Compositions and methods for treating biofilm disorders and infections

Abstract

The present disclosure relates to organic sulfur-containing compositions, particularly 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. Such compositions are useful for treating infections and reducing or degrading biofilms both in vivo and in vitro. In particular, such compositions are useful in the treatment of biofilm-related disorders, including but not limited to mastitis, digital dermatitis, and chronic wound infections.

Description

[Technical field]

[0001] The present disclosure relates to organic sulfur-containing compositions, particularly 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. Such compositions are useful for treating infections and reducing or degrading biofilms both in vivo and in vitro. In particular, such compositions are useful in the treatment of biofilm-related disorders, including but not limited to mastitis, digital dermatitis, and chronic wound infections. [Background technology]

[0002] Udder health plays a key role in dairy animals, both from a health and wellness perspective, and from an economic perspective. Infections in the mammary gland of dairy animals, such as cows, known as mastitis, have a significant economic impact on dairy farms worldwide. Annual losses worldwide are estimated at 4-5 billion euros. In dairy mastitis, the udder is unable to mount an effective defense response against invading microorganisms. Several factors are known that disrupt the balance at the udder level, potentially compromising the ability of dairy animals to kill mastitis-causing microorganisms. As a result, host response mechanisms are unable to mount an efficient defense response to eliminate the invading pathogen, which may lead to bacterial colonization of the udder and the development of clinical or subclinical mastitis.

[0003] Bacterial colonization, especially the formation of bacterial reservoirs, in the udder of dairy cows is generally difficult to combat and leads to chronic infections that may persist despite specific treatment strategies with or without antibiotics. Important factors associated with the persistence of bacterial infections in the udder of dairy cows are epithelial adhesion, biofilm production, and bacterial susceptibility to phagocytosis.

[0004] To control or prevent mastitis in dairy cows, several strategies have been used, such as antibiotic treatments and vaccinations, to name a few, to reduce the clinical and economic impact of the disease. However, most treatments and strategies have little or no effect on ameliorating the disease. There are several reasons for this lack of effectiveness. First, although several virulence factors have been suggested as potential antigens for single-component vaccines, experimental trials have demonstrated that induction of immunity against single factors is not sufficient to confer strong protection against bacteria causing mastitis in dairy cows. Second, bacterial antigens are poorly immunogenic and require appropriate adjuvantion. Third, a major challenge in controlling mastitis is an effective antibiotic that can reach the bacterial pool in the udder, for example as a result of the formation of a biofilm by the bacteria.

[0005] Microorganisms such as bacteria do not necessarily have to produce biofilms, but are much more likely to survive in the host if they are able to adhere, for example, to epithelial cells. Adhesion is an active process involving a series of attachments and detachments, resulting in the formation of biofilms, which are accompanied by significant genetic and subsequent physiological changes in the microorganisms, resulting, among other things, in the loss of susceptibility to virtually all classes of antibiotics. Thus, the management of bacterial udder infections has become increasingly difficult due to the emergence and increasing prevalence of bacterial pathogens resistant to antibiotics. In some cases, low doses of antibiotics can even promote biofilm formation, suggesting a natural defense mechanism of bacteria in avoiding the lethal effects of antibiotics. Due to this complex and problematic situation, mastitis remains one of the most important diseases of dairy cows, despite progress made in recent years in improving general udder health.

[0006] Furthermore, when cattle are frequently or continuously treated with antibiotics, the antibiotics and their decomposition products are also found in the manure. The manure containing the antibiotics and their decomposition products is spread to the soil. The presence of antibiotics has been shown to affect the bacterial diversity of the soil, which is an undesirable effect for the environment. An alternative composition for treating infections in cattle prevents the spread of antibiotics to the environment and restores the soil microbiome.

[0007] Although descriptions of bacterial biofilms can be found in the scientific literature much earlier, the meaning of biofilm became known in 1982 when Costerton observed Staphylococcus aureus forming biofilms on cardiac pacemaker leads. Subsequent research and clinical observations revealed that bacterial biofilms can be found on implants and catheters, prosthetic devices, and other implanted biomaterials. Even more importantly, it was observed that microbial biofilms can also form on biological surfaces of human and animal tissues, such as the periodontal mucosa in the oral cavity (dental plaque), the paranasal sinuses (chronic sinusitis), the inner ear (latent labyrinthitis), blood vessels and heart valves (endocarditis), alveolar surfaces (multiple lung diseases), or the biliary tract and bladder.

[0008] The first stage of biofilm formation involves the attachment of cells to a surface. In the second stage, cell proliferation occurs with the formation of a mature structure with many layers of cells. A slime layer is also formed to further protect the bacteria. See, for example, Melchior et al. Veterinary Journal 2006 171:398-407. Upon reaching a critical mass, the outermost cell layer of the biofilm may release "planktonic organisms". These organisms can further colonize other surfaces. Biofilms can form on a wide variety of surfaces, including living tissue, indwelling medical devices, industrial or potable water piping, or natural water systems. As will be appreciated by those skilled in the art, not all infections result in the formation of biofilms.

[0009] Research over the past two decades has revealed that collective biofilm formation is driven by a bacterial communication system, denoted quorum sensing (QS). QS occurs using small chemical molecules (so-called auto-inducers, AIs) that are permanently excreted by bacteria into their environment. These signaling molecules (e.g., oligopeptides (AIPs) or N-acetylhomoserine lactones (AHLs)) are recognized and monitored via specific receptors by other bacteria in their vicinity. Once a certain density of AIs is reached (quorum), bacterial cells collectively change their gene expression and either produce virulence factors to attack somatic cells or activate metabolic pathways to form a biofilm on tissue surfaces. Biofilm formation involves the formation of an extracellular matrix, initially composed of large polymers, mainly polysaccharides, which upon maturation are stabilized by proteins and lipids, resulting in a three-dimensional structure.

[0010] Once a biofilm infection is established, it can be very difficult to eradicate. Mature biofilms intermittently shed planktonic cells. This can lead to chronic infections with intermittent exacerbations. While antibiotics or the host's immune response can resolve symptoms caused by planktonic cells, mature biofilms may persist.

[0011] Microbial cells under the protection of biofilms are often more resistant to antibiotics and the body's natural immune response. Dormant bacteria are metabolically inactive and therefore do not express typical targets of many antibiotics, such as synthesis of bacterial cell wall components (targets of β-lactam antibiotics such as penicillins and cephalosporins and vancomycin), rapid proteins (targets of aminoglycosides, tetracyclines, macrolides, and linezolid), and DNA synthesis (fluoroquinolones and rifampicin) or folate synthesis (sulfonamides, aminopyrimidines (e.g. trimethoprim)). Thus, antibiotic treatment alone is generally not sufficient to eradicate biofilm infections. (See Wu et al. Int J Oral Sci. 2015 Mar; 7(1): pp. 1-7). Antibiotics can be effective against dispersed (planktonic) bacteria, but it is difficult to reach the minimum concentration of antibiotics required to eradicate the microorganisms in biofilms. Various in vitro experiments have demonstrated that bacteria growing in biofilms are 10-1000 times more resistant to various antibacterial agents when compared to planktonic bacteria of the same strain (see, for example, Amorena et al. 1999 J. Antimicrob. Chemother. 44:43-55; Ceri et al., 1999 J. Clin. Microbiol. 37:1771-1776; and Olson et al., 2002 Can. J. Vet. Res. 66:86-92). Olsen et al. have reviewed various mechanisms of antibiotic resistance and tolerance induced by biofilms (Eur J Clin Microbiol Infect Dis 2015 34:877-886).

[0012] In the case of bovine mastitis, it has been reported in a study with a field strain of Staphylococcus aureus (Melchior, Gaastra & Fink-Gremmels, J. Vet. Med. 2006 53:326-332). The MIC50, minimum bacterial concentration (BMIC) and MBEC (minimal biofilm eradicating concentration) were determined for seven strains isolated from mastitis-infected cows. Antibiotics tested included those commonly used to treat bovine mastitis, such as penicillin, amoxicillin, cloxacillin, cephalothin, cefoperazone, cefquinome, cloxacillin / penicillin combination, lincomycin, pirlimycin, tyrosine, neomycin, gentamicin, trimethoprim / sulfamethoxazole, florfenicol and danofloxacin. For all antibiotics tested, the difference between MIC (planktonic bacteria) and MBEC (biofilm eradication concentration) was more than 256-fold and in the vast majority of cases more than 2048-fold.

[0013] There is also evidence that antibiotics can stimulate biofilm formation. For example, some antibiotics (such as tetracyclines, quinupristin-dalfopristin, and erythromycin) can stimulate the expression of genes in bacteria that promote bacterial adhesion (e.g., ica genes) (Melchior et al., see above). Interestingly, ica genes have been shown to be prevalent among S. aureus mastitis isolates (reviewed in Melchior et al., see above). These results support the hypothesis that udder infections are associated with biofilm formation.

[0014] Biofilms can also contain dormant bacteria. Biofilms use several mechanisms to evade the 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 also transition from dormancy to become active. In general, the immune system only acts against active bacteria, and dormant bacteria can therefore escape the immune system of an individual. Dormant bacteria can detach from the biofilm and quickly become active and harmful to the host.

[0015] Fungal-associated biofilms are also known to be more resistant to antifungal drugs compared to planktonic cells (see, for example, Fanning and Mitchell PLOS Pathog 2012 8:e1002585 for a review). Therefore, compounds with antibacterial effects are not necessarily suitable for the treatment or prevention of biofilms.

[0016] In mature biofilms, the dormancy stage is adopted by downregulation (gene shift) of primary metabolism. Dormancy involves biofilm bacteria almost completely suppressing the expression of typical targets of antibiotics, such as protein and DNA synthesis and cell wall remodeling. Biofilms are subsequently essentially insensitive to antibiotics, often exhibiting upwards of 1000-fold higher resistance to them than planktonic (free-moving) bacteria. Furthermore, the high cell density in biofilms considerably increases the likelihood of horizontal gene transfer, thereby increasing the likelihood of the emergence of strains with enhanced resistance or altered virulence profiles. The clinical outcome is phenotypic resistance to common (even the most current) antibiotics.

[0017] In such a protective biofilm environment, bacterial survival time is increased. Moreover, the self-assembled, inert, polysaccharide-rich matrix is ​​non-immunogenic, protecting the biofilm-embedded bacteria from recognition (via PAMPS-pathogen-associated molecular patterns) and phagocytosis by the host's immune cells.

[0018] Biofilm formation can have serious adverse effects in medical, industrial, and natural environments. In particular, biofilm-associated infections (i.e., biofilm-associated disorders) are a serious problem in both humans and animals. Such disorders can be characterized by a chronic inflammatory response with recurrent acute episodes and resistance to antimicrobial therapy and / or host defense. Wound biofilms delay tissue repair, resulting in chronic wounds. Biofilm infections are currently suggested to account for up to 80% of all human microbial infections (Bartell JA et al., 20 Evolutionary highways to persistent bacterial infection. Nature Communications (2019) 10:629 and Sharma et al. 2019 Antimicrobial Resistance and Infection Control 8:76). Jamal et al. reported that the National Institutes of Health showed that 65% of microbial infections and 80% of chronic infections are associated with biofilm formation (Journal of Chinese Medical Association 81:7-11). Biofilms and biofilm-related disorders have been widely discussed in the literature; see, for example, Sharma et al. 2019 Antimicrobial Resistance and Infection Control 8:76; Roy et al. 2018 Virulence 9:522-554; and Jamal et al. 2018 Journal of the Chinese Medical Association 81:7-11. Vestby et al. have reviewed bacterial biofilms and their role in disease (Antibiotics (Basel). 2020 Feb; 9(2): 59).A comprehensive overview of biofilm infections was presented by the European Clinical Society of Microbiology and Infectious Disease in 2014; see also David Lebeaux et al. Microbiol. Mol. Biol. Rev. 2014; doi:10.1128 / MMBR.00013-14.

[0019] For example, Pseudomonas aeruginosa, an organism that causes hospital-acquired infections, forms biofilms on a wide variety of surfaces, including cystic fibrosis lung tissue, contact lenses, and catheter lines. Pseudomonas aeruginosa growing as biofilms has also been found in chronic wounds, which can result in impaired wound healing. Biofilms, particularly P. aeruginosa biofilms, also cause chronic infections in respiratory diseases such as bronchiectasis, chronic obstructive pulmonary disease, and chronic rhinosinusitis. Biofilms formed on medical devices act as reservoirs of bacteria that can be shed into the body, resulting in chronic systemic infections. Candida albicans (yeast) is the most common fungal biofilm found in hospitals, but it is extremely difficult to treat and does not respond well to typical antifungal treatments.

[0020] Pioneering studies of bacterial biofilm formation focused on Staphylococcus aureus (gram-positive) and Pseudomonas aeruginosa (gram-negative) due to their involvement in recurrent mastitis and complicated wound infections in dairy cows. In addition, biofilm formation of Streptococcus ssp., avian pathogenic E. coli (APEC), and Campylobacter jejuni biofilms attracted attention due to the public health implications of these bacterial species. Subsequently, other important animal pathogens were recognized as causing biofilm infections, including, for example, Actinobacillus pleuropneumoniae (severe pulmonary infections in pigs that can be fatal), E. coli (local and systemic infections, as well as frequently fatal systemic septicemia in poultry), canine and feline skin and intestinal diseases, and equine wound infections and endometritis. This list is non-exclusive, as biofilm formation is a general property of almost all microorganisms. [Prior art documents] [Non-patent literature]

[0021] [Non-Patent Document 1] Melchior et al. Veterinary Journal 2006 171:398-407 [Non-Patent Document 2] Wu et al. Int J Oral Sci. 2015 Mar; 7(1): 1-7 [Non-Patent Document 3] Amorena et al. 1999 J. Antimicrob. Chemother. 44:43-55 [Non-Patent Document 4] Ceri et al., 1999 J. Clin. Microbiol. 37:1771~1776 [Non-Patent Document 5] Olson et al., 2002 Can. J. Vet. Res. 66:86-92 [Non-Patent Document 6] Eur J Clin Microbiol Infect Dis 2015 34:877~886 pages

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[0022] Thus, there is a need for antimicrobial compounds and alternative treatments for biofilm-associated disorders. [Means for solving the problem]

[0023] The present disclosure provides the following preferred embodiments.

[0024] - a compound according to formula I, or a composition comprising a compound according to formula I, for use in the treatment of a biofilm-associated disorder:

[0025] [ka]

[0026] (Wherein, n is 2, one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O)2-; R 1 and R 2 are independently selected from optionally substituted alkyl or optionally substituted aryl; and the compound according to formula I is selected from the group consisting of di-n-butyl disulfide, dimethyl disulfide, diethyl disulfide, di-n-propyl disulfide, diethyl thiosulfinate, di-n-propyl thiosulfinate, di-n-butyl thiosulfinate, di-n-propyl thiosulfonate, di-methyl thiosulfonate, and di-phenyl thiosulfonate.

[0027] - a compound according to formula I, or a composition comprising a compound according to formula I, for use in the treatment of a biofilm-associated disorder:

[0028] [ka]

[0029] (Wherein, n is 2, one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O)2-; R 1 and R 2are independently selected from optionally substituted alkyl or optionally substituted aryl.

[0030] - the compound or composition for use according to any one of the preceding embodiments, wherein the biofilm-associated disorder is a chronic and / or persistent infection.

[0031] - the compound or composition for use according to any one of the preceding embodiments, wherein the biofilm-associated disorder is digital dermatitis, udder clefts, or chronic wound infections.

[0032] - the compound or composition for use according to any one of the preceding embodiments, wherein the biofilm comprises bacteria, yeast, fungi, microalgae, or a combination thereof.

[0033] A compound according to formula I, or a composition comprising a compound according to formula I, for use in the treatment or prevention of a microbial infection, preferably a bacterial infection or a fungal infection:

[0034] [ka]

[0035] (Wherein, n is 2, one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O)2-; R 1 and R 2 are independently selected from optionally substituted alkyl or optionally substituted aryl.

[0036] - the compound or composition for use according to any one of the preceding embodiments, wherein the use further comprises the administration of an antibacterial agent, preferably selected from an antifungal or an antibiotic.

[0037] - the compound or composition for use according to any one of the preceding embodiments, wherein the use further comprises administration of an anti-inflammatory agent.

[0038] - a compound according to formula I:

[0039] [ka]

[0040] (Wherein, n is 2, one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O)2-; R 1 and R 2 is independently selected from optionally substituted alkyl or optionally substituted aryl; The composition is a pharmaceutical composition, a functional food, or a cleaning product.

[0041] - an in vitro method for preventing or reducing the formation or growth of a biofilm on a surface, or for degrading or reducing a biofilm on a surface, comprising: The method includes: or applying a composition to a surface to degrade or reduce biofilm on the surface, the composition comprising a compound according to formula I:

[0042] [ka]

[0043] (Wherein, n is 2, one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O)2-; R 1 and R 2 is independently selected from optionally substituted alkyl or optionally substituted aryl.

[0044] - the compound or composition, the method, or the composition for use according to any one of the preceding embodiments, wherein the composition is essentially free of diallyl thiosulfinate.

[0045] - the compound or composition, the method or the composition for use according to any one of the preceding embodiments, wherein the composition further comprises an antibacterial agent, preferably selected from an antifungal agent or an antibiotic.

[0046] - the compound or composition, method, or composition for use according to any one of the preceding embodiments, wherein the composition further comprises an anti-inflammatory agent.

[0047] - a compound according to formula I:

[0048] [ka]

[0049] (Wherein, n is 2, one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O)2-; R 1 and R 2 is independently selected from optionally substituted alkyl or optionally substituted aryl); preferably, the article is a medical device or a surgical instrument.

[0050] - R 1 and R 2 But independently, C 1~6 alkyl and phenyl; preferably, R 1 and R 2 But independently, C 1~4 The compound or composition, method, article, or composition for use according to any one of the embodiments, wherein:

[0051] - R 1 and R 2are identical.

[0052] - each X is -S- and R 1 and R 2 But independently, C 1-4 The compound or composition, method, article, or composition for use according to any one of the embodiments, wherein:

[0053] - one X is -S- and the other X is selected from the group consisting of -S(O)- and -S(O)2-; R 1 and R 2 is independently selected from alkyl, aryl, alkylaryl, and arylalkyl.

[0054] - When one X is -S- and the other X is -S(O)-, R 1 and R 2 is independently selected from the group consisting of n-propyl, isopropyl, butyl, benzyl, and p-tolyl; and / or when the other X is -S(O)-, R 1 and R 2 is independently selected from the group consisting of methyl, phenyl, benzyl, 4-methylbenzene, and n-propyl.

[0055] Preferably, where one X is -S- and the other X is -S(O)-, then R 1 and R 2 is independently selected from the group consisting of isopropyl, butyl, benzyl, and p-tolyl; where when the other X is -S(O)-, R 1 and R 2 is independently selected from the group consisting of methyl, phenyl, benzyl, 4-methylbenzene, and n-propyl.

[0056] The compound or composition, method, article, or composition for use according to any one of the embodiments, wherein 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, di-n-propyl thiosulfinate, di-n-butyl thiosulfinate, di-n-propyl thiosulfonate, di-methyl thiosulfonate, and di-phenyl thiosulfonate.

[0057] The compound or composition, method, article, or composition for use according to any one of the embodiments, wherein 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-methylbenzenethiosulfonate, di-isopropyl thiosulfinate, di-benzyl thiosulfinate, di-benzyl thiosulfonate, di-n-propyl thiosulfinate, and di-n-propyl thiosulfonate.

[0058] The compound or composition, method, article, or composition for use according to any one of the embodiments, wherein the compound according to formula I is selected from the group consisting of di-n-butyl thiosulfinate, di-methyl thiosulfonate, and di-phenyl thiosulfonate.

[0059] Methods of treatment are also disclosed, comprising administering a compound of formula I to an individual in need thereof. Such methods are useful for treating biofilm disorders as disclosed herein. Such methods are also useful for treating infectious diseases as disclosed herein. [Brief description of the drawings]

[0060] [Figure 1] FIG. 1 shows somatic cell counts in cattle 60 days after treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0061] The present disclosure provides compounds of formula I:

[0062] [ka]

[0063] (Wherein, n is 2, one X is -S- and the other X is selected from the group consisting of -S-, -S(O)-, and -S(O)2-; 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) are useful for reducing, degrading, and / or preventing the formation of biofilms.

[0064] As used herein, "alkyl" refers to a saturated aliphatic hydrocarbyl group. Unless otherwise stated, the alkyl group can be linear or branched. Preferably, the alkyl group is linear. As used herein, the alkyl group can be substituted or unsubstituted. Preferably, the alkyl group is unsubstituted. In a preferred embodiment, in formula I, the alkyl group is selected from the group consisting of C 1~6 Alkyl, more preferably C 1~4 It is an alkyl.

[0065] As used herein, "aryl" refers to an aromatic hydrocarbon ring system containing 6 to 24 carbon atoms, more preferably 6 to 12 carbon atoms, and may include monocyclic and polycyclic structures. When the aryl group is a polycyclic structure, it is preferably a bicyclic structure. Optionally, the aryl group is substituted with one or more substituents as further defined herein. Preferably, the aryl group is unsubstituted. Examples of aryl groups are phenyl, benzyl, and naphthyl. Most preferably, the aryl group is phenyl.

[0066] As used herein, "substituted" indicates that a group contains one or more substituents. Preferably, the substituents are independently selected from halogen, -C(O)OH, -C(O)NH, -OH, =O, C(O)NH2, -C(O)NH3, -C(O)NH4, -C(O)NH5, -C(O)NH6, -C(O)NH7, -C(O)NH8, -C(O)NH9, -C(O)NH10, -C(O)NH2, -C(O)NH10, -C(O)NH2, -C(O)NH3, -C(O)NH4, -C(O)NH5, -C(O)NH6, -C(O)NH2, -C(O)NH2, -C(O)NH4 ... 1~3 Preferably, the halogen is selected from the group consisting of -Cl, -F, -Br, and -I. Most preferably, the halogen is -Cl. In a preferred embodiment, the groups disclosed herein contain at most three substituents, more preferably at most two substituents, and most preferably at most one substituent.

[0067] In a preferred embodiment, R 1 and R 2 is independently selected from alkyl and aryl. More preferably, R 1 and R 2 is independently 1~6 More preferably, R is selected from alkyl and phenyl. 1 and R 2 is independently 1-4 Most preferably, R 1 and R 2 are independently linear C 1~4 Alkyl, that is, methyl, ethyl, n-propyl, or n-butyl.

[0068] In a preferred embodiment, R 1 and R 2 are identical.

[0069] In a preferred embodiment, each X is -S-. In a preferred embodiment, one X is -S- and the other X is -S(O)-. In another preferred embodiment, one X is -S- and the other X is -S(O)-.

[0070] In a preferred embodiment, in formula I, each X is -S- and R 1 and R 2 is independently 1~4 Alkyl, preferably linear C1~4 Alkyl, i.e. methyl, ethyl, n-propyl, or n-butyl. Preferably, compositions comprising these particular compounds according to formula I further comprise one or more antibacterial agents.

[0071] In a preferred embodiment of formula I, one X is -S- and the other X is selected from the group consisting of -S(O)- and -S(O)-; R 1 and R 2 is independently selected from alkyl, aryl, alkylaryl, and arylalkyl.

[0072] Preferably, where one X is -S- and the other X is -S(O)-, then R 1 and R 2 is independently selected from the group consisting of n-propyl, isopropyl, butyl, benzyl, and p-tolyl; and / or where when the other X is -S(O)-, R 1 and R 2 is independently selected from the group consisting of methyl, phenyl, benzyl, 4-methylbenzene, and n-propyl.

[0073] In a preferred embodiment, the compound of formula I is not propyl-propanethiosulfonate (PTSO, also known as di-n-propylthiosulfonate) or propyl-propane-thiosulfinate (PTS, also known as di-n-propylthiosulfinate).

[0074] Preferably, where one X is -S- and the other X is -S(O)-, then R 1 and R 2 is independently selected from the group consisting of isopropyl, butyl, benzyl, and p-tolyl; where when the other X is -S(O)-, R 1 and R 2 is independently selected from the group consisting of methyl, phenyl, benzyl, 4-methylbenzene, and n-propyl.

[0075] In a preferred embodiment, 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 called di-n-propyl thiosulfinate), n-butyl butane thiosulfinate (also called di-n-butyl thiosulfinate), n-propyl propane-1-thiosulfonate (also called di-n-propyl thiosulfonate), methyl methane thiosulfonate (also called di-methyl thiosulfonate), and phenyl benzene thiosulfonate (also called di-phenyl thiosulfonate). In a preferred embodiment, 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 disulfide. In a preferred embodiment, 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 a preferred embodiment, the compound according to formula I is selected from the group consisting of di-n-propyl thiosulfonate, di-methyl thiosulfonate, and di-phenyl thiosulfonate. In a preferred embodiment, 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-methylbenzenethiosulfonate, di-isopropyl thiosulfinate, di-benzyl thiosulfinate, and di-benzyl thiosulfonate.

[0076] In a preferred embodiment, the compound according to formula I is dimethyl disulfide. In a preferred embodiment, the compound according to formula I is diethyl sulfide. In a preferred embodiment, the compound according to formula I is di-n-propyl disulfide. In a preferred embodiment, the compound according to formula I is di-n-butyl disulfide. In a preferred embodiment, the compound according to formula I is diphenyl disulfide. In a preferred embodiment, the compound according to formula I is diethyl thiosulfinate. In a preferred embodiment, the compound according to formula I is di-n-propyl thiosulfinate. In a preferred embodiment, the compound according to formula I is n-butyl butane thiosulfinate. In a preferred embodiment, the compound according to formula I is di-n-propyl thiosulfonate. In a preferred embodiment, the compound according to formula I is di-methyl thiosulfonate. In a preferred embodiment, the compound according to formula I is di-phenyl thiosulfonate.

[0077] It will be understood that the terms "sulphide" and "sulfide" are used interchangeably herein.

[0078] In some embodiments, the compounds are obtained from natural sources, such as plants. The compounds can be extracted from plant materials in a variety of ways. The appropriate method depends on the chemical nature of the compound. For example, extraction can begin with a non-polar solvent, followed by increasing the polarity of the solvent. The compounds can also be commercially available or prepared as described in Example 1.

[0079] The present disclosure provides compositions comprising the compounds disclosed herein. Preferably, such compositions are substantially free of diallyl thiosulfinate (also called allyl-2-propene-1-sulfinothioate). Diallyl thiosulfinate is commonly known as allicin. Allicin is an organosulfur compound. When raw garlic is chopped or crushed, the enzyme alliinase converts alliin to allicin, which is responsible for the aroma of raw garlic. The resulting allicin is unstable and quickly transforms into a series of other sulfur-containing compounds, such as diallyl disulfide.

[0080] As used herein, "substantially free" refers to a composition that contains less than 5 wt% of diallyl thiosulfinate. In some embodiments, the composition contains less than 1 wt% of diallyl thiosulfinate, preferably less than 0.5 wt% of diallyl thiosulfinate. In some embodiments, the composition contains a weight ratio of the compound having formula I to diallyl thiosulfinate that is at least 10:1, more preferably at least 100:1.

[0081] The composition of the present disclosure is also preferably substantially free of diallyl disulfide. In some embodiments, the composition comprises less than 1 wt% of diallyl disulfide, preferably less than 0.5 wt% of diallyl disulfide. In some embodiments, the composition comprises a weight ratio of the compound having formula I to diallyl disulfide that is at least 10:1, more preferably at least 100:1.

[0082] In some embodiments, compositions are provided in which at least 50% by weight, preferably at least 90% by weight, of the active ingredient is a compound according to formula I as disclosed herein. In some embodiments, compositions are provided in which the only active ingredient is a compound according to formula I, optionally including an additional antibacterial and / or anti-inflammatory agent. In some embodiments, compositions are provided in which at least 50% by weight, preferably at least 90% by weight, of the active ingredient is a compound according to formula I as disclosed herein. In some embodiments, compositions are provided in which the only active ingredient is a compound according to formula I, optionally including an additional antibacterial and / or anti-inflammatory agent.

[0083] The compounds disclosed herein and compositions containing them are useful for treating or preventing infections. For example, particular uses are for treating or preventing respiratory infections, intestinal infections, breast infections, udder infections, skin infections, bladder infections, ear infections, systemic infections, joint infections, and brain infections.

[0084] As used herein, "infection" refers to, for example, a pathogenic infection that can result in disease. In particular, such an infection is a bacterial infection or a fungal infection. Preferably, the infection is a microbial infection. In a preferred embodiment, the infection is a bacterial infection. In a preferred embodiment, the infection is a fungal infection (including a yeast infection).

[0085] Bacteria and fungi are found in most places and exist in a great variety of forms. Most are not harmful and are in fact essential to life on earth and are essential for the health of plants, animals and humans. For example, the microbiome in the intestinal tract of humans and animals, where bacteria and fungi live with their host as symbionts, is the so-called gut microbiota. Bacteria also naturally reside on the skin and form part of the immune system. Another example is soil biology, which consists mostly of bacteria and fungi. Some bacteria and fungi can cause pathogenic infections, for example in animals or humans. These pathogenic infections can result in diseases and illnesses in the infected individuals.

[0086] As used herein, "treatment of an infectious disease" refers to reducing the severity and / or duration of an infectious disease and / or reducing the severity and / or duration of symptoms from an infectious disease. Preferably, said treatment leads to restoration of the health of the individual. Preferably, the individual experiences less severe or shorter duration of disease symptoms. As used herein, "prevention of an infectious disease" refers to preventing or alternatively delaying the onset of an infectious disease or one or more symptoms associated with an infectious disease.

[0087] Some microorganisms, such as bacteria, microalgae, fungi, etc., can form biofilms. The compounds disclosed herein are also useful for preventing or reducing the formation or growth of biofilms and / or for decomposing or reducing biofilms. Preferably, the compounds and compositions comprising them are useful for treating or preventing biofilm-associated disorders. As those skilled in the art will recognize, not all compounds that can treat microbial (e.g., bacterial) infections can treat biofilm-associated disorders.

[0088] The term biofilm was originally used in industrial and environmental microbiology to describe communities of sessile bacteria and other microorganisms attached to natural or artificial surfaces. Microbial biofilm formation is initiated by bacterial colonization of a surface to which they attach and produce a slimy film composed of organic polymers. This initial bacterial film attracts other microorganisms, such as algae and protozoa, fungi and protozoa, resulting in the formation of a visible multi-species biofilm. Such three-dimensional biofilms are ubiquitous in nature and are found on all surfaces in contact with water. A public health concern are microbial biofilms in municipal water supplies and domestic water pipelines and equipment.

[0089] As used herein, the term "biofilm" refers to a population of microorganisms concentrated at an interface (usually solid / liquid) and typically surrounded by an extracellular polymeric slime matrix. Biofilms form on living or non-living surfaces and can be found in natural, industrial, and hospital environments. Biofilms can include various types of microorganisms, such as bacteria, archaea, protozoa, fungi, and algae. Preferably, such biofilms include bacteria, microalgae (such as Prototheca spp.), or fungi.

[0090] As used herein, "treatment of a biofilm-related disorder", also referred to herein as a "biofilm associated disorder", refers to a reduction in the severity and / or duration of the disorder and / or a reduction in the severity and / or duration of symptoms from the disorder, in particular symptoms of an infection. Preferably, said treatment leads to a restoration of the health of the individual. Preferably, the individual experiences a milder or shorter duration of disease symptoms.

[0091] As used herein, "preventing or reducing biofilm formation or growth" refers to preventing, slowing, or reducing biofilm formation or growth. As will be appreciated by those skilled in the art, such reduction of biofilm formation or growth can slow the growth of biofilms compared to untreated biofilm growth. Preferably, the composition is useful for reducing biofilm formation or growth. As used herein, "degradation or reduction of biofilm" refers to partial or complete removal of biofilm. As will be appreciated by those skilled in the art, planktonic bacteria may still be present after such treatment.

[0092] The compounds disclosed herein may be capable of disrupting the structure of biofilms, such as the extracellular mucous matrix. In some embodiments, the compounds are useful for inhibiting cell adhesion. In particular, the compounds can prevent the adhesion of all cell types encountered in microbial biofilms, particularly free-living microorganisms, to static or live surfaces (without killing the bacteria).

[0093] Without wishing to be bound by theory, the present disclosure proposes that the therapeutic compounds disclosed herein may exert some of their effects by affecting quorum sensing. Quorum sensing (QS) signaling plays an important role, for example, in controlling the expression of bacterial virulence factors. QS is involved in the accumulation of signaling molecules in the surrounding environment, which allows single cells to sense the number of bacteria and the density of signaling molecules, so that the bacterial population as a whole can respond in a coordinated manner. These cell-cell communication systems regulate various bacterial functions, such as motility, virulence, sporulation, antibiotic production, DNA exchange, and the development of more complex multicellular structures such as biofilms. Thus, interference with the QS signaling system may suggest a new strategy to combat persistent (chronic) bacterial infections. The ability of certain substances, such as naturally occurring compounds with quorum quenching (QQ) capabilities, can be used as anti-adhesion compounds and as compounds that disrupt biofilm formation.

[0094] The compounds disclosed herein are particularly useful for treating biofilm-associated disorders, which are characterized by chronic and / or persistent infections. The compounds disclosed herein are particularly useful for treating chronic and / or persistent infections. The terms persistent and chronic infections are often used interchangeably, but are based on different mechanisms. Persistent infections are usually suppressed by immune defenses, but can become activated when such immune defenses weaken. Persistent infections are often asymptomatic and only become clinically evident when immune defenses are no longer able to control the pathogens. Persistent infections are often asymptomatic, but those skilled in the art are familiar with the means to detect such persistent infections, including, for example, detecting microorganisms in patient samples (e.g., blood or urine). In chronic infections, the pathogens remain in groups of cells / parts of tissues (e.g., joints or lung tissue). The patient always has symptoms of the disease, but these may be milder than those in the acute phase of infection.

[0095] A prominent example of a biofilm disease is bovine mastitis, a clinical term for infection of the mammary gland of cows that can be caused by multiple pathogens, the most prevalent forms of which are Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, as well as Serratia marescens and other facultative pathogenic Enterobacteriaceae and Prototheca species, the latter of which is considered an emerging pathogen causing aggressive and incurable mastitis in many parts of the world. The present invention encompasses treating biofilms containing such microorganisms with the compounds and compositions disclosed herein.

[0096] In some embodiments, wounds treated with the compounds of the present invention include bacteria such as Staphylococcus aureus; Streptococci; Gram-negative bacteria, such as Treponema spp., Escherichia coli, Yersiania pestis, Pseudomonas aeruginosa; or yeast / fungi, such as Candida spp. (albicans), Cladosporidium herbarum, Trichosporum, Rhodosporidium, and Malassezia.

[0097] It is now generally recognized that biofilm-associated microorganisms cause many infections, including endocarditis, osteomyelitis, sinusitis, urinary tract infections, chronic prostatitis, periodontitis, chronic lung infections in cystic fibrosis patients, middle ear infections, and a variety of hospital-acquired infections, especially those associated with all known indwelling devices (catheters, implants). The burden of biofilm diseases is large and is a major concern in healthcare. Although it is known that almost all bacterial species are capable of forming biofilms under conditions of stress, in current clinical practice some bacterial and fungal species are of great interest because the associated infections are almost completely resistant to treatment, even though non-biofilm, planktonic forms of the same species and strains show very good sensitivity to common therapeutic agents (antibiotics and antifungals / fungicides).

[0098] Examples of pathogens that have become major clinical problems due to their biofilm-associated therapy resistance include: Aspergillus fumigatus - Pulmonary aspergillosis (fungal disease) Burkholderia cepacian-pulmonary cystic fibrosis superinfection Candida species (yeast) - mucosal surfaces of the gastrointestinal and genitourinary tracts Gardnerella vaginalis (urogenital tract) E. coli (multiple organ and sepsis) Pseudomonas aeruginosa (multisystem and pulmonary infections, including cystic fibrosis) Staphylococcus aureus (multiple tissue and wound infections, hospital-acquired infections) Staphylococcus epidermidis (multiple tissue and wound infections) Stenotrophomonas maltophilia (chronic airway disease).

[0099] The treatment / treatment of such biofilm-associated disorders and biofilm-associated microorganisms is encompassed by the present invention. In a preferred embodiment, the biofilm-associated disorder is a breast cleft. Others that may be treated with the compounds of formula I include ulcers (e.g., diabetic foot ulcers (DFU), venous leg ulcers (VLU), and pressure ulcers (PU)); mortellaro (digital dermatitis); and eczema.

[0100] Microbial biofilms are not only formed by bacteria, but also by other microorganisms, especially pathogenic fungi (Aspergillus fumigatus, the main cause of multiple Aspergillus-associated lung diseases) and yeasts (Candida spp.) colonizing mucosal surfaces of the gastrointestinal and genitourinary tracts. The most prevalent implant-associated biofilms are formed by Staphylococcus aureus (MSSA and MRSA), Candida albicans, Pseudomonas aeruginosa, Klebsiella pneumonia, and Enterococcus faecalis. Furthermore, microalgae, e.g. Prototheca spp., can form biofilms and are the cause of disease (protothecia) in humans and animals.

[0101] In some embodiments, the biofilm is selected from the group consisting of Treponema spp., Yersinia pestis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Serratia marcescens, Trueperella pyogenes, Mannheimia haemolytica, Pasteurella multocida, Pseudomonas aeruginosa, Burkholderia cepacia, Streptococcus neumoniae, Hemophilus influenza, Legionella neumophila, Fusobacterium necrophorum, and the like. The bacteria include bacteria selected from one or more of: Corynebacterium pseudotuberculosis, Streptococcus spp., Porphyromonas gingivalis, Pseudomonas aeruginosa, Enterococcus faecalis, Neisseria gonorrhoeae, Escherichia coli, Salmonella enteritidis, and Pseudomonas aeruginosa. In some embodiments, the biofilm is selected from the group consisting of 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. spp., Paecilomyces spp., Paracoccidioides brasiliensis, Penicillium spp., Phymatotrichum spp., Phytophthora spp., Pythium spp., Piedraia hortai, Rhizoctonia spp., Rhizopus spp., Rhodosporidium spp., Saccharomyces spp., Scerotium spp. spp.), Sclerotinia spp., Torulopsosis spp.), and Trichophyton spp. Many medically important fungi are known to produce biofilms, such as Candida, Aspergillus, Cryptococcus, Trichosporon, Coccidioides, and Pneumocystis. In some embodiments, the biofilm comprises microalgae, such as Prototheca spp.

[0102] In certain embodiments, 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. In certain embodiments, the bacterial infection or biofilm-associated disorder is caused by a multi-drug resistant bacterium. In certain embodiments, the bacterial infection is a Methicillin-resistant Staphylococcus aureus (MRSA)-associated infection or a Staphylococcus epidermidis (e.g., MRSE)-associated infection.

[0103] In a preferred embodiment, the biofilm causing bacterium is Escherichia coli and preferably the biofilm infection is a recurrent urinary tract infection, a catheter associated urinary tract infection, or a biliary tract infection.

[0104] In a preferred embodiment, the biofilm causing bacterium is Pseudomonas aeruginosa and 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 associated keratitis.

[0105] In a preferred embodiment, the biofilm causing bacteria is Staphylococcus aureus and preferably the biofilm infection is chronic osteomyelitis, chronic rhinosinusitis, endocarditis, chronic otitis media, or of an (orthopedic) implant.

[0106] In a preferred embodiment, the biofilm causing bacteria is Staphylococcus epidermidis and preferably the biofilm infection is of a central venous catheter, an orthopedic implant, or chronic osteomyelitis.

[0107] In a preferred embodiment, the biofilm causing bacterium is Streptococcus pneumoniae, and preferably the biofilm infection is an infection of the nasopharynx, chronic rhinosinusitis, chronic otitis media, or an infection in chronic obstructive pulmonary disease.

[0108] In a preferred embodiment, the biofilm causing bacteria is Streptococcus pyogenes and preferably the biofilm infection is an infection of the oral cavity and nasopharynx, recurrent tonsillitis.

[0109] The clinical signs of biofilm infection are known to physicians (see, for example, Table 1 in Wu et al. Int J Oral Sci. 2015 Mar; 7(1): 1-7). Such biofilm disorders can result in chronic infections. The determination of acute versus chronic infections is also known to physicians. For example, according to the Mayo Clinic, the occurrence of four or more yeast infections within a year indicates a chronic yeast infection, while the occurrence of two or more bladder infections within a six-month period indicates a chronic bladder infection (also called a recurrent urinary tract infection).

[0110] The most common method of treating bacterial pathogenic infections is the use of antibiotics. Current antibiotics act primarily through growth-dependent mechanisms and target rapidly dividing bacteria. However, non-replicating or slowly growing bacteria (e.g., dormant persister cells, biofilms) exhibit high levels of antibiotic resistance and / or resistance, contributing to persistent and recurrent infections. The compounds disclosed herein are suitable for use in infections or biofilms involving antibiotic resistant bacteria, antibiotic tolerant bacteria, and antibiotic persistent bacteria. The compounds disclosed herein are also suitable as second-line therapy, or rather in individuals who have not responded to previous treatments (e.g., antibacterial treatments) or whose disorder has recurred within, for example, one year or six months.

[0111] The present disclosure further provides compounds disclosed herein and compositions comprising the same for treating any biofilm-induced or biofilm-associated disorder. Biofilm-induced or biofilm-associated disorders are well known to those skilled in the art. In particular, such disorders are biofilm-associated infections. Disorders suitable for treatment include, for example, bacterial prostatitis, bacterial vaginosis, biliary infections, chronic sinusitis, chronic lung disease, dental caries, endocarditis, kidney stones, laryngitis, lung infections in cystic fibrosis, gingivitis, mastitis, middle ear infections, hospital-acquired (bloodstream) infections, obstructive lung disease, osteomyelitis, otitis media, periodontitis, pneumonia, prostatitis, rhinosinusitis, sinusitis, tonsillitis, tuberculosis, urinary tract infections, and wound infections. For example, Mycoplasma bovis is known to cause breast infections and joint infections. Biofilm-associated 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, particularly not for colitis.

[0112] In some embodiments, the compounds and compositions disclosed herein are also useful for treating and preventing infections of implanted medical devices, such as artificial joints and heart valves, as further disclosed herein.

[0113] In some embodiments, the compounds and compositions disclosed herein are also useful for preventing or reducing inflammation in response to bacterial infections or biofilms. Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, and is a defense response involving immune cells and molecular mediators. The function of inflammation is to eliminate pathogens.

[0114] In a preferred embodiment, treatment of an individual with a compound disclosed herein or a composition comprising the same prevents or reduces clinical inflammation in an animal, e.g., a cow. Preferably, the treatment prevents or reduces (clinical) inflammation of the udder. In another embodiment, treatment of an individual with the compound and a composition comprising the same prevents or reduces (clinical) inflammation in a human. For example, the treatment prevents or reduces inflammation of the skin, preferably preventing eczema.

[0115] Without wishing to be bound by theory, treatment of an individual with the compounds disclosed herein or compositions comprising the same reduces the formation or growth of biofilms and / or causes the degradation or reduction of biofilms. Thus, the individual no longer develops an inflammatory response in the presence of pathogens. In other words, removal of biofilms and pathogens within these biofilms reduces the inflammatory response and prevents clinical inflammation.

[0116] During the treatment of biofilms, microorganisms (such as bacteria and fungi) are released from the biofilm. In some cases, the immune system of the individual reacts to the active microorganisms. This can result in inflammation of the tissue. Activated immune cells and inflammatory responses can also damage tissue, for example, in the mammary gland. Thus, suppression of the inflammatory response can prevent or reduce damage to tissue.

[0117] Furthermore, after tissue is damaged and inflammation subsides, the body begins to repair. The macrophages that are still present stimulate the generation of new blood vessels. They also ensure that fibroblasts are attracted. These fibroblasts ultimately cause the formation of granulation tissue. For example, in the case of treatment of dairy cows, scar tissue may form instead of milk-producing tissue. Thus, the cow may produce less milk than before the inflammation.

[0118] In addition to the compounds described herein, additional anti-inflammatory drugs can be administered to suppress inflammatory responses and reduce tissue damage. In a preferred embodiment, the treatments (both therapeutic and prophylactic) disclosed herein further include administration of an anti-inflammatory agent. 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, capsicum, cinnamon, clove, sage, rosemary, black pepper, natural aspirin, Boswellia, Sanguinaria, and / or green tea, can also be used. In some embodiments, the methods and uses disclosed herein include combination treatment of a therapeutic organosulfur compound disclosed herein with an anti-inflammatory agent. The compounds may be administered together or separately. In some embodiments, compositions are provided comprising a therapeutic organosulfur compound disclosed herein and an anti-inflammatory agent.

[0119] In some embodiments, the methods include administering to an individual in need thereof a composition comprising a compound disclosed herein, preferably to prevent or reduce biofilm formation or growth, to degrade or reduce biofilm, and / or to treat or prevent infection, particularly bacterial or fungal infection. In some embodiments, the composition can be administered to an individual for treatment (e.g., therapeutic) or prevention (e.g., prophylactic) of a disease, or disorder, or infection. In some embodiments, the individual has or is at risk of developing a biofilm-associated disease.

[0120] The composition can be administered to any individual, particularly an animal. Preferably, the animal is a ruminant (such as cows and goats), more preferably a cow. In some embodiments, the animal is not a cow. Preferably, the animal is a non-ruminant, such as a monogastric, rodent, non-human primate, pig, horse, dog, cat, or bird. In a preferred embodiment, the animal is a human. In some embodiments, the animal is a non-human animal. In some embodiments, the animal is not an aquatic animal, such as a fish, mollusc, or crustacean. Preferably, the animal is a mammal or a bird.

[0121] Without wishing to be bound by theory, the present disclosure provides that the compounds disclosed herein have beneficial effects after a single administration. In a preferred embodiment, the effects are achieved by a single oral administration of the compounds disclosed herein. Such oral administration can be, for example, as a tablet that provides sustained release of the compounds disclosed herein.

[0122] The present disclosure also provides for multiple administrations. For example, the composition can be provided more than once a day, daily, weekly, or monthly. In an exemplary embodiment, the composition can be provided once a day for a week or until symptoms are alleviated.

[0123] The actual dosage level of the pharmaceutical formulations described herein can be varied to obtain an amount of active ingredient that is effective for achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being harmful to the patient.The dosage level selected depends on various factors, including the activity of the particular compound, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination, the age, sex, weight, condition, overall health, and previous medical history of the patient being treated, and similar factors well known in the medical field.A physician or veterinarian having ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required.

[0124] Those skilled in the art recognize that smaller animals have higher metabolic rates and therefore require larger drug doses on a body weight basis. Dose conversion between animals and between humans and animals is reviewed in Nair and Jacob (J Basic Clin Pharm. March 2016-May 2016; 7(2): 27-31) and Holliday, et al. (1967 The Relation of Metabolic Rate to Body Weight and Organ Size. A Review. Pediat.Res. 1: 185-195).

[0125] In some embodiments of the methods and uses disclosed herein, at least 5 mg / day of the compound disclosed herein is provided to a human (e.g., by oral administration). Preferably, at least 10 mg / day of the compound is provided. In some embodiments, the compound disclosed herein is provided to a human at a dose of 0.1 mg / kg to 100 mg / kg. Such amounts of the compound are particularly useful when the compound is provided systemically (e.g., orally). One skilled in the art will recognize that smaller amounts can be used when administered locally (e.g., to skin, gums, wounds). The compositions disclosed herein are preferably provided for at least one week, or until symptoms are alleviated. Such compositions may be provided several times (e.g., once a week, once a month, twice a year, etc.), although prophylactic and therapeutic effects are observed after a single use.

[0126] In some embodiments, 1-50 g of a compound disclosed herein is administered to a cow, for example, for the treatment of mastitis. Preferably, at least 5 g of the compound is administered.

[0127] In some embodiments, compounds are provided that include a compound disclosed herein together with one or more additional agents, such as antibiotics (e.g., antibacterial, antiviral, antifungal), anti-inflammatory agents, antipyretics, and analgesics.

[0128] In some embodiments, the compounds disclosed herein are used with antibacterial agents, such as antifungals or antibiotics. Without wishing to be bound by theory, the present disclosure provides that the compounds disclosed herein target biofilms. The antibacterial agent can then exert its effect on the microbial cells in the disintegrated biofilm as well as on the remaining planktonic cells. As will be appreciated by those skilled in the art, the combination of the antibacterial agent with the compounds described herein can reduce the dosage and / or frequency of the antibacterial agent.

[0129] Exemplary antibacterial agents that may be used in combination therapy include antifungals such as miconazole, ketoconazole, econazole, terbinafine, ciclopirox, tolnaftate, sertaconazole, sulconazole, amphotericin b, chloroxylenol, clioquinol, butenafine, naftifine, nystatin, and clotrimazole. Exemplary antibiotics include penicillins, tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulfonamides, glycopeptides, aminoglycosides, and carbapenems.

[0130] The present disclosure provides compositions comprising a compound disclosed herein together with an antimicrobial agent. As will be appreciated by those skilled in the art, the compound and the antimicrobial agent can be provided separately. In some embodiments, the compound and the antimicrobial therapy overlap. In some embodiments, therapy with a compound of the present invention precedes the antimicrobial therapy.

[0131] In some embodiments, the compositions disclosed herein are provided as or in a food or functional food. The term "functional food" as used herein refers to foods that are prepared not only for their nutritional properties but also to perform a specific function, such as promoting health or reducing the risk of contracting a disease. Such functional foods may also be called dietary supplements or (animal) food additives. For this purpose, biologically active compounds such as minerals, vitamins, fatty acids, bacteria with beneficial effects, dietary fiber, and antioxidants are added thereto. Such foods may be in any form suitable for oral ingestion, for example, liquid, gel, powder, pill, tablet, or gel capsule.

[0132] Functional foods may also include animal digest, e.g., any material resulting from chemical and / or enzymatic hydrolysis of clean, undecomposed animal tissue. Functional foods may also include dried brewer's yeast, e.g., a dried inert body that is a by-product of the brewing industry. Animal digest and dried brewer's yeast have been found to increase the palatability of functional foods. When present in functional foods, the animal digest comprises about 10% to about 90% of the functional food, and the dried brewer's yeast comprises about 1% to about 30% of the functional food.

[0133] In some embodiments, the present disclosure provides compositions comprising the therapeutic organosulfur compounds disclosed herein together with at least one pharma- ceutically 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). As used herein, the term "pharma-ceutically acceptable" refers to those compositions, or combinations of agents, materials, or compositions, and / or dosage forms thereof, that are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problem or complication, and commensurate with a reasonable benefit / risk ratio. Furthermore, the term "pharma-ceutically acceptable diluent or carrier" refers to pharma-ceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, involved in carrying or transporting peptides from one organ or part of the body to another organ or part of the body.

[0134] The pharmaceutical composition can be administered by any suitable route and mode. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. The pharmaceutical composition can be formulated according to routine procedures for administration by any route, such as parenteral, topical (including intraocular), oral, sublingual, transdermal, or by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intracoronary, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and substernal injection and infusion. The preferred route is oral or topical administration.

[0135] The composition may be in any suitable form, such as liquid, semi-solid, and solid dosage forms. The composition may be in the form of a tablet, capsule, powder, granule, lozenge, cream, or liquid preparation (particularly for administration to the skin or eyes), such as a sterile parenteral solution or suspension, or a spray, aerosol, or other conventional method for inhalation. Pharmaceutical compositions of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. In certain embodiments, the composition is a topical composition in the form of a cream, gel, ointment, lotion, foam, suspension, spray, aerosol, or powder aerosol. This composition is particularly useful for administration to the skin. Suitable compositions also include oral care compositions, such as toothpaste, dentifrices, tooth powders, tooth gels, subgingival gels, mouth rinses / mouth washes, artificial saliva, denture products, mouth sprays, lozenges, oral tablets, and chewing gums.

[0136] The present disclosure also provides an in vitro use of the compositions disclosed herein for preventing or reducing the formation or growth of biofilm on a surface and / or for decomposing or reducing biofilm on a surface. Preferably, the method is for reducing biofilm or biofilm formation on a surface. In some embodiments, the method comprises contacting a biofilm attached to a surface with a composition disclosed herein. The composition comprises, for example, a cleaning composition. Compositions for cleaning (or rather removing or reducing) biofilm are known in the art and may comprise surfactants and enzymes (e.g., proteases and polysaccharidases).

[0137] Any surface can be treated with the compositions disclosed herein to coat such surfaces. The surface can be, for example, sprayed, dipped, or soaked into the composition. Some surfaces include glass, metal, porous, and non-porous surfaces. It also relates to the exterior and interior and surfaces of equipment that may be contaminated, such as those found in the food industry or medical equipment found in hospitals and medical facilities, as well as plumbing systems (e.g., sink drains), countertops, building materials, plumbing, and clean rooms. Some surfaces also refer to the interior or exterior of pipes, such as drains, as well as surfaces of swimming pools, aquariums (e.g., for aquaculture), purification filters, toilets, washbasins, and greenhouses. Some surfaces also include water, such as from drinking tubs.

[0138] In some embodiments, the surface is a surface of a medical device such as a prosthetic (hip implant, dental implant, artificial joint, voice prosthetic, penile prosthetic), mechanical heart valve, cardiac pacemaker, arteriovenous shunt, scleral buckle, catheter (e.g., central venous catheter, intravascular catheter, urinary catheter, Hickman catheter, peritoneal dialysis catheter, endotracheal catheter), tympanostomy tube, tracheostomy tube, surgical suture, bone anchor, bone screw, intraocular lens, contact lens, intrauterine contraceptive device, aortofemoral graft, or vascular graft. Other infections from medical devices include those from abdominal drains, biliary stents, breast implants, cardiac pacemakers, cerebrospinal fluid shunts, contact lenses, defibrillators, dentures, electrodialysis machines, endotracheal tubes, indwelling urinary catheters, intrauterine contraceptive devices, intravenous catheters, artificial joints, mechanical heart valves, nephrostomy tubes, orthopedic implants, peritoneal dialysis catheters, artificial heart valves, artificial joints, allosplastic orthopedic devices, tissue fillers, urethral stents, vascular grafts, ventilator-associated pneumonia, ventricular assist devices, ventricular derivations, ventricular shunts, and voice prostheses.

[0139] In some embodiments, the surface is the surface of a surgical instrument such as a clamp, forceps, scissors, skin hook, tube, needle, retractor, scaler, drill, chisel, file, or saw.

[0140] As used herein, the term "to comprise" and its conjugations are used in an open-ended sense, meaning that the items following the word are included, but items not specifically mentioned are not excluded. Additionally, the verb "to consist" can be replaced with "to consist essentially of," meaning that the compound or sub-compound defined herein may contain additional components than those specifically stated that do not alter the unique properties of the invention.

[0141] 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. By way of example, "an element" means one element or more than one element.

[0142] The words "approximately" or "about" when used in connection with a numerical value (such as approximately 10, about 10) preferably mean that the value may be 10% greater or less than the given value.

[0143] The compounds and compositions disclosed herein are useful as therapies and in therapeutic treatments, and thus are useful as medicaments and may be used in methods of preparing medicaments. In some embodiments, the disclosure provides methods that are not treatments of the human or animal body and / or that do not involve altering the genetic identity of the human germline, where the cells are not human germline.

[0144] All patent and literature references cited herein are hereby incorporated by reference in their entirety.

[0145] The present invention will be further described in the following examples, which are not intended to limit the scope of the invention but are merely intended to clarify the invention. EXAMPLES

[0146] Example 1 Chemical synthesis of thiosulfinates General procedures for the synthesis of symmetrical aliphatic thiosulfinates are well known (see, for example, J Am Chem Soc; 1947; 69; pp. 1710-1713). The reaction scheme is shown below, where in compound 1, both R are ethyl; in compound 2, both R are propyl; in compound 3, both R are butyl.

[0147] [ka]

[0148] Starting materials were obtained from the following suppliers: disulfides (diethyl disulfide, dipropyl disulfide, and dibutyl disulfide) were purchased from TCI, m-CPBA and magnesium sulfate from Sigma Aldrich, sodium hydrogen bicarbonate from Thermo Fisher, and dichloromethane from Rathburn.

[0149] The disulfide (16.36 mmol) was dissolved in dry dichloromethane (DCM, 30 mL) at -78 °C under N2 atmosphere, then meta-chloroperbenzoic acid (m-CPBA, 16.36 mmol) dissolved in dry DCM (30 mL) was added dropwise. Once the addition was complete, the reaction was allowed to stir for 3 h and slowly warmed to 0 °C. The reaction was quenched with saturated sodium bicarbonate and the resulting aqueous solution was extracted three times with DCM. The combined organic fractions were then dried over anhydrous MgSO4 and the solvent was evaporated under reduced pressure. The crude product was then purified by automated flash chromatography (40 g silica, 30:70% ethyl acetate:heptane, 20 min, 28 mL / min). The product was detected by thin layer chromatography (TLC) using KMnO4 stain. The product-containing fractions were combined and the solvent was evaporated in vacuo at 40 °C. 1 Pure products (compounds 1-3) were obtained as analyzed by H-NMR.

[0150] Compound 1: S-Ethyl ethanesulfinothioate (diethyl thiosulfinate) Yield: 54%, as a yellow oil; 1H-NMR δ (400 MHz, CDCl3): 3.25-3.05 (4H, m, O=SCH2, SCH2), 1.46 (3H, t, J = 7.2 Hz, O=SCH2CH3), 1.40 (3H, t, J = 7.6 Hz, SCH2CH3) ppm.

[0151] Compound 2: S-propylpropane-1-sulfinothioate (di-n-propylthiosulfinate) Yield: 49%, as a yellow oil; 1H-NMR δ (400 MHz, CDCl3): 3.18-3.00 (4H, m, O=SCH 2, SCH2), 1.90-1.72 (4H, m, O=SCH2CH2, SCH2CH2), 1.06 (3H, t, J = 7.2 Hz, (O=SCH2CH2CH3), 1.01 (3H, t, J = 7.6 Hz, SCH2CH2CH3) ppm.

[0152] Compound 3: S-Butyl butane-1-sulfinothioate (di-n-butyl thiosulfinate) Yield: 51%, as a yellow oil; 1H-NMR δ (400 MHz, CDCl3): 3.10-2.90 (4H, m, O=SCH2, SCH2), 1.75-1.58 (4H, m, O=SCH2CH2, SCH2CH2), 1.45-1.28 (4H, m, O=SCH2CH2CH2CH3, SCH2CH2CH2CH3), 0.84 (3H, t, J = 7.2 Hz, O=SCH2CH2CH2CH3), 0.81 (3H, t, J = 7.6 Hz, SCH2CH2CH2CH3) ppm.

[0153] Example 1b Experimental Design: Oxidation of disulfides leads to the formation of the corresponding thiosulfinates and thiosulfonates. Commercially available disulfides were used in the oxidation reactions, and mcpba (meta-chloroperbenzoic acid) was used as an oxidizing agent in organic synthesis, which leads to the formation of the corresponding thiosulfinates.

[0154] General procedure for the synthesis of symmetrical thiosulfinates (J Am Chem Soc; 1947; 69; pp. 1710-1713). The disulfide (16.36 mmol) was dissolved in dry DCM (=dichloromethane, 30 mL) at -78 °C under N2 atmosphere and m-CPBA (meta-chloroperbenzoic acid 16.36 mmol) dissolved in dry DCM (30 mL) was added dropwise. Once the addition was complete, the reaction was allowed to stir for 3 h and slowly warmed to 0 °C. The reaction was quenched with saturated sodium bicarbonate and the resulting aqueous solution was extracted three times with DCM. The combined organic fractions were subsequently dried over anhydrous MgSO4 and the solvent was evaporated under reduced pressure. The crude product was then purified by autoflash (supplier: Teledyne Isco). The product was detected by thin layer chromatography (TLC) using KMnO4 staining. The product containing fractions were combined and the solvent was evaporated in vacuo at 40° C. 1Pure products were obtained that were analyzed by H-NMR. Note: For aliphatic, F-aryl, and heterocyclic analogs, take special care when evaporating the solvent. Keep the temperature low (around 25°C) to prevent decomposition or evaporation due to volatility.

[0155] General procedure for the synthesis of symmetrical thiosulfonates (Chem. Eur. J. 2015, 21, 8105). At 30°C, the disulfide (0.012 mol) was added to acetic acid (2.34 ml), followed by hydrogen peroxide (30% Wt, 10.89 ml) over approximately 5 min. The temperature was raised to 55°C and the reaction was stirred at this temperature for approximately 4 h. The reaction was cooled to room temperature and treated with 5 M NaOH until the pH was basic. The raw material was then extracted three times with DCM. The combined organic extracts were dried over anhydrous MgSO4 and the solvent was evaporated under reduced pressure to give the crude mixture. Purification was then performed by automated flash (Teledyne ISCO) (tables 1 and 2). The product was detected by TLC using KMnO4 staining, and the fractions containing the product were combined and the solvent was evaporated in vacuo. 1 A pure product was obtained which was analyzed by H-NMR. Note: For aliphatic and heterocyclic, the reaction is set to 40°C after adding the oxidizing agent.

[0156] S-Propyl-4-methylbenzenethiosulfonate was prepared according to Angew.Chem. Int. Ed., 2018, 57, pp. 12290-12293.

[0157] Results. When hydrogen peroxide was used as oxidizing agent, the corresponding thiosulfonates were formed in the presence of acetic acid. Most of the compounds were synthesized under standard reaction conditions with good to moderate yields as summarized in the overview (Table 1). Some, especially aliphatic and heterocyclic, required milder reaction conditions in thiosulfonation to obtain reaction products compared to aryl substituted analogues. The benzyl substituted analogue was more reactive compared to other aryl substituted analogues, which may be the reason for the low reaction products under the attempted reaction conditions. The heterocyclic analogue (dibenzothiazolyl disulfide) did not undergo reaction due to solubility issues and produced tars with insoluble solids. The reaction of its thiosulfinate was attempted in two different solvents (dichloromethane and chloroform) with the same results. The reaction of the corresponding thiosulfonate was attempted at elevated temperature but was also unsuccessful. The oxidation of furfuryl disulfide was also difficult due to instability. It gave low amounts of thiosulfinate, but the reaction of the corresponding thiosulfonate produced tars. The synthesized F-aryl thiosulfinates / thiosulfonates were first purified using reverse phase chromatography and analyzed by 1H NMR (tables 1 and 2).

[0158] The compounds were not stable at high temperatures and decomposed during work-up, therefore they were resynthesized and purified by silica chromatography.

[0159] [Table 1A]

[0160] [Table 1B]

[0161] [Table 2]

[0162] Example 2 MIC and biofilm assays The MIC assay in this example measures the growth inhibition of planktonic microorganisms. As shown herein, several of the compounds tested showed efficacy against planktonic bacteria.

[0163] As will be appreciated by those skilled in the art, the effect of a compound on planktonic microorganisms is not indicative of the effect of such a compound on biofilms. This has been demonstrated with several antimicrobial agents as previously discussed herein and is well known in the literature. See, for example, Roy et al. (2018 Virulence 9:522-554), who show that bacteria in biofilms are approximately 1000 times more resistant to conventional antibiotics.

[0164] In contrast to studies on planktonic microorganisms, the biofilm eradication assays of this example demonstrate the effect of compounds on biofilms.

[0165] The primary mechanism of action of compounds in biofilm assays is believed to involve inhibition of the collective genetic switches that lead to quorum sensing and subsequent bacterial adhesion, or the synthesis or assembly of the extracellular matrix that forms the three-dimensional structure of the biofilm. These mechanisms are completely distinct from a pure antibacterial / antimicrobial "killing" effect.

[0166] If a compound is effective in biofilm assay but does not have antibacterial effect, the compound can be combined with antibiotics to ensure that bacteria released from biofilm are killed.This is especially true when the immune system of the animal is compromised.However, such compounds are also particularly useful for specifically targeting biofilms, while having little or no effect on, for example, healthy gut flora.Such compounds are particularly preferred.

[0167] [Table 3]

[0168] 1.1 Compound dissolution and dilution Into an Eppendorf tube, 10-20 mg of the compound shown in table 3 was weighed. The exact weight was recorded and the volume of solvent was calculated to reach an 80 mM dilution. First, half the volume was added by adding Tween 80. In the second step, another half volume of DMSO was added and the capped tube was inverted 5-10 times and then thoroughly vortexed for at least 30 seconds. If undissolved material was still visible after this procedure, the tube was placed on a shaker at 37°C and 150 rpm for at least 30 minutes. The dissolved and clear dilution was at a concentration of 80 mM and was diluted 10-fold in cation-adjusted Mueller Hinton Broth 2 (MHB-II) for the MIC assay (Method 1.2). For the MBEC assay (Method 1.3), the solution needs to be diluted in 0.9% saline to a final concentration of 2 mM, then further diluted in saline to 1 mM, 0.5 mM, 0.25 mM, and 0.125 mM. These dilutions were added as treatments to 96-well plates containing grown biofilms (see Method 3).

[0169] 1.2 MIC assay Frozen aliquots of Staphylococcus epidermis ATCC 35984 and Pseudomonas aeruginosa ATCC 27853 were used to inoculate tryptic soy agar (TSA) plates and incubated overnight at 37°C. After incubation, 3–5 well-separated colonies of the same morphology were selected from the TSA plates, resuspended in 2 mL of 0.9% saline with 3–6 glass beads, and vortexed. The optical density at 600 nm (OD) of the bacterial suspension was measured. 600 ) was measured using a spectrophotometer (Evolution (registered trademark) 201 / 220 UV-Visible Spectrophotometer, Thermo Fisher Scientific Co., Ltd.) and 6 OD of 0.0008 similar to CFU / mL 600 was diluted to .

[0170] MIC assays were performed in flat-bottom 96-well microtiter plates. 50 μL of MHB-II was added to columns 2-11, and 100 μL was added to column 12 as a sterility control. 100 μL of 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 μL of the 8 mM solution from column 1 in column 2. This process was repeated up to column 10. The respective concentrations of Tween 80 and DMSO in column 1 were 2.5%, which were diluted two-fold across the plate. 50 μL of the bacterial suspension was added to columns 1-11. The microtiter plate was sealed with adhesive polyethylene film for sealing microplates (Diversified Biotech) and incubated at 37° C. for 24 hours. After incubation, OD was measured at 600 nm using a Varioskan (Thermo Fisher Scientific). The minimal concentrations that reduced the final OD600 of the bacterial culture by 50% (MIC50) and 90% (MIC90) were calculated after normalizing the obtained OD values ​​to the growth control in column 11. The assay was repeated three times in three independent experiments.

[0171] 1.3 MBEC assay Frozen aliquots of Staphylococcus epidermidis ATCC 35984 and Pseudomonas aeruginosa ATCC 27853 were used to inoculate 20 mL of tryptic soy broth (TSB) in 100 mL Erlenmeyer flasks and incubated overnight at 37°C and 150 rpm (Infors). The OD of the overnight cultures was 600 was measured using a spectrophotometer (Evolution (registered trademark) 201 / 220 UV-Visible Spectrophotometer, Thermo Fisher Scientific Co., Ltd.) and 8 OD of 0.2 similar to CFU / mL 600 The OD was measured by pipetting 100 µL per well in columns 1-11. 600A cell suspension of 0.2 was seeded in a U-bottom 96-well plate, and column 12 was filled with 100 μL of medium as a sterility control. The 96-well plate was sealed with an adhesive polyethylene film for sealing microplates and incubated at 37°C for 48 hours in a stationary state. After the biofilm growth phase, 100 μL of treatment solution (method 1) was added to the vials to give treatment concentrations of 1 mM, 0.5 mM, 0.25 mM, and 0.125 mM, with Tween 80 and DMSO concentrations of 0.63%, 0.32%, 0.16%, and 0.08%, respectively. The same concentrations were applied to four different wells of the same bacterial strain, resulting in four replicates for each concentration and strain. The treatments were incubated at 37°C for 20 hours in a stationary state. After the treatments, the supernatant was carefully removed and the wells with the biofilms were carefully washed once with 200 μL of 0.9% saline. 200 μL of saline was added and the biofilm was thoroughly resuspended. After thorough homogenization, 20 μL was used for the first dilution in 180 μL of 0.9% saline, which was prepared in a second plate. This dilution was continued up to a dilution step of 10E-5, and 100 μL of the 10E-4 and 10E-5 dilutions were plated on TSA plates. The TSA plates were incubated at 37° C. for 24 hours. The colonies on the plates were counted and the colony forming units per mL of dispensed biofilm and their average value, as well as the log reduction compared to untreated samples from the same well plate, were calculated. The reduction in colony forming units occurs in relation to the thickness of the biofilm and therefore the number of bacteria present in the biofilm. Therefore, a significant reduction in colony forming units of the biofilm was defined as a log reduction value of ≧1. The assay was repeated twice in two independent experiments.

[0172] 1.4 MBIC assay Frozen aliquots of Staphylococcus epidermidis ATCC 35984 and Pseudomonas aeruginosa ATCC 27853 were used to inoculate tryptic soy agar (TSA) plates and incubated overnight at 37°C. After incubation, 3–5 well-separated colonies with the same morphology were selected from the TSA plates, resuspended in 2 mL of 0.9% saline with 3–6 glass beads, and vortexed. The optical density of the bacterial suspension was measured at 600 nm (OD 600 ) and measured with a spectrophotometer (Evolution® 201 / 220 UV-Visible Spectrophotometer, Thermo Fisher Scientific) to obtain an OD of 0.2. 600 The MBIC assay was performed in a 96-well microtiter plate. 50 μL of TSB was added to columns 2-11, and 100 μL was added to column 12 as a sterility control. 100 μL of 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 μL of the 8 mM solution from column 1 in column 2. This process was repeated up to column 10. The respective concentrations of Tween 80 and DMSO in column 1 were 2.5%, which were diluted two-fold across the plate. 50 μL of the bacterial suspension was added to columns 1-11. The microtiter plate was sealed with adhesive polyethylene film for sealing microplates (Diversified Biotech) and incubated at 37°C for 48 hours. After incubation, the supernatant was carefully removed and the wells containing the biofilms were washed once with 100 μL of 0.9% saline. 100 μL of 0.1 M HCl was added and incubated at room temperature for 1 h to fix the biofilm. After incubation, HCl was removed and 100 μL of crystal violet (0.1% v / v in water) was added and incubated at room temperature for 30 min. Unbound crystal violet was removed and wells were washed once with 100 μL of demineralized water. 100 μL of 30% acetic acid was added and incubated at 37° C. and 150 rpm for 1 h. The solution was resuspended and transferred to a flat-bottom 96-well plate and the absorbance was measured in a spectrophotometer at 540 nm. The assay was repeated twice in two independent experiments.

[0173] Results and Discussion. Table 4 shows the results of the study.

[0174] When biofilms are eradicated or decomposed, microorganisms are released into the environment. When biofilms are present in vivo, for example in humans or animals, the released microorganisms may be attacked by the immune system. The advantage of this in vitro system is that it gives the opportunity to investigate the effect of drugs on biofilms and their respective microorganisms without adding the influence of the environment (e.g., immune system).

[0175] In table 4, several organosulfur compounds showed low MIC values, while their MBEC values ​​were high. Table 4 also shows some compounds with high MIC values ​​and very low MBEC values, which 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, these compounds have a strong effect on biofilms.

[0176] [Table 4A]

[0177] [Table 4B]

[0178] Example 3 In vivo test - Mastitis To demonstrate the in vivo efficacy of the compounds disclosed herein, several organosulfur-containing compositions were tested in vivo against several biofilm-associated disorders. Bovine mastitis is a disease that affects millions of cows worldwide each year. The disease is caused by a variety of widely different bacteria, and in some cases even yeasts, that invade the mammary gland of lactating cows, causing persistent infection and inflammatory responses. Bovine mastitis can be caused by a variety of gram-positive and gram-negative pathogens, and the prevalence of individual pathogens can vary to some extent between countries and continents. The most prominent clinical sign of all forms of mastitis is an undesirable increase in the number of somatic cells in milk intended for human consumption, caused by a (chronic) inflammatory response in the mammary tissue. This secondary inflammation reduces milk production and therefore causes serious economic losses for farmers. Over the last few decades, numerous pharmaceutical products, including different classes of antibiotics, have been developed and administered alone or in combination to treat the main infectious agents of mastitis. These pharmaceutical products are administered systemically (by injection) or locally via the teat channel to combat bacterial infections, but despite these extensive efforts, bovine mastitis remains the most prevalent disease in dairy cows, antibiotic therapy is generally only temporarily effective, and in many cases somatic cell counts (SCC), a clinical marker of mastitis, remain elevated.

[0179] Mastitis is known as a biofilm-associated disorder. The formation of bacterial biofilms has been demonstrated in in vitro experiments where mastitis pathogens are cultured under conditions that favor bacterial biofilm formation (quantifiable after staining, by measuring genes that drive biofilm formation) and in situ by staining the biofilm matrix in infected bovine mammary tissue. See, for example, Figure 1 in Schonborn S and Kromker V (2016 Journal Veterinary Microbiology, 30;196:126-128), which shows the biofilm matrix from mammary tissue of a bovine mastitis.

[0180] Biomedical research in the last decade has revealed that biofilm formation does not occur only in cases of udder infections and bovine mastitis, but is a general feature of microorganisms that invade human and animal tissues and cause persistent infections and inflammation. To date, microbial biofilms are one of the main unsolved problems in modern therapy of human and animal infectious diseases. Clinical evidence provided in the examples below reveals the unexpected therapeutic effect of several organosulfur-containing compositions in the treatment of important biofilm diseases such as bovine mastitis and infected chronic wounds (e.g., UCD). Several field experiments are described that demonstrate that application of the compounds is directly associated with a long-term stable reduction in somatic cell counts in treated cows, suggesting bacterial healing and tissue regeneration. Further examples describe the effect on biofilm-associated chronic wounds (i.e., Udder Cleft Dermatitis (UCD)). These findings are noteworthy because it is generally recognized that common antibiotics are not effective against biofilm infections. These observations on antibiotics are documented in numerous scientific papers in human and veterinary medicine, all of which suggest that biofilm formation is a major cause of recurrent and persistent infections, as well as tissue inflammation and damage. The presented experimental (in vitro) and in vivo (clinical observations) efficacy of the compounds described herein against prototypic biofilm infections demonstrates that these compounds can be effectively used in clinical practice to prevent and resolve biofilm infections. Biofilm formation and resolution occurs as a direct molecule-microorganism interaction and is therefore host (animal or human) independent. As the mechanisms involved in biofilm formation are highly conserved among bacterial species, it is also presumed that the compounds will be effective against a wide range of biofilm infections in humans and animals. The examples also demonstrate that the compounds can be administered orally and exert their effects at different sites (e.g., mammary gland and UCD).

[0181] range Experiments were conducted to examine the effect of compounds on cell counts or somatic cell counts (SCC) in milk. The compounds tested and the total dosage administered are shown in Table 5.

[0182] Somatic cell count (SCC) is related to the amount of pathogens in a quarter that the immune system can recognize. The cell count of each cow is measured at the milk production registration (MPR), which is done regularly every 30-40 days on every farm. The SCC is the main indicator of milk quality. The majority of somatic cells are leukocytes (white blood cells) that become more and more present in milk, usually as an immune response to pathogens that cause mastitis, and a small number are epithelial cells, which are milk-producing cells that are shed from the inside of the udder when an infection occurs.

[0183] Inflammation often occurs in one of the quarters of the udder, resulting in an increased cell count in this udder. However, due to dilution in the other quarters of the udder, the overall increase in cell count is lower. SCC is quantified as the number of cells per ml of milk. SCC indicates the presence of (subclinical) udder infection, e.g. with pathogens causing mastitis, and is a main indicator of milk quality. The relationship between SCC and mastitis is reviewed in Sharma et al., 2011 Asian-Aust J Anim Sci 24:429-438.

[0184] Data on SCC were extracted from the Milk Production Register (MPR). The MPR is a database showing all details (e.g. SCC) of milk produced by each individual cow. The MPR for selected farms was available on a regular basis. SCC is quantified as the number of cells per ml of milk. In general, individual cows with SCC values ​​below 100,000 indicate "uninfected" cows, without significant production losses due to subclinical mastitis. A threshold SCC above 250,000 indicates infection, while numbers between 100,000 and 250,000 indicate a high risk of infection.

[0185] [Table 5]

[0186] Study design The experiment was carried out on a farm with 90 cows. The tablets were used to test for cell counts for 60 days.

[0187] For administration of one tablet, cows with a SCC above 250.000 cells / ml before treatment were selected.

[0188] The milk somatic cell counts and mean somatic cell counts of the cows are shown in Figure 1. The SCC of the reference cows remained relatively high (see Figure 1; compare approximately 673 on day 0 to 587 on day 60). In contrast, SCC was significantly reduced in cows receiving a single treatment of di-n-propylthiosulfinate, di-methylthiosulfonate, or di-phenylthiosulfonate. Since clinically significant effects were observed in mammary tissue, this suggests that the compounds not only cross the rumen (indicating stability to ruminal microorganisms), but also reach the post hepatic blood stream (indicating minor or insignificant hepatic biotransformation and inactivation), ultimately reaching the mammary gland in therapeutically effective concentrations.

[0189] Example 4 In vivo test - Mammary cleft dermatitis One classification of chronic infections is wounds infected with biofilm-forming microorganisms. When a wound becomes infected, the microorganisms begin to form a biofilm that remains attached to the wound. The production of microbial EPS (extracellular polymeric substances) helps the biofilm to form complex three-dimensional structures within hours. These complex structures are resistant to the defense mechanisms of the wound. When antibiotics are applied to attack the bacteria, it may only partially eradicate the biofilm, and the wound and underlying tissues may remain infected. These biofilms are known to result in chronic infections and non-healing wounds. Approximately 16 million new cases of biofilm-based infections are diagnosed each year in the United States. Hence, biofilms are a major barrier to wound healing. Examples of pathogenic microorganisms that infect such wounds are bacteria (Gram-positive bacteria, e.g., Staphylococcus aureus; Streptococcus; Gram-negative bacteria, e.g., Treponema spp., Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa; yeasts / fungi, e.g., Candida spp. (albicans spp.), Cladosporium herbarium, Trichosporon spp., Rhodosporidium spp., and Malassezia spp.).

[0190] An example of digital dermatitis is clawed digital dermatitis (synonyms are hairy heel wart, strawberry foot rot, mortellaro disease, Italian foot rot, and papillomatous digital dermatitis), an infection that causes lameness in cattle.

[0191] An example of a biofilm-associated disorder is uncut udder dermatitis (UCD), which is associated with chronic wounds. In this example, di-alkyl thiosulfonates, di-alkyl thiosulfinates, and di-alkyl disulfides were tested for their effect on cows with UCD.

[0192] UCD is a skin lesion located at the anterior junction between the udder and abdominal wall or the front quarters of the udder. The lesions may vary in appearance and size, but thickened skin, crusts, pus, and wounds that bleed easily are common findings. Udder cleft dermatitis can be difficult to detect due to its anatomical location and the fact that affected cows rarely show general signs of the disease. Few studies on UCD prevalence have been published, and most of them include only one or a few herds, which are primarily classified as problematic herds. Within-herd prevalence in these studies varied between 0 and 22%. However, a recent Dutch study included 20 herds, of which three were not UCD, while within-herd prevalence in the other herds varied between 2.5 and 13% (Amersfort et al., 2012). The cause of UCD is unknown, but several factors such as udder morphology and udder edema have been suggested to play a role. Bovine factors such as parity and DIM (days in milk) are also associated with UCD (Beattie and Taylor, 2000, J. Brit. Cattle Vet. Assoc. 8, 377-380).

[0193] Lesions are most commonly identified on the planter aspect of the interdigital cleft of the hind limbs. Treponema species are routinely present in numerous active lesions. Lesions are tender to the touch and clinical lameness may result. The nature of this infection generally leads to endemic infection of cattle herds and causes significant economic losses.

[0194] Study design Experiments were conducted to investigate the effect of various propellants containing water (control), test compound, or a reference compound (CTC propellant containing chlortetracycline hydrochloride, obtained from Dechra veterinary products).

[0195] 0.35 mL of spray was sprayed into the mammary cleft per treatment. Treatments were administered once daily at t=0, t=3 days, and t=6 days. The results of these experiments are shown in Table 7. Table 6 describes the evaluation qualifications used in Table 7.

[0196] [Table 6]

[0197] As shown in Table 7 below, a rapid reduction in inflammation was observed immediately after treatment with di-n-propyl disulfide, di-n-propyl thiosulfinate, di-n-butyl thiosulfinate, di-methyl thiosulfonate, di-n-propyl thiosulfonate (PTSO), and S-di-phenyl thiosulfonate, followed by a reduction in the intensity of infection and wound healing.

[0198] In contrast, the control treatment with clean water did not improve the infection and the wounds became even larger. The reference treatment with chlortetracycline hydrochloride-based CTC spray did not show any effect on the wounds.

[0199] In summary, this example clearly demonstrates that the claimed compounds are capable of treating biofilm-associated disorders.

[0200] [Table 7A]

[0201] [Table 7B]

[0202] [Table 7C]

Claims

1. A compound according to formula I, or a composition comprising a compound according to formula I, for use in the treatment of a biofilm-associated disorder: 【Chemical 1】 (wherein n is 2, One X is -S- and the other X is -S(O)- and -S(O) 2 - selected from the group consisting of; R 1 and R 2 are independently selected from optionally substituted alkyl or optionally substituted aryl).

2. 2. The compound or composition for use according to claim 1, wherein the biofilm-associated disorder is a chronic infection and / or a persistent infection.

3. 2. The compound or composition for use according to claim 1, wherein the biofilm-associated disorder is digital dermatitis, breast clefts, or chronic wound infection.

4. 2. The compound or composition for use according to claim 1, wherein the biofilm comprises bacteria, yeast, fungi, microalgae, or a combination thereof.

5. A compound according to formula I, or a composition comprising a compound according to formula I, for use in the treatment or prevention of a microbial infection, including a bacterial or fungal infection: 【Chemistry 2】 (wherein n is 2, One X is -S- and the other X is -S(O)- and -S(O) 2 - selected from the group consisting of; R 1 and R 2 are independently selected from optionally substituted alkyl or optionally substituted aryl).

6. 10. The compound or composition for use according to claim 1 or 5, wherein the use further comprises the administration of an antibacterial agent selected from an antifungal agent or an antibiotic.

7. 10. The compound or composition for use according to claim 1 or 5, wherein the use further comprises the administration of an anti-inflammatory agent.

8. Compounds according to formula I: 【Chemistry 3】 (wherein n is 2, One X is -S- and the other X is -S(O)- and -S(O) 2 - selected from the group consisting of; R 1 and R 2 is independently selected from optionally substituted alkyl or optionally substituted aryl; The composition is a pharmaceutical composition, a functional food, or a cleaning product.

9. 1. An in vitro method for preventing or reducing the formation or growth of a biofilm on a surface, or for degrading or reducing a biofilm on a surface, comprising: The method includes: or applying a composition to a surface to degrade or reduce biofilm on the surface, said composition comprising a compound according to formula I: 【Chemistry 4】 (wherein n is 2, One X is -S- and the other X is -S(O)- and -S(O) 2 - selected from the group consisting of; R 1 and R 2 are independently selected from optionally substituted alkyl or optionally substituted aryl.

10. 10. The compound or composition for use or composition according to claim 1, 5 or 8, wherein the composition is essentially free of diallyl thiosulfinate.

11. 10. The compound or composition for use or composition according to claim 1, 5 or 8, wherein the composition further comprises an antibacterial agent selected from an antifungal or an antibiotic.

12. 10. The compound or composition for use or composition according to claim 1, 5 or 8, wherein the composition further comprises an anti-inflammatory agent.

13. Compounds according to formula I: 【Chemistry 5】 (wherein n is 2, One X is -S- and the other X is -S(O)- and -S(O) 2 - selected from the group consisting of; R 1 and R 2 is independently selected from optionally substituted alkyl or optionally substituted aryl; An article that is a medical device or surgical instrument.

14. R 1 and R 2 14. The compound or composition, article, or composition for use of claim 1, 5, 8, or 13, wherein is independently selected from alkyl, aryl, alkylaryl, and arylalkyl.

15. R 1 and R 2 But independently, C 1-6 14. The compound or composition, article, or composition for use according to claim 1, 5, 8 or 13, wherein said alkyl is selected from alkyl and phenyl.

16. 14. The compound or composition for use, article, or composition according to claim 1, 5, 8, or 13, wherein the compound according to formula I is not di-n-propyl thiosulfinate or di-n-propyl thiosulfonate.

17. When the other X is -S(O)-, R 1 and R 2 are independently selected from the group consisting of isopropyl, butyl, benzyl, and p-tolyl; and the other X is -S(O) 2 -When R 1 and R 2 is independently selected from the group consisting of methyl, phenyl, benzyl, 4-methylbenzene, and n-propyl.

18. 14. The compound or composition, article, or composition for use according to claim 1, 5, 8, or 13, wherein 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, di-isopropyl thiosulfinate, di-benzyl thiosulfinate, di-benzyl thiosulfonate, S-propyl-4-methylbenzenethiosulfonate, di-n-propyl thiosulfinate, and di-n-propyl thiosulfonate.

19. R 1 and R 2 14. The compound or composition, article, or composition for use according to claim 1, 5, 8, or 13, wherein:

20. 14. The compound or composition, article, or composition for use according to claim 1, 5, 8, or 13, wherein 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-methylbenzenethiosulfonate, di-isopropyl thiosulfinate, di-benzyl thiosulfinate, and di-benzyl thiosulfonate.

21. 21. The compound or composition, article, or composition for use according to claim 20, wherein the compound according to formula I is selected from the group consisting of di-n-butyl thiosulfinate, di-methyl thiosulfonate, and di-phenyl thiosulfonate.

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