Effect of 4 days of mouth rinsing with delmopinol or chlorhexidine on the vitality of plaque bacteria

Jan Rundegren, Eva Bondesson Hvid, Maria Johansson and Mikael Astrom Biosurface AB, Box 839, S-20180 Malmo, Sweden :

Rundegren J, Hvid EB, Johansson M and Astrom M: Effect of 4 days of mouth rinsing with delmopinol or chlorhexidine on the vitality of plaque bacteria. J Clin Periodontol 1992; 19: 322-325. Abstract. Delmopinol is a new surface active anti-plaque agent that has demonstrated a low antimicrobial effect in vitro. By use of a vitahty staining technique, the antimicrobial effect on bacteria in plaque samples was tested after rinsing with delmopinol or chlorhexidine. 6 healthy male subjects volunteered to rinse for 4 days using a double-blind cross-over study design with a wash-out period between the rinsing regimens. No oral hygiene measures were allowed during the test periods and each test period started with a professional tooth cleaning procedure 2 days before the start of rinsing to allow for plaque formation. Rinsing was performed with 0.2% delmopinol hydrochloride or 0.2% chlorhexidine digluconate 2 x a day. Small samples of plaque were collected from the buccai surfaces of premoiars and 1 st molars before the first rinse on day 1 and then before and 1, 2, 4, 7, and 24 h after the last rinse on the 4th day. The plaque samples were immediately stained with propidium iodide and fluoresceine diacetate to visualize dead and vital microorganisms respectively. The vitality of the mieroflora was evaluated using a fluorescence microscope. The baseline vitality values were 91% for chlorhexidine and 86% for delmopinol. At day 4, the plaque vitality for chlorhexidine was approximately 40% up to 4 h and 50% at 7 h and 60% at 24 h after the last rinse. Corresponding values for plaque vitality after delmopinol rinsing were between 70 and 80% on all sampling occasions. The differences in plaque vitality between delmopinol and chlorhexidine were statistically significant on all sampling occasions ( / J < 0 . 0 5 ) except for the baseline values. The low antimicrobial profile of delmopinol confirmed in vivo suggests a low risk for an ecological shift of the oral microfiora due to cidal effects but also that delmopinol has other modes of action as an anti-plaque agent than an antimicrobial action.

Delmopinol, (+ )-3-(4-propylheptyl)-4morpholineethanol hydrochloride, is a highly surface active compound that has shown promising effects as an antiplaque agent in introductory in vivo studies (CoUaert et al. 1992, Klinge et al. 1989). On-going elinieal trials with a delmopinol mouth rinse are concerned with an evaluation of the substance as a prescribed drug against gingivitis. In vitro delmopinol has proven to be effective both against developing plaque and against established plaque when evaluated in an artificial mouth (Simonsson et al. 1991a, b). For a number of baeterial strains representing oral and nonoral species, the antimicrobial effect of delmopinol measured as minimal inhibi-

tory concentrations (MIC) was 5-125 x lower than that of chlorhexidine (Simonsson et al. 1991b). For prevalent oral species like Streptococcus sanguis and Actinomyces viscosus, the MICvalues showed that delmopinol was at least 25 x less antibacterial than chlorhexidine suggesting that on an equimolar basis the risk of alterations of the oral mierofiora would be reduced if delmopinol was used. However, MICvalues may not accurately describe the in vivo antimicrobial effect of a drug (Brian 1982). Thus in the present study, plaque vitality was tested after use of delmopinol as a mouth rinse. Rinsing was carried out 2 x a day for 4 days during which time no other oral hy-

Key words: plaque; mouthrinse; bacteria vitality. Accepted for publication 8 March 1991

giene measures were undertaken. Plaque vitality was evaluated by fluorescence microscopy (Netuschil et al. 1989) and delmopinol was compared to chlorhexidine by cross-over design. Material and iVIetiiods Study population

A total of 6 healthy volunteers working at Biosurface AB were selected. The volunteers all had complete dentitions except for 3rd molars or 1 premolar per quadrant extracted for orthodontic reasons. The premoiars and 1st molars had no crowns or bridge work, nor buccai or extensive interproximal restorations. Precautions were taken against con-

Vitality of delmopinol plaque founding treatment with antibiotics or antifiogistics. The subjects were told to restrict their intake of sucrose-containing food or beverages but otherwise not to change their normal eating habits. Clinical procedures

At the start of the study, the teeth were professionally cleaned and all oral hygiene measures were discontinued. After 2 days ofno oral hygiene, rinsing started with either delmopinol or chlorhexidine. Rinsing was performed for 60 s 2 x a day for 4 days followed by a 24-day wash-out period before the 2nd rinsing period. The rinsing was not supervised except for the first and the last rinse. The study design was double-blind cross-over. Plaque samples were collected on the 1 st and 4th day of rinsing and also one day after the last rinse. On day 1 of rinsing, samples were collected prior to the first rinse. On day 4, samples were collected just before the last rinse, which took place in the morning, and then 1, 2, 4, and 7 h after the rinse. On day 5, the final plaque sample was collected in the morning 24 h after the last rinse. The last plaque sample collection was followed by a professional cleaning of the teeth. Drug administration

The volunteers were assigned to treatment groups according to a computergenerated randomization list that was kept secret to the clinical trial manager and his staff until all study data were collected. Half of the subjects started with delmopinol and the other half with chlorhexidine. The volunteers received their code number in consecutive order. Solutions of 0.2% (6.4 mM) delmopinol hydrochloride (Biosurface AB, Kabi Pharmacia Therapeutics, Malm5, Sweden) and 0.2% (2.2 mM) chlorhexidine digluconate (Hibitane, ICI, Maeclesfield, England) were selected for the study. Mouth rinsing was performed with 10 ml of a solution of delmopinol or chlorhexidine for 60 s b.i.d. Morning doses were taken between 7 a.m. and 9 a.m. and evening doses between 7 p.m. and 10 p.m. Intake of food or beverages was not allowed for 30 min after rinsing. The different test solutions were dispensed in identical bottles with coded labels. Labeling and dispensing was carried out by the pharmaceutical department of Pharmacia AB, Uppsala, Sweden.

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Vitality of the plaque microfiora

Results

Small samples of supragingival plaque were collected from the buceal surfaces of premoiars or first molars by means of a Nystrom approximal dental carver (Nordenta, Enkoping, Sweden). The order of sampling surfaces was the same in the 2 treatment periods and on each sampling occasion, 2 samples were collected from 1 surface. Immediately after collection, the plaque samples were stained with fiuorescent dyes according to a modification of the technique described by Netuschil et al. (1989). This technique uses fiuoresceine diaeetate, that stains living cells green and ethidium bromide (EB) that gives dead cells a red stain. In the present study, EB was exchanged for propidium iodide (P4170, Sigma, St. Louis, MO, USA) at a concentration of 10 ^g per ml of a 0.15 M sodium chloride solution for visualizing of dead cells. The plaque samples were read at a 200-fold magnification in a fiuoreseent microscope (Nikon, Tokyo, Japan) equipped with a counting grid containing 25 squares for a visual field. 5 different visual fields were read and each of the squares was given a score from 1 to 5 depending on the degree and type of stain (Table 1). From the scoring of numbers (1 's to 5's), the % of vital micro-organisms was computed. The analysis was based on the mean of the 2 samples collected from each surface. A photograph was taken from each sample using a diapositive Ektaehrome 200 daylight film (Kodak). All samples were collected by one person and scored by a second person.

The baseline samples collected just before the first rinse showed a vitality of plaque micro-organisms of 86% for delmopinol and 91% for chlorhexidine (Fig. 1). The sample collected just before the last rinse on day 4 demonstrated a vitality of 71% for delmopinol and 35% for chlorhexidine. 1 h after the last rinse, the difference in vitality between the plaque samples for the 2 substances was smaller, but at 4 h after the last rinse, the difference was considerable, with a vitality scoring of 77% for delmopinol and 36% for chlorhexidine. After this point of time, the difference in vitality became less, and at 24 h after the last rinse the vitality for delmopinol was 76% and 61% for chlorhexidine. All mean values for chlorhexidine showed more variability compared with delmopinol. The difference between delmopinol and ehlorhexidine was statistically significant (p

Effect of 4 days of mouth rinsing with delmopinol or chlorhexidine on the vitality of plaque bacteria.

Delmopinol is a new surface active anti-plaque agent that has demonstrated a low antimicrobial effect in vitro. By use of a vitality staining techniqu...
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