Rutamycin b and its derivatives for treatment of infections caused by neuropathogens

Abstract

Provided herein are methods for treating or preventing an infection including administering to a subject in need thereof a composition including a therapeutically effective amount of rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof. The method may include administering a therapeutically effective amount of amphotericin B along with rutamycin B to the subject in need thereof.

Description

[0001] Attorney Docket No. 15670-0455WO1

[0002] RUTAMYCIN B AND ITS DERIVATIVES FOR TREATMENT OF INFECTIONS CAUSED BY NEUROPATHOGENS

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Application Serial No.

[0005] 63 / 770,472, filed on March 12, 2025. The entire contents of the foregoing are incorporated herein by reference.

[0006] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with Government support under EY034294 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] The present disclosure relates to methods of treating infections. More specifically, the present disclosure relates to a method of treating infections caused by neuropathogens using rutamycin B and its derivatives.

[0010] BACKGROUND

[0011] Naegleria fowleri (hereinafter “TV. fowlerf") is a free-living amoeba that lives in warm freshwater resources, such as rivers, lakes, canals, and hot springs. It can be present in untreated or undertreated water supplies and swimming pools or spas. N. fowleri trophozoites enter the host from contaminated water through the nasal mucosa to access the central nervous system via the olfactory neuroepithelia. N. fowleri has been reported in water used for nasal cleansing, such as sinus irrigation with neti pots or ablution for religious practices. It causes severe primary amoebic meningoencephalitis (PAM) and disproportionately affects males (>75%) and children (85%, median 12 years old). PAM kills over 97% of infected people. The high mortality rate and lack of effective therapy make PAM a particularly tragic infection for many families. To date, there are only five documented survivors out of 167 known infected individuals in the United States and seven confirmed survivors out of a total of 488 global cases of PAM. N. fowleri infection is not a notifiable disease in the US. Thus, it is possible that the infection is underreported. Although misdiagnoses may also contribute to the underestimation of number of cases and the CDC is working onAttorney Docket No. 15670-0455WO1

[0012] improving the diagnostic methods, identification of safer drugs and a drop-in fatality rate would promote development and implementation of rapid and non-invasive diagnostic practices worldwide. N. fowl er i has been listed by the NIAID as a category B priority7biodefense pathogen. PAM has a rapid clinical course with symptoms usually7starting within 1-9 days (median 5 days) after exposure and death occurring within median 5 days after symptoms begin.

[0013] An optimal treatment regimen for PAM has not been established. The current standard of antimicrobial treatment recommended by the CDC is deoxycholate formulation of amphotericin B intravenously (IV) and intrathecally, an azole drug (posaconazole) IV or by mouth (PO), azithromycin IV or PO, rifampin IV or PO, and miltefosinePO. Each of these drugs presents individual complications, such as potential toxicity7, minimum inhibitory7concentrations (MICs) dependent on isolates, and accessibility. The documented survivors also received management of elevated intracranial pressure using dexamethasone IV to reduce the inflammation of the brain. In some cases, hypothermia was also induced. While 71% of PAM patients worldwide were treated with amphotericin B, only 9% received miltefosine. All confirmed PAM survivors received amphotericin B, but miltefosine has not shown consistent results. Amphotericin B deoxycholate used for the treatment of PAM is dose-limited by nephrotoxicity. Newer lipid-based formulations of amphotericin B are less toxic, but previous studies suggest that the N. fowler i MIC for deoxy cholate amphotericin B is 10 times lo7er than for the liposomal formulation. Despite the use of amphotericin B, it is not FDA-approved for PAM and no more than seven people with a confirmed diagnosis worldwide have been successfully treated with amphotericin B, either alone or in combination with other drugs.

[0014] SUMMARY

[0015] In one aspect of the present disclosure, a method for treating or preventing an infection is provided. The method includes administering to a subject in need thereof a composition comprising a therapeutically effective amount of rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, the infection is an amoeba infection.

[0017] In some embodiments, the infection is an N. fowleri infection.Attorney Docket No. 15670-0455WO1

[0018] In some embodiments, administering includes contacting at least a portion of the rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof with a brain of the subject.

[0019] In some embodiments, administering includes delivering the therapeutically effective amount of the rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof to the subject via intravenous injection, intrathecal injection, intranasal injection, oral administration, or combinations thereof.

[0020] In some embodiments, administering includes delivering the therapeutically effective amount of rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof at a concentration of at least 0.002 pM to the subject.

[0021] In some embodiments, the concentration is from 0.002 pM to 2 pM.

[0022] In some embodiments, administering the therapeutically effective amount of the rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof reduces a concentration of A'. fowleri trophozoites in the subject.

[0023] In some embodiments, administering the therapeutically effective amount of rutamycin B, derivative thereof, or a pharmaceutically acceptable salt thereof to the subject occurs while the therapeutically effective amount of rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present in a pharmaceutically acceptable carrier.

[0024] In some embodiments, the derivative thereof is an ester derivative of rutamycin B.

[0025] In some embodiments, the ester derivative comprises an ester formed at a hydroxyl group of rutamycin B.

[0026] In some embodiments, the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbon atoms.

[0027] In some embodiments, the saturated or unsaturated fatty acid ester is selected from the group consisting of formate, acetate, propionate, butyrate, linoleate, and linolenate.

[0028] In another aspect, a method is provided for treating or preventing an infection. The method includes administering to a subject in need thereof a therapeutically effective amount of rutamycin B and amphotericin B, derivatives thereof, or pharmaceutically acceptable salts thereof.

[0029] In some embodiments, the infection is an amoeba infection.Attorney Docket No. 15670-0455WO1

[0030] In some embodiments, the infection is an N. fowleri infection.

[0031] In some embodiments, administering includes contacting at least a portion of rutamycin B and amphotericin B, the derivatives thereof, or the pharmaceutically acceptable salts thereof with a brain of the subject.

[0032] In some embodiments, administering includes the therapeutically effective amount of the rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof at a concentration of at least 0.002 pM to the subject.

[0033] In some embodiments, a concentration of amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is greater than the concentration of rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof.

[0034] In some embodiments, a ratio of amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof to rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is from 1:8 to 1:32.

[0035] In some embodiments, the derivative thereof of rutamycin B is an ester derivative of rutamycin B.

[0036] In some embodiments, the ester derivative comprises an ester formed at a hydroxyl group of rutamycin B.

[0037] In some embodiments, the ester is a saturated or unsaturated fatty7acid ester having 1 to 22 carbon atoms.

[0038] In some embodiments, the derivative thereof of amphotericin B is an ester derivative of amphotericin B.

[0039] In some embodiments, the ester derivative comprises an ester formed at a hydroxyl group of amphotericin B.

[0040] In some embodiments, the ester is a saturated or unsaturated fatty7acid ester having 1 to 22 carbon atoms.

[0041] In an aspect, a composition for treating or preventing an infection of a subject is provided. The composition includes a therapeutically effective amount of rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable earner, excipient, or a combination thereof.

[0042] In some embodiments, rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present in the composition in an amount of at least 0.002 pM.Attorney Docket No. 15670-0455WO1

[0043] In some embodiments, rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present in the composition in an amount from 0.002 pM to 2 pM.

[0044] In some embodiments, the derivative thereof is an ester derivative of rutamycin B.

[0045] In some embodiments, the ester derivative comprises an ester formed at a hydroxyl group of the rutamycin B.

[0046] In some embodiments, the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbon atoms.

[0047] In some embodiments, the saturated or unsaturated fatty acid ester is selected from the group consisting of formate, acetate, propionate, butyrate, linoleate, and linolenate.

[0048] In some embodiments, the composition includes amphotericin B, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0049] In some embodiments, amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present at a concentration that is greater than a concentration of rutamycin B, the derivative thereof or the pharmaceutically acceptable salt thereof.

[0050] In some embodiments, a ratio of amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof to rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is from 1 :8 to 1 :32.

[0051] In some embodiments, the ester derivative comprises an ester formed at a hydroxyl group of amphotericin B.

[0052] In some embodiments, the ester derivative comprises an ester formed at a hydroxyl group of amphotericin B.

[0053] In some embodiments, the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbon atoms.

[0054] In some embodiments, the saturated or unsaturated fatty acid ester is selected from the group consisting of formate, acetate, propionate, butyrate, linoleate, and linolenate.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the presentAttorney Docket No. 15670-0455WO1

[0056] invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0057] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0058] DESCRIPTION OF DRAWINGS FIG. 1 is a graph illustrating grow th inhibition curves of rutamycin B at different time points against N. fowleri. Different concentrations of compounds w ere tested in triplicate for activity’ against KUL trophozoites (ATCC 30808). ECso curves were generated from mean values of percentage growth inhibition ± SEM of rutamycin B against N. fowleri. Negative control contained 0.5% DMSO and positive control contained 50 pM amphotericin B.

[0059] FIG. 2 is a graph illustrating growth inhibition curves of amphotericin B at different time points against N. fowleri. Different concentrations of compounds were tested in triplicate for activity against KUL trophozoites. ECso curves w ere generated from mean values of percentage growth inhibition ± SEM of amphotericin B against N. fowleri. Negative control contained 0.5% DMSO and positive control contained 50 pM amphotericin B.

[0060] FIG. 3 is a graph illustrating accumulation of intracellular ROS in N. fowleri after exposure to rutamycin B. Relative fluorescence unit (RFU) of N. fowleri detecting ROS within trophozoites following 2 h treatment with 0.78, 0.39, and 0.2 pM of rutamycin B and amphotericin B. Control trophozoites were treated with 1% DMSO alone. *P < 0.05 by Student’s t test compared to DMSO-treated N. fowleri.

[0061] FIG. 4 is a graph illustrating the effect of rutamycin B on N. fowleri mitochondrial FiFo ATP synthase activity7. Amphotericin B (50 pM) and different concentrations of rutamycin B w ere tested against isolated mitochondria of N. fowleri strain KUL. ATP synthase activity was measured following decrease in absorbance at 340 nm over time. 2% (v / v) DMSO was used as a solvent control. *P < 0.05, ****P < O.0001 by Student’s t test compared to DMSO-treated A. fowleri.

[0062] FIG. 5 is a graph illustrating a synergistic effect of rutamycin B and amphotericin B, as analyzed by CompuSyn. Isobolograms indicate effective doses forAttorney Docket No. 15670-0455WO1

[0063] each drug to induce 50% (Fa = 0.5), 75% (Fa = 0.75), and 90% (Fa = 0.9) growth inhibition of N. fowleri trophozoites at a combination of 8: 1 ratio of rutamycin B (Dose A) and amphotericin B (Dose B). Synergism is indicated by the pairing of dosing of each drug plotted as a point (symbol) below the respective Fa isobole or line.

[0064] FIG. 6 is a graph illustrating a synergistic effect of rutamycin B and amphotericin B, as analyzed by CompuSyn. Isobolograms indicate effective doses for each drug to induce 50% (Fa = 0.5), 75% (Fa = 0.75), and 90% (Fa = 0.9) growth inhibition of N. fowleri trophozoites at a combination of 32: 1 ratio of rutamycin B (Dose A) and amphotericin B (Dose B). Synergism is indicated by the pairing of dosing of each drug plotted as a point (symbol) below the respective Fa isobole or line.

[0065] DETAILED DESCRIPTION

[0066] Definitions

[0067] As used herein, the terms ‘'subject,” “individual,” or “patient,” used interchangeably, refer to any animal, including mammals such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human.

[0068] As used herein, the terms “treat” or “treatment” refer to therapeutic or palliative measures. Beneficial or desired clinical results include, but are not limited to, alleviation, in whole or in part, of symptoms associated with a disease or disorder or condition, diminishment of the extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state (e.g., one or more symptoms of the disease), and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0069] The term “preventing” as used herein means the prevention of the onset, recurrence or spread, in whole or in part, of the disease or condition as described herein, or a symptom thereof.

[0070] The term “administration” or “administering"’ refers to a method of giving a dosage of a compound or pharmaceutical composition to a vertebrate or invertebrate, including a mammal, a bird, a fish, or an amphibian. The preferred method of administration can vary depending on various factors, e.g., the components of the pharmaceutical composition, the site of the disease, and the severity of the disease.Attorney Docket No. 15670-0455WO1

[0071] By “therapeutically effective amount” or “pharmaceutically effective amount” of a compound as provided herein is an amount which is sufficient to achieve the desired effect and can vary according to the nature and severity' of the disease condition, and the potency of the compound. A therapeutic effect is the relief, to some extent, of one or more of the symptoms of the disease, and can include curing a disease. “Curing” means that the symptoms of active disease are eliminated. However, certain long-term or permanent effects of the disease can exist even after a cure is obtained (such as, e.g., extensive tissue damage).

[0072] As used herein, the term “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the subject compound and exhibits minimal undesired toxicological effects. The pharmaceutically acceptable salt may be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively. In some embodiments, a pharmaceutically acceptable salt can be preferred over the respective free base or free acid because such a salt imparts greater stability or solubility' to the molecule thereby facilitating formulation into a dosage form. Basic compounds are generally capable of forming pharmaceutically acceptable acid addition salts by treatment with a suitable acid. Suitable acids include pharmaceutically acceptable inorganic acids and pharmaceutically acceptable organic acids. Non-limiting examples of a pharmaceutically acceptable acid addition salt include hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, sulfamate, phosphate, acetate, hydroxyacetate, phenylacetate, propionate, but rate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p-aminosalicy elate, glycollate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate, glutarate, glutamate, estolate, methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate, benzenesulfonate (besylate), p-aminobenzenesulfonate, p-toluenesulfonate (tosylate),napthalene-2-sulfonate, ethanedisulfonate, and 2,5-dihydroxy benzoate.

[0073] As used herein, the tenn “alkyl” refers to a saturated acyclic hydrocarbon radical that may be a straight chain or branched chain, containing the indicated number ofAttorney Docket No. 15670-0455WO1

[0074] carbon atoms. For example, C1-C22 indicates that the group may have from 1 to 22 (inclusive) carbon atoms in it. Non-limiting examples include methyl, ethyl, / iso-propyl, tert-butyl, tt-hexyl. The term “saturated’’ as used in this context means only single bonds present between constituent carbon atoms and other available valences occupied by hydrogen and / or other substituents as defined herein.

[0075] As used herein, “alkenyl” refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more double bonds.

[0076] As used herein, the terms “ester” and “C-carboxy” refer to a -C(=O)OR group in which R can be hydrogen, alkyd, or alkenyl.

[0077] As used herein, the term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10%, 5%, or 1%.

[0078] Method of treatment

[0079] The present disclosure provides methods of treating or preventing an infection in a subject. In some embodiments, the infection is an amoeba infection, such as amoebic meningoencephalitis. In some embodiments, the infection is caused by N. fowleri, which is a free-living amoeba that lives in warm freshwater resources, such as rivers, lakes, canals, and hot springs.

[0080] In some embodiments, the method provided herein includes administering to a subject in need thereof a composition that includes a therapeutically effective amount of rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, rutamycin B is a compound with the following formula:Attorney Docket No. 15670-0455WO1

[0081]

[0082] In some embodiments, the derivative thereof of rutamycin B is an ester derivative of rutamycin B. For example, the derivative thereof of rutamycin B can include an ester formed at a hydroxyl group of rutamycin B. In some embodiments, the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbons. For example the saturated or unsaturated fatty acid ester may have at least 1 carbon (e.g., at least 2 carbons, at least 3 carbons, at least 4 carbons, at least 5 carbons, at least 10 carbons, at least 15 carbons) and / or at most 22 carbons (e.g., at most 21 carbons, at most 20 carbons, at most 19 carbons, at most 18 carbons, at most 17 carbons, at most 16 carbons). In some embodiments, the derivative thereof of rutamycin B has the following formula:Attorney Docket No. 15670-0455WO1

[0083]

[0084] wherein R is hydrogen, C-carboxy-Ci-22 alkyl, or C-carboxy-C2-22 alkenyl. In some embodiments, R is C-carboxy-Ci-3 alkyl, C-carboxy-Ci-6 alkyl, C-carboxy-Ci-9 alkyl, C-carboxy-Ci-12 alkyl, C-carboxy-Ci-i6 alkyl, C-carboxy-C3-22 alkyl, C-carboxy-Ce-22 alkyl, C-carboxy-C9-22 alkyl, C-carboxy-Ci2-22 alkyl, C-carboxy-Ci6-22 alkyl. In some embodiments, R is C-carboxy-C2-3 alkenyl, C-carboxy-C2-6 alkenyl, C-carboxy-C2-9 alkenyl, C-carboxy-C2-i2 alkenyl, C-carboxy-C2-i6 alkenyl, C-carboxy-C3-22 alkenyl, C-carboxy-Ce-22 alkenyl, C-carboxy-C9-22 alkenyl. C-carboxy-C 12-22 alkenyl, C-carboxy-C16-22 alkenyl.

[0085] Administration of rutamycin B, and / or the derivative thereof, and / or the pharmaceutically acceptable salt thereof, can be via any accepted mode of administration. For example, the method can include administering the rutamycin B,Attorney Docket No. 15670-0455WO1

[0086] the derivative thereof, or the pharmaceutically acceptable salt thereof via intravenous injection, intrathecal injection, intranasal injection, oral administration, or combinations thereof. In some embodiments, administering includes contacting rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof with the brain of the subject. In some embodiments, administering the therapeutically effective amount of rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof reduces a concentration of N. fowleri trophozoites in the subject.

[0087] In some embodiments, the method includes administering a combination of rutamycin B and one or more derivatives thereof. In some embodiments, the methods includes administering a combination of rutamycin B and one or more pharmaecutically acceptable salts thereof. In some embodiments, the method includes adminstering a combination of one or more derivatives of rutamycin B and one or more pharmeceutically acceptable salts thereof of rutamycin B. In some embodiments, the method includes administering a combination of rutamy cin B, one or more derivatives thereof, and one or more pharamecutically acceptable salts thereof.

[0088] In some embodiments, administering rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof to the subject can include delivering the therapeutically effective amount of rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof at a concentration at least about 0.002 pM to the subject. In some embodiments, the concentration is at least about 0.002 pM (e.g.. at least about 0.01 pM, at least about 0.05 pM, at least about 0.1 pM, at least about 0.2 pM, at least about 0.3 pM, at least about 0.4 pM, at least about 0.5 pM, at least about 0.6 pM, at least about 0.7 pM, at least about 0.8 pM, at least about 0.9 pM, or at least about 1.0 pM) and / or at most 2 pM (e.g., at most about 1.9 pM. at most about 1.8 pM, at most about 1.7 pM, at most about 1.6 pM, at most about 1.5 pM, at most about 1.4 pM, at most about 1.3 pM, at most about 1.2 pM, at most about 1.1 pM, at most about 1 pM, at most about 0.9 pM, at most about 0.8 pM, at most about 0.7 pM, at most about 0.6 pM).

[0089] In some embodiments of any of the methods described herein, rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof, is administered in combination with a therapeutically effective amount of at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent includesAttorney Docket No. 15670-0455WO1

[0090] amphotericin B. In some embodiments, amphotericin B is a compound with the following formula:

[0091]

[0092] In some embodiments, the derivative thereof of amphotericin B includes an ester derivative of amphotericin B. For example, the derivative thereof of amphotericin B can include an ester formed at a hydroxyl group of amphotericin B. In some embodiments, the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbons. For example the saturated or unsaturated fatty acid ester may have at least 1 carbon (e.g., at least 2 carbons, at least 3 carbons, at least 4 carbons, at least 5 carbons, at least 10 carbons, at least 15 carbons) and / or at most 22 carbons (e.g., at most 21 carbons, at most 20 carbons, at most 19 carbons, at most 18 carbons, at most 17 carbons, at most 16 carbons). In some embodiments, the derivative thereof of amphotericin B includes an amine derivative of amphotericin B. For example, the derivative thereof of amphotericin B can include an amide formed at an amine group of amphotericin B. In some embodiments, the amide is a saturated or unsaturated fatty acid amide having 1 to 22 carbons. For example the saturated or unsaturated fatty acid amide may have at least 1 carbon (e.g., at least 2 carbons, at least 3 carbons, at least 4 carbons, at least 5 carbons, at least 10 carbons, at least 15 carbons) and / or at most 22 carbons (e.g., at most 21 carbons, at most 20 carbons, at most 19 carbons, at most 18 carbons, at most 17 carbons, at most 16 carbons). In some embodiments, the derivative thereof of amphotericin B has the following formula:Attorney Docket No. 15670-0455WO1

[0093]

[0094] wherein R is hydrogen, C-carboxy-Ci-22 alkyl, or C-carboxy-C2-22 alkenyl. In some embodiments, R is C-carboxy-Ci-3 alkyd, C-carboxy-C 1-6 alkyl, C-carboxy-C 1-9 alkyl, C-carboxy-Ci-12 alkyl, C-carboxy-Ci-i6 alkyl, C-carboxy-C3-22 alkyl, C-carboxy-Ce-22 alkyd. C-carboxy-C9-22 alkyl, C-carboxy-Ci2-22 alkyl, C-carboxy-Ci6-22 alkyl. In some embodiments, R is C-carboxy-C2-3 alkenyl, C-carboxy-C2-6 alkenyl, C-carboxy-C2-9 alkenyl, C-carboxy-C2-t2 alkenyl, C-carboxy-C2-t6 alkenyl, C-carboxy-Cs-22 alkenyl, C-carboxy-Ce-22 alkenyl, C-carboxy-Cg-22 alkenyl, C-carboxy-C 12-22 alkenyl, C-carboxy-C16-22 alkenyl.

[0095] In some embodiments, Ri and R2 are each independently selected from hydrogen, C-carboxy-Ci-22 alkyl, or C-carboxy-C2-22 alkenyl. In some embodiments, R is C-carboxy-C 1-3 alkyl, C-carboxy-C 1-6 alkyl, C-carboxy-Ci-9 alkyl, C-carboxy-Ci-12 alkyl, C-carboxy-Ci-i6 alkyl, C-carboxy-Cs-22 alkyl, C-carboxy-Ce-22 alkyl, C-carboxy-C9-22 alkyl, C-carboxy-Ci2-22 alkyl, C-carboxy-Ci6-22 alkyl. In some embodiments, R is C-carboxy-C2-3 alkenyl, C-carboxy-C2-6 alkenyl, C-carboxy-C2-9 alkenyl, C-carboxy-C2-12 alkenyl, C-carboxy-C2-t6 alkenyl, C-carboxy-C’3-22 alkenyl, C-carboxy-Ce-22 alkenyl, C-carboxy-C9-22 alkenyl, C-carboxy-Ct2-22 alkenyl, C-carboxy-C 16-22 alkenyl.

[0096] In some embodiments, amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present in the composition at a concentration that is greater than a concentration of rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof. In some embodiments, amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present in the composition to rutamycin B. the derivative thereof, or the pharmaceutically acceptableAttorney Docket No. 15670-0455WO1

[0097] salt thereof at a ratio from 1 : 8 to 1 : 32. In some embodiments, the ratio is at least about 1 :8 (e.g., at least about 1:9, at least about 1:10, at least about 1 : 11, at least about 1:12, at least about 1:13, at least about 1:14, at least about 1:15, at least about 1:20) and / or at most about 1:32 (e.g., at most about 1:31, at most about 1:30, at most about 1:29, at most about 1:28, at most about 1:27, at most about 1:26, at most about 1:25, at most about 1:24. at most about 1:23, at most about 1:22, at most about 1:21, at most about 1:20).

[0098] Composition

[0099] Also provided herein are compositions for treating or preventing an infection in a subject. Any of the compositions described herein can be administered to the subject to treat the infection as described herein.

[0100] In some embodiments, the composition includes a therapeutically effective amount of rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof. In some embodiments, the composition further includes a therapeutically effective amount of amphotericin B, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0101] In some embodiments, the composition can include a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof. In some embodiments, the pharmaceutically acceptable carrier or excipient includes a solid, a semi-solid, a liquid, solutions, a colloid, a liposome, an emulsion, a suspension, a complex, a coacervate, and an aerosol, or a combination thereof. The composition can also include dosage forms, such as, e.g., tablets, capsules, powders, liquids, suspensions, suppositories, aerosols, implants, controlled release or the like. The rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof and / or amphotericin B, a derivative thereof, or a pharmaceutically acceptable salt thereof, can also be administered in sustained or controlled release dosage forms, including depot injections, osmotic pumps, pills (tablets and or capsules), transdermal (including electrotransport) patches, implants and the like, for prolonged and / or timed, pulsed administration at a predetermined rate.

[0102] In some embodiments, the composition is a tablet. In some embodiments, the composition is a film-coated tablet.Attorney Docket No. 15670-0455WO1

[0103] The rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof and / or amphotericin B, a derivative thereof, or a pharmaceutically acceptable salt thereof, can be administered either alone or in combination with a pharmaceutically acceptable carrier, excipient or the like. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, selfemulsifying drug delivery systems (SEDDS) such as d-a-tocopherol, polyethylene glycol 1000, succinate, surfactants used in pharmaceutical dosage forms such as Tweens, poloxamers or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, tris, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty' acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium-chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat. Cyclodextrins can also be used to enhance delivery of compounds described herein.

[0104] In some embodiments, a composition described herein will take the form of a unit dosage form such as a pill or tablet and thus the composition may contain, along with The rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof and / or amphotericin B, a derivative thereof, or a pharmaceutically acceptable salt thereof, a diluent such as lactose, sucrose, dicalcium phosphate, or the like; a lubricant such as magnesium stearate or the like; and a binder such as starch, gum acacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives or the like. In another solid dosage form, a powder, marume, solution or suspension (e.g. in propylene carbonate, vegetable oils, PEGs, poloxamer 124 or triglycerides) is encapsulated in a capsule (gelatin or cellulose base capsule). Unit dosage forms in which one or more rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof and / or amphotericin B, a derivative thereof, or a pharmaceutically acceptable salt thereof, provided herein or additional active agents are physically separated are also contemplated, e.g, capsules with granules (or tablets in a capsule) of each drug, two-layer tablets, two-compartment gel caps, etc. Enteric coated or delayed release oral dosage forms are also contemplated.Attorney Docket No. 15670-0455WO1

[0105] In some embodiments, the composition includes one or more excipients selected from the group consisting of: hypromellose, magnesium stearate, microcrystalline cellulose, and sodium starch glycolate.

[0106] In some embodiments, the composition is a liquid. Liquid administrable compositions can, for example, be prepared by dissolving, dispersing, etc. a compound provided herein and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution, colloid, liposome, emulsion, complexes, coacervate or suspension. If desired, the pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, co-solvents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like).

[0107] EXAMPLES

[0108] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0109] EXAMPLE 1: Treating or preventing an infection using rutamycin B Material and Methods:

[0110] N. fowleri cell culture

[0111] N. fowleri strains European KUL (ATCC 30808), US genotype I Davis, US genotype II CAMP, US genofype III TY, and Australian CDC:V1005 were axenically cultured in Nelson’s medium supplemented with 10% FBS at 37°C. US and Australian strains were acquired from CDC, USA. Experiments were conducted using trophozoites harvested at 48 hours when they were at the logarithmic phase of growth. Trophozoites were counted using a hemocytometer.

[0112] Culture of mammalian epithelial cells

[0113] HEK-293 and HT-29 epithelial cells were maintained in DMEM medium, supplemented with penicillin (100 U / mL), streptomycin (100 pg / mL). and 10% FBS. All experiments were performed using human cells harvested during the logarithmic phase of growth. Mammalian cells were counted using a hemocytometer.Attorney Docket No. 15670-0455WO1

[0114] Isolation of rutamycin B

[0115] For isolation of rutamycin B. actinomycete strain CNH301 was cultivated, extracted, and fractionated to produce the compound at 5-10 mg / L. Actinomycete strain CNH301 was selected, and the strain was cultivated in a2.8L Fembach flask containing 1 L of liquid medium (10% starch, 4% yeast extract, 2% peptone, 75% seawater, 25% DI water) at 28°C and 200 rpm for 7 days. It was then extracted with 1 L of ethyl acetate twice, and the extraction solvent was dried in vacuo. The crude extract was loaded onto a small silica gel column for fractionation with hexane, ethyl acetate, and methanol into 10 fractions. Fractions 4 and 5 were found to contain the targeted compound by HR-LCMS and NMR analysis. LCMS system utilized electrospray ionization in negative mode with a linear gradient of 10-100% aqueous MeCN at a flow rate of 0.7 mL / min over 20 min on a reversed-phase Cl 8 column. Rutamycin B was eluted with te = 20.0 min, with a measured mass m / z 761.5124, which corresponds to [M+H]+of the compound ion with the theoretical m / z 761.5204. It was further purified by reversed-phase HPLC with a linear gradient of 10-100% aqueous MeCN at a flow rate of 3 mL / min over 60 min eluting at 59 min.

[0116] Activity of rutamycin B on N. fowleri strains

[0117] Pure compound rutamycin B was evaluated on the reference European KUL strain, and against Australian CDC:V1005, US genotype I Davis. US genotype II CAMP and US genotype III TY strains. ECso of rutamycin B on multiple strains was determined by serially diluting the compound two-fold to achieve concentrations ranging from 50 to 0.002 pM and 0.5 pL of each compound was added to a Greiner Bio-One Cellstar white, 96-well flat bottom microplate. Thereafter, 99.5 pL of complete growth media containing 10 x 103trophozoites was added in each well of the plate. The plates were then incubated at 37°C and 5% CO2 for 48 h and at the end of incubation, 50 pL of CellTiter-Glo luminescence cell viability assay (Promega) was added to each well. The plates were shaken on an orbital microplate shaker (VWR) at 360 rpm for 10 minutes and kept at room temperature for additional 10 minutes before measuring luminescence on an EnVision 2104 Multilabel Reader (Perkin Elmer). The ATP -bioluminescence data were processed through Excel to determine the inhibition percentages of growth of N. fowleri trophozoites. Data from a minimum of threeAttorney Docket No. 15670-0455WO1

[0118] independent experiments (biological replicates) conducted in triplicate were analyzed on GraphPad Prism 10 to determine the ECso value.

[0119] Effect of rutamycin B on growth inhibition at different time points

[0120] To determine the rate of killing of N. fowler i by rutamycin B, we measured the growth inhibition of KUL strain at different time points. For this, KUL trophozoites were incubated with different concentrations of rutamycin B ranging from 50 to 0.002 pM, in triplicate for 4, 10, 16, and 24 h in 96-well microplates. Trophozoites were also treated with same concentrations of the control drug amphotericin B at 4, 10, 16, and 24 h. EC50 at different timepoints was determined by the CellTiter-Glo Luminescent Cell Viability’ Assay following the same protocol as described above. Experiment was done in triplicate in three independent biological replicates.

[0121] Mammalian cytotoxicity assay

[0122] For mammalian cytotoxicity assays, 0.5 pL of rutamycin B was transferred into a 96-well screen plate followed by addition of 99.5 pL of HEK-293 or HT-29 cells (10,000 cells) to yield concentrations ranging from 50 to 0.002 pM with a final DMSO concentration of 0.5%. The negative and positive controls contained 0.5% DMSO and 100 pM of staurosporine (BioVision), respectively. The assay was performed in triplicate using the CellTiter-Glo Luminescent Cell Viability Assay.

[0123] Results

[0124] Activity of rutamycin B on N. fowleri strains and mammalian cells

[0125] To determine if pure rutamycin B is active against N. fowleri, we first measured its EC50 against the reference European KUL strain, and once found active, we tested on other clinical strains. Rutamycin B was equally potent on European, Australian, and US genotypes strains with EC50 ranging between 0.02 to 0.3 pM (Table 1). Rutamycin B was equipotent or more potent than amphotericin B against different strains of N. fowleri. When tested on two mammalian cell lines HEK293 and HT-29 for 48 h, rutamycin B provided selectivity' indices of about 200-300 based on KUL and mammalian cell types examined (Table 1).Attorney Docket No. 15670-0455WO1

[0126]

[0127] CL. confidence limit

[0128] Effect of rutamycin B on growth inhibition at different time points

[0129] To establish how fast rutamycin B kills N. fowleri, we measured dose-response of the compound at different time points between 4 and 24 h of exposure and compared the effect with amphotericin B. Grow th inhibition curves generated at different time points (FIGS. 1-2) with the KUL strain demonstrated rutamycin B required only 4 h to achieve nanomolar potency. This is faster than amphotericin B which required 16 h to reach higher ECso concentration (0.2 pM) than rutamycin B.

[0130] DiscussionAttorney Docket No. 15670-0455WO1

[0131] Both plants and microbial secondary metabolites are sources of compounds that display antiparasitic activity. Although natural products have been used for the treatment of parasitic diseases, none of them have come from marine sources. Prior research identifying new natural product-based anti -A. fowleri agents was limited to the marine alga Laurencia sp., but the compounds isolated from Laurencia did not exhibit good potency against N. fowleri. Marine microorganisms, especially actinobacteria, produce a host of bioactive secondary metabolites including antibiotics, immunosuppressive, and antitumor agents. These actinobacteria also provide an additional advantage of obtaining continuous supply of required compounds compared to seasonal occurrence of metabolites associated with other natural sources such as plants. Typical actinobacteria genera used in such research are the Salinispora and Marinospora, which have produced unique anticancer drugs, plinabulin and marizomib, and these drugs entered late Phase 3 clinical trials.

[0132] Example 1 identified rutamycin B exhibiting nanomolar potency on multiple genotypes of N fowleri and it was 15- or 2500-times more potent than the recommended drugs amphotericin B or miltefosine. Rutamycin also provided selectivity indices of about 200-300 when tested on N. fowleri and two mammalian cells.

[0133] Since PAM has a rapid clinical course, it is important to identify a drug that is fast-acting. Example 1 shows that rutamycin B is fast-acting and it reached nanomolar potency in 4 hours. While rutamycin B was a highly potent compound identified in this Example, derivatives of rutamycin B are conceived to be an excellent source for N. fowleri infection treatments.

[0134] EXAMPLE 2: Treating or preventing an infection using rutamycin B and amphotericin B

[0135] Effect of rutamycin B on accumulation of reactive oxygen species (ROS) Changes in ATP synthase can regulate mitochondrial ROS formation. In this Example, the effect of rutamycin B on the accumulation of ROS was investigated. Briefly, 10,000 KUL trophozoites were treated with 0.4 mM of 2', 7'- dichlorodihydrofluorescein diacetate for 30 min in the dark, washed and incubated with 1% DMSO, and 0.78, 0.39, and 0.2 pM of rutamycin B or amphotericin B at 37°C forAttorney Docket No. 15670-0455WO1

[0136] only 2 h. Fluorescence was measured with an excitation at 485 nm and emission at 535 nm. The experiment was performed in triplicate. A brief treatment of N. fowleri with rutamycin B accumulated significantly more ROS (P < 0.05) at these three concentrations than DMSO-treated trophozoites (FIG. 3), but the same concentrations of amphotericin B did not accumulate significantly more intracellular ROS than DMSO-treated trophozoites.

[0137] Effect of rutamycin B on mitochondrial FIFO ATP synthase activity N. fowleri To understand the target of rutamycin B in N. fowleri, Example 2 undertook the biochemical studies with mitochondria isolated from KUL strain of N. fowleri trophozoites. Briefly, 200 x 106 trophozoites were harvested and mitochondria were prepared using the Mitochondria Isolation Kit for Cultured Cells (Thermo Scientific™) in presence of lx protease inhibitor cocktail. Protein concentration of mitochondrial samples was measured using the BCA Protein Assay Kit (BioVision). The isolated mitochondria were subjected to colorimetric FIFO ATP synthase activity assay by ELISA-based ATP Synthase Enzyme Activity Microplate Assay Kit (abeam®). The isolated mitochondria underwent a minimum of three freeze-thaw cycles and subsequent addition of buffer solution and detergent as described in the assay kit (abeam®). The assay was conducted in triplicate in a 96-well plate with each well containing 10 pg of mitochondria in presence of 2% DMSO, or 50 pM of amphotericin B, or 3.13, 1.56, 0.78, and 0.39 pM of rutamycin B in a volume of 50 pL of buffer solution and incubated at room temp, for 3 h. Following incubation and immobilization of the mitochondrial enzyme by the bound monoclonal antibody to the wells, the wells were washed with the buffer solution. Subsequently, 40 pL of lipid mix and 200 pL of reagent mix were added to each well following the protocol described in the kit and absorbance was measured at 340 nm at 30°C for 180 min. While DMSO and high concentration (50 pM) of amphotericin B did not reduce mitochondrial ATP synthase activity of F fowleri, both 1.56 and 3.13 pM of rutamycin B significantly reduced the mitochondrial ATP synthase activity (P < 0.05) of N. fowleri trophozoites, confirming FIFO ATP synthase as a target of rutamycin B (FIG. 4).

[0138] Synergistic effect of rutamycin B and amphotericin B on N. fowleriAttorney Docket No. 15670-0455WO1

[0139] The inhibitory' effects of rutamycin-amphotericin B pairing at fixed concentration ratios (in triplicate in three independent biological replicates) were investigated. The dose-effect relationships were assessed by the CompuSyn software to calculate Chou-Talalay combination indices (CI) and dose-reduction indices (3). Thus, combination of rutamycin B with amphotericin B at 1:8 (CI = 0.6 ± 0.09) and 1:32 (CI = 0.5 ± 0.03) caused 95% growth inhibition with 10- to 30-fold dose reduction for rutamycin B and 2-fold dose reduction for amphotericin B (FIGS. 5-6).

[0140] Evaluation of mammalian cytotoxicity of combination of rutamycin B and amphotericin B

[0141] To test the effect of the combination of rutamycin B and amphotericin B on a mammalian cell, a cytotoxicity' experiment of a synergistic combination (1:32) on HT-29 mammalian cells for 72 h in triplicate was conducted. A combination of 0.012 pM rutamycin B and 0.4 pM amphotericin B (1:32) caused about 11.5% growth inhibition of HT-29 cells. Thus, it appears while the concentrations used in the combination study exhibited synergistic effect on N. fowleri with 95% growth inhibition of trophozoites, those were less toxic to human intestinal cells.

[0142] References

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[0145] 2. Yoder JS, Eddy BA, Visvesvara GS. Capewell L, Beach MJ. The epidemiology' of primary' amoebic meningoencephalitis in the USA, 1962-2008. Epidemiol Infect. 2010;138(7):968-75. Epub 2009 / 10 / 23. doi:

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[0148] Diagnosis, Clinical Course, and Treatment of Primary Amoebic Meningoencephalitis in the United States, 1937-2013. J Pediatric Infect Dis Soc. 2015;4(4):e68-75. Epub 2015 / 11 / 20. doi: 10.1093 / jpids / piul03. PubMed PMID: 26582886.

[0149] 4. Gharpure R, Bliton J, Goodman A. Ali IKM, Yoder J, Cope JR. Epidemiology and Clinical Characteristics of Primary Amebic Meningoencephalitis Caused byAttorney Docket No. 15670-0455WO1

[0150] Naegleria fowleri'. A Global Review. Clin Infect Dis. 2020. Epub 2020 / 05 / 06. doi: 10.1093 / cid / ciaa520. PubMed PMID: 32369575.

[0151] 5. Bellini NK, Santos TM, da Silva MT A, Thiemann OH. The therapeutic strategies against Naegleria fowleri. Exp Parasitol. 2018;187:1-11. Epub 2018 / 03 / 05. doi: 10.1016 / j.exppara.2018.02.010. PubMed PMID: 29501696.

[0152] 6. Gallis HA, Drew RH, Pickard WW. Amphotericin B: 30 years of clinical experience. Rev Infect Dis. 1990;12(2):308-29. PubMed PMID: 2184499.

[0153] 7. Laniado-Laborin R, Cabral es-Vargas MN. Amphotericin B: side effects and toxicity. Rev Iberoam Micol. 2009;26(4):223-7. doi: 10.1016 / j.riam.2009.06.003. PubMed PMID: 19836985.

[0154] 8. Visvesvara GS. Amebic meningoencephalitides and keratitis: challenges in diagnosis and treatment. Curr Opin Infect Dis. 2010:23(6): 590-4. Epub 2010 / 08 / 31. doi: 10.1097 / QCO.0b013e32833ed78b. PubMed PMID: 20802332.

[0155] 9. Escrig, J. I.; Hahn, H. J.; Debnath, A. Activity of Auranofin against Multiple Genotypes of Naegleria fowleri and Its Synergistic Effect with Amphotericin B In Vitro. ACSChem Neurosci 2020, 11 (16), 2464-2471. DOI:

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[0157] 10. Shing, B.; Balen, M.; Fenical, W.; Debnath, A. Development of a Machine Learning-Based Cysticidal Assay and Identification of an Amebicidal and Cysticidal Marine Microbial Metabolite against Acanthamoeba. Microbiol Spectr 2022. 10 (3), e0007722. DOI: 10.1128 / spectrum.00077-22.

[0158] 11. Ndjonka D, Rapado LN, Silber AM, Liebau E, Wrenger C. Natural products as a source for treating neglected parasitic diseases. Int J Mol Sci. 2013;14(2):3395-439. Epub 2013 / 02 / 08. doi: 10.3390 / ijmsl4023395. PubMed PMID: 23389040; PubMed Central PMCID: PMC3588050.

[0159] 12. Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. JNat Prod. 2012;75(3):311-35. Epub 2012 / 02 / 10. doi: 10.1021 / np200906s. PubMed PMID: 22316239; PubMed Central PMCID:

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[0163] 14. Kayser O, Kiderlen AF, Croft SL. Natural products as antiparasitic drugs. Parasitol Res. 2003;90 Suppl 2:S55-62. Epub 2003 / 08 / 26. doi: 10.1007 / s00436-002- 0768-3. PubMed PMID: 12937967.

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[0168] 17. Arberas-Jimenez I, Garcia-Davis S, Rizo-Li endo A, Sifaoui I. Reyes-Batlle M, Chiboub O, et al. Laurinterol from Laurencia johnstonii eliminates Naegleria fowleri triggering PCD by inhibition of ATPases. Sci Rep. 2020;10(l):17731. Epub 2020 / 10 / 22. doi: 10.1038 / s41598-020-74729-y. PubMed PMID: 33082417; PubMed Central PMCID: PMC7576160.

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[0180] OTHER EMBODIMENTS

[0181] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 15670-0455WO1WHAT IS CLAIMED IS:

1. A method of treating or preventing an infection comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the infection is an amoeba infection.

3. The method of claim 2, wherein the infection is an N. fowleri infection.

4. The method of claim 1, wherein administering includes contacting at least a portion of the rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof with a brain of the subject.

5. The method of claim 1, wherein administering includes delivering the therapeutically effective amount of the rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof to the subject via intravenous injection, intrathecal injection, intranasal injection, oral administration, or combinations thereof.

6. The method of claim 1, wherein administering includes delivering the therapeutically effective amount of rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof at a concentration of at least 0.002 pM to the subj ect.

7. The method of claim 6, wherein the concentration is from 0.002 pM to 2 pM.

8. The method of claim 3, wherein administering the therapeutically effective amount of the rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof reduces a concentration of A. fowleri trophozoites in the subject.

9. The method of claim 1, wherein administering the therapeutically effective amount of rutamycin B, derivative thereof, or a pharmaceutically acceptable salt thereof to the subject occurs while the therapeutically effective amount of rutamycinAttorney Docket No. 15670-0455WO1B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present in a pharmaceutically acceptable carrier.

10. The method of claim 1, wherein the derivative thereof is an ester derivative of rutamycin B.

11. The method of claim 10, wherein the ester derivative comprises an ester formed at a hydroxyl group of rutamycin B.

12. The method of claim 11, wherein the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbon atoms.

13. The method of claim 11, wherein the saturated or unsaturated fatty acid ester is selected from the group consisting of formate, acetate, propionate, butyrate, linoleate, and linolenate.

14. A method of treating or preventing an infection comprising administering to a subject in need thereof a therapeutically effective amount of rutamycin B and amphotericin B, derivatives thereof, or pharmaceutically acceptable salts thereof.

15. The method of claim 14, wherein the infection is an amoeba infection.

16. The method of claim 15, wherein the infection is an N. fowler i infection.

17. The method of claim 14, wherein administering includes contacting at least a portion of rutamycin B and amphotericin B, the derivatives thereof, or the pharmaceutically acceptable salts thereof with a brain of the subj ect.

18. The method of claim 14, administering includes the therapeutically effective amount of the rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof at a concentration of at least 0.002 pM to the subject.Attorney Docket No. 15670-0455WO119. The method of claim 18, wherein a concentration of amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is greater than the concentration of rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof.

20. The method of claim 19, wherein a ratio of amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof to rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is from 1 :8 to 1 :32.

21. The method of claim 14, wherein the derivative thereof of rutamycin B is an ester derivative of rutamycin B.

22. The method of claim 21, wherein the ester derivative comprises an ester formed at a hydroxyl group of rutamycin B.

23. The method of claim 22, wherein the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbon atoms.

24. The method of claim 14, wherein the derivative thereof of amphotericin B is an ester derivative of amphotericin B.

25. The method of claim 24, wherein the ester derivative comprises an ester formed at a hydroxyl group of amphotericin B.

26. The method of claim 25, wherein the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbon atoms.

27. A composition for treating or preventing an infection of a subject, the composition comprising:a therapeutically effective amount of rutamycin B, a derivative thereof, or a pharmaceutically acceptable salt thereof; anda pharmaceutically acceptable carrier, excipient, or a combination thereof.Attorney Docket No. 15670-0455WO128. The composition of claim 27, rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present in the composition in an amount of at least 0.002 pM.

29. The composition of claim 28, wherein rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present in the composition in an amount from 0.002 pM to 2 pM.

30. The composition of claim 27, wherein the derivative thereof is an ester derivative of rutamycin B.

31. The composition of claim 30, wherein the ester derivative comprises an ester formed at a hydroxyl group of the rutamycin B.

32. The composition of claim 31, wherein the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbon atoms.

33. The composition of claim 31, wherein the saturated or unsaturated fatty' acid ester is selected from the group consisting of formate, acetate, propionate, butyrate, linoleate, and linolenate.

34. The composition of claim 27 further comprising:amphotericin B, a derivative thereof, or a pharmaceutically acceptable salt thereof.

35. The composition of claim 34, wherein amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is present at a concentration that is greater than a concentration of rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof.

36. The composition of claim 35, wherein a ratio of amphotericin B, the derivative thereof, or the pharmaceutically acceptable salt thereof to rutamycin B, the derivative thereof, or the pharmaceutically acceptable salt thereof is from 1:8 to 1:32.Attorney Docket No. 15670-0455WO137. The composition of claim 34, wherein the ester derivative comprises an ester formed at a hydroxyl group of amphotericin B.

38. The composition of claim 37, wherein the ester derivative comprises an ester formed at a hydroxyl group of amphotericin B.

39. The composition of claim 38, wherein the ester is a saturated or unsaturated fatty acid ester having 1 to 22 carbon atoms.

40. The composition of claim 39, wherein the saturated or unsaturated fatty’ acid ester is selected from the group consisting of formate, acetate, propionate, butyrate, linoleate, and linolenate.

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