High-efficiency antimicrobial composition

Abstract

New high-efficiency synergistic antimicrobial composition comprising a combination of Triazolo(4,5-d)pyrimidine derivative together with one antibacterial agent selected from the group consisting of fluoroquinolone and pharmaceutical acceptable salts thereof, tetracycline and pharmaceutical acceptable salts thereof or azole and pharmaceutical acceptable salts thereof. Medical device, biomaterial implants or bioprosthesis comprising the new high- efficiency antimicrobial composition incorporated in a coating or bulk distributed.

Description

[0001] NEW HIGH-EFFICIENCY ANTIMICROBIAL COMPOSITION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a new high-efficiency antimicrobial composition and application thereof.

[0004] The present invention also relates to a medical device, biomaterial implants or bioprosthesis comprising the new high-efficiency antimicrobial composition.

[0005] The present invention also relates to a method of microbial killing or prevention of microbial growth on a surface.

[0006] BACKGROUND OF INVENTION

[0007] Antimicrobial composition is used against pathogenic microorganisms including bacteria or fungi or a mixture thereof, for prevention or treatment of various infection and disease to host mammal (human and animal).

[0008] But microorganisms are becoming more and more resistant to antimicrobial composition used either as antibacterial or antifungal agent.

[0009] According to The Lancet dated May 23, 2024; each year, an estimated 7-7 million deaths are associated with bacterial infections, 1-27 million of which are caused by bacterial pathogens resistant to the antibiotics available to treat them.

[0010] Besides human medicine, companion animals, such as cats, dogs, and horses, can also be colonized and infected by microorganism such as MRSA (methicillin resistant), without host adaptation, and therefore may act as reservoirs for human infections. Bacteria can also develop distinct resistance when hosted by animals.

[0011] Fungal pathogen such as C. albicans, Aspergillus fumigatus and Cryptococcus neoformans are also becoming resistant to antifungal agent. Particularly C. albicans is inherently resistant to the majority of known antifungal drugs comprising various azole derivatives (fluconazole, isavuconazile, itraconazole, Posaconazole, voriconazole), but also to polyenes such as amphotericin B and even chlorhexidine (CHX) belonging to the Bisbiguanide antifungal agents having both antifungal and antibacterial activity.

[0012] Current antifungal treatments are therefore limited to a small number of drug classes, primarily azoles, echinocandins and polyenes, and the efficacy of these drugs is increasingly challenged by the emergence of resistant strains. The relatively small number of antifungal drug targets compared to antibacterial drugs contributes to the limited treatment options.

[0013] Moreover, some antifungals can have significant toxicities. For instance, azoles, including voriconazole is known to cause hepatotoxicity.

[0014] Finally, antifungal drugs also tend to cause more and more worse side effect among more people than other antimicrobials. Common side effects are abdominal pain, nausea, vomiting and diarrhea.

[0015] The increasing resistance of microorganisms to antimicrobial agent, their toxicity, and the undesired side effects haves increased the demand for new high efficiency antimicrobial agent exhibiting both antimicrobial efficacy and anti-antimicrobial resistance. As this demand is not easily met by a conventional one-target-one molecule approach, other approaches are required such as multi-target antimicrobials.

[0016] Particularly for antifungal, there is an urgent need for novel therapeutic strategies

[0017] WO2024062235 describes antimicrobial combinations of three or four existing antimicrobial agents. But, none of such combinations is active against a broad microorganisms spectra covering gram positive bacteria, gram negative bacteria and yeast or fungi. Moreover, a combination of three or four antimicrobial agents may be difficult to combine in the same composition for solubility reason or for toxicity reason when overdoses of antimicrobials are used to obtain a broad-spectrum antimicrobial effect or when the antimicrobials induce liver or renal injury. SUMMARY OF INVENTION

[0018] It has been surprisingly found that Triazolo(4,5-d)pyrimidine derivative, particularly fluometacyl or triafl uocy I combined with only one antimicrobial agent selected from the group consisting of fluoroquinone and pharmaceutical salts thereof or tetracycline and pharmaceutical acceptable salts thereof or azole and pharmaceutical acceptable salts thereof; may generate a synergistic antimicrobial effect at low concentration of antimicrobials and more surprisingly generate an antimicrobial effect on a microorganism even if none of both compounds has an antimicrobial effect on such microorganism.

[0019] Advantageously, the antimicrobial combination provides a large synergistic antimicrobial activity covering gram-positive bacteria, gram-negative bacteria, and also yeast or fungi.

[0020] Particularly for yeast or fungi, the antimicrobial combination of Triazolo(4,5- d)pyrimidine derivative, such as Fluometacyl or triafluocyl together with azole or pharmaceutical acceptable salts thereof, as for example fluconazole or voriconazole and pharmaceutical acceptable salts thereof, overcome the problem of fungal resistance already met by C. albicans against fluconazole and voriconazole.

[0021] The new combination Triazolo(4,5-d)pyrimidine derivative, with an azole antimicrobial or pharmaceutical acceptable salts thereof requires a low concentration of azole molecules, has a MIC value at least 2-fold lower than the MIC value of azole antifungal used alone. With a lower concentration of azole antifungal, dual therapy with the antimicrobial composition significantly reduces side effect of medical treatment or prevention. Moreover the new combination of antimicrobial with Triazolo(4,5- d)pyrimidine derivative, significantly broaden the spectrum beyond antimicrobial activity of the antimicrobial used alone.

[0022] The object of the present invention is to provide a high-efficiency synergistic antimicrobial combination and its application, particularly its use in prevention or treatment of microbial infection on human and animal, most particularly for prevention or treatment of systemic or superficial infection caused by gram-positive-bacteria, gram-negative bacteria and fungi or a mixture thereof.

[0023] The object of the invention is also to provide a medical device, biomaterial implants or bioprosthesis, particularly a catheter comprising the synergistic antimicrobial combination and method of coating thereof. The method of coating totally or partially, an internal or external surface of the medical device includes applying the Triazolo(4,5- djpyrimidine derivative and the antimicrobial agent of the antimicrobial composition either simulltanously or separatly to at least a portion of the medical device, biomaterial implants or bioprosthesis either by impregnation or in a polymeric coating or bulk distributed. The method of coating generally involves applying a polymer or nanogel comprising the Triazolo(4,5-d)pyrimidine derivative after a preliminary impregnation with the antimicrobial agent on the surface of the medical device.

[0024] Finally, the object of the invention is also to provide a method of killing or prevention of microbial growth in biofilm formation on a surface

[0025] DETAILLED DESCRIPTION

[0026] According to a first aspect of the invention, there is provided an antimicrobial composition comprising a synergistic combination of:

[0027] Triazolo(4,5-d)pyrimidine derivative of formula (I) wherein Ri is C 3-5 alkyl optionally substituted by one or more halogen atoms; R? is a phenyl group, optionally substituted by one or more halogen atoms; R3 and R4are both hydroxyl; R is XOH, wherein X is CH?, OCH2CH2, or a bond; or a pharmaceutical acceptable salt or solvate thereof, or a solvate of such a salt provided that when X is CH? or a bond, Ri is not propyl; when X is CH? and Ri CH2CH2CF3, butyl or pentyl, the phenyl group at Rj must be substituted by fluorine; when X is OCH2CH2 and Ri is propyl, the phenyl group at R2 must be substituted by fluorine, together with one antimicrobial agent selected from the group consisting of fluoroquinolone and pharmaceutically acceptable salts thereof, tetracycline and pharmaceutically acceptable salts thereof or azole and pharmaceutically acceptable salts thereof.

[0028] The antimicrobial composition may also comprise a synergistic combination of Triazolo(4,5-d)pyrimidine derivative of formula (I) together with two or three antimicrobial agents selected from the group consisting of a fluoroquinolone, tetracycline and pharmaceutically acceptable salts thereof or azole and pharmaceutically acceptable salts thereof.

[0029] The term derivative as used herein refers to a similar structurally Triazolo(4, 5- d)pyrimidine that exhibits same functional characteristics of the identified analogues. The derivative may be structurally similar by lacking one or more atoms or by been substituted by one or more chemical group.

[0030] The term synergistic as used herein refers to a combination of at least two antimicrobial compounds to produce a combined antimicrobial effect greater than the sum of their separate antimicrobial effects.

[0031] The term pharmaceutically acceptable salt as used herein refers to suitable acid addition or base salts of compounds from the antimicrobial combination. A review of suitable pharmaceutically salts may be found in Berge et al, J.Pharm SCi, 66, 1- 19(1977).

[0032] The term Fluoroquinolone or quinolone as used herein refers to cinoxacin, flumequine, oxolinic acid, piromidic acid, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, perfloxacin, rufloxacin, balofloxacin, grepafloxacin, levofloxacin, pazufloxacin, mesilate, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, moxifloxacin, gatifloxacin, sitafloxacin, trovafloxacin, ecinofloxacin, prulifloxacin, delafloxacin, clinofloxacin, balofloxacin, prulifloxacin, trovafloxacin, sitafloxacin, difloxacin, marbofloxacin, orbifloxacin, sarafloxacin, danofloxacin, enrofloxacin, gatifloxacinand a mixture thereof.

[0033] The term Tetracycline as used herein refers to tetracycline, chlortetracycline, oxitetracycline, demeclocycline, doxycycline, lymecycline, meclocycline, minocycline, methacycline, rolitetracycline, penimepicycline, eravacycline, omadacycline, serecycline and a mixture thereof.

[0034] The term Azole or imidazole as used herein refers to ketoconazole, fluconazole, miconazole, clotrimazole, tioconazole, sulconazole, econazole, itraconazole, voriconazole, posaconazole, isavuconazole, and a mixture thereof.

[0035] The antimicrobial composition of the present invention is used against pathogenic microorganisms including bacteria, archaea, protozoa, yeast, or fungi or a mixture thereof, for prevention or treatment of various infection and disease to host mammal (human and animal), particularly for systemic and superficial infections caused by grampositive bacteria, gram-negative bacteria and fungi or a mixture thereof.

[0036] The Microorganisms as used herein refers to small but not always microscopic organism. Bacteria may be for example gram positive bacteria such as methicillin- resistant 5. aureus (MRSA), methicillin-resistant 5. epidermidis (MRSE), glycopeptide intermediate 5. aureus (GISA), Coagulase-negative staphylococci (CoNS), Vancomycin- resistant enterococci (VRE), beta-hemolytic Streptococcus agalactiae (Group B Streptococcus, GBS); but also gram negative bacteria such as Acinetobacter spp., such as Acinetobacter baumannii, Bordetella pertussis, Campylobacter spp.;

[0037] Enterobacteriaceae such as Citrobacter spp., Enterobacter spp., Escherichia coli, Klebsiella spp., Salmonella spp., Serratia marcescens, Shigella spp., Yersinia spp.; Haemophilus influenza, Helocobacter pylorilegionella pneumophila, Neisseria spp., Pseudomonas aeruginosa, Vibrio cholera and the like; and to yeast or fungi such as for example C. albicans, Aspergillus fumigatus, Cryptococcus neoformans, C.tropicalis, C.krusei, C.parapsilosis, C.glabrata, A.terreus, A.niger, A.nidulans Sporothrix

[0038] Trichophyton, Microsporum, and Epidermophyton (causing Ringworm (tinea)) Madurella mycetomatis, Madurella grisea, Exophiala jeanselmei, Leptosphaeria senegalensis, and Pyrenochaeta species (causing eumycetoma), Candida auris

[0039] The systemic and superficial infections as used herein refers to tuberculosis, anthrax, abscesses, acne vulgaris, actinomycosis, asthma, bacilliary dysentery, bacterial conjunctivilis, bacterial keratitis, bacterial vaginosis, botulism, Buruli ulcer, bone and joints infections, bronchitis, brucellosis, burn wounds, cat scratch fever, cellulitis, chancroid, cholangitis, cholecystitis, cutaneous diphtheria, cystic fibrosis, cystitis, diffuse panbronchiolitis, diphtheria, dental caries, upper respiratory tract diseases, eczema, empyema, endocartitis, endometritis, enteritis, epididymitis, epiglottitis, erysipelis, erysipelas, erysipeloid, erythrasma, eye infections, ears infections, furuncles, gastrointestinal infections, genital infections, gingivitis, gonorrhoes, granuloma inguinale, infected burns, infections associated with prostheses, infections associated with cardiovascular devices, catheters and the like, intraabdominal abscesses, Legionaire'sdisease, leprosy, leptospirolisis, listeriosis, Lyme disease, lymphogranuloma venerium, mastitis, mastoiditis, meningitis, infections of the central nervous system, infections of urinary tract, mycetoma, nocardiosis, opthalmia, osteomyelitis, otitis, orchitis, pancreatitis, paronychia, pelveoperitonis, peritonitis, pharyngitis, phlegmons, pinta, plague, pneumonia, prostatitis, psittacosis, emphysema, pyelonephritis, pyoderma, reticulosis, Ritter's disease, salmonellosis, salpingitis, septic arthritis, septicaemia, sinusitis, skin infections, syphilis, tonsillitis, trachoma, tularaemia, typhoid, typhus, thrush, urethritis, yaws, asperigillosis, candidiasis, cryptococcosis, favus, histoplasmosis, intertrigo, mucomycosis, tinea, onychomycosis, pityriasis versicolor, ringworm, sporotrichosis, wounds and all Infections caused by Escherichia coli, Klebsiella spp., Acinobacter spp., Pseudomonas aeruginosa, Serratia spp. Proteus, MRSA, Staphylococcus aureus, C. albicans,

[0040] In a preferred embodiment, the antimicrobial composition comprises a synergistic combination of Triazolo(4,5-d)pyrimidine derivative of formula (I) together with levofloxacin or minocycline or fluconazole or pharmaceutical acceptable salts thereof or voriconazole or pharmaceutical acceptable salts thereof.

[0041] The antimicrobial composition may also comprise a synergistic combination of Triazolo(4,5-d)pyrimidine derivative of formula (I) together with two or three antimicrobial agents selected from the group consisting of a levofloxacin, minocycline or fluconazole or voriconazole or pharmaceutical acceptable salts thereof.

[0042] The antimicrobial composition may also comprise in addition to the synergistic combination of Triazolo(4,5-d)pyrimidine derivative of formula (I) together with fluoroquinolone and pharmaceutically acceptable salts thereof or tetracycline and pharmaceutically acceptable salts thereof or azole and pharmaceutically acceptable salts thereof, other bioactive molecules, therapeutic molecules or drugs including antibiotics such as chlorhexidine, alexidine, anti-biofilm formation agents, anti-platelet agents, anti-coagulants, anti -thrombotic agents, and anti-calcification agents.

[0043] Bioactive agents may include any agent which is desired to be delivered to molecules, cells, tissues or organs for modulating or otherwise modifying molecule or cell function, including for therapeutic effects. Bioactive agents include, but are not limited to, pharmaceutically active compounds or diagnostic compounds. Bioactive compounds include, but are not limited to, nucleotides (aptamers, RNAi, antisense oligonucleotides), peptides, oligopeptides, proteins, apoproteins, glycoproteins, antigens and antibodies or antibody fragments thereto, receptors and other membrane proteins, retro-inverso oligopeptides, protein analogs in which at least one non-peptide linkage replaces a peptide linkage, enzymes, coenzymes, enzyme inhibitors, amino acids and their derivatives, hormones, lipids, phospholipids, liposomes, ricin or ricin fragments; toxins such as aflatoxin, digoxin, xanthotoxin, rubratoxin; analgesics such as aspirin, ibuprofen and acetaminophen; bronchodilators such as theophylline and albuterol; beta-blockers such as propranolol, metoprolol, atenolol, labetolol, timolol, penbutolol and pindolol; antimicrobial agents such as those described above and chlorhexidine, alexidine, ciprofloxacin, cinoxacin and norfloxacin; antihypertensive agents such as clonidine, methyldopa, prazosin, verapamil, nifedipine, aptopril and enalapril; cardiovascular agents including antiarrhythmics, cardiac glycosides, antianginals and vasodilators; central nervous system agents including stimulants, psychotropics, antimanics and depressants; antiviral agents; antihistamines such as chlorphenirmine and brompheniramine; cancer drugs including chemotherapeutic agents, such as chlorambucil, carboplatin, derivatives of busulfan, doxorubicin, etoposide, topotecan (TPT); tranquilizers such as diazepam, chordiazepoxide, oxazepam, alprazolam and triazolam, anti-depressants such as fluoxetine, amitriptyline, nortriptyline and imipramine; H-2 antagonists such as nizatidine, cimetidine, famotidine and ranitidine; anticonvulsants; a ntina useants; prostaglandins; muscle relaxants; anti-inflammatory substances; stimulants; decongestants; antiemetics; diuretics; antispasmodics; antiasthmatiics; anti-Parkinson agents; expectorants; cough suppressants; mucolytics; vitamins; and mineral and nutritional additives. Other molecules include nucleotides; oligonucleotides; polynucleotides; and their art- recognized and biologically functional analogs and derivatives including, for example, methylated polynucleotides and nucleotide analogs having phosphorothioate linkages; plasmids, cosmids, artificial chromosomes, other nucleic acid vectors; antisense polynucleotides including those substantially complementary to at least one endogenous nucleic acid or those having sequences with a sense opposed to at least portions of selected viral or retroviral genomes; promoters; enhancers; inhibitors; other ligands for regulating gene transcription and translation.

[0044] The bioactive agent may be an anti-infective agent. Anti-infective agents include, but are not limited to antibiotics, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, dorpenem, imipenem / cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cephalexin, cefaclor, cefamandole, cefoxitin, cefproxil, cefuroxime, cefixime, cefdinir, cedfitoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, daibavancin, oritavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, linezolid, amoxicillin, ampicillin, piperacillin, ticarcillin, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levoflaxicin, lomefloxacilin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, mafenide, sulfacetamide, sulfadizine, silver sulfadizine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfisoxazole, trimethoprimsulfamethoxazole, sulfonamidochrysoidine, demeclocyline, doxycycline, minocycline, oxytetracycline, tetracycline, clofazimine, dapsone, rifampicin, rifabutin, arspehnamine, chloramphenicol, fosfomycin, metronidazole, thiamphenicol, tigecycline, tinidazole, chlorhexidine, alexidine and trimethoprim.

[0045] Anti-biofilm formation agents include, but are not limited to naturally occurring peptides such as human cathelicidin LL-37 or the bovine peptide indolicidin, or synthetic peptides such as 1018, natural compounds with 2-aminoimidazole moiety, 2- aminoimidazole based inhibitors, benzimidazoles analogs, indole-triazo-amide analogs, plant-derived biofilm inhibitors such as emodin, phloretin, casbane diterpene, resveratrol and its oligomers, sulphur derivatives, brominated furanone analogs, bromopyrrole alkaloids, skyllamycins and (-)-ageloxime D structures, cembranoids, N- acyl homoserine lactone analogs, carolacton, molecules that interfere with the formation of amyloid-like fibres, fatty acids, nitric oxide donors, ionic liquids as 1-alkyl- 3-methyl imidazolium chloride, 1-alkylquinolinium bromide, all these agents can be used in combination with conventional antibiotics.

[0046] Anti-coagulants include, but are not limited, to acenocoumarol, coumatetralyl, dicoumarol, ethyl biscoumacetate, phenprocoumon, warfarin, clorindione, dipjenadione, phenindione, ticlomarol, bemiparin, certoparin, ardeparin, dalteparin, enoxaparin, nadroparin, parnaparin, reviparin, dabigatran, apixaban, betrixabaan, darexaban, edoxaban, otamixaban, rivaroxaban, alteplase, danaparoid, tinzaparin, and fondaparinux. Anti-calcification agents include, but are not limited to, bisphosphonates, aluminium salts, glutaraldehyde, amino oleic acid, and metalloproteinase inhibitors.

[0047] Anti-platelet agents include, but are not limited to, irreversible cyclooxygenase inhibitors such as aspirin and triflusa I (Disgren), adenosine diphosphate (ADP) receptor inhibitors such as clopidogrel (Plavix), prasugrel (Effient), ticagrelor (Brilinta), ticlopidine (Ticlid), Phosphodiesterase inhibitors such as cilostazol (Pletal), Protease- activated receptor-1 (PAR-1) antagonists such as vorapaxar (Zontivity), glycoprotein IIB / IIIA inhibitors (intravenous use only) such as abciximab (ReoPro), eptifibatide (Integrilin), tirofiban (Aggrastat), Adenosine reuptake inhibitors such as dipyridamole (Persantine), thromboxane inhibitors, thromboxane synthase inhibitors and thromboxane receptor antagonists such as terutroban, glycoprotein VI inhibitors such as Revacept, glycoprotein lb inhibitors, and von Willebrand factor inhibitors.

[0048] Levofloxacin as used herein refers to : is (S)isomer of ofloxacin and is an antibiotic used to treat bacterial infections, particularly acute bacterial sinusitis, pneumonia, urinary tract infections, chronic prostatis. It is available by oral or intravenous form.

[0049] Minocycline as used herein refers to: It is generally used to treat bacterial infection in many parts of the body. It is also used to treat acne vulgaris. It is used to treat anthrax infection.

[0050] Fluconazole as used herein refers to:

[0051] Fluconazole is an antifungal medicine to treat infections caused by different kind of fungus such as Candida albicans; brain infection called cryptococcol meningitis.

[0052] Voriconazole as used herein refers to Voriconazole is an antifungal drug with large spectrum of action.

[0053] Some compounds in the antimicrobial combination may exist as optical isomers e.g. may possess one or more chiral carbon atom. The present invention includes the enantiomers and tautomers that may be isolated by methods known in the art.

[0054] Some compounds in the antimicrobial combination may exists with an asymetric center. The present invention includes stereoisomers provided that they retain the appropriate functional activity but not necessary with the same intensity.

[0055] The antimicrobial combination according to the invention also includes compounds or pharmaceutically acceptable salts thereof with at least one isotopic atom. Compounds with Isotopic atoms that may be radioactive or not, refers to a compound wherein at least one atom is replaced by an atom having the same atomic number but a different atomic mass. For example, hydrogen has an isotope deuterium; Carbon has14C and13C; Oxigen,17O and18O, Fluor18F.

[0056] In a preferred embodiment, the antimicrobial composition comprises a synergistic combination of Triazolo(4,5-d)pyrimidine derivative of formula (I) wherein Ri is C 3-5 alkyl; R2 is a phenyl group, substituted by one or more halogen atoms; R3 and R4 are both hydroxyl; R is OH, or, OCH2CH2OH; or a pharmaceutical acceptable salt, together with levofloxacin or minocycline or fluconazole or pharmaceutically acceptable salts thereof.

[0057] In a more preferred embodiment the antimicrobial composition with levofloxacin or minocycline or fluconazole or pharmaceutically acceptable salts thereof together with Triazolo(4,5-d)pyrimidine derivative, has a Mie value reduced at least 2-fold compared to the Mic value of levofloxacin or minocycline or fluconazole or pharmaceutically acceptable salts thereof used alone.

[0058] In a most preferred embodiment, the Triazolo(4,5-d)pyrimidine derivatives are the ones including R2 as 4-fluorophenyl or 3,4-difluorophenyl and or R as OCH2 CH2OH.

[0059] Most preferred Triazolo(4,5-d)pyrimidine derivatives is (lS,2S,3R,5S)-3-[7-[(lR,2S)-2- (3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[l,2,3]-triazolo[4,5- d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-l,2-cyclopentanediol as defined in formula (II) and also called triafluocyl hereafter;

[0060] (ID and a pharmaceutical acceptable salt or solvate thereof, or a solvate of such a salt.

[0061] Another most preferred Triazolo(4,5-d)pyrimidine derivative is (lS,2R,3S,4R)-4-[7-

[0062] [[(lR,2S)-2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-l,2,3- triazolo[4,5-d]pyrimidin-3-yl]-l,2,3-cyclopentanetriol as defined in formula (III) and also called Fluometacyl hereafter and a pharmaceutical acceptable salt or solvate thereof, or a solvate of such a salt.

[0063] It has been observed that Fluometacyl or triafl uocyl when combined with another antimicrobial agent synergistically enhance the antimicrobial activity of the other agent.

[0064] Moreover, when Fluometacyl or triafluocyl have no activity against a microorganism such as for example against fungi, their combination with another antimicrobial agent such as for example minocycline or fluconazole or voriconazole enhances activity of the combination against fungi. Similar synergy is observed with the combination of two or three antimicrobial agents.

[0065] In a preferred embodiment, the antimicrobial composition comprises a combination of Fluometacyl and levofloxacin or a pharmaceutically acceptable salt thereof. In another preferred embodiment the antimicrobial composition comprises a combination of Fluometacyl and minocycline or a pharmaceutically acceptable salt thereof.

[0066] In another preferred embodiment the antimicrobial composition comprises a combination of Fluometacyl and fluconazole or a pharmaceutically acceptable thereof.

[0067] In another preferred embodiment the antimicrobial composition comprises a combination of Fluometacyl with levofloxacin and minocycline and optionally fluconazole or pharmaceutically acceptable salts thereof.

[0068] In another preferred embodiment the antimicrobial composition comprises a combination of Fluometacyl with voriconazole or a pharmaceutically acceptable salt thereof.

[0069] In another preferred embodiment the antimicrobial composition comprises a combination of triafluocyl with voriconazole or a pharmaceutically acceptable salt thereof.

[0070] In another preferred embodiment the antimicrobial composition comprises a combination of triafluocyl and fluconazole or a pharmaceutically acceptable salt thereof.

[0071] According to a second aspect of the invention, there is provided a pharmaceutical composition comprising the antimicrobial combination defined herein with a pharmaceutical acceptable adjuvant, diluent and carrier. The pharmaceutical composition is for use in treatment of infection caused by gram-negative or grampositive bacteria or yeast or fungi.

[0072] The pharmaceutical composition may be administered either in separate formulations or as a single combined formulation. The pharmaceutical composition may be administered by oral, nasal, parenteral such as for example subcutaneous, intrathecal, intramuscular or intravenous injection; by topical, sublingal, rectal administration or a combination thereof. Administration by inhalation or insufflation are also possible. Preferably, the pharmaceutical composition of the invention is formulated for oral administration via galenic compositions including tablets, capsules, powders, pills, syrups, chewing, granules, and the like. The galenic composition may be produced through well-known technique and with use of typical additives such as excipients, auxiliary substances, preservatives, solvent and / or viscosity modulating agents, flavorings, sweeteners, buffering agent, lubricants, binders and the like.

[0073] By solvent, one means for example water, saline or any other physiological solution, ethanol, glycerol, oil such as vegetable oil or a mixture thereof. By viscosity modulating agent on means for example carboxymethylcellulose.

[0074] By sweetening agent, one means saccharin, aspartame, acesulfame, inulin, isomalt, dextrose, fructose, galactose, maltitol, sorbitol, trehalose, xylitol, mannitol, sucrose, glucose, stevia, alitame and the like.

[0075] Suitable auxiliary substances and pharmaceutical compositions are described in Remington's Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al.+

[0076] The concentration of each antimicrobial agent in the synergistic combination is equal to or lower than the Minimum Inhibitory Concentration (MIC) in monotherapy for the bacteria against which the combination is being used. This reduces toxicity issues and antimicrobial resistance of one or more agents in the combination.

[0077] By Minimum Inhibitory Concentration (MIC) one means the lowest concentration of antimicrobial that prevents visible growth of a microorganism (determined according to ISO standard 20776-1).

[0078] According to a third aspect of the invention, there is provided an antimicrobial composition comprising a synergistic combination of:

[0079] Triazolo(4,5-d)pyrimidine derivative of formula (I) wherein Ri is C 3-5 alkyl optionally substituted by one or more halogen atoms; R2 is a phenyl group, optionally substituted by one or more halogen atoms; R3 and R4 are both hydroxyl; R is XOH, wherein X is CH2, OCH2CH2, or a bond; or a pharmaceutical acceptable salt or solvate thereof, or a solvate of such a salt provided that when X is CH2 or a bond, Ri is not propyl; when X is CH2 and Ri CH2CH2CF3, butyl or pentyl, the phenyl group at R2 must be substituted by fluorine; when X is OCH2CH2 and Ri is propyl, the phenyl group at R2 must be substituted by fluorine, together with one antimicrobial agent selected from fluoroquinolone and pharmaceutical salts, or tetracyclin and pharmaceutical salts or with azole and pharmaceutical salts or with a combination thereof; for use in prevention or treatment of infection on human or animal.

[0080] The antimicrobial agents for use according to the invention may be administered separately or in a single combined formulation. They may be administered by oral, nasal, parenteral such as for example subcutaneous, intrathecal, intramuscular or intravenous injection; by topical, sublingal, rectal administration or a combination thereof. Administration by inhalation or insufflation are also possible. Topical administration is preferred.

[0081] In a preferred embodiment, the Triazolo(4,5-d)pyrimidine derivatives are the ones including R2 as 4-fluorophenyl or 3,4-difluorophenyl and or R as OCH2 CH2OH.

[0082] Most preferred Triazolo(4,5-d)pyrimidine derivatives is (lS,2S,3R,5S)-3-[7-[(lR,2S)-2- (3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[l,2,3]-triazolo[4,5- d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)-l,2-cyclopentanediol as defined in formula (II) and also called triafluocyl hereafter; and a pharmaceutical acceptable salt or solvate thereof, or a solvate of such a salt.

[0083] Another most preferred Triazolo(4,5-d)pyrimidine derivative is (lS,2R,3S,4R)-4-[7-

[0084] [[(lR,2S)-2-(3,4-Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-l,2,3- triazolo[4,5-d]pyrimidin-3-yl]-l,2,3-cyclopentanetriol as defined in formula (III) and also called Fluometacyl hereafter and a pharmaceutical acceptable salt or solvate thereof, or a solvate of such a salt.

[0085] In a preferred embodiment, the antimicrobial composition for use in prevention or treatment of infection, comprises a combination of Fluometacyl and levofloxacin or a pharmaceutically acceptable salt thereof, particularly for treatment or prevention of infection caused by Staphylococcus aureus.

[0086] The ratio of Fluometacyl to levofloxacin (weight to weight ratio) is preferably 1.25:1 for treatment or prevention of infection caused by Staphylococcus aureus. Moreover, Levofloxacin combined with Fluometacyl, has a MIC value reduced 2 to 4- fold compared to the MIC value of Levofloxacine used alone against Staphylococcus aureus. In another preferred embodiment the antimicrobial composition for use in prevention or treatment of infection, comprises a combination of Fluometacyl and minocycline or a pharmaceutically acceptable salt thereof, particularly for treatment or prevention of infection caused by MRSA; Pseudomonas aerigunosa and Candida albicans.

[0087] The ratio of Fluometacyl to minocycline (weight to weight ratio) is preferably 1.25:1 for treatment or prevention of infection caused by Candida albicans.

[0088] Moreover, minocycline combined with Fluometacyl, has a MIC value reduced at least 2- fold compared to the MIC value of minocycline used alone against Pseudomonas aerigunosa or against Candida albicans .

[0089] In another preferred embodiment the antimicrobial composition for use in prevention or treatment of infection, comprises a combination of Fluometacyl and fluconazole or a pharmaceutically acceptable thereof, particularly for treatment or prevention of infection caused by Candida albicans.

[0090] The ratio of Fluometacyl to fluconazole (weight to weight ratio) is preferably between 10:1 to 5:1 for treatment or prevention of infection caused by Candida albicans.

[0091] The combination of fluconazole with Fluometacyl allows a 2-fold reduction of its MIC value compared to the MIC value of fluconazole uses alone against Candida albicans.

[0092] In another preferred embodiment the antimicrobial composition for use in prevention or treatment of infection comprises a combination of Fluometacyl with levofloxacin and minocycline and optionally fluconazole or pharmaceutically acceptable salts thereof particularly for treatment or prevention of infection caused by Pseudomonas aeruginosa.

[0093] In another preferred embodiment the antimicrobial composition for use in prevention or treatment of infection comprises a combination of t riafl uocyl with fluconazole or a pharmaceutically acceptable thereof, for use in prevention or treatment of infection caused by Candida albicans. The ratio of triafluocyl to fluconazole (weight to weight ratio) is preferably between 20 / 1 and 40 / 1.

[0094] The combination of fluconazole with triafluocyl allows a 4-fold reduction of its MIC value compared to the MIC value of fluconazole uses alone against Candida albicans.

[0095] In another preferred embodiment the antimicrobial composition for use in prevention or treatment of infection comprises a combination of triafluocyl with voriconazole or a pharmaceutically acceptable thereof, for use in prevention or treatment of infection caused by Candida albicans.

[0096] The combination of voriconazole with triafluocyl allows a 2-fold reduction of its MIC value compared to the MIC value of voriconazole used alone against Candida albicans.

[0097] The ratio of Triafluocyl to voriconazole (weight to weight ratio) may be very high since only a very small quantity of voriconazole is necessary (between 320 / 1 and 640 / 1)

[0098] In another preferred embodiment the antimicrobial composition for use in prevention or treatment of infection, comprises a combination of Fluometacyl and voriconazole or a pharmaceutically acceptable thereof, particularly for treatment or prevention of infection caused by Candida albicans.

[0099] The combination of voriconazole with Fluometacyl allows a 4-fold reduction of its MIC value compared to the MIC value of voriconazole used alone against Candida albicans.

[0100] The ratio of Fluometacyl to voriconazole (weight to weight ratio) may be very high since only a very small quantity of voriconazole is necessary (between 640 / 1 and 2560 / 1).

[0101] According to a fourth aspect of the invention, there is also provided a medical device, biomaterial implants or bioprosthesis comprising the antimicrobial composition according to the invention incorporated in a coating or bulk distributed. The medical device, biomaterial implants or bioprosthesis may also be impregnated with antimicrobial composition according to the invention. The medical device, biomaterial implant or bioprosthesis is prepared by applying the Triazolo(4,5-d)pyrimidine derivative and the antimicrobial agent of the antimicrobial composition either simultanously or separatly to at least a portion of the medical device, biomaterial implants or bioprosthesis, either by impregnation or in a polymeric or nanogel coating. The method generally involves applying a polymer or nanogel comprising the Triazolo(4,5-d)pyrimidine derivative such as triafl uocy I or fluometacyl, following a preliminary impregnation of the antimicrobial agent on at least a portion of the surface of the medical device.

[0102] The medical device as used herein includes, but is not limited to, any device, tool, instrument, implant, or the like, relating to medicine or the practice of human or veterinary medicine, or intended for use to heal or treat a disease or condition. A medical device may include all natural and synthetic materials and both fibrous and non-fibrous materials. For example, the materials may be comprised of a metal, plastic, glass, ceramic, textile, rubber, polymer, composite material or any other material or combination of materials. Exemplary medical devices include, but are not limited to, any kind of catheter; cannulae; needles; stents of any size, shape, or placement; coils of any size, shape, or placement; contact lenses; IUDs; peristaltic pump chambers; endotracheal tubes; gastroenteric feeding tubes; arteriovenous shunts; condoms; oxygenator and kidney membranes; gloves; pacemaker leads; wound dressings; metallic pins, plates and screws; metallic artificial hips; artificial knees; and gels; creams and ointments.

[0103] The biomaterials, or biomaterial implant as used herein refers to all implantable foreign material for clinical use in human or animal such as for prosthetic joints, pacemakers, implantable cardioverter-defibrillators, catheters such as intravascular or urinary catheters, stent including coronary stent, prosthetic heart valves, bioprosthesis, intraocular lens, dental implants, breast implants, endotracheal tubes, gastrostomy tubes and the like.

[0104] The bioprothesis, as used herein refers to a prosthesis made of biological material. Examples include heart valves, pericardium, vascular grafts, urinary bladder prostheses, tendon prostheses, hernia patches, surgical mesh and skin substitutes.

[0105] In preferred embodiment the medical device, biomaterial implant or bioprosthesis is a catheter or a cardiovascular device.

[0106] The medical device, biomaterial implant or bioprosthesis is treated, coated, impregnated or bulk-distributed on at least part of its surface, with the synergistic antimicrobial composition according to the invention.

[0107] According to a fifth aspect, the invention provides a method of producing the medical device, biomaterial implant or bioprosthesis comprising the synergistic antimicrobial composition upon at least part of its external or internal surface and comprising the following steps: i) contacting the surface to be treated with the antimicrobial solution either by dipping, spraying, soaking or wiping ii) drying the resulting surface obtained at step i)

[0108] Alternatively, the method of producing the medical device, biomaterial implant or bioprosthesis is carried out by applying a polymer coating on at least part of the internal or external surface, with the antimicrobial composition; wherein the coating is obtained by the following in-sequence steps: i) dipping the surface to be coated in a buffer solution of dopamine; ii) dipping the surface coated with dopamine at step i); in a solution of polymer bearing primary or secondary amine groups; then iii) dipping the resulting coated surface obtained at step ii) in a mixture of a poly(methacrylamide)-bearing quinone groups of formula (1)

[0109] wherein x is an integer > 1, preferably x is between 1 and 100 with the synergistic antimicrobial composition; iv)drying the crosslinked coated surface obtained at step iii) to obtain a coated surface comprising the antimicrobial composition; v)optionally repeating steps ii) to iv) to obtain a surface coated with several layers of polymers comprising the synergic or synergistic antimicrobial composition.

[0110] Alternatively, the method of producing the medical device, biomaterial implant or bioprosthesis is carried out by applying a polymer coating on at least part of the internal or external surface, with the antimicrobial composition; wherein the coating is obtained by the following in-sequence steps: i) dipping the surface to be coated in a buffer solution of dopamine; ii) dipping the surface coated with dopamine at step i); in a solution of polymer bearing primary or secondary amine groups; then iii) dipping the resulting coated surface obtained at step ii) in a mixture of a poly(vinylquinone) of formula (6) wherein n is an integer >1, preferably between 1 and 100 with the synergistic antimicrobial composition; iv)drying the crosslinked coated surface obtained at step iii) to obtain a coated surface comprising the antimicrobial composition; v)optionally repeating steps ii) to iv) to obtain a surface coated with several layers of polymers comprising the synergic or synergistic antimicrobial composition.

[0111] In a preferred embodiment, as described In W02018 / 122318, the method of producing the medical device, biomaterial implant or bioprosthesis by applying a coating also called nanogel. The nanogel is formed through a reaction between two polymers. The first polymer is bearing one or more catechol moieties and the second polymer comprising primary or secondary amines. The nanogel further comprises the synergistic antimicrobial composition that is release progressively from within the nanogel.

[0112] In a most preferred embodiment, the method of producing the medical device, particularly a catheter, comprising the synergistic antimicrobial composition upon at least part of its external and / or internal surface comprises the following steps: i) dipping the surface to be coated in one or more solutions of antibacterial either sequentially or simultaneously (impregnation step) ii) dipping the surface impregnated at step i) in a buffer solution of dopamine; iii) dipping the resulting surface coated with dopamine at step ii); in a solution of poly(allylamine hydrochloride (PAH) then iv) dipping the resulting surface obtained at step iii) with an antimicrobial- loaded-nanogel prepared with Fluometacyl or triafluocy I and D-a- tocophenyl polyethylene glycol succinate (TPGS); v) optionally repeating steps iii) to iv) to obtain a surface coated with several layers of crosslinked nanogels comprising the synergistic antimicrobials composition. In still another preferred embodiment, the method of producing the medical device, particularly a catheter, comprising the synergistic antimicrobial composition upon at least part of its external or internal surface comprises the following steps: i) dipping the surface to be coated in one or more solutions of antimicrobial either sequentially or simultaneously (impregnation step); ii) dipping the surface impregnated at step i) in a buffer solution of dopamine; iii) dipping the resulting surface coated with dopamine at step ii); in a solution of poly(allylamine hydrochloride (PAH); then iv) dipping the resulting surface obtained at step iii) with an antimicrobials- loaded-nanogel prepared with Fluometacyl or triafluocy I and D-a- tocophenyl polyethylene glycol succinate (TPGS) or dipping the resulting surface obtained at step iii) with a polymer solution made of oxidated poly(vinylquinone), Fluometacyl or triafluocyl and TPGS, but without crosslinking agent; v) optionally repeating steps iii) to iv) to obtain a surface coated with several layers of crosslinked nanogels comprising the synergistic antimicrobial composition; vi) optionally dipping the resulting surface obtained at step v) in a solution of antimicrobial. vii) drying the crosslinked coated surface obtained at step iii) to obtain a coated crosslinked nanogel surface comprising the synergistic antimicrobial composition.

[0113] In still another preferred embodiment, the method of producing the medical device, particularly a catheter, comprising the synergistic antimicrobial composition upon at least part of its external or internal surface comprises the following steps: i)di pping the surface to be coated in one or more solutions of antimicrobial either sequentially or simultaneously (impregnation step); ii)di ppi ng the surface impregnated at step i) in a buffer solution of dopamine; iii)di pping the resulting surface coated with dopamine at step ii); in a solution of poly(allylamine hydrochloride (PAH); then iv)di pping the resulting surface obtained at step iii) with an antimicrobials-loaded- nanogel prepared with Fluometacyl or triafl uocyl and D-a-tocophenyl polyethylene glycol succinate (TPGS) or dipping the resulting surface obtained at step iii) with a polymer solution also called PTF made of Pox(mDOPA), Fluometacyl or triafl uocyl and TPGS, but without crosslinking agent; v)optionally repeating steps iii) to iv) to obtain a surface coated with several layers of crosslinked nanogels comprising the synergistic antimicrobial composition; vi)optionally dipping the resulting surface obtained at step v) in a solution of antimicrobial. vii)drying the crosslinked coated surface obtained at step iii) to obtain a coated crosslinked nanogel surface comprising the synergistic antimicrobial composition.

[0114] According to a sixth aspect, the invention provides a method ex-vivo of killing or prevention of microbial growth in biofilm formation comprising using , by applying on a surface, an effective amount of the antimicrobial composition according to the invention.

[0115] By surface one means any type of surface such as rubber or plastic surface as for example surface made of polyethylene, polypropylene, polyurethane, polyvinyl chloride, polyvinylpyrrolidone, polytetrafluoroethylene, silicone or the like, or copolymers but also and preferably metallic surface such as stainless steel, silver, gold, titanium, metallic alloys pyrolitic carbon, and the like. It can also be used on bioabsorbable or biomaterial surface such as biological prosthesis or devices which are made of biological material such as for example porcine or bovine pericardium

[0116] By microbial killing one means reduction of microbial biofilm formation either of bacteria or of fungi. 1

[0117] By prevention of microbial growth, one means prevention or an inhibition of adherence of microorganism on the surface at the first step of biofilm formation, but also and mainly an inhibition in microorganism grow, multiplication, and formation of microcolonies on the surface at their progressing step(2). By inhibition of microbial biofilm, one means inhibition of the matrix at their maturation step (3) and inhibition of microorganism dispersion from the matrix in a colonisation step (4). By inhibition of microbial biofilm, one also means killing microorganisms at all steps of the biofilm formation.

[0118] The method of microbial reduction or prevention of microorganism growth on a surface is generally applied to biomaterials or medical devices, preferably on implantable foreign material for clinical use in human or animal such as prosthetic devices, pacemakers, implantable cardioverter-defibrillators, all types of catheters, coronary stent, heart valves, intraocular lens and the like but could be extended to other medical devices requesting no microbial contamination such as for example wound dressings, soft tissue fillers containing local anaesthetics, root canal fillers with ancillary medicinal substances and the like.

[0119] The method of microbial killing or prevention of microbial growth could also be applied to surface of experimental device in need of such antimicrobial treatment.

[0120] The method of microbial killing or prevention of microbial growth on a surface or part of it comprises contacting the surface to be treated with the antimicrobial composition of the invention either by dipping, spraying, soaking or wiping.

[0121] Alternatively the method of microbial reduction or prevention of microbial growth in biofilm formation comprises using , by applying a polymeric coating on a surface, an effective amount of the composition according to the invention.

[0122] The coating may be made of any polymeric support susceptible to incorporate the synergic or synergistic antimicrobial composition such as for example polyurethane, polyester, polycarbonate, polydimethylsiloxane, poly(N-methacryloyl-3,4dihydroxy-L- phenylalenine methyl ester) (also called Pm(DOPA)), polyallylamine, polyvinylamines, polyvinylamides, polyvinylalcohol, poly(meth)acrylates, poly(meth)acrylamide, Polyethylene glycol(PEG) or a polyelectrolyte (cationic, anionic or zwitterionic) or a hydrophilic biopolymer such as a polysaccharide such as chitosan or hyaluronan.

[0123] Preferably the coating is made of a first polymer and a second polymer, the first polymer is bearing one or more catechol moieties; and the second polymer comprises a hydrophilic backbone with one or more reactive moieties as described in WO2018 / 122318.

[0124] Most preferably the coating is made according to one of the method of producing a medical device described above, according to the fifth aspect.

[0125] The invention will be further described by means of non-limiting examples only, with references to the following figures and experimental examples.

[0126] Figure 1 shows microbial Inhibition zone (diameter expressed in mm) of a catheter impregnated with a combination of minocycline (lOmg / ml), fluconazole and levofloxacin (15 mg / ml) and coated with a nanogel comprising Fluometacyl and TPGS called NFT (0.2:0.4; 3 layers) or non-coated catheter, said inhibition zone being obtained against Methicillin-resistant 5. aureus (MRSA) for 13 days.

[0127] Figure 2 shows microbial Inhibition zone (diameter expressed in mm) of a catheter impregnated with a combination of minocycline (lOmg / ml), fluconazole and levofloxacin (15 mg / ml) and coated with a nanogel comprising Fluometacyl and TPGS called NFT (0.2:0.4; 3 layers) or non-coated catheter, said inhibition zone being obtained against P. aeruginosa for 20 days.

[0128] Figure 3 shows microbial Inhibition zone (diameter expressed in mm) of catheter impregnated with a combination of minocycline (lOmg / ml), fluconazole and levofloxacin (15 mg / ml) and coated with a nanogel comprising Fluometacyl and TPGS called NFT (0.2:0.4; 3 layers) or non-coated catheter, said inhibition zone being obtained against C. albicans for 7 days.

[0129] To evaluate synergy between compounds of an antimicrobial composition, two methods are used: For both methods, a checkerboard assay is used. The difference between method 1 and 2 is that for method 1, MIC values are used to determine the FIC and FIC index, while for method 2 differences in absorbance (delta OD600 values) are used as a read out of partial growth inhibition for FIC index calculation. The second method is useful when there is no MIC for the compounds on the tested microorganism.

[0130] Method 1) is a synergy measurement by checkerboard analysis developed by Emery Pharma. It allows to determine the impact on antibiotics combination in comparison to their individual activities. The comparison is represented as a Fractional Inhibitory Concentration (FIC) index value. The FIC index value takes into account the combination of antimicrobials that produces the greatest change from the individual antimicrobial's MIC.

[0131] The FIC index is the sum of the FICs of each antimicrobial when used in combination (formula below). The FIC of an antimicrobial in a combination, is the MIC of the antimicrobial in the combination divided by the MIC of the same antimicrobial when used alone. Minimum inhibitory concentrations (MIC) are defined as the lowest concentration of an antimicrobial that, under defined in vitro conditions, prevents visible growth of bacteria within a defined period of time" (ISO 20776-1) and practically it is the lowest concentration that inhibits visible growth of a microorganism after overnight incubation

[0132] Regarding fungi, it is slightly different. As mentioned by EUCAST in Journal Compilation 2008 European Society of Clinical Microbiology and Infectious Diseases 14, 398-405, the MIC of an antifungal is the lowest antifungal concentration giving rise to an inhibition of growth of >50% of that of the antifungal-free control."

[0133] To quantify the interactions between the antimicrobials being tested (the FIC index), the following equation is used:

[0134] A + B = FICA+ FICB = FIC Index MICAMICB wherein A and B are the MIC of respectively A and B in combination (in a single well), and MIC and MICB are the MIC of A and B individually.

[0135] Method 2) is a synergy masurement by checkerboard analysis described in Bellio P, Fagnani L, Nazzicone L, Celenza G. New and simplified method for drug combination studies by checkerboard assay. MethodsX. 2021 Oct ll;8:101543. doi: 10.1016 / j.mex.2021.101543. PMID: 34754811; PMCID: PMC8563647.

[0136] The Synergy is calculated from AOD600 values as follows:

[0137] AOD600 of antimicrobial A in combination / AOD600 of antimicrobial A alone + AOD600 of antimicrobial B in combination / AOD600 of antimicrobial B alone

[0138] The FIC index is the sum of the FICs of each antimicrobial when used in combination. The FIC of an antimicrobial in a combination, is the AOD600 of the antimicrobial in the combination divided by the AOD600 of the same antimicrobial when used alone; wherein AOD600 is the substraction of the OD600 measured at time 0 and after 24h growth in the presence of the tested antimicrobials (A and B), alone or in combination.

[0139] In tables (a) of the following examples values representing AOD600 (substraction of OD measured at time 0 and after 24h growth) are reported according to the following protocol;

[0140] Antimicrobials mixes were co-incubated with the microorganisms (5 x 104 - 5 x 105 CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between OD600 at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table. Gray scale indicates the gradient of the growth, namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of bacterial growth inhibition. In tables (b) of the following examples values representing the FIC index for each antimicrobial combination at the indicated concentrations (microg / mL) are calculated following the following equation:

[0141] AOD600 of antimicrobial A in combination / AOD600 of antimicrobial A alone + AOD600 of antimicrobial B in combination / AOD600 of antimicrobial B alone

[0142] The FIC Index value is then used to categorize the interaction of both antimicrobials tested.

[0143] IF FIC < 0.5, there is synergy;

[0144] IF FIC is between 0.5 and 1.09, there is partial synergy;

[0145] IF FIC is between 1.1 and 4, there is additive effect or indifference;

[0146] IF FIC > 4, there is antagonism. when the combination of compounds results in an FIC value of <0.5, then the combination of the compounds increases the inhibitory activity (decrease in MIC) of one or both compounds than the compounds alone.

[0147] Additive or indifference: when the combination of compounds results in an FIC value of 0.5 - 4, the combination has no increase in inhibitory activity or a slight increase in inhibitory activity from the additive effect of both compounds combined.

[0148] Antagonism: when the combination of compounds results in an FIC value of >4, the combination of compounds increases the MIC, or lowers the activity of the compounds. criteria for the FIC index have been defined

[0149] - Lorian 5th edition, Chapter 9 (2005) Antimicrobial Combinations, in Antibiotics In

[0150] Laboratory Medicine, pp. 365-441. Lippincott Williams and Wilkins, Philadelphia, PA.

[0151] - Valderrama MJ, Alfaro M, Rodriguez-Avial I, Baos E, Rodriguez-Avial C, Culebras E.

[0152] Synergy of Linezolid with Several Antimicrobial Agents against Linezolid-Methicillin- Resistant Staphylococcal Strains. Antibiotics (Basel). 2020 Aug 9; 9(8): 496. doi: 10.3390 / antibiotics9080496. PMID: 32784878; PMCID: PMC7460281.

[0153] Methicillin-Resistant Staphylococcal Strains. Antibiotics (Basel). 2020 Aug 9; 9(8): 496. doi: 10.3390 / antibiotics9080496. PMID: 32784878; PMCID: PMC7460281.

[0154] Example 1. Synergic effect of fluometacyl and levofloxacin against gram-positive bacteria (Staphylococcus aureus {MRSA). Synergy Checkerboard assay.

[0155] Protocol. Staphylococcus aureus {MRSA, ATCC 6538) was grown overnight (19h) in TSB (tryptic soy broth) medium. Subsequently the culture was lOOx diluted in 4 mL of TSB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Bacterial culture was then diluted lOOx in TSB which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test antibacterials in 96-well plate according to the following scheme:

[0156] Wells with combination of two antibacterials: 100 pl of bacteria + 50pl of fluometacyl / TSB + 50pl of levofloxacin / TSB

[0157] Wells with one antibacterial only: lOOpI of bacteria + 50pl of the antibacterial + 50pl of TSB

[0158] Growth control: lOOpI of bacteria + 50pl of TSB + 50pl of TSB

[0159] - BLANK: TSB only

[0160] Antimicrobials were prepared as follows:

[0161] Fluometacyl (4 mg / mL in 100% EtOH) served as a master stock. To obtain the final concentration of 40 pg / ml in the bacterial suspension, initial stock of 160 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0162] (1): 40pg / ml (160pg / ml, prepared in TSB)

[0163] (2): 20pg / ml (80pg / ml, dilution in TSB)

[0164] (3): lOpg / ml (40pg / ml, dilution in TSB)

[0165] (4): 5pg / ml (20pg / ml, dilution in TSB)

[0166] (5): 2.5 pg / ml (lOpg / ml, dilution in TSB)

[0167] Levofloxacin (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 4 pg / ml in the bacterial suspension, initial stock of 16 pg / ml in TSB was prepared and further 2- fold diluted using the following serial dilution scheme:

[0168] (1): 4pg / ml (16 pg / ml, prepared in TSB) (2): 2pg / m I (8pg / ml, dilution in TSB)

[0169] (3): lpg / ml (4pg / ml, dilution in TSB)

[0170] Subsequently the antimicrobials mixes were co-incubated with MRSA (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0171] Table la shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of S. aureus with increasing amounts of fluometacyl or levofloxacin separately or with the combination of both. Gray scale indicates the gradient of the growth, namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of bacterial growth inhibition.

[0172] Concentrations of levofloxacin presented in the table are 1 pg / mL, 2 pg / mL and 4 pg / mL, whereas for fluometacyl 2.5 pg / mL, 5 pg / mL, 10 pg / mL and 20 pg / mL.

[0173] Table lb shows the FIC values resulted from the table la exclusively for the combination of fluometacyl with levofloxacin tested against S. aureus as an example. Concentrations of levofloxacin presented in the table are 1 pg / mL, 2 pg / mL and 4 pg / mL, whereas for fluometacyl 2.5 pg / mL, 5 pg / mL, 10 pg / mL and 20 pg / mL.

[0174] For the concentrations of fluometacyl ranging from 2.5 to 10 pg / mL there is no measurable impact on bacterial growth, whereas 20 pg / mL reached MIC value (table 1). Levofloxacin at the concentrations 1 and 2 pg / mL didn't show any effect, whereas for 4 pg / mL an effect was moderate. This effect was further potentiated in the combination with fluometacyl, showing a noticeable partial synergic effect for the concentrations fluometacyl / levofloxacin 5 / 4 (giving the ratio of 1.25 / 1). For the other ratios of the antibacterials there is additive or indifferent effect of the combination.

[0175] Table la. The results reported in the table are OD600 values determined as described herein Table lb

[0176] Example 2. Synergic effect of Fluometacyl and minocycline against gram-positive bacteria (Staphylococcus aureus {MRSA). Synergy Checkerboard assay.

[0177] Protocol. Staphylococcus aureus {MRSA. ATCC 6538) was grown overnight (19h) in TSB (tryptic soy broth) medium. Subsequently the culture was lOOx diluted in 4 mL of TSB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Bacterial culture was then diluted lOOx in TSB which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test antibacterials in 96-well plate according to the following scheme:

[0178] Wells with combination of two antibacterials: 100 pl of bacteria + 50pl of fluometacyl / TSB + 50pl of minocycline / TSB

[0179] Wells with one antibacterial only: lOOpI of bacteria + 50pl of the antibacterial + 50pl of TSB

[0180] Growth control: lOOpI of bacteria + 50pl of TSB + 50pl of TSB

[0181] - BLANK: TSB only

[0182] Antimicrobials were prepared as follows:

[0183] Fluometacyl (4 mg / mL in 100% EtOH) served as a master stock. To obtain the final concentration of 40 pg / ml in the bacterial suspension, initial stock of 160 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0184] (1): 40pg / ml (160pg / ml. prepared in TSB)

[0185] (2): 20pg / ml (80pg / ml. dilution in TSB)

[0186] (3): lOpg / ml (40pg / ml. dilution in TSB)

[0187] (4): 5pg / ml (20pg / ml. dilution in TSB)

[0188] (5): 2.5pg / ml (lOpg / ml. dilution in TSB)

[0189] (6): 1.25pg / ml (5pg / ml. dilution in TSB)

[0190] (7): 0.625pg / ml (2.5pg / ml. dilution in TSB) Minocycline (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 4 pg / ml in the bacterial suspension, initial stock of 16 pg / ml in TSB was prepared and further 2- fold diluted using the following serial dilution scheme:

[0191] (1): 4pg / ml (16 pg / ml. prepared in TSB)

[0192] (2): 2pg / ml (8pg / ml. dilution in TSB)

[0193] (3): lpg / ml (4pg / ml. dilution in TSB)

[0194] (4): 0.5pg / ml (2pg / ml. dilution in TSB)

[0195] (5): 0.25pg / ml (lpg / ml. dilution in TSB)

[0196] (6): 0.125pg / ml (0.5pg / ml. dilution in TSB)

[0197] Subsequently the antimicrobials mixes were co-incubated with MRSA (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0198] Table 2a. The results reported in the present table OD600 values determined as described above:

[0199] Table 2b

[0200] Example 3. Synergic effect of Fluometacyl and minocycline against gram-negative bacteria {Pseudomonas aeruginosa). Synergy Checkerboard assay.

[0201] Protocol. Pseudomonas aeruginosa {ATCC 15442) was grown overnight (19h) in TSB (tryptic soy broth) medium. Subsequently the culture was lOOx diluted in 4 mL of TSB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Bacterial culture was then diluted lOOx in TSB which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test drugs in 96-well plate according to the following scheme:

[0202] Wells with combination of two antimicrobials: 100 pl of bacteria + 50pl of fluometacyl / TSB + 50pl of minocycline / TSB

[0203] Wells with one antimicrobial only: lOOpI of bacteria + 50pl of the drug + 50pl of TSB Growth control: lOOpI of bacteria + 50pl of TSB + 50pl of TSB

[0204] - BLANK: TSB only

[0205] Antimicrobials were prepared as follows:

[0206] Fluometacyl (4 mg / mL in 100% EtOH) served as a master stock. To obtain the final concentration of 40 pg / ml in the bacterial suspension, initial stock of 160 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0207] (1): 40pg / ml (160pg / ml. prepared in TSB)

[0208] (2): 20pg / ml (80pg / ml. dilution in TSB)

[0209] (3): lOpg / ml (40pg / ml. dilution in TSB)

[0210] (4): 5pg / ml (20pg / ml. dilution in TSB)

[0211] (5): 2.5pg / ml (lOpg / ml. dilution in TSB)

[0212] (6): 1.25pg / ml (5pg / ml. dilution in TSB)

[0213] (7): 0.625pg / ml (2.5pg / ml. dilution in TSB)

[0214] Minocycline (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 8 pg / ml in the bacterial suspension, initial stock of 32 pg / ml in TSB was prepared and further 2- fold diluted using the following serial dilution scheme:

[0215] (1): 8pg / ml (32 pg / ml. prepared in TSB)

[0216] (2): 4pg / ml (16 pg / ml. prepared in TSB)

[0217] (3): 2pg / ml (8pg / ml. dilution in TSB)

[0218] (4): lpg / ml (4pg / ml. dilution in TSB)

[0219] (5): 0.5pg / ml (2pg / ml. dilution in TSB)

[0220] Subsequently the antimicrobials mixes were co-incubated with P. aeruginosa (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table. Table 3a shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of S. aureus with increasing amounts of Fluometacyl or minocycline separately or with the combination of both. Gray scale indicates the gradient of the growth. Namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of bacterial growth inhibition. Concentrations of minocycline presented in the table 5 are 0.5 pg / mL. 1 pg / mL. 2 pg / mL. 4 pg / mL and 8 pg / mL whereas for fluometacyl 0.625 pg / mL. 1.25 pg / mL. 2.5 pg / mL. 5 pg / mL and 10 pg / mL.

[0221] Table 3b shows the FIC values resulted from the table 3a exclusively for the combination of fluometacyl with minocycline tested against P. aeruginosa as an example. Concentrations of minocycline presented in the table are 2 pg / mL and 4 pg / mL. whereas for fluometacyl 0.625 pg / mL. 1.25 pg / mL. 2.5 pg / mL. 5 pg / mL and 10 pg / mL.

[0222] Fluometacyl in the shown range of concentrations doesn't impact bacterial growth on its own. Minocycline at the concentrations 0.5 and lpg / mL didn't show any effect, for 8 pg / mL MIC value is reached, for 2 and 4 pg / mL an effect is moderate which in the combination with Fluometacyl 10 pg / mL results in a partial synergy. For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0223] Table 3a. The results reported in the table are OD600 values determined as described above

[0224] Table 3b Example 4. Synergic effect of fluometacyl and minocycline against fungi {Candida albicans).

[0225] Synergy Checkerboard assay.

[0226] Protocol. Candida albicans 3147 {ATCC 10231D) was grown overnight (24h) in MEB (malt extract broth) medium. Subsequently the culture was 50x diluted in 4 mL of MEB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Fungal culture was then diluted lOOx in TSB which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test drugs in 96-well plate according to the following scheme:

[0227] Wells with combination of two antimicrobials: 100 pl of fungi + 50pl of Fluometacyl / MEB + 50pl of minocycline / MEB

[0228] Wells with one antimicrobial only: lOOpI of fungi + 50pl of the antimicrobial + 50pl of MEB

[0229] Growth control: lOOpI of fungi + 50pl of MEB + 50pl of MEB

[0230] - BLANK: MEB only

[0231] Antimicrobials were prepared as follows:

[0232] Fluometacyl (4 mg / mL in 100% EtOH) served as a master stock. To obtain the final concentration of 40 pg / ml in the fungal suspension, initial stock of 160 pg / ml in MEB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0233] (1): 40pg / ml (160pg / ml. prepared in MEB)

[0234] (2): 20pg / ml (80pg / ml. dilution in MEB)

[0235] (3): lOpg / ml (40pg / ml. dilution in MEB)

[0236] (4): 5pg / ml (20pg / ml. dilution in MEB)

[0237] (5): 2.5pg / ml (lOpg / ml. dilution in MEB)

[0238] (6): 1.25pg / ml (5pg / ml. dilution in MEB)

[0239] (7): 0.625pg / ml (2.5pg / ml. dilution in MEB)

[0240] Minocycline (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 32 pg / ml in the fungal suspension, initial stock of 128 pg / ml in MEB was prepared and further 2- fold diluted using the following serial dilution scheme:

[0241] (1): 32pg / ml (128 pg / ml. prepared in MEB)

[0242] (2): 16pg / ml (64 pg / ml. prepared in MEB)

[0243] (3): 8pg / ml (32 pg / ml. prepared in MEB)

[0244] (4): 4pg / ml (16 pg / ml. prepared in MEB) (5): 2pg / ml (8pg / ml. dilution in MEB)

[0245] (6): lpg / ml (4pg / ml. dilution in MEB)

[0246] (7): 0.5pg / ml (2pg / ml. dilution in MEB)

[0247] Subsequently the antimicrobial mixes were co-incubated with C. albicans (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0248] Table 4a shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of C. albicans with increasing amounts of Fluometacyl or minocycline separately or with the combination of both. Gray scale indicates the gradient of the growth, namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of bacterial growth inhibition. Concentrations of minocycline presented in the table 7 are 0.5 pg / mL. 1 pg / mL. 2 pg / mL. 4 pg / mL. 8 pg / mL. 16 pg / mL and 32 pg / mL whereas for fluometacyl 0.625 pg / mL. 1.25 pg / mL. 2.5 pg / mL. 5 pg / mL. 10 pg / mL. 20 pg / mL and 40 pg / mL.

[0249] Table 4b shows the FIC values resulted from the table 4a exclusively for the combination of Fluometacyl with minocycline tested against C. albicans as an example. Concentrations of minocycline presented in the table are 0.5 pg / mL. 1 pg / mL. 2 pg / mL. 4 pg / mL. 8 pg / mL. 16 pg / mL and 32 pg / mL whereas for Fluometacyl 0.625 pg / mL. 1.25 pg / mL. 2.5 pg / mL. 5 pg / mL. 10 pg / mL. 20 pg / mL and 40 pg / mL.

[0250] Fluometacyl and minocycline used separately in the broad range of concentrations don't impact bacterial growth. Minocycline at the concentrations of 16 pg / mL together with Fluometacyl 40 pg / mL does show some moderate effect however not yet synergistic. Using minocycline 32 pg / mL in the combination with Fluometacyl ranging from 5 pg / mL to 40 pg / mL there is increasing growth inhibition observed with a partial synergy reached at the highest concentration of fluometacyl (resulting in the ratio Fluometacykminocycline 1.25:1). For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0251] Table 4a. The results reported in the table are OD600 values determined as described herein

[0252] Table 4b

[0253] Example 5. Synergic effect of Fluometacyl and fluconazole against fungi {Candida albicans). Synergy Checkerboard assay.

[0254] Protocol. Candida albicans 3147 {ATCC 10231D) was grown overnight (24h) in MEB (malt extract broth) medium. Subsequently the culture was 50x diluted in 4 mL of MEB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Fungal culture was then diluted lOOx in TSB which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test drugs in 96-well plate according to the following scheme:

[0255] Wells with combination of two antimicrobials: 100 pl of fungi + 50pl of Fluometacyl / MEB + 50pl of fluconazole / MEB

[0256] Wells with one antimicrobial only: lOOpI of fungi + 50pl of the antimicrobial + 50pl of MEB

[0257] Growth control: lOOpI of fungi + 50pl of MEB + 50pl of MEB

[0258] - BLANK: MEB only

[0259] Antimicrobials were prepared as follows:

[0260] Fluometacyl (4 mg / mL in 100% EtOH) served as a master stock. To obtain the final concentration of 40 pg / ml in the fungal suspension, initial stock of 160 pg / ml in MEB was prepared and further 2-fold diluted using the following serial dilution scheme: (1): 40pg / ml (160pg / ml. prepared in MEB)

[0261] (2): 20pg / ml (80pg / ml. dilution in MEB)

[0262] (3): lOpg / ml (40pg / ml. dilution in MEB)

[0263] (4): 5pg / ml (20pg / ml. dilution in MEB)

[0264] (5): 2.5pg / ml (lOpg / ml. dilution in MEB)

[0265] (6): 1.25pg / ml (5pg / ml. dilution in MEB)

[0266] (7): 0.625pg / ml (2.5pg / ml. dilution in MEB)

[0267] Fluconazole (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 64 pg / ml in the fungal suspension, initial stock of 256 pg / ml in MEB was prepared and further 2- fold diluted using the following serial dilution scheme:

[0268] (1): 64pg / ml (256 pg / ml. prepared in MEB)

[0269] (2): 32pg / ml (128 pg / ml. prepared in MEB)

[0270] (3): 16pg / ml (64 pg / ml. prepared in MEB)

[0271] (4): 8pg / ml (32 pg / ml. prepared in MEB)

[0272] (5): 4pg / ml (16 pg / ml. prepared in MEB)

[0273] (6): 2pg / ml (8pg / ml. dilution in MEB)

[0274] (7): lpg / ml (4pg / ml. dilution in MEB)

[0275] (8): 0.5pg / ml (2pg / ml. dilution in MEB)

[0276] Subsequently the antimicrobial mixes were co-incubated with C. albicans (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0277] Table 5a shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of C. albicans with increasing amounts of Fluometacyl or fluconazole separately or with the combination of both. Gray scale indicates the gradient of the growth, namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of fungal growth inhibition. Concentrations of fluconazole presented in the table 5a are 1 pg / mL. 2 pg / mL. 4 pg / mL. 8 pg / mL. whereas for fluometacyl 0.625 pg / mL. 1.25 pg / mL. 2.5 pg / mL. 5 pg / mL. 10 pg / mL. 20 pg / mL and 40 pg / mL.

[0278] Table 5b shows the FIC values resulted from the table 5a exclusively for the combination of Fluometacyl with fluconazole tested against C. albicans as an example. Concentrations of fluconazole presented in the table are 1 pg / mL. 2 pg / mL. 4 pg / mL and 8 pg / mL whereas for fluometacyl 0.625 pg / mL. 1.25 pg / mL. 2.5 pg / mL. 5 pg / mL. 10 pg / mL. 20 pg / mL and 40 pg / mL.

[0279] Fluometacyl used separately in the entire range of concentrations doesn't impact fungal growth.

[0280] Fluconazole alone at the concentrations equal to 8 pg / mL and more reaches a MIC value. Use of 4 pg / mL of fluconazole didn't display an effect on fungal growth while in the combination with

[0281] Fluometacyl 10-40 pg / mL the growth of C. albicans was affected to reach a partial synergistic effect for 20-40 pg / mL of Fluometacyl. For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0282] Table 5a. The results reported in the table are OD600 values determined as described herein

[0283] Table 5b

[0284] Example 6. Synergic effect of minocycline and levofloxacin against gram-positive bacteria (Staphylococcus aureus {MRSA). Synergy Checkerboard assay. Protocol. Staphylococcus aureus {MRSA. ATCC 6538) was grown overnight (19h) in TSB (tryptic soy broth) medium. Subsequently the culture was lOOx diluted in 4 mL of TSB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Bacterial culture was then diluted lOOx in TSB which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test drugs in 96-well plate according to the following scheme:

[0285] Wells with combination of two antimicrobials: 100 pl of bacteria + 50pl of Fluometacyl / TSB + 50pl of minocycline / TSB

[0286] Wells with one antimicrobial only: lOOpI of bacteria + 50pl of the antimicrobial + 50pl of TSB

[0287] Growth control: lOOpI of bacteria + 50pl of TSB + 50pl of TSB

[0288] - BLANK: TSB only

[0289] Antimicrobials were prepared as follows:

[0290] Minocycline (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 1 pg / ml in the bacterial suspension, initial stock of 4 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0291] (1): lpg / ml (4pg / ml. prepared in TSB)

[0292] (2): 0.5pg / ml (2pg / ml. dilution in TSB)

[0293] (3): 0.25pg / ml (lpg / ml. dilution in TSB)

[0294] (4): 0.125pg / ml (0.5pg / ml. dilution in TSB)

[0295] (5): 0.0625 pg / ml (0.25pg / ml. dilution in TSB)

[0296] (6): 0.03125 pg / ml (0.125pg / ml. dilution in TSB)

[0297] Levofloxacin (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 4 pg / ml in the bacterial suspension, initial stock of 16 pg / ml in TSB was prepared and further 2- fold diluted using the following serial dilution scheme:

[0298] (1): 4pg / ml (16 pg / ml. prepared in TSB)

[0299] (2): 2pg / ml (8pg / ml. dilution in TSB)

[0300] (3): lpg / ml (4pg / ml. dilution in TSB)

[0301] (4): 0.5pg / ml (2pg / ml. dilution in TSB)

[0302] Subsequently the antimicrobial mixes were co-incubated with MRSA (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table. Table 6a shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of S. aureus with increasing amounts of minocycline or levofloxacin separately or with the combination of both. Gray scale indicates the gradient of the growth. Namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of bacterial growth inhibition.

[0303] Concentrations of levofloxacin presented in the table 6a are 0.5 pg / mL. 1 pg / mL and 2 pg / mL. whereas for minocycline 0.03125 pg / mL. 0.0625 pg / mL and 0.125 pg / mL.

[0304] Table 6b shows the FIC values resulted from the table 6a exclusively for the combination of minocycline with levofloxacin tested against S. aureus as an example. Concentrations of levofloxacin presented in the table are 0.5 pg / mL. 1 pg / mL and 2 pg / mL. whereas for minocycline 0.03125 pg / mL and 0.0625 pg / mL.

[0305] For the concentration of minocycline at 0.03125 pg / mL there is no measurable impact on bacterial growth, whereas for 0.125 pg / mL M IC value is observed (table 11). At the concentration of 0.0625 pg / mL antibacterial effect of minocycline is observed which is further potentiated (partial synergy) by addition of levofloxacin at the concentrations 0.5 pg / mL. 1 pg / mL and 2 pg / mL. For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0306] Table 6a. The results reported in the table are OD600 values determined as described herein

[0307] Table 6b

[0308] Example 7. Synergic effect of minocycline and levofloxacin against gram-negative bacteria {Pseudomonas aeruginosa). Synergy Checkerboard assay. Protocol. Pseudomonas aeruginosa {ATCC 15442) was grown overnight (19h) in TSB (tryptic soy broth) medium. Subsequently the culture was lOOx diluted in 4 mL of TSB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Bacterial culture was then diluted lOOx in TSB which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test drugs in 96-well plate according to the following scheme:

[0309] Wells with combination of two antimicrobials: 100 pl of bacteria + 50pl of minocycline / TSB + 50pl of levofloxacin / TSB

[0310] Wells with one antimicrobial only: lOOpI of bacteria + 50pl of the antimicrobial + 50pl of TSB

[0311] Growth control: lOOpI of bacteria + 50pl of TSB + 50pl of TSB

[0312] - BLANK: TSB only

[0313] Antimicrobials were prepared as follows:

[0314] Levofloxacin (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 40 pg / ml in the bacterial suspension, initial stock of 160 pg / ml in TSB was prepared and further 2- fold diluted using the following serial dilution scheme:

[0315] (1): 4pg / ml (16 pg / ml. prepared in TSB)

[0316] (2): 2pg / ml (8pg / ml. dilution in TSB)

[0317] (3): lpg / ml (4pg / ml. dilution in TSB)

[0318] (4): 0.5pg / ml (2pg / ml. dilution in TSB)

[0319] (5): 0.25pg / ml (lpg / ml. dilution in TSB)

[0320] (6): 0.125pg / ml (0.5pg / ml. dilution in TSB)

[0321] Minocycline (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 16 pg / ml in the bacterial suspension, initial stock of 64 pg / ml in TSB was prepared and further 2- fold diluted using the following serial dilution scheme:

[0322] (1): 16pg / ml (64 pg / ml. prepared in TSB)

[0323] (2): 8pg / ml (32 pg / ml. prepared in TSB)

[0324] (3): 4pg / ml (16 pg / ml. prepared in TSB)

[0325] (4): 2pg / ml (8pg / ml. dilution in TSB)

[0326] (5): lpg / ml (4pg / ml. dilution in TSB)

[0327] (6): 0.5pg / ml (2pg / ml. dilution in TSB) Subsequently the antimicrobial mixes were co-incubated with P. aeruginosa (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0328] Table 7a shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of P. aeruginosa with increasing amounts of minocycline or levofloxacin separately or with the combination of both. Gray scale indicates the gradient of the growth, namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of bacterial growth inhibition. Concentrations of minocycline presented in the table 7a are 0.5 pg / mL. 1 pg / mL. 2 pg / mL and 4 pg / mL. whereas for levofloxacin 0.125 pg / mL and 0.25 pg / mL.

[0329] Table 7b shows the FIC values resulted from the table 7a exclusively for the combination of Fluometacyl with minocycline tested against P. aeruginosa as an example. Concentrations of minocycline presented in the table are 0.5 pg / mL. 1 pg / mL. 2 pg / mL. whereas for levofloxacin 0.125 pg / mL.

[0330] Minocycline at the concentrations of 0.5-1 pg / mL doesn't show any significant effect, whereas for 2 pg / mL bacterial growth is somewhat affected with the MIC value reached at 4 pg / mL. Levofloxacin at the concentration of 0.25 pg / mL results in MIC value. Combination of minocycline ranging from 0.5 pg / mL - 2 pg / mL with levofloxacin 0.125 pg / mL results in a significant synergistic effect seen as a full growth inhibition. For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0331] Table 7a. The results reported in the table are OD600 values determined as described herein

[0332] Table 7b

[0333] Example 8. Synergy between Fluometacyl. minocycline, levofloxacin and fluconazole against gram-positive bacteria Staphylococcus aureus {MRSA). Synergy Checkerboard assay.

[0334] Protocol. Staphylococcus aureus {MRSA. ATCC 6538) was grown overnight (19h) in TSB (tryptic soy broth) medium. Subsequently the culture was lOOx diluted in 4 mL of TSB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Bacterial culture was then diluted lOOx in TSB which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test drugs in 96-well plate according to the following scheme:

[0335] Wells with combination of four antimicrobials: 68 pl of bacteria + 33pl of minocycline / TSB + 33pl of levofloxacin / TSB + 33pl of fluconazole / TSB + 33pl of Fluometacyl / TSB Wells with 3 antimicrobials: 68pl of bacteria + 33pl of minocycline / TSB + 33pl of levofloxacin / TSB + 33pl of fluoconazole / TSB + 33pl of TSB

[0336] Wells with 2 antimicrobials: 68pl of bacteria + 33pl of minocycline / TSB and 33pl of levofloxacin / TSB + 33pl of TSB+ 33pl of TSB

[0337] Wells with 1 antimicrobial only: 68pl of bacteria + 33pl of fluconazole / TSB + 33pl of

[0338] TSB + 33pl of TSB + 33pl of TSB

[0339] Growth control: 68pl of bacteria + 33pl of TSB + 33pl of TSB + 33pl of TSB+ 33pl of TSB

[0340] - BLANK: TSB only

[0341] Antimicrobials were prepared as follows:

[0342] Fluometacyl (stock of 4 mg / mL in EtOH 100%) served as a master stock. To obtain the final concentration of 40 pg / ml in the bacterial suspension, initial stock of 240 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0343] (1): 40pg / ml (240pg / ml. prepared in TSB)

[0344] (2): 20pg / ml (120pg / ml. dilution in TSB)

[0345] (3): lOpg / ml (60pg / ml. dilution in TSB)

[0346] (4): 5pg / ml (30pg / ml. dilution in TSB)

[0347] (5): 2.5 pg / ml (15pg / ml. dilution in TSB)

[0348] (6): 1.25 pg / ml (7.5pg / ml. dilution in TSB)

[0349] (7): 0.625 pg / ml (3.75pg / ml. dilution in TSB) Minocycline hydrochloride (stock of 2 mg / mL in H2O mQ) served as a master stock. To obtain the final concentration of 4 pg / ml in the bacterial suspension, initial stock of 24 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0350] (1): 4pg / ml (24 pg / ml) in TSB

[0351] (2): 2pg / ml (12 pg / ml) in TSB

[0352] (3): lpg / ml (6pg / ml) in TSB

[0353] (4): 0.5pg / ml (3pg / ml) in TSB

[0354] (5): 0.25pg / ml (1.5pg / ml) in TSB

[0355] - (6): 0.125pg / ml (0.75pg / ml) in TSB

[0356] - (7): 0.0625pg / ml (0.375pg / ml) in TSB

[0357] Levofloxacin (stock of 2 mg / mL in H2O mQ) served as a master stock. To obtain the final concentration of 4 pg / ml in the bacterial suspension, initial stock of 24 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0358] (1): 4pg / ml (24pg / ml. prepared in TSB)

[0359] (2): 2pg / ml (12pg / ml. prepared in TSB)

[0360] (3): lpg / ml (6pg / ml. prepared in TSB)

[0361] (4): 0.5pg / ml (3pg / ml. prepared in TSB)

[0362] (5): 0.25pg / ml (1.5pg / ml. prepared in TSB)

[0363] (6): 0.125pg / ml (0.75pg / ml. dilution in TSB)

[0364] Fluconazole (stock of 2 mg / mL in H2O mQ) served as a master stock. To obtain the final concentration of 32 pg / ml in the bacterial suspension, initial stock of 192 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0365] (1): 32pg / ml (192pg / ml. dilution in TSB)

[0366] (2): 16pg / ml (96pg / ml. dilution in TSB)

[0367] (3): 8pg / ml (48pg / ml. dilution in TSB)

[0368] Subsequently the antimicrobial mixes were co-incubated with MRSA (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0369] Table 8a shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of S. aureus with fixed amounts amounts of minocycline and levofloxacin used as a backbone for the combination therapy. Following concentrations of minocycline / levofloxacin were used: M0.0625 / 0.125. M0.0625 / 0.25. M0.0625 / 0.5. M0.125 / L0.25 and M0.5 / L0.25. This 2-mer therapy was combined with increasing amount of fluconazole 8 -32 pg / mL and with or without fluometacyl at the concentrations ranging from 0.625 - 5 pg / mL. Gray scale indicates the gradient of the growth, namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of bacterial growth inhibition. Test was run in two separate replicates.

[0370] Table 8b shows the FIC values resulted from the table 8a, tested against S. aureus as an example. Concentrations of the 2-mer mixture of M0.125 / L0.25 and M0.5 / L0.25 were excluded from the FIC calculations since full inhibitory effect was obtained for these concentrations (MIC values).

[0371] From the total FIC values (table 8b) it's noticeable that addition of Fluometacyl at the concentrations of 2.5 - 5 pg / mL to minocycline / levofloxacin M0.0625 / L0.125. M0.0625 / 0.25 and M0.0625 / 0.5 (ratios M / L 1 / 2 to 1 / 8) with fluconazole between 8-32 pg / mL results in the partial synergies. For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0372] Table 8a. The results reported in the table are OD600 values determined as described herein

[0373] Replicate 1:

[0374] Replicate 2: Table 8b

[0375] FIC values for the replicate 1:

[0376] FIC values for the replicate 2: Example 9. Synergy between Fluometacyl. minocycline, levofloxacin and fluconazole against gram-negative bacteria (Pseudomonas aeruginosa). Synergy Checkerboard assay. Protocol. Pseudomonas aeruginosa {ATCC 15442) was grown overnight (19h) in TSB (tryptic soy broth) medium. Subsequently the culture was lOOx diluted in 4 mL of TSB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Bacterial culture was then diluted lOOx in TSB which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test drugs in 96-well plate according to the following scheme:

[0377] Wells with combination of four antimicrobials: 68 pl of bacteria + 33pl of minocycline / TSB + 33pl of levofloxacin / TSB + 33pl of fluconazole / TSB + 33pl of fluometacyl / TSB Wells with 3 antimicrobials: 68pl of bacteria + 33pl of minocycline / TSB + 33pl of levofloxacin / TSB + 33pl of fluoconazole / TSB + 33pl of TSB

[0378] Wells with 2 antimicrobials: 68pl of bacteria + 33pl of minocycline / TSB and 33pl of levofloxacin / TSB + 33pl of TSB+ 33pl of TSB

[0379] Wells with 1 antimicrobial only: 68pl of bacteria + 33pl of fluconazole / TSB + 33pl of

[0380] TSB + 33pl of TSB + 33pl of TSB

[0381] Growth control: 68pl of bacteria + 33pl of TSB + 33pl of TSB + 33pl of TSB+ 33pl of TSB

[0382] - BLANK: TSB only

[0383] Antimicrobials were prepared as follows:

[0384] Fluometacyl (stock of 4 mg / mL in EtOH 100%) served as a master stock. To obtain the final concentration of 40 pg / ml in the bacterial suspension, initial stock of 240 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0385] (1): 40pg / ml (240pg / ml. prepared in TSB)

[0386] (2): 20pg / ml (120pg / ml. dilution in TSB)

[0387] (3): lOpg / ml (60pg / ml. dilution in TSB)

[0388] (4): 5pg / ml (30pg / ml. dilution in TSB)

[0389] (5): 2.5 pg / ml (15pg / ml. dilution in TSB)

[0390] (6): 1.25 pg / ml (7.5pg / ml. dilution in TSB)

[0391] (7): 0.625 pg / ml (3.75pg / ml. dilution in TSB)

[0392] Minocycline hydrochloride (stock of 2 mg / mL in H2O mQ) served as a master stock. To obtain the final concentration of 4 pg / ml in the bacterial suspension, initial stock of 24 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0393] (1): 4pg / ml (24 pg / ml) in TSB

[0394] (2): 2pg / ml (12 pg / ml) in TSB (3): lpg / ml (6pg / ml) in TSB

[0395] (4): 0.5pg / ml (3pg / ml) in TSB

[0396] (5): 0.25pg / ml (1.5pg / ml) in TSB

[0397] - (6): 0.125pg / ml (0.75pg / ml) in TSB

[0398] - (7): 0.0625pg / ml (0.375pg / ml) in TSB

[0399] Levofloxacin (stock of 2 mg / mL in H2O mQ) served as a master stock. To obtain the final concentration of 4 pg / ml in the bacterial suspension, initial stock of 24 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0400] (1): 4pg / ml (24pg / ml. prepared in TSB)

[0401] (2): 2pg / ml (12pg / ml. prepared in TSB)

[0402] (3): lpg / ml (6pg / ml. prepared in TSB)

[0403] (4): 0.5pg / ml (3pg / ml. prepared in TSB)

[0404] (5): 0.25pg / ml (1.5pg / ml. prepared in TSB)

[0405] (6): 0.125pg / ml (0.75pg / ml. dilution in TSB)

[0406] Fluconazole (stock of 2 mg / mL in H2O mQ) served as a master stock. To obtain the final concentration of 32 pg / ml in the bacterial suspension, initial stock of 192 pg / ml in TSB was prepared and further 2-fold diluted using the following serial dilution scheme:

[0407] (1): 32pg / ml (192pg / ml. dilution in TSB)

[0408] (2): 16pg / ml (96pg / ml. dilution in TSB)

[0409] (3): 8pg / ml (48pg / ml. dilution in TSB)

[0410] Subsequently the antimicrobial mixes were co-incubated with P. aeruginosa (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0411] Table 9a shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of P. aeruginosa with fixed amounts amounts of minocycline and levofloxacin used as a backbone for the 4-mer combination therapy. Following concentrations of minocycline / levofloxacin were used: M0.0625 / 0.25. M0.0625 / 0.5 and M0.5 / L0.25. This 2-mer therapy was combined with increasing amount of fluconazole 8 - 32 pg / mL and with or without Fluometacyl at the concentrations ranging from 0.625 - 5 pg / mL. Gray scale indicates the gradient of the growth. namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of bacterial growth inhibition.

[0412] Table 9b shows the FIC values resulted from the table 9a, tested against P. aeruginosa as an example.

[0413] From the total FIC values (table 9b) it's noticeable that for the following 2-mer therapy minocycline / levofloxacin M0.0625 / 0.5. for which there is no visible effect on bacterial growth, addition of Fluometacyl at the concentrations ranging from 2.5 to 5 pg / mL results in synergistic or partial synergistic effects. Fluconazole used at the concentrations of 8-32 pg / mL doesn't seemingly impact the observations. Combining duotherapy M0.0625 / L0.5 with fluconazole 16 pg / mL gave an unexpected result which is considered as an error and excluded from the other data. For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0414] Table 9a. The results reported in the table are OD600 values determined as described herein. Monotherapy (mono) and duotherapy (duo) indicate respectively X-axis for fluconazole and Y- axis for minocycline / levofloxacin.

[0415] *Error

[0416] Table 9b. FIC values Example 10 Summary

[0417] MIC values were determined for antimicrobials alone and in combination as described above.

[0418] Mino refers to minocycline; Levo for Levofloxacin; Fluo for Fluometacyl; Fluco for Fluconazone. For some combinations, a partial bacterial growth inhibition was observed which did not correspond to MIC definition (indicated as not MIC). In this case, FIC index values were calculated based on OD600 values (see Tables 1-9) and they are not reported in the Table. For fungi, the tested agents did not inhibit fungal growth when tested alone (no MIC), and FIC values were not included either.

[0419] S. aureus:

[0420] A FIC index of 0.375 was calculated for the combination of minocycline with levofloxacin, which indicates a synergy.

[0421] A FIC index of 0.28 was calculated for the combination of Fluometacyl with minocycline, which indicates a synergy.

[0422] Combining levofloxacin (4mg / L) with Fluometacyl (5mg / L) potentiated bacterial growth inhibition by Fluometacyl . FIC index= 0.75.

[0423] P. aeruginosa:

[0424] A FIC index of 0.625 was calculated for the combination of minocycline with levofloxacin, which indicates a partial synergy.

[0425] The combination of Fluometacyl (10 mg / L) with minocycline (4 mg / L) partially inhibited bacterial growth, while the antimicrobials alone did not affect bacterial growth (Fluometacyl up to 100 mg / L and minocycline up to 8 mg / L).

[0426] Candida albicans:

[0427] The combination of Fluometacyl (40 mg / L) with minocycline (32 mg / L) partially inhibited fungal growth, while the antimicrobials alone had no effect (Fluometacyl up to 100 mg / L and minocycline up to 32 mg / L).

[0428] The combination of Fluometacyl (20-40 mg / L) with fluconazole (4 mg / L) potentiated fluconazole activity (2-fold reduced MIC), , while Fluometacyl alone had no effect (Fluometacyl up to 100 mg / L ).

[0429] Example 11a: Method of production of an antimicrobial catheter comprising the antimicrobial composition of the present invention by impregnation followed by nanogel coating

[0430] 1.1 Catheter impregnation with one antimicrobial: Minocycline, was dissolved in 100% ethanol (Analytical reagent grade, Fisher Scientific). Polyurethane catheters (20 cm-long central venous catheters and 55 cm-long peripherally inserted central catheters from Teleflex) were first impregnated in a solution of minocycline (lOmg / ml) for 30min. After impregnation, catheters were air flushed to remove any excess of solution from the lumens of the catheters, dried at 37°C during 48h, washed with water, and dried again.

[0431] 1.2 Preparation of the nanogel called NFT with P(mDOPA) Fluometacyl® and TPGS.

[0432] The Nanogel is prepared in liquid solution. After crosslinking the nanogel remains suspended in the liquid solution.

[0433] A homopolymer of methacrylamide bearing 3,4-dihydroxy-L-phenylalanine (P(mDOPA) is synthetized according to Faure & al in Adv Funct. Mater. 2012; 22:5271-5282, and is oxidized in aqueous media under basic conditions for 12 hours to form hydrosoluble Pox(mDOPA). Oxidized catechol moieties of Pox(mDOPA) are necessary for the covalent interaction of PAH through amine / quinone reaction and / or Schiff base formation at room temperature, and consequently for the preparation of stable cross-linked nanogel in water suspension.

[0434] P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL) and NaOH (0.1 M) was slowly added in order to raise the pH above 10 and to promote the oxidation of catechol groups of P(mDOPA).

[0435] 1.2.2 preparation of Fluometacyl / TPGS mixture in ethanol

[0436] Fluometacyl (prepared according to Eur J Med Chem 2020 Dec 15:208:112767) and TPGS provided by MedChemExpress LLC were dissolved separately in ethanol to prepare stocks solution of 3,33 mg / mL. 160 pL of Fluometacyl solution and 160 pl of TPGS solution were mixed under stirring (300 rpm) with a magnetic stirrer and then concentrated to about 160pl under vacuum at RT.

[0437] 1.2.3 preparation of the NFT nanogel with Fluometacyl / TPGS and PAH

[0438] Pox(mDOPA) (5ml, 0.5mg / ml) was added under stirring (300 rpm) with a magnetic stirrer, to the concentrated mixture obtained at point 1.2.2 After one hour of homogenization at RT, an aqueous solution of PAH provided from Sigma Aldrich (0.5 mL, 0.5 mg / ml) at pH 10 was slowly added to the mixture solution. The solution was allowed to react for one hour at room temperature under vigorous stirring (500 rpm) with a magnetic stirrer. Nanogels with a diameter ranging from lOOnm to 350 nm were observed in water suspension by Dynamic Light Spectrometry (Zetasizer Advance Pro, Malvern).

[0439] 1.3 Coating of the impregnated catheters with the NET nanogel

[0440] Impregnated polyurethane catheter prepared in the point 1.1, was coated according to the method of the invention.

[0441] In Step 1: catheters were dipped into a Dopamine Tris buffer solution pH=7,4 from Aldrich (0.2 g L-l) for 3h.

[0442] In Step 2: After rinsing twice with water, the modified catheter substrate was dipped into an aqueous solution of PAH (pH>10) for 30min and rinsed twice with water.

[0443] In step 3: The modified catheter substrate obtained in step 2 was dipped into an aqueous solution of the bioactive molecule-loaded nanogels prepared according to point 1.2 for 18h and rinsed twice with water.

[0444] Steps 2 to 3 were repeated to build-up a multilayer assembly of nanogels on the surface of coated device (five times to obtain a five-layer assembly of cross-linked nanogels).

[0445] Example lib: Method of production of an antimicrobial catheter comprising the antimicrobial composition of the present invention by impregnation followed by nanogel coating

[0446] 1.1 Catheter impregnation with three antimicrobials:

[0447] Minocycline, Fluconazole and Levofloxacin were dissolved in 100% ethanol (Analytical reagent grade, Fisher Scientific). Polyurethane catheters (20 cm-long central venous catheters and 55 cm-long peripherally inserted central catheters from Teleflex) were first impregnated in a solution of minocycline (lOmg / ml) for 30min and then continued to be impregnated with a mixture of Fluconazole (lOmg / ml) and Levofloxacin (10 mg / ml) for an additional 30min. After impregnation, catheters were air flushed to remove any excess of solution from the lumens of the catheters, dried at 37°C during 48h, washed with water, and dried again.

[0448] 1.2 Preparation of the nanogel called NET with P(mDOPA) Fluometacyl® and TPGS.

[0449] The Nanogel is prepared in liquid solution. After crosslinking the nanogel remains suspended in the liquid solution. A homopolymer of methacrylamide bearing 3,4-dihydroxy-L-phenylalanine (P(mDOPA) is synthetized according to Faure & al in Adv Funct. Mater. 2012; 22:5271-5282, and is oxidized in aqueous media under basic conditions for 12 hours to form hydrosoluble Pox(mDOPA). Oxidized catechol moieties of Pox(mDOPA) are necessary for the covalent interaction of PAH through amine / quinone reaction and / or Schiff base formation at room temperature, and consequently for the preparation of stable cross-linked nanogel in water suspension.

[0450] P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL) and NaOH (0.1 M) was slowly added in order to raise the pH above 10 and to promote the oxidation of catechol groups of P(mDOPA).

[0451] 1.2.2 preparation of Fluometacyl / TPGS mixture in ethanol

[0452] Fluometacyl (prepared according to Eur J Med Chem 2020 Dec 15:208:112767) and TPGS provided by MedChemExpress LLC were dissolved separately in ethanol to prepare stocks solution of 3,33 mg / mL. 160 pL of Fluometacyl solution and 160 pl of TPGS solution were mixed under stirring (300 rpm) with a magnetic stirrer and then concentrated to about 160pl under vacuum at RT.

[0453] 1.2.3 preparation of the NFT nanogel with Fluometacyl / TPGS and PAH

[0454] Pox(mDOPA) (5ml, 0.5mg / ml) was added under stirring (300 rpm) with a magnetic stirrer, to the concentrated mixture obtained at point 1.2.2 After one hour of homogenization at RT, an aqueous solution of PAH provided from Sigma Aldrich (0.5 mL, 0.5 mg / ml) at pH 10 was slowly added to the mixture solution. The solution was allowed to react for one hour at room temperature under vigorous stirring (500 rpm) with a magnetic stirrer.

[0455] Nanogels with a diameter ranging from lOOnm to 350 nm were observed in water suspension by Dynamic Light Spectrometry (Zetasizer Advance Pro, Malvern).

[0456] 1.3 Coating of the impregnated catheters with the NFT nanogel

[0457] Impregnated polyurethane catheter prepared in the point 1.1, was coated according to the method of the invention.

[0458] In Step 1: catheters were dipped into a Dopamine Tris buffer solution pH=7,4 from Aldrich (0.2 g L-l) for 3h.

[0459] In Step 2: After rinsing twice with water, the modified catheter substrate was dipped into an aqueous solution of PAH (pH>10) for 30min and rinsed twice with water. In step 3: The modified catheter substrate obtained in step 2 was dipped into an aqueous solution of the bioactive molecule-loaded nanogels prepared according to point 1.2 for 18h and rinsed twice with water.

[0460] Steps 2 to 3 were repeated to build-up a multilayer assembly of nanogels on the surface of coated device (five times to obtain a five-layer assembly of cross-linked nanogels).

[0461] Example 12: Method of Production of an antimicrobial catheter comprising the antimicrobial composition of the present invention by impregnation followed by polymer coating

[0462] 2.1 Catheters impregnation: Impregnation of polyurethane catheters (Teleflex, CVCs and PICCs) with a novel combination of three antimicrobial compounds. Briefly, Minocycline, Fluconazole and Levofloxacin were dissolved in ethanol (Analytical reagent grade, Fisher Scientific). Polyurethane catheters (CVCs (20 cm) and PICCs (55cm) in length) were first impregnated in a solution of Minocycline (lOmg / ml) for 30min and then continued to be impregnated with a mixture of Fluconazole (lOmg / ml) and Levofloxacin (10 mg / ml) for an additional 30min. After impregnation, catheters were air flushed to remove any excess coating solution from the lumens of the catheters, dried at 37°C during 48h, washed with water, and dried again.

[0463] 2.2 Preparation of the polymer solution with P(mDOPA) Fluometacyl® and TPGS (PFT)

[0464] The PFT solution is prepared in aqueous media similar to NFT but without adding a crosslinking agent (PAH).

[0465] A homopolymer of methacrylamide bearing 3,4-dihydroxy-L-phenylalanine (P(mDOPA) is synthetized according to Faure & al in Adv Funct. Mater. 2012; 22:5271-5282, and is oxidized in aqueous media under basic conditions for 12 hours to form hydrosoluble Pox(mDOPA). Oxidized catechol moieties of Pox(mDOPA) are necessary for the covalent interaction of PAH through amine / quinone reaction and / or Schiff base formation at room temperature, and consequently for the preparation of stable cross-linked layer-by-layer (LBL) coating on the surface of medical devices.

[0466] P(mDOPA) (2.5 mg) was dissolved in distilled water (5 mL) and NaOH (0.1 M) was slowly added in order to raise the pH above 10 and to promote the oxidation of catechol groups of P(mDOPA).

[0467] 2.2.2 preparation of Fluometacyl / TPGS mixture in ethanol Fluometacyl (prepared according to Eur J Med Chem 2020 Dec 15:208:112767) and TPGS provided by MedChemExpress LLC were dissolved separately in ethanol to prepare stocks solution of 3,33 mg / mL. 160 pL of Fluometacyl solution and 160 pl of TPGS solution were mixed under stirring (300 rpm) with a magnetic stirrer and then concentrated to about 160pl under vacuum at RT.

[0468] 2.2.3 preparation of the PFT solution with Pox(mDOPA) and Fluometacyl / TPGS

[0469] Pox(mDOPA) (5ml, 0.5mg / ml) was added under stirring (300 rpm) with a magnetic stirrer, to the concentrated mixture obtained at point 2.2.2. The solution was allowed to react for one hour at room temperature under vigorous stirring (500 rpm) with a magnetic stirrer.

[0470] 2.3 Coating of the impregnated catheters with the PFT solution

[0471] Impregnated polyurethane catheter prepared in the point 2.1, was coated according to the method of the invention.

[0472] In Step 1: catheters were dipped into a Dopamine Tris buffer solution pH=7,4 from Aldrich (0.2 g L-l) for 3h.

[0473] In Step 2: After rinsing twice with water, the modified catheter substrate was dipped into an aqueous solution of PAH (pH>10) for 30min and rinsed twice with water.

[0474] In step 3: The modified catheter substrate obtained in step 2 was dipped into a PFT solution prepared according to point 2.2 for 18h and rinsed twice with water.

[0475] Steps 2 to 3 were repeated to build-up a multilayer assembly of PFT loaded bioactive molecules on the surface of coated device (five times to obtain a five-layer assembly of cross-linked LBL coating).

[0476] Example 13: Method of Production of an antimicrobial catheter comprising the antimicrobial composition of the present invention by impregnation followed by polymer or nanogel coating then dipping in a solution of Minocycline

[0477] 3.1 Catheters impregnation:

[0478] Impregnation of polyurethane catheters (Teleflex, CVCs and PICCs) with a novel combination of three antimicrobial compounds. Briefly, Minocycline, Fluconazole and Levofloxacin were dissolved in ethanol (Analytical reagent grade, Fisher Scientific). Polyurethane catheters (CVCs (20 cm) and PICCs (55cm) in length) were first impregnated in a solution of minocycline(10mg / ml) for 30min and then continued to be impregnated with a mixture of Fluconazole (lOmg / ml) and Levofloxacin (10 mg / ml) for an additional 30min. After impregnation, catheters were air flushed to remove any excess coating solution from the lumens of the catheters, dried at 37°C during 48h, washed with water, and dried again.

[0479] 3.2 Coating of the impregnated catheters with NFT or PFT

[0480] Impregnated polyurethane catheter prepared in the point 3.1, was coated according to the method of the invention.

[0481] In Step 1: catheters were dipped into a Dopamine Tris buffer solution pH=7,4 from Aldrich (0.2 g L-l) for 3h.

[0482] In Step 2: After rinsing twice with water, the modified catheter substrate was dipped into an aqueous solution of PAH (pH>10) for 30min and rinsed twice with water.

[0483] In step 3: The modified catheter substrate obtained in step 2 was dipped into an aqueous solution of the bioactive molecule-loaded nanogels prepared according to point 1.2 or into a PFT solution prepared according to point 2.2 for 18h and rinsed twice with water.

[0484] Steps 2 to 3 were repeated to build-up a multilayer assembly on the surface of coated device (five times to obtain a five-layer assembly of cross-linked coating).

[0485] 3.3 dipping of Coated catheters in a solution of minocycline

[0486] 3.3.1 preparation of the solution of minocycline in H2O Minocycline provided by Merck was dissolved in H2O to prepare a stock solution of lOmg / mL.

[0487] 3.3.1 clipping of coated catheters in the solution of minocycline

[0488] Impregnated and coated polyurethane catheter prepared in the point 3.2, was dipped into the minocycline solution during 5 min at room temperature without any agitation. After dipping, catheters were air flushed to remove any excess solution from the lumens of the catheters, dried at room temperature during 48h.

[0489] Example 14: Inhibition of bacterial and fungal growth by catheters impregnated with a combination of minocycline, fluconazole and levofloxacin and coated with nanogel NFT.

[0490] Tryptic soy agar (TSA) - Agar plates are prepared following the provider's instructions in sterile Petri dishes prior to testing. The optimal thickness of the solid agar medium is 3-4 mm.

[0491] Bacterial cultures - Individual bacterial colonies are picked up and inoculated in 3 mL of TSB using an inoculation loop. Bacteria are grown at 37°C for 18 h (overnight culture) in tryptic soy broth (TSB; prepared following the provider instructions) to a concentration of 108-109CFU / mL. Working suspension of bacteria 103-104CFU / mL used in this test is made by dilution of the overnight culture in TSB medium.

[0492] Fungal (C. albicans) cultures- Individual fungal colonies are picked up and inoculated in 3 mL of malt extract broth (MEB; prepared following the provider instructions) using an inoculation loop. Fungi are grown at 37°C for 24 h (overnight culture) MEB; prepared following the provider instructions) to a concentration of 108-109CFU / mL.

[0493] Working suspension of fungi 102CFU / mL used in this test is made by dilution of the overnight culture in MEB medium.

[0494] Test

[0495] 1. Petri dishes with tryptic soy agar (TSA; for bacteria) or malt extract agar (MEA; for fungi) are air dried under the laminar hood.

[0496] 3. Diluted bacterial suspension (S aureus: lOOOx; P. aeruginosa:10 OOOx) or fungal suspension (C. albicans: lOOx) are homogenized by vortexing.

[0497] 4. 3 mL or 4 mL of bacterial / fungal suspension is plated on agar medium (10- cm dishes and 12xl2-cm dishes, respectively).

[0498] 5. Bacteria or fungi suspension are left few seconds before aspirating the excess of liquid.

[0499] 6. 0.5-cm segments of catheters are placed vertically into the agar using sterile tweezers. This is done in a one firm action to avoid redundant side moving of the catheter in the agar.

[0500] 7. Bacterial / fungal plates with the catheters are incubated at 37 °C for 24 h.

[0501] 8. The size of the zone of inhibition (ZOI) for each of the tested catheter is assessed by measuring the diameter of the clear zone perpendicular to the long axis of the catheter segment. Data represents mm of zone of microbial growth inhibition.

[0502] 9. The catheters are then transferred into a new agar plate with freshly seeded bacteria / fungi and incubated at 37 °C for an additional 24h.

[0503] 10. Steps 8 and 9 are repeated until the zones of inhibition around the catheter are not visible (diameter of the clear zone reduced to 0 mm) to monitor the duration of catheter antimicrobial activity. Example 15: Inhibition of bacterial and fungal growth by catheters impregnated with a combination of minocycline, fluconazole and levofloxacin and coated with nanogel comprising Fluometacyl and TPGS (NFT).

[0504] In this example, catheters were impregnated with minocycline (lOmg / ml prepared in ethanol), fluconazole and levofloxacin (15 mg / ml prepared in ethanol) and coated with NFT (3 layers of nanogels prepared with 0.2 mg / mL Fluometacyl and 0.4 mg / mL TPGS) as described in example 11.

[0505] In Figure 1, the zone of inhibition of growth of methicillin-resistant S. aureus (MRSA) is expressed as diameter of the clear zone perpendicular to the long axis of the catheter segment in mm. The diamonds stand for the antimicrobial activity of impregnated / coated catheters at indicated time, and squares represent control uncoated catheter.

[0506] Impregnated / coated catheters exhibit a zone of inhibition for up to 12 days, indicating antibacterial activity. On the first day the zone of inhibition had a size of 10 mm, which slowly decreased over time until reaching around 2 mm after 12 days. No activity was detected at 13 days.

[0507] Figure 2 shows the zones of inhibition obtained with P. aeruginosa. The diamonds stand for the antimicrobial activity of impregnated / coated catheters at indicated time, and squares represent control uncoated catheter.

[0508] Impregnated / coated catheters exhibit a zone of inhibition for at least 20 days. On the first day the zone of inhibition had a size of 16.5 mm, which remained stable over time until reaching around 13 mm after 20 days.

[0509] Figure 3 shows the zone of inhibition obtained with C. albicans. The diamonds stand for the antimicrobial activity of impregnated / coated catheters at indicated time, and squares represent control uncoated catheter.

[0510] Impregnated / coated catheters exhibit a zone of inhibition for up to 6 days. On the first day the zone of inhibition had a size of 7 mm, which decreased over time until reaching around 2.5 mm after 6 days. No activity was detected at 7 days.

[0511] Overall, these data show that catheters containing the antimicrobial composition of the present invention inhibit the growth of methicillin-resistant S. aureus, P. aeruginosa and C. albicans.

[0512] Example 16 Synergic effect of triafluocyl and fluconazole against fungi {Candida albicans). Synergy Checkerboard assay. Protocol. Candida albicans 3147 {ATCC 10231D) was grown overnight (24h) in MEB (malt extract broth) medium. Subsequently the culture was 50x diluted in 4 mL of MEB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Fungal culture was then diluted lOOx in RPMI which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test antimicrobials in 96-well plate according to the following scheme:

[0513] Wells with combination of two antimicrobials: 100 pl of fungi + 50pl of triafluocyl / RPMI + 50pl of fluconazole / RPMI

[0514] Wells with one antimicrobial only: lOOpI of fungi + 50pl of the antimicrobial + 50pl of RPMI

[0515] Growth control: lOOpI of fungi + 50pl of RPMI + 50pl of RPMI

[0516] - BLANK: RPMI only

[0517] Antimicrobials were prepared as follows:

[0518] Triafluocyl(4 mg / mL in 100% EtOH) served as a master stock. To obtain the final concentration of 40 pg / ml in the fungal suspension, initial stock of 160 pg / ml in RPMI was prepared and further 2-fold diluted using the following serial dilution scheme:

[0519] (1): 40pg / ml (160pg / ml. prepared in RPMI)

[0520] (2): 20pg / ml (80pg / ml. dilution in RPMI)

[0521] (3): lOpg / ml (40pg / ml. dilution in RPMI)

[0522] (4): 5pg / ml (20pg / ml. dilution in RPMI)

[0523] (5): 2.5pg / ml (lOpg / ml. dilution in RPMI)

[0524] Fluconazole (2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 64 pg / ml in the fungal suspension, initial stock of 256 pg / ml in RPMI was prepared and further 2- fold diluted using the following serial dilution scheme:

[0525] (1): 64pg / ml (256 pg / ml. prepared in MEB)

[0526] (2): 32pg / ml (128 pg / ml. prepared in MEB)

[0527] (3): 16pg / ml (64 pg / ml. prepared in MEB)

[0528] (4): 8pg / ml (32 pg / ml. prepared in MEB)

[0529] (5): 4pg / ml (16 pg / ml. prepared in MEB)

[0530] (6): 2pg / ml (8pg / ml. dilution in MEB)

[0531] (7): lpg / ml (4pg / ml. dilution in MEB) Subsequently the antimicrobial mixes were co-incubated with C. albicans (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0532] Table 11 shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of C. albicans with increasing amounts of triafluocyl or fluconazole separately or with the combination of both. Gray scale indicates the gradient of the growth, namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of fungal growth inhibition. Concentrations of fluconazole presented in the table 19 are

[0533] 1 pg / mL. 2 pg / mL, 4 pg / mL and 8 pg / mL, whereas for triafluocyl 2.5 pg / mL. 5 pg / mL. 10 pg / mL. 20 pg / mL and 40 pg / mL.

[0534] Table 12 shows the FIC values resulted from the table 19 exclusively for the combination of trafluocyl with fluconazole tested against C. albicans as an example. Concentrations of fluconazole presented in the table are 1 pg / mL. 2 pg / mL (as for 4 pg / mL and 8 pg / mL inhibitory effect was observed), whereas for triafluocyl 2.5 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL and 40 pg / mL.

[0535] Triafluocyl used separately in the entire range of concentrations doesn't impact fungal growth. Fluconazole alone at the concentrations equal to 4 pg / mL and more reaches a MIC value. Use of

[0536] 2 pg / mL of fluconazole didn't display a strong effect on fungal growth while in the combination with triafluocyl 20-40 pg / mL the growth of C. albicans was affected to reach a partial synergistic effect for 40 pg / mL of triafluocyl. Similarly, the strong synergistic effect (partial synergy) was obtained for fluconazole 1 pg / mL combined with triafluocyl at 40 pg / mL. For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0537] Table 11. The results in the table are reported as OD600 values determined as described herein Table 12. FIC values. Combinations with synergistic effects are indicated.

[0538] Example 17. Synergic effect of triafluocyl (Tria)and voriconazole against fungi {Candida albicans). Synergy Checkerboard assay.

[0539] Protocol. Candida albicans 3147 {ATCC 10231D) was grown overnight (24h) in MEB (malt extract broth) medium. Subsequently the culture was 50x diluted in 4 mL of MEB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Fungal culture was then diluted lOOx in RPMI which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test antimicrobials in 96-well plate according to the following scheme:

[0540] Wells with combination of two antimicrobials: 100 pl of fungi + 50pl of triafluocyl / RPMI + 50pl of voriconazole / RPMI

[0541] Wells with one antimicrobial only: lOOpI of fungi + 50pl of the antimicrobial + 50pl of RPMI

[0542] Growth control: lOOpI of fungi + 50pl of MEB + 50pl of RPMI

[0543] - BLANK: RPMI only

[0544] Antimicrobials were prepared as follows:

[0545] Triafluocyl (4 mg / mL in 100% EtOH) served as a master stock. To obtain the final concentration of 40 pg / ml in the fungal suspension, initial stock of 160 pg / ml in RPMI was prepared and further 2-fold diluted using the following serial dilution scheme:

[0546] (1): 40pg / ml (160pg / ml. prepared in RPMI)

[0547] (2): 20pg / ml (80pg / ml. dilution in RPMI)

[0548] (3): lOpg / ml (40pg / ml. dilution in RPMI)

[0549] (4): 5pg / ml (20pg / ml. dilution in RPMI)

[0550] (5): 2.5pg / ml (lOpg / ml. dilution in RPMI)

[0551] Voriconazole (VZL, 2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 4 pg / ml in the fungal suspension, initial stock of 16 pg / ml in RPMI was prepared and further 2-fold diluted using the following serial dilution scheme: (1): 4pg / ml (16 pg / ml prepared in RPMI)

[0552] (2): 2pg / ml (8pg / ml dilution in RPMI)

[0553] (3): lpg / ml (4pg / ml dilution in RPMI)

[0554] (4): 0.5pg / ml (2pg / ml dilution in RPMI)

[0555] (5): 0.25pg / ml (lpg / ml dilution in RPMI)

[0556] (6): 0.125pg / ml (0.5pg / ml dilution in RPMI)

[0557] (7): 0.0625pg / ml (0.25pg / ml dilution in RPMI)

[0558] (8): 0.03125pg / ml (0.125pg / ml dilution in RPMI)

[0559] Subsequently the antimicrobial mixes were co-incubated with C. albicans (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0560] Table 13 shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of C. albicans with increasing amounts of triafluocyl or voriconazole separately or with the combination of both. Gray scale indicates the gradient of the growth, namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of fungal growth inhibition. Concentrations of voriconazole presented in the table 20 are 0.03125 pg / mL. 0.0625 pg / mL, 0.125 pg / mL, 0.25 pg / mL and 0.5 pg / mL, whereas for triafluocyl 2.5 pg / mL. 5 pg / mL. 10 pg / mL. 20 pg / mL and 40 pg / mL.

[0561] Table 14 shows the FIC values resulted from the table 23 exclusively for the combination of triafluocyl with voriconazole tested against C. albicans as an example. Concentrations of voriconazole presented in the table are 0.03125 pg / mL and 0.0625 pg / mL (as for 0.125 pg / mL MIC was reached), whereas for triafluocyl 2.5 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL and 40 pg / mL.

[0562] Triafluocyl used separately in the entire range of concentrations doesn't impact fungal growth. Voriconazole alone at the concentration equal to 0.125 pg / mL reaches a MIC value. Use of 0.0625 pg / mL of voriconazole didn't display a strong effect on fungal growth while in the combination with triafluocyl 20-40 pg / mL the growth of C. albicans was affected to reach a synergistic effect (partial synergy) for 20 pg / mL of triafluocyl. Further increase of the concentration of triafluocyll to 40 pg / mL potentiated the synergistic effect approaching the threshold for the synergy. For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0563] Table 13. The results in the table are reported as OD600 values determined as described herein

[0564] Table 14. FIC values. Combinations with synergistic effects are indicated.

[0565] Example 18: Synergic effect of Fluometacyl and voriconazole against fungi (Candida albicans). Synergy Checkerboard assay.

[0566] Protocol. Candida albicans 3147 (ATCC 10231D) was grown overnight (24h) in MEB (malt extract broth) medium. Subsequently the culture was 50x diluted in 4 mL of MEB and grown at 37C under agitation 200 rpm until OD600 reached 0.5. Fungal culture was then diluted lOOx in RPMI which corresponds to the range of 5 x 104- 5 x 105CFU / ml and mixed together with test anntimcrobials in 96-well plate according to the following scheme:

[0567] Wells with combination of two antimicrobials: 100 pl of fungi + 50pl of Fluometacyl / RPMI + 50pl of voriconazole / RPMI

[0568] Wells with one antimicrobial only: lOOpI of fungi + 50pl of the antimicrobial + 50pl of RPMI

[0569] Growth control: lOOpI of fungi + 50pl of MEB + 50pl of RPMI

[0570] - BLANK: RPMI only antimicrobials were prepared as follows:

[0571] Fluometacyl (4 mg / mL in 100% EtOH) served as a master stock. To obtain the final concentration of 40 pg / ml in the fungal suspension, initial stock of 160 pg / ml in RPMI was prepared and further 2-fold diluted using the following serial dilution scheme:

[0572] (1): 40pg / ml (160pg / ml prepared in RPMI)

[0573] (2): 20pg / ml (80pg / ml dilution in RPMI)

[0574] (3): lOpg / ml (40pg / ml dilution in RPMI)

[0575] (4): 5pg / ml (20pg / ml dilution in RPMI)

[0576] (5): 2.5pg / ml (lOpg / ml dilution in RPMI)

[0577] Voriconazole (VZL, 2 mg / mL in H2O) served as a master stock. To obtain the final concentration of 4 pg / ml in the fungal suspension, initial stock of 16 pg / ml in RPMI was prepared and further 2-fold diluted using the following serial dilution scheme:

[0578] (1): 4pg / ml (16 pg / ml prepared in RPMI)

[0579] (2): 2pg / ml (8pg / ml dilution in RPMI)

[0580] (3): lpg / ml (4pg / ml dilution in RPMI)

[0581] (4): 0.5pg / ml (2pg / ml dilution in RPMI)

[0582] (5): 0.25pg / ml (lpg / ml dilution in RPMI)

[0583] (6): 0.125pg / ml (0.5pg / ml dilution in RPMI)

[0584] (7): 0.0625pg / ml (0.25pg / ml dilution in RPMI)

[0585] (8): 0.03125pg / ml (0.125pg / ml dilution in RPMI)

[0586] - (9): 0.015625pg / ml (0.03125pg / ml dilution in RPMI)

[0587] Subsequently the antimicrobial mixes were co-incubated with C. albicans (5 x 104- 5 x 105CFU / ml) and growth was measured at the endpoint after incubation at 37°C for 24 hours under shaking at 200 rpm. The difference between ODgoo at timepoint 0 h and after 24 hours was determined as AOD600 and presented in the table.

[0588] Tablel5 The results in the table are reported as OD600 values determined as described herein.

[0589] Tablel6 FIC values. Combinations with synergistic effects are indicated.

[0590] Table 15 shows the heat map of AOD600 for the measurement at t=Oh and t=24h of growth of C. albicans with increasing amounts of Fluometacyl or voriconazole separately or with the combination of both. Gray scale indicates the gradient of the growth, namely strong gray stands for normal unaffected growth whereas the gradually decreasing intensity of gray color indicates increase of fungal growth inhibition. Concentrations of voriconazole presented in the table 20 are 0.015625 pg / mL, 0.03125 pg / mL, 0.0625 pg / mL, 0.125 pg / mL, and 0.25 pg / mL, whereas for Fluometacyl 2.5 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL and 40 pg / mL.

[0591] Table 16 shows the FIC values resulted from the table 15 exclusively for the combination of Fluometacyl with voriconazole tested against C. albicans as an example. Concentrations of voriconazole presented in the table are 0.015625 pg / mL and 0.03125 pg / mL (as for 0.0625 pg / mL MIC was reached), whereas for Fluometacyl 2.5 pg / mL, 5 pg / mL, 10 pg / mL, 20 pg / mL and 40 pg / mL.

[0592] Fluometacyl used separately in the entire range of concentrations does not impact fungal growth. In this example, voriconazole alone at the concentration equal to 0.0625 pg / mL reaches a MIC value. Use of 0.015625 pg / mL of voriconazole did not display a strong effect on fungal growth while in the combination with Fluometacyl 20-40 pg / mL the growth of C. albicans was affected to reach a synergistic effect for 40 pg / mL and a partial synergy for 20 pg / mL Fluometacyl. In the presence of 0.03125 pg / mL of voriconazole, Fluometacyl displayed a synergistic effect at 20-40 pg / mL. For the other ratios of the antimicrobials there is additive or indifferent effect of the combination.

[0593] Example 19: summary for Candida albicans

[0594] Table 17 below shows superiority of using a combination therapy over a monotherapy seen as decrease of MIC values of individual antimicrobials used in the combination with the other antimicrobial. This finds an implication in combining many known antimicrobials together rather than discovering new chemical entities.

[0595] Table 17. Summary of MIC values of antifungal {Candida albicans) used in monotherapy and in combination therapies. Fluo refers to Fluometacyl, Fluco to Fluconazole, Mino to minocycline and vorico to voriconazole.

[0596] Wherein >100 corresponds to NO MIC or no antibacterial activity according to the EUCAST

[0597] And N / A signifies not applicable since there is no MIC for one of the antimicrobials.

[0598] One can therefore obtain a synergistic antimicrobial combination against C. albicans by mixing voriconazole with triafluocyl in an antimicrobial composition containing triafluocyl at a final concentration of 10 to 20 mg / L and voriconazole at a final concentration corresponding to half of its MIC. Similarly, one can obtain a synergistic antimicrobial combination against C. albicans by mixing voriconazole with Fluometacyl in an antimicrobial composition containing Fluometacyl at a final concentration of 20 to 40 mg / L and voriconzole at a final concentration corresponding to a quarter of its MIC. One can therefore obtain a synergistic antimicrobial combination against C. albicans by mixing fluconazole with triafluocyl in an antimicrobial composition containing triafluocyl at a final concentration of 40 mg / L and fluconazole at a final concentration corresponding to a quarter of its MIC.

[0599] Similarly, one can therefore obtain a synergistic antimicrobial combination against C. albicans by mixing fluconazole with Fluometacyl in an antimicrobial composition containing Fluometacyl at a final concentration of 20 to 40 mg / L and fluconazole at a final concentration corresponding to half of its MIC.

Claims

73Claims :

1. An antimicrobial composition comprising a combination ofTriazolo(4,5-d)pyrimidine derivative of formula (I)wherein Ri is C 3-5 alkyl; R? is a phenyl group, substituted by one or more halogen atoms; R3 and R4are both hydroxyl; R is OH or OCH2CH2OH; or a pharmaceutical acceptable salt, together with an antimicrobial agent selected from the group consisting of azole and pharmaceutical acceptable salt thereof .

2. The antimicrobial composition according to claim 1 with a MIC value reduced at least 2-fold compared to the MIC value of the antimicrobial agent taken alone.

3. The antimicrobial composition according to claim 1 or 2 wherein the Triazolo(4,5- d)pyrimidine derivative is (lS,2R,3S,4R)-4-[7-[[(lR,2S)-2-(3,4- Difluorophenyl)cyclopropyl]amino]-5-(propylthio)-3H-l,2,3-triazolo[4,5-d]pyrimidin-3-yl]-1,2,3-cyclopentanetriol as defined in formula (III) and also called Fluometacyl.

4. The antimicrobial composition according to claim 1 or 2 wherein theTriazolo(4,5- d)pyrimidine derivative is (lS,2S,3R,5S)-3-[7-[(lR,2S)-2-(3,4- difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[l,2,3]-triazolo[4,5-d]pyrimidin-3-74 yl]-5-(2-hydroxyethoxy)-l,2-cyclopentanediol as defined in formula (II) and also calledTriafluocyl;CD5. The antimicrobial composition according to claim 3 wherein Fluometacyl is combined with the azole fluconazole or a pharmaceutical acceptable salt thereof, and wherein the antimicrobial composition has a 2-fold MIC reduction compared to fluconazole alone6. The antimicrobial composition according to claim 5 wherein the concentration ratio of Fluometacyl to fluconazole is between (weight to weight ratio) 10 / 1 to 5 / 1.

7. The antimicrobial composition according to claim 3 wherein Fluometacyl is combined with the azole voriconazole or a pharmaceutical acceptable salt thereof and wherein the antimicrobial composition has a 4-fold MIC reduction compared to voriconazole alone8. The antimicrobial composition according to claim 7 wherein the concentration ratio Fluometacyl to voriconazole (weight to weight ratio) is between 1280 / 1 to 2560 / 19. The antimicrobial composition according to claim 4 wherein Triafluocyl is combined with the azole fluconazole or a pharmaceutical acceptable salt thereof and wherein the antimicrobial composition has a 4-fold MIC reduction compared to fluconazole alone.

10. The antimicrobial composition according to claim 9 wherein the concentration ratio Triafluocyl to fluconazole (weight to weight ratio) is between 20 / 1 to 40 / 1.

11. The antimicrobial composition according to claim 4 wherein Triafluocyl is combined with the azole voriconazole or a pharmaceutical acceptable salt thereof and wherein the antimicrobial composition has a 2-Fold MIC reduction compared to voriconazole alone.

12. The antimicrobial composition according to claim 11 wherein the concentration ratio Triafluocyl to voriconazole is (weight to weight ratio) between 1 / 160 to 1 / 320.7513.The antimicrobial composition according to anyone of claims 1 to 12 for use in prevention or treatment of infection on human or animal.14.The antimicrobial composition for use according to claim 13 wherein the infection is caused by yeast or fungi.

15. A pharmaceutical composition comprising the antimicrobial combination according to any one of claims 1 to 12 and a pharmaceutical acceptable adjuvant, diluent or carrier16.The pharmaceutical composition according to claim 15 for use in prevention or treatment of infection caused by fungi.17.The composition for use according to anyone of claims 13 or 14 , or the pharmaceutical composition for use according to claim 15 or 16 wherein the infection is a systemic or superficial infection, or wherein the infection is caused by one or more of C. albicans, Aspergillus fumigatus, Cryptococcus neoformans, C.tropicalis, C.krusei or a mixture thereof, or preferably wherein the infection is caused by Candida albicans, C.parapsilosis, C.glabrata, A.terreus, A.niger, A.nidulans, Candida auris , Sporothrix, Trichophyton, Microsporum, and Epidermophyton (causing Ringworm (tinea))Madurella mycetomatis, Madurella grisea, Exophiala jeanselmei, Leptosphaeria senegalensis, and Pyrenochaeta species (causing eumycetoma), preferably C. albicans .

18. A medical device, biomaterial implants or bioprosthesis impregnated with antimicrobial agent selected from the group consisting of azole and pharmaceutical acceptable salt thereof and further coated with a polymer or nanogel comprising the Triazolo(4,5-d)pyrimidine derivative of formula (I) as defined in claim 119.The medical device, biomaterial implants or bioprosthesis according to claim 18 wherein the Triazolo(4,5-d)pyrimidine derivative of formula (I) is Fluometacyl as defined in claim 2 or Triafluocyl as defined in claim 3.

20. The medical device, biomaterial implants or bioprosthesis according to claim 18 or 19 wherein the antimicrobial agent is fluconazole or voriconazole.21.The medical device, biomaterial implants or bioprosthesis according to any one of claim 18 to 20 which is a catheter or a cardiovascular device.7622. A method of ex-vivo microbial killing or prevention of microbial growth in biofilm formation comprising using , by applying on a surface, an effective amount or concentration of the composition according to any one of claims 1 to 12 .

23. A method of microbial killing or prevention of microbial growth in biofilm formation comprising, by applying in a polymeric coating on a surface, an effective amount or concentration of the composition according to any one of claims 1 to 12.

24. A coating for medical device, biomaterial implants or bioprosthesis comprising the antimicrobial composition according to any one of claims 1 to 12.25.The coating according to claim 24, wherein the medical device, biomaterial implants or bioprosthesis is a catheter or a cardiovascular device.

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