Effects of topical application of metronidazole-- containing mucoadhesive lipogel in periodontal pockets

Introduction. Metronidazole is the drug of choice in the treatment of periodontal pockets up to 5 mm in depth. It is topically applied – directly into the periodontal pockets. There are no registred trademark preparations for this purpose in the market of Serbia and Montenegro. The aim of our latest research was to test the efficacy of newly formulated preparation containing 25% metronidazole suspended in a lipogel in vitro – in anaerobic cultures isolated from the periodontal pockets, and in vivo – by the direct application into periodontal pockets. Methods. Preparation efficacy was tested in a randomized controlled study involving 25 patients, and was confirmed by the membrane-free agar diffusion method on the anaerobic strains isolated from the periodontal pockets. The duration of the testing was 30 days. The preparation was applied twice – immediately after the taking of the first swab, and on day 15, when the control swab was taken for the assessment of the effects of the applicd preparation. Results. Seven anaerobic strains were isolated and tested, and each was confirmed as highly susceptible to metronidazole. Anaerobic strains were not isolated in any of the pockets treated with metronidazole-containing lipogel. The strains isolated in the control pockets were the same as were those at the beginning of the study. Conclusion. Metronidazole, in a lipogel-type base applied in the concentration of 25%, provided an efficient treatment of anaerobic infection in the periodontal pockets.


Introduction
Much attention is paid presently to the conservative treatment of periodontal disease.Firstly, the periodontal pocket conservative treatment is practiced, and the best results are achieved if the periodontal pockets are not deeper than 5 mm.
Beside the periodontal pocket debridement (subgingival scaling and curettage), topical application of metronidazole seems to be very efficacious.Metronidazole efficiently inhibited anaerobic microorganisms in the periodontal pockets (1-5).The basic problem in manufacturing preparations applied to periodontal pockets is to obtain the adequate mucoadhesivity, i.e., the maintenance of the drug at the site of application (6)(7)(8).
Biomucoadhesive preparations can be formulated as hydrogels or as lipogels.Lipogels can contain lipids and emulsifiers of both types (producing oil-in-water and water-in-oil type emulsions).In contact with mucus, they form an amphiphilic preparation that is gradually washed down.In our earlier research, metronidazole-containing hydrogel preparations (9)(10)(11) were formulated and their efficacy was tested in vitro (9)(10)(11).The aim of this study was to evaluate the efficacy of the preparations with metronidazole formulated in a lipogel basis -a precursor of an amphiphilic preparation in inhibiting the anaerobic flora isolated from periodontal pockets.

Formulating the preparation
The original formulation of organic lipogel, containing 25% metronidazole, was made (12).Metronidazole was compounded into the base according to the principles of manufacturing the suspension-type ointments (13).Pure metronidazole base was used, with the particle size of 0.15 (14).
The medium consisted of a complex emulsifier, sesame oil and an appropriate jellifying agent with the bioadhesive properties.As the complex emulsifier with the hydrophylic lipophilic balance 10.2 (15), a preparation with amphiphilic properties was created ex tempore, at body temperature and in contact with mucus.The advantage of such formulations were their slow release characteristics, since only the layer in contact with mucus gradually eluted.The preparation had thixotropic rheological properties, enabling the periodontal pockets to be well-filled, and the amphiphilic layer created to express good mucoadhesivity.

Preliminary testing of the preparation
In vitro testing was performed, using the membranefree diffusion method on blood agar with haemin and K vitamin.A Peptococcus anaerobius strain, with the inoculum density 10 6 ml -1 was inoculated on a standard plate, standardized according to McFarland's standard No. 0.25.Twenty-five milligrams of the preparation, which corresponded to 6.25 mg of metronidazole, were measured (on Mettler AT-100 scales) into a circular opening (4 mm in radius) in the plate.Following the 48-hour incubation, the width of the obvious bacterial culture growth inhibition zone was 56 mm, demonstrating the efficacy of preparation and the release of metronidazole from the vehicle.

Experimental group
A total of 25 patients of both sexes, who had the diagnosis of periodontal disease, and were not treated within the previous six months, were randomly chosen.In each patient 2 periodontal pockets were chosen, up to 5 mm in depth, of which one was experimental and the other was the control.The study was performed with the informed consent of the patients.
Before the standard periodontal pocket treatment, swabs were taken from both pockets by a culturette (anaerobe transport system).Next, the preparation described above was applied into the experimental pocket, whereas the control pocket was without treatment.Patients were checked-up at days 15 and 30, by taking swabs from both experimental and control periodontal pockets.Immediately after taking the swab, on day 15 affter the first application of the metronidasole containing gel, the preparation was reapplied into the experimental pockets (10).

Results
The isolated strains are presented in Table 1.It shows the number of positive cultures, and the maximal values of the obvious bacterial culture growth inhibition zone width (millimeters).  2 for days 0, 15 and 30, as representative for the swabs taken from both experimental and control pockets in 4 patients.
Table 2. Anaerobic bacterial strains isolated from experimental treated by 25% metronidazole lipogel and control periodontal pockets without the treatment on days 0, 15 and 30

Discussion
All the swabs taken by culturette (both from the experimental and the control periodontal pockets) were processed by standard bacteriological procedures for isolating anaerobic bacteria.A total of 7 strains were isolated.After isolating and cultivating, every strain was reinoculated onto plates by the above mentioned method, to test the efficacy of the preparation in vivo, by measuring the width of the obvious bacterial culture growth inhibition zone.We ap- As it can be seen from the results of in vitro testing, presented in Table 1, a total of 7 bacterial strains was isolated (Eubacterium spp., Peptostreptococcus spp., Bifidobacterium spp., Bacteroides spp., Prevotella melaninogenica, Veillonella parvula, Peptococcus anaerobius) from both treated and untreated pokets.Each of the isolated strains showed a high susceptibility to metronidazole with the growth inhibition zone of 42-76 mm width.This was a direct confirmation that 25 mg of the preparation (i.e., 6.25 mg metronidazole) was sufficient to inhibit the growth of the strains referred to.
The results of in vivo testing, performed during 30 days in 25 patients, also confirmed the efficacy of the lipogel preparations.The control pockets were with the same bacteriological findings on days 0, 15, and 30, while in the pokets treated by the tested formulation, the bacteriological finding was negative on day 30 -no bacterial strains were isolated (Table 2).In most cases, a negative bacteriological finding in the pockets examined was already achieved on day 15.

Conclusion
Our original formulation -metronidazole-containing lipogel -was shown to be efficient in the eradication of periodontal pocket's anaerobic strains within the period of 30 days.In all of the patients, no anaerobic strains were isolated from the treated pockets on day 30, whereas anaerobic cultures were always confirmed in the control, untreated pockets.

Table 1 In vitro efficacy of 25% metronidazol lipogel against anaerobic cultures isolated from periodontal pockets
Results for 25 studied patients are presented in Table