Novel efficient antibacterial composition

Abstract

The invention relates to a novel efficient cooperative or synergistic antibacterial composition and application thereof. Medical devices, biomaterial implants or bioprostheses comprising a novel, highly effective antibacterial composition incorporated with a coating or integrally distributed.

Description

[0001] This application is a divisional application of application number 202480035088.2, filed on August 9, 2024, entitled "Novel Highly Effective Antibacterial Composition". Technical Field

[0002] This invention relates to a novel, highly effective synergistic or cooperative antimicrobial composition and its application, particularly for the treatment or prevention of infections or inflammation in humans or animals, and more specifically for use on the skin of humans or animals.

[0003] The present invention also relates to a medical device, biomaterial implant or bioprosthetic comprising a novel, highly effective antibacterial composition incorporated in or integrally distributed within a coating.

[0004] The present invention also relates to a method for killing or preventing the growth of microorganisms on a surface, particularly on a medical device, and more specifically on a catheter. Background Technology

[0005] Antimicrobial compositions are used to combat pathogenic microorganisms, including bacteria, viruses, or fungi, or mixtures thereof, to prevent or treat various infections and diseases in a host animal (humans and animals).

[0006] However, microorganisms are becoming increasingly resistant to antimicrobial compositions used as antibacterial or antifungal agents.

[0007] For example, bacteria are developing resistance to antibacterial agents belonging to the classes of penicillins, methicillins, carbapenems, cephalosporins, quinolones, aminoglycosides, and glycopeptides, and an increasing number of infections are becoming difficult to cure.

[0008] In particular, coagulase-negative staphylococci (CoNS) are the most common bacteria in the normal skin flora. These bacteria are common contaminants in clinical specimens and are recognized as pathogens of clinically significant infections, including bacteremia and endocarditis. Patients particularly susceptible to CoNS infection include those with prosthetic devices, pacemakers, intravascular catheters, and immunocompromised hosts.

[0009] In intensive care units, coagulase-negative staphylococci account for about one-third of bloodstream isolates, making these organisms the most common cause of bloodstream infections in hospitals.

[0010] Enterococci can cause a variety of infections, including urinary tract infections, bacteremia, endocarditis, and meningitis. Enterococci are relatively resistant to cell wall activators (penicillin, ampicillin, and vancomycin) and do not readily penetrate aminoglycosides.

[0011] Vancomycin-resistant enterococci (VRE) are an increasingly common and difficult-to-treat cause of hospital-acquired infections.

[0012] Multiple VRE infection outbreaks have been described in different hospital settings (e.g., internal medicine and surgical intensive care units, and internal medicine and pediatric wards), and like methicillin-resistant Staphylococcus aureus, VRE is endemic in many large hospitals.

[0013] Beyond human medicine, pets (such as cats, dogs, and horses) can also be colonized and infected by microorganisms such as MRSA (methicillin-resistant substituents) without host adaptation, thus potentially serving as reservoirs for human infections. Bacteria can also develop unique drug resistance within animals.

[0014] Fungal pathogens such as Candida albicans, Aspergillus fumigatus, and Cryptosporidium neoformans also develop resistance to antifungal agents.

[0015] In particular, Candida albicans is inherently resistant to most known antifungal drugs, including various azole derivatives (fluconazole, isavuconazile, itraconazole, posaconazole, voriconazole), but also polyenes such as amphotericin B, and even chlorhexidine (CHX), a biguanide antifungal agent with both antifungal and antibacterial activity.

[0016] Studies have reported that chlorhexidine exhibits resistance in different bacterial species when used at low concentrations.

[0017] Furthermore, chlorhexidine is insoluble in water and needs to be formulated with gluconic acid or acetic acid to form water-soluble digluconate or diacetate. Chlorhexidine also requires high concentrations to be effective and may cause skin irritation or allergic reactions in humans or animals.

[0018] Chlorhexidine may be ineffective against some microorganisms such as Gram-negative bacteria or fungi, and may not kill microorganisms rapidly, as shown in the table below, published by TM KARPINSKI and AK SZKARADKIEWICZ in European Review for Medical and Pharmacological Sciences 2015; 19:1321-1326.

[0019] Table 1: Minimum Inhibitory Concentration (MIC) of Chlorhexidine against Various Microorganisms

[0020] As reported in EP2968677B1, treatment of medical devices with chlorhexidine may cause allergic reactions, including life-threatening ones.

[0021] Despite these drawbacks, CHX has been and continues to be widely used for the disinfection of medical device surfaces and for direct application to human and animal skin. However, there is a need for a highly effective antimicrobial composition with broad-spectrum activity and lower toxicity to humans or animals.

[0022] Wound care requires highly effective antibacterial compositions to prevent or treat wound infections and promote wound healing.

[0023] Antimicrobial compositions can be applied to the skin of humans or animals for many reasons, including after surgery, injury, burns, ulcers, and mucous membranes.

[0024] Antimicrobial compositions can also be applied to the surface of medical devices, biomaterial implants, or bioprosthetics to prevent infection during or after insertion into a human or animal.

[0025] Preventive measures may include coating medical devices, biomaterial implants, or bioprosthetics with an antimicrobial coating containing sufficient antimicrobial agent to maintain adequate antimicrobial efficacy during implantation or insertion into a human or animal.

[0026] In 2017, C. Oury and P. Lancellotti described a novel use of triazolo(4,5-d)pyrimidine derivatives for the prevention and treatment of bacterial infections in EP3509598B1. However, triazolo(4,5-d)pyrimidine derivatives such as triafluocyl or fluometacyl are generally insoluble in aqueous environments or aqueous media and require high concentrations to achieve highly effective antibacterial or bactericidal effects.

[0027] The increasing resistance of microorganisms to antimicrobial agents has created a growing demand for novel, highly effective antimicrobial agents that possess both antimicrobial efficacy and resistance to antimicrobial resistance. Since traditional "single-target-single-molecule" approaches are insufficient to meet this need, alternative approaches such as multi-target antimicrobial agents are required. Summary of the Invention

[0028] Surprisingly, triazolo(4,5-d)pyrimidine derivatives, when combined with biguanides, particularly chlorhexidine and alexiidine, provided synergistic antibacterial activity even at low concentrations.

[0029] The object of this invention is to provide a highly effective synergistic or co-operative antimicrobial composition and its application, particularly its use in the prevention or treatment of microbial infections or inflammation on the skin of humans or animals, more specifically. The synergistic or co-operative antimicrobial composition exhibits broad-spectrum activity at low concentrations, reduces allergic reactions, and effectively kills pathogenic microorganisms on surfaces and human or animal skin, particularly Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa. The synergistic or co-operative antimicrobial composition advantageously has a lower risk of antimicrobial resistance.

[0030] A further objective of this invention is to provide a medical device, biomaterial implant, or bioprosthetic comprising the aforementioned cooperative or synergistic antimicrobial composition, and a method thereof for coating the same. The coating method includes applying the antimicrobial composition to at least a portion of the medical device, biomaterial implant, or bioprosthetic, whether as a coating or integrally distributed.

[0031] The present invention also aims to provide a medical device, biomaterial implant or bioprosthetic with antibacterial properties for the prevention or treatment of microbial infections or inflammation on the skin of humans or animals, especially humans or animals.

[0032] Finally, the present invention also relates to a coating of a medical device, biomaterial implant or bioprosthetic comprising the antimicrobial composition, and a wound dressing comprising the antimicrobial composition and its use in the prevention or treatment of microbial infections or inflammation in humans or animals. Detailed Implementation

[0033] According to a first aspect of the present invention, an antibacterial composition is provided, comprising a synergistic or cooperative combination of the following substances: Triazolo(4,5-d)pyrimidine derivatives of formula (I) (I) Where R 1 It is a C that is optionally substituted with one or more halogen atoms. 3-5 Alkyl; R 2 It is a phenyl group that is optionally substituted with one or more halogen atoms; R 3 and R 4 All are hydroxyl groups; R is XOH, where X is CH2, OCH2CH2 or a bond; Or a pharmaceutically acceptable salt or solvate thereof, or a solvate thereof, provided that when X is CH2 or a bond, R 1 Not propyl; when X is CH2 and R 1 When it is CH2CH2CF3, butyl, or pentyl, R 2 The phenyl group at the position must be replaced by fluorine; when X is OCH2CH2 and R 1 When it is propyl, R 2 The phenyl group at the position must be replaced by fluorine; And biguanides, preferably biguanides selected from the group consisting of chlorhexidine, alexiidine or polyhexamethylene biguanide; Used for the prevention or treatment of infections or inflammations on the skin of humans or animals, especially humans or animals.

[0034] As used herein, the term "derivative" refers to structurally similar triazolo(4,5-d)pyrimidines that exhibit the same functional properties as the identified analogues. The derivatives can be structurally similar by the absence of one or more atoms or by substitution with one or more chemical groups.

[0035] The term “synergy” as used in this article refers to a combination of at least two antimicrobial compounds that produces a combined antimicrobial effect greater than the sum of their individual antimicrobial effects.

[0036] The biguanide or HN(C(NH)NH2)2 used in this article refers to... ; The biguanides used in this article refer to chlorhexidine, alexiidine, and polyhexamethylene biguanide; The chlorhexidine used in this article refers to chlorhexidine base.

[0037] However, it can also refer to chlorhexidine salts, such as chlorhexidine diphosphate aniline salt, chlorhexidine digluconate, chlorhexidine diacetate, chlorhexidine dinitrate, chlorhexidine dihydrochloride, chlorhexidine dichloride, chlorhexidine acetate, chlorhexidine dipropionate, chlorhexidine maleate, chlorhexidine succinate, chlorhexidine thiosulfate, chlorhexidine diphosphate, chlorhexidine malate, chlorhexidine dibenzoate, chlorhexidine diisophthalate, chlorhexidine dilaurate, chlorhexidine distearate, etc.

[0038] The alexidin used in this article refers to alexidin base. ; However, it can also refer to alexidine hydrochloride, alexidine dihydrochloride, alexidine monoacetate, alexidine diacetate, alexidine gluconate, alexidine digluconate, and mixtures thereof.

[0039] The antimicrobial compositions of the present invention are used to combat pathogenic microorganisms, including bacteria, viruses, archaea, protozoa, yeasts, or fungi, or mixtures thereof, to prevent or treat various infections and diseases in a host animal (human or animal), particularly for use on the skin of humans or animals. Microorganisms as used herein refer to small, but not necessarily microscopic, organisms. Bacteria can be, for example, Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), glycopeptide-intermediate Staphylococcus aureus (GISA), coagulase-negative staphylococci (CoNS), vancomycin-resistant enterococci (VRE), and β-hemolytic agalactococci (Group B Streptococci, GBS); also include Gram-negative bacteria such as Acinetobacter spp., for example, Acinetobacter baumannii (… Acinetobacter baumannii Bordetella pertussis ( ) Bordetella pertussis), Campylobacter spp.; Enterobacteriaceae family, such as Citrobacter spp., Enterobacter spp., Escherichia coli, Klebsiella spp., Salmonella spp., Serratia marcescens, Shigella spp., Yersinia spp. ( Yersinia spp. ); Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Neisseria spp., Pseudomonas aeruginosa, Vibrio cholerae, etc.; and yeasts or fungi, such as Candida albicans, Aspergillus fumigatus, Cryptosporidium neoformans, Candida tropicalis, Candida crocephalosporinus, or mixtures thereof.

[0040] As used in this article, the term "skin" refers to the thin layer of tissue that forms the outer covering of the human or animal body, or to the ear canal of a human or animal. The term "skin" also refers to mucous membranes, such as the oral mucosa in the mouth or the nasal mucosa in the nasal cavity.

[0041] In a preferred embodiment, the antibacterial composition comprises a co- or synergistic combination of triazolo(4,5-d)pyrimidine derivatives of formula (I). (I) Where R 1 It is C 3-5 Alkyl; R 2 It is a phenyl group substituted with one or more halogen atoms; R 3 and R 4 All are hydroxyl groups; R is OH or OCH2CH2OH; Or a pharmaceutically acceptable salt thereof, and chlorhexidine, for the prevention or treatment of infection or inflammation of the skin of humans or animals, particularly humans or animals.

[0042] The antimicrobial composition of the present invention for the prevention or treatment of infections or inflammation on the skin of humans or animals, particularly humans or animals, has several advantages, such as lower cytotoxicity, lower or no allergic reactions, and a lower risk of antimicrobial resistance, because the antimicrobial composition has multiple targets and a faster killing effect on microorganisms. Therefore, the likelihood of microorganisms developing mechanisms to protect themselves from antimicrobial action is lower.

[0043] In the most preferred embodiment, the triazolo(4,5-d)pyrimidine derivative is one that contains R as a 4-fluorophenyl or 3,4-difluorophenyl. 2 And / or as a derivative of R in O CH2CH2OH.

[0044] The most preferred triazolo(4,5-d)pyrimidine derivative is (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol, hereinafter also referred to as Terifol; (II) And its pharmaceutically acceptable salts or solvates, or solvates of such salts.

[0045] Another preferred triazolo(4,5-d)pyrimidine derivative is (1S,2R,3S,4R)-4-[7-[[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, hereinafter also referred to as fluoromethol. (III) And its pharmaceutically acceptable salts or solvates, or solvates of such salts.

[0046] Synergistic or combined antibacterial compositions of triazolo(4,5-d)pyrimidine derivatives and chlorhexidine exhibit broad-spectrum activity at low concentrations of both the triazolo(4,5-d)pyrimidine derivatives and chlorhexidine.

[0047] In fact, reports have shown that tereflux at concentrations of 40 µg / ml to 0.625 µg / ml, preferably 20 µg / ml to 10 µg / ml, and most preferably 20 µg / ml, combined with chlorhexidine at concentrations of 4 µg / ml to 0.25 µg / ml, preferably 1 µg / ml to 0.5 µg / ml, exhibited synergistic or co-inhibitory effects.

[0048] Similarly, it has been reported that flumetex at concentrations of 40 µg / ml to 0.625 µg / ml, preferably 20 µg / ml to 5 µg / ml, and most preferably 10 µg / ml to 5 µg / ml, in combination with chlorhexidine at concentrations of 4 µg / ml to 0.125 µg / ml, preferably 4 µg / ml to 0.5 µg / ml, showed synergistic or co-inhibitory effects.

[0049] In a preferred embodiment, the antimicrobial composition for the prevention or treatment of infection or inflammation on the skin of humans or animals, particularly humans or animals, has a triazolo(4,5-d)pyrimidine derivative to chlorhexidine in a weight ratio (by weight) of 2.5 to 80:1, preferably 2.5 to 20:1.

[0050] In a specific embodiment, the antimicrobial composition for the prevention or treatment of infection or inflammation on the skin of humans or animals, particularly humans or animals, has a toleroxetine to chlorhexidine ratio (by weight) of 2.5 to 40:1, preferably 2.5 to 20:1.

[0051] In another specific embodiment, an antimicrobial composition for the prevention or treatment of infection or inflammation on the skin of a person or animal, particularly a person or animal, has a ratio (by weight) of flumethasone to chlorhexidine of 2.5 to 20:1.

[0052] Collaborative or synergistic antimicrobial compositions containing triazolo(4,5-d)pyrimidine derivatives and low concentrations of chlorhexidine can reduce allergic reactions and skin irritation, and can effectively prevent or reduce microbial pathogens on object surfaces and human or animal skin, especially Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0053] In one most specific embodiment, the antimicrobial composition having a toleroxate to chlorhexidine ratio (by weight) of 2.5:1 to 5:1 is used for the prevention or treatment of infection or inflammation caused by Candida albicans.

[0054] Advantageously, the combination of chlorhexidine at a concentration of only 4 µg / ml with tereflux at a concentration of 10 µg / ml to 20 µg / ml can prevent and reduce Candida albicans.

[0055] In another most specific embodiment, the antimicrobial composition contains flumethasone to chlorhexidine in a weight-to-weight ratio of 2.5:1 to 5:1 for the prevention or treatment of infection or inflammation caused by Candida albicans.

[0056] Advantageously, the combination of chlorhexidine at a concentration of only 4 µg / ml and flumethasone at a concentration of 20 µg / ml can also prevent and reduce Candida albicans.

[0057] In another most specific embodiment, the antimicrobial composition contains flumethasone to chlorhexidine in a weight-to-weight ratio of 5:1 to 20:1 for the prevention or treatment of infection or inflammation caused by Staphylococcus aureus (MRSA).

[0058] Advantageously, the combination of chlorhexidine at a concentration of only 0.5 to 1 µg / ml and flumethasone at a concentration of 5 to 10 µg / ml can also prevent and reduce Staphylococcus aureus (MRSA), while Table 1 above discloses that the minimum inhibitory concentration (MIC) of chlorhexidine against Staphylococcus aureus (MRSA) ranges from 1 to 8 µg / ml.

[0059] In yet another specific embodiment, the antimicrobial composition comprises a flumetrexed to chlorhexidine ratio (by weight) of 2.5:1 to 5:1, for the prevention or treatment of infection or inflammation caused by Pseudomonas aeruginosa.

[0060] In another preferred embodiment, the antibacterial composition comprises a co- or synergistic combination of triazolo(4,5-d)pyrimidine derivatives of formula (I). (I) Where R 1 It is C 3-5 Alkyl; R 2 It is a phenyl group substituted with one or more halogen atoms; R 3 and R 4 All are hydroxyl groups; R is OH or OCH2CH2OH; Or a pharmaceutically acceptable salt thereof, in combination with alexidin, for the prevention or treatment of infection or inflammation of the skin of humans or animals, particularly humans or animals.

[0061] Advantageously, antibacterial compositions containing a combination of alexicon and a triazol(4,5-d)pyrimidine derivative have stronger antibacterial effects and lower allergic reactions than those containing chlorhexidine; especially when combined with flumethasone.

[0062] In the most preferred embodiment, the triazolo(4,5-d)pyrimidine derivative is one that contains R as a 4-fluorophenyl or 3,4-difluorophenyl. 2 And / or as a derivative of R in OCH2CH2OH.

[0063] The most preferred triazolo(4,5-d)pyrimidine derivative is (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol, hereinafter also referred to as Terifol; (II) And its pharmaceutically acceptable salts or solvates, or solvates of such salts.

[0064] Another preferred triazolo(4,5-d)pyrimidine derivative is (1S,2R,3S,4R)-4-[7-[[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also referred to herein as fluoromethol. (III) And its pharmaceutically acceptable salts or solvates, or solvates of such salts.

[0065] The synergistic combination of the triazolo(4,5-d)pyrimidine derivative of formula (I) and alexicon is advantageously soluble in aqueous environments or aqueous media, and imparts effective antibacterial effects at low levels of the triazolo(4,5-d)pyrimidine derivative and low levels of alexicon.

[0066] In fact, reports have shown that tereflux at concentrations of 10 µg / ml to 20 µg / ml, preferably 20 µg / ml, combined with alexidin at concentrations of 0.25 µg / ml to 1 µg / ml, preferably 1 µg / ml, exhibited synergistic or co-inhibitory effects.

[0067] Similarly, reports have shown that 20 µg / ml flumethasone, in combination with 2 µg / ml to 0.0625 µg / ml, preferably 2 µg / ml alexidin, exhibits synergistic or co-inhibitory effects on microorganisms.

[0068] In a further preferred embodiment, the ratio of the triazolo(4,5-d)pyrimidine derivative of formula (I) to alexidin in the antimicrobial composition is from 5:1 to 320:1 (by weight); most preferably from 5:1 to 40:1.

[0069] In a particular embodiment, an antimicrobial composition for the prevention or treatment of infection or inflammation on the skin of a person or animal, particularly a person or animal, has a toleroxetine to alexicon ratio (by weight) of 5:1 to 80:1.

[0070] In another specific embodiment, an antimicrobial composition for the prevention or treatment of infection or inflammation on the skin of a person or animal, particularly a person or animal, has a fluorometholone to alexicon ratio (by weight) of 10:1 to 320:1.

[0071] In one most specific embodiment, the antimicrobial composition contains tereflux to alexicon in a weight-to-weight ratio of 10:1, for the prevention or treatment of infection or inflammation caused by Pseudomonas aeruginosa.

[0072] In another, most specific embodiment, the antimicrobial composition contains tereflux to alexicon in a weight-to-weight ratio of 20:1, for the prevention or treatment of infection or inflammation caused by Candida albicans.

[0073] In one most specific embodiment, the antimicrobial composition contains 40:1 fluorometholone to alexiconate (by weight) for the prevention or treatment of infection or inflammation caused by Staphylococcus aureus (MRSA).

[0074] Collaborative or synergistic antimicrobial compositions for the prevention or treatment of microorganisms on the skin of humans or animals, particularly humans or animals, can be applied to wounds, burns, ulcers, mucous membranes, and / or infections and / or inflammations that may occur on the skin of humans or animals for a variety of reasons, including surgery, medical device implantation (such as cardiovascular devices), medical device insertion (such as catheters, cannulas, valves, or needles), including wounds caused by bacteria, fungi, viruses, or bites from venomous animals (such as snakes or arthropods). Wound care is crucial for preventing microbial infections from bacteria, fungi, or viruses.

[0075] Collaborative or synergistic antimicrobial compositions used to prevent or treat microorganisms on the skin of humans or animals, particularly humans or animals, can also reduce inflammation along with infections, such as erythema, phlebitis, hyperplasia, etc.

[0076] Collaborative or synergistic antimicrobial compositions for the prevention or treatment of infections or inflammations on the skin of humans or animals, particularly humans or animals, may be administered in vitro or in vivo, for example, by topical, parenteral, rectal, nasal, transdermal, oral, sublingual, or combinations thereof.

[0077] Collaborative or synergistic antimicrobial compositions used for the prevention or treatment of microbial infections or inflammation on the skin of humans or animals, particularly humans or animals, may be administered alone or in combination with an acceptable drug carrier.

[0078] Collaborative or synergistic antimicrobial compositions used for the prevention or treatment of skin infections or inflammation may be applied directly to the skin to clean wounds, or impregnated onto dressings, bandages, cotton, rayon, polyester fabrics, polyethylene fabrics, activated carbon, polyurethane, polyester polyurethane, polycarbonate polyurethane, polydimethylsiloxane polyurethane, polyurethane foam, cellulose fibers, patches, etc.

[0079] Collaborative or synergistic antimicrobial compositions may be incorporated into gels, creams, ointments, foams, aqueous or solvent solutions, aerosols, or other pharmaceutically acceptable carriers that can be applied to wounds, oral infections, or inflammation.

[0080] Chlorhexidine or alexiconazole or their pharmaceutically acceptable salts may be used in amounts of 0.01-5% of the total weight of the antimicrobial composition.

[0081] Triazolo(4,5-d)pyrimidine derivatives or pharmaceutically acceptable salts thereof may be used in amounts ranging from 5 to 20% of the total weight of the antimicrobial composition.

[0082] The synergistic or co-active antimicrobial compositions according to the present invention may further comprise various pharmaceutically acceptable excipients, such as adjuvants, preservatives, solvents and / or viscosity modifiers, flavoring agents, sweeteners, buffers, etc.

[0083] Solvents include, for example, water, saline or any other physiological solution, ethanol, glycerol, oils (such as vegetable oils), or mixtures thereof. Viscosity modifiers include, for example, carboxymethyl cellulose.

[0084] Sweeteners include saccharin, aspartame, acetylsupan, inulin, isomaltitol, dextrose, fructose, galactose, maltitol, sorbitol, trehalose, xylitol, mannitol, sucrose, glucose, stevia, alitane, etc.

[0085] Suitable adjuvants and pharmaceutical compositions are described in Remington's Pharmaceutical Sciences, 16th edition, 1980, Mack Publishing Co., edited by Oslo et al.

[0086] According to a second aspect of the invention, a synergistic or co-active antibacterial composition is also provided, comprising a combination of fluorometholone (also known as (1S,2R,3S,4R)-4-[7-[[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol) represented by formula (III), (III) And its pharmaceutically acceptable salts or solvates, or solvates of such salts; in combination with chlorhexidine or alexiidine.

[0087] Synergistic or combined antimicrobial compositions of flumetex with chlorhexidine or alexicon have broad-spectrum activity at low concentrations of flumetex and chlorhexidine or alexicon.

[0088] The cooperative or synergistic antimicrobial composition has several advantages, such as lower cytotoxicity, lower or no allergic reactions, and lower risk of antimicrobial resistance, because the combination has multiple targets and faster killing kinetics that make microorganisms unable to resist the antimicrobial composition.

[0089] According to a specific implementation scheme, the ratio (by weight) of flumethasone to chlorhexidine in the antibacterial composition is 2.5:1 to 20:1.

[0090] Collaborative or synergistic antimicrobial compositions containing flumethasone and low concentrations of chlorhexidine can reduce allergic reactions or skin irritation and effectively prevent or reduce microbial pathogens on object surfaces and human or animal skin, especially Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0091] Advantageously, antimicrobial compositions comprising chlorhexidine at a concentration of only 0.5 to 1 µg / ml and flumethasone at a concentration of 5 to 10 µg / ml are also capable of preventing and killing Staphylococcus aureus (MRSA), and Table 1 above discloses that the minimum inhibitory concentration (MIC) of chlorhexidine against Staphylococcus aureus (MRSA) ranges from 1 to 8 µg / ml.

[0092] According to another specific embodiment, the ratio (by weight) of fluorometholone to alexicon in the antimicrobial composition is from 10:1 to 320:1.

[0093] Antibacterial compositions containing alexicin are effective at relatively low concentrations of alexicin, thus producing fewer allergic reactions.

[0094] Advantageously, it has been reported that flumetex at a concentration of 20 µg / ml, in combination with alexicon at a concentration of 0.5 µg / ml to 0.0625 µg / ml, most preferably 0.5 µg / ml, exhibits synergistic or co-synergistic antibacterial inhibitory effects.

[0095] Antimicrobial compositions containing alexidin at concentrations from 0.5 µg / ml to 0.0625 µg / ml, combined with flumethasone at a concentration of 20 µg / ml, can also kill or prevent Staphylococcus aureus (MRSA).

[0096] The synergistic or cooperative antimicrobial composition may be incorporated into gels, creams, ointments, foams, aqueous or solvent solutions, aerosols, or other pharmaceutically acceptable carriers.

[0097] Chlorhexidine or alexiconazole or their pharmaceutically acceptable salts may be used in amounts of 0.01-5% of the total weight of the antimicrobial composition.

[0098] Triazolo(4,5-d)pyrimidine derivatives or pharmaceutically acceptable salts thereof may be used in amounts ranging from 5 to 20% of the total weight of the antimicrobial composition.

[0099] The synergistic or co-active antimicrobial compositions according to the present invention may further comprise various pharmaceutically acceptable excipients, such as adjuvants, preservatives, solvents and / or viscosity modifiers, flavoring agents, sweeteners, buffers, etc.

[0100] Solvents include, for example, water, saline or any other physiological solution, ethanol, glycerol, oils (such as vegetable oils), or mixtures thereof. Viscosity modifiers include, for example, carboxymethyl cellulose.

[0101] Sweeteners include saccharin, aspartame, acetylsupan, inulin, isomaltitol, dextrose, fructose, galactose, maltitol, sorbitol, trehalose, xylitol, mannitol, sucrose, glucose, stevia, alitane, etc.

[0102] Suitable excipients and pharmaceutical compositions are described in Remington Pharmaceutical Science, 16th edition, 1980, Mack Publishing, edited by Oslo et al.

[0103] The synergistic or co-operative antimicrobial composition can be administered in vitro or in vivo, for example, by topical, parenteral, rectal, nasal, transdermal, oral, sublingual, or combinations thereof.

[0104] According to a third aspect of the invention, a medical device, biomaterial implant, or bioprosthetic is provided comprising a cooperative or synergistic antimicrobial composition according to the invention, the antimicrobial composition being incorporated into a coating or integrally distributed for the prevention or treatment of infections and / or inflammation caused by microorganisms in human or animal, particularly human or animal skin.

[0105] As used in this article, "bulk distributed" refers to the uniform or non-uniform distribution of the antimicrobial composition throughout the medical device. The antimicrobial composition may be present on the outer surface, for example, by adhering to the outer surface of the medical device; but it may also optionally be present at points deeper than the outer surface.

[0106] US 4,925,668, US 5,165,952, and US 5,707,366 describe the overall distribution of points deeper than the outer surface of a medical device.

[0107] As used herein, "microorganisms" refers to small, but not necessarily microscopic, organisms, such as bacteria, archaea, protozoa, yeast, and fungi. This invention targets pathogenic microorganisms that can cause disease in humans or animals. Bacteria can be, for example, Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), glycopeptide-intermediate Staphylococcus aureus (GISA), coagulase-negative staphylococci (CoNS), vancomycin-resistant enterococci (VRE), and β-hemolytic agalactococci (Group B Streptococci, GBS); also including Gram-negative bacteria such as Acinetobacter spp., for example Acinetobacter baumannii, Bordetella pertussis, Campylobacter spp.; Enterobacteriaceae such as Citrobacter spp., Enterobacter spp., Escherichia coli, Klebsiella spp., Salmonella spp., Serratia marcescens, Shigella spp., Yersinia spp.; Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Neisseria spp., Pseudomonas aeruginosa, Vibrio cholerae, etc.; and yeasts or fungi such as Candida albicans, Aspergillus fumigatus, Cryptosporidium neoformans, Candida tropicalis, Candida krusei, or mixtures thereof.

[0108] The term "medical device" as used herein includes, but is not limited to, any device, tool, instrument, implant, etc., relating to or intended for the cure or treatment of a disease or condition, whether in medical, human, or veterinary practice. Medical devices can include all natural and synthetic materials, as well as fibrous and non-fibrous materials. For example, materials may be made of metals, plastics, glass, ceramics, textiles, rubber, polymers, composite materials, or any other material or combination of materials. Exemplary medical devices include, but are not limited to, any kind of catheter; cannula; needle; clamp; scalpel; tube; syringe; curette; support of any size, shape, or placement; coil of any size, shape, or placement; contact lens; intrauterine device (IUD); peristaltic pump chamber; endotracheal tube; gastrointestinal feeding tube; arteriovenous shunt; condom; oxygenator and renal membrane; gloves; pacemaker leads; wound dressings; metal needles, plates, and screws; metal artificial hip joints; artificial knee joints; and gels; creams and ointments.

[0109] The term "biomaterials" or "biomaterial implants" as used in this article refers to all implantable foreign materials used in human or animal clinical practice, such as prosthetic joints, pacemakers, implantable cardioverter defibrillators, catheters (such as intravascular or urethral catheters in pigs), stents (including coronary artery stents), artificial heart valves, bioprosthetics, intraocular lenses, dental implants, breast implants, endotracheal tubes, gastrostomy tubes, etc.

[0110] As used in this article, "biological prosthesis" refers to a prosthesis made of biological materials. Examples include heart valves, pericardial grafts, vascular grafts, bladder prostheses, tendon prostheses, hernia patches, surgical mesh, and skin substitutes.

[0111] In a preferred embodiment, the medical device, biomaterial implant, or bioprosthetic is a catheter or cardiovascular device.

[0112] The medical device, biomaterial implant, or bioprosthetic is treated, coated, impregnated, or integrally distributed on at least a portion of its surface with a synergistic or cooperative antimicrobial composition according to the invention or with an antimicrobial composition according to the invention for the prevention or treatment of microbial infections or inflammation on the skin of humans or animals, particularly humans or animals.

[0113] According to a fourth aspect of the present invention, a method for manufacturing a medical device, biomaterial implant, or bioprosthetic is provided, wherein the device, implant, or prosthesis comprises the cooperative or synergistic antimicrobial composition on at least a portion of its external or internal surface, the method comprising the following steps: i) Contact the surface to be treated with the antibacterial solution by immersion, spraying, soaking or wiping; ii) Dry the surface obtained in step i).

[0114] Alternatively, the method of producing medical devices, biomaterial implants, or bioprosthetics is carried out by applying a coating of the antimicrobial composition to at least a portion of an internal or external surface; wherein the coating is obtained by the following sequential steps: i) Immerse the surface to be coated in a dopamine buffer solution; ii) Immerse the dopamine-coated surface from step i) into a polymer solution containing primary or secondary amine groups; then iii) Immerse the resulting coated surface obtained in step ii) in a mixture of the quinone-based poly(methacrylamide) of formula (1) and the said cooperating or synergistic antibacterial composition. (1) Where x is an integer greater than 1, preferably x is between 1 and 100; iv) Dry the cross-linked coated surface obtained in step iii) to obtain a coated cross-linked nanogel surface containing the antibacterial composition; v) Optionally repeat steps ii) to iv) to obtain a surface coated with several layers of cross-linked nanogel containing the collaborative or synergistic antimicrobial composition.

[0115] More generally, as described in WO2018 / 122318, the method of producing medical devices, biomaterial implants, or bioprosthetics by applying a coating (sometimes also referred to as a nanoreservoir) includes applying a first polymer and a second polymer to their surfaces. The first polymer has one or more catechol moieties, and the second polymer comprises a hydrophilic backbone having one or more reactive moieties. The nanoreservoir also comprises a cooperative or synergistic antimicrobial composition that is gradually released from within the nanoreservoir.

[0116] According to a fifth aspect, the present invention provides a method for killing microorganisms or preventing microbial growth in biofilm formation, the method comprising applying an effective amount or effective concentration of an antimicrobial composition according to the invention or an antimicrobial composition for the prevention or treatment of infection or inflammation on the skin of humans or animals, particularly humans or animals, to a surface.

[0117] In a preferred embodiment, the effective amount or effective concentration of the triazolo(4,5-d)pyrimidine derivative is 0.5-20 mg / L, and the effective concentration of chlorhexidine is 0.1-5 mg / L.

[0118] In a preferred embodiment, the effective amount or effective concentration of the triazolo(4,5-d)pyrimidine derivative is 0.5-20 mg / L, and the effective concentration of the alexicon is 0.01-5 mg / L.

[0119] A surface refers to any type of surface, such as rubber or plastic surfaces, for example, surfaces made of polyethylene, polypropylene, polyurethane, polyvinyl chloride, polyvinylpyrrolidone, polytetrafluoroethylene, silicone, etc., or copolymers, but also preferably includes metallic surfaces, such as stainless steel, silver, gold, titanium, metal alloys, pyrolytic carbon, etc. It can also be used on bioabsorbable or biomaterial surfaces, such as bioprosthetics or devices made of biomaterials (e.g., porcine or bovine pericardium).

[0120] Killing microorganisms refers to inhibiting the formation of microbial biofilms (whether they are biofilms of bacteria, yeast, fungi, or any other microorganisms).

[0121] Preventing microbial growth refers to preventing or inhibiting microbial adhesion to the surface in the first step of biofilm formation, but primarily refers to inhibiting microbial growth, reproduction, and microcolony formation on the surface in step 2. Inhibiting microbial biofilm refers to inhibiting the substrate in the maturation step 3 and inhibiting the dispersion of microorganisms from the substrate in the colonization step. Inhibiting microbial biofilm also refers to killing microorganisms in all steps of biofilm formation.

[0122] Methods for killing or preventing the growth of microorganisms on a surface are commonly applied to biomaterials or medical devices, preferably to implantable foreign body materials for clinical use in humans or animals, such as prosthetic devices, pacemakers, implantable cardioverter defibrillators, all types of catheters, coronary artery stents, heart valves, intraocular lenses, etc., but can be extended to other medical devices that require no microbial contamination, such as wound dressings, soft tissue fillers containing local anesthetics, root canal fillers containing adjuvant pharmaceutical substances, etc.

[0123] Methods for killing or preventing the growth of microorganisms can also be applied to the surfaces of experimental devices that require such antimicrobial treatment.

[0124] Methods for killing or preventing the growth of microorganisms on a surface or a portion thereof include contacting the surface to be treated with the antimicrobial composition of the present invention or a composition for the prevention or treatment of infection or inflammation of a person or animal (particularly the skin of a person or animal), said contact being made by impregnation, spraying, soaking or wiping.

[0125] Alternatively, methods for killing microorganisms or preventing microbial growth during biofilm formation include applying an effective amount or concentration of the composition according to the invention or a composition for the prevention or treatment of infection or inflammation in humans or animals (particularly human or animal skin) to a polymer coating on the surface.

[0126] The coating can be made from any polymer carrier capable of incorporating a cooperative or synergistic antimicrobial composition, such as polyurethane, polyester, polycarbonate, polydimethylsiloxane, poly(N-methacryloyl-3,4-dihydroxy-L-phenylalanine methyl ester) (also known as Pm(DOPA)), polyallylamine, polyethyleneamine, polyvinylamide, polyvinyl alcohol, poly(meth)acrylate, poly(meth)acrylamide, polyethylene glycol (PEG), or polyelectrolytes (cationic, anionic, or amphoteric) or hydrophilic biopolymers, such as polysaccharides, such as chitosan or hyaluronic acid.

[0127] Preferably, the coating is made of a first polymer and a second polymer, the first polymer having one or more catechol moieties; and the second polymer comprising a hydrophilic backbone having one or more reactive moieties, as described in WO2018 / 122318.

[0128] According to a sixth aspect of the invention, a coating for medical devices, biomaterial implants or bioprosthetics is provided, comprising a synergistic or cooperative antimicrobial composition according to the invention or a composition for the prevention or treatment of infection or inflammation in humans or animals (particularly human or animal skin).

[0129] The coating for use in medical devices is made of a polymer or copolymer capable of incorporating a synergistic or co-antimicrobial composition and may contain, for example, polyurethane, polyester, polycarbonate, polydimethylsiloxane, poly(N-methacryloyl-3,4-dihydroxy-L-phenylalanine methyl ester) (also known as Pm(DOPA)), polyallylamine, polyethyleneamine, polyvinylamide, polyvinyl alcohol, poly(meth)acrylate, poly(meth)acrylamide, polyethylene glycol (PEG), or polyelectrolytes (cationic, anionic, or amphoteric) or hydrophilic biopolymers, such as polysaccharides, such as chitosan or hyaluronic acid.

[0130] According to a seventh aspect of the invention, a wound dressing is provided comprising a synergistic or cooperative antimicrobial composition according to the invention or a composition for the prevention or treatment of infection or inflammation in a person or animal (particularly the skin of a person or animal).

[0131] The wound dressing can be any type of dressing used to cover a wound to prevent or treat skin infections and / or inflammation in humans or animals. Wound dressings can be in the form of gels (hydrogels), foams, hydrocolloids, gauze, bandages, patches, films, etc.

[0132] Wound dressings can be made from woven cotton fabrics, elastomers, coated polyurethanes, hydrophilic polymers, and water fibers (such as calcium alginate, carboxymethyl cellulose, etc.).

[0133] Wound dressings containing synergistic or cooperative antimicrobial compositions are prepared using techniques well known in the art, such as soaking, wetting, impregnation, spraying, etc.

[0134] The present invention will be further described with reference to the following figures and experimental embodiments, which are for non-limiting purposes only.

[0135] Figure 1 Real-time microcalorimetric measurements of bacterial metabolic activity, expressed as heat flux, are shown, using *Pseudomonas aeruginosa* as an example of a Gram-negative strain. Line A represents control bacteria using the vector (mQH2O), while lines B, C, D, E, F, and G represent bacteria treated with 1, 2, 4, 6, 12, and 24 mg / L chlorhexidine, respectively.

[0136] Figure 2 The effect of the combination of chlorhexidine and tereflux on the proliferation of *Pseudomonas aeruginosa* is shown using a microcalorimetric assay of bacterial metabolic activity. Line A represents the carrier control bacteria containing 0.66% ethanol in TSB, while line B represents bacteria treated with 20 mg / L tereflux; C - 1 mg / L chlorhexidine; D - 1 mg / L chlorhexidine and 20 mg / L tereflux; E - 2 mg / L chlorhexidine; F - 4 mg / L chlorhexidine; G - 6 mg / L chlorhexidine; H - 12 mg / L chlorhexidine; I - 4 mg / L chlorhexidine and 20 mg / L tereflux; J - 6 mg / L chlorhexidine and 20 mg / L tereflux; and K - 12 mg / L chlorhexidine and 20 mg / L tereflux.

[0137] Figure 3 Real-time microcalorimetric measurements of bacterial metabolic activity, expressed as heat flux, are shown, using Staphylococcus aureus as an example of a Gram-positive strain. Line A represents a carrier control bacterium containing 0.66% ethanol in TSB, while lines B represent bacteria treated with 1 mg / L tereflux; C - 1 mg / L chlorhexidine; D - 2 mg / L chlorhexidine; and E - 1 mg / L chlorhexidine with 1 mg / L tereflux.

[0138] Figure 4 Real-time microcalorimetric measurements of bacterial metabolic activity, expressed as heat flux, are shown, using Staphylococcus aureus as an example. Line A represents control bacteria using a carrier (ethanol), while lines B represent bacteria treated with 0.25 μg / mL chlorhexidine; C - 0.5 μg / mL chlorhexidine; D - 1 μg / mL chlorhexidine; E - 0.25 μg / mL alexiconazole; F - 0.5 μg / mL alexiconazole; and G - 1 μg / mL alexiconazole.

[0139] Figure 5Real-time microcalorimetric measurements of bacterial metabolic activity, expressed as heat flux, are shown, using Staphylococcus aureus as an example. Line A represents control bacteria using a carrier (ethanol), while lines B represent bacteria treated with 0.25 μg / mL alexiconine; C - 0.25 μg / mL alexiconine and 0.25 μg / mL flumethasone; D - 0.5 μg / mL alexiconine; and E - 0.5 μg / mL alexiconine and 0.5 μg / mL flumethasone.

[0140] Figure 6 The effect of the combination of alexidin and flumetrexed on biofilm formation in *Pseudomonas aeruginosa* was shown using microcalorimetry. Line A represents control bacteria treated with a carrier (ethanol), while lines B and C represent bacteria treated with 4 μg / mL alexidin and a combination of 4 μg / mL alexidin and 5 μg / mL flumetrexed, respectively.

[0141] Figure 7 Real-time microcalorimetric measurements of fungal metabolic activity, expressed as heat flow, are shown, using Candida albicans as an example. Line A represents control growth using a carrier (ethanol), while line B represents fungi treated with 1 μg / mL alexidin; C – 1 μg / mL alexidin and 5 μg / mL flumethasone.

[0142] To assess the synergistic effects between compounds in antimicrobial compositions, a checkerboard assay developed by Emery Pharma was performed to measure synergistic effects. This method determines the effect of antibiotic combinations compared to their individual activities. This comparison is then expressed as a partial inhibitory concentration (FIC) index value. The FIC index value takes into account the antibiotic combination that produces the largest change from the MIC of any individual antibiotic.

[0143] To quantify the interactions between the tested antibiotics (FIC index), the following equation is used:

[0144] Where A and B are the MICs of A and B in the combination (in a single hole), respectively, and MIC A and MIC B These are the individual MICs for A and B, respectively.

[0145] The interaction between the two tested antibiotics was then classified using the FIC index value.

[0146] If FIC < 0.5, a synergistic effect exists; If FIC > 4, there is an antagonistic effect; If FIC is between 0.5 and 4, there may be additive or unrelated effects.

[0147] Synergistic effect When a combination of compounds results in an FIC value < 0.5, the combination of compounds increases the inhibitory activity of one or two compounds (MIC decreases) compared to the individual compounds.

[0148] Additive or unrelated effects When the combination of compounds results in an FIC value of 0.5-4, the combination does not increase the inhibitory activity, or it slightly increases the inhibitory activity due to the additive effect of the two compounds.

[0149] Antagonistic effect When a combination of compounds results in an FIC value > 4, the combination of compounds increases the MIC or decreases the activity of the compounds.

[0150] Example 1: Synergistic or combined effects of tereflux and chlorhexidine on Gram-negative bacteria (Pseudomonas aeruginosa).

[0151] The following describes the synergistic or cooperative effects of the tereflux / chlorhexidine combination against Pseudomonas aeruginosa compared to the antibacterial activity of tereflux or chlorhexidine alone.

[0152] Terifol or (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol was supplied by Polpharma in Poland, while chlorhexidine was chlorhexidine gluconate supplied by Merck.

[0153] Pseudomonas aeruginosa (ATCC 15442) was cultured overnight in TSB (trypsin-soybean broth) medium, and then diluted 1x10 in fresh TSB. 7 The bacterial suspension was then diluted 300 μL aliquots with chlorhexidine (gluconate solution, 20% H2O, Merck) at concentrations of 1, 2, 4, 6, 12, and 24 mg / L, respectively. The aqueous chlorhexidine solution was refrigerated according to the manufacturer's instructions. Chlorhexidine or mQH2O as a carrier was added to the bacterial suspension to achieve the given concentration, followed by a brief vortexing. The bacteria were then aliquoted into dedicated inactivated inserts in 48-well plates and incubated statically at 37°C for 24 hours or longer, with bacterial growth and metabolic activity measured in real-time using Calscreener technology, as described at https: / / cordis.europa.eu / project / id / 784514.

[0154] To investigate the combination of chlorhexidine and tereflux, the above-diluted bacterial suspensions were supplemented with chlorhexidine alone (1, 2, 4, 6, 12 mg / L) or mixed with tereflux (20 mg / L, Falma, Poland). Tereflux was stored refrigerated in anhydrous ethanol at a concentration of 3 mg / mL. Tereflux, chlorhexidine, or both, along with ethanol as a carrier, were added to the bacterial suspension to obtain a given concentration.

[0155] Figure 1 This illustrates the real-time microcalorimetric measurement of bacterial metabolic activity, expressed as heat flux, using *Pseudomonas aeruginosa* as an example. Line A represents control bacteria using the carrier (mQH2O), while lines B, C, D, E, F, and G represent bacteria treated with 1, 2, 4, 6, 12, and 24 mg / L chlorhexidine, respectively.

[0156] Chlorhexidine completely inhibited bacterial growth at a concentration of 24 mg / L.

[0157] Figure 2 This study illustrates the effect of the combination of chlorhexidine and tereflux on the proliferation of *Pseudomonas aeruginosa* using microcalorimetry to measure bacterial metabolic activity. Line A represents the carrier control bacteria containing 0.66% ethanol in TSB, while line B represents bacteria treated with 20 mg / L tereflux; C - 1 mg / L chlorhexidine; D - 1 mg / L chlorhexidine and 20 mg / L tereflux; E - 2 mg / L chlorhexidine; F - 4 mg / L chlorhexidine; G - 6 mg / L chlorhexidine; H - 12 mg / L chlorhexidine; I - 4 mg / L chlorhexidine and 20 mg / L tereflux; J - 6 mg / L chlorhexidine and 20 mg / L tereflux; and K - 12 mg / L chlorhexidine and 20 mg / L tereflux.

[0158] Terifolium (20 mg / L) did not inhibit the growth of Pseudomonas aeruginosa, but the combination of 20 mg / L Terifolium with various concentrations of chlorhexidine (1-12 mg / L) showed a strong synergistic effect against Gram-negative bacteria. Complete bactericidal effect was achieved with 20 mg / L Terifolium and 12 mg / L chlorhexidine.

[0159] Example 2: Synergistic effect of tereflux and chlorhexidine on Gram-positive bacteria (Staphylococcus aureus (MRSA, ATCC6538)).

[0160] The following describes the synergistic or cooperative effects of the tereflux / chlorhexidine combination against Staphylococcus aureus compared to the antibacterial activity of tereflux or chlorhexidine alone.

[0161] Terifol or (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol was supplied by Pharma in Poland, while chlorhexidine was chlorhexidine gluconate supplied by Merck.

[0162] Staphylococcus aureus (MRSA, ATCC 6538) was cultured overnight in TSB (trypsin-soy broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 6 The bacterial suspension was then diluted 300 mL aliquots with 1 mg / L and 2 mg / L chlorhexidine (gluconate solution, 20% H2O, Merck). To investigate the synergistic or co-active effect of tereflux, 1 mg / L chlorhexidine was combined with 1 mg / L tereflux (Polamar, Poland). Aqueous solutions of chlorhexidine and ethanol-based solutions of tereflux were refrigerated. Chlorhexidine, tereflux, or both, along with ethanol as a carrier, were added to the bacterial suspension to obtain a given concentration, followed by brief vortexing. The bacteria were then aliquoted into dedicated inactive inserts in 48-well plates and incubated statically at 37°C for 24 hours or longer. Bacterial growth and metabolic activity were measured in real-time using Calscreener technology, as described at https: / / cordis.europa.eu / project / id / 784514.

[0163] exist Figure 3 The image shows real-time microcalorimetric measurements of bacterial metabolic activity, expressed as heat flow, with Staphylococcus aureus as an example of a Gram-positive strain. Line A represents the carrier control bacteria containing 0.66% ethanol in TSB, while lines B represent bacteria treated with 1 mg / L tereflux; C - 1 mg / L chlorhexidine; D - 2 mg / L chlorhexidine; and E - 1 mg / L chlorhexidine and 1 mg / L tereflux.

[0164] Chlorhexidine exhibits complete growth inhibition or bactericidal activity at 2 mg / L, while a strong shift in metabolic activity was observed at 1 mg / L, indicating reduced bacterial proliferation. Terifolium (1 mg / L) does not affect the growth of MRSA. However, the combination of 1 mg / L terifolium with an equal amount of chlorhexidine showed a strong synergistic effect against Gram-positive bacteria, leading to complete bacterial growth inhibition.

[0165] Example 3: Alexidine showed stronger antibacterial activity than chlorhexidine.

[0166] Staphylococcus aureus (MRSA, ATCC 6538) was cultured overnight in TSB (trypsin-soy broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 6 The bacterial suspension was then diluted 300 μL aliquots with 0.25, 0.5, and 1 µg / mL of chlorhexidine (gluconate solution, 20% H2O, Merck) or alexidin dihydrochloride (stock solution was 3 mg / mL ethanol solution, Merck).

[0167] Aqueous solutions of chlorhexidine and ethanol-based solutions of alexiidine were refrigerated. Chlorhexidine, alexiidine, and ethanol as a carrier were added to the bacterial suspension to obtain a given concentration, followed by brief vortexing. The bacteria were then aliquoted into dedicated inactive inserts in 48-well plates and incubated at 37°C statically for 24 hours or longer. Bacterial growth and metabolic activity were measured in real time using Calscreener technology, as described at https: / / cordis.europa.eu / project / id / 784514.

[0168] exist Figure 4 In this study, real-time microcalorimetric measurements of bacterial metabolic activity are expressed as heat flux, using Staphylococcus aureus as an example. Line A represents control bacteria using a carrier (ethanol), while lines B represent bacteria treated with 0.25 μg / mL chlorhexidine; C - 0.5 μg / mL chlorhexidine; D - 1 μg / mL chlorhexidine; E - 0.25 μg / mL alexiidine; F - 0.5 μg / mL alexiidine; and G - 1 μg / mL alexiidine.

[0169] Chlorhexidine at a concentration of 1 μg / mL induced a strong shift in the metabolic activity of Staphylococcus aureus. Alexidine, at concentrations of 0.25 and 0.5 μg / mL, showed significantly stronger effects. Compared to chlorhexidine, alexidine at a concentration of 1 μg / mL caused complete growth inhibition.

[0170] Example 4: Synergistic or combined effects of flumethasone and alexiconol on Gram-positive bacteria.

[0171] Staphylococcus aureus (MRSA, ATCC 6538) was cultured overnight in TSB (trypsin-soy broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 6The bacterial suspension was then diluted 300 μL aliquots with 0.25 and 0.5 μg / mL alexidin dihydrochloride (stock solution was 3 mg / mL ethanol, Merck). To investigate the synergistic or co-operating effects of flumetrexed, 0.25 μg / mL and 0.5 μg / mL alexidin were combined with 0.25 μg / mL and 0.5 μg / mL flumetrexed (ULiege Pharmacy), respectively. The ethanol-based solutions of alexidin and flumetrexed were stored at -20°C. Alexidin, alone or with flumetrexed, along with ethanol as a carrier, was added to the bacterial suspension to obtain a given concentration, followed by a brief vortexing. The bacteria were then aliquoted into dedicated inactive inserts in 48-well plates and incubated at 37°C static conditions for 24 hours or longer, with bacterial growth and metabolic activity measured in real time using Calscreener technology.

[0172] exist Figure 5 In this study, real-time microcalorimetric measurements of bacterial metabolic activity are expressed as heat flow, using Staphylococcus aureus as an example. Line A represents control bacteria using a carrier (ethanol), while lines B represent bacteria treated with 0.25 μg / mL alexiconine; C represents bacteria treated with 0.25 μg / mL alexiconine and 0.25 μg / mL flumethasone; D represents bacteria treated with 0.5 μg / mL alexiconine; and E represents bacteria treated with 0.5 μg / mL alexiconine and 0.5 μg / mL flumethasone.

[0173] The combination of flumetrexed and alexiconol at equivalent concentrations showed a significant synergistic or cooperative effect, resulting in strong bacterial growth inhibition at a concentration of 0.5 μg / mL of the two antimicrobial compounds.

[0174] Example 5: Synergistic or combined effects of flumethasone and alexiconol on Gram-negative bacteria.

[0175] Pseudomonas aeruginosa (ATCC 15442) was cultured overnight in TSB (trypsin-soy broth) medium, and then diluted 1×10⁻⁶ in fresh TSB. 7The bacterial suspension was then diluted 300 μL aliquots with 4 μg / mL alexidin dihydrochloride (stock solution was 3 mg / mL ethanol, Merck). To investigate the synergistic or co-operating effects of flumetrexed, 4 μg / mL alexidin (concentration that significantly alters the microcalorimetric signal) was combined with 5 μg / mL flumetrexed (ethanol stock solution, ULiege Pharmacy). The stock solutions of alexidin and flumetrexed were cryopreserved according to the manufacturer's instructions. Alexidin, alone or with flumetrexed, along with ethanol as a carrier, was added to the bacterial suspension to obtain a given concentration, followed by brief vortexing. The bacteria were then aliquoted into dedicated inactive inserts in 48-well plates and incubated at 37°C statically for 24 hours or longer, with bacterial growth and metabolic activity measured in real time using Calscreener technology, as described at https: / / cordis.europa.eu / project / id / 784514.

[0176] exist Figure 6 The image shows the effect of the combination of alexidin and flumetrexed on biofilm formation in *Pseudomonas aeruginosa* using microcalorimetry. Line A represents control bacteria treated with a carrier (ethanol), while lines B and C represent bacteria treated with 4 μg / mL alexidin and a combination of 4 μg / mL alexidin and 5 μg / mL flumetrexed, respectively.

[0177] Alexiconidine showed strong bacterial growth inhibition at a concentration of 4 μg / mL.

[0178] The addition of flumethasone (5 μg / mL) to the alexicon (4 μg / mL) mixture further enhanced the antibacterial effect, highlighting a strong synergistic effect against Gram-negative bacteria.

[0179] Example 6: Synergistic or combined effects of flumethasone and alexiconazole on Candida albicans.

[0180] Candida albicans (3147 ATCC 10231D) was cultured overnight in MEB (malt extract broth) medium, and then diluted 1×10⁻⁶ in fresh MEB. 4The concentrations were then adjusted. Subsequently, 300 μL aliquots of the diluted fungal suspension were supplemented with 1 μg / mL alexiconil dihydrochloride (stock solution: 3 mg / mL ethanol, Merck). To investigate the synergistic effect of flumetrexed, 1 μg / mL alexiconil was combined with 5 μg / mL flumetrexed (ULiege Pharmacy). Ethanol-based solutions of alexiconil and flumetrexed were stored at -20°C. Alexiconil, alone or with flumetrexed, along with ethanol as a carrier, was added to the fungal suspension to obtain a given concentration, followed by brief vortexing. Aliquots were then dispensed into dedicated inactivated inserts in 48-well plates and incubated statically at 37°C for 24 hours or longer, with fungal growth and metabolic activity measured in real-time using Calscreener technology.

[0181] exist Figure 7 In this study, real-time microcalorimetric measurements of fungal metabolic activity are expressed as heat flow, using Candida albicans as an example. Line A represents control growth using a carrier (ethanol), while line B represents fungi treated with 1 μg / mL alexidin; C – 1 μg / mL alexidin and 5 μg / mL flumethasone.

[0182] The combination of flumethasone and alexiconazole at given concentrations showed a significant synergistic effect, resulting in strong growth inhibition of Candida albicans.

[0183] Example 7: Synergistic or cooperative effects of flumethasone and chlorhexidine on Gram-positive bacteria. Synergistic checkerboard analysis.

[0184] Protocol: Staphylococcus aureus (MRSA, ATCC 6538) was cultured overnight (19 hours) in TSB (trypsin-soy broth). The culture was then diluted 100-fold in 4 mL of TSB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The bacterial culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl bacteria + 50 µl flumethasone / TSB + 50 µl chlorhexidine / TSB - Wells containing only one antimicrobial compound: 100µl bacteria + 50µl antimicrobial compound + 50µl TSB - Growth control: 100µl bacteria + 50µl TSB + 50µl TSB - Blank: TSB only Antimicrobial compounds are prepared as follows: Flumetex (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the bacterial suspension, an initial stock solution of 160 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40 µg / ml (160 µg / ml, prepared in TSB) - (2): 20 µg / ml (80 µg / ml, diluted in TSB) - (3): 10 µg / ml (40 µg / ml, diluted in TSB) - (4): 5 µg / ml (20 µg / ml, diluted in TSB) - (5): 2.5 µg / ml (10 µg / ml, diluted in TSB) - (6): 1.25 µg / ml (5 µg / ml, diluted in TSB) Chlorhexidine acetate (2 mg / mL, soluble in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the bacterial suspension, an initial stock solution of 16 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in TSB) - (2): 2 µg / ml (8 µg / ml, diluted in TSB) - (3): 1 µg / ml (4 µg / ml, diluted in TSB) - (4): 0.5 µg / ml (2 µg / ml, diluted in TSB) - (5): 0.25 µg / ml (1 µg / ml, diluted in TSB) Subsequently, the antibacterial compound mixture was combined with MRSA (5 × 10⁻⁶). 4 - 5 × 10 5 The culture was co-incubated with the antimicrobial agent (CFU / ml) and the endpoint growth was measured after 24 hours of shaking at 37°C and 200 rpm. The difference between OD600 at 0 hours and 24 hours determined ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it represents the MIC value of the antimicrobial agent used.

[0185] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activities with the individual activities of each compound, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the combination of molecules that produces the largest variation from the minimum inhibitory concentration (MIC) of any individual drug. Antimicrobial compounds are also referred to as drugs in this document.

[0186] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0187] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 2 shows the FIC values ​​for the combination of flumetrexed and chlorhexidine in the test for Staphylococcus aureus. The chlorhexidine concentrations presented in the table are 0.25 μg / mL, 0.5 μg / mL, and 1 μg / mL, while the flumetrexed concentrations are 2.5 μg / mL, 5 μg / mL, and 10 μg / mL.

[0188] The combination of flumetrexed and chlorhexidine showed significant synergistic or co-functional effects at concentrations of 10 / 0.5, 10 / 1, and 5 / 1, and a partial synergistic effect at a concentration of 10 / 0.25. For other ratios of the antimicrobial compounds, the combination exhibited additive or unrelated effects.

[0189] Table 2

[0190] Example 8: Synergistic or cooperative effects of tereflux and chlorhexidine on Gram-positive bacteria. Synergistic checkerboard analysis.

[0191] Protocol: Staphylococcus aureus (MRSA, ATCC 6538) was cultured overnight (19 hours) in TSB (trypsin-soy broth). The culture was then diluted 100-fold in 4 mL of TSB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The bacterial culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl bacteria + 50 µl tereflux / TSB + 50 µl chlorhexidine / TSB - Wells containing only one antimicrobial compound: 100µl bacteria + 50µl antimicrobial compound + 50µl TSB - Growth control: 100µl bacteria + 50µl TSB + 50µl TSB - Blank: TSB only Antimicrobial compounds are prepared as follows: Ticagrelor (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the bacterial suspension, an initial stock solution of 160 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40 µg / ml (160 µg / ml, prepared in TSB) - (2): 20 µg / ml (80 µg / ml, diluted in TSB) - (3): 10 µg / ml (40 µg / ml, diluted in TSB) - (4): 5 µg / ml (20 µg / ml, diluted in TSB) - (5): 2.5 µg / ml (10 µg / ml, diluted in TSB) - (6): 1.25 µg / ml (5 µg / ml, diluted in TSB) Chlorhexidine acetate (2 mg / mL, soluble in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the bacterial suspension, an initial stock solution of 16 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in TSB) - (2): 2 µg / ml (8 µg / ml, diluted in TSB) - (3): 1 µg / ml (4 µg / ml, diluted in TSB) - (4): 0.5 µg / ml (2 µg / ml, diluted in TSB) - (5): 0.25 µg / ml (1 µg / ml, diluted in TSB) Subsequently, the antibacterial compound mixture was combined with MRSA (5 × 10⁻⁶). 4 - 5 × 10 5 Co-incubate with (CFU / ml) culture medium and shake at 37°C and 200 rpm for 24 hours, then measure the endpoint growth. The difference between OD600 at 0 hours and 24 hours determines ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it expresses the MIC value of the antimicrobial agent used.

[0192] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0193] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0194] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 3 shows the FIC values ​​of the combination of tereflux and chlorhexidine for testing against Staphylococcus aureus. The chlorhexidine concentrations presented in the table are 0.25 μg / mL, 0.5 μg / mL, and 1 μg / mL, while the tereflux concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0195] The combination of tereflux and chlorhexidine showed significant partial synergistic or co-synergistic effects at concentrations of 10 / 1, 20 / 1, and 20 / 0.5 (a 40 / 1 ratio is given). For other ratios of the antimicrobial compounds, the combination had additive or unrelated effects.

[0196] Table 3

[0197] Example 9: Synergistic or cooperative effects of flumethasone and alexiconazole on Gram-positive bacteria. Synergistic checkerboard analysis.

[0198] Protocol: Staphylococcus aureus (MRSA, ATCC 6538) was cultured overnight (19 hours) in TSB (trypsin-soy broth). The culture was then diluted 100-fold in 4 mL of TSB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The bacterial culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl bacteria + 50 µl flumethasone / TSB + 50 µl alexiconazole / TSB - Wells containing only one antimicrobial compound: 100µl bacteria + 50µl antimicrobial compound + 50µl TSB - Growth control: 100µl bacteria + 50µl TSB + 50µl TSB - Blank: TSB only Antimicrobial compounds are prepared as follows: Flumetex (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the bacterial suspension, an initial stock solution of 160 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40µg / ml (160µg / ml, prepared in TSB) - (2): 20µg / ml (80µg / ml, diluted in TSB) - (3): 10µg / ml (40µg / ml, diluted in TSB) - (4): 5µg / ml (20µg / ml, diluted in TSB) - (5): 2.5 µg / ml (10 µg / ml, diluted in TSB) - (6): 1.25µg / ml (5µg / ml, diluted in TSB) Alexidine (2 mg / mL, dissolved in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the bacterial suspension, an initial stock solution of 16 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in TSB) - (2): 2µg / ml (8µg / ml, diluted in TSB) - (3): 1µg / ml (4µg / ml, diluted in TSB) - (4): 0.5µg / ml (2µg / ml, diluted in TSB) - (5): 0.25µg / ml (1µg / ml, diluted in TSB) - (6): 0.125µg / ml (0.5µg / ml, diluted in TSB) - (7): 0.0625µg / ml (0.25µg / ml, diluted in TSB) Subsequently, the antibacterial compound mixture was combined with MRSA (5 × 10⁻⁶). 4 - 5 × 10 5Co-incubate with (CFU / ml) culture medium and shake at 37°C and 200 rpm for 24 hours, then measure the endpoint growth. The difference between OD600 at 0 hours and 24 hours determines ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it expresses the MIC value of the antimicrobial agent used.

[0199] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0200] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0201] FIC values ​​are classified as follows (based on Emery Pharma's data). https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing (As described) - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 4 shows the FIC values ​​of the flumetrexed and alexiconine combination for testing against Staphylococcus aureus. The alexiconine concentrations presented in the table are 0.0625 μg / mL, 0.125 μg / mL, 0.25 μg / mL, and 0.5 μg / mL, while the flumetrexed concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0202] The combination of flumetrexed and alexidin showed significant synergistic or co-synergistic effects at concentrations of 20 / 0.5, and partial synergistic effects at concentrations of 10 / 0.5, 20 / 0.25, 20 / 0.125, and 20 / 0.0625. For other ratios of the antimicrobial compounds, the combination exhibited additive or unrelated effects.

[0203] Table 4

[0204] Example 10: Synergistic or cooperative effects of tereflux and alexiconol against Gram-positive bacteria. Synergistic checkerboard analysis.

[0205] Protocol: Staphylococcus aureus (MRSA, ATCC 6538) was cultured overnight (19 hours) in TSB (trypsin-soy broth). The culture was then diluted 100-fold in 4 mL of TSB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The bacterial culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl bacteria + 50 µl Terifolium / TSB + 50 µl Alexidine / TSB - Wells containing only one antimicrobial compound: 100µl bacteria + 50µl antimicrobial compound + 50µl TSB - Growth control: 100µl bacteria + 50µl TSB + 50µl TSB - Blank: TSB only Antimicrobial compounds are prepared as follows: Terifolium (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the bacterial suspension, an initial stock solution of 160 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40µg / ml (160µg / ml, prepared in TSB) - (2): 20µg / ml (80µg / ml, diluted in TSB) - (3): 10µg / ml (40µg / ml, diluted in TSB) - (4): 5µg / ml (20µg / ml, diluted in TSB) - (5): 2.5 µg / ml (10 µg / ml, diluted in TSB) Alexidine (2 mg / mL, dissolved in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the bacterial suspension, an initial stock solution of 16 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in TSB) - (2): 2µg / ml (8µg / ml, diluted in TSB) - (3): 1µg / ml (4µg / ml, diluted in TSB) - (4): 0.5µg / ml (2µg / ml, diluted in TSB) - (5): 0.25µg / ml (1µg / ml, diluted in TSB) - (6): 0,125µg / ml (0.5µg / ml, diluted in TSB) Subsequently, the antibacterial compound mixture was combined with MRSA (5 × 10⁻⁶). 4 - 5 × 10 5 Co-incubate with (CFU / ml) culture medium and shake at 37°C and 200 rpm for 24 hours, then measure the endpoint growth. The difference between OD600 at 0 hours and 24 hours determines ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it expresses the MIC value of the antimicrobial agent used.

[0206] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0207] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0208] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 5 shows the FIC values ​​of the combination of tereflux and alexicon for testing against Staphylococcus aureus. The alexicon concentrations presented in the table are 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, and 1 μg / mL, while the tereflux concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0209] The combination of tereflux and alexidin showed significant partial synergistic or co-synergistic effects at concentrations of 20 / 1, 20 / 0.5, and 20 / 0.25, respectively. For other ratios of the antimicrobial compounds, the combination had additive or unrelated effects.

[0210] Table 5

[0211] Example 11: Synergistic or cooperative effects of flumethasone and chlorhexidine acetate against Gram-negative bacteria. Synergistic checkerboard analysis.

[0212] Protocol: *Pseudomonas aeruginosa* (ATCC 15442) was cultured overnight (19 hours) in TSB (trypsin-soybean broth). The culture was then diluted 100-fold in 4 mL of TSB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The bacterial culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl bacteria + 50 µl flumethasone / TSB + 50 µl chlorhexidine / TSB - Wells containing only one antimicrobial compound: 100µl bacteria + 50µl antimicrobial compound + 50µl TSB - Growth control: 100µl bacteria + 50µl TSB + 50µl TSB - Blank: TSB only Antimicrobial compounds are prepared as follows: Terifolium (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the bacterial suspension, an initial stock solution of 160 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40µg / ml (160µg / ml, prepared in TSB) - (2): 20µg / ml (80µg / ml, diluted in TSB) - (3): 10µg / ml (40µg / ml, diluted in TSB) - (4): 5µg / ml (20µg / ml, diluted in TSB) - (5): 2.5 µg / ml (10 µg / ml, diluted in TSB) Chlorhexidine (2 mg / mL, dissolved in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the bacterial suspension, an initial stock solution of 16 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in TSB) - (2): 2µg / ml (8µg / ml, diluted in TSB) - (3): 1µg / ml (4µg / ml, diluted in TSB) - (4): 0.5µg / ml (2µg / ml, diluted in TSB) Subsequently, the mixture of antimicrobial compounds was reacted with Pseudomonas aeruginosa (5 × 10⁻⁶). 4 - 5 × 10 5 Co-incubate with (CFU / ml) culture medium and shake at 37°C and 200 rpm for 24 hours, then measure the endpoint growth. The difference between OD600 at 0 hours and 24 hours determines ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it expresses the MIC value of the antimicrobial agent used.

[0213] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0214] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A=MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0215] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 6 shows the FIC values ​​for the flumetrexed / chlorhexidine combination for testing against Pseudomonas aeruginosa. The chlorhexidine concentrations presented in the table are 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, while the flumetrexed concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0216] The combination of flumetrexed and chlorhexidine showed significant partial synergistic or co-operating effects at concentrations of 5 / 2 and 10 / 2, respectively. For other ratios of the antimicrobial compounds, the combination exhibited additive or unrelated effects.

[0217] Table 6

[0218] Example 12: Synergistic or cooperative effects of tereflux and chlorhexidine acetate against Gram-negative bacteria. Synergistic checkerboard analysis.

[0219] Protocol: *Pseudomonas aeruginosa* (ATCC 15442) was cultured overnight (19 hours) in TSB (trypsin-soybean broth). The culture was then diluted 100-fold in 4 mL of TSB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The bacterial culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl bacteria + 50 µl tereflux / TSB + 50 µl chlorhexidine / TSB - Wells containing only one antimicrobial compound: 100µl bacteria + 50µl antimicrobial compound + 50µl TSB - Growth control: 100µl bacteria + 50µl TSB + 50µl TSB - Blank: TSB only Antimicrobial compounds are prepared as follows: Terifolium (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the bacterial suspension, an initial stock solution of 160 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40µg / ml (160µg / ml, prepared in TSB) - (2): 20µg / ml (80µg / ml, diluted in TSB) - (3): 10µg / ml (40µg / ml, diluted in TSB) - (4): 5µg / ml (20µg / ml, diluted in TSB) - (5): 2.5 µg / ml (10 µg / ml, diluted in TSB) Chlorhexidine (2 mg / mL, dissolved in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the bacterial suspension, an initial stock solution of 16 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in TSB) - (2): 2µg / ml (8µg / ml, diluted in TSB) - (3): 1µg / ml (4µg / ml, diluted in TSB) - (4): 0.5µg / ml (2µg / ml, diluted in TSB) Subsequently, the mixture of antimicrobial compounds was combined with Pseudomonas aeruginosa (5 × 10⁻⁶). 4 - 5 × 10 5The culture was co-incubated with the antimicrobial agent (CFU / ml) and the endpoint growth was measured after 24 hours of shaking at 37°C and 200 rpm. The difference between OD600 at 0 hours and 24 hours determined ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it represents the MIC value of the antimicrobial agent used.

[0220] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0221] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0222] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 7 shows the FIC values ​​of the tereflux and chlorhexidine combination for testing against Pseudomonas aeruginosa. The chlorhexidine concentrations presented in the table are 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, while the tereflux concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0223] The combination of tereflux and chlorhexidine showed significant partial synergistic or co-synergistic effects at concentrations of 10 / 2, 20 / 2, and 20 / 1, respectively. For other ratios of the antimicrobial compounds, the combination exhibited additive or unrelated effects.

[0224] Table 7

[0225] Example 13: Synergistic or cooperative effects of flumethasone and alexiconazole against Gram-negative bacteria. Synergistic checkerboard analysis.

[0226] Protocol: *Pseudomonas aeruginosa* (ATCC 15442) was cultured overnight (19 hours) in TSB (trypsin-soybean broth). The culture was then diluted 100-fold in 4 mL of TSB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The bacterial culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl bacteria + 50 µl flumethasone / TSB + 50 µl alexiconazole / TSB - Wells containing only one antimicrobial compound: 100µl bacteria + 50µl antimicrobial compound + 50µl TSB - Growth control: 100µl bacteria + 50µl TSB + 50µl TSB - Blank: TSB only Antimicrobial compounds are prepared as follows: Flumetex (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the bacterial suspension, an initial stock solution of 160 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40µg / ml (160µg / ml, prepared in TSB) - (2): 20µg / ml (80µg / ml, diluted in TSB) - (3): 10µg / ml (40µg / ml, diluted in TSB) - (4): 5µg / ml (20µg / ml, diluted in TSB) - (5): 2.5 µg / ml (10 µg / ml, diluted in TSB) Alexidine (2 mg / mL, dissolved in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the bacterial suspension, an initial stock solution of 16 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in TSB) - (2): 2µg / ml (8µg / ml, diluted in TSB) - (3): 1µg / ml (4µg / ml, diluted in TSB) - (4): 0.5µg / ml (2µg / ml, diluted in TSB) - (5): 0.25µg / ml (1µg / ml, diluted in TSB) Subsequently, the mixture of antimicrobial compounds was combined with Pseudomonas aeruginosa (5 × 10⁻⁶). 4 - 5 × 10 5 The culture was co-incubated with the antimicrobial agent (CFU / ml) and the endpoint growth was measured after 24 hours of shaking at 37°C and 200 rpm. The difference between OD600 at 0 hours and 24 hours determined ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it represents the MIC value of the antimicrobial agent used.

[0227] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0228] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0229] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 8 shows the FIC values ​​of the flumetrexed and alexiconin combination for testing against Pseudomonas aeruginosa. The alexiconin concentrations presented in the table are 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, while the flumetrexed concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0230] The combination of flumetrexed and alexidin showed significant synergistic or co-synergistic effects at a concentration of 20 / 2, and partial synergistic or co-synergistic effects at concentrations of 10 / 2, 20 / 1, 20 / 0.5, and 20 / 0.25. For other ratios of the antimicrobial compounds, the combination had additive or unrelated effects.

[0231] Table 8

[0232] Example 14: Synergistic or cooperative effects of tereflux and alexiconazole against Gram-negative bacteria. Synergistic checkerboard analysis.

[0233] Protocol: *Pseudomonas aeruginosa* (ATCC 15442) was cultured overnight (19 hours) in TSB (trypsin-soybean broth). The culture was then diluted 100-fold in 4 mL of TSB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The bacterial culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl bacteria + 50 µl Terifolium / TSB + 50 µl Alexidine / TSB - Wells containing only one antimicrobial compound: 100µl bacteria + 50µl antimicrobial compound + 50µl TSB - Growth control: 100µl bacteria + 50µl TSB + 50µl TSB - Blank: TSB only Antimicrobial compounds are prepared as follows: Terifolium (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the bacterial suspension, an initial stock solution of 160 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40µg / ml (160µg / ml, prepared in TSB) - (2): 20µg / ml (80µg / ml, diluted in TSB) - (3): 10µg / ml (40µg / ml, diluted in TSB) - (4): 5µg / ml (20µg / ml, diluted in TSB) - (5): 2.5 µg / ml (10 µg / ml, diluted in TSB) Alexidine (2 mg / mL, soluble in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the bacterial suspension, an initial stock solution of 16 µg / mL TSB was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in TSB) - (2): 2µg / ml (8µg / ml, diluted in TSB) - (3): 1µg / ml (4µg / ml, diluted in TSB) - (4): 0.5µg / ml (2µg / ml, diluted in TSB) Subsequently, the mixture of antimicrobial compounds was reacted with Pseudomonas aeruginosa (5 × 10⁻⁶). 4 - 5 × 10 5 Co-incubate with (CFU / ml) culture medium and shake at 37°C and 200 rpm for 24 hours, then measure the endpoint growth. The difference between OD600 at 0 hours and 24 hours determines ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it expresses the MIC value of the antimicrobial agent used.

[0234] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0235] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0236] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 9 shows the FIC values ​​of the combination of tereflux and alexicon for testing against Pseudomonas aeruginosa. The alexicon concentrations presented in the table are 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, while the tereflux concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0237] The combination of tereflux and alexidin showed significant synergistic or co-synergistic effects at a concentration of 20 / 2, and partial synergistic effects at concentrations of 20 / 4 and 20 / 1. For other ratios of the antimicrobial compounds, the combination had additive or unrelated effects.

[0238] Table 9

[0239] Example 15: Synergistic or cooperative effects of flumethasone and chlorhexidine acetate on fungi. Synergistic chessboard analysis.

[0240] Protocol: Candida albicans 3147 (ATCC 10231D) was cultured overnight (24 hours) in MEB (malt extract broth). The culture was then diluted 50-fold in 4 mL of MEB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The fungal culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl fungicide + 50 µl flumethasone / MEB + 50 µl chlorhexidine / MEB - Pores containing only one antimicrobial compound: 100µl fungus + 50µl antimicrobial compound + 50µl MEB - Growth control: 100µl fungus + 50µl MEB + 50µl MEB - Blank: MEB only Antimicrobial compounds are prepared as follows: Flumetex (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the fungal suspension, an initial MEB stock solution of 160 µg / mL was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40 µg / ml (160 µg / ml, prepared in MEB) - (2): 20µg / ml (80µg / ml, diluted in MEB) - (3): 10µg / ml (40µg / ml, diluted in MEB) - (4): 5µg / ml (20µg / ml, diluted in MEB) - (5): 2.5 µg / ml (10 µg / ml, diluted in MEB) Chlorhexidine (2 mg / mL, soluble in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the fungal suspension, an initial MEB stock solution of 16 µg / mL was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in MEB) - (2): 2µg / ml (8µg / ml, diluted in MEB) - (3): 1µg / ml (4µg / ml, diluted in MEB) - (4): 0.5µg / ml (2µg / ml, diluted in MEB) Subsequently, the mixture of antibacterial compounds was combined with Candida albicans (5 × 10⁻⁶). 4 - 5 × 10 5 The culture was co-incubated with the antimicrobial agent (CFU / ml) and the endpoint growth was measured after 24 hours of shaking at 37°C and 200 rpm. The difference between OD600 at 0 hours and 24 hours determined ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it represents the MIC value of the antimicrobial agent used.

[0241] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0242] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0243] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 10 shows the FIC values ​​for the combination of flumetrexed and chlorhexidine in the test for Candida albicans. The chlorhexidine concentrations presented in the table are 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, while the flumetrexed concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0244] The combination of flumetrexed and chlorhexidine showed significant synergistic or co-synergistic effects at a concentration ratio of 20 / 4, and partial synergistic effects at a concentration ratio of 10 / 4. For other ratios of antimicrobial compounds, the combination had additive or unrelated effects.

[0245] Table 10

[0246] Example 16: Synergistic or cooperative effects of terefluxate and chlorhexidine acetate on fungi. Synergistic chessboard analysis.

[0247] Protocol: Candida albicans 3147 (ATCC 10231D) was cultured overnight (24 hours) in MEB (malt extract broth). The culture was then diluted 50-fold in 4 mL of MEB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The fungal culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl fungicide + 50 µl tereflux / MEB + 50 µl chlorhexidine / MEB - Pores containing only one antimicrobial compound: 100µl fungus + 50µl antimicrobial compound + 50µl MEB - Growth control: 100µl fungus + 50µl MEB + 50µl MEB - Blank: MEB only Antimicrobial compounds are prepared as follows: Terifolium (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the fungal suspension, an initial MEB stock solution of 160 µg / mL was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40 µg / ml (160 µg / ml, prepared in MEB) - (2): 20µg / ml (80µg / ml, diluted in MEB) - (3): 10µg / ml (40µg / ml, diluted in MEB) - (4): 5µg / ml (20µg / ml, diluted in MEB) - (5): 2.5 µg / ml (10 µg / ml, diluted in MEB) Chlorhexidine (2 mg / mL, soluble in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the fungal suspension, an initial MEB stock solution of 16 µg / mL was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in MEB) - (2): 2µg / ml (8µg / ml, diluted in MEB) - (3): 1µg / ml (4µg / ml, diluted in MEB) - (4): 0.5µg / ml (2µg / ml, diluted in MEB) Subsequently, the mixture of antibacterial compounds was combined with Candida albicans (5 × 10⁻⁶). 4 - 5 × 10 5 Co-incubate with (CFU / ml) culture medium and shake at 37°C and 200 rpm for 24 hours, then measure the endpoint growth. The difference between OD600 at 0 hours and 24 hours determines ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it expresses the MIC value of the antimicrobial agent used.

[0248] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0249] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0250] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 11 shows the FIC values ​​for the combination of tereflux and chlorhexidine in the test for Candida albicans. The chlorhexidine concentrations presented in the table are 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, while the tereflux concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0251] The combination of tereflux and chlorhexidine showed significant synergistic or additive effects at concentrations of 10 / 4 and 20 / 4. For other ratios of the antimicrobial compounds, the combination exhibited additive or indifferent effects.

[0252] Table 11

[0253] Example 17: Synergistic or cooperative effects of flumethasone and alexiconyl acetate on fungi. Synergistic checkerboard analysis.

[0254] Protocol: Candida albicans 3147 (ATCC 10231D) was cultured overnight (24 hours) in MEB (malt extract broth). The culture was then diluted 50-fold in 4 mL of MEB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The fungal culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl Fungus + 50 µl Flumethasone / MEB + 50 µl Alexidine / MEB - Pores containing only one antimicrobial compound: 100µl fungus + 50µl antimicrobial compound + 50µl MEB - Growth control: 100µl fungus + 50µl MEB + 50µl MEB - Blank: MEB only Antimicrobial compounds are prepared as follows: Flumetex (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the fungal suspension, an initial MEB stock solution of 160 µg / mL was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40 µg / ml (160 µg / ml, prepared in MEB) - (2): 20µg / ml (80µg / ml, diluted in MEB) - (3): 10µg / ml (40µg / ml, diluted in MEB) - (4): 5µg / ml (20µg / ml, diluted in MEB) - (5): 2.5 µg / ml (10 µg / ml, diluted in MEB) Alexidine (2 mg / mL, soluble in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the fungal suspension, an initial MEB stock solution of 16 µg / mL was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in MEB) - (2): 2µg / ml (8µg / ml, diluted in MEB) - (3): 1µg / ml (4µg / ml, diluted in MEB) - (4): 0.5µg / ml (2µg / ml, diluted in MEB) - (5): 0.25µg / ml (1µg / ml, diluted in MEB) Subsequently, the mixture of antibacterial compounds was combined with Candida albicans (5 × 10⁻⁶). 4 - 5 × 10 5 Co-incubate with (CFU / ml) culture medium and shake at 37°C and 200 rpm for 24 hours, then measure the endpoint growth. The difference between OD600 at 0 hours and 24 hours determines ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it expresses the MIC value of the antimicrobial agent used.

[0255] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0256] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0257] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 12 shows the FIC values ​​for the combination of flumetrexed and alexiconin for testing against Candida albicans. The alexiconin concentrations presented in the table are 0.25 μg / mL, 0.5 μg / mL, and 1 μg / mL, while the flumetrexed concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0258] The combination of flumetrexed and alexidin showed partial synergistic or co-active effects at a concentration of 20 / 1. For other ratios of antimicrobial compounds, the combination had additive or unrelated effects.

[0259] Table 12

[0260] Example 18: Synergistic or cooperative effects of terefluxate and alexiconazole acetate on fungi. Synergistic checkerboard analysis.

[0261] Protocol: Candida albicans 3147 (ATCC 10231D) was cultured overnight (24 hours) in MEB (malt extract broth). The culture was then diluted 50-fold in 4 mL of MEB and incubated at 37°C with shaking at 200 rpm until an OD600 of 0.5 was achieved. The fungal culture was then diluted 100-fold in TSB, corresponding to 5 × 10⁻⁶ ppm. 4 - 5 × 10 5 The CFU / ml range was used, and the antimicrobial compound was mixed in a 96-well plate according to the following protocol: - Wells containing a combination of two antimicrobial compounds: 100 µl Fungus + 50 µl Terifolide / MEB + 50 µl Alexidine / MEB - Pores containing only one antimicrobial compound: 100µl fungus + 50µl antimicrobial compound + 50µl MEB - Growth control: 100µl fungus + 50µl MEB + 50µl MEB - Blank: MEB only Antimicrobial compounds are prepared as follows: Terifolium (4 mg / mL, dissolved in 100% EtOH) was used as the master stock solution. To obtain a final concentration of 40 µg / mL in the fungal suspension, an initial MEB stock solution of 160 µg / mL was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 40 µg / ml (160 µg / ml, prepared in MEB) - (2): 20µg / ml (80µg / ml, diluted in MEB) - (3): 10µg / ml (40µg / ml, diluted in MEB) - (4): 5µg / ml (20µg / ml, diluted in MEB) - (5): 2.5 µg / ml (10 µg / ml, diluted in MEB) Alexidine (2 mg / mL, soluble in H2O) was used as the master stock solution. To obtain a final concentration of 4 µg / mL in the fungal suspension, an initial MEB stock solution of 16 µg / mL was prepared and further diluted 2-fold using the following serial dilution protocol: - (1): 4 µg / ml (16 µg / ml, prepared in MEB) - (2): 2µg / ml (8µg / ml, diluted in MEB) - (3): 1µg / ml (4µg / ml, diluted in MEB) - (4): 0.5µg / ml (2µg / ml, diluted in MEB) Subsequently, the mixture of antibacterial compounds was combined with Candida albicans (5 × 10⁻⁶). 4 - 5 × 10 5 The culture was co-incubated with the antimicrobial agent (CFU / ml) and the endpoint growth was measured after 24 hours of shaking at 37°C and 200 rpm. The difference between OD600 at 0 hours and 24 hours determined ΔOD600, which represents the microbial growth rate. If this value is close to zero after subtracting the blank (no microbial medium), it represents the MIC value of the antimicrobial agent used.

[0262] The synergistic effect of a combination of antimicrobial compounds, assessed by comparing their activity with that of each individual drug, is expressed as a partial inhibitory concentration (FIC) index. The FIC index considers the molecular combination that produces the largest change from the minimum inhibitory concentration (MIC) of any individual drug.

[0263] FIC is calculated as follows: ΣFICs = FIC A + FIC B FIC A =MIC A+B / MIC A And FIC B =MIC B+A / MIC B MIC A+B The value is the MIC of the antibiotic combination (mixed in a single well), while the MIC A and MIC B It is the MIC for each drug individually.

[0264] FIC values ​​are categorized as follows (based on Emery Pharma's description at https: / / emerypharma.com / solutions / cell-microbiology-services / antimicrobial-synergy-study-checkerboard-testing): - Synergistic effect <0.5 - Partial synergistic effect 0.5 - 1.09 - Additive or unrelated effects 1.1 - 4.0 - Antagonistic effect >4.0 Table 13 shows the FIC values ​​of the combination of tereflux and alexicon for testing against Candida albicans. The alexicon concentrations presented in the table are 0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL, while the tereflux concentrations are 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL.

[0265] The combination of tereflux and alexidin showed synergistic or additive effects at a concentration of 20 / 1, and partial synergistic effects at concentrations of 20 / 2, 20 / 4, and 10 / 1. For other ratios of the antimicrobial compounds, the combination exhibited additive or unrelated effects.

[0266] Table 13

Claims

1. A synergistic antibacterial composition comprising the following: Triazolo(4,5-d)pyrimidine derivatives of formula (I) (I) Where R 1 It is C 3-5 Alkyl; R 2 It is a phenyl group that is optionally substituted with one or more halogen atoms; R 3 and R 4 All are hydroxyl groups; R is XOH, where X is OCH2CH or a bond; Or its pharmaceutically acceptable salt; And alexidine.

2. The synergistic antibacterial composition according to claim 1, characterized in that, The weight ratio of the triazolo(4,5-d)pyrimidine derivative to alexidin is 5:1 to 320:1; preferably 5:1 to 40:

1.

3. The synergistic antibacterial composition according to claim 1 or 2, characterized in that, The triazolo(4,5-d)pyrimidine derivative is (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-1,2-cyclopentanediol, also known as terefrox, with a concentration of 10 µg / ml to 20 µg / ml, preferably 20 µg / ml, and a concentration of alexiconol from 0.25 µg / ml to 1 µg / ml, preferably 1 µg / ml.

4. The synergistic antibacterial composition according to any one of claims 1 to 3, characterized in that, Terifolium to alexiconium weight ratio of 5:1 to 20:1 is used for infections or inflammation caused by Pseudomonas aeruginosa, or 5:1 to 20:1 is used for infections or inflammation caused by Candida albicans.

5. The synergistic antibacterial composition according to claim 1, characterized in that, The triazolo(4,5-d)pyrimidine derivative is (1S,2R,3S,4R)-4-[7-[[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also known as flumetex, which is used to prevent or treat infections or inflammation in a weight ratio of 5:1 to 320:1 with alexiconine.

6. The synergistic antibacterial composition according to claim 5, characterized in that, The concentration of fluorometholone is 20 µg / ml, and the concentration of alexiconine is from 2 µg / ml to 0.0625 µg / ml, preferably 2 µg / ml.

7. The synergistic antibacterial composition according to claim 5 or 6, characterized in that, Flumetex to alexiconium weight ratio of 40:1 is used for the prevention or treatment of infections or inflammation caused by Staphylococcus aureus (MRSA), or 10:1 is used for the prevention or treatment of infections or inflammation caused by Pseudomonas aeruginosa.

8. A medical device, biomaterial implant, or bioprosthetic, particularly a catheter or cardiovascular device, comprising the antimicrobial composition as defined in any one of claims 1 to 7, incorporated into a coating or integrally distributed therein.

9. A method for killing microorganisms or preventing microbial growth in vitro during biofilm formation, the method comprising applying an effective amount or effective concentration of the composition as defined in any one of claims 1 to 7 to a surface.

10. A method for killing microorganisms or preventing microbial growth in vitro during biofilm formation, the method comprising applying an effective amount or effective concentration of the composition as defined in any one of claims 1 to 7 to a polymer coating on a surface.

11. The method for killing microorganisms or preventing microbial growth in biofilm formation according to claim 9 or 10, wherein the effective amount or effective concentration is 0.5-20 mg / L of triazolo(4,5-d)pyrimidine derivative and 0.01-5 mg / L of alexiidine.

12. The method according to any one of claims 9 to 11, wherein the method is a non-therapeutic method.

13. A synergistic antibacterial composition comprising the following: Triazolo(4,5-d)pyrimidine derivatives of formula (I) (I) Where R 1 It is C 3-5 Alkyl; R 2 It is a phenyl group substituted with one or more halogen atoms; R 3 and R 4 All are hydroxyl groups; R is OH; or a pharmaceutically acceptable salt thereof; And alexidine.

14. The synergistic antibacterial composition according to claim 13, characterized in that, The triazolo(4,5-d)pyrimidine derivative is (1S,2R,3S,4R)-4-[7-[[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol, also known as flumetrexed, with a weight ratio of flumetrexed to alexicon of 5:1 to 320:

1.

15. The synergistic antibacterial composition according to claim 14, characterized in that, The concentration of fluorometholone is 20 µg / ml, and the concentration of alexiconine is from 2 µg / ml to 0.00625 µg / ml, preferably 2 µg / ml, and most preferably 0.5 µg / ml.

16. A wound dressing comprising the antimicrobial composition according to any one of claims 13 to 15.

17. Use of a synergistic antimicrobial composition according to any one of claims 1 to 7 in the preparation of a medicament for the prevention or treatment of infection or inflammation of the skin of a person or animal, preferably a person or animal.

18. The use of the synergistic antibacterial composition according to claim 1 in the preparation of a medicament for the prevention or treatment of infection or inflammation, characterized in that, The infection or inflammation is caused by one or more of the following: methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis (MRSE), glycopeptide-intermediate Staphylococcus aureus (GISA), coagulase-negative staphylococci (CoNS), vancomycin-resistant enterococci (VRE), β-hemolytic agalactococci (Group B streptococci, GBS); Acinetobacter spp., Acinetobacter baumannii, Bordetella pertussis, Campylobacter spp.; Enterobacteriaceae, such as Citrobacter spp., Enterobacter spp., Escherichia coli, Klebsiella spp., Salmonella spp., Serratia marcescens, Shigella spp., Yersinia spp.; Haemophilus influenzae, Helicobacter pylori, Legionella pneumophila, Neisseria spp., Pseudomonas aeruginosa, Vibrio cholerae, etc.; Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Candida tropicalis, Candida krusei, or mixtures thereof.

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