SI23311A - Pharmaceutical combination of acetylsalicylic acid and antifungal substance for destroying or inhibition of growth and replication of fungi - Google Patents

Abstract

The invention presented relates to a pharmaceutical combination of an antifungal substance and salicylic or acetylsalicylic acid and salts or derivatives of a compound for an improved fungicidal and fungistatic effect of the antifungal substance for treatment or prevention of fungal infections. The invention refers to a medicine or a phitopharmaceutical preparation which contains the pharmaceutical combination and to a method of prevention of fungal infections in vitro for disinfection of implant surfaces.

Description

A pharmaceutical combination of acetylsalicylic acid and an antifungal substance to kill or inhibit the growth and replication of fungi

FIELD OF THE INVENTION

The subject invention is a pharmaceutical combination of acetylsalicylic acid and an antifungal substance for the destruction or inhibition of fungal growth and replication. The invention relates to the field of antifungal substances, to the field of methods of killing fungi or inhibiting the growth and replication of fungi and to the field of drugs for the treatment and prevention of fungal infections in vivo and in vitro.

The state of the art

Fungi including pathogens are widespread worldwide. They are important pests in agriculture and food production. Health care is a problem due to the increased number of severe and difficult to cure infections resulting from the lack of new antifungal substances, increased resistance of fungi to older antifungals, and an increasing number of immunocompromised patients more susceptible to fungal infections. Fungi in humans cause three types of clinical conditions: infections, allergic conditions and toxicosis. Immunosuppression in organ transplantation and AIDS, burns and chemotherapy, and diabetic ketoacidosis are the major predisposing factors for the development of infections. All clinical conditions of fungal infections may occur in local or systemic form. Of all filamentous fungi, Aspergillus is the second most common cause of opportunistic mycosis, most commonly caused by Candida. The genus Aspergillus in Europe is the most common cause of aspergillosis of A. fumigatus, followed by A. flavus. There are few known antifungal antifungals. Their use is limited mainly by very common side effects, poor passage of the drug into different tissues, a narrow antifungal spectrum, and insensitivity of some fungi to certain antifungals (Groll and Kolve, 2004; Groll et al., 1998; Klich, 2007).

There are currently four classes of antifungal agents in clinical use: polyene antifungals, azole derivatives, allylamines, thiocarbamates and fluoropyrimidines. The target site of the first three classes is ergosterol (Groll et al. 1998). Azoles inhibit lanosterol-demethylase, older azoles (imidazoles) inhibit many other membrane-bound enzymes, and membrane lipid biosynthesis. (Sheehan et al., 1999). A group of polyene antifungals consist of about 200 natural macrolide antibiotics, of which only amphotericin B and nystatin have been developed for the systemic treatment of fungal infections. Polyenes are amphipathic molecules and are embedded in the cell membrane and form channels through which altered permeability, loss of vital cytoplasmic components and subsequent death of the microorganism (Georgopapadakou and Walsh, 1996). Clinically, two allylamine antifungal substances naphthyne and terbinafine are used, and tolnaftate, which is thiocarbamate. All three are reversible, non-competitive squalene-2,3-epoxidase inhibitors. Synthetically produced morpholines are agricultural fungicides, with the exception of amorolfine, which is used as a topical treatment for nail infections. Echinocandins are a new class of antifungal lipopeptides. They inhibit the synthesis of l, 3-β-D-glucan, which is a polysaccharide in the cell wall of many pathogenic molds and fungi, and is not present in the mammalian cell wall (Groll et al. 2004).

A key weakness of antifungals is the resistance of fungi to antifungal substance. Acquired resistance to fluconazole among C. albicans strains has been reported especially in HIV-infected patients. Acquired amphotericin resistance has been reported in Cryptococcus neoformans and Candida spp., Among others C. lusitaniae, C. glabrata and C. krusei. However, filamentous fungi (Peudalesheria boydii, Scopulariopsis, Fusarium) are known, as well as some yeasts (Trichosporon beigelii, C. lusitanii, C. guiliermondii) that have congenital resistance to amphotericin B (Richter et al., 2005; Matos and Beovic, 2001 ).

Antifungal treatment is often accompanied by side effects. Ketoconazole, which is very lipophilic, accumulates in adipose tissue. Clinical use of amphotericin B is associated with toxicity issues such as: nephrotoxicity, azotemia, decreased glomerular filtration, decreased urine concentration, renal loss of sodium and potassium, renal tubulam acidosis, hypokalemia, hypomagnesemia, low blood pressure, headache, bone marrow depression leukopenia, hepatitis, hyperlipidemia and hyponatremia, fever, chills, muscle pain, anemia and thrombocytopenia.

The above clearly demonstrates the need for new products having antifungal activity, or for products that improve antifungal activity and thereby reduce the required therapeutically effective amount of antifungal substance. The operation of the new product is expected to affect a wide range of fungi. The products should be useful in combination with other antifungal substances, especially if the activity of the accompanying antifungal substance is improved, as this improves the effect of therapy and reduces the potential toxicity of the antifungal substance.

The problem of acquired resistance to antifungals is solved by the use of a combination of several different antifungals. The resistance of fungi to a particular antifungal agent is also resolved by combining it with other substances that do not themselves or have weak antifungal activity, such as curcumin or FK506 (Sharma et al., 2009; Heitman, 2005).

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The inventors have discovered that salicylic acid and its acetylsalicylic acid derivative effectively improve the action of antifungal substances.

References:

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

Groll A.H., Kolve H. 2004. Antifungal agents: In vitro susceptibility testing, pharmacodynamics, and prospects for combination therapy. European Joumal of Clinical Microbiology & Infectious Diseases, 23, 256-270.

Groll A.H., Piscitelli S.C, Walsh T.J. 1998. Clinical pharmacology of systemic antifungal agents: a comprehensive review of agents in clinical use, current investigational compounds, and putative targets for antifungal drug development. Advances in Pharmacology, 44, 343500.

Heitman J. 2005 A fungal achilles' Heel. Science 309, 2175-2176.

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

Matos T, Beovich B. 2001. Antifungal drug resistance. Medical Views, 40, S2, 65-72.

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 Ciin Microbiol 43, 2155–2162.

Sharma M., Manoharlal R., Shukla S., Puri N., Prasad T., Ambudkar S.V., Prasad R. 2009 Curcumin modulates efflux mediated by yeast ABC multidrug transporters and is synergistic with antifungals. Antimicrob Agent Chemoter 53: 3256-3265.

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

Summary of the Invention

The invention is a pharmaceutical combination containing an antifungal substance and salicylic or acetylsalicylic acid, a salt or a derivative of the compounds, and the pharmaceutical combination of the invention is used to kill fungi or inhibit growth and replicate fungi, preferably pathogenic and • · · ·

opportunistic pathogenic fungi. The pharmaceutical combination of the invention is for the preparation of a medicament for in vivo and in vitro prevention and treatment of fungal infections in animals and humans, and the medicament comprises a therapeutically effective amount of a pharmaceutical combination. The following is a pharmaceutical combination for the preparation of a plant protection product for in vivo and in vitro prevention and treatment of fungal infections in plants, and the preparation contains a fungicidally or fungistatically effective amount of a pharmaceutical combination.

The discovery is based on the realization that a combination of an antifungal substance and salicylic or acetylsalicylic acid, a salt or a derivative of the compounds, is fungicidally or fungistatically more effective in combating fungal infections than the antifungal substance itself. The pharmaceutical combination according to the invention, whose components act synergistically, results in reduced consumption of antifungal substance, improved therapeutically more effective action of antifungal substance, reduced treatment time, and in many cases achieved the fungicidal action of the pharmaceutical combination, which could not be achieved with individual components. .

The present invention is based on the discovery that the synergistic action of salicylic or acetylsalicylic acid and an antifungal substance can be used to prepare a medicament for treating a fungus-infected subject and providing a way of treating such infections. An advantage of the invention is the ability to treat fungal diseases that are considered difficult to cure. An additional advantage of the invention is the treatment of fungal infections that have acquired resistance to known antifungal agents. The following is an advantage of treatment with the pharmaceutical combination according to the invention in order to reduce the amount of antifungal substance having adverse side effects. The advantage of treatment with the pharmaceutical combination of the invention is the acquisition of quality of life as it reduces the duration of therapy.

The invention further includes the preparation of (i) a medicament or (ii) a plant protection product containing a pharmaceutical combination for the prevention and treatment of fungal infections in plants, animals and humans. The pharmaceutical combination of the invention can be used in the form of a medicament for the treatment of fungal infections or for decontamination of surfaces, sterilization of medical equipment and implants. The medicament of the invention containing the pharmaceutical combination is prepared in a suitable pharmaceutical form and contains all necessary pharmaceutical additives and, if necessary, additional antifungal agents. The pharmaceutical combination of the invention in the form of a plant protection product with suitable, acceptable additives is used to treat or prevent fungal infections in plants. According to the invention, the medicament containing the pharmaceutical combination is in a suitable pharmaceutical form suitable for topical, intravenous, oral, • intramuscular, subcutaneous, intraperitoneal, intraocular, transpulmonary, transdermal, aerosol administration and the active substances are in the form of a mixture or as a separate individual components, but are used in such a way as to achieve a synergistic effect of the mixture and the active components are separated in order to improve the properties of the individual components.

In addition, the invention provides a method of destroying or inhibiting the growth of fungi, which contains direct contact of the drug or plant protection product containing the pharmaceutical combination with the fungi. The method is used in vitro for decontamination of surfaces and sterilization of medical equipment and implants, in situ sterilization of catheters and intravenous implants. The method of the invention involves either the co-administration of the active components of the pharmaceutical combination or the separate antifungal and separate salicylic or acetylsalicylic acid, salt or derivative of the compounds, but in such a way that the effect is the same or better than when co-administered and a separate application is necessary due to the improved properties of the individual components .

The invention provides a medicament with a therapeutically effective amount of a pharmaceutical combination for the therapeutic or preventive treatment of patients infected with fungi.

Index images

Figure 1. Metabolic activity of A. niger in the presence of a combination of acetylsalicylic acid and the antifungal agent of amphotericin B, fluconazole, ketoconazole or clotrimazole. The combination of acetylsalicylic acid (final concentrations of 0, 0.5, 2 mM) and amphotericin (final concentrations of 0, 0.125, 0.25 mg / l) (A), fluconazole (final concentrations of 0, 0.5, 1 g / l) (B) were used in the test (B ), ketoconazole (final concentrations 0, 5, 10 mg / l), (C) and clotrimazole (final concentrations 0, 0.4, 1 mg / l) (D).

Figure 2. Inhibition of S. cerevisiae yeast growth. Biomass increment in the presence of a combination of acetylsalicylic acid (0, 2, 5 mM) and clotrimazole (0, 0.2, 0.5 mg / l) [A].

Figure 3. Microscopic images of A. niger in the presence of antifungal substance and acetylsalicylic acid. Growth of the fungus in the culture medium without the addition of a pharmaceutical combination [A]; with the addition of 25 μΜ amiodarone and 0.5 mM acetylsalicylic acid [B]; with the addition of 0.4 mg / l clotrimazole and 0.5 mM acetylsalicylic acid [C].

Figure 4. Metabolic activity of A. niger in the presence of salicylic acid (A) and acetylsalicylic acid (B).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is generally accepted by one of skill in the art. The terminology used in the description of the invention is intended to describe a particular part of the invention and not to limit the invention. In the description of the invention and in the claims, the description is in the singular, but the invention also includes a plural, which is not emphasized for ease of understanding.

The present invention relates to the discovery that salicylic or acetylsalicylic acid, salts and derivatives of a compound enhance the action of an antifungal substance and can be used as a pharmaceutical combination of an antifungal substance and salicylic or acetylsalicylic acid for the preparation of (i) a drug or (ii) of a plant protection product for the treatment of a subject: a human, animal or plant infected with fungi and providing a way of treating such infections. According to the invention, a synergistic pharmaceutical combination of antifungal substance and salicylic or acetylsalicylic acid is useful for the preparation of a medicament for the in vivo prevention and treatment of fungal infections, preferably pathogenic and opportunistic pathogens and in vitro for decontamination of surfaces and sterilization of medical equipment and implants. In the following, the synergistic pharmaceutical combination of an antifungal substance and salicylic or acetylsalicylic acid is useful for the preparation of a plant protection product for the in vivo prevention and treatment of fungal infections in plants.

The term antifungal substance refers to an antifungal or fungicide 'and is an antifungal substance used to prevent and treat fungal infections. The term antifungal refers to a substance that inhibits the growth and replication of fungi or causes destruction of a fungal cell and the substance is selected from the group (i) of polyene antifungal agents such as: natamycin, rimocidin, Filipin, nystatin, amphotericin B, candicine; (ii) azole antifungals such as: miconazole, ketoconazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxyconazole, sertaconazole, sulconazole, thioconazole, pramiconazole; (iii) triazole antifungals such as: fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole; (iv) allylamine antifungals such as: terbinafine, amorolfin, naphtidine, butenafine; or (v) echinocandins, such as: anidulafungin, caspofungin, mikaiungin; and others, such as: amiodarone, terbinafine, cyclopyrox, pimaricin, flucytosine, allylamintiocarbamates, grizofulvin, haloprogin. The term antifungal substance also refers to proteins that inhibit the growth of fungi, such as: PAF, AFP, plant defensins and other antifungal peptides and proteins. The term fungicide refers to chemical agents used to control fungi in plant protection products, which may be contact, semi-systemic and systemic.

The present invention preferably relates to the discovery that salicylic acid or acetylsalicylic acid, which is a derivative of salicylic acid, salts or derivatives of compounds, improves the fungicidal or fungistatic action of an antifungal substance, as evidenced by the reduced therapeutically effective amount of an antifungal substance in the prevention and treatment of fungi infections.

The term corresponding salts, derivatives, refers to salts or salicylic acid and acetylsalicylic acid derivatives, which is also a salicylic acid derivative that does not substantially alter the function of the compound, e.g. increase solubility, accessibility to tissue, improve or modify metabolism, breakdown or elimination from the body, and more. The function of salicylic acid and acetylsalicylic acid according to the invention is a synergistic action together with an antifungal substance in the treatment and prevention of fungal infections.

The term synergistic represents an improvement in the fungicidal or fungistatic therapeutic efficacy of an antifungal substance in the presence of salicylic or acetylsalicylic acid. The term therapeutic efficacy refers to a successful clinical outcome and does not necessarily require the pharmaceutical combination to destroy 100% of the organisms involved in the infection. Success depends on the intensity of the fungal infection at the site of infection and refers to the improvement of the infection status. The therapeutic efficacy also depends on the condition of the subject and in a healthy subject a smaller amount of the pharmaceutical combination is required than in a subject having a weakened immune response. According to the invention, the therapeutically effective amount of the antifungal substance in the presence of salicylic or acetylsalicylic acid in the pharmaceutical combination of the invention is lower than in the case of treatment with the antifungal substance alone.

The invention relates to a pharmaceutical combination for the preparation of a medicament or a plant protection product for the prevention or treatment of infections caused by pathogenic and opportunistic pathogenic yeasts and filamentous fungi.

The term fungal infection refers to infections with filamentous fungi and yeasts in plants, animals and humans, as well as in materials, liquids and surfaces. The term fungus refers to microorganisms from the kingdom of fungi, preferably yeast and filamentous fungi, which are plant or animal pathogens and opportunistic pathogens. The pathogenic and opportunistic pathogenic fungi include fungi of the genus 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. sydowi, A. glaucus, A. oryzae, A. ustus, A. versicolor; Histoplasma, preferably Histoplasma capsulatum; Paracoccidioides, preferably P. brasiliensis; Sporothrix, preferably Sporothrix schenckii; Blastomyces, preferably B. dermatitidis; Paecilomyces preferably P. lilacinus, P. variotii; Pneumocystis, preferably P. jirovecii; Stachybotrys, preferably S. chartarum; Malassezia, preferably M. jurfur; 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. soudanum, T. arucoscosense, T. arucoscosense, T. arucoscosense T. concentricum, T. equinum, T. erinacei, T. flavescens, T. gloriae, T. interdigitale, T. megnini, T. phaseoliforme, T. schoenleini, T. simii, T. terrestre, T. tonsurans, T. vanbreuseghemii, T. violaceum, T. yaoundei ·, Microsporum, preferably M. canis, M. gypseum, M. audouini, M. gallinae, M. ferrugineum, M. distortum, M. nanum, M. cookie, M. vanbreuseghemii · , Epidermophyton, preferably E. floccosum, E. stockdaleae; Trichosporon, Fusarium, preferably F. oxysporum, F. scholi, 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; Chaetomium preferably C. atrobrunneum, C. funicola, C. globosum, C. strumarium. The term fungus refers to yeast and filamentous fungi that do not cause disease in a healthy subject, but in immunocompromised subjects, these fungi can cause disease. The term fungus also refers to fungi that are plant pathogens and are Fusarium spp., Thielaviopsis spp., Verticillium spp., Rhizoctonia spp., Phakospora pachyrhizi Sydow, 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 condition caused by yeast infections and filamentous fungi. The term disease refers to superficial diseases that are restricted to infection of the skin, nails and hair; subcutaneous diseases such as dermatitis, which are infections of subcutaneous tissues and adjacent structures; systemic diseases that are infections of the internal organs: lungs, liver, heart, central nervous system, gastrointestinal tract and opportunistic diseases, infections of immunocompromised patients with non-pathogenic fungi. The diseases are expressed in the form of mycoses, candidiasis, systemic mycoses, histoplasmosis, blastomycosis, coccidiomycosis and paracoccidiomycosis, inflammations, redness, dandruff, athletic foot, candidiasis of the mouth and vagina, dermatitis, eczema, onychomycosis, aspergillosis, aspergillosis, aspergillosis , asthma. The term disease refers to a disease caused by fungi in animals and humans, preferably in bees and vertebrates; mainly birds, poultry, mammals; especially equidae, rabbits, beasts, cats, primates, man.

The term treatment refers to preventive and therapeutic treatment. The in vitro method of treatment relates to the destruction and inhibition of the growth and replication of fungi by transferring a pharmaceutical combination of the invention to a fungal infected site.

The invention provides a medicament containing a pharmaceutical combination of the invention for the treatment of fungal infections, and a medicament comprising a pharmaceutical combination in effective amounts to inhibit or destroy fungal cells.

One skilled in the art will determine the therapeutically effective amount of a pharmaceutical combination of an antifungal substance and a proton homeostasis disruptor. The therapeutically effective amount of the pharmaceutical combination according to the invention contains a smaller amount of antifungal substance due to the simultaneous or sequential administration of salicylic or acetylsalicylic acid. The pharmaceutical combination may contain, in addition to the pharmaceutical combination of the antifungal substance and salicylic acid or acetylsalicylic acid, salts or derivatives of the compound.

The more effective action of an antifungal substance in a pharmaceutical combination is seen by the reduced effective amount of the antifungal substance required for complete removal or inhibition of fungal replication and growth. The use of certain antifungal substances is limited by their systemic toxicity or the high cost of treating such antifungal substance. Reducing the concentration of antifungal substances with salicylic or acetylsalicylic acid, salt or derivative of the compounds is necessary in order to reduce the side effects and costs, thus making the pharmaceutical combination more widely used. The medicament of the invention is formulated to provide faster or more effective fungistatic or fungicidal effects than would be achieved with the use of the antifungal substance itself. A medicament containing the pharmaceutical combination of the invention may degrade the fungal resistance of the antifungal substance or change the fungistatic action of the antifungal substance into a fungicidal one. In this case, in the pharmaceutical combination, the effective amount of the antifungal substance is reduced due to the synergistic action of the components of the pharmaceutical combination.

The drug may contain active substances: an antifungal substance and salicylic or acetylsalicylic acid in the pharmaceutical combination of the invention, in the form of a mixture or individual active component, provided that the drug substances are used in a manner that ensures that the separate substances achieve effects at the fungal site infections equal to the synergistic effects of the mixture. The active components in the drug are separated because of the improved stability of the separate components and their ease of use especially in separate routes. The individual components of the pharmaceutical combination of the invention, i.e. the antifungal substance and salicylic or acetylsalicylic acid, may be present in different pharmaceutical forms. A pharmaceutical combination that provides concomitant administration of an antifungal substance and acetylsalicylic acid is expected to be more effective, with a stronger fungistatic or fungicidal effect in vivo than administration of a single substance of the pharmaceutical combination of the invention. The term fungistatic effect "describes the inhibited growth and reproduction of fungi, and the term fungicidal effect" refers to the destruction of fungi.

The invention, a pharmaceutical combination, enables the destruction or inhibition of fungal growth by direct contact of the pharmaceutical combination with the fungus. This method can be used in vitro, such as in plant protection, decontamination of fluids and surfaces, or for the sterilization of surgical and other medical tools and implants, including prosthetics, in situ sterilization of catheters, intravenous implants.

The invention further comprises the preparation of a medicament comprising a pharmaceutical combination for the treatment of fungal infection. It is in any pharmaceutical form and contains the necessary pharmaceutical additives.

The pharmaceutical form is the form in which the active ingredient, the pharmaceutical combination of the invention is incorporated, and relates to powder, suspension, emulsion, solution, paste, cream, ointment, gel, lotion, liniment, pharmaceutical dosage forms, infusion dosage forms, tablet, capsule. , suppository, transdermal patch, therapeutic system, tincture, percolate, extract. The term pharmaceutical additives refers to additives in a pharmaceutical formulation or drug that facilitate, enhance the uptake of active substances, pharmaceutical combinations of the invention at the site of infection, and form part of a pharmaceutical form. Pharmaceutical supplements include suitable diluents, adjuvants, carriers. Administration of the active substance may be topical, intravenous, oral or intramuscular, via aerosol, subcutaneous, intraperitoneal, intraocular, transpulmin, transdermal.

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The pharmaceutical combination of the invention may be administered together with other medicaments. The pharmaceutical combination may contain one or more antifungal substances with salicylic or acetylsalicylic acid.

The invention also relates to a plant protection product in a suitable form and with appropriate additives and is intended to prevent or treat fungal infections in plants. The invention relates to a method of using a pharmaceutical combination in the form of a drug or plant protection product for the destruction of fungi or inhibiting the growth and reproduction of fungi, and is used in the plant protection and decontamination of liquids and surfaces or for the sterilization of surgical and other medical tools and implants, including prosthetics, in situ sterilization of catheters, intravenous implants.

Below are examples of procedures designed to illustrate innovation. Case descriptions are not intended to limit innovation and should be understood in terms of demonstrating innovation.

Performance examples

Example 1. Growth inhibition test

The effect of the pharmaceutical combination was tested on Aspergillus fungi in liquid medium. The spores were grown overnight (from 16 to 24 h) in a synthetic liquid medium at Vogel at 30 ° C. The Vogel medium contained various combinations of antifungal and acetylsalicylic acid. For positive control, we used a fungus grown in culture medium without the addition of a pharmaceutical combination. The method ΧΤΤ used to determine the metabolic activity of fungal cells and their survival is described below.

Preparation of suspension of spores and media

Spore suspensions were prepared in 0.1% (w / v) Tween 80 solution. Spores of final concentrations of 10 ⁵ spores / ml were used to inoculate liquid medium according to Vogel. The Vogel medium contains: 8.5 mM sodium citrate dihydrate (Ε ^^ ΡΕΟγ)), 36.8 mM KH2PO4, 25.0 mM NH4NO3, 0.7 mM CaCl ₂ .2H ₂ O, 0.8 mM MgSO4.7H ₂ O, pH 6.0, 0.2 ml metal trace (34.3 mM EDTA, 15.3 mM ZnSO ₄ .7H ₂ O, 8 mM MnCl ₂ .4H ₂ 0, 1.6 mM CoC1 ₂ .6H ₂ O, 1.3 mM CuSO ₄ .5H ₂ O, 0.2 mM (NH4) 6Mo ₇ O ₂ 4.4H ₂ 0, 10 mM CaCl ₂ .2H ₂ O, 3.6 mM FeSO ₄ .7H ₂ O; pH 6.5), 20 μΜ D-biotin and 2% (w / v) glucose. Antifungals (amiodarone 2 mM in 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 in water were dissolved in the respective solvents v • c appropriate concentrations. Pipette 0.2 ml of culture medium spores into the wells of a microtiter plate (96-well plates were used). A pharmaceutical combination was added to the spore solution. Dispute medium without pharmaceutical combination was used as growth control (100% growth). This was followed by 24 μm incubations at 37 ° C.

The presence of live cells was determined by the ΧΤΤ method. ΧΤΤ The reagent crosses the cell membrane and is converted to the colored compound in active mitochondria. Solutions and menadione were prepared. ΧΤΤ was dissolved in hot water at a final concentration of 1 g / l. Menadion was dissolved in acetone at a concentration of 10 mM, and then diluted 1:10 with saline. The final solution contains a reagent (concentration 0.6 g / l; final concentration in the hole 0.2 g / l) and menadion (concentration 75 μ koncent, final concentration in the hole 25 μΜ).

100 μΐ ΧΤΤ of solution was added to each well. The plate was then incubated at 30 ° C, this time for 4 hours. After incubation, 100 μΐ of the solution was pipetted from each well into a new clear microtiter plate, whereupon the absorbance of the solution was measured at a wavelength of 450 nm on a Bio-Tek® PowerWave XS micro reader.

Figure 1 presents the results of the combination of acetylsalicylic acid (0, 0.5, 2 and 4 mM) and the antifungals of amphotericin, fluconazole, ketoconazole and clotrimazole. Although amphotericin and acetylsalicylic acid act synergistically, in none of the tested combinations did fungicidal activity be achieved. A combination of 0.5 mM acetylsalicylic and 1 g / l fluconazole inhibits growth of 99%. Higher concentrations of the pharmaceutical combination are fungicidal. The same applies to the pharmaceutical combination of acetylsalicylic acid and clotrimazole, which is fungicidal at a concentration of 2 mM acetylsalicylic acid and 1 mg / l of clotrimazole. Somewhat worse results were achieved with the combination of acetylsalicylic acid and ketoconazole, which still works synergistically.

Figure 4 presents the inhibition of the metabolism of A. niger in the presence of salicylic and acetylsalicylic acid. The results show that both salicylic and acetylsalicylic acid, a derivative of salicylic acid, are capable of inhibiting fungal growth.

A combination of acetylsalicylic acid and clotrimazole on A. flavus (Table 1). Both combinations at the concentrations used act on the growth of the fungus synergistically with 99% growth inhibition.

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Table 1. Inhibition of the A. flavus fungus by pharmaceutical combinations of acetylsalicylic acid and clotrimazole. For comparison, the medium without components and with the individual components of the pharmaceutical combination was used.

Acetyl-salicylic acid - 4 mM - 4 mM Clotrimazole - - 1.3 mg / l 1.3 mg / l Growth of A. flavus (%) 100 ± 5 22 ± 1 14 ± 2 Oil

Example 2. Confocal microscopy

Microscopy was used to evaluate the effect of the pharmaceutical combination on the growth of the test fungus. Cells were stained with SynaptoRed and SYTOX dye. The first dye stains the cell membrane, whereas SYTOX poorly crosses the intact cell wall and fluoresces only after DNA binding. Detection of SYTOX dye inside the cell indicates that the cell membrane is damaged.

Combinations of amiodarone and acetylsalicylic acid, clotrimazole and acetylsalicylic acid were tested. A. niger fungus without the addition of a pharmaceutical combination was used as a control. The results are shown in Figure 3. Magnifications of images are equal to magnification of the control preparation. Comparison of growth and staining with SYTOX clearly indicate that fungal growth is impaired in the presence of the pharmaceutical combination. It is observed that the growth of the control preparation is more intense and does not stain within the cell contents with SYTOX dye. Pharmaceutical combinations strongly inhibit growth. The exact implementation of the individual steps of the experiment is described below.

We used a Leica TCS SP5 confocal microscope at work. This is a laser microscope mounted on a Leica DMI 6000 CS inverted microscope (Leica Microsystems, Germany). We used an immersion lens with HCX plan apo 63x oil (NA 1.4). SynaptoRed 2 (Biotium) dye excited at 514 nm with an argon laser was used for dyeing, and emission was collected between 650 and 700 nm and SYTOX Green excited at 488 nm and emitted at 520 to 540 nm.

A 10 ⁵ spore / ml spore suspension was prepared in a Vogel medium containing an antifungal substance and a proton homeostasis disruptor. 0.2 ml of said suspension was applied to a glass slide. The preparation was incubated for 14 hours at 30 ° C in a humidified chamber. The preparations were stained with Synapta Red (cell membrane dyes) and 10 μΐ 0.5 mM Sytox Green dye for 15 minutes at 30 ° C.

Example 3. Inhibition of yeast growth

The pharmaceutical combinations were tested on the yeast Saccharomyces cerevisiae in liquid medium. The yeast was grown overnight (16 to 24 h) in a synthetic liquid medium at 28 ° C. The medium contained various pharmaceutical combinations. Yeast control cells were grown in culture medium without pharmaceutical combination. Yeast cells were diluted to optical density at 600 nm 0.04 (OD600). The growth was monitored spectrophotometrically. The results presented are measured after 8 h of incubation at 28 ° C. We added pharmaceutical combinations to the yeasts and monitored their growth. The results are presented in Figure 2.

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

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

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

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Claims (11)

Claims A pharmaceutical combination comprising an antifungal substance and salicylic or acetylsalicylic acid, a salt or derivative of a compound for killing fungi or inhibiting the growth and replication of fungi. Pharmaceutical combination according to claim 1, characterized in that the antifungal substance is selected from the group of antifungals or fungicides. Pharmaceutical combinations according to any one of claims 1 to 2, characterized in that the fungicidal or fungistatic therapeutic efficacy of the antifungal substance in the pharmaceutical combination in the presence of salicylic or acetylsalicylic acid, salt or derivative of the compound is superior to the therapeutic efficacy of the antifungal substance itself. Pharmaceutical combinations according to any one of claims 1 to 3 for the preparation of a medicament or plant protection product for in vivo and in vitro prevention or treatment of infections caused by fungi, preferably pathogenic and opportunistic pathogenic yeasts and filamentous fungi. A medicament comprising a pharmaceutical combination according to any one of claims 1 to 4 for the prevention or treatment of fungal infections that cause diseases in animals or humans, and those in animals or humans are superficial, limited to the skin, nails and hair; subcutaneous, subcutaneous tissue infections and nearby structures; systemic, these are infections of the internal organs. Medicament according to claim 5, characterized in that it contains a therapeutically effective amount of a pharmaceutical combination according to any one of claims 1 to 4 in a suitable pharmaceutical form with appropriate pharmaceutical additives. Medicament according to any one of claims 5 to 6, characterized in that the medicament is in pharmaceutical form suitable for topical, intravenous, oral, intramuscular, subcutaneous, intraperitoneal, intraocular, transpulmonary, transdermal, aerosol administration. Medicament according to any one of claims 5 to 7, characterized in that it contains a pharmaceutical combination in the form of separately containing an antifungal substance and separately salicylic or acetylsalicylic acid, salts or derivatives of a compound, which are used in a manner to achieve synergistic effects. the effect is characteristic of the pharmaceutical combination and are separated by improved stability of the individual components of the pharmaceutical combination or a separate route of administration thereof. A plant protection product comprising a pharmaceutical combination according to any one of claims 1 to 4 in a suitable pharmaceutical form with suitable acceptable additives and intended to prevent or treat fungal infections in plants. 10. A method of destroying fungi or inhibiting the growth and replication of fungi, preferably pathogenic and opportunistically pathogenic fungi by transferring a pharmaceutical combination according to any one of claims 1 to 4 in the form of a medicament according to any one of claims 5 to 8 or a plant protection product according to claim 9 onto a fungus contaminated site and used in plant protection products, decontamination of fluids and surfaces, or for sterilization of surgical and other medical tools and implants, including prosthetics, in situ sterilization of catheters, intravenous implants. A method according to claim 10, characterized in that the active substances of the pharmaceutical combination according to any one of claims 1 to 4 are used separately, sequentially, but still in such a way that the synergistic effect obtained is characteristic of simultaneous use.