[go: up one dir, main page]

WO2016014779A1 - Dérivés macrolides de polyènes antifongiques à toxicité réduite pour les mammifères - Google Patents

Dérivés macrolides de polyènes antifongiques à toxicité réduite pour les mammifères Download PDF

Info

Publication number
WO2016014779A1
WO2016014779A1 PCT/US2015/041710 US2015041710W WO2016014779A1 WO 2016014779 A1 WO2016014779 A1 WO 2016014779A1 US 2015041710 W US2015041710 W US 2015041710W WO 2016014779 A1 WO2016014779 A1 WO 2016014779A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
moiety
mycosaminyl
epi
polyene macrolide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2015/041710
Other languages
English (en)
Inventor
Martin D. Burke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Illinois at Urbana Champaign
University of Illinois System
Original Assignee
University of Illinois at Urbana Champaign
University of Illinois System
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Illinois at Urbana Champaign, University of Illinois System filed Critical University of Illinois at Urbana Champaign
Publication of WO2016014779A1 publication Critical patent/WO2016014779A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/08—Hetero rings containing eight or more ring members, e.g. erythromycins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin

Definitions

  • amphotericin B The polyene macrolide natural product, amphotericin B (AmB), is the archetype for small molecules that form ion channels in living cells 1 and antibiotics that are inherently refractory to microbial resistance. 2 AmB is also, unfortunately, highly toxic, 3 which often limits its effective utilization as the last line of defense against life-threatening systemic fungal infections. Because the incidence of such fungal infections and resistance to all other classes of antifungals are both on the rise, 2 finding a way to improve the therapeutic index of AmB has become an increasingly important problem. Some progress has been made with liposomal formulations, but they are often prohibitively expensive, 4 and substantial toxicity still remains. 5 Despite 50 years of extensive efforts worldwide, a clinically viable derivative of AmB with an improved therapeutic index has yet to emerge. 6
  • the invention is based, at least in part, on the remarkable and surprising discovery that substitution of C2-epi-mycosamine for mycosamine in an antifungal mycosamine- bearing polyene macrolide results in a C2'-epi-polyene macrolide with dramatically improved therapeutic index (i.e., preserved desirable anti-fungal activity and dramatically reduced undesirable toxic side effects).
  • An aspect of the invention is a compound or a pharmaceutically acceptable salt thereof, represented by A-O-M; wherein A-0 is a polyene macrolide aglycone moiety; O is an oxygen atom; M is a C2-epi-mycosaminyl moiety; O-M is a glycosidic bond between the oxygen atom and the anomeric carbon of the C2-epi-mycosaminyl moiety; and said compound has antifungal activity; provided that the compound is not C2'epiAmB or a pharmaceutically acceptable salt thereof.
  • An aspect of the invention is a pharmaceutical composition, comprising a compound of the invention; and a pharmaceutically acceptable carrier.
  • An aspect of the invention is a method of inhibiting growth of a yeast or fungus, comprising contacting the yeast or fungus with an effective amount of a compound or a pharmaceutically acceptable salt thereof represented by A-O-M; wherein A-0 is a polyene macrolide aglycone moiety; O is oxygen; M is a C2-epi-mycosaminyl moiety; O-M is a glycosidic bond between a hydroxyl moiety of the polyene aglycone moiety and the anomeric carbon of the C2-epi-mycosaminyl moiety; and said compound has antifungal activity; provided that the compound is not C2'epiAmB or a pharmaceutically acceptable salt thereof.
  • A-0 is a polyene macrolide aglycone moiety
  • O oxygen
  • M is a C2-epi-mycosaminyl moiety
  • O-M is a glycosidic bond between a
  • An aspect of the invention is a method of treating a fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof represented by A-O-M; wherein A-0 is a polyene macrolide aglycone moiety; O is oxygen; M is a C2-epi-mycosaminyl moiety; O-M is a glycosidic bond between a hydroxyl moiety of the polyene aglycone moiety and the anomeric carbon of the C2-epi-mycosaminyl moiety; and said compound has antifungal activity; provided that the compound is not C2'epiAmB or a pharmaceutically acceptable salt thereof.
  • A-0 is a polyene macrolide aglycone moiety
  • O oxygen
  • M is a C2-epi-mycosaminyl moiety
  • O-M is a glycosidic bond between a hydroxy
  • FIG. 1 depicts a scheme for synthesis of C2'epiAmB.
  • FIG. 2 depicts a scheme for synthesis of C2'epiAmB.
  • FIG. 3 is a table of binding affinity, anti- fungal activity, and toxicity data for C2'epiAmB.
  • Amphotericin B an example of an antifungal polyene macrolide, comprises a polyhydroxylated, polyunsaturated macrolactone ring core, decorated with a mycosamine "appendage".
  • Mycosamine is 3-amino-3,6-dideoxy-P-D-mannopyranose.
  • the mycosamine portion of the molecule plays a key role in the desired biological effects and the undesirable side-effects of Amphotericin B.
  • the compounds shown above can be generally characterized as comprising a C25- C37 cyclic polyene macrolide aglycone moiety, linked via an oxygen atom to the anomeric carbon of a mycosaminyl moiety.
  • the C2'-OH and C3'- NH 2 have cis relative stereochemistry between the C2' and C3' of the mycosaminyl moiety.
  • compounds of the invention comprise a polyene macrolide aglycone moiety (A), linked via an oxygen atom (O) to a C2-epi-mycosaminyl moiety (M), wherein the absolute stereochemistry at the C2'-hydroxyl is inverted, and the C2'-OH and C3'-NH 2 have trans relative stereochemistry between C2' and C3' of the C2-epi-mycosaminyl moiety.
  • A polyene macrolide aglycone moiety
  • O oxygen atom
  • M C2-epi-mycosaminyl moiety
  • An aspect of the invention is a compound or a pharmaceutically acceptable salt thereof, represented by A-O-M; wherein A-0 is a polyene macrolide aglycone moiety; O is an oxygen atom; M is a C2-epi-mycosaminyl moiety; O-M is a glycosidic bond between the oxygen atom and the anomeric carbon of the C2-epi-mycosaminyl moiety; and said compound has antifungal activity; provided that the compound is not C2'epiAmB or a pharmaceutically acceptable salt thereof.
  • C2'epiAmB refers to a compound represented by
  • Amphotericin B is a clinically vital antimycotic, but its use is limited by its toxicity. Whereas binding ergosterol, independent of channel formation, is the primary mechanism by which AmB kills yeast, binding cholesterol may primarily account for its toxicity to human cells. A leading structural model predicts that the C2' hydroxyl group on the mycosamine appendage is key to binding to both sterols.
  • AmB is generally obtained from a strain of Streptomyces nodosus. It is currently approved for clinical use in the United States for the treatment of progressive, potentially life -threatening fungal infections, including infections such as systemic candidiasis, aspergillosis, cryptococcosis, blastomycosis, coccidioidomycosis, histoplasmosis, and mucormycosis. AmB is generally formulated for intravenous injection. Amphotericin B is commercially available, for example, as Fungizone® (Squibb), Amphocin® (Pfizer), Abelcet® (Enzon), and Ambisome® (Astellas). Due to its unwanted toxic side effects, dosing is generally limited to a maximum of about 1.0 mg/kg/day, and total cumulative doses not to exceed about 3 g in humans.
  • C2'epiAmB A new efficacious non-toxic AmB derivative, C2'epiAmB, was recently discovered that has shown the great potential as a clinically viable therapeutic replacement for AmB.
  • C2'epiAmB retains the zwitterionic character of AmB, and differs only in the inversion of a single stereocenter.
  • amphotericin B is the sterol binding domain of amphotericin B (i.e., the portion of the molecule that directly binds ergosterol and cholesterol).
  • the inventor has previously used a structure-based ligand-selective allosteric effects model rationally to predict that epimerization of the C2' stereogenic center of amphotericin B would lead to retained ergosterol binding and, thus, maintained antifungal action but dramatically reduced or no cholesterol binding and thus little or no toxicity. Testing of this prediction proved it to be correct. See U.S. Provisional Patent Application No. 61/994,450, filed May 16, 2014, and PCT Application No. US2015/030965, filed May 15, 2015, the entire contents of both of which are incorporated by reference.
  • mycosamine -bearing polyene macrolides will lead to retained ergosterol binding and thus maintained antifungal action but dramatically reduced or no cholesterol binding and thus little or no undesired toxicity.
  • a compound of the invention is purified, i.e., isolated from other compounds and components including the corresponding polyene macrolide comprising a mycosaminyl moiety.
  • a compound of the invention is present in a mixture together with the corresponding polyene macrolide comprising a mycosaminyl moiety.
  • a compound of the invention represents at least 50 percent of the polyene macrolide present in such mixture.
  • a compound of the invention represents at least 50 percent, at least 60 percent, at least 70 percent, at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent of the polyene macrolide present in such mixture.
  • the binding avidity for ergosterol of a compound of the invention is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In various individual embodiments, the binding avidity for ergosterol of a compound of the invention is at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In an embodiment, the binding avidity for ergosterol of a compound of the invention is at least 100 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety.
  • the binding avidity for cholesterol of a compound of the invention is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In various individual embodiments, the binding avidity for cholesterol of a compound of the invention is less than or equal to 20 percent, less than or equal to 15 percent, less than or equal to 10 percent, or less than or equal to 5 percent, of the binding avidity for cholesterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In an embodiment, a compound of the invention has essentially no binding avidity for cholesterol.
  • the binding avidity for ergosterol of a compound of the invention is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety; and the binding avidity for cholesterol of the compound of the invention is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide comprising a mycosaminyl moiety.
  • the polyene macrolide aglycone moiety is selected from the group consisting of the polyene macrolide aglycone moieties of amphotericin A, arenomycin B, candicidin D, candidin, candidoin, CE-108, etruscomycin, eurocidin D, eurocidin E, FR-008-VI, HA-2-91, hamycin A, levorin AO, levorin A3, mycoheptin, natamycin (pimaricin), nystatin Al, nystatin A2, nystatin A3, partricin A, polyfungin B, rimocidin, tetramycin A, tetramycin B, tetrin A, tetrin B, tetrin C, trichomycin A, trichomycin B, vacidin A, YS-822A, 3874 HI, 3874 H2, 3874 H3, and 67- 121 -A.
  • Certain compounds of the invention are C2'-epi-amphotericin A, C2'-epi- arenomycin B, C2'-epi-candicidin D, C2'-epi-candidin, C2'-epi-candidoin, C2'-epi-CE-108, C2'-epi-etruscomycin, C2'-epi-eurocidin D, C2'-epi-eurocidin E, C2'-epi-FR-008-VI, C2'- epi-HA-2-91, C2'-epi-hamycin A, C2'-epi-levorin AO, C2'-epi-levorin A3, C2'-epi- mycoheptin, C2'-epi-natamycin (pimaricin), C2'-epi-nystatin Al, C2'-epi-nystatin A2, C
  • a compound of the invention can be made using any suitable method.
  • a compound of the invention is made using so-called sugar swap technology to replace natural sugars (mycosamine) with C2-epi-mycosamine.
  • This technique involves the use of certain glycosyltransferases and a source of C2-epi-mycosamine to catalyze substitution of C2'-epi-mycosamine for mycosamine on any mycosamine-bearing polyene macrolide. See, for example, U.S. Patent Nos. 7,479,385 to Thorson; 8,093,028 to Thorson et al; and 8,637,287 to Thorson et al; and U.S. Published Patent Application Nos.
  • the compounds of the invention may also be prepared by a synthetic approach in which a polyene macrolide aglycone is glysosylated with a glycosyl donor form of C2-epi- mycosamine. See Croatt MP et al, Org. Lett., 2011, 13(6), 1390-1393.
  • the synthesis of the C2-epi-mycosamine glycosyl donor commences with the conversion of 2-acetylfuran to an allylic alcohol in five steps. This resulting alcohol is engaged in a directed epoxidation reaction and subsequently protected to afford a protected epoxide.
  • the synthesis of the mycosamine donor analog is completed by epoxide opening with azide, acylation of the resultant alcohol, and generation of the corresponding trichloroacetimidate.
  • the synthetic route is relatively short and, more importantly, allows for facile manipulation of different groups of the sugar.
  • the detailed experimental procedure is presented in Example 5 in the Exemplification section below. A semisynthetic route to C2'-epi-mycosamine-containing polyene macrolides that commences with the counterpart polyene macrolide may be relied upon to access a suitably protected aglycone.
  • AmB may be subjected to amine protection (Fmoc); esterification (CI6-CO 2 H ⁇ C16-C0 2 Me); and methyl ketal formation to yield a partially protected polyene macrolide.
  • Fmoc amine protection
  • esterification CI6-CO 2 H ⁇ C16-C0 2 Me
  • methyl ketal formation CI6-CO 2 H ⁇ C16-C0 2 Me
  • methyl ketal formation a partially protected polyene macrolide.
  • the polyene macrolide aglycone may then be glysosylated with the glycosyl donor form of C2-epi-mycosamine to provide a compound of the invention.
  • An aspect of the invention is a pharmaceutical composition, comprising a compound of the invention; and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means one or more compatible solid or liquid filler, diluent or encapsulating substances which are suitable for administration to a human or other subject.
  • the pharmaceutical composition is formulated for systemic administration.
  • the pharmaceutical composition is formulated for intravenous administration.
  • the pharmaceutical composition is formulated for oral administration.
  • the pharmaceutical composition is formulated for topical administration.
  • the pharmaceutical composition is formulated for local administration.
  • the pharmaceutical composition is formulated for intraperitoneal administration.
  • the pharmaceutical composition is formulated for intrathecal administration.
  • An aspect of the invention is a method of inhibiting growth of a yeast or fungus.
  • the method includes the step of contacting the yeast or fungus with an effective amount of a compound or a pharmaceutically acceptable salt thereof represented by A-O-M; wherein A-0 is a polyene macrolide aglycone moiety; O is oxygen; M is a C2-epi-mycosaminyl moiety; O-M is a glycosidic bond between a hydroxyl moiety of the polyene aglycone moiety and the anomeric carbon of the C2-epi-mycosaminyl moiety; and said compound has antifungal activity; provided that the compound is not C2'epiAmB or a
  • Yeasts are eukaryotic organisms classified in the kingdom Fungi. Yeasts are typically described as budding forms of fungi. Of particular importance in connection with the invention are species of yeast that can cause infections in mammalian hosts. Such infections most commonly occur in immunocompromised hosts, including hosts with compromised barriers to infection (e.g., burn victims) and hosts with compromised immune systems (e.g., hosts receiving chemotherapy or immune suppressive therapy, and hosts infected with HIV).
  • Pathogenic yeast include, without limitation, various species of the genus Candida, as well as of Cryptococcus . Of particular note among pathogenic yeasts of the genus Candida are C albicans, C tropicalis, C stellatoidea, C. glabrata, C. krusei, C. parapsilosis, C. guilliermondii, C. viswanathii, and C. lusitaniae.
  • Cryptococcus specifically includes Cryptococcus neoformans.
  • Yeast can cause infections of mucosal membranes, for example oral, esophageal, and vaginal infections in humans, as well as infections of bone, blood, urogenital tract, and central nervous system. This list is exemplary and is not limiting in any way.
  • Fungi include, in addition to yeasts, other eukaryotic organisms including molds and mushrooms.
  • a number of fungi can cause infections in mammalian hosts. Such infections most commonly occur in immunocompromised hosts, including hosts with compromised barriers to infection (e.g., burn victims) and hosts with
  • Pathogenic fungi include, without limitation, species of Aspergillus, Rhizopus, Mucor, Histoplasma, Coccidioides,
  • Blastomyces, Trichophyton, Microsporum, and Epidermophyton are A. fumigatus, A.flavus, A. niger, H. capsulatum, C. immitis, and B.
  • Fungi can cause deep tissue infections in lung, bone, blood, urogenital tract, and central nervous system, to name a few. Some fungi are responsible for infections of the skin and nails.
  • the phrase “effective amount” refers to any amount that is sufficient to achieve a desired biological effect.
  • “inhibit” or “inhibiting” means reduce by an objectively
  • inhibit or inhibiting means reduce by at least a statistically significant amount compared to control. In one embodiment, inhibit or inhibiting means reduce by at least 5 percent compared to control. In various individual embodiments, inhibit or inhibiting means reduce by at least 10, 15, 20, 25, 30, 33, 40, 50, 60, 67, 70, 75, 80, 90, or 95 percent compared to control.
  • the binding avidity for ergosterol of a compound of the invention is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In various individual embodiments, the binding avidity for ergosterol of a compound of the invention is at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In an embodiment, the binding avidity for ergosterol of a compound of the invention is at least 100 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety.
  • the binding avidity for cholesterol of a compound of the invention is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In various individual embodiments, the binding avidity for cholesterol of a compound of the invention is less than or equal to 20 percent, less than or equal to 15 percent, less than or equal to 10 percent, or less than or equal to 5 percent, of the binding avidity for cholesterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In an embodiment, a compound of the invention has essentially no binding avidity for cholesterol.
  • the binding avidity for ergosterol of a compound of the invention is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety; and the binding avidity for cholesterol of the compound of the invention is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide comprising a mycosaminyl moiety.
  • the polyene macrolide aglycone moiety is selected from the group consisting of the polyene macrolide aglycone moieties of amphotericin A, arenomycin B, candicidin D, candidin, candidoin, CE-108, etruscomycin, eurocidin D, eurocidin E, FR-008-VI, HA-2-91, hamycin A, levorin AO, levorin A3, mycoheptin, natamycin (pimaricin), nystatin Al, nystatin A2, nystatin A3, partricin A, polyfungin B, rimocidin, tetramycin A, tetramycin B, tetrin A, tetrin B, tetrin C, trichomycin A, trichomycin B, vacidin A, YS-822A, 3874 HI, 3874 H2, 3874 H3, and 67- 121 -A.
  • An aspect of the invention is a method of treating a fungal infection, comprising administering to a subject in need thereof a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof represented by A-O-M; wherein A-0 is a polyene macrolide aglycone moiety; O is oxygen; M is a C2-epi-mycosaminyl moiety; O-M is a glycosidic bond between a hydroxyl moiety of the polyene aglycone moiety and the anomeric carbon of the C2-epi-mycosaminyl moiety; and said compound has antifungal activity; provided that the compound is not C2'epiAmB or a pharmaceutically acceptable salt thereof.
  • A-0 is a polyene macrolide aglycone moiety
  • O oxygen
  • M is a C2-epi-mycosaminyl moiety
  • O-M is a glycosidic bond between a hydroxy
  • treating and “treat” refer to performing an intervention that results in (a) preventing a condition or disease from occurring in a subject that may be at risk of developing or predisposed to having the condition or disease but has not yet been diagnosed as having it; (b) inhibiting a condition or disease, e.g., slowing or arresting its development; or (c) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease.
  • treating and “treat” refer to performing an intervention that results in (a) inhibiting a condition or disease, e.g., slowing or arresting its development; or (b) relieving or ameliorating a condition or disease, e.g., causing regression of the condition or disease.
  • yeast or fungal infection refers to an infection with a yeast or fungus as defined herein.
  • a subject refers to a living mammal.
  • a subject is a non-human mammal, including, without limitation, a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, cat, dog, pig, horse, cow, or non-human primate.
  • a subject is a human.
  • a "subject having a yeast or fungal infection” refers to a subject that exhibits at least one objective manifestation of a yeast or fungal infection.
  • a subject having a yeast or fungal infection is a subject that has been diagnosed as having a yeast or fungal infection and is in need of treatment thereof.
  • administering has its usual meaning and encompasses
  • administering by any suitable route of administration, including, without limitation, intravenous, intramuscular, intraperitoneal, intrathecal, subcutaneous, direct injection (for example, into a tumor), mucosal, inhalation, oral, and topical.
  • routes of administration including, without limitation, intravenous, intramuscular, intraperitoneal, intrathecal, subcutaneous, direct injection (for example, into a tumor), mucosal, inhalation, oral, and topical.
  • terapéuticaally effective amount refers to any amount that is sufficient to achieve a desired therapeutic effect, e.g., to treat a yeast or fungal infection.
  • the binding avidity for ergosterol of a compound of the invention is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In various individual embodiments, the binding avidity for ergosterol of a compound of the invention is at least 80 percent, at least 85 percent, at least 90 percent, or at least 95 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In an embodiment, the binding avidity for ergosterol of a compound of the invention is at least 100 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety.
  • the binding avidity for cholesterol of a compound of the invention is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In various individual embodiments, the binding avidity for cholesterol of a compound of the invention is less than or equal to 20 percent, less than or equal to 15 percent, less than or equal to 10 percent, or less than or equal to 5 percent, of the binding avidity for cholesterol of the counterpart polyene macrolide comprising a mycosaminyl moiety. In an embodiment, a compound of the invention has essentially no binding avidity for cholesterol.
  • the binding avidity for ergosterol of a compound of the invention is at least 75 percent of the binding avidity for ergosterol of the counterpart polyene macrolide comprising a mycosaminyl moiety; and the binding avidity for cholesterol of the compound of the invention is less than or equal to 25 percent of the binding avidity for cholesterol of the counterpart polyene macrolide comprising a mycosaminyl moiety.
  • the polyene macrolide aglycone moiety is selected from the group consisting of the polyene macrolide aglycone moieties of amphotericin A, arenomycin B, candicidin D, candidin, candidoin, CE-108, etruscomycin, eurocidin D, eurocidin E, FR-008-VI, HA-2-91, hamycin A, levorin AO, levorin A3, mycoheptin, natamycin (pimaricin), nystatin Al, nystatin A2, nystatin A3, partricin A, polyfungin B, rimocidin, tetramycin A, tetramycin B, tetrin A, tetrin B, tetrin C, trichomycin A, trichomycin B, vacidin A, YS-822A, 3874 HI, 3874 H2, 3874 H3, and 67- 121 -A.
  • the compound is administered systemically.
  • the compound is administered intravenously.
  • the compound is administered orally.
  • the compound is administered topically.
  • the compound is administered locally.
  • the compound is administered intraperitoneally.
  • the compound is administered intrathecally.
  • an "effective amount” refers to any amount that is sufficient to achieve a desired biological effect.
  • an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound of the invention being administered, the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular compound of the invention and/or other therapeutic agent without necessitating undue experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to some medical judgment. Multiple doses per day may be contemplated to achieve appropriate systemic levels of compounds.
  • Appropriate systemic levels can be determined by, for example, measurement of the patient's peak or sustained plasma level of the drug. "Dose” and “dosage” are used interchangeably herein.
  • intravenous administration of a compound of the invention may typically be from 0.1 mg/kg/day to 20 mg/kg/day. In one embodiment, intravenous administration of a compound of the invention may typically be from 0.1 mg/kg/day to 2 mg/kg/day.
  • intravenous administration of a compound of the invention may typically be from 0.5 mg/kg/day to 5 mg/kg/day. In one embodiment, intravenous administration of a compound of the invention may typically be from 1 mg/kg/day to 20 mg/kg/day. In one embodiment, intravenous administration of a compound of the invention may typically be from 1 mg/kg/day to 10 mg/kg/day. Intravenous dosing thus may be similar to, or advantageously, may exceed maximal tolerated doses of a given
  • daily oral doses of active compounds will be, for human subjects, from about 0.01 milligrams/kg per day to 1000 milligrams/kg per day. It is expected that oral doses in the range of 0.5 to 50 milligrams/kg, in one or more administrations per day, will yield therapeutic results. Dosage may be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, it is expected that intravenous administration would be from one order to several orders of magnitude lower dose per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds.
  • the therapeutically effective amount can be initially determined from animal models.
  • a therapeutically effective dose can also be determined from human data for compounds of the invention which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration.
  • the applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
  • compositions of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • an effective amount of the compound of the invention can be administered to a subject by any mode that delivers the compound of the invention to the desired surface.
  • Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection (for example, into a tumor or abscess), mucosal (e.g., topical to eye), inhalation, and topical.
  • a compound of the invention generally may be formulated similarly to the corresponding polyene macrolide comprising a mycosaminyl moiety.
  • C2'epiAmB can be formulated as a lyophilized preparation with desoxycholic acid, as a lyophilized preparation of liposome - intercalated or -encapsulated active compound, as a lipid complex in aqueous suspension, or as a cholesteryl sulfate complex.
  • Lyophilized formulations are generally reconstituted in suitable aqueous solution, e.g., in sterile water or saline, shortly prior to administration.
  • the compounds of the invention can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or
  • polyvinylpyrrolidone PVP
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the oral formulations may also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions or may be administered without any carriers.
  • oral dosage forms of the above component or components may be chemically modified so that oral delivery of the derivative is efficacious.
  • the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine.
  • the increase in overall stability of the component or components and increase in circulation time in the body examples include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
  • the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.
  • the stomach the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine.
  • One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine.
  • the release will avoid the deleterious effects of the stomach environment, either by protection of the compound of the invention (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.
  • a coating impermeable to at least pH 5.0 is essential.
  • examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed films.
  • a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow.
  • Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell may be used.
  • the shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.
  • the therapeutic can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
  • the therapeutic could be prepared by compression.
  • Colorants and flavoring agents may all be included.
  • the compound of the invention (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
  • diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
  • Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
  • Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500,
  • Emcompress and Avicell Emcompress and Avicell.
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form.
  • Materials used as disintegrates include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate,
  • Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
  • disintegrants are the insoluble cationic exchange resins.
  • Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
  • Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin.
  • MC methyl cellulose
  • EC ethyl cellulose
  • CMC carboxymethyl cellulose
  • PVP polyvinyl pyrrolidone
  • HPMC hydroxypropylmethyl cellulose
  • Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000. Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents which can be used and can include benzalkonium chloride and benzethonium chloride.
  • Non-ionic detergents that could be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compound of the invention or derivative either alone or as a mixture in different ratios.
  • compositions which can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • compounds of the invention for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • pulmonary delivery of a compound of the invention is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • inhaled molecules include Adjei et al, Pharm Res 7:565-569 (1990); Adjei et al, Int J Pharmaceutics 63:135-144 (1990) (leuprolide acetate); Braquet et al, J
  • Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
  • Ultravent nebulizer manufactured by Mallinckrodt, Inc., St. Louis, Mo.
  • Acorn II nebulizer manufactured by Marquest Medical Products, Englewood, Colo.
  • the Ventolin metered dose inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina
  • the Spinhaler powder inhaler manufactured by Fisons Corp., Bedford, Mass.
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
  • Chemically modified compound of the invention may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.
  • Formulations suitable for use with a nebulizer will typically comprise compound of the invention (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active compound of the invention per mL of solution.
  • the formulation may also include a buffer and a simple sugar (e.g., for compound of the invention stabilization and regulation of osmotic pressure).
  • the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the compound of the invention caused by atomization of the solution in forming the aerosol.
  • Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the compound of the invention (or derivative) suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a
  • hydrochlorofluorocarbon a hydrofluorocarbon, or a hydrocarbon, including
  • Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
  • Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing compound of the invention (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.
  • the compound of the invention (or derivative) should advantageously be prepared in particulate form with an average particle size of less than 10 micrometers ( ⁇ ), most preferably 0.5 to 5 ⁇ , for most effective delivery to the deep lung.
  • Nasal delivery of a pharmaceutical composition of the present invention is also contemplated.
  • Nasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
  • Formulations for nasal delivery include those with dextran or cyclodextran.
  • a useful device is a small, hard bottle to which a metered dose sprayer is attached.
  • the metered dose is delivered by drawing the pharmaceutical composition of the present invention solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed.
  • the chamber is compressed to administer the pharmaceutical composition of the present invention.
  • the chamber is a piston arrangement.
  • Such devices are commercially available.
  • a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used.
  • the opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation.
  • the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.
  • the compounds when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.
  • a suitable vehicle e.g., sterile pyrogen- free water
  • the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • compounds of the invention may also be formulated as a depot preparation.
  • Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto
  • the pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249: 1527- 33 (1990).
  • a compound of the invention and optionally other therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.
  • Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic.
  • such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5%) w/v); and phosphoric acid and a salt (0.8- 2%> w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03%) w/v);
  • chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
  • compositions of the invention contain an effective amount of a compound of the invention and optionally other therapeutic agents included in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • the therapeutic agent(s), including specifically but not limited to compounds of the invention, may be provided in particles.
  • Particles as used herein means nanoparticles or microparticles (or in some instances larger particles) which can consist in whole or in part of the compound of the invention or the other therapeutic agent(s) as described herein.
  • the particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating.
  • the therapeutic agent(s) also may be dispersed throughout the particles.
  • the therapeutic agent(s) also may be adsorbed into the particles.
  • the particles may be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc.
  • the particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof.
  • the particles may be microcapsules which contain the compound of the invention in a solution or in a semi-solid state.
  • the particles may be of virtually any shape.
  • Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s).
  • Such polymers may be natural or synthetic polymers.
  • the polymer is selected based on the period of time over which release is desired.
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney H S et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein.
  • polyhyaluronic acids casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • the therapeutic agent(s) may be contained in controlled release systems.
  • controlled release is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release
  • sustained release also referred to as “extended release”
  • extended release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period.
  • delayed release is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug there from. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.”
  • Long-term sustained release implant may be particularly suitable for treatment of chronic conditions.
  • Long-term release as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days.
  • Long-term sustained release implants are well- known to those of ordinary skill in the art and include some of the release systems described above.
  • C2'epiAmE was modified to allow access to the deprotected material. Specifically, a readily removable allyl ester was employed at the C41 -position. Employing a previously reported route to the fully protected aglycone 5.5 with the mycosamine donor and glycosylation conditions previously used in the construction of C3'deNHAmB, C2'epiAmB was synthesized (FIG. 1).
  • AmB aglycone 5.5 (2.19 g, 1.34 mmol, 1.0 equiv.) was azeotripically dried with benzene (3 x 10 mL) and left on high vac overnight in a 500 mL round bottom flask.
  • Trichloroacetimidate 5.4 (944 mg, 1.93 mmol, 1.44 equiv.) was added to the flask containing 5.5 as a solution in benzene and concentrated down. Hexanes (70 mL) was added and subsequently cooled to 0 °C after the system was placed under an N 2
  • the precipitate was filtered through a 5" pipette containing a small piece of a kim-wipeTM as a filter.
  • the filter cake was then washed with additional Et 2 0 then eluted through the filter with DMSO.
  • the filter was washed further with minimal DMSO.
  • the combined DMSO fractions were lyophilized to yield 5.9 (68.9 mg, 0.714 mmol, 69%) as a yellow powder and taken on to the next reaction without additional purification. By analytical HPLC full conversion to a single peak was observed.
  • etruscomycin eurocidin D, eurocidin E, FR-008-VI, HA-2-91 , hamycin A, levorin AO, levorin A3, mycoheptin, natamycin (pimaricin), nystatin Al , nystatin A2, nystatin A3, partricin A, polyfungin B, rimocidin, tetramycin A, tetramycin B, tetrin A, tetrin B, tetrin C, trichomycin A, trichomycin B, vacidin A, YS-822A, 3874 HI , 3874 H2, 3874 H3, and 67- 121-A are treated with a glycosyltransferase, e.g., a polyene glycosyltransferase, in the presence of C2'-epi-mycosamine, for example in accordance with any of U.S.
  • the resulting compounds are optionally purified by HPLC and characterized using NMR.
  • the resulting compounds are characterized for binding to ergosterol and cholesterol using methods similar to those described in Example 3.
  • the resulting compounds are characterized for antifungal activity using methods similar to those described in Example 4.
  • the resulting compounds are also characterized for toxicity to human cells using methods similar to those described in Example 5.
  • C2'epiAmB binding capability of C2'epiAmB was investigated to determine whether epimerization at C2' impacts the capacity of AmB to bind ergosterol.
  • C2'epiAmB binds to ergosterol, but not cholesterol, within the limits of the binding assay, as indicated by FIG. 3, row A.
  • ITC isothermal titration calorimetry
  • LUVs large unilamellar vesicles
  • POPC l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • the titration was repeated using POPC LUVs containing 10% ergosterol.
  • a significant increase in net exotherm was observed when switching to ergosterol-containing LUVs, indicating a direct AmB-sterol binding interaction.
  • the titration was repeated using C2'epiAmB.
  • a significant increase in net exotherm indicated a retained capacity for the epimeric derivative to bind ergosterol.
  • the ITC assay was also conducted with cholesterol in place of ergosterol. C2'epiAmB was not found to bind to cholesterol.
  • Palmitoyl oleoyl phosphatidylcholine (POPC) was obtained as a 20 mg/mL solution in CHCI 3 from Avanti Polar Lipids (Alabaster, AL) and was stored at -20 °C under an atmosphere of dry argon and used within 1 month.
  • a 4 mg/mL solution of ergosterol in CHCI 3 was prepared monthly and stored at 4 °C under an atmosphere of dry argon. Prior to preparing a lipid film, the solutions were warmed to ambient temperature to prevent condensation from contaminating the solutions.
  • a 13 x 100 mm test tube was charged with 800 POPC and 230 of the ergosterol solution.
  • a 13 x 100 mm test tube was charged with 800 ⁇ , POPC and 224 ⁇ ⁇ of the ergosterol solution.
  • a 13 x 100 mm test tube was charged with 800 ⁇ , POPC.
  • the solvent was removed with a gentle stream of nitrogen and the resulting lipid film was stored under high vacuum for a minimum of eight hours prior to use.
  • the film was then hydrated with 1 mL of K buffer and vortexed vigorously for approximately 3 minutes to form a suspension of multilamellar vesicles (MLVs).
  • MLVs multilamellar vesicles
  • the resulting lipid suspension was pulled into a Hamilton (Reno, NV) 1 mL gastight syringe and the syringe was placed in an Avanti Polar Lipids Mini-Extruder.
  • the lipid solution was then passed through a 0.20 ⁇ Millipore (Billerica, MA) polycarbonate filter 21 times, the newly formed large unilamellar vesicle (LUV) suspension being collected in the syringe that did not contain the original suspension of MLVs to prevent the carryover of MLVs into the LUV solution.
  • LUV large unilamellar vesicle
  • Ergosterol content was determined spectrophotometrically.
  • a 50 portion of the LUV suspension was added to 450 ⁇ , 2: 18:9 hexane:isopropanol:water (v/v/v).
  • Three independent samples were prepared and then vortexed vigorously for approximately one minute.
  • the solutions were then analyzed by UV/Vis spectroscopy and the concentration of ergosterol in solution was determined by the extinction coefficient of 10400 L mol "1 cm "1 at the UV max of 282 nm and was compared to the concentration of phosphorus to determine the percent sterol content.
  • the extinction coefficient was determined independently in the above ternary solvent system. LUVs prepared by this method contained between 7 and 14%) ergosterol.
  • NanoAnalyze software (TA Instruments) was used for baseline determination and integration of the injection heats, and Microsoft Excel was used for subtraction of dilution heats and the calculation of overall heat evolved. To correct for dilution and mixing heats, the heat of the final injection from each run was subtracted from all the injection heats for that particular experiment. See, for example, te Welscher, YM et al. (2008) J. Biol. Chem. 283:6393. By this method, the overall heat evolved during the experiment was calculated using the following formula:
  • C. albicans represents the most common cause of life-threatening systemic fungal infections in humans.
  • Exemplary methods for antifungal activity assays are as follows: Growth Conditions for S. cerevisiae
  • yeast peptone dextrose (YPD) growth media consisting of 10 g/L yeast extract, 20 g/L peptone, 20 g/L dextrose, and 20 g/L agar for solid media.
  • the media was sterilized by autoclaving at 250 °F for 30 min.
  • Dextrose was subsequently added as a sterile 40% w/v solution in water (dextrose solutions were filter sterilized).
  • Solid media was prepared by pouring sterile media containing agar (20 g/L) onto Corning (Corning, NY) 100 x 20 mm polystyrene plates. Liquid cultures were incubated at 30 °C on a rotary shaker and solid cultures were maintained at 30 °C in an incubator. Growth Conditions for C. albicans
  • C. albicans was cultured in a similar manner to S. cerevisiae except both liquid and solid cultures were incubated at 37 °C.
  • the solution was diluted 10-fold with YPD, and 195 aliquots of the dilute cell suspension were added to sterile Falcon (Franklin Lakes, NJ) Microtest 96-well plates in triplicate.
  • Compounds were prepared either as 400 ⁇ (AmB, C2'deOAmB) or 2 mM (AmdeB) stock solutions in DMSO and serially diluted to the following concentrations with DMSO: 1600, 1200, 800, 400, 320, 240, 200, 160, 120, 80, 40, 20, 10 and 5 ⁇ . 5 ⁇ aliquots of each solution were added to the 96-well plate in triplicate, with each column representing a different concentration of the test compound.
  • the concentration of DMSO in each well was 2.5% and a control well to confirm viability using only 2.5% DMSO was also performed in triplicate. This 40-fold dilution gave the following final concentrations: 50, 40, 30, 20, 10, 8, 6, 4, 1, 0.5, 0.25 and 0.125 ⁇ .
  • the plates were covered and incubated at 30 °C (S. cerevisiae) or 37 °C (C. albicans) for 24 hours prior to analysis.
  • the MIC was determined to be the concentration of compound that resulted in no visible growth of the yeast.
  • the experiments were performed in duplicate and the reported MIC represents an average of two experiments.
  • Example 5 C2'epiAmB Is Not Toxic to Human Cells In Vitro
  • the activity of AmB, C2'deOAmB, and C2'epiAmB was probed against human cells.
  • Two of the most important toxic side effects associated with AmB are anemia and nephrotoxicity caused by damage to red blood cells and renal proximal tubule cells, respectively.
  • 5a ' 12 Consistent with literature precedent, AmB causes 90% hemolysis of human red blood cells at a concentration of 8.5 ⁇ . This is defined as the minimum hemolysis concentration (MHC).
  • MHC minimum hemolysis concentration
  • erythrocyte pellet was suspended in 1 mL of RBC buffer (10 mM NaH 2 P0 4 , 150 mM NaCl, 1 mM MgCl 2 , pH 7.4) to form the erythrocyte stock suspension.
  • MHC Minimum Hemolysis Concentration
  • Positive and negative controls were prepared by adding 1 ⁇ , of DMSO to MilliQ water or RBC buffer, respectively to 0.2 mL PCR tube. To each PCR tube, 0.63 ⁇ , of the erythrocyte stock suspension was added and mixed by inversion. The samples were incubated at 37 °C for 2 hours. The samples were mixed by inversion and centrifuged at 10,000 g for 2 minutes. 15 ⁇ ⁇ of the supernatant from each sample was added to a 384-well place. Absorbances were read at 540 nm using a Biotek HI Synergy Hybrid Reader (Winooski, VT). Experiments were performed in triplicate and the reported MHC represents an average of three experiments.
  • % hemolysis A _ x
  • Concentration vs. percent hemolysis was plotted and fitted to 4-parameter logistic (4PL) dose response fit using OriginPro 8.6. Sebaugh, JL (2011) Pharmaceut. Statist. 10: 128- 134.
  • the MHC was defined as the concentration to cause 90% hemolysis.
  • RPTECs Primary human renal proximal tubule epithelial cells
  • ATCC Manassas, VA
  • Complete growth media was prepared using renal epithelial cell basal medium (ATCC, PCS-400-030), renal epithelial cell growth kit (ATCC, PCS-400-040), and penicillin-streptomycin (10 units/mL and 10 ⁇ g/mL).
  • Complete media was stored at 4 °C in the dark and used within 28 days.
  • Primary RPTECs were grown in C0 2 incubator at 37 °C with an atmosphere of 95% air/5% C0 2 .
  • WST-8 cell proliferation assay kit (10010199) was purchased from Cayman
  • a suspension of primary RPTECs in complete growth media was brought to a concentration of 1 x 10 5 cells/mL.
  • a 96-well plate was seeded with 99 ⁇ ⁇ of the cell suspension and incubated at 37 °C with an atmosphere of 95% air/5% C0 2 for 3 hours.
  • Positive and negative controls were prepared by seeding with 100 ⁇ , of the cell suspension or 100 ⁇ ⁇ of the complete media.
  • the 96-well plate was incubated at 37 °C with an atmosphere of 95% air/5% C0 2 for 24 hours. After incubation, the media was aspirated and 100 ⁇ , of serum- free media was added and 10 ⁇ , of the WST-8 reagent solution was added to each well.
  • the 96-well plate was mixed in a shaking incubator at 200 rpm for 1 minute and incubated at 37 °C with an atmosphere of 95%) air/5%) C0 2 for 2 hours. Following incubation, the 96-well plate was mixed in a shaking incubator at 200 rpm for 1 minute and absorbances were read at 450 nm using a Biotek HI Synergy Hybrid Reader (Winooski, VT). Experiments were performed in triplicate and the reported cytotoxicity represents an average of three experiments. Data Analysis
  • Percent hemolysis was determined according to the following equation:
  • Concentration vs. percent hemolysis was plotted and fitted to 4-parameter logistic (4PL) 8 dose response fit using OriginPro 8.6.
  • the MTC was defined as the concentration to cause 90%) loss of cell viability.
  • reaction was quenched at 0 °C with saturated aqueous sodium bicarbonate (100 mL), diluted with Et 2 0 (50 mL), and the layers were separated. The organic layer was washed with brine (100 mL), the aqueous layers were extracted with Et 2 0 (3 x 50 mL), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to yield lactol S2 (628 mg, 4.91 mmol, 100% yield) as a clear, colorless oil. An aliquot was removed and matched previous data. Guo H et al., Angew Chem Int Ed 46: 5205 (2007). This material was used in the next reaction without further purification.
  • the reaction was warmed to 0 °C and quenched with saturated aqueous sodium bicarbonate (20 mL), the layers were separated, and the organic layer was washed with brine (20 mL). The combined aqueous layers were extracted with ether (3 x 25 mL), the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure.
  • triphenylphosphine (0.056 g, 0.22 mmol, 2.1 mol %) and Pd(dba) 2 (0.031 g, 0.054 mmol, 5.0 mol %).
  • the solution turned colors from dark red/purple upon addition of Pd(dba) 2 to green over 45 min, and was then warmed to ambient temperature and washed with saturated aqueous sodium bicarbonate (10 mL) and Et 2 0 (20 mL). The layers were separated, the aqueous layer was extracted with Et 2 0 (3 x 10 mL), and the combined organic layers were dried over sodium sulfate, filtered, concentrated under reduced pressure.
  • Example 8 Deprotection of Protected Polyene Macro lide Including C2'-Epi-Mycosamine Glycoside Comprising an Azide Moiety

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Wood Science & Technology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Agronomy & Crop Science (AREA)
  • Epidemiology (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Dentistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention concerne des macrolides antifongiques C2'-épi-polyènes à indice thérapeutique considérablement amélioré par rapport aux macrolides de polyènes correspondant qui portent de la mycosamine. L'invention concerne également des procédés de fabrication des macrolides antifongiques C2'-épi-polyènes, des compositions pharmaceutiques comprenant les macrolides antifongiques C2'-épi-polyènes, des procédés d'inhibition de la croissance d'une levure ou d'un champignon avec les macrolides antifongiques C2'-épi-polyènes, et des procédés de traitement d'une infection fongique avec les macrolides antifongiques C2'-épi-polyènes.
PCT/US2015/041710 2014-07-23 2015-07-23 Dérivés macrolides de polyènes antifongiques à toxicité réduite pour les mammifères Ceased WO2016014779A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201462028068P 2014-07-23 2014-07-23
US62/028,068 2014-07-23

Publications (1)

Publication Number Publication Date
WO2016014779A1 true WO2016014779A1 (fr) 2016-01-28

Family

ID=55163750

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/041710 Ceased WO2016014779A1 (fr) 2014-07-23 2015-07-23 Dérivés macrolides de polyènes antifongiques à toxicité réduite pour les mammifères

Country Status (1)

Country Link
WO (1) WO2016014779A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9957290B2 (en) 2015-04-15 2018-05-01 Sfunga Therapeutics, Inc. Derivatives of amphotericin B
CN114502173A (zh) * 2019-08-08 2022-05-13 伊利诺伊大学评议会 具有降低的毒性的混合两性霉素b衍生物
WO2024010968A3 (fr) * 2022-07-08 2024-03-07 The Board Of Trustees Of The University Of Illinois Transfert de sucres épimérisés en c2 à l'amphotéricine b aglyconique

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020165250A1 (en) * 1996-07-12 2002-11-07 Holt Dennis A. Materials and method for treating or preventing pathogenic fungal infection
US20090186838A1 (en) * 2006-02-23 2009-07-23 Eidgenössische Technische Hochschule Zürich Amphotericin Derivatives
US20090221520A1 (en) * 2005-03-23 2009-09-03 Francisco Malpartida Romero Polyene Antibiotics, Compositions Containing Said Antibiotics, Method and Micro-Organisms Used to Obtain Same and Applications Thereof
WO2013132014A1 (fr) * 2012-03-09 2013-09-12 Blirt S.A. Dérivés semi-synthétiques de nystatine a1
WO2014059436A1 (fr) * 2012-10-12 2014-04-17 The Board Of Trustees Of The University Of Illinois Ajustement électronique de sélectivité de site
WO2014165676A1 (fr) * 2013-04-03 2014-10-09 The Board Of Trustees Of The University Of Illinois Dérivé d'amphotéricine b à toxicité réduite

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020165250A1 (en) * 1996-07-12 2002-11-07 Holt Dennis A. Materials and method for treating or preventing pathogenic fungal infection
US20090221520A1 (en) * 2005-03-23 2009-09-03 Francisco Malpartida Romero Polyene Antibiotics, Compositions Containing Said Antibiotics, Method and Micro-Organisms Used to Obtain Same and Applications Thereof
US20090186838A1 (en) * 2006-02-23 2009-07-23 Eidgenössische Technische Hochschule Zürich Amphotericin Derivatives
WO2013132014A1 (fr) * 2012-03-09 2013-09-12 Blirt S.A. Dérivés semi-synthétiques de nystatine a1
WO2014059436A1 (fr) * 2012-10-12 2014-04-17 The Board Of Trustees Of The University Of Illinois Ajustement électronique de sélectivité de site
WO2014165676A1 (fr) * 2013-04-03 2014-10-09 The Board Of Trustees Of The University Of Illinois Dérivé d'amphotéricine b à toxicité réduite

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MATSUMORI ET AL.: "Mycosamine Orientation of Amphotericin B Controlling Interaction with Ergosterol:Sterol-Dependent Activity of Conformation-Restricted Derivatives with an Amino- Carbonyl Bridge", J AM CHEM SOC., vol. 127, no. 30, 3 August 2005 (2005-08-03), pages 10667 - 10675 *
MITCHELL ET AL.: "Probing the Role of the Mycosamine C2'-OH on the Activity of Amphotericin B", ORG. LETT., vol. 13, no. 6, 2011, pages 1390 - 1393, XP055090264, DOI: doi:10.1021/ol2000765 *
WILCOCK ET AL.: "The C2'-OH of Amphotericin B Plays an Important Role in Binding the Primary Sterol of Human But Not Yeast Cells", J AM CHEM SOC., vol. 1 35, no. 23, 12 June 2013 (2013-06-12), pages 8488 - 8491, XP055090253, DOI: doi:10.1021/ja403255s *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9957290B2 (en) 2015-04-15 2018-05-01 Sfunga Therapeutics, Inc. Derivatives of amphotericin B
US10597420B2 (en) 2015-04-15 2020-03-24 Sfunga Therapeutics, Inc. Derivatives of amphotericin B
US10882883B2 (en) 2015-04-15 2021-01-05 Sfunga Therapeutics, Inc. Derivatives of amphotericin B
CN114502173A (zh) * 2019-08-08 2022-05-13 伊利诺伊大学评议会 具有降低的毒性的混合两性霉素b衍生物
JP2022543421A (ja) * 2019-08-08 2022-10-12 ザ ボード オブ トラスティーズ オブ ザ ユニヴァーシティ オブ イリノイ 毒性が低減されたハイブリッドアムホテリシンｂ誘導体
WO2024010968A3 (fr) * 2022-07-08 2024-03-07 The Board Of Trustees Of The University Of Illinois Transfert de sucres épimérisés en c2 à l'amphotéricine b aglyconique

Similar Documents

Publication Publication Date Title
EP3142672B1 (fr) Dérivé d'amphotéricine b à toxicité réduite
EP3055030B1 (fr) Dérivés d'amphotéricine b ayant un indice thérapeutique amélioré
WO2014165676A1 (fr) Dérivé d'amphotéricine b à toxicité réduite
US20230016424A1 (en) Hybrid amide derivatives of amphotericin b
WO2016014779A1 (fr) Dérivés macrolides de polyènes antifongiques à toxicité réduite pour les mammifères
WO2007096137A1 (fr) Dérivés d'amphotéricine
KR20030031479A (ko) 포유동물에서 세균 감염 또는 원생생물 감염을 치료 또는 예방하기 위한 아잘라이드 항생제 조성물
US10683318B2 (en) Scalable synthesis of reduced toxicity derivative of amphotericin B
AU2016205187A1 (en) Concise synthesis of urea derivatives of amphotericin B
AU2011255633B2 (en) Methods of treating recurring bacterial infection
CN101166749B (zh) 6-11桥联的肟乙基琥珀酸红霉素酯衍生物
WO2016040779A1 (fr) Dérivés d'urée d'antibiotiques macrolides polyéniques
WO2014049356A1 (fr) Dérivés de kétolides d'érythromycine portant des modifications en c-10
Masood-ur-Rahman et al. Synthesis and antimicrobial activity of triazolyl analogs of diosgenin
US9126983B2 (en) Extracts from kibdelos porangium as antibacterial agents
Амангелді et al. Polyene Antibiotics Used in Antifungal Therapy
WO2013178622A1 (fr) Nouveaux dérivés de monosaccharide et applications biologiques de ceux-ci
JP2008513486A (ja) 抗生物質化合物
Endo Synthesis-enabled understanding of the mechanism of action of amphotericin B and the development of increased therapeutic derivatives
Ibrahim Studies towards the efficient isolation and purification of analogues of amphotericin B from Streptomyces nodosus
MX2007013730A (es) Derivados de eritromicina oxima con puente 6-11.
NZ757647B2 (en) Amphotericin B derivatives with improved therapeutic index

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15824552

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15824552

Country of ref document: EP

Kind code of ref document: A1