DD240669C2 - PROCESS FOR PREPARING PREPARATIONS FOR THE BIOLOGICAL CARE PROPHYLAXIS - Google Patents

Description

Field of application of the invention

The invention relates to a process for the preparation of preparations for biological Kanesprophylaxe, which in local or oral administration of concentrations that trigger a measurable attachment competition

- On the one hand displaced Str mutans from the biotope and thus prevent the caries in the short term and

on the other hand, over the temporarily high "monoclonalizing-simulated" antigen levels, which stimulate local boostability-reactive immunity, thereby significantly reducing the incidence of Kanes

Characteristic of the known technical solutions

Caries is an infectious disease manifested by dietary habits The destruction of enamel caused by several serotypes in particular of Str mutans affect the deciduous dentition (with subsequent damage in the Dentition) and the permanent teeth of the adult with a maximum between the 10 and 20 years (LEHNER, Bnt dental J 149, [1980], 318-325)

Mutans streptococci apparently attach themselves by means of protein receptors (VAN HOUTE, in Dental Plaque and Surface Interactions in the Oral Cavity, IRL Press Ltd, [1980], 69-100, McGHEE & MICHALEK, Ann Rev Microbiol 35 [1981], 595-638, GIBBONS, J Dent Res 63, [1984], 378-385) to complementary structures of glycoproteins which deposit from saliva to enamel

However, the attachement pnnzip is not widely known in glucans or lipoteichosaurs. Overall, however, little is known about the molecular processes leading to irreversible germ and plaque anchorage. An important role is played by the binding of glucosyltransferase (GTF) to glucan (FIGURES u EDWARDS, Carbohydr Res 88 [1981] , 107-117, KOGA & INOUE, Infect & Immun 19, [1978], 402-410, HAMADA et al J Dent Res 63, [1984], 407-411, TSUMORI et al Microbiol Immunol 26, [1982] 677 -688, STINSON et al. Infect Immun 32, [1981], 583-591) and cocci and adherence of glucan to the pathogen and enamel. The cocci now adhering to the enamel and growing into the microcolony synthesize from sucrose with the aid of mainly (possibly plasmid GTF a hyperbranched glucan (mutan) polymer which rests as a plaque on the enamel (McGHEE u MICHALEK, Ann Rev Microbiol 35 [1981], 595-638) and of further sugar splits This Plaqueflora caused by carbohydrate degradation in the Plaquebereich effective lowering of H * ion concentration below the critical threshold of pH 5.5 which leads to Entminerahsierung the enamel Accordingly, the quantitative formation of insoluble glucans correlates with the canogenic activity or virulence of the various Str mutans strain (MICHALEK et al Proc Soc Exp Biol Med 150, [1975], 498-502) Currently discussed or practice-effective Kanesprophylaxe

- Low-sucrose or free diet Such a prophylaxis fails because of our nutritional standards

- Dentifrice with plaque-dissolving enzymes (DE 3 248 541, DT 2 265 327) or the bakterel ι cell walls or their Mureingerust splitting lysozyme (DE 2 852 792)

- Fluoridation of drinking water or food and / or local application of fluorine (HOROWITZ, Bnt dental J 149 [1980] 311-318) This prophylaxis requires a remineralization of the enamel (also is accumulating in the border area for enzyme inhibition fluorine enrichment in plaque for discussion (MARQUIS J Dental Res 56, [1977], 6, 704) The performance limit of this prophylaxis is about 40% caries reduction

Predominantly animal studies on immunoprophylaxis to detect attachment blockade caused by secretory antibody (BROWN et al J Dental Res 57, [1978], 882-893, MICHALEK et al Infect & Immun 19, [1978], 217-224), or / and GTF inhibition to prevent plaque formation

Parenteral administration of inactivated mutans streptococci requires a detectable local immune response and caricidal action (OE Patent Specification No. 320 848, LINZER et al Infect & Immun. 31, [1981], 345-351, CALDWELL, J Med Microbiol

* In addition to attachment-blocking specific and inactive s IGA of a different specificity, there are further attachment (demanding and) inhibitory components in saliva (GAHNBERG et al., Infect & Immun., 37, (1982), 401-406), such as that the growth inhibiting lactofins (ARNOLD et al Infect & Immun 28 [1980] 893-898)

10, [1977], 213-224) In this connection, it is worth mentioning that other antibody spectra occur upon immunization with living germs (BRATHALL and PETTERSON J Dental Res 55, [1976], 60-64).

Immunization with the enzyme GTF mediates a serotype-independent protection against Kanesbefall (SMITH et al. Infect & Immun 37, [1982], 656-661, TAUBMAN, United States Patent [19], 4, 150, 116, Apr 17 1979 McGHEEu MICHALEK Ann Rev Microbiol 35 [1981], 595-638)

• Oral immunization with whole germs or / and stimulated in the culture supernatant locally or via 'homing', ie by immigration of s IgA forming cells from the Peyer plaques in salivary Ddrusen (MONTGOMERY et al., McGHEE et al Proceedings of the International Symposium on the Secretory Immune System and Caries Immunity, Plenary Press, New York and London 1978 p 113-122, HANSON et al. Ib 165-176, MONTGOMERY et al ibid 113-122, LEHTONEN et al Infect Immun 43, 1984], 308-313) - in the oral area protective secretory antibodies which after booster rapid and high IgA synthesis occurs (McGHEE u MICHALEK, Ann Rev Microbiol 35 [1981], 505-638, LINZER et al Infect & Immun 31, [ 1981], 345-351, MESTECKY et al Jelin Invest 61, [1978], 731-737, McGHEE et al., McGHEE et al., Procedures of the International Symposium on the Secretory Immune System and Canes Immunity, Plenum Press, New York and London 1978 ρ 177-184)

Recently, a "ribosome preparation" (EVERHART et al Med Microbiol Immunol 172 [1984], 215-222) is presented as an immunogen, which reacts as an antigen with the sera of all str mutans serotypes. The authors expect this "ribosome vaccine" three

A caries-protective effect covering all serotypes (whereby u a is overlooked, that intracellular antigens are not relevant for an antibody-mediated attachment blockade)

- No formation of cross-reacting antibodies to human heart and muscle tissue

It is interesting to note that the titre of humoral precipitins against Teichonsauren oral bacteria - as an expression of the normal flora emanating permanent immune stimulation - with a least not increased to significantly reduced Kanesanfalligkeit correlated (LEVINE et al Arch oral Biol 29, [1984], 191-194)

This active immunoprophylaxis, which has hitherto not yet been developed for practical relevance, in principle only leads to an increase or temporal advance * of the natural local basic immunity which ζ B is stimulated in humans by> 10 "/ day swallowed oral cavity germs (Mc GHEE u MICHALEK, Ann Rev Microbiol 35 [1981], 595-638) and presumably the decreasing incidence of caries as one grows older. This correlates with the observation that people with low rates of high kanesanfallness differ in their increased antigen-specific IgA in saliva (LEHTONEN et al Infect Immun 43 , [1984], 308-313, CLANCY u BEESENSTOCK, in Ferguson et al. Immunological aspects of the liver and gastrointestinal tract pp 121-152, Baltimore, Univ Park Press, 1976, McNABB, Ann Rev Microbiol 35, [1981], 477-496, LAMM, Advances Immunol 22, [1976], 223-290, MESTECKI et al J CIm Invest 61, [1978], 731-737) The immune response found in the experimental animal against a colonizing s Antigen (FITZGERALD u BIRDSELL, J pendontal Res 17, [1982], 237-246) is therefore valid mutatis mutandis for the colonizing - or additionally administered in high doses - Str. mutans in humans Difficult or interpretable with caution in this context the reported finding (MICHALEK et al Infect & Immun 19, [1978], 217-224) that in rats 10 ⁷ and 10 ⁸ formalin-inactivated str mutans CFU / gram diets elicit a significant S-IgA titer, but 10 ⁹ / CFU of the same antigen apparently no immune response in saliva drove the current focus in the microbiological caries research or prophylaxis (Abstracts of Papers 62 ^nd General session International Association for Dental research, March 15-18, 1984 in J. Dental research 63, [1984] , Special Issue), gear up

- Imputations (colonization) Experiments on animal models with non-cariogenic streptococci for the purpose of competetive displacement of str mutans in the biotope and the aim to reduce caries frequency (In this sense, the investigations on the settlement of defined str mutans or sanguis strains in the oral cavity can also be interpreted (SVANBERG et al Infect Immun 43, [1984], 817-821, WESTERGREN and SVANBERG Arch Oral Biol 28, [1983], 729-733)}

- Studies on the correlation between the titer of antigen-specific secretory IgA in saliva and the Kanesanfalligkeit of humans, including appropriate immunization experiments in rats This law is the patent (DE 3 427 477) fair, which the addition of - produced by immunization of animals antibodies proposes against the acidic glycoproteins of Str mutans cell surface to dentifrices

- Studies on the substrate of the attachment, where the reported data indicate proteins or glycoproteins (Fussy coat)

For the sake of objectivity, it should be mentioned that in parenterally hyperimmunized rabbits humoral antibodies with cross reactivity to cardiac tissue were found (FERRETTI et al Infect & Immun 30, [1980], 69-73), but on the other hand such animals were less susceptible to experimental streptococci Endocarditis (McGHEE u MICHALEK, Ann Rev Microbiol 35, [1981], 595-638)

Further, there are indications (HUGHPS et al Infect Immun 27, [1980], 576-588, VAN DE RIGN et al J Dent Res 55, [1976], 59-14, STINSON et al Infect Immun 27, [1980], 604-613), in which after immunization of humans with Str mutans full-seed vaccines with heart and muscle tissue cross-reactive antibodies may occur In this context, it is necessary to note that the lawful occurrence of humoral antibodies against orally fed or colonizing antigens (including PIERCE, J exper Med 148, [1978], 195-205 CUSHING, Develop Biol Standard VoI 53, pp 107-111) can not be classified as clinically questionable since about 70% of all adolescents in the serum have antibodies against milk protein (cited in ROTHBERG et al J Immunol 98, [1967], 386-395)

If one pursued the idea "possible side effects" with all consequences, this could lead to a questioning of ultimately any therapy or immunoprophylaxis

For example

Penicillin always induces the absorption of large quantities of biologically complex peptidoglycans from gram-positive cocci (PARK et al., Infect. Immun. 43, [1984], 139-142).

* Similar conditions are found in the meningococci, pneumococci and Hamophilus influenzae polysaccharide vaccines, which induce protective titers from the end of the 2nd year onwards. Unvaccinated populations do not acquire such titers until the age of 5 via mapparente infections or colonization (Linde et al Zschr arztl Fortbild 75 [1981] 669-674)

  In individual cases, virus vaccinations mediate the allergic reaction to a later natural infection (KARZON, in Human Immunity to Viruses Symp on Human Immune Responses to Viruses Recent Developments, Ed. Orlando, F A, Academic Press 1983, ρ 111-130).

Object of the invention

The aim of the invention is to develop etn methods for the preparation of such preparations for biological caries prophylaxis, which have a high effectiveness in the Kanesprophylaxe without the demand for unrealistic dietary habits and lead to immunity even in childhood and adolescence, resulting in consistent use of a high health policy and economic benefits

Essence of the invention

The invention has for its object to provide a method for the preparation of preparations for biological Kanesprophylaxe, resulting in the preparation of preparations which are suitable for inducing a local immunization against caries by local or oral application of these preparations in concentrations that simulate monocolonization ( Displacement of str mutans from the biotope) and therefore the immunological switch on tripping invention Streptokokkus mutans wildstamme or GTF mutants are used. It has been found that improved Kanes prophylaxis by temporary application of and temporary boosting with attachment-active inactivated streptococcus mutans preparations or the living GTF Mutants are reached in concentrations that lead to a competitive displacement (simulated an immunological switch on tripping monopolization) of Str mutans wild strain population in the biotope

The process according to the invention is characterized by the following process steps Streptococcus mutans wildstems or GTF mutants are propagated by means of processes known per se in high-yielding methods in mass culture. The bacterial material thus obtained is used either as a resuspended sediment or as a milk preparation

- gently inactivated by known methods in such a way that the Attachmentaktivitat is not affected, or

- the attachment principle extracted from pathogen and / or cultural supernatant or

- leave the GTF mutant as a survivor

A preparation which activates attachment for local or oral application or immunization is obtained. This preparation must be concentrated if necessary. Subsequently, it is further processed in forms suitable for application or immunization by means of techniques known per se

The preparation obtained for local or oral application or immunization is advantageously used at concentrations until the oral cavity mutans streptococci are displaced from the biotope via an attachment competition and as a result of this "simulated monocolonization" an immunological switch on occurs The resulting and correspondingly concentrated preparation is further characterized in that it is administered as a booster in a primary vaccination and at intervals. The primary immunization may be about 4 weeks followed by boostering at regular intervals For example, these can in 2 to 4 weeks distance over each one day for oral or local application the preparation is prepared in the form of dentifrices, gels, sweets, milk preparations, etc

The multiplication by mass culture is carried out expediently in that during the incubation the pH is neutral korregiert It is likewise expedient to carry out the careful inactivation ζ B at 56 ⁰ C or with 0.35% formalin at full germ preparations of St. mutans wild strains

The preparations produced by the method according to the invention ensure a high effectiveness in caries prophylaxis by advancing the increasing (adult) age naturally developing - more or less protective immunity to tooth decay in the child and adolescence

Concept for the biological Kanesprophylaxe

went out

The locus flora of the mucous membranes, or the pathogens entering this biotope, are anchored specifically to surface host receptors by means of special surface structures, thus preventing their dilution by the juice flow and allowing subsequent colonization to microcolony (colonization). Examples are the colonization factor antigens or fimbriae (Plasmid and / or chromosomally encoded) of Enterobacteriaceae, gonococcal grafts, etc., etc. (ELWELL, Ann. Rev. Microbiol., 34, [198O]) 465-496, BARON et al. SAZ, J. Bacteriol. 133, [1978], 972-986; RABSCH et al Adhasme in Gram-Negative Bacteria, Special Issue on Animal Hygiene Information 17, [1985], ISSN 0138 1881) Isolated direct and indirect indications of the prophylactic or therapeutic efficacy of a substitution treatment based on attachment competition, inter alia with colicimals The scientific recognition of such an effect principle based on attachment competition and biotope filling with apathogenic "normal flora" germs has failed to date on an irreproducible ("50% of the Enterobacteriaceae flora)" Monoclonalization "by such" substitution strains "or because quantitatively too small amounts of antigen result in no significant attachment competition. Therapeutically or prophylactically relevant attachment competition, however, implies antigen concentrations corresponding to nonphysiological monocolonization n and therefore result in a local immune response (Schroder et al unpublished)

Such conditions in the biotope are roughly comparable to the traveler disease by enterotoxic coIi The susceptible host has no specific local defense against the so far unknown pathogen, so that there is - associated with corresponding clinical symptoms - de facto (Enterobacteriaceae) monocolonization occurring here, a critical threshold exceeding antigen concentrations now stimulate a measurable local immunity (which terminates the apparent infection)

From a molecular biological point of view - within the complex pathomechanism of Kanesinfektion - the attachment of the mutans streptococci to the enamel surface is of essential importance Specifically to the GTF mutant regarding colonization and caries prevention (DE STOPPELAAR Arch Oral Biol 16 [1971], 791-975), Attachment suppressed but unfulfilled expectations is to say that as a consequence of not conclusive consequences, quantum aspects of the attachment blockade with subsequent stimulation of the local immunity elaborated and evaluated experiments continued

Equally surprisingly and logically, we have found that simulated monoclonalization considering a quantitative aspect using a gently inactivated attachment active mutant antigen competitively interferes with the attachment of the str mutans wild strain population to the enamel and subsequently recognizes a local immunity ζB on the rat model due to lack of detection of str mutans in the biotope plaque reduction and caries prevention during the four-week antigen challenge and the persistence of caries resistance, after completion of this oral immunization period 'with temporary boosting

According to our conception, using carefully inactivated antigen (at a later date, possibly an active colonizing GTF mutant would be possible [possible solution of the problem of "reproducible colonization" with a GTF mutant, inter alia, see DD 218 834 Methods for Producing Mutant Bacteria with Altered Performance Parameters) competitively displaces the str mutans wild strain population from the attachment site and thus prevents plaque formation in the short-term rat experiment or prevents caries formation. At the same time, as a result of oral immunization with high antigenic inputs-which exceed the critical threshold for the draw of immunological reactivity-prevention via attachment site is suppressed increasingly relieved by secretory IgA mediated immunological parameters of the attachment blockade

"From the point of view of a biological caries prophylaxis, B were also to discuss colonial mutant mutant experiments as a natural symbiont because these germs can reduce kanesanfallness via increased lactic acid degradation (VAN DER HOEVEN et al Caries Res 12 [1978] 142-147).

Exemplary embodiments

The invention is explained below with reference to exemplary embodiments A caries rats model

Attitude of the animals and evaluation of the experiments

a) At the beginning of the test series, the OSBORNE MENDEL rats were not older than 21 days. Each animal received 15 g of feed daily, as well as aqua dest ad libitum. The animals were fed in the morning. The average weight gain per animal during the trial period was 70 grams

b) The fissure caries was evaluated according to the modified scheme of KÖNIG (possibilities of caries prophylaxis in humans and their investigation in short-term rat expresion, Hans Huber Verlag, Bern 1966) which divides the infestation of the fissures into 4 degrees of severity.

Grade A (1) Color reaction on the enamel limited with intact enamel surface and uncolored enamel dentin boundary

Grade T (2) Color reaction to enamel Dentin Limit Grade B (3) Color reaction at and beyond the enamel dentin border incipient dentin involvement and low spread

in the depth and after occlusal degree C (4) intensive Dentinfarbung occlusally and pulpawarts, d h advanced caries with incipient lifting of the enamel and incipient cavitation

5 fissures (1 and 2 molar) were evaluated, and for each fissure the number of totally staged stages was evaluated. On special diagnostic sheets, the carious lesions of each tooth were recorded, the total number of staged stages was summed up separately and the mean value was calculated (max 20)

B The plaque index was determined by planimetry. After removal of the soft tissue, the vestibular lower half of the mandible was stained for 15 seconds with a fuchsia solution consisting of 3 parts of distilled water and 1 part of saturated alcoholic basic fuchsia solution, then rinsed briefly with tap water and the flat expanse of the stained plaque Stereomicroscope SMXX (VEB Carl Zeiss) measured by incident light The stadiums were evaluated

0 = no staining

1 = staining only in the lower third of the vestibular molar surface (ν Μ)

2 = stain extends to two-thirds of ν Μ

3 = complete red coloration of ν Μ

The numbers (stadia) values determined on the first and second molars of a lower jaw half were summed and the mean value (maximum number = 6) was formed (STOOKEY Plaque Scoring Evaluation in Living Animals, in Proceeding Symposium on Animal Models in Biology Ed Tanzer JM Sp Supp Microb Abstracts [ 1981] 205-214 Int Retrieval Inc London) C Semiquantitative determination of total streptococcus CFU and str mutans in approximately 1 mg plaque material from buccal tooth surfaces

The plaque material taken with sterile myrtle leaves was suspended in 1 ml of sterile buffered saline and homogenized by means of glass beads and 15 min vibration vibrating treatment. From this "germ suspension" a logarithmic dilution series was prepared using buffered saline and for each dilution step 0 1 ml on Mitis Sahvarius agar and without 0 1% sulfisomidine (MSA B

* Standard food = negative control

pure Kanesdiat (Stephan 580 Konig et al see under Ab) = positive control Kariesdiat with appropriate concentrations of the biological preparation = test series

238, Difco) and incubated anaerobically for 24 hours and aerobically 37 ° C. for a further 24 hours. The colonies grown on the medium without sulfisomidine were referred to as streptococcal species, while the bacteria growing on the medium with inhibiting substrate were paid as streptococcus mutans (KLIMM, Zahn- Mund u Kieferheiikunde 66, [1978], 771-775, KLIMM, Diss Prom B Leipzig [1981]). The colonies grown on the sulfisomidine medium were randomly compared with the str mutans identity with the str mutans reference strain NCTC 10449 colony-morphologically and by means of Gramfarbung D used culture medium and modalities of Bebrutung

a) Commercially available thioglycollate broth with 0.5% dextrose. Bred the inoculated medium for 24 hours at 37 ° C and readjust the pH to 7.4 after 10 and 24 hours

The pure yield achieved was 2 × 10 ⁹ CFU / ml

b) Supplemental H-milk

The H milk supplemented with 0.1% yeast extract (VEB Bramsch, Dresden) and adjusted to pH 7 4 was incubated at 37 ^° C. for 18 hours after inoculation. At this time, 0 25% dextrose was added and adjusted to pH 7.4 incubated at 37 ° C for a further 12 hours. This allowed the total yield of 4 χ 10 ⁹ CFU / ml H-milk to be increased to 2 χ 10 'CFU / ml supplemented and pH corrected H milk. Preparation of effective (and ineffective) preparations biological caries prophylaxis

a) The angezuchteten as D 1 in Thioglykollat Bouillon nuclei were centrifuged, the pellet resuspended in _^1/0 of the original volume in physiological saline solution and then for 30 minutes at core temperatures of 45 ⁰ C, 5O ⁰ C, 56 ⁰ C, 65 ⁰ C , 75 ⁰ C and 85 ° C inactivated Another batch was killed for 15 minutes at 100 ⁰ C inactivation with 0.35% formalin was carried out for 24 hours at 4 ⁰ C, then washed by centrifuging the free formalin The GTF mutant was used as a liaison

b) The germs grown as in D 2 in supplemented H milk were inactivated in the milk for 30 minutes at 56 ⁰ C.

c) Extraction and preparation of the active principle according to methods known per se, such as B in DE 3 427 477.

example 1

Quantitative relationships between locally / orally induced different concentrations of a caries-prophylactic preparation and the realized simulated "monoclonalization" (as a threshold for an immunological switch on), measurable at caries prevention in the short-term caries rat model

To demonstrate the concentration-dependent activity of the biological principle "competitive attachment displacement" was the 56 ° C inactivated Str mutans antigen - or the H-milk after partial enrichment with bred in thioglycollate broth antigen - in graded CFU Keimaquivalenten of 10 ⁸ , 5 \ (fi, 2 and 5 10 ⁹ 10 ⁹ / gram caries feed used is the quantitative dependence of the Kanesreduktion the concentration of the biological active preparation recorded in Table 1

Table 1 (see page 6)

As Table 1 shows, increases with increasing or decreasing concentrations of the active principle, the Kanesanf susceptibility progressively from or to at 10 ^A CFU-Keimaquivalente / gram caries feed the effectiveness drops to the "maximum values of the fluorine prophylaxis" (see Table 4), 5 χ 10 ⁹ CFU germ equivalents prevent any caries infestation in the rat model

Example 2

Demonstration of the realized attachment competition in the wake of a simulated monocolonization via

- The specific displacement of Str mutans germs in plaque compared to the other oral streptococci as well

- the altered plaque index

During and after the prophylaxis with 2 × 10 ⁹ CFU-equivalent / gram of caries, the CFU of str mutans and the other oral streptococci was determined semiquantitatively as described under C and the plaque index was determined as described under B.

The summary mean values are summarized in Table 2.

Table 2 (see page 6)

Table 2 illustrates that during a realized "monocolonization" in accordance with the extent of caries reduction

In the positive control and after discontinuation of the effective biological preparation this pathogen is found at a frequency of - 10 ³ CFU / ~ mg plaque material, the values in the negative control sicti move by 10 ¹ CFU

The specificity of this attachment displacement is evident from the experimental-independent unchanged> 10 * CFU of other oral streptococci

The plaque index at about 4.3 moves between the negative control value of ~ 3.5 and the positive control value of ~ 5.0

Example 3

Indirect indication of a more effective by 4wochiger simulated Monokolonisierung - with inactivated at 56 ⁰ C Str mutans 2 χ 10 ⁹ CFU Keimaquivalenten / gram Kariesdiat - additionally becomes effective kariespreventive, immunological component for the indirect detection of such immunologically becoming effective component was the 2, 3 and 4 weeks antigen application of a 28-day trial compared with an 8-week "long-term trial" in which after continuous caries prevention in the first 4 weeks then pure caries date, or pure caries date with a daily booster at the end of the 6 week, or two daily boosts spread to one day at the end of the 5 and 7 weeks The incidence of Kanes within these different experimental groups is recorded in Table 3 Table 3 (see page 7)

As Table 3 shows, a two- and three-week prophylaxis to the Kanesinzidenz at the end of the 28-day trial de facto without influence In four weeks continuous administration of caries prophylactic biological preparation remains a clear, only immunologically explainable, protective effect and the unique booster after 14 days At the end of the 6-week trial, the 8-week trial guarantees optimal caries prevention

Example 4

Comparison of the efficacy of various preparations of 2 x 10 ⁹ CFU Keimaquivalenten of Str mutans with each other and with the effectiveness of fluoroprophylaxis on short-term caries rat mode

The preparations grown as described under D a and D b and processed according to E a and E b were added to the Str mutans wild strain as inactivated antigen or to the GTF mutant as live germs in the concentration of 2 × 10 ⁹ CFU keima equivalents / gram of caries feed

The influence of these preparations on the Kanesreduktion compared to the positive u. Negative control and the effectiveness of fluoroprophylaxis are shown in Table 4

Table 4 (see page 7)

As shown in Table 4, 2 x 10 ⁹ CFU microbial counts of the live GTF mutant or inactivated 56 ⁰ C or formalin antigen significantly reduce caries by a factor> 10 to factor - 1. Significant caries reduction is also observed with respect to optimal fluoroprophylaxis Inaktivierungstemperaturen decreases the caries prophylactic efficacy, at> 85 ° C inactivated antigens are de facto ineffective

Table 1

Dosisabhangigkeit the simulated Monokolonisierung or biological caries prophylaxis on short-term caries rat model, detectable by addition of - (angezuchteten in H milk and / or Thioglykollat broth) at 56 ⁰ C inactivated - stepped Str mutans CFU Keimaquivalent concentrations ( "antigen") / gram Kanesdiat (KD ) Trial period 4 weeks, experimental groups of 26 rats each

feed concentration Mean of the 5x10 ⁹ significance of the "antigen" Kanesbefalls 2x10 ⁹ CFU equivalents max 20 at CFU too five fissures 5 xiO ⁸ 1 χ 10 ⁸ KD 1 10 ⁸ 30 α 0.1 KD 5 10 ⁸ 1.9 KD 2 10 ⁹ 08 α 0.01 α 0 01 KD 5 10 ⁹ 0.0 KD Positivkontr 12.3 standard Negativkontr 01 dardf

Table 2

Plaque Indices and Semiquantitative Determination of CFU of Str mutans and Other Oral Streptococci (Cultivation on Mitis Sahvarius Agar with and Without Sulfisomidine) / ~ mg Plaquematenal During and After Prophylactic Administration of Strutanes 2 - 10 ⁹ CFU Keima Equivalents - Inactivated at 56 ⁰ C ( "Antigen") / grams Kanesdiat (KD) at short-term caries rat model

feed Caries CFU Str mutans -10 ³ other Str plaque infestation while to with or without indices please refer "Antigen' 'Gift "Antigen" gift Tab. 4 - 10 ' KD plus "Antigen" 1.0 <10 ° ~ 10 ³ a 10 « -43 KD Positive- kontr 12.0 a 10 « -50 standard dardf Negative- 0.1 ⁴ -3.5 kontr

Table 3

Indirect evidence of after 4 weeks application of - at 56 ⁰ C inactivated - Str.mutans 2 χ 10 ⁹ CFU germ equivalents ( "antigen") / gram caries diet (KD) becomes effective kariespreventiven immunological component at the caries rat model test duration 4 to 8 weeks, experimental groups each 26 rats

Duration of the trial feed "A Ga Bo the 1 ¹ Need:> e-> i easter X.W 2 -Appl berX \ above oche 3 katior W och a 4 7 8th Positive Control Negative Control L , L L ' Positive Control Negative Control Mean value of caries infestation: max. 20 at five fissures four weeks KD KD KD => 7.0 6.8 1.0 KD standard! r. Perr en bzv day ar 5 11.8 0.1 KD KD KD KD 2.0 1.0 1.2 1? eight weeks nanen V. η end 6 KD standard f. 12.0 0.1

Table 4

Influence of different inactivation temperatures as well as the formalin killing on the (attachment competition conditioned) caries prophylactic activity of 2 χ 10 ⁹ CFU Str. Mutans germ equivalents / gram caries diet compared to the living GTF mutant as "antigen" and the effectiveness of a fluoroprophylaxis on short-term caries rat model

feed biological Mean of the significance to preparation Caries: 2x10 ⁹ CFU Max. 20 at positive Fluorpro five fissures kontr. prophylaxis KD Wildst. 56 ⁰ C 0.8 α 0.01 a 0.01 KD Wildst. 65 ⁰ C 3.6 KD Wildst. 75 ⁰ C 3.8 KD Wildst. 85 ⁰ C 7.8 KD Wildst. 100 ° C 8.0 KD Wildst. formalin 1.0 α 0.01 α 0.01 KD GTF mutants 0.7 α 0.01 α 0.01 KD Positivkontr. 12.0 Was standing- Negativkontr. 0.2 dardf. KD Silica fluorine 5.0 KD Elmex gel 3.1

Claims (4)

invention claim 1 method for the preparation of preparations for biological Kanesprophylaxe characterized in that streptococcus mutans wildstorms or GTF mutants by means of known per se a high yield of guaranteeing methods in mass culture propagates and the resulting bacterial material as resuspended sediment or as a milk preparation either is gently inactivated by known methods in such a way that the Attachmentaktivitat is not affected, or - extracted the Attachmentpnnzip from pathogen and / or cultural supernatant or - The GTF mutant will be left as a lifelong and obtaining an attachment-active preparation for the local application and immunization, if necessary, it is concentrated and further processed in a form suitable for the application or immunization by means of techniques known per se 2. Method according to item 1, characterized in that the preparation obtained for the local application and immunization is concentrated until the mutans streptococci of the oral cavity are displaced via an attachment competition from the biotope and as a consequence of this "simulated monocolonization" an immunological switch on is drawn 3 Method according to items 1 and 2, characterized in that a preparation to be administered for a period of the primary immunization and the subsequent regular boostings is obtained 4. The method according to item 1 to 3, characterized in that em is prepared for the local or oral application in the form of dentifrices, gels, sweets, milk preparations, etc. preparable and administered preparation