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 <BR> <BR> in these patients (Herbrecht, Eur. J. Haematol. , 56: 12,1996 ; Cox et al. , Curr.<BR> <P>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., Drugs, 54: 657, 1997). The most common pathogens associated with invasive fungal infections are the opportunistic yeast, Candida albicans, and the filamentous <BR> <BR> fungus, Aspergillusfumigatus (Bow, Br. J. Haematol. , 101: 1, 1998 ; Wamock,<BR> J. Antimicrob. Chemother. , 41: 95,1998). There are an estimated 200,000 patients per year who acquire nosocomial fungal infections (Beck-Sague et al., <BR> <BR> 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 neofomrcans, Pneumocystis carinii, and C. albicans (HIV/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. krusei, 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"gold 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 an antifungal agent, terbinafine, and a manganese compound brings about substantial inhibition of growth in yeast. This combination of compounds strongly inhibited growth of Candida glabrata, but can also be used to treat fungal infections.

Moreover, based on the shared action among antifungal family members, other antifungal agents can be used in the combinations of the inventions.

Accordingly, the invention features a method for treating a patient who has, or is at risk for developing, a fungal infection by administering to the patient an antifungal agent and the manganese compound in amounts that treat the patient. The antifungal agent and a manganese compound may be administered separately or as components of a pharmaceutical composition. In one embodiment, the antifungal agent and the manganese compound are administered in the absence of a chelator. In another embodiment, the manganese compound is administered topically, intravenously, or intrathecally.

The antifungal agent and the manganese compound can be administered <BR> <BR> within fourteen days of each other (e. g. , within ten days, five days, twenty-four hours, or one hour of each other, or even simultaneously). Administration of each compound can occur 1-4 times each day, or as necessary to alleviate symptoms.

The invention also features a method of preventing, stabilizing, or inhibiting the growth of fungal cells by contacting fungal cells (or a site susceptible to growth of fungal cells) with an antifungal agent and a manganese compound in amounts sufficient to prevent, stabilize, or inhibit the growth of the fungal cells.

The invention also features a composition that includes an antifungal agent and the manganese compound in amounts sufficient to inhibit the growth of fungal cells.

Desirably, the methods and compositions of the invention are more effective or safe, better tolerated, or provide more treatment satisfaction when compared to a method or composition employing either component alone.

The methods and compositions of the invention can be used for the preservation of food, beverages, cosmetics, deodorants, contact lens products, food ingredients, enzyme compositions, grains, or animal feed.

In other uses, the combinations are 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 each component compound that are administered depend on variety of factors, such as the specific combination of the components (e. g. , the specific antifungal agent and/or manganese compound) and the mode of administration. Those skilled in the art of treating and preventing the various fungal infections will be able to apply the teachings herein to determine the proper dosing and administration.

Generally, when orally, topically or intravenously administered to humans, the antifungal agent is be administered at a dosage between 0.00001 mg to 2000 mg/day, desirably between 0.001 mg to 2000 mg per day, more desirably between 1 mg to 1600 mg per day, and most desirably between 50 mg to 800 mg per day. The manganese compound is typically administered at a dosage between 0.000001 to 2400 mg per day, desirably between 0.00001 mg to about 2400 mg per day, more desirably between 0.0001 to 1600 mg per day, even more desirably between 0. 001 to 1600 mg or even 0. 01 to 1200 mg/day, and more desirably between 0.5 mg to 1600 mg per day, and most desirably between 10 mg to 1200 mg per day. In one desirable dose combination, the ratio of an antifungal agent to a manganese compound is about 100,000 : 1, 50, 000 : 1, 10, 000 : 1, 5000: 1,1000 : 1, 500: 1, 100: 1,50 : 1, 10: 1 5 : 1 or 1: 1 by weight. In another aspect, depending on patient's needs, the dose combination of a manganese compound to an antifungal agent is about 100,000 : 1,50, 000: 1, 10, 000 : 1, 5000: 1, 1000: 1,500 : 1, 100: 1,50 : 1, 10: 1 or 5: 1 by weight.

When the combinations of the invention are used in non-pharmaceutical applications, higher doses of each component can 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 <BR> <BR> bred for food or kept as pets (e. g. , horses, cows, sheep, poultry, fish, pigs, cats, and dogs).

The fungal infection to be targeted by a combination of the invention may be caused, for example by a fungus selected from the group consisting of Absidia corymbifera, Acremonium falciforme, 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. <BR> <BR> glabrata, C. guillermondii, C. krusei, C. parapsilosis, C. kefyr, C. tropicalis), Cryptococcus neoformans, Cunninghamella elegans, Emmonsia parva, <BR> <BR> 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, Microspoum spp, Neotestudifza rosatii, Paracoccidioides brasiliensis, Penicillium marneffei, Phialophora verrucosa, Piedraia hortae, Pneumocystis carinii, Pseudallescheria boydii, <BR> <BR> Pyre710chaeta romeroi, Rhizomucor pusillus, Sporothrix schenckli,<BR> Trichophyton spp, Trichosporon beigelii, and Xylohypha bantiana.

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

The invention also features a method for identifying compounds useful for treating a fungal infection. The method includes the steps of : contacting fungal cells in vitro with an antifungal agent, a manganese compound, and a candidate compound, and determining whether the growth of the fungal cells is reduced relative to (a) fungal cells contacted with the antifungal agent and manganese compound but not contacted with the candidate compound, or (b) fungal cells contacted with the candidate compound but not with the an antifungal agent and a manganese compound.

A candidate compound that, when combined with the antifungal agent and a manganese compound, inhibits the growth of fungal cells to a greater degree than the antifungal agent or the manganese compound is a compound that is useful for treating 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.

The combination of an antifungal agent with a manganese compound for the treatment of fungal infections allows for the administration of a low dose of each compound and less total active compound, thus providing greater efficacy.

Another advantage is that the combinations of the invention are effective against Candida spp.

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, most preferably a human.

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 amount sufficient"is meant the amount of a compound, in a combination of the invention, sufficient to treat or prevent a microbial pathogen infection in a clinically relevant manner, such as in an animal, on a surface, or in a solution or product. An amount of an active compound sufficient to practice the present invention for therapeutic treatment of conditions caused by or contributing to a microbial infection in an animal varies depending upon the manner of the administration, the age, body weight, the general health of the patient. Ultimately, a prescriber will decide the appropriate amount and dosage regimen.

By a"low dosage"is meant at least 5% less (e. g. , at least 10%, 20%, 30%, 50%, 70%, 80%, or 90%) than the lowest standard recommended dosage of a particular compound for treatment of any human disease or condition.

By a"high dosage"is meant at least 5% (e. g. , at least 10%, 20%, 50%, 100%, 200%, or 300%) more than the highest standard recommended dosage of a particular compound for treatment of any human disease or condition By a"moderate dosage"is meant the dosage between the low dosage and the high dosage.

"Compounds"or"agents"useful in the invention also 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 and pure isomers of the compounds described herein.

A"patient"or"individual"are used interchangeably herein and refer to a vertebrate, preferably a mammal.

By"antifungal agent"is meant any compound that inhibits growth of a species of fungus by at least 25%.

Exemplary routes of administration for the various embodiments can include, but are not limited to, topical, transdermal, and systemic administration (such as, intravenous, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ophthalmic or oral administration). As used herein, "systemic administration"refers to all nondermal routes of administration, and specifically excludes topical and transdermal routes of administration.

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

Detailed Description The invention features methods and compositions employing (i) an antifungal agent, and (ii) a manganese compound, to treat a fungal infection or otherwise inhibit, prevent, or reduce fungal growth.

The combination of an antifungal agent with a manganese compound 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. Another advantage is that the combinations of the invention are effective against different strains of yeast.

The manganese compound may enhance the efficacy of the antifungal agent such that the dosage of the antifungal agent is lowered to achieve the same therapeutic benefit, thereby moderating any unwanted side effects.

Alternatively, the manganese compound may be used to augment the efficacy of an antifungal agent at its normal dose, such that an increased therapeutic benefit is obtained. In addition, when used with an additional antifungal agent, combinations of the invention may be useful in improving the ability of that agent to overcome drug resistance.

We have demonstrated the antifungal activity of terbinafine in combination with manganese sulfate. Based on known properties that are shared among antifungal agents and among manganese compounds,

it is likely that mechanistically or structurally related compounds can be substituted for terbinafine and/or manganese sulfate in the methods and compositions of the invention. Information regarding each of the compounds and its analogs and metabolites is provided below.

Terbinafine and Allylamine Antifungal Agents Terbinafine is a synthetic antifungal agent that inhibits ergosterol biosynthesis via inhibition of squalene epoxidase, an enzyme part of the fungal sterol synthesis pathway that creates the sterols needed for the fungal cell membrane. In vitro, terbinafine has activity against most Candida spp., Aspergillus spp., Sporothrix schenckii, Penicillium marneffei, Malassezia furfur, Cyptococcus neoformans, Trichosporon spp. and Blastoschizomyces.

The standard recommended dose of terbinafine is between 200-300 mg/day. Terbinafine formulations can be administered orally and topically.

Although terbinafine alone is effective against infections caused by azole- resistant Candida spp, terbinafine is more effective when used in combination with an azole (e. g., fluconazole) to treat such infections.

In addition to terbinafine, allylamines include amorolfine, butenafine, naftifine, N- (5, 5-dimethylhex-3-yn-1-yl)-N-methyl-l- naphthalenemethanamine, (E)-N- (6, 6-dimethyl-2-hepten-4-ynyl) -N- (iminomethyl)-l-naphthalenemethanamine, (E)-N- (6, 6-dimethyl-2-hepten-4- ynyl)-N- (l-iminoethyl)-l-naphthalenemethanamine, (Z)-N- (3-chloro-6, 6- dimethyl-2-hepten-4-ynyl)-N-methyl-1-naphthalenemethanamine, and N- methyl-N-propargyl-2-aminotetralin, some of which are shown in Table 1.

Table 1 H3 i H3 H3 H C \/ 3 Terbinafine N U-L13 /CHs \ Naftifine Cl3 H3 N H3C CH3 N- (5, 5-Dimethylhex-3-yn-1-yl)-N-methyl-1-naphthalenemethanamine CH3 |/CH3 NEZ N-Methyl-N-propargyl-2-aminotetralin OH CH3 _ o C22 H25 N 02

Other allylamine or allylamine analogs that can be used in the methods, kits, and compositions of the invention are described in U. S. Patent Nos. 4,202, 894 ; 4, 282, 251; 4,751, 245; 4,755, 534; 5,021, 458; 5,132, 459; 5,234, 946; 5,334, 628; 5,935, 998 ; and 6,075, 056.

Other Antifungal Agents Other antifungal agents suitable for use in the methods, compositions, and kits of the invention are described below. The antifungal azoles are preferred. Antifungal azoles are generally within in two classes, the imidizoles, such as miconazole, ketoconazole, and clotrimazole; and the triazoles, such as fluconazole, voriconazole, and ravuconazole. Other azoles are azaconazole, bromuconazole bitertanol, propiconazole, difenoconazole, diniconazole, cyproconazole, epoxiconazole, fluquinconazole, flusilazole, flutriafol, hexaconazole, itraconazole, imazalil, imibenconazole, ipconazole, tebuconazole, tetraconazole, fenbuconazole, metconazole, myclobutanil, perfurazoate, penconazole, posaconazole, pyrifenox, prochloraz, terconazole, triadimefon, triadimenol, triflumizole, and triticonazole.

Preferred antifungal agents are selected from fluconazole, itraconazole, ketoconazole, posaconazole, ravuconazole, voriconazole, clotrimazole, econazole miconazole, oxiconazole, sulconazole, terconazole, tioconazole, nikkomycin Z, caspofungin, micafungin (FK463), anidulafungin (LY303366), amphotericin B (AmpB), AmpB lipid complex, AmpB colloidal dispersion, liposomal AmpB, liposomal nystatin, nystatin, pimaricin, lucensomycin, griseofulvin, ciclopirox olamine, haloprogin, tolnaftate, undecylenate, gentamicin, amikacin, kanamycin, framycetin, neomycin, netilmicin, streptomycin, tobramycin, silver sulfadiazine, sodium sulfacetamide, sulfamethoxazole, sulfanilamide sulfasalazine, sulfisoxazole, trimethoprim, sulfamethoxazole, triple sulfa, amrolfine, fenpropimorph, butenafine, and flucytosine.

Manganese Compounds As used herein, a"manganese compound"is any salt or a complex of manganese. By"manganese salt"is meant any compound that results from replacement of part or all of the acid hydrogen of an acid by manganese.

Manganese salts include, without limitation, acetate, adipate, alginate, ascorbate, aspartate, benzoate, bicarbonate, borate, butyrate, camphorate, carbonate, chlorate, clorite, citrate, cyanate, digluconate, fumarate, glucoheptanoate, glutamate, glycerophosphate, heptanoate, hexanoate, hydroxide, hypochlorite, lactate, maleat, nicotinate, nitrate, nitrite, oxalate, oxide, palmitate, pamoate, pectinate, perchlorate, peroxide, 3- phenylpropionate, phosphate, hydrogen phosphate, dihydrogen phosphate, phosphite, picrate, pivalate, propionate, salicylate, suberate, succinate, tartrate, triiodide, bromide, chloride, fluoride, and iodide. The salt can be the manganese salt of a metal complex, e. g. manganese (II) zinc bis (dithiocarbamate) (also known as Mancozeb). Preferred manganese salts are those of sulfur-containing anions including, without limitation, sulfide, sulphite, sulfate, bisulfate, bisulfite, persulfate, thiosulfate, hyposulfite, undecanoate sulfate, thiocyanate, benzenesulfonate, 2-hydroxyethanesulfonate, dodecylsulfate, hemisulfate, methanesulfonate, 2-naphthalenesulfonate, tosylate, ethanesulfonate, and camphorsulfonate. Desirably, the manganese compound is manganese sulfate or manganese chloride. Specifically excluded from the definition of"manganese compound"is manganese when present in food.

By"manganese complex"is meant a manganese compound including one or more chelate rings wherein the ring includes a manganese atom.

Desirably, the complex is a macrocyclic or polydentate complexes of manganese. Manganese complexes include, without limitation, complexes of phenanthroline, 8-quinolinol, 2,6-diaminopyridine, bipyridine, diethylenetriamine, DPDP, EDDA, EDTA, EDTP, EDTA-BMA, DTPA,

DOTA, D03A, acetylacetonate, azamacrocycles, porphyrins, and Schiff-base complexes. Manganese complexes include those complexes described in U. S.

Patent Nos. 6,541, 490,6, 525, 041, 6, 204, 259, 6, 177, 419, 6, 147, 094, 6, 084, 093, 5, 874, 421, 5, 637, 578, 5, 610, 293,5, 246, 847, 5,155, 224, 4, 994, 259, 4, 978, 763, 4, 935, 518,4, 654, 334, and 4, 478,935. Binuclear, trinuclear, andtetranuclear complexes of manganese can also be used. Preferably, the manganese complex is a complex of ethylene-bis-dithiocarbamate. Most preferably, the manganese complex is manganese (II) ethylene bis (dithiocarbamate) (also known as Maneb). Methods for preparing manganese complexes are described in, for example, U. S. Patent No. 5, 155, 224 and by F. A. Cotton and G. Wilkinson "Advanced Inorganic Chemistry, "John Wiley & Sons, 5th Ed. (1988).

The manganese compounds described herein can be selected from any oxidation state (e. g. , Mn (0) to Mn (VII) ). Preferably the manganese compound<BR> is a manganous (e. g., Mn (II) compounds) or manganic (e. g. , Mn (III)) salt or complex.

Manganese compounds are available orally as part of a multivitamin/mineral combination. Manganese compounds are also administered parenterally as manganese chloride or manganese sulfate to prevent or treat manganese deficiency, such a improper formation of bone and cartlidge, decreased body's ability to use sugar properly and growth problems.

Standard recommended dosages adults usually range from 2 to 5 mg.

Additional Agents When the manganese compound is incorporated as an enhancer in the formulation of an antifungal compound, it is desirable to include additional agents. The term"enhancer"as used herein refers to heightened or increased, especially, increased or improved quality or desirability of the combination of compounds. Thus, in some of the instances, manganese may act as an enhancer of antifungal activity of a combination of antifungal agents.

For example, when manganese is used in combination with an allylamine- derived antifungal agent, such as terbinafine, or an azole-derived antifungal agent, such as fluconazole, itraconazole, or caspofungin, manganese enhances the antifungal activity of these compounds against C. glabrata, thereby acting as an enhancer.

The additional agent administered may be any compound that is suitable for intravenous, rectal, oral, topical, intravaginal, ophthalmic, or inhalation administration. Preferably, such agents are administered to alleviate other symptoms of the disease or for co-morbid conditions. In general, this includes: antibacterial agents (e. g. , sulfonamides, antibiotics, tetracyclines, aminoglycosides, macrolides, lincosamides, ketolides, fluoroquinolones, glycopeptide antibiotics, and polymyxin antibiotics); analgesic agents; antidiarrheals; antihelminthics; anti-infective agents such as antibiotics and antiviral agents; antifungal agents; antinauseants; antipruritics; antitubercular agents; antiulcer agents; antiviral agents; cough and cold preparations, including decongestants; diuretics; genetic materials; herbal remedies; nutritional agents, such as vitamins, essential amino acids and fatty acids; ophthalmic drugs such as antiglaucoma agents. Administration of the antifungal agent and manganese compound can be administered before, during, or after administration of one or more of the above agents.

For example, administration of a combination of the invention can be administered before, during, or after administration of one or more antibacterial agents. Exemplary antibacterial agents that can be administered in the methods <BR> <BR> of the invention are P-lactams such as penicillins (e. g. , penicillin G, penicillin V, methicillin, oxacillin, cloxacillin, dicloxacillin, nafcillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, piperacillin, azlocillin, and temocillin), cephalosporins (e. g. , cepalothin, cephapirin, cephradine, cephaloridine, cefazolin, cefamandole, cefuroxime, cephalexin, cefprozil, cefaclor,. loracarbef, cefoxitin, cefinatozole, cefotaxime, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefixime, cefpodoxime, ceftibuten,

cefdinir, cefpirome, cefepime, BAL5788, and BAL9141), carbapenams (e. g., imipenem, ertapenem, and meropenem), and monobactams (e. g. , astreonam);<BR> P-lactamase inhibitors (e. g. , clavulanate, sulbactam, and tazobactam);<BR> tetracyclines (e. g. , tetracycline, chlortetracycline, demeclocycline, minocycline, oxytetracycline, methacycline, and doxycycline); macrolides (e. g., erythromycin, azithromycin, and clarithromycin); ketolides (e. g. , telithromycin,<BR> ABT-773); lincosamides (e. g. , lincomycin and clindamycin); glycopeptides<BR> (e. g. , vancomycin, oritavancin, dalbavancin, and teicoplanin); streptogramins<BR> (e. g., quinupristin and dalfopristin) ; sulphonamides (e. g. , sulphanilamide, para- aminobenzoic acid, sulfadiazine, sulfisoxazole, sulfamethoxazole, and <BR> <BR> sulfathalidine); oxazolidinones (e. g. , linezolid); quinolones (e. g. , nalidixic acid, oxolinic acid, norfloxacin, perfloxacin, enoxacin, ofloxacin, ciprofloxacin, temafloxacin, lomefloxacin, fleroxacin, grepafloxacin, sparfloxacin, trovafloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, and sitafloxacin); metronidazole; daptomycin; garenoxacin; ramoplanin; faropenem; polymyxin ; tigecycline, AZD2563; and trimethoprim. These antibacterial agents can be used in the dose ranges currently known and used for these agents. Different concentrations may be employed depending, e. g., on the clinical condition of the patient, the goal of therapy (treatment or prophylaxis), the anticipated duration, and the severity of the infection for which the drug is being administered. Additional considerations in dose selection include the type of infection, age of the patient (e. g. , pediatric, adult, or geriatric), general health, and comorbidity. Determining what concentrations to employ are within the skills of the pharmacist, medicinal chemist, or medical practitioner. Typical dosages and frequencies are provided, e. g. , in the Merck Manual of Diagnosis & Therapy (17th Ed. MH Beers et al., Merck & Co.).

If desired, a combination of the invention may also be administered in conjunction with one or more anti-inflammatory agents (e. g. , non-steroidal<BR> anti-inflammatory drugs (NSAIDs ; e. g. , detoprofen, diclofenac, diflunisal,

etodolac, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenameate, mefenamic acid, meloxicam, nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin, celecoxib, rofecoxib, aspirin, choline salicylate, salsalte, and sodium and magnesium salicylate).

A combination of the invention can also be administered along with an antiprotozoal agent, such as pentamidine, propamidine, butamidine, heptamidine, nonamidine, dibrompropamidine, 2,5-bis (4-amidinophenyl) furan, 2,5-bis (4-amidinophenyl) furan-bis-O-methylamidoxime, 2,5-bis (4- amidinophenyl) furan-bis-0-4-fluorophenyl, 2,5-bis (4-amidinophenyl) furan- bis-0-4-methoxyphenyl, 2,4-bis (4-amidinophenyl) furan, 2,4-bis (4- amidinophenyl) furan-bis-O-methylamidoxime, 2,4-bis (4-amidinophenyl) furan- bis-0-4-fluorophenyl, 2,4-bis (4-amidinophenyl) furan-bis-0-4-methoxyphenyl, 2,5-bis (4-amidinophenyl) thiophene, 2,5-bis (4-amidinophenyl) thiophene-bis- O-methylamidoxime, 2,4-bis (4-amidinophenyl) thiophene, or 2,4-bis (4- amidinophenyl) thiophene-bis-O-methylamidoxime.

Cheating agents can also be used with an antifungal agent and a manganese compound in the methods, compositions, and kits of the invention.

Chelating agents include phosphonic acids, methylenglycine diacetic acid, iminodisuccinate, glutamate, N, N-bis (carboxymethyl, S, S'-ethylenediamine disuccinic acid (EDDS), P-alaninediacetic acid, ethylenediamine-N, N, N', N',- <BR> <BR> tetraacetic acid, ethylenediamine-N, N, N', N', -tetraacetic acid, disodium salt,<BR> dihydrate, ethylenediamine-N, N, N', N', -tetraacetic acid, trisodium salt, trihydrate, ethylenediamine-N, N, N', N'-tetraacetic acid, tetrasodium salt, tetrahydrate, ethylenediamine-N, N, N', N'-tetraacetic acid, dipotassium salt, dihydrate, ethylenediamine-N, N, N', N'-tetraacetic acid, dilithium salt, monhydrate, ethylenediamine-N, N, N', N'-tetraacetic acid, diammonium salt, ethylenediamine-N, N, N', N'-tetraacetic acid, tripotassium salt, dihydrate, ethylenediamine-N, N, N', N'-tetraacetic acid, ethylenediamine-N, N, N', N'- tetraacetic acid, calcium chelate, ethylenediamine-N, N, N', N'-tetraacetic acid, cerium chelate, ethylenediamine-N, N, N', N'-tetraacetic acid, ethylenediamine-

N, N, N', N'-tetraacetic acid, ethylenediamine-N, N, N', N'-tetraacetic acid, dysprosium chelate, ethylenediamine-N, N, N', N'-tetraacetic acid, europium chelate, ethylenediamine-N, N, N', N'-tetraacetic acid, iron chelate, ethylenediamine-N, N, N', N'-tetraacetic acid, ethylenediamine-N, N, N', N'- tetraacetic acid, ethylenediamine-N, N, N', N'-tetraacetic acid, ethylenediamine- N, N, N', N'-tetraacetic acid, ethylenediamine-N, N, N', N'-tetraacetic acid, ethylenediamine-N, N, N', N'-tetraacetic acid, samarium chelate, ethylenediamine-N, N, N', N'-tetraacetic acid, ethylenediamine-N, N, N', N'- tetraacetic acid, zinc chelate, trans-1, 2-diaminocyclohexane-N, N, N', N'- tetraaceticacid, monohydrate, N, N-bis (2-hydroxyethyl) glycine, 1, 3-diamino-2- hydroxypropane-N, N, N', N'-tetraacetic acid, 1,3-diaminopropane-N, N, N', N'- tetraacetic acid, ethylenediamine-N, N'-diacetic acid, ethylenediamine-N, N'- dipropionic acid dihydrochloride, ethylenediamine-N, N'- bis (methylenephosphonic acid), hemihydrate, N- (2- hydroxyethyl) ethylenediamine-N, N, N', N'-triacetic acid, ethylenediamine- N, N, N', N'-tetrakis (methylenephosponic acid), O, O'-bis (2- aminoethyl) ethyleneglycol-N, N, N', N'-tetraacetic acid, N, N-bis (2- hydroxybenzyl) ethylenediamine-N, N-diacetic acid, 1,6- hexamethylenediamine-N, N, N', N'-tetraacetic acid, N- (2- hydroxyethyl) iminodiacetic acid, iminodiacetic acid, 1, 2-diaminopropane- N, N, N', N'-tetraacetic acid, nitrilotriacetic acid, barium chelate, cobalt chelate, copper chelate, indium chelate, lanthanum chelate, magnesium chelate, nickel chelate, strontium chelate, nitrilotripropionic acid, dimercaprol (2,3- dimercapto-1-propanol), nitrilotris (methylenephosphoric acid), trisodium salt, 7,19, 30-trioxa-1, 4,10, 13,16, 22,27, 33-octaazabicyclo [l 1, 11, 1l] pentatriacontane hexahydrobromide, and triethylenetetramine-N, N, N', N", N"', N"'-hexaacetic acid. When the chelating agent is used in combination with an antifungal agent and a manganese compound, there is desirably a decrease in the consumption of either the antifungal agent or the manganese compound, or both.

Any of the foregoing compounds may be administered separately to the patient, surface, device or animals as needed or may be included in a composition of the invention.

In any of the foregoing aspects, treatment with the aforementioned antimicrobial compositions can be directed to a microbial infection of one or more of Cryptococcus spp., Candida spp., Aspergillus spp., Histoplasma spp., Coccidioides spp., Paracoccidioides spp. Blastomyces spp., Fusarium spp., Sporothrix spp., Trichosporon spp., Rhizopus spp., Pseudallescheria spp., dermatophytes, Paeciliomyces spp., Alternaria spp., Curvularia spp., Exophiala spp., Wangiella spp., Penicillium spp., Saccharomyces spp., Dematiaceous fungi and Pneumocystis cariyaii. Fungus selected from the group consist of Absidia corymbifera, Acremonium falciforme, A. kilieyzse, A. recifei, Ajellomyces dermatitidis, A. capsulata, Aspergillus spp., (e. g., A. flavus, A. <BR> <BR> fumigatus, A. nidulans, A. niger, A. terreus), Candida spp. (e. g., C. albicans, C. glabrata, C. guillermondii, C. krusei, C. parapsilosis, C. kefyr, C. tropicalis), Cryptococcus neoformans, Cunninghamella elegans, Emmonsia parva, Epidermophytofz, floccosum, Exophialia dermitidis, E. werneckii, E. jeanselmei, E. spiyaifera, 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 of the above fungi, among others.

Other Uses Combinations of the invention can be incorporated into, for example, unpreserved food, beverages, contact lens products, food ingredients, or cosmetics, such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouthwash, contact lens products, enzyme formulations, in an amount effective for killing or inhibiting the growth of fungal pathogens.

Thus, a combination of the invention may be useful as a disinfectant, e. g. , in the treatment of acne, eye infections, mouth infections, fingernail infections, top nail infections, skin infections, wounds, or in treating infections caused by the insertion of stents. Combinations of the invention are also useful for cleaning, disinfecting, or inhibiting fungal growth on any hard surface.

Examples of surfaces which may advantageously be contacted with a combination 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).

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 and dental implants, prosthetic devices, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, stents, cerebrospinal fluid shunts, urinary catheters, and continuous ambulatory peritoneal dialysis (CAPD) catheters. 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 micro-organisms shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in body time of the device.

Moreover, the compositions of the invention are useful as veterinary products for treating socially and/or economically valuable non-human vertebrate animals, such as pets and laboratory animals (horses, dogs, cats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes), livestock, fish, captive aquatic mammals, and birds. For land based animals, the compositions can be incorporated into feed, drinking water, or administered by IV, topically, or by other methods suitable for treating the particular animal in need of such treatment. For fish and other aquatic animals, the compositions may be administered by, e. g. , food, or added to aquarium or tank water, and may be combined with other agents usually administered to aquatic animals, or the tank or aquarium, e. g. anti-algae agents, antimolluscides, and antihelmintics (e. g. , niclosamide).

The compounds of the invention are also useful as screening tools.

Combinations of the invention can be employed in antiproliferative or mechanistic assays to determine whether other combinations or single agents are as effective as the combination in inhibiting yeast or fungal cell proliferation, using assays generally known in the art, e. g. anti-proliferative assays, specific, non-limiting examples of which are described in the Examples section. Candidate compounds are combined with a compound from either class of agent, applied to fungal cells, and after a suitable time, the cells are examined for antiproliferative activity. The relative effects of the combinations versus each other, and versus the single agents are compared, and effective compounds and combinations are identified.

The combinations of the invention are also useful tools in elucidating mechanistic information about the biological pathways involved in microbial cell metabolism, and proliferation. Such information can lead to the development of new combinations or single agents for inhibiting fungal cell proliferation or overcoming drug resistance. Methods known in the art to determine biological pathways can be used to determine the pathway,

or network of pathways affected by contacting microbial cells with the compounds of the invention. Such methods can include, analyzing cellular constituents that are expressed or repressed after contact with the compounds of the invention as compared to untreated, positive or negative control compounds, and/or new single agents and combinations, or analyzing some other metabolic activity of the cell such as enzyme activity, nutrient uptake, reproduction and proliferation. Cellular components analyzed can include gene transcripts, and protein expression. Suitable methods can include standard biochemistry techniques, radiolabeling the compounds of the invention (e. g., 14C or 3H labeling), and observing the compounds binding to proteins, e. g. using 2d gels, gene expression profiling, and the use of knock out strains of fungal cells.

The antifungal combinations of the invention can be used for a variety of domestic or industrial applications, e. g. , to reduce fungal populations on a surface or object or in a body or stream of water. The compositions can be applied in a variety of areas including kitchens, bathrooms, factories, hospitals, dental offices and food plants, and can be applied to a variety of hard or soft surfaces having smooth, irregular or porous topography. Suitable hard surfaces <BR> <BR> include, for example, architectural surfaces (e. g. , floors, walls, windows, sinks, tables, counters and signs); eating utensils; hard-surface medical or surgical instruments and devices; and hard-surface packaging. Such hard surfaces can be made from a variety of materials comprising, for example, ceramic, metal, glass, wood or hard plastic. Suitable soft surfaces include, for example paper; filter media, hospital and surgical linens and garments; soft-surface medical or surgical instruments and devices; and soft-surface packaging. Such soft surfaces can be made from a variety of materials comprising, for example, paper, fiber, woven or nonwoven fabric, soft plastics and elastomers. The compositions of the invention can also be applied to soft surfaces such as food and skin.

The compositions are also suitable for application to growing or harvested plant material including leaves, stems, tubers, roots, and seeds. Once identified, such compounds can be used in in-vivo models to further validate the tool or develop new anti-microbial agents.

The antifungal compositions of the invention can be included in products such as sterilants, sanitizers, disinfectants, preservatives, deodorizers, antiseptics, fungicides, germicides, sporicides, virucides, detergents, bleaches, hard surface cleaners, hand soaps and pre-or post-surgical scrubs. The compositions have particular utility as cold or hot aseptic packaging treatments.

The antifungal compositions can also be used in veterinary products such as mammalian skin treatments or in products for sanitizing or disinfecting animal enclosures, pens, watering stations, and veterinary treatment areas such as inspection tables and operation rooms.

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 on an outpatient basis. If the treatment becomes prolonged or that the disease becomes chronic or that the patient's condition starts deteriorating, treatment may then be extended to that patient in-house in a hospital. Alternatively, in some cases, for example, if the patient is suffering from a case of systemic mycosis, treatment may also begin 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.

Combinations of the invention preferably are formulated as pharmaceutical compositions. The combinations are admixed with a pharmaceutically acceptable excipient, and the first and second compounds are each present in amounts that, when administered together to a patient, kills or inhibits the development of a fungal infection.

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 The administration of a combination of the invention may be by any suitable means that results in suppression of inhibition of microbial growth target region. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for the oral, parenteral (e. g., intravenously, intramuscularly), rectal, cutaneous, nasal, vaginal, inhalant, skin (patch), or ocular administration route. Thus, the composition may be in the form of, e. g. , tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e. g., Remington: The Science and Practice of Pharmacy, 20th edition, 2000, ed.

A. R. Gennaro, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Each compound of the combination may be formulated in a variety of ways that are known in the art. For example, the first and second agents may be formulated together or separately. Desirably, the first and second agents are formulated together for the simultaneous or near simultaneous administration of the agents. Such co-formulated compositions can include, for example, the terbinafine and the manganese compound formulated together in the same pill, capsule, liquid, etc. It is to be understood that, when referring to the <BR> <BR> formulation of"terbinafine/manganese compound, "the formulation technology employed is also useful for the formulation of the individual agents of the <BR> <BR> combination, as well as other combinations of the invention (e. g. , analogs of terbinafine/manganese compound combination). By using different formulation strategies for different agents, the pharmacokinetic profiles for each agent can be suitably matched.

The individually or separately formulated agents can be packaged together as a kit. Non-limiting examples include kits that contain, e. g. , two pills, a pill and a powder, a suppository and a liquid in a vial, two topical creams, etc. The kit can include optional components that aid in the administration of the unit dose to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one patient, multiple uses for a particular patient (at a constant dose or in which the individual compounds may vary in potency as therapy progresses) ; or the kit may contain multiple doses suitable for administration to multiple patients ("bulk packaging"). The kit components may be assembled in cartons, blister packs, bottles, tubes, and the like.

Controlled/Extended Release Formulations Administration of any one of the combinations of this invention, for example, the terbinafine/manganese compound combination in which one or both of the active agents is formulated for controlled/extended release is useful where the terbinafine or the manganese compound, has (i) a narrow therapeutic <BR> <BR> 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 (EDso)) ; (ii) a narrow absorption window in the gastro-intestinal tract; (iii) a short biological half-life ; or (iv) the pharmacokinetic profile of one or both components must be modified to maximize the contribution of each agent, when used together, to an amount of that is therapeutically effective for cytokine suppression.

Accordingly, a sustained release formulation may be used to avoid frequent dosing that may be required in order to sustain the plasma levels of both agents at a therapeutic level. Many strategies can be pursued to obtain controlled/extended release in which the rate of release outweighs the rate of metabolism of the therapeutic compound. For example, controlled/extended release can be obtained by the appropriate selection of formulation parameters <BR> <BR> and ingredients (e. g. , appropriate controlled/extended release compositions and coatings). Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes. The release mechanism can be controlled/extended such that the antifungal agent and/or manganese compound (e. g. , terbinafine and/or manganese sulfate) are released at period intervals, the release could be simultaneous, or a delayed release of one of the agents of the combination can be affected, when the early release of one particular agent is preferred over the other.

Controlled/extended release formulations may include a degradable or nondegradable polymer, hydrogel, organogel, or other physical construct that modifies the bioabsorption, half-life or biodegradation of the agent. The controlled/extended release formulation can be a material that is painted or otherwise applied onto the afflicted site, either internally or externally. In one example, the invention provides a biodegradable bolus or implant that is surgically inserted at or near a site of interest (for example, proximal to an arthritic joint). In another example, the controlled/extended release formulation implant can be inserted into an organ, such as in the lower intestine for the treatment inflammatory bowel disease.

Hydrogels can be used in controlled/extended release formulations for the combinations of this invention (e. g., terbinafine/manganese compound combination). Such polymers are formed from macromers with a polymerizable, non-degradable, region that is separated by at least one degradable region. For example, the water soluble, non-degradable, region can form the central core of the macromer and have at least two degradable regions which are attached to the core, such that upon degradation, the non-degradable regions (in particular a polymerized gel) are separated, as described in U. S.

Patent No. 5,626, 863. Hydrogels can include acrylates, which can be readily polymerized by several initiating systems such as eosin dye, ultraviolet or visible light. Hydrogels can also include polyethylene glycols (PEGs), which are highly hydrophilic and biocompatible. Hydrogels can also include oligoglycolic acid, which is a poly (a-hydroxy acid) that can be readily degraded by hydrolysis of the ester linkage into glycolic acid, a nontoxic metabolite. Other chain extensions can include polylactic acid, polycaprolactone, polyorthoesters, polyanhydrides or polypeptides. The entire network can be gelled into a biodegradable network that can be used to entrap and homogeneously disperse various combinations of the invention (for example, terbinafine/manganese compound combination) for delivery at a controlled/extended rate.

Chitosan and mixtures of chitosan with carboxymethylcellulose sodium (CMC-Na) have been used as vehicles for the sustained release of drugs, as described by Inouye et al. , Drug Design and Delivery 1: 297-305, 1987.

Mixtures of these compounds and agents of the combinations described above, when compressed under 200 kg/cm2, form a tablet from which the active agent is slowly released upon administration to a subject. The release profile can be changed by varying the ratios of chitosan, CMC-Na, and active agent (s). The tablets can also contain other additives, including lactose, CaHP04 dihydrate, sucrose, crystalline cellulose, or croscarmellose sodium.

Baichwal, in U. S. Patent No. 6,245, 356, describes a sustained release oral solid dosage forms that includes agglomerated particles of a therapeutically active medicament (e. g. , terbinafine/manganese compound combination or component thereof of the present invention) in amorphous form, a gelling agent, an ionizable gel strength enhancing agent and an inert diluent. The gelling agent can be a mixture of a xanthan gum and a locust bean gum capable of cross-linking with the xanthan gum when the gums are exposed to an environmental fluid. Preferably, the ionizable gel-enhancing agent acts to enhance the strength of cross-linking between the xanthan gum and the locust bean gum and thereby prolonging the release of the medicament component of the formulation. In addition to xanthan gum and locust bean gum, acceptable gelling agents that may also be used include those gelling agents well known in the art. Examples include naturally occurring or modified naturally occurring gums such as alginates, carrageenan, pectin, guar gum, modified starch, hydroxypropylmethylcellulose, methylcellulose, and other cellulosic materials or polymers, such as, for example, sodium carboxymethylcellulose and hydroxypropyl cellulose, and mixtures of the foregoing.

In another formulation useful for the combinations of the invention, Baichwal and Staniforth in U. S. Patent No. 5,135, 757 describe a free-flowing slow release granulation for use as a pharmaceutical excipient that includes from about 20 to about 70 percent or more by weight of a hydrophilic material

that includes a heteropolysaccharide (such as, for example, xanthan gum or a derivative thereof) and a polysaccharide material capable of cross-linking the heteropolysaccharide (such as, for example, galactomannans, and most preferably locust bean gum) in the presence of aqueous solutions, and from about 30 to about 80 percent by weight of an inert pharmaceutical filler (such as, for example, lactose, dextrose, sucrose, sorbitol, xylitol, fructose or mixtures thereof). After mixing the excipient with an antifungal agent/manganese compound (e. g., terbinafine/manganese sufate) combination, or combination agent, of the invention, the mixture is directly compressed into solid dosage forms such as tablets. The tablets thus formed slowly release the medicament when ingested and exposed to gastric fluids. By varying the amount of excipient relative to the medicament, a slow release profile can be attained.

In another formulation useful for the combinations of the invention, Shell, in U. S. Patent No. 5,007, 790, describes sustained-release oral drug- dosage forms that release a drug in solution at a rate controlled/extended by the solubility of the drug. The dosage form comprises a tablet or capsule that includes a plurality of particles of a dispersion of a limited solubility drug (such as, for example, terbinafine, terbinafine, or any other agent of the combination of the present invention) in a hydrophilic, water-swellable, crosslinked polymer that maintains its physical integrity over the dosing lifetime but thereafter rapidly dissolves. Once ingested, the particles swell to promote gastric retention and permit the gastric fluid to penetrate the particles, dissolve drug and leach it from the particles, assuring that drug reaches the stomach in the solution state which is less injurious to the stomach than solid-state drug. The programmed eventual dissolution of the polymer depends upon the nature of the polymer and the degree of crosslinking. The polymer is nonfibrillar and substantially water soluble in its uncrosslinked state, and the degree of crosslinking is sufficient to enable the polymer to remain insoluble for the desired time period, normally at least from about 4 hours to 8 hours up to 12

hours, with the choice depending upon the drug incorporated and the medical treatment involved. Examples of suitable crosslinked polymers that may be used in the invention are gelatin, albumin, sodium alginate, carboxymethyl cellulose, polyvinyl alcohol, and chitin. Depending upon the polymer, crosslinking may be achieved by thermal or radiation treatment or through the use of crosslinking agents such as aldehydes, polyamino acids, metal ions and the like.

Silicone microspheres for pH-controlled/extended gastrointestinal drug delivery that are useful in the formulation of the combinations of the invention <BR> <BR> have been described by Carelli et al. , Int. J. Pharmaceutics 179: 73-83,1999.

The microspheres so described are pH-sensitive semi-interpenetrating polymer hydrogels made of varying proportions of poly (methacrylic acid-co- methylmethacrylate) (Eudragit L100 or Eudragit S100) and crosslinked polyethylene glycol 8000 that are encapsulated into silicone microspheres in the 500 to 1000 Zm size range.

Slow-release formulations can include a coating which is not readily water-soluble but which is slowly attacked and removed by water, or through which water can slowly permeate. Thus, for example, the terbinafine/manganese compound combination of the invention can be spray- coated with a solution of a binder under continuously fluidizing conditions, <BR> <BR> such as describe by Kitamori et al. , U. S. Patent No. 4,036, 948. Examples of<BR> water-soluble binders include pregelatinized starch (e. g. , pregelatinized corn starch, pregelatinized white potato starch), pregelatinized modified starch, water-soluble celluloses (e. g. hydroxypropyl-cellulose, hydroxymethyl- cellulose, hydroxypropylmethyl-cellulose, carboxymethyl-cellulose), polyvinylpyrrolidone, polyvinyl alcohol, dextrin, gum arabicum and gelatin, <BR> organic solvent-soluble binders, such as cellulose derivatives (e. g. , cellulose acetate phthalate, hydroxypropylmethyl-cellulose phthalate, ethylcellulose).

Combinations of the invention, or a component thereof, with sustained release properties can also be formulated by spray drying techniques.

In one example, as described by Espositio et al., Pharm. Dev. Technol. 5: 267- 78, 2000, terbinafine was encapsulated in methyacrylate microparticles (Eudragit RS) using a Mini Spray Dryer, model 190 (Buchi, Laboratorium Technik AG, Flawil, Germany). Optimal conditions for microparticle formation were found to be a feed (pump) rate of 0.5 mL/min of a solution containing 50 mg terbinafine in 10 mL of acetonitrile, a flow rate of nebulized air of 600 L/hr, dry air temperature heating at 80°C, and a flow rate of aspirated drying air of 28 m3/hr.

Yet another form of sustained release antifungal agent/manganese <BR> <BR> compound (e. g. , terbinafine/manganese sulfate) combinations can be prepared by microencapsulation of combination agent particles in membranes which act as microdialysis cells. In such a formulation, gastric fluid permeates the microcapsule walls and swells the microcapsule, allowing the active agent (s) to dialyze out (see, for example, Tsuei et al. , U. S. Patent No. 5,589, 194). One commercially available sustained-release system of this kind consists of microcapsules having membranes of acacia gum/gelatine/ethyl alcohol. This product is available from Eurand Limited (France) under the trade name Diffucaps. Microcapsules so formulated might be carried in a conventional gelatine capsule or tabletted.

Extended-and/or controlled/extended-release formulations of both antifungal agent/manganese compound (e. g. , terbinafine/manganese sulfate) can be performed following the examples of U. S. Patent Nos. 4, 839, 177, 5,102, 666, and 5,422, 123, which has an enteric coat to delay the start of drug release until after the tablets have passed through the stomach. Other examples include U. S. Pat. No. 5,102, 666 which describe a polymeric controlled/extended release composition comprising a reaction complex formed by the interaction of (1) a calcium polycarbophil component which is a water- swellable, but water insoluble, fibrous cross-linked carboxy-functional polymer, the polymer containing (a) a plurality of repeating units of which at least about 80% contain at least one carboxyl functionality, and (b) about 0.05

to about 1.5% cross-linking agent substantially free from polyalkenyl polyether, the percentages being based upon the weights of unpolymerised repeating unit and cross-linking agent, respectively, with (2) water, in the presence of an active agent selected from the group consisting of antifungal agent such as terbinafine. The amount of calcium polycarbophil present is from about 0.1 to about 99% by weight, for example about 10%. The amount of active agent present is from about 0.0001 to about 65% by weight, for example between about 5 and 20%. The amount of water present is from about 5 to about 200% by weight, for example between about 5 and 10%. The interaction is carried out at a pH of between about 3 and about 10, for example about 6 to 7. The calcium polycarbophil is originally present in the form of a calcium salt containing from about 5 to about 25% calcium.

Other extended-release formulation examples are described in U. S.

Patent No. 5,422, 123. Thus, a system for the controlled/extended release of an active substance which is an antifungal agent such as terbinafine, comprising (a) a deposit-core comprising an effective amount of the active substance and having defined geometric form, and (b) a support-plafform applied to the deposit-core, wherein the deposit-core contains at least the active substance, and at least one member selected from the group consisting of (1) a polymeric material which swells on contact with water or aqueous liquids and a gellable polymeric material wherein the ratio of the swellable polymeric material to the gellable polymeric material is in the range 1: 9 to 9: 1, and (2) a single polymeric material having both swelling and gelling properties, and wherein the support-platform is an elastic support, applied to said deposit-core so that it partially covers the surface of the deposit-core and follows changes due to hydration of the deposit-core and is slowly soluble and/or slowly gellable in aqueous fluids. The support-platform may comprise polymers such as hydroxypropylmethylcellulose, plasticizers such as a glyceride, binders such as polyvinylpyrrolidone, hydrophilic agents such as lactose and silica, and/or hydrophobic agents such as magnesium stearate and glycerides.

The polymer (s) typically make up 30 to 90% by weight of the support- platform, for example about 35 to 40%. Plasticizer may make up at least 2% by weight of the support-platform, for example about 15 to 20%. Binder (s), hydrophilic agent (s) and hydrophobic agent (s) typically total up to about 50% by weight of the support-platform, for example about 40 to 50%.

To make these low dose levels of active substance possible, the active substance, i. e. the antifungal, such as terbinafine or manganese compound, such as manganese compound, is micronized, suitably mixed with known diluents, such as starch and lactose, and granulated with PVP (polyvinylpyrrolidone). Further, the granulate is laminated with a sustained release inner layer resistant to a pH of 6. 8 and a sustained release outer layer resistant to a pH of 1.0. The inner layer is made of Eudragit'DRL (copolymer of acrylic and methacrylic esters with a low content of quaternary ammonium groups) and the outer layer is made of Eudragit'OL (anionic polymer synthesized from methacrylic acid and methacrylic acid methyl ester).

A bilayer tablet can be formulated for the combinations described above (e. g. , terbinafine/manganese compound combination) in which different custom granulations are made for each agent of the combination and the two agents are compressed on a bi-layer press to form a single tablet. For example, terbinafine, formulated for a controlled/extended release that may be combined in the same tablet with manganese compound, which is formulated such that the tl/2 approximates that of terbinafine. In addition to controlling the rate of manganese compound release in vivo, an enteric or delayed release coat may be included that delays the start of drug release such that the Tax of manganese compound approximate that of terbinafine. The times and dosages are illustrative of various tablet formulations; one of skill in the art will know hoe to formulate the tablet for the appropriate release rate for various dosages.

Cyclodextrins are cyclic polysaccharides containing naturally occurring D (+) -glucopyranose units in an a- (1, 4) linkage. Alpha-, beta-, and gamma- cyclodextrins, which contain, respectively, six, seven or eight glucopyranose

units, are most commonly used and suitable examples are described in PCT Publication Nos. W091/11172, W094/02518 and W098/55148. Structurally, the cyclic nature of a cyclodextrin forms a torus or donut-like shape having an inner apolar or hydrophobic cavity, the secondary hydroxyl groups situated on one side of the cyclodextrin torus and the primary hydroxyl groups situated on the other. The side on which the secondary hydroxyl groups are located has a wider diameter than the side on which the primary hydroxyl groups are located.

The hydrophobic nature of the cyclodextrin inner cavity allows for the inclusion of a variety of compounds. (Comprehensive Supramolecular Chemistry, Volume 3, J. L. Atwood et al. , eds. , Pergamon Press (1996); Cserhati, Analytical Biochemistry 225: 328-32, 1995; Husain et al., Applied Spectroscopy 46: 652-8, 1992. Cyclodextrins have been used as a delivery vehicle of various therapeutic compounds by forming inclusion complexes with various drugs that can fit into the hydrophobic cavity of the cyclodextrin or by forming non-covalent association complexes with other biologically active molecules. U. S. Patent No. 4,727, 064 describes pharmaceutical preparations consisting of a drug with substantially low water solubility and an amorphous, water-soluble cyclodextrin-based mixture in which the drug forms an inclusion complex with the cyclodextrins of the mixture.

Formation of a drug-cyclodextrin complex can modify the drug's solubility, dissolution rate, bioavailability, and/or stability properties. For example, cyclodextrins have been described for improving the bioavailability <BR> <BR> of terbinafine, as described by Uekama et al. , J. Pharm Dyn. 6: 124-7, 1983. A P-cyclodextrin/terbinafine complex can be prepared by adding both components to water and stirring at 25°C for 7 days. The resultant precipitate recovered is a 1: 2 terbinafine/cyclodextrin complex.

Sulfobutylether-ß-cyclodextrin (SBE-ß-CD, commercially available from CyDex, Inc, Overland Park, KA, USA and sold as CAPTISOL) can also be used as an aid in the preparation of sustained-release formulations of agents of the combinations of the present invention.

For example, a sustained-release tablet has been prepared that includes terbinafine and SBE-ß-CD compressed in a hydroxypropyl methylcellulose <BR> <BR> matrix (see Rao et al. , J. Pharm. Sci. 90: 807-16, 2001). In another example of the use of various cyclodextrins, EP 1109806 B1 describes cyclodextrin complexes of terbinafine, where a-, y-, or (3-cyclodextrins (including eptakis (2- 6-di-O-methyl)-ß-cyclodextrin, (2,3, 6-tri-0-methyl)-p-cyclodextrin, monosuccinyl eptakis (2, 6-di-O-methyl)-ß-cyclodextrin, or 2-hydroxypropyl- (3- cyclodextrin) in anhydrous or hydrated form formed complex ratios of agent to cyclodextrin of from 1: 0.25 to 1 : 20 can be obtained.

Polymeric cyclodextrins have also been prepared, as described in U. S.

Patent Application Publication Nos. 2003/0017972 Al and 2003/0008818 Al.

The cyclodextrin polymers so formed can be useful for formulating agents of the combinations of the present invention. These multifunctional polymeric cyclodextrins are commercially available from Insert Therapeutics, Inc., Pasadena, CA, USA.

As an alternative to direct complexation with agents, cyclodextrins may be used as an auxiliary additive, e. g. as a carrier, diluent or solubiliser.

Formulations that include cyclodextrins and other agents of the combinations of the present invention can be prepared by methods similar to the preparations of the cyclodextrin formulations described herein.

Liposomal Formulations One or both components of the combinations of the invention or mixtures of the two components together, can be incorporated into liposomal carriers for administration. The liposomal carriers are composed of three general types of vesicle-forming lipid components. The first includes vesicle- forming lipids that will form the bulk of the vesicle structure in the liposome.

Generally, these vesicle-forming lipids include any amphipathic lipids having hydrophobic and polar head group moieties, and which (a) can form spontaneously into bilayer vesicles in water, as exemplified by phospholipids,

or (b) are stably incorporated into lipid bilayers, with its hydrophobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its polar head group moiety oriented toward the exterior, polar surface of the membrane.

The vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a polar head group. Included in this class are the phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. The above-described lipids and phospholipids whose acyl chains have a variety of degrees of saturation can be obtained commercially, or prepared according to published methods. Other lipids that can be included in the invention are glycolipids and sterols, such as cholesterol.

The second general component includes a vesicle-forming lipid that is derivatized with a polymer chain that will form the polymer layer in the composition. The vesicle-forming lipids that can be used as the second general vesicle-forming lipid component are any of those described for the first general vesicle-forming lipid component. Vesicle forming lipids with diacyl chains, such as phospholipids, are preferred. One exemplary phospholipid is PE, which provides a reactive amino group that is convenient for coupling to the activated polymers. An exemplary PE is distearyl PE (DSPE).

The preferred polymer in the derivatized lipid is polyethyleneglycol (PEG), preferably a PEG chain having a molecular weight between 1,000- 15,000 daltons, more preferably between 2,000 and 10,000 daltons, most preferably between 2,000 and 5,000 daltons. Other hydrophilic polymers that may be suitable include polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses, such as hydroxymethylcellulose or hydroxyethylcellulose.

Additionally, block copolymers or random copolymers of these polymers, particularly including PEG segments, may be suitable. Methods for preparing lipids derivatized with hydrophilic polymers, such as PEG, are well known e. g. , as described in U. S. Patent No. 5,013, 556.

A third general vesicle-forming lipid component, which is optional, is a lipid anchor by which a targeting moiety is anchored to the liposome, through a polymer chain in the anchor. Additionally, the targeting group is positioned at the distal end of the polymer chain in such a way so that the biological activity of the targeting moiety is not lost. The lipid anchor has a hydrophobic moiety which serves to anchor the lipid in the outer layer of the liposome bilayer surface, a polar head group to which the interior end of the polymer is covalently attached, and a free (exterior) polymer end which is or can be activated for covalent coupling to the targeting moiety. Methods for preparing lipid anchor molecules of these types are described below.

The lipids components used in forming the liposomes are preferably present in a molar ratio of about 70-90 percent vesicle forming lipids, 1-25 percent polymer derivatized lipid, and 0.1-5 percent lipid anchor. One exemplary formulation includes 50-70 mole percent underivatized PE, 20-40 mole percent cholesterol, 0.1-1 mole percent of a PE-PEG (3500) polymer with a chemically reactive group at its free end for coupling to a targeting moiety, 5- 10 mole percent PE derivatized with PEG 3500 polymer chains, and 1 mole percent alpha-tocopherol.

The liposomes are preferably prepared to have substantially homogeneous sizes in a selected size range, typically between about 0.03 to 0.5 microns. One effective sizing method for REVs and MLVs involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2 microns.

The pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane.

Homogenization methods are also useful for down-sizing liposomes to sizes of 100 nm or less.

The liposomal formulations of the present invention include at least one surface-active agent. Suitable surface-active agents useful for the formulation of the combinations described herein (e. g., terbinafine/manganese compound combination) include compounds belonging to the following classes: polyethoxylated fatty acids, PEG-fatty acid diesters, PEG-fatty acid mono-ester and di-ester mixtures, polyethylene glycol glycerol fatty acid esters, alcohol-oil transesterification products, polyglycerized fatty acids, propylene glycol fatty acid esters, mixtures of propylene glycol esters and glycerol esters, mono-and diglycerides, sterol and sterol derivatives, polyethylene glycol sorbitan fatty acid esters, polyethylene glycol alkyl ethers, sugar esters, polyethylene glycol alkyl phenols, polyoxyethylene-polyoxypropylene block copolymers, sorbitan fatty acid esters, lower alcohol fatty acid esters, and ionic surfactants.

Commercially available examples for each class of excipient are provided below.

Polyethoxylated fatty acids may be used as excipients for the formulation of the combinations described herein. Examples of commercially available polyethoxylated fatty acid monoester surfactants include: PEG 4-100 monolaurate (Crodet L series, Croda), PEG 4-100 monooleate (Crodet O series, Croda), PEG 4-100 monostearate (Crodet S series, Croda, and Myrj Series, Atlas/ICI), PEG 400 distearate (Cithrol 4DS series, Croda), PEG 100,200, or 300 monolaurate (Cithrol ML series, Croda), PEG 100,200, or 300 monooleate (Cithrol MO series, Croda), PEG 400 dioleate (Cithrol 4DO series, Croda), PEG 400-1000 monostearate (Cithrol MS series, Croda), PEG-1 stearate (Nikkol MYS-lEX, Nikko, and Coster Kl, Condea), PEG-2 stearate (Nikkol MYS-2, Nikko), PEG-2 oleate (Nikkol MYO-2, Nikko), PEG-4 laurate

(Mapeg 200 ML, PPG), PEG-4 oleate (Mapeg 200 MO, PPG), PEG-4 stearate (Kesscot PEG 200 MS, Stepan), PEG-5 stearate (Nikkol TMGS-5, Nikko), PEG-5 oleate (Nikkol TMGO-5, Nikko), PEG-6 oleate (Algon OL 60, Auschem SpA), PEG-7 oleate (Algon OL 70, Auschem SpA), PEG-6 laurate (Kessco (R) PEG300 ML, Stepan), PEG-7 laurate (Lauridac 7, Condea), PEG-6 stearate (Kessco PEG300 MS ; Stepan), PEG-8 laurate (Mapeg (g) 400 ML, PPG), PEG-8 oleate (Mapeg 400 MO, PPG), PEG-8 stearate (Mapeg 400 MS, PPG), PEG-9 oleate (Emulgante A9, Condea), PEG-9 stearate (Cremophor S9, BASF), PEG-10 laurate (Nikkol MYL-10, Nikko), PEG-10 oleate (Nikkol MYO-10, Nikko), PEG-12 stearate (Nikkol MYS-10, Nikko), PEG-12 laurate (Kessco PEG 600 ML, Stepan), PEG-12 oleate (Kessco PEG 600 MO, Stepan), PEG-12 ricinoleate (CAS # 9004-97-1), PEG-12 stearate (Mapegi) 600 MS, PPG), PEG-15 stearate (Nikkol TMGS-15, Nikko), PEG-15 oleate (Nikkol TMGO-15, Nikko), PEG-20 laurate (Kessco PEG 1000 ML, Stepan), PEG-20 oleate (Kessco PEG 1000 MO, Stepan), PEG-20 stearate (Mapeg 1000 MS, PPG), PEG-25 stearate (Nikkol MYS-25, Nikko), PEG-32 laurate (Kessco PEG 1540 ML, Stepan), PEG-32 oleate (Kessco PEG 1540 MO, Stepan), PEG-32 stearate (Kessco PEG 1540 MS, Stepan), PEG-30 stearate (Myrj 51), PEG-40 laurate (Crodet L40, Croda), PEG-40 oleate (Crodet 040, Croda), PEG-40 stearate (Emerest (D 2715, Henkel), PEG-45 stearate (Nikkol MYS-45, Nikko), PEG-50 stearate (Myrj 53), PEG-55 stearate (Nikkol MYS- 55, Nikko), PEG-100 oleate (Crodet O-100, Croda), PEG-100 stearate (Ariacel <BR> <BR> 165, ICI), PEG-200 oleate (Albunol 200 MO, Taiwan Surf. ), PEG-400 oleate (LACTOMUL, Henkel), and PEG-600 oleate (Albunol 600 MO, Taiwan Surf.).

Formulations of one or both components of the combinations according to the invention may include one or more of the polyethoxylated fatty acids above.

Polyethylene glycol fatty acid diesters may also be used as excipients for the combinations described herein. Examples of commercially available polyethylene glycol fatty acid diesters include: PEG-4 dilaurate (Mapeg 200 DL, PPG), PEG-4 dioleate (Mapeg 200 DO, PPG), PEG-4 distearate

(Kessco 200 DS, Stepan), PEG-6 dilaurate (Kessco PEG 300 DL, Stepan), PEG-6 dioleate (Kesscot) PEG 300 DO, Stepan), PEG-6 distearate (Kessco (g PEG 300 DS, Stepan), PEG-8 dilaurate (Mapeg 400 DL, PPG), PEG-8 dioleate (Mapeg (g) 400 DO, PPG), PEG-8 distearate (Mapeg 400 DS, PPG), PEG-10 dipalmitate (Polyaldo 2PKFG), PEG-12 dilaurate (Kessco PEG 600 DL, Stepan), PEG-12 distearate (Kessco PEG 600 DS, Stepan), PEG-12 dioleate (Mapeg 600 DO, PPG), PEG-20 dilaurate (Kessco PEG 1000 DL, Stepan), PEG-20 dioleate (Kessco (E PEG 1000 DO, Stepan), PEG-20 distearate (Kessco PEG 1000 DS, Stepan), PEG-32 dilaurate (Kessco PEG 1540 DL, Stepan), PEG-32 dioleate (Kessco PEG 1540 DO, Stepan), PEG-32 distearate (Kessco PEG 1540 DS, Stepan), PEG-400 dioleate (Cithrol 4DO series, Croda), and PEG-400 distearate Cithrol 4DS series, Croda). Formulations of the combinations according to the invention may include one or more of the polyethylene glycol fatty acid diesters above.

PEG-fatty acid mono-and di-ester mixtures may be used as excipients for the formulation of the combinations described herein. Examples of commercially available PEG-fatty acid mono-and di-ester mixtures include: PEG 4-150 mono, dilaurate (Kessco PEG 200-6000 mono, Dilaurate, Stepan), PEG 4-150 mono, dioleate (Kessco PEG 200-6000 mono, Dioleate, Stepan), and PEG 4-150 mono, distearate (Kesscog 200-6000 mono, Distearate, Stepan). Formulations of the combinations according to the invention may include one or more of the PEG-fatty acid mono-and di-ester mixtures above.

In addition, polyethylene glycol glycerol fatty acid esters may be used as excipients for the formulation of the combinations described herein. Examples of commercially available polyethylene glycol glycerol fatty acid esters include: PEG-20 glyceryl laurate (Tagat L, Goldschmidt), PEG-30 glyceryl laurate (Tagat L2, Goldschmidt), PEG-15 glyceryl laurate (Glycerox L series, Croda), PEG-40 glyceryl laurate (Glycerox L series, Croda), PEG-20 glyceryl stearate (Capmul EMG, ABITEC), and Aldo MS-20 KFG, Lonza), PEG-20

glyceryl oleate (Tagat RO O, Goldschmidt), and PEG-30 glyceryl oleate (agate 02, Goldschmidt). Formulations of the combinations according to the invention may include one or more of the polyethylene glycol glycerol fatty acid esters above.

Alcohol-oil transesterification products may also be used as excipients for the formulation of the combinations described herein. Examples of commercially available alcohol-oil transesterification products include: PEG-3 castor oil (Nikkol CO-3, Nikko), PEG-5,9, and 16 castor oil (ACCONON CA series, ABITEC), PEG-20 castor oil, (Emalex C-20, Nihon Emulsion), PEG-23 castor oil (Emulgante EL23), PEG-30 castor oil (Incrocas 30, Croda), PEG-35 castor oil (Incrocas-35, Croda), PEG-38 castor oil (Emulgante EL 65, Condea), PEG-40 castor oil (Emalex C-40, Nihon Emulsion), PEG-50 castor oil (Emalex C-50, Nihon Emulsion), PEG-56 castor oil (Eumulgin PRT 56, Pulcra SA), PEG-60 castor oil (Nikkol CO-60TX, Nikko), PEG-100 castor oil, PEG-200 castor oil (Eumulgin (b PRT 200, Pulcra SA), PEG-5 hydrogenated castor oil (Nikkol HCO-5, Nikko), PEG-7 hydrogenated castor oil (Cremophor W07, BASF), PEG-10 hydrogenated castor oil (Nikkol HCO-10, Nikko), PEG-20 hydrogenated castor oil (Nikkol HCO-20, Nikko), PEG-25 hydrogenated castor oil (Simulsol 1292, Seppic), PEG-30 hydrogenated castor oil (Nikkol HCO- 30, Nikko), PEG-40 hydrogenated castor oil (Cremophor RH 40, BASF), PEG- 45 hydrogenated castor oil (Cerex ELS 450, Auschem Spa), PEG-50 hydrogenated castor oil (Emalex HC-50, Nihon Emulsion), PEG-60 hydrogenated castor oil (Nikkol HCO-60, Nikko), PEG-80 hydrogenated castor oil (Nikkol HCO-80, Nikko), PEG-100 hydrogenated castor oil (Nikkol HCO- 100, Nikko), PEG-6 corn oil (Labrafilg M 2125 CS, Gattefosse), PEG-6 almond oil (Labrafil (V M 1966 CS, Gattefosse), PEG-6 apricot kernel oil (Labrafil (R) M 1944 CS, Gattefosse), PEG-6 olive oil (Labrafil M 1980 CS, Gattefosse), PEG-6 peanut oil (Labrafil M 1969 CS, Gattefosse), PEG-6 hydrogenated palm kernel oil (Labrafil (g) M 2130 BS, Gattefosse), PEG-6 palm kernel oil (Labrafil M 2130 CS, Gattefosse), PEG-6 triolein (Labrafil M

2735 CS, Gattefosse), PEG-8 corn oil (Labrafil WL 2609 BS, Gattefosse), PEG-20 corn glycerides (Crovol M40, Croda), PEG-20 almond glycerides (Crovol A40, Croda), PEG-25 trioleate (TAGATX TO, Goldschmidt), PEG-40 palm kernel oil (Crovol PK-70), PEG-60 corn glycerides (Crovol M70, Croda), PEG-60 almond glycerides (Crovol A70, Croda), PEG-4 caprylic/capric triglyceride (Labrafac Hydro, Gattefosse), PEG-8 caprylic/capric glycerides (Labrasol, Gattefosse), PEG-6 caprylic/capric glycerides (SOFTIGEN (g) 767, Huls), lauroyl macrogol-32 glyceride (GELUCIRE 44/14, Gattefosse), stearoyl macrogol glyceride (GELUCIRE 50/13, Gattefosse), mono, di, tri, tetra esters of vegetable oils and sorbitol (SorbitoGlyceride, Gattefosse), pentaerythrityl tetraisostearate (Crodamol PTIS, Croda), pentaerythrityl distearate (Albunol <BR> <BR> DS, Taiwan Surf. ), pentaerythrityl tetraoleate (Liponate PO-4, Lipo Chem.),<BR> pentaerythrityl tetrastearate (Liponate PS-4, Lipo Chem. ), pentaerythrityl tetracaprylate tetracaprate (Liponate PE-810, Lipo Chem.), and pentaerythrityl tetraoctanoate (Nikkol Pentarate 408, Nikko). Also included as oils in this category of surfactants are oil-soluble vitamins, such as vitamins A, D, E, K, etc. Thus, derivatives of these vitamins, such as tocopheryl PEG-1000 succinate (TPGS, available from Eastman), are also suitable surfactants.

Formulations of the combinations (e. g. , Terbinafine/manganese sulfate) according to the invention may include one or more of the alcohol-oil transesterification products above.

Polyglycerized fatty acids may also be used as excipients for the formulation of the combinations described herein. Commercially available polyglycerized fatty acids include: polyglyceryl-2 stearate (Nikkol DGMS, Nikko), polyglyceryl-2 oleate (Nikkol DGMO, Nikko), polyglyceryl-2 isostearate (Nikkol DGMIS, Nikko), polyglyceryl-3 oleate (Caprolt) 3GO, ABITEC), polyglyceryl-4 oleate (Nikkol Tetraglyn 1-O, Nikko), polyglyceryl- 4 stearate (Nikkol Tetraglyn 1-S, Nikko), polyglyceryl-6 oleate (Drewpol 6-1- O, Stepan), polyglyceryl-10 laurate (Nikkol Decaglyn 1-L, Nikko), polyglyceryl-10 oleate (Nikkol Decaglyn 1-O, Nikko), polyglyceryl-10 stearate

(Nikkol Decaglyn 1-S, Nikko), polyglyceryl-6 ricinoleate (Nikkol Hexaglyn PR-15, Nikko), polyglyceryl-10 linoleate (Nikkol Decaglyn 1-LN, Nikko), polyglyceryl-6 pentaoleate (Nikkol Hexaglyn 5-O, Nikko), polyglyceryl-3 dioleate (Cremophor G032, BASF), polyglyceryl-3 distearate (Cremophor GS32, BASF), polyglyceryl-4 pentaoleate (Nikkol Tetraglyn 5-O, Nikko), polyglyceryl-6 dioleate (Caprol 6G20, ABITEC), polyglyceryl-2 dioleate (Nikkol DGDO, Nikko), polyglyceryl-10 trioleate (Nikkol Decaglyn 3-O, Nikko), polyglyceryl-10 pentaoleate (Nikkol Decaglyn 5-O, Nikko), polyglyceryl-10 septaoleate (Nikkol Decaglyn 7-O, Nikko), polyglyceryl-10 tetraoleate (Caprol 1OG40, ABITEC), polyglyceryl-10 decaisostearate (Nikkol Decaglyn 10-IS, Nikko), polyglyceryl-101 decaoleate (Drewpol 10-10- O, Stepan), polyglyceryl-10 mono, dioleate (Caprol PGE 860, ABITEC), and polyglyceryl polyricinoleate (Polymuls, Henkel). Formulations of the combinations according to the invention may include one or more of the polyglycerized fatty acids above.

In addition, propylene glycol fatty acid esters may be used as excipients for the formulation of the combinations described herein. Examples of commercially available propylene glycol fatty acid esters include: propylene glycol monocaprylate (Capryol 90, Gattefosse), propylene glycol monolaurate (Lauroglycol 90, Gattefosse), propylene glycol oleate (Lutrol OP2000, BASF), propylene glycol myristate (Mirpyl), propylene glycol monostearate (LIPO PGMS, Lipo Chem. ), propylene glycol hydroxystearate, propylene glycol ricinoleate (PROPYMULS, Henkel), propylene glycol isostearate, propylene glycol monooleate (Myverol P-06, Eastman), propylene glycol dicaprylate dicaprate (Captex (g) 200, ABITEC), propylene glycol dioctanoate (Captex0 800, ABITEC), propylene glycol caprylate caprate (LABRAFAC PG, Gattefosse), propylene glycol dilaurate, propylene glycol distearate (Kesseog PGDS, Stepan), propylene glycol dicaprylate (Nikkol Sefsol 228, Nikko), and propylene glycol dicaprate (Nikkol PDD, Nikko).

Formulations of the combinations to the invention may include one or more of the propylene glycol fatty acid esters above.

Mixtures of propylene glycol esters and glycerol esters may also be used as excipients for the formulation of the combinations described herein. One preferred mixture is composed of the oleic acid esters of propylene glycol and glycerol (Arlacel 186). Examples of these surfactants include: oleic (ATMOS 300, ARLACEL 186, ICI), and stearic (ATMOS 150). Formulations of the combinations according to the invention may include one or more of the mixtures of propylene glycol esters and glycerol esters above.

Further, mono-and diglycerides may be used as excipients for the formulation of the combinations described herein. Examples of commercially available mono-and diglycerides include: monopalmitolein (C16 : 1) (Larodan), monoelaidin (C18 : 1) (Larodan), monocaproin (C6) (Larodan), monocaprylin (Larodan), monocaprin (Larodan), monolaurin (Larodan), glyceryl monomyristate (C 14) (Nikkol MGM, Nikko), glyceryl monooleate (C18 : 1) (PECEOL, Gattefosse), glyceryl monooleate (Myverol, Eastman), glycerol monooleate/linoleate (OLICINE, Gattefosse), glycerol monolinoleate (Maisine, Gattefosse), glyceryl ricinoleate (Softigen 701, Huls), glyceryl monolaurate (ALDOO MLD, Lonza), glycerol monopalmitate (Emalex GMS-P, Nihon), glycerol monostearate (Capmul GMS, ABITEC), glyceryl mono-and dioleate (Capmul@ GMO-K, ABITEC), glyceryl palmitic/stearic (CUTINA MD-A, ESTAGEL-G18), glyceryl acetate (Lamegin EE, Grunau GmbH), glyceryl laurate (Imwitor 312, Huls), glyceryl citrate/lactate/oleate/linoleate (Imwitor 375, Huls), glyceryl caprylate (Imwitor 308, Huls), glyceryl caprylate/caprate (Capmulg MCM, ABITEC), caprylic acid mono-and diglycerides (Imwitor 988, Huls), caprylic/capric glycerides (Imwitor 742, Huls), Mono-and diacetylated monoglycerides (Myvacet 9-45, Eastman), glyceryl monostearate (Aldo MS, Arlacel 129, ICI), lactic acid esters of mono and diglycerides (LAMEGIN GLP, Henkel), dicaproin (C6) (Larodan), dicaprin (C10) (Larodan), dioctanoin (C8) (Larodan), dimyristin (C14)

(Larodan), dipalmitin (C16) (Larodan), distearin (Larodan), glyceryl dilaurate (C12) (Capmult) GDL, ABITEC), glyceryl dioleate (Capmul GDO, ABITEC), glycerol esters of fatty acids (GELUCIRE 39/01, Gattefosse), dipalmitolein (C16: 1) (Larodan), 1,2 and 1,3-diolein (C18 : 1) (Larodan), dielaidin (C18 : 1) (Larodan), and dilinolein (C18 : 2) (Larodan). Formulations of the combinations according to the invention may include one or more of the mono-and diglycerides above.

Sterol and sterol derivatives may also be used as excipients for the formulation of the combinations described herein. Examples of commercially available sterol and sterol derivatives include: cholesterol, sitosterol, lanosterol, PEG-24 cholesterol ether (Solulan C-24, Amerchol), PEG-30 cholestanol (Phytosterol GENEROL series, Henkel), PEG-25 phytosterol (Nikkol BPSH- 25, Nikko), PEG-5 soyasterol (Nikkol BPS-5, Nikko), PEG-10 soyasterol (Nikkol BPS-10, Nikko), PEG-20 soyasterol (Nikkol BPS-20, Nikko), and PEG-30 soyasterol (Nikkol BPS-30, Nikko). Formulations of the combinations according to the invention may include one or more of the sterol and sterol derivatives above.

Polyethylene glycol sorbitan fatty acid esters may also be used as excipients for the formulation of the combinations described herein. Examples of commercially available polyethylene glycol sorbitan fatty acid esters include: PEG-10 sorbitan laurate (Liposorb L-10, Lipo Chem. ), PEG-20 sorbitan monolaurate (Tween 20, Atlas/ICI), PEG-4 sorbitan monolaurate (Tween 21, Atlas/ICI), PEG-80 sorbitan monolaurate (Hodag PSML-80, Calgene), PEG-6 sorbitan monolaurate (Nikkol GL-1, Nikko), PEG-20 sorbitan monopalmitate (Tween RO 40, Atlas/ICI), PEG-20 sorbitan monostearate (Tween 60, Atlas/ICI), PEG-4 sorbitan monostearate (Tween 61, Atlas/ICI), PEG-8 sorbitan monostearate (DACOL MSS, Condea), PEG-6 sorbitan monostearate (Nikkol TS 106, Nikko), PEG-20 sorbitan tristearate (Tween 65, Atlas/ICI), PEG-6 sorbitan tetrastearate (Nikkol GS-6, Nikko), PEG-60 sorbitan tetrastearate (Nikkol GS-460, Nikko), PEG-5 sorbitan

monooleate (Tween (R) 81, Atlas/ICI), PEG-6 sorbitan monooleate (Nikkol TO- 106, Nikko), PEG-20 sorbitan monooleate (Tween 80, Atlas/ICI), PEG-40 sorbitan oleate (Emalex ET 8040, Nihon Emulsion), PEG-20 sorbitan trioleate (TweenS 85, Atlas/ICI), PEG-6 sorbitan tetraoleate (Nikkol GO-4, Nikko), PEG-30 sorbitan tetraoleate (Nikkol GO-430, Nikko), PEG-40 sorbitan tetraoleate (Nikkol GO-440, Nikko), PEG-20 sorbitan monoisostearate (Tween 120, Atlas/ICI), PEG sorbitol hexaoleate (Atlas G-1086, ICI), polysorbate 80 (Tween 80, Pharma), polysorbate 85 (Tween 85, Pharma), polysorbate 20 (Tween 20, Pharma), polysorbate 40 (Tween 40, Pharma), polysorbate 60 (Tween 60, Pharma), and PEG-6 sorbitol hexastearate (Nikkol GS-6, Nikko). Formulations of the combinations according to the invention may include one or more of the polyethylene glycol sorbitan fatty acid esters above.

In addition, polyethylene glycol alkyl ethers may be used as excipients for the formulation of the combinations described herein. Examples of commercially available polyethylene glycol alkyl ethers include: PEG-2 oleyl ether, oleth-2 (Brij 92/93, Atlas/ICI), PEG-3 oleyl ether, oleth-3 (Volpo 3, Croda), PEG-5 oleyl ether, oleth-5 (Volpo 5, Croda), PEG-10 oleyl ether, oleth-10 (Volpo 10, Croda), PEG-20 oleyl ether, oleth-20 (Volpo 20, Croda), PEG-4 lauryl ether, laureth-4 (Brij 30, Atlas/ICI), PEG-9 lauryl ether, PEG-23 lauryl ether, laureth-23 (Brij 35, Atlas/ICI), PEG-2 cetyl ether (Brij 52, ICI), PEG-10 cetyl ether (Brij 56, ICI), PEG-20 cetyl ether (BriJ 58, ICI), PEG-2 stearyl ether (Brij 72, ICI), PEG-10 stearyl ether (Brij 76, ICI), PEG-20 stearyl ether (Brij 78, ICI), and PEG-100 stearyl ether (Brij 700, ICI). Formulations of the combinations according to the invention may include one or more of the polyethylene glycol alkyl ethers above.

Sugar esters may also be used as excipients for the formulation of the combinations described herein. Examples of commercially available sugar esters include: sucrose distearate (SUCRO ESTER 7, Gattefosse), sucrose distearate/monostearate (SUCRO ESTER 11, Gattefosse), sucrose dipalmitate,

sucrose monostearate (Crodesta F-160, Croda), sucrose monopalmitate (SUCRO ESTER 15, Gattefosse), and sucrose monolaurate (Saccharose monolaurate 1695, Mitsubisbi-Kasei). Formulations of the combinations according to the invention may include one or more of the sugar esters above.

Polyethylene glycol alkyl phenols are also useful as excipients for the formulation of the combinations described herein. Examples of commercially available polyethylene glycol alkyl phenols include: PEG-10-100 nonylphenol series (Triton X series, Rohm & Haas) and PEG-15-100 octylphenol ether series (Triton N-series, Rohm & Haas). Formulations of the combinations to the invention may include one or more of the polyethylene glycol alkyl phenols above.

Polyoxyethylene-polyoxypropylene block copolymers may also be used as excipients for the formulation of the combinations described herein. These surfactants are available under various trade names, including one or more of Synperonic PE series (ICI), Pluronic series (BASF), Lutrol (BASF), Supronic, Monolan, Pluracare, and Plurodac. The generic term for these copolymers is"poloxamer" (CAS 9003-11-6). These polymers have the formula shown below: HO (C2H40), (C3H60) b (C2H40), H where"a"and"b"denote the number of polyoxyethylene and polyoxypropylene units, respectively. These copolymers are available in molecular weights ranging from 1000 to 15000 daltons, and with ethylene oxide/propylene oxide ratios between 0.1 and 0.8 by weight. Formulations of the combinations according to the invention may include one or more of the polyoxyethylene-polyoxypropylene block copolymers above.

Polyoxyethylenes, such as PEG 300, PEG 400, and PEG 600, may be used as excipients for the formulation of the combinations described herein.

Sorbitan fatty acid esters may also be used as excipients for the formulation of the combinations described herein. Examples of commercially sorbitan fatty acid esters include: sorbitan monolaurate (Span-20, Atlas/ICI), sorbitan monopalmitate (Span-40, Atlas/ICI), sorbitan monooleate (Span-80, Atlas/ICI), sorbitan monostearate (Span-60, Atlas/ICI), sorbitan trioleate (Span-85, Atlas/ICI), sorbitan sesquioleate (Arlacel-C, ICI), sorbitan tristearate (Span-65, Atlas/ICI), sorbitan monoisostearate (Crill 6, Croda), and sorbitan sesquistearate (Nikkol SS-15, Nikko). Formulations of the combinations according to the invention may include one or more of the sorbitan fatty acid esters above.

Esters of lower alcohols (C2 to C4) and fatty acids (C8 to C18) are suitable surfactants for use in the invention. Examples of these surfactants include: ethyl oleate (Crodamol EO, Croda), isopropyl myristate (Crodamol IPM, Croda), isopropyl palmitate (Crodamol IPP, Croda), ethyl linoleate (Nikkol VF-E, Nikko), and isopropyl linoleate (Nikkol VF-IP, Nikko).

Formulations of the combinations according to the invention may include one or more of the lower alcohol fatty acid esters above.

In addition, ionic surfactants may be used as excipients for the formulation of the combinations described herein. Examples of useful ionic surfactants include: sodium caproate, sodium caprylate, sodium caprate, sodium laurate, sodium myristate, sodium myristolate, sodium palmitate, sodium palmitoleate, sodium oleate, sodium ricinoleate, sodium linoleate, sodium linolenate, sodium stearate, sodium lauryl sulfate (dodecyl), sodium tetradecyl sulfate, sodium lauryl sarcosinate, sodium dioctyl sulfosuccinate, sodium cholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, sodium taurodeoxycholate, sodium glycodeoxycholate, sodium ursodeoxycholate, sodium chenodeoxycholate, sodium taurochenodeoxycholate, sodium glyco cheno deoxycholate, sodium

cholylsarcosinate, sodium N-methyl taurocholate, egg yolk phosphatides, hydrogenated soy lecithin, dimyristoyl lecithin, lecithin, hydroxylated lecithin, lysophosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylcholine, phosphatidyl ethanolamine, phosphatidic acid, phosphatidyl glycerol, phosphatidyl serine, diethanolamine, phospholipids, polyoxyethylene-10 oleyl ether phosphate, esterification products of fatty alcohols or fatty alcohol ethoxylates, with phosphoric acid or anhydride, ether carboxylates (by oxidation of terminal OH group of, fatty alcohol ethoxylates), succinylated monoglycerides, sodium stearyl fumarate, stearoyl propylene glycol hydrogen succinate, mono/diacetylated tartaric acid esters of mono-and diglycerides, citric acid esters of mono-, diglycerides, glyceryl-lacto esters of fatty acids, acyl lactylates, lactylic esters of fatty acids, sodium stearoyl-2-lactylate, sodium stearoyl lactylate, alginate salts, propylene glycol alginate, ethoxylated alkyl sulfates, alkyl benzene sulfones, a-olefin sulfonates, acyl isethionates, acyl taurates, alkyl glyceryl ether sulfonates, sodium octyl sulfosuccinate, sodium undecylenamideo-MEA-sulfosuccinate, hexadecyl triammonium bromide, decyl trimethyl ammonium bromide, cetyl trimethyl ammonium bromide, dodecyl ammonium chloride, alkyl benzyldimethylammonium salts, diisobutyl phenoxyethoxydimethyl benzylammonium salts, alkylpyridinium salts, betaines (trialkylglycine), lauryl betaine (N-lauryl, N, N-dimethylglycine), and ethoxylated amines (polyoxyethylene-15 coconut amine). For simplicity, typical counterions are provided above. It will be appreciated by one skilled in the art, however, that any bioacceptable counterion may be used. For example, although the fatty acids are shown as sodium salts, other cation counterions can also be used, such as, for example, alkali metal cations or ammonium.

Formulations of the combinations according to the invention may include one or more of the ionic surfactants above.

The excipients present in the formulations of the invention are present in amounts such that the carrier forms an aqueous dispersion of the antifungal agent or manganese compound (e. g., terbinafine or manganese sulfate),

or the combination thereof, sequestered within the liposome. The relative amount of a surface active excipient necessary for the preparation of liposomal or solid lipid nanoparticulate formulations is determined using known methodology. For example, liposomes may be prepared by a variety of techniques. Multilamellar vesicles (MLVs) can be formed by simple lipid-film hydration techniques. In this procedure, a mixture of liposome-forming lipids of the type detailed above dissolved in a suitable organic solvent is evaporated in a vessel to form a thin film, which is then covered by an aqueous medium.

The lipid film hydrates to form MLVs, typically with sizes between about 0.1 to 10 microns.

Other established liposomal formulation techniques can be applied as needed. For example, the use of liposomes to facilitate cellular uptake is described in U. S. Patent Nos. 4,897, 355 and 4,394, 448.

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., sodium 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.

The following examples are to illustrate the invention. They are not meant to limit the invention in any way.

Examples Using the methods described below, we tested the ability of combinations of various concentrations of terbinafine and manganese sulfate to inhibit the proliferation of different strains of C. glabrata using reduction of Alamar Blue as an indicator of cell number. The data are depicted in Tables 3- 8 as the percent inhibition, compared to untreated controls.

Table 3 % Inhibition of C. glabrata ATCC #32312 Manganese Sulfate (M) 0 0. 0023 0. 0046 0. 0092 0. 018 0. 037 0. 074 0. 15 0. 3 0-1. 28 0. 53-4. 92-3. 47-5. 73-2. 36 19. 6 26. 1 63. 9 0. 12 0. 66-3. 97-5. 19-5. 24-7. 82-6. 02-3. 82 28. 7 61. 9 0. 24 4. 78-1. 05-5. 65-0. 77-4. 34 0. 37 18. 1 56. 0 52. 6 0. 48-5. 56-7. 05-1. 37-1. 62-4. 14 6. 07 15. 4 43. 5 73. 5 0. 95-4. 88-5. 40-7. 84-4. 55-2. 32 3. 76 19. 9 34. 8 77. 0 1. 9-2. 76-6. 79-0. 05-2. 84 0. 24 13. 0 51. 5 75. 2 92. 4 EH 3. 8 13. 1 12. 7 22. 9 23. 3 44. 2 58. 2 80. 1 91. 3 95. 3 7. 6 18. 6 55. 4 81. 9 59. 0 78. 0 80. 4 91. 3 95. 5 96. 2 15 49. 9 62. 2 66. 5 73. 0 83. 5 91. 8 93. 2 95. 7 96. 4 Table 4 % Inhibition of C. glabrata ATCC #48435 Manganese Sulfate 0 0. 0023 0. 0046 0. 0092 0. 018 0. 037 0. 074 0. 15 0. 3 0 0. 11-0. 17-2. 32-2. 61-4. 93 4. 26 13. 4 29. 3 67. 4 : L 0. 12-1. 17-3. 52-1. 26-2. 03-1. 16-1. 81 17. 9 35. 5 55. 1 0. 24-0. 71-2. 58-2. 94 1. 24 4. 42-1. 56 4. 45 37. 5 51. 7 0. 48-3. 71-1. 82-0. 98 2. 30-1. 04 0. 10 11. 3 33. 6 57. 1 0. 95-2. 05-4. 78-2. 34-1. 12-1. 62 2. 46 25. 1 38. 4 44. 5 Cd 1. 9-4. 10-2. 59-2. 30-3. 08 5. 48 20. 6 41. 2 66. 8 87. 2 3. 8 6. 98 6. 41 5. 70 6. 00 39. 9 34. 1 63. 0 70. 9 91. 3 7. 6 7. 43 16. 1 3. 49 16. 7 37. 2. 45. 9 61. 4 87. 5 91. 6 15 30. 1 23. 5 41. 0 63. 2 48. 4 79. 1 81. 7 91. 0 94. 1

Table 5 Inhibition of C. labrata ATCC #38326 Manganese Sulfate ( 0 0. 0023 0. 0046 0. 0092 0. 018 0. 037 0. 074 0. 15 0. 3 0-6. 42-0. 79-7. 49-10. 4-10. 1-8. 56 43. 8 67. 5 86. 7 0. 12-8. 13-9. 24-10. 7-6, 00-11. 6 20. 9 48. 0 65. 1 88. 3 0. 24-6. 23-5. 72-9. 98 28. 4-11. 0-11. 5 22. 7 71. 0 88. 2 0. 48-10. 5-12. 4-5. 69-7. 60-9. 80-6. 46 32. 4 72. 8 87. 8 0. 95-11. 7-14. 3-13. 9-10. 0-9. 33 20. 1 56. 7 62. 5 88. 3 b 1. 9-10. 6-10. 5-13. 9-11. 6-9. 91-12. 1 51. 8 67. 5 86. 9 . 8-10. 9-11. 2-14. 2-15. 9-2. 18 16. 5 43. 5 77. 5 93. 4 7. 6-14. 4-15. 5-15. 7 3. 42 49. 1 56. 4 83. 9 83. 9 93. 6 15 33. 1 0. 51 39. 7 48. 0 52. 6 68. 5 88. 8 93. 7 96. 1 Table 6 % Inhibition of C. labrata ATCC #90876 Manganese Sulfate 0 0. 0023 0. 0046 0. 0092 0. 018 0. 037 0. 074 0. 15 0. 3 0 0. 74-6. 42-5. 93-4. 96-5. 63 6. 74 45. 8 75. 4 42. 7 0. 12-2. 67-4. 72-5. 55 2. 88-4. 50 12. 0 36. 8 74. 8 89. 8 0. 24-3. 97-4. 96-2. 94 2. 01 0. 69 14. 1 28. 0 75. 7 87. 2 0. 48-4. 12-3. 12-0. 93 1. 08 5. 24 26. 1 46. 4 77. 6 87. 4 0. 95 1. 57-5. 37-1. 13 4. 43 7. 04 34. 9 56. 7 80. 2 45. 2 b 1. 9 16. 6 23. 4 31. 1 47. 3 54. 0 58. 8 81. 9 89. 4 95. 8 3. 8 60. 4 64. 7 70. 8 73. 3 79. 7 88. 2 92. 4 95. 2 92. 8 7. 6 79. 2 82. 1 81. 2 85. 7 90. 4 91. 0 95. 2 96. 4 91. 8 15 86. 8 88. 4 89. 1 91. 9 92. 3 94. 2 96. 1 96. 2 92. 7 Table 7 % Inhibition of C. elabrata ATCC #32554 v Manganese Sulfate ( 0 0. 0023 0. 0046 0. 0092 0. 018 0. 037 0. 074 0. 15 0. 3 0-4. 82 2. 69 0. 55 30. 2-0. 05 53. 2 67. 4 84. 3 50. 0 0. 12 13. 8 13. 6 12. 5 31. 5 42. 0 54. 1 73. 4 85. 8 93. 1 0. 24 12. 2 12. 2 27. 2 20. 4 39. 4 66. 2 71. 0 84. 4 93. 2 0. 48 13. 6 31. 9 29. 8 49. 7 50. 5 67. 1 80. 4 88. 6 94. 4 0. 95 40. 1 36. 0 45. 7 51. 8 59. 8 71. 7 83. 4 91. 1 64. 3 1. 9 61. 0 61. 6 61. 5 76. 8 77. 5 84. 5 91. 8 94. 9 95. 9 . 8 76. 7 79. 8 82. 1 86. 1 88. 8 91. 8 95. 2 95. 9 94. 6 7. 6 86. 5 89. 2 90. 9 92. 0 93. 5 94. 2 95. 8 96. 0 93. 8 15 91. 2 91. 2 91. 3 92. 9 94. 1 95. 6 96. 0 96. 2 95. 3 Table 8 Table 6 % Inhibition of A. fumigatus ATCC #46645 Manganese SuIfate ( 0. 00 0. 18 0. 37 0. 74 1. 5 3 6 12 24 0. 00 0. 90 2. 10-0. 40-20. 0-3. 15-8. 65-2. 20 3. 40-2. 75 0. 48 27. 1 20. 1 25. 1 55. 5 14. 6 51. 4 12. 5 51. 6 56. 2 0. 95 45. 1 55. 8 47. 8 50. 0 45. 3 53. 6 57. 4 50. 1 52. 4 1. 9 79. 8 82. 5 75. 0 73. 2 75. 3 75. 7 70. 2 75. 2 72. 6 3. 8 72. 7 70. 2 64. 0 69. 4 62. 2 71. 0 69. 3 69. 6 66. 5 ; a 7. 6 81. 9 81. 7 78. 2 64. 3 74. 3 76. 5 76. 2 78. 5 74. 7 15 74. 5 71. 2 66. 4 68. 0 69. 5 72. 0 70. 2 68. 0 69. 6 30 85. 3 84. 2 77. 1 75. 8 72. 9 75. 8 80. 6 79. 2 79. 2 61 78. 7 78. 8 69. 4 67. 4 69. 2 72. 7 74. 5 73. 7 71. 9

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

Fungal Strains Clinical Candida glabrata ATCC Number 32312 isolate was obtained from the culture collection of the Seattle Biomedical Research Institute (Seattle, WA). Additional fungal strains were obtained from the American Type Culture Collection (C. glabrata : ATCC Numbers; 48435; 38326; 90876; and 32554). Yeast strains were stored in 15% glycerol at-70°C. Isolates were cultured in growth medium (RPMI; 2% glucose w/o Nacra an phenol red, buffered with 0.165M MOPS to pH 7.0) at 35°C for 24 hours prior to in vitro susceptibility testing.

Compounds and Compound Preparation The following compounds were used: terbinafine (Sequoia Research Products, LTD, United Kingdom), manganese sulfate (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 terbinafine (0-7 pL) and 10X manganese sulfate (0-7 pL) were then plated in 45 ! 1L 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. Final concentration ranges were 0- 15 p. g/mL terbinafine and 0-0.3 p. g/mL manganese sulfate.

Susceptibility All fungal 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 103 cells/mL and a final concentration of 11% 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 cell number.

After the incubation period, the RFU of each well was determined fluorometrically using a microplate readed (Fusion, Packard Bioscience, Meriden, CT) equipped with a 540 nm excitation filter and a 590 nm emission filter.

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.

All publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication was specifically and individually incorporated by reference.

We claim: