Method for treating or preventing locally periodontal disease

A method for treating or preventing periodontal disease which comprises administering ofloxacin or salts thereof locally to periodontal tissue are disclosed. The method is excellent on the treatment of periodontal disease in small dose without disturbance of bacterial flora.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method for treating or preventing 
locally periodontal disease. In particular, the present invention relates 
to a method for treating or preventing periodontal disease which comprises 
administering ofloxacin 
(9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido 
[1,2,3-de][1,4]benzoxazine-6-carboxylic acid) or salts thereof to 
periodontal tissue of a patient having periodontal disease. 
2. Description of the Prior Art 
Periodontal disease and dental caries are the two most common diseases in 
the dental field. In most cases, tooth loss in adults is caused by 
periodontal disease. Periodontal disease is an inflammatory disease on 
periodontal tissue such as the gingiva, and includes various types which 
vary depending on the progress degree of the disease or the age of the 
patient. Generally, those types can be mainly classified into gingivitis 
and periodontitis. Further, periodontitis can be classified into adult 
periodontitis and juvenile periodontitis. 
Previously, it was hypotherized that periodontal disease was caused by 
non-specific stimulation derived from dental calculus or dental plaque. 
However, recent intensive bacteriological and immunochemical investigation 
have made it apparent that some specific bacteria among more than 200 
kinds of bacteria, which exist in the dental plaque, relate to the 
occurrence of periodontal disease. 
Particularly, as those bacteria, Actionmyces group, Spirochetes group, and 
gram negative bacteria such as Bacteroides group, Actinobacillus group, 
Fusobacterium group, Capnocytophaga group, Eikenella group are noted. 
Further, relationships between some bacteria and some periodontal diseases 
have been reported. For example, Bacteroides gingivalis is supposed to 
relate to occurrence of adult periodontitis and Actinobacillus 
actinomycetemcomitans is supposed to relate to occurrence of juvenile 
periodontitis. 
Periodontal disease has been treated by the following methods; 
(1) Scaling for the purpose of removing sub-gingival plaque and dental 
calculus from periodontal pockets, 
(2) Gingivectomy for the purpose of removing the inflammatory tissue or 
periodontal pocket, and, 
(3) Root planning for the purpose of mechanically grinding the surface of 
the dental roots to accelerate the adhesion of the gingiva on the dental 
roots. 
These methods are effective to some extent. However, some patients with 
specific internal diseases, for example a circulatory disease cannot 
undergo those surgical operations. 
Further, as treating agents for periodontal disease, dentifrices containing 
a germicide or antiinflammatory agent, and pastes for massaging the 
gingiva have been used. However, these agents are not yet satisfactory for 
the treatment of periodontal disease. Under these circumstances, a method 
for treating periodontal disease, which is more effective than hose 
mechanical or surgical methods, is strongly desired. 
As described above, specific bacteria relate to the occurrence of 
periodontal disease. Therefore, it is supposed to be effective for the 
treatment of periodontal disease to apply an antibacterial agent such as 
antibiotics in a suitable form to treat at an effective concentration the 
lesions of periodontal disease. However, this method has not been 
conducted for the following reasons: 
(i) The bacteria which cause periodontal disease have not been identified. 
(ii) Periodontal disease is usually classified into a chronic inflammatory 
disease and, it takes a long time to treat that disease. Therefore, when 
an antibacterial agent is given over a long period, a side effect of the 
agent may appear, or an opportunistic infection may appear by a 
disturbance of normal bacterial flora in the oral cavity and intestines. 
(iii) No suitable administration method has been proposed. The bacteria 
which cause periodontal disease exists in the periodontal pockets between 
the teeth and the gingiva. Since the pockets are anatomically outer parts 
of the body, antibacterial agents are not delivered effectively to them 
when it is administered orally or by injection. Furthermore, even when 
they are administered in the form of a dentifrice, mouth wash or gingival 
massaging agent, they are not substantially delivered to the periodontal 
pockets and further, they are removed rapidly by the washing effect of 
saliva. Therefore, an effective concentration of antibacterial agents in 
the lesions of periodontal disease cannot be maintained. 
After intensive investigations to solve these problems, the inventors have 
found that ofloxacin or salts thereof exhibit strong antibacterial effects 
against those bacteria and that when they are administered locally to 
lesions of periodontal disease, specifically to the periodontal pockets, 
remarkable effects on the treatment of periodontal disease are obtained. 
SUMMARY OF THE INVENTION 
This invention relates to a method for treating or preventing periodontal 
disease which comprises administering ofloxacin or salts thereof locally 
to periodontal tissue.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Examples of the salts of ofloxacin include acid addition salts thereof with 
an inorganic acid such as hydrochloric and sulfuric acid or an organic 
acid, and carboxylate with alkali metals or alkaline earth metals such as 
sodium, potassium and calcium. 
Typical examples of the pharmaceutical preparations for administering 
ofloxacin or salts thereof locally to periodontal tissue include gels for 
the oral cavity, ointments adhesive to the membrane of the oral cavity, 
preparations to be inserted into periodontal pockets and tapes to be 
adhered to the gingiva. 
The preparations for local administration can be prepared by mixing an 
active ingredient, that is, ofloxacin or salts thereof with excipients or 
vehicles used for preparations to the oral cavity by using conventional 
methods. 
The preferred excipients and vehicles for those preparations include 
hydroxypropylcellulose, methylcellulose, hydroxymethylcellulose, sodium 
carboxymethylcellulose, hydroxypropylmethylcellulose, liquid paraffin, 
white petrolatum, plastibase, Eudragit L, sodium alginate, propylene 
glycol alginate, pullulan, tragantha, xanthane gum, chitosan, polyvinyl 
pyrrolidone, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, 
ethyl methacrylate, dimethylamino acetate, cellulose acetate, 
polyethyleneglycol, collagen and atelocollagen. 
These substances can be used either respectively or as a combination of two 
or more kinds thereof. The combination is suitably selected depending on 
the preparation form. Those preparations may contain a coloring agent or a 
perfume as an additive. Typical examples of those preparations are shown 
below. 
On the administration of those preparations, the gels or ointments are 
spread to the gingiva, the tapes are adhered to the gingiva and the 
preparations to be inserted into the pockets are inserted therein. 
The dose varies depending on the degree of periodontal disease. Usually, a 
suitable amount of the preparation containing at least 0.01 wt % of 
ofloxacin is applied to the lesions of periodontal disease. 
Ofloxacin has already been used clinically as an excellent synthetic 
antibacterial agent by oral administration. Acute toxicity (LD.sub.50 in 
oral administration) of olfoxacin is 5,450 mg/kg in mice, 200 mg/kg or 
more in dogs and 500 to 1,000 mg/kg in monkeys. 
According to the present invention, a method for treating or preventing 
periodontal disease, which exhibits very excellent effects without 
disturbance of bacterial flora even in a small dose, can be obtained. 
Also, according to the present invention, the excellent effective 
concentrations of ofloxacin or salts thereof in periodontal tissue are 
kept compared to that obtained by administering those compound orally or 
in the form of injections. 
The following examples will further illustrate the present invention, but 
by no means limit the invention. 
EXAMPLES 
Example 1 
Gels for the oral cavity: 
945 g of water was added to 50 g of high molecular weight 
hydroxypropylcellulose to prepare a gel. 5 g of ofloxacin was added to the 
gel to produce a homogeneous mixture to be used as the gels for the oral 
cavity. 
Example 2 
Ointments adhesive to the mucous membrane of the oral cavity: 
313 g of liquid paraffin was added in small portions to 30 g of ofloxacin 
to obtain a mixture. 115 g of white petrolatum and 229 g of plastibase 
were added to the mixture and the obtained mixture was kneaded. Further, 
313 g of sodium carboxymethylcellulose was added thereto and the mixture 
was thoroughly kneaded to obtain a homogeneous ointment. 
Example 3 
Preparations to be inserted into the periodontal pockets: 
250 g of Eudragit L30D-55 (30% dispersion), 5 g of ofloxacin and 20 g of 
Tween 80 were mixed together. By using a casting method a film having a 
thickness of 300 .mu.m was prepared from the mixture. The film was cut 
into strips having a width of 1 mm and a length of 10 mm to be inserted 
into the periodontal pockets. 
Example 4 
Tapes to be adhered to the gingiva: 
85 g of a low molecular weight hydroxypropylcellulose was dissolved in 
1,000 ml of water to form a gel. 5 g of ofloxacin and 10 g of polyethylene 
glycol 400 were mixed therein. The mixture was formed into a film having a 
thickness of 300 .mu.m by the casting method. The film was cut into strips 
having a width of 10 mm and a length of 100 mm to be used as tapes to be 
adhered to the gingiva. 
Test Example 1 
Effects of gels containing ofloxacin on experimental periodontitis: 
The effects of the gel preparation containing ofloxacin on experimental 
periodontitis of hamsters infected with Actinomyces viscosus (hereinafter 
referred to as A. viscosus) were examined. 
18 male golden Syrian hamsters (3-week-old) were fed with an ordinary solid 
diet, CE-2 (CLEA Japan Co.) for two weeks, and were given penicillin G 
solution (4000 U/ml) freely for three days before the infection so as to 
control the indigenous bacterial flora and to facilitate the infection. 
The hamsters were then randomly assigned to one of the following three 
groups: an infected group to be treated, an infected group not to be 
treated and a non-infected group, each group comprising six hamsters. The 
oral cavity of each animal of the infected groups was inoculated with 0.25 
ml of culture broth (2.5.times.10.sup.8 colonyl forming unit/ml; 
hereinafter CFU/ml) of A. viscosus ATCC 15987. The bacterial inoculation 
was performed daily for five days. From the first day of the infection, 
all animals were fed with Diet 2050 in place of feed CE-2. Diet 2050 was a 
powdery periodontitis-inducing feed comprising 28% of sucrose, 28% of corn 
starch, 28% of skim milk powder, 6% of flour, 4% of beer yeast, 3% of 
alfalfa powder, 1% of liver powder and 2% of common salt. After two weeks, 
0.1 ml/day of the gels prepared in Example 1 was spread to the gingiva of 
each hamster everyday. 
Eleven weeks after the first bacterial inoculation, the salivary occult 
blood degree of each animal was evaluated with occult blood test papers 
(Hemasticks III; Miles Sankyo Co.) under anesthesia, and the results were 
classified into five ranks of 0 to 4 according to the color standard. The 
gingival index was evaluated and assigned into 4 ranks of 0 to 3 
macroscopically according to the criteria of Rosenberg et al. (J. 
Periodontol, 37: 208, 1966). Then, all the hamsters were decapitated. 
Dental plaque was scratched off throughly from the first molar of the left 
maxilla of each hamster, and was immediately suspended in an anaerobic 
Ringer's solution. A 10 fold dilution series was prepared in an anaerobic 
glove box. The total number of the bacteria was enumerated on a GAM agar 
culture medium (Nissui Seiyaku Co.), the number of A. viscosus was 
enumerated on a selective medium for A. viscosus (J. Clin. Microbiol., 15: 
253, 1982) and the number of black-pigmented Bacteroides was enumerated on 
Kanamycin-Vancomycinmenadion-blood agar medium (BBL Co.). The plaque index 
was evaluated by the method of Regolati and Hotz (Helv. Odonto. Acta, 16: 
13, 1972) after staining the teeth with a colorant (Red-Cote; Buttler 
Co.). After evaluation of the plaque index, the jawbones were taken out. 
The soft tissue and debris were removed from the left jawbone to make a 
bone specimen. A macrograph of the each jaw was taken with a 
stereomicroscope along the lingual aspect. Measurements of the distances 
from the cement-enamel junction to alveolar crest were done for all 
lingual roots of molars on the macrograph. The sum of these distances in 
each hamster was calculated to determine the alveolar bone loss vale. On 
the other hand, the right jaw was fixed in formalin, dehydrated, 
decalcified and embedded in paraffin. It was then sectioned and stained 
with haematoxylin and eosin. Histological observation of the samples was 
conducted with a microscope. 
As shown in Table 1, the degree of the periodontitis in the treated group 
was lower than that in the untreated, infected group with respect to the 
salivary occult blood which indicated hemorrhage from the gingival 
crevice, gingival index indicating the degree of inflammation of the 
gingiva, the plaque index indicating the quantity of the plaque, and the 
alveolar bone loss which is especially peculiar to periodontitis. The 
condition of the periodontal tissue in the treated group was similar to 
that of the non-infected group. 
The counts of bacteria in the plaque in the respective groups are shown in 
Table 2. In the treated group, the counts of A. viscosus used for the 
infection and black-pigmented Bacteroides, whose pathogenicity in 
periodontitis has been noted, were quite small, though the total counts of 
bacteria was only slightly lower than that in the infected group. 
In the histological observation, the deposition of a large amount of the 
plaque and emigration of polymorphonuclear leucocytes were observed in the 
infected group, proving presence of periodontitis, though these are not 
shown in the tables. On the contrary, they were only slight in the treated 
group and the degrees thereof were similar to those of the non-infected 
group. 
These results indicate that the local administration of ofloxacin gel is 
effective on periodontal disease, particularly, periodontitis. 
Test Example 2 
Comparison of the concentrations of ofloxacin in the oral cavity by oral 
administration and local administration: 
The preparations of Example 3 containing ofloxacin to be inserted into the 
periodontal pockets were locally administered to the pockets of volunteers 
and the concentration of ofloxacin in the periodontal fluid in the pockets 
was determined and compared with that in saliva obtained by oral 
administration thereof. In the test, the preparations of Example 3 having 
a thickness of 0.3 mm and width of 1 mm was cut into strips having a 
length of 4 mm (each containing 0.06 mg of ofloxacin). The strip was 
inserted into a periodontal pocket of each volunteer. The probing depth of 
the pocket was 5 mm. After a given time, a filter paper having a width of 
1 mm was inserted into the pocket to take the periodontal fluid out of the 
pocket. The amount of ofloxacin in the periodontal fluid was determined 
from the diameter of an inhibition circle according to an agar plate 
diffusion method with E. coli K 12. The quantity of the periodontal fluid 
thus taken out was determined from a calibration curve prepared from the 
quantity of a previously prepared liquid sample and the wetted area of the 
filter paper. The concentration of ofloxacin was thus determined. The time 
profile of the concentration of ofloxacin in saliva in the oral 
administration was taken from a report of Morihana et al. (Chemotherapy, 
32: Sl, 1070, 1984). 
The results are shown in Table 3. When 0.06 mg of ofloxacin was locally 
inserted into the periodontal pocket, the concentration thereof in the 
pocket was maintained above 70 .mu.g/ml for longer than 5 h and 16.6 
.mu.g/ml even after 24 hours, while the maximum concentration in saliva 
was 1.8 .mu.g/ml in 3 hours after the oral administration of 200 mg 
thereof. Namely, when ofloxacin in an amount of less than 1/3,000 of that 
given by the oral administration was inserted in the periodontal pocket 
according to the local administration, the concentration thereof in the 
pocket could be kept higher than that obtained by the oral administration. 
The concentration of ofloxacin in the periodontal pockets can be 
maintained for a longer period of time by varying the composition of the 
high molecular vehicles. 
TABLE 1 
__________________________________________________________________________ 
Results of experiment on periodontitis of hamsters 
Weight Gain.sup.a 
Salivary Occult 
Gingival 
Plaque Alveolar Bone Loss 
Group (g) Blood Degree.sup.b 
index.sup.b 
Index.sup.a 
Value.sup.a 
(mm) 
__________________________________________________________________________ 
Treated Group 
87.1 2.67 1.50 37.8 7.59 
.+-.6.2 
.+-.0.21 .+-.0.22 
.+-.3.7 
.+-.0.50 
** * *** * 
Infected 
87.5 3.83 2.67 71.2 9.54 
Group .+-.6.9 
.+-.0.17 .+-.0.21 
.+-.6.2 
.+-.0.51 ** 
** ** ** 
Non-infected 
91.7 3.00 1.00 32.0 5.04 
Group .+-.5.6 
.+-.0.41 .+-.0.00 
.+-. 3.7 
.+-.0.27 
__________________________________________________________________________ 
Each value is the mean .+-. standard error. 
a: Examined by Student's t test. ***; p .ltoreq. 0.0001, **; 0.001 
.ltoreq. p .ltoreq. 0.01, *; 0.01 .ltoreq. p .ltoreq. 0.05 
b: Examined by MannWhitney's U test. **; 0.001 .ltoreq. p .ltoreq. 0.01, 
*; 0.01 .ltoreq. p .ltoreq. 0.05 
TABLE 2 
______________________________________ 
Comparison of numbers of bacteria in dental plaques 
Logarithmic number of bacteria (CFU/ml) 
Black-pigmented 
Group Total Bacteria 
A. Viscosus 
Bacteroides 
______________________________________ 
Treated Group 
6.12.+-. 0.43 
4.76.+-. 0.53 
&lt;3.00 * 
Infected Group 
6.86 .+-. 0.18 
6.29 .+-. 0.31 
5.53 .+-. 0.16 
Non-infected 
4.23 .+-. 0.81 
&lt;3.00 * &lt;3.00 * 
Group 
______________________________________ 
* No colony was formed at all from the Ringer's solution diluted to 
1/10.sup.3 or less. The other values are logarithmic number of bacteria i 
the Ringer's solution, and are expressed in the form of the mean 
.+-.standard error (CFU/ml). 
TABLE 3 
______________________________________ 
Influence of the administration method on 
change of concentration of ofloxacin in 
the oral cavity 
______________________________________ 
Administration 
Oral administration* 
Local administration** 
method 
Dose 200 mg/body 0.06 mg/pocket 
Subject 8 subjects 8 periodontal pockets 
(part) (average body of 4 subjects (average 
weight: 60.6 kg) 
depth of the pockets: 
5 mm) 
Preparation 
Ofloxacin powder 
Preparations of 
given Example 3 
Time after 
1 1.18 72.1 
administra- 
2 1.59 88.1 
tion (h) 
3 1.80 84.0 
4 1.65 83.3 
5 1.40 76.5 
24 ND 16.6 
______________________________________ 
*According to the report of Morihana et al. (Chemotherapy 32: S1, 1070 to 
1083, 1984). The numerals are the average concentrations (.mu.g/ml) of 
ofloxacin in saliva given by the oral administration method. 
**The numerals are the average concentration (.mu.g/ml) of ofloxacin in 
the periodontal fluid in the pockets after insertion of the preparations 
of Example 3 (1 mm width .times. 0.3 mm thickness .times. 4 mm length int 
the periodontal pockets.