A PHARMACEUTICAL PREPARATION AND A CONJUGATE OF A BACTERICIDE AND AN ANTIBODY DIRECTED AGAINST PLAQUE FORMING OR CARIES-INDUCING BACTERIA

Abstract: A conjugated of a bactericide and an antibody directed against at least a certain type of dental plaque-forming and/or caries-forming bacteria, such as Streptococcus mutans is described. The bactericide of the conjugate is preferably an antibiotic, e.g. Netilmicin. Moreover, pharmaceutical preparations comprising a conjugate of the invention and a method of treating dental plaque and/or caries are described.

Description: A pharmaceutical preparation and a conjugate of a bactericide and an antibody directed against plaque forming or caries-inducing bacteria. The invention relates to a conjugate of a bactericide and an antibody directed against at least a certain type of dental plaque-forming and/or caries-forming bacteria, and to a pharmaceutical preparation for the treatment of dental plaque and/or caries. Furthermore, it relates to a method of treating dental plaque and/or caries. Background It is generally accepted that dental caries and plaque result from the action of certain bacteria in the saliva. Some of these species produce adhesive sub¬ stances that enable the bacteria to adhere to the tooth surfaces. Bacteria in plaque convert sugars in the diet into acids that dissolve mineral components of the teeth and produce cavities. The plaque-forming bacteria can also colonize crevices between the teeth and gums, moving down along the roots of the teeth, enlarging the crevices and destroying gums, ligaments and bone holding the teeth in the jaw. The disease is a major cause of tooth loss, and the treatment can be both painful and costly. The most important bacterium causative of human dental caries and plaque is Streptococcus mutans, but there are several other bacteria which may participate in the formation of dental plaque and caries and in causing tooth-loosening. The common way of preventing the formation of plaque and caries is tooth-brushing and mechanical cleaning of the teeth by a dentist. Also various agents incorpo¬ rated into tooth-pastes and mouth-washes help, but they are far from being completely effective. Antibiotics have previously been used for the treat¬ ment of caries, but the treatment has required long treatment periods (hours) during which the antibiotic has to be in contact with the infected areas to be effec- tive. The present invention provides a new method of treating dental plaque and caries, in which use is made of a conjugate of a bactericide and an antibody direc¬ ted against at least a certain type of dental plaque- forming and/or caries-forming bacteria. It has previously been shown that if an antibody is binding to an antigen this will take place in but a few seconds. The conju¬ gate of the invention will attach rapidly to the bacteria against which the antibody of the conjugate is directed, thus keeping the conjugate in place while the bacteri¬ cide of the conjugate rapidly acts on the target bacteria. Description of the invention The present invention provides a new conjugate of a bactericide and an antibody directed against at least a certain type of dental plaque-forming and/or caries-forming bacteria. The conjugate, preferably com¬ prised in a pharmaceutical preparation, is useful in the local treatment of an infection caused by said certain type of dental plaque-forming and/or caries forming bacteria. Thus, the invention also provides a method of trea¬ ting dental plaque and/or caries comprising local app¬ lication of a bactericidal amount of a conjugate of the invention or a pharmaceutical preparation of the invention, to a surface infected by dental plaque-forming and/or caries-forming bacteria. The bactericide of the conjugate is preferably an antibiotic selected with regard to effectiveness against a certain target bacterium. In a preferred embo- diment of the invention, the antibiotic is Netilmicin (0-3-deoxy-4-C-methyl-3(methyl-amino)-β-L-arabinopyra- nosyl-(l→-4)-0-[2,6-diamino-2,3,4,6-tetradeoxy-α-D-gly- cero-hex-4-enopyranosyl-(l→-6 )-2-deoxy-N -ethyl-L-strept- amine sulfate (2:5) salt). The antibody of the conjugate is either polyclonal or monoclonal and is directed against at least a certain type of dental plaque-forming and/or caries-forming bacteria. In said preferred embodiment of the invention, the antibody of the conjugate is preferably directed against at least a certain type of bacteria selected from the group consisting of Streptococcus mutans, Strep¬ tococcus sanguis, Bacteroides gingivalis, Actinobacillus actinomycetemcomitans, Eikenella corrodens, Veronella alcalescens and Actinomyces viscosus. In a particularly preferred embodiment of the invention, the antibody is directed against Streptococcus mutans. The invention further provides a pharmaceutical preparation comprising a conjugate of a bactericide and an antibody directed against at least a certain type of dental plaque-forming and/or caries-forming bacteria, together with a pharmaceutically acceptable carrier and/or diluent and, optionally, other pharmaceutically acceptable additives. The concentration of the conjugate in the pharmaceutical preparation is preferably such that a single local treatment of an infection caused by a certain type of dental plaque-forming and/or caries- -forming bacteria against which the antibody of the conjugate is directed, will provide a bactericidal effect. The pharmaceutically acceptable carrier and diluent which can be used in the pharmaceutical preparation of the invention are not critical, and examples of useful carriers and diluents can be found in the U.S. Pharma¬ copoeia. The pharmaceutical preparation according to the invention is preferably in the form of a paste, a tooth- paste, a suspension, a solution or a mouth-wash. The optional pharmaceutically acceptable additives are such as are needed for the above listed forms of the pharma¬ ceutical preparation. Preferred embodiments of the pharmaceutical prepa¬ rations of the invention comprise at least one of the preferred conjugates of the invention. In practical use it is possible that a pharmaceutical preparation according to the invention will comprise several conju¬ gates of perhaps different bactericides and different antibodies each directed against at least a certain type of dental plaque-forming and/or caries-forming bacteria. Furthermore, it may be necessary to mecha- nically, e.g. by tooth-brushing, break the polysaccaride capsule of the target bacteria in order to lay bare the immunologically active parts of said bacteria so that the conjugate can attach to and act on the bac¬ teria. As stated above, the most important bacterium causa¬ tive of dental plaque and caries is Streptococcus mutans, and the invention will therefore be illustrated by a conjugate of an antibiotic and antibodies directed against Streptococcus mutans. Description of the preparation and testing of a preferred conjugate of the invention. Bacterial_strain A large number of bacteria were cultivated in conven¬ tional B-medium (the medium was autoclaved before use, and one litre of medium contained 10 g tryptone soya broth (Oxoid code CM 129), 5 g yeast extract (Oxoid code L 21), 10 g NaCl, pH adjusted to 7.5 with 1 M NaOH, and the balance water) . The cultivation was performed in an incubator at 37°C and constant agitation overnight. The bacteria for the immunization and affinity purifi¬ cation were washed repeatedly with phosphate buffered saline solution (PBS). The bacteria for testing were stored in a refrigerator until use (bacterial medium) . Antibodies were produced by repeated immunization of hens (White Leghorn line) with formaline-killed Strep¬ tococcus mutans (strain AHT) bacteria. The bacterial strain was received from the Department of Oral Micro¬ biology, Huddinge Hospital, Sweden. The IgG fraction of hyperimmune serum from these animals was received from Immun System AB, Uppsala, Sweden. 2y-i-i2§£i2Q_2f_§2£i 2^i? _ Y_§££i2i£Y_2l}ϊ25!i£22ϊ§El}Y Ultrasonicated Streptococcus mutans bacteria of the same strain where coupled to BrCN-activated Sepha- rose 4B (Pharmacia, Uppsala, Sweden) in a column in conventional manner, and the antibodies were applied onto the column, and then the column was washed with PBS. Bound antibodies were eluted with 0.1 M glycine pH 2.25. The bound specific antibodies were concentrated by precipitation with 50% ammonium sulphate and dialysed against PBS (without azide) for three days. Pr garation_of_conjH2§t§ The conjugation of an antibody to a bactericide can be performed in any conventional manner. The method selected was a one-step method using glutaraldehyde. 200 μl Netilmicin (ESSEX Lakemedel, Box 27190, Stockholm, Sweden) in PBS (5 mg/ml ) was mixed with 50 μl of 0.2% glutaraldehyde. The mixture was incubated for 1 hour at room temperature. To the mixture was added 100 μl of antibodies in water (0.4 mg antibodies in 0.6 ml H-O). The mixture was further incubated for two hours at room temperature, whereupon 100 μl 1 M lysine, pH 7.0, was added. As a control, a non-specific conjugate of bovine serum albumin (BSA), and Netilmicin was pro¬ duced. Excess Netilmicin was removed by dialysis against PBS in four stages in two days. The success of the conju- 125 gation was verified by using isotope labelled ( I) Netilmicin. For comparison, also a solution in which Netilmicin was conjugated to itself, was prepared. ϊ?22_ § D3=iιbacterial_effect_of_the_con;iugates I. The anti-bacterial effect of the conjugates was tested using a conventional technique where the conjugate is placed in wells punched in agar and the size of the inhibition zones is measured. 5 ml of LB agar (LB medium to which 0.7% agar-agar has been added) was poured into a petri dish and allowed to solidify. An additional 5 ml LB agar were heated to 45°C. The stored bacterial medium in a final dilution —3 —6 of 10 -10 was added to the warm LB agar and rapidly mixed therewith, whereupon the mixture was immediately poured onto the LB agar in the petri dish. Wells were punched in the agar layer, and 20 μl each of the different samples of conjugate were added to separate wells. The plate was incubated overnight at 37°C, and the next day the diameter of the inhibition zones was measured. Results: Conjugate of Inhibition zone (mm) Antibody-Netilmicin 8 Netilmicin-Netilmicin 7 BSA-Netilmicin 4 II. In order to make conditions as realistic as possible, a system was used where the bacteria grew on a solid surface (simulated tooth), and this surface was subjected to test solutions for a short period of time (simulated mouth-wash) . Nitrocellulose paper strips were incubated for 3-4 hours at room temperature in LB medium containing the bacterial medium diluted 10,000-1,000,000. The paper strips were washed four times with PBS to remove bac¬ teria which had not adhered to the paper. The strips were incubated in A: a specific conjugate (Netilmicin- antibody), B: a non-specific conjugate (Netilmicin-bovine serum albumin), C: Netilmicin conjugated to itself, and D: buffer, for the shortest possible reproducible time, one minute, whereupon they were washed four times with PBS. The incubation solutions A, B and C contained approximately equimolar amounts of Netilmicin. The paper strips were placed on LB agar plates prepared in the same way as above, with the exception that no bacterial agar layer was added. The plates were incubated overnight at 37°C and read the next day. The strips were evaluated as growth or no growth. Results: Conjugate of Growth (+) No growth (-) A. Antibody-Netilmicin B. BSA-Netilmicin +++ C. Netil icin-Netilmicin +++ D. Buffer (blank) +++ From the above results, it can be established that the conjugate of antibody-antibiotic has a clearly better anti-bacterial effect on Streptococcus mutans than the same antibiotic conjugated to a non-specific protein. The major advantage of using a conjugate according to the invention for the treatment of dental caries and plaque is the short period of exposure needed for suffi¬ cient anti-bacterial effect.

Claims: CLAIMS 1. A conjugate of a bactericide and an antibody, c h a r a c t e r i s e d in that the antibody is directed against at least a certain type of dental plaque-forming and/or caries-forming bacteria. 2. A conjugate according to claim 1, c h a r a c ¬ t e r i s e d in that the antibody is directed against at least a certain type of bacteria selected from the group consisting of Streptococcus mutans, Streptococcus sanguis, Bacteroides gingivalis, Actinobacillus actino- mycetemcomitans, Eikenella corrodens, Veronella alcalescens and Actinomyces viscosus. 3. A conjugate according to claim 1 or 2, c h a ¬ r a c t e r i s e d in that the bactericide is an anti¬ biotic. 4. A conjugate according to claim 2 or 3, c h a ¬ r a c t e r i s e d in that the bactericide is Netil¬ micin and the antibody is directed against Streptococcus mutans. 5. A pharmaceutical preparation, c h a r a c t e - r i s e d in that it comprises a conjugate of a bac¬ tericide and an antibody directed against at least a certain type of dental plaque-forming and/or caries-forming bacteria together with a pharmaceutically acceptable carrier and/or diluent and, optionally, other pharmaceuti- cally acceptable additives. 6. A pharmaceutical preparation according to claim 5, c h a r a c t e r i s e d in that the antibody is directed against at least a certain type of bacteria selected from the group consisting of Streptococcus mutans, Streptococcus sanguis, Bacteroides gingivalis, Actinobacillus actinomycetemcomi ans, Eikenella corrodens, Veronella alcalescens and Actinomyces viscosus. 7. A pharmaceutical preparation according to claim 5 or 6, c h a r a c t e r i s e d in that the bacteri- cide is an antibiotic. 8. A pharmaceutical preparation according to claim 6 or 7, c h a r a c t e r i s e d in that it comprises a conjugate of Netilmicin and an antibody directed against Streptococcus mutans. 9. A pharmaceutical preparation according to any one of claims 5-8, c h a r a c t e r i s e d in that it is in the form of a paste, a tooth-paste, a suspen¬ sion, a solution or a mouth-wash. 10. A method of treating dental plaque and/or caries comprising local application of a bactericidal amount of a conjugate according to any one of claims 1-4 or a pharmaceutical preparation according to any one of claims 5-9, to a surface infected by dental plaque-forming and/or caries-forming bacteria.

Inventors: BERGRYD MATS, FREDRIKSSON GUNNAR, BERGRYD, MATS, FREDRIKSSON, GUNNAR
Applicants: RAMA BIO LINK AB, RAMA BIO LINK AKTIEBOLAG
Application year: 1989
Main classification: A61K39
IPC codes: A61K8, A61K39, A61K47, A61Q11