Compositions and methods for treating fungal infections

Abstract

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

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to Australian Provisional Patent Application No. 2022903688, the contents of which are incorporated herein in their entirety.

[0002] Technical Field The present invention relates to compositions and methods for treating fungal infections. The present invention also relates to rendering certain fungi susceptible to antifungal agents. [Background technology]

[0003] Fungal infections are common and can range in severity from mere annoyance to immediate life-threatening illness. In fact, fungal infections are reported to cause over 1.5 million deaths and affect over 1 billion people annually.

[0004] Fungal infections occur when fungal spores enter a patient's body, usually through the respiratory tract (i.e., via inhalation) or a wound. Fungal infections include candidiasis (caused by Candida), cryptococcosis (caused by Cryptococcus neoformans), histoplasmosis (caused by Histoplasma), pneumocystis (caused by Pneumocystis jirovecii), and mucormycosis (caused by Mucormycetes).

[0005] Many antifungal agents are prescribed to treat various fungal infections. However, the toxicity of such agents often limits their tolerability (especially in severely ill patients) and reduces their effectiveness. Furthermore, antifungal resistance is becoming increasingly common and is a particularly serious problem for some invasive fungi. Some fungi are naturally resistant to antifungal agents (e.g., fluconazole is inactive against Aspergillus), while acquired resistance can develop over time in other fungi, particularly Aspergillus and Candida.

[0006] It would be beneficial to provide new therapeutic options for treating fungal infections. Summary of the Invention

[0007] In a first aspect, the present invention provides a method for treating a fungal infection in a patient, the method comprising administering to the patient a therapeutically effective combination of a glycoprotein-acting protease and an antifungal agent.

[0008] In a second aspect, the present invention provides a method for sensitizing a fungus causing a fungal infection in a patient to an antifungal agent, wherein the fungus has a fungal cell wall that contains glycoproteins, the method comprising administering to the patient a protease that acts on the glycoprotein.

[0009] In a third aspect, the present invention provides a method for rendering a fungus having a fungal cell wall containing glycoproteins susceptible to an antifungal agent, the method comprising contacting the fungus with a protease that acts on the glycoprotein.

[0010] In a fourth aspect, the present invention provides a method for treating a fungal infection in a patient, the method comprising administering to the patient a therapeutically effective combination of a protease that acts on glycoproteins and an agent that cleaves disulfide bonds.

[0011] In a fifth aspect, the present invention provides the use of proteases that act on glycoproteins to sensitize fungi having fungal cell walls that contain glycoproteins, thereby increasing the effectiveness of antifungal agents.

[0012] In a sixth aspect, the present invention provides a composition comprising a protease that acts on a glycoprotein and an antifungal agent.

[0013] In a seventh aspect, the present invention provides a synergistic combination of a protease that acts on glycoproteins and an antifungal agent.

[0014] In an eighth aspect, the present invention provides a synergistic combination of a glycoprotein-acting protease and an antifungal agent when used to render a fungus having a glycoprotein-containing fungal cell wall susceptible to the antifungal agent.

[0015] In a ninth aspect, the present invention provides the use of a combination of a protease that acts on a glycoprotein and an antifungal agent for the preparation of a medicament for the treatment of a fungal infection in a patient.

[0016] In a tenth aspect, the present invention provides a combination of a protease that acts on glycoproteins and an antifungal agent for use in medicine.

[0017] In an eleventh aspect, the present invention provides a combination of a glycoprotein-acting protease and an antifungal agent for use in treating a fungal infection in a patient.

[0018] The use of bromelain (a specific protease that acts on glycoproteins) as an antifungal agent has been reported to limit the growth of certain plant fungi. However, this effect has not been reported to be widespread across many fungi. Indeed, experiments conducted by the present inventors, some of which are described below, have shown that bromelain alone does not appear to have measurable antifungal activity against Rhizopus microspores, Fusarium solani cx, Rhizopus arrhizus, and Candida krusei QC. In fact, the use of bromelain in some of these fungi stimulated their growth under the experimental conditions used.

[0019] It was therefore surprising to the inventors that the combination of bromelain with many types of antifungal agents was found to have a synergistic effect against the fungi that cause mucormycosis. The inventors are not aware of any suggestion in the literature that bromelain may improve the effectiveness of antifungal agents or otherwise sensitize fungi, particularly those capable of infecting humans.

[0020] Subsequent experiments conducted by the inventors following their initial findings demonstrated that co-administration of bromelain with various antifungal agents resulted in increased susceptibility to the antifungal agents (in some cases, apparent synergistic effects were observed) across a wide range of Mucor isolates and various types of Candida. As will be appreciated, because Candida is a yeast and Mucor is a mold, the present invention is believed to have applicability to multiple types of fungi. The inventors observed different levels of susceptibility among different types of fungi and (without wishing to be bound by theory) speculate that the fungal cell wall may be responsible for these differences. As will be appreciated by those skilled in the art, fungal cell walls are complex cellular organelles composed of glucans, chitin, chitosan, and other glycosylated proteins. The ratios of these components vary among different types of fungi, and the inventors believe this may be the reason for the differences in susceptibility observed in preliminary experiments.

[0021] Broadly speaking, and based solely on our preliminary data, we speculate that the variations in efficacy observed when practicing various aspects of the invention in different fungi (some of which are described below) depend on the proportion of glycoproteins in the cell wall of a particular fungus, and similarly, on the accessibility of those glycoproteins.

[0022] As will be appreciated by those skilled in the art, cell wall proteins have a variety of functions, including maintaining cell shape, adhesion processes, protecting cells against various substances, absorbing molecules, signal transduction, and synthesizing and reorganizing cell wall components. Glycoproteins have been 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 varies depending on the morphological phase of the fungus, and has been reported to represent 1-2% of the dry weight of the yeast cell wall and up to 10-20% of the dry weight of the filamentous fungi. Other fungi, such as Lomentaspora and Scedosporium, have cell walls with a protective layer of melanin.

[0023] Furthermore, without wishing to be bound by theory, the inventors believe that the observed improvement in fungal susceptibility to antifungal agents is due to bromelain acting on the fungal cell wall, which contains glycoproteins (at least) and glycans, and dissolving or otherwise degrading the cell wall allows the antifungal agent to have greater access to the cell, thereby enhancing its therapeutic effect.The inventors predict that the mechanism of action of bromelain in fungal susceptibility is likely to be related to the dissolution of glycoproteins in the fungal wall (although, to the best of the inventors' knowledge, this has never been studied before), and therefore the present invention (i.e., the use of any protease that acts on glycoproteins) is likely to be widely applicable to any fungi that have a fungal cell wall containing glycoproteins.

[0024] As noted above, invasive fungal diseases have a high mortality rate and limited treatment options. Treatment according to the present invention may increase or at least maintain efficacy using reduced doses of antifungal agents, thereby limiting side effects.

[0025] The inventors believe that the results of their preliminary experiments, some of which are described below, lead to the reasonable prediction that, in addition to bromelain, proteases that act on glycoproteins, including those described herein, may also be effective in therapeutic applications such as those described herein. Further experiments, some of which are currently underway, will support their prediction.

[0026] Due to the promising results obtained from the inventors' initial experiments, the antifungal activity of a combination of a protease acting on glycoproteins and an agent that cleaves disulfide bonds was reexamined, specifically in the form of a combination of bromelain and N-acetylcysteine ​​(a known therapeutic agent under the trade name BromAc®). The inventors were surprised to find that this combination showed antifungal effects, especially in fungi whose cell walls contain a relatively high proportion of glycoproteins and polysaccharides. Without wishing to be bound by theory, the inventors believe that contact between the fungal cell wall and BromAc results in the degradation of glycoproteins and other components of the cell wall. Such degradation weakens the cell wall either to the extent that the fungus can no longer survive or to the extent that it is sufficient to allow bromelain and / or N-acetylcysteine ​​(and optionally the antifungal agent) to enter the cell, thereby killing the cell.

[0027] In some embodiments, the fungal infection may be caused by a fungus from a fungal genus selected from one or more of Rhizopus, Mucor, Rhizomucor, Cryptococcus, Candida, Syncephalastrum, Cunninghamella, Apophysomyces, Aspergillus, Histoplasma, Pneumocystis, and Lichtheimia.

[0028] In some embodiments, the fungal infection is caused by Rhizopus arrhizopus, Rhizopus microsporus, Aspergillus fumigatis, Aspergillus flavus, Aspergillus niger, Fusarium solani, Candida krusei, Candida auris, Candida parapslosis, Candida tropicalis, Candida glabrata, Candida albicans, Cryptococcus gattii, Cryptococcus neoformans, or the like. The fungus may be caused by one or more of the following fungi: Neoformans, Cunninghamella bertholletiae, Lichtheimia corymbifera, Mucor circinelloides, Histoplasma capsulatum, Pneumocystis jirovecii, and Microsporidia.

[0029] In some embodiments, the fungal infection may be selected from, for example, one or more of mucormycosis (e.g., cutaneous mucormycosis, rhinocerebral mucormycosis, and pulmonary mucormycosis), histoplasmosis, cryptococcosis, Pneumocystis pneumonia, candidiasis, and aspergillosis. More broadly, the present invention may 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 within the respiratory tract. It is possible that any respiratory infection caused by a fungus may also be treatable using the present invention, noting that Aspergillus, Candida, and Mucorales are particularly pulmonary invasive.

[0030] In some embodiments, the protease acting on glycoprotein and the antifungal agent can be administered to a patient simultaneously or sequentially.The protease acting on glycoprotein and the antifungal agent can, for example, be administered to a patient simultaneously.The protease acting on glycoprotein and the antifungal agent can, for example, be administered to a patient via different administration routes.Possible administration routes include, for example, locally, systemically, intravenously, by inhalation, by nebulization, by intratracheal injection, by intracavitary injection, by intraperitoneal injection, by intrapleural injection, by intraocular injection, by intraparenchymal injection, or by injection into the cerebrospinal fluid via intraventricular or intrathecal (cisternomagna or lumbar) route.

[0031] In some embodiments, the protease acting on glycoproteins may be a cysteine ​​protease, or may be selected from one or more of bromelain, papain, ficain, actinidyne, zingibain, fastuosain, and ananain.

[0032] In some embodiments, the antifungal agent may be selected from one or more of the group consisting of polyenes (e.g., amphotericin B and nystatin), echinocandins (e.g., caspofungin and micofungin), azoles (e.g., isavuconazole, posaconazole, fluconazole, ketaconazole, voriconazole, and itraconazole), allylamines (e.g., terbinafine), and orthomides. Newer antifungal agents, including inhibitors of calcineurin, trehalose pathway inhibitors, and inhibitors of sphingolipid synthesis, may also find use in the present invention.

[0033] In some embodiments, the present invention may further comprise administering to the patient an agent that cleaves disulfide bonds (which may also function as a thiol donor) in combination with a protease that acts on glycoproteins and an antifungal agent (or, as in embodiments of the fourth aspect of the present invention, only a protease that acts on glycoproteins). A synergistic effect between a protease that acts on glycoproteins, such as bromelain, and an agent that cleaves disulfide bonds, such as N-acetylcysteine ​​and cysteamine, has previously been observed, for example, by some of the present inventors.

[0034] In such embodiments, the agent that cleaves disulfide bonds can be selected from, for example, one or more of N-acetylcysteine, cysteamine, erdosteine, s-carboxymethylcysteine, glutathione, dithiothreitol, nasisterine, mercaptoethanesulfonic acid, carbocysteine, dornase alfa, gelsolin, thymosin P4, dextran, bucillamine, dithiobutylamine (DTBA), and heparin. In such embodiments, the protease that acts on glycoproteins and the agent that cleaves disulfide bonds can be administered simultaneously or sequentially.

[0035] In some embodiments, the present invention may further comprise administering to the patient an additional agent (e.g., DNase or collagenase), which may help to even further degrade components of the fungal cells and thus even further enhance the effectiveness of the antifungal agent.

[0036] 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 antiviral agents, antibacterial agents, bronchodilators, and expectorants.

[0037] It will be understood that any features and embodiments described in detail herein with respect to a particular aspect of the invention are applicable to other aspects of the invention as well. Other aspects, features, and advantages of the invention are described below. [Brief explanation of the drawings]

[0038] Embodiments of the invention will now be described in further detail with reference to the following figures: In the figures, p-values ​​are used to assess significant killing above growth controls, with *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 in the graphs. All doses given are in μg / ml, except for acetylcysteine, which is in mg / ml.

[0039] [Figure 1A] Figure 1 (A) shows a dose-response graph depicting the growth rate (%) of C. glabrata when exposed to bromelain alone and to bromelain in combination with amphotericin B, and (B) shows a dose-response graph depicting the growth rate (%) of C. glabrata when exposed to amphotericin B alone (GC = growth control, B = bromelain, and A = amphotericin B). [Figure 1B] See legend to Figure 1A. [Figure 2] A dose-response graph showing the percent growth of C. glabrata when exposed to amphotericin B alone is shown (GC = growth control, A = amphotericin B). [Figure 3] FIG. 1 shows a dose-response graph depicting the percent growth of C. 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 the use of higher and lower concentrations of bromelain. [Figure 4] A dose-response graph showing the percent growth of C. glabrata when exposed to posaconazole alone is shown (GC = growth control and P = posaconazole). [Figure 5]Dose-response graphs depicting the percent growth of C. glabrata when exposed to posaconazole, BromAc, and BromAc with posaconazole (GC = growth control, P = posaconazole, B = bromelain, and N = acetylcysteine). (a) and (b) represent the use of higher and lower concentrations of bromelain. [Figure 6] A dose-response graph showing the percent growth of C. glabrata when exposed to fluconazole alone is shown (GC = growth control and P = posaconazole). [Figure 7] Dose-response graphs depicting the percent growth of C. glabrata when exposed to fluconazole, BromAc, and BromAc with fluconazole (GC = growth control, F = fluconazole, B = bromelain, and N = acetylcysteine). (a) and (b) represent the use of higher and lower concentrations of bromelain. [Figure 8] A dose-response graph showing the percent growth of C. krusei when exposed to amphotericin B alone is shown (GC = growth control and A = amphotericin B). [Figure 9] FIG. 1 shows a dose-response graph depicting the percent growth of C. 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 the use of higher and lower concentrations of bromelain. [Figure 10] Figure 10(a) shows a dose-response graph depicting the growth rate (%) of C. krusei when exposed to posaconazole alone at various doses, and (b) shows a dose-response graph depicting the growth rate (%) of C. krusei when exposed to posaconazole, BromAc, and BromAc together with posaconazole (GC = growth control, P = posaconazole, B = bromelain, and N = acetylcysteine). [Figure 11] FIG. 1 is a dose-response graph showing the percent growth of C. krusei when exposed to fluconazole alone (GC=growth control and F=fluconazole). [Figure 12] Dose-response graphs depicting the percent growth of C. 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 the use of higher and lower concentrations of bromelain. [Figure 13] Figure 13(a) shows a dose-response graph depicting the percent growth of Aspergillus fumigatus when exposed to voriconazole alone at various doses, and (b) shows a dose-response graph depicting the percent growth of Aspergillus fumigatus when exposed to voriconazole, BromAc, and BromAc together with voriconazole (GC = growth control, V = voriconazole, B = bromelain, and N = acetylcysteine). [Figure 14] Figure 14(a) shows a dose-response graph depicting the percent growth of Aspergillus fumigatus when exposed to caspofungin alone at various doses, and (b) shows a dose-response graph depicting the percent growth of Aspergillus fumigatus when exposed to caspofungin, BromAc, and BromAc together with caspofungin (GC = growth control, C = caspofungin, B = bromelain, and N = acetylcysteine). DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description of the Invention As noted above, in its broadest form, the present invention provides a method for treating a fungal infection in a patient, wherein the patient is administered a therapeutically effective combination of a protease acting on glycoproteins and an antifungal agent. The present invention also provides a method for rendering a fungus having a fungal cell wall containing glycoproteins susceptible to an antifungal agent, wherein the fungus is contacted with a protease acting on glycoproteins. The present invention also provides a method for rendering a fungus causing a fungal disease or infection in a patient susceptible to an antifungal agent, wherein the fungus has a fungal cell wall containing glycoproteins, wherein the patient is administered a protease acting on glycoproteins. Finally, the present invention also provides a method for treating a fungal infection in a patient, wherein the patient is administered a therapeutically effective combination of a protease acting on glycoproteins and an agent that cleaves disulfide bonds.

[0041] Fungal infections anticipated to be treatable according to the present invention include mucormycosis (e.g., cutaneous mucormycosis, rhinocerebral mucormycosis, and pulmonary mucormycosis), histoplasmosis, cryptococcosis, Pneumocystis pneumonia, candidiasis, and aspergillosis. The present invention may also be used to treat systemic fungal infections, such as systemic fungal sepsis, as well as localized fungal infections, such as those found on prostheses. Noting that Aspergillus, Candida, and Cryptococcus are particularly pulmonary invasive, any respiratory infection caused by a fungus may also be treatable using the present invention.

[0042] Such fungal infections can be caused by fungi from the genera Fungi, including Rhizopus, Mucor, Rhizomucor, Cryptococcus, Candida, Syncephalastrum, Cunninghamella, Apophysomyces, Aspergillus, Histoplasma, Pneumocystis, and Lictheimia. Specific fungi causing 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 bertholechiae, Lictheimia corymbifera, Mucor circinelloides, Histoplasma capsulatum, Pneumocystis jirovecii, and Microsporidia.

[0043] In some embodiments, the patient is a mammalian subject. Typically, the patient is a human patient, although other subjects may benefit from the present invention. For example, the subject may be a pig, mouse, rat, dog, cat, cow, sheep, horse, or any other mammal of social, economic, or research importance.

[0044] As mentioned above, the inventors predict that the mechanism of action of bromelain in the observed increased susceptibility to antifungal drugs is likely related to the dissolution of glycoproteins (and possibly other components such as glucans and chitin) in the fungal wall, which, to the best of the inventors' knowledge, has not been previously studied. The inventors believe that the results of their preliminary experiments, some of which are described below, support the reasonable prediction of the broad therapeutic applications disclosed herein. The inventors also believe that the results of their preliminary experiments support the reasonable prediction that other proteases acting on glycoproteins will have utility in the present invention. Further experiments along the lines of those described herein support the inventors' prediction.

[0045] Proteases that act on glycoproteins The present invention includes the use of proteases that act on glycoproteins. Proteases that act on glycoproteins are proteolytic enzymes that cause proteolysis of glycoproteins. In light of the inventors' preliminary data on bromelain, a protease enzyme that acts on glycoproteins by hydrolyzing peptide and glycosidic bonds within glycoproteins, the inventors believe that any protease that acts on glycoproteins may be used in the present invention, and that only routine trial and experimentation (in light of the teachings contained herein) is required to determine the suitability of any particular protease that acts on glycoproteins. As used herein, the term "acting on glycoproteins" should be understood to mean acting on glycoproteins (e.g., chitin) in fungal cell walls in any therapeutically effective manner, such as by digesting, liquefying, or otherwise disrupting or degrading glycoproteins (as well as other peptide bonds) in fungal cell walls. Proteases that act on glycoproteins may be effective, for example, to disrupt glycoproteins. Proteases that act on glycoproteins can be effective, for example, in hydrolyzing peptide and glycosidic bonds of glycoproteins.

[0046] As mentioned above, glucan, especially β-glucan, is another major component in fungal cell walls.Therefore, when the present invention also includes an agent that acts on glucan, enhanced therapeutic effects can be achieved.In some embodiments, the protease that acts on glycoproteins can also be a protease that acts on glucan (for example, as in the case of bromelain).In alternative embodiments, an agent that acts on glucan, such as poisic acid and echinocandin, can be used in combination with a protease that acts on glycoproteins, for example.

[0047] The protease acting on glycoproteins may be, for example, a cysteine ​​protease. Cysteine ​​proteases (also known as thiol proteases) degrade proteins by a common catalytic mechanism and are commonly found in fruits, including papaya, pineapple, fig, and kiwifruit. Examples of cysteine ​​proteases include bromelain, papain (extracted from papaya), and ananain, a plant cysteine ​​protease belonging to the papain superfamily of cysteine ​​proteases.

[0048] Other plant-derived proteolytic enzymes exhibit the same properties as bromelain, and the inventors anticipate that any plant-derived protease enzyme or recombinant enzyme active against glycoproteins can be used in the present invention. Again, routine experimentation should confirm the suitability of any particular plant-derived protease enzyme. In some embodiments, for example, the plant-derived protease enzyme may be selected from one or more of the group consisting of bromelain, papain (extracted from papaya), ficain (extracted from fig), actinidain (extracted from fruits including kiwifruit, pineapple, mango, banana, and papaya), zingibain (extracted from ginger), and fastuosein (a cysteine ​​proteinase derived from Bromelia fastuosa). Proteases from asparagus, mango, other kiwifruit, and papaya may also be useful.

[0049] It should be noted that an active fraction of a protease acting on glycoproteins may be used in the present invention, and that not all of the material in a protease extract may need to be included, provided that the fraction itself acts on glycoproteins. It is anticipated that recombinantly obtained protease enzymes acting on glycoproteins may also be used in the present invention.

[0050] As used herein, "bromelain" is understood to encompass one or more substances present in extracts of the pineapple plant (Ananas Comosus) that act on glycoproteins and, optionally, are therapeutically active in other ways. Bromelain is a mixture of substances (including various thiol endopeptidases, as well as other components such as phosphatases, glucosidases, peroxidases, cellulases, esterases, and some protease inhibitors), and it may not be necessary to include all of these substances in the combination, as long as at least some of the substances in the combination can act on glycoproteins.

[0051] The bromelain used in the experiments described herein was obtained commercially from the Enzybel Group, with any further extraction and purification processing carried out by Mucpharm Pty Ltd.

[0052] The amounts and relative proportions of glycoprotein-acting proteases and antifungal agents in the present invention may vary depending on factors such as the type of glycoprotein-acting protease and antifungal agent, their intended use, as well as patient factors such as body weight and other health factors, including the severity of the infection. However, broadly speaking, it is expected that a combination or composition for administration will contain between about 5 μg / mL and about 2 mg / mL of glycoprotein-acting protease. While these amounts are greater than may be required to achieve a therapeutic effect via some routes of administration, the inventors note that only a small portion of the agent administered via some routes (e.g., nebulization) may become bioavailable. Because drug loss may vary between treatment regimens and devices, the amounts of glycoprotein-acting proteases described below refer to the amount a patient would receive, as can be measured using routine techniques.

[0053] Amounts of proteases acting on glycoproteins below about 5 μg / mL may be ineffective, and amounts above about 1,000 μg / mL are more likely to cause systemic undesirable side effects. In some embodiments, a combination of proteases acting on glycoproteins may be administered, e.g., 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, or 1,000 μg / mL.

[0054] It is contemplated that for some therapies and / or patients, repeat treatment may be beneficial (or necessary) to complete effective treatment.

[0055] The protease acting on glycoproteins and the antifungal agent may be administered to a patient in any manner that provides the intended therapeutic effect, for example, they may be administered simultaneously (e.g., in a single composition), sequentially (e.g., in separate compositions, one after the other), or separately, possibly via different routes of administration (e.g., in separate compositions).

[0056] antifungal agents Any antifungal agent may be used in the present invention, and only routine trial and experimentation is required to determine the suitability of a given antifungal agent.Examples of potentially suitable antifungal agents include polyenes (e.g., amphotericin B, liposomal amphotericin, and nystatin), echinocandins (e.g., caspofungin and mycofungin), azoles (e.g., posaconazole, isavuconazole, fluconazole, ketaconazole, voriconazole, and itraconazole), allylamines (e.g., terbinafine), and orthomides.It is understood that some fungal infections are treated with multiple classes of antifungal agents (e.g., azoles in addition to polyenes), and this may also be the case in the present invention.

[0057] New antifungal agents, including calcineurin inhibitors, trehalose pathway inhibitors, and sphingolipid synthesis inhibitors, may also be potentially used in the present invention. Other antifungal agents, such as fungal cytochrome P450 enzyme inhibitors, B-glucan synthesis inhibitors, chitin synthase inhibitors, ergosterol binders, squalene epoxidase inhibitors, alcineurin signaling inhibitors, DNA synthesis inhibitors, Hsp90 inhibitors, protein synthesis inhibitors, microtubule assembly agents, and ROS (reactive oxygen species) generators, may provide synergistic effects in treatment.

[0058] In use, the antifungal agent achieves its indicated therapeutic effect (at least for experiments performed by the inventors, see below), but a synergistic effect has been observed in which the amount of antifungal agent required for the same therapeutic outcome is significantly reduced. As noted above, given that many antifungal agents have undesirable side effects, reducing the dosage can be highly advantageous.

[0059] As noted above, the amounts and relative proportions of glycoprotein-acting proteases and antifungal agents in the present invention may vary depending on several factors. However, broadly speaking, it is expected that a combination or composition for administration will contain significantly less antifungal agent than would be required to achieve its therapeutic effect if the antifungal agent were administered alone (i.e., conventionally). It is within the ability of one of ordinary skill in the art to determine the appropriate amount of antifungal agent based on the teachings contained herein and information available from standard medical sources. For example, amphotericin is indicated for the treatment of Aspergillus, Candida, and / or Cryptococcus species at a dosage rate of 3-5 mg / kg IV once daily (qDay).

[0060] Disulfide bond cleaving agents In some embodiments, the invention may further comprise administering to the patient, in combination with the protease acting on glycoproteins and the antifungal agent (or, according to such aspects of the invention, only the protease acting on glycoproteins), an agent that cleaves disulfide bonds. Disulfide bond cleaving agents are species that cleave disulfide bridges that help define the tertiary structure of proteins.

[0061] In such embodiments, the agent that cleaves disulfide bonds may be selected from, for example, one or more of N-acetylcysteine, cysteamine, erdosteine, S-carboxymethylcysteine, glutathione, dithiothreitol, nacysterine, mercaptoethanesulfonic acid, carbocysteine, N-acysterine, dornase alfa, gelsolin, thymosin P4, dextran, bucillamine, dithiobutylamine (DTBA), and heparin. In such embodiments, the protease that acts on glycoproteins and the agent that cleaves disulfide bonds may be administered simultaneously or sequentially.

[0062] 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 an agent that cleaves disulfide bonds, such as N-acetylcysteine, may be included in the combination or composition for administration.

[0063] The term "BromAc" as used herein refers to a combination of bromelain and acetylcysteine, a drug combination developed by some of the present inventors for the treatment of mucinous cancer. While neither drug worked alone, BromAc was found to rapidly dissolve and remove tumor mucin.

[0064] Additional agents that degrade fungal cell walls In some embodiments, the invention may further comprise administering to the patient one or more additional agents (e.g., DNase or collagenase) that can contribute to the degradation of fungal cell walls (i.e., in combination with the protease that acts on glycoproteins and the antifungal agent (or, in such aspects of the invention, with the protease that acts on glycoproteins and the agent that cleaves disulfide bonds)). Such additional agents may serve to even further degrade (or more rapidly degrade) the fungal cell walls (e.g., DNase can degrade any DNA within the fungal cell), thereby even further enhancing the effectiveness of the antifungal agent.

[0065] In compositions that include an additional agent that degrades fungal cell walls, the agent may be present in the composition in any amount that provides a beneficial effect, for example, in the case of DNase, an amount of about 5-200 μg / mL is expected to provide the beneficial effects described herein.

[0066] Additional therapeutic agents The present invention may also include an additional therapeutic agent. Any additional therapeutic agent having appropriate indications in the context of treating fungal infections may also be co-administered to patients. Examples of additional therapeutic agents include antiviral agents, antibacterial agents, bronchodilators, and / or expectorants. The additional therapeutic agent may be administered simultaneously or (more likely) sequentially with the protease acting on glycoproteins and / or the antifungal agent.

[0067] In the composition containing additional therapeutic agent, any amount of agent that produces beneficial effect can be used.Determining the appropriate amount of any such additional therapeutic agent is within the ability of those skilled in the art.In some embodiments, two or more additional therapeutic agents can provide beneficial effect, especially when their therapeutic effect is mediated by different mechanisms.

[0068] The present invention may also include other components that do not necessarily have a direct therapeutic effect. Such components include pharmaceutically acceptable excipients and carriers.

[0069] Administration The glycoprotein-acting protease and the antifungal agent may be administered to a patient in any manner that provides the intended therapeutic effect. Any administration route that allows the glycoprotein-acting protease (and other components of the combination) to access the fungus is expected to be effective. Possible administration routes include, for example, local, systemic, intravenous, by inhalation, by nebulization, by intratracheal injection, by pulmonary lavage, by intracavitary injection, by intraperitoneal injection, by intrapleural injection, by intraocular injection, by intraparenchymal injection, or by injection into the cerebrospinal fluid via an intraventricular or intrathecal (cisternomagna or lumbar) route.

[0070] In some embodiments, the protease acting on a glycoprotein and the antifungal agent may be administered simultaneously or sequentially. The protease acting on a glycoprotein and the antifungal agent may, for example, be co-administered to a patient. The protease acting on a glycoprotein and the antifungal agent may, for example, be administered to a patient via different routes of administration.

[0071] When administered to a patient's lungs (e.g., after nebulization), for example, to treat mucormycosis, the combination may be sprayed into the trachea or bronchi using specialized medical equipment such as a bronchoscope. Alternatively (or additionally), when the patient inhales via nebulization, the combination may be sprayed into the patient's nose, mouth, or trachea. Alternatively (or additionally), the combination may be nebulized and delivered to the air surrounding the patient, such as in a closed tent or other enclosed environment for treatment.

[0072] Nebulization is a widely used method for delivering drugs into the respiratory tract. A nebulizer is a delivery device used to administer drugs in the form of a mist that is inhaled into the lungs, and can use oxygen, compressed air, or ultrasonic power to break solutions and suspensions into small aerosol droplets that are inhaled through the mouthpiece of the device.

[0073] Another widely used method for delivering medications into the respiratory tract is the use of dry powder inhalers or metered dose inhalers, which are well known in the art and deliver a specific amount of medication to the lungs, usually in the form of a short burst of aerosolized medication that is self-administered by the patient via inhalation.

[0074] 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, etc. It will be within the ability of one of ordinary skill in the art to determine the appropriate dosage (and dosing regimen) based on the teachings contained herein and experience.

[0075] Pharmaceutical Compositions The combination of a protease acting on glycoproteins, an antifungal agent, and any additional agent used in the present invention may, in some embodiments, be provided in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0076] Such pharmaceutically acceptable carriers are determined by the route of administration of the composition.Liquid form preparations can include solutions, suspensions, and emulsions, such as water or water-propylene glycol solutions, for making aerosols for delivery to the respiratory tract (intranasal or intratracheal).Pharmaceutically acceptable carriers suitable for use in the pharmaceutical compositions of the present invention include physiologically buffered saline, normal saline, hypertonic saline, dextrose solution, Ringer's solution, etc.As mentioned above, powder formulations for inhalation are also contemplated.

[0077] Pharmaceutical compositions suitable for delivery to a patient may be prepared immediately prior to delivery to the patient's body, or may be prepared in advance and appropriately pre-stored.

[0078] Pharmaceutical compositions and medicaments for use in the present invention may contain pharmaceutically acceptable carriers, adjuvants, excipients, and / or diluents. Carriers, diluents, excipients, and adjuvants must be "acceptable" in terms of being compatible with the other ingredients of the composition or medicament and the delivery method, and generally not deleterious to the recipient thereof.

[0079] It will be understood that some of the components in the combinations or pharmaceutical compositions described herein may, where appropriate, be provided in the form of their metabolites, pharmaceutically acceptable salts, solvates, or prodrugs. A "metabolite" of a component of the invention refers to intermediates and products of metabolism.

[0080] "Pharmaceutically acceptable," such as pharmaceutically acceptable carrier, excipient, etc., means that a particular compound is pharmacologically tolerable and substantially non-toxic to the subject to which it is administered.

[0081] "Pharmaceutically acceptable salt" refers to conventional acid 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 bases. Exemplary 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 those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, and fumaric acid. Exemplary 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 is a technique well known to pharmaceutical chemists to improve the physical and chemical stability, hygroscopicity, flowability, and solubility of the compounds. See, for example, H. Ansel et. al., Pharmaceutical Dosage Forms and Drug Delivery Systems (6th ed., 1995), pp. 196 and 1456-1457, incorporated herein by reference.

[0082] "Prodrugs" and "solvates" of some components are also contemplated. The term "prodrug" refers to a compound (e.g., a drug precursor) that is converted in vivo to produce a compound required by the present invention, or a metabolite, pharmaceutically acceptable salt, or solvate thereof. The conversion can occur by various mechanisms (e.g., metabolic or chemical processes). A discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, "Prodrugs as Novel Delivery Systems," Vol. 14 of the ACS Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987. [Example]

[0083] Experimental results Experiments performed by the inventors to demonstrate the effectiveness of certain aspects of the present invention will now be described.

[0084] material The bromelain used in the experiments described herein was obtained commercially from the Enzybel Group, with any further processing performed by MUCPharma Pty Ltd, and provided as a sterile powder. Bromelain was diluted either with phosphate-buffered saline (PBS) when used alone, or directly with acetylcysteine ​​solution when used in combination (often referred to as "BromAc" in the examples) to prepare formulations of various concentrations. Acetylcysteine ​​(often referred to as "Ac" in the examples) 200 mg mL -1 was purchased from Link Pharma (Australia) and prepared by diluting the solution with PBS.

[0085] Amphotericin B was obtained in aqueous solution (Gibco, Thermo Fisher), while isavuconazole and posaconazole were obtained in powder form, 3.2 mg mL -1 The antifungal agents were dissolved in DMSO at a concentration of 0.01%. Other antifungal agents listed below were obtained in liquid or powder form and dissolved / diluted according to the guidelines. All agents were diluted to the appropriate concentration from DMSO or aqueous stock solutions in RPMI 1640 (Sigma-Aldrich) containing L-glutamine but without sodium bicarbonate, and buffered to pH 7.0 with 0.165 M MOPS (Sigma-Aldrich).

[0086] Antifungal susceptibility testing - checkerboard broth microdilution method 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 established by the Clinical Laboratory Standards Institute (CLSI). The minimum inhibitory concentration (MIC) was obtained for each experiment, indicating the lowest concentration of drug that resulted in complete inhibition of growth.

[0087] Unless otherwise stated, fungal cultures were grown to maturity (appearance of conidia) on potato dextrose agar plates at 35°C and used to prepare suspensions in sterile water. The suspensions were diluted 1:60 with RPMI. These suspensions were then added in equal volumes to solutions containing the candidate species listed above and incubated at 35°C for 24 hours. Plates were then analyzed for fungal growth inhibition and MIC values ​​were determined.

[0088] The Fractional Inhibitory Concentration (FIC), which indicates the nature of drug interaction between drug A and drug B (an FIC of 0.5 or less indicates synergy, an FIC of greater than 0.5 to 1 indicates additive effect, an FIC of 1 to 4 is "irrelevant", and an FIC of greater than 4 is "antagonism"), is calculated using the formula: [MICA (combination) / MICA] + [MICB (combination) / MICB] It can be calculated using:

[0089] MICA (combination) 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 individually. Similarly, MICB (combination) is the MIC of drug B when both drugs are present, and MICB is the MIC of drug B used individually.

[0090] In the bromelain / antifungal synergy assay described below, 12.5, 25, 50, 100, and 250 μg mL -1Representative strains of various fungi were tested using the checkerboard broth microdilution method at bromelain concentrations of 0.5, 1, 2, and 5 mg mL in experiments containing acetylcysteine. -1 A concentration of 1000 mg / ml was used.

[0091] Example 1 Four fungi from the Mucormycete group (Rhizopus microspores, Fusarium solani cx, Rhizopus arizus, and Candida krusei QC (Quality Control), all obtained clinically) were tested with bromelain and acetylcysteine ​​alone. In these experiments, all fungi were observed to grow well at all concentrations of bromelain tested, with MICs above 250 μg / mL. Indeed, bromelain individually was found to be effective at 500 μg / mL by susceptibility assays. -1 No inhibitory effect was observed at concentrations up to 1000 mg / kg.

[0092] The individual MIC of acetylcysteine ​​required to inhibit growth of Mucorales was 10 mg / mL by susceptibility assay. -1 Initial testing was performed on one strain of Rhizopus microsporus, one strain of Rhizopus arizus, and one strain of Candida krusei QC, all clinically obtained.

[0093] NAC at 5 mg / mL was used in combination with various concentrations of bromelain (12.5–500 μg / mL) to determine whether there was a synergistic, or at least additive, effect between these two agents. Antifungal effects against Rhizopus arizus and Rhizopus microsporus were observed at 12.5 μg / mL and 25 μg / mL bromelain. These data indicate that bromelain itself has no antifungal effect against these fungal species, while NAC does have some antifungal effect. However, the combination of bromelain and NAC appeared to be more effective. These data allowed us to calculate MICs, which were then further tested with antifungal agents to determine whether additional inhibitory effects were observed.

[0094] Example 2A Two Rhizopus strains (Rhizopus arizus and Rhizopus microspores) were also tested with dilutions of bromelain (250, 100, 50, and 25 μg / mL) in combination with amphotericin B (2, 1, 0.5, 0.25, 0.06, 0.03, 0.008, and 0.004 mg / L) or posaconazole (2, 1, 0.5, 0.25, 0.06, 0.03, 0.008, and 0.004 mg / L), both of which are indicated antifungal agents. The results shown below are very encouraging in that a synergistic effect was demonstrated, reducing the amount of antifungal agent required when bromelain was added.

[0095] From the experimental data, the following calculations can be made: TIFF2025538895000001.tif150128

[0096] In summary, fungal cultures of R. arrhizus and R. microsporus were grown in multiwell plates and treated with a series of 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 was ineffective at concentrations of 250 and 500 μg / mL, and amphotericin alone was ineffective at 0.25 mg / L. However, when combined with 25 μg / mL bromelain, complete inhibition of R. arrhizus was observed at 0.3 mg / L amphotericin, and similar results were observed for R. microsporus. Synergistic effects were also observed between bromelain and posaconazole in both fungal types.

[0097] Example 2B Candida glabrata (ATCC) was also tested with dilutions of bromelain (31.2, 15.6, 7.8, and 3.9 μg / mL), both individually and in combination with amphotericin B (0.125, 0.06, 0.03, and 0.01 mg / L). The results, shown in Figures 1A and 1B, indicate that the MICs were significantly reduced, possibly synergistically, when bromelain and amphotericin B were used in combination.

[0098] Example 3: A combination of bromelain and acetylcysteine ​​("BromAc") acts synergistically with azole antifungals to inhibit the growth of mucoromycetes From the data obtained by the inventors, there is a significant difference between bromelain and acetylcysteine, with a difference of 12.5 μg mL -1 , and 5 or 10 mg mL -1 There appeared to be an additive effect at concentrations of 100 mg / kg bromAc. This combination was used for further investigation in combination with amphotericin B, isavuconazole, and posaconazole (three common antifungal agents used to treat Mucor infections). MIC values ​​for these three antifungals were calculated individually and compared with the corresponding MICs for antifungal activity in the presence of BromAc using standard susceptibility assays.

[0099] To obtain a representative view of the efficacy of these agents in combination, eight Rhizopus microsporus and eight Rhizopus arizus strains (all sourced from various clinical isolates from Australian hospitals) were used in these studies. Each drug combination was tested against all 16 Mucorales strains, and these studies were performed in duplicate.

[0100] BromAc in combination with amphotericin B For R. microsporus strains, the MIC for amphotericin B is 0.5–1 μg mL -1 range (average 0.7 μg mL -1 ) and the MIC in the presence of BromAc was 0.004–0.125 μg mL -1 range (average 0.034 μg mL -1 ) (Table I).

[0101] For R. arrhizus strains, the MIC for amphotericin B is 0.25–1 μg mL -1 range (average 0.6 μg mL -1 ) and the MIC with BromAc was 0.008 to 0.125 μg mL -1 range (average 0.05 μg mL -1 ) (Table I).

[0102] Two assays confirmed this trend.

[0103] BromAc in combination with isavuconazole For R. microsporus strains, the MIC for isavuconazole is 0.5–1 μg mL -1 range (average 0.9 μg mL -1 ) and the MIC with BromAc was 0.03 to 0.25 μg mL -1 range (average 0.13 μg mL -1 ) (Table I).

[0104] For R. arrhizus strains, the MIC for isavuconazole is 0.5–2 μg mL -1 range (average 0.8 μg mL-1 ) and the MIC with BromAc was 0.06–0.5 μg mL -1 range (average 0.17 μg mL -1 ) (Table I).

[0105] Two assays confirmed this trend.

[0106] BromAc in combination with posaconazole For R. microsporus strains, the MIC for posaconazole is 0.25–1 μg mL -1 range (average 0.5 μg mL -1 ) and the MIC with BromAc was 0.008-0.08 μg mL -1 range (average 0.03 μg mL -1 ) (Table I).

[0107] For R. arrhizus strains, the MIC for posaconazole is 0.25–1 μg mL -1 range (average 0.8 μg mL -1 ) and the MIC with BromAc was 0.004 to 0.25 μg mL -1 range (average 0.06 μg mL -1 ) (Table I).

[0108] Two assays confirmed this trend.

[0109] Table 1. MIC values ​​against R. arrhizus and R. microsporus isolates treated with amphotericin B, posaconazole, and isavuconazole alone and in combination with BromAc. TIFF2025538895000002.tif66170

[0110] As can be seen from the results shown in Table 1, when the exemplary antifungal agents are combined with BromAc, MICs are observed to decrease by one to three orders of magnitude. This clearly demonstrates increased susceptibility of the tested antifungal agents. Because the BromAc concentration was constant relative to the antifungal agent concentration, we cannot conclude that a synergistic effect occurred, but we can say that the MICs of the antifungal agents were reduced by many fold.

[0111] Example 4: A combination of bromelain and acetylcysteine ​​("BromAc") acts synergistically with amphotericin B, posaconazole, isavuconazole, and fluconazole to inhibit the growth of Mucorales BromAc (5 mg / ml acetylcysteine ​​+ 12.5 μg / ml bromelain) was used in combination with amphotericin B, isavuconazole, posaconazole, and fluconazole. MIC values ​​of these four antifungal agents against the fungi, described below, were calculated and compared with their corresponding MIC values ​​in the presence of BromAc using the standard susceptibility assay described above.

[0112] The fungal strains shown in Table 2, all sourced from various clinical isolates from Australian hospitals, were used in these experiments to gain further understanding of the efficacy of BromAc in combination with amphotericin B, isavuconazole, posaconazole, and fluconazole.

[0113] Table 2: Fungal strains tested TIFF2025538895000003.tif154128

[0114] (Table 3) Observed inhibitory effects (strong = 5-10 fold (or higher) inhibitory effect; moderate = 1-5 fold inhibitory effect; possible = notable fungal cell changes (morphological); and N / A = not tested). TIFF2025538895000004.tif85168

[0115] Data from these experiments show that: Candida auris - a greater than 10-fold increase in 2 of 5 strains for amphotericin and 4 of 5 for posaconazole and isobuconazole. Cryptococcus - greater than 10-fold in 5 of 5 strains for amphotericin, posaconazole, isavuconazole, and fluconazole. Candida glabrata – A greater than 10-fold increase in susceptibility in 5 of 5 isolates to amphotericin, 5 of 5 to posaconazole, and 5 of 5 to isavuconazole. Candida krusei - more than 10 times greater for amphotericin and posaconazole. Candida tropicalis - A greater than 10-fold increase in susceptibility in 4 of 5 strains to amphotericin and 4 of 5 strains to posaconazole. Candida parapsilosis - nearly 10-fold increased for amphotericin and more than 10-fold increased for posaconazole. · Cunninghamella - Some synergy is seen with posaconazole. Mucor circinelloides - 2 of 2 strains for amphotericin, 1 of 2 for posaconazole, and 2 of 2 for isavuconazole. Rhizopus arizos - 10-fold in 8 of 8 strains for amphotericin, 7 of 8 for posaconazole, and 6 of 8 for isavuconazole. Rhizopus microsporus - >10-fold increase in 9 out of 10 strains for amphotericin and >10-fold increase in 9 out of 10 strains for posaconazole and isavuconazole.

[0116] Example 5: Determination of the minimum inhibitory concentration (MIC) in Candida glabrata for BromAc alone and in the presence of antifungals using broth dilution susceptibility testing These experiments were conducted to determine the efficacy of BromAc alone and in combination with existing antifungal agents against Candida glabrata (clinical isolates).

[0117] The methods used in these experiments are summarized below: A. Preparation of seeding material : 1. Candida glabrata was grown on Sabouraud dextrose agar (SDA) at 37°C for 24–48 hours. 2. Then, five colonies with a diameter of 1 mm were selected, suspended in 5 mL of sterile saline (0.9% saline), and vortexed for 15 seconds. 3. A working suspension is prepared from the stock suspension as a 1:100 dilution in RPMI 1640 culture medium. 4. The cell density is adjusted using a spectrophotometer to a transmittance produced by an OD of 0.5 McFarland standard (0.08-0.1) at a wavelength of 625 nm.

[0118] B. Preparation of Bromelain and N-acetylcysteine ​​Dilutions: 1. For bromelain, a bromelain solution at a stock concentration of 1 mg / ml was prepared in RPMI medium and diluted to test concentrations of 62.4, 31.2, 15.6, and 7.8 μg / ml in RPMI 1640 medium. 2. The stock concentration of acetylcysteine ​​is 200 mg / ml and is diluted to concentrations of 40, 20, 10, and 5 mg / ml in RPMI 1640 medium. 3. These concentrations are prepared at twice the concentrations required in the final assay plate.

[0119] C. Preparation of antifungal stock solution: (Amphotericin B / Posaconazole / Fluconazole) The antifungal stock concentrations are as follows: 1. Amphotericin B, stock concentration: 250 μg / ml in water. 2. Posaconazole, original concentration: 300 mg in 16.7 ml, working stock concentration: 1 mg / ml in DMSO. 3. Fluconazole, original concentration: 100 mg in 50 ml, working stock concentration: 1 mg / ml in DMSO. 4. Final concentrations were prepared in RPMI 1640 medium, with concentration ranges of 0.01-0.5 μg / mL for amphotericin B, 0.01-2 μg / mL for posaconazole, and 0.01-64 μg / mL for fluconazole. 5. These concentrations were prepared at twice the concentrations required in the final assay plate.

[0120] D. Antifungal assay using 96-well microtiter plates (MIC): 1. Appropriate concentrations and amounts of drugs were placed in the wells of a 96-well plate. 2. Sterility and growth controls are included on each plate. 3. Each 96-well plate (except control wells) contains 100 μL of double strength (2x final antifungal drug) concentration. 4. Add 100 μL of fungal suspension to the 96-well plate (except the sterility control wells). 5. Growth control wells contained 100 μL of sterile drug-free medium and 100 μL of inoculum suspension, and sterility control wells contained 200 μL of sterile drug-free medium. 6. The plate is then incubated at 37°C and read at wavelengths of 625, 430, and 530 nm. 7. The absorbance data at 24 hours and 625 nm were used for analysis.

[0121] result A dose-dependent effect was observed with amphotericin B alone against C. glabrata, as shown in Figure 2. The MIC was found to be 1 μg / m (MIC for amphotericin is determined at 90% growth inhibition).

[0122] As shown in Figure 3, at the concentrations tested, BromAc by itself had either a reduced or similar effect on C. glabrata viability compared to amphotericin B alone (Figures 3a and 3b, respectively). The addition of amphotericin B to BromAc significantly enhanced its effect on C. glabrata compared to either treatment alone. Overall, all treatment groups were observed to inhibit fungal cell growth compared to the growth control.

[0123] As shown in Figure 4, posaconazole had a weaker antifungal effect against C. glabrata compared to amphotericin (i.e., Figure 2). In fact, no dose response was observed across the concentrations tested.

[0124] The MIC for posaconazole, representing 50% growth inhibition, was determined to be 2 μg / ml; however, at this concentration, only a 25% decrease in viability of C. glabrata was observed compared to the growth control (Figure 4). Lower concentrations also showed a slight decrease compared to GC and were further used to test efficacy in combination with BromAc.

[0125] As can be seen in Figure 5, BromAc showed a moderate reduction of C. glabrata compared to posaconazole alone at the concentrations tested (Figures 5a and 5b). The addition of posaconazole to BromAc (higher and lower concentrations) showed a significant enhancement (>25%) of efficacy against C. glabrata over posaconazole alone.

[0126] As shown in Figure 6, similar to posaconazole, the MIC for fluconazole was determined to be 50% growth inhibition of C. glabrata at concentrations >32 μg / ml. Fluconazole treatment showed significant (30-40%) inhibition compared to the growth control at concentrations of 32 μg / ml and 64 μg / ml. Lower concentrations of fluconazole showed no change compared to the growth control (Figure 6).

[0127] As can be seen in Figure 7, BromAc showed no change in C. glabrata viability, similar to posaconazole alone at the doses tested (Figures 7a and 7b). However, similar to posaconazole, the combination of fluconazole with BromAc (at higher and lower concentrations) showed significant activity (>30%) against C. glabrata compared with the fluconazole-treated group (Figures 7a and 7b).

[0128] In conclusion, BromAc itself exhibited effective antifungal properties against C. glabrata. Furthermore, BromAc was found to enhance the efficacy of antifungal drugs such as amphotericin B, posaconazole, and fluconazole at concentrations several hundred times lower than the MIC values ​​measured against C. glabrata.

[0129] Example 6: Determination of the minimum inhibitory concentration (MIC) in Candida krusei for BromAc alone and in the presence of antifungals using broth dilution susceptibility testing These experiments were conducted to determine the efficacy of BromAc alone and in combination with existing antifungal agents against Candida krusei (ATCC 6258 strain).

[0130] The methods used in these experiments were essentially the same as those already described in Example 5.

[0131] result As shown in Figure 8, treatment with amphotericin B achieved a MIC of nearly 90% at 1 μg / ml against C. krusei. Significant inhibition of the fungus was also observed at lower doses of amphotericin B.

[0132] As shown in Figure 9, BromAc by itself showed reduced viability of C. krusei compared to GC at both the higher (Figure 9a) and lower (Figure 9b) concentrations of bromelain tested. The addition of amphotericin B to BromAc significantly enhanced (>25%) the effect against C. krusei compared to BromAc and amphotericin B treatment alone at the concentrations tested.

[0133] As shown in Figure 10, posaconazole showed significant (>50%) activity at its MIC of 1 μg / ml. Lower concentrations of posaconazole, 0.03 μg / ml, did not show significant inhibition compared to GC (Figure 10a).

[0134] BromAc by itself showed significant inhibition compared to GC. Addition of posaconazole to BromAc did not significantly alter the effect compared to BromAc alone; however, it was significant compared to posaconazole-only treatment at the concentrations tested, and also compared to GC (Figures 10a and 10b).

[0135] As shown in Figure 11, the MIC for fluconazole at 32 μg / ml achieved 50% growth inhibition against C. krusei. This level of MIC is classified as an ineffective agent. Lower concentrations of fluconazole showed no efficacy compared to the growth control.

[0136] BromAc by itself showed significant activity compared to GC, as shown in Figure 12. The addition of fluconazole did not alter the effect compared to treatment with BromAc alone; however, the effect was significant compared to treatment with fluconazole alone at the concentrations tested (*p<0.05, **p<0.01), and was also significant compared to GC (Figures 11a and 11b).

[0137] In conclusion, BromAc by itself exhibited effective antifungal properties against C. krusei. Combinations of certain antifungal agents with BromAc also demonstrated superior activity compared to the concentrations of each agent alone.

[0138] Example 7: Determination of the minimum inhibitory concentration (MIC) in Aspergillus fumigatus for BromAc alone and in the presence of antifungal drugs These experiments were conducted to determine the efficacy of BromAc against Aspergillus fumigatus (ATCC 13697 strain) alone and in combination with existing antifungal agents.

[0139] The methods used in these experiments were essentially the same as those previously described in Example 5. However, in these experiments, echinocandins such as voriconazole and caspofungin were used instead of the azole antifungals described in the previous examples.

[0140] result As shown in Figure 13a, a MIC of 2 μg / mL was observed for Aspergillus fumigatus after treatment with voriconazole. However, at lower concentrations (e.g., 1 μg / mL and 0.5 μg / mL), little inhibitory effect was observed (Figure 13a).

[0141] As can be seen in Figure 13b, BromAc by itself showed a slight decrease in viability of Aspergillus fumigatus compared to GC. However, the combination of BromAc with voriconazole (even at low doses) significantly enhanced the efficacy against Aspergillus fumigatus compared to single treatments of BromAc and voriconazole at the concentrations tested (p<0.01).

[0142] Referring now to Figures 14a and 14b, a similar effect is seen with caspofungin. As seen in Figure 13a, MICs of >1 μg / ml were observed against Aspergillus fumigatus following treatment with caspofungin. However, at lower concentrations (e.g., 0.25 μg / ml and 0.125 μg / ml), little inhibitory effect was observed.

[0143] As can be seen in Figure 14b, BromAc by itself showed a slight decrease in survival rate of Aspergillus fumigatus compared to GC. However, the combination of BromAc with caspofungin (even at a low dose) significantly enhanced the efficacy against Aspergillus fumigatus compared to the single treatments of BromAc and caspofungin at the concentrations tested (p<0.01).

[0144] These data indicate the applicability of this invention to other classes of antifungal agents, including echinocandins, and clearly demonstrate that lower concentrations of antifungal agents can be used when added in combination with BromAc. For a systemic approach, administering drugs (with known side effects) at lower concentrations is important. The limitations of antifungal agents include the balance between efficacy and toxicity, particularly with voriconazole.

[0145] Although data are not shown, we have observed similar efficacy using these antifungal agents (as well as micafungin) against Candida auris and Aspergillus flavus.

[0146] The inventors believe that these data, showing the strong inhibitory effects of combinations according to aspects of the present invention across many fungal types, lead to reasonable predictions for the therapeutic indications described herein. Further experiments, some of which are already underway, will confirm the inventors' predictions.

[0147] As described herein, the present invention provides compositions and methods for treating fungal infections / diseases in which a protease that acts on glycoproteins, such as bromelain, is administered. Aspects of the present invention offer many advantages over existing therapies, some of which are described above.

[0148] Those skilled in the art will recognize that many modifications may be made without departing from the spirit and scope of the invention, and all such modifications are intended to fall within the scope of the following claims.

[0149] In the claims that follow and in the preceding description of the invention, unless the context requires otherwise by clear language or necessary implication, the term "comprise" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., to specify the presence of stated features but do not exclude the presence or addition of further features in various aspects of the invention.

Claims

1. A method for treating a fungal infection in a patient, comprising administering to the patient a therapeutically effective combination of a protease that acts on a glycoprotein and an antifungal agent.

2. 10. The method of claim 1, wherein the fungal infection is caused by a fungus of a fungal 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 fungal infection may be caused by Rhizopus arrhizus, Rhizopus microsporus, Aspergillus fumigatis, Aspergillus flavus, Aspergillus niger, Fusarium solani, Candida krusei, Candida auris, Candida parapslosis, Candida tropicalis, Candida glabrata, Candida albicans, Cryptococcus gattii, Cryptococcus neoformans, or the like.

3. The method of claim 1 or claim 2, wherein the infection is caused by a fungus selected from one or more of the group consisting of: Pneumocystis jirovecii, Pneumocystis neoformans, Pneumocystis jirovecii, Pneumocystis jirovecii, Pneumocystis jirovecii, Pneumocystis circinelloides ...

4. 4. The method of 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 protease acting on glycoproteins and the antifungal agent are administered to the patient simultaneously or sequentially.

6. The method of any one of claims 1 to 4, wherein the protease acting on glycoproteins and the antifungal agent are administered simultaneously to the patient.

7. The method according to any one of claims 1 to 6, wherein the protease acting on glycoproteins and the antifungal agent are administered to the patient via different routes of administration.

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

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

10. 10. The method of any one of claims 1 to 9, wherein the protease acting on glycoproteins is selected from one or more of the group consisting of bromelain, papain, ficain, actinidyne, zingibain, fastuosain, and ananain.

11. 11. The method of 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, micofungin, isavuconazole, posaconazole, ketaconazole, itraconazole, voriconazole, fluconazole, and terbinafine.

12. administering to the patient an agent that cleaves disulfide bonds in combination with the protease that acts on the glycoprotein and the antifungal agent. The method of any one of claims 1 to 11, further comprising:

13. 13. The method of claim 12, wherein the agent that cleaves disulfide bonds is selected from one or more of the group consisting of N-acetylcysteine, cysteamine, carbocysteine, bucillamine, dithiobutylamine (DTBA), and glutathione.

14. 14. The method of claim 12 or 13, wherein the protease acting on the glycoprotein and the agent that cleaves disulfide bonds are administered simultaneously or sequentially.

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

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

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

18. 18. The method of claim 17, wherein the one or more additional therapeutic agents are selected from the group consisting of antiviral agents, antibacterial agents, bronchodilators, and expectorants.

19. 1. A method for rendering a fungus responsible for a fungal infection in a patient susceptible to an antifungal agent, comprising: the fungus has a fungal cell wall containing glycoproteins; The method comprises administering to the patient a protease that acts on glycoproteins, method.

20. A method for rendering a fungus having a fungal cell wall containing glycoproteins susceptible to an antifungal agent, the method comprising contacting the fungus with a protease that acts on glycoproteins.

21. A method for treating a fungal infection in a patient, comprising administering to the patient a therapeutically effective combination of a protease that acts on glycoproteins and an agent that cleaves disulfide bonds.

22. 1. The use of a protease that acts on glycoproteins to sensitize fungi having fungal cell walls that contain glycoproteins, thereby increasing the effectiveness of antifungal agents.

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

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

25. A synergistic combination of a glycoprotein-acting protease and an antifungal agent when used to render fungi having a glycoprotein-containing fungal cell wall susceptible to the antifungal agent.

26. Use of a combination of a protease acting on glycoproteins and an antifungal agent for the preparation of a medicament for the treatment of a fungal infection in a patient.

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

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

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