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WO2000063240A9 - AGENTS ANTIFONGIQUES ISOLES A PARTIR DU $i(PSEUDOMONAS SYRINGAE) - Google Patents

AGENTS ANTIFONGIQUES ISOLES A PARTIR DU $i(PSEUDOMONAS SYRINGAE)

Info

Publication number
WO2000063240A9
WO2000063240A9 PCT/US2000/008724 US0008724W WO0063240A9 WO 2000063240 A9 WO2000063240 A9 WO 2000063240A9 US 0008724 W US0008724 W US 0008724W WO 0063240 A9 WO0063240 A9 WO 0063240A9
Authority
WO
WIPO (PCT)
Prior art keywords
syringae
depsidecapeptide
compnses
decapeptide
synngae
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/US2000/008724
Other languages
English (en)
Other versions
WO2000063240A1 (fr
Inventor
Palaniappan Kulanthaivel
Matthew David Belvo
James William Martin
Douglas Joseph Zeckner
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.)
Eli Lilly and Co
Original Assignee
Eli Lilly and Co
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
Priority to AU41886/00A priority Critical patent/AU4188600A/en
Priority to BR0009732-2A priority patent/BR0009732A/pt
Priority to EA200101086A priority patent/EA200101086A1/ru
Priority to CA002371363A priority patent/CA2371363A1/fr
Priority to JP2000612328A priority patent/JP2002542258A/ja
Priority to MXPA01010244A priority patent/MXPA01010244A/es
Application filed by Eli Lilly and Co filed Critical Eli Lilly and Co
Priority to EP00921592A priority patent/EP1173473A1/fr
Publication of WO2000063240A1 publication Critical patent/WO2000063240A1/fr
Publication of WO2000063240A9 publication Critical patent/WO2000063240A9/fr
Priority to NO20014938A priority patent/NO20014938L/no
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K11/00—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K11/02—Depsipeptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof cyclic, e.g. valinomycins ; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to P. syringae depsidecapeptides, method for making such peptide, and methods employing antifungal activity of these peptides.
  • Fungal infections are a significant cause of disease, degradation of quality of life, and mortality among humans, particularly for immune compromised patients.
  • the incidence in fungal infections in humans has increased greatly m the past 20 years. This is m part due to increased numbers of people with immune systems weakened or threatened by organ transplants, cancer chemotherapy, AIDS, age. and other similar disorders or conditions.
  • Such patients are prone to attack by fungal pathogens that are prevalent throughout the population but are kept in check by a functioning immune system.
  • These pathogens are difficult to control because some existing antifungal agents are either highly toxic or only inhibit fungal activity
  • the polyenes are fungicidal but toxic; whereas, the azoles are much less toxic but only fungistatic. More importantly, there have been recent reports of azole and polyene resistant strains of Candida which severely limits therapy options against such strains.
  • Pseudomonas sMringae produce several classes of antifungal or antibiotic agents, such as the pseudomycms, sy ⁇ ngomycins, sy ⁇ ngotoxins, and sy ⁇ ngostatins. which are podepsmonapeptides. Natural strains and transposon generated mutants of P. syringae produce these hpodepsmonapeptides Several of the pseudomycms. sy ⁇ ngomycins and other hpodepsipeptide antifungal agents have been isolated, chemically characte ⁇ zed.
  • the pseudomycms, the sy ⁇ ngomycins. the sv ⁇ ngotoxms, and the sy ⁇ ngostatins represent structurally distinct families of antifungal compounds
  • the present invention provides a depsidecapeptide produced by P syringae which contains the unusual amino acids homose ⁇ ne (Hse), dehydroam obuty ⁇ c acid (Dhb) and dehydroalanine (Dha) as part of a depsidecapeptide ⁇ ng More specifically, the P syringae depsidecapeptide includes a depsidecapeptide ⁇ ng having the amino acids, argmme, threonine, homose ⁇ ne, dehydroam obuty ⁇ c acid, and dehydroalanine.
  • the depsidecapeptide is a lipodepsidecapeptide a cyclic peptide coupled to a lipophi c moiety
  • the hpophilic moiety is a fatt ⁇ acid moiety coupled to the amino group of the threonine by an amide bond
  • the fatty acid moiety is an rc-dodecanoic acid moiety
  • the lipodepsidecapeptide is represented by formula I
  • the lipophi c moiety includes C -C ⁇ 5 alkyl, C 9 -C ⁇ 5 hydroxyalkyl, C -C[ 5 dihydroxyalkyl, Cg-C ⁇ 5 alkenyl, C -C
  • the lipophihc moiety is Cn alkyl
  • the alkyl, hydroxyalkyl, dihydroxyalkyl, alkenyl, hydroxyalkenyl. or dihydroxyalkenyl groups may be branched or unbranched.
  • the amino acid sequence of the depsidecapeptide ⁇ ng is threonine-alanine-threonine-glutamme-homose ⁇ ne-dehydroaminobuty ⁇ c acid-alanme- dehydroalanine-threonine-argm e, referred to herein as "25-B1 decapeptide" or "Thr-Ala-Thr-Gln-Xaa-Xaa-Ala-Xaa-Thr-Arg (SEQ ID NO: 1)"
  • the invention also relates to methods employing a P syringae depsidecapeptide for inhibiting fungal activity or for reducing the symptoms ot a fungal infection in a patient in need thereof.
  • Such methods can kill the fungus, decrease the burden of a fungal infection, reduce fever and increase general well being ot a patient
  • Consequentlv the P syringae depsidecapeptides mav be used in the manufacture ot a medicament for treatment of a patient as desc ⁇ bed herein
  • the methods and medicaments ot the invention are effective against fungi such as Candida paraps ⁇ osis, Candida albicans, Crvptococcus neoformans or Histoplasma capsulatum
  • the invention provides using microorganisms in a method for producing an antifungal agent, such as the P syringae depsidecapeptides desc ⁇ bed above and including a 25-B 1 decapeptide
  • the method involves cultu ⁇ ng Pseudomonas syringae in media including three or fewer am o acids and recove ⁇ ng one or more P syringae depsidecapeptides from the culture
  • P syringae culture is in medium including glycme and a pid, a potato product, or a combination thereof at a pH of about 4 to 6 5 until one or more P syringae depsidecapeptides is produced at a concentration of at least about 10 ⁇ g/mL
  • the invention provides P syringae depsidecapeptides prepared by the method desc ⁇ bed above
  • the invention also provides a method for treating or preventing fungal growth in a plant whereby a fungus is
  • lipodepsidecapeptide antifungal agent refers to an antifungal agent having a cyclic decapeptide ⁇ ng closed by a lactone group and having an appended hydrophobic group, such as a fatty acid moiety
  • Lipodepsidecapeptide antifungal agents are produced by Pseudomonas syringae A representative of this class of compounds, 25- Bl decapeptide antifungal agent A, has been pu ⁇ fied and its structure determined
  • P syringae lipodepsidecapeptide ' refers to a lipodepsidecapeptide antifungal agent produced by P syringae. and includes 25-B1 decapeptide antifungal agent A and related analogs
  • each ot these antifungal agents includes the unusual amino acids homose ⁇ ne (Hse) dehydroaminobuty ⁇ c acid (Dhb) and dehydroalanine (Dha) as part of a depsidecapeptide ⁇ ng
  • Hse dehydroaminobuty ⁇ c acid
  • Dha dehydroalanine
  • a carboxyl group of an argimne residue linked to the hydroxyl group of the N-termmal threonine forms a lactone that closes the depsidecapeptide ⁇ ng.
  • the sequence of the depsidecapeptide ⁇ ng of the P syringae lipodepsidecapeptide can be represented as:
  • the hpodepsidecapeptides of the present invention do not contain chlorothreomne which is suspected to be the cause for ir ⁇ tation at the injection site of pharmaceutical formulations containing pseudomycm compounds.
  • the depsidecapeptide ⁇ ng is linked to a lipophihc moiety, such as a fatty acid, through an amide bond with an amino group of the N-termmal threonine.
  • the fatty acid generally includes 10, 12, 14, or 16 carbons, typically bea ⁇ ng zero, one or two hydroxyl groups.
  • the fatty acid may be branched or unbranched and may also contain at least one unsaturation.
  • Preferred fatty acid moieties include an rc-decanoic acid moiety, an n- decanoic acid moiety substituted with one or two hydroxyl groups, an n-dodecanoic acid moiety, an / ⁇ -dodecano ⁇ c acid moiety substituted with one or two hydroxyl groups, an ..- tetradecanoic acid moiety, or an rc-tetradecanoic acid moiety substituted with one or two hydroxyl groups.
  • 25-Bl decapeptide antifungal agent refers to one or more members of a family of antifungal agents that has been isolated from the bacte ⁇ um Pseudomonas syringae.
  • a 25-Bl decapeptide antifungal agent is a P. syringae lipodepsidecapeptide.
  • a 25-Bl decapeptide antifungal agent is a P. syringae lipodepsidecapeptide having a depsidecapeptide ⁇ ng with the sequence:
  • Each of the 25-Bl decapeptide antifungal agents has the same cyclic peptide nucleus, but they differ in the hydrophobic side chain attached to this nucleus.
  • the 25-Bl decapeptide antifungal agents include 25-B 1 decapeptide antifungal agent A
  • the 25-Bl decapeptide antifungal agents include a fatty acid linked through an amide bond with the ammo group of the N-terminal threonine.
  • the fatty acid moiety ot 25-Bl decapeptide antifungal agent A is an ⁇ -dodecanoic acid moiety
  • a P. syringae lipodepsidecapeptide. such as 25-B 1 decapeptide antifungal agent A. has several biological activities including killing and inhibiting activity of va ⁇ ous fungi, such as fungal pathogens of plants and animals.
  • a 25-Bl decapeptide antifungal agent is an active antimycotic agent against fungi that cause opportunistic infections in immune compromised individuals. These fungi include Cryptococcus neoformans, Histoplasma capsulatum and va ⁇ ous species of Candida including C. paraps ⁇ osis and C. albicans.
  • Pseudomonas synnsae Pseudomonas synngae include a wide range of bacte ⁇ a that are generally associated with plants. Some of the P. synngae are plant pathogens, while others are only weakly pathogenic or are saprophytes. Many different isolates of P. synngae produce one or more cytotoxic agents that can help this bacte ⁇ um survive in the wild where it must compete with fungi and other bacte ⁇ a. The cytotoxic agents produced by P. syringae include anti-fungal agents such as the P.
  • synngae hpodepsidecapeptides including 25-Bl decapeptide antifungal agent A, the pseudomycms, the sy ⁇ ngomycins, the sy ⁇ ngotoxins. and the sy ⁇ ngostatms.
  • Strains of P. syringae that are suitable for production of one or more P. syringae Hpodepsidecapeptides can be isolated from environmental sources including plants such as barley plants, citrus plants, and lilac plants, and from forest floor litter, soil, water, air, and dust.
  • the present invention includes a strain, an isolate, and a biologically-purified culture of P. syringae that produce one or more P.
  • the biologically-purified culture of a microorganism is of Pseudomonas syringae strains MSU 16H, 25-Bl, 67H1, 7H9-1, or a pseudomycin-producing mutant, variant, isolate, or recombinant of these strains.
  • Cultures of MSU 16H are on deposit at Montana State University (Bozeman, Montana, USA) and available from the American Type Culture Collection (Parklawn Drive, Rockville, MD, USA) Accession No. ATCC 67028.
  • a strain of P. syringae that is suitable for production of one or more P. syringae Hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, can be isolated from environmental sources including plants, such as barley plants, citrus plants, and Hlac plants, and also from sources such as soil, water, air, and dust.
  • a preferred strain is isolated from plants.
  • These environmental isolates of P. syringae can be referred to as wild type.
  • wild type refers to a dominant genotype which naturally occurs in the normal population of P. syringae (i.e., strains or isolates of P. syringae that are found in nature and not produced by laboratory manipulation).
  • wild type refers to a dominant genotype which naturally occurs in the normal population of P. syringae (i.e., strains or isolates of P. syringae that are found in nature and not produced by laboratory manipulation).
  • RECTIFIED SHEET (RULE 91) ISA/EP 25-Bl. and 7H9 are subject to va ⁇ ation.
  • progeny of these strains e.g.. recombmants, mutants and va ⁇ ants, may be obtained by methods well-known to those skilled in the art.
  • Mutant strains of P. synngae are also suitable for production of one or more P. syringae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A
  • mutant refers to a sudden he ⁇ table change in the phenotype of a strain, which can be spontaneous or induced by known mutagenic agents, including radiation and va ⁇ ous chemicals.
  • Mutant P. syringae of the present invention can be produced using a va ⁇ ety of mutagenic agents including radiation such as ultraviolet light, and x-rays: chemical mutagens; site-specific mutagenesis; and transposon mediated mutagenesis. Examples of chemical mutagens are ethyl methyl sulfonate (EMS), diepoxyoctane. N- methyl-N-nitro-N'-mtrosoguanine (NTG), and nitrous acid.
  • EMS ethyl methyl sulfonate
  • P syringae suitable for producing one or more P. syringae hpodepsidecapeptides such as 25-B l decapeptide antifungal agent A.
  • P syringae suitable for producing one or more P. syringae hpodepsidecapeptides can be generated by treating the bacte ⁇ a with an amount of a mutagenic agent effective to produce mutants that overproduce one or more P. syringae hpodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A. or that produce one or more P. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, under advantageous growth conditions.
  • mutants of the invention are those that overproduce 25-B 1 decapeptide antifungal agent A, and grow in minimal medium.
  • the mutants overproduce a P. synngae lipodepsidecapeptide. such as 25-B 1 decapeptide antifungal agent A, preferably from at least about 10 ⁇ g/mL to about 50 ⁇ g /mL.
  • synngae can be subjected to selection for desirable traits of growth habit, growth medium, nut ⁇ ent source, carbon source, growth conditions, and ammo acid requirements
  • a strain of P synngae producing P. syringae lipodepsidecapeptide such as 25-B l decapeptide antifungal agent A. is selected for growth on minimal defined medium
  • Preferred strains exhibit the characte ⁇ stics of producing one or more P. synngae hpodepsidecapeptides. such as 25-Bl decapeptide antifungal agent A. when grown on a medium including glycine plus, optionally, a hpid. a potato product, or both
  • Recombinant strains can be developed by transforming the P synngae strains, using procedures well-known to those skilled in the art. Through the use of recombinant technology, the P. synngae strains can be transformed to express a va ⁇ ety of gene products in addition to the antibiotics these strains produce. For instance, one can transform the strains with a recombinant vector that confers resistance to an antibiotic to which the strains are normally sensitive.
  • Transformants thus obtained will produce not only a P synngae lipodepsidecapeptide, such as 25-Bl decapeptide antifungal agent A, but also the resistance-confemng enzyme that allows selection of the transformed from wild type cells Furthermore, using similar techniques, one can modify the present strains to introduce multiple copies of the endogenous hpodepsidecapeptide-biosynthesis genes to achieve greater lipodepsidecapeptide yield. Progeny, i.e. natural and induced va ⁇ ants, mutants and recombinants, of the P. synngae strains 25-Bl, 67H1, and 7H9-1 which retain the characte ⁇ stic of lipodepsidecapeptide production are part of this invention
  • aqueous nut ⁇ ent media refers to a water-base composition including minerals and organic compounds and their salts necessary for growth of the bacte ⁇ um used in the present invention.
  • Preferred nut ⁇ ent media contain an effective amount of three or fewer amino acids, preferably, glutamic acid, glycine. histidine, or a combination thereof.
  • the medium contains an effective amount of glycine and, optionally, one or more of a potato product and a pid.
  • Glycme can be provided as a single ammo acid or as part of a mixture of amino acids, such as hydrolyzed protein Suitable hpids include soybean oil, fatty acids, or fatty acid esters.
  • Suitable potato products include potato dextrose broth, potato dext ⁇ n, potato protein, or a commercial mashed potato mix food product.
  • Preferred minerals in the nut ⁇ ent medium include salt mixtures typically used in cell culture and fermentation, such as Czapek mineral salts, which includes KC1. MgS0 , and FeSOj..
  • Organic compounds in the nut ⁇ ent media preferably includes glucose and can optionally include soluble starch; other like organic compounds can also be included.
  • the pH of the medium is preferably between about 4 and 6.5, more preferably about 4.5 to about 5 7, most preferably about 5.2.
  • a preferred amount of glycine is about 0.1 g/L to about 10 g/L, more preferably about 0.3 g/L to about 3 g/L, most preferably about 1 g/L.
  • a preferred amount of hpid is about 1 g/L to about 10 g/L of an oil product such as soybean oil, more preferably about 0.5 g/L to about 2 g/L of soybean oil.
  • a preferred amount of a fatty acid or fatty acid ester is about 0.5 g L to about 5 g/L.
  • Preferred amounts of potato products include about 12 g/L to about 36 g L, more preferably about 24 g/L of potato dextrose broth; about 5 g/L to about 50 g/L, preferably about 30 g/L of a commercial mashed potato mix; about 1 g L to about 30 g/L, preferably about 20 g/L of potato dext ⁇ n; and/or about 1 g/L to about 10 g/L, preferably about 4 g/L of potato protein.
  • a preferred nut ⁇ ent medium includes minerals, preferably, KC1 at about 0.02 to about 2 g/L, more preferably about 0.2 g/L; MgSO 4 , preferably MgSO 4 »7H 2 O, at about 0.02 to about 2 g/L, more preferably about 0.2 g/L; and FeSO 4 , preferably FeSO »7H 2 O, at about 0.4 to about 40 mg/L. more preferably about 4 mg/L.
  • soluble starch is preferably at about 0.5 to about 50 g/L, more preferably about 5 g/L.
  • Glucose is preferably present at about 2 to about 80 g/L, more preferably about 20 g L.
  • P. syringae are typically grown in the media desc ⁇ bed under conditions of controlled or regulated pH, and temperature.
  • P. synngae grow and produce one or more cytotoxic agents at temperatures between about 15 °C and about 35 °C, preferably about 20 °C to about 30 °C, more preferably about 25 °C.
  • P. synngae grow and produce one or more cytotoxic agents at pH between about 4 and about 9, more preferably between about 4 and about 6, most preferably from about 4.5 to about 5.5.
  • growth of P. synngae does not occur when the temperature is above about 37° C or below 10° C or when the pH is above about 9 or below about 4 Method for Producing a P. synngae Lipodepsidecapeptide
  • P. synngae hpodepsidecapeptides such as 25-Bl decapeptide antifungal agent A
  • the organism is cultured with agitation in an aqueous nutrient medium including an effective amount of three or fewer amino acids.
  • the three or fewer amino acids are preferably glutamic acid, glycine, histidine, or a combination thereof.
  • the amino acids include glycine and, optionally, one or more of a potato product and a lipid. Cultu ⁇ ng is conducted under conditions effective for growth of P. synngae and production of a desired P.
  • syringae lipodepsidecapeptide such as 25-Bl decapeptide antifungal agent A.
  • Effective conditions include a temperature of about 22° C to about 27° C, and a duration of about 36 hours to about 96 hours.
  • P. synngae can grow at cell densities up to about 10-15 g/L dry weight and produce a P. synngae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, in a total amount at least about 10 ⁇ g/mL, preferably at least about 50 ⁇ g/mL.
  • Controlling the concentration of oxygen in the medium du ⁇ ng cultu ⁇ ng of P. syringae is advantageous for production of a P syringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A
  • oxygen levels are maintained at about 5% to about 50% saturation, more preferably about 30% saturation. Sparging with air, with pure oxygen, or with gas mixtures including oxygen can regulate the concentration of oxygen in the medium. Further, adjustment of the agitation rate can be used to adjust the oxygen transfer rate.
  • Controlling the pH of the medium du ⁇ ng cultu ⁇ ng of P. synngae is advantageous for production of a P. syringae lipodepsidecapeptide, such as 25-Bl decapeptide antifungal agent A.
  • the pH of the culture medium can be maintained at less than about 6 and above about 4.
  • P syringae can produce a P synngae lipodepsidecapeptide. such as 25-Bl decapeptide antifungal agent A, when grown in batch culture.
  • fed-batch or semi-contmuous feed of glucose and. optionally, an acid or base, such as ammonium hydroxide, to control pH. enhances production of a P. synngae lipodepsidecapeptide. such as 25-Bl decapeptide antifungal agent A.
  • Production of a P. syringae lipodepsidecapeptide, such as 25-Bl decapeptide antifungal agent A. by P. syringae can be further enhanced by using continuous culture methods in which glucose and. optionally, an acid or base, such as ammonium hydroxide, to control pH, are fed automatically.
  • the pH is preferably maintained at a pH of about 5 to about 5.4. more preferably about 5.0 to about 5.2.
  • Choice of P. syringae strain can affect the amount and dist ⁇ bution of a P. syringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, produced by cultu ⁇ ng under the conditions described herein.
  • a P. syringae lipodepsidecapeptide such as 25-B 1 decapeptide antifungal agent A
  • strain 25 Bl can produce predominantly 25-Bl decapeptide antifungal agent A.
  • the cyclic decapeptide nucleus of the P. syringae hpodepsidecapeptides can be prepared by cleaving off the lipophihc moiety, such as by deacylation. Cleavage and deacylation methods are well-known to those skilled in the art, such as the use of deacylase enzymes.
  • a P. syringae lipodepsidecapeptide. such as 25-Bl decapeptide antifungal agent A. shows in vitro and in vivo activity and is useful in combating either systemic fungal infections or fungal skin infections. Accordingly, the present invention provides a method of inhibiting fungal activity including contacting a P. syringae lipodepsidecapeptide, such as a 25-Bl decapeptide antifungal agent, or a pharmaceutically acceptable salt thereof, with a fungus.
  • a preferred method includes inhibiting growth or activity of va ⁇ ous fungi such as Crvptococcus neoformans, Histoplasma capsulatum, and species of Candida including C. parapsilosis and C.
  • contacting a compound of the invention with a parasite or fungus refers to a union or junction, or apparent touching or mutual tangency of a compound of the invention with a parasite or fungus. However, the term contacting does not imply any mechanism of inhibition.
  • the present invention further provides a method of treating a fungal infection which includes admmiste ⁇ ng an effective amount of a P. syringae lipodepsidecapeptide, such as a 25-B 1 decapeptide antifungal agent, or a pharmaceutically acceptable salt thereof, to a host in need of such treatment.
  • a preferred method includes treating an infection by va ⁇ ous fungi such as Crvptococcus neoformans. Histoplasma capsulatum. and strains of Candida including C. parapsilosis and C. albicans.
  • a formulation of one or more P. synngae hpodepsidecapeptides such as 25-Bl decapeptide antifungal agent A, reduces the burden of a fungal infection, reduces symptoms associated with the fungal infection, and can result in elimination of the fungal infection.
  • antifungal therapy typically includes intravenous administration, of a formulation of one or more P. synngae hpodepsidecapeptides (e.g.. the 25-Bl decapeptide antifungal agents) over several days to halt or retard the infection.
  • the term "effective amount" means an amount of a compound of the present invention which is capable of inhibiting fungal growth or activity, or reducing symptoms of the fungal infection.
  • the compositions will be administered to a patient (human or other animal, including mammals such as. cats, horses and cattle and avian species) in need thereof, in an effective amount to inhibit the fungal infection.
  • the dose administered will vary depending on such factors as the nature and seventy of the infection, the age and general health of the host and the tolerance of the host to the antifungal agent.
  • the particular dose regimen likewise may vary according to such factors and may be given in a single daily dose or in multiple doses du ⁇ ng the day. The regimen may last from about 2-3 days to about 2-3 weeks or longer.
  • a typical daily dose (administered in single or divided doses) will contain a dosage level of from about 0.01 mg/kg to about 100 mg/kg of body weight of an active compound of this invention.
  • Preferred daily doses generally will be from about 0.1 mg/kg to about 60 mg/kg and ideally from about 2.5 mg/kg to about 40 mg/kg
  • the compound can be administered by intravenous infusion using, for example, 0.01 to 10 mg/kg/hr of the active ingredient.
  • the present invention also provides pharmaceutical formulations useful for administe ⁇ ng the antifungal compounds of the invention. Accordingly, the present invention also provides a pharmaceutical formulation including one or more pharmaceutically acceptable earners, diluents, vehicles, excipients, or other additives and one or more P. synngae hpodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A.
  • the active ingredient in such formulations includes from 0.1% to 99.9% by weight of the formulation, more generally from about 10% to about 30% by weight.
  • pharmaceutically acceptable it is meant that the earner, diluent or excipient is compatible with the other ingredients of the formulation and not delete ⁇ ous to the recipient thereof.
  • the formulation can include additives such as va ⁇ ous oils, including those of petroleum, animal, vegetable or synthetic o ⁇ gin, for example, peanut oil, soybean oil. mineral oil, and sesame oil.
  • Suitable pharmaceutical excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, magnesium stearate, sodium stearate, glycerol monostearate. sodium chlo ⁇ de, d ⁇ ed skim milk, glycerol, propylene glycol, water, and ethanol.
  • the compositions can be subjected to conventional pharmaceutical expedients.
  • ste ⁇ hzation can contain conventional pharmaceutical additives, such as preservatives, stabilizing agents, wetting, or emulsifying agents, salts for adjusting osmotic pressure, and buffers.
  • suitable pharmaceutical earners and their formulations are desc ⁇ bed in Martin. "Remington's Pharmaceutical Sciences,” 15th Ed.; Mack Publishing Co., Easton (1975): see. e.g., pp. 1405-1412 and pp. 1461-1487
  • pharmaceutically acceptable salt refers to salts of the compounds of the above formula that are substantially non-toxic to living organisms.
  • Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base. Such salts are known as acid addition and base addition salts. Acids commonly employed to form acid addition salts are mineral acids such as hydrochlo ⁇ c acid, hydrobromic acid, hydroiodic acid, sulfunc acid, and phosphonc acid, and organic acids such as p-toluenesulfonic. methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citnc acid, benzoic acid, and acetic acid.
  • mineral acids such as hydrochlo ⁇ c acid, hydrobromic acid, hydroiodic acid, sulfunc acid, and phosphonc acid
  • organic acids such as p-toluenesulfonic. methanesulfonic acid, oxalic acid, p- bromophenylsulf
  • salts examples include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chlonde, bromide, iodide, acetate, propionate. decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate. propiolate. oxalate, malonate, succmate, suberate, sebacate, fumarate, maleate, butyne- 1.4-d ⁇ oate.
  • hexyne-l,6-d ⁇ oate benzoate, chlorobenzoate, methylbenzoate, dmitrobenzoate. hydroxybenzoate, methoxybenzoate, phthalate. sulfonate, xvlenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma -hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1- sulfonate. napththalene-2-sulfonate, and mandelate.
  • Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochlonc acid and hydrobromic acid, and those formed with organic acids such as maleic acid and methanesulfonic acid.
  • Base addition salts include those denved from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, and bicarbonates.
  • bases useful m prepa ⁇ ng the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, and calcium carbonate.
  • the potassium and sodium salt forms are particularly preferred.
  • any salt of this invention is not of a c ⁇ tical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the countenon does not cont ⁇ bute undesired qualities to the salt as a whole.
  • a P. synngae lipodepsidecapeptide such as 25-B 1 decapeptide antifungal agent A. may be administered parenterally, for example using intramuscular, subcutaneous, or mtra-pe ⁇ toneal injection, nasal, or oral means.
  • a P. synngae lipodepsidecapeptide such as 25-Bl decapeptide antifungal agent A, may be applied topically for superficial skin infections or to inhibit fungal growth m the mucus.
  • the formulation includes one or more P. svnngae hpodepsidecapeptides. such as 25-Bl decapeptide antifungal agent A. and a physiologically acceptable diluent such as deionized water, physiological saline, 5% dextrose and other commonly used diluents.
  • the formulation may contain a cyclodextnn and/or a solubihzing agent such as a polyethylene glycol or polypropylene glycol or other known solubihzing agent.
  • Such formulations may be made up ste ⁇ le vials containing the antifungal and excipient a dry powder or lyophihzed powder form. P ⁇ or to use, a physiologically acceptable diluent is added and the solution withdrawn via sy ⁇ nge for administration to the patient.
  • the active ingredient is generally admixed with a earner, or diluted by a earner, or enclosed with a earner which may be in the form of a capsule, sachet, paper or other container
  • a earner When the earner serves as a diluent, it may be a solid, semi-solid or liquid mate ⁇ ai which acts as a vehicle, excipient or medium for the active ingredient
  • the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositones, stenle mjectable solutions, or stenle packaged powders.
  • the antifungal compound is filled into gelatin capsules or formed into tablets. Such tablets may also contain a binding agent, a dispersant or other suitable excipients suitable for prepanng a proper size tablet for the dosage and a P. syringae lipodepsidecapeptide, such as 25-Bl decapeptide antifungal agent A
  • a P. syringae lipodepsidecapeptide such as 25-Bl decapeptide antifungal agent A
  • the antifungal compound may be formulated into a flavored liquid suspension, solution or emulsion.
  • a preferred oral formulation is l oleic acid. cremophor RH-60 and water and preferably in the amount (by volume) of 8% lmoleic acid. 5% cremophor RH-60, 87% stenle water and a P. synngae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, in an amount of from about 2 5 to about 40 mg
  • the antifungal compound may be formulated with a dry powder for application to the skin surface or it may be formulated in a liquid formulation including a solubihzing aqueous liquid or non-aqueous liquid, e.g., an alcohol or glycol.
  • the present invention also encompasses a kit including the present pharmaceutical compositions and to be used with the methods of the present invention.
  • the kit can contain a vial which contains a formulation of the present invention and suitable earners, either dned or in liquid form.
  • the kit further includes instructions in the form of a label on the vial and or in the form of an insert included in a box m which the vial is packaged, for the use and administration of the compounds.
  • the instructions can also be p ⁇ nted on the box in which the vial is packaged.
  • the instructions contain information such as sufficient dosage and administration information so as to allow a worker in the field to administer the drug It is anticipated that a worker in the field encompasses any doctor, nurse, or technician who might administer the drug.
  • the present invention also relates to a pharmaceutical composition including a formulation of one or more P. syringae hpodepsidecapeptides. such as 25-B l decapeptide antifungal agent A, and that is suitable for administration by injection.
  • a formulation of one or more P svnngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A. can be used for manufactunng a composition or medicament suitable for administration by injection.
  • the invention also relates to methods for manufactunng compositions including a formulation of one or more P svnngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, in a form that is suitable tor oral or topical administration.
  • a liquid or solid formulation can be manufactured in several ways, using conventional techniques.
  • a liquid formulation can be manufactured by dissolving the one or more P. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, in a suitable solvent, such as water, at an approp ⁇ ate pH, including buffers or other excipients
  • Agricultural Uses Antibiotics produced from P. synngae NRRL B-12050 have been demonstrated to effectively treat Dutch elm disease, (see, e.g., U.S. Patent Nos. 4,342,746 and 4,277,462)
  • P. syringae MSU 16H has been shown to confer a greater protection than the wild-type strain in elms infected with Ceratocvstis ulmi, the causal agent of Dutch elm disease, (see e.g., Lam et al, Proc. Natl. Sci. USA, 84, 6447-6451 (1987)). More extensive tests on field-grown elms confirmed the phenomenon of biocontrol at the prophylactic level.
  • the hpodepsidecapeptides of the present invention may be useful as a preventative treatment for Dutch Elm disease.
  • the pseudomycms have been shown to be toxic to a broad range of plant-pathogenic fungi including Rvnchosponum secahs, Ceratocvstis ulmi, Rizoctonia solani, Sclerotinia sclerotwrum, Verticillium albo- atrum, Venicillium dahhae, Thielavwpis basicola, Fusa um oxysporum and Fusanum culmomm.
  • Pseudomycms a family of novel peptides from Pseudomonas svnngae possessing broad-spectrum antifungal activity, " J. General Microbiology, 7, 2857-2865 (1991) ) Consequently, one or more P. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A (including hydrates, solvates.
  • the infected plants are treated bv injecting or spraying an aqueous suspension of the lipodepsidecapeptide compounds into or onto the plant.
  • Means of injection are well-known to those skilled in the art (e.g , gouge pistol). Any means of spraying the suspension may be used that dist ⁇ butes an effective amount of the active matenal onto the plant surface.
  • the suspension may also include other additives generally used by those skilled in the art. such as solubi zers, stabilizers, wetting agents, and combinations thereof
  • Treatment of the plant may also be accomplished using a dry composition containing one or more P. svnngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A.
  • the dry formulation may be applied to the plant surface by any means well-known to those skilled in the art, such as spraying or shaking from a container.
  • the present invention may be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.
  • P. syringae MSU 16H is publicly available from the Amencan Type Culture Collection, Parklawn D ⁇ ve, Rockville, MD, USA as Accession No. ATCC 67028. P synngae strains 25-Bl, 7H9-1, and 67 HI were deposited with the Amencan Type Culture Collection on March 23, 2000 and were assigned the following Accession Nos.: 25-Bl Accession No. PT A- 1622
  • Example 1 Production of 25-Bl Antifungal Agent A Fermentation methods were developed for producing a lipodepsidecapeptide antifungal agent, 25-Bl decapeptide antifungal agent A, in the fermentation broth of a Pseudomonas svnngae strain. Mate ⁇ als and Methods
  • Fermentation broth was composed of (g/L): dextrose (20), soluble starch (5), Basic Amencan Foods Country Style Potato Pearls instant mashed potatoes (30), glycine (1), MgSO 4 7H 2 0 (0.2), KC1 (0.2), and FeSO 4 7H 2 0 (0.004) m tap water.
  • the pH was adjusted to 5.2 before ste ⁇ hzation. Fermentation was earned out at 25°C for 68 hr. Dissolved oxygen was maintained at or above 30% of air saturation by continuous adjustment of air flow and impeller agitation rate. The pH was maintained between 4.0 and 5.4 through the addition of either H 2 SO 4 or NaOH.
  • Dextrose may be fed to the fermentors starting 24 hours after initial inoculation at a rate of 60 mL per hour. Feeding may be continued throughout the course of the fermentation.
  • a process has been used where dissolved oxygen is maintained at 5% of air saturation starting 24 hours after inoculation and continuing until the end of the fermentation penod. Maintenance of dissolved oxygen at 5% was achieved through addition of inert nitrogen gas (N ) to the air supply leading to the fermentor. In all cases, gas was supplied through a single submerged sparger tube with an opening positioned just below the bottom agitator turbine in the fermentor.
  • N inert nitrogen gas
  • the ESEV1S data showed a possible [M+H] ⁇ peak at m/z 1165.7. which is different from the known antifungal agents that have been found thus far from P. Svnngae.
  • the ⁇ NMR spectrum showed signals reminiscent of pseudomycin-hke hpopeptide but indicated the presence of more than one compound
  • an additional broth from 4X100 L fermentation was processed as desc ⁇ bed above and in addition, the final pu ⁇ fication was earned out on a reversed-phase column [Raimn C ⁇ 8 , 6 ⁇ m. 24X250 mm. 0.
  • HPLC methods similar to those used to pu ⁇ fy other hpodepsipeptide antifungal agents resulted in pu ⁇ fication of 25-Bl decapeptide antifungal agent A from fermentation broth.
  • Mass spectrometry and NMR determined the structure of a lipodepsidecapeptide antifungal agent. 25-Bl decapeptide antifungal agent A.
  • the molecular formula of 25-Bl decapeptide antifungal agent A was determined by high resolution FABMS as C 52 H g8 N ⁇ 4 Oi 6 [m z 1165.6581 for C 52 H 89 N 14 O 16 (M+H) + , ⁇ +0 9 ppm].
  • the lj C and DEPT NMR spectra showed 50 distinct resonances, which included twelve carbonyl carbons, five olefimc carbons, four oxygenated sp J carbons, eight typical ammo acid ⁇ -carbons, fifteen methylene carbons and six methyl carbons.
  • one of the methyl carbon signals at ⁇ 16.7 and one of the methylene carbon signals at ⁇ 28.9 each constituted a set of degenerate carbons, thus accounting for the total number of 52 carbons observed in the molecular formula.
  • Thr adjacent to Ala did not show a long range ⁇ - l j C correlation to the carbonyl of Arg residue, instead it showed a correlation to a carbonyl assigned to the dodecanoyl side chain.
  • the absence of Thr-NH/Arg-CO correlation and presence of a co ⁇ elation between the Thr- ⁇ -H ( ⁇ n 4.94, ⁇ c 70.5)/Arg-CO clearly established an ester linkage between the Thr- ⁇ -OH and Arg-COOH. Consistent with these assignments are the ROESY correlations that were observed between the amide protons and the adjacent amino acid ⁇ -protons (see Scheme II below).
  • Compound 25-Bl decapeptide antifungal agent A represents a novel class of lipodepsipeptide which possesses several ammo acid residues that are not present in any of the pseudomycms. sy ⁇ ngomycins. sy ⁇ ngotoxm and sy ⁇ ngostatins produced by different isolates of P. syringae.
  • the new depsipeptide is composed of ten amino acids which is also a departure from the pseudomycms and synngomycins which possess only nine amino acid residues.
  • the new depsipeptide does not include chlorothreonme which is suspected to be the cause for lrntation at the injection site of pharmaceutical formulations containing pseudomycm compounds.
  • Example 4 Antifungal activity of 25-Bl Decapeptide Antifungal Agent A
  • the antifungal studies were conducted using a microtiter broth dilution assay according to National Committee for Clinical Laboratory Standards guidelines 96 well microtiter plates. Sabourauds and dextrose broth was adjusted to contain 2.5 X 10 4 conida/ml. Test compound was dissolved in water and tested in two-fold dilutions starting with the highest concentration of 20 ⁇ g/ml. Plates were incubated at 35°C for 48 hr. The results in Table 2 show the minimal inhibitory concentration (MIC) of the compound that completely inhibited growth compared to untreated growth controls.
  • MIC minimal inhibitory concentration
  • Organism MIC ( ⁇ g/ml)
  • the presence or amount of one or more P. syringae hpodepsipeptides can be determined by measu ⁇ ng the antifungal activity of a preparation.
  • Antifungal activity can be determined in vitro by obtaining the minimum inhibitory concentration (MIC) of the preparation using a standard agar dilution test or a disc diffusion test.
  • a preparation of one or more P. syringae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A can be an extract of a cell culture, or a more punfied mixture.
  • a typical fungus employed in testing antifungal activity is C. albicans.
  • Antifungal activity is considered significant when the test preparation causes 10-12 mm diameter zones of inhibition on Candida albicans x657 seeded agar plates.
  • Example 5 Isolation, Characterization and Mutagenesis of Pseudomonas syrineae
  • strains subjected to mutagenesis include MSU 174, MSU 16H, and 25-Bl.
  • the strain to be mutagenized was grown in a medium containing potato product, then divided into the medium including 0, 1, 2. 4, 16, or 32 ⁇ M of the chemical mutagen l-methyl-3-n ⁇ tro-l-n ⁇ trosoguan ⁇ dme (NTG or MNNG) These cells were then frozen for future screening and selection
  • Mutagenized cells were selected for desirable growth conditions and/or production of one or more P. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A Chemically mutagenized cells of P. syringae, such as mutagenized strain 25-Bl, were thawed and diluted to 6 cells/mL in N21SM medium (Table 3) This medium sometimes contained one or more components for selection, such as varying concentrations of phosphate.
  • an aliquot, typically 5 ⁇ L, from each well was se ⁇ ally diluted (e.g. 1:56, 1: 196, 1:320, 1:686, and/or 1: 1715) and evaluated for activity against Candida albicans in a liquid microtiter plate bioassay.
  • the plates were incubated at 37 °C overnight and the wells were scored for inhibition of C. albicans growth. Suitable strains were picked, inoculated into CSM medium (Table 4), and grown for 1 to 3 days at 25 °C
  • the selected strains were preserved and inoculated into fermentation bottles containing 13mL of N21SM medium and grown for approximately 66 hours at 25 °C. An aliquots was removed from this fermentation, extracted for 1 hour with a volume of acetonit ⁇ le equal to the volume of the aliquot, centnfuged, and decanted for HPLC analysis of one or more P. syringae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, as desc ⁇ bed m Examples 1-3. Strains producing one or more P. syringae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, were reisolated, refermented, and prepared for growth on a larger scale.
  • the selection methods and cnte ⁇ a disclosed herein are effective for producing strains of P. synngae that grow on minimal medium and produce one or more P. syringae hpodepsidecapeptides. such as 25-Bl decapeptide antifungal agent A
  • active ingredient means a P. syringae lipodepsidecapeptide or a pharmaceutically acceptable salt thereof.
  • Hard gelatin capsules are prepared using the following ingredients:
  • a tablet is prepared using the ingredients below. The components are blended and compressed to form tablets each weighing 665 mg.
  • An aerosol solution is prepared containing the following components.
  • the active compound is mixed with ethanol and the mixture added to a portion of the propellant 22. cooled to - 30° C. and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remainder of the propellant. The valve units are then fitted to the container.
  • Propellant 22 (Chlorodifluoromethane ) 74.00
  • Tablets each containing 60 mg of active ingredient, are made as follows:
  • the active ingredient, starch and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly.
  • the aqueous solution containing polyvmyl-pyrro done is mixed with the resultant powder, and the mixture then is passed through a No. 14 mesh U.S. sieve.
  • the granules so produced are d ⁇ ed at 50o C. and passed through a No. 18 mesh U.S. sieve.
  • the sodium carboxymethyl starch, magnesium stearate and talc previously passed through a No. 60 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 150 mg
  • Capsules each containing 80 mg of active ingredient, are made as follows Active ingredient 80 mg
  • the active ingredient, cellulose, starch and magnesium stearate are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules in 200 mg quantities
  • Supposito ⁇ es each containing 225 mg of active ingredient, are made as follows:
  • the active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycendes previously melted using the minimum heat necessary
  • the mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool
  • Suspensions each containing 50 mg of active ingredient per 5 ml dose, are made as follows-
  • An intravenous formulation may be prepared as follows. The solution of these ingredients generally is administered intravenously to a subject at a rate of 1 ml per minute.

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Abstract

L'invention concerne des depsidécapeptides du P. syringae, une technique de préparation de ces peptides, et des techniques utilisant l'activité antifongique desdits peptides. Ces depsidécapeptides P. syringae comprennent un composé représenté par la formule (a), où R représente une fraction lipophile, ou un ester, et un sel pharmaceutiquement acceptable, ou un hydrate de ceux-ci.
PCT/US2000/008724 1999-04-15 2000-04-14 AGENTS ANTIFONGIQUES ISOLES A PARTIR DU $i(PSEUDOMONAS SYRINGAE) Ceased WO2000063240A1 (fr)

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BR0009732-2A BR0009732A (pt) 1999-04-15 2000-04-14 Agentes antifúngicos isolados de pseudomonas syringae
EA200101086A EA200101086A1 (ru) 1999-04-15 2000-04-14 Антигрибковые агенты, выделенные из pseudomonas syringae
CA002371363A CA2371363A1 (fr) 1999-04-15 2000-04-14 Agents antifongiques isoles a partir du pseudomonas syringae
JP2000612328A JP2002542258A (ja) 1999-04-15 2000-04-14 シュードモナス・シリンガエから単離された抗真菌性物質
MXPA01010244A MXPA01010244A (es) 1999-04-15 2000-04-14 Agentes antifungosos aislados de pseudomonas syringae.
AU41886/00A AU4188600A (en) 1999-04-15 2000-04-14 Antifungal agents isolated from (pseudomonas syringae)
EP00921592A EP1173473A1 (fr) 1999-04-15 2000-04-14 Agents antifongiques isoles a partir du pseudomonas syringae
NO20014938A NO20014938L (no) 1999-04-15 2001-10-10 Antifungale stoffer isolert fra pseudomonas syringae

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EP1369426B1 (fr) * 2001-02-08 2011-08-24 Toyo Suisan Kaisha, Ltd. Peptides, derives de ceux-ci, procede de production de ceux-ci, souche produisant ceux-ci, et agent antiviraux comprenant ceux-ci en tant qu'ingredient actif
AU2005273638B2 (en) * 2004-08-18 2011-09-29 Novabiotics Limited Antimicrobial peptides comprising an arginine- and/or lysine-containing motif
CN101473226B (zh) * 2005-08-02 2012-08-08 密苏里大学管委会 针对豆薯层锈菌和疣顶单胞锈菌的噬菌体展示植物防御肽
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