WO2000047175A1

WO2000047175A1 - Oral compositions and methods of production thereof

Info

Publication number: WO2000047175A1
Application number: PCT/US2000/003681
Authority: WO
Inventors: John A. Budny; Matthew J. Budny
Original assignee: Pharmacal Biotechnologies, Llc
Priority date: 1999-02-12
Filing date: 2000-02-11
Publication date: 2000-08-17
Also published as: AU3489700A

Abstract

A composition for controlling bacterial growth/colonization is provided. The composition comprises an enzyme, an anchor molecule coupled to the enzyme to form an enzyme-anchor complex, with the anchor being capable of attaching to a substrate proximal to a bacterial colony. The composition is produced using recombinant DNA techniques. In one form, the composition is a fusion protein coded for by a polynucleotide segment. The attachment to the substrate permits prolonged retention time of the enzyme-anchor complex where the bacterial colony is present to increase the effectiveness of the complex. The invention is also for a method of controlling colonization of bacterial plaque in the oral cavity, as well as a method of forming a composition for controlling the proliferation of bacterial colonies in the oral cavity.

Description

ORAL COMPOSITIONS AND METHODS OF PRODUCTION THEREOF

FIELD OF THE INVENTION

The invention relates to compositions for controlling bacterial colonization, particularly, but not restricted to, an oral application for reducing dental plaque. The invention is also for an oral therapeutic treatment which will limit or restrict the extent of bacterial colonization in the oral cavity thus reducing the quantity of dental plaque. By controlling the extent or size of plaque structures with enzymes, bacterial colony proliferation and their invasion into gingival tissue can be limited. The invention also relates to methods of manufacturing such compositions.

BACKGROUND OF THE INVENTION

Periodontal disease is one of the oldest and most common diseases of man. It is apparent in human fossil remains and occurs in otherwise healthy individuals. Today, periodontal disease represents a major worldwide health problem. The disease is a result of the accumulation of dental plaque at the gingival margin. There are two broad classes of periodontal disease which roughly approximates the degree or severity of the pathology: gingivitis and periodontitis .

SUMMARY OF THE INVENTION

A principal aspect of the invention lies in two concepts, both of which are necessary for a successful therapy for the prevention of periodontal disease. The first of these is the regulation of the amount and architecture of the plaque structure within the oral cavity by using enzymes; the second is the means of retaining the enzymes in the oral cavity. Both of these concepts must preferably be implemented for effective control of periodontal disease to occur.

In one aspect, the present invention modifies selected enzymes in a manner that they will have the capability of limiting plaque or its components. The enzymes selected are preferably ones that specifically degrade polysaccharides . In this way, the backbone structure of the plaque matrix may be limited without either selective or broad-spectrum kill of bacteria, thus avoiding any bacterial imbalances.

The modified enzyme is preferably attached to selected "anchor" molecule(s) to be retained in the oral cavity. The retention of the enzymes in the oral cavity is preferably maximized by coupling the enzymes to specific molecules that will adhere to the structures and existing biofilms within the oral cavity. Enzymatic activity should be maintained after the coupling. It is important that the process of connecting the selected enzymes to the specific "anchor" molecules does not wholly destroy the enzymatic activity, although it is possible that such activity may be reduced by reason of the coupling. However, at least a minimum effective amount of enzymatic activity should be present after coupling.

According to one aspect of the invention, there is provided a polydeoxyribonucleotide segment comprising a first portion which codes for an enzyme selected for its ability to degrade a colonization matrix and a second portion which codes for an anchor selected for its ability to attach to a substrate proximal to a bacterial colony.

In another aspect, the invention is for a fusion protein comprising an enzyme portion selected for its ability to degrade a colonization matrix and an anchor portion coupled directly or indirectly to the enzyme, the anchor being selected for its ability to attach to a substrate proximal to a bacterial colony.

According to other aspects of the invention, there is provided fusion proteins comprising: an enzyme and spacer, enzyme and anchor, and/or anchor and spacer; nucleotide sequences for such proteins; and recombinant DNA techniques and processes for forming such proteins and sequences.

According to yet a further aspect, the invention is for a polydeoxyribonucleotide segment comprising a first portion which codes for an enzyme selected for its ability to degrade a colonization matrix and a second portion which codes for a spacer molecule having a preferably terminal functionality which can be coupled to an anchor selected for its ability to attach to a substrate proximal to a bacterial colony. In one embodiment, the polydeoxyribonucleotide segment may also code for the anchor itself. In another embodiment, the anchor may be a non-protein and chemically or otherwise coupled to the spacer in a separate process.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic view of the enzyme-anchor complex of the invention, when attached to a tooth; Figure 2 is a schematic view of the enzyme-anchor complex of the invention, when attached to a pellicle or other surface in the oral cavity;

Figure 3 is a schematic view of a further embodiment of the enzyme-anchor complex of the invention, when attached in the oral cavity;

Figure 4 is a schematic view of the enzyme-anchor complex of the invention, when attached to a bacterial colony matrix in the oral cavity;

Figure 5 is a schematic view of the enzyme-anchor complex of the invention, when attached to a bacterium in the bacterial colony matrix in the oral cavity;

Figure 6 is a schematic view of an enzyme-anchor complex identifying the interactions between enzyme, anchor and surface;

Figure 7 is a schematic view of an enzyme-anchor complex identifying the interactions between enzyme, anchor, intra- anchor and surface;

Figure 8 is a schematic view of an enzyme-anchor complex identifying multiple enzymes;

Figure 9 is a schematic view of an enzyme-anchor complex incorporating a spacer molecule; and

Figure 10 is a schematic view of an enzyme-anchor complex which is a fusion protein.

DETAILED DESCRIPTION The present invention proposes to retain selected enzymes in the oral cavity. Unlike incorporating free and nascent enzymes in a dentifrice or oral rinse (where the effects are only transient), enzymes are allowed to have a prolonged opportunity to carry out their desired biochemical reactions and beneficial effects by modifying them so that they can be retained within the oral cavity. In addition, the specific enzymes are preferably selected to minimize toxic responses in the bacteria so as to maintain the normal bacterial balance and at the same time not adversely affect other necessary and protective biofilms, for example, the "acquired pellicle".

Certain polysaccharide degrading enzymes are modified so that they are able to adsorb to surfaces and structures in the oral cavity, and inhibit the proliferative bacterial colonization associated with the plaque matrix. The enzymes are derivatized or coupled to "anchor" molecules. The "anchor" portion of the enzyme-anchor complex can then adhere to structures in the oral cavity, inhibiting the buildup of plaque. Streptococcus mutans and plaque are recognized as being intimately involved in the formation of dental caries . This cariogenic bacterium utilizes sucrose to produce substrates for metabolism for the entire microbial population in the oral cavity. The end products of this sucrose-supported metabolism are organic acids which initiate the sequence of steps involved in the formation of dental caries. In addition, Streptococcus mutans also uses sucrose to enhance colonization of the oral flora by using the sucrose-supported substrate pool to produce polysaccharides that are complex and water insoluble. This scenario most likely takes place with many other bacteria that are colonized with the dental plaque.

The insoluble polysaccharide structures provide the backbone for extended bacterial colonization which, when aggregated, is the observable film recognized as plaque. While polysaccharides are not a requirement for initial attachment of the "pioneering" bacteria to the tooth's surface, the colonization and perpetuation of colonies requires these insoluble polysaccharides. It is likely that complex polysaccharides, by their insoluble nature, not only cause colonization and proliferation of the initial bacteria, but may also shield the bacteria from therapeutic agents. Consequently, this invention may be used in conjunction with agents that result in bacterial kill, either specific or non-specific. Restricting and controlling the amount of insoluble polysaccharides, and ultimately bacterial colonization into plaque, has a beneficial effect for the prevention and progression of periodontal disease. One of these complex, insoluble polysaccharides is glucan. The enzymatic degradation of glucan is therefore one of the objects of this invention. The invention provides for a composition and method to immobilize certain glucan degrading enzymes to surfaces and structures in the oral cavity. This inhibits the buildup of plaque which is a necessary precursor step to periodontal disease. Inhibiting proliferative bacterial colonization may well avoid any distortion of the microbial ecology or balance among the various bacterial strains. In general, avoiding bacterial population shifts is desirable because of the potential for over-growth of opportunistic bacteria, some of which may be pathogenic. The composition of the invention seeks to retain the normal relative ratios of the various bacterial strains in the oral cavity. However, the absolute numbers of at least certain strains of the bacteria will be reduced because the colonies thereof will be smaller.

The development of a mechanism to increase the enzyme ' s residence time in the oral cavity provides the opportunity for increased clinical efficacy. To achieve this goal, effective enzymes must remain in the oral cavity longer to accomplish their intended action. The increased retention time of the enzymes in the oral cavity will control plaque by limiting the polysaccharide backbone of the plaque matrix. The composition of the invention is thus designed to facilitate a longer residence time for the enzymes in the oral cavity. This approach involves derivatizing, or coupling, the appropriate enzyme(s) with an "anchor" molecule which will bind to structures in the oral cavity with the "anchor" portion of the derivatized enzyme-anchor complex. The anchor molecule will be specifically chosen to bind to, for example, existing plaque or the acquired pellicle that covers the tooth. Due to the relatively rapid turnover of epithelial tissue, the mucosal tissue layer within the oral cavity is a less preferred choice of a binding site than either the existing plaque or pellicle. Reference is made to Figures 1 and 2 of the drawings, which schematically illustrate the anchor-enzyme complex of the invention. The drawings are diagrammatic representations, are not intended to be to scale and are for illustrative purposes only. In Figure 1, there is shown a tooth 10 having a surface 12. On the surface 12, a colony 14 of bacteria within a matrix is attached to the tooth 10. Also attached on the surface 12 of the tooth is an anchor molecule 16, which may be an adhesion peptide. An immobilized enzyme 18 is attached to the anchor molecule 16, and the anchor molecule 16 and immobilized enzyme 18 together form the anchor-enzyme complex 20. The anchor-enzyme complex 20 competes with the colony 14 for attachment to the surface 12 of the tooth 10 and thus reduces the potential substrate sites for colony 14 attachment. Additionally, and most importantly, the enzyme 18 exercises its catalytic effect on the colony 14, degrading the plaque matrix and/or polysaccharide backbone. In Figure 1, the termination 22 of the matrix by the enzyme 18 can be seen. The colony 14 will thus be severely impaired in its ability to expand. Furthermore, the anchor- enzyme complex 20 has significant retention time on the tooth surface 12, thus providing more than a temporary obstacle to plaque matrix and colony 14 proliferation.

Another embodiment of the invention is shown in Figure 2. In this figure, elements corresponding to those in Figure 1 have been accorded the same reference numeral. In the embodiment shown in Figure 2, the tooth surface 12 has thereon a pellicle 24 to which the enzyme attaches. The pellicle, which includes peptides, proteins and the like, may provide or constitute the anchor, or a separate anchor molecule preattached to the enzyme may be used.

In Figure 4, a detail of a bacterial colony matrix 14 is shown, including individual bacteria 44. In this embodiment, the anchor molecule 16 of the complex 20 attaches to the bacterial matrix, and the termination 22 of the matrix can be clearly seen. In Figure 5, the anchor 16 of the complex 20 attaches directly on to a bacterium 44 within the matrix 14.

Figure 6 shows schematically an enzyme 50 which has an interaction 52 with an anchor 54, which in turn has an interaction 56 with substrate 58. The figure identifies the different types of covalent or noncovalent interactions which are possible. Figure 7 shows a complex which is similar to that shown in Figure 6, but the anchor comprises a pair of proteins 60 and 64 coupled by an intra-anchor interaction 64. Multiple anchors are therefore possible. Figure 8 shows the corresponding situation where multiple enzymes 66 and 68 are present, coupled by an intra-enzyme interaction 70.

Figure 9 adds the feature of the spacer molecule 72 between the enzyme 74 and anchor 76. The spacer may offer a number of advantages including the prevention or reduction of steric hindrance between enzyme and anchor, as well as the ability of the enzyme to have effect in an area spaced or remote from the substrate to which the complex is attached.

Figure 10 shows schematically a fused protein including an antibody to a surface in the oral cavity, 78, and an antibody to an enzyme, 80.

The invention provides compositions and methods to retain enzymes in the oral cavity so that they can restrict or limit plaque structures. It follows that an interaction between the enzyme(s), anchor(s) and the oral cavity itself must occur. The nature of these interactions, which constitutes an important aspect of the invention, centers around these elements and their working together and interacting with each other. Further, the invention is not limited to either the anchor or enzyme to when present as a single component. Consequently, the anchor can be an anchor complex and an enzyme can be an enzyme complex, each having more than one component.

In order to retain an enzyme in the oral cavity for an extended period, the interactions between the various elements (enzyme(s), anchor(s) and oral surface or substrate) must be strong. The nature of these interactions typically fall into one of two groups, namely interactions which are bonding or non- bonding in character. Bonding interactions are covalent interactions formed by a chemical bond. The following are examples of such bonding interactions: carbon - carbon; carbon - nitrogen; carbon - oxygen; carbon - sulfur; sulfur -sulfur; and the like. An example of compositions or materials which have bonding interactions is a fusion protein where individual proteins or parts of individual proteins are joined during the process of protein synthesis (translation).

Non bonding interactions are non covalent interactions . The following are examples of non-bonding interactions: hydrogen bonding; Van der Waals; ionic bonding; hydrophobic interactions; metal chelation; metal coordination and complexation, and the like. One example of a non-bonding interaction is antibody recognition of an antigen, which is a strong and non-covalent interaction. Another is glutathione-S-transferase (GST) binding to glutathione, which is also very strong but nevertheless, non- covalent. (See Figure 6 for a schematic representation of such interactions . )

The anchor's function is to retain the enzyme in the oral cavity. In performing this function, there are two primary interactions : the enzyme with the anchor and the anchor with surfaces in the oral cavity. It is important to note that the anchor's composition can be varied and not limited to a single anchor molecule. An anchor is a structure, structural complex or composite with components that can have bonded or non-bonded interactions within the anchoring structure. A specific protein that interacts with an enzyme can be fused to a different protein that interacts with the oral cavity. Figure 7 illustrates this arrangement which shows two proteins, with their own internal interaction to form an anchor complex.

It may be beneficial to anchor more than one enzyme in the oral cavity. Just as the anchor complex may have more than one component resulting in an intra-anchor interaction to form an anchor complex, so too enzymes can be configured to form an enzymatic complex with more than one enzyme. As above, the possible kinds interactions between an enzyme and another enzyme are the same as the anchor-to-anchor interactions. Figure 8 illustrates this arrangement.

It also may be desirable, under certain circumstances, to separate the amino acid sequence of one enzyme from another (if more than one enzyme is used) or to separate the amino acid sequence of the enzyme from the amino acid sequence of the anchor portion of the fused protein. Also, ways to separate or space these components from each other are provided. This can readily be accomplished by known processes in genetic engineering and recombinant DNA technology. The fused DNA sequence can produce a single protein that has the amino acid sequences of an enzyme and anchor, or multiple enzymes and/or multiple anchors separated by the amino acid sequence of the "spacer". Consequently, this procedure can produce a single protein with multiple enzymatic and anchoring capabilities . Figure 4 shows the enzyme and anchor separated by a spacer molecule 72.

Genetic Engineering Technology

Gene fusions, created using technologies from molecular biology and biotechnology, have been used to produce unique proteins from recombinant DNA segments. The DNA sequences that code for specific proteins or polypeptides can be obtained from cDNA libraries or prepared by methods described. Using recombinant DNA technology, implemented through standard protocols, or one of many other available literature sources, the DNA which codes for specific proteins are incorporated into plasmid vectors. These fusion proteins which are the DNA products of the fused DNA segments, are expressed in bacteria. Using the techniques described and referenced above, a DNA sequence which specifically codes for the synthesis of a protein enzyme (DNA-E) and a second DNA sequence which specifically codes for the synthesis of a polypeptide anchor (DNA-A) are joined together to form a single synthetic DNA segment (DNA-EA) . This fused DNA segment, which contains the capability to produce a single fusion protein, has the amino acid sequence of both enzyme and anchor in a single protein molecule. Consequently, the resulting fused protein (Protein-EA) coded by DNA-EA has the capability to perform an enzymatic function as well as an anchoring function. It is clear that this process can be repeated to produce a DNA segment that would code for more than one enzyme or more than one anchor. In addition, a DNA sequence coding for a spacer, as described and illustrated with reference to Figure 9, can also be inserted into the synthetic DNA, producing a DNA-ESA segment.

EXAMPLES Example 1. Glutathione/Glutathione-S-transferase

Interaction

The glutathione-S-transferase (GST) gene fusion system provides a complete system for expressing fusion proteins in E. coli. The system utilizes plasmid vectors designed for inducible, high- level expression of genes or gene fragments as fusion proteins which is described by Smith in U.S. Patent No. 5,654,176. Glutathione-S-transferase can be derived from any animal species. U.S. Patent No. 5,817,497 describes the polynucleotide sequence with codes for human glutathione-S-transferase. This system utilizes immobilized glutathione for affinity purification of expression products. Expressed fusion proteins are purified from bacterial lysates by affinity chromatography using glutathione sepharose 4B. The glutathione-S-transferase (GST) is used as an affinity handle to separate the desired gene product from the rest of the solution. The mild elution condition during purification maintains protein antigenicity and functional activity.

After the fusion protein is isolated the GST can be removed. Cleavage of the desired protein from the fusion product is achieved using a site-specific protease whose recognition sequences are located immediately upstream from the MCS (multiple cloning site). The GST System has been used successfully in many applications such as molecular immunology, the production of vaccines and studies involving protein-protein and DNA-protein interactions .

In the present invention, the GST/glutathione interaction is used to anchor an enzyme in the oral cavity. In one or more

iO steps, an enzyme-GST fusion protein is created. Additionally, glutathione is allowed to interact with a surface or substrate in the oral cavity. In one embodiment of the invention, glutathione may be used to attach to sugars in the oral cavity. The glutathione interaction with GST achieves the intent and objective of the invention by allowing increased retention times of an enzyme(s) in the oral cavity.

In this embodiment, the anchor is an anchor complex and is composed of glutathione and GST. The intra-anchor interaction is a non-bonded one while the glutathione portion of the anchor complex maintains a bonded interaction with the surface in the oral cavity. The interface of the GST portion of the anchor complex with the enzyme is also a bonded one and is created during the process of protein synthesis.

Example 2 ♦ Avidin and Streptavidin/Biotin Interaction Avidin is a glycoprotein, which has a molecular mass of approximately 68 kd and has four identical subunits, which bind one molecule of biotin to each subunit. Streptavidin, while similar to avidin in its basic structure and binding properties with biotin, does not contain any carbohydrate residues. The molecular mass of streptavidin is approximately 60 kd. The binding sites on avidin and streptavidin are associated with the tryptophan and lysine residues on the individual subunits that comprise the complete protein.

Biotin is a small (244d) vitamin which binds in a non- covalent interaction with these two proteins , resulting in the strongest protein-ligand interaction that is known to occur in biologic systems (K_a = lOiSM-¹). The combination of biotin ' s size and its strong interaction with these two proteins has proven useful in purification processes. Biotin is coupled to a target molecule, peptide or protein with a covalent bond. Subsequent to the biotinylation step, avidin or streptavidin is introduced which allows for the separation of the biotinylated material from the rest of the components .

In one aspect of the present invention, surfaces within the oral cavity are biotinylated by a process similar those describe by Rothenberg and Wilchek (1988), Ngo, et al. (1982) and Bodansky and Fagen (1977). Avidin and/or streptavidin is fused to the specific selected enzyme that is to be retained in the oral cavity by the genetic engineering and fusion protein procedures described previously. As a consequence, the desired enzyme is retained in the oral cavity by the interaction of the avidin or streptavidin with the biotin on surface of the oral cavity.

When implemented in this fashion, the anchor is comprised of the avidin or streptavidin protein and biotin, having a non bonded interaction between them, forming a two component anchor complex. At the biotin portion of the anchor complex, the interaction between the anchor complex and the surface within the oral cavity is a bonded interaction. So too, the interaction between the avidin or streptavidin portion of the anchor complex and the enzyme is a bonded interaction because the bonded interaction was created during the process of protein synthesis.

Example 3. Antigen and Antibody Interaction Antigen and antibody interactions have been well established. Many antibodies to specific antigens have been identified and characterized. While some antibodies are generic and interact with a wide variety of antigens, many are specific to discrete antigens. In the context of the present invention, specificity, or lack thereof, is of little consequence to the objective and intent of extended retention of specific enzymes in the oral cavity to achieve the prevention of periodontal disease.

In this additional aspect of the invention, antibodies to either unknown or known antigens such as plaque, plaque components and structures, bacterial cells, bacterial cell walls, bacterial cell fragments, ghost cells (either bacterial or host cells ) , are f sed to enzymes to create an enzyme anchor complex wherein the anchor is the antibody and the specific surface in the oral cavity is the antigen to the selected antibody. These techniques for separation and analysis have been described previously both herein and in publications (U. S. Patent 5,100,788; Belts and Burd, 1989; Harlow and Lane, 1988). In this example, the antibody is the anchor. The interaction between the anchor and the enzyme is a bonded one while the interaction between the anchor and the surface in the oral cavity is non bonded, specifically the well-established antigen-antibody interaction.

Example 4. Use of Recombinant Peptide as an Anchor Molecule.

The gene segment encoding all or part of the nucleotide sequence described, for example, by Kelly et al is coupled to the nucleotide sequence which codes for a specific enzyme to produce an expressed fused protein that serves as the enzyme- anchor complex. The anchor peptide portion of the anchor-enzyme complex can be comprised of either the complete versions of one or two peptides (or selected fragments thereof) or both peptides or fragments from both. The peptides, pl025 and pi125, have the following amino acid sequence respectively: pl025: QLKTADLPAGRDETTSFLV P1125: AYGIKSNWRVTTPGKPNDP

Example 5. Use of Enzyme "Domains". Plaque serves two functions : ( 1 ) as the structural framework for organized bacterial proliferation and colony growth; and (2) as a food source for the bacteria during times when sugars are absent from the oral cavity. It is known that while glucose can be transported across the bacterial cell wall, with the input of energy, sucrose being a disaccharide cannot. It follows that if bacteria are to regulate plaque polysaccharides, they must do so by synthesizing the appropriate enzymes that dictate the balance, and then express or secrete these. The two enzymes that regulate the function of plaque are bacterial dextranase (for use as a food source) and glucosyl transferase (for using excess glucose to build more plaque structure). Fructonase (levanase) and fructosyl transferase operate in a similar mode with respect to fructose.

These enzymes which are expressed by the bacteria are retained in the oral cavity. The enzymes have, in their amino acid sequences, and when properly "folded" or oriented, areas or "domains" that bind to plaque. Such areas or domains may, within the context of the present invention, be fused or otherwise coupled to selected enzymes, operating as an anchor within the anchor enzyme complex.

It is within the scope of this invention to expand the enzyme-anchor complex to incorporate polysaccharide-degrading enzymes other than those which hydrolyze or degrade glucans e.g., enzymes that degrade fructose-based polysaccharide enzymes that hydrolyze glycoproteins etc. The complex could also extend to cover ligand-based "anchor" molecules that mimic exterior cell surfaces of bacteria so as to create direct competitive binding between bacteria and "anchor" enzyme complexes. Further, the complex may include receptor-based "anchor" molecules that mimic the bacterial attachment sites so that "anchored" enzymes can be adsorbed onto bacterial surfaces that are already adhering to plaque. Finally, anchor molecules comprised of polypeptides that are known adhesion molecules may be used.

Purification of potentially suitable hydrolytic enzymes (polysaccharide hydrolases, glycoprotein degrading enzymes, etc.) may be carried out to achieve higher specific activity and a more focused specific type of reaction.

Thereafter, procedures for determining the extent or degree of coupling between the enzyme and "anchor" molecules may also be carried out, thus establishing the number of "anchor" molecules attached to the enzyme that will provide the best combination of enzymatic activity and degree of binding.

It will be appreciated that any effective enzyme which prevents or reduces bacterial colonization may be used in this invention. Preferably, a group of enzymes which have a hydrolytic action, or hydrolases, are used since they are particularly effective. This group facilitates the hydrolysis of chemical bonds that link moieties, which after the hydrolysis reaction occurs, can exist as separate chemical entities. Preferred enzymes which may be used in this invention may be selected from one or more of the following: esterases - those enzymes that cleave ester bonds; glycolytic cleavage enzymes - those enzymes that cleave bonds that are found in oligo- and polysaccharides; ether bond cleavage enzymes; peptide bond cleaving enzymes where proteins are the substrate (reactant); carbon-nitrogen bond cleavage where the substrate (reactant) is not a protein; acid anhydride cleaving enzymes; carbon-carbon bond cleavage; halide bond cleavage; phosphorus-nitrogen bond cleavage; sulfur-nitrogen bond cleavage; and carbon-phosphorus bond cleavage.

Anchor molecules and structures for anchoring the enzymes in the oral cavity may be selected from a number of different categories, as set out below: A. proteins, protein fragments and polypeptides a. naturally-occurring b. naturally-occurring, but modified c. synthetic polypeptides i. using naturally occurring amino acids ii. using synthetic, non-naturally occurring amino acids e.g. D-amino acids, β-substituted amino acids, alpha, alpha-disubstituted etc . d. charge prevalence i. cationic (basic amino acids) ii. anionic (acidic amino acids) iii. neutral (aliphatic amino acids) e. any combination of the above B. saccharides and oligosaccharides a. naturally occurring e.g. glucose, mannose, galactose, rhamnose, fucose, fructose, sucrose etc. b. naturally occurring amino sugars e.g. glucosamine, galactosamine, N-actylglucosamine, N-acetylgalactosamine, neuramenic acid, sialic acid, etc. c. synthetic or non-naturally occurring saccharides and amino sugars i. esters of sugars e.g. sugar-organic acid esters etc ii. chemically combined sugars and proteins/polypeptides e.g. synthetic glycoproteins C. Glycoproteins/proteoglycans a. naturally occurring e.g. elastin, lectins etc. b. synthetic e.g. modified naturally occurring glycoproteins/proteoglycans

D. Glycolipids a. naturally occurring e.g. sphingomyelin, cerebroside, gangliosides etc b. synthetic e.g. modified natural glycol; lipids through some chemical procedure such as esterification, amidation or similar chemical process E. Lipoprotein e.g. chylomicron, Very Low Density Lipoproteins (VLDL), Low Density Lipoproteins (LDL) , High Density Lipoproteins (HDL), etc.

F. Lipids a. non-polar, natural or synthetic e.g. triglycerides , cholesterol or other plant or animal sterols , etc b. polar, natural or synthetic e.g. phospholipids (phosphatidyl serine), etc

G. Cell fragments and cell ghosts - segments or portions of exterior bacterial or animal cell walls or membranes that would mimic live and viable bacterial or animal cells for the purpose of securing an enzyme to the surface within the oral cavity. H. Non-biologic, polymeric materials a. homopolymers e.g. polyethylene glycol (PEG), etc b. copolymers e.g. styrene-butadiene polymers etc.

The connections between the anchor molecules and the enzymes may also take a number of forms . These connections may thus be chemical, chemisorption, or covalent bonds, including: amide (peptide); ester; glycosidic (sugar linkages); and/or ether. The connections may also be physical, physisorption such as: van der Waals attractive forces, including lipophilicity; charge-charge attractions/interactions, including electrostatic interactions; and/or hydrogen bonding, including hydrophilicity The connections between the anchor of the anchor-enzyme complex and the surface substrate within the oral cavity would typically be the same as those listed in the preceding paragraph. With reference to Figure 3 of the drawings, there is shown in schematic form of a substrate 30 which is a surface in the oral cavity such as a tooth, existing plaque, an appliance or mucosal tissue, and an anchor-enzyme complex 32 attached thereto. The anchor-enzyme complex 32 comprises an anchor portion 34 and an enzyme portion 36. There is an anchor-surface interface 38 between the complex 32 and the substrate 30 and an anchor-enzyme connection 40. It is believed that there will be a greater tendency for the connection between the enzyme portion 36 and the anchor portion 34 to be of the chemical type, while the interaction between the anchor portion 34 of the anchor- enzyme complex 32 and the substrate 30 is more likely to be of the physical type.

There will be a greater tendency for the connection between the enzyme and anchor to be of the chemical type. The interaction of the anchor portion of the anchor-enzyme complex will more likely be of the physical type.

Example 6.

An embodiment of the invention involves selection of two enzymes known to have activity on degrading the polysaccharide backbone of the dental plaque matrix. Two such enzymes are:

1) α-Glucosidase EC 3.2.1.20 ; [(1→3) 3-glucanohydrolase] . α-Glucosidase is commercially available. While the enzyme shows greatest activity toward -1,4 glucose linkage, it will also hydrolyze -1,2 and α-1,3 linkages. The enzyme will also hydrolyze α-1,6 linkages, but only at a very slow rate.

2) Dextranase EC 3.2.1.11 ; [ (1→6) 6-glucanohydrolase] . Dextranase is also commercially available. This enzyme cleaves glucose molecules from polysaccharides that are linked α-1,6.

Many researchers describe the glucan structure as α-l→3 and α-l-→ 6. Glucan has also been described as having -l→4 and -l→2 linkages. From a structural perspective, α-l→6 linkages give the glucan its length and the α-l→3, α-l-→4, and α-l→2 linkages gives the glucan its branching characteristics. It is not known whether glucan length or glucan branching is important for bacterial colonization. For this reason, the two commercially available enzymes were selected: α-Glucosidase, providing cleavage activity for α-l→4, α-l-→2 and α-l-→3 i.e., cleaving at branching points in the glucan structure; and Dextranase, which will provide cleavage of α-l→6 linkage i.e., cleavage at lengthening linkages . These enzymes will be separately coupled with each of the following "anchor" molecules:

1) a basic polypeptide e.g., Lys-Lys-Glu-Lys-Lys or some similar basic polypeptide;

2) an acidic polypetide e.g., Glu-Glu-Lys-Glu-Glu or some similar acidic polypeptide.

Teichoic acids and lipoteichoic are important bacterial cell wall components for binding. These components are also associated with phosphate esters which would present an anionic character to the exterior portion of the bacterial cell surface. For this reason, the "anchor" molecule, Lys-Lys-Glu-Lys-Lys, which is a cationic species, would be attracted to the bacterial cell wall.

Since available evidence suggests that the bacterial cell surface is anionic in character, it is reasonable to suspect the colonization of bacteria on to portions of plaque that are principally cationic in character. Indeed, if there are regions or areas of cationic character associated with plaque, the "anchor" molecule, Glu-Glu-Lys-Glu-Glu, which is an anionic species that would be attracted to the cationic regions of plaque, would be a good choice.

Additionally, or alternately, any other densely arranged lipid character such as micelles may serve as either a substrate in the oral cavity or the anchor molecule to which the enzyme- anchor complex attaches. The rationale of charge attractions, as the basis for anchoring selected enzymes to various organic structures in the oral cavity, may be one factor for bacterial attachment. However, bacterial adhesion in the colonization of plaque also may involve factors other than charge attraction alone. Thus, specific proteins may be responsible for the binding of oral bacteria to polysaccharide (glucan) and plaque. However, the actual mechanism for bacterial binding in plaque does not preclude other binding mechanisms for enzymes that are connected to specific "anchor" molecules, and would be encompassed by this invention.

The enzymes and anchors set out in this example will produce six derivatized enzymes with the potential for a broad charge-binding capability.

Synthesis

The synthesis part of the derivatized enzyme-anchor complexes involves coupling of each "anchor" molecule to the two individual enzymes. The basic polypeptide Lys-Lys-Glu-Lys-Lys is coupled to the two enzymes through the free carboxyl group of the Glu residue and there is some coupling through the "C" terminus of the polypeptide. The acidic polypeptide Glu-Glu-Lys-Glu-Glu is coupled through the free amino group of the Lys residue and there is some coupling through the "N" terminus of the polypeptide to the two enzymes . Purification of the six derivatized enzyme reaction products may be carried out by molecular size exclusion on column chromatography. The purified coupled enzymes may be assayed and compared to the underivatized enzymes to determine any changes in enzymatic activity as a consequence of the coupling procedure.

The six anchor-enzyme complexes produced in this example, or complexes of other enzymes and anchors , may further be tested in the in vitro system prior to clinical application. Any suitable procedure for testing may be used. The basic and acidic polypeptides, which are commercially available, for example from Peptides International, Louisville, Kentucky, are synthesized, for example, by a variation of the solid-phase method. These starting materials may be used without purification; however, a retained portion of each starting material should preferably be assayed for purity, as necessary e.g., to describe unexpected reaction products, etc. The enzymes, which are also commercially available and may be purchased from United States Biochemical, Cleveland, OH and Worthington Biochemical, Freehold, NJ, may also be used without purification. Other enzymes which can be used and which may not be commercially available can be isolated and purified from tissues and organisms, using standard procedures. A retained portion of each enzyme, too, should be analyzed, only if necessary to determine purity. Such purification analyses may be important depending upon the results of the in vitro experimentation. These analyses may be conducted using the retained portions of the enzymes .

The enzymatic activity should preferably be determined both before and after the derivatization (coupling) reaction and this can readily be accomplished using, for example, 4-nitrophenyl-α-D-glucose in a standard assay procedure. The basic polypeptide, Lys-Lys-Glu-Lys-Lys may be coupled to each of the enzymes. This procedure uses l-ethyl-3-[-3-dimethylaminopropyl]-carbodiimide (EDC) as the coupling agent. The EDC-activated carboxyl group of Glu in the polypeptide (as well as the carboxyl group from the "C" terminus end of the polypeptide) will be coupled to free amine groups on the enzymes, forming covalent amide bonds.

The acidic polypeptide, Glu-Glu-Lys-Glu-Glu, may be coupled to each of the enzymes. In this procedure, the free amine group of Lys (as well as the free amine group from the "N" terminus of the polypeptide) is converted to an aldehyde and then coupled to the free amine groups on the enzymes.

In both of the coupling or derivativization reactions involving the polypeptide "anchor" molecules, there will be a wide variety of by-products produced; however, there will also be a wide diversity among the sizes of the molecules (molecular weights) which will allow a clean-up procedure using, for example, HPLC with a 3000 PW column for a separation based on molecular size.

The purpose of this separation step is a "clean-up" of the reaction. The clean-up removes unreacted polypeptide "anchor" molecules, polypeptide mixtures resulting from the

"anchor" molecules that reacted with each other, and the desired product of enzyme-"anchor" complexes. There may also be a number of desired enzyme-"anchor" complexes, depending upon the number of "anchor" molecules attached to the enzyme. It is not considered necessary to separate enzyme-"anchor" complexes into discrete fractions depending upon the number of "anchor" molecules; rather, all types of enzyme-"anchor" complexes may be tested and clinically applied collectively. Separating the types of enzyme-"anchor" complexes into discrete molecular entities may, however, be carried out where it is considered appropriate. Where desired or considered necessary, the clean up procedure may be validated by defining and setting the column (HPLC) operating conditions. Sample runs may be made with: 1) the enzyme alone; 2) the anchor molecule alone; and 3) the reaction mixture without the addition of enzyme. Retention time/fraction number for total protein will be determined under the defined operating conditions that will allow separation of free "anchor" molecules, reaction products among "anchor" molecules , free enzyme and derivatized or coupled enzymes .

In Vitro Assay

Prior to clinical application, the effectiveness of any synthesised enzyme-anchor complexes may be determined in an in vitro assay. One such assay is described below. Subjects are screened for salivary output and a high level of Streptococcus mutans and Actlnomyces viscosus (plate counts) which are recognized as high plaque-forming bacteria. Salivary output from the selected population may be stimulated by chewing an inert material such as parafilm or carbowax. The collected saliva will serve as the stock inoculum solution. This stock solution will be prepared by combining the saliva samples with the greatest population of the identified stains (20 - 25% of the total samples taken).

Thereafter, the following solutions are prepared: a) Enriched Sucrose Broth. b) Positive control solution of 20mg/ml of chlorohexidine, a known inhibitor of plaque formation. c) The two test-related controls may be the underivatized enzyme i.e., enzymes without "anchor" molecules. d) The 8 treatment solutions (6 test solution and 2 test-related controls) may be prepared with Enriched Sucrose Broth as the solvent, giving stock solutions with concentrations of 10, 1.0, and O.lmg/ml.

Procedure

Sterile glass slides are placed in 50 ml test tubes containing 39 ml of Enriched Sucrose Broth. The tubes are inoculated with 1 ml of stock inoculum (saliva) solution. The tubes are incubated at 37°C under 5% C0₂ for 24 to 48 hours, until visual evidence of plaque formation appears . The slides are removed, transferred to dosing solutions of fresh Enriched Sucrose Broth (39 ml in 50 ml test tubes) to which 1 ml of the appropriate test solution is added. The dosing solutions may have the following composition:

1) No treatment control - Enriched Sucrose broth

2) Positive control - 20 mg/ml chlorohexidine 3) Control related to treatments 1A, IB and 1C - 1.0 mg/ml un-"anchored" α-Glucosidase

4) Control related to treatments 2A, 2B and 2C - 1.0 mg/ml un-"anchored" Dextranase

5) Test treatments 2A, 2B and 2C (3 Dextranase-"anchor" ) : 10, 1.0 and 0.1 mg/ml.

6) Test treatments 2A, 2B and 2C (3 Dextranase-"anchor" ) : 10, 1.0 and 0.1 mg/ml.

The glass slides remain in their respective dosing solutions for approximately one hour. They are then removed and rinsed by dipping in a clean Enriched Sucrose Broth.

The slides may then be placed in fresh Enriched Sucrose Broth and the tubes incubated in the same manner for 24 to 48 hours. The amount of plaque is recorded (photographed) for each treatment and the plaque from each slide is harvested, dried and weighed. The enzymatic activity of both enzymes before and after the derivatization is determined, as well as the efficiency of the reaction clean-up. Visual observation is made of each test; photographs are taken of each treatment (combined triplicate test of each treatment as a single photograph) , and the amount (weight) of plaque formed in each test is determined.

In the selection of enzymes, anchors and the coupling methods and procedures, a number of factors should be taken into account to provide the most effective enzyme-anchor complexes . Some of these are as follows : The enzymes and anchor molecules selected should always be the most appropriate for limiting a bacterial colonization matrix. More than one enzyme may be necessary to cause a critical limitation of the polysaccharide backbone for plaque formation.

The potential advantages of this invention are threefold: 1) it does not require bactericidal activity, 2) normal microbial balance in the oral cavity will be maintained, and 3) the likelihood of adverse effects in the host at sites removed from the oral cavity are minimized or eliminated.

Claims

CLAIMS :

1. A process for producing an anchor-enzyme complex for controlling bacterial growth/colonization, the process comprising: formulating a polydeoxyribonucleotide segment DNA-E which codes for a selected enzyme, formulating a polydeoxyribonucleotide segment DNA-A which codes for a selected anchor molecule, the anchor being selected for its ability to attach to a substrate proximal to a bacterial colony; coupling directly or indirectly the polydeoxyribonucleotide segment DNA-E to the polydeoxyribonucleotide segment DNA-A to form a fused polydeoxyribonucleotide segment DNA-EA, incorporating said fused DNA-EA segment into a plasmid vector, isolating the anchor-enzyme complex expressed by the fused DNA-EA segment, wherein the attachment of the anchor to the substrate permits prolonged retention time of the enzyme-anchor complex where the bacterial colony is present.

2. A polydeoxyribonucleotide segment comprising a first portion which codes for at least one enzyme selected for its ability to degrade a colonization matrix and a second portion which codes for at least one anchor selected for its ability to attach to a substrate proximal to a bacterial colony.

3. A polydeoxyribonucleotide segment as claimed in claim 2 which codes for multiple enzymes .

4. A polydeoxyribonucleotide segment as claimed in claim 2 which codes for multiple anchors.

5. A recombinant plasmid adapted for transformation of a microbial host, said plasmid comprising a plasmid vector into which has been inserted a polydeoxyribonucleotide segment comprising a first portion which codes for at least one enzyme selected for its ability to degrade a colonization matrix and a second portion which codes for at least one anchor selected for its ability to attach to a substrate proximal to a bacterial colony.

6. A transformed microorganism which includes a recombinant plasmid, said plasmid comprising a plasmid vector into which has been inserted a polydeoxyribonucleotide segment comprising a first portion which codes for an enzyme selected for its ability to degrade a colonization matrix and a second portion which codes for an anchor selected for its ability to attach to a substrate proximal to a bacterial colony.

7. A process for producing an anchor-enzyme complex for controlling bacterial growth/colonization, the process comprising: formulating a polydeoxyribonucleotide segment DNA-E which codes for a selected enzyme, formulating a polydeoxyribonucleotide segment DNA-S which codes for a spacer molecule, the spacer molecule having a terminus for coupling to an anchor; coupling the polydeoxyribonucleotide segment DNA-E to the polydeoxyribonucleotide segment DNA-S to form a fused polydeoxyribonucleotide segment DNA-ES; incorporating said fused DNA-ES segment into a plasmid vector, isolating the enzyme-spacer molecule expressed by the fused DNA-ES segment; and coupling the enzyme-spacer molecule to an anchor at the terminus of the spacer molecule, the anchor being selected for its ability to attach to a substrate proximal to a bacterial colony; wherein the attachment of the anchor to the substrate permits prolonged retention time of the enzyme-anchor complex where the bacterial colony is present.

8. The method as claimed in claim 7 wherein the anchor is comprised of non-protein material.

9. The method as claimed in claim 7 further comprising formulating a polydeoxyribonucleotide segment DNA-A which codes for a selected anchor molecule, the anchor being selected for its ability to attach to a substrate proximal to a bacterial colony, and coupling the polydeoxyribonucleotide segment DNA-A to the polydeoxyribonucleotide segment DNA-ES, whereby an enzyme-spacer-anchor complex is expressed by the plasmid vector.

10. A polydeoxyribonucleotide segment comprising a first portion which codes for an enzyme selected for its ability to degrade a colonization matrix and a second portion which codes for a spacer molecule having a terminal functionality which can be coupled to an anchor selected for its ability to attach to a substrate proximal to a bacterial colony.

11. A fusion protein comprising an enzyme selected for its ability to degrade a colonization matrix and a spacer molecule coupled directly or indirectly to the enzyme, the spacer having a terminal functionality which can be coupled to an anchor selected for its ability to attach to a substrate proximal to a bacterial colony.

12. A fusion protein as claimed in claim 11 wherein the anchor is a non-protein material.

13. A fusion protein comprising an enzyme portion selected for its ability to degrade a colonization matrix and an anchor portion coupled directly or indirectly to the enzyme, the anchor being selected for its ability to attach to a substrate proximal to a bacterial colony.

14. A fusion protein as claimed in claim 13 further comprising a spacer portion located between the enzyme and anchor.

15. A fusion protein as claimed in claim 13 wherein the anchor portion comprises glutathione-S-transferase.

16. A fusion protein as claimed in claim 13 wherein the anchor portion comprises glutathione-S-transferase and/or glutathione.

17. A fusion protein as claimed in claim 13 wherein the anchor portion is selected from the group consisting of: avidin, streptavidin, avidin and biotin, streptavidin and biotin, and any combination of the aforegoing.

18. A fusion protein as claimed in claim 13 wherein the anchor portion comprises an antibody.

19. A fusion protein as claimed in claim 18 wherein the antibody is an antibody to an antigen selected from the group consisting of plaque, plaque components and structures, bacterial cells, bacterial cell walls, bacterial cell fragments, and ghost cells (either bacterial or host cells).

20. A fusion protein as claimed in claim 13 wherein the anchor comprises at least a domain or area of an enzyme produced by oral bacteria, the domain having characteristics which enable it to bind to structures in the oral cavity.

21. A fusion protein as claimed in claim 13 wherein the enzyme selected is a hydrolase having hydrolytic activity.

22. A fusion protein as claimed in claim 13 wherein the enzyme is selected from the group consisting of: esterases, for cleaving ester bonds; glycolytic cleavage enzymes, for cleaving bonds that are found in oligo- and polysaccharides; ether bond cleavage enzymes; peptide bond cleavage enzymes where proteins are the substrate (reactant); carbon-nitrogen bond cleavage enzymes where the substrate (reactant) is not a protein; acid anhydride cleavage enzymes; carbon-carbon bond cleavage enzymes; halide bond cleavage enzymes; phosphorus-nitrogen bond cleavage enzymes; sulfur-nitrogen bond cleavage enzymes; and carbon- phosphorus bond cleavage enzymes.

23. A fusion protein as claimed in claim 13 where the enzyme is α-Glucosidase.

24. A fusion protein as claimed in claim 13 where the enzyme is Dextranase.

25. A fusion protein as claimed in claim 13 wherein the anchor molecule is a basic polypeptide.

26. A fusion protein as claimed in claim 25 where in the basic polypeptide is Lys-Lys-Glu-Lys-Lys.

27. A fusion protein as claimed in claim 13 wherein the anchor molecule is an acidic polypeptide.

28. A fusion protein as claimed in claim 27 wherein the acidic polypeptide is Glu-Glu-Lys-Glu-Glu.

29. A fusion protein as claimed in claim 13 wherein the anchor molecule is selected from the group consisting of proteins, protein fragments and polypeptides.

30. A fusion protein as claimed in claim 12 wherein the anchor molecules are saccharides and/or oligosaccharides selected from the group consisting of: glucose, mannose, galactose, rhamnose, fucose, fructose, sucrose, glucosamine, galactosamine, N-actylglucosamine, N-acetylgalactosamine, neuramenic acid, sialic acid, amino sugars, esters of sugars, sugar-organic acid esters, chemically combined sugars and proteins/polypeptides and synthetic glycoproteins

31. A fusion protein as claimed in claim 13 wherein the anchor molecules are glycoproteins/proteoglycans, selected from the group consisting of: elastins, lectins, glycoproteins/proteoglycans .

32. A fusion protein as claimed in claim 12 wherein the anchor molecules are glycolipids selected from the group consisting of: sphingomyelin, cerebroside, gangliosides, glycol, and lipids .

33. A fusion protein as claimed in claim 13 wherein the anchor molecules are lipoproteins selected from the group consisting of chylomicron, Very Low Density Lipoproteins (VLDL), Low Density Lipoproteins (LDL), and High Density Lipoproteins (HDL).

34. A fusion protein as claimed in claim 12 wherein the anchor molecules are lipids selected from the group consisting of: triglycerides , cholesterol or other plant or animal sterols, and phospholipids (phosphatidyl serine) .

35. A fusion protein as claimed in claim 12 wherein the anchor molecules are cell fragments, cell ghosts or segments or portions of exterior bacterial or animal cell walls or membranes that mimic live and viable bacterial or animal cells for the purpose of securing an enzyme to the surface within the oral cavity.

36. A fusion protein as claimed in claim 12 wherein the anchor molecules are non-biologic, polymeric materials selected from the group consisting styrene-butadiene polymers.

37. A process of producing and selectively isolating a protein or polypeptide as a fusion product, the fusion product controlling bacterial growth/colonization and being retained in the oral cavity, the process comprising: constructing a DNA vector comprising a DNA sequence coding for an enzyme selected for its ability to degrade a colonization matrix with a DNA sequence coding for an anchor selected for its ability to attach to a substrate proximal to a bacterial colony; transforming a compatible host with the DNA vector; culturing the transformed host in a suitable growth medium to produce the fusion product; and selectively isolating the fusion product.

38. A process as claimed in claim 37 wherein the fusion product is purified from bacterial lysates by affinity chromatography using glutathione sepharose 4B.

39. A process as claimed in claim 37 wherein the fusion product includes glutathione-S-transferase (GST), the GST forming an affinity handle to facilitate separation of the fusion product from the remainder of the solution.

40. An isolated and purified fusion product prepared according to the method of claim 37.

41. A fusion protein as claimed in claim 13 wherein the anchor is selected from a ligand and a receptor.