Bacterial Anti-adhesion compound and use thereof

Abstract

The invention relates to a compound and use thereof for preventing and / or inhibiting bacterial adhesion to a non-living substrate or a living surface as well as for preventing and / or treating bacterial infection.

Description

[0001] BACTERIAL ANTI-ADHESION COMPOUND AND USE THEREOF

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a compound and use thereof for preventing or inhibiting bacterial adhesion to a non-living substrate or a living surface as well as for preventing or treating bacterial infection.

[0004] BACKGROUND OF THE INVENTION

[0005] Bacteria commonly transition from a planktonic to surface attached-lifestyle. This represents a signal during host colonization, allowing them to physiologically adapt and initiate infection. Upon host contact, pathogens become more virulent by secreting toxins and forming antibiotic- tolerant structures called biofilms.

[0006] The biofilm formation on abiotic surfaces in food, dental and medical sectors constitutes great public health concern. In fact, biofilms present a persistent source for pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which lead to severe infections such as foodbome and nosocomial infections. Such biofilms are also a source of material deterioration and failure. The environmental conditions, commonly met in food, dental and medical areas seem also to enhance the biofilm formation and their resistance to disinfectant agents.

[0007] The concept of anti-attachment on pathogens has been previously explored in a few occurrences, for example against Escherichia coli in the scope of urinary tract infections. However, developing anti-attachment molecules for some pathogens, such as P. aeruginosa, poses a significant challenge due to the low specificity of attachment of these pathogens. Unlike many pathogens that cause diseases in only a few host species due to long coevolutionary histories, some pathogens, such as P. aeruginosa, can attach to and infect various hosts, including plants, vertebrate and nonvertebrate animals. This low specificity and complex attachment mechanism significantly complicate target-based drug discovery for anti-adhesive therapies.

[0008] Thus there is still a need for effective anti-adhesive therapies against virulent pathogens. SUMMARY OF THE INVENTION

[0009] An aspect of the present invention provides a compound of the formula: or a pharmaceutically acceptable salt thereof.

[0010] A further aspect of the present invention provides a composition for preventing or inhibiting bacterial adhesion to a non-living substrate comprising a carrier and an effective amount of the compound of the invention, or a pharmaceutically acceptable salt thereof.

[0011] Another aspect of the present invention provides a compound of the invention or a pharmaceutically acceptable salt thereof for use in a method for preventing or inhibiting bacterial adhesion to a living surface, comprising contacting the living surface with an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0012] Another aspect of the present invention provides a method for preventing or inhibiting bacterial adhesion to a non-living substrate comprising contacting the non-living substrate with an effective amount of the compound of the invention, or a pharmaceutically acceptable salt thereof. Another aspect of the present invention provides a use of the compound of the invention or a pharmaceutically acceptable salt thereof for preventing or inhibiting bacterial adhesion on a non-living substrate.

[0013] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0014] Another aspect of the present invention provides an oral care composition comprising an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof, and an orally acceptable carrier.

[0015] Another aspect of the present invention provides a compound of the invention or a pharmaceutically acceptable salt thereof for use as a medicament.

[0016] Another aspect of the present invention provides a compound of the invention, or a pharmaceutically acceptable salt thereof, or an oral care composition of the invention, for use in a method of preventing and / or inhibiting bacterial adhesion to an oral cavity surface of a mammal.

[0017] Another aspect of the present invention provides a compound of the invention or a pharmaceutically acceptable salt thereof for use in a method of preventing and / or treating a bacterial infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0018] BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1 shows validation of the anti-attachment effect of the compound of the invention (MB1) against abiotic surface. (A) Phase contrast microscope images, obtained using the image-based assay, demonstrate a reduction in attachment when bacteria are treated with 25 pM MB1, compared to the control DMSO group. (B) The figure presents the dose-response curve of the compound MB 1. The x-axis represents the concentration, while the y-axis shows the cell attachment ratio compared to the negative control. Figure 2 shows anti-attachment properties of the compound of the invention (MB1) against both Gram-negative bacteria and Gram-positive. Phase contrast microscope images taken after bacteria attached and the unattached cells were removed by washing step. For Gram-negative bacteria, MB1 reduced the number of attached Pseudomonas aeruginosa by 40%, Vibrio cholerae by 65%, and of Acinetobacter baumannii by 83% after wash. For Gram-positive bacteria, MB1 reduced the number of attached Staphylococcus aureus cells by 60 %, Enterococcus faecalis cells by 65 % and Streptococcus mutans by 45 %. These results show that 25 pM MB1 represses attachment of broad-spectrum of bacteria species compared to the control DMSO group in a similar manner to P. aeruginosa.

[0020] Figure 3 shows validation of the anti-attachment effect of the compound of the invention (MB1) against biotic surface with MDCK cell model. (A) Confocal microscopy images show a marked reduction in P. aeruginosa attachment following treatment with 25 pM MB1 compared to the DMSO control. MDCK epithelial cells are shown in magenta, forming an intact monolayer, while P. aeruginosa cells appear in green. (B) A bar graph presents the dosedependent reduction in bacterial attachment to MDCK cells by MB1. The x-axis shows the MB1 treatment concentrations, and the y-axis represents the quantified bacterial signal per field of view.

[0021] Figure 4 shows the compound of the invention (MB1) anti-virulence efficacy against P. aeruginosa in a THP1 macrophage cytotoxicity assay. (A) Living THP1 cells were stained with CMFDA dye (green). Upon cell death, the Draq7 dye (purple) stains the cells. The four images display results at 0 h and 6 h post-treatment and infection. (B) The x-axis represents time, while the y-axis denotes the cytotoxicity percentage of the THP1 cells. Triangles represent 25 pM, whereas squares indicate 0 pM as a control.

[0022] Figure 5 shows the compound of the invention (MB1) bactericidal effect. (A) Growth curve of P. aeruginosa when incubated with MB1. The x-axis represents time, while the y-axis signifies the OD600 measurement. The chart includes four different concentrations. (B) Doubling time of the exponential phase growth for each group was calculated, and significance was analyzed using a two-paired t-test. The asterisk (*) indicates a P-value less than 0.05. MB1 reduces growth at high concentration, without abolishing it. Figure 6 illustrates the inhibitory effect of the compound of the invention (MB1) on biofilm formation. Biofilms were quantified using crystal violet staining after 24 hours of incubation in 96-well plates containing a mixture of MB1 and bacterial cells. (A) Pseudomonas aeruginosa (Gram-negative) and (B) Streptococcus mutans (Gram-positive) both exhibited a dosedependent reduction in biofilm formation in response to MB1 treatment, indicating the compound's broad-spectrum anti-biofilm activity.

[0023] Figure 7 demonstrates the synergistic effect of the compound of the invention (MB1) in combination with antibiotics, exemplified by polymyxin B (PMX). (A) shows bacterial growth curves under four conditions: untreated control (blue), sub-MIC PMX alone (green), 25 pM MB1 alone (orange), and the combination treatment (red). The peak time point, defined as the time of maximum OD600 in the untreated control, was used for comparison. (B) presents a checkerboard assay displaying normalized OD600 values at the peak time across a range of MB1 and PMX concentrations. The results indicate that the growth inhibition observed with the combination treatment is dose-dependent, confirming a synergistic interaction between MB1 and PMX.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] All, documents, patents, patent applications, publications, product descriptions, and protocols which are cited throughout this application are incorporated herein by reference in their entireties for all purposes. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0026] In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0027] The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Also as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of "consisting of “ and / or "consisting essentially of’.

[0028] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0029] As used in the specification and claims, the term "and / or" used in a phrase such as "A and / or B" herein is intended to include "A and B", "A or B", "A", and "B".

[0030] As used herein, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and / or duration of bacterial infection, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of bacterial infection resulting from the administration of one or more therapies (e.g. a compound of the invention). In specific embodiments, the terms "treat", "treatment" and "treating" refer to the amelioration of at least one measurable physical parameter of bacterial infection. In other embodiments the terms "treat", "treatment" and "treating" refer to the inhibition of the progression of bacterial infection, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms "treat", "treatment" and "treating" refer to the reduction or stabilization of bacterial infection.

[0031] As used herein, the terms “prevent”, “prevention”, “preventing” or “prevented” refer to the prevention of the onset, recurrence or spread of bacterial adhesion, bacterial contamination and / or bacterial infection, or of one or more symptoms thereof. The terms also refer to the reduction in the risk of acquiring or developing bacterial infection, or the reduction or inhibition of the recurrence or bacterial infection. In certain embodiments, the terms refer to the treatment with or administration of a compound provided herein prior to the onset of symptoms, particularly to subjects at risk of being infected by bacteria. The terms encompass the inhibition or reduction of a symptom of bacterial infection. In other embodiments, a compound of the invention is administered as a preventative measure to a subject in order to avoid bacterial infection. In this context, the term “prevention” may be interchangeably used with the term “prophylactic treatment”. As used herein, the terms "subject", "patient" and "mammal" are used interchangeably. The terms "subject" and "patient" refer to an animal (e.g., a bird such as a chicken, quail or turkey, or a mammal), preferably a mammal including a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, and mouse) and a primate (e.g., a monkey, chimpanzee and a human), and more preferably a human. In one embodiment, the subject is a non-human animal such as a farm animal (e.g., a horse, cow, pig or sheep), or a pet (e.g., a dog, cat, guinea pig or rabbit). In a preferred embodiment, the subject is human.

[0032] As used herein, a "therapeutically effective amount" refers to an amount sufficient to elicit the desired biological response. In the present invention the desired biological response is to reduce or ameliorate the severity, duration, progression, or onset of bacterial infection, prevent the advancement of bacterial infection, cause the regression of bacterial infection, prevent the recurrence, development, onset or progression of a symptom associated with bacterial infection, or enhance or improve the prophylactic or therapeutic effect(s) of another antibacterial therapy. The precise amount of compound administered to a subject will depend on the mode of administration, the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of bacterial infection, and the mode of administration. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. When co-administered with other agents, e.g., when co-administered with other agent that impacts the growth and / or attachment and / or virulence of the bacteria, a "pharmaceutically effective amount" of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of condition(s) being treated and the amount of compound of the invention being used. In cases where no amount is expressly noted, an effective amount should be assumed.

[0033] Bacteria, such as P. aeruginosa, possess surface-specific physiological adaptation strategies promoting pathogenicity, including surface motility, contact-dependent virulence secretion, and biofilm formation. Inventors have discovered that P. aeruginosa mechanosenses surfaces to deploy an arsenal stimulating pathogenicity via stimulation of virulence gene expression. By extension, perturbing surface-bacteria interactions has the potential to mitigate the deployment of P. aeruginosa's surface-associated pathogenic arsenal and ultimately inhibiting the progress of infection. Indeed, the bacterium P. aeruginosa uses two main systems to adhere to surfaces. The first is Psi polysaccharide, which acts as a glue facilitating cell body adhesion and is also the primary component of the biofilm matrix. The second is type IV pili (T4P), which helps cells anchor to the surface upon contact. A compound that can disrupt these structures could have anti-adhesive properties.

[0034] Inventors have focused on targeting the attachment process and anti-virulence as potential strategies to combat bacterial infections, such as P. aeruginosa infections. To this end, inventors have developed a specific image-based anti-attachment screening method, which has identified multiple compounds that appeared to reduce P. aeruginosa attachment. Through rigorous validation, inventors have identified the compound of the invention that limits the attachment of P. aeruginosa to surfaces. By preventing the attachment of P. aeruginosa, the pathogen remains away from its host and stays in a low virulence state, reducing its ability to cause severe infections. The compound of the invention does not affect bacterial growth and therefore has the potential to provide a novel therapeutic option to mitigate the devastating impact of resistant bacterial infections, such as P. aeruginosa infections.

[0035] Thus an aspect of the present invention provides a compound of the formula: or a pharmaceutically acceptable salt thereof. The compound of the present invention is also designated as MB1 in the present disclosure. According to an embodiment, the chemical name of the compound of the invention is a-L- Rhamnose 1-6 [a-L-Rhamnose (2,3,4 Ac) 1-2] a-D-Glucose a-unsaturated alkene lipid.

[0036] The compound of the invention can exist in free form or where appropriate, as a pharmaceutically acceptable salt.

[0037] As used herein, the term "pharmaceutically acceptable salt" refers to salts of a compound which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue side effects, such as, toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio.

[0038] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al, describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1- 19, incorporated herein by reference. Pharmaceutically acceptable salts of the compound of the invention include those derived from suitable inorganic and organic acids and bases. These salts can be prepared in situ during the final isolation and purification of the compounds. Acid addition salts can be prepared by 1) reacting the purified compound in its free-based form with a suitable organic or inorganic acid and 2) isolating the salt thus formed. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, palmoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Base addition salts can be prepared by 1) reacting the purified compound in its acid form with a suitable organic or inorganic base and 2) isolating the salt thus formed. Salts derived from appropriate bases include alkali metal (e.g., sodium, lithium, and potassium), and alkaline earth metal (e.g., magnesium and calcium).

[0039] It should be understood that the invention includes mixtures / combinations of different pharmaceutically acceptable salts and also mixtures / combinations of compounds in free form and pharmaceutically acceptable salts.

[0040] In addition to the compound of the invention, pharmaceutically acceptable derivatives or prodrugs of the compound of the invention may also be employed in preventing or inhibiting bacterial adhesion and in preventing or treating bacterial infection. As used herein and unless otherwise indicated, the term "prodrug" means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of this invention. Prodrugs may become active upon such reaction under biological conditions, or they may have activity in their unreacted forms. Examples of prodrugs contemplated in the invention include, but are not limited to, analogs or derivatives of the compound of the invention that comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Prodrugs can typically be prepared using well-known methods, such as those described by BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5th ed).

[0041] A "pharmaceutically acceptable derivative" is an adduct or derivative which, upon administration to a subject, is capable of providing, directly or indirectly, a compound as otherwise described herein, or a metabolite or residue thereof. Examples of pharmaceutically acceptable derivatives include, but are not limited to, esters and salts of such esters.

[0042] A "pharmaceutically acceptable derivative or prodrug" includes any pharmaceutically acceptable ester, salt of an ester or other derivative or salt thereof of a compound of the invention which, upon administration to a subject, is capable of providing, either directly or indirectly, a compound of the invention. Preferred derivatives or prodrugs are those that increase the bioavailability of the compound of the invention when such compounds are administered to a subject (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system) relative to the parent species. Pharmaceutically acceptable prodrugs of the compounds of this invention include, without limitation, esters, amino acid esters, phosphate esters, metal salts and sulfonate esters.

[0043] Another aspect of the present invention provides a composition for preventing or inhibiting bacterial adhesion to a non-living substrate comprising a carrier and an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0044] The non-living substrate can be any material or surface where bacterial adhesion is to be prevented or inhibited. For example, the material is typically selected from the group comprising plastic materials, silicone, metals, textile fibers, glass, wood, paper, foams, and bricks. The surface is typically selected from the group comprising work surfaces, surgical or other instruments, surgical implants or prostheses, dental implants, contact lenses, foods, crops, industrial plant, floors and walls (both internal and external), bedding, furniture, clothing and many other surfaces (non-living substrates or surfaces). In preferred embodiments, the nonliving substrate is selected from the group comprising surgical instruments and tools, surgical implants, surgical prostheses, catheters, wound dressing, dental implants, contact lenses, building floors, building walls (both internal and external), bedding, furniture, and clothing.

[0045] In an embodiment, the suitable carrier is selected from the group comprising stabilizers, fragrance, colorants, emulsifiers, thickeners, wetting agents, and mixtures thereof.

[0046] In another embodiment, the composition can be in the form of a liquid, a gel, a foam, a spray or an emulsion.

[0047] According to an embodiment, the composition of the invention is a coating composition whereby when applied to a surface of the non-living substrate, an effective amount of the compound of the invention is left deposited on that substrate to prevent or inhibit bacterial adhesion on the surface of the non-living substrate. Examples of coating compositions include, without limitation, paints, stains, sealants, waxes, cleaning products and disinfectants.

[0048] The coating composition can be formulated for the specific surface on which the compound of the invention is to be delivered (surface of the non-living substrate). In one embodiment, the coating composition is formulated to adhere to or be absorbed by silicone. In another embodiment, the coating composition is formulated to adhere to or be absorbed by a solid polymer (e.g. to a polymeric substrate such as polyvinyl chloride). In another embodiment, the coating composition is formulated to adhere to a metal or a metallic surface. Metallic and other such surfaces include, without limitation, maritime vehicles and equipment, equipment used in water purification, transport and storage, oil and gas pipelines, cooling towers, heat exchangers, warm water systems, filter systems, water treatment membranes.

[0049] As known to those of skill in the art, depending on the particular type of surface or surface environment, the mode of applying the coating may vary. In some embodiments, the composition may be applied to a surface using a brush or mechanical sprayer. In other embodiments, the surface may be dipped, submerged, or infused with the coating.

[0050] In some embodiments, the effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof is the amount which is effective to prevent, suppress and / or inhibit the adhesion, attachment and / or settlement of bacteria on the surface, e.g. the surface of the non-living substrate. In some embodiments of the present invention, the effective amount ranges from 1 nM to 1 mM (molar concentration of the compound of the invention), in preferred embodiments, the effective amount ranges from 10 nM to 100 pM, in a preferred embodiment, the effective amount ranges from 100 nM to 10 pM.

[0051] Another aspect of the present invention provides a product which is generated by application of the compositions of the invention, such as the coating composition described herein, to a non-living substrate. As such, the present invention encompasses such non-living substrates described herein which have an effective amount of the compound of the invention deposited on or absorbed to their surface, following application of the composition of the invention, such as the coating composition described herein. This includes, without limitation, metal substrates, silicone, textile fibers, glass, wood, paper and other polymeric or plastic substrates. This also includes non-living substrates designed for specific products which are particularly susceptible to bacterial adhesion, e.g, products designed for contact and / or implantation into the subject's body, including without limitation, catheters, dental implants, medical implants, surgery tools, endoscopes, contact lenses, wound dressings. It further includes non-living substrates designed for products such as components of cooling towers, heat exchanger or warm water systems, pipelines (e.g., oil, gas, water). Another aspect of the present invention provides a method for preventing or inhibiting bacterial adhesion to a non-living substrate comprising contacting the non-living substrate with an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0052] Another aspect of the present invention provides a use of the compound of the invention or a pharmaceutically acceptable salt thereof for preventing or inhibiting bacterial adhesion on a non-living substrate.

[0053] Thus the compound of the invention or a pharmaceutically acceptable salt thereof may be used to disinfect work surfaces, surgical, dental or other instruments (including implants or prostheses) or other devices against bacteria. It may be used to treat protective clothing such as surgical gloves, clothing or bedding. In an embodiment, it may be used to treat an implant or other device which is intended for use within the subject's body.

[0054] Another aspect of the present invention provides a compound of the invention or a pharmaceutically acceptable salt thereof for use in a method for preventing or inhibiting bacterial adhesion to a living surface, comprising contacting the living surface with an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0055] An embodiment of the present invention provides a method for preventing or inhibiting bacterial adhesion to a living surface, comprising contacting the living surface with an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0056] In some embodiments, the contacting occurs with an animal, preferably a mammal, most preferably a human.

[0057] In other embodiments, the contacting occurs in the presence of an additional agent that impacts the growth and / or attachment and / or virulence of the bacteria.

[0058] In preferred embodiments, the additional agent is selected from a microbiocidal agent, bacteriostatic agent, antibacterial agent, and combinations thereof. In an embodiment the additional agent is polymyxin B. In some embodiments, the living surface is a portion of a subject's body selected from the group comprising an interior surface of a subject's body, an external surface of a subject's body (such as skin, hair, and nails), a mucous membrane of a subject's body, and solid parts of a subject's body (such as teeth and bones). In a preferred embodiment, the living surface is a mucous membrane of the subject's body. In another preferred embodiment, the living surface is a tooth of the subject's body.

[0059] Another aspect of the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

[0060] The pharmaceutically acceptable carrier, adjuvant, or vehicle, as used herein, includes any and all solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compound of the present invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention.

[0061] Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, or potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0062] Another aspect of the present invention provides an oral care composition comprising an effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof, and an orally acceptable carrier.

[0063] As used herein, the term "oral care composition" refers to a composition that is delivered to one or more surfaces of the oral cavity of a mammal, preferably a human. Generally, during the normal course of use, the composition is not swallowed, but rather it is retained in the oral cavity for a time sufficient to contact one or more surfaces of the oral cavity, such as teeth, and provide the desired benefit. Examples of such compositions include, but are not limited to, toothpaste, mouthwash or mouth rinse, oral gel, denture cleanser, and the like.

[0064] In an embodiment, the oral care composition of the present invention is in a form selected from a group comprising a mouthwash, a toothpaste, oral gel and an oral spray.

[0065] As used herein, the term "mouthrinse" or "mouthwash" refers to oral care compositions that are substantially liquid in character. In such preparation, the orally acceptable carrier typically has an aqueous phase comprising water or a water and alcohol mixture. In various embodiments, the alcohol is typically ethyl alcohol.

[0066] In another embodiment of the oral care composition of the present invention, the orally acceptable carrier is one or more compounds selected from the group comprising a humectant, a flavoring, and a surfactant. In some embodiments, the surfactant is selected form the group comprising anionic surfactants, cationic surfactants, nonionic surfactants, amphoteric surfactants and zwitterionic surfactants. In further embodiments, the oral care compositions of the present invention may further comprise one or more fluoride ion sources, e.g., soluble fluoride salts. In further embodiments, the oral care composition of the present invention may further comprise a tartar control (anticalculus) agent. In further embodiments, the oral care compositions of the present invention may also comprise one or more chelating agents able to complex calcium found in the cell walls of the bacteria. Binding of this calcium weakens the bacterial cell wall and augments bacterial lysis.

[0067] The compound of the invention or the pharmaceutically acceptable salt thereof inhibits bacterial adhesion (attachment) and is therefore effective to treat bacterial infections and / or provides a prophylactic treatment (prevention) to avoid bacterial infections. Specifically, the compound of the invention reduces or suppresses the ability of bacteria to deploy cytotoxic components (resulting in reduced or suppressed cytotoxicity) following a dose-dependent trend and does not impede bacterial growth, only their virulence.

[0068] Another aspect of the present invention provides a compound of the invention, or a pharmaceutically acceptable salt thereof, or an oral care composition of the invention for use in a method for preventing and / or inhibiting bacterial adhesion to an oral cavity surface of a mammal, preferably human.

[0069] According to an embodiment, the present invention provides a method for preventing or inhibiting bacterial adhesion to an oral cavity surface of a mammal, preferably human, comprising contacting the oral cavity surface of a mammal, preferably human, with an effective amount of the compound of the invention, or a pharmaceutically acceptable salt thereof, or with an oral care composition of the invention.

[0070] According to another embodiment, the present invention provides a use of the compound of the invention or a pharmaceutically acceptable salt thereof for manufacturing an oral care composition for preventing and / or inhibiting bacterial adhesion to an oral cavity surface of a mammal, preferably human.

[0071] Another aspect of the present invention provides a compound of the invention or a pharmaceutically acceptable salt thereof for use as a medicament. A further aspect of the present invention provides a compound of the invention or a pharmaceutically acceptable salt thereof for use in a method of preventing and / or treating a bacterial infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0072] In some embodiments of the present invention, the therapeutically effective amount of the compound of the invention ranges from 1 to 100 mg / kg of subject (patient) per day.

[0073] An embodiment of the present invention provides a method for preventing and / or treating a bacterial infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of the invention or a pharmaceutically acceptable salt thereof.

[0074] In some embodiments, the method of preventing and / or treating further comprises administering an additional agent that impacts the growth and / or attachment and / or virulence of the bacteria. In some preferred embodiments, the additional agent is selected from a microbiocidal agent, bacteriostatic agent, antibacterial agent, and combinations thereof. In an embodiment the additional agent is polymyxin B.

[0075] The additional agent may be present in the same pharmaceutical composition or may be administered separately to the subject.

[0076] In some embodiments, a regiment for preventing and / or treating a bacterial infection includes administration of a therapeutically effective amount over a period of several days, up to and including between one week and about six months.

[0077] The compound of the invention or a pharmaceutically acceptable salt thereof and the pharmaceutical composition of the invention may be administered topically (including ophthalmic, vaginal, rectal, intranasal, epidermal, and transdermal), orally or parenterally. Parenteral administration includes intravenous, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g. by inhalation or insufflation, or intracranial, e.g. intrathecal or intraventricular administration. The compound of the invention or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the invention are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered can be determined by the skilled practitioner for each individual.

[0078] An embodiment of the present invention provides a use of the compound of the invention or a pharmaceutically acceptable salt thereof for manufacturing a medicament for preventing and / or treating a bacterial infection in a subject.

[0079] In some embodiments, the bacteria which adhesion is prevented or inhibited by the compound of the present invention or the pharmaceutically acceptable salt thereof and the bacteria which cause the bacterial infections are biofilm producing bacteria. In some other embodiments, the bacteria are Gram-positive bacteria or Gram-negative bacteria.

[0080] In preferred embodiments, the bacteria which adhesion is prevented or inhibited by the compound of the present invention or the pharmaceutically acceptable salt thereof and the bacteria which cause the bacterial infections, are selected from the group comprising P. aeruginosa, Acinetoacter baumannii, Escherichia coli, Enterococcus faecalis. Vibrio cholerae, Klebsiella pneumoniae, Clostridium difficile, and Salmonella enterica.

[0081] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein. The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the application and the scope of the invention.

[0082] EXAMPLES

[0083] The compound of the invention (MB1) can be obtained by any suitable methods known in the art. In an embodiment, the compound of the invention can be extracted from Dalbergia duperreana according to methods known in the art. According to another embodiment, the compound of the invention can be synthetized by chemical methods known in the art.

[0084] It was found that the compound of the invention (MB1) inhibits P. aeruginosa attachment in a dose-dependent manner. An image-based attachment analysis showed that MB1 can reduce bacterial attachment, with a half maximum effective concentration (EC50) of approximately 12 pM after 30 min of incubation (Fig. 1).

[0085] To test the applicability of this molecule to other infection types, it was explored how MB1 impacts the attachment of both Gram-negative and Gram-positive pathogens. For Gramnegative pathogens, apart from P. aeruginosa, the enteric pathogen Vibrio cholerae, causative agent of cholera, and the emerging drug resistant pathogen Acinetobacter baumannii were tested. In both cases, MB1 reduced the attachment of single cells to the surface (Fig. 2). MB1 reduced the number of attached V. cholerae cells by 65%. For A. baumannii, it was necessary to perform an extra washing step to fully demonstrate efficacy as this bacterium is non-motile. The results show that MB1 alone reduced baumannii attachment by 32% without wash, and by 83% after wash. The following pathogens were also tested: the Gram-positive pathogen Staphylococcus aureus, a major cause of skin infections and systemic diseases; Enterococcus faecalis, a commensal-turned-opportunist frequently associated with hospital-acquired infections; and Streptococcus mutans, a key contributor to dental caries and biofilm formation in the oral cavity. In all cases, MB1 reduced the attachment of single cells to the surface (Fig. 2). MB1 reduced the number of attached S. aureus cells by 60 %, E. faecalis cells by 65 % and S. mutans by 45 %. This demonstrates that MB1 has a broad spectrum which makes it potentially widely applicable as an anti-infective. To extend the investigation beyond abiotic surfaces, it was also examined whether MB1 could inhibit P. aeruginosa attachment to mammalian cells. The MDCK cell model was employed, which consists of canine kidney epithelial cells forming a confluent monolayer at the bottom of the culture plate. Microscopy revealed distinct signals: MDCK cells were visualized in magenta, indicating an intact epithelial layer, while Pseudomonas appeared in green. The results show that the treatment of wild-type Pseudomonas with MB1 resulted in a clear reduction of bacterial association with the epithelial surface. These results demonstrate that MB1 effectively inhibits Pseudomonas attachment to mammalian cells. (Fig. 3).

[0086] Then, MBl's efficacy in inhibiting pathogenicity was evaluated using a macrophage killing assay with a high P. aeruginosa multiplicity of infection (10: 1). It was found that MB1 decreased macrophage killing in a dose-dependent manner compared to the untreated control. While the bacteria quickly infected and killed the macrophages, resulting in 50% cytotoxicity at 6 h post-infection, cells incubated with 12.5 pM MB1 showed less than 20% cytotoxicity in the same timeframe. These promising results suggest that MB1 could be a potential therapeutic agent as an anti-virulence drug (Fig. 4).

[0087] To examine MBl's antimicrobial effect, a minimum inhibitory concentration (MIC) evaluation was conducted and a growth curve tracking study. P. aeruginosa's growth in Mueller-Hinton Broth (MHB) was monitored for 24 h and found that the MIC is higher than 100 pM, at least an order of magnitude higher than the dose required for inhibiting attachment. Moreover, MB 1 did not influence the growth rate of P. aeruginosa at concentrations of 25 pM and below. This was determined by identifying the doubling time ofP. aeruginosa growth. These results suggest that MB 1 acts as an anti -virulence molecule rather than an antimicrobial, aligning with the goal of finding an anti-virulence drug that applies less selection pressure and reduces the likelihood of bacterial resistance development (Fig. 5).

[0088] Next, the efficacy of MB1 was evaluated in inhibiting bacteria biofilm formation, including P. aeruginosa and S. mutans. Bacteria were incubated with MB1 for 24 hours, followed by washing and crystal violet staining to assess biofilm biomass on the bottom surface of the plate. (Fig. 6). The crystal violet staining revealed a marked reduction in biofilm formation in the presence of MB1. Compared to the untreated control, MB1 showed dose-dependent inhibition of biofilm formation: at 25 pM MB1, P. aeruginosa biofilm formation was reduced by approximately 40%, while S. mutans showed a 70% reduction. These findings indicate that MB1 disrupts biofilm development by interfering with stable surface attachment, highlighting its potential as a broad-spectrum anti-biofilm agent.

[0089] Additionally, it was found that MB 1 exhibits a synergistic effect with polymyxin B in inhibiting bacterial growth. Polymyxin B acts by disrupting the outer and inner membranes of Gramnegative bacteria through binding to lipopolysaccharides (LPS). When bacteria were treated with a sub-inhibitory concentration of polymyxin B (0.5 pg / mL), a delay in entry into the exponential growth phase was observed. This delay was further prolonged with co-treatment of MB1 and polymyxin B. Checkerboard assays performed across a range of polymyxin B and MB1 concentrations confirmed that the delayed growth phenotype is dose-dependent. The most pronounced reduction was observed with 25 pM MB1 combined with 0.5 pg / mL polymyxin B, where the OD at the peak time point (normalized to the untreated control) dropped from 59.7% with polymyxin B alone to just 8% with the combination treatment. Overall, co-treatment with MB1 and polymyxin B resulted in significant growth inhibition, supporting a synergistic interaction between the two compounds (see Fig. 7).

Claims

CLAIMS1. A compound of the formula:or a pharmaceutically acceptable salt thereof.

2. A composition for preventing or inhibiting bacterial adhesion to a non-living substrate comprising a carrier and an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.

3. A compound of claim 1 or a pharmaceutically acceptable salt thereof for use in a method for preventing or inhibiting bacterial adhesion to a living surface, comprising contacting the living surface with an effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

4. The compound for use of claim 3, wherein the contacting occurs with an animal, preferably a mammal, most preferably a human.

5. The compound for use of any one of claims 3-4, wherein the contacting occurs in the presence of an additional agent that impacts the growth and / or attachment and / or virulence of the bacteria.

6. The compound for use of claim 5, wherein the additional agent is selected from a microbiocidal agent, bacteriostatic agent, antibacterial agent, and combinations thereof.

7. A method for preventing or inhibiting bacterial adhesion to a non-living substrate comprising contacting the non-living substrate with an effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof.

8. A use of the compound of claim 1 or a pharmaceutically acceptable salt thereof for preventing or inhibiting bacterial adhesion on a non-living substrate.

9. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

10. An oral care composition comprising an effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof, and an orally acceptable carrier.

11. A compound of claim 1 or a pharmaceutically acceptable salt thereof for use as a medicament.

12. A compound of claim 1, or a pharmaceutically acceptable salt thereof, or an oral care composition of claim 10, for use in a method of preventing and / or inhibiting bacterial adhesion to an oral cavity surface of a mammal.

13. A compound of claim 1 or a pharmaceutically acceptable salt thereof for use in a method of preventing and / or treating a bacterial infection in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

14. The compound for use of claim 13, wherein the method further comprises administering an additional agent that impacts the growth and / or attachment and / or virulence of the bacteria.

15. The compound for use of claim 14, wherein the additional agent is selected from a microbiocidal agent, bacteriostatic agent, antibacterial agent, and combinations thereof.

16. The composition of claim 2, the compound for use of claims 3-6, the method of claim 7, the use of claim 8, or the compound for use of claims 12-15, wherein the bacteria are selected from the group comprising P. aeruginosa, Acinetoacter baumannii, Escherichia coli, Enterococcus faecalis. Vibrio cholerae, Klebsiella pneumoniae, Clostridium difficile,Salmonella enterica, Staphylococcus aureus, Streptococcus mutans and Porphyromonas gingivalis.

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