ulcer was evaluated by subtracting the most recent reading from the initial ulcer measurement at each observation interval. Thus, clinical healing was defined using the decrease in the ulcer area: the higher the amount of the difference between the initial measurement and the observation day, the better the clinical healing.
In each group, three animals were killed on days 3, 6, 9 and 12 for histological evaluation. 36 rabbits were included (12 rabbits in three groups representing three animals in each test group for each observation period) ( Fig. 2 ).
The treatment started 24 h after ulcer initiation (day 1). The dose of Bnz HCl was 1 mg/kg, with 0.1 g gel formulation applied to the ulcer using a syringe without a needle. The applications were performed twice daily.
Clinical photographs of the ulcers were recorded with a digital camera. In each photograph, a filter paper disc of 5 mm diameter was included to provide calibration of the ulcer area measurement between the images. A free share computer program (Image J, NIH, USA) ( , accessed on 21 May 2009) was used to measure the area of ulceration as cm 2 ( Fig. 3 ). All measurements were performed by the same researcher (P.G.) in a blinded fashion three times on the same day and a mean ulcer area was calculated.
For histological examination, three animals selected randomly from each group were killed with an excess dose of pentobarbital on each observation day. The ulcers were excised on days 3, 6, 9, and 12 by an experienced oral surgeon and fixed in 10% neutral buffered formalin. In the laboratory, they were embedded in paraffin blocks and 4 μm thick tissue sections prepared. The specimens were stained with haematoxylin–eosin and were examined in a blinded fashion by the same pathologist using a pre-established histological healing score ( Fig. 4 , Table 1 ).
