EP4398891A1 - Compositions et méthodes de traitement de troubles et d'infections liés au biofilm - Google Patents

Compositions et méthodes de traitement de troubles et d'infections liés au biofilm

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Publication number
EP4398891A1
EP4398891A1 EP22769406.4A EP22769406A EP4398891A1 EP 4398891 A1 EP4398891 A1 EP 4398891A1 EP 22769406 A EP22769406 A EP 22769406A EP 4398891 A1 EP4398891 A1 EP 4398891A1
Authority
EP
European Patent Office
Prior art keywords
composition
compound
biofilm
thiosulfinate
formula
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22769406.4A
Other languages
German (de)
English (en)
Inventor
Lex De Boer
Gerrit Jan STREEFLAND
Anna Kristina WRONSKA
Anne Myrthe SCHUTTEVAAR
Shaista Sheroze KHOKHAR
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Ahv International BV
Original Assignee
Ahv International BV
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Filing date
Publication date
Application filed by Ahv International BV filed Critical Ahv International BV
Priority claimed from PCT/NL2022/050507 external-priority patent/WO2023038522A1/fr
Publication of EP4398891A1 publication Critical patent/EP4398891A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N41/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom
    • A01N41/02—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom containing a sulfur-to-oxygen double bond
    • A01N41/04—Sulfonic acids; Derivatives thereof
    • A01N41/08—Sulfonic acid halides; alpha-Hydroxy-sulfonic acids; Amino-sulfonic acids; Thiosulfonic acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N41/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom
    • A01N41/12—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom not containing sulfur-to-oxygen bonds, e.g. polysulfides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution

Definitions

  • compositions and methods for treating biofilm disorders and infection FIELD OF THE INVENTION
  • the disclosure relates to organosulfur containing compositions, in particular Di-n-butyl thiosulfinate, Di-methyl thiosulfonate, Di -phenyl thiosulfonate, Bis (p-tolyl) thiosulfinate, Di-isopropyl thiosulfinate, Di-benzyl thiosulfinate, Di-benzyl thiosulfonate, S-propyl-4-methylbenzene thiosulfonate, Di -n-propyl thiosulfinate, and Di-n-propyl thiosulfonate.
  • compositions are useful for treating infection and reducing or degrading biofilms both in vivo and in vitro.
  • such compositions are useful in the treatment of biofilm-related disorders, including but not limited to mastitis, digital dermatitis, and chronic wound infections.
  • biofilm-related disorders including but not limited to mastitis, digital dermatitis, and chronic wound infections.
  • mastitis The infection in mammary glands of dairy animals, such as cows, known as mastitis, has a significant economic impact on dairy farms worldwide. The overall global loss per year is estimated to be four to five billion Euros. In dairy mastitis, the udder is incapable of mounting an efficacious defence response to invading microorganisms.
  • mastitis a major challenge in the control of mastitis are effective antibiotics able to reach bacterial pools in the udder for instance as a result of the formation of biofilms by bacteria.
  • Microorganisms such as bacteria, do not necessarily need to produce a biofilm, but they have much better possibilities to survive in the host if they can adhere, for instance, to epithelial cells.
  • Adhesion is an active process, involving a series of attachments and detachments, with resulting biofilm formation which is accompanied by significant genetic and subsequent physiological changes in the microorganisms resulting, inter alia, in a loss of sensitivity to virtually all classes of antibiotics.
  • microbial biofilms can be formed also on biological surfaces is human and animal tissues such as the periodontal mucosa in the oral cavity (dental plaque), nasal sinuses (chronic sinusitis), the inner ear (otitis media), blood vessels and heart valves (endocarditis), the alveolar surface (multiple lung diseases) or the biliary and urinary bladder.
  • the first stage of biofilm formation comprises the attachment of cells to a surface.
  • cell multiplication occurs accompanied by the formation of mature structures have many layers of cells.
  • a slime layer is also formed which further protects the bacteria. See, e.g., Melchior et al. Veterinary Journal 2006171:398-407.
  • Biofilms may form on a wide variety of surfaces, including living tissues, indwelling medical devices, industrial or potable water system piping, or natural aquatic systems. As will be understood by a skilled person, not all infections lead to the development of biofilms. Research in the last 20 years revealed that collective biofilm formation is facilitated by bacterial communication system, denoted as quorum sensing (QS). QS occurs by means of small chemical molecules (so called auto-inducers, AI) permanently excreted by bacteria into their environment.
  • QS quorum sensing
  • oligopeptides AIP
  • AHL N-acetyl homoserine lactones
  • bacterial cells collectively alter gene expression and either produce virulence factors to attack body cells, or to activate metabolic pathways to form a biofilm at tissue surfaces.
  • Biofilm formation involves the formation of an extracellular matrix consisting of large polymers, initially predominantly polysaccharides, which upon maturation are stabilized by proteins and lipids, resulting in three-dimensional structure. Once a biofilm infection has been established, it can be very difficult to eradicate. Mature biofilms will intermittently release planktonic cells.
  • Dormant bacteria are metabolically inactive and hence do not express the typical targets of many antibiotics such as synthesis of bacterial cell wall constituents (target for beta-lactam antibiotics such as penicillins and cephalosporins and Vancomycin) and rapid protein (target for Aminoglycosides, Tetracyclines, Macrolides and Linezolid) and DNA synthesis (Fluoroquinolones and Rifampicin) or Folic acid synthesis (Sulfonamides, aminopyrimidines (such as Trimethoprim). Therefore, antibiotic treatment alone is generally not sufficient to eradicate biofilm infections (see also Wu et al. Int J Oral Sci. 2015 Mar; 7(1): 1–7).
  • MIC planktonic bacteria
  • MBEC biofilm eradication concentrations
  • antibiotics such as tetracyclines, quinopristine-dalfopristins, and erythromycin
  • genes e.g, the ica genes
  • Biofilms employ a number of mechanisms to evade a host’s immune response including activating regulators/suppressors that affect immune cell activity and acting as a physical barrier to immune cells (Gonzalez Pathog Dis. 2018 Apr; 76(3)), but can transit out of dormancy and become active. In general, the immune system only acts against active bacteria and therefore the dormant bacteria can escape the immune system of an individual. Dormant bacteria are able to detach from the biofilm and quickly become active and harmful to the host. Fungal related biofilms are also known to be more resistant to antifungal drugs as compared to planktonic cells (see, e.g., Fanning and Mitchell PLOS Pathog 2012 8:e1002585 for a review).
  • biofilm-associated infections i.e., biofilm related disorders
  • Such disorders may be characterized by a chronic inflammatory response with recurrent acute episodes and resistance to antimicrobial therapy and/or host defenses.
  • Biofilms in particular, P. aeruginosa biofilms, also cause chronic infections in the respiratory diseases such as bronchiectasis, chronic obstructive pulmonary disease and in chronic rhinosinusitis.
  • Biofilms formed on medical devices serve as a reservoir of bacteria that can be shed into the body, leading to a chronic systemic infection.
  • Candida albicans a yeast is the most common fungal biofilm found in hospitals, but is extremely difficult to treat and do not respond well to typical antifungal treatments.
  • R 1 and R 2 are independently selected from optionally substituted alkyl or optionally substituted aryl; for use in the treatment of a biofilm-related disorder.
  • compositions comprising a compound according to Formula I: Formula I, wherein n is 2; wherein one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O) 2 -; and R 1 and R 2 are independently selected from optionally substituted alkyl or optionally substituted aryl; wherein the composition is a pharmaceutical composition, functional food, or a cleaning product.
  • An in vitro method for preventing or reducing biofilm formation or growth on a surface or for degrading or reducing biofilms on a surface comprising applying a composition to the surface such as to prevent or reduce biofilm formation or growth on a surface, or such as to degrade or reduce biofilms on a surface, wherein said composition comprises a compound according to Formula I: Formula I, wherein n is 2; wherein one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O) 2 -; and R 1 and R 2 are independently selected from optionally substituted alkyl or optionally substituted aryl.
  • composition for use, the method, or the composition according to any one of the preceding embodiments, wherein the composition is essentially free of diallyl thiosulfinate.
  • composition further comprises an antimicrobial agent, preferable selected from an antifungal or an antibiotic.
  • composition further comprises anti- inflammatory agent.
  • R 1 and R 2 are independently selected from C1-6 alkyl, and phenyl; preferably R 1 and R 2 are independently C 1-4 alkyl.
  • R 1 and R 2 are identical.
  • each X is -S-, and R 1 and R 2 are independently C 1-4 alkyl.
  • one X is -S- and the other X is selected from the group consisting of -S(O)- and -S(O) 2 -; and R 1 and R 2 are independently selected from alkyl, aryl, alkylaryl, and arylalkyl.
  • R 1 and R 2 are independently selected from the group consisting of isopropyl, butyl, benzyl, and p-tolyl; and wherein when the other X is -S(O) 2 -, R 1 and R 2 are independently selected from the group consisting of methyl, phenyl, benzyl, 4-methylbenzene, and n-propyl.
  • the disclosure relates to compounds according to Formula I: Formula I, wherein n is 2; wherein one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O) 2 -; and R 1 and R 2 are independently selected from optionally substituted alkyl and optionally substituted aryl.
  • Such compounds also referred to herein as organosulfur compounds
  • organosulfur compounds are useful for reducing, degrading, and/or preventing the formation of biofilms.
  • alkyl relates to a saturated aliphatic hydrocarbyl group.
  • an alkyl group can be linear or branched. Preferably, alkyl groups are linear. As used herein, alkyl groups can be substituted or unsubstituted. Preferably, alkyl groups are unsubstituted. In preferred embodiments, in Formula I an alkyl is a C 1-6 alkyl, more preferably a C 1-4 alkyl.
  • aryl refers to an aromatic hydrocarbon ring system that comprises six to twenty-four carbon atoms, more preferably six to twelve carbon atoms, and may include monocyclic and polycyclic structures. When the aryl group is a polycyclic structure, it is preferably a bicyclic structure.
  • the aryl group is substituted by one or more substituents further specified in this document.
  • an aryl group is unsubstituted.
  • aryl groups are phenyl, benzyl, and naphthyl.
  • an aryl group is phenyl.
  • substituted indicates that a group contains one or more substituents.
  • halogens are selected from the group consisting of -Cl, -F, -Br, and -I. Most preferably, a halogen is -Cl.
  • the groups as disclosed herein contain at most three substituents, more preferably at most two substituents, and most preferably at most one substituent.
  • R 1 and R 2 are independently selected from alkyl and aryl. More preferably, R 1 and R 2 are independently selected from C1-6 alkyl, and phenyl. Even more preferably, R 1 and R 2 are independently C 1-4 alkyl. Most preferably, R 1 and R 2 are independently linear C 1-4 alkyl, i.e.
  • each X is -S-.
  • one X is -S- and the other X is -S(O)-.
  • one X is -S- and the other X is -S(O) 2 -.
  • R 1 and R 2 are independently C 1-4 alkyl, preferably linear C 1-4 alkyl, i.e. methyl, ethyl, n-propyl, or n-butyl.
  • compositions comprising these particular compounds according to Formula I further comprise one or more antimicrobial agents.
  • one X is -S- and the other X is selected from the group consisting of -S(O)- and -S(O) 2 - and R 1 and R 2 are independently selected from alkyl, aryl, alkylaryl, and arylalkyl.
  • R 1 and R 2 are independently selected from the group consisting of n-propyl, isopropyl, butyl, benzyl, and p-tolyl; and/or wherein when the other X is -S(O) 2 -, R 1 and R 2 are independently selected from the group consisting of methyl, phenyl, benzyl, 4-methylbenzene, and n-propyl.
  • the compound of Formula I is not propyl-propane thiosulfonate (PTSO, also referred to as Di-n-propyl thiosulfonate) or propyl-propane- thiosufinate (PTS, also referred to as Di-n-propyl thiosulfinate).
  • PTSO propyl-propane thiosulfonate
  • PTS propyl-propane- thiosufinate
  • R 1 and R 2 are independently selected from the group consisting of isopropyl, butyl, benzyl, and p-tolyl; and wherein when the other X is -S(O) 2 -, R 1 and R 2 are independently selected from the group consisting of methyl, phenyl, benzyl, 4-methylbenzene, and n-propyl.
  • the compound according to Formula I is selected from the group consisting of dimethyl disulfide, diethyl disulfide, di-n-propyl disulfide, di-n-butyl disulfide, diethyl thiosulfinate, n-propyl propane-1-thiosulfinate (also referred to as Di-n- propyl thiosulfinate), n-butyl butane thiosulfinate (also referred to as Di-n-butyl thiosulfinate), n-propyl propane-1-thiosulfonate (also referred to as Di-n-propyl thiosulfonate) , methyl methane thiosulfonate (also referred to as Di-methyl thiosulfonate), and phenyl benzene thiosulfonate (also referred to as Di-phenyl thiosulfonate).
  • the compound according to Formula I is selected from the group consisting of dimethyl disulfide, diethyl disulfide, di-n-propyl disulfide, and di-n-butyl disulphide. In preferred embodiments, the compound according to Formula I is selected from the group consisting of diethyl thiosulfinate, Di-n-propyl thiosulfinate, and Di-n-butyl thiosulfinate. In preferred embodiments, the compound according to Formula I is selected from the group consisting of Di-n-propyl thiosulfonate, Di-methyl thiosulfonate, and Di-phenyl thiosulfonate.
  • the compound according to Formula I is selected from the group consisting of Di-n-butyl thiosulfinate, Di-methyl thiosulfonate, Di -phenyl thiosulfonate, Bis (p-tolyl) thiosulfinate, S-propyl-4-methylbenzene thiosulfonate, Di-isopropyl thiosulfinate, Di- benzyl thiosulfinate, and Di-benzyl thiosulfonate.
  • the compound according to Formula I is dimethyl disulphide.
  • the compound according to Formula I is diethyl sulphide.
  • the compound according to Formula I is di-n-propyl disulfide. In preferred embodiments, the compound according to Formula I is di-n-butyl disulfide. In preferred embodiments, the compound according to Formula I is diphenyl disulfide. In preferred embodiments, the compound according to Formula I is diethyl thiosulfinate. In preferred embodiments, the compound according to Formula I is Di-n-propyl thiosulfinate. In preferred embodiments, the compound according to Formula I is n-butyl butane thiosulfinate. In preferred embodiments, the compound according to Formula I is Di-n-propyl thiosulfonate.
  • the compound according to Formula I is Di-methyl thiosulfonate. In preferred embodiments, the compound according to Formula I is Di-phenyl thiosulfonate. It will be understood that the terms “sulphide” and “sulfide” are used interchangeably herein.
  • the compounds are obtained from natural sources such as plants. Compounds can be extracted from plant material in various ways. The appropriate method depends on the chemical properties of the compounds. For example, the extraction can start with a non-polar solvent and follow that with solvents of increasing polarity. The compounds are also commercially available or can be prepared as described in example 1. The disclosure provides compositions comprising the compounds as disclosed herein.
  • compositions are substantially free of diallyl thiosulfinate (also referred to as allyl-2-propene-1-sulfinothioate).
  • Diallyl thiosulfinate is better known under the name Allicin. Allicin is an organosulfur compound. When fresh garlic is chopped or crushed, the enzyme alliinase converts alliin into allicin, which is responsible for the aroma of fresh garlic. The allicin generated is unstable and quickly changes into a series of other sulfur-containing compounds such as diallyl disulfide.
  • substantially free refers to compositions comprising less than 5 wt% of diallyl thiosulfinate.
  • compositions comprise less than 1 wt% of diallyl thiosulfinate, preferably less than 0.5 wt% diallyl thiosulfinate.
  • the composition comprises a ratio of a compound having formula I to diallyl thiosulfinate by weight of at least 10:1, more preferably of at least 100:1.
  • the compositions of the present disclosure are also preferably substantially free of diallyl-disulfide.
  • compositions comprise less than 1 wt% of diallyl disulfide, preferably less than 0.5 wt% diallyl disulfide.
  • the composition comprises a ratio of a compound having formula I to diallyl-disulfide by weight of at least 10:1, more preferably of at least 100:1.
  • compositions are provided wherein at least 50%, preferably at least 90% by weight of the active ingredients are compounds according to formula I as disclosed herein.
  • compositions are provided wherein the only active ingredients are compounds according to formula I, optionally including further antimicrobial agents and/or anti-inflammatory agents.
  • compositions are provided wherein at least 50%, preferably at least 90% by weight of the active ingredients are compounds according to formula I as disclosed herein.
  • compositions are provided wherein the only active ingredients are compounds according to formula I, optionally including further antimicrobial agents and/or anti-inflammatory agents.
  • the compounds disclosed herein and compositions comprising same are useful in the treatment or prevention of infection.
  • particular uses are for the treatment or prevention of respiratory infection, bowel infection, breast infection, udder infection, skin infection, bladder infection, ear infection, systemic infection, joint infection, brain infection.
  • infection refers to, e.g., pathogenic infections which can lead to disease.
  • infections are bacterial or fungal infections.
  • the infection is a microbial infection.
  • the infection is a bacterial infection.
  • the infection is a fungal infection (including yeast infection).
  • Bacteria and fungi are found almost everywhere and exist in very diverse forms. Most are not harmful and are actually indispensable for life on earth and essential for plant, animal and human health.
  • the microbiome in the intestines of humans and animals where bacteria and fungi live as symbionts with their host is the so-called gut flora.
  • bacteria are naturally present on the skin, which form part of the immune system.
  • the soil biology which for the most part consists of bacteria and fungi.
  • Some bacteria and fungi can cause pathogenic infections, for example in animals, or humans. These pathological infections can lead to disease and illness of the infected individual.
  • treatment of infection refers to a reduction in the severity and/or duration of the infection and/or a reduction of the severity and/or duration of symptoms from the infection. Preferably, said treatment results in restoration of the health of an individual. Preferably, the individual has less disease symptoms or for a shorter time.
  • prevention of infection refers to the prevention of or alternatively delaying the onset of infection or of one or more symptoms associated with infection. Some microorganisms, such as bacteria, microalgae, fungi, etc., can form biofilms. The compounds disclosed herein are also useful in the prevention or reduction of biofilm formation or growth and/or for degradation or reduction of biofilms.
  • the compounds, and compositions comprising same are useful in the treatment or prevention of biofilm-related disorders.
  • microbial e.g., bacterial
  • biofilm was initially used in technical and environmental microbiology to describe a community of sessile bacteria and other microorganisms attached to natural or artificial surfaces.
  • the formation of a microbial biofilms is initiated by the colonization of bacteria on a surface to which they attach and produce a slimy film consisting of organic polymers. This primary bacterial film attracts other microorganisms such as algae and protozoa, fungi and protozoa resulting in the formation of a visible multispecies biofilm.
  • biofilms are ubiquitous in nature and found on all surfaces that are in contact with water. Of public health concern are microbial biofilms in municipal water supplies and household water pipelines and devices.
  • biofilm refers to a population of microorganisms that are concentrated at an interface (usually solid/liquid) and typically surrounded by an extracellular polymeric slime matrix. Biofilms may form on living or non-living surfaces and are found in natural, industrial and hospital settings. Biofilms can contain many different types of micro-organisms, e.g. bacteria, archaea, protozoa, fungi and algae. Preferably, such biofilms comprise bacteria, microalgae (such as Prototheca spp.) or fungi.
  • treatment of a biofilm-related disorder refers to a reduction in the severity and/or duration of the disorder and/or a reduction of the severity and/or duration of symptoms from the disorder, in particular the symptoms of infection.
  • said treatment results in restoration of the health of an individual.
  • the individual has less disease symptoms or for a shorter time.
  • prevention or reduction of biofilm formation or growth refers to the prevention, delay, or reduction of biofilm formation or growth. As will be understood by a skilled person, such reduction of biofilm formation or growth may slow the growth of biofilms as compared to the growth of untreated biofilms.
  • the compositions are useful for reducing biofilm formation or growth.
  • “degradation or reduction of biofilms” refers to the elimination, either partially or completely, of a biofilm.
  • planktonic bacteria may still be present.
  • the compounds disclosed herein may be capable of disrupting the structure of the biofilm, for example the extracellular mucous matrix.
  • the compounds are useful for inhibiting cell adhesion.
  • the compounds may prevent the adhesion (without killing bacteria) to a static or live surface of all cell types encountered in microbial biofilms in particular free living microbes.
  • Quorum Sensing (QS) signalling plays an important role in the control of e.g. the expression of bacterial virulence factors.
  • QS involves the accumulation of signalling molecules in the surrounding environment which enables a single cell to sense the density of the number of bacteria and the signalling molecules, and therefore the bacterial population as a whole, can make a coordinated response.
  • These cell-cell communication systems regulate various functions of the bacteria such as motility, virulence, sporulation, antibiotic production, DNA exchange, and development of more complex multicellular structures such as biofilm.
  • the interference with QS signalling systems might offer a new strategy to combat persisting (chronic) bacterial infections.
  • the ability of certain substances, such as naturally occurring compounds that have Quorum Quenching (QQ) abilities can be used as anti-adhesive compounds and as compounds that interfere with biofilm formation.
  • the compounds disclosed herein are particularly useful for treating biofilm-related disorders, wherein the disorder is characterized by a chronic and/or persistent infection.
  • the compounds disclosed herein are particularly useful for the treatment of a chronic and/or persistent infection.
  • the terms persistent infection and chronic infection are often used interchangeably, but are based on different mechanisms. Persistent infections are normally held in check by immune defenses but may be activated when such immune defenses are weakened.
  • Persistent infections are often asymptomatic and become clinically visible only when the immune defense fails to control the pathogen.
  • persistent infections are often asymptomatic, a skilled person is well aware of means to detect such persistent infections, including e.g., detecting microorganisms from patient samples (e.g., blood or urine).
  • patient samples e.g., blood or urine
  • pathogens remain in a group of cells / parts of tissue (e.g., joints or lung tissue).
  • the patient always has symptoms of disease, although these might be milder that in the acute phase of infection.
  • a prominent example of a biofilm disease is bovine mastitis.
  • Bovine mastitis is the clinical term for infections of the mammary gland of cows and can be caused by multiple pathogens, the most prevalent forms are Staphylococcus aureaus, Streptococcus uberis, Streptococcus agalactia, Streptococcus dysgalactiae as well as Serratia marescens and other facultative pathogenic Enterobacteriaceae and Prototheca spp, the latter considered as an emerging pathogen causing an aggressive, non-curable mastitis in many regions of the world.
  • the invention encompasses treating biofilms comprising such microorganisms with the compounds and compositions disclosed herein.
  • the wounds treated by the compound of the invention comprise, for example, Staphylococcus aureus; Streptococci; gram negative bacteria, for example Treponema spp., Escherichia coli, Yersiania pestis, Pseudomonas aeruginosa; or yeast/fungi, for example Candida spp (albicans), Cladosporidium herbarum, Trichosporum, Rhodosporidium, and Malassezia.
  • Staphylococcus aureus Streptococci
  • gram negative bacteria for example Treponema spp., Escherichia coli, Yersiania pestis, Pseudomonas aeruginosa
  • yeast/fungi for example Candida spp (albicans), Cladosporidium herbarum, Trichosporum, Rhodosporidium, and Malassezia.
  • biofilm-associated microorganisms cause a large number of infections including endocarditis, osteomyelitis, sinusitis, urinary tract infections, chronic prostatitis, periodontitis, chronic lung infection in cystic fibrosis patients, middle ear infections, and various nosocomial infections, especially those related to all known indwelling devices (catheters, implants).
  • endocarditis osteomyelitis
  • sinusitis urinary tract infections
  • chronic prostatitis chronic prostatitis
  • periodontitis chronic lung infection in cystic fibrosis patients
  • middle ear infections middle ear infections
  • various nosocomial infections especially those related to all known indwelling devices (catheters, implants).
  • the burden of biofilm- disease is significant and represents a major concern in medical care.
  • pathogens which are of major clinical concern due to their biofilm associated therapy-resistance are listed below: Aspergillus fumigatus – Lung aspergillosis (fungal disease) Burkholderia cepacian – pulmonary cystic fibrosis superinfections Candida spp (Yeast) – mucosal surfaces of the gastro-intestinal and urogenital tract Gardnerella vaginalis (urogenital tract) Escherichia coli (multiple organs and septicaemia) Pseudomonas aeruginosa (multiple organs and lung infections including cystic fibrosis) Staphylococcus aureus (multiple tissues and wound infections, nosocomial infections) Staphylococcus epidermidis (multiple tissues and wound infections) Stenotrophomonas maltophilia (chronic respiratory tract diseases).
  • the biofilm related disorder is udder cleft.
  • Other include which may be treated with a compound of formula I include ulcers (e.g., diabetic foot ulcers (DFUs), venous leg ulcers (VLUs), and pressure ulcers (PUs)); mortellaro (digital dermatitis); and eczema.
  • Microbial biofilms are not only formed by bacteria, but also by other microorganisms, particularly pathogenic fungi (Aspergillus fumigatus a major cause of multiple Aspergillus-related lung diseases) and yeasts (Candida spp) colonizing of mucosal surfaces of the gastro-intestinal and uro-genital tract.
  • pathogenic fungi Aspergillus fumigatus a major cause of multiple Aspergillus-related lung diseases
  • yeasts Candida spp colonizing of mucosal surfaces of the gastro-intestinal and uro-genital tract.
  • the most prevalent implant- related biofilms are formed by Staphylococcus aureus (MSSA and MRSA), Candida albicans, Pseudomonas aeruginosa, Klebsiella pneumonia, and Enterococcus faecalis.
  • microalgae such as, e.g., Prototheca spp can form biofilms and are a source of disease in humans and animals (Protothecosis).
  • the biofilm comprises bacteria selected from one or more of Treponema spp, Yersiania pestis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Serratia marescens, Trueperella pyogenes, Mannheimia haemolytica, Pasteurella multocida, Pseudomonas aeruginosa, Burkolderia cepacia, Streptococcus neumoniae, Hemophilus influenza, Legionella neumophila, Fusobacterium necrophorum, Corynebacterium pseudotuberculosis, Streptococcus spp., Porphyromonas
  • the biofilm comprises fungi selected from Absidia spp., Actinomyces spp., Aspergillus spp., Botrytis spp., Candida spp., Centrospora spp., Cephalosporium spp., Ceratocystis spp., Chaetoconidium spp., Chaetomium spp., Cladosporium spp., Colletotrichum spp, Conidiobolus spp., Fulvia spp., Fusarium spp., Geotrichum spp., Guignardia spp., Helminthosporium spp., Histoplasma spp., Lecythophora spp., Malassezia spp., Nectria spp., Nocardia spp., Oospora spp., Ophiobolus spp., Paecilomyces s
  • biofilm comprises microalgae, e.g., Prototheca spp.
  • the bacterial infection or biofilm associated disorder is caused by a Gram-negative bacterium. In certain embodiments, the bacterial infection or biofilm associated disorder is caused by a Gram-positive bacterium.
  • the bacterial infection or biofilm associated disorder is caused by a multidrug-resistant bacterium.
  • the bacterial infection is a methicillin-resistant Staphylococcus aureus (MRSA)-related infection or a Staphylococcus epidermidis (e.g., MRSE) related infection.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MRSE Staphylococcus epidermidis
  • the biofilm causing bacteria is Escherichia coli, preferably the biofilm infection is recurrent urinary tract infection, catheter-associated urinary tract infection, or biliary tract infection.
  • the biofilm causing bacteria is Pseudomonas aeruginosa, preferably the biofilm infection is Cystic fibrosis lung infection, chronic wound infection, catheter-associated urinary tract infection, chronic rhinosinusitis, chronic otitis media, bronchiectasis, chronic obstructive pulmonary disease or contact lens-related keratitis.
  • the biofilm causing bacteria is Staphylococcus aureus, preferably the biofilm infection is Chronic osteomyelitis, chronic rhinosinusitis, endocarditis, chronic otitis media, or of (orthopaedic) implants.
  • the biofilm causing bacteria is Staphylococcus epidermidis, preferably the biofilm infection is Central venous catheter, orthopaedic implants, or chronic osteomyelitis.
  • the biofilm causing bacteria is Streptococcus pneumoniae, preferably the biofilm infection is infection of nasopharynx, chronic rhinosinositis, chronic otitis media, or infection in chronic obstructive pulmonary disease.
  • the biofilm causing bacteria is Streptococcus pyogenes, preferably the biofilm infection is infection of oral cavity and nasopharynx, recurrent tonsilitis.
  • biofilm infections are known to the medial practitioner (see, e.g., Table 1 of Wu et al. Int J Oral Sci. 2015 Mar; 7(1): 1–7). Such biofilm disorders may lead to chronic infections.
  • the determination of acute versus chronic infection is also known to the practitioner. For example, according to the Mayo Clinic, the occurrence of a yeast infection 4 or more times within a year indicates the presence of a chronic yeast infection whereas the occurrence of two or more bladder infections during a 6-month period indicates the presence of a chronic bladder infection (also referred to as recurrent urinary tract infection).
  • the most common method to treat a pathological infection of bacteria is the use of antibiotics. Current antibiotics operate primarily through growth-dependent mechanisms and target rapidly-dividing bacteria.
  • non-replicative or slower growing bacteria display high levels of antibiotic tolerance and/or resistance contributing to persistent and recurring infection.
  • the compounds disclosed herein are suitable for the use in infections or biofilms comprising antibiotic resistant bacteria, antibiotic tolerant bacteria, and antibiotic persistent bacteria.
  • the compounds disclosed herein are also suitable as a second-line therapy, or rather for individuals that did not response to previous treatment (e.g., antimicrobial treatment) or the disorder returned within e.g., one year or 6 months.
  • the disclosure further provides the compounds as disclosed herein and compositions comprising same for treating any disorder induced by or relating to biofilms. Disorders induced by or relating to biofilms are well-known to a skilled person.
  • disorders are biofilm-related infections.
  • Suitable disorders for treatment include, for example, bacterial prostatitis, bacterial vaginosis, biliary tract infections, chronic sinusitis, chronic lung disease, dental caries, endocarditis, kidney stones, laryngitis ,lung infection in cystic fibrosis, gingivitis mastitis, middle ear infections, nonsocomial (bloodstream) infections, obstructive pulmonary diseases, osteomyelitis, otitis media, periodontitis, pneumonia prostatitis, rhinosinusitis, sinusitis, tonsillitis, tuberculosis, urinary tract infections, and wound infections.
  • Mycoplasma Bovis is known to cause udder infection and joint infection.
  • Biofilm-related disorders also include disorders caused by biofilms formed on indwelling devices (e.g., medical implants, catheters, etc.). Generally, such disorders are treated by removing/replacing the implant. In a preferred embodiment the disorder is mastitis. In some embodiments, the disorder is not mastitis. In some embodiments the treatment is not for inflammatory bowel disease and in particular is not colitis. In some embodiments, the compounds and compositions as disclosed herein are also useful for treating and preventing infections of implanted medical devices such as joint prosthesis and heart valves as disclosed further herein. In some embodiments, the compounds and compositions as disclosed herein are also useful for preventing or reducing inflammation in response to bacterial infection or biofilms.
  • Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, and is a protective response involving immune cells and molecular mediators.
  • a function of inflammation is to eliminate the pathogens.
  • treatment of an individual with the compounds as disclosed herein or compositions comprising same prevents or reduces a clinical inflammation in an animal, e.g., a cow.
  • the treatment prevents or reduces a (clinical) inflammation of the udder.
  • treatment of an individual with the compounds and compositions comprising same prevents or reduces a (clinical) inflammation in a human.
  • the treatment prevents or reduces inflammation of the skin, preferably prevents eczema.
  • treatment of an individual with the compounds as disclosed herein, or compositions comprising same reduces biofilm formation or growth and/or causes degradation or reduction of biofilms. Therefore, the individual no longer responds with an inflammatory reaction in the presence of the pathogens. With other words, clearance of the biofilms and pathogens within these biofilms reduces the inflammation response and prevents a clinical inflammation.
  • the microorganisms such as bacteria and fungi
  • the individual’s immune system will respond to the active microorganism. This may result in inflammation of the tissue.
  • the activated immune cells and the inflammatory response can also damage the tissue, for example in the milk gland.
  • Suppression of the inflammatory response may therefore prevent or reduce damage to the tissue.
  • the body starts the repair.
  • the macrophages still present stimulate the production of new blood vessels. They also ensure the attraction of fibroblasts. These fibroblasts ultimately cause the formation of granulation tissue.
  • scar tissue may be formed instead of milk producing tissue. The milk production of the cow may therefore be lower than before the inflammation.
  • further anti-inflammatory drugs can be administered to suppress the inflammatory response and reduce the tissue damage.
  • the treatments disclosed herein both therapeutic and prophylactic
  • Anti- inflammatory agents include, for example, nonsteroidal anti-inflammatory agents (cox/lox inhibitors) such as ibuprofen, paracetamol, aspirin, diclofenac, ketoprofen, tolmetin, etodolac, and fenoprofen.
  • Natural anti-inflammatory agents such as Curcumin, Ginger, Spirulina, Cayenne, Cinnamon, Clove, Sage, Rosemary, Black Pepper, natural aspirins, Boswelia, Sanguinaria, and/or Green Tea may also be used.
  • the methods and uses disclosed herein comprise the combined treatment of the therapeutic organosulfur compounds disclosed herein with an anti-inflammatory agent. The compounds may be administered together or separately.
  • compositions comprising the therapeutic organosulfur compounds disclosed herein with an anti-inflammatory agent.
  • the methods comprise administering to an individual in need thereof compositions comprising the compounds disclosed herein, preferably such as to prevent or reduce biofilm formation or growth, degrade or reduce biofilms, and/or treat or prevent infection (in particular bacterial or fungal infection).
  • the composition can be administered to an individual for the treatment (e.g., therapeutic agent) or prevention (e.g., prophylactic agent) of a disease or disorder or infection.
  • the individual has or is at risk of developing a biofilm-related infection.
  • the compositions can be administered to any individual, in particular to animals.
  • the animal is a ruminant (such as cows and goats), more preferably a cow. In some embodiments, the animal is not a cow.
  • the animal is a non-ruminant, such as a monogastric, a rodent, non-human primate, porcine, equine, canine, feline, or avian.
  • the animal is a human.
  • the animal is a non-human animal.
  • the animal is not an aquatic animal such as fish, mollusks, and crustaceans.
  • the animal is a mammal or bird. While not wishing to be bound by theory, the disclosure provides that the compositions disclosed herein can have advantageous effects after a single administration.
  • effects are achieved by providing a single oral administration of the composition disclosed herein.
  • Such oral dosing may be, e.g., as a tablet which provides an extended release of the compounds disclosed herein.
  • the disclosure also provides for multiple administrations.
  • the compositions may be provided more than once per day, daily, weekly, or monthly.
  • the composition may be provided once daily for a week or until symptoms are alleviated.
  • Actual dosage levels of the pharmaceutical preparations described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a skilled person is aware that as smaller animals have higher metabolic rates and thus smaller animals require a larger drug dose on weight basis. Dose conversions between animals, and between humans and animals, are reviewed in Nair and Jacob (J Basic Clin Pharm.
  • At least 5mg/day of compounds disclosed herein are provided to a human (such as by oral administration).
  • at least 10mg/day of the compounds are provided.
  • a compound as disclosed herein is provided to a human at a dose of between 0.1 mg/kg to 100 mg/kg. Such amounts of the compounds are particularly useful when providing the compounds systemically (e.g., orally).
  • compositions disclosed herein are preferably provided for at least one week or until symptoms are alleviated. While such compositions may be provided several times (e.g., one a week, once a month, twice a year, etc.), prophylactic and therapeutic effects are observed after a single use.
  • between 1-50 g of a compound as disclosed herein is administered to a cow, e.g., for the treatment of mastitis.
  • Preferably, at least 5g of the compound is administered.
  • a composition comprising the compounds as disclosed herein together with one or more additional agents, such as, antibiotics (e.g., antibacterial agents, antiviral agents, anti-fungal agents), anti-inflammatory agents, anti-pyretic agents, and pain-relieving agents.
  • antibiotics e.g., antibacterial agents, antiviral agents, anti-fungal agents
  • anti-inflammatory agents e.g., anti-pyretic agents, and pain-relieving agents.
  • the compounds disclosed herein are used together with an antimicrobial agent such as antifungal drugs or antibiotics. While not wishing to be bound by theory, the disclosure provides that the compounds disclosed herein target the biofilms. The antimicrobial drugs can then exert their effect on remaining planktonic cells as well as the microbial cells in the disrupted biofilm.
  • the combination of an antimicrobial with the compounds described herein can reduce the dosage and/or dosage frequency of the antimicrobial.
  • antimicrobials which may be used in the combination treatment include antifungals such as miconazole, ketoconazole, econazole, terbinafine, ciclopirox, tolnaftate, sertaconazole, sulconazole, amphotericin b, cholorxylenol, clioquinol, butenafine, naftifine, nystatin, and clotrimazole.
  • antibiotics include Penicillins, Tetracyclines, Cephalosporins, Quinolones, Lincomycins, Macrolides, Sulfonamides, Glycopeptides, Aminoglycosides, and Carbapenems.
  • the disclosure provides compositions comprising a compound disclosed herein together with an antimicrobial.
  • the compound and an antimicrobial may also be provided separately.
  • the compound and an antimicrobial therapy overlap.
  • the therapy with a compound of the invention precedes antimicrobial therapy.
  • the compositions disclosed herein are provided as or in a food product or a functional food product.
  • the term "functional food” as used herein, refers to those foods that are prepared not only for their nutritional characteristics, but also to fulfil a specific function, such as improving health or reducing the risk of contracting diseases. Such functional foods may also be referred to as dietary supplements or (animal) food additive. To this end, biologically active compounds, such as minerals, vitamins, fatty acids, bacteria with beneficial effects, dietary fibre and antioxidants, etc., are added thereto. Such food products may be in any form suitable for oral consumption, e.g., in the form of a liquid, gel, powder, pill, tablet, or in gel capsules.
  • the functional food may also include animal digest, e.g., any material that results from chemical and/or enzymatic hydrolysis of clean and undecomposed animal tissue.
  • the functional food may also include dried brewers yeast, e.g., the dried, inactive agent that is a byproduct of the brewing industry.
  • the animal digest and dried brewers yeast have been found to enhance the palatability of the functional food.
  • the animal digest comprises from about 10% to about 90% of the functional food and the dried brewers yeast comprises from about 1% to about 30% of the functional food.
  • the disclosure provides compositions comprising the therapeutic organosulfur compounds as disclosed herein together with at least one pharmaceutically acceptable carrier, diluent and/or excipient. (See e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997).
  • the term “pharmaceutically acceptable” refers to those compositions or combinations of agents, materials, or compositions, and/or their dosage forms, which are within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable diluent or carrier refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the peptide from one organ, or portion of the body, to another organ, or portion of the body.
  • the pharmaceutical composition may be administered by any suitable route and mode. As will be appreciated by the person skilled in the art, the route and/or mode of administration will vary depending upon the desired results.
  • the pharmaceutical compositions may be formulated in accordance with routine procedures for administration by any routes, such as parenteral, topical (including ocular), oral, sublingual, transdermal, or by inhalation.
  • Parenteral administration includes, e.g., intravenous, intramuscular, intra-arterial, intracoronary, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • Preferred routes are oral or topical administration.
  • the compositions may be in any suitable forms, such as liquid, semi-solid and solid dosage forms.
  • compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations (in particular for administration to the skin or eye), such as sterile parenteral solutions or suspensions or in the form of a spray, aerosol or other conventional method for inhalation.
  • the pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the composition is a topical composition in the form of a cream, gel, ointment, lotion, foam, suspension, spray, aerosol, or powder aerosol.
  • the compositions are particularly useful for administration to the skin.
  • compositions also include oral care compositions, e.g., toothpaste, dentifrice, tooth powder, tooth gel, subgingival gel, mouthrinse/mouthwash, artificial saliva, denture product, mouthspray, lozenge, oral tablet, and chewing gum.
  • oral care compositions e.g., toothpaste, dentifrice, tooth powder, tooth gel, subgingival gel, mouthrinse/mouthwash, artificial saliva, denture product, mouthspray, lozenge, oral tablet, and chewing gum.
  • the disclosure also provides the in vitro use of the compositions as disclosed herein for preventing or reducing biofilm formation or growth on a surface, and/or for degradation or reduction of biofilms on a surface.
  • the methods are for reducing biofilm or biofilm formation on a surface.
  • the method comprises contacting a biofilm attached to a surface with the compositions disclosed herein.
  • the compositions include, e.g., cleaning compositions.
  • compositions for cleaning (or rather removing or reducing) biofilms are known in the art and may include surfactants and enzymes (e.g., proteases and polysaccharidases). Any surface may be treated with the compositions disclosed herein so as to coat such surfaces. The surfaces may be, e.g., sprayed, dipped, or soaked in the compositions.
  • a surface includes glass, metal, porous, and non-porous surfaces. It also pertains to exterior and interior and surfaces of equipment that can be contaminated, such as those found in the food industry or the medical equipment found in hospitals and health care facilities, as well as plumbing systems (e.g., sink drain), countertops, building materials, ductwork, clean rooms.
  • a surface also refers to the interior or exterior of pipes, for example drains, as well as swimming pools, tanks (e.g., for aquaculture), purification filters, toilet bowl, sinks, surfaces in the greenhouse.
  • a surface also includes water, such as from a drinking trough.
  • the surface is of a medical device, such as prosthetics (hip implants, dental implants, prosthetic joint, a voice prosthetic, a penile prosthetic) a mechanical heart valve, a cardiac pacemaker, an arteriovenous shunt, a schleral buckle, catheters (e.g., central venous catheter, an intravascular catheter, an urinary catheter, a Hickman catheter, a peritoneal dialysis catheter, an endrotracheal catheter), tympanostomy tube, a tracheostomy tube, a surgical suture, a bone anchor, a bone screw, an intraocular lens, a contact lens, an intrauterine device, an aortofemoral graft, or a vascular graft.
  • prosthetics hip implants, dental implants, prosthetic joint, a voice prosthetic, a penile prosthetic
  • a mechanical heart valve e.g., a mechanical heart valve, a cardiac pacemaker, an arteriovenous shunt, a
  • infections from medical devices include those from abdominal drains, biliary tract stents, breast implants, cardiac pacemakers, cerebrospinal fluid shunts, contact lenses, defibrillators, dentures, electrical dialyzers, endotracheal tubes, indwelling urinary catheters, intrauterine devices, intravenous catheters, joint prostheses, mechanical heart valves, nephrostomy tubes, orthopedic implants, peritoneal dialysis catheters, prosthetic heart valves, prosthetic joints allosplastic orthopedic devices, tissue fillers, urethral stents, vascular prostheses, ventilator-associated pneumonia, ventricular assist devices, ventricular derivations, ventricular shunts, and voice prostheses.
  • the surface is of a surgical device, such as clamp, forceps, scissor, skin hook, tubing, needle, retractor, scaler, drill, chisel, rasp, or saw.
  • a surgical device such as clamp, forceps, scissor, skin hook, tubing, needle, retractor, scaler, drill, chisel, rasp, or saw.
  • to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • verb “to consist” may be replaced by “to consist essentially of” meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • the word “approximately” or “about” when used in association with a numerical value (approximately 10, about 10) preferably means that the value may be the given value of 10 more or less 1% of the value.
  • the compounds and compositions disclosed herein are useful as therapy and in therapeutic treatments and may thus be useful as medicaments and used in a method of preparing a medicament.
  • the disclosure provides methods which are not a treatment of the human or animal body and/or methods that do not comprise a process for modifying the germ line genetic identity of a human being. wherein the cell is not a human germ cell line.
  • All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.
  • the disulphide (16.36 mmol) was dissolved in dry dichloromethane (DCM, 30 mL) under a N2 atmosphere at -78 °C and meta-chloroperoxybenzoic acid (m-CPBA, 16.36 mmol), dissolved in dry DCM (30 mL), was then added dropwise. Once the addition was complete, the reaction was left to stir for 3 h, slowly warming to 0 °C. The reaction was quenched with saturated sodium hydrogen bicarbonate, and the resulting aqueous solution was extracted 3 times with DCM. The combined organic fractions were then dried over anhydrous MgSO4 and solvent was evaporated under reduced pressure.
  • DCM dry dichloromethane
  • m-CPBA meta-chloroperoxybenzoic acid
  • Example 1b Experimental design: Oxidation of a disulfide leads to the formation of the corresponding thiosulfinate and thiosulfonate.
  • mcpba metal-chloroperoxybenzoic acid
  • mcpba metal-chloroperoxybenzoic acid
  • the benzyl substituted is more reactive in comparison with other aryl substituted analogues and that might be the reason for low reaction product in tried reaction conditions.
  • the heterocyclic analogues did not undergo a reaction due to solubility issue and resulted in tar with unsoluble solid.
  • the reaction for its thiosulfinate was attempted in 2 solvents (dichloromethane and chloroform) with the same outcome.
  • the reaction for the corresponding thiosulfonate was attempted with increased temperature also without success.
  • Example 2 MIC and biofilm assays
  • the MIC assay in this example measures growth inhibition of planktonic microorganisms.
  • a number of compounds tested show an effect on planktonic bacteria.
  • the effect of a compound against planktonic microorganisms is not indicative of the effect of such compounds against a biofilm. This has been demonstrated for a number of antimicrobials as previously discussed herein and is also well-known in the literature. See, e.g., Roy et al. which indicates that bacteria in biofilms have increased resistance against conventional antibiotics by around 1000-fold (2018 Virulence 9:522-554).
  • the Biofilm eradication assay in this example demonstrates the effect of compounds against a biofilm.
  • the main mechanisms of action of a compound in a biofilm assay are believed to be related to either the inhibition of quorum sensing and the following collective gene switch resulting in adhesion of bacteria and the synthesis or to the assembly of extracellular matrix to form the 3-dimensional structure of a biofilm. These mechanisms are entirely different from a pure antibacterial/antimicrobial “killing” effect. If a compound tested effectively in a biofilm assay, but has no antibacterial effect, then the compound may be combined with an antibiotic to ensure that the bacteria released from the biofilm are killed. This is especially the case if the immune system of the animal is compromised. However, such compounds are also particularly useful at specifically targeting biofilms, while have little or no effect on, e.g., healthy gut flora. Such compounds are particularly preferred.
  • Table 3 Compounds with CAS number and supplier used in this study.
  • the dissolved and transparent dilutions had a concentration of 80 mM and were diluted 10 times with Mueller Hinton Broth 2 cation adjusted (MHB-II) for the MIC assay (method 1.2).
  • MBE-II Mueller Hinton Broth 2 cation adjusted
  • the solution needs to be diluted to a final concentration of 2 mM in 0.9% saline solution and then further diluted to 1 mM, 0.5 mM, 0.25 mM and 0.125 mM in saline.
  • Those dilutions were added as treatment to the 96 well plates with the grown biofilm (see method 3).
  • the optical density at 600 nm (OD 600 ) of the bacterial suspension was measured with a spectrophotometer (EvolutionTM 201/220 UV-Visible Spectrophotometer, Thermo Fisher Scientific) and diluted to an OD 600 of 0.0008, which resembles 10 6 CFU/mL.
  • the MIC assay was performed in a flat-bottom 96-microtiter plate.50 ⁇ L of MHB-II was added into columns 2-11, and 100 ⁇ L to column 12 for sterility control. 100 ⁇ L of the 8 mM test compound solution (method 1) was added to column 1.
  • a two-fold dilution series of the solution in MHB-II was achieved by resuspending 50 ⁇ L of the 8 mM solution from column 1 to column 2. This step was repeated until column 10. The respective concentration of Tween 80 and DMSO in column 1 was 2.5%, which is two- fold diluted throughout the plate. 50 ⁇ L of the bacterial suspension was added to column 1-11.
  • the microtiter plates were sealed with adhesive polyethylene film for sealing microplates (Diversified Biotech) and incubated for 24 h at 37 °C. After incubation the OD was measured at 600 nm with the Varioskan (Thermo Fisher Scientific).
  • MIC50 The minimal concentration which causes reduction of final OD600 of the bacterial culture by 50% (MIC50) and by 90% (MIC90) was calculated after normalization of the obtained OD values to the growth control in column 11. This assay was repeated three times in three independent experiments.
  • 1.3 MBEC assays Frozen aliquots of Staphylococcus epidermis ATCC 35984 and Pseudomonas aeruginosa ATCC 27853 were used to inoculate 20 mL of Tryptic Soy Broth (TSB) in a 100 mL Erlenmeyer flask at 37 °C and 150 rpm and incubated overnight (Infors).
  • TTB Tryptic Soy Broth
  • the OD 600 of the overnight culture was measured with a spectrophotometer (EvolutionTM 201/220 UV-Visible Spectrophotometer, Thermo Fisher Scientific) and diluted to an OD 600 of 0.2, which resembles 10 8 CFU/mL.
  • the 96 well plates were sealed with adhesive polyethylene film for sealing microplates and incubated for 48 h at 37 °C at static conditions.
  • treatment solutions 100 ⁇ L of treatment solutions (method 1) were added to the vials resulting in treatment concentrations of 1 mM, 0.5 mM, 0.25 mM and 0.125 mM and with the respective concentration of Tween 80 and DMSO each at 0.63%, 0.32%, 0.16% and 0.08%.
  • the same concentration was applied to four different wells of the same bacterial strain, resulting in four replicates for each concentration and strain.
  • the treatment was incubated for 20 h at 37 °C at static conditions.
  • the optical density of the bacterial suspension was measured at 600 nm (OD 600 ) with a spectrophotometer (EvolutionTM 201/220 UV-Visible Spectrophotometer, Thermo Fisher Scientific) and diluted to an OD 600 of 0.2.
  • the MBIC assay was performed in a 96-microtiter plate.50 ⁇ L of TSB was added into columns 2-11, and 100 ⁇ L to column 12 for sterility control. 100 ⁇ L of the 8 mM test compound solution (method 1) was added to column 1. A two- fold dilution series of the solution in TSB was achieved by resuspending 50 ⁇ L of the 8 mM solution from column 1 in column 2. This step was repeated until column 10.
  • the respective concentration of Tween 80 and DMSO in column 1 is at 2.5%, which is two- fold diluted throughout the plate.
  • 50 ⁇ L of the bacterial suspension was added to column 1-11.
  • the microtiter plates were sealed with adhesive polyethylene film for sealing microplates (Diversified Biotech) and incubated for 48 h at 37 °C. After incubation the supernatant is carefully removed and the well with the biofilm washed for one time with 100 ⁇ L 0.9% saline solution.100 ⁇ L 0.1 M HCl was added and incubated for 1h at room temperature to fix the biofilm. After incubation the HCl was removed and 100 ⁇ L crystal violet (0.1% v/v in water) was added and incubated for 30 min at room temperature.
  • the advantage of the present in vitro system is that it gives the opportunity to investigate the effects of agents on a biofilm and the respective microorganisms as such, without additional effects of the environment (e.g., the immune system).
  • a number of organosulfur compounds show low MIC-values, whereas the MBEC values were high.
  • Table 4 also depict a number of compounds that exhibit high MIC-values and very low MBEC-values. This is a very surprising result. This suggests that the growth of S. epidermis and P. aeruginosa is not affected by these organosulfur compounds, but rather that the compounds have a strong effect against biofilms.
  • Bovine mastitis is a disease affecting worldwide millions of cows annually. The disease is caused by a variety of very different bacteria and in some cases also yeasts that invade the mammary gland of lactating cows, causing a persistent infection and an inflammatory response. Bovine mastitis can be caused various Gram-positive as well as Gram-negative pathogens and the prevalence of individual pathogens may vary to some extent between countries and continents.
  • Mastitis is a known biofilm-related disorder.
  • the formation of bacterial biofilms has been demonstrated in in vitro experiments cultivating mastitis pathogen under conditions favoring bacterial biofilm formation (quantifiable after staining and by measuring the genes that drive biofilm formation) as well as in situ by staining the biofilm matrix in the infected bovine udder tissue.
  • Figure 1 of Schönborn S and Krömker V 2016 Journal Veterinary Microbiology, 30;196:126-128 which depicts a biofilm matrix from udder tissue from cows suffering from mastitis.
  • Biomedical research of the last decennia revealed that biofilm formation is not only occurring in cases of udder infection and bovine mastitis but is a general trait of microorganisms invading human and animal tissues and causing persistent infections and inflammation.
  • microbial biofilms are one of the major unresolved challenges in modern therapy of infectious diseases in humans and animals.
  • Clinical evidence provided in the examples below revealed an unexpected therapeutic effect of a number of organosulfur containing compositions in the treatment of important biofilm diseases such as bovine mastitis and infected chronic wounds (e.g., UCD).
  • Somatic cell counts is related to the amount of pathogens that are in the quarter visible for the immune system.
  • the cell count of each cow was measured with the milk production registration (MPR). This MPR is done periodically every 30-40 days at all farms.
  • the Somatic Cell Count (SCC) is a main indicator of milk quality.
  • the majority of somatic cells are leukocytes (white blood cells) - which become present in increasing numbers in milk usually as an immune response to a mastitis-causing pathogen - and a small number of epithelial cells, which are milk-producing cells shed from inside of the udder when an infection occurs.
  • the SCC is quantified as the number of cells per ml of milk. SCC gives an indication of the presence of an (subclinical) udder infection with pathogens causing for instance mastitis and a main indicator of milk quality. The relationship of SCC and mastitis is reviewed in Sharma et al., 2011 Asian-Aust J Anim Sci 24:429-438.
  • the SCC data was extracted from milk production registrations (MPRs). MPRs are databases showing all details (e.g. SCC) of the produced milk per individual cow.
  • SCC is quantified as the number of cells per ml of milk. In general terms: an individual cow with a value for SCC of 100,000 or less indicates an 'uninfected' cow and there were no significant production losses due to subclinical mastitis. A threshold SCC above 250,000 indicates infection and counts between 100,000 to 250,000 indicate a high risk of infection. Table 5. Compounds and corresponding total amounts that were orally administered to cows. n.a.: not applicable Study Design The experiments were performed on a farm with 90 cows. Examination was performed on cell count with the above described tablets during 60 days. Cows with a SCC of more as 250.000 cells/ml before treatment were selected for administration of a single tablet.
  • the counts and average somatic cell counts of the milk of the cows are shown in Fig. 1.
  • the SCC of the reference cows remained relatively high (see Fig 1; compare around 673 on day 0 to 587 at day 60).
  • the SCC was significantly decreased in cows receiving a single treatment of di-n-propyl thiosulfinate, di-methyl thiosulfonate, or di-phenyl thiosulfonate.
  • Example 4 in vivo tests – Udder Cleft Dermatitis
  • a category of chronical infections are wounds that are infected by biofilm-forming microorganisms. Once the wound is infected, the microorganisms start to form a biofilm that remains attached to the wounds.
  • microbial EPS Extracellular Polymeric Substances
  • EPS Extracellular Polymeric Substances
  • wounds infecting pathogenic microorganisms are bacteria (Gram positive bacteria, for example Staphylococcus aureus; Streptococci; gram negative bacteria, for example Treponema spp., Escherichia coli, Yersiania pestis, Pseudomonas aeruginosa; yeast/fungi, for example Candida spp (albicans), Cladosporidium herbarum, Trichosporum, Rhodosporidium, Malassezia.
  • bacteria Gram positive bacteria, for example Staphylococcus aureus; Streptococci
  • gram negative bacteria for example Treponema spp., Escherichia coli, Yersiania pestis, Pseudomonas aeruginosa
  • yeast/fungi for example Candida spp (albicans), Cladosporidium herbarum, Trichosporum, Rhodosporidium, Malassezia.
  • the lesions may vary in appearance and size, but thickened skin, crusts, pus, and wounds that easily bleed are common findings.
  • Udder cleft dermatitis can be difficult to detect due to its anatomical position and the fact that affected cows seldom show general signs of disease.
  • Few studies on UCD prevalence have been published, and most have included only one or a few herds, mainly categorized as problem herds. The within- herd prevalence in those studies varied between 0 and 22%. In a recent Dutch study, however, 20 herds were included, of which 3 had no UCD, whereas the within-herd prevalence in the other herds varied between 2.5 and 13% (Amersfort et al., 2012).
  • UCD The etiology of UCD is unclear, but several factors, such as udder conformation and udder edema have been suggested to play a role. Cow factors such as parity and DIM (days in milk) have also been associated with UCD (Beattie and Taylor, 2000, J. Brit. Cattle Vet. Assoc. 8, 377-380). Lesions are most commonly identified on the plantar aspect of the interdigital cleft of the hind limbs. Treponema spp are routinely present in large numbers of active lesions. Lesions are painful to the touch and can result in clinical lameness. The infectious nature generally results in endemic infection of cattle herds and is responsible for large economical losses.

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Abstract

L'invention concerne des compositions contenant de l'organosoufré, en particulier le Di-n-butyl thiosulfinate, le Di-méthyl thiosulfonate, le Di-phényl thiosulfonate, le Bis (p-tolyl) thiosulfinate, le Di-isopropyl thiosulfinate, le Di-benzyl thiosulfinate, le Di-benzyl thiosulfonate, le S-propyl-4-méthylbenzène thiosulfonate, le Di-n-propyl thiosulfinate, et le Di-n-propyl thiosulfonate. De telles compositions sont utiles pour traiter une infection et réduire ou dégrader des biofilms à la fois in vivo et in vitro. En particulier, de telles compositions sont utiles dans le traitement de troubles liés à un biofilm, comprenant, mais sans s'y limiter, la mammite, la dermatite digitale, et les infections cutanées chroniques.
EP22769406.4A 2021-09-10 2022-09-08 Compositions et méthodes de traitement de troubles et d'infections liés au biofilm Pending EP4398891A1 (fr)

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