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Home , Amphotericin B, Anidulafungin

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date Χ t it t o A t 22 September 2011 (22.09.2011) 2U1 1/1 15585 Al

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 31/4164 (2006.01) A61K 31/60 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 31/4196 (2006.01) A61K 31/7084 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, A61K 31/496 (2006.01) A61P 31/10 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, PCT/SI20 10/00005 1 KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (22) International Filing Date: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 7 September 2010 (07.09.2010) NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, (25) Filing Language: English TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, P-201 000095 19 March 2010 (19.03.2010) SI GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicant (for all designated States except US): K I- TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, JSKI INSTITUT [SI/SI]; Hajdrihova 19, 1000 Ljubljana EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, ΓΓ, LT, LU, (SI). LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, (72) Inventors; and GW, ML, MR, NE, SN, TD, TG). (75) Inventors/ Applicants (for US only): BENCINA, Mojca [SI/SI]; Ul. Franca Nebca 8, 1000 Ljubljana (SI). Published: BAGAR, Tanja [SLSI]; Gornji Petrovci 56, 9203 Petro- — with international search report (Art. 21(3)) vci (SI). MEDVED, Natasa [SI/SI]; Gubceva ulica 1, 1270 Litija (SI). (74) Agent: ITEM d.o.o.; Resljeva 16, 1000 Ljubljana (SI).

(54) Title: PHARMACEUTICAL COMBINATION OF ACETYLSALICYLIC ACID AND COMPOUND FOR DESTROYING OR GROWTH INHIBITION AND REPRODUCTION OF FUNGI (57) Abstract: The presented invention relates to a pharmaceutical combination of an antifungal substance and salicylic or acetyl - , salt or derivative thereof to enhance fungicidic or fungistatic efficiency of the antifungal substance for treating or preventing fungal . The invention refers to a medicament or a plant protection product, which contains the pharmaceuti cal combination and a method for preventing fungal in vitro for the disinfection of surfaces and implants. Pharmaceutical combination of acetylsalicylic acid and antifungal compound for destroying or growth inhibition and reproduction of fungi

Field of invention

The invention relates to a pharmaceutical combination of acetylsalicylic acid and an antifungal compound for destroying or growth inhibition and reproduction of fungi. Invention is classified as an improvement of antifungal compound; the invention refers to a method for destroying or growth inhibition and reproduction of fungi, and treatment and prevention of fungal infections in vivo in in vitro.

State of the art

Fungi, including pathogenic species can be found all over the world. They are important pests in agriculture and food production. They also represent a serious threat in medicine, due to the increasing number of severe and difficult to cure fungal infections that result from a lack of novel antifungal substances, increased resistance of fungi to existing and growing number of immunocompromised patients, who are susceptible to fungal infections. Fungi in humans cause three types of clinical situations: infections, allergic reactions and toxicosis. Immunosuppression after transplantation of organs and patients affected by AIDS, extensive burns, diabetic ketoacidosis and chemotherapy are the main predisposing factors for the development of infections. Clinical conditions resulting after fungal infections can occur in a local or systemic form. Of all the filamentous fungi, Aspergillus is the second most common cause of opportunistic , the first being . In Europe, the most common cause of are A. fumigatus, followed by A. flavus. There are only a few antimycotics known to work against fungi. Their use is restricted mainly due to very common side effects, poor transition in various tissues, narrow range of antimycotic activity and insensitivity of some fungi to certain antifungals (Groll and Kolva River, 2004, 1998, lich, 2007).

Currently there are four classes of antifungal substances in clinical use: polyene antifungals, derivatives of , alilamines, tiokarbamats and fluoropyrimidine. The target of the first three classes is . Azoles inhibit lanosterol-demethylase, older azoles () inhibit a number of other enzymes, membrane bound enzymes and the biosynthesis of membrane lipids. (Sheehan et al., 1999). Polyene antifungal group is composed of nearly 200 natural , of which only and were developed for systemic treatment of fungal infections. Polyens are amphipatic molecules that incorporate into the membrane of cells, thereby forming channels that change the permeability, cause the loss of vital cytoplasmic components and consequently lead to the death of the micro-organism (Georgopapadakou and Walsh, 1996). Currently there are two alilamin antifungal substances in clinical use, naftifin and and tolnaftat, which is a tiokarbamat. All three are reversible, non-competitive inhibitors of squalane-2 ,3-epoksidase. Synthetically prepared morpholines are agricultural fungicides, except for the amorpholine, which is used topically for treatment of nail infections. are a new class of antifungal lipopeptides. They inhibit the synthesis of 1,3- -D-glucan, a polysaccharide present in the cell wall of many pathogenic fungi and fungi, but absent in the cell wall of mammals (Groll et al., 2004).

The key drawback of antimycotics is the developing resistance of fungi to the antifungal

substances. The acquired resistance to among strains of C. albicans has been reported particularly in patients infected with the HIV virus. Acquired resistance to amphotericin has been reported in and Candida spp., and also in C. lusitaniae, C. glabrata and C. krusei. Among filamentous fungi (Peudalesheria boydii, Scopulariopsis, Fusarium), as well as some yeasts {Trichosporon beigelii, C. Lusitania, C. guiliermondii), some species have inherited resistance to amphotericin B (Richter et al., 2005).

Treatment with antifungals is often accompanied by adverse side effects. accumulates in fatty tissues due to its high lipophilicity. Clinical use of amphotericin B is associated with toxicity in the form of , azotemia, reduced glomerular filtration rate, decreased ability to concentrate , renal loss of sodium and potassium, renal tubular acidosis, hypocalaemy, hypomagnesaemia, low blood pressure, , bone marrow depression, leucopenia, hepatitis, hyperlipidemia and hyponatremia, , chills, muscle aches, anemia and .

It is evident from the above-mentioned that there is a dire need for new products, with antifungal activity or products, which would improve function of the antifungal substances and thus lower required therapeutically effective amount of antifungal substance. Ideally, the antifungal activity of the new product would affect a broad spectrum of fungi. Products would be useful in combination with other antifungal substances especially if the activity of the antifungal substance is improved, since this improves the effectiveness of therapy and reduces the potential toxicity of the antifungal substance. The problem of acquired resistance to the antifungals can be resolved by using a combination of various antimycotics. The resistance of fungi to certain antifungal drugs can also be addressed through combinations with other substances that by themselves have weak or no antifungal activity, such as curcumin or FK506.

The inventors have come to the discovery that salicylic acid and its derivative acetylsalicylic acid effectively improves the performance of antifungal substances.

References:

Georgopapadakou N.H., Walsh T.J. 1996. Minireview, Antifungal agents: Chemoterapeutic targets and immunologic strategies. Antimicrobial agents and chemoterapy, 40, 279-291.

Groll A.H., Kolve H. 2004. Antifungal agents: In vitro susceptibility testing, pharmacodynamics, and prospects for combination therapy. European journal of clinical microbiology & infectious diseases, 23, 256-270.

Klich M.A. 2007. : the major producer of aflatoxin. Molecular Plant Pathology, 8(6), 713-722.

Richter S.S., Galask R.P., Messer S.A., Hollis R.J., Diekema D.J., Pfaller M.A. 2005 Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases. J Clin Microbiol 43, 2155-2162.

Sheehan D.J., Hitchcock C.A., Sibley CM. 1999. Current and emerging antifungal agents. Clinical Microbiology Reviews, 12, 40-79.

Summary of invention

An invention is a pharmaceutical combination containing an antifungal substance and salicylic acid or acetylsalicylic acid, salt or derivative of salicylic acid or acetylsalicylic acid, where the pharmaceutical combination according to the invention is used to destroy or inhibit the growth and replication of fungi, preferably pathogenic and opportunistic fungi. The invention refers to the pharmaceutical combination to be used for developing a drug for in vivo and in vitro prevention and treatment of fungal infections in animals and humans and the drug contains an effective therapeutic amount of the pharmaceutical combination. Invention refers to the pharmaceutical combination for the prevention and treatment of fungal infections in vivo and in vitro in plants, where the plant protection product contains a fungicidic or fungistatic amount of the pharmaceutical combination.

The discovery is based on the realization that a combination of antifungal substance and salicylic or acetylsalicylic acid, a salt or derivative increases the fungicidic or fungistatic effect of antifungal substance against fungal infections compared to the antifungal substance itself. Within the pharmaceutical combination, according to the invention, the active ingredients act synergistically and reduce amount of the antifungal substance, increase the efficiency of therapeutic action of the antifungal substance, shorten the treatment time, lower the costs of treatment and in many cases achieve a fungicidal effect with the pharmaceutical combination, which could not be achieved with individual components.

The presented invention is based on the discovery that the synergistic effect of salicylic or acetylsalicylic acid with antifungal substances can be used to prepare products for the treatment of a subject that is infected with fungi and provides a novel way of treating such infections. The advantage of this invention is the ability to treat fungal infections that are considered difficult to treat. An additional advantage of the invention is the possibility to treat infections with fungi that have acquired resistance to known antifungal substance. Below we present the advantage of the combination therapy according to the invention, where there is a marked reduction of the amount of antifungal substances, thereby also a reduction of undesirable side effects. The advantage is also an improved quality of life, since the novel treatment considerably reduces the duration of therapy.

The invention also includes the preparation of (i) a drug or (ii) a plant protection product containing the pharmaceutical combination for prevention and treatment of fungal infections in plants, animals and humans. The pharmaceutical combination, according to the invention, may be used in the form of a drug to treat fungal infections or for decontamination of surfaces, sterilization of medical equipment and implants. Drug according to the invention, which contains the pharmaceutical combination, is prepared in an appropriate pharmaceutical form, containing all the necessary pharmaceutical additives and, if necessary, also additional antifungal substances. The pharmaceutical combination, according to the invention, in a form of plant protection product with relevant and acceptable additives is used to prevent or treat fungal infections in plants. According to the invention the product contains the pharmaceutical combination in an appropriate pharmaceutical form. The pharmaceutical combination is prepared in such a manner to be suitable for topical, intravenous, oral, intramuscular, subcutaneous, intraperitoneal, intraocular, transpulmular, transdermal, and aerosol use. The active ingredients of the pharmaceutical combination are presented in mixed form or used in a form containing active ingredients separate, but used in a manner that achieves desired synergistic effect.

Additionally, the invention represents a method for destroying or inhibiting the growth of fungi, which includes direct contact of the medicine or the protection product containing the pharmaceutical combination with the fungi. The method can be used in vitro for decontamination of surfaces and sterilization of medical equipment and implants, for in situ sterilization of intravenous catheters and implants.

The invention refers to a method of sequential co-administration of active ingredients of the pharmaceutical combination; however, the active ingredients are in individual forms. The therapeutic effect of separate forms is equal to or better than the concurrent administration and the separate application is applied because of the improved properties of the individual components.

The invention presented here is a product with an effective therapeutic amount of a pharmaceutical combination for treatment of patients infected with fungi.

Figure legend

Figure 1. Metabolic activity of the fungus A. niger in the presence of the combination; acetylsalicylic acid and antifungal amphotericine B, fluconazole, ketoconazole or . Acetylsalicylic acid (final concentrations of 0, 0.5, 2 mM) and amphotericine (final

concentrations of 0, 0125, 0.25 g / 1) (A), fluconazole (final concentrations of 0, 0.5, 1 g / 1)

(B), ketoconazole (final concentrations of 0, 5, 10 mg / 1), (C) and clotrimazole (final

concentrations of 0, 0.4, 1 mg / 1) (D) were used.

Figure 2. Growth inhibition of yeast S. cerevisiae. Increase in biomass in the presence of acetylsalicylic acid (0, 2, 5 mM) and clotrimazole (0, 0.2, 0.5 mg/1) [A].

Figure 3. Microscopic images of fungus A. niger in the presence of the antifungal substance and acetylsalicylic acid. Growth of fungi in the medium without the pharmaceutical combination [A], with 25 µΜ amiodarone and 0.5 mM acetylsalicylic acid [B], with 0.4 mg/1 clotrimazole and 0.5 mM acetylsalicylic acid [C]. Figure 4. Metabolic activity of the fungus A. niger in the presence of salicylic acid (A) and acetylsalicylic acid (B).

A detailed description of the invention

Unless defined otherwise, technical and scientific terms have the same meaning as it is generally accepted among experts. The terminology in the description of the invention is intended to describe a certain part of the invention and not to limit the invention. The description of the invention and claims are in singular form, but the invention also includes the plural, which is not highlighted for ease of understanding.

The presented invention refers to the discovery that salicylic or acetylsalicylic acid, salt and derivative thereof improves function of an antifungal substance and could be used as a pharmaceutical combination of the antifungal substance and salicylic or acetylsalicylic acid, salt and derivative thereof to prepare (i) a medicament or (ii) a plant protection product for treatment of subject: human, animal or plant; which is infected with fungi and provides means for treating such infection. According to the invention, a synergistic pharmaceutical combination of the antifungal substance and salicylic or acetylsalicylic acid is used to prepare a medicament, which is applied in vivo (i) for prevention and treatment of fungal infections, preferentially of pathogenic and opportunistic pathogenic fungi and in vitro (ii) for decontamination of surfaces and sterilizing medical equipment and implants. According to the invention, the pharmaceutical combination of the antifungal substance and salicylic or acetylsalicylic acid could be used for preparation of a plant protection product and in vivo prevention and treatment of fungal infection in plant.

The term "antifungal substance" refers to "antifungal drug" or "fungicide" and represents the antifungal substance that is used to prevent and treat fungal infection. The term "antimycotic or antifungal drug" refers to a substance that inhibits the growth and replication of the fungus or destroys fungal cell and the drug is selected from a group of: (i) polyene antimycotics such as: natamicin, rimocidin, philipine, nystatin, amphotericin B, candicin (ii) azole antimycotics, such as: , ketoconazole, clotrimazole, ekonazol, bifonazol, butokonazol, fentikonazol, izokonazol, oksikonazol, sertaconazol, sulkonazol, tiokonazol, pramikonazol, (iii) antimycotics, such as: fluconazole, , izavukonazol, ravukonazol, posakonazol, , terkonazol, (iv) alilamine antimycotics, such as: terbinafine, amorolfin, naftidin, butenafin, or (v) echinocandins, such as: , kaspofungin, mikafungin, and others, such as: amiodarone, terbinafine, ciklopiroks, pimaricin, flucitosin, allylamin-tiokarbamati, grizeofulvin, . The term "antifungal substance" also refers to proteins that inhibit the growth of fungi, such as PAF, AFP, and other plant defensins, antifungal proteins and peptides. The term "fungicide" refers to a chemical agent used to control fungal infection and could function as contact, half-systemic or systemic drug.

The invention refers to the discovery that salicylic acid or acetylsalicylic acid, which is a derivative of salicylic acid, salt or derivative thereof, improves fungicidic or fungistatic activity of the antifungal substance, and the improvement is expressed as a reduction of therapeutically effective amount of the antifungal substance in combination with salicylic acid or acetylsalicylic acid and the combination could be used for prevention and treatment of fungal infection.

The term "appropriate salt, derivative" refers to salt or derivative of salicylic acid and acetylsalicylic acid, which is also a derivative of salicylic acid. The salt and derivative of compound does not substantially alter function of compound, but improves solubility, availability in tissue, improves or alters metabolism, degradation or elimination from the body.

The function of salicylic acid and acetylsalicylic acid according to the invention is an enhancement of therapeutic efficiency of the antifungal substance for treatment and prevention of fungal infections and the enhancement is expressed synergistically.

The term "synergistic" describes the improved fungicidic or fungistatic therapeutic efficacy of the antifungal substance in the presence of proton homeostasis modifier. The term "therapeutic efficacy" refers to the successful clinical outcome, which not necessarily means that the pharmaceutical combination eliminates all fungi involved in infection. The success depends on intensity of fungal infection at the site of infection and refers to the improvement of the infection state. The therapeutic effectiveness depends also upon the condition of treated subject. A healthy subject requires less of the combination compared to the subject with a weakened immune response. According to the invention the therapeutically effective amount of the antifungal substance in the presence of proton homeostasis modifier in the combination according to the invention is reduced compared to the treatment with only the antifungal substance. The invention refers to the pharmaceutical combination for preparation of a medicament or a plant protection product for prevention or treatment of infection caused by opportunistic pathogens and pathogenic yeasts and filamentous fungi.

The term "fungal infection" refers to infection of plant, animal and human as well as material, liquid and surface with filamentous fungi and yeasts. The term "fungus" refers to micro-organism from the kingdom of fungi, preferentially yeasts and filamentous fungi, which are plant or animal pathogens or opportunistic pathogens. The group of pathogenic and opportunistic pathogenic fungi are selected from: Candida, preferably C. albicans, C. glabrata,

C. krusei, C. laurentii, C . parapsilosis, C. tropicalis, C. lusitaniae, C. kefyr, C. guilliermondii, C. dubliniensis, Cryptococcus, preferably C. neoformans, C. albidus, C. gattii, Aspergillus, preferably A. flavus, A. fumigatus, A . clavatus, A. niger, A. nidulans, A. terreus, A. Sydow, A. glaucus, A. oryzae, A. ustus, A. versicolor, Histoplasma, Histoplasma capsulatum priority, Paracoccidioides, preferably P. brasiliensis, Sporothrix, schenckii Sporothrix priority, Blastomyces, preferably B. dermatitidis, Paecilomyces preferably P. lilacinus, P. variotii, pneumonia, preferably P. jirovecii, Stachybotrys, preferably S. chartarum, Malassezia, preferably M. furfuryl, Pityrisporum, preferably P. ovalae, Coccidioides, Fonsecaea, Cladosporium, Basidiobolus, Conidiobolus, Rhizopus, Rhizomucor, Mucor, Absidia, Mortierella, Cunninghamella, Saksenaea, Trychophyton, preferably T. rubrum, T. tonsurans, T. mentagrophytes, T. soudanense, T. verrucosum, T. ajelloi, T. concentricum, T. equinum, T. erinacei, T.flavescens, T. gloriae, T. interdigitale, T. megnini, T. phaseoliforme, T. schoenleini, T. simia, T. terrestre, T. tonsurans, T. vanbreuseghemii, T. violaceum, T. Yaounde, Microsporum, preferably M. canis, M. gypseum, M. audouini, M. gallinae, M. ferrugineum, M. distortum, M. nanum, M. cookie, M. vanbreuseghemii, Epidermophyton, preferentially E. floccosum, E. stockdaleae, Trichosporon, Fusarium, preferably F. oxysporum, F. solani, F. semitectum, Scytalidium, preferably S. dimidiatum, S. hyalinum, S. infestans, S. japonicum, S. lignicola, Penicillium, Actinomyceees, Cochliobolus, Scopulariopsis, preferably S. brevicaulis, S. Candida, S. koningii, S. acremonium, S. flava, S. cinerea, S. trigonospora, S . brumptii, S. chartarum, S. fusca, S. asperula, Alternaria, preferably A. alternate, A. chartarum, A . dianthicola, A. geophilia, A. infectoria, A. stemphyloides, A. teunissima, Curvularia, preferably

C. brachyspora, C. clavata, C. geniculata, C. lunata, C. pallescens, C. senegalensis, C .

verruculosa, C. Chaetomium priority atrobrunneum, C. funicola, C. globosum, C. strumarium. The term "fungi" refers to yeasts and filamentous fungi which in healthy subject do not cause a disease, but in immunocompromised patient the same fungi can cause a disease. The term "fungi" refers also to fungi, which are plant pathogens such as: Fusarium spp., Thielaviopsis spp., Verticillium spp., Rhizoctonia spp. Pachyrhizi Sydow Phakospora, Puccinia spp. Magnaporthe spp. e.g. Magnaporthe grisea, Ustilago spp. e.g. Ustilago mydis, Uncinula necator, Plasmopara viticola, Pseudopezicula tracheiphila, Botrytis spp. e.g. Botrytis cinerea, Phomopsis viticola.

The term "disease" refers to a disease caused by infection with yeasts and filamentous fungi. The term "disease" refers to a disease caused by (a) surface infection of fungi and is limited to infection of the skin, nails and hair, (b) disease caused by subcutaneous infection such as dermatitis and is infection of subcutaneous tissue and surrounding structures, (c) systemic disease is infections of internal organs: lungs, liver, heart of the central nervous system, and opportunistic diseases, (d) infection of immune compromised patient with non-pathogenic fungi. Disease is expressed in the form of mycosis, candidosis, systemic mycosis, , , cocidiomycosis and paracocidiomycosis, inflammation, redness, dandruff, athletic feet, mouth and vaginal candidosis, dermatitis, eczema, onikomycosis, aspergillosis, histoplasmosis, pneumonia, respiratory damage and , asthma. The term "disease" refers to illness caused by fungi in animal or human, preferably in honeybees and vertebrates, particularly birds, poultry, mammals, especially ungulates, rabbits, carnivores, cats, primates, and man.

The term "treatment" refers to preventive and therapeutic treatment. In vitro method of treatment refers to the killing and inhibiting of fungal growth and replication through the transfer of the pharmaceutical combination according to the invention to the site of infection with fungi, which is a surface of material.

The invention refers to a medicament, which contains the pharmaceutical combination according to the invention to treat fungal infection and the medicament contains the pharmaceutical combination in effective amount to inhibit or destroy fungal cells.

Techniques of assessment a therapeutically effective amount of the pharmaceutical combination with the antifungal substance and salicylic acid are well known to the experts in the field.

The therapeutically effective amount of the pharmaceutical combination according to the invention includes quantitatively reduced amount of the antifungal substance due to the synergistic action of salicylic or acetylsalicylic acid which is administrated/incorporated into the pharmaceutical combination as a mixture or separate component. A drug of the pharmaceutical combination could contain other medicaments. A synergistic effect of the salicylic or acetylsalicylic acid with the antifungal substance is expressed with a less of the antifungal substance needed for elimination or inhibition of replication and growth of fungi.

These is important since application of some antifungal substances is limited by their systemic toxicity or costs related with treatment with antifungal substance. Reducing the concentration of antifungal substance with the addition of salicylic or acetylsalicylic acid, salt or derivate thereof will decrease side effects of treatment and its cost, which enables wider use of the pharmaceutical combination. A medicament according to the invention ensures a faster or more efficient fungistatic or fungicidic effect compared to antifungal substance. A drug that contains the pharmaceutical combination according to the invention overcomes fungal resistance to the antifungal substance, or change the fungistatic mode of action of antifungal substance into fungicidic. In the pharmaceutical combination a therapeutically effective amount of the antifungal substance is reduced due to the synergistic activity of components of the pharmaceutical composition.

According to the invention a medicament may contain active ingredients of the pharmaceutical combination: antifungal substance and salicylic or acetylsalicylic acid either in a form of a mixture of these two active ingredients or each active component is in separate form. However, the medicament containing separate forms of active ingredient is used in a manner, which ensures that the effect of substances in separate form at the site of fungal infections is identical to their mixture. Active components in the medicament are in separate forms due to improved stability of the individual components and due to the easier administration when using different routes of administration. Individual components of the pharmaceutical combination of the invention, the antifungal substance and salicylic or acetylsalicylic acid can be in various pharmaceutical forms. The pharmaceutical combination that provides simultaneous administration of the antifungal substance and salicylic or acetylsalicylic acid expected to be as efficient as co-application, with fungistatic or fungicidal effects in vivo compared with the administration of individual components of the combination of the invention. The term "fungistatic effect" describes inhibited growth and reproduction of fungi, the term "fungicidic effect" refers to the death of fungi.

According to the invention, the pharmaceutical combination destroys or inhibits fungal growth in the way of direct contact of fungus with the pharmaceutical combination. This method can be used in vitro, as for plant protection products, for decontamination of fluids and surfaces or to sterilize surgical and other medical tools and implants, including prostheses, in situ sterilization of catheters, intravenous implants.

The invention refers to the preparation of a medicinal product that contains the pharmaceutical combination for treatment of fungal infection. The medicament is in any pharmaceutical form and contains the required pharmaceutical additives.

The term "pharmaceutical form" refers to a form in which the pharmaceutical combination in a form of powder, suspension, emulsion, solution, paste, cream, ointment, gel, lotion, liniment, dosage forms for , infusion formulation, tablet, capsules, suppositories, transdermal patch, a therapeutic system, tinctures, percolat extract. The term "pharmaceutical supplement" refers to additives in pharmaceutical formulation. The function of pharmaceutical supplement is to facilitate and enhance the entry of the pharmaceutical combination of the invention to the infection site and is part of the pharmaceutical form. The pharmaceutical supplement includes appropriate dilutants, adjuvants and carriers. Administration of the pharmaceutical form containing the combination may be topic, intravenous, oral or intramuscular, via an aerosol, subcutaneous, intraperitoneal, intraocular, transpulmonary, transdermal.

The pharmaceutical combination according to the invention may be administrated in conjunction with other drugs. The pharmaceutical combination may contain one or more antifungal substances in addition to salicylic or acetylsalicylic acid.

The invention also relates to a plant protection product in an appropriate form and with appropriate additives and is used to prevent or treat fungal infection in plant. The invention relates to a method for use the combination in form of a medicament or a plant protection product for elimination or inhibition of fungal growth and reproduction and is used as plant protection product and for decontamination of fluids and surfaces or to sterilize surgical and other medical tools and implants, including prostheses, and in situ sterilization of catheters, intravenous implants.

Below are examples to illustrate the innovation. Descriptions of cases are not intended to limit innovation and to be understood in terms of innovation demonstrations.

Examples

Example 1. Growth inhibition test Effect of the pharmaceutical combination was tested on the fungi Aspergillus spp. in liquid media. Spores were grown overnight (16 to 24 h) in a synthetic liquid Vogel's medium at 30 oC. The Vogel's medium was supplemented with the combination of the antifungal compound and the modifier of proton homeostasis. For positive control the medium without the active ingredients of the combination was used. The XTT method was used for measuring the metabolic activity of fungi.

Preparation of spore suspension and media. Spore suspension was prepared in 0.1% (w/v) Tween 80. Spore suspension in the final concentration of 105 spores/ml was used to inoculate the liquid Vogel's medium. Vogel's medium contains: 8.5 mM sodium citrate dihydrate of (Na3(C6H507)), 36.8 mM KH2P04, 25 mM NH4N03, 0.7 mM CaC12.2H20, 0.8 mM MgS04.7H20, pH 6.0, 0.2 ml of trace elements (34.3 mM EDTA, 15.3 mM ZnS04.7H20, 8 MnC12.4H20 mM, 1.6 mM CoC12.6H20, 1.3 mM CuS04.5H20, 0.2 mM (NH4)6Mo7O24.4H20, CaC12.2H20 10 mM, 3.6 mM FeS04.7H20, pH 6.5) and 2% (w/v) glucose. Antifungal substance (amiodarone 2 mM DMSO, amphotericin 0.04 g/1 DMSO, clotrimazole 1 g/1 DMSO, fluconazole 40 g/1 DMSO, ketoconazole 5 g/1 methanol) and acetylsalicylic acid were dissolved in an appropriate solvent in adequate concentration. 0.2 ml of spore solution with supplements was placed in the wells of microtitre plates (we used 96 well plates). The test was performed after 24-hour incubation at 37 °C.

The XTT method was used to determine the presence of living cells. XTT reagent is internalised in the cell and is converted to a coloured compound in active mitochondria. Solution of XTT and menadione was freshly prepared. XTT was dissolved in hot water at a final concentration of 1 g/1. Menadione was dissolved in acetone at a concentration of 10 mM, and then it was further diluted with in the ratio 1:10. Final XTT reagent contains the XTT reagent (concentration 0.6 g/1 final concentration in the well is 0.2 g/1) and menadione (concentration 75 µΜ , the final concentration in the well 25 µΜ).

After adding 0.1 ml of XTT reagent in each well, the plate was incubated at 30 °C for 4 hours. After incubation we transferred 0.1 ml from each well to a new transparent microtiter plate, where the absorbance of the solutions was measured at a wavelength of 450 nm on a Bio- Tek ® XS PowerWave reader.

Figure 1 presents results of the combination of acetylsalicylic acid (0, 0.5, 2 and 4 mM) and antimycotics amphotericin, fluconazole, ketoconazole and clotrimazole. Although amphotericin and acetylsalicylic acids work synergistically, we did not achieve fungicidic activity in any of the tested concentrations. The combination of 0.5 mM acetylsalicylic acid and 1 g/1 fluconazole inhibits the growth 99%. Higher concentrations of the pharmaceutical combinations were fungicidic. The same is true for the pharmaceutical combination of acetylsalicylic acid and clotrimazole, which is fungicidic at a concentration of 2 mM acetylsalicylic acid and 1 mg/1 clotrimazole. Somewhat lower inhibition was achieved with a combination of acetylsalicylic acid and ketoconazole, but a synergistic effect is demonstrated.

Figure 4 presents the inhibition of metabolism of the fungus A. niger in the presence of salicylic and acetylsalicylic acid. The results show that both salicylic and acetylsalicylic acid, which is a derivative of salicylic acid, are able to inhibit growth of fungi.

Effect of the combination of acetylsalicylic acid and clotrimazole on the fungus A. flavus (Table 1). The combination acts synergistically on the growth of fungi with a 99% growth inhibition.

Table 1. Growth inhibition of A. flavus with the pharmaceutical combination; acetylsalicylic acid and clotrimazole. For comparison, growth inhibition in media without and with single active ingredient was determine. Acetylsalicylic acid - 4 mM - 4 mM clotrimazole - - 1.3 mg/1 1.3 mg/1 Growth A.flavus (%) 100 ±5 22 ± 1 14 ±2 0 ± 1

Example 2. Confocal microscopy

With microscopy we evaluated an impact of the combination on the growth of the tested fungi. Cells were stained with the SynaptoRed and SYTOX dye. SynaptoRed dye stains the , while SYTOX cell membrane impermeable and emits fluorescence only after binding to DNA. Visualization of the SYTOX dye within the cell is an indication of damaged cell membrane.

We tested the pharmaceutical combination of amiodarone and acetylsalicylic acid, clotrimazole and acetylsalicylic acid. As a control, the fungus A . niger, without any pharmaceutical combinations. Results are shown in Figure 3. Magnification is similar for all images. Comparison of growth and staining with SYTOX clearly shows impaired growth of fungi in presence of the pharmaceutical combinations. It is obvious that growth of A . niger in media without active ingredients is more intensive and the intracellular content is not stained with SYTOX dye. The pharmaceutical combination strongly inhibited the growth. Detailed description of the performed experiment is presented below.

We used confocal microscope Leica TCS SP5. This is a laser microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany). Immersion lens with HCX Plan Apo 63x oil (NA 1.4) was used. The SynaptoRed 2 (Biotium) is excited with the argon laser at 514 nm and the emission is collected between 650 and 700 ran, and SYTOX Green is excited at 488 nm and emits light at 520 to 540 nm.

A spore suspension with the concentration of 105 spores/ml Vogel's medium was prepared. The media was supplemented with the active ingredients of the combination. 0.2 ml of the spore suspension was inoculated on cover slip and incubated for 4 hours at 30 °C in a humid chamber. Preparations were stained with Synapto Red (dyes the cell membrane) and 10 µΐ of 0.5 mM Sytox Green dye for 15 minutes at 30 °C.

Example 3. Inhibition of yeast growth

The effect of the pharmaceutical combinations was tested on the yeast Saccharomyces cerevisiae in liquid media. Yeast culture was grown overnight (16 to 24 h) in a synthetic liquid medium at 28 °C. The medium contained different pharmaceutical combinations. For a control, yeast cells were grown in medium free of any active ingredient of the pharmaceutical combinations. Initially yeast cells were diluted to optical density 0.04 at 600 nm. Growth speed was monitored spectrophotometrically in 8 h incubation at 28 °C. Results are presented in Figure 2.

The fastest growth was observed for yeast cultivated in medium without supplements, positive control. Results are calculated as a percentage of growth relative to positive control. The results show (Figure 2) that the pharmaceutical combination of acetylsalicylic acid and amphotericin inhibits the growth of S. cerevisiae and that the inhibition of growth is much better than inhibition of growth in the presence of individual components. Similar results were obtained with the yeast C. albicans (Table 3).

Table 3. The growth of the yeast C. albicans in the presence of the pharmaceutical combination of amiodarone and acetylsalicylic acid.

amiodarone (2 mikroM) 0 + 0 + acetylsalicylic acid (2 mM) 0 0 + + Growth (%) 100 ±6 58 ±4 64 ±3 10 ±2 Claims

1 A pharmaceutical combination that includes an antifungal substance and salicylic or acetylsalicylic acid, a salt or derivative thereof to destroy or inhibit the growth of fungi and fungal replication.

2 The pharmaceutical combination according to claim 1 characterised by that the antifungal substance is selected from a group of antimycotics or fungicides.

3 The pharmaceutical combinations according to any claim 1 or 2 characterised by that a fungicidic or a fungistatic therapeutic efficient amount of the antifungal substance in combination with salicylic or acetylsalicylic acid, salt or derivative is improved compared to the therapeutically efficient amount of the individual antifungal substance.

4 The pharmaceutical combinations according to any claim from 1 to 3 for the preparation of a medicament or a plant protection product for in vivo and in vitro prevention or treatment infections caused by fungi, preferentially pathogenic and opportunistic pathogenic yeasts and filamentous fungi.

5 A medicament that contains the pharmaceutical combination according to any claim from 1 to 4, designed to prevent or to treat fungal infection that cause disease in animal or human and the infection in animal or human is (i) superficial, limited to the skin, nails and hair, (ii) subcutaneous infections of tissues and surrounding structures, (iii) systemic infections of internal organs.

6 The medicament according to claim 5 characterised by that it contains a therapeutically effective amount of the pharmaceutical combination according to any claim from 1 to 4 in a suitable pharmaceutical form with appropriate pharmaceutical additives.

7 The medicament according to any claim from 5 to 6 characterised by that the medicament is in the pharmaceutical form suitable for topical, intravenous, oral, intramusculature, subcutaneous, intraperitoneal, intraocular, transpulmonary, transdermal, aerosol use.

8 The medicament according to any claim from 5 to 7 characterised by that it contains active ingredients of the pharmaceutical combination separated; divided individual active components, antifungal substance and salicylic or acetylsalicylic acid, a salt or derivate thereof; of the pharmaceutical combination are used in a manner to attain a synergistic effect characteristic for the pharmaceutical combination; the individual active components of the pharmaceutical combination are as separate forms to improve stability of each component or to be administrated differently.

A plant protection product containing the pharmaceutical combination according to any claim from 1 to 4 in an acceptable form with the appropriate additives and is intended to prevent or treat fungal infections in plants.

The method of destroying fungi or inhibiting fungal growth and replication, preferentially pathogenic and opportunistic pathogenic fungi, through the transfer of the pharmaceutical combinations according to any claim from 1 to 4 in the form of medicament according to any claim from 5 to 8 or a plant protection product of claim 9 to the region infected with the fungus and is used for plant protection, for decontamination of fluids and surfaces or to sterilize surgical and other medical tools and implants, including prostheses, in situ sterilization of catheters, intravenous implants.

The method according to claim 10 characterised by that the active ingredients of the pharmaceutical combination according to any claim from 1 to 4 are used sequentially, being presented as individual forms, but still the result of sequential application of these individual forms is as good as when the pharmaceutical combination is used as a mixture of active ingredients, antifungal substance and salicylic or acetylsalicylic acid, a salt or derivate thereof.

A. CLASSIFICATION OF SUBJECT MATTER INV . A61K31/4164 A61K31/4196 A61K31/496 A61K31/60 A61K31/7084 A61P31/10 ADD. According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) A61K A61P

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

EPO-Internal , BIOSIS , EMBASE , WPI Data

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

WO 2007/131253 A2 (PHARMACON FORSCHUNG UND 1-11 BERATU [AT] ; NOE CHRISTIAN [AT] ; NOE MARION [A) 22 November 2007 (2007-11-22) page 11 , paragraph 2 page 16

DEVA RUPAL ET AL : "Invol vement of 1-11 aspiri n-sensitive oxyl ipi ns i n vul vovaginal candidi asi s " , FEMS MICROBIOLOGY LETTERS , vol . 198, no. 1 , 20 Apri l 2001 (2001-04-20) , pages 37- XP002615413, ISSN: 0378-1097 figure 3 -/--

Further documents are listed in the continuation o Box C. See patent family annex.

* Special categories of cited documents : "T" later document published after the international filing date or priority date and not in conflict with the application but "A" document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance invention "E" earlier document but published on or after the international "X" document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to "L" document which may throw doubts on priority claim(s) or involve an inventive step when the document is taken alone which is cited to establish the publication date of another ■Υ document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the Ό " document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu other means ments, such combination being obvious to a person skilled "P" document published prior to the international filing dale but in the art. later than the priority date claimed ■&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

29 December 2010 13/01/2011

Name and mailing address of the ISA/ Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 NL - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70) 340-3016 Biittner, Ul f

Form PCT/ISA 2 0 (second sheet) (April 2005) C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X WO 2009/051840 A2 (UNIV YALE [US] ; MEHAL 5-9 WAJAHAT [US] ; AVLIN IMAEDA [US]) 23 Apri l 2009 (2009-04-23) c l aims 1 , 14

X WO 2009/057835 Al (LEE JINSIK [KR] ) 10, 11 7 May 2009 (2009-05-07) Y examples 4 , 5 1-9

X DE 101 13 045 Al (TARES GMBH [DE] ) 1-11 19 September 2002 (2002-09-19) paragraph [0017] paragraphs [0020] - [0022]

X DATABASE WPI 1-11 Week 200361 Thomson Scientific , London , GB; AN 2003-643867 XP002615414, & NL 1 019 441 C6 (ROVERS C G M) 2 June 2003 (2003-06-02) * abstract

X DATABASE MEDLINE [Onl i ne] 1-8, 10, US NATIONAL LIBRARY OF MEDICINE (NLM) , 11 BETHESDA, MD, US; 2006, C0RRADINI C ET AL: "Amphotericin B and lysine acetyl sal icyl ate i n the combi ned treatment of nasal polyposi s associ ated with mycotic i nfecti on . " , XP002615415 , Database accessi on no . NLM16784013 * abstract & JOURNAL OF INVESTIGATIONAL ALLERGOLOGY & CLINICAL IMMUNOLOGY : OFFICIAL ORGAN OF THE INTERNATIONAL ASSOCIATION OF ASTHMOLOGY ( INTERASMA) AND S0CIEDAD LATINOAMERICANA DE ALERGIA E INMUN0L0GIA 2006 LNKD- PUBMED: 16784013, vol . 16, no . 3 , 2006, pages 188-193, ISSN: 1018-9068

X SCOTT EILEEN M ET AL : "Demonstrati on of 1-11 synergy with fluconazole and either ibuprofen , sodium sal icyl ate, or propylparaben agai nst Candida al bicans i n vitro" , ANTIMICROBIAL AGENTS AND CHEMOTHERAPY , vol . 39, no. 12, 1995 , pages 2610-2614, XP002615416 , ISSN: 0066-4804 table 4 -/--

Form PCT/ISA 2 0 (continuation of second sheet) (April 2005) C(Continuation). DOCUMENTS CONSIDERED TO B E RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

Y WO 00/28821 Al (ASTAN INC [US] ) 1-11 25 May 2000 (2000-05-25) c l aims

Y TROFA D ET AL: "Acetyl sal icy! c acid 1-11 () reduces damage t o reconstituted human t i ssues infected with Candida species by i nhibiti ng extracel lul ar fungal l ipases" , MICROBES AND INFECTION , ELSEVIER, PARIS, FR , vol . 11 , no. 14-15, 1 December 2009 (2009-12-01 ) , pages 1131-1139, XP026789718, ISSN: 1286-4579, DOI : DOI : 10 . 1016/ . MICINF . 2009 . 08 . 007 [retrieved on 2009-08-22] page 1136, col umn 2 , paragraph 2

Y ELENA RUSU ET AL : "The conventi onal 1-11 identi ficati on and effect of dicl ofenac and aspi r i n on the Candida species" , ROMANIAN BI0TECHN0L0GICAL LETTERS , vol . 14, no . 5 , September 2009 (2009-09) , pages 4720-4727, XP009142889 , ISSN: 1224-5984 * abstract

Y STEPANOVIC S ET AL : "Infl uence of 1-11 acetyl sal icyl i c acid (aspiri n ) on bi ofi l m producti on by Candida species" , JOURNAL OF CHEMOTHERAPY, vol . 16, no. 2 , Apri l 2004 (2004-04) , pages 134-138, XP009142890 , ISSN : 1120-009X page 138, l ast paragraph

Y KIM JONG H ET AL: "Enhancement of 1-11 f l udi oxoni l fungicidal acti vity by di srupting cel lul ar gl utathione homeostasi s with 2,5-dihydroxybenzoic acid" , FEMS MICROBIOLOGY LETTERS, vol . 270, no. 2 , May 2007 (2007-05) , pages 284-290, XP002615417, ISSN: 0378-1097 page 270, l ast paragraph

Y KOCK J0HAN L F ET AL: "Oxyl ipin studies 1-11 expose aspiri n as anti fungal " , FEMS YEAST RESEARCH, vol . 7 , no. 8 , December 2007 (2007-12) , pages 1207-1217, XP002615418, ISSN: 1567-1356 page 1213 , l ast paragraph

Form PCT/ISA/210 (continuation of second sheet) (April 2005) Patent document Publication Patent family Publication cited in search report date member(s) date

WO 2007131253 A2 22--11--2007 AU 2007250502 A l 22-11-2007 CA 2651726 A l 22-11-2007 CN 101484162 A 15-07-2009 EP 2018164 A2 28-01-2009 JP 2009536926 T 22-10-2009 RU 2008148977 A 20-06-2010 US 2009208558 A l 20-08-2009

o 2009051840 A2 23--04--2009 EP 2211852 A2 04-08-2010 US 2009239831 A l 24-09-2009 US 2010297271 A l 25-11-2010

o 2009057835 A l 07--05--2009 AU 2007360838 A l 07-05-2009 CN 101842015 A 22-09-2010 EP 2214492 A l 11-08-2010

DE 10113045 A l 19--09--2002 NONE

NL 1019441 C6 NONE

W0 0028821 A l 25-05-2000 AT 309032 T 15-11-2005 AU 771851 B2 01-04-2004 AU 1725700 A 05-06-2000 BR 9915394 A 05-03-2002 CA 2351536 A l 25-05-2000 CN 1359262 A 17-07-2002 DE 69928278 D l 15-12-2005 DE 69928278 T2 14-06-2006 E 1130964 A l 12-09-2001 IL 143156 A 20-11-2005 P 2003535020 T 25-11-2003 MX PA01004908 A 10-03-2003 US 6159977 A 12-12-2000 US 6391879 B l 21-05-2002