EP1773313A2 - Steuerung von biofilmbildung - Google Patents

Steuerung von biofilmbildung

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Publication number
EP1773313A2
EP1773313A2 EP05791709A EP05791709A EP1773313A2 EP 1773313 A2 EP1773313 A2 EP 1773313A2 EP 05791709 A EP05791709 A EP 05791709A EP 05791709 A EP05791709 A EP 05791709A EP 1773313 A2 EP1773313 A2 EP 1773313A2
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EP
European Patent Office
Prior art keywords
acid
compound
expression
tissue
compound modulates
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EP05791709A
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English (en)
French (fr)
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EP1773313A4 (de
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Gary R. Eldridge
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Individual
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Individual
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Priority claimed from US11/085,279 external-priority patent/US7604978B2/en
Priority claimed from US11/133,858 external-priority patent/US20060264411A1/en
Application filed by Individual filed Critical Individual
Publication of EP1773313A2 publication Critical patent/EP1773313A2/de
Publication of EP1773313A4 publication Critical patent/EP1773313A4/de
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention generally relates to methods and compounds useful for reducing or preventing invasion of a bacterium into a tissue comprising modulating the expression of a cysB gene in the bacterium.
  • the present invention also relates to an in vivo method for reducing or preventing the formation of a biof ⁇ lm in a tissue and to a method for controlling or preventing a chronic bacterial infection.
  • biofilms are involved in 65% of human bacterial infections. Biofilms are involved in prostatitis, biliary tract infections, urinary tract infections, cystitis, lung infections, sinus infections, ear infections, acne, rosacea, dental caries, periodontitis, nosocomial infections, open wounds, and chronic wounds.
  • Bacterial biofilms exist in natural, medical, and engineering environments.
  • the biofilms offer a selective advantage to a microorganism to ensure its survival, or allow it a certain amount of time to exist in a dormant state until suitable growth conditions arise.
  • this selective advantage poses serious threats to animal health, especially human health.
  • the inhibitors may also be used to cure, treat, or prevent a variety of conditions, such as, but are not limited to, arterial damage, gastritis, urinary tract infections, pyelonephritis, cystitis, otitis media, otitis externa, leprosy, tuberculosis, benign prostatic hyperplasia, chronic prostatitis, chronic lung infections of humans with cystic fibrosis, osteomyelitis, bloodstream infections, skin infections, open or chronic wound infections, cirrhosis, and any other acute or chronic infection that involves or possesses a biofilm.
  • conditions such as, but are not limited to, arterial damage, gastritis, urinary tract infections, pyelonephritis, cystitis, otitis media, otitis externa, leprosy, tuberculosis, benign prostatic hyperplasia, chronic prostatitis, chronic lung infections of humans with cystic fibrosis, osteomyelitis, bloodstream infections, skin infections,
  • biofilms Using the protection of biofilms, microbes can resist antibiotics at a concentration ranging from 1 to 1.5 thousand times higher than the amount used in conventional antibiotic therapy.
  • bacteria surrounded by biofilms are rarely resolved by the immune defense mechanisms of the host.
  • Costerton, Stewart, and Greenberg, leaders in the field of biofilms have proposed that in a chronic infection, a biofilm gives bacteria a selective advantage by reducing the penetration of an antibiotic into the depths of the tissue needed to completely eradicate the bacteria's existence.
  • Biofilm inhibitors can provide an alternative mechanism of action from conventional antibiotics. For example, successful treatment of nosocomial infections currently requires an administration of a combination of products, such as amoxicillin/clavulanate and quinupristin/dalfopristin, or an administration of two antibiotics simultaneously. In one study of urinary catheters, rifampin was unable to eradicate methicillin-resistant Staphylococcus aureus in a biofilm but was effective against planktonic, or suspended cells (Jones, S. M., et. al., "Effect of vancomycin and rifampicin on methicillin-resistant Staphylococcus aureus biofilms", Lancet. 357:40- 41, 2001). Biofilm inhibitors act on the biological mechanisms that provide bacteria protection from antibiotics and from a host's immune system. Biofilm inhibitors may be used to "clear the way" for the antibiotics to penetrate the affected cells and eradicate the infection.
  • biofilm inhibitors are not likely to trigger growth-resistance mechanisms or affect the growth of the normal human flora. Thus, biofilm inhibitors could potentially extend the product life of antibiotics.
  • Biofilm inhibitors can also be employed for the treatment of acne.
  • Acne vulgaris is the most common cutaneous disorder.
  • Propionibacterium acnes which is the predominant microorganism occurring in acne, reside in biofilms. Its existence in a biofilm matrix provides a protective, physical barrier that limits the effectiveness of antimicrobial agents (Burkhart, CN. et. al., "Microbiology's principle of biofilms as a major factor in the pathogenesis of acne vulgaris", International J. of Dermatology. 42:925-927, 2003).
  • Biofilm inhibitors may be used to effectively prevent, control, reduce, or eradicate P. acnes biofilms in acne.
  • Plaque biofilms contribute to cavities and and periodontitis. Plaque biofilms accumulate due to bacterial colonization of Streptococci spp. such as S. mutans, S. sobrinas, S. gordonii, and Porphyromonas gingivalis, and Actinomyces spp (Demuth, D. et al. Discrete Protein Determinant Directs the Species- Species Adherence of Porphyromonas gingivalis to Oral Streptococci, Infection and Immunity, 2001, 69(9) p5736-5741; Xie, H., et al. Intergeneric Communication in Dental Plaque Biofilms. J. Bacteriol.
  • Biofilm inhibitors can be employed to prevent microorganisms from adhering to surfaces that may be porous, soft, hard, semi-soft, semi-hard, regenerating, or non-regenerating.
  • These surfaces can be teeth, the polyurethane material of central venous catheters, or metal, alloy, or polymeric surfaces of medical devices, or regenerating proteins of cellular membranes of mammals, or the enamel of teeth.
  • These inhibitors can be coated on or impregnated into these surfaces prior to use, or administered at a concentration surrounding these surfaces to control, reduce, or eradicate the microorganisms adhering to these surfaces.
  • Diabetic foot ulcers are examples of the most common types of chronic wounds. Diabetic foot ulcers appear to be the most prevalent. These wounds are typically colonized by multiple species of bacteria including Staphylococcus spp., Streptococcus spp., Pseudomonas spp. and Gram- negative bacilli (Lipsky, B. Medical Treatment of Diabetic Foot Infections. Clin. Infect. Dis. 2004, 39, p.S104-14). Based on clinical evidence , researchers know that multiple microorganisms can cause or contribute to chronic wound infections.
  • Biofilm inhibitors in a combination therapy with antibiotics may provide an alternative to the treatment of chronic wounds.
  • Gram-negative bacteria share conserved mechanisms of bacterial pathogenesis involving cellular invasion and biofilms.
  • Haemophilus influenzae invade epithelial cells and form biofilms (Hardy, G. et al., Methods MoI. Med., 2003, 71, p.1-18; Greiner, L. et al, Infection and Immunity, 2004, 72(7) p.4249-4260).
  • Burkholderia spp. invade epithelial cells and form biofilm (Utaisincharoen, P, et al. Microb Pathog. 2005, 38(2-3) p.107-112; Schwab, U. et al.
  • Yersinia pestis invade epithelial cells and form biofilms (Cossart, P. Science, 2004, 304, p.242-248; Jarrett, C. et al. J. Infect. Dis., 2004, 190, p.783-792).
  • Neisseria gonorrhea invade epithelial cells and form biofilms (Edwards, J. et al., Cellular Micro., 2002, 4(9), p.585- 598;Greiner, L. et al. Infection and Immunity. 2004, 73(4), p.1964-1970).
  • E. coli and P. aeruginosa are two of the most widely studied Gram-negative pathogens. researchers believe that the pathogenesis of these bacteria involves invasion of host cells and formation of biofilms. These models have enabled those skilled in the art to understand the pathogenesis of other Gram-negative bacteria.
  • Gram-positive bacteria also share conserved mechanisms of bacterial pathogenesis involving cellular invasion and biofilms.
  • Staphylococcus aureus invade epithelial cells and form biofilms (Menzies, B, et al. Curr Opin Infect Dis, 2003, 16, p.225-229; Ando, E, et al. Acta Med Okayama, 2004, 58(4), p.207-14).
  • Streptococcus pyogenes invade epithelial cells and form biofilms (Cywes, C. et al., Nature, 2001,414, p.648-652; Conley, J, et al. J. Clin. Micro., 2003, 41(9), p.4043- 4048).
  • the present invention provides a method for reducing or preventing the invasion of a bacterium into a tissue comprising modulating the expression of a cysB gene in the bacterium.
  • the present invention further provides an in vivo method for reducing or preventing the formation of a biofilm in a tissue comprising modulating expression of a cysB gene in a cell capable of biofilm formation.
  • the present invention also provides a method for controlling or preventing a chronic bacterial infection in a subject in need thereof comprising modulating the expression of a cysB gene in a bacterium that causes or contributes to the chronic bacterial infection.
  • Figure 1 shows the chemical synthesis of an analog of ursolic acid.
  • Figure 2 shows a confocal microscopy image of an IBC of E coli from a bladder of a control mouse inoculated with E. coli UTI89.
  • Figure 3 shows a confocal microscopy image of a small collection of E coli from a bladder of a mouse inoculated with E. coli UTI89 and corosolic acid.
  • Figure 4 shows a confocal microscopy image of an IBC from a bladder of a mouse inoculated with wild type E. coli UTI89.
  • Figure 5 shows a confocal microscopy image of a loose collection of E coli from a bladder of a mouse inoculated with 50:50 E. coli UTI89 cysB ⁇ I wild type E. coli UIT89.
  • Figure 6 shows a confocal microscopy image of a loose collection of E coli from a bladder of a mouse inoculated with 50:50 E. coli UTI89 cysB ⁇ I wild type E. coli UIT89.
  • Acceptable carrier refers to a carrier that is compatible with the other ingredients of the formulation and is not deleterious to the recipient thereof.
  • Reducing or inhibiting in reference to a biofilm refers to the prevention of biof ⁇ lm formation or growth, reduction in the rate of biofilm formation or growth, partial or complete inhibition of biofilm formation or growth.
  • Modulates or “modulating” refers to up-regulation or down-regulation of a gene's replication or expression.
  • the present invention provides a method for reducing or preventing the invasion of a bacterium into a tissue comprising modulating the expression of a cysB gene in the bacterium.
  • the cysB gene may be modulated in a number of ways. For example, N- acetyl-serine and sulfur limitation up-regulate cysB. Lochowska, A. et al., Functional Dissection of the LysR-type CysB Transcriptional Regulator. J. Biol. Chem. 2001, 276, 2098-2107. In addition, like other LysR type regulators, cysB can repress itself. Lilic, M. et al., Identification of the CysB-regulated gene, hslJ, related to the Escherichia coli novobiocin resistance phenotype, FEMS Micro. Letters. 2003, 224:239-246.
  • a tissue is contacted with a composition comprising a compound selected from the group consisting of ursolic acid or asiatic acid, or a pharmaceutically acceptable salt of such compound, or hydrate of such compound, or solvate of such compound, an N-oxide of such compound, or combination thereof.
  • the compound is corosolic acid, 30-hydroxyursolic acid, 20- hydroxyursolic acid, 2-hydroxyoleanolic acid, and madecassic acid.
  • the compound is pygenic acid (A, B, or C), euscaphic acid, and tormentic acid.
  • the compounds used in the present invention may be isolated from a plant as previously described or prepared semi-synthetically (Eldridge, G, et al; Anal. Chem. 2002, 74, p. 3963-3971 ⁇ ). If prepared semi-synthetically, atypical starting material may be ursolic acid, oleanolic acid, corosolic acid, asiatic acid, madecassic acid or other compound used in the present invention. In designing semi-synthetic strategies to prepare analogs, certain positions of the scaffold of the compounds are important for modulating biofilm inhibition, while other positions improve bioavailability of the compounds, which could expand the therapeutic range of the compounds by reducing certain cellular toxicities in mammals.
  • Herbal preparations of Centella asiatica plant extracts which contain hundreds to thousands of compounds, have been used throughout history in numerous countries for the treatment of dermatological conditions, including wound healing, such as burns and scar reduction.
  • Herbal preparations of Centella asiatica plant extracts have also be used to treat asthma, cholera, measles, diarrhea, epilepsy, jaundice, syphilis, and cystitis. These herbal preparations are commercially available.
  • the preparations may include asiaticoside, madecassoside, brahmoside, brahminoside, asiatic acid, and madecassic acid.
  • ursolic acid and asiatic acid modulate the expression of a cysB gene in E. coll
  • the compound could also modulate the expression of genes under the control or within the same biochemical pathway as cysB.
  • the cysB protein is a transcriptional regulator of the Ly sR family of genes. The transcriptional regulators of this family have helix-turn-helix DNA binding motifs at their amino-terminus.
  • the cysB protein is required for the full expression of the cys genes, which are involved in the biosynthesis of cysteine.
  • cysDIJK The family of genes, cysDIJK are under the transcriptional control of the cysB gene.
  • cysD, cysl, cys J, and cysK are proteins involved in the biosynthesis of cysteine. CysK has been shown to respond to extracellular signals in bacteria (Sturgill, et al. J. Bacteriol. 2004, 186(22) p. 7610-7617).
  • YbiK is under the direct control of cysB and participates in glutathione intracellular transport.
  • bO829 is involved in glutathione transport.
  • bl729 is suspected to be a carboxylate transporter based upon sequence homology.
  • bl729 is conserved amongst Gram-negative and Gram-positive bacteria (http.7/www.ncbi.nlm.nih.gov/ sutils/genomtable.cgi). Accordingly, preferably, the compound used in the present invention modulates the expression of cysD, cysl, cys J, cysK,ybiK, B0829, bl729,yeeD, and/or yeeE.
  • LysR transcriptional regulators like CysB, have been demonstrated to regulate diverse metabolic processes.
  • cysB exhibits direct control of the biosynthesis of cysteine (Verschueren et al., at p. 260).
  • the cysB gene is involved, directly or indirectly, in glutathione intracellular transport, carbon source utilization, alanine dehydrogenases, and the arginine dependent system.
  • glutathione intracellular transport carbon source utilization
  • alanine dehydrogenases alanine dehydrogenases
  • arginine dependent system There is also recently published evidence that suggests that cysB responds directly or indirectly to extracellular signals (Sturgill, et al. J. Bacteriol. 2004,186(22) p. 7610-7617).
  • CysB regulates the expression of CysK, cysM, cysA, which are closely linked to err, ptsl, and ptsH (Byrne, et al. J. Bacteriol. 170(7) p. 3150-3157). Ptsl has been implicated in the sensing of external carbohydrates (Alder, et al. PNAS, 1974, 71, ⁇ .2895-2899).
  • the cysB gene in a Gram-negative bacterium is modulated.
  • the bacterium is Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae.
  • Gram-positive and Gram-negative bacteria invade their cellular hosts through conserved mechanisms of bacterial pathogenesis. The process enables the bacteria to evade the hosts' immune responses to allow the bacteria to increase their population. Therefore, compounds which can reduce bacterial invasion would significantly assist the immune system in the eradication of these pathogens. A reduction in bacterial invasion into cells would also increase the efficiency and potency of conventional antibiotics. Niels Moller-Frimodt demonstrated that antibiotics efficiently killed bacteria in the urine in a urinary tract infection, but were less effective in killing the bacteria in the bladder or tissues (Moller-Frimodt, N. Int. J. of Antimicrob Agents, 2002, 19, ⁇ .546-553).
  • cysB The cysB gene is genetically conserved among different species of bacteria, such as Gram-negative bacteria. Verschueren, et at, Acta Cryst. (2001) D57, 260- 262; Byrne et al., J. Bacteriol. 1988 170(7):3150-3157. In fact, cysB is conserved among Pseudomonas sp. including, but not limited to, P. aeruginosa, P. putida, and P. syringae. (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi).
  • the cysB gene is also genetically conserved among the following species of bacteria: Vibrio sp. ⁇ e.g. V. harveyi and V. cholera), Proteus mirablis, Burkholderia sp. (e.g. B. fongorum, B. mallei, and B.
  • cysB gene is also genetically conserved among the Gram-positive bacteria of Bacillus sp. including, but not limited to, B. subtilis, B. cereus, and B. anthracis.
  • the cysB gene is involved in the invasion of a bacterium into a cell.
  • the cell may be mammalian cells, preferably epithelial cells.
  • epithelial cells As demonstrated in the examples herein, the removal of a cysB gene from E. coli resulted in a significant reduction in invasion of E. coli into bladder epithelial cells as compared to wild-type E. coli.
  • the method reduces or prevents the invasion of a bacterium into a mammalian tissue.
  • the mammalian tissue is a murine tissue. More preferably, the mammalian tissue is a human tissue. Still preferably, the human tissue is a bladder, a kidney, or a prostate.
  • E. coli invades the kidney and prostate of humans. E. coli causes pyelonephritis and prostatitis, which are infectious diseases that can lead to death. (Russo, T., et al. Medical and economic impact of extraintestinal infections due to Escherichia coli: focus on an increasingly important endemic problem. Microbes and Infection.
  • the method reduces or prevents the invasion of a bacterium into a plant tissue.
  • Gram-negative bacteria invade and colonize plants.
  • the compounds of the invention that modulate cysB can be isolated from a very few plants, but to date it has not been shown that they can be isolated from commercial food crops or ornamental plants.
  • Pseudomonas putida a Gram-negative bacterium, forms biofilms on plants (Arevalo-Ferro, C; Biofilm formation of Pseudomonas putida IsoF: the role of quorum sensing as assessed by proteomics. Syst. Appl. Microbiol. 2005, 28(2) p.87-114.) Plants that produce the compounds used in the present invention have probably evolved to make these compounds to reduce, prevent, or control the invasion of bacteria and the formation of biofilms.
  • the present invention further provides an in vivo method for reducing or preventing the formation of a biofilm in a tissue comprising modulating expression of a cysB gene in a cell capable of biofilm formation.
  • cysB plays a significant role in the formation of biofilms and the invasion of bacteria into mammalian cells. Therefore, the cysB gene is vital for the pathogenesis of bacteria.
  • Compounds used in the present invention reduce the formation of biofilms and reduce or prevent the invasion of bacteria into mammalian cells.
  • the compounds modulate the expression of a cysB gene in a cell capable of biofilm formation.
  • the in vivo method comprises contacting the tissue with a composition comprising a compound selected from the group of ursolic acid, or asiatic acid, or a pharmaceutically acceptable salt of such compound, or hydrate of such compound, or solvate of such compound, an N-oxide of such compound, or combination thereof.
  • Example 5 show that asiatic acid, corosolic acid and madecassic acid, along with an antibiotic, can reduce the sustainability of pre-formed biofilms. Because biofilm contributes to many chronic bacterial infections, these examples strongly support the use of the compounds of the present invention to treat chronic bacterial infections, such as lung and ear infections and diabetic foot ulcers. The results of the examples demonstrate the distinct difference between the methods used to discover biofilm inhibitors and the NCCLS methods used to discover conventional antibiotics. Not surprisingly, the NCCLS method fails to identify antibiotics that can effectively treat chronic infections involving biofilms.
  • the compound is corosolic acid, 30-hydroxyursolic acid, 20-hydroxyursolic acid, 2-hydroxyoleanolic acid, and madecassic acid, hi another preferred embodiment, the compound is pygenic acid (A, B, or C), euscaphic acid, and tormentic acid.
  • the compound By modulating the cysB gene, the compound could also modulate the expression of genes under the control or within the same biochemical pathway as cysB. Preferably, the compound modulates the expression of cysD, cysl, cysJ, cysK, ybiK, bO829, b 1729, yee D, and/or yeeE.
  • the cysB gene in a Gram-negative bacterium is modulated.
  • the bacterium is Escherichia coli, Pseudomonas aeruginosa, Haemophilus influenzae.
  • Examples 1, 4, 5, and 6 demonstrate that the compounds of the present invention serve as biofilm inhibitors by reducing the attachment of Pseudomonas aeruginosa, Escherichia coli, Streptococcus mutans, and Streptococcus sobrinas to surfaces.
  • the compounds prevent, reduce or inhibit biofilm across a broad spectrum of bacteria.
  • the present invention demonstrates that asiatic acid, corosolic acid, madecassic acid exhibit inhibition or reduction of biofilm of bacteria that are genetically diverse from each other. These bacteria may be Gram-positive or Gram- negative and may beclinical or laboratory strains.
  • the examples also specifically demonstrate that asiatic acid, corosolic acid and madecassic acid can reduce a mature biofilm with antibiotic.
  • the method reduces or prevents formation of a biofilm in a mammalian tissue.
  • the mammalian tissue is a murine tissue. More preferably, the mammalian tissue is a human tissue. Still preferably, the human tissue is a bladder, a kidney, or a prostate.
  • the method reduces or prevents formation of a biofilm in a plant tissue.
  • the present invention also provides a method for controlling or preventing a chronic bacterial infection in a subject in need thereof comprising modulating the expression of a cysB gene in a bacterium that causes or contributes to the chronic bacterial infection.
  • Biofilm inhibitors will have a substantial medical impact by treating many chronic infections, reducing catheter- and medical device-related infections, and treating lung and ear infections. Biofilm inhibitors may be used to control microorganisms existing extracellularly or intracellularly of living tissues.
  • ⁇ may be used to cure, treat, or prevent a variety of conditions, such as, but are not limited to, arterial damage, gastritis, urinary tract infections, otitis media, leprosy, tuberculosis, benign prostatic hyperplasia, cystitis, pyeolonephritis, prostatitis, lung, ear, and sinus infections, periodontitis, cirrhosis, osteomyelitis, bloodstream infections, skin infections, acne, rosacea, open or chronic wound infections, and any other acute or chronic infection that involves or possesses a biofilm.
  • conditions such as, but are not limited to, arterial damage, gastritis, urinary tract infections, otitis media, leprosy, tuberculosis, benign prostatic hyperplasia, cystitis, pyeolonephritis, prostatitis, lung, ear, and sinus infections, periodontitis, cirrhosis, osteomyelitis, bloodstream infections, skin infections, acne
  • the modulation of the cysB gene comprises administering to a subject in need thereof with an effective amount of a composition comprising a compound selected from the group consisting of ursolic acid or asiatic acid, or a pharmaceutically acceptable salt of such compound, or hydrate of such compound, or solvate of such compound, an N-oxide of such compound, or combination thereof.
  • the compound is corosolic acid, 30-hydroxyursolic acid, 20-hydroxyursolic acid, 2-hydroxyoleanolic acid, and madecassic acid.
  • the compound is pygenic acid (A, B, or C), euscaphic acid, and tormentic acid.
  • the compound By modulating the cysB gene, the compound could also modulate the expression of genes under the control or within the same biochemical pathway as cysB. Preferably, the compound modulates the expression of cysD, cysl, cysJ, cysK, ybiK, bO829, bl729,yeeD, and/or yeeE.
  • the chronic bacterial infection is selected from the group consisting of urinary tract infection, gastritis, lung infection, ear infection, cystitis, pyelonephritis, arterial damage, leprosy, tuberculosis, benign prostatic hyperplasia, prostatitis, osteomyelitis, bloodstream infection, cirrhosis, skin infection, acne, rosacea, open wound infection, chronic wound infection, and sinus infection.
  • Example 7 demonstrates how the compounds of the present invention interrupt, delay, or prevent the cycle of pathogenesis of other E. coli infections such as, but not limited to, pyelonephritis, prostatitis, meningitis, sepsis, and gastrointestinal infections.
  • the chronic bacterial infection results from an infection of a bacterium.
  • the bacterium is a Gram-negative bacterium. More preferably, the bacterium is Escherichia coli, Pseudomonas aeruginosa, or Haemophilus influenzae.
  • the chronic bacterial infection causes an autoimmune disease in a mammal.
  • the mammal is a human.
  • the bacteria may also play a critical role in other autoimmune diseases such as CRST syndrome (calcinosis, Raynaud's phenomenon, sclerodactyly, telangiectasia), the sicca syndrome, autoimmune thyroiditis, or renal tubular acidosis, ankylosing spondylitis, antiphospholipid syndrome, Crohn's disease, ulcerative colitis, insulin dependent diabetes, fibromyalgia, Goodpasture syndrome, Grave's disease, lupus, multiple sclerosis, myasthenia gravis, myositis, pemphigus vulgaris, rheumatoid arthritis, sarcoidosis, scleroderma, or Wegener's granulomatosis.
  • CRST syndrome calcinosis, Raynaud's phenomenon, sclerodactyly, telangiectasia
  • the sicca syndrome autoimmune thyroiditis
  • renal tubular acidosis ankylosing spondylitis
  • the present invention may be used to treat autoimmune diseases caused by bacteria that invade and live within a protective biofilm.
  • Biofilm inhibition experiments were conducted using an assay adapted from the reported protocol described in Pratt and Kolter, 1998, Molecular Microbiology, 30: 285-293; Li et al., 2001, J. Bacterid., 183: 897-908.
  • E. coli clinical strain UTI89 was grown in LB in 96 well plates at room temperature for one or two days without shaking.
  • E. coli laboratory strain JMl 09 was grown in LB plus 0.2% glucose in 96 well plates at room temperature for one day without shaking.
  • To quantify the biofilm mass the suspension culture was poured out and the biofilm was washed three times with water. The biofilm was stained with 0.1% crystal violet for 20 minutes. The plates were then washed three times with water.
  • OD reading at 540 nm was measured to quantify the biofilm mass at the bottom of the wells. Then 95% ethanol was added to dissolve the dye at the bottom and on the wall and the OD reading at 540 nm was measured to quantify the total biofilm mass. To study the overall effect of the compounds (3.6 mg/mL in 100% ethanol as stock solution), it was added with the inoculation and a time course of biofilm mass was measured. Appropriate amounts of 100% ethanol were added to each sample to eliminate the effect of solvent. Each condition had 3-4 replicates on each plate and was performed over multiple days.
  • Madecassic acid inhibited biofilm formation of the JM109 strain by about 75% and 60% as compared to the controls at 10 and 5 ug/ml, respectively.
  • An isogenic cysB deletion mutant was prepared from E. coli clinical strain UTI89. Briefly, the construction of a cysB deletion strain was prepared as follows: the red-recombinase method was utilized (Murphy, K. C, and K. G. Campellone. 2003. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC MoI Biol 4:11).
  • Peg lids were rinsed three times in sterile water, placed onto flat-bottom microtiter plates containing biofilm inhibitors at 5 ug/ml in 100 ⁇ l of CAMHB per well and incubated for approximately 40 hours at 37°C.
  • Pegs were rinsed, placed in a 0.1% (wt/vol) crystal violet solution for 15 min, rinsed again, and dried for several hours. To solubilize adsorbed crystal violet, pegs were incubated in 95% ethanol (150 ⁇ lper well of a flat-bottom microtiter plate) for 15 min. The absorbance was read at 590 nm on a plate reader. The wells containing asiatic acid were compared to negative controls. Negative controls were prepared as stated above but without asiatic acid.
  • Asiatic acid caused an average detachment of mature biofilms of approximately 50% at 5 ug/ml compared to the negative controls against eighteen clinical isolates of P. aeruginosa. The range of detachment of mature biofilms against all eighteen clinical isolates was 25% to 74%. This example demonstrates the ability of asiatic acid and the compounds of the invention to reduce mature biofilms in clinical isolates of P. aeruginosa.
  • Biofilm formation of P. aeruginosa was evaluated using a standardized biofilm method with a rotating disk reactor (RDR).
  • RDR rotating disk reactor
  • the rotating disk reactor consists of a one-liter glass beaker fitted with a drain spout.
  • the bottom of the vessel contains a magnetically driven rotor with six 1.27 cm diameter coupons constructed from polystyrene.
  • the rotor consists of a star-head magnetic stir bar upon which a disk was affixed to hold the coupons.
  • the vessel with the stir bar was placed on a stir plate and rotated to provide fluid shear.
  • a nutrient solution (AB Trace Medium with 0.3 mM glucose, see Table 1 below for composition) was added through a stopper in the top of the reactor at a flow rate of 5 ml/min.
  • the reactor volume was approximately 180 ml and varied slightly between reactors depending on the placement of the drain spout and the rotational speed of the rotor. At a volume of 180 ml, the residence time of the reactors was 36 minutes. The reactors were operated at room temperature (c.a. 26 0 C). [96] Table 1. Composition of the AB Trace Medium used for the RDR test.
  • Control reactors received 10 ml of ethanol. The reactors were then incubated for an additional 24 hours in batch (no flow) mode. After this incubation period, the six coupons were removed from each reactor and placed in 12-well polystyrene tissue culture plates with wells containing either 2 ml of a 100 ⁇ g/ml tobramycin solution or 2 ml of phosphate-buffered saline (PBS). These plates were incubated at room temperature for two hours. The coupons were then rinsed by three transfers to plates containing 2 ml of fresh PBS.
  • PBS phosphate-buffered saline
  • the resulting bacterial suspensions were then serially diluted in PBS and plated on tryptic soy agar plates for enumeration of culturable bacteria. The plates were incubated for 24 hours at 37° C before colony forming units (CFU) were determined.
  • CFU colony forming units
  • Asiatic acid was tested against S. mutans 25175 and S. sobrinus 6715 at a concentration of 40 ug/ml using the method described in Example 1. The use of 1 mL polycarbonate tubes were used in place of 96 well polysterene microtiter plates. [107] Testing asiatic acid at 40 ⁇ g/mL against S. mutans 25175 and S. sobrinus 6715 showed greater than 75 % biofilm growth inhibition.
  • Binding was assessed at time zero and invasion was assessed at approximately 5, 15, 30, or 60 minutes from completing the mixture of compound, bacteria, and epithelial cells. As a control ethanol was added to cells to a final concentration of 0.1%. The effect of bacterial viability and bacterial adherence during the infection period was evaluated according to the methods described in Martinez et al., 2000, EMBO J., 19:2803-2812. The test compounds did not affect the binding of E. coli to bladder epithelial cells. The test compounds reduced the invasion of E. coli into bladder epithelial cells.
  • the present invention demonstrates that corosolic acid, asiatic acid, ursolic acid, and other compounds of the present invention reduce invasion of E. coli into bladder epithelial cells and therefore interrupt the pathogenesis of E. coli in bladder epithelial cells.
  • the cycle of pathogenesis of E. coli in recurrent urinary tract infections involves repeated invasions allowing the bacteria to survive and persist in the host.
  • the invasion of E. coli into the bladder epithelial cells enables them to resist the mammalian immune response, which allows the bacteria to re-invade deeper into host's tissues.
  • the compounds interrupt a key point in the bacteria's life cycle.
  • Example 6 The method described in Example 6 was used to examine the binding and invasion of E. coli UTI89 cysB ' (described in Example 2) into bladder epithelial cells.
  • E. coli UTI89 cysB ⁇ exhibited about 93% reduction of invasion into bladder epithelial cells as compared to wild type. The invasion of E. coli UTI89 cysB ⁇ into bladder epithelial cells was slightly restored by plasmid complementation of cysB demonstrating only a 70% reduction of invasion as compared to wild type.
  • Asiatic acid and madecassic acid were evaluated at 50 mg/kg (oral). Two animals were assigned to each group. Prior to dosing, a baseline blood sample was taken from each animal. At time zero, asiatic acid and madecassic acid, a single bolus dose in 50% Labrasol (Gattefosse) was given to each animal. Bladders were analyzed at 24 hours. Concentrations of both asiatic acid and madecassic acid in the bladder were approximately 30 ⁇ g/g at 24 hours. Asiatic acid and madecassic acid significantly reduced bacterial invasion within 15 minutes of administration.
  • E. coli UTI89[pCOMGFP] was prepared after retrieval from frozen stocks by inoculating in LB medium statically for approximately 20 hours. Cells were harvested and suspended in 1 ml of PBS. Cells were diluted to achieve approximately a 10 CFU or 10 7 CFU input into C3H/HeN mice (2 mice per group). [125] Mice were deprived of water for approximately two hours. In experiment 1, all mice were anesthetized with 0.15 cc ketamine cocktail.
  • mice were anesthetized with isofluorane.
  • urine was dispelled from the bladders and approximately 40 ⁇ g/ml of test compound or an appropriate amount of ethanol as control was introduced into the bladders via catheterization of the urethra using a tubing coated tuberculin syringe. 30 minutes was allowed to elapse.
  • bladders were not pre-incubated with test compounds. Bladders were then expelled and an inoculum of 10 CFU (Experiment 1) or 10 CFU (Experiment 2) of E. coli containing 40 ⁇ g/ml of test compound or equivalent amount of ethanol as controls were introduced into the bladders as indicated above.
  • mice were anesthetized and sacrificed.
  • the bladders were removed, bisected, stretched, and fixed in 3% paraformaldehyde for 1 hour at room temperature.
  • Bladders were then permeabilized in 0.01% Triton/PBS for 10 minutes and counter stained with TOPRO3 (Molecular Probes) for 10 minutes for visualization by confocal microscopy. Bladders were mounted on Prolong antifade (Molecular Probes).
  • the compounds of the present invention can work in combination with the mammalian immune system and/or an antibiotic to reduce, prevent, treat, or eradicate the bacterial infections involving biofilms.
  • This animal model is representative of chronic lung, ear, and sinus infections, acne, rosacea, and chronic wounds. It is also representative of the cycle of pathogenesis of other E. coli infections such as, but not limited to, pyelonephritis, prostatitis, meningitis, sepsis, and gastrointestinal infections.
  • coli enables the bacteria to form biof ⁇ lms in the tissues of bladders.
  • the cysB gene is genetically conserved amongst Gram-negative bacteria. Therefore, it is contemplated that modulation of this gene by the compounds of the present invention would also reduce the formation of biofilms in chronic infections caused by other bacteria besides E. coli.
  • a topical gel was prepared containing 2% of madecassic acid by weight with azithromycin for use in treating acne, rosacea, and skin infections
  • Solutions were prepared comprising 2 mg/ml and 10 mg/ml of madecassic acid in ethanol/propylene glycol/water (85:10:5). These solutions were nebulized separately by a ProNeb Ultra nebulizer manufactured by PARI. The nebulized solutions were collected in a cold trap, processed appropriately, and detected by mass spectrometry. Madecassic acid was recovered from both formulations demonstrating that nebulization can be used to deliver this compound to patients with lung infections.
  • Toothpaste preparations were prepared containing 2% madecassic acid with and without antibiotic and with and without polymer.
  • polymer Gantrez S-97, was added to improve retention of madecassic acid and antibiotic on teeth.
  • Formulations A and B were prepared and stored for thirty days at 2°C to 8°C, room temperature (approximately 22°C), and at 30°C.
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US11/133,858 US20060264411A1 (en) 2005-05-20 2005-05-20 Control of biofilm formation
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Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7604978B2 (en) 2004-07-14 2009-10-20 Sequoia Sciences, Inc. Inhibition of biofilm formation
CA2580078A1 (en) 2004-09-14 2006-03-23 Gary R. Eldridge Compounds, compositions and methods for controlling biofilms and bacterial infections
TWI413646B (zh) * 2010-09-03 2013-11-01 Univ China Medical 用於抑制基質金屬蛋白酶活性及／或生成、抑制有絲分裂原活化蛋白激酶磷酸化、及／或促進膠原蛋白生成之醫藥組合物及其用途
CN102784096B (zh) * 2011-05-18 2016-10-12 上海现代药物制剂工程研究中心有限公司 一种积雪草酸自微乳化给药系统及其制备方法
US8324264B1 (en) 2011-07-22 2012-12-04 Sequoia Sciences, Inc. Inhibitors of bacterial biofilms and related methods
WO2013021258A1 (en) * 2011-08-05 2013-02-14 Council Of Scientific & Industrial Research Anti-tubercular compounds
EP2712863B1 (de) * 2012-09-28 2014-10-22 Sequoia Sciences, Inc. Neue Hemmer bakterieller Biofilme und zugehörige Verfahren
EP2928451B1 (de) * 2012-12-06 2017-07-26 Colgate-Palmolive Company Orales gel zur linderung von zahnschmerzen
CN108358991B (zh) * 2018-02-01 2020-11-10 云南中烟工业有限责任公司 一种澄广花中的三萜类化合物及其制备方法和应用
CN110141572B (zh) * 2019-06-20 2020-05-12 牡丹江医学院 一种治疗哮喘的药物及其制备方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5985601A (en) * 1995-06-05 1999-11-16 Human Genome Sciences, Inc. DNA encoding human cystatin E
FR2750326B1 (fr) * 1996-06-28 1998-07-31 Oreal Composition cosmetique et/ou dermatologique acide contenant un poly(acide 2-acrylamido 2-methylpropane sulfonique) reticule et neutralise a au moins 90 %
US6264926B1 (en) * 1999-02-12 2001-07-24 Council Of Scientific And Industrial Research Formulation useful as a natural herbal tooth powder
US6669929B1 (en) * 2002-12-30 2003-12-30 Colgate Palmolive Company Dentifrice containing functional film flakes

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
BELOIN C ET AL: "Finding gene-expression patterns in bacterial biofilms" TRENDS IN MICROBIOLOGY, ELSEVIER SCIENCE LTD., KIDLINGTON, GB, vol. 13, no. 1, January 2005 (2005-01), pages 16-19, XP004705447 ISSN: 0966-842X *
DONLAN RODNEY M ET AL: "Biofilms: Survival mechanisms of clinically relevant microorganisms" CLINICAL MICROBIOLOGY REVIEWS, vol. 15, no. 2, April 2002 (2002-04), pages 167-193, XP002438976 ISSN: 0893-8512 *
HALL-STOODLEY LUANNE ET AL: "Bacterial biofilms: From the natural environment to infectious diseases" NATURE REVIEWS MICROBIOLOGY, vol. 2, no. 2, February 2004 (2004-02), pages 95-108, XP002438975 ISSN: 1740-1526 *
KAPLAN J B: "Methods for the treatment and prevention of bacterial biofilms" EXPERT OPINION ON THERAPEUTIC PATENTS, ASHLEY PUBLICATIONS, GB, vol. 15, no. 8, 2005, pages 955-965, XP002383461 ISSN: 1354-3776 *
LOCHOWSKA A ET AL: "Functional dissection of the LysR-type CysB transcriptional regulator. Regions important for DNA binding, inducer response, oligomerization, and positive control" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 276, no. 3, 19 January 2001 (2001-01-19), pages 2098-2107, XP002295378 ISSN: 0021-9258 *
MAH THIEN-FAH C ET AL: "Mechanisms of biofilm resistance to antimicrobial agents" TRENDS IN MICROBIOLOGY, vol. 9, no. 1, January 2001 (2001-01), pages 34-39, XP002438506 ISSN: 0966-842X *
REN ET AL: "Differential gene expression for investigation of Escherichia coli biofilm inhibition by plant extract ursolic acid" APPLIED AND ENVIRONMENTAL MICROBIOLOGY, WASHINGTON,DC, US, vol. 71, no. 7, July 2005 (2005-07), pages 4022-4034, XP002365546 ISSN: 0099-2240 *
See also references of WO2006010147A2 *
STANLEY NICOLA R ET AL: "Identification of catabolite repression as a physiological regulator of biofilm formation by Bacillus subtilis by use of DNA microarrays" JOURNAL OF BACTERIOLOGY, WASHINGTON, DC, US, vol. 185, no. 6, March 2003 (2003-03), pages 1951-1957, XP002301705 ISSN: 0021-9193 *

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