BRPI0806928A2 - COMPOSITION FOR INHIBITION OF ORAL CAVITY GROWTH, METHOD FOR INHIBITION OF ORAL CAVITY BACTERIA, METHODS FOR TREATMENT OR PREVENTION OF DENTAL CARIES, AND DENTAL PLACEMENT, AND INFLAMENTAL TREATMENT PRESENCE OF DENTAL BOARD, AND FUSION PROTEIN. - Google Patents

Description

“COMPOSITION FOR INHIBITION OF BACTERIAL GROWTH IN ORAL CAVITY, METHOD FOR INHIBITION OF BACTERIA IN ORAL CAVITY, METHODS FOR TREATMENT OR PREVENTION OF DENTAL CARIES, PERIODONAL DISEASE, AND DENTAL DISEASE PLACEMENT ASSOCIATED WITH THE PRESENCE OF DENTAL PLATE, AND FUSION PROTEIN ”CLAIMING PRIORITY

This application claims priority for US Interim Application No. 60 / 880,753, filed January 16, 2007, the presentation of which is incorporated herein by reference in its entirety. BACKGROUND OF THE INVENTION

For many years researchers have been trying to develop chemotherapeutic agents that may be useful in fighting infections in an oral cavity. Back in the mid-44's, with the introduction of antibiotics it became clear that while all of these potent agents had the potential to be effective, conical problems in the oral cavity interfered with their utility. Over time, the development of plaque in the oral cavity became better understood. With that better understanding, it was clear that agents administered to the oral cavity needed to be adhered to oral tissues to resist removal through saliva flows and oral chewing forces. It was widely accepted that for antimicrobial agents to be effective in the oral cavity, the agent needed to be maintained in that environment over time. An experiment illustrating this principle compared the effectiveness of two important antimicrobial agents, tetracycline and penicillin. Penicillin has been shown to be a potent agent compared to tetracycline when tested in vitro. Thus, penicillin was shown to be effective against significantly lower oral bacteria compared to tetracycline when tested against bacterial growth in a broth medium (1 μg / ml for penicillin versus 10-20 μg / ml). ml for tetracycline). In addition, penicillin is a bactericidal agent and therefore promotes bacterial death, while tetracycline is bacteriostatic and therefore reduces bacterial growth and development. However, despite these dramatic differences in potency, tetracycline proves to be a more effective antimicrobial agent when applied topically to the oral cavity. Therefore, when tetracycline is added to a saliva-coated hydroxylapatite (SHA) disc, a substitute for the tooth / varnish surface, and then this SHA tooth counterpart is treated with tetracycline and washed and then placed in a bacterial culture, it was seen that very few of these organisms were able to adhere to SHA. On the other hand, when SHA was treated similarly with penicillin, there was no effect. Thus, despite the reduced potency of tetracycline as compared to penicillin, tetracycline was superior in an assay for adherence and release of a drug from a saliva-coated tooth analog. Because the two most common dental diseases, tooth decay and periodontal disease, result from oral bacteria adhering to the surface of the teeth, tests for antimicrobial efficacy in the oral cavity need to be focused on ways to prevent or reduce bacterial attack on the teeth. tooth surfaces.

With that understanding, research in the late 1960s and early 1970s examined agents with the characteristic of substantivity, the ability of an agent to bind and to be released from a toothlike surface. One of the first agents studied that had this characteristic was chlorhexidine, a bisbiguanide antimicrobial that showed, both in vitro and in vivo, that it reduced the adhesion of plaque to teeth. The agents known as Peridex® and Perioguard® have been investigated and are currently commercially available oral mouth rinsing agents that have been tested and shown to adhere to oral tissues. This property of substantivity combined with antimicrobial activity results in reduced levels of both gingivitis and caries when used in clinical studies. Since that time, there have been 5 attempts to develop agents that work effectively in the oral cavity. So far clinical trials have shown very little success. One such success used the agent, triclosan, the active ingredient, in Colgate Total® toothpaste. This toothpaste has been tested enough to receive acceptance from federal agencies such as the ADA and the 10 FDA. Commercially available dentifrice containing triclosan is produced by Colgate Palmolive Company. Total toothpaste was developed using a known antimicrobial, triclosan, a phenolic compound, which was used in shampoos and deodorants, and by binding it to an agent that allows it to bind to oral tissues. The top 15 industrial companies, Procter Gamble, Unilever and Colgate Palmolive Company, all used triclosan as a possible agent in the oral cavity at about the same time. Each company had its own strategy that was designed to make the active agent triclosan bind in oral tissues so that it was active against the adhesion of oral microorganisms to the teeth. To confirm this it was found that triclosan itself did not bind to oral tissues and was therefore ineffective in the oral cavity despite its potent antimicrobial properties. Colgate Palmolive Company mixed igicare or gantrex with triclosan to form a polymeric compound which when administered into the oral cavity was bound such that it made triclosan effective. This combination product, in addition to chlorhexidine containing products and other agents containing phenolic compounds, are among the only effective agents developed in the last 40 years.

Cavities are the single most common chronic childhood disease, five times more common than asthma. More than 70% of children over 17 have cavities. More than 108 million Americans lack dental insurance, so treatment for this infection does not exist. S. mutans is considered one of the microbes most associated with dental caries. In 5 studies of children with localized juvenile periodontitis, now known as aggressive localized periodontitis, several of these children were found to have minimal cavities. In addition, it was found in these studies that children with this form of periodontal disease had a variant on a saliva protein, lactoferrin (Lf). when this Lf variant, the 10 lysine variant, was expressed in an insect vector and tested it was found to kill Streptococcus mutans. In contrast, another more common Lf variant, the arginine variant, present in healthy children without periodontal disease, had little or no effect on S. mutans survival.

We recently demonstrated that a synthetic lysine-shaped peptide of this lactoferrin protein kills Streptococcus mutans. Others have shown that lactoferrin peptides have antimicrobial activity, but no one has yet shown that the antibacterial activity derived from this peptide is useful against organisms in the oral cavity. We also determined that the peptide will have limited utility in the oral cavity unless it is bound to something that allows the peptide to attach to the surface of the tooth. Therefore, we have developed a fusion peptide in which the active peptide is bound to a small saliva peptide, staterin, so that it can retain both its antimicrobial activity and also be retained on the tooth surface to reduce S bonding. mutans on that surface.

Lactoferrin (Lf) is a multifunctional protein that is found in mammary secretion, tears and saliva, and in the gastrointestinal tract. Its main function is to capture the surrounding iron to prevent oral microbes from reaching iron, a necessary mineral for survival. In addition, at its N terminus, Lf has a region that has antimicrobial properties. This region is called the defensin region and it has been demonstrated since 1980 that Lf can kill many types of bacteria, including oral bacteria. It has also been shown that within the 5-terminus N region of amino acid position 11 to 31 there are at least two polymorphic forms. For the most part, variation in this defensin region at amino acid position 29 may contain arginine or lysine. Then recently in 1998, it was demonstrated that these two amino acid position 29 variants had different antimicrobial activities and that peptides 10 designated with those forms could have antimicrobial activity. In fact, in 2003 our group produced full-length lactoferrin secreted by insect cells. This secreted lactoferrin was produced with two polymorphic forms (lysine and arginine) and was introduced via a baculovirus vector. The secreted protein had all the characteristic properties of human lactoferrin and we tested these two forms for activity against Gram + and Gram- organisms. The form of lysine was found to be most active against a range of Gram + and Gram- organisms, and specifically more active against S. mutans, the known cause of dental caries. As the Lf form of lysine was found more frequently 20 in patients who had localized aggressive periodontitis (LAP), a disease in which children had fewer caries than the combined controls, we speculated that this form of lactoferrin lysine could produce an ecological advantage for these children, which could explain the reduction in caries seen in these children. In addition, we then determined that pure lactoferrin would have limited utility in the oral cavity unless it was attached to something that allowed it to attach to the surface of the teeth. Staterin is a small saliva phosphoprotein found in saliva at concentrations of 200 μg / ml in stimulated saliva but at concentrations around ten times lower in unstimulated saliva. Adsorption of salivary proteins onto the varnish forms a coating of varnish film in which bacteria in the mouth adhere to the initial bacterial components of plaque. The initial salivary proteins that are adsorbed are mucins, amylase, IgA, lysozyme, cystatins, acidulated proline rich proteins (PRPs), and agglutinins. Film adsorption on the varnish or surfaces of hydroxylapatite (HA) promotes the adhesion of S. mutans. In contrast, salivary proteins such as staterin and histanins appear to have the opposite effect, and thus inhibit S. mutans adhesion (Gibbons and Hay 1989). Staterin is a tyrosine 10 rich phosphoprotein containing 43 amino acids (Hay and Moreno 1989). Staterin is most noted for its ability to reduce stone formation through calcium binding (Hay and Moreno 1987). Recently, Shimotoyodeme et al (2006) suggested that staterin competes with high molecular weight salivary proteins to reduce the binding of S. mutans on the tooth surface. They 15 further demonstrated that a small peptide consisting of the first 26 amino acids also has this ability. To date, there is no evidence to determine whether these amino acid residues could bind to another salivary protein or whether a fusion protein consisting of staterin and lactoferrin would still retain its ability to compete with other salivary proteins for space on the surface of the proteins. teeth. Furthermore, it is not known whether a staterin-containing fusion peptide would be retained on the varnish and whether this fusion peptide would retain its antimicrobial activity.

Described herein is a fusion peptide in which the antibacterial peptide (in the form of lactoferrin) is attached to a small salivary peptide (staterin) which allows the antibacterial peptide to bind to the surface of the teeth and thus interfere with the ability of S Mutans attach to the surface of the teeth. SUMMARY OF THE INVENTION The present invention is directed to the treatment of dental diseases caused by oral bacteria using lactoferrin and staterin fusion proteins, and compositions thereof.

In certain embodiments, the present invention is directed to a composition for inhibiting bacterial growth in the oral cavity consisting of a fusion protein composed of a lysine form of a lactoferrin polypeptide or a functionally equivalent peptide molecule and a staterin polypeptide or functionally equivalent polypeptide molecule thereof.

In other embodiments, the present invention is directed to a fusion protein having SEQ ID NO. 10; SEQ ID NO. 11; or SEQ ID NO. 13, or a functionally equivalent peptide molecule thereof. In still other embodiments, the present invention is directed to a composition consisting of a fusion protein having a SEQ ID NO. 10; SEQ ID NO. 11; or SEQ ID NO. 13, or a functionally equivalent peptide molecule thereof.

In other embodiments, the present invention is directed to a method of inhibiting oral cavity bacteria, treating or preventing tooth decay, treating or preventing periodontal disease, treating or preventing dental plaque, or treating inflammation of the tooth. oral cavity associated with the presence of dental plaques, by administering an effective amount of a composition consisting of a fusion protein composed of a lysine form and a lactoferrin polypeptide or a functionally equivalent peptide molecule, and a staterin polypeptide or a functionally equivalent peptide molecule thereof to a patient in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1: Lactoferrin peptide sequences, staterin, and STAT-LF fusion proteins.

FIGURE 2: Substantivity test illustrating the ability of peptides to bind to hydroxyapatite (HA) or tooth analog.

FIGURE 3: Diffusion assay showing that only one form of lactoferrin has activity. In all panels, the circle is a disc of filter paper impregnated with different agents (PBS = phosphate buffered saline which is a control); LF "R" = at 11 mer of lactoferrin with arginine (R) at amino acid position 29; LF "K" to 11 mer of lactoferrin with lysine (K) at amino acid position 29. The disc containing a standard concentration of one of the agents is placed on a confluent culture of Streptococcus mutans. A clear zone around the discs shows that the agent was diffused into agar medium and inhibited S. mutans culture growth and therefore indicates that there is antibacterial activity and that S. mutans cannot grow in that area.

Left panel: PBS impregnated disc = control showing no inhibition.

Center Panel: Disc impregnated with the arginine variant of lactoferrin showing no clear zone and therefore no inhibition of S. mutans.

Right panel: disk impregnated with the lysine variant of a lactoferrin showing a clear zone indicating that the agent has diffused into the agar and is inhibiting S. mutans growth.

FIGURE 4: Diffiasing assay showing that the most active fusion peptide is LF and 6-mer staterin. Five panels are presented, each evaluating the activity of the agent tested.

In all panels, the circle is a disk impregnated with different agents. The disc is placed over a confluent culture of Streptococcus mutans. A clear zone around the discs shows that there is antibacterial activity and that S. mutans does not grow in that area.

Top left panel: lactoferrin (Ilmer) where lysine is present. This formulation inhibits the growth of S. mutans seen as a clear zone around the disc.

The right side panels in the middle and top are two staterin peptides, one 6 mer and one 15 mer. In none of these formulations is there any antibacterial activity seen by the fact that no zone of inhibition is seen adjacent to the disc.

Bottom left panel: Lactoferrin 11 mere noted as

fused with LF on the 6 mere staterine. This fusion protein retains the activity of mere 11 lactoferrin.

Bottom panel on the right: Lactoferrin 11 mer noted as fused with LF in Staterin 15 mer. This fusion protein has less activity than when LF is fused to Stat 6 mer.

The two panels at the bottom show the level of interference when LF is added to both staterinas. LF activity when the smaller staterin peptide is fused to its largest is compared to when the larger peptide is fused to the LF.

FIGURE 5: S. mutans recovery from SHA squares

after 2h of peptide treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions for inhibiting bacterial colony formation. For purposes of this invention, the term "inhibition" means reducing or killing existing bacteria, or preventing further bacterial growth. The invention is also for an oral therapeutic treatment that inhibits the formation of bacterial colonies in the oral cavity, thereby reducing the amount of dental plaque and caries. One embodiment of the invention is a substantially purified fusion protein consisting of lactoferrin and staterin (ST AT-LF). Figure 1 shows nucleic and amino acid sequences for STAT-LF. In general, "purified" refers to a protein or peptide composition that has been fractionated to remove various other components, and whose composition substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a composition in which the protein or peptide forms the major component of the composition, as constituting about 50%, about 60%, about 70%, about 80%. %, about 10 to 90%, about 95% or more of the proteins in the composition. In certain embodiments, the protein or polypeptide of the invention may be operably linked to a second polypeptide sequence.

Functionally equivalent nucleic acid molecules

Certain embodiments of the invention include nucleic acid molecules that are functional equivalents of all or part of the STAT-LF sequence. (A nucleic acid molecule may also be referred to as a DNA sequence or a nucleotide sequence in this application. All of these terms have the same meaning as the nucleic acid molecule.) Functionally equivalent nucleic acid molecules are 20 DNA and RNA (such as genomic DNA, complementary DNA, synthetic DNA, and messenger RNA) that encode peptides having the same STAT-LF-like activity as the STAT-LF peptide shown in the figure. 1. Functionally equivalent nucleic acid molecules may encode peptides containing a region having a sequence identity to a region of a STAT-LF peptide or, more preferably, the entire STAT-LF peptide. The mutations described in this request preferably do not destroy the reading structure of the coding sequence. Nucleic acid molecules functionally equivalent to STAT-LF sequences may occur in various forms as described below. Nucleic acid molecules may encode conservative amino acid changes in the STAT-LF peptide. Certain embodiments of the invention include functionally equivalent nucleic acid molecules that encode conservative amino acid changes within a STAT-LF amino acid sequence and produce silent amino acid changes in STAT-LF.

Nucleic acid molecules may encode non-conservative amino acid substitutions, additions or deletions in the STAT-LF peptide. Some embodiments of the invention include functionally equivalent nucleic acid molecules that make non-conservative amino acid changes within the STAT-LF amino acid sequence (Figure I). Functionally equivalent nucleic acid molecules include DNA and RNA encoding peptides, peptides and proteins having non-conservative amino acid substitutions (preferably substitution of a chemically similar amino acid), additions, or deletions but which also retain the same or an activity. similar to STAT-LF than STAT-LF peptide. DNA or RNA may encode fragments of STAT-LF variants. STAT-LF or STAT-LF-like activity of such fragments and variants is identified by assays as described below. Preferably, the STAT-LF fragments and variants covered by the present invention should have at least about 40%, 60%, 80% or 95% sequence identity to the STAT-LF nucleic acids encoding the molecule, or a region of the sequence, such as the coding sequence or one of the conserved domains of the nucleic acid molecule. Nucleic acid molecules functionally equivalent to the

STAT-LF molecule in Figure 1 will be apparent from the following description. For example, the sequence shown in [figure 1 (d)] may have its length altered through natural or artificial mutations, such as a nucleotide insertion or partial deletion, so that when the entire length of the coding sequence in [figure 1 (d)] is considered to be 100%, the functionally equivalent nucleic acid molecule preferably having a length of about 60-120% thereof, more preferably about 80-110% thereof. Fragments 5 may be less than 60%.

Nucleic acid molecules containing partial natural or artificial mutations (usually 80% or less, preferably 60% or less, more preferably 40% or less of the entire length) such that some codons in these sequences encode different amino acids, but where 10 the resulting peptide retains the same or similar activity as STAT-LF. The mutated DNAs thus created should preferably encode a peptide having at least about 40%, preferably at least about 60%, at least about 80%, and more preferably at least about 90%. or 95% sequence identity with the 15 amino acid sequence of the STAT-LF peptide in [Figure 1 (d)].

Because the genetic code is degenerate, the nucleic acid sequence in [Figure 1 (d)] is not the only sequence that could be encoded by a peptide having STAT-LF activity. This invention includes nucleic acid molecules having the same essential genetic information as that of the nucleic acid molecule described in [Figure I (d)]. Nucleic acid molecules (including RBA) having one or more nucleic acid alterations of the sequences described in this application and resulting in the production of a peptide shown in [Figure 1 (d)] are within the scope of various embodiments of the invention.

Other equivalent functional forms of the nucleic acids of

STAT-LF encoding can be isolated using standard DNA-DNA or DNA-RNA hybridization techniques. Accordingly, certain embodiments of the present invention also include nucleic acid molecules which are hybridized to one or more of the sequences in [Figure 1 (d)] or to their complementary sequence, and which encode the expression of peptides, peptides and proteins that present same or similar activity to that of the STAT-LF peptide produced by DNA in [Figure 1 (d)] or its variants. Such nucleic acid molecules preferably are hybridized to the sequence in [Figure 1 (d)] under moderate to highly stringent conditions (see Sambrook et al. Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY). Highly stringent washes are low in salt (preferably about 0.2% SSC), and low stringent washes 10 are high in salt (preferably around 2% SSC). A temperature around 37 ° C or about 42 ° C is considered to be of low accuracy, and a temperature of about 50 - 65 ° C. It is of high rigor. Some embodiments of the invention also include a method of identifying nucleic acid molecules encoding a STAT-LF activating peptide (preferably a mammalian peptide), including contacting a sample containing nucleic acid molecules, including all or part of [Figure 1 (d)] (preferably at least about 13 to 20 nucleotides of [Figure 1 (d)] under moderate to highly stringent hybridization conditions, and identifying nucleic acid molecules that are hybridized to 20 nucleic acid molecules including all or part of [Figure 1 (d)] Similar methods are described in U.S. Patent No. 5,851,788.

Certain embodiments of the present invention also include methods of using all or part of the nucleic acid molecules that are hybridized to all or part of [Figure 1 (d)], for example as probes or assays for the identification of peptide antagonists or inhibitors produced by nucleic acid molecules (described below). Some embodiments of the present invention include methods of using nucleic acid molecules having sequence identity with the STAT-LF nucleic acid molecule (as described below) in similar methods.

Certain embodiments of the invention also include a nucleic acid molecule detection kit, preferably in a suitable container medium or attached to a surface, a nucleic acid molecule as set forth herein by modifying the STAT-LF of [Figure 1 ( d)] or a peptide having STAT-LF activity and a detection reagent (as a detectable marker).

Pharmaceutical Compositions and Treatment Methods

STAT-LF is also useful when combined with a carrier in a pharmaceutical composition. The compositions are useful when administered in methods of medical treatment or prophylaxis of a disease, disorder or abnormal physical condition caused by S. mutans. Certain embodiments of the invention also include methods of medical treatment of a disease, disorder or abnormal physical condition characterized by excess S. mutans, for example by administering a pharmaceutical composition including a carrier and STAT-LF. Caries are an example of a disease that can be treated or prevented by administering STAT-LF [Figure I (d)]. The compositions are also useful when administered in methods of medical treatment or prophylaxis of a disease, disorder or abnormal physical condition caused by another dental plaque generating Gram + or Gram- bacteria including but not limited to Actinomyces spp. and others Streptococci spp.

The pharmaceutical compositions may be administered to humans or animals by methods such as in food, food additives, dentifrices, gels, toothpaste, mouth lotions, dental floss, denture wash, denture adhesives, chewing gum, candy, cookies, soda or sports drinks in medical treatment methods. STAT-LF may be coupled to lipids or carbohydrates. This increases their ability to adhere to teeth, extending the duration of adhesion or increasing their affinity, or both. They may also be coupled to polymers, for example, in dental work (eg crowns, braces, fillings) or in dental floss. The pharmaceutical compositions may be administered to humans or animals. The dosages to be administered depend on the individual condition of the patient, the indication of the drug, the physical and chemical stability of the drug, the toxicity of the desired effect and the route of administration chosen (Robert Rakel, ed., Conn1S Current Therapy (1995, WB Saunders Company, USA) Pharmaceutical compositions are used for the treatment of 10 dental plaque diseases forming bacterial infections such as dental caries, periodontal disease and endocarditis In a preferred embodiment, the pharmaceutical compositions are used for the treatment of Diseases Caused by Actinomyces spp. and Streptococci spp. In another preferred embodiment, the pharmaceutical compositions are used for the treatment of diseases as used to treat Streptococci infections caused but not limited to: S. mutans, S. sobrinus. , S. oralis, S. sanguis, S. mitis, S. gordonii, S. pneumonide, S. pyogenes, and S. agalactiae.

Nucleic acid molecules and STAT-LF [Figure 1 (d)] may be introduced into cells using in vivo delivery vehicles such as liposomes. They may also be introduced into these cells using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation or the use of liposomes. In some cases it will be desirable to use liposomes directed to the bacterium of interest.

In a preferred embodiment, the pharmaceutical compositions according to the invention are prepared using one or more STAT-LF peptides capable of inhibiting biofilm formation in dental plaque that is associated with the bacterium. Pharmaceutical compositions consisting of STAT-LF peptides are especially useful for methods of treatment or prevention (prophylaxis) of a disease, disorder or abnormal physical condition caused by Streptococci infection. Such pharmaceutical compositions are especially useful for treating infections with one or more oral Streptococci bacteria, such as S. mutans, S. sobrinus, S. oralis, S. sanguis, S. mitis, or S. gordonii. The pharmaceutical compositions are also useful for treating and preventing other types of Streptococci infections, such as S. pneumoniae, S. pyogenes, and S. agalactiae.

Pharmaceutical compositions may be prepared by

of known methods for the preparation of pharmaceutically acceptable compositions, which may be administered to patients, such that an effective amount of nucleic acid or peptide molecule is combined in a mixture with a pharmaceutically acceptable carrier. Acceptable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa, USA). Vehicles include saline and D5W (5% dextrose and water). Excipients include additives such as a buffer, solubilizer, surfactant, suspending agent, emulsifying agent, viscosity control agent, taste, lactose filler, antioxidant, preservative or dye. Preferred excipients are for peptide stabilization for parenteral and other administration. Excipients include serum albumin, glutamic or aspartic acid, phospholipids and fatty acids.

In other preferred embodiments, surfactants are included in the

compositions to aid penetration of the biofilm. Surfactants that could be used in the present invention generally include pharmaceutically acceptable anionic surfactants, cationic surfactants, amphoteric (amphipathic / amphophilic) surfactants, and nonionic surfactants. Suitable pharmaceutically acceptable anionic surfactants include, for example, monovalent alkyl carboxylates, acyl lactylates, alkyl ether carboxylates, N-acyl sarcosinates, polyvalent alkyl carbonates, N-acyl glutamates, fatty acid polypeptide condensates, sulfuric acid esters, alkyl sulfates ( including sodium lauryl sulfate (SLS)), ethoxylated alkyl sulfates, ester-linked sulfonates (including sodium docusate or sodium dioctyl succinate (DSS)), alpha olefin sulfonates, and phosphate ethoxylated alcohols. Such suitable pharmaceutically acceptable cationic surfactants include, for example, quaternary monoalkyl ammonium salts, quaternary dialkyl ammonium compounds, amidoamines and aminoimides. Suitable pharmaceutically acceptable amphoteric (amphipathic / amphophilic) surfactants include, for example, N-substituted alkyl amides, N-alkyl betaines, sulfobetaines, and N-alkyl beta aminopropionates.

In even more preferred embodiments, the composition is comprised of about

1 to about 10%, about 0.3 to about 5%, or about 0.5 to about 2% by volume sodium lauryl sulfate. Alternatively, a substituted sequence on one of the lysine residues that incorporates activities that produce activities such as sodium lauryl sulfate may be inserted into the fusion peptide. For example, the Lys side chain may be modified with undecanoic acid: this will introduce a long alkyl chain similar to the dodecyl radical in SLS.

aminoundecanoic acid: This will introduce a long alkyl chain similar to the dodecyl radical in SLS and a positive charge (NH2) at the end.

In preferred embodiments, sodium lauryl sulfate is used. ο

The Lys side chain may also be modified, for example, with aminoundecanoic acid and coupled aspartic acid at the end to introduce a dipolar ion at the end.

Based on this, the pharmaceutical compositions could include an active compound or substance, such as a STAT-LF peptide, associated with one or more pharmaceutically acceptable carriers or diluents, and contained in buffered solutions of a suitable pH and isosmotic with physiological fluids. A pharmaceutical carrier will depend on the intended route of administration. Methods of combining the active molecules with vehicles or combining them with diluents are well known to those skilled in the art. The compositions may also contain additives such as antioxidants, buffer solutions, bacteriostats, and bactericidal antibiotics and solutes that cause the formulation to become isotonic in the intended container; and sterile aqueous and non-aqueous suspensions which may include suspending agents and thickening agents. The composition could include a targeting agent for transporting the active compound to specified sites.

The pharmaceutical compositions according to the invention may be administered by any suitable route known in the art. In cases where the infection is localized, the pharmaceutical composition may be administered topically to the infected area. In cases where the infection is systemic, the pharmaceutical composition may be administered orally, intravenously, or parenterally. In some cases, it may be desirable to administer the pharmaceutical compositions according to the invention with a known antibacterial agent, such as an antibiotic. In some cases, pharmaceutical compositions that suppress biofilm formation are also useful for making bacterial cells more susceptible to antibiotics.

The term "antibiotic" used herein refers to any compound known to the person of ordinary skill in the art that inhibits growth, or kills the bacterium. The term "antibiotic" includes, but is not limited to, beta-lactams (penicillins and cephalosporins), vancomycin, bacitracins, macrolides (erythromycin), lincosamides (clindomycin), chloramphenicol, tetracyclines, aminoglycosides (gentamicins), amphotericins, cephoterines, clindamycin, mupirocin, sulfonamide and trimethoprim, rifanpicin, metronidazole, quinolone, novobiocin, polymyxin, gramicidin or any salts or variants thereof. The antibiotic used will depend on the type of bacterial infection.

A therapeutically effective dosage of the pharmaceutical compositions according to the invention will depend upon the type and severity of the infection and whether the pharmaceutical composition is comprised of another active ingredient, such as an antibiotic. Generally, the therapeutically effective dose is the minimum amount sufficient to control biofilm or tooth decay formation and is non-toxic to the treated human or animal. Methods for determining effective dosages and toxicity are known in the art.

According to another aspect of the present invention, it is

A process for producing genetically modified cells for the production of STAT-LF peptides that inhibit biofilm or tooth decay formation is presented. Another aspect is the administration to a patient of genetically engineered cells for the production of STAT-LF peptides that inhibit the formation of biofilm or dental caries.

Another aspect of the present invention provides use in the preparation of a medicament for administration to a mammalian patient to improve oral health or to alleviate dental caries of genetically modified viable transfected cells for production of STAT-LF peptides that inhibit biofilm formation.

Antimicrobial Compositions

The STAT-LF peptides described above for use in the preparation of pharmaceutical compositions may also be used for the preparation of antimicrobial compositions, such as a liquid disinfectant solution. Such solutions may further comprise antimicrobial or antifungal agents such as alcohol, povidone iodine solution and antibiotics as well as preservatives. These solutions may be used, for example, as disinfectants of the skin or surrounding area prior to insertion or implantation of a device such as a catheter, a catheter fixer and / or lavage solutions such as antiseptic rinsing to any medical device, including but not limited to catheter components such as needles, Leur-Lok connectors, needleless connectors and hubs, as well as other implant devices. These solutions may also be used for coating or disinfecting surgical instruments, including, but not limited to, clamps, forceps, scissors, skin clips, tubes, needles, retractors, scalers, perforators, chisels, scrapers and saws.

Antimicrobial compositions for inhibiting biofilm formation may be comprised of any STAT-LF peptide, antisense peptides and antibodies described above.

An antimicrobial composition may further be comprised of additional ingredients, including but not limited to: a surfactant, an antiseptic and an antibiotic (see examples listed above). STAT-LF production in eukaryotic and prokaryotic cells

The nucleic acid molecules presented herein may be obtained from a cDNA collection. Nucleotide molecules may also be obtained from other sources known in the art, such as by directed sequence analysis of expressed in vitro synthesis. The DNA described in this application (including variants that are functional equivalents) may be introduced and expressed in a variety of eukaryotic and prokaryotic host cells. A recombinant nucleic acid molecule for STAT-LF contains suitable operably linked transcriptional or translational regulatory elements. Suitable regulatory elements are derived from various sources, and they can be readily chosen by one of ordinary skill in the art (Sambrook, J. Fritsch, E.E. & Maniatis, T. (Latest Edition). Molecular Cloning: a Laboratory Cold Spring Harbor Laboratory Press, New York; Ausubel et al (Latest Edition) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). For example, if a person needs to over-regulate the expression of the nucleic acid molecule, he could insert a sense sequence or silence the appropriate RNA and promoter in the vector. Promoters may be inducible or constitutive, environmentally regulated or developing, or cell or tissue specific. Transcription is enhanced with promoters known in the art for expression. CMV and SV40 promoters are commonly used to express desired peptides in cells. Other promoters known in the art may also be used (various suitable promoters and vectors are described in the patent applications referred to in this application).

If a person were to downregulate the expression of the nucleic acid molecule, he could insert the antisense sequence and appropriate promoter into the vehicle. A nucleic acid molecule may be isolated from a native source (in sense or antisense orientations), synthesized, or it may be a mutated or synthetic native sequence or a combination thereof.

Examples of regulatory elements include a transcriptional enhancement promoter or RNA polymerase binding sequence, a ribosomal binding sequence, including a translational initiation signal. Additionally, depending on the vector used, other genetic elements such as selectable markers may be incorporated into the recombinant molecule. Other regulatory regions that could be used include a promoter domain and a termination region. Regulatory elements may be of bacterial, fungal, viral or avian origin. Similarly, reporting elements may originate from plants, yeasts, insects, or other sources, including synthetically produced elements and mutated elements.

In addition to using the expression vectors described above,

A peptide may be expressed by inserting a recombinant nucleic acid molecule into a known expression system derived from bacteria, viruses, yeast, mammal, insect, fungus or bird. A recombinant molecule may be introduced into cells by techniques such as Agrobacterium tumefaciens-mediated transformation, particle bombardment-mediated transformation, direct absorption, microinjection, co-precipitation, transfection and electroporation, depending on the cell type. Retroviral vectors, adenoviral vectors, Adeno-associated virus (AAV) vectors, DNA virus vectors, and liposomes could all be used. Suitable grafts are inserted into an expression vector, which may also include markers for choosing transformed cells. A graft may be inserted into a site created by restriction enzymes.

In one embodiment of the invention, a cell is transfected with a nucleic acid molecule of the invention inserted into an expression vector to produce cells expressing a peptide encoded by the nucleic acid molecule.

Another embodiment of the invention relates to a method of transfecting a cell with a nucleic acid molecule set forth herein in an expression vector to produce a cell expressing the STAT-LF peptide [Figure 1] or another peptide of the invention. . In accordance with certain embodiments of the invention, a method for expressing the peptides presented in a cell is presented. A preferred process would include culturing a cell including a recombinant DNA vector including a STAT-LF encoding nucleic acid molecule [Figure 1] (or other nucleic acid molecule of the invention) in a culture medium such that the peptide is expressed. The process preferably further includes recovering the peptide from cells or the culture medium.

Probes

Certain embodiments of the present invention include probe probes.

oligonucleotides made from the cloned STAT-LF peptide molecules described in this application or from other nucleic acid molecules presented here (see Materials and Methods section). Probes may be 15 to 20 peptides in length. A preferred probe has at least 15-20 STAT-LF peptides in [Figure 1]. Certain embodiments of the invention also include at least 15 consecutive nucleotides of [Figure I]. Waves are useful for identifying nucleic acids encoding STAT-LF peptides as well as functionally equivalent STAT-LF peptides. Oligonucleotide probes are capable of hybridization of the sequence shown in [Figure 1] under stringent hybridization conditions. A nucleic acid molecule encoding a peptide presented herein may be isolated from other organisms by selecting a collection under moderate to high stringency hybridization conditions with a labeled probe. The activity of the peptide encoded by the nucleic acid molecule is evaluated by cloning and DNA expression. After the expression product is isolated, the peptide is tested for STAT-LF activity as described in this application.

Functionally equivalent STAT-LF peptide molecules from other cells, or cDNAs or synthetic DNAs encoding equivalent STAT-LF, can also be isolated by amplification using polymerase chain reaction (PCR) methods. Oligonucleotide primers, such as degenerate primers, based on [Figure 1] can be prepared and used with PCR and reverse transcriptase (ES 10 Kawasaki (1990), in Innis et al., Eds., PCR Protocols, Academic Press, San Diego, Chapter 3, p. 21) to amplify the functional equivalent DNAs of cDNA or genomic collections of other organisms. Oligonucleotides may also be used as probes for selecting cDNA collections.

Functionally equivalent peptides, peptides and proteins

The present invention includes not only the peptides encoded by the sequences presented herein, but also functionally equivalent peptides, peptides and proteins that exhibit the same or similar activity as the STAT-LF peptide as well as mimetics (Figure 1).

EXAMPLES

METHODS:

Two different experiments were performed to demonstrate the antimicrobial activity of the substances we tested. The first one was a disk diffusion choice essay that we developed in our lab. In that assay we created an overnight culture of S. mutans in a broth consisting of trypticase soybean and 20% sucrose. Cells were grown overnight and then germinated in confluence on Mitis Salivarius Bacitracin (MSB) agar. In addition, we used a 25 cm sterile filter paper disc and added 200 μΐ of whole saliva, the lysine-containing saliva or the lactoferrin arginine variant, this saliva was allowed to dry on the filter paper for 1 hour and The added protein-containing filter paper was then placed over the center of the S. mutans confluent culture on the MSB agar. This was then incubated at 37 ° C for 24h after which we examined the plate for a S. mutans growth inhibition zone around the filter paper disc. This assay was used to identify potential antimicrobial activity.

The second set of experiments consisted of growing an overnight S. mutans stock culture in a triticase soybean broth containing 20% sucrose (TSBS) which should produce approximately 1 x 108 cells per ml within a period of time. 24h We then intensely centrifuged this dense culture so that it was homogeneous and then removed a 100 μΐ portion of this culture and placed it in a new tube containing 9.8 ml of sterile TSBS into which we added 100 μΐ / ml of a single dose. 2 μg / ml of a) Lf lysine variant, b) Lf arginine variant, c) chlorhexidine [positive control], d) PBS [negative control]. This mixture was allowed to grow overnight. The following morning, a 100 μΐ portion was removed and placed on MSB agar, seeded on this agar which is then grown overnight and examined the next day for colony formation in units / ml. This set of experiments was used to compare and quantify the antimicrobial activity of Lf variants by standard MIC growth standards. These experiments were also performed by mixing bacterial cultures and test agents for 1 to 2h and then seeding overnight to determine agent activity after a short contact time period.

The third set of experiments used the substantivity assay, which demonstrates the ability of peptides to bind to hydroxyapatite (HA) (Figure 4). The test results are shown in figure 5.

Peptide Synthesis Method

5 The method used to synthesize peptides involves a

stepwise addition of protected amino acids that were added to a solid resin particle via covalent bonding. The procedure uses Fmoc synthetic chemistry. The average cycle time for an amino acid addition took about 60 minutes. The added amino acids are protected. The protecting groups used to protect the side chain functional groups are labile acids, while the amino acid N-terminal amino acid function is protected by the vulnerable Fmoc group at the base. The resin is in the form of 200 - 400 mesh porous beads which allows reagents to penetrate the gel mesh. Addition of amino acids 15 is done in a protected environment and then reagents and by-products are removed by filtration and recrystallization. The cultured peptide chain is completely protected in the insoluble solid phase. The peptide formed is identical to the samples prepared by the standard p-nitrophenyl ester procedure.

RESULTS:

The disk diffusion assay allows specific proteins to be added to a filter paper disk through which the protein can then be diffused into the agar to determine antimicrobial activity. The following peptides were added to the disc; R = 25 arginine form of Lf (Figure I (a)). K = lysine form of Lf (I (a). PBS = phosphate buffered saline which is used as the control solution and in which the R form or Lf form K is added. In Figure 1 below we add the R form, or K form at a concentration of 1 μg / ml to 200 μΐ PBS and then we add it to the surface of a 25 cm strip of filter paper and allow the strip to dry for 1 hour. a S. mutans culture for confluence on a tripicase soy agar plate containing 20% sucrose and then place the dry disc over the center of the plate.The cultures were allowed to grow in a 37 ° C regulated incubator and then 24 or 48h after inoculation.A zone of inhibition can be seen around the K-discs, where the lysine variant was suspended in PBS, and on K-disk, where the lysine was suspended in non-saliva. (which had an antibacterial activity prior to the addition of K). No zone was seen on the R / PB S or R / saliva disc where the arginine variant was replaced by the lysine variant (Figure 3). The R and K form of the Lf peptide was added at a concentration around 15 μg / ml.

Figure 4 shows that ST6 and STl 5 have no antimicrobial activity. In the bottom row of Figure 4, from left to right, it can be seen that the formulation of LF11 (Figure 1) inhibited S. mutans growth, seen as a clear zone around the disc. The fused LF that is fused to staterin 6, this fusion protein retains the activity of the mere 11 lactoferrin (Figure 4). Lf combined with ST15 has less activity than STAT-LF 6.

Claims (24)

Composition for inhibiting bacterial growth in the oral cavity, characterized in that it consists of a fusion protein composed of: i. a lysine form of a lactoferrin polypeptide or a functionally equivalent peptide molecule thereof; and ii. a staterin polypeptide or a functionally equivalent peptide molecule thereof. Composition according to Claim 1, characterized in that the fusion protein is substantially purified. Composition according to Claim 1, characterized in that the composition further comprises a pharmaceutically acceptable excipient. Composition according to Claim 1, characterized in that the composition further comprises a surfactant or a surfactant-like molecule. Composition according to Claim 4, characterized in that the surfactant is sodium lauryl sulphate. Composition according to Claim 5, characterized in that sodium lauryl sulfate is present in an amount effective to allow the composition to penetrate the biofilm. Composition according to Claim 1, characterized in that the fusion protein is further composed of an additional polypeptide sequence. Composition according to Claim 3, characterized in that the composition is a dosage form chosen from the group consisting of a food, a food additive, toothpaste, gels, toothpaste, mouthwash, dental floss, denture wash, denture adhesives, chewing gum, candy, chewing tablets and solution. Composition according to Claim 1, characterized in that it is further composed of an antibacterial agent, an antimicrobial agent, an antifungal agent or a combination thereof. A method for inhibiting bacteria in the oral cavity, which comprises administering an effective amount of a composition composed of a fusion protein comprising: i. a lysine form of a lactoferrin polypeptide or a functionally equivalent peptide molecule thereof; and ii. a staterin polypeptide or a functionally equivalent peptide molecule thereof to a patient in need thereof. A method for the treatment or prevention of dental caries, which comprises administering an effective amount of a composition consisting of a fusion protein composed of: i. a lysine form of a lactoferrin polypeptide or a functionally equivalent peptide molecule thereof; and ii. a staterin polypeptide or a functionally equivalent peptide molecule thereof to a patient in need thereof. A method for treating or preventing periodontal disease, which comprises administering an effective amount of a composition composed of a fusion protein consisting of: i. a lysine form of a lactoferrin polypeptide or a functionally equivalent peptide molecule thereof; and ii. a staterin polypeptide or a functionally equivalent peptide molecule thereof to a patient in need thereof. A method for treating or preventing dental plaque, characterized in that it is comprised of administering an effective amount of a composition consisting of a fusion protein composed of: a lysine form of a lactoferrin polypeptide or a functionally equivalent peptide molecule thereof; and ii. a staterin polypeptide or a functionally equivalent peptide molecule thereof to a patient in need thereof. A method for treating oral cavity inflammation associated with the presence of dental plaque, which comprises administering an effective amount of a composition composed of a fusion protein consisting of: i. a lysine form of a lactoferrin polypeptide or a functionally equivalent peptide molecule thereof; and ii. a staterin polypeptide or a functionally equivalent peptide molecule thereof to a patient in need thereof Method according to any one of claims 10 to 14, characterized in that the fusion protein is substantially purified. A method according to any one of claims 10 to 14, characterized in that the composition further comprises a pharmaceutically acceptable excipient. A method according to any one of claims 10 to 14, characterized in that the composition further comprises a surfactant or a surfactant-like molecule. Method according to claim 17, characterized in that the surfactant is sodium lauryl sulfate. Method according to claim 18, characterized in that the sodium lauryl sulfate is in an amount effective to allow the composition to penetrate the biofilm. Method according to any of claims a14, characterized in that the composition is administered by a dosage form chosen from the group consisting of a food, a food additive, toothpastes, gels, toothpaste, buccal solutions, dental floss, denture wash solution, denture adhesives, chewing gum, candy, chewable tablets and solution. A method according to any one of claims 10a14, characterized in that the composition further comprises an antibacterial agent, an antimicrobial agent, an antifungal agent or a combination thereof. Method according to any of claims a14, characterized in that the fusion protein is linked to an additional polypeptide sequence. 23. Composition for the inhibition of bacterial growth in the oral cavity, characterized in that it consists of a fusion protein having a SEQ ID NO. 10; SEQ ID NO. 11; or SEQ ID NO. 13, or a functionally equivalent peptide molecule thereof. 24. Fusion protein, characterized in that it has a SEQ ID NO. 10; SEQ ID NO. 11; or SEQ ID NO. 13, or a functionally equivalent peptide molecule thereof. <formula> formula see original document page 33 </formula> <213> Staterin peptide <400> 6 Gly Ala Thr Gly Ala Thr Gly Ala Thr Gly Ala Gly Ala Ala Ala Ala Ala <210> 7 <211> 15 <212 > PRT <213> Staterin Peptide <400> 7 Asp Asp Asp Glu Glu Lys Phe Leu Arg Arg Ile Gly Arg Phe Gly <210> 8 <211> 45 <212> Staterin Peptide <400> 8 gatgatgatg aagagaaatt tttgcgtaga attggaagat tcggt <210> 9 <211> 17 <212> PRT <213> Iactoferrin-Staterin fusion peptide <400> 9 Asp Asp Asp Glu Glu Lys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg <210> 10 <211 > 51 <212> DNA <213> Lactoferrin-Staterin fusion peptide <400> 10 gatgatgatg aagagaaatt ccaatggcaa aggaatatga gaaaagtgcg t <210> 11 <211> 51 <212> DNA <213> Lactoferrin-Staterin fusion peptide <400> 11 gatgatgatg aagaaaaatt ccaatggcaa aggaatatga gaaaagtgcg t <210> 12 <211> 26 <212> PRT <213> Lactoferrin-Staterin Fusion Peptide <400> 12 Asp Asp Asp Glu Glu Lys Phe Leu Arg Ar g Ile Gly Arg Phe Gly Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg <210> IB <211> 45 <212> DNA <213> Lactoferrin-Staterin Fusion Peptide <400> 13 gatgatgatg aagagaaatt tttgcgtaga attggaagat tcggt