JPH1077218A - Product for oral cavity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a product effective for prevention or reduction of the formation of dental plaque and/or dental caries and prevention or reduction of foul breath. SOLUTION: This product contains at least one kind of excipient. In the excipients, polypeptides which possess a specific bond affinity for a target part in plexues of microorganisms existing on the surface of teeth and enzymes which execute a function increasing peripheral pH are contained. The enzymes exist adhesively on the polypeptides or possess a means which combines the enzyme with the polypeptides at least in using. When using, the enzymes such as urease and the like are carried by polypeptides such as an antibody and a fragment of antibody and combined with dental plaque or its peripheral target part. The enzymes achieve a function increasing the pH nearby the part and consequently the formation of dental plague and/or dental caries is prevented or reduced. In addition, the increase of the pH promotes decomposition of H2 S and consequently prevents or reduces foul breath.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to oral medicine, and more particularly, to prevention of dental plaque and caries, and prevention of bad breath.

[0002]

2. Description of the Related Art The oral microflora is a complex ecosystem containing a variety of bacteria (species). Oral bacteria such as Streptococcus mutans (Streptococcus mutans)
At least one of the ococus mutans bacteria has the ability to utilize dietary carbohydrates for the synthesis of an insoluble polysaccharide matrix that promotes attachment to hard surfaces and colonization of hard surfaces.

[0003] Dental plaque is composed of this matrix polysaccharide and oral microorganisms present in and on the matrix polysaccharide.

[0004] It has been confirmed that Streptococcus mutans, a bacterium, contributes greatly, if not exclusively, to dental plaque.
It has been shown that caries can be induced in sterile animals.

[0005] Other bacteria found in substantial proportions in the bacteria normally present in dental plaque include Streptococus sanguis and Nestlund actinomycetes (Ac).
tinomyces naeslundii).

[0006] Dental plaque is an acidic microenvironment. Its acidity is produced by oral bacteria and their extracellular enzymes, which convert dietary carbohydrates into a polysaccharide matrix. Acidity has damaged teeth with oral bacteria and plaque. However, the bacteria themselves are resistant to acidity. Extracellular enzymes are more active under acidic conditions than in neutral or alkaline environments.

[0007] There have been many proposals for delivering biologically active agents such as enzymes that exhibit cytotoxicity at the target site by using antibodies having specific affinity for the target site.

The use of antibodies to attach cytotoxic agents to target sites in the mouth has already been proposed. For example, WO 89/11866 [Rama Biolink] discloses a conjugate of a drug having a bactericidal action with an anti-Streptococcus mutans antibody.

It has been proposed to deliver enzymes as cytotoxic agents. For example, Okuda et al., In Infection and Immunity, 27, 690 (1980), described two enzymes chemically linked to antibodies to target cells, namely, xanthine oxidase and lactoperoxidase; It describes the use of 90
Over a minute they have achieved in vitro a reduction of living Candida albicans cells by one to two orders of magnitude better than that achieved with lactoperoxidase in solution.

Other disclosures relating to the use of antibodies or antibody fragments to target cytotoxic agents include WO 90/03185 [Ideon] and 91/91.
00112 [Brunswick].

EP-A-450,800, EP-A-451,972, EP-A-453,097 and EP-A-47.
No. 9,600 [All Unilever] is an antibody and an antibody fragment, which act in cooperation with such an antibody and the like, and an antibody for attaching a cytotoxic enzyme to a target site, particularly an oral bacterium. Disclose the use of antibody fragments.

[0012]

SUMMARY OF THE INVENTION An object of the present invention is to provide a product used in the oral cavity which is effective for preventing or reducing the formation of dental plaque and caries, and for preventing or reducing bad breath.
The product is a composition comprising an excipient, a particular polypeptide and a particular enzyme.

[0013] Another object of the present invention is to provide a method for preventing or reducing the formation of dental plaque and caries and a method for preventing or reducing bad breath by using a specific product locally in the oral cavity. And

Furthermore, the present invention relates to the use of specific polypeptides and specific enzymes as drugs (agents) for preventing or reducing the formation of dental plaque and caries, and for preventing or reducing bad breath. The purpose is to provide.

[0015]

SUMMARY OF THE INVENTION According to the present invention, there is provided a product for topical use in the oral cavity, the product comprising at least one excipient, wherein the excipient (s) (I) a polypeptide having a specific binding affinity for a target site in a microflora present on the tooth surface, which is distributed in the same excipient or in a plurality of excipients; )
An enzyme that functions to increase the pH of the surroundings is included, and the enzyme is attached to the polypeptide, or the product binds the enzyme to the polypeptide at least during its use. A product having means is provided.

With the product of the invention in the mouth,
The enzyme is carried by the polypeptide, thereby binding to the target site at or near the dental plaque. The enzyme, in its immediate vicinity, serves to raise the pH. Since it is the acidity that leads to the erosion of the tooth surface and consequently the formation of tooth decay, reducing the acidity by the action of enzymes is directly beneficial for teeth with plaque.

It is also beneficial, but less direct, that reducing acidity reduces the activity of extracellular enzymes that cause acidity.

Furthermore, the ability to create a less acidic microenvironment can reduce the relative advantage of acid-resistant oral bacteria and reduce bacterial colonization at the tooth surface by bacteria that are less harmful to the tooth. Colonies).

In another aspect, the present invention provides a method for preparing at least one excipient comprising a polypeptide as described above and an enzyme.
A use for the prevention or reduction of the formation of dental plaque / caries by topical use in the mouth is provided.

The present invention also provides (i) a polypeptide having a specific binding affinity for a target site in a microflora present on a tooth surface; and (ii) an alkaline product.
Preventing the formation of dental plaque / caries, including topically using at least one excipient that can be used in the mouth, including an enzyme that serves to raise the pH thereby. Or the method of reducing, wherein the polypeptide and the enzyme are attached together and the excipient (s) attach the enzyme to the polypeptide at least during its use. Also provided is a method that includes the means.

An important factor in halitosis or breath is H ₂ S, which is produced by organisms, including streptococci, such as Sanguis streptococci. The volatility of H ₂ S depends on the pH. H ₂ S is, pK _a is about 7, and hydrogen ions, is highly reactive with soluble HS ^- dissociates into the ions.
At pH 8.0, more than 90% of the H ₂ S dissociates. Therefore, the effect of increasing the pH by the use of the enzyme in the product of the present invention also promotes the dissociation of H ₂ S in the oral cavity, thereby preventing or reducing bad breath.

Accordingly, the present invention provides (i) a polypeptide having a specific binding affinity for a target site in a microflora present on a tooth surface, and (ii) an alkaline product. A method for preventing or reducing bad breath, comprising topically using at least one excipient that can be used in the mouth, including an enzyme that functions to increase the pH of the mouth. The polypeptide and the enzyme are attached together, or the excipient (s) comprises means for attaching the enzyme to the polypeptide at least during its use. Also provide.

In another embodiment, the present invention provides a method for producing an alkaline product comprising the steps of: (i) generating an alkaline product, thereby increasing pH; and (ii) a target site in a microflora present on the tooth surface. Includes the use of polypeptides that have a specific binding affinity for and that also serve to attach the enzyme at least during its use as a drug to prevent or reduce bad breath.

Enzymes generally perform the function of producing alkaline products and / or of converting alkaline substrates into more alkaline products.
Thereby, it is expected that the pH at the very close place is increased. Alternatively, the enzyme is a precursor,
The material can then be converted in situ into the alkaline product which undergoes the next transformation. Such further conversion may be performed spontaneously by bacteria naturally present in the dental plaque. Increasing the concentration of the precursor will increase the concentration of alkaline products that naturally occur from the precursor.

[0025]

DETAILED DESCRIPTION OF THE INVENTION A polypeptide having a specific binding affinity for a target site may be an intact protein or, rather, a smaller polypeptide. It will usually be an antibody to the target site or an antibody fragment which has the specific binding characteristics of an antibody.

The binding of the polypeptide to the target site
It involves a ligand-receptor type interaction between the polypeptide and the substance bound to the polypeptide. An equilibrium exists between bound and unbound material, which can be represented by the following formula:

[0027]

Embedded image Here, A represents an unbound polypeptide, S represents an unbound target site, and AS represents a complex of the bound polypeptide and target.

The concentration is related by the following equation:

Embedded image Here, K _a is referred constant and affinity constant.

The specific binding affinity between the polypeptide and target, often have ¹⁰⁶ liters / mole or K _a values greater than it. Its binding affinity, may have a value of at least ¹⁰⁷ liters / mol, or may even have at least 10 ⁸ liters / mole of value, for example, binding affinity between antibody and antigen It may have a value in the range of 10 ⁷ to 10 ⁹ liters / mole, which is typical of sex. However, even higher affinities, above 10 ⁹ l / mol, are possible,
It can be utilized within the scope of the present invention.

The present invention can be carried out using a polyclonal antibody obtained by a conventional method of immunizing a host animal with a target antigen (eg, oral bacteria) as a binding polypeptide.

The present invention can also utilize monoclonal antibodies that can be obtained by standard techniques from B lymphocytes that elicit a polyclonal antibody response.

Further suitable for use as binding polypeptides are antibody fragments. The fragment used may be a Fab or Fv fragment, or may be a heavy chain (Dab fragment)
Alternatively, it may be a variable region of a light chain. Some antibody fragments, especially Fab fragments, can be obtained by enzymatic digestion of whole antibodies. A preferred method for obtaining antibody fragments is through recombinant DNA technology, wherein the fragments are expressed by a genetically transformed organism. These techniques begin with DNA or mRNA from a hybridoma producing a monoclonal antibody. However, other methods use immunoglobulin V region gene libraries from immunized or unimmunized animals.

Antibody fragments produced by recombinant DNA technology need not be the same as antibody fragments produced by other methods. For example, they may include a sequence of amino acids that differs from the sequence of amino acids found in antibodies produced by other methods, especially at the end of the fragment. Somewhat analogously, antibody fragments produced through recombinant DNA technology can include extra amino acid sequences at their termini that have no corresponding site in antibodies produced by other methods. .

[0034] Techniques for making antibody fragments are well known in the literature. Appropriate disclosures include Saiki et al., Science, 230, 13
50-54 (1985); Orlandy
i) et al., Proc. Natl. Acad. Sci.
A), 86, 3833-7 (1989); WO 89/9825 [Celltech], EP 368,684 [Medical Research Council], and International Publication No. 91/8482 [Unilever] is available.

Through the use of recombinant DNA technology, it is possible to produce polypeptides that contain more than one fragment of an antibody and thus have specific binding affinity for more than one antigen. Such an arrangement can be used in the present invention.

One possibility is that the polypeptide binding agent used in the present invention is a synthetic polypeptide that mimics the specific binding activity of the antigen binding determinant of a natural antibody. Such a polypeptide is effectively a fragment of an artificial antibody.

Enzymes that are particularly contemplated for use in the present invention include:
Urease. This enzyme converts urea to ammonium hydroxide and carbon dioxide. Enzymes are found in many bacteria, yeasts and plants. Urease of various origins is found in 6317, Missouri, USA
8, Sigma Chemical Company in St. Louis, PO Box 14508, and Sigma Chemical El, Poole, Dorset, UK
Available from TD (Sigma Chemical Ltd.). Species that may be the source of urease include Bacillus
Pasturi (Bacillus pasteurii), metofiras (Me
thophilus) and Helicobacte
r pylori).

Another enzyme that may be used is called asparaginase, which converts L-asparagine to L-aspartic acid and ammonia, thereby increasing the overall alkalinity, more fully L-asparagine amidinohydrolase. Things. It has the International Union of Biochemistry number 3.5.1.1 and is also available from Sigma.

Still other suitable enzymes are arginase,
More specifically, L-arginine amidinohydrolase (International Union for Biochemistry No. 3.5.3.1). It is also available from Sigma and converts arginine to ornithine (which is more alkaline) and urea.

As mentioned above, the enzyme may instead serve the function of producing an intermediate which is not itself alkaline, but which undergoes further reactions in situ to produce alkaline products.

[0041] Attachment of the enzyme to the polypeptide, which serves to attach the enzyme to the target site, can be accomplished by a variety of methods. The conjugation may be direct covalent conjugation to an antibody or antibody fragment that exhibits a specific binding affinity for the oral microorganism.

Techniques for chemically bonding one polypeptide to another are known. In general, these techniques use a set of coupling agents that react separately with each polypeptide and, when mixed, react with each other to link the polypeptides.

One example of polypeptide conjugation is found in Knowles et al., J. Clinical Investigation, 52, 1443 (1973). Knowles et al., Dutton et al., Biochemistry / Biophysics research papers (Bioche
m. Biophys. Res. Commun.), 23, 730 (19)
1966). Another example is Lal (La
l) etc., Journal of Immunology Methods (Journal of Immunologica
Methods, 79, 307 (1985).

In the chemical bond to the antibody fragment,
Fab fragment or F expressed with an additional "tail" as one extension of the peptide chain
It is appropriate to use the v fragment.

Also suitable are antibodies or antibody fragments, which do not bind directly to one species (bacteria) of the oral microbiota, but instead bind to other proteins or antibody fragments which bind to the target protein. Is to bind an enzyme to what binds to. This method utilizes a chemical bond. However, this method does not require that the antibody or antibody fragment that binds to the target protein undergoes a chemical reaction that might adversely affect it.

[0046] More suitably, the enzyme and the antibody fragment that binds to the oral microorganism are made as separate parts of a single polypeptide that is expressed as a fusion protein by the genetically transformed microorganism. .

Instead of direct chemical bonding, antibody-antigen bonding is also possible. A number of such techniques are disclosed in Applicant's published European patent applications, i.e., above-mentioned EP-A-450,800;
No. 3,097, No. 451,972 and No. 479,6
No. 00, the disclosures of which are incorporated herein. In particular, European Patent Publication No. 453,097 discloses a link between an antibody having a specific binding affinity for a target site and an antibody having a specific binding affinity for an enzyme, wherein a bridge is formed between the antibodies. Ligation using a third antibody to form is described. The European Patent Publication,
EP-A-479,60, also assigned by the applicant.
Almost the same configuration can be achieved using the antibody fragments described in No. 0.

Another method by antibody-serotype binding is to utilize a polypeptide comprising the two antibody fragments described above. One of the fragments is
Binds to the target site and the other binds to the enzyme. Suitably both fragments are Fv fragments.

International Publication No. 91/08482, cited above.
Describe the production of polypeptides comprising two _VH chains with different specific binding affinities. International Publication 94
/ 09131 shows two Fvs with different specific binding affinities
The preparation of a construct comprising an antibody fragment is described.

Holliger et al., Proceedings of the American Scientific Association (Proc. Natl. Acad. Sci. USA), 90, 6444.
Page (1993) also describes the production of polypeptides, each containing two Fv fragments.

In the present invention, the target site may be one of bacteria present in dental plaque. The effect of targeting one of these bacteria is to raise the pH both in the vicinity of that organism and in the area around the dental plaque where other bacteria may also be present. It is in. The plaque matrix polysaccharide itself can be used as a target, and the antibody or antibody fragment exhibits a specific binding affinity for its target matrix polysaccharide.

The products of the present invention include a polypeptide having a specific binding affinity for a target (eg, an antibody or antibody fragment) and an enzyme whose action is to increase pH. , At least one excipient.

If the enzyme is attached directly to the binding polypeptide, as a fusion protein or conjugate, it can be contained in a single excipient. However, if the enzyme is attached to the antibody or antibody fragment with specific binding affinity for the target through antibody-antigen binding, with at least one further antibody or antibody fragment, In that case, the enzyme and the antibody or antibody fragment having specific binding affinity for the target are distributed in the plurality of excipients,
It is quite desirable that the complex of the enzyme and the antibody or antibody fragment is formed only when the excipient is mixed (eg, during use). In this latter case, some of the conjugates can be linked together beforehand and if they are in the same excipient, some of the conjugates can be linked together beforehand, Only when all of the components of are combined together will complete complex formation occur.
Delaying the formation of the complete complex until the time of use may be advantageous to avoid loss of activity during storage.

The complex between the large enzyme and the polypeptide tends to be unstable and tends to precipitate during storage. They are also slower to spread. For this reason, if whole antibodies are used, it may be advantageous to partition the enzyme and antibody between different excipients. Since polyclonal antibodies tend to form larger complexes than monoclonal antibodies, it may be advantageous to partition the enzyme and antibody between different excipients, especially if the antibody is polyclonal.

Antibody fragments have the advantage of being smaller and form smaller complexes that are expected to retain their activity during storage better than the complexes formed with whole antibodies.

Obviously, for the purposes of the present invention, the excipient (s) used should be suitable for ingestion.
There are many suitable ones.

The simplest and most suitable are sterile aqueous solutions for use as mouthwashes. this is,
It can be a one-component mouthwash containing a binding polypeptide chemically linked to an enzyme. Alternatively, if the enzyme is not directly attached to the binding polypeptide, it can be a multi-component mouthwash consisting of multiple solutions including (i)-(iv) below.

(I) an antibody or antibody fragment having specific binding affinity to a site in dental plaque; (ii) an enzyme such as urease; and (iii) an enzyme such as urease having a specific binding affinity. The antibody or antibody fragment, and (iv) having specific binding affinity for the other plurality of antibodies or antibody fragments, and forming a cross-link between the other plurality of antibodies or antibody fragments. Antibodies or bivalent antibody fragments.

For example, there can be one solution containing (i) and (iii) and a second solution containing (ii) and (iv).

These individual aqueous solutions or suspensions are mixed by the user before use as a mouthwash, so that in the case of whole antibodies EP-A-453,097 by the applicant. As mentioned in the issue,
A complex consisting of anti-target antibody: cross-linked antibody: anti-enzyme antibody: enzyme is formed.

Other possible forms of excipient include toothpaste,
There are lozenges, chewing gum and dental floss that dissolve in the mouth. These forms of the product can be employed even when multiple excipients are required. Toothpaste, consisting of two components, where the two components are stored separately in a toothpaste container and, as a result, the two components are separated until the toothpaste is extruded from the container, is known per se. It is a concept. A lozenge licked in the mouth may include various excipients contained in separate areas of the lozenge so that when the lick is licked the excipients are mixed. Two different excipient forms can be used in combination, such as a method of providing multiple excipients. An example is toothpaste, which is used as a mouthwash or lozenge after use as a toothpaste.

The products of the present invention can include an enzyme substrate or, alternatively, can utilize an enzyme substrate present at the target site.

For urease, the substrate is urea. The present inventors have found that saliva contains a certain amount of urea, and that the amount in vivo is high enough to change the pH. The effect of the pH change has been proven in a test tube.

The development of the present invention provides for the production of other enzymes with beneficial effects that are also carried by polypeptides having specific binding affinity for target sites in the microbiota present on the tooth surface. To be incorporated into This further enzyme is suitable for exhibiting its beneficial effects in the alkaline environment created by the first enzyme. A particularly anticipated enzyme as the second enzyme is ureate oxidase, which serves to convert uric acid into carbon dioxide and allantoin, while at the same time generating hydrogen peroxide. Hydrogen peroxide is rapidly degraded in vivo. However, if an enzyme that generates hydrogen peroxide is present in the vicinity of the cell to be attacked, hydrogen peroxide can have cytotoxic effects.

[0065]

【Example】

Example 1 A mouse monoclonal antibody having specific binding affinity for S. aureus was made by known techniques.

Urease-antibody conjugate (conjugate) was purchased from Sigma Chemical LTD. It was one in which urease was chemically conjugated to a goat anti-mouse polyclonal antibody. The supplied solution of the conjugate was diluted at various dilution ratios using sterile physiological saline as a diluent as described below.

The Sanguis streptococci cells were suspended in sterile physiological saline containing 5% w / v glucose and 0.1% w / v urea to a concentration of about 10 ⁹ cells / ml.

One sample of this suspension was used as a control. The urease-antibody conjugate was added to another sample (suspension) at a dilution of 1/100.

The urease-antibody conjugate was added to another sample (suspension) at the same dilution or a higher dilution, together with a mouse anti-Sanguis streptococcal antibody (at a concentration of about 5 μg / ml).

The mouse anti-Sanguis streptococcal antibody served as a cross-linking antibody that allowed the urease-antibody conjugate to bind to the Sanguis streptococcal cells. The complex formed consisted of a urease-antibody conjugate bound to a mouse antibody bound to S. aureus cells.

The pH of the medium was adjusted to 1 using a pH meter.
/ 2, 1, 2, 3, and 24 hours later. The results are listed in Table 1.

[0072]

[Table 1]

As can be seen from these results, Streptococcus sanguis, by itself, gradually made the medium more acidic. This was prevented by adding urease-antibody conjugate.

For 3 hours, the pH is kept stable,
Thereafter, it gradually became alkaline. Add anti-Sanguis streptococcal antibodies (enzyme attaches to cells)
Almost immediately, even when the concentration of urease was reduced, the pH became alkaline.

In the mouth in vivo, the urease-antibody conjugate that did not adhere to cells or other targets was washed away by the flow of saliva and had little effect, while it adhered to cells of the oral microflora. It could be expected from these results that urease could remain in the mouth and reduce or overcome acidity production.

Example 2 In the headspace above the stable S. aureus streptococcus culture (the part filled with gas above the bacterial cell suspension in the bacterial culture flask; the same applies hereinafter). Using H ₂ S content as an indicator of biomass, H ₂ as a model of the effect of using the product of the present invention in the oral cavity
To demonstrate the effect of treatment with urease and antibody on S content, an in vitro test was performed.

Below pH 4.0, H ₂ S is only present in the headspace. However, as the medium becomes more basic, H ₂ S dissociates into soluble and highly reactive HS ⁻ ions, as shown in the following equation ^:

[0078]

Embedded image

In fact, at pH 8.0, 90% of H ₂ S
% Are dissociated and dissolved in the medium. In pH10.0 greater, HS ^- becomes S ^2- further dissociated. Importantly, in the presence of metal salts, the dissociation of H ₂ S is essentially irreversible, as the highly reactive HS ⁻ ion forming an insoluble sulfide precipitate. In a preliminary study, H from the headspace in the presence of metal or ammonium salts
Some irreversible loss of ₂ S was demonstrated in the experiment (data not shown).

After treatment as outlined below, the S. aureus, whose biomass was in a stable state, was resuspended in a medium (containing no amino acids other than cysteine) as described below.

The composition of the medium used for the growth of oral microorganisms was as follows:
Basic media (g / l): KNO ₃ (0.1); KH ₂ PO
₄ (1.0); K ₂ HPO ₄ (1.5); NaCl (0.
1); (NH ₄ ) ₂ PO ₄ (2.0).

Vitamins (mg / l): Nicotinic acid (1.0); Nicotinamide (1.0); NADH (1.
0); folic acid (1.0); calcium pantothenate (1.
0); riboflavin (1.0); thiamine hydrochloride (1.
0); spermine tetrahydrochloride (1.0); spermine trihydrochloride (1.0); putrescine dihydrochloride (1.0); pimelic acid (0.1); D, L-mevalonate lactone (0) .
1); D-mevalonic acid lactone (0.1); biotin (0.1); p-aminobenzoic acid (0.1); lipoic acid (0.1); β-alanine (10.0); Inositol (10.0); choline chloride (50.0).

Pyridoxals (mg / l): pyridoxal (1.0); pyridoxal hydrochloride (1.0); pyridoxamine dihydrochloride (1.0).

Purines / pyrimidines (mg / l): adenine (10.0); guanine (10.0); cytosine (10.0); thymine (10.0);
0); hypoxanthine (10.0); uracil (10.
0).

Salts (mg / l): KI (4.0); Cu
SO ₄ (1.0); H ₃ BO ₃ (0.4); FeSO ₄ (5.
0); MnSO ₄ (50.0); Na ₂ MoO ₄ (5.
0).

Other additives: hemin (5.0 mg / l);
Glucose (4.0 g / l); CaCl ₂ (10.0 mg
/ G); MgSO ₄ (0.7 g / l); NaHCO ₃ (1.
0 g / l).

Amino acids (mg / l): cysteine (5
00).

The first antibody used in the study (1st Ab)
Is 0.1 mg / ml final concentration (in blocking buffer)
Was a murine anti-Sanguis streptococcal immunoglobulin G (IgG) monoclonal antibody used in Example 1. The first antibody binds to a bacterial cell. Second antibody (2nd
Ab) is a commercially available (from Sigma) Jack
Goat anti-mouse I conjugated to Bean urease
gG preparation. The second antibody was 1/5 before use.
Diluted to a concentration of 0 (in blocking buffer). The second antibody binds to the first antibody and catalyzes the hydrolysis of urea. The blocking buffer (Tris buffered saline containing Tween; hereinafter, referred to as TBS-Tween) is 29 mM Tris-HCl (pH 6.8), NaC
1 contained 0.15 M, 12.5 μM EDTA, and 0.5% Tween 20. Blocking buffers are also used to prevent non-specific binding and to wash away unbound antibody.

Before resuspension in medium, each cell pellet was subjected to one of the following treatments, all performed at 30 ° C. with agitation: (i) Untreated control (Ii) culturing for 1 hour in TBS-Tweem; (iii) culturing for 1 hour in the first antibody and then culturing TBS-Tweem.
Perform two 15 minute washes in Tween; (iv) Incubate in the second antibody for 1 hour, followed by TBS-
Two washes of 15 minutes in Tween are performed; (v) Culture in primary antibody for 1 hour, TBS-Twee
2x15 min washes in the second antibody.
Culture for an hour, followed by two washes in TBS-Tweem for 15 minutes.

The amount of volatile sulfur compounds in the headspace (hereinafter referred to as VSC amount) was measured after 22 hours,
After 3 hours, cells are harvested and 10 mM urea / 12.5 μl
M EDTA (with stirring at 30 ° C. for 1 hour).
Culture for 5 minutes). At this time, the amount of VSC in the headspace was measured again, and the pH value of the suspension was recorded. Table 2 shows the results.

[0091]

[Table 2]

The data in Table 2 clearly show that H ₂ S produced by microorganisms is removed from the headspace as the pH increases with ammonia produced from urea catalyzed by urease. . Urease - when bound second antibody was added alone, because meaningful change in pH (or H ₂ S removal) was not observed, the effect is clearly due to the fact that the antibody binds to a target. Similarly, both the first antibody and the blocking buffer
Individually had no effect. Importantly, the effect was also long lasting, since the removal of H ₂ S was observed for more than 23 hours after exposing the cells to the antibody, which is an important factor for use as an oral deodorant. This is an important prerequisite.

A further requirement for active ingredients for suppressing bad breath is that they can function after a short contact. Thus, a second experiment was performed to determine whether the antibody / target binding urease system could remove H ₂ S in the headspace with only 5 minutes indirect access to the S. aureus cells. Was.

For this experiment, S. aureus, whose biomass was in a stable state, was treated as outlined below and then resuspended in medium (containing no amino acids other than cysteine) as described above. did. The first and second (urease-bound) antibodies used the same amounts as outlined previously.

Prior to resuspension, the following treatments were performed: (i) Incubation in TBS-Tweem for 10 minutes; (ii) Incubation in the first antibody for 5 minutes, TBS-Twee
(iii) Incubate for 5 minutes in the second antibody, and wash with TBS-Twee.
(iv) Incubate in the first antibody and the second antibody (mixture) for 5 minutes and wash in TBS-Tweem for 5 minutes.

The amount of VSC in the headspace was measured after 18 hours, and after 19 hours the cells were harvested and resuspended in 10 mM urea / 12.5 μM EDTA (at 30 ° C. with stirring). Culture for 10 minutes). At this time, the amount of VSC in the headspace was measured again, and the pH value of the suspension was recorded. Twenty hours later, the cells were pelleted and a second resuspension in urea / EDTA was performed. At this time, before the suspension is cultured for 10 minutes as described above, the inside of the culture flask is degassed, and oxygen-free nitrogen containing chemically generated H ₂ S (about 2000 ng / vial) is added to the flask. To the headspace. At this time, the VSC amount and the pH value were recorded again. Table 3 shows the results.

[0097]

[Table 3]

The data in Table 3 shows that the antibody / target binding urease system can remove H ₂ S in the headspace with only 5 minutes indirect access to the S. aureus cells. In addition, the activity of the system was retained even after the addition of fresh medium and chemically generated H ₂ S 20 hours after the S. aureus was in contact with the antibody for only 5 minutes. This confirms that, despite the dilution of the remaining active substance, it is effective continuously for a long time. It was also confirmed that the activity of the system was dependent on the binding of the antibody to the target.

As shown in Tables 2 and 3, the results of this study indicate that antibody / target binding to prevent (or reverse) the volatilization of H ₂ S generated by microorganisms in the oral cavity. The possibility of using urease activity is clearly shown. Their potential for success as breath fresheners is evidenced by the rapid, targeted, effective and long-lasting responses they elicit. In situ, these actives can affect pH elevation localized to specific locations, thereby causing S. sanguis and other oral bacteria (antibodies also to these other oral bacteria). it is expected that it is possible to suppress the generation of volatile H ₂ S by which can) be.

 ──────────────────────────────────────────────────続 き Continuing the front page (72) Inventor Tony Mainhas Great Britain and Northern Ireland United Kingdom, Kent Dea 1.2 and 2 Eidget, Dartford, King Edward Avenue 30 (72) Inventor John Casey Great Britain and Northern Ireland United Kingdom, Northamptonshire NN 9.6 Pell, Wellingboro, Stanwick, Manor Gardens 2 (72) Inventor Della Hillian's Great Britain and Northern Ireland United Kingdom, Northampton NN 5.5 5.5 Gordon James, Great James, Seymour Street, St. James, 29 (72) Liten and Northern Ireland United Kingdom, Northamptonshire N. 10.8 Elge, Hyam Fairways, Philippe Way 16 (72) Inventor Gary Mickok Great Britain and Northern Ireland United Nations, Northamptonshire N. 10.8 LH, Higham Fairs, Pressland Drive 8

Claims (14)

[Claims] 1. A product for topical use in the oral cavity, the product comprising at least one excipient,
In the same or separate excipient (s), a polypeptide having a specific binding affinity for a target site in the microflora present on the tooth surface and a function of increasing the surrounding pH are included. A product comprising an enzyme to be performed, wherein the enzyme is attached to the polypeptide, or the product comprises means for binding the enzyme to the polypeptide at least during its use. 2. The product of claim 1, wherein said polypeptide has a specific binding affinity for oral bacteria. 3. The product according to claim 1, wherein the polypeptide is an antibody or an antibody fragment. 4. The polypeptide comprises two antibody fragments, one fragment having specific binding affinity for the target site and the other fragment specific for the enzyme. 4. The product according to claim 3, having a high binding affinity. 5. The product of claim 4, wherein said fragments are both Fv fragments. 6. The excipient (s) having a second antibody or antibody fragment having specific binding affinity for the enzyme, and the antibody or antibody fragment (first antibody or antibody fragment) 4. The product of claim 3, comprising a third antibody or antibody fragment capable of binding both the second antibody or antibody fragment. 7. The product of claim 1, wherein said enzyme is directly attached to said polypeptide. 8. The product of claim 7, wherein said polypeptide having specific binding affinity for said enzyme and said target site is a separate part of a fusion protein. 9. The method according to claim 9, wherein the enzyme has a second polypeptide having a specific binding affinity for the polypeptide having a specific binding affinity for the target site (the first polypeptide). The product according to any one of claims 1 to 3, which is directly attached. 10. The excipient (s) also includes an enzyme (second enzyme) that functions to generate hydrogen peroxide in an alkaline environment, wherein the second enzyme is present on the tooth surface. Is attached to a polypeptide that has a specific binding affinity for a target site in the microbiota, or the product, at least during its use, binds the second enzyme to such a polypeptide. 10. A product according to any one of the preceding claims, comprising means for attaching. 11. A polypeptide having a specific binding affinity for a target site in a microflora present on the tooth surface, and (ii) producing an alkaline product, thereby increasing the pH. At least one excipient that can be used in the mouth, including an enzyme that performs the function of causing plaque, in a method of preventing or reducing plaque / caries formation, including the topical use in the mouth. Thus, the polypeptide and the enzyme are attached together, or the excipient (s) includes means for attaching the enzyme to the polypeptide at least during its use. Method. (I) producing an alkaline product;
An enzyme that functions to raise the pH thereby, and (ii) has a specific binding affinity for a target site in the microflora present on the tooth surface and attaches the enzyme at least during its use Of the polypeptide which also plays a role,
Use as a medicament for preventing or reducing plaque / cavity formation. 13. A polypeptide having a specific binding affinity for a target site in a microflora present on the tooth surface, and (ii) producing an alkaline product, thereby increasing the pH. A method of preventing or reducing bad breath, comprising topically using at least one excipient that can be used in the mouth, including an enzyme that functions to cause Whether the peptide and the enzyme are attached together and
The method wherein the excipient (s) comprises means for attaching the enzyme to the polypeptide at least during its use. 14. (i) producing an alkaline product;
An enzyme that functions to raise the pH thereby, and (ii) has a specific binding affinity for a target site in the microflora present on the tooth surface and attaches the enzyme at least during its use Of the polypeptide which also plays a role,
Use as a medicine to prevent or reduce bad breath.