Compositions and methods for treating fungal infections

Abstract

Disclosed herein is a method for treating a fungal infection in a patient comprising administering to the patient a therapeutically effective combination of a protease affecting a glycoprotein and an antifungal agent. Also disclosed is a method for sensitizing a fungus having a fungus cell wall containing a glycoprotein to an antifungal agent. The method comprises contacting the fungus with a protease that affects a glycoprotein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims priority from Australian Provisional Patent Application No. 2022903688, the contents of which are incorporated herein in their entirety. Technical Field

[0003] The present invention relates to compositions and methods for treating fungal infections. The present invention also relates to sensitizing certain fungi to antifungal agents. Background Art

[0004] Fungal infections are common and can vary in severity from a minor nuisance to an immediately life-threatening illness. In fact, fungal infections are reported to cause over 1.5 million deaths each year and affect over a billion people.

[0005] Fungal infections usually develop when fungal spores enter a person's body through the respiratory tract (that is, by inhalation) or through a wound. Fungal infections include candidiasis (caused by Candida species), cryptococcosis (caused by Cryptococcosis neoformans), histoplasmosis (caused by Histoplasma species), pneumocystis (caused by Pneumocystis jirovecii), and mucormycosis (caused by mucormycetes).

[0006] Many antifungal agents are used to treat a variety of fungal infections. However, the toxicity of these agents often limits the tolerated doses, especially in critically ill patients, thereby reducing their effectiveness. Furthermore, antifungal resistance is becoming increasingly common and is a particular problem for some invasive fungi. Some fungi are naturally resistant to antifungal agents (for example, fluconazole is inactive in Aspergillus), while others, particularly Aspergillus and Candida species, develop acquired resistance over time.

[0007] It would be advantageous to provide new therapeutic options for treating fungal infections. Summary of the Invention

[0008] In a first aspect, the present invention provides a method for treating a fungal infection in a patient, comprising administering to the patient a therapeutically effective combination of a glycoprotein-affecting protease and an antifungal agent.

[0009] In a second aspect, the present invention provides a method for sensitizing a fungus that is the cause of a fungal infection in a patient to an antifungal agent, wherein the fungus has a fungal cell wall containing a glycoprotein. The method comprises administering to the patient a protease that affects the glycoprotein.

[0010] In a third aspect, the present invention provides a method for sensitizing a fungus having a fungal cell wall containing a glycoprotein to an antifungal agent, wherein the method comprises contacting the fungus with a protease that affects the glycoprotein.

[0011] In a fourth aspect, the present invention provides a method for treating a fungal infection in a patient, comprising administering to the patient a therapeutically effective combination of a glycoprotein-affecting protease and a disulfide bond cleaving agent.

[0012] In a fifth aspect, the present invention provides a use of a protease that affects glycoproteins for sensitizing fungi having fungal cell walls containing glycoproteins, thereby improving the efficacy of antifungal agents.

[0013] In a sixth aspect, the present invention provides a composition comprising a protease that affects glycoproteins and an antifungal agent.

[0014] In a seventh aspect, the present invention provides a synergistic combination of a protease that affects glycoproteins and an antifungal agent.

[0015] In an eighth aspect, the present invention provides a synergistic combination of a protease affecting glycoproteins and an antifungal agent, wherein the synergistic combination is used to sensitize a fungus having a fungal cell wall containing glycoproteins to the antifungal agent.

[0016] In a ninth aspect, the present invention provides a use of a combination of a glycoprotein-affecting protease and an antifungal agent for the preparation of a medicament for treating a fungal infection in a patient.

[0017] In a tenth aspect, the present invention provides a combination of a protease affecting glycoproteins and an antifungal agent, for use in medicine.

[0018] In an eleventh aspect, the present invention provides a combination of a glycoprotein-affecting protease and an antifungal agent for use in treating a fungal infection in a patient.

[0019] Bromelain (a specialized protease that affects glycoproteins) has been reported as an antifungal agent in the context of limiting the growth of certain plant fungi. However, this effect is not reportedly prevalent in many fungal species. In fact, experiments conducted by the present inventors, some of which are described below, have shown that bromelain alone appears to have no significant antifungal activity against Rhizopus microsporus, Fusarium solani cx, Rhizopus arrhizus, and Candida krusei QC. Instead, bromelain was found to stimulate fungal growth in some of these fungi under the experimental conditions used.

[0020] Therefore, the inventors were surprised to find that the combination of bromelain and various antifungal agents had a synergistic effect against fungi that cause mucormycosis. The inventors are not aware of any suggestion in the literature that bromelain might improve the effectiveness of antifungal agents or otherwise sensitize fungi, particularly fungi that can infect humans.

[0021] Experiments conducted by the inventors following their initial discovery showed that co-administration of bromelain with various antifungal agents resulted in increased susceptibility to the antifungals in various Mucor isolates and different types of Candida (in some cases, significant synergy was observed). As will be appreciated, Candida is a yeast, and Mucor is a mold, so the present invention appears to be applicable to a wide range of fungi. The inventors observed varying levels of sensitivity among different fungi types and speculated (without wishing to be bound by theory) that the fungal cell wall may be responsible for this variation. As will be appreciated by those skilled in the art, the fungal cell wall is a complex organelle composed of glucan, chitin, chitosan, and other glycosylated proteins. The proportions of these components vary between different fungal types, which the inventors believe may be responsible for the varying susceptibilities observed in their preliminary work.

[0022] In general, and based solely on their preliminary data, the inventors speculate that the variation in efficacy they observe when performing various embodiments of the invention with different fungi (some of which are described below) depends on the proportions of glycoproteins in the cell wall of the particular fungus, as well as the accessibility of those glycoproteins.

[0023] As will be appreciated by those skilled in the art, cell wall proteins have diverse functions, including maintenance of cell shape, adhesion processes, cellular protection against various substances, absorption of molecules, signaling, and the synthesis and reorganization of wall components. Glycoproteins are reported to comprise 30%-50% of the dry weight of the fungal wall in yeast and 20%-30% of the dry weight of the wall in filamentous fungi. The chitin content of fungal cell walls is reported to vary depending on the morphological stage of the fungus, comprising 1%-2% of the dry weight of the yeast cell wall and up to 10%-20% in filamentous fungi. Other fungi, such as lomentaspora and scedosporium, have cell walls with a protective melanin coating.

[0024] Again, without wishing to be bound by theory, the inventors believe that the observed increased sensitivity of fungi to antifungal agents is due to bromelain affecting glycoproteins (at least) and glycans in the cell walls of fungi containing such agents, solubilizing or otherwise degrading the cell walls, allowing the antifungal agents to more easily enter the cells and thereby enhancing their therapeutic effects. The inventors predict that the mechanism of action of bromelain in fungal sensitization may be related to the solubilization of glycoproteins in the fungal wall (although, to the best of their knowledge, this has never been previously investigated), and therefore the present invention may be generally applicable to any fungus having a fungal cell wall containing glycoproteins (i.e., utilizing any protease that affects glycoproteins).

[0025] As mentioned above, invasive fungal diseases have a high mortality rate and limited therapies. Treatment according to the present invention has the potential to improve efficacy or at least maintain efficacy using reduced doses of the antifungal agent, thereby limiting its side effects.

[0026] The inventors believe that the results of their preliminary experiments, some of which are described below, lead to a reasonable prediction that proteases other than bromelain, including those described herein, that affect glycoproteins may be effective for therapeutic applications as described herein. Further experiments, some of which are currently underway, should confirm the inventors' predictions.

[0027] Encouraging results from earlier experiments by the present inventors prompted a re-examination of the antifungal activity of glycoprotein-affecting proteases in combination with disulfide bond breakers, particularly in the form of bromelain in combination with N-acetylcysteine, a therapeutic agent known under the brand name BromAc. ®The inventors were surprised to find that this combination does exhibit antifungal activity, particularly against fungi with cell walls containing a relatively high proportion of glycoproteins and polysaccharides. Again, without wishing to be bound by theory, the inventors believe that contact between the fungal cell wall and BromAc causes degradation of glycoproteins and other components of the cell wall. This degradation weakens the cell wall, either rendering the fungus inviable or allowing bromelain and / or N-acetylcysteine ​​(and optionally the antifungal agent) to enter the cell and kill it.

[0028] In some embodiments, the fungal infection may be caused by a fungus from a fungal genus selected from one or more of the following: Rhizopus, Mucor, Rhizomucor, Cryptococcus, Candida, Syncephalastrum, Cunninghamella, Apophysomyces, Aspergillus, Histoplasma, Pneumocystis, and Lichtheimia.

[0029] In some embodiments, the fungal infection may be caused by a fungus selected from one or more of the following: Rhizopus arrhizus, Rhizopus microsporus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Fusarium solani, Candida krusei, Candida auris, Candida parapslosis, Candida tropicalis, Candida glabrata, Candida albicans, Cryptococcus gattii, Cryptococcus neoformans, Cunninghamella bertholletiae, Lichtheimiacorymbifera, Mucor circinelloides, circinelloides), Histoplasma capsulatum, Pneumocystis jiroveci, and Microsporidia.

[0030] In some embodiments, the fungal infection can be, for example, selected from one or more of the following: mucormycosis (e.g., cutaneous mucormycosis, rhinocerebral mucormycosis, and pulmonary mucormycosis), histoplasmosis, cryptococcosis, pneumocystis pneumonia, candidiasis, and aspergillosis. More generally, the present invention can be used to treat systemic fungal infections, such as systemic fungal sepsis, as well as localized fungal infections, such as those found on prostheses or in the respiratory tract. Any respiratory tract infection caused by a fungus can also be treated using the present invention, with the caveat that Aspergillus, Candida, and Mucor being particularly pulmonary invasive.

[0031] In some embodiments, the protease that affects glycoproteins and the antifungal agent can be administered to the patient simultaneously or sequentially. For example, the protease that affects glycoproteins and the antifungal agent can be administered to the patient together. For example, the protease that affects glycoproteins and the antifungal agent can be administered to the patient via different routes of administration. For example, potential routes of administration include topical, systemic, intravenous, by inhalation, by aerosolization, by intratracheal injection, by intracavitary injection, by intraperitoneal injection, by intrapleural injection, by intraocular injection, by injection into the parenchyma, or by injection into the cerebrospinal fluid via an intraventricular or intrathecal (cisternal or lumbar) route.

[0032] In some embodiments, the protease affecting glycoproteins may be a cysteine ​​protease. In some embodiments, the protease affecting glycoproteins may be selected from one or more of the group consisting of bromelain, papain, ficin, taurantine, zingiberenin, fastuosain, and ananain.

[0033] In some embodiments, the antifungal agent can be selected from one or more of the group consisting of polyenes (e.g., amphotericin B and nystatin), echinocandins (e.g., caspofungin and micafungin), azoles (e.g., isavuconazole, posaconazole, fluconazole, ketaconazole, voriconazole, and itraconazole), propionamides (e.g., terbinafine), and ortomides. Emerging antifungal agents, including inhibitors of calcineurin, trehalose pathway inhibitors, and inhibitors of sphingolipid synthesis, may also be potentially useful in the present invention.

[0034] In some embodiments, the present invention may further comprise administering to the patient a disulfide bond cleaving agent (which may also function as a thiol donor) in combination with a protease that affects glycoproteins and an antifungal agent (or in embodiments of the fourth aspect of the invention, only a protease that affects glycoproteins). Some of the present inventors have previously observed, for example, a synergistic effect between proteases that affect glycoproteins (such as bromelain) and disulfide bond cleaving agents (such as N-acetylcysteine ​​and cysteamine).

[0035] In such embodiments, the disulfide bond cleaving agent can be, for example, selected from one or more of the following: N-acetylcysteine, cysteamine, erdosteine, s-carboxymethylcysteine, glutathione, dithiothreitol, nacystelyn, mercapto-ethanesulfonate, carbocysteine, dornase alpha, gelsolin, thymosin P4, dextran, bucillamine, dithiobutylamine (DTBA) and heparin. In such embodiments, the protease that affects the glycoprotein and the disulfide bond cleaving agent can be administered simultaneously or sequentially.

[0036] In some embodiments, the present invention may further comprise administering to the patient an additional agent (e.g., DNase or collagenase). Such additional agents may help further degrade fungal cell components, thereby even further enhancing the effect of the antifungal agent.

[0037] In some embodiments, the present invention may further comprise administering to the patient one or more additional therapeutic agents. In such embodiments, the one or more additional therapeutic agents may be selected from the group consisting of: an antiviral agent, an antibacterial agent, a bronchodilator, and an expectorant.

[0038] It should be understood that any features or embodiments described in detail herein with respect to a particular aspect of the invention are also applicable to other aspects of the invention. Other aspects, features and advantages of the invention will be described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The embodiments of the present invention will now be described in more detail with reference to the following figures, wherein p values ​​are used to measure significant killing of growth control and are shown in the figures: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. All doses presented are in μg / ml, with the exception of acetylcysteine, which is in mg / ml.

[0040] Figure 1 shows (A) a dose-response graph showing the percent growth of Candida glabrata (C. glabrata) when exposed to bromelain alone and in combination with bromelain and amphotericin B; and (B) a dose-response graph showing the percent growth of Candida glabrata when exposed to amphotericin B alone (GC = Growth Control, B = Bromelain, and A = Amphotericin B).

[0041] Figure 2 Shown is a dose response graph showing the percentage growth of Candida glabrata when exposed to amphotericin B alone (GC = Growth Control; A = Amphotericin B).

[0042] Figure 3 Shown are dose-response graphs showing the percentage growth of Candida glabrata when exposed to amphotericin B, BromAc, and BromAc with amphotericin B (GC = Growth Control, A = Amphotericin B, B = Bromelain, and N = Acetylcysteine). a) and b) represent higher and lower concentrations of bromelain used.

[0043] Figure 4 Shown is a dose response graph showing the percent growth of C. glabrata when exposed to posaconazole alone (GC = growth control, and P = posaconazole).

[0044] Figure 5 Shown are dose-response graphs showing the percentage growth of Candida glabrata when exposed to posaconazole, BromAc, and BromAc with posaconazole (GC = Growth Control, P = Posaconazole, B = Bromelain, and N = Acetylcysteine). a) and b) represent higher and lower concentrations of bromelain used.

[0045] Figure 6 Shown is a dose response graph showing the percent growth of C. glabrata when exposed to fluconazole alone (GC = growth control, and P = posaconazole).

[0046] Figure 7 Shown are dose-response graphs showing the percentage growth of Candida glabrata when exposed to fluconazole, BromAc, and BromAc with fluconazole (GC = Growth Control, F = Fluconazole, B = Bromelain, and N = Acetylcysteine). a) and b) represent higher and lower concentrations of bromelain used.

[0047] Figure 8Shown is a dose response graph showing the percent growth of C. krusei when exposed to amphotericin B alone (GC = Growth Control, and A = Amphotericin B).

[0048] Figure 9 Shown are dose-response graphs showing the percentage growth of Candida krusei when exposed to amphotericin B, BromAc, and BromAc with amphotericin B (GC = Growth Control, A = Amphotericin B, B = Bromelain, and N = Acetylcysteine). a) and b) represent higher and lower concentrations of bromelain used.

[0049] Figure 10 Shown are (a) a dose-response graph showing the percent growth of Candida krusei when exposed to different doses of posaconazole alone, and (b) a dose-response graph showing the percent growth of Candida krusei when exposed to posaconazole, BromAc, and BromAc with posaconazole (GC = growth control, P = posaconazole, B = bromelain, and N = acetylcysteine).

[0050] Figure 11 Shown is a dose response graph showing the percent growth of Candida krusei when exposed to fluconazole alone (GC = Growth Control, and F = Fluconazole).

[0051] Figure 12 Shown are dose-response graphs showing the percentage growth of Candida krusei when exposed to fluconazole, BromAc, and BromAc with amphotericin B (GC = Growth Control, F = Fluconazole, B = Bromelain, and N = Acetylcysteine). a) and b) represent higher and lower concentrations of bromelain used.

[0052] Figure 13 Shown are (a) a dose-response graph showing the percent growth of Aspergillus fumigatus when exposed to different doses of voriconazole alone, and (b) a dose-response graph showing the percent growth of Aspergillus fumigatus when exposed to voriconazole, BromAc, and BromAc with voriconazole (GC = growth control, V = voriconazole, B = bromelain, and N = acetylcysteine).

[0053] Figure 14Shown are (a) a dose-response graph showing the percentage growth of Aspergillus fumigatus when exposed to different doses of caspofungin alone, and (b) a dose-response graph showing the percentage growth of Aspergillus fumigatus when exposed to caspofungin, BromAc, and BromAc with caspofungin (GC = growth control, C = caspofungin, B = bromelain, and N = acetylcysteine). DETAILED DESCRIPTION

[0054] As described above, in its most general form, the present invention provides a method for treating a fungal infection in a patient, wherein a therapeutically effective combination of a protease that affects glycoproteins and an antifungal agent is administered to the patient. The present invention also provides a method for sensitizing a fungus having a fungal cell wall containing glycoproteins to an antifungal agent, wherein the fungus is contacted with a protease that affects glycoproteins. The present invention also provides a method for sensitizing a fungus that is the cause of a fungal disease or infection in a patient, wherein the fungus has a fungal cell wall containing glycoproteins, wherein the protease that affects glycoproteins is administered to the patient. Finally, the present invention also provides a method for treating a fungal infection in a patient, wherein a therapeutically effective combination of a protease that affects glycoproteins and a disulfide bond cleaving agent is administered to the patient.

[0055] Fungal infections contemplated for treatment according to the present invention include mucormycosis (e.g., cutaneous, rhinocerebral, and pulmonary), histoplasmosis, cryptococcosis, pneumocystis pneumonia, candidiasis, and aspergillosis. The present invention is also potentially useful for treating systemic fungal infections, such as systemic fungal sepsis, as well as localized fungal infections, such as those found on prostheses. Any respiratory tract infection caused by a fungus may also be treated using the present invention, with the caveat that Aspergillus, Candida, and Cryptococcus species are particularly pulmonary-invasive.

[0056] Such fungal infections may be caused by fungi from the genera including Rhizopus, Mucor, Rhizomucor, Cryptococcus, Candida, Coprosporus, Cunninghamella, Lepidoptera, Aspergillus, Histoplasma, Pneumocystis, and Trachoderma. Specific fungi that cause these fungal infections may include: Rhizopus arrhizus, Rhizopus microsporus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Fusarium solani, Candida krusei, Candida auris, Candida parapsilosis, Candida tropicalis, Candida glabrata, Candida albicans, Cryptococcus gattii, Cryptococcus neoformans, Cunninghamella griseus, Trachoderma corymbifera, Mucor circinelloides, Histoplasma capsulatum, Pneumocystis jiroveci, and microsporidia.

[0057] In some embodiments, the patient can be a mammalian subject. Typically, the patient will be a human patient, although other subjects may benefit from the present invention. For example, the subject can be a pig, mouse, rat, dog, cat, cow, sheep, horse, or any other mammal of social, economic, or research importance.

[0058] As described above, the inventors predict that the mechanism of action of bromelain in the observed antifungal sensitization may be related to the solubilization of glycoproteins (and potentially other components such as glucans and chitin) in the fungal wall, although to their knowledge, this has not been previously investigated. The inventors believe that their preliminary experimental results, some of which are described below, support the reasonable prediction of broad therapeutic applications disclosed herein. The inventors also believe that the results of their preliminary experiments support the reasonable prediction that other proteases that affect glycoproteins will have utility in the present invention. Further experiments along the lines described herein should confirm the inventors' predictions.

[0059] Proteases that affect glycoproteins

[0060] The present invention relates to the use of proteases that affect glycoproteins. Proteases that affect glycoproteins are proteolytic enzymes that cause proteolysis of glycoproteins. In view of the inventors' preliminary data on bromelain, a protease that affects glycoproteins by hydrolyzing peptides and glycosidic bonds within glycoproteins, the inventors believe that any protease that affects glycoproteins can be used in the present invention, and that only routine testing and experimentation (based on the teachings contained herein) are required to determine the suitability of any particular protease that affects glycoproteins. As used herein, the term "affecting glycoproteins" is understood to mean affecting glycoproteins (e.g., chitin) in the fungal cell wall in any therapeutically effective manner, for example, by digesting, liquefying, or otherwise causing the disintegration or degradation of glycoproteins (and other peptide bonds) in the cell wall. For example, a protease that affects glycoproteins can effectively disintegrate glycoproteins. For example, a protease that affects glycoproteins can effectively hydrolyze peptides and glycosidic bonds in glycoproteins.

[0061] As mentioned above, glucans (particularly β-glucans) are another major component of fungal cell walls. Therefore, if the present invention also includes an agent that affects glucans, an enhanced therapeutic effect can be achieved. In some embodiments, the protease that affects glycoproteins can also be a protease that affects glucans (e.g., as in the case of bromelain). In alternative embodiments, agents that affect glucans, such as poacic acid and echinocandins, can be used in combination with proteases that affect glycoproteins.

[0062] For example, a protease that affects glycoproteins can be a cysteine ​​protease. Cysteine ​​proteases (also known as thiol proteases) degrade proteins through a common catalytic mechanism and are commonly derived from fruits, including papaya, pineapple, fig, and kiwi. Examples of cysteine ​​proteases include bromelain, papain (extracted from papaya), and bromelain, a plant cysteine ​​protease in the papain superfamily of cysteine ​​proteases.

[0063] Other plant-derived proteolytic enzymes expressing the same characteristics as bromelain and the inventors' expectation that any plant-derived protease or recombinant that glycoprotein is affected can be used in the present invention.Again, routine tests should be able to confirm the applicability of the protease of any specific plant origin.For example, in some embodiments, the protease of plant origin can be selected from one or more of the following groups: bromelain, papain (extracting from papaya), ficin (extracting from fig), kiwifruit protease (extracting from fruits including kiwifruit, pineapple, mango, banana and papaya), ginger protease (extracting from ginger) and schizonepeta protease (cysteine ​​protease from bromelia fastuosa).Asparagus, mango and other kiwifruit and papain can also be useful.

[0064] The active portion of the protease that affects glycoprotein can be used in the present invention, it should be noted that it may not be necessary to include all substances in the protease extract, provided that the portion itself affects glycoprotein. It is expected that the protease that affects glycoprotein obtained using genetic recombination can also be used in the present invention.

[0065] As used herein, "bromelain" is understood to encompass the substances that affect glycoproteins and, optionally, one or more other therapeutically active substances present in extracts of the pineapple plant (Ananas Comosus). Bromelain is a mixture of substances (including different thiol endopeptidases and other components such as phosphatases, glucosidases, peroxidases, cellulases, esterases, and several protease inhibitors), and it may not be necessary to include all of these substances in the combination, provided that at least some of the substances in the combination can affect glycoproteins.

[0066] Bromelain used in the experiments described herein was commercially sourced from Enzybel Group, and any further extraction and purification processing was performed by Mucpharm Pty Ltd.

[0067] The amounts and relative proportions of the glycoprotein-affecting protease and antifungal agent used in the present invention can vary depending on a variety of factors, such as the type of glycoprotein-affecting protease and antifungal agent, its intended use, and patient factors, such as its weight and other health factors, including the severity of the infection. However, in general, the combination or composition for administration is expected to include about 5 μg / mL to about 2 mg / mL of the glycoprotein-affecting protease. These amounts are higher than those required for therapeutic efficacy with some routes of administration, but the inventors note that only a small portion of the drug administered via some routes (e.g., nebulization) may be bioavailable. Because drug losses may vary depending on the treatment regimen and device, the amounts of the glycoprotein-affecting protease described below refer to the amount of the glycoprotein-affecting protease that will be received by the patient, as can be measured using conventional techniques.

[0068] Amounts below about 5 μg / mL of the glycoprotein-affecting protease may be ineffective, and amounts above about 1,000 μg / mL will be more likely to cause systemic adverse side effects. In some embodiments, for example, about 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, 550 μg / mL, 600 μg / mL, 650 μg / mL, 700 μg / mL, 750 μg / mL, 800 μg / mL, 850 μg / mL, 900 μg / mL, 950 μg / mL, 1,000 μg / mL of a glycoprotein-affecting protease can be administered in combination.

[0069] It is envisioned that for some therapies and / or patients, repeated treatments may be beneficial (or necessary) to achieve effective treatment.

[0070] The protease that affects glycoproteins and the antifungal agent can be administered to the patient in any manner that provides the desired therapeutic effect. For example, the protease that affects glycoproteins and the antifungal agent can potentially be administered simultaneously (e.g., in a single composition), sequentially (e.g., in separate compositions, one after the other), or separately (e.g., in separate compositions) via different routes of administration.

[0071] antifungal agents

[0072] Any antifungal agent may be used in the present invention, and the suitability of any given antifungal agent can be determined using only routine testing and experimentation. Examples of potentially suitable antifungals include polyenes (e.g., amphotericin B, liposomal amphotericin, and nystatin), echinocandins (e.g., caspofungin and micafungin), azoles (e.g., posaconazole, isavuconazole, fluconazole, ketoconazole, voriconazole, and itraconazole), propionamides (e.g., terbinafine), and ortomides. As will be appreciated, some fungal infections are treated with multiple classes of antifungal agents (e.g., polyenes plus azoles), and this may also be the case in the present invention.

[0073] Emerging antifungal agents, including calcineurin inhibitors, trehalose pathway inhibitors, and sphingolipid synthesis inhibitors, may also potentially be used in the present invention. Other antifungal agents, such as fungal cytochrome P450 enzyme inhibitors, beta-glucan synthesis inhibitors, chitin synthase inhibitors, ergosterol binders, squalene epoxidase inhibitors, calcineurin signaling inhibitors, DNA synthesis inhibitors, Hsp90 inhibitors, protein synthesis inhibitors, microtubule assembly inhibitors, and ROS (reactive oxygen species) generators, may potentially produce synergistic effects in treatment.

[0074] In use, the antifungal agent achieved its indicated therapeutic effect (at least for experiments performed by the inventors, see below), but a synergistic effect was observed, and the amount of antifungal agent required for the same therapeutic result was significantly reduced. As described above, given that many antifungal agents have undesirable side effects, lower dosages can be very advantageous.

[0075] As described above, the amounts and relative proportions of the glycoprotein-affecting protease and antifungal agent used in the present invention may vary depending on a variety of factors. However, in general, the combination or composition for administration is expected to include an amount of antifungal agent that is significantly less than the amount required to achieve its therapeutic effect if administered alone (i.e., conventionally). Based on the teachings contained herein and information available from standard medical sources, one skilled in the art will be able to determine the appropriate amount of antifungal agent. For example, amphotericin is used to treat Aspergillus, Candida, and / or Cryptococcus species at a dosage rate of 3-5 mg / kg IV once daily (qDay).

[0076] Disulfide bond cleaving agents

[0077] In some embodiments, the present invention may further comprise administering to the patient a disulfide bond cleaving agent in combination with a protease that affects glycoproteins and an antifungal agent (or, according to such aspects of the invention, in combination only with a protease that affects glycoproteins). Disulfide bond cleaving agents are species that disrupt the disulfide bridges that help define the tertiary structure of proteins.

[0078] In such embodiments, the disulfide bond cleaving agent can be, for example, selected from one or more of the following: N-acetylcysteine, cysteamine, erdosteine, s-carboxymethylcysteine, glutathione, dithiothreitol, nacystelyn, mercapto-ethanesulfonate, carbocysteine, N-acystelyn, dornase alpha, gelsolin, thymosin P4, dextran, bucillamine, dithiobutylamine (DTBA), and heparin. In such embodiments, the protease that affects the glycoprotein and the disulfide bond cleaving agent can be administered simultaneously or sequentially.

[0079] In some embodiments, for example, about 2.5 mg / ml (0.25% w / v), 5 mg / ml (0.5% w / v), 10 mg / ml (1.0% w / v), 15 mg / ml (1.5% w / v), or 20 mg / ml (2.0% w / v) of a disulfide bond breaker, such as N-acetylcysteine, may be included in the combination or composition for administration.

[0080] As used herein, the term "BromAc" is a combination of bromelain and acetylcysteine, a drug combination developed by some of the present inventors for the treatment of mucinous cancers. BromAc has been found to rapidly dissolve and remove tumor mucin, whereas neither drug alone is effective.

[0081] Additional agents that degrade fungal cell walls

[0082] In some embodiments, the present invention may further comprise administering to the patient one or more additional agents (e.g., DNA enzymes or collagenases) that can help degrade the fungal cell wall (i.e., in combination with a protease that affects glycoproteins and an antifungal agent (or in such aspects of the invention, with a protease that affects glycoproteins and a disulfide bond cleaving agent). Such additional agents can help further degrade (or degrade more rapidly) the fungal cell wall (e.g., a DNA enzyme can degrade any DNA in the fungal cell), thereby even further enhancing the antifungal effect.

[0083] In compositions comprising an additional fungal cell wall degrading agent, the agent may be present in the composition in any amount that produces a beneficial effect. For example, in the case of DNA enzymes, an amount of about 5 μg / mL to 200 μg / mL is expected to provide the beneficial effects described herein.

[0084] Additional therapeutic agents

[0085] The present invention may also include additional therapeutic agents. Any additional therapeutic agent with appropriate indications in the context of treating fungal infections may also be co-administered to the patient. Examples of additional therapeutic agents include antivirals, antibacterials, bronchodilators, and / or expectorants. The additional therapeutic agent may be administered simultaneously with the glycoprotein-affecting protease and / or antifungal agent or, more likely, sequentially.

[0086] In the composition that includes additional therapeutic agents, any amount of the agent that produces a beneficial effect can be used. Those skilled in the art are capable of determining the appropriate amount of any such additional therapeutic agent. In some embodiments, two or more additional therapeutic agents may provide a beneficial effect, especially if their therapeutic effects are through different mechanisms.

[0087] Other components that do not necessarily have a direct therapeutic effect may be included in the present invention. Such components include pharmaceutically acceptable excipients and carriers.

[0088] Application

[0089] The protease that affects glycoproteins and the antifungal agent can be administered to the patient in any manner that provides the desired therapeutic effect. Any route of administration that brings the protease that affects glycoproteins (and the other components of the combination) into proximity with the fungus is expected to be effective. For example, potential routes of administration include topical, systemic, intravenous, by inhalation, by aerosolization, by intratracheal injection, by lung lavage, by intracavitary injection, by intraperitoneal injection, by intrapleural injection, by intraocular injection, by injection into the parenchyma, or by injection into the cerebrospinal fluid via an intraventricular or intrathecal (cistern or lumbar) route.

[0090] In some embodiments, the protease that affects glycoproteins and the antifungal agent can be administered simultaneously or sequentially. For example, the protease that affects glycoproteins and the antifungal agent can be administered to the patient together. For example, the protease that affects glycoproteins and the antifungal agent can be administered to the patient via different routes of administration.

[0091] If administered to a patient's lungs (e.g., after nebulization), for example, to treat mucormycosis, the combination can be inhaled into the trachea or bronchi using specialized medical equipment, such as through a bronchoscope. Alternatively (or additionally), the combination can be inhaled into the patient's nose or mouth or trachea during nebulization. Alternatively (or additionally), the combination can be aerosolized and delivered to the patient's surroundings, such as a closed system tent or other enclosed environmental space, for treatment.

[0092] Nebulization is a common method of delivering medications into the respiratory tract. A nebulizer is a delivery device used to administer medications in the form of a mist that is inhaled into the lungs, and can use oxygen, compressed air, or ultrasonic power to break up solutions and suspensions into small aerosol droplets that are inhaled from the device's mouthpiece.

[0093] Another common method of delivering medication to the respiratory tract is the use of a dry powder inhaler or metered dose inhaler. Such inhalers are well known in the art and deliver a specific amount of medication to the lungs in the form of a short burst of aerosolized medication, typically self-administered by the patient through inhalation.

[0094] The dosage of the combination of the present invention administered to a patient will depend on factors such as the route of administration, the severity of the infection, and the patient's weight. It is within the capabilities of those skilled in the art to determine appropriate dosages (and administration regimens) based on the teachings contained herein and the experience of those skilled in the art.

[0095] Pharmaceutical composition

[0096] In some embodiments, the combination of a glycoprotein-affecting protease, an antifungal agent, and optional additional agents used in the present invention can be provided in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0097] Such pharmaceutically acceptable carriers will depend on the route of administration of the composition. Liquid form formulations may include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions, for generating aerosols for airway (intranasal or intratracheal) delivery. Suitable pharmaceutically acceptable carriers for use in the pharmaceutical compositions of the present invention include physiologically buffered saline, normal saline, hypertonic saline, dextrose solution, and Ringer's solution, among others. As described above, powder formulations for inhalation are also contemplated.

[0098] Pharmaceutical compositions suitable for delivery to a patient may be prepared immediately prior to delivery to the patient, or may be prepared in advance and appropriately stored in advance.

[0099] The pharmaceutical compositions and medicaments used in the present invention may contain pharmaceutically acceptable carriers, adjuvants, vehicles and / or diluents. The carriers, diluents, vehicles and adjuvants must be "acceptable" in the sense of being compatible with the other ingredients of the composition or medicament and the delivery method, and generally not harmful to the recipient thereof.

[0100] It will be appreciated that, where appropriate, some of the components of the combinations or pharmaceutical compositions described herein may be provided in the form of metabolites, pharmaceutically acceptable salts, solvates, or prodrugs thereof. "Metabolites" of components of the present invention refer to intermediates and products of metabolism.

[0101] "Pharmaceutically acceptable," such as pharmaceutically acceptable carriers, excipients, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered.

[0102] "Pharmaceutically acceptable salts" refers to conventional acid addition salts or base addition salts that retain the biological effectiveness and properties of the components and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Example acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, as well as salts derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Example base addition salts include those derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides such as tetramethylammonium hydroxide. Chemically modifying pharmaceutical compounds (i.e., drugs) into salts to improve the physical and chemical stability, hygroscopicity, flowability, and solubility of the compounds is a technique well known to pharmaceutical chemists. See, e.g., H. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th ed. 1995) at pp. 196 and 1456-1457, incorporated herein by reference.

[0103] Also contemplated are "prodrugs" and "solvates" of some components. The term "prodrug" means a compound (e.g., a drug precursor) that is transformed in vivo to produce the desired compound of the invention or a metabolite, pharmaceutically acceptable salt, or solvate thereof. The transformation can occur by various mechanisms (e.g., by metabolic or chemical processes). A discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, "Prodrugs as Novel Delivery Systems," Volume 14 of the ACS Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0104] Experimental results

[0105] Experiments conducted by the present inventors to confirm the effects of specific embodiments of the present invention will now be described.

[0106] Material

[0107] Bromelain used in the experiments described herein was commercially sourced from Enzybel, and any further processing was performed by MUCPharm Pte Ltd and provided as a sterile powder. Bromelain was diluted in phosphate-buffered saline (PBS) when used as a single agent, or directly diluted in acetylcysteine ​​solution when used in combination (sometimes referred to as "BromAc" in the examples) to prepare formulations with varying concentrations. Acetylcysteine ​​(sometimes referred to as "Ac" in the examples) 200 mg / mL -1 It was purchased from Link Pharma (Australia) and solutions were prepared by dilution in PBS.

[0108] Amphotericin B was obtained as an aqueous solution (Gibco, Thermo Fisher), while isavuconazole and posaconazole were obtained as powders and diluted at 3.2 mg mL -1 The other antifungal drugs described below were obtained in liquid or powder form and dissolved / diluted according to the instructions. All drugs were diluted from DMSO or aqueous stock solutions to the appropriate concentration in RPMI 1640 (Sigma-Aldrich) containing L-glutamine but lacking sodium bicarbonate, buffered to pH 7.0 with 0.165 M MOPS (Sigma-Aldrich).

[0109] Antifungal Susceptibility Testing - Checkerboard Broth Microdilution Method

[0110] The in vitro antifungal properties of bromelain, acetylcysteine, BromAc, amphotericin B, isavuconazole, fluconazole, and posaconazole were determined using the broth microdilution method in round-bottom 96-well plates according to standard procedures defined by the Clinical Laboratory Standards Institute (CLSI). The minimum inhibitory concentration (MIC) was obtained for each assay, showing the lowest drug concentration that caused complete growth inhibition.

[0111] Unless otherwise noted, fungal cultures were grown on potato dextrose agar plates at 35°C until maturity (conidia appeared) and used to prepare suspensions in sterile water. The suspensions were diluted 1:60 in RPMI. These suspensions were then added in equal volumes to solutions containing the candidate species described above and incubated at 35°C for 24 hours. The plates were then analyzed for fungal growth inhibition and the MIC values ​​were determined.

[0112] The fractional inhibitory concentration (FIC), which indicates the nature of the drug interaction between drug A and drug B, can be calculated using the following formula (where FIC ≤ 0.5 indicates synergy, FIC > 0.5-1 indicates additive effect, FIC of 1-4 is 'no difference', and FIC > 4 is 'antagonism'):

[0113] [MICA (combined) / MICA] + [MICB (combined) / MICB]

[0114] MICA (combined) is the MIC of drug A when both drugs (i.e., drug A and drug B) are present, and MICA is the MIC of drug A used alone. Similarly, MICB (combined) is the MIC of drug B when both drugs are present, and MICB is the MIC of drug B used alone.

[0115] For the bromelain / antifungal synergy assay described below, representative strains of various fungi were tested using the checkerboard broth microdilution method with bromelain at a concentration of 12.5 µg mL -1 , 25 µgmL -1 , 50 µgmL -1 , 100 µg mL -1 and 250 µg mL -1 In experiments involving acetylcysteine, 0.5 mg mL -1 , 1mgmL -1 , 2 mgmL -1 and 5 mg mL -1 concentration.

[0116] Example 1

[0117] Four fungi from the Mucorales group (Rhizopus microsporus, Fusarium solani complex, Rhizopus arrhizus, and Candida krusei QC (quality control), all of clinical origin) were tested with bromelain alone and acetylcysteine. In these experiments, the fungi were observed to grow well in the presence of all concentrations of bromelain tested, with MICs greater than 250 µg / mL. In fact, susceptibility assays showed that the fungi grew well in the presence of bromelain at concentrations up to 500 µg / mL. -1 No inhibitory effect of bromelain alone was observed at concentrations below 1.

[0118] Susceptibility assays showed that the MIC of acetylcysteine ​​required to inhibit the growth of Mucorales was 10 mg / mL -1 A Rhizopus microsporus strain, a Rhizopus arrhizus strain, and a Candida krusei QC strain, all of clinical origin, were initially tested.

[0119] NAC was combined with various concentrations of bromelain (12.5–500 µg / mL) to determine whether there was a synergistic or at least additive effect between the two agents. Antifungal activity was observed against Rhizopus arrhizus and Rhizopus microsporus at 12.5 and 25 µg / mL bromelain. These data suggest that bromelain alone has no antifungal effect against the fungal types described above, while NAC exhibits some antifungal activity. However, the combination of bromelain and NAC appears to be more effective. These data enabled the calculation of the MIC, which was then further tested with antifungal agents to examine whether additional inhibitory effects would be observed.

[0120] Example 2A

[0121] Dilutions of bromelain (250 µg / mL, 100 µg / mL, 50 µg / mL, 25 µg / mL) were also tested in combination with amphotericin B (2 mg / L, 1 mg / L, 0.5 mg / L, 0.25 mg / L, 0.06 mg / L, 0.03 mg / L, 0.008 mg / L, 0.004 mg / L) or posaconazole (2 mg / L, 1 mg / L, 0.5 mg / L, 0.25 mg / L, 0.06 mg / L, 0.03 mg / L, 0.008 mg / L, 0.004 mg / L) (both of which are designated as antifungals) against two strains of Rhizopus (Rhizopus arrhizus and Rhizopus microsporus). The following results are very encouraging, as a synergistic effect was demonstrated, and the amount of antifungal agent required was reduced with the addition of bromelain.

[0122] Based on the experimental data, the following calculations can be performed:

[0123] Amphotericin B and bromelain

[0124] ● Rhizopus arrhizus (80-21-015-4777)

[0125] Amphotericin: 0.5 mg / L

[0126] Bromelain: > 500 mg / L

[0127] Amphotericin B + Bromelain: 0.03 mg / L amphotericin B + 25 mg / L bromelain

[0128] Σ FIC 0.113 (synergy)

[0129] ● Rhizopus microsporus (80-21-006-5340)

[0130] Amphotericin B: 0.5 mg / L

[0131] Bromelain: > 500 mg / L

[0132] Amphotericin B + Bromelain: 0.125 mg / L amphotericin B + 25 mg / L bromelain

[0133] Σ FIC 0.3 (synergistic effect)

[0134] Posaconazole and bromelain

[0135] ● Rhizopus arrhizus (80-21-015-4777)

[0136] Posaconazole: 0.5 mg / L

[0137] Bromelain: > 500 mg / L

[0138] Posaconazole + bromelain: 0.125 mg / L posaconazole + 50 mg / L bromelain

[0139] Σ FIC 0.35 (synergistic effect)

[0140] ● Rhizopus microsporus (80-21-006-5340)

[0141] Posaconazole: 1 mg / L

[0142] Bromelain: > 500 mg / L

[0143] Posaconazole + bromelain: 0.25 mg / L posaconazole + 25 mg / L bromelain

[0144] Σ FIC 0.3 (synergistic effect)

[0145] In summary, fungal cultures of R. arrhizus and R. microsporus were grown in multiwell plates and treated with decreasing concentrations of amphotericin, with or without bromelain at concentrations ranging from 25 to 250 µg / mL (equivalent to 25 to 250 mg / L). Bromelain alone had no effect at 250 µg / mL and 500 µg / mL, and amphotericin alone had no effect at 0.25 mg / L. However, when combined with 25 µg / mL bromelain, complete inhibition of R. arrhizus was seen at 0.3 mg / L amphotericin, and similar results were seen for R. microsporus. Bromelain and posaconazole also demonstrated synergistic effects in both fungal species.

[0146] Example 2B

[0147] Candida glabrata (ATCC) was also tested with dilutions of bromelain (31.2 µg / mL, 15.6 µg / mL, 7.8 µg / mL, and 3.9 µg / mL) alone and in combination with amphotericin B (0.125 mg / L, 0.06 mg / L, 0.03 mg / L, and 0.01 mg / L). Figure 1A and 1B and demonstrate that when bromelain and amphotericin B are used in combination, the MIC is significantly reduced, possibly even synergistically.

[0148] Example 3 - Bromelain combined with acetylcysteine ​​("BromAc") acts synergistically with azole antifungals to Inhibit the growth of Mucorales

[0149] According to the data obtained by the present inventors, the -1 and 5 mg mL -1 or 10 mg / mL -1 At concentrations of bromelain and acetylcysteine, an additive effect appears to exist between bromelain and acetylcysteine. This combination was further studied in combination with amphotericin B, isavuconazole, and posaconazole—three common antifungal agents used to treat Mucor infections. MIC values ​​for these three individual antifungal agents were calculated and compared to the corresponding MICs of the antifungal agents in the presence of BromAc using a standard susceptibility assay.

[0150] Eight strains of Rhizopus microsporus and eight strains of Rhizopus arrhizus (all from a collection of clinical isolates at a hospital in Australia) were used in these studies to obtain a representative view of the efficacy of these drugs when combined. Each drug combination was tested on all 16 Mucor strains, and the studies were performed in duplicate.

[0151] BromAc combined with amphotericin B

[0152] For Rhizopus microsporus strains, the MIC range of amphotericin B is 0.5-1 µgmL -1 (average 0.7 µgmL -1 ), and the MIC range of BromaC is 0.004–0.125 µg mL -1 (average 0.034 µgmL -1 ) (Table I).

[0153] For Rhizopus arrhizus strains, the MIC range of amphotericin B is 0.25–1 µg mL -1 (average 0.6 µg mL -1 ), and the MIC range of BromaC is 0.008–0.125 µg mL -1(average 0.05 µg mL -1 ) (Table I).

[0154] Duplicate assays confirmed this trend.

[0155] BromAc and isavuconazole combination

[0156] For Rhizopus microsporus strains, the MIC range of isavuconazole is 0.5-1 µgmL -1 (average 0.9 µgmL -1 ), and the MIC range of BromaC is 0.03–0.25 µg mL -1 (average 0.13 µgmL -1 ) (Table I).

[0157] For Rhizopus arrhizus strains, the MIC range of isavuconazole is 0.5-2 µgmL -1 (average 0.8 µgmL -1 ), and the MIC range of BromaC is 0.06-0.5 µgmL -1 (average 0.17 µg mL -1 ) (Table I).

[0158] Duplicate assays confirmed this trend.

[0159] BromAc and posaconazole combination

[0160] For Rhizopus microsporus strains, the MIC range of posaconazole is 0.25-1 µgmL -1 (average 0.5 µg mL -1 ), and the MIC range of BromaC is 0.008–0.08 µg mL -1 (average 0.03 µgmL -1 ) (Table I).

[0161] For Rhizopus arrhizus strains, the MIC range of posaconazole is 0.25–1 µg mL -1 (average 0.8 µgmL -1 ), and the MIC range of BromaC is 0.004–0.25 µg mL -1 (average 0.06 µg mL -1 ) (Table I).

[0162] Duplicate assays confirmed this trend.

[0163]

[0164] Table 1 - MIC values ​​for R. arrhizus and R. microsporus isolates treated with amphotericin B, posaconazole and isavuconazole alone and in combination with BromAc.

[0165] As can be seen from the results listed in Table 1, when BromAc was combined with the exemplary antifungal agents, a reduction in MIC of 1-3 orders of magnitude was observed. This clearly demonstrates a sensitizing effect of the antifungal agents tested. Since the concentration of BromAc was constant relative to the concentration of the antifungal agents, the inventors cannot conclude that a synergistic effect is occurring, but they can say that the MIC of the antifungal agents was reduced many-fold.

[0166] Example 4 - Combination of Bromelain and Acetylcysteine ​​("BromAc") and Amphotericin B, Posaconazole, and Ephedrine Saperconazole and fluconazole act synergistically to inhibit the growth of Mucorales

[0167] BromAc (5 mg / ml acetylcysteine ​​+ 12.5 ug / ml bromelain) was used in combination with amphotericin B, isavuconazole, posaconazole, and fluconazole. MIC values ​​for these four antifungal agents against the fungi described below were calculated using the standard susceptibility assay described above and compared to the corresponding MIC values ​​in the presence of BromAc.

[0168] Fungal strains listed in Table 2, all from a range of clinical isolates collected in Australian hospitals, were used in these experiments to further understand the efficacy of BromAc in combination with amphotericin B, isavuconazole, posaconazole, and fluconazole.

[0169] Table 2 - Fungal strains tested

[0170]

[0171] Table 3 - Observed Inhibition (Strong = 5-10 fold (or greater) inhibition; Moderate = 1-5 fold inhibition; Possible = fungal cell changes (morphology) noted; and N / A = not tested)

[0172]

[0173] Data from these experiments showed that:

[0174] Candida auris - increased more than 10-fold in 2 of 5 patients taking amphotericin and in 4 of 5 patients taking posaconazole and isavuconazole

[0175] Cryptococcus - more than 10 times the risk for 5 of 5 with amphotericin, posaconazole, isavuconazole, and fluconazole

[0176] Candida glabrata - 5 of 5 isolates with amphotericin, 5 of 5 with posaconazole, and 5 of 5 with isavuconazole showed a greater than 10-fold increase in susceptibility

[0177] Candida krusei – more than 10 times with amphotericin and posaconazole

[0178] Candida tropicalis - susceptibility increased more than 10-fold in 4 of 5 isolates with amphotericin and 4 of 5 isolates with posaconazole

[0179] Candida parapsilosis - almost 10 times more likely with amphotericin and more than 10 times more likely with posaconazole

[0180] Cunninghamella parvum - some synergistic effects seen with posaconazole

[0181] Circinelloides - 2 of 2 for amphotericin, 1 of 2 for posaconazole, and 2 of 2 for isavuconazole

[0182] Rhizopus arrhizus - 10-fold in 8 of 8 patients taking amphotericin, 7 of 8 patients taking posaconazole, and 6 of 8 patients taking isavuconazole

[0183] Rhizopus microsporus - more than 10 times more common with 9 of 10 amphotericin users and 9 of 10 with posaconazole and isavuconazole

[0184] Example 5 - Determination of BromAc Alone and in the Presence of Antifungal Agents Using Broth Dilution Susceptibility Testing Minimum inhibitory concentration (MIC) in Candida glabrata.

[0185] These experiments were performed to determine the effects of BromAc alone and in combination with existing antifungal drugs against C. glabrata (clinical isolates).

[0186] The methods used in these experiments are summarized below:

[0187] A. Preparation of inoculum

[0188] 1. Grow C. glabrata on Sabouraud dextrose agar (SDA) at 37°C for 24-48 hours.

[0189] 2. Then, pick 5 colonies with a diameter of 1 mm and suspend them in 5 mL of sterile saline (0.9% saline) and vortex for 15 seconds.

[0190] 3. Prepare a 1:100 dilution of the working suspension from the stock suspension in RPMI 1640 medium.

[0191] 4. Using a spectrophotometer, adjust the cell density to the transmittance produced by a 0.5 McFarland standard (0.08 to 0.1) OD at 625 nm.

[0192] B. Preparation of Bromelain and N-acetylcysteine ​​dilutions:

[0193] 1. For bromelain, prepare a stock solution of 1 mg / ml bromelain in RPMI medium and dilute to test concentrations of 62.4 µg / ml, 31.2 µg / ml, 15.6 µg / ml, and 7.8 µg / ml in RPMI 1640 medium.

[0194] 2. The concentration of acetylcysteine ​​stock solution is 200 mg / ml. Dilute it to 40 mg / ml, 20 mg / ml, 10 mg / ml and 5 mg / ml in RPMI 1640 medium.

[0195] 3. These concentrations are prepared at 2x the desired concentration in the final assay plates.

[0196] C. Preparation of antifungal stock solution:

[0197] (amphotericin B / posaconazole / fluconazole)

[0198] The concentrations of antifungal stock solutions are as follows-

[0199] 1. Amphotericin B, stock solution concentration: 250 μg / ml in water.

[0200] 2. Posaconazole, original concentration: 300 mg in 16.7 ml, working stock concentration: 1 mg / ml in DMSO.

[0201] 3. Fluconazole, original concentration: 100 mg in 50 ml, working stock concentration: 1 mg / ml in DMSO.

[0202] 4. Prepare final concentrations in RPMI 1640 medium with amphotericin B concentration range of 0.01-0.5 µg / mL, posaconazole 0.01-2 µg / mL, and fluconazole 0.01-64 µg / mL.

[0203] 5. These concentrations are prepared at 2x the desired concentration in the final assay plates.

[0204] D. Antifungal assays (MIC) were performed using 96-well microtiter plates.

[0205] 1. Inoculate the wells of a 96-well plate with the appropriate concentration and volume of drug.

[0206] 2. Include sterility and growth controls on each plate.

[0207] 3. Each 96-well plate (except control wells) contains 100 µL of duplicate concentration (twice the final antifungal drug concentration).

[0208] 4. Add 100 µL of fungal suspension to each well of a 96-well plate (except for the sterile control wells).

[0209] 5. Growth control wells contain 100 µL of sterile drug-free medium and 100 µL of inoculum suspension, while sterility control wells contain 200 µL of sterile drug-free medium.

[0210] 6. The plate was then incubated at 37°C and read at 625 nm, 430 nm, and 530 nm wavelengths.

[0211] 7. Perform analysis using 24 hours of data and absorbance at 625 nm.

[0212] result

[0213] like Figure 2 As shown, a dose-dependent effect of amphotericin B alone was observed against C. glabrata, with an MIC of 1 μg / ml (the MIC for amphotericin is determined at 90% growth inhibition).

[0214] like Figure 3 As shown, BromAc alone showed reduced or similar effects on the viability of C. glabrata compared to amphotericin B alone at the concentrations tested ( Figure 3 (a and 3b). Adding amphotericin B to BromAc significantly enhanced the effect against C. glabrata compared to either treatment alone. Overall, inhibition of fungal cell growth was observed in all treatment groups compared to the growth control.

[0215] like Figure 4 As shown, with amphotericin (i.e. Figure 2 Compared to safflower, posaconazole showed a weaker antifungal effect against Candida glabrata. In fact, no dose response was observed within the concentration range tested.

[0216] The MIC of posaconazole, 50% growth inhibition, was determined to be 2 µg / ml; however, at this concentration, Candida glabrata viability was reduced by only 25% compared to the growth control ( Figure 4 Lower concentrations also showed a slight decrease relative to GC and were further used to test the efficacy of the BromAc combination.

[0217] If you can Figure 5As seen in the results, BromAc showed a slight decrease in C. glabrata compared to posaconazole alone at the concentrations tested ( Figure 5 The addition of posaconazole to BromAc (at higher or lower concentrations) showed a significant enhancement of the effect against C. glabrata (> 25%) compared to posaconazole alone.

[0218] like Figure 6 As shown, like posaconazole, the MIC of fluconazole was determined as 50% growth inhibition of C. glabrata, observed at concentrations >32 µg / ml. Significant (30% to 40%) inhibition was observed with fluconazole treatment at concentrations of 32 µg / ml and 64 µg / ml compared to the growth control. Lower concentrations of fluconazole showed no change compared to the growth control ( Figure 6 ).

[0219] If you can Figure 7 As seen in Figure 2, BromAc showed no alteration in C. glabrata viability and was similar to posaconazole alone at the doses tested ( Figure 7 a and 7b). However, like posaconazole, the combination of fluconazole and BromAc (at higher or lower concentrations) showed significant activity (> 30%) against C. glabrata over that of the fluconazole-treated group ( Figure 7 a and 7b).

[0220] In conclusion, BromAc was shown to possess potent antifungal properties on its own against C. glabrata. Furthermore, BromAc was found to enhance the effects of antifungal agents such as amphotericin B, posaconazole and fluconazole at concentrations hundreds of times lower than the MIC values ​​for C. glabrata.

[0221] Example 6 - Determination of BromAc Alone and in the Presence of Antifungal Agents Using Broth Dilution Susceptibility Testing Minimum inhibitory concentration (MIC) in Candida krusei.

[0222] These experiments were performed to determine the effects of BromAc alone and in combination with existing antifungal drugs against Candida krusei (ATCC strain 6258).

[0223] The methods used in these experiments were essentially the same as those described in Example 5 above.

[0224] result

[0225] like Figure 8 As shown, treatment of Candida krusei with amphotericin B achieved an MIC of nearly 90% at 1 μg / ml. Significant inhibition of the fungus was also observed at lower doses of amphotericin B.

[0226] like Figure 9 As shown, at the higher ( Figure 9 a) and lower ( Figure 9 b) BromAc alone showed decreased viability against C. krusei compared to GC at both bromelain concentrations. At the concentrations tested, the addition of amphotericin B to BromAc significantly enhanced the effect against C. krusei (> 25%) compared to BromAc and amphotericin B alone.

[0227] like Figure 10 As shown in Figure 2, posaconazole showed significant (> 50%) activity at its MIC of 1 ug / ml. Compared with GC, a low concentration of posaconazole, 0.03 µg / ml, did not show significant inhibition ( Figure 10 a).

[0228] BromAc alone showed significant inhibition compared to GC. Adding posaconazole to BromAc did not significantly alter the effect compared to BromAc alone; however, the effect was significant compared to posaconazole alone and GC at all concentrations tested ( Figure 10 a and 10b).

[0229] like Figure 11 As shown, the MIC of fluconazole at 32 µg / ml achieved 50% growth inhibition against Candida krusei. This MIC is classified as ineffective. Lower concentrations of fluconazole showed no efficacy compared to the growth control.

[0230] like Figure 12 As shown, BromAc alone showed significant activity compared to GC. Addition of fluconazole did not modify the effect compared to BromAc treatment alone, however, at the concentrations tested, the effect was significant (*p < 0.05, **p < 0.01) compared to fluconazole treatment alone and compared to GC ( Figure 11 a and b).

[0231] In conclusion, BromAc was shown to have effective antifungal properties on its own against Candida krusei. Combinations of certain antifungals and BromAc also showed superior activity compared to their respective individual concentrations.

[0232] Example 7 - Determination of Minimum Inhibitory Concentration in Aspergillus fumigatus with BromAc Alone and in the Presence of Antifungal Agents (MIC)

[0233] These experiments were conducted to determine the efficacy of BromAc alone and in combination with existing antifungal drugs against Aspergillus fumigatus (ATCC strain 13697).

[0234] The methods used in these experiments were essentially the same as those described above in Example 5. However, in these experiments, echinocandins such as voriconazole and caspofungin were used instead of the azole antifungal agents described in the earlier examples.

[0235] result

[0236] like Figure 13 As shown in a, the MIC of voriconazole for Aspergillus fumigatus treatment was observed to be 2 µg / mL. However, at lower concentrations (e.g., 1 µg / ml and 0.5 µg / ml), little inhibition was observed ( Figure 13 a).

[0237] If you can Figure 13 As seen in (b), BromAc alone showed a slight decrease in viability against A. fumigatus compared to GC. However, the combination of BromAc and voriconazole (even at low voriconazole doses) significantly enhanced the effect against A. fumigatus compared to BromAc and voriconazole alone at all concentrations tested (p < 0.01).

[0238] Now refer to Figure 14 a and 14b, similar effects can be seen for caspofungin. Figure 13 As seen in Figure 2a, MICs > 1 µg / ml were observed for caspofungin treatment of A. fumigatus. However, at lower concentrations (e.g., 0.25 µg / ml and 0.125 µg / ml), little inhibition was observed.

[0239] If you can Figure 14 As seen in (b), BromAc alone showed a slight decrease in viability against A. fumigatus compared to GC. However, the combination of BromAc and caspofungin (even at low caspofungin doses) significantly enhanced the effect against A. fumigatus compared to BromAc and caspofungin alone at all concentrations tested (p < 0.01).

[0240] These data demonstrate that the present invention is applicable to other classes of antifungal agents, including the echinocandins, and clearly indicate that lower concentrations of antifungal agents can be used when added in combination with BromAc. Administration of lower concentrations of drugs with known side effects is important for systemic approaches. Limitations of antifungal agents include the balance between efficacy and toxicity, particularly as seen with voriconazole.

[0241] Data not shown, but the present inventors have observed similar effects against C. auris and A. flavus using these antifungal drugs (as well as micafungin).

[0242] The inventors believe that these data show a strong inhibitory effect of the combination according to the embodiments of the present invention on a variety of fungal types, which reasonably predicts the therapeutic indications described herein. Further experiments, some of which are already underway, will confirm the inventors' expectations.

[0243] As described herein, the present invention provides compositions and methods for treating fungal infections / diseases in which a protease that affects glycoproteins, such as bromelain, is administered. Embodiments of the present invention provide numerous advantages over existing therapies, some of which are described above.

[0244] It will be understood by those skilled in the art that many modifications may be made without departing from the spirit and scope of the invention. All such modifications are intended to fall within the scope of the following claims.

[0245] In the appended claims and foregoing description of the invention, unless the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e., specifying the presence of the recited features but not excluding the presence or addition of additional features in various embodiments of the invention.

Claims

1. A method for treating a fungal infection in a patient, said method comprising administering to said patient a therapeutically effective combination of a glycoprotein-affecting protease and an antifungal agent.

2. The method of claim 1 , wherein the fungal infection is caused by a fungus from a genus selected from one or more of the group consisting of: Rhizopus, Mucor, Rhizomucor, Cryptococcus, Candida, Syncephalastrum, Cunninghamella, Apophysomyces, Aspergillus, Histoplasma, Pneumocystis, and Lichtheimia.

3. The method of claim 1 or claim 2, wherein the fungal infection is caused by a fungus selected from one or more of the group consisting of: Rhizopus arrhizus, Rhizopus microsporus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Fusarium solani, Candida krusei, Candida auris, Candida parapslosis, Candida tropicalis, Candida glabrata, Candida albicans, Cryptococcus gattii, Cryptococcus neoformans, Cunninghamella grisei, and the like. bertholletiae), Lichtheimia corymbifera, Mucor circinelloides, Histoplasma capsulatum, Pneumocystis jirovecii, and Microsporidia.

4. The method according to any one of claims 1 to 3, wherein the fungal infection is selected from one or more of the group consisting of mucormycosis (cutaneous mucormycosis, rhinocerebral mucormycosis and pulmonary mucormycosis), histoplasmosis, cryptococcosis, pneumocystis pneumonia, candidiasis and aspergillosis.

5. The method according to any one of claims 1 to 4, wherein the glycoprotein-affecting protease and the antifungal agent are administered to the patient simultaneously or sequentially.

6. The method according to any one of claims 1 to 4, wherein the glycoprotein-affecting protease and the antifungal agent are co-administered to the patient.

7. The method according to any one of claims 1 to 6, wherein the glycoprotein-affecting protease and the antifungal agent are administered to the patient by different routes of administration.

8. The method according to any one of claims 1 to 7, wherein the glycoprotein-affecting protease and the antifungal agent are administered to the patient topically, systemically, intravenously, by inhalation, by nebulization, by intratracheal injection, by intracavitary injection, by intraperitoneal injection, by intrapleural injection, by intraocular injection, by injection into the parenchyma or by injection into the cerebrospinal fluid via the intraventricular or intrathecal (cisternal or lumbar) route.

9. The method according to any one of claims 1 to 8, wherein the protease affecting glycoproteins is a cysteine ​​protease.

10. The method according to any one of claims 1 to 9, wherein the protease affecting glycoproteins is selected from one or more of the group consisting of bromelain, papain, ficin, thaumain, zingiberen, fastuosain and ananain.

11. The method according to any one of claims 1 to 10, wherein the antifungal agent is selected from one or more of the group consisting of amphotericin B, nystatin, caspofungin, micafungin, isavuconazole, posaconazole, ketaconazole, itraconazole, voriconazole, fluconazole and terbinafine.

12. The method of any one of claims 1 to 11, further comprising administering to the patient a disulfide bond cleaving agent in combination with the glycoprotein-affecting protease and the antifungal agent. 13 . The method of claim 12 , wherein the disulfide bond cleaving agent is selected from one or more of the group consisting of N-acetylcysteine, cysteamine, carbocysteine, bucillamine, dithiobutylamine (DTBA), and glutathione.

14. The method of claim 12 or claim 13, wherein the glycoprotein-affecting protease and the disulfide bond cleaving agent are administered simultaneously or sequentially.

15. The method of any one of claims 1 to 14, further comprising administering to the patient one or more additional agents that degrade fungal cell walls or cellular DNA components.

16. The method of claim 15, wherein the one or more additional therapeutic agents is a DNase.

17. The method of any one of claims 1 to 16, further comprising administering one or more additional therapeutic agents to the patient.

18. The method of claim 17, wherein the one or more additional therapeutic agents are selected from the group consisting of an antiviral agent, an antibacterial agent, a bronchodilator, and an expectorant.

19. A method for sensitizing a fungus to an antifungal agent, said fungus being the cause of a fungal infection in a patient, wherein said fungus has a fungal cell wall comprising glycoproteins, The method comprises administering to the patient a protease that affects a glycoprotein.

20. A method for sensitizing a fungus to an antifungal agent, said fungus having a fungal cell wall containing a glycoprotein, The method comprises contacting the fungus with a protease that affects glycoproteins.

21. A method for treating a fungal infection in a patient, the method comprising administering to the patient a therapeutically effective combination of a glycoprotein-affecting protease and a disulfide bond cleaving agent.

22. Use of a glycoprotein-affecting protease for sensitizing a fungus having a fungal cell wall containing glycoproteins, thereby improving the efficacy of an antifungal agent.

23. A composition comprising a protease that affects glycoproteins and an antifungal agent.

24. A synergistic combination of a protease affecting glycoproteins and an antifungal agent.

25. A synergistic combination of a protease affecting a glycoprotein and an antifungal agent for use in sensitizing a fungus having a fungal cell wall containing a glycoprotein to the antifungal agent.

26. Use of a glycoprotein-affecting protease in combination with an antifungal agent for the preparation of a medicament for treating a fungal infection in a patient.

27. A combination of a protease affecting glycoproteins and an antifungal agent for use in medicine.

28. A combination of a glycoprotein-affecting protease and an antifungal agent for use in treating a fungal infection in a patient.

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