Methods of increasing sensitivity of bacteria resistant to antibiotics and inhibiting biofilm formation

Abstract

The present application provides methods of increasing the sensitivity of antibiotic-resistant bacteria to an antibiotic to which the bacteria are resistant using stabilized amorphous calcium carbonate and therefore allows more efficient treatment of diseases caused by these bacteria. In addition, the application provides methods for inhibiting or preventing the formation of a biofilm. Further, the application is directed to methods of treating an oral bacterial infection using stabilized ACC, while the stabilized ACC is an active agent used.

Description

[0001] METHODS OF INCREASING SENSITIVITY OF BACTERIA RESISTANT TO ANTIBIOTICS AND INHIBITING BIOFILM FORMATION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates in part to methods of increasing sensitivity (reducing resistance) of bacteria resistant to antibiotics, methods for inhibiting a biofilm formation, and applications thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] The rapid emergence of resistant bacteria occurs s worldwide, endangering the efficacy of antibiotics, which have transformed medicine and saved millions of lives. Many decades after the first patients were treated with antibiotics, bacterial infections have again become a threat. The antibiotic resistance crisis has been attributed to the overuse and misuse of these medications, as well as a lack of new drug development by the pharmaceutical industry due to reduced economic incentives and challenging regulatory requirements. Infections associated with multidrug-resistant (MDR) bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Pseudomonas aeruginosa, and extended- spectrum beta-lactamase (ESBL)-producing Escherichia coli, still represent a major challenge because of the limited treatment options.

[0006] Several studies explored the effect of calcium comprising composition on bacterial growth and biofilm formation. Al-Azzawi (The Egyptian Journal of Hospital Medicine, January 2023, Vol. 90 (2), Page 2275-2282) discusses both the antibacterial and anti-biofilm formation by aggregated nanometric crystalline calcium carbonate (calcite, CaCOs) in the range of 30 to 40nm. Goctii et al (ACS Appl. Nano Mater. 2023, 6, 16286-16296) refer to selenite-incorporated magnesium-stabilized amorphous calcium carbonate (ACMC) nanoparticles as a sustainable antibacterial material.". US5882631 describes that the addition of porous calcium carbonate to the oral compositions makes it possible to prevent the decrease in the bactericidal activity of water-insoluble noncationic bactericides such as triclosan and improve the stability thereof while exerting excellent effects of eliminating dental plaque, preventing halitosis and eliminating tooth-staining substances. US2016015737 and WO2011152819 describe oral care composition comprising calcium carbonate.

[0007] Nevertheless, novel and effective means to treat antibiotic-resistant bacteria and improvement of oral care are needed. SUMMARY OF THE INVENTION

[0008] According to one aspect, the present invention provides a method of preventing or treating an oral infection, wherein the method comprises contacting the oral cavity with a composition comprising a stabilized amorphous calcium carbonate (stabilized ACC), wherein the stabilized ACC is the only active agent in the composition. In some examples, the present invention provides a topical oral composition comprising a stabilized amorphous calcium carbonate (stabilized ACC), for use in preventing or treating a disease of an oral cavity caused by an infection, wherein the method comprises contacting the oral cavity with the topical oral composition and wherein the stabilized ACC is the main active agent in the composition. According to some examples, stabilized ACC is the only active agent. According to some examples, the stabilized ACC acts as an anti-bacterial agent. According to some examples, the stabilized ACC provides a bacteriostatic effect. According to some examples, the stabilized ACC provides a bacteriocidic effect. According to some examples, the disease of the oral cavity is gum infection. According to some examples, the disease of the oral cavity is periodontitis. In some examples, the use comprises contacting the oral cavity with the topical oral composition for at least 30 seconds. In some examples, the topical oral composition is selected from an oral gel, ointment, and oral wash. According to some examples, the use further comprises a systemic administration of a composition comprising stabilized ACC. In some examples, the use prevents or attenuates the formation of a bacterial biofilm e.g. formation of a biofilm on teeth. According to some examples, topical oral composition in a form or liquid or semi-liquid composition comprising from 2 to 20 wt% of the stabilized ACC.

[0009] According to another aspect, the present invention provides a method of preventing or treating a disease of an oral cavity caused by an infection, wherein the method comprises contacting the oral cavity with the topical oral composition and wherein the stabilized ACC is the main active agent in the composition.

[0010] According to another aspect, the present invention provides a composition comprising a stabilized amorphous calcium carbonate (stabilized ACC) for use in treating an infection caused by antibiotic -resistant bacteria in a subject in need thereof, the use comprises administering to said subject composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent. In some examples, stabilized ACC is the only active agent. In further examples, the use comprises co- administering the composition and an antibiotic to which the antibiotic -resistant bacteria is resistant.

[0011] According to another aspect, the present invention provides a composition comprising a stabilized amorphous calcium carbonate (stabilized ACC), for use in improving the efficacy of the treatment of an infection caused by antibiotic-resistant bacteria, the use comprises administering a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent to a subject. In some examples, stabilized ACC is the only active agent. In further examples, the use comprises co-administering the composition and an antibiotic to which the antibiotic-resistant bacteria is resistant.

[0012] According to any one of the aspects, examples and embodiments of the invention, the composition may be an oral care composition or a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0013] According to one aspect, the present invention provides a method of enhancing the sensitivity of an antibiotic-resistant bacteria to an antibiotic to which the bacteria is resistant, the method comprises contacting the antibiotic -resistant bacteria with stabilized amorphous calcium carbonate (stabilized ACC).

[0014] According to another aspect, the present invention provides a method of treating an infection caused by antibiotic -resistant bacteria in a subject in need thereof, the method comprises administering to said subject an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent. According to some examples, the method comprises co-administering the stabilized ACC and an antibiotic to which the antibiotic-resistant bacteria is resistant.

[0015] According to a further aspect, the present invention provides a method of inhibiting the growth of antibiotic-resistant bacteria, the method comprises contacting the antibioticresistant bacteria with a stabilized amorphous calcium carbonate (stabilized ACC).

[0016] According to one aspect, the present invention provides a method of inhibiting or preventing the formation of a biofilm by bacteria, the method comprises contacting the bacteria with a composition comprising stabilized amorphous calcium carbonate (stabilized ACC). According to some examples, the bacteria is an antibiotic-resistant bacteria. According to one embodiment, the bacteria are located in the oral cavity.

[0017] According to any one of the above aspects and examples, the antibiotic-resistant bacteria is selected from multiantibiotic-resistant S. aureus, methicillin-dependent S. aureus, and Pseudomonas BAA2108. BRIEF DESCRIPTION OF DRAWINGS

[0018] Fig. 1 shows well- and HA-associated biofilm formation in the presence of ACC. Formed biofilms were stained with MTT and photographed.

[0019] Fig. 2. shows well- and HA-associated biofilm formation in the presence of ACC.

[0020] Fig. 3 shows the calculation of biofilm formation in the presence of ACC or CCC at different concentrations.

[0021] Fig. 4 shows the effect of ACC, CCC, and CaCh concentrations (mM) on the planktonic growth of MDRSA CI-M.

[0022] Fig. 5 shows the effect of CaCh, CCC, and ACC concentrations (mM) on the planktonic activity of MDRSA Cl-M. There is a clear correlation between the reduction of the ATP content and increased concentrations of ACC.

[0023] Fig. 6 shows the effect of ACC concentrations (mM) on the planktonic growth and biofilm formation of MDRSA CI-M. The ACC’s initial pH after mixing 4 stock solutions was 10.

[0024] Fig. 7 shows the effect of ACC concentrations (mM) on the planktonic growth and biofilm formation of MDRSA CI-M. ACC’s initial pH after mixing 4 stock solutions was adjusted to 7.

[0025] Fig. 8 shows the effect of CaCh concentrations (mM) on the planktonic growth and biofilm formation of MDRSA CI-M. The pH of CaCh was adjusted to 10. It is possible that at this pH the CaCh is converted to Ca(OH)2

[0026] Fig. 9 shows the effect of CaCh concentrations (mM) on the planktonic growth and biofilm formation of MDRSA CI-M. The initial pH of the CaCh was 5.

[0027] Fig. 10 shows the effect of CaCh concentrations (mM) on the planktonic growth and biofilm formation of MDRSA CI-M. CaCh initial pH 5 and TSBG adjusted to pH 9.

[0028] Fig. 11 shows the effect of CaCh, CCC and ACC on the biofilm formation of MDRSA CI- M.

[0029] Fig. 12 shows the effect of Density Kids™ and pHDirect™ (produced by Amorphical LTD) at different concentrations (mg / ml) on the planktonic growth of MDRSA CI-M.

[0030] Fig. 13 shows the effect of Density Kids and PH Direct suspensions at different concentrations (mg / ml) on biofilm formation of MDRSA CI-M.

[0031] Fig. 14 shows the effect of ACC at 2 and 5mM concentrations on the planktonic growth of MRS A in the presence of different Methicillin concentrations.

[0032] Fig. 15 shows a schematic representation of a tooth affected or not with parodontids and measured parameters. Figs. 16A and 16B show the effect of the treatment with ACC on CEJ-ABC distance (Fig. 16A) and Alveolar Bone Resorption (Fig. 16B). The figures show an average of all 3 molars (Ml, M2 and M3) in all experiments (i.e. 9 mice per group).

[0033] Fig. 16C and 16D show alveolar bone resorption area under M2 (Ligated Molar) (Fig. 16C shows results per each experiment and Fig. 16D shows the average of the experiments).

[0034] Fig. 16E shows CEJ-ABC distance by Molar (Avg of Exp#l+2+3).

[0035] Fig. 17 shows known CEJ-ABC Distance in untreated mice.

[0036] Fig. 18 shows an arrangement of an experiment demonstrating the effect of ACC in the treatment of Periodontitis on 28 mice using Periodontitis Model (Ligature).

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of conflict, the patent specification, including definitions, will control.

[0039] The present invention is based on an unexpected observation that stabilized amorphous calcium carbonate (stabilized ACC) increases the sensitivity of antibiotic -resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), to antibiotics to which they are resistant (in the case of MRSA to methicillin). This allows to revive and use these antibiotics, what significantly increases the chances to cure infections caused by these bacteria. An additional unexpected observation is that stabilized ACC inhibited the formation of a biofilm formed by bacteria, which makes it a potent disinfectant. Further, it was shown that a topical oral formulation of stabilized ACC (a gel) effectively treated periodontitis in a mouse model, much more effectively than crystalline calcium carbonate (CCC) or sodium carbonate.

[0040] According to one aspect, the present invention provides a method of enhancing the sensitivity of an antibiotic-resistant bacteria to an antibiotic, the method comprises exposing the antibiotic -resistant bacteria to a stabilized amorphous calcium carbonate (stabilized ACC). According to one embodiment, the antibiotic is an antibiotic to which the bacteria is resistant.

[0041] Therefore, according to some embodiments, the present invention provides a method of enhancing sensitivity of antibiotic -resistant bacteria to an antibiotic to which the bacteria is resistant, the method comprises contacting the antibiotic -resistant bacteria with stabilized amorphous calcium carbonate (stabilized ACC). All terms, embodiments and definitions disclosed in any one of the above or below aspects apply and are encompassed herein as well.

[0042] The term "antibiotic -resistant bacteria" is used herein as well-known in the art and refers to bacteria acquired or exhibiting resistance to a particular antibiotic. According to some embodiments, the antibiotic -resistant bacteria is selected from methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Staphylococcus aureus (MDRSA), Pseudomonas BAA2108, multidrug-resistant Pseudomonas aeruginosa and extended- spectrum beta-lactamase (ES BL) -producing Escherichia coli. According to some embodiments, the antibiotic-resistant bacteria is as described in any one of the aspects and embodiments of the invention. According to some embodiments, the antibiotic -resistant bacteria is methicillin-resistant Staphylococcus aureus (MRSA). According to some embodiments, the antibiotic -resistant bacteria is multidrug-resistant Staphylococcus aureus.

[0043] As used herein, the terms "antibiotic sensitivity" and "antibiotic susceptibility" are used herein interchangeably and refer to the degree to which a target microorganism is killed or inhibited by antibiotics, and microorganisms with low or no antibiotic sensitivity, that is, microorganisms with antibiotic resistance, are of public health or clinical importance. The antibiotic includes, for example, amikacin, amoxicillin, ampicillin, aztreonam, benzylpenicillin, calburanic acid (Clavulanic Acid), cefazolin), Cefepime, Cefotaxime, Cefotetan, Cefoxitin, Cefpodoxime, Ceftazidime, Ceftriaxone, Cefuroxime (Cefuroxime), Ciprofloxacin, Dalfopristin, Doripenem, Daptomycin, Ertapenem, Arithromycin,

[0044] Gentamicin Imipenem), Levofloxacin, Linezolid, Meropenem, Minocycline, Moxifloxacin, Nitrofurantoin, Norfloxacin, Piperacillin, Quinupristin, Rifampicin, Streptomycin,

[0045] Sulbactam, Sulfamethoxazole, Telithromycin, Tetracycline, Ticarcillin (Ticarcillin), tigecycline, tobramycin, trimethoprim, and vancomycin, but is not limited thereto. In some embodiments, the terms antibiotic sensitivity and antibiotic susceptibility are used interchangeably. According to some embodiments, enhancing sensitivity to an antibiotic comprises reducing the minimal inhibitory concentration (MIC) of the antibiotic.

[0046] As used herein, the term "minimal inhibitory concentration" (MIC) refers to the lowest concentration of an antimicrobial agent (e.g., a peptide as described herein) required to prevent growth or otherwise modify a function of a microorganism under certain conditions, for example in liquid broth medium, and can be determined for a number of different microorganisms according to standard techniques well known in the art. The MIC may be determined by any known method, e.g. as described above with respect to determining the sensitivity of bacteria to antibiotics. The term “enhancing” as used herein refers to promoting, improving, augmenting, typically increasing the parameters, i.e. sensitivity or susceptibility to an antibiotic, and is measured relatively to a control sample. The sensitivity of bacteria to antibiotics may be measured by any method known in the art. Non-limiting examples of such a method are Disk Diffusion Method (Kirby-Bauer Test), Broth Microdilution Method, Agar Dilution Method, E-test (Epsilometer Test), and Time-Kill Assay. The terms "enhancing sensitivity" and "reducing resistance" may be used herein interchangeably.

[0047] According to one embodiment, the enhancing sensitivity comprises enhancing by from 10% to 100%, from 20% to 90%, from 30% to 80%, from 40% to 70% or from 50 to 60% the sensitivity of the antibiotic. According to another embodiment, the enhancing sensitivity comprises enhancing from 5% to 25%, from 25% to 50% from 50% to 75% or from 75% to 100% sensitivity of the antibiotic. According to a further embodiment, the enhancing sensitivity comprises conferring at least 1.5, at least 2, at least 2.5, at least 3, at least 5, or at least 10-fold higher sensitivity to the antibiotic.

[0048] According to yet another aspect, the present invention provides a method of treating an infection caused by antibiotic-resistant bacteria in a subject in need thereof, the method comprises administering to said subject a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent. According to some embodiments, the present invention provides stabilized amorphous calcium carbonate (stabilized ACC) for use in treating an infection caused by antibiotic-resistant bacteria in a subject in need thereof, the method comprises administering to said subject a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent. According to some embodiments, the present invention provides a composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC) for use in treating a bacterial infection, the method comprises administering to said subject a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent. All terms, embodiments and definitions disclosed in any one of the above or below aspects apply and are encompassed herein as well.

[0049] The term “therapeutically effective amount” of a drug or agent is an amount of a drug or an agent that, when administered to a subject will have the intended therapeutic effect, e.g. antibacterial effect. The full therapeutic effect does not necessarily occur by the administration of one dose and may occur only after the administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The precise effective amount needed for a subject will depend upon, for example, the subject's size, health and age, the nature and extent of the cognitive impairment, and the therapeutics or combination of therapeutics selected for administration, and the mode of administration. The skilled person can readily determine the effective amount for a given situation by routine experimentation.

[0050] According to some embodiments, the antibiotic -resistant bacteria is selected from methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Staphylococcus aureus (MDRSA), multidrug-resistant Pseudomonas aeruginosa and extended- spectrum beta-lactamase (ES BL) -producing Escherichia coli. According to some embodiments, the antibiotic-resistant bacteria is a methicillin-resistant Staphylococcus aureus (MRSA) and the antibiotic is methicillin. According to some embodiments, the antibiotic -resistant bacteria is multidrug-resistant Staphylococcus aureus (MDRSA) and the antibiotic is selected from methicillin, penicillin, erythromycin, gentamicin and norfloxacin.

[0051] The term “treating” a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, ameliorating, abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and / or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).

[0052] According to some embodiments, the use and method comprises administering the stabilized ACC to the subject. The term "administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. According to some embodiments, the compound or an agent can be administered locally to the mouth cavity and has a local effect. According to some embodiments, the compound or an agent may be administered systemically and have a systemic effect. For example, a compound or an agent can be administered intravenously, arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), topically, intranasally (by inhalation), intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). According to some embodiments, stabilized ACC is administered orally. According to some embodiments, stabilized ACC is administered sublingually. According to some embodiments, stabilized ACC is administered via inhalation. According to some embodiments, stabilized ACC is administered buccally. According to some embodiments, stabilized ACC is administered via enteric encapsulation (i.e., passing the stomach without being decomposed by the gastric acid). According to some embodiments, stabilized ACC is administered by a combination of two of oral, sublingual, buccal and via inhalation modes. A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. According to some embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a day. According to other embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a month. In some embodiments, the administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and / or who provides a patient with a prescription for a drug is administering the drug to the patient.

[0053] According to some embodiments, the use and method comprise co-administering the stabilized ACC or the composition comprising stabilized ACC and an antibiotic to which the antibiotic -resistant bacteria is resistant.

[0054] Co-administration of the compounds is performed in a regimen selected from a single combined composition, separate individual compositions administered substantially at the same time, and separate individual compositions administered under separate schedules and include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “sequential manner” refers to an administration of two compounds at different times, and optionally in different modes of administration. The agents can be administered sequentially in either order. The term “coadministration” encompasses the administration of a first and second agent in an essentially simultaneous manner, such as in a single dosage form, e.g., a capsule or tablet having a fixed ratio of first and second amounts, or in multiple dosage forms for each. The agents can be administered sequentially in either order. When coadministration involves the separate administration of each agent, the agents are administered sufficiently close in time to have the desired effect (e.g., complex formation). The term “substantially simultaneous manner” refers to the administration of two compounds with only a short time interval between them. In some embodiments, the time interval is in the range of from 0.5 to 60 minutes. According to some embodiments, the present invention provides a composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC) for use in treating an infection caused by antibiotic -resistant bacteria in a subject in need thereof, the use comprises administering to said subject a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent and administering an antibiotic to which the bacteria is resistant.

[0055] In some embodiments, the present invention provides a composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC), for use in improving the efficacy of a treatment of an infection caused by antibiotic -resistant bacteria, the use comprises administering a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent to a subject. In some embodiments, the use comprises co-administering the composition and an antibiotic to which the antibiotic -resistant bacteria is resistant.

[0056] According to some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0057] According to another embodiment, the present invention provides stabilized amorphous calcium carbonate (stabilized ACC) for use in a method for improving the efficacy of the treatment of an infection caused by antibiotic-resistant bacteria, the method comprises administering to a subject a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0058] According to any one of the above aspects and embodiments, the antibiotic -resistant bacteria is selected from multiantibiotic-resistant S. aureus, methicillin-resistant S. aureus, vancomycin-resistant Enterococcus (VRE), multi-drug-resistant Mycobacterium tuberculosis (MDR-TB), carbapenemase-producing Enterobacterales (CPE), and Pseudomonas BAA2108, multiantibiotic -resistant Clostridioides difficile, multiantibiotic - resistant Neisseria gonorrhoeae, multiantibiotic-resistant Acinetobacter baumannii, multiantibiotic -resistant Streptococcus pneumoniae, third-generation cephalosporin- resistant Enterobacterales, Carbapenem-resistant Acinetobacter baumannii, Rifampicin- resistant Mycobacterium, tuberculosis, Fluoroquinolone -resistant Salmonella Typhi, Fluoroquinolone-resistant Shigella spp., Vancomycin-resistant Enterococcus faecium, Carbapenem-resistant Pseudomonas aeruginosa, Fluoroquinolone-resistant non- typhoidal Salmonella, Third-generation cephalosporin Neisseria gonorrhoeae, fluoroquinolone-resistant Neisseria gonorrhoeae, Macrolide-resistant Group A Streptococci, Macrolide-resistant Streptococcus pneumoniae, Ampicillin-resistant Haemophilus influenzae, Penicillin -resistant Group B Streptococci, Vancomycin- resistant Enterococcus (VRE), Extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales, Multidrug-resistant (MDR) Pseudomonas aeruginosa, Penicillin- resistant Enterococcus, Linezolid-resistant Enterococcus.

[0059] According to some embodiments, the antibiotic -resistant bacteria is methicillin- resistant S. aureus and the antibiotic is methicillin. According to some embodiments, the antibiotic-resistant bacteria is vancomycin-resistant Enterococcus (VRE) and the antibiotic is vancomycin.

[0060] According to another aspect, the present invention provides a method of inhibiting the growth of antibiotic-resistant bacteria, the method comprises contacting or exposing the antibiotic-resistant bacteria with stabilized amorphous calcium carbonate (stabilized ACC). All terms, embodiments, and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.

[0061] According to yet another aspect, the present invention provides a method of inhibiting or preventing the formation of a biofilm by bacteria, the method comprises contacting the bacteria with stabilized amorphous calcium carbonate (stabilized ACC). In some embodiments, the present invention provides stabilized amorphous calcium carbonate (stabilized ACC) for use in inhibiting or preventing the formation of a biofilm by bacteria. In some embodiments, the present invention provides a composition comprising stabilized amorphous calcium carbonate (stabilized ACC) for use in inhibiting or preventing the formation of a biofilm by bacteria. The content of the composition is as described in the present invention.

[0062] All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well. The term "biofilm", as used herein, refers to microbial growth formed by the attachment of microorganisms to surfaces and the subsequent development of multiple layers of cells. According to some embodiment, bacteria in the biofilm secretes extracellular polysaccharides. According to some embodiments, the stabilized ACC or composition comprising same inhibits the secretion of the polysaccharides. According to some embodiments, the bacteria is an antibiotic -resistant bacteria. According to some embodiments, the method is in vitro method. According to some embodiments, the bacteria are located in an oral cavity. According to some embodiments, the method comprises inhibiting or preventing the formation of a biofilm by bacteria on a dental surface. According to some embodiments, the prevention of biofilm formation comprises inhibition of the formation of the organic polymeric binding compositions that are critical to the adhesion of the bacteria to a tissue or teeth and provide a growing bed for enhanced culturing and activity of the bacteria.

[0063] According to yet another aspect, the present invention provides a method of preventing or treating an oral infection, wherein the method comprises contacting the oral cavity with a composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC), wherein the stabilized ACC is the main active agent. In some embodiments, the present invention provides a stabilized amorphous calcium carbonate (stabilized ACC), for use in a method of preventing or treating an oral infection, wherein the method comprises contacting the oral cavity with a composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC), wherein the stabilized ACC is the main active agent in the composition. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well. According to further embodiments, the present invention provides a topical oral composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC), for use in preventing or treating a disease of an oral cavity caused by an infection, wherein the method comprises contacting the oral cavity with the topical oral composition and wherein the stabilized ACC is the main active agent in the composition.

[0064] The terms "topical oral composition" and "oral care composition" are used herein interchangeably. Typically, the term “topical oral composition” is a formulation comprising an active agent that is applied to the interior of the mouth, though is not swallowed. The composition according to this invention may therefore be any appropriate type of topical oral formulation, including a solution, suspension, dispersion, mouthwash, oral gel, paste, ointment, chewable tablets, dissolvable tablets, capsules, stickers, small towels including the same and the like. According to some embodiments, the active agent is an antibiotic agent and therefore, the topical oral composition is a topical oral antibiotic composition.

[0065] As used herein, an “oral care composition” refers to a composition for which the intended use includes oral care, oral hygiene, and / or oral appearance, or for which the intended method of use comprises administration to the oral cavity, and refers to compositions that are palatable and safe for topical administration to the oral cavity, and for providing a benefit to the teeth and / or oral cavity. The term “oral care composition” thus specifically excludes compositions which are highly toxic, unpalatable, or otherwise unsuitable for administration to the oral cavity. In some embodiments, an oral care composition is not intentionally swallowed but is rather retained in the oral cavity for a time sufficient to affect the intended utility.

[0066] According to some embodiments, the topical oral composition comprises an orally acceptable carrier. As used herein, the term “orally acceptable carrier” refers to any vehicle useful in formulating the oral care compositions disclosed herein. The orally acceptable carrier is not harmful to a mammal in amounts disclosed herein when retained in the mouth, without swallowing, for a period sufficient to permit effective contact with a dental surface as required herein. In general, the orally acceptable carrier is not harmful even if unintentionally swallowed. As used herein, the term “orally acceptable” refers to a material that is safe and palatable at the relevant concentrations for use in an oral care formulation, such as a mouthwash or dentifrice. Suitable orally acceptable carriers include, for example, one or more of the following: water, a thickener, a buffer, a humectant, a surfactant, an abrasive, a sweetener, a flavorant, a pigment, a dye, an anti-caries agent, an anti-bacterial, a whitening agent, a desensitizing agent, a vitamin, a preservative, an enzyme, and mixtures thereof.

[0067] The oral care composition is further defined as a product which, during the normal course of usage, is not for the purpose of systemic administration of particular therapeutic agents intentionally swallowed, but is rather retained in the oral cavity for a time sufficient to contact substantially all of the dental surfaces and / or oral tissues for the purposes of oral activity. Examples of such oral care compositions include, but are not limited to dentifrice, mouthwash, mouth rinse, topical oral gel, denture cleanser, dental strips, beads, varnish, chewing gum, lozenges, toothpowder and the like.

[0068] As used herein, the terms “mouthwash” or "mouth rinse" refer to oral care compositions that are substantially liquid in character, such as a spray, or rinse. In such a preparation, the oral care composition typically has an aqueous phase comprising water or a water and alcohol mixture. Further, in various embodiments, the oral care composition includes a humectant and surfactant as described below.

[0069] As used herein, the term “topical oral gel” refers to a mixture prepared and immediately transferred into a retaining tray, such as those used in holding whitening gels, and the person can wear the tray for an effective period of time. The teeth that are in contact with the mixture will be treated. For use with retaining trays, the mixture can be in the form of a low- viscosity liquid or a gel. In certain embodiments, the whitening agent of the disclosure is formulated in an oral care composition comprising crosslinked copolymer of polyacrylic acid (Carbopol® polymer), glycerin and water. According to some embodiments, the disease of an oral cavity is caused by an infection. According to some embodiments, the disease of the oral cavity is caused by a bacterial infection. Any known bacterial infection that causes oral disease is contemplated. Non-limiting examples of such bacteria are Streptococcus mutans, Streptococcus sobrinus, Lactobacillus species, and Actinomyces species causing Tooth Decay (Dental Caries); Periodontal Diseases (Gum Diseases) are typically caused by Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, or Fusobacterium nucleatum. Additional examples of bacteria that cause oral diseases are Enterococcus faecalis, Prevotella intermedia, Pep to streptococcus species, Staphylococcus aureus, Streptococcus species, Streptococcus species (e.g., S. viridans), Staphylococcus aureus, Bacteroides species, and fusobacterium species. Thus, according to some embodiments, the present invention provides a topical oral composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC), for use in preventing or treating a disease of an oral cavity caused by a bacterial infection, wherein the method comprises contacting the oral cavity with the topical oral composition and wherein the stabilized ACC is the main active agent in the composition. The term "contacting the oral cavity" has also the meaning of applying the composition to any of the parts of the oral cavity such as gums or teeth.

[0070] The term “oral cavity” is understood to mean the cavity of the mouth, and includes the inner upper and lower lips, all parts of the inner cheek, the sublingual area under the tongue, the tongue itself, the upper and lower gums, the hard and soft palate and teeth. According to some embodiments, treatment of the disease in oral cavity comprises treatment or prevention of the disease of teeth. According to some embodiments, treatment or prevention of the disease in oral cavity comprises treatment or prevention of the disease of gums. According to some embodiments, the treatment or prevention of the disease in oral cavity comprises treatment or prevention of periodontitis.

[0071] The term "oral infection" or "oral disease" and "disease of an oral cavity" as used herein refers to oral conditions considered to be unhealthy and can be dental caries; dental plaque; gingivitis; periodontal diseases; mucosal infections; oral and pharyngeal cancers; and precancerous legions. According to some embodiments, oral disease is selected from dental caries, dental plaque, Dental calculus gingivitis, peri-implant mucositis and periimplantitis and periodontal diseases. According to some embodiments, the disease of the oral cavity is periodontitis. The term “dental plaque” refers to a fine film comprised of remains of food, bacteria and saliva. If dental plaque is not eliminated regularly, it sticks to the surface of the enamel, attacking tooth structures and producing a series of substances that cause gum swelling, giving rise to gingivitis (inflammation of the gum), redness and probable gingival bleeding.

[0072] The term “dental calculus” or “tartar” refers to a form of hardened dental plaque. It is caused by the precipitation of minerals from saliva and gingival crevicular fluid in plaque on the teeth. This process of precipitation kills the bacterial cells within dental plaque, but the rough and hardened surface that is formed provides an ideal surface for further plaque formation. This leads to calculus buildup, which compromises the health of the gingiva (gums). Calculus can form both along the gumline, where it is referred to as supragingival, and within the narrow sulcus that exists between the teeth and the gingiva, where it is referred to as subgingival.

[0073] The term “gingivitis” refers to an inflammation of the gums that gives rise to an increase in sensitivity and irritation and which in many cases is accompanied by bleeding. Its most frequent trigger is dental plaque or biofilm, consisting of a fine film comprised of remains of food, bacteria and saliva. If it is not treated in time, it can evolve to periodontitis (pyorrhoea), and even enter the tooth and damage its bone structure, leading to the irreversible loss of the tooth.

[0074] The term “periodontitis” refers to an inflammatory gingival process that spreads to the periodontal ligament which holds and fixes the tooth, and even the alveolar bone, destroying both tissues. Periodontal bags appear, which are an increase in the gap or space between the surface of the tooth and the internal part of the gum. This destructive process spreads until tooth stability is compromised. Due to the loss of fixation, in the end stages the teeth become separated and loose, losing their normal location, and finally, the tooth irremediably falls out. Streptococci, spirochetes and bacteroides are found to be the possible pathogens responsible for the disease. According to some embodiments, treating periodontitis comprises killing or inhibiting at least one of the following bacteria: Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Parvimonas micra, Campylobacter rectus, Eikenella corrodens, Streptococcus intermedius or Prevotella intermedia.

[0075] The term “caries” or “cavities” refers to the breakdown of tooth structure due to acids produced by bacteria. Streptococcus mutans is considered one of the main bacteria involved in this process. The term “peri-implant mucositis” refers to an inflammatory lesion of the soft tissues surrounding an endo-osseous implant in the absence of loss of supporting bone or continuing marginal bone loss. Peri-implant mucositis is caused by bacteria from dental biofilms, which disrupts the host-microbe homeostasis at the implant-mucosa interface, resulting in an inflammatory lesion. Biofilm removal is a prerequisite for the prevention and management of peri-implant mucositis. Peri-implant mucositis is considered a precursor for peri- implantitis.

[0076] The term “periimplantitis” refers to an inflammatory lesion of the mucosa that affects the surrounding tissue of the implant, predominately the supporting bone with loss of osseointegration. As peri-implant mucositis, it is also caused by the adhesion of pathogenic bacterial biofilms on the implant surface and peri-implant tissues.

[0077] According to some embodiments, the use comprises contacting the oral cavity with the topical oral composition or applying the topical oral composition to a surface of the oral cavity for at least 30 seconds. According to some embodiments, the use comprises contacting the oral cavity with the topical oral composition for at least 45 seconds. According to some embodiments, the use comprises contacting the oral cavity with the topical oral composition for at least 1, 2 or 5 minutes. According to some embodiments, the use comprises applying the oral cavity with the topical oral composition 1, 2, 3, 4, 5 or 6 times a day. According to some embodiments, the use comprises applying the oral cavity with the topical oral composition for from 1, 2, 3, 4, 5, 6 or 7 days, for 1, 2, 3, or 4 weeks, for 1, 2, 3, 4, 5 or 6 months.

[0078] According to some embodiments, the topical oral composition may be any known topical oral composition. According to some embodiments, the topical oral composition is liquid such as oral wash. According to some embodiments, the topical oral composition is semi-liquid such as gels and ointments. According to some embodiments, the topical oral composition is solid such as powders (e.g. powder rub). Thus, in some embodiments, the topical oral composition is selected from an oral gel, ointment, powder and oral wash. According to some embodiments, the topical oral composition is a gel. According to some embodiments, the topical oral composition is an adhesive gel, e.g. as described in the Examples.

[0079] As a function of the formulation, the topical oral composition may comprise from 2 to 100 wt% of stabilized ACC. Typically, solid compositions comprise a higher content of stabilized ACC. According to some embodiments, the stabilized ACC constitutes at least 50 wt% of the composition. According to some embodiments, the stabilized ACC constitutes at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt% at least 75 wt%, at least 80 wt%, at least 85 wt%, at least 90 wt% or at least 95 wt% of the composition. The liquid or semi-liquid composition comprises lower amount of stabilized ACC. According to some embodiment, the liquid or semi-liquid composition topical oral composition such as gels comprises from 2 to 30 wt% of the stabilized ACC. According to some embodiment, the topical oral composition comprises from 2 to 25 wt% of the stabilized ACC. According to some embodiment, the topical oral composition comprises from 2 to 20 wt% of the stabilized ACC. According to some embodiment, the topical oral composition comprises from 5 to 15 wt% of the stabilized ACC. According to some embodiment, the topical oral composition comprises from 7 to 12 wt% of the stabilized ACC. According to some embodiment, the topical oral composition comprises about 10 wt% of the stabilized ACC. According to some embodiment, the topical oral composition comprises from 20 to 50 wt% of the stabilized ACC. According to some embodiment, the topical oral composition comprises from 25 to 45 wt% of the stabilized ACC. According to some embodiment, the topical oral composition comprises from 30 to 40 wt% of the stabilized ACC.

[0080] According to some embodiments, the stabilized ACC is the main active agent. According to some embodiments, the stabilized ACC is the only active agent in the composition. According to some embodiments, the stabilized ACC is the only antibacterial active agent in the composition. According to some embodiments, the composition consists of stabilized ACC. According to some embodiments, the moiety is an active moiety. The terms "active agent" and “active moiety” are used herein interchangeably and refer to an agent that has biological activity, pharmacological effects and / or therapeutic utility. In some embodiments, the active agent has an antibacterial activity. According to some embodiments, antibacterial activity refers to bacteriostatic and / or bacteriocidic activity. The term "main active agent" has a meaning that stabilized ACC is accounted for at least 50% of the effect. According to some embodiments, stabilized ACC is responsible for at least 70% of the effect. According to some embodiments, stabilized ACC is responsible for at least 80% of the effect. According to some embodiments, stabilized ACC is responsible for at least 90% of the effect. In other examples, the term "main active agent" has the meaning that the amount of the active agent is present in the largest amount of other active agents. According to some embodiments, stabilized ACC constitutes at least 50% of all active agents. According to some embodiments, stabilized ACC constitutes at least 60% of all active agents. According to some embodiments, stabilized ACC constitutes at least 70% of all active agents. According to some embodiments, stabilized ACC constitutes at least 80% of all active agents. According to some embodiments, stabilized ACC constitutes at least 85% of all active agents. According to some embodiments, stabilized ACC constitutes at least 90% of all active agents.

[0081] According to some embodiments, the use of the topical oral composition further comprises a systemic administration of a composition comprising stabilized ACC. According to some embodiments, such systemic administration comprises oral administration, administration via inhalation or any other known methods of administering. Therefore, according to some embodiments, the present invention provides a topical oral composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC), for use in preventing or treating a disease of an oral cavity caused by a bacterial infection, wherein the method comprises contacting the oral cavity with the topical oral composition, wherein the stabilized ACC is the main active agent in the composition, wherein the method further comprises oral systemic administration of a composition comprising stabilized ACC.

[0082] According to some embodiments, the use prevents or attenuates the formation of a bacterial biofilm on the surface of the oral cavity. According to some embodiments, the use prevents or attenuates the formation of a bacterial biofilm on the surface of teeth. According to some embodiments, the disease or condition is periodontitis and the use prevents or attenuates the formation of a bacterial biofilm on the surface of teeth.

[0083] According to any one of the aspects and embodiments of the present invention, the terms "stabilized ACC" and "stabilized amorphous calcium carbonate" refer to an amorphous calcium carbonate comprising a stabilizing agent as part of the stabilized ACC and wherein the calcium carbonate is present in an amorphous form. The term "stable" as used herein indicates that the calcium carbonate is maintained in the amorphous form for a long period of time, for example for about at least 7 days in the solid form having less than or about 30% crystalline calcium carbonate. According to any one of the above embodiments, the composition is stable for at least 7 days. According to some embodiments, the composition is stable for at least 1 month. According to other embodiments, the composition is stable for at least 3 months. According to a further embodiment, the composition is stable for 6 months. According to certain embodiments, the composition is stable for at least 1 year. According to a particular embodiment, the composition is stable for at least 2 years. According to some embodiments, ACC is stable in amorphous form for at least 7 days, at least 1 month, at least 3 months or 6 months in an aqueous solution. ACC Stabilizers

[0084] The stabilizer may comprise an organic molecule having one or more functional groups selected from, but not limited to, hydroxyl, carboxyl, ester, amine, phosphino, phosphono, phosphate, sulfonyl, sulfate or sulfino groups. The hydroxy bearing compounds, combined with the hydroxide, optionally also bear other functions like carboxyl, etc. but with the hydroxyl not being esterified.

[0085] According to some embodiments, the stabilizer has low toxicity or no toxicity to mammalian cells or organism, and in particular to a human being. According to some embodiment, the stabilizer is of food, nutraceutical or pharmaceutical grade.

[0086] In certain embodiments, the ACC stabilizing agent is independently at each occurrence, an organic acid, phosphorylated, phosphonated, sulfated or sulfonated organic compound, phosphoric or sulfuric ester of a hydroxyl carboxylic acid, an organoamine compound, an organic compound comprising a hydroxyl, an organophosphorous compound or salts thereof, phosphorylated amino acids and derivatives thereof, a bisphosphonate, triphosphonate, or tetraphosphonate compound, an organophosphate compound (e.g., phytic acid, AMP, ADP and ATP), an organophosphonate compound, an inorganic phosphorous acid, an organic compound having multiple functional groups as defined above, an inorganic phosphate and polyphosphate compound, an organic compound having a polyphosphate chain, an organic surfactant, a bio-essential inorganic ion, salts thereof or any combination thereof.

[0087] According to some embodiments, the stabilizer is an organic acid or salt thereof. According to certain embodiments, the organic acid is selected from ascorbic acid, citric acid, lactic acid, acetic acid, oxalic acid, malonic acid, glutaconic acid, succinic acid, maleic acid, lactic acid, aconitic acid, or salts thereof and optionally include compounds having at least two carboxylic groups and molecular weight not larger than 250g / mol, such as citric acid, tartaric acid, malic acid, etc. According to one particular embodiment, the stabilizer is citric acid or a citrate salt

[0088] In another embodiment, the phosphoric ester of hydroxyl carboxylic acids is a phosphoenolpyruvate. In another embodiment, the phosphoric or sulfuric esters of hydroxyl carboxylic acids comprise amino acids. Examples of such esters are phosphoserine, phosphothreonine, sulfoserine, sulfothreonine and phosphocreatine.

[0089] The hydroxyl bearing compounds combined with hydroxide may comprise, for example, mono-, di- tri-, oligo-, and polysaccharides like sucrose or other polyols like glycerol. The hydroxyl bearing compounds may further comprise hydroxy acids like citric acid, tartaric acid, malic acid, etc., or hydroxyl-bearing amino acids such as serine or threonine and salts thereof. Each possibility represents a separate embodiment, of the present invention.

[0090] Some specific unlimited examples of such ACC stabilizers that include phytic acid, citric acid, and salts thereof, sodium pyrophosphate dibasic, adenosine 5 '-monophosphate (AMP) sodium salt, adenosine 5 '-diphosphate (ADP) sodium salt and adenosine 5'- triphosphate (ATP) disodium salt hydrate, phosphoserine, phosphorylated amino acids, food-grade surfactants, sodium stearoyl lactylate, and combinations thereof.

[0091] According to some embodiments, the stabilizer comprises at least one component selected from phosphoric or sulfuric esters of hydroxyl carboxylic acids, such as phosphoenolpyruvate, phosphoserine, phosphothreonine, sulfoserine or sulfothreonine and hydroxyl bearing organic compounds, selected from mono-, di-, tri-, oligo- and polysaccharides, for example, sucrose, mannose, glucose.

[0092] The hydroxyl bearing compound may further comprise at least one alkali hydroxide, such as sodium hydroxide, potassium hydroxide and the like. The phosphorylated acids may be present in oligopeptides and polypeptides. In other embodiments, of the invention, the stabilizer is an organic acid selected from monocarboxylic acid or multiple carboxylic acid, e.g., dicarboxylic acid or tricarboxylic acid. Each possibility represents a separate embodiment of the invention. The organic acid may be as defined above.

[0093] In some embodiments of the invention, the ACC stabilizer is selected from phosphorylated amino acids, polyols and combinations thereof. In some embodiments, the stable ACC comprises a phosphorylated compound as a stabilizer wherein the phosphorylation is performed on the hydroxyl group of an organic compound. In some embodiments, the stable ACC comprises a stabilizer selected from the group consisting of citric acid, phosphoserine, phospho threonine and combinations thereof. The non-limiting examples of stabilizers containing phosphate, phosphite, phosphonate groups and salts or esters thereof include phytic acid, dimethyl phosphate, trimethyl phosphate, sodium pyrophosphate, tetraethyl pyrophosphate, ribulose bisphosphate, etidronic acid and other medical bisphosphonates, 3 -phosphoglyceric acid salt, glyceraldehyde 3-phosphate, 1- deoxy-D-xylulose-5-phosphate sodium salt, diethylene triamine pentakis(methylphosphonic acid), nitrilo tri(methylphosphonic acid), 5-phospho-D-ribose 1-diphosphate pentasodium salt, adenosine 5 '-diphosphate sodium salt, adenosine 5 '-triphosphate disodium salt hydrate, a-D-galactosamine 1-phosphate, 2-phospho-L-ascorbic acid trisodium salt, a-D-galactose 1- phosphate dipotassium salt pentahydrate, a-D-galactosamine 1-phosphate, O- phosphorylethanolamine, disodium salt hydrate, 2,3-diphospho-D-glyceric acid pentasodium salt, phospho(enol)pyruvic acid monosodium salt hydrate, D -glyceraldehyde 3-phosphate, sn-glycerol 3-phosphate lithium salt, D-(-)-3-phosphoglyceric acid disodium salt, D-glucose 6-phosphate sodium salt, phosphatidic acid, ibandronate sodium salt, phosphonoacetic acid, DL-2-amino-3-phosphonopropionic acid or combinations thereof.

[0094] In some embodiments, the stabilizers can be bio-essential inorganic ions including, inter alia, Na, K, Mg, Zn, Fe, P, S, N, P, or S in the phase of oxides, or N as ammonia or nitro groups.

[0095] The stabilized ACC may be stabilized by more than one stabilizer, e.g., 2, 3, or more stabilizers. The stabilizers can be added during the synthesis and precipitation of the ACC primary particles and they are defined as “internal stabilizer”. Stabilizers can be added after the synthesis and bind to the external surface of the particles. They are defined as “external stabilizers”. In some embodiments, where both internal and external stabilizers are used, the internal stabilizer and the external stabilizer are similar. In other embodiments, the internal stabilizer and the external stabilizer are different stabilizers. The internal and the external stabilizers may be each independently as defined hereinabove and each can be a combination of more than one type of stabilizer.

[0096] The stable ACC can comprise more than two stabilizers, wherein one or more stabilizers are added to the ACC during the formation and precipitation of the ACC, or added after the precipitation, or added in both steps.

[0097] According to some embodiments, the stabilizer is selected from the group consisting of a phosphate, polyphosphate, polyphosphonate, bisphosphonate, phosphorylated amino acid, citric acid, salts thereof and any combination thereof. In some embodiments, more than one stabilizer, e.g., 2, 3, or 4 stabilizers are added.

[0098] According to one embodiment, ACC is stabilized by a combination of phosphoserine and citric acid. According to another embodiment, ACC is stabilized by a combination of tripolyphosphate (also defined as triphosphate) and citric acid.

[0099] According to some embodiments, the stabilizer is phosphate and / or a polyphosphate or pharmaceutically acceptable salts thereof. According to some embodiments, the polyphosphate is physiologically compatible, water-soluble polyphosphate salt selected from the group consisting of sodium, potassium, and any other essential cation of polyphosphate. In one embodiment, the polyphosphate is organic or inorganic polyphosphate. The term “polyphosphate” as used herein refers to (1) polymeric unhydrates of PO4 or a (2) phosphorylated organic compound containing more than 1 phosphorylated groups via C-O-P (ether) bonding An example for Type 2 polyphosphate is phytic acid or salts thereof, which contains 6-phosphorylated groups.

[0100] According to some embodiments, the polyphosphate is a physiologically compatible water-soluble polyphosphate salt selected from the group consisting of sodium and potassium polyphosphate. In some embodiments, the polyphosphate is an inorganic polyphosphate or pharmaceutically acceptable salts thereof. Not-limiting examples of such salt are Na, K, Mg, Mn, and Zn. According to some embodiments, the polyphosphate such as an inorganic polyphosphate comprises 2 to 10 phosphate groups, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate groups. According to some embodiments, the inorganic polyphosphate is selected from pyrophosphate, triphosphate, and hexametaphosphate. According to one embodiment, the stabilizer is pyrophosphate or pharmaceutically acceptable salts thereof such as sodium pyrophosphate. According to another embodiment, the stabilizer is triphosphate (tripolyphosphate) or pharmaceutically acceptable salts thereof such as sodium triphosphate. The term “triphosphate” and “tripolyphosphate” are used herein interchangeably. According to a further embodiment, the stabilizer is hexametaphosphate or a pharmaceutically acceptable salt thereof such as sodium hexametaphosphate.

[0101] According to some embodiments, the stabilizer is a polyphosphonate such as bisphosphonate or tetraphosphonate or pharmaceutically acceptable salts thereof. The nonlimiting examples of salt are Na, K, Mg, Mn and Zn.

[0102] The term “bisphosphonate” as used herein refers to organic compounds having two phosphonate (PO(OH)2) groups. The term further relates to compounds having a backbone of POs-organic-POs. Most typical is a series of bisphosphonates that are used as pharmaceuticals for treating osteoporosis. According to some embodiments, the bisphosphonate is selected from the group consisting of etidronic acid, zoledronic acid, medronic acid, alendronic acid, and a pharmaceutically acceptable salt thereof. According to some embodiments, the stabilizer is an etidronic acid or a pharmaceutically acceptable salt thereof. According to another embodiment, the stabilizer is a zoledronic acid or a pharmaceutically acceptable salt thereof. According to a further embodiment, the stabilizer is a medronic acid or a pharmaceutically acceptable salt thereof. According to certain embodiments, the stabilizer is alendronic acid or a pharmaceutically acceptable salt thereof.

[0103] According to certain embodiments, the stabilizer is a phosphorylated amino acid. According to one embodiment, the phosphorylated amino acid is phosphoserine. According to another embodiment, the phosphorylated amino acid is phospho threonine.

[0104] According to certain embodiments, the stabilizer is phytic acid or salts thereof. According to some embodiments, the ACC composition comprises a combination of the stabilizers disclosed above.

[0105] According to some embodiments, the stabilizer is an inorganic polyphosphate or a bisphosphonate as defined hereinabove, and the molar ratio between P atoms of the stabilizer and Ca atoms of the ACC (P:Ca molar ratio) is about 1:90 to 1:1. In one embodiment, the P:Ca molar ratio is about 1 :40 to about 1 : 1. In a further embodiment, the P:Ca molar ratio is about 1:35 to about 1:2. In certain embodiments, the P:Ca molar ratio is about 1:30 to about 1:3. In certain embodiments, the P:Ca molar ratio is about 1:28 to about 1:3. In other embodiments, the P:Ca molar ratio is about 1:25 to about 1:4. In further embodiment, the P:Ca molar ratio is about 1:20 to about 1:5. In another embodiment, the P:Ca molar ratio is about 1:20 to about 1:6. In a particular embodiment, the P:Ca molar ratio is about 1:15 to about 1:5. In another particular embodiment, the P:Ca molar ratio is about 1:25 to about 1:5. According to some embodiments, such inorganic polyphosphate is pyrophosphate, triphosphate, hexametaphosphate or a pharmaceutically acceptable salt thereof. According to another embodiment, the bisphosphonate is alendronic acid, etidronic acid, zoledronic acid or medronic acid and the P:Ca molar ratio is as defined hereinabove.

[0106] According to some embodiments, the calcium content (Ca content) of such compositions comprising stabilizers is about 1 wt% to about 39 wt%, about 5 wt% to about 39 wt%, about 10% to about 39 wt%, about 15% to about 39 wt%, about 20 wt% to about 38 wt%, about 25 wt% to about 38 wt%, or about 30 wt% to about 38 wt% of the dry ACC particles The terms “Ca content” and “calcium content” is used herein interchangeably and refer to the content of calcium of the ACC in the final composition.

[0107] In certain embodiments, the P:Ca molar ratio is about 1:40 to about 1:1, and the Ca content is about 20 wt% to about 39 wt%. In some embodiments, the molar ratio is 1:28 to about 1:3, and the Ca content is about 30 wt% to about 38 wt% of the dry ACC particles. In another embodiment, the molar ratio is 1 :25 to about 1:5, and the Ca content is about 30 wt% to about 36 wt% of the dry ACC particles.

[0108] According to some embodiments, the stabilizer is an inorganic polyphosphate or a bisphosphonate as defined hereinabove, and the molar ratio between P atoms of the stabilizer and Mg atoms of amorphous magnesium carbonate (AMC) (P:Mg molar ratio) is about 1:90 to 1:1. In one embodiment, the P:Mg molar ratio is about 1:40 to about 1:1. In a further embodiment, the P:Mg molar ratio is about 1:35 to about 1:2. In certain embodiments, the P:Mg molar ratio is about 1:30 to about 1:3. In certain embodiments, the P:Mg molar ratio is about 1 :28 to about 1 :3. In other embodiments, the P:Mg molar ratio is about 1 :25 to about 1:4. In further embodiment, the P:Mg molar ratio is about 1:20 to about 1:5. In another embodiment, the P:Mg molar ratio is about 1:20 to about 1:6. In a particular embodiment, the P:Mg molar ratio is about 1 : 15 to about 1 :5. In another particular embodiment, the P:Mg molar ratio is about 1:25 to about 1:5. According to some embodiments, such inorganic polyphosphate is pyrophosphate, triphosphate, hexametaphosphate or a pharmaceutically acceptable salt thereof. According to another embodiment, the bisphosphonate is alendronic acid, etidronic acid, zoledronic acid or medronic acid and the P:Mg molar ratio is as defined hereinabove.

[0109] According to some embodiments, the stabilizer is selected from the group consisting of a polyphosphate, phosphorylated amino acid, bisphosphonate, citric acid, tartaric acid and any combination thereof. According to one embodiment, the polyphosphate is selected from the group consisting of triphosphate, pyrophosphate, and hexametaphosphate, the phosphorylated amino acid is phosphoserine or phosphothreonine, and the bisphosphonate is selected from the group consisting of alendronate, etidronic acid, zoledronic acid and medronic acid. According to some embodiments, the polyphosphate is an inorganic polyphosphate.

[0110] According to one embodiment, the stabilizer is selected from the group consisting of organic acids, phosphorylated, phosphonated, sulfated or sulfonated organic compound, phosphoric or sulfuric esters of hydroxy carboxylic acids, phosphorylated amino acids, bisphosphonate, organic polyphosphate, hydroxyl bearing organic compounds, derivatives thereof, proteins and any combinations thereof.

[0111] According to another embodiment, the stabilizer is selected from the group consisting of phosphoserine, adenosine triphosphate, adenosine diphosphate, phytic acid, citric acid, etidronic acid, pyrophosphate, polyphosphate, inorganic triphosphate, hexamethaphosphate, ethanol, and any combination thereof.

[0112] According to some embodiments, the wherein the stabilizer is selected from the group consisting of polyphosphates, organic acids, phosphorylated amino acids, phosphorylated, phosphonated, sulfated or sulfonated organic compounds, phosphoric or sulfuric esters of hydroxy carboxylic acids, bisphosphonates, organic polyphosphates, polyphosphates, hydroxyl bearing organic compounds, derivatives thereof, proteins and any combinations thereof.

[0113] According to some embodiments, wherein the stabilizer is selected from the group consisting of tripolyphosphate or a salt thereof, phosphoserine, citric acid, sodium triphosphate and citric acid, adenosine triphosphate, adenosine diphosphate, phytic acid, etidronic acid, pyrophosphate, polyphosphate, hexamethaphosphate, a salt thereof, ethanol, and any combination thereof.

[0114] Therefore, according to some embodiments, the present invention provides a method of treating of an infection caused by antibiotic-resistant bacteria in a subject in need thereof, the method comprises administering to said subject an amorphous calcium carbonate comprising a stabilizer as described above as an active agent. According to some embodiments, the method comprises co-administering the ACC with an antibiotic to which the bacteria is resistant.

[0115] The invention will now be illustrated by the following non-limiting Examples.

[0116] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

[0117] The terms “a,” “an,” and “the” are used herein interchangeably and mean one or more.

[0118] The term “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes, (A and B) and (A or B).

[0119] The term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination if the combination is not mutually exclusive.

[0120] The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.

[0121] As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / - 10%, or + / -5%, + / -!%, or even + / -0.1% from the specified value. EXAMPLES

[0122] Example 1. Effect of ACC on S. mutans ACC effect on bacterial growth

[0123] Materials and Methods:

[0124] All of the experiments described with S. mutans were done using freshly prepared stabilized ACC suspensions. A typical procedure for making such suspensions is as follows: An amount of 36 ml of 3% Calcium chloride solution is mixed with 10ml of 0.5406% pentasodium-triphosphate solution. Then, 40 ml of 1.9485% Sodium carbonate solution is added to instantaneously form an ACC suspension. Finally, an additional amount of 10ml of the stabilizing solution containing 0.5406% pentasodium-triphosphate is added to the ACC suspension creating the final stabilized ACC suspension. The formed stabilized ACC suspension consists of small aggregates with a mean size in the range of 10 microns, formed from primary nanoparticles of ACC having a particle size range of 10 to 100 nanometers and a mean size range of 50 nm.

[0125] Bacterial growth

[0126] S. mutans: Dilutions of ACC or CCC in bacterial growth media of BHI broth final concentrations (from 1.5 mM to 20 mM in ACC or 3.75, 7.5 and 15 mM of crystalline calcium carbonate (CCC)were prepared in a polystyrene flat bottomed 24-well microplate with hydroxyapetite (HA) tablets (one tablet per well). Wells with bacteria but without the addition of ACC or CCC, served as positive controls. Wells with no bacteria but with ACC or CCC served as blanks. An equal volume (1 ml) of the various tested bacterial suspensions at optical density (OD)595=0.02 was added to each well. The bacteria growth was monitored by recording the turbidity of the media at optical density (OD) at 595 nm, using a Tecan plate reader Spectrophotometer after a 24-h incubation at 37°C under aerobic conditions, in an atmosphere of 5% CO2.

[0127] Effect of ACC on biofilm formation on hydroxyapatite (HA) tablets and in polystyrene microplate wells

[0128] The assay was performed as described above for S. mutans bacterial growth except for the growth medium used, which was BHI concentrated 2 times supplemented with 2% sucrose. ACC was applied at 1.5-20 mM doses on HA tablets in microplate wells. After incubation for 24 h, spent media and free-floating bacteria were removed by aspiration and the wells and HA tablets were washed twice with phosphate -buffered saline (PBS, pH 7.4), before quantifying biofilm by Metabolic Assay, using tetrazolium salt_(MTT) staining.

[0129] Metabolic Assay (MTT)

[0130] The assay measuring the cell proliferation rate was performed as follows. Briefly, biofilms were be overlaid with 100 mM MTT and incubated for 1 h at 37°C. Under these conditions, the lightly yellowish MTT is reduced to an insoluble blue tetrazolium salt accumulated within the cells. The amount of intracellular tetrazolium salts was quantified spectrophotometrically by measuring the absorbance of the solution at 570 nm. The accumulation of tetrazolium salt by the reduction of MTT via cellular dehydrogenases is proportional to the number of viable cells growing in the biofilm.

[0131] Results:

[0132] ACC inhibited both well- and HA-associated biofilm

[0133] As shown in Fig.l and Fig. 2, ACC efficacy is highly dose-dependent in its ability to inhibit S. mutans biofilm formation either in wells or at HA surface. Already at 10 mM, ACC inhibited HA-associated biofilm by more than 90%, thus exhibiting minimal biofilm inhibitory concentration of 90% (MBIC90) at 10 mM (Fig. 2). ACC at lowest tested dose of 1.25 mM inhibited more than 50% of HA-associated biofilm, thus exhibiting minimal biofilm inhibitory concentration of 50% (MBIC50) at 1.25 mM (Fig. 2). MBIC50 for well- associated biofilm was detected at 5 mM of ACC (Fig.2).

[0134] No effect on bacterial growth was detected (data not shown). Without being bound to any particular theory, it is assumed that the major effect on the biofilm formation must be associated with altering the bacteria metabolic pathways or preventing the coalescence of the biofilm constituents.

[0135] In an additional experiment, the formation of a biofilm by S. mutans in the presence of ACC or CCC was tested. Dilutions of ACC (from 2.5 mM to 10 mM) and CCC (from 3.75 to 15 mM) in bacterial growth media of BHI broth + 1% sucrose were prepared in a polystyrene flat bottomed 24-well microplate. CCC and ACC were added in equal volumes to the final solution. Wells with no ACC / CCC and with bacteria served as positive controls. Wells with no bacteria and with ACC / CCC served as blanks. An equal volume (1 ml) of each tested bacterial suspension at optical density (OD)s95=0.02 was added. After a 24h incubation at 37°C under aerobic conditions, in an atmosphere of 5% CO2, growth was monitored after 24h by recording the bacteria growth by turbidity of the media at optical density (OD) at 595 nm using a Tecan plate reader Spectrophotometer.

[0136] For S. mutans, the assay was performed as described above for S. mutans bacterial growth except that the used growth medium was BHI concentrated 2 times and supplemented with 2% sucrose. After incubation for 24h, the spent media and free-floating bacteria were removed by aspiration and the wells were washed twice with phosphate -buffered saline (PBS, pH 7.4), prior to quantifying the biofilm by MTT staining.

[0137] The assay measuring the cell proliferation rate was performed by MTT assay.

[0138] As shown in Figs. 3A and 3B, ACC in dose-dependent manner was able to inhibit S. mutans biofilm formation, while at the highest tested dose of 10 mM ACC inhibited the biofilm formation by 84%. In contrast, CCC, has no significant effect on the biofilm formation.

[0139] Conclusion: ACC is much more potent in reducing planktonic growth and biofilm formation than CCC.

[0140] Example 2. ACC affects extracellular polysaccharides (EPS) production.

[0141] EPS are important constituents of the formed oral bacterial biofilm. Confocal laser scanning microscopy (CLSM) was performed to examine the EPS production in the biofilm after treatment with the ACC formulations. The biofilms were grown on hydroxyapatite tablets in the presence or absence of the ACC formulations in a 24-well tissue culture plate for 24 h, washed twice with PBS, and then each well was dyed with 300 pl of 20 pg / ml of Alexa Fluor647- labeled dextran conjugated solution (which dyes the EPS). The stained biofilms were washed with DDW and fixated using 4% paraformaldehyde and 50% glycerol. Samples were visualized under a Nikon Confocal Microscope, resulting in an EPS distribution within the deposited biofilm, using the NIS -Element AR software. At least three random fields were scanned.

[0142] Results

[0143] The CLSM assay detected intact and strong biofilm formed at the surface of the untreated control with large amounts of produced EPS (data not shown). In contrast, already at a concentration of 5 mM of ACC, a notable decrease in biomass and EPS production was observed as compared to the control (CCC). In the presence of 20 mM of ACC the EPS was almost totally eliminated, while only non-bacterial mineral aggregates, probably induced by the ACC formulation were detected instead of the biofilm. Conclusions

[0144] The ACC was able to inhibit S. mutans biofilm formation on hydroxyapatite (HA) tablets in a dose-dependent manner.

[0145] The amount of Extracellular polysaccharides produced by S. mutans and immobilized at the HA surface was significantly reduced in direct correlation to the ACC concentration.

[0146] Example 2 - Effect of ACC on Multi Antibiotic-resistant S. aureus (MDRSA)

[0147] Objective: Assess the antibacterial efficacy of ACC in comparison with CCC and CaCh on the planktonic growth or biofilm formation of a clinically isolated MDRSA (MDSA CI-M).

[0148] Materials and Methods:

[0149] The following samples were used in this experiment:

[0150] 1. ACC suspension made by mixing “4-pack” stock solutions (the same in example 1) (pH 10)

[0151] 2. CalCh solution (pH 5)

[0152] 3. CCC suspension (pH 7) - this suspension immediately formed sediments, even after vortex.

[0153] 4. ACC powder (200 mg calcium in a 1.25 g powder formulation, Density Kidstm(currently sold as pHKidstm) suspension lOmg / ml double distilled water (DDW) - pH 7

[0154] 5. ACC powder (300 mg calcium in 1.60 g, pHDirecttm) suspension lOmg / ml double distilled water (DDW) - pH 10. This powder is defined in the figures as “Amorphicure”

[0155] Growth medium for planktonic culture:

[0156] Tryptic soy broth (TSB) supplemented with 1% D-glucose (TSBG).

[0157] The culturing was performed in 96 flat-bottomed tissue culture plates, in triplicates, and in a volume of 200 pl / well.

[0158] The bacteria viability was assessed by turbidimeter. The background OD600nm, measured in the absence of bacteria, were subtracted from the OD600nm obtained with bacteria.

[0159] In addition, ATP assay was performed using BacTiter-Glo microbial Cell viability assay (Promega), 100 pl of assay buffer to 200 pl of bacterial culture.

[0160] The biofilm viability assay was analyzed by MTT. As a first step, the effect of ACC, CCC and CalCh on the pH of the media was evaluated.

[0161] The results are presented in Table 1.

[0162] Table 1. pH measurements of the tested solutions in TSBG at their final concentrations.

[0163] The pH values of the suspensions were measured before the incubation with bacteria. Due to the low concentrations, the changes were small. However, it was important to notice that ACC gradually increased the pH in direct correlation with the increased concentration (indicating extended solubility). In contrast, the CCC hardly increased the pH. The CaCh slightly reduces the pH, as its concentrated solutions have a typical pH range around 5.

[0164] The effect ACC, CCC and CaCh concentrations (mM) on the planktonic growth of MDRSA CI-M was evaluated. The results are presented in Figs. 4 and 5.

[0165] Viability assessment was done according to turbidity at OD600nm. (* p<0.05). A significant change in viability was observed at 10 mM and higher of ACC and at 25 mM and above for CaCh. There is a trend for reducing viability as a function of both ACC while at CaCh a viability increase was observed. The increase in ATP in the presence of CaCh may indicate that the bacteria are in stress and need more energy to acclimate to the new environmental surroundings. The CCC does not have any effect on the viability at any concentration.

[0166] Conclusion 1: ACC at high concentrations (10 mM and more) attenuated the growth (planktonic) of MDRSA CI-M. Also, ACC reduced the metabolic activity as measured by reduced ATP content at the higher concentrations (5-25mM).

[0167] Further, the effect of the initial pH of the medium comprising ACC, CaCh and CCC on the planktonic growth and biofilm formation of MDRSA CI-M was tested. The results are presented in Figs. 6 and 7 for ACC, and in Figs. 8, 9, and 10 for CaCh.

[0168] Conclusion 2: The growth of MDRSA is not affected by the initial pH of the medium. It was therefore concluded that the observed effect of ACC is not due to the higher pH. ACC partially reduces viability and biofilm formation both at initial pH of 10 and initial pH of 7.

[0169] Further, the effect of ACC, CCC and CaCh on viability when grown at a surface and subsequently film formation of MDRSA was measured.

[0170] The results are shown in Fig. 11.

[0171] Conclusion 3: ACC significantly (statistically) reduced MDRSA biofilm formation at concentrations of 10 and 25 mM of elemental calcium, whereas CaCh and CCC reduced MDRSA biofilm production to a lesser degree.

[0172] It was further tested and shown that commercial products Density Kids™ (currently pHKids™) and pHDirect™ (Amorphicure), in which ACC is present as a premade stabilized powder, significantly reduced the viability of MDRSA grown at the surface and subsequently inhibited the biofilm formation, as shown in Fig. 13. The effect on the planktonic growth of MDRSA CI-M was seen only in higher concentrations of the products. (Fig. 12). The units in Figs. 12 and 13 are given in mg / ml of the commercial formulation. For pHDirect - 1 mg / ml means 1.6 mM of calcium, and for Density Kids -1 mg / ml means 1.9 mM of calcium. What is remarkable in the results presented in Fig. 12 and partially in Fig 13, is that the efficacy of pHDirect is consistently a bit higher than Density Kids, which is easily explained by the 18% higher active doses in the pHDirect product.

[0173] Conclusion 4: The growth of MDRSA at the surface was significantly affected by pHDirect and Density Kids suspensions.

[0174] Example 3 - Effect of ACC on Methicillin Resistant S. aureus (MRSA)

[0175] In this experiment, MRSA was cultured in TSB to which 100, 120 and 140 pg / ml of Methicillin was added. The MRSA bacteria continued to grow at these Methicillin concentrations. In addition, ACC prepared form 4 stock solutions (as in examples 1 and 2) was added at concentrations of 2 and 5mM elemental calcium in ACC.

[0176] Viability of MRSA was assessed by turbidity in colony forming units (CFU) / ml. The results are presented on Fig. 14.

[0177] Conclusion 5: ACC at a concentration of 5mM has reduced MRSA growth when cultured in the presence of 120 and 140pg / ml Methicillin compared to MRSA at the same concentration of Methicillin without ACC. It is shown for the first time that ACC is capable of increasing sensitivity of MRSA to methicillin.

[0178] Summarizing all above examples, it can be seen that:

[0179] ACC at high concentrations (10-25 mM) attenuated the growth of MDRSA CI-M. ACC at 5mM concentration increased MRSA sensitivity to 120 and 140|ig / ml Methicillin.

[0180] ACC at least partially prevents biofilm formation of MDRSA. pHDirect and Density Kids suspensions reduced biofilm formation (0.15-1 mg powder / ml).

[0181] The results of these experiments are unexpected since ACC was not considered as effective for affecting the sensitivity an antibacterial agent. The effect of ACC on preventing the growth of the biofilm and reducing the rates of metabolism, is significant also at low concentrations of ACC, while the viability effect becomes significant only at the high concentrations.

[0182] In a further experiment, the effect of ACC on the susceptibility of MRSA to Methicillin was compared to the effect of CCC. In a similar experimental arrangement, the effect of ACC and CCC on susceptibility following antibiotic resistant bacteria to antibiotic to which the bacteria are resistant is tested. The examined bacteria are multidrug-resistant Staphylococcus aureus (MDRSA), Pseudomonas BAA2108, multidrug-resistant Pseudomonas aeruginosa and extended- spectrum beta-lactamase (ESBL)-producing Escherichia coli.

[0183] Example 4. Evaluation the effect of ACC on antibiotic-resistant bacteria

[0184] Aim:

[0185] The purpose of this study is to evaluate whether the addition of ACC to antibioticresistant bacteria, will result in increased sensitivity of the bacteria to the antibiotics to which it is resistant.

[0186] Study Design:

[0187] The study is performed on one or more antibiotic-resistant bacteria and evaluates at least one antibiotic to which it is resistant.

[0188] The following treatments are evaluated on multidrug-resistant bacteria: ACC + an antibiotic at different concentrations:

[0189] 1. CCC + an antibiotic at different concentrations

[0190] 2. CaCh + an antibiotic at different concentrations

[0191] 3. Nano-CCC from eggshells + an antibiotic at different concentrations

[0192] 4. Negative control + antibiotic at different concentrations

[0193] 5. Negative control no antibiotics at different concentrations Bacteria that are tested and antibiotics to which they confer resistance are specified below:

[0194] 1. MRS A - resistant to methicillin / penicillin

[0195] 2. MDRSA - resistant to methicillin / penicillin / erythromycin / gentamicin / norfloxacin

[0196] 3. Pseudomonas BAA2108 - resistant to: Amoxicillin-clavulanate; Ampicillin;

[0197] Cefalotin; Cefotaxime; Cefoxitin; Cefpodoxime; Cefuroxime; Cefuroxime axetil;

[0198] Imipenem; Nitrofurantoin; Tetracycline; Tigecycline; Trimethoprimsulfamethoxazole

[0199] The study is performed in two parts:

[0200] (1) The first part is aimed on evaluating different concentrations of the given treatment groups on the selected bacteria in order to find out, which concentrations provide the maximal enhancement in sensitivity of bacteria and subsequently confer most damage to bacteria and at which concentrations of antibiotics. In this part the viability of bacteria is performed with the following assays: MTT or XTT, ATP, turbidity

[0201] (2) The second part is a repetition of the best results oobtained in the first part. Best results are defined as conditions (ACC and other treatments concentration and antibiotics concentration), which are most damaging to the bacteria. In this part we quantify the population of the bacteria in addition to the viability assays described above. This is performed by either colony forming unit, or live / dead florescent stains.

[0202] Example 5. ACC for the treatment of periodontitis.

[0203] The efficacy of ACC for treating periodontitis was studied in a mouse model using ligature-induced periodontitis model based on the methods described in Lin et al., 2021; Kaboosaya et al., 2017; and Abe et al., 2013. Shortly, A 5-0 silk suture was looped around the second molar, with the contralateral molar left unligated as a control. Mice were sacrificed after 9 days to evaluate alveolar bone loss. Alveolar bone loss was assessed by measuring the distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC). Three experiments were performed. Three mice were assigned to each experimental group (ACC = 3, CCC = 3).

[0204] Treatment:

[0205] Two treatments were tested in two experiments: ACC (10%) or CCC (7.8%) were administered in a gum adhesive gel comprising 3% sodium carboxymethylcellulose (SCMC) + 1% Carbopol 980. The gel was administered twice a day for 9 days. In addition, ACC and CCC were provided mixed with the food (0.5% Ca as ACC (for the group receiving the ACC treatment) or CCC (for the group that received CCC). The calcium dose of ACC and CCC were equivalent with respect to the amount of calcium and carbonate.

[0206] In the 3rdexperiment, 3 treatments were assessed: ACC (10%), CCC (7.8%), and NaHCOs (6.6%) were administered in a gum adhesive gel comprising 3% sodium carboxymethylcellulose (SCMC) + 1% Carbopol 980. The gel was administered twice a day for 9 days. In addition, ACC and CCC were provided mixed with the food [0.5% Ca as ACC (group received ACC treatment) or CCC (group that received CCC or NaHCCh)].

[0207] The content of calcium in different treatment groups is detailed in Table 2.

[0208] Table 2. Content of calcium in different treatment groups

[0209] At the end of the 9-day treatment period, mice were euthanized, and maxillae were harvested for analysis. Bone specimens were defleshed, bleached, stained with methylene blue, and imaged under a binocular microscope with a standardized scale. Several parameters were tested: CEJ-ABC distance and alveolar bone resorption area. Alveolar bone resorption was quantified using ImageJ software. CEJ-ABC distance - refers to Cemento- Enamel Junction to Alveolar Bone Crest - (see Fig. 15, bold arrow).

[0210] Further bacterial analysis was performed for samples of the 3rdexperiment. The analysis was performed as follows:

[0211] • Silk sutures were recovered from the mouse oral cavity 7 days after ligature placement.

[0212] • Each suture was vortexed in 500 pL of sterile PBS for 3 minutes. • For CFU analysis, bacterial suspension was serially diluted, plated on the blood agar, and incubated for 48 hours in aerobic and anaerobic conditions.

[0213] The results are presented in Figs. 16A-16E. It can be seen from the figures that treatment with ACC in a gum adhesive gel and diet profoundly reduced CEJ-ABC distance and alveolar bone resorption, the hallmark of periodontitis, compared to CCC treatment in the gum disease model. It can be seen in Figs. 16A and 16B that the average alveolar bone loss was at least 2 times lower than in the case of treatment with CCC. Figs 16C-16E show more detailed results of specific molars. The treatment with gum adhesive gel comprising CCC resembles the results of untreated periodontitis. For example, Pereira et al. / MethodsX 6 (2019) 2156-2165 shows that the average CEJ-ABC distance of ligated teeth is about 0.7 mm (see Fig. 17). For the CCC treated groups the average CEJ-ABC distance was 0.69 mm.

[0214] A detailed analysis reveals that the treatment with ACC significantly attenuated the alveolar bone resorption compared to CCC. Image analysis demonstrated a reduction in the cemento-enamel junction for the alveolar bone crest (CEJ-ABC) distance in the ACC-treated group (0.33 mm ± 0.03) compared to the CCC-treated group (0.69 mm ± 0.06 mm) (p value = 0.005). Furthermore, the total alveolar bone resorption area was substantially lower in the ACC-treated group (0.74 ± 0.25 mm2) compared to CCC (2.02 ± 0.29 mm2) (p value = 0.009). When focusing on the ligated second molar (M2), alveolar bone resorption area was 0.27 mm2± 0.10 mm2in ACC-treated mice, whereas the resorption area in the CCC group reached 0.71 ± 0.06 mm2(p value = 0.005). Notably, NaHCCE treatment resulted in severe alveolar bone loss, leading to complete loss of the second and third molars in two out of three mice.

[0215] These findings highlight the protective effect of ACC in mitigating alveolar bone resorption in a ligature-induced periodontitis model.

[0216] An additional experiment for evaluating the effect of ACC in the treatment of Periodontitis on 28 mice using the Periodontitis Model (Ligature) is performed according to the arrangement as described in Fig. 18. It is also shown that the treatment allows to restore and repair of the damaged tissue of the bone, tooth and gum.

[0217] Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

CLAIMS1. A topical oral composition comprising an effective amount of stabilized amorphous calcium carbonate (stabilized ACC), for use in preventing or treating a disease of an oral cavity caused by an infection, wherein the method comprises contacting the oral cavity with the topical oral composition and wherein the stabilized ACC is the main active agent in the composition.

2. The topical oral composition for use of claim 1, wherein the stabilized ACC is the only active agent.

3. The topical oral composition for use of claim 1, wherein the infection is a bacterial infection.

4. The topical oral composition for use of claim 1, wherein the stabilized ACC acts as an anti-bacterial agent.

5. The topical oral composition for use of claim 1 or 2, wherein the disease or condition is periodontitis.

6. The topical oral composition for use of any one of claims 1 to 5, wherein the use comprises contacting the oral cavity with the topical oral composition for at least 30 seconds.

7. The topical oral composition for use of any one of claims 1 to 6, wherein the composition is selected from an oral gel, ointment, powder, and oral wash.

8. The topical oral composition for use of any one of claims 1 to 7, wherein the use further comprises a systemic administration of a composition comprising stabilized ACC.

9. The topical oral composition for use of any one of claims 1 to 7, wherein the use prevents or attenuates the formation of a bacterial biofilm.

10. The topical oral composition for use of claim 9, wherein the disease or condition is periodontitis and the use prevents or attenuates the formation of a bacterial biofilm on the surface of teeth.

11. A composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC) for use in treating an infection caused by antibiotic-resistant bacteria in a subject in need thereof, the use comprises administering to said subject an effective amount of stabilized amorphous calcium carbonate (stabilized ACC) as an active agent.

12. A composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC), for use in improving the efficacy of a treatment of an infection caused by antibiotic-resistant bacteria, the use comprises administering an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent to a subject.

13. The composition for use of claim 11 or 12, wherein the stabilized ACC is the only active agent.

14. The composition for use of any one of claims 11 to 13, wherein the use comprises coadministering the composition and an antibiotic to which the antibiotic-resistant bacteria is resistant.

15. A composition comprising an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC) for use in treating a bacterial infection, the method comprises administering to said subject an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC) as an active agent.

16. The composition for use of claim 15, wherein the stabilized ACC is the only active agent.

17. The composition for use of claim 15 or 16, wherein the use further comprises administering a composition comprising an additional antibacterial agent.

18. The composition for use according to any one of claims 1 to 17, wherein the composition is a pharmaceutical composition.

19. A method of enhancing sensitivity of an antibiotic-resistant bacteria to an antibiotic, the method comprises exposing the antibiotic -resistant bacteria to an effective amount of stabilized amorphous calcium carbonate (stabilized ACC).

20. The method of claim 19, wherein the antibiotic is an antibiotic to which the bacteria is resistant.

21. A method of inhibiting the growth of antibiotic -resistant bacteria, the method comprises contacting the antibiotic -resistant bacteria with an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC).

22. A method of inhibiting or preventing the formation of a biofilm by bacteria, the method comprises contacting the bacteria with an effective amount of a stabilized amorphous calcium carbonate (stabilized ACC).

23. The method of claim 22, wherein the bacteria is an antibiotic -resistant bacteria.

24. The method of claim 22 or 23, wherein the bacteria are located in an oral cavity.

25. The composition for use or a method of any one of claims 1 to 18 or the method of any one of claims 19 to 24, wherein the antibiotic -resistant bacteria is selected from multiantibiotic -resistant S. aureus, methicillin restistant S. aureus, vancomycin- resistant Enterococcus (VRE), multi-drug-resistant Mycobacterium tuberculosis (MDR-TB), carbapenemase-producing Enterobacterales (CPE), and Pseudomonas BAA2108, multiantibiotic -resistant Streptococcus pneumoniae, multiantibiotic - resistant Clostridioides difficile, multiantibiotic-resistant Neisseria gonorrhoeae, and multiantibiotic -resistant Acinetobacter baumannii.