Technical field

The present invention refers to compositions useful for prophylactic, therapeutic treatment and/or diagnosis in connection with infections. This includes among other things dental plaque formation and illnesses caused thereby such as caries, gingivit and peridontoclausia.

Background of the invention

It is known that microorganisms have developed special adherence mechanisms for their colonization. The microbes carries on their surface adhesive proteins having a special fit for special molecules (receptors) on either the surface of the host (bacteria-host binding) or on other bacteria (bacteria-bacteria binding). Animal tissue surfaces and bacteria are to a substantial part covered by carbohydrates in different forms, which explains the large number of known carbohydrate receptors. In the mouth there are carbohydrate receptors on the saliva-coated tooth surface and the oral mucous membrane as well as on the surface of other plague bacteria. In plaque formation the bacteria-bacteria binding is dominating and it is possibly this binding that the present invention interfers with.

It has previously been proved that carbohydrates such as lactose (Galβl-4Glc) can counteract bacteria-bacteria bind¬ ing between several different plaque bacteria. In case of binding of a certain strain of Actinomyces naeεlxxndii to streptococci it has proved that GalBl-3GalNAc is a conside¬ rably stronger inhibitor than lactose. Other tests with the same bacteria strain (J. of Biol. Chem. , 1990, 265, 11251- 11258) has, on the other side, proved a strong binding to glycolipids containing the sequence GalNAcfi, and that Gal- NAcβ-3Galα-0-etyl inhibits this binding considerably better than lactose.

Dental plaque is characterized by a great variety of strains (totally about 250 different bakteria strains), and of a big intra- and inter-individual variation in the content of bacteria. Certain pattern in the plaque formation can howe¬ ver be notioced. Thus it is started by that the saliva film on the teeth are colonized by non-patogen streptococci, mainly S. sangxxis and S. mitis . The variety of streptococci are then colonized by different so called secondary colo- nizers, including Act±nomyces, Veillonella, Hemofilus, Ne±ssera, Staphylococcus , Rothia, etc. This secondary flora is believed to consitiute a necessary connection link for growth of different anaerobic bacteria, e.g. Bacteroideε, Peptostreptok~ ocker and Vol±nella which are associated with development of peridontoclausia. Dental plaque formation is thus a very complicated process and the effect of the elimi¬ nation of one plaque bacteria can therefore hardly be fore¬ seen.

Summary of the invention

An object of the invention is to provide a composition or a substance which effectively prevents and forestalls dental plaque formation, as well as decomposes plaque already formed. The invention also includes sugar substitutes (swee- teners) containing the active substance according to the invention and which completely or partly can replace con¬ ventional sweeteners. A furhter purpose is to provide a composition or a substance that can be used for diagnosis either of microorganisms or patients, where either the compositions according to the invention are the diagnostic instrument or be used for the preparation of diagnostics.

The active substance in the composition according to the invention has the following structural formula:

wherein R ₁ comprises the structure

and R ₂ is chosen from the following group of substituents: acetamide group, propyl, butyl and lower acyl group and R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ are the same or different and are hydro- gen or an organic group, e.g. lower alkyl-, lower acyl- or a carbohydrate group or an inorganic group such as sulphate or phosphate.

According to a preferred embodiment OR _α , 0R ₃ and OR< are in β-configuration, but α-configuration is by that not exclu¬ ded.

The substituent R _x is preferably chosen from the following group of structures α-D-Galg-l-0-R ₅ , β-D-Galp-l-0-R ₅ , β-D- GalNAc£-l-0-R ₅ and α-D-GalNAcp-l-0-R ₅ .

Concerning the substituent R ₅ it is preferably chosen from the group: hydrogen, lower alkyl, -0C ₆ H _s , -0C ₆ H ₄ N0 ₂ or another optional group like e.g.

Concerning the substituent R ₃ it can i.a. be a carbohydrate group preferably having the following structure:

where R _g , R _l ' ₀ , R _X1 and R ₁₂ are the same or different and are hydrogen or an organic group, e.g. lower alkyl, lower acyl or a carbohydrate group or an inorganic group such as sulph- ate or phosphate.

The structure for R ₃ according to above is in the preferred embodiment a GalBl-structure, most preferably β-D-GalNAc£-l

The substituent R _. * can i.a. be a carbohydrate group, which preferably has the following structure:

where R ₁₃ , R ₁₄ , R ₁₅ and R ₁₆ are the same or different and are hydrogen or an organic group, e.g. lower alkyl, lower acyl or a carbohydrate group or an inorganic group such as sulph¬ ate or phosphate.

The substituent R ₄ is according to another preferred em- bodiment α-D-Galf-1.

Especially preferred compounds according to the invention are: β-D-GalNAcp(1-3 )α-D-Gal-l-0-C ₂ H ₅ , β-D-Galp(1-3)β-D-GalNAcp( 1-3)-α-D-Gal-l-0-C ₂ H ₅ , β-D-Galp(l-3)β-D-GalNAcp or β-D-Galf( 1-6)β-D-GalNAcp( 1-3)-α-D-Gal-l-0-C ₂ H ₅

The invention also refers to use of the receptor analogue linked to a macromolecular carrier via an optional coupling arm. As carriers there can be used synthetic or naturally occurring polypeptides ^' such as bovine serum albumin, poly-

saccharides or other types of polymers or micro particles.

The invention further refers to use of the receptor analogue in a method for preparation of a substance or a composition having affinity for compounds on biological surfaces or in biological liquids. Such an example is preparation of mono¬ clonal antibodies having affinity for receptors on strepto¬ cocci, mucous membranes, tooth or in saliva. Such antibodies could be used for identification as well as for blocking of the microbial receptor sequence. Receptor blocking could be achieved by rinsing solutions with e.g. generated antibodi¬ es. Detection of receptor structures from the own body could be a possibility for prediction of patients having an in¬ creased risk for developing dental diseases. These examples are however not limiting. The methods for detection of receptor structures from the own body or generation of monoclonal antibodies are based on commonly known immunolo- gical technique and a more detailed description therefore seems to be superfluous.

A further use of the invention is in a method for identifi¬ cation, preparation or preparation in pure form of molecules having affinity for the compound. To this molecule group counts the microbial adhesive proteins and their use for the production of vaccine against microorganisms. The latter is however not limiting.

The specific compounds described are previously known in basic research, and can be prepared according to known procedures. They have however not previously been used for medical purposes.

Description of the figures Figure 1 shows how different saliva glycoproteins have been separated in individual fractions by gel filtration on Sephacryl S-200 (la). In figure lb selective binding of

A. naeslundii 12104 to certain of the fractions is shown, and especially to the fraction a but also certain binding to the fractions c-e. In figure lc is shown that these binding properties are determined by Actinomyces "GalNAcβ"-binding property when GalNAcβl-3Galα-0-etyl inhibits the bacteria binding to these fractions.

Figure 2 shows the plaque-reducing effect with GalNAcβl- 3Galα-0-etyl (2a and c) in relation to placebo (2b and d) . Despite that the substance was only applied on the right side of the upper jaw a general plaque reduction was seen. The figure also shows a dramatic change of the exterior and thickness of the plaque.

Description of the invention

It was mentioned above that lactose (Galβl-4Glc) is a rela¬ tively weak inhibitor of several bacteria-bacteria bindings between different bacteria that occur in dental plaque. We have examined whether GalNAcβ-based receptor analogues would be better inhibitors than lactose in certain cases of inter- bacterial interactions.

We have surprisingly found that it is the β-configuration at the carbon 1 ( ₁ ) , the acetamid group at C ₂ and the axial hydroxyl group at C_* that strongly influences the bacterial binding properties of "GalNAcβ". The stronger binding with GalNAcβl-3Galα-0-ethyl than with GalNAcβl-O-ethyl shows besides that parts of the binding activity of GalNAcβ is found in the neighbour group. Chemical modification of Gal- NAcβ would from this knowledge lead to more potent receptor analogues. As can be seen from the claims the inventions also covers such analogues of "GalNAcβ" where the acetamide group at C ₂ is replaced with a propyl-, butyl- or lower acyl group.

It has previously been proved that Galβl-3GalNAc is a stron¬ ger inhibitor than lactose, and equally strong as GalNAcβl-

3Galα-0-ethyl, of the binding reaction between Actinomyceε 12104 and streptococci. An explanation could be that also Galβl-3 is involved in the receptor function and that GalBl- 3GalNAcβl-3Gal in this case contains the natural receptor sequence. Receptor activity in the polysaccharide capsule of streptococci is found in a hexasaccharide that contains the sequence GalBl-6GalNAcBl-3Gal. Potent receptor analogues therefore ought to be similar to these structures. The invention is however not bound or limited to these theories.

In the following examples GalNAcβ-based receptor analogues have been examined with respect to their effect on Actinomy- ces aggregation of other bacteria, saliva and red corpuscles and to their binding to saliva-coated hydroxyapatite and oral mucosal cells. The effect of receptor analogues on formation of dental plaque has besides been studied in clinical human tests.

Example 1

Blocking of Actinomyceε binding to other plaque bacteria with GalNAcβl-3Galα-0-ethyl

In Table 1 below the ability of A. naeεlundi to aggregate oral streptococci and Bacteroides in the absense and presen¬ ce of GalNAcβl-3Galα-0-ethyl is shown. Equal volumes of 0.5 ml cell suspensions (5«10 ⁹ /ml) of the bacteria were mixed. Presence of coaggregation was read visually.

Table 1

A. naeslundi A. naeslundi+

GalNAcβl-3Galα-0-etyl

S. εangiuε + S. mitiε +

S. mutanε

B . gingivaliε

Table 1 shows that A . naeεlimdi binds to plaque streptococci such as S. εangiuε and S. mitis but not to __.. mutans, and

that GalNAcβl-3Galα-0-ethyl (2mg/ml) blocks this interac¬ tion. The' interaction between B. gingivaliε and Actinomyceε is ended if B. gingivaliε is heat-treated, but is uneffected 5 if Actinomyceε is treated in a corresponding way. This indicates that the reaction is mediated by a protein on the surface of B. gingivaliε and that it thus is completely unrelated to the "GalNAcβ"-interaction.

0 Example 2

Multiple binding reactions mediated via different GalNAcβ- containing glycoconjugates

We have recently shown that GalNAcβl-3Galα-0-ethyl besides the above described blocking effect on coaggregation also 5 prevents Actinomyceε binding to the buccal mucous membrane and saliva-coated tooth surface in vitro (Table 2) . The receptors on the tooth surface, vhich are not previously known, have been identified as different low- and high- molecular saliva glycoproteins, which also induces aggrega- 0 tion of Actinomyceε 12104 (Fig. 1).

Table 2 Inhibiting effect of lactose and GalNAcβl-3Gal-0- ethyl on different adhesion related properties of

A.naeεlundi 12104 5

Substance Hemaggl ³ Coaggr ⁵ S-HA ^C Buccal Saliva aggr ^e

(S.mitis) cells ^d

30 lactose 500 10.000 50% 5% 2000

GalNABl-

3Galα-0-ethyl 62.5 250 100% 100% 200 ) Minimum inhibiting concentration (μg/ml) of hemagglutination.

Minimum inhibiting concentration (μg/ml) of coaggration between R.naeεlundii and S.mitiε

CCUG 2238. ^c Percent reduction of adhesion to εaliva-coated hydroxyapatite (S-HA) after F-" ^eln _* ^<:u ' ^:| ati<-r _* gf 12104 with 2mg/ml of the substances. ^U Percent reduction of adhesion to buccal epitheliuπ* cells after incubation c 12104 with

2 mg/irl cf substances. ^e Minimum inhibiting concentration (μg/ml) of saliva-induced aggregation ot A. naeεlundii

12104.

45

Example 3

Inhibition of Actinomycesinduced hemagglutination with the receptor analogue

In Table 3 below is shown the ability of uni- and multi- valent derivatives of GalNAcβ to more effectively than other mono- and disaccharides block Actinomyceε agglutination of human erythrocytes. For the hemagglutination tests lOμl each the bacteria (10 ⁹ cells/ml) and erythrocytes (4%) were mixed, both suspended in PBS, pH 7.2, and applied on a glass disc. Occurrence of agglutination was examined visually during gentle mixing for 5 minutes at room temperature.

Table 3

Saccharide Min. blocking cone, (μg/ml)

GΪc •= Gal 1000 GlcNAc

GalNAc 500

Galβl-4Glc 500

Galβl-4Glcβ-0-ethyl 500 Galαl-4Gal-0-ethyl 500

GalNAcβ-O-ethyl 500 (Galβl-4Glcβ0) ₂₅ -BSA

GalNAcβ-0-(P-nitrophenyl) 500

GalNAcβl-3Galα-0-ethyl 62.5 (GalNAcβl-3Galα-0) ₂₅ -BSA 0.12

Table 3 shows that GalNAcβl-3Galα-0-ethyl inhibits hemagglu¬ tination already at a concentration of 62.5 μg/ml, which makes it a considerably more effective inhibitor than Gal- NAcβ-O-ethyl. It also shows that if the receptor analogue is conjugated with bovine serumalbumin (BSA) the inhibiting ability is increased more than 500 times. Structures that are ineffective at 2000 μg/ml have been marked with a minus sign.

There are many other examples of macromolecular carrier with which the receptor analogue according to the invention can

be conjugated. As carriers can thus be used synthetic or naturally occurring polypeptides, polysaccharides or other types of polymers or micro particles.

The linking arm between the receptor analogue and the macro¬ molecular carrier can be any of the following, but this selection is not limiting:

Structural element -0-<§ — HCSNH-carrier " -O—< ^ - N=N-carrier

-NH-(CH ₂ ) _n -C0NH-carrier

-NH-carrier " -0-CH ₂ -CH ₂ -S-CH ₂ -CH ₂ -CONH-carrier

In the above examples n can vary between 1 and 15. The linking of the receptor analogue to the macromolecular carrier via a linking arm per se is known technique, and a detailed description therefore seems to be superfluous.

Example 4

Occurrence of GalNAc-sensitive binding of plaque strains

Studies of Actinomyces-strains from different locals in the mouth, such as buccal, tongue and plaque at five persons have proved that GalNAcB-sensitive binding, measured as aggregation of streptococci and GalNAcβ-sensitive hem¬ agglutination, is common among plaque strains. Table 4 shows the result from one individual, which also corresponds to the results from the other four individuals.

3+

Example 5

Different GalNAcβ-binding bacteria types

Examination of the above bacteria strains (Table 4) has proved that the GalNAcβ-binding can be of two different receptor-binding types which differ from one another by different binding patterns to glycolipids, plaque strepto¬ cocci, soluble sugar structures, and to saliva-coated tooth surface. One of the receptor-binding types is overrepre- sentated in plaque strains, while the other is more common in strains from the buccal mucous membrane. This means that one could construct receptor analogues with selective effect on the plaque strains.

Table 5. Occurrence and properties of two different receptor-binding types of Actinomyces.

Binding to Natural local

Strain glycolipid strepto- free S-HA for strains cocci saccharides that resembles 12104 och Y 7

12104 broad broad Plaque reactivity reactivity

LY 7 narrow narrow buccal mu¬ reactivity reactivity cous membr.

The type strains 12104 and LY7 differ with respect to binding to the receptors on artificial surfaces as well as to natural membranes. While 12104 binds to GalNAcE- containing glycolipids LY7 seems to prefer receptor structures on membrane glycoproteins.

With a broad reactivity is meant that 12104 has an ability to recognize a larger numβer of streptococci strains, as well as to be inhibited by a larger number of soluble saccharides.

The receptor-binding type seems to be linκeό to the local in which the bacteria grows in the moutn.

Example 5 Clinical plaque inhibition with GalNAcβl-3Galα-0-ethyl Clinical studies with GalNAcβl-3Galα-0-ethyl concerning its plaque-inhibiting effect have been performed on five in- dividual. The test were performed in such a way that 80μl placebo, PBS (pH 7.2), or 2mg/ml GalNAcβl-3Galα-0-ethyl dissolved in PBS were applied on four teeth 16 times during 48 hours. The surprisingly big reduction of the plaque amount and reduction in number of AJ tinomyceε is shown in Table 6 and figure 2.

Table 6

Individual %Actinomyces Quantitative

Placebo GalNAcβl-3Galα-0-ethyl plaque reduction

very strong strong strong weak very strong

The study shows that GalNAcBl-3Galα-0-ethyl compared to placebo gives a strong general reduction of the total amount of plaque, which is illustrated in fig. 1 with the individu¬ als one and three. It was further shown a displacement to- wards an actinomyces-poor plaque. Despite that the substan¬ ce was applied only on four teeth one could notice a plaque- reduction on all teeth. The exterior and the thickness of the plaque was also substantially changed, which can be interpreted that the bacteria layers on top of the strep- tococci layer is being eliminated.

The surprisingly strong plaque-reducing effect for the Gal- NAc ^β -analogue which is shown indicates that it can block several plaque bacteria. This assumption is supported by the fact that lactose besides Actinomyceε also has been proved to be able to inhibit other secondary plaque colonizers.

The above described examples are limited to the use of the GalNAcβ-analogue as a plaque-inhibiting agent. The invention however also refers to the use thereof for prophylactic, therapeutic or dignostic purposes for other infections caused by microorganisms, where the substance according to the invention has an ability to replace the normal receptor- function with respect to the microorganism in question.