WO2003066049A1

WO2003066049A1 - Combinations for the treatment of fungal infections

Info

Publication number: WO2003066049A1
Application number: PCT/US2003/003039
Authority: WO
Inventors: George N. Serbedzija; Benjamin A. Auspitz; Grant R. Zimmermann; Curtis Keith; M. James Nichols
Original assignee: Combinatorx, Incorporated
Priority date: 2002-02-06
Filing date: 2003-01-31
Publication date: 2003-08-14
Also published as: AU2003212888A1

Abstract

The invention features methods and reagents for treating a patient who has a fungal infection, or is at risk for developing a fungal infection, by administering to the patient a triazole and an aminopyridine simultaneously or within 14 days of each other in amounts sufficient to reduce or inhibit fungal growth. The invention also features methods and reagents for preventing, stabilizing, or inhibiting the growth of fungal cells in or on a mammal, or on a surface.

Description

COMBINATIONS FOR THE TREATMENT OF FUNGAL INFECTIONS

Background of the Invention

The invention relates to the treatment of fungal infections.

In animals, fungal infections (mycoses) may be superficial or systemic. Superficial mycoses include tinea capitis, tinea corporis, tinea pedis, onychomycosis, perionychomycosis, pityriasis versicolor, oral thrush, and other candidoses such as vaginal, respiratory tract, biliary, eosophageal, and urinary tract candidoses. Systemic mycoses include systemic and mucocutaneous candidosis, cryptococcosis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, and sporotrichosis.

The need for novel antifungal treatments is significant, and is especially critical in the medical field. Immunocompromised patients provide perhaps the greatest challenge to modern health care delivery. During the last three decades there has been a dramatic increase in the frequency of fungal infections in these patients (Herbrecht, Eur. J. Haematol., 56: 12, 1996; Cox et al., Curr. Opin. Infect. Dis., 6:422, 1993; Fox, ASM News, 59:515, 1993). Deep-seated mycoses are increasingly observed in patients undergoing organ transplants and in patients receiving aggressive cancer chemotherapy (Alexander et al., Drags, 54:657, 1997). The most common pathogens associated with invasive fungal infections are the opportunistic yeast, Candida albicans, and the filamentous fungus, Aspergillus fumigatus (Bow, Br. J. Haematol., 101 :1, 1998; Wamock, J. Antimicrob. Chemother., 41 :95, 1998). There are an estimated 200,000 patients per year who acquire nosocomial fungal infections (Beck-Sague et al., J. Infect. Dis., 167:1247, 1993). Also adding to the increase in the numbers of fungal infections is the emergence of Acquired Immunodeficiency Syndrome (AIDS) where virtually all patients become affected with some form of mycoses during the course of the disease (Alexander et al., Drugs, 54:657, 1997; Hood et al, J. Antimicrob. Chemother., 37:71, 1996). The most common organisms encountered in these patients are Cryptococcus neoformans, Pneumocystis carinii, and C. albicans (HIN/AIDS Surveillance Report, 1996, 7(2), Year-End Edition; Polis, M. A. et al., AIDS: Biology,

Diagnosis, Treatment and Prevention, fourth edition, 1997). New opportunistic fungal pathogens such as Penicillium marneffei, C. Jcrusei, C. glabrata, Histoplasma capsulatum, and Coccidioides immitis are being reported with regularity in immunocompromised patients throughout the world. The development of antifungal treatment regimens has been a continuing challenge. Currently available drugs for the treatment of fungal infections include amphotericin B, a macrolide polyene that interacts with fungal membrane sterols, flucytosine, a fluoropyrimidine that interferes with fungal protein and DNA biosynthesis, and a variety of azoles (e.g., ketoconazole, itraconazole, and fluconazole) that inhibit fungal membrane- sterol biosynthesis (Alexander et al., Drugs, 54:657, 1997). Even though amphotericin B has a broad range of activity and is viewed as the standard of antifungal therapy, its use is limited due to infusion-related reactions and nephrotoxicity (Wamock, J. Antimicrob. Chemother., 41 :95, 1998). Flucytosine usage is also limited due to the development of resistant microbes and its narrow spectrum of activity. The widespread use of azoles is causing the emergence of clinically-resistant strains of Candida spp. Due to the problems associated with the current treatments, there is an ongoing search for new treatments.

Summary of the Invention We have discovered that the combination of a triazole, either fluconazole, or itraconazole, and a diaminopyridine, phenazopyridine (PZP), brings about substantial inhibition of growth of fmcanazole-resistant and fluconazole-susceptible strains of C. albicans in vitro. These combinations of compounds also inhibited growth of two other Candida species (C. krusei and C. galbrata) and C. neoformans. Thus, these combinations can be used to treat fungal infections. Moreover, based on the mechanism of action shared among triazoles family members and among aminopyridine family members, fluconazole or itraconazole and/or PZP can be substituted with a family member in the combination.

Accordingly, the invention features a method for treating a patient who has, or is at risk for developing, fungal infection, by administering to the patient (i) a triazole; and (ii) an aminopyridine, in amounts that treat the patient. The triazole and the aminopyridine may be administered separately or as components of a pharmaceutical composition.

The triazole and aminopyridine can be administered within ten days of each other (e.g, within five days, twenty- four hours, or one hour of each other, or simultaneously). Administration of each compound can occur 1-4 times each day, or as necessary to produce a therapeutic effect. The invention also features a method of preventing, stabilizing, or inhibiting the growth of fungal cells. The method includes contacting fungal cells (or a site susceptible to growth of fungal cells) with a combination of compounds of the invention in amounts sufficient to prevent, stabilize, or inhibit the growth of the fungal cells. The method of contacting fungal cells with a combination of the invention can be used, for example, for the preservation of food, beverages, cosmetics, deodorants, contact lens products, food ingredients, enzyme compositions, grains, or animal feed. In other uses, the compound is incorporated into cleaning compositions or disinfectants for hard surface cleaning or water treatment. In yet other uses, the combinations can be applied to bioimplants, such as in-dwelling catheters, surgical implants, prosthetic devices, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, and continuous ambulatory peritoneal dialysis (CAPD) catheters. The specific amounts of the triazole and aminopyridine administered depend on the specific combination of components (i.e., the specific triazole/aminopyridine combination) and the mode of administration. Generally, when orally, topically or intravenously administered to a human, fluconazole is administered at a dosage of 0.001 mg to 2000 mg per day, desirably 1 mg to 1600 mg per day, and most desirably 50 mg to 800 mg per day. For the same modes of administration, itraconazole is administered at a dosage of 0.001 mg to 1600 mg/ day, desirably 1 mg to 1200 mg per day, and most desirably 25 mg to 800 mg per day. PZP is desirably administered at a dosage of 0.001 mg to about 2400 mg per day, desirably 0.5 mg to 1600 mg per day, and most desirably 10 mg to 1200 mg per day. In a preferred dose combination, the ratio of fluconazole to PZP is 4:1 by weight, while the ratio of itraconazole to PZP is desirably 10:1 to 20:1 by weight. When the combinations of the invention are used in other applications, higher doses of each component may be used.

A combination of the invention can also be used to treat a fungal infection in a vertebrate animal, particularly a domestic animal, such as those bred for food or kept as pets (e.g., horses, cows, sheep, poultry, fish, pigs, cats, and dogs). In yet other uses, the combinations can be applied as a cleaning agent, a treatment, or impregnated into or on surgical tools and endoscopy equipment.

The fungal infection or mycosis to be targeted by a combination of the invention may be caused, for example by a fungus selected from the group consisting oϊAbsidia corymbifera, Acremoniumfalciforme, A. kiliense, A. recifei, Ajellomyces dermatitidis, A. capsulata, Aspergillus spp., (e.g., A.flavus, A. fumigatus ', A. nidulans, A. niger, A. terreus), Candida spp. (e.g., C. albicans, C. glabrata, C. guillermondii, C. krusei, C. parapsilosis, C. ke yr, C. tropicalis), C. neoformans, Cunninghamella elegans, Emmonsia parva, Epidermophyton floccosum, Exophialia dermitidis, E. werneckii, E. jeanselmei, E. spinifera, E. richardsiae, Filobasidiella neoformans, Fonsecaea compacta, F. pedrosoi, Histoplasma capsulatum, Leptoshaeria senegarlensis, Madurella mycetomatis, M. grisea, Malassezia furfur, Microsporum spp, Neotestudina rosatii, Paracoccidioides brasiliensis, Penicillium marneffei, Phialophora verrucosa, Piedraia hortae, Pneumocystis carinii, Pseudallescheria boydii, Pyrenochaeta romeroi, Rhizomucor pusillus, Sporothrix schenckii, Trichophyton spp, Trichosporon beigelii, and Xylohypha bantiana.

Accordingly, the invention discloses a method of treating infections by the above fungi, among others.

The invention also features a method for identifying compounds useful for treating a patient having a fungal infection. The method includes the steps of: contacting fungal cells in vitro with (i) a triazole and/or an aminopyridine; and (ii) a candidate compound, and determining whether the growth of the fungal cells is modulated relative to (a) fungal cells contacted with the triazole and/or aminopyridine but not contacted with the candidate compound, and (b) fungal cells contacted with the candidate compound but not with the triazole and/or aminopyridine. A candidate compound that, when combined with the triazole and/or aminopyridine, modulates the growth of fungal cells to a greater degree than controls, is a compound that is potentially useful for treating a patient having a fungal infection.

Compounds useful in the invention include those described herein in any of their pharmaceutically acceptable forms, including isomers such as diastereomers and enantiomers, salts, solvates, and polymorphs thereof, as well as racemic mixtures of the compounds described herein.

By "triazole" is meant any member of the class of anti- fungal compounds having a five-membered ring of two carbon atoms and three nitrogen atoms. A compound is considered "antifungal" if it inhibits growth of a species of fungus by at least 25%. Exemplary triazoles include, for example, fluconazole, terconazole, itraconazole, posaconazole (SCH 56592), ravuconazole (BMS 207147), and voriconazole (UK- 109,496), the structures of which are depicted in Table 1, below. By "aminopyridine" is meant any pyridine ring-containing compound in which the pyridine has one, two, or three amino group substituents. Other substituents may optionally be present. Exemplary aminopyridines include, for example, phenazopyridine, 4-aminopyridine, 3,4-diaminopyridine, 2,5- diamino-4-methylpyridine, 2,3,6-triaminopyridine, 2,4,6-triaminopyridine, and 2,6-diaminopyridine, the structures of which are depicted in Table 2, below.

Table 1

Table 2

By "treating" is meant administering a pharmaceutical composition for prophylactic and/or therapeutic purposes, wherein the growth of fungal cells is prevented, stabilized, or inhibited, or wherein fungal cells are killed. To "prevent disease" refers to prophylactic treatment of a subject who is not yet infected, but who is susceptible to, or otherwise at risk of, a particular infection. To "treat disease" or use for "therapeutic treatment" refers to administering treatment to a subject already suffering from an infection to improve the subject's condition. By "subject" is meant any animal.

By "fungal infection" is meant the invasion of a host animal by fungal cells. For example, the infection may include the excessive growth of fungi that are normally present in or on the animal, or growth of fungi that are not normally present in or on the animal. More generally, a fungal infection can be any situation in which the presence of a fungal population is detrimental or damaging to a host animal. Thus, an animal is "suffering" from a fungal infection when an excessive amount of a fungal population is present in or on the animal, or when the presence of a fungal population is damaging the cells or tissue of the animal. In one embodiment, the number of a particular genus or species of fungus is at least 2, 4, 6, or 8 times the number normally found in the plant or animal. By "an effective amount" is meant the amount of a compound, in a combination of the invention, required to treat or prevent a fungal infection. The effective amount of active compound(s) used to practice the present invention for therapeutic or prophylactic treatment of conditions caused by or contributed to by a fungal infection varies depending upon the manner of administration, the age, body weight, and general health of the subject.

Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

The combination of a triazole with an aminopyridine for the treatment of fungal infections allows for the administration of a low dose of each compound and less total active compound, thus providing similar efficacy with less toxicity, and reduced costs. Another advantage is that the combinations of the invention are effective against fluconazole-resistant strains of Candida spp.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Detailed Description

We have discovered that the combination of triazoles, such as fluconazole, and itraconazole, with an aminopyridine, such as phenazopyridine (PZP), had substantial antifungal activity. Concentrations that substantially inhibited fungal growth were not unacceptably toxic to normal cells. Thus, combinations of triazoles and aminopyridines are useful for the treatment of fungal infections.

Antifungal triazoles as described herein refers to any member of the class of anti- fungal compounds having a five-membered ring of two carbon atoms and three nitrogen atoms. Exemplary triazoles include, for example, fluconazole, terconazole, itraconazole, posaconazole (SCH 56592), ravuconazole (BMS-207147), and voriconazole (UK-109,496) (Table 1).

By "aminopyridine" is meant any pyridine ring-containing compound in which the pyridine has one, two, or three amino group substituents. Other substituents may optionally be present. Exemplary aminopyridines include, for example, phenazopyridine, 4-amino-pyridine, 3,4-diaminopyridine, 2,5- diamino-4-methylpyridine, 2,3,6-triaminopyridine, 2,4,6-triaminopyridine, and 2,6-diaminopyridine (Table 2).

Therapy

Combination therapy according to the invention may be performed alone or in conjunction with another therapy and may be provided at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital. Treatment generally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed. The duration of the combination therapy depends on the type of disease or disorder being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient responds to the treatment. Additionally, a person having a greater risk of developing a fungal infection (e.g., a person who is to undergo a surgical procedure) may receive prophylactic treatment to inhibit or delay the onset of symptoms. The dosage, frequency and mode of administration of each component of the combination can be controlled independently. For example, one compound may be administered orally three times per day, while the second compound may be administered topically once per day. Combination therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to recovery from any as yet unforeseen side-effects. The compounds may also be formulated together such that one administration delivers both compounds.

Formulation of Pharmaceutical Compositions

Suitable modes of administration include oral, rectal, intravenous, topical or transdermal, vaginal, and ophthalmic. Administration of each compound of the combination may be by any suitable means that results in a concentration of the compound that, combined with the other compound, is effective. Each compound can be admixed with a suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A.R. Gennaro, 2000, Lippencott Williams & Wilkens, Philadelphia, PA, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time period after administration, using controlled release formulations.

Administration of compounds in controlled release formulations is useful where the compound, either alone or in combination, has (i) a narrow therapeutic index (e.g., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; generally, the therapeutic index, TI, is defined as the ratio of median lethal dose (LD₅₀) to median effective dose (ED₅₀)); (ϋ) a narrow absorption window in the gastro-intestinal tract; or (iii) a short biological half-life, so that frequent dosing during a day is required in order to sustain the plasma level at a therapeutic level. Many strategies can be pursued to obtain controlled release in which the rate of release outweighs the rate of metabolism of the therapeutic compound. For example, controlled release can be obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.

Topical Compositions

Therapeutic compositions suitable for topical application include conventional anhydrous or aqueous preparations including ointments, lotions, creams, pastes, jellies, sprays, aerosols, and oils. There preparations can include oleaginous, aqueous, or emulsion-type bases. Optionally, topically applied formulations can be covered with an occlusive or semi-occlusive dressing.

Solid Dosage Forms For Oral Use

Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). The two compounds may be mixed together in a tablet or other vehicle, or may be partitioned. In one example, the first compound is contained on the inside of the tablet, and the second compound is on the outside, such that a substantial portion of the second compound is released prior to the release of the first compound.

Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium.

Dosages

The dosage of each compound of the^" claimed combinations used in any given therapeutic method depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect dosage used.

As is described above, the compound(s) may be administered orally in the form of tablets, capsules, elixirs or syrups, or rectally in the form of suppositories. Parenteral administration of a compound is suitably performed, for example, in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound in itself is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied. Below, the dosages for triazoles and aminopyridines are described.

Generally, when orally, topically or intravenously administered to a human, fluconazole is administered at a dosage of 0.001 mg to 2000 mg per day, desirably 1 mg to 1600 mg per day, and most desirably 50 mg to 800 mg per day. For the same modes of administration, itraconazole is administered at a dosage of 0.001 mg to 1600 mg/ day, desirably 1 mg to 1200 mg per day, and most desirably 25 mg to 800 mg per day. PZP is desirably administered at a dosage of 0.001 mg to about 2400 mg per day, desirably 0.5 mg to 1600 mg per day, and most desirably 10 mg to 1200 mg per day. In a preferred dose combination, the ratio of fluconazole to PZP is 4:1 by weight, while the ratio of itraconazole to PZP is desirably 10:1 to 20:1 by weight. When the combinations of the invention are used in other applications, higher doses of each component may be used.

Other Uses

Combinations of the invention may also be used for the preservation of food, beverages, cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouthwash, contact lens products, enzyme formulations, or food ingredients. Methods for use as a preservative include incorporating a compound of the invention into, for example, unpreserved food, beverages, cosmetics, contact lens products, or food ingredients in an amount effective for killing or inhibiting the growth of fungi.

Thus, a compound of the invention may be useful as a disinfectant, e.g., in the treatment of acne, eye infections, mouth infections, fingernail infections, toenail infections, skin infections, or wounds. It is also contemplated that a compound of the invention is useful for cleaning, disinfecting, or inhibiting fungal growth on any hard surface. Examples of surfaces which may advantageously be contacted with a compound of the invention are surfaces of process equipment used in dairies, chemical or pharmaceutical process plants, water sanitation systems, paper pulp processing plants, water treatment plants, cooling towers, cooking utensils, or surfaces in any area in which food is prepared (e.g., hospitals, nursing homes, or restaurants). The composition of the invention should be used in an amount that is effective for cleaning, disinfecting or inhibiting microbial growth on the relevant surface.

In addition, combinations of the invention are useful for cleaning, disinfecting, or inhibiting fungal growth on or in an in-dwelling device in a patient. In-dwelling devices include, but are not limited to, surgical implants, prosthetic devices, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, and continuous ambulatory peritoneal dialysis (CAPD) catheters. In yet other uses, the combinations can be applied as a cleaning agent, a treatment, or impregnated into or on surgical tools and endoscopy equipment. A combination of the invention may be used to bathe an in-dwelling device immediately before insertion. Alternatively, the combination may be administered by injection to achieve a local or systemic effect against relevant fungi shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device.

The following example is to illustrate the invention. It is not meant to limit the invention in any way.

Example

Using the methods described below, we tested the ability of combinations of various concentrations of fluconazole and PZP to inhibit the proliferation of fluconazole-resistant C. albicans using reduction of Alamar Blue as an indicator of cell number. The data are depicted in Tables 3 and 4. A high concentration of fluconazole (65 μM) alone resulted in no inhibition of proliferation. In contrast, the combination of 20 μM PZP with this amount of fluconazole resulted in 80% and 92% inhibition of proliferation in strain 17 and MYA 573, respectively, relative to control cultures grown in the absence of both fluconazole and PZP. Low dose PZP (5 μM) inhibits 33% of C. albicans strain 17 proliferation. This inhibition was increased to 63 % by the addition of 65 μM fluconazole. Table 3: percent inhibition of proliferation of fluconazole-resistant C. albicans strain 17

Fluconazole (μM)

130.50 65.25 32.63 16.31 8.16 4.08 2.04 1.02 0.00

40.00 83.24 82.28 79.48 69.97 65.27 69.89 61.78 70.67 72.68

20.00 81.94 80.42 62.58 59.29 63.73 57.22 64.55 65.24 65.04

10.00 80.45 76.04 59.33 53.29 44.14 41.82 57.03 51.06 48.52

5.00 78.34 63.25 49.76 47.99 43.07 46.61 41.85 40.99 33.58

N 2.50 72.49 24.22 15.86 31.29 30.55 26.43 30.56 31.00 25.91

1.25 61.15 28.08 0.00 1.36 0.00 0.78 17.99 0.68 4.52

0.63 44.69 15.07 0.00 0.00 0.00 16.08 13.35 0.00 0.00

0.31 57.55 9.97 0.00 3.89 0.00 0.00 2.83 0.00 0.00

0.16 49.83 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 51.88 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Table 4: percent inhibition of proliferation of fluconazole-resistant C. albicans strain MYA 573

Fluconazole (μM)

130.50 65.25 32.63 16.31 8.16 4.08 2.04 1.02 0.00

40.00 96.92 96.69 92.09 86.14 76.37 71.47 72.70 67.23 58.21

20.00 96.14 91.97 76.50 45.69 39.77 43.04 41.29 49.38 45.34

10.00 91.66 57.19 27.39 9.91 0.00 7.83 13.91 2.33 5.85

5.00 60.89 4.08 15.68 0.00 0.00 1.55 3.45 0.00 0.00

2.50 5.28 6.96 0.00 0.00 0.00 0.00 0.00 0.00 0.00

N 1.25 30.77 0.00 4.13 0.00 0.00 0.00 0.00 0.00 0.00

0.63 10.62 9.57 2.65 0.00 1.40 1.76 0.00 0.00 0.00

0.31 26.56 2.78 5.79 0.00 0.00 0.00 0.00 7.72 0.00

0.16 11.06 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 9.35 6.95 9.68 0.00 0.00 0.00 6.68 0.00 0.00

Similar results were obtained when combining itraconazole with phenazopyridine. As shown in Table 5, high concentration of itraconazole (0.25 μM) alone resulted in a 46% inhibition of proliferation. In contrast, the combination of 5 μM PZP with this amount of itraconazole resulted in 70% inhibition of proliferation in strain 17, relative to control cultures grown in the absence of both itraconazole and PZP. Low dose PZP (2.5 μM) inhibits 30% of C. albicans strain 17 proliferation. This inhibition was increased to 57% by the addition of 0.25 μM itraconazole.

Table 5: percent inhibition of proliferation of fluconazole-resistant C. albicans strain 17

Itraconazole (μM)

4.00 2.00 1.00 0.50 0.25 0.13 0.06 0.03 0.02 0.00

40.00 94.78 94.72 94.65 91.84 83.23 75.06 74.83 71.38 72.57 72.20

20.00 94.52 94.42 93.97 91.46 78.02 68.37 70.33 69.99 66.46 64.56

10.00 93.33 93.24 92.72 89.90 76.19 59.97 65.94 61.20 60.69 53.54

5.00 91.33 92.57 91.43 84.35 69.67 50.19 43.47 54.16 51.69 43.93

2.50 89.61 90.20 89.80 77.95 57.24 31.55 22.08 32.53 37.47 30.01

N

Pπ 1.25 88.77 88.83 88.83 79.88 56.48 24.11 20.40 20.69 16.74 23.40

0.63 88.16 90.01 88.53 79.19 56.24 23.25 17.30 3.59 7.74 5.21

0.31 89.80 89.30 89.83 79.86 57.70 29.96 15.90 4.28 3.49 7.50

0.16 89.79 87.70 89.08 78.26 57.17 15.12 -3.46 -4.36 -5.90 9.05

0.00 89.18 88.51 89.13 65.58 46.11 12.93 7.09 4.63 2.21 5.58

The foregoing results were obtained with the following materials and methods.

Fungal Strains

Fluconazole-resistant C. albicans strain 17 was obtained from the culture collection of the Seattle Biomedical Research Institute (Seattle, WA). Fluconazole-resistant C. albicans strain MYA 573 was obtained from the American Type Culture Collection (Manassas, NA). Yeast were stored in 15% glycerol at -70°C. Isolates were cultured in growth medium (RPMI; 2% glucose w/o ΝaC0₂ and phenol red, buffered with 0.165M MOPS to pH 7.0) at 35°C for 24 hours prior to in vitro susceptibility testing. All media reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Compounds and compound preparation

The following compounds were used: fluconazole (Interchem Corp.,

Paramus, NJ), itraconazole (Interchem Corp.), PZP and amphotericin B (Sigma

Chemical Co.). In these experiments, amphotericin B was used as a positive control for antifungal activity. Stock solutions of each compound were prepared and stored at -20°C. Prior to use, stock solutions were diluted in growth medium to produce 10X solutions. 10X fluconazole or itraconazole (0- 7 μL) and 10X PZP (0-7 μL) were then plated in 45 μL of test medium (growth medium with 18% Alamar Blue; BioSource Intl., Camerillo, CA) in 384 well microtiter plates to form a matrix of compound combinations, as indicated in Tables 3-5. Final concentration ranges were 0-80 μg/mL fluconazole, 0-2.8 μg/mL itraconazole, and 0-10 μg/ml PZP. Amphotericin B was added as indicated at a final concentration of 4 μg/mL.

Susceptibility

All antifungal susceptibility testing was performed according to document M-27A as published by the National Committee for Clinical Laboratory Standards (Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts (1997), Wayne, PA). Briefly, yeast inocula from overnight cultures were standardized to a turbidity equivalent to a 0.5 McFarland Standard using a spectrophotometer at 530 nm, giving rise to a stock solution of 1 x 10⁶ cells/mL. Each yeast suspension was further diluted in growth medium to yield a final inoculum concentration of approximately 4 x 10³ cells/mL. Twenty microliters of this inoculum was then inoculated into each well, resulting in a final concentration of 1 x 10 cells/mL and a final concentration of 1 l%Alamar Blue. Drug-free controls were included on each plate. Plates were incubated for 16 hours at 35°C. Alamar Blue is metabolically reduced by the yeast mitochondria to produce a fluorescent product, the amount of which (referred to as the "RFU") being proportional to the live cell number. After the incubation period, the RFU of each well was determined fiuorometrically using a microplate reader (Fusion, Packard Bioscience, Meriden, CT) equipped with a 540 nm excitation filter and a 590 nm emission filter. In some experiments, the number of cells in a well were determined using by cell counting using a hemocytometer. Determination of inhibition

Percent inhibition was determined using the following formula: [(RFU control-RFU test)/RFU control] * 100 = percent inhibition.

Other Embodiments

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in cellular and molecular biology, pharmacology, endocrinology, or related fields are intended to be within the scope of the invention.

What is claimed is:

Claims

1. A method for treating a patient who has a fungal infection, or inhibiting the development of a fungal infection in a patient who is at risk for developing a fungal infection, said method comprising administering to said patient a triazole and an aminopyridine, wherein the triazole and the aminopyridine are administered simultaneously or within 10 days of each other, in amounts sufficient to treat or inhibiting the development of a fungal infection in said patient.

2. The method of claim 1, wherein said triazole is fluconazole, terconazole, itraconazole, voriconizole, posuconizole, or ravuconazole.

3. The method of claim 2, wherein said triazole is fluconazole.

4. The method of any one of claims 1-3, wherein said aminopyridine is phenazopyridine, 4-amino-pyridine, 3,4-diaminopyridine, 2,5-diamino-4- methylpyridine, 2,3,6-triaminopyridine, 2,4,6-triaminopyridine, or 2,6- diaminopyridine .

5. The method of claim 4, wherein said aminopyridine is phenazopyridine.

6. The method of claim 1, wherein said triazole is fluconazole and said aminopyridine is phenazopyridine.

7. The method of any one of claims 1-6, wherein said triazole and said aminopyridine are administered within five days of each other.

8. The method of claim 7, wherein said triazole and said aminopyridine are administered within 24 hours of each other.

9. The method of claim 8, wherein said triazole and said aminopyridine are administered within one hour of each other.

10. The method of claim 9, wherein said triazole and said aminopyridine are administered simultaneously.

11. The method of any one of claims 1-10, wherein said patient has tinea capitis, tinea corporis, tinea pedis, onychomycosis, perionychomycosis, pityriasis versicolor, oral thrush, vaginal candidosis, respiratory tract candidosis, biliary candidosis, eosophageal candidosis, urinary tract candidosis, systemic candidosis, mucocutaneous candidosis, cryptococcosis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, or sporotrichosis.

12. The method of any one of claims 1-10, wherein said fungal infection is an infection of Candida albicans, Candida krusei, Candida glabrata, or Cryptococcus neoformans.

13. The method of any one of claims 1-12, wherein each of said triazole and said aminopyridine is, independently, administered to said patient by intravenous, rectal, oral, topical, intravaginal, ophthalmic, or inhalation administration.

14. The method of any one of claims 1-13, wherein said triazole is administered in an amount between 0.001 and 2000 mg per day, and said aminopyridine is administered in an amount between 0.001 mg per day per day and 2400 mg per day.

15. The method of claim 14, wherein said triazole is administered in an amount between 25 and 800 per day, and said aminopyridine is administered in an amount between 10 and 1000 mg per day.

16. The method of claim 14, wherein said triazole is itraconazole, said aminopyridine is phenazopyridine, and said itraconazole and phenazopyridine are administered at a ratio of 10 parts by weight of itraconazole to one part by weight of phenazopyridine.

17. The method of claim 14, wherein said triazole is fluconazole, said aminopyridine is phenazopyridine, and said fluconazole and phenazopyridine are administered at a ratio of 4 parts by weight of fluconazole to one part by weight of phenazopyridine.

18. A pharmaceutical composition comprising a triazole, an aminopyridine, and a pharmaceutically acceptable carrier, wherein said triazole and said aminopyridine are each present in amounts that, when administered together to a patient, inhibit or reduce fungal growth.

19. The pharmaceutical composition of claim 18, wherein said triazole is fluconazole, terconazole, itraconazole voriconizole, posuconizole, or ravuconazole.

20. The pharmaceutical composition of claim 18, wherein said triazole is fluconazole.

21. The pharmaceutical composition of any one of claims 18-20, wherein said aminopyridine is phenazopyridine, 4-amino-pyridine, 3,4- diaminopyridine, 2,5-diamino-4-methylpyridine, 2,3,6-triaminopyridine, 2,4,6- triaminopyridine, or 2,6-diaminopyridine.

22. The pharmaceutical composition of claim 21, wherein said aminopyridine is phenazopyridine.

23. The pharmaceutical composition of claim 18, wherein said triazole is fluconazole and said aminopyridine is phenazopyridine.

24. The pharmaceutical composition of any one of claims 18-23, wherein said triazole and said aminopyridine are suitable for intravenous, rectal, oral, topical, intravaginal, ophthalmic, or inhalation administration.

25. A pharmaceutical pack comprising a triazole and an aminopyridine.

26. The pharmaceutical pack of claim 25, wherein said triazole is fluconazole, terconazole, itraconazole voriconizole, posuconizole, or ravuconazole.

27. The pharmaceutical pack of claim 25 or 26, wherein said aminopyridine is phenazopyridine, 4-amino-pyridine, 3,4-diaminopyridine, 2,5- diamino-4-methylpyridine, 2,3,6-triaminopyridine, 2,4,6-triaminopyridine, or 2,6-diaminopyridine.

28. The pharmaceutical pack of any one of claims 25-21, wherein said triazole and said aminopyridine are formulated separately and in individual dosage amounts.

29. The pharmaceutical pack of any one of claims 25-27, wherein said triazole and said aminopyridine are formulated together and in individual dosage amounts.

30. A method of preventing, stabilizing, or inhibiting the growth of fungal cells, said method comprising contacting said fungal cells with a triazole and an aminopyridine of the invention in amounts that, in combination, are sufficient to prevent, stabilize, or inhibit the growth of the fungal cells.

31. The method of claim 30, wherein said triazole is fluconazole, terconazole, itraconazole, voriconizole, posuconizole, or raviconazole.

32. The method of claim 30 or 31 , wherein said aminopyridine is phenazopyridine, 4-amino-pyridine, 3,4-diaminopyridine, 2,5-diamino-4- methylpyridine, 2,3,6-triaminopyridine, 2,4,6-triaminopyridine, or 2,6- diaminopyridine .

33. A method for identifying combinations of compounds useful for treating a patient having a fungal infection, said method comprising the steps of:

(a) contacting fungal cells in vitro with (i) a triazole and/or an aminopyridine, and (ii); a candidate compound; and

(b) determining whether the combination of said triazole and/or aminopyridine and said candidate compound reduces growth of said fungal cells relative to fungal cells contacted with said triazole and/or aminopyridine but not contacted with said candidate compound, or fungal cells contacted with said candidate compound but not with said triazole and/or aminopyridine, wherein a decrease in fungal growth identifies said combination as a combination that is useful for treating a patient having a fungal infection.

34. A method for preventing, stabilizing, or inhibiting the growth of fungal cells on a surface, said method comprising contacting said surface with an amount of a combination of a triazole and an aminopyridine sufficient to prevent stabilize, or inhibit growth of said fungal cells.

35. The method of claim 34, wherein said surface is selected from the group of process equipment, a water sanitation system, a cooking utensil, a food preparation area, and a medical device.

36. The method of claim 35 wherein said medical device is selected from the group consisting of a surgical tool, endoscopy equipment, a surgical implant, a prosthetic device, an artificial joint, a heart valve, a pacemaker, a vascular graft, a vascular catheter, a cerebrospinal fluid shunt, a urinary catheter, and a continuous ambulatory peritoneal dialysis catheter.

37. Use of a triazole and an aminopyridine for the manufacture of a medicament for the treatment of a patient who has a fungal infection.

38. The use of claim 37, wherein said triazole is fluconazole, terconazole, itraconazole, voriconizole, posuconizole, or ravuconazole.

39. The use of claim 38, wherein said triazole is fluconazole.

40. Use of an aminopyridine for the manufacture of a medicament for the treatment of a patient who has a fungal infection.

41. The use of any one of claims 37-40, wherein said aminopyridine is phenazopyridine, 4-amino-pyridine, 3,4-diaminopyridine, 2,5-diamino-4- methylpyridine, 2,3,6-triaminopyridine, 2,4,6-triaminopyridine, or 2,6- diaminopyridine .

42. The use of claim 40, wherein said aminopyridine is phenazopyridine .

43. The use of claim 37, wherein said triazole is fluconazole and said aminopyridine is phenazopyridine.

44. The use of any one of claims 37-39 and 41-43, wherein said triazole and said aminopyridine are administered within five days of each other.

45. The use of claim 44, wherein said triazole and said aminopyridine are administered within 24 hours of each other.

46. The use of claim 45, wherein said triazole and said aminopyridine are administered within one hour of each other.

47. The use of claim 46, wherein said triazole and said aminopyridine are administered simultaneously.

48. The use of any one of claims 37-47, wherein said patient has tinea capitis, tinea corporis, tinea pedis, onychomycosis, perionychomycosis, pityriasis versicolor, oral thrush, vaginal candidosis, respiratory tract candidosis, biliary candidosis, eosophageal candidosis, urinary tract candidosis, systemic candidosis, mucocutaneous candidosis, cryptococcosis, aspergillosis, mucormycosis, paracoccidioidomycosis, North American blastomycosis, histoplasmosis, coccidioidomycosis, or sporotrichosis.

49. The use of any one of claims 37-47, wherein said fungal infection is an infection of Candida albicans, Candida krusei, Candida glabrata, or Ciyptococcus neoformans.