Topical minocycline and tetracycline rinses in treatment of recurrent aphthous stomatitis: a randomized cross-over study
Published Web Locationhttps://doi.org/10.5070/D31658k5fq
Topical minocycline and tetracycline rinses in treatment of recurrent aphthous stomatitis: a randomized cross-over study1. Department of Oral Pathology and Oral Medicine the Maurice and Gabriella Goldschleger School of Dental Medicine Tel Aviv
University, Israel. 2. Department of Oral Medicine and Diagnostic sciences, College of Medicine, University of Illinois, Chicago,
Illinois, USA 3. Department of Oral Pathology and Oral Medicine the Maurice and Gabriella Goldschleger School of Dental Medicine
Tel Aviv University and the Department of Oral Surgery at the Sheba Medical Center, Israel. Keywords: Aphthous stomatitis,
Meir Gorsky DMD1, Joel B Epstein DMD MSD FRCD(C) 2, Shira Rabenstein DMD1, Hanita Elishoov DMD MSD1 , Noam Yarom DMD3
Dermatology Online Journal 13 (2): 1
Recurrent aphthous stomatitis (RAS) is a common ulcerative condition of the oral mucosa. We assessed minocycline and tetracycline oral rinses in patients with frequent episodes of RAS in a clinical randomized crossover trial. Seventeen patients with high frequency recurrent minor RAS were randomly allocated to a cross-over topical therapy with 0.2 percent minocycline or 0.25 percent tetracycline aqueous solution mouthwash. The minimum interval between the cross-over was 30 days. The intensity of pain was recorded daily using a visual analogue scale. Minocycline mouthwashes as compared to topical tetracycline rinses resulted in significantly improved pain control, by reducing the severity and duration of pain. Topical minocycline rinse may be a potential treatment for other mucosal diseases that result in mucosal ulceration and pain.
Recurrent Aphthous Stomatitis (RAS) is a common disease affecting the oral mucosa. It is a painful inflammatory ulcerative condition that affects 5-25 percent of the general population, and in selected groups such as students during school exams, it may reach a prevalence of over 50 percent . Although it is commonly accepted that RAS is found more in women, there are studies that report higher prevalence in men . It is also believed that the involvement is significantly greater among high socioeconomic groups . The onset of RAS is usually in childhood with a decrease in frequency and severity with increasing age .
Clinically, RAS is characterized by a prodrome of localized burning or pain, usually lasting 24 to 48 hours, that precedes the classical clinical appearance of the ulceration. The classic RAS lesions involve the non-keratinized oral mucosa presenting as a self-limited, painful, clearly defined shallow round or oval 1-3 ulcers, each with a shallow necrotic center. The ulcers are covered with a yellow-gray pseudomembrane surrounded by minimally raised margins and an erythematous halo representing superficial vasculitis . The most common ulcers, (Minor RAS), usually persist for 7-14 days with severe pain for a few days until there is a thicker fibrinous cover or early epithelialization.
The specific etiology of RAS is unknown and the patients who suffer from this disorder are usually otherwise healthy individuals. Most evidence suggests that RAS is a non-infectious inflammatory mucosal disease with an immunologic mechanism. The mucosa usually serves as the first mechanical and immunologic line of defense against physical injuries and infectious agents.
Although there is no definitive treatment for RAS, management using topical immunosuppressive anti-inflammatory medications, particularly corticosteroids, analgesic modalities, or systemic corticosteroids are the primary approaches [6, 7]. The major complication of this immunosuppressive therapy is adrenal suppression with long-term or repeated use, and local oral complications including secondary infections. Thalidomide is probably one of the most effective agents for the control of RAS , particularly when associated with HIV , but its use is restricted by severe adverse effects . There continues to be a need for effective prevention and treatment with minimum adverse effects.
Biopsies of RAS show non-specific ulceration with lymphocytic infiltration in the ulcer periphery. High levels of collagenase are seen in the connective tissue adjacent to the ulceration, originating from neutrophils (PMNs) in the connective tissue cells immediately beneath the necrotic epithelial layer , which suggests that collagenase may play a role in ulcer formation.
Systemic and local tetracycline regimens have been used for RAS because of an antibacterial effect . Tetracyclines, in addition to their antimicrobial effect have been shown to reduce collagen breakdown by collagenase activity [11, 12]. Minocycline is a semi-synthetic tetracycline with broad-spectrum antibacterial activity with indications for systemic treatment of acne, leprosy and rheumatoid inflammation . In addition to antimicrobial activity, minocycline exerts immunomodulatory effects via suppression of T lymphocytes and PMNs leading to reduction of tissue destruction attributed to active T cells and PMNs . Minocycline delays T cell proliferation associated with IL-2 activity  and suppresses T cell production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), which play a role in the epithelial damage . Minocycline in vitro delayed the migration of PMNs  and reduced phagocytic activities of the PMNs . It also was reported that incubation of PMNs with minocycline suppressed the tissue damage linked to PMN activity  and that the inhibitory potential of minocycline is directly associated with the dosage of the medication . Hence, it is speculated that because of its immunomodulatory effects minocycline may reduce the epithelial damage associated with RAS.
The effect of topical tetracyclines, other than minocycline, in RAS also has been assessed in a number of studies and reduction in the discomfort associated with RAS was reported [18, 19, 20 21, 22, 23].
The purpose of this study was to compare the clinical efficacy of a minocycline mouth rinse to tetracycline mouth rinse in reducing the severity and the duration of pain and mucosal ulceration associated with RAS.
Material and methods
A crossover study was initially intended to include 17 patients (10 men and 7 women) with a mean age of 39 years (range 17-70 years) with RAS. One of the 17 patients did not complete both arms. All patients were seeking treatment for RAS in the oral medicine clinic of Tel Aviv University from January 2003 through December 2004 and had not participated in any previous RAS study. All subjects (or parents when less than 18 years old) signed institutional approved informed consent and were clinically diagnosed with minor RAS with a high recurrence rate (over six recurrences in the prior year). The clinical diagnosis was made on the basis of data collected by the investigators that included history of years of recurrent painful ulcers on the non-keratinized oral mucosa that resolve within 7-14 days and typical clinical appearance of the lesions. All subjects were otherwise healthy individuals as documented in health history and with normal complete blood counts, iron, B-12, folate and blood glucose. None had any contraindication to use of tetracycline medications (allergy, breast-feeding, pregnancy and age less than 12 years).
Using a random numbers computer program, qualified patients were randomly allocated to a cross-over topical therapy starting the treatment with minocycline or tetracycline rinses. Randomization was completed prior to the first arm of the crossover treatment; then they were seen to supply them the other medication to use when new recurrence occurred.
The rinses were prepared based upon the minimum commercial dosage of the medications; subjects used 2mg/1ml (0.2 %) aqueous solution of minocycline and 2.5mg/1ml (0.25 %) aqueous solution of tetracycline as mouthwash without added flavoring; the preparations were of similar consistency without color. The subjects were instructed to begin mouthwashes as randomly assigned, the first day following the development of the first symptomatic ulcer of RAS. They were instructed to rinse their mouth with tap water prior to the administration of the study rinse and then to rinse with 5ml of the solution for one minute and spit out, 4 times a day avoiding any food or drink for at least ½ hour after the rinses.
Therapy continued for up to 10 days or until the lesions and symptoms resolved, during which subjects completed a daily follow-up form that included data about the daily intensity of pain caused by oral RAS. Pain intensity was recorded using a visual analogue scale (VAS), with anchors of 0 (no pain) to 10 (the worst pain possible) following the 4th daily rinse. The minimum time interval between the cross-over was 30 days. At the end of the treatment each patient was asked to indicate the subjective duration, in days, of the oral ulcers and to state if recurrent ulcers appeared during the period while on each treatment (up to 10 days) and any possible side effects.
Statistical analyses were conducted using the two-way analysis of variance, ANOVA test with repeated measures and the T-test for independent samples.
Forty-one patients were initially interviewed for the study. Five patients were disqualified due to low serum iron, low folic acid (2 patients), pregnancy (2 patients) and leukemia (1 patient). Nineteen patients did not complete the study because of personal reasons unrelated to the study protocol or failed to complete a follow-up form of a minimum of one arm as prescribed. Sixteen patients followed the study protocol completing both arms of the study. One patient, who had to leave the country for an extended period, completed the tetracycline arm only. Of the 17 patients who initially participated in the study, three (18 %) had a history of smoking, ten (59 %) reported family history of RAS and ten (59 %) recognized an association between stress and recurrence of oral lesions.
Tables I and II show the daily pain level of each patients treated with each one of the two drugs (Table I - minocycline, Table II tetracycline). The mean daily pain during the study is presented in Fig. 1. Although a higher mean score of pain was recorded prior to the initiation of the minocycline arm (6.3 vs. 5.8 prior to rinsing with tetracycline), the difference was not significant (p > 0.05). The decrease of mean level of pain was in favor of minocycline starting on day 2 and for the remaining days of the study (p < 0.05). The mean score on day 4 with minocycline was similar to that of day 10 with tetracycline.
As illustrated in Fig. 2, 18.8 percent of the patients on minocycline reported complete remission of pain on day 3 compared to none of those on tetracycline. Over 50 percent of the patients on minocycline were free of pain on the fourth day of treatment. For the patients on tetracycline, 11.8 percent experienced complete reduction of pain only on day five. The extent of the daily decrease of mean pain following the use of minocycline was significantly superior to the outcome with tetracycline for each one of the treatment days 1-10 (Fig. 3). On day 3, patients on minocycline experienced 61 percent reduction of mean pain compared to only 23 percent when on tetracycline. On day 6 the level of the mean pain was reduced by 91 percent when on minocycline compared to less than 50 percent when on tetracycline (Fig. 3).
When the mean level of pain was calculated for the whole period of treatment, a highly significant difference was found between the effect of minocycline and that of tetracycline (p = 0.001; mean pain 1.5 and 3.66, respectively).
The mean duration of pain while on minocycline was significantly shorter than that of tetracycline (p = 0.002; mean days 5.8 and 8.2, respectively). Participants were asked to state subjectively the duration of the oral ulcers and significantly lower mean duration was recorded when on minocycline compared to the treatment with tetracycline (p = 0.001; mean days 6.9 and 8.7, respectively).
Significantly fewer patients reported onset of recurrent ulcers while on the minocycline regimen (2 patients) than when on tetracycline (8 patients) (p = 0.031). No side effects were reported and the order of use of the medications had no influence on the outcome (p > 0.05). Statistically, no difference was noted in the outcome between genders. Although no specific examination of patient compliance was conducted, all patients reported use of medications exactly as instructed.
Recurrent Aphthous Stomatitis has been described as presenting in three forms including minor aphthae, major aphthae and herpetiform aphthae . Minor aphthae account for 75-85 percent of all RAS cases and can involve any non-keratinized mucosa of the oral cavity. The characteristic lesions are smaller than 10 millimeters and heal within 7-14 days without scarring. Major aphthae, comprising 10-15 percent of RAS cases, are larger, of longer duration, are more intense than minor aphthae, and tend to leave scars after healing. The herpetiform ulcers represent only 5-10 percent of RAS cases and consist of multiple 1-3 mm painful ulcers resembling herpes simplex, but involving non-keratinized mucosa.
Although no data on the mean and range of interval time between episodes of RAS have been recorded, we believe that because the order of use of the medications did not influence the outcome, that the minimum time interval of 30 days between the cross-over was sufficient washout time. More men than women participated in the current study, and the prevalence in this limited number of subjects does not represent the prevalence in the general population. Although the individuals in the present study were diagnosed with minor aphthae, we believe that the effect of minocycline mouthwashes in the management of RAS as compared to that of tetracycline may apply to all three clinical presentations of RAS, but additional studies are needed to support our speculation.
In a previous study by our group (submitted for publication) significant pain relief was seen with minocycline mouthwash compared to placebo. A small sub-group in that study participated in a crossover trial and the differences in the outcomes of minocycline vs. placebo were statistically significant in favor of minocycline. More individuals were included in the present crossover study in which minocycline was compared to tetracycline acqueous rinse. Tetracycline is accepted among clinicians as a potential treatment modality for RAS [18, 19, 24] and it was demonstrated that minocycline created greater pain relief compared to tetracycline in patients with RAS.
The present crossover design eliminates the possible impact of other potential risk factors for RAS including age, gender, stress, family history, and food components.
Although the mean pretreatment level of pain in the minocycline arm was higher (VAS = 6.3) than that in the tetracycline arm (VAS = 5.8), the difference was non significant. Significantly greater pain relief was reported with minocycline by day 2 (p < 0.05). No effect of the order of use of minocycline or tetracycline in the cross-over was noted, and therefore, no carry-over effects were seen.
Relative daily change of pain may be of more importance than the absolute VAS pain score. Changes in pain score are shown in Figs. 1, 2 and 3. When on tetracycline, several patients (patients 3, 4, 5, 6, 9, 15, 16, Table II) reported some increase in the level of pain after initial reduction in pain, which may be due to the onset of new aphthae as subjects in this study experienced high recurrence rate of oral ulcers. Unfortunately, validation of this is not possible with this data. The same pattern was not seen when on minocycline (Tables I and II). These findings, although not statistically significant, further support the increased effect of minocycline. Significantly fewer recurrences of aphthous lesions were documented in patients when on minocycline than when on tetracycline rinses (2 vs. 8 cases respectively; p = 0.031). These findings suggest possible use of minocycline for prevention of RAS.
Even though the participants were not asked to provide any personal statement at the close of the trial, 6 patients reported that one treatment, later identified as minocycline, was superior. Four of these subjects indicated that no new lesions occurred while on minocycline, which is atypical given the regular high rate of recurrence of RAS. No side effects were reported with either of the oral rinses.
The concentration of minocycline rinse in the current trial (0.2%) was lower than that of Tetracycline (0.25 %). It was based upon the minimum dosage of the medications available in capsule or tablets and as used in a previous study in which minocycline was compared to placebo in RAS patients (submitted for publication). It is possible that higher concentrations may result in improved reduction of pain related to RAS, and dose ranging studies remain to be conducted.
The previously reported topical use of tetracycline, combined with systemic dosing was based on the assumption that a bacterial etiology accompanies the onset of RAS . However, the impact of tetracycline upon cytokine production, cellular degranulation and collagenase activity may represent the mechanisms of action in RAS as has been documented in periodontal disease . Although tetracycline can inhibit collagenase activity in vitro, such benefit was not demonstrated in vivo . The current study, strongly suggests that topical minocycline may have greater impact upon the tissue damage and healing than tetracycline in vivo.
Although no subjects reported any side effect using minocycline or tetracycline mouthwashes, some subjects may have swallowed the medication. However, even if the entire dose of rinse provided was swallowed, this is below the standard systemic dose for infection. In high doses, systemic tetracyclines may be associated with changes in cutaneous pigmentation and accumulation of derivates may occur in bones and developing teeth. Gastrointestinal disturbances, allergies, oral candidiasis and erythema nodosum have also been reported [13, 25]. Prior reports of topical tetracyclines for RAS also report a lack of major side effects [18, 20 21].
Other topical approaches for the management of RAS have been studied. Topical steroids such as triamcinolone acetonide in carboxymethylcellulose paste have been used for years . Topical betamethasone  and fluocinonide  are also suggested for management of RAS. Transient relief of pain was reported with benzydamine hydrochloride mouthwash, but it was not more effective than placebo on ulcer healing . Hunter et al.  reported that chlorhexidine gluconate mouthwash was not better than placebo and no significant difference in pain was recorded. Similar results were also reported by others [19, 29]. The use of Listerine mouth rinse for RAS  did not show benefit compared to the control. Topical sucralfate was effective in managing discomfort associated with RAS .
A trial of chlortetracycline mouthwashes on RAS  reported significant reduction in duration of the ulcers with no side effects; however, pain was not assessed in this study. Graykowski and Kingman  reported a significant reduction in RAS ulcer duration, size, and pain with tetracycline suspension combined with systemic tetracycline. The combination of topical-systemic treatment prevented an evaluation of the effect of topical application. Another trial , using a combination of amphotericin and tetracycline hydrochloride mouthwash, reported reduction in mean pain score. The efficacy of topically applied doxymycine-cyanoacrylate in RAS was studied  and a significant reduction in pain intensity was reported in days 2-5 out of 10 days of follow up. It was speculated that effective pain relief in that study  was associated with the tetracycline effect upon tissue collagenase preventing tissue destruction.
The results of the present cross-over study show that minocycline rinses are significantly more effective than tetracycline for management of RAS as assessed by pain report. Further studies of the mechanisms of action of minocycline in RAS are indicated. It is also speculated that topical minocycline may represent a potential treatment for other mucosal diseases that result in mucosal ulceration and pain. Further investigations for the most effective dosage and the most appropriate minocycline management method for RAS, and possibly for other immune related diseases involving the oral cavity are indicated.
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