WO2008020830A1

WO2008020830A1 - Method of treating an aspergillus or cryptococcus infection by administering ambruticin vs-3, vs-4, or vs-5

Info

Publication number: WO2008020830A1
Application number: PCT/US2006/031590
Authority: WO
Inventors: Leonard Katz
Original assignee: Kosan Biosciences Incorporated
Priority date: 2006-08-14
Filing date: 2006-08-14
Publication date: 2008-02-21

Abstract

A method of treating or reducing the probability of an Aspergillus or Cryptococcus infection in a subject in need of such treatment or reduction of probability, comprising administering to the subject a pharmaceutical composition comprising ambruticin VS-3, VS-4, or VS-5.

Description

METHOD OF TREATING AN ASPERGILLUS OR CRYPTOCOCCUS INFECTION BY ADMINISTERING AMBRUTICIN VS-3, VS-4, OR VS-5

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION

[0001] This invention relates to the treating of diseases and conditions caused by a Aspergillus spp. or Cryptococcus spp. infection by administering compounds having antifungal activity.

2. DESCRIPTION OF RELATED ART

[0002] Aspergillosis refers to a spectrum of disease conditions caused by the infection of a fungus of the genus Aspergillus. Aspergillus is ubiquitous in nature. Infection is initiated by the inhalation of Aspergillus spores or mycelial fragments. Allergic bronchopulmonary aspergillosis is an infection of the bronchial tree by an Aspergillus spp. Invasive aspergillosis usually occurs in immunocompromised patients. Aspergollosis can also occur with non-invasive colonization of exposed tissue, such as the pulmonary canal, external ear canal, or cornea. The most common pathogenic species is Aspergillus fumigatus.

[0003] Current treatment for aspergillosis involves administering voriconazole, amphotericin B (deoxycholate and lipid preparations), and itraconazole (Denning et al., Rev. Infέct. Dis. 12:1147-1201 (1990), Herbrecht et al, 41st Intersci. Conf. Antimicrob. Agents Chemother., Abstract No. J-680 (2001), Stevens et al, Clin. Infect. Dis. 30:696- 709 (2000)). Other new investigational drugs include posaconazole, ravuconazole, caspofungin, FK463, and anidulafungin (Maertens et al., 41st Intersci. Conf. Antimicrob. Agents Chemother, Abstract No. 1103 (2000)).

[0004] Cryptococcosis is caused by the infection with an encapsulated yeast of the genus Cryptococcus. Cryptococcus is found in nature in the soil and in avian feces. Infection can be initiated by inhalation of the yeasts. The organisms can metastasize from the lungs to nearly any organ in the body, where invasive of the central nervous system appears most common. The most common pathogenic species is Cryptococcus neoformans.

[0005] Current treatment for cryptococcosis involves administering fluconazole, itraconazole or amphotericin B (Saag et al., Clin. Infect. Dis. 30:710-718 (2000)). [0006] Ambruticin S (also referred to as Acid S, W 7783, (5S, άR)-5,6-dihydroxypoly- angioic acid, or, sometimes, simply as ambruticin) is an antifungal compound isolated from cultures of Polyangium cellulosum var. fulvum and has the structure shown below. See Strandtmann et al, US 3,804,948 (1974); Barnes et al, Tetrahedron Letters 22 (18), 1751-1754 (1981); Kende et al, J. Am. Chem. Soc. 112 (26), 9645-9646 (1990).

Ambruticin S

[0007] Subsequently, another research group isolated from cultures of Sorangium cellulosum strain Se celO a series of structurally closely related compounds having at C5 an amino group instead of a hydroxyl group. Bedorf et al, WO 91/00860 (1991); Hδfle et al., Liebigs Ann. Chem. 1991, 941-945. These compounds include ambruticin VS-3 (or (5)S,(5i?)-5-(dimethylamino)-6-hydroxypolyangioic acid), VS-4 (or (5S,6R)-5- (methylamino)-6-hydroxypolyangioic acid), and VS-5 (or (56'₎5i?)-5-(amino)-6- hydroxypolyangioic acid), and have the structures shown below.

[0008] Other disclosures relating to the chemistry or mechanism of action of these and other ambruticin analogs include: Connor et al, US 3,932,620 (1976); Connor et al, US 3,932,621 (1976); Connor et al, US 4,001,398 (1977); Connor et al, US 4,009,261 (1977); Connor et al, US 4,016,257 (1977); Connor et al, US 4,098,998 (1978); Connor et al, US 4,107,429 (1978); Connor et al, US 4,138,550 (1979); Connor et al, US 4,191,825 (1979); Connor et al, US RE 30,339 (1980); Connor e al, DE 2,659,575 (1978) (Chem. Abs. 89:109030); Connor et al, J. Med. Chem. 22 (9), 1055-1059 (1979); Connor et al, J. Med. Chem. 22 (9), 1144-1147 (1979); Knauth et al, J. Antibiotics 53 (10), 1182-1190 (2000); and, US Patent Application Ser. No. 11/305,802, filed Dec. 16, 2005.

[0009] It is disclosed that ambruticin S has in vitro antifungal activity against Aspergillus flavus (Ringel, J. Antibiotics 30:371-375 (1977)) and Aspergillus fumigatus (Ringel, Recent Trends in the Discovery, Development and Evaluation of Antifungal Agents R.A. Fromtling (Ed.), J.R. Prous Publishers, S.A. (1987); Shadomy et ah, Antimicrob. Ag. Chemothera. 14:99-104). It is also disclosed that ainbruticin S has in vitro antifungal activity against Cryptococcus neoformans (Ringel (1977)). Further, it is disclosed that ambruticin S has a low animal toxicity (Ringel, Antimicrob. Agents Chemother. 13:762-769 91978); Levine et al Chest 73:202-206 (1978)).

[0010] The disclosures of the foregoing documents and the other documents cited in this BACKGROUND OF THE INVENTION section are incorporated herein by reference.

BRIEF SUMMARY OF THE INVENTION

[0011] This invention provides for a method of treating or reducing the probability of an. Aspergillus or Cryptococcus infection in a subject in need of such treatment or reduction of probability, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of ambruticin VS-3, VS-4, or VS-5 and optionally a pharmaceutically acceptable carrier. This invention also provides for the use of ambruticin VS-3, VS-4, or VS-5 for the preparation of a medicament for treating an Aspergillus or Cryptococcus infection.

[0012] This invention also provides for the method such that the growth of an Aspergillus or Cryptococcus cell is inhibited. The inhibition of the Aspergillus or Cryptococcus cell includes the reduction in the growth of the Aspergillus or Cryptococcus cell. The reduction of growth includes one or more of the following: a decrease in the growth of the Aspergillus or Cryptococcus cell, a decrease in the rate of cell division of the Aspergillus or Cryptococcus cell, and the killing of the Aspergillus or Cryptococcus cell. This invention also provides for the method such that the subject is cleared of the Aspergillus or Cryptococcus infection, or is relieved of a symptom caused by the Aspergillus or Cryptococcus infection. This invention also provides for the method such that the subject, who but for the administering of the pharmaceutical composition to the subject avoids an Aspergillus or Cryptococcus infection.

[0013] In one embodiment, the subject in need of such treatment or reduction of probability of the Aspergillus or Cryptococcus infection is one who has a high chance of acquiring thoAspergillus or Cryptococcus infection. Such subjects include healthy or immunocompromised individuals. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0014] "Pharmaceutically acceptable ester" means an ester that hydrolyzes in vivo (for example in the human body) to produce the parent compound or a salt thereof or has per se activity similar to that of the parent compound. Suitable ester groups include, without limitation, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety preferably has no more than six carbon atoms. Illustrative esters include formates, acetates, propionates, butyrates, acrylates, citrates, succinates, and ethylsuccinates.

[0015] "Pharmaceutically acceptable salt" means a salt of a compound suitable for the pharmaceutical formulation. Where a compound has one or more basic functionalities, the salt can be an acid addition salt, such as a sulfate, hydrobromide, tartrate, mesylate, maleate, citrate, phosphate, acetate, pamoate (embonate), hydroiodide, nitrate, hydrochloride, lactate, methylsulfate, fumarate, benzoate, succinate, mesylate, lactobionate, suberate, tosylate, and the like. Where a compound has one or more acidic moieties, the salt can be a salt such as a calcium salt, potassium salt, magnesium salt, meglumine salt, ammonium salt, zinc salt, piperazine salt, tromethamine salt, lithium salt, choline salt, diethylamine salt, 4-phenylcyclohexylamine salt, benzathine salt, sodium salt, tetramethylammonium salt, and the like.

[0016] "Therapeutically effective amount" means that amount of ambruticin VS-3, VS-4, or VS-5 that elicit the biological or medicinal response in a tissue system, animal or human sought by a researcher, veterinarian, medical doctor or other clinician, which response includes alleviation of the symptoms of the disease or disorder being treated. The specific amount of ambruticin VS-3, VS-4, or VS-5 needed to elicit the biological or medicinal response will depend on a number of factors, including but not limited to the disease or disorder being treated, the ambruticin VS-3, VS-4, or VS-5 being administered, the method of administration, and the condition of the patient.

Ambruticin VS-3, VS-4 and VS-5

[0017] The present invention includes within its scope prodrugs of ambruticin VS-3, VS-4, or VS-5. Such prodrugs are in general functional derivatives of ambruticin VS-3, VS-4, or VS-5 that are readily convertible in vivo into the required compound. Thus, in the methods of treatment of the present invention, the term "administering" shall encompass the treatment of the various disorders described with the compound specifically disclosed or with a compound which may not be specifically disclosed, but which converts to the specified compound in vivo after administration to a subject in need thereof. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Wermuth, "Designing Prodrugs and Bioprecursors," in Wermuth, ed., The Practice of Medicinal Chemistry, 2nd Ed., pp. 561- 586 (Academic Press 2003). Prodrugs include esters that hydrolyze in vivo (for example in the human body) to produce a compound of this invention or a salt thereof. Suitable ester groups include, without limitation, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety preferably has no more than six carbon atoms. Illustrative esters include formates, acetates, propionates, butyrates, acrylates, citrates, succinates, and ethylsuccinates.

[0018] The present invention also includes within its scope pharmaceutically acceptable salts of ambruticin VS-3, VS-4, or VS-5.

Aspergillus infection

[0019] The Aspergillus infection is caused by a cell of the genus Aspergillus. Preferably, the fungus is Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus., Aspergillus clavatus, Aspergillus glaucus, or Aspergillus versicolor. Aspergillus fumigatus strains include strain ATCC 204305. Aspergillus flavus strains include strain ATCC 204304. In one embodiment, the cell of the genus Aspergillus is resistant to one or more antibiotics, wherein none is an ambruticin. Such antibiotics include, but are not limited, voriconazole, amphotericin B (deoxycholate and lipid preparations), itraconazole posaconazole, ravuconazole, caspofungin, FK463, and anidulafungin (LY303366).

[0020] In one embodiment, the Aspergillus infection is aspergillosis. In one embodiment, the aspergillosis is allergic bronchopulmonary aspergillosis, pulmonary aspergilloma, or invasive aspergillosis. In one embodiment, the site of the allergic bronchopulmonary aspergillosis is in one or more of the following: sinuses and lungs. In one embodiment, the site of the pulmonary aspergilloma is in a lung cavity. In one embodiment, the invasive aspergillosis is one or more of the following infections: pulmonary aspergillosis, central nervous system (CNS) aspergillosis, sinonasal aspergillosis, osteomyelitis, endophthalmitis, endocarditis, renal abscess, and cutaneous infection. In one embodiment, the Aspergillus infection is cutaneous (resulting from a trauma, such as a burn, a post-surgical wound, or a intravenous insertion site), otomycosis, exogenous endophthalmitis, allergic fungal sinusitis, or a urinary infection.

[0021] In one embodiment, the site of Aspergillus infection is in the subject's respiratory system. In one embodiment, the site of Aspergillus infection is in the subject's lungs. In another embodiment, the site of Aspergillus infection is in the subject's gastrointestinal tract, brain, liver, kidney, heart, skin, and/or eye.

[0022] In one embodiment, the aspergillosis that is an allergic form of aspergillosis, non-invasive colonization aspergillosis, or invasive aspergillosis, hi one embodiment, the allergic form of aspergillosis is asthma, allergic bronchopulmonary aspergillosis, or extrinsic allergic alveolitis, hi one embodiment, non-invasive colonization aspergillosis is aspergilloma or a non-pulmonary local infection, hi one embodiment, invasive aspergillosis is pulmonary or disseminated.

Cryptococcus infection

[0023] The Cryptococcus infection is caused by a cell of the genus Cryptococcus. Preferably, the cell is Cryptococcus neoformans, Cryptococcus albidus var. albidus, Cryptococcus albidus var. diffluens, Cryptococcus luteolus, Cryptococcus laurentii, Cryptococcus uniguttulatus, Cryptococcus terreus, or Cryptococcus gastricus. More preferably, Cryptococcus neoformans is Cryptococcus neoformans var. neoformans, Cryptococcus neoformans var. gattii, or Cryptococcus neoformans var. grubii. hi one embodiment, the Cryptococcus neoformans is strain 97-14, 11239, or 11240. hi one embodiment, the cell of the genus Cryptococcus is resistant to one or more antibiotics, wherein none is an ambruticin. Such antibiotics include, but are not limited to, fluconazole, amphotericin B (deoxycholate and lipid preparations), itraconazole, and 5- flurocytosine.

[0024] In one embodiment, the Cryptococcus infection is cryptococcosis, hi one embodiment, the cryptococcosis is localized or disseminated, hi one embodiment, the localized cryptococcosis is a pulmonary cryptococcosis. The pulmonary cryptococcosis is an acute infection or is chronic. The disseminated cryptococcosis is acute or chronic. In one embodiment, the cryptococcosis is cryptococcal meningitis. The site of the Cryp- tococcus infection can be in the CNS, or in the respiratory system, such as in the lungs.

Patients Diagnosed with an Aspergillus or Cryptococcus infection and Immunocompromised Patients

[0025] hi another embodiment, the subject is one who is diagnosed with an Aspergillus or Cryptococcus infection, and/or is immunocompromised. Examples of immunocompromised subjects include, but are not limited to, patients infected with Human Immunodeficiency Virus, organ transplant recipients, patients undergoing chemotherapy (such as cancer patients), patients undergoing corticosteroids therapy, cancer patients, patients with diabetes mellitus, chronic obstructive pulmonary disease, cirrhosis, rheumatoid arthritis, or systemic lupus erythematous, pregnant women, and patients having undergone a splenectomy. Cancer patients include, but are not limited to, patients afflicted with chronic lymphatic leukemia, Hodgkin's disease, chronic myelogenous leukemia, myeloma, lymphosarcoma, acute lymphoblastic leukemia, or lung cancer.

Modes of Administration and Pharmaceutical Formulations

[0026] Suitable modes of administration of the pharmaceutical composition include, but are not limited to, oral, topical, aerosol, inhalation by spray, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration. The term parenteral, as used herein, includes subcutaneous injections, and intravenous, intrathecal, intramuscular, and intrasternal injection or infusion techniques. A preferred mode of administration is one that brings ambruticin VS-3, VS-4, or VS-5 to the actual or potential site(s) of infection in the subject. The pharmaceutical composition can be in a solid, semi-solid, or liquid form.

[0027] The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well known to those who are skilled in the art and are readily available. Preferably, the carrier is chemically inert to ambruticin VS-3, VS-4, or VS-5 and has no detrimental side effects or toxicity under the conditions of use. Preferably, the pharmaceutically acceptable carrier is free of pyrogen. The pharmaceutically acceptable carriers which can be used include, but are not limited to, water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, and urea. [0028] Arnbruticin VS-3, VS-4, or VS-5 may be combined with the pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the subject treated and the particular mode of administration. Suitable dosage levels of ambruticm VS-3, VS-4, or VS-5 are of the order from about 0.01 mg to about 100 mg per kg body weight per day, preferably from about 0.1 mg to about 50 mg per kg body weight per day. Dosage unit forms will generally contain from about 0.1 mg to about 500 mg of ambruticin VS-3, VS-4, or VS-5. For external administration, ambruticin VS-3, VS-4, or VS-5 may be formulated within the range of, for example, 0.00001% to 60% by weight, and preferably from 0.001% to 10% by weight. In addition, the pharmaceutical composition can be administered on an intermittent basis, i.e., at twice daily, daily, semi- weekly, or weekly intervals. It will be understood, however, that the specific dose level for a particular subject will depend on a variety of factors. These factors include the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the subject; the time and route of administration and the rate of excretion of the drug; whether a drug combination is employed in the treatment; and, the severity of the particular disease or condition for which therapy is sought.

[0029] The pharmaceutical compositions suitable for oral administration include, but are not limited to, (a) liquid formulations; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, and optionally a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscar- mellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and the like. The tablet can further comprise one or more colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, or flavoring agents.

[0030] The pharmaceutical composition, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants (such as dichlorodifluoromethane, propane, nitrogen, and the like) or non-pressured preparations (such as in a nebulizer or an atomizer). When the site(s) of infection of a subject is the lungs, a preferred mode of administration is inhalation of an aerosol formulation either orally or nasally. Preferably, the aerosol formulation comprises particles of a respirable size, including, but not limited to, mean particle sizes of 5 μm to 500 μm.

[0031] The pharmaceutical composition can be an injectable formulation. The requirements for effective carriers for injectable compositions are well known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company, Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)). Preferably, injectable compositions are administered intravenously. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0032] The pharmaceutical composition can further comprise an excipient. Exci- pients that may be used include one or more carriers, surface active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients is taught in Geraiaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0033] The practice of this invention can be further understood by reference to the following examples, which are provided by way of illustration and not of limitation.

Example 1

[0034] The antifungal activities of ambruticin VS-3, VS-4, and VS-5 against two different Aspergillus species were determined by microdilution methods according to the National Committee for Clinical Laboratory Standards (NCCLS) reference method no. M38-A, Vol.. 22, No. 16, "Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi; Approved Standard", herein incorporated by reference. Results are presented in Table A and include comparative data for ambruticin S, and the structurally unrelated fungicides amphotericin B, itraconazole, and ketoco- nazole. Where multiple assays were performed and a range of values was obtained, the minimum and maximum values are reported. The multiple inhibitory concentration (MIC) is that for which the growth of the fungi is inhibited by 90% to 100%.

Table A — Antifungal Activity

MIC (μg/mL)

Compound

A. βavus ATCC 204304 A. fumigatus ATCC 204305

Amphotericin B 1 — 8 0.5 — 8

Itraconazole 0.0625 — 1 0.0625 — 1

Ketoconazole 2 — 4 0.0625 — 8

Ambruticin S 8 — 32 8 —32

Ambruticin VS-3 0.0625 — 1 0.125 —1

Ambruticin VS-4 0.125 — 1 0.25 — 4

Ambruticin VS-5 0.25 — 2 0.25 — 4

Example 2

[0035] The in vivo susceptibility of Aspergillus fumigatus to ambruticin VS-3, VS-4, and VS-5 is tested according to the method of Petraitis et al., "Comparative Antifungal Activities and Plasma Pharmacokinetics of Micafungin (FK463) against Disseminated Candiasis and invasive Pulmonary Aspergillosis in Persistently Neutropenic Rabbits," Antibicrob. Agents Chemother. 46:1857-1869 (2002)), which is incorporated herein by reference.

[0036] Pulmonary aspergillosis is established in female New Zealand white rabbits (Hazleton Research Products, Inc., Denver PA) as previously described (Francis et ah, J. Infect. Dis. 169:356-368 (1994); Lee et al., Antimicrob. Agents Chemother. 38:713-718 (1994); both herein incorporated by reference). Prior to each experiment, an A. fumigatus inoculum is prepared fresh from a frozen isolate (stored at -70°C), which is then subcultured onto Sabouraud dextrose slants (BBL Microbiology Systems, Cockeysville, MD). These slants are incubated for 24 hours at 37⁰C and then kept at room temperature for 5 days before use. The conidia are harvested under a laminar airflow hood with a solution of 10 niL of 0.025% Tween 20 (Fisher Scientific, Fair Lawn, NJ) in sterile normal saline, transferred to a 50 mL conical tube, washed, and counted with a hemacytometer. The concentration is adjusted in order to give each rabbit an inoculum of 1 X 10⁸ to 1.5 X 10⁸ conidia of A. fumigatus in a volume of 250 to 350 μL. The rabbits are inoculated on day 2 of the experiments while the rabbits are under general anesthesia. On day 2 of the experiment, a Flagg O straight-blade laryngoscope (Welch Allyn Inc., Skaneateles Falls, NY) is inserted into the oral cavity of each rabbit under general anesthesia until the vocal cords are clearly visible. The A. fumigatus inoculum is then administered intratracheally with a tuberculin syringe attached to a 5.25-inch 16-gauge Teflon catheter (Becton Dickinson Infusion Therapy Systems Inc., Sandy, UT).

[0037] To simulate the conditions of persistent neutropenia in invasive pulmonary aspergillosis, therapy with cytarabine is initiated intravenously on day 1 (i.e., before the endotracheal inoculation of the animals.) Profound and persistent neutropenia (a granulocyte concentration of <100 granulocytes/ μL) is achieved by an initial course of 525 mg of cytarabine per m² for 5 consecutive days. A maintenance dose of 484 mg of cytarabine per m² is administered for 4 additional days (days 8, 9, 13, and 14) of the experiment. Concomitant thrombocytopenia is present in a range of 30,000 to 50,000 platelets/μL. Methylprednisolone (5 mg/kg) is administered on days 1 and 2 of the experiment to inhibit macrophage activity against conidia in order to facilitate establishment of invasive pulmonary aspergillosis. All rabbits with invasive pulmonary aspergillosis receive ceftazidime, gentamicin, and vancomycin (according to the protocol of Petraitis et al. (2002) in order to prevention of opportunistic bacterial infections (including Clostridium spiriforme) during neutropenia.

[0038] There are three treatment groups. The first group is the untreated control rabbits. The second group consists of rabbits treated with amphotericin administered by intravenously at 5 mg/kg/day (0.1 mL every 10 seconds). The third group consists of rabbits treated with ambruticin VS-3, VS-4, or VS-5 administered as an intravenous bolus over 4 minutes at dosages of 0.25 mg/kg/day, 0.5 mg/kg/day, 1 mg/kg/day, or 2 mg/kg/day (0.1 mL every 10 seconds). Antifungal therapy is initiated 24 hours after inoculation. Treatment is continued throughout the course of the experiments for a maximum of 12 days.

[0039] The in vivo antifungal efficacy of ambruticin VS-3, VS-4, and VS-5 against experimental invasive pulmonary aspergillosis is assessed by measuring the following: residual fungal burden in lung tissue (CFU/g), pulmonary infarct score, lung weight, and survival. These measurements are performed according to the protocol described in Petraitis et al. (2002). [0040] Based on the in vitro results in Example 1, the rabbits treated with ambruticin VS-3, VS-4, or VS-5 are expected to have a lower mean pulmonary log A. fumigatus CFU/g lung tissue than the rabbits treated with amphotericin B. The rabbits treated with ambruticin VS-3, VS-4, or VS-5 are expected to have a lower mean infarct score than the rabbits treated with amphotericin B. The rabbits treated with ambruticin VS-3, VS-4, or VS-5 are expected to have a lower mean total lung weight than the rabbits treated with amphotericin B. The rabbits treated with ambruticin VS-3, VS-4, or VS-5 are expected to longer post-inoculation survival than the rabbits treated with amphotericin B.

Example 3

[0041] The antifungal activities of ambruticin VS-3, VS-4, and VS-5 against three different Cryptococcus neoformans strains were determined by microdilution methods according to the National Committee for Clinical Laboratory Standards (NCCLS) reference method no. M27-A2, "Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts", herein incorporated by reference. Results are presented in Table B and include comparative data for ambruticin S, and the structurally unrelated fungicides amphotericin B and itraconazole. Where multiple assays were performed and a range of values was obtained, the minimum and maximum values are reported. The MIC is that for which the growth of the fungi is inhibited by 90% to 100%.

Table B — Antifungal Activity

MIC (μg/mL)

Compound C. neoformans C. neoformans C. neoformans

97-14 11239 11240

Amphotericin B 0.25 — 2 0.25 — 2 0.25 — 2

Itraconazole 0.25 0.0625 — 0.25 0.25 — 0.5

Ambruticin S 32 16 >32

Ambruticin VS-3 0.125 — 1 0.0625 — 1 0.125 — 1

Ambruticin VS-4 O.0625 — 0.125 O.0625 — 0.25 O.0625 — 0.25

Ambruticin VS-5 0.125 — 0.5 0.25 — 0.5 0.25 — 0.5

Example 4

[0042] The in vivo susceptibility of C. neoformans to ambruticin VS-3, VS-4, and VS-5 is tested according to the method of Miller et al., "In Vitro and In Vivo Efficacy of the New Triazole Albaconazole against Cryptococcus neoformans " Antimicrob. Agent Chemother. 48:384-387 (2004)), which is incorporated herein by reference.

[0043] C. neoformans is grown at 35⁰C on Sabourand agar plates with chloramphenicol, harvested, and suspended in 0.015 M phosphate-buffered saline at pH 7.4 to a density of 1.0 x 10⁹ CFU/mL. One day prior to the inoculation with C. neoformans, and during the duration of the experiment, the test rabbits (New Zealand White) receive a daily intramuscular injection of hydrocortisone acetate (5.0 mg/kg; Sigma, St. Louis MO). The rabbits are sedated and inoculated intracisternally with 0.3 mL of the C. neoformans inoculum. Treatment regimens of 5 mg/kg/day, 20 mg/kg/day, and 80 mg/kg/day (or any other appropriate amount) of ambruticin, VS-3, VS-4 or VS-5 is administered to the rabbits beginning 2 days after initial inoculation. There are an untreated control and a fluconazole control. Intracisternal taps are performed on the rabbits to aspirate 0.5 mL of cerebrospinal fluid (CSF) on days 4, 8, 12, 16, and 20 days after inoculation. The CSF is serially diluted with the phosphate-buffered saline and cultured on Sabourand agar plates with chloramphenicol to determine the CFU of C. neoformans per mL CSF.

[0044] Based on the in vitro results in Example 3, ambruticin VS-3, VS-4, and VS-5 are expected to show in vivo efficacy against C. neoformans.

[0045] The foregoing detailed description of the invention includes passages that are chiefly or exclusively concerned with particular parts or aspects of the invention. It is to be understood that this is for clarity and convenience, that a particular feature may be relevant in more than just the passage in which it is disclosed, and that the disclosure herein includes all the appropriate combinations of information found in the different passages. Similarly, although the various figures and descriptions herein relate to specific embodiments of the invention, it is to be understood that where a specific feature is disclosed in the context of a particular figure or embodiment, such feature can also be used, to the extent appropriate, in the context of another figure or embodiment, in combination with another feature, or in the invention in general.

[0046] Further, while the present invention has been particularly described in terms of certain preferred embodiments, the invention is not limited to such preferred embodiments. Rather, the scope of the invention is defined by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating or reducing the probability of an Aspergillus infection in a subject in need of such treatment or reduction of probability, comprising administering to the subject a pharmaceutical composition comprising ambruticin VS-3, VS-4, or VS-5, and optionally a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the Aspergillus infection is aspergillosis.

3. The method of claim 2, wherein said aspergillosis is invasive aspergillosis.

4. The method of claim 1 , wherein the pharmaceutical composition comprises ambruticin VS-3.

5. The method of claim 1 , wherein the pharmaceutical composition comprises ambruticin VS-4.

6. The method of claim 1 , wherein the pharmaceutical composition comprises ambruticin VS-5.

7. The method of claim 1 , wherein the subject is immunocompromised.

8. The method of claim 1, such that said administering results in the inhibition of the growth of an Aspergillus cell in the subject.

9. The method of claim 8, where said Aspergillus cell is Aspergillus fumigatus or Aspergillus flavus.

10. The use of ambruticin VS-3, VS-4, or VS-5 for the preparation of a medicament for treating or reducing the probability of an Aspergillus infection.

11. A method of treating or reducing the probability of a Cryptococcus infection in a subject in need of such treatment or reduction of probability, comprising administering to the subject a pharmaceutical composition comprising ambruticin VS-3, VS-4, or VS-5, and optionally a pharmaceutically acceptable carrier.

12. The method of claim 11 , wherein the Cryptococcus infection is cryptococcosis.

13- The method of claim 12, wherein said cryptococcosis is pulmonary cryptococcosis, disseminated cryptococcosis, or cryptococcal meningitis.

14. The method of claim 11 , wherein the pharmaceutical composition comprises ambruticin VS-3.

15. The method of claim 11 , wherein the pharmaceutical composition comprises ambruticin VS-4.

16. The method of claim 11 , wherein the pharmaceutical composition comprises ambruticin VS-5.

17. The method of claim 11 , wherein the subj ect is immunocompromised.

18. The method of claim 11, such that said administering results in the inhibition of the growth of a Cryptococcus cell in the subject.

19. The method of claim 18, where said Cryptococcus spp. is Cryptococcus neoformans.

20. The use of ambruticin VS-3, VS-4, or VS-5 for the preparation of a medicament for treating or reducing the probability of a Cryptococcus infection.

- 15 -