WO2000063240A1

WO2000063240A1 - ANTIFUNGAL AGENTS ISOLATED FROM $i(PSEUDOMONAS SYRINGAE)

Info

Publication number: WO2000063240A1
Application number: PCT/US2000/008724
Authority: WO
Inventors: Palaniappan Kulanthaivel; Matthew David Belvo; James William Martin; Douglas Joseph Zeckner
Original assignee: Eli Lilly And Company
Priority date: 1999-04-15
Filing date: 2000-04-14
Publication date: 2000-10-26
Also published as: CN1347421A; EA200101086A1; NO20014938L; WO2000063240A9; MXPA01010244A; CA2371363A1; EP1173473A1; AU4188600A; BR0009732A; NO20014938D0; JP2002542258A; HUP0200897A2

Abstract

The invention relates to P. syringae depsidecapeptides, method for making such peptide, and methods employing antifungal activity of these peptides. The P. syringae depsidecapeptides include a compound having formula (a) where R is a lipophilic moiety, or a pharmaceutically acceptable salt, ester, or hydrate thereof.

Description

ANTIFUNGAL AGENTS ISOLATED FROM PSEUDOMONAS SYRINGAE

FIELD OF THE INVENTION The present invention relates to P. syringae depsidecapeptides, method for making such peptide, and methods employing antifungal activity of these peptides.

BACKGROUND 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 in the past 20 years. This is in part due to increased numbers of people with immune systems weakened or devastated 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. For example, 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 syringae produce several classes of antifungal or antibiotic agents, such as the pseudomycms, syπngomycms, syπngotoxins, and syπngostatms. which are podepsmonapeptides. Natural strains and transposon generated mutants of P. svringae produce these hpodepsmonapeptides. Several of the pseudomycms, syπngomycins and other podepsipeptide antifungal agents have been isolated, chemically characteπzed. and shown to possess wide spectrum antifungal activity, including activity against important fungal pathogens in both humans and plants The pseudomycms, the syπngomycins. the syπngotoxms. and the syπngostatms represent structurally distinct families of antifungal compounds.

None of the P. syringae hpodepsmonapeptides has been brought to market for antifungal therapy. Discovery of undesirable side effects, making formulations, scaling up production, and other development problems have thus far prevented exploitation of the P. syringae hpodepsmonapeptides against the full range of fungal infections that affect animals, humans and plants. There remains a need for an antifungal agent that can be used against infections not treated by existing antifungal agents and for application against infections m animals, humans, or plants.

SUMMARY OF THE INVENTION The present invention provides a depsidecapeptide produced by P. syringae which contains the unusual ammo acids homoseπne (Hse), dehydroammobutyπc acid (Dhb) and dehydroalanme (Dha) as part of a depsidecapeptide πng. More specifically, the P. syringae depsidecapeptide includes a depsidecapeptide πng having the ammo acids, arginine, threonine, homoseπne, dehydroaminobutyπc acid, and dehydroalanme. and a lactone formed from a carboxyl group of the arginine and a hydroxyl group of the threonme. As isolated from R. svnngae, the depsidecapeptide is a hpodepsidecapeptide. a cyclic peptide coupled to a lipophilic moiety. Typically the lipophilic moiety is a fatty acid moiety coupled to the ammo group of the threonine by an amide bond. Preferably, the fatty acid moiety is an rc-dodecanoic acid moiety. The hpodepsidecapeptide is represented by formula I

where R is a lipophilic moiety The lipophilic moiety includes C₉-Cι₅ alkyl, C₉-Cι₅ hydroxyalkyl, C -Cι₅ dihydroxyalkyl, C₉-Cι₅ alkenyl, C₉-C_{! 5} hydroxyalkenyl, or C₉-Cι₅ dihydroxyalkenyl Preferably, the lipophilic moiety is Cn alkyl The alkyl, hydroxyalkyl, dihydroxyalkyl, alkenyl, hydroxyalkenyl. or dihydroxyalkenyl groups may be branched or unbranched. Preferably, the amino acid sequence of the depsidecapeptide πng is threonme-alanine-threonme-glutarmne-homoseπne-dehydroammobutyπc acid-alamne- dehydroalanine-threomne-argmme, referred to herein as "25-B1 decapeptide" or "Thr-Ala-Thr-Gln-Xaa-Xaa-Ala-Xaa-Thr-Arg (SEQ ID NO: 1)". As used herein, the term "25-B 1 decapeptide antifungal agent A" refers to the specific depsidecapeptide having the preferred ammo acid sequence SEQ LD NO: 1 and R = unbranched Cn alkyl (i.e , R = -(CH₂)ιoCH-) The invention also relates to methods employing a P svringae depsidecapeptide for inhibiting fungal activity or for reducing the symptoms of 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 of a patient. Consequently, the P. syringae depsidecapeptides may be used in the manufacture ot a medicament for treatment of a patient as descπbed herein. The methods and medicaments of the invention are effective against fungi such as Candida parapsύosis, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum.

The invention provides using microorganisms in a method for producing an antifungal agent, such as the R. syringae depsidecapeptides descπbed above and including a 25-B 1 decapeptide. The method involves cultuπng Pseudomonas syringae in media including three or fewer amino acids and recoveπng one or more R. syringae depsidecapeptides from the culture. In one embodiment, R. syringae culture is in medium including glycine and a pid, a potato product, or a combination thereof at a pH of about 4 to 6.5 until one or more R. syringae depsidecapeptides is produced at a concentration of at least about 10 μg/mL. In addition, the invention provides R. syringae depsidecapeptides prepared by the method descπbed above. The invention also provides a method for treating or preventing fungal growth in a plant whereby a fungus is contacted with a one or more of the R. syringae depsidecapeptides descπbed above.

DETAILED DESCRIPTION Lipodepsidecapeptide Antifungal Agents

As used herein "hpodepsidecapeptide antifungal agent" refers to an antifungal agent having a cyclic decapeptide πng 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- B 1 decapeptide antifungal agent A, has been puπfied and its structure determined. As used herein the term "R. syringae podepsidecapeptide" refers to a podepsidecapeptide antifungal agent produced by R. syringae, and includes 25-B 1 decapeptide antifungal agent A and related analogs

R syringae podepsidecapeptides share several structural features. For example. each of these antifungal agents includes the unusual amino acids homoseπne (Hse), dehydroaminobutyπc acid (Dhb) and dehydroalanme (Dha) as part of a depsidecapeptide πng. In each of the R syringae podepsidecapeptides. a carboxyl group of an arginine residue linked to the hydroxyl group of the N-termmal threonine forms a lactone that closes the depsidecapeptide πng. The sequence of the depsidecapeptide πng of the P. syringae podepsidecapeptide can be represented as:

Thr-Xaa-Xbb-Xcc-Hse-Dhb-Xdd-Dha-Xee-Arg in which each of Xaa. Xbb, Xcc, Xdd, and Xee are individually naturally occurπng ammo acids. Unlike the pseudomycin natural products, the hpodepsidecapeptides of the present invention do not contain chlorothreonine which is suspected to be the cause for irπtation at the injection site of pharmaceutical formulations containing pseudomycin compounds. The depsidecapeptide πng is linked to a lipophilic moiety, such as a fatty acid, through an amide bond with an amino group of the N-terminal threonine. The fatty acid generally includes 10, 12, 14, or 16 carbons, typically beaπng 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 n-decanoic acid moiety, an n- decanoic acid moiety substituted with one or two hydroxyl groups, an n-dodecanoic acid moiety, an n-dodecanoic acid moiety substituted with one or two hydroxyl groups, an n- tetradecanoic acid moiety, or an n-tetradecanoic acid moiety substituted with one or two hydroxyl groups.

25-Bl Decapeptide Antifungal Agents

As used herein, "25-Bl decapeptide antifungal agent" refers to one or more members of a family of antifungal agents that has been isolated from the bacteπum Pseudomonas synngae. A 25-B 1 decapeptide antifungal agent is a P. syringae hpodepsidecapeptide. Specifically, a 25-Bl decapeptide antifungal agent is a R. syringae hpodepsidecapeptide having a depsidecapeptide πng with the sequence:

Thr-Ala-Thr-Gln-Hse-Dhb-Ala-Dha-Thr-Arg (SEQ LD NO: 1) 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-termmal threonine. The fatty acid moiety ot 25-Bl decapeptide antifungal agent A is an rc-dodecanoic acid moiety.

Biological Activities of R svnnzae Lipodepsidecapeptides

A P. synngae hpodepsidecapeptide, such as 25-Bl decapeptide antifungal agent A. has several biological activities including killing and inhibiting activity of vaπous fungi, such as fungal pathogens of plants and animals. In particular, 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 vaπous species of Candida including C. parapsύosis and C. albicans.

Pseudomonas syringae Pseudomonas syringae include a wide range of bacteπa that are generally associated with plants. Some of the R. synngae are plant pathogens, while others are only weakly pathogenic or are saprophytes. Many different isolates of R. synngae produce one or more cytotoxic agents that can help this bacteπum survive m the wild where it must compete with fungi and other bacteπa. The cytotoxic agents produced by R synngae include anti-fungal agents such as the R synngae hpodepsidecapeptides, including 25-Bl decapeptide antifungal agent A, the pseudomycms, the syπngomycins, the syπngotoxins. and the synngostauns

Isolated strains of R. synngae that produce one or more pseudomycms, syπngomycins, syπngotoxins, syπngostatins are well-known to those skilled in the art Wild type strain MSU 174 and a mutant of this strain generated by transposon mutagenesis, MSU 16H (ATCC 67028) have been descπbed in U.S. Patent No 5.576.298, issued November 19, 1996 to G. Strobel et al., Harπson et al., "Pseudomycms, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity." J Gen Microbiology 137, 2857-2865 (1991); and Lamb et al , "Transposon mutagenesis and tagging of fluorescent pseudomonas Antimycotic production is necessary for control of Dutch elm disease." Proc. Natl. Acad. Sci. USA 84. 6447-6451 (1987). Methods for growth of vaπous strains of R. syringae and their use in production of antifungal agents such as pseudomycms are also disclosed in U S. Patent Application Seπal No. by Matthew D. Hilton, et al. entitled "Pseudomycin Production By Pseudomonas Syringae" submitted evendate herewith and descπbed below. The disclosures of the references cited m this paragraph are incorporated herein by reference.

Strains of R. synngae that are suitable for production of one or more R. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, 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-puπfied culture of R. synngae that produce one or more R. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A. in amounts greater than about 10 μg/mL, preferably from at least about 10 μg/mL to about 50 μg/mL. Preferably, the biologically-puπfied culture of a microorganism is of Pseudomonas synngae strains MSU 16H, 25-Bl, 67H1, 7H9-1, or a pseudomycm-producing mutant, vaπant, isolate, or recombinant of these strains. Cultures of MSU 16H are on deposit at Montana State University (Bozeman, Montana, USA) and available from the Ameπcan Type Culture Collection (Parklawn Dπve, Rockville. MD, USA) Accession No. ATCC 67028.

A strain of P. syringae that is suitable for production of one or more P. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, can be isolated from environmental sources including plants, such as barley plants, citrus plants, and lilac plants, and also from sources such as soil, water, air, and dust. A preferred strain is isolated from plants. These environmental isolates of P. synngae can be referred to as wild type As used herein, "wild type" refers to a dominant genotype which naturally occurs in the normal population of P synngae (i.e.. strains or isolates of P. synngae that are found in nature and not produced by laboratory manipulation). As is the case with other organisms, the characteπstics of the hpodepsidecapeptide-producmg cultures employed in this invention. P. syringae .strains such as MSU 174. MSU 16H. MSU 206. 25-Bl. and 7H9 are subject to vaπation. Thus, progeny of these strains, e.g.. recombmants, mutants and vanants, 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. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A. As used herein, "mutant" refers to a sudden heπtable change in the phenotype of a strain, which can be spontaneous or induced by known mutagenic agents, including radiation and vaπous chemicals. Mutant R. syringae of the present invention can be produced using a vaπety 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'-nitrosoguamne (NTG), and nitrous acid.

R. synngae suitable for producing one or more R. syringae hpodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, according to the present invention can be generated by treating the bacteπa with an amount of a mutagenic agent effective to produce mutants that overproduce one or more R. syringae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A. or that produce one or more P. syringae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A. under advantageous growth conditions. While the type and amount of mutagenic agent to be used can vary, a preferred method is to seπally dilute NTG to levels ranging from about 1 to about 100 μg/mL. Preferred mutants of the invention are those that overproduce 25-Bl decapeptide antifungal agent A, and grow minimal medium. The mutants overproduce a R. synngae podepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, preferably from at least about 10 μg/mL to about 50 μg /mL. Environmental isolates, mutant strains, and other desirable strains of P. synngae can be subjected to selection for desirable traits of growth habit, growth medium, nutπent source, carbon source, growth conditions, and amino acid requirements. Preferably, a strain of P. synngae producing P. syringae hpodepsidecapeptide, such as 25-Bl decapeptide antifungal agent A. is selected for growth on minimal defined medium. Preferred strains exhibit the charactenstics of producing one or more R. synngae hpodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A. when grown on a medium including glycme plus, optionally, a pid. 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 vaπety 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 R. synngae podepsidecapeptide, 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 hpodepsidecapeptide yield. Progeny, i.e. natural and induced vaπants, mutants and recombinants, of the P. synngae strains 25-Bl, 67H1, and 7H9-1 which retain the characteπstic of podepsidecapeptide production are part of this invention.

Growth of Pseudomonas synngae

As descπbed herein, "aqueous nutπent media" refers to a water-base composition including minerals and organic compounds and their salts necessary for growth of the bacteπum used m the present invention. Preferred nutπent media contain an effective amount of three or fewer amino acids, preferably, glutamic acid, glycme. histidine, or a combination thereof. In one embodiment, the medium contains an effective amount of glycme and, optionally, one or more of a potato product and a hpid. Glycine can be provided as a single am o acid or as part of a mixture of am o acids, such as hydrolyzed protein. Suitable pids include soybean oil, fatty acids, or fatty acid esters. Suitable potato products include potato dextrose broth, potato dextπn, potato protein, or a commercial mashed potato mix food product. Preferred minerals in the nutπent medium include salt mixtures typically used in cell culture and fermentation, such as Czapek mineral salts, which includes KC1, MgSO₄. and FeSO₄. Organic compounds m the nutπent 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.

Although the amount of each ingredient in the nutπent broth is not typically cπtical to growth of the bacteπa or to production of a R. synngae hpodepsidecapeptide. such as 25-Bl decapeptide antifungal agent A, certain levels of nutπents are advantageous. 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 dextπn; and/or about 1 g/L to about 10 g/L, preferably about 4 g/L of potato protein. A preferred nutπent medium includes minerals, preferably, KC1 at about 0.02 to about 2 g/L, more preferably about 0.2 g/L; MgSO₄, preferably MgSO₄«7H₂O, at about 0.02 to about 2 g/L, more preferably about 0.2 g/L; and FeSO , preferably FeSO *7H O, at about 0.4 to about 40 mg/L. more preferably about 4 mg/L. When present, 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 descπbed under conditions of controlled or regulated pH, and temperature. R. 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. syringae 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. Typically growth of R. 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 R. synngae Lipodepsidecapeptide

To produce one or more R. synngae hpodepsidecapeptides. such as 25-B 1 decapeptide antifungal agent A, from a wild type or mutant strain of R. synngae, 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. In one preferred embodiment, the amino acids include glycine and, optionally, one or more of a potato product and a lipid. Cultuπng is conducted under conditions effective for growth of P. synngae and production of a desired R. synngae podepsidecapeptide, 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. When cultivated on media such as those descπbed herein, R. synngae can grow at cell densities up to about 10-15 g/L dry weight and produce a R. synngae podepsidecapeptide, such as 25-Bl decapeptide antifungal agent A, in a total amount at least about 10 μg/mL, preferably at least about 50 μg/mL.

Controlling the concentration of oxygen in the medium duπng cultuπng of R. synngae is advantageous for production of a R. svnngae podepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A. Preferably, 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 m the medium. Further, adjustment of the agitation rate can be used to adjust the oxygen transfer rate.

Controlling the pH of the medium duπng cultuπng of R. synngae is advantageous for production of a P. syringae podepsidecapeptide, 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.

R synngae can produce a R. synngae lipodepsidecapeptide. such as 25-Bl decapeptide antifungal agent A, when grown in batch culture. However, fed-batch or semi-continuous feed of glucose and. optionally, an acid or base, such as ammonium hydroxide, to control pH. enhances production of a R svnngae podepsidecapeptide. such as 25-Bl decapeptide antifungal agent A Production of a R syringae hpodepsidecapeptide. such as 25-Bl decapeptide antifungal agent A. by P. synngae 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 R. synngae strain can affect the amount and distπbution of a R. syringae hpodepsidecapeptide, such as 25-Bl decapeptide antifungal agent A, produced by cultuπng under the conditions descπbed herein. For example, strain 25 BI can produce predominantly 25-Bl decapeptide antifungal agent A.

The cyclic decapeptide nucleus of the R syringae hpodepsidecapeptides can be prepared by cleaving off the lipophilic moiety, such as by deacylation. Cleavage and deacylation methods are well-known to those skilled m the art, such as the use of deacylase enzymes.

Formulation and Antifungal Action of R syringae Lipodepsidecapeptides

A R. synngae podepsidecapeptide. such as 25-B 1 decapeptide antifungal agent A, shows in vitro and in vivo activity and is useful in combating either systemic fungal infections or fungal sk infections. Accordingly, the present invention provides a method of inhibiting fungal activity including contacting a R. synngae podepsidecapeptide, 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 vaπous fungi such as Crvptococcus neoformans, Histoplasma capsulatum, and species of Candida including C. parapsilosis and C. albicans. As used herein "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 admmisteπng an effective amount of a R. syringae lipodepsidecapeptide, such as a 25-B l 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 vaπous fungi such as Crvptococcus neoformans. Histoplasma capsulatum. and strains of Candida including C. parapsilosis and C. albicans. When administered in an effective antifungal amount, a formulation of one or more R. syringae 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.

Some patients in need of antifungal therapy with a formulation of one or more R synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, have severe symptoms of infection, such as high fever, and are likely to be m intensive or cπtical care. Vaπous fungi can cause such seπous infections. Candida spp., for example, causes mucosal and seπous systemic infections and may exist as azole- or polyene- resistant strains. Aspergύlus causes life-threatening systemic infections. Crvptococcus is responsible for meningitis. Such seπous fungal infections may occur in immune compromised patients, such as those receiving organ or bone marrow transplants, undergoing chemotherapy for cancer, recoveπng from major surgery, or suffeπng from HTV infection. For such patients, antifungal therapy typically includes intravenous administration, of a formulation of one or more R. synngae hpodepsidecapeptides (e.g., the 25-B 1 decapeptide antifungal agents) over several days to halt or retard the infection. With respect to antifungal activity, 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. For most fungal infections reduction of symptoms of the infection includes reduction of fever, return to consciousness, and increased well being of the patient. Preferably, symptoms are reduced by killing the fungus to eliminate the infection or to bπng the infection to a level tolerated by the patient or controlled by the patient's immune system. As used herein "inhibiting" refers to inhibiting fungal activity, including stopping, retarding or prophylactic ally hindeπng or preventing the growth or any attending characteπ sties and results from the existence of a fungus. Typically, 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 duπng 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. For seπous infections, 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 administeπng 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 R. svnngae hpodepsidecapeptides, such as 25-Bl 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. By "pharmaceutically acceptable" it is meant that the earner, diluent or excipient is compatible with the other ingredients of the formulation and not deletenous to the recipient thereof.

The formulation can include additives such as vanous oils, including those of petroleum, animal, vegetable or synthetic ongin, 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 chloπde, dned skim milk, glycerol, propylene glycol, water, and ethanol. The compositions can be subjected to conventional pharmaceutical expedients. such as stenhzation. and 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 descnbed in Martin. "Remington's Pharmaceutical Sciences," 15th Ed.; Mack Publishing Co.. Easton (1975); see. e.g., pp. 1405-1412 and pp. 1461-1487. The term "pharmaceutically acceptable salt", as used herein, 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 hydrochloπc acid, hydrobromic acid, hydroiodic acid, sulfunc acid, and phosphoπc acid, and organic acids such as p-toluenesulfomc, methanesulfomc acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succimc acid, citnc acid, benzoic acid, and acetic acid. Examples of such pharmaceutically acceptable salts are 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-dιoate, hexyne-l,6-dιoate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate. hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma -hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1- sulfonate. napththalene-2-sulfonate, and mandelate. Prefened 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 methanesulfomc acid.

Base addition salts include those denved from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, and bicarbonates. Such bases useful in prepanng 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 prefened.

It should be recognized that the particular countenon forming a part of any salt of this invention is not of a cπtical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the countenon does not contnbute undesired qualities to the salt as a whole.

A R. synngae hpodepsidecapeptide. such as 25-Bl decapeptide antifungal agent A. may be administered parenterally, for example using intramuscular, subcutaneous, or tra-pentoneal injection, nasal, or oral means. In addition to these methods of administration, a R. syringae podepsidecapeptide, such as 25-Bl decapeptide antifungal agent A. may be applied topically for superficial skin infections or to inhibit fungal growth in the mucus.

For parenteral administration the formulation includes one or more P. syringae 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 in stenle vials containing the antifungal and excipient in a dry powder or lyophihzed powder form. Pnor to use, a physiologically acceptable diluent is added and the solution withdrawn via synnge for administration to the patient.

The present pharmaceutical formulations are prepared by known procedures using known and readily available ingredients. In making the compositions of the present invention, the active ingredient is generally admixed with a earner, or diluted by a earner, or enclosed withm a earner which may be in the form of a capsule, sachet, paper or other container. When the earner serves as a diluent, it may be a solid, semi-solid or liquid matenal which acts as a vehicle, excipient or medium for the active ingredient. Thus, 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 ι. ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositones, steπle mjectable solutions, or stenle packaged powders.

For oral administration, 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 prepaπng a proper size tablet for the dosage and a R syringae podepsidecapeptide, such as 25-Bl decapeptide antifungal agent A. For pediatnc or genatπc use the antifungal compound may be formulated into a flavored liquid suspension, solution or emulsion. A preferred oral formulation is hnoleic acid, cremophor RH-60 and water and preferably in the amount (by volume) of 8% hnoleic acid. 5% cremophor RH-60, 87% stenle water and a P. synngae podepsidecapeptide, such as 25-Bl decapeptide antifungal agent A, in an amount of from about 2.5 to about 40 mg/ml.

For topical use 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.

Uses of Formulations of a P. svnngae Lipodepsidecapeptide

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 dπed or in liquid form. The kit further includes instructions in the form of a label on the vial and/or m the form of an insert included in a box in which the vial is packaged, for the use and administration of the compounds. The instructions can also be pπnted 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. svnngae hpodepsidecapeptides, such as 25-B l decapeptide antifungal agent A. and that is suitable for administration by injection. According to the invention, a formulation of one or more R. svnngae hpodepsidecapeptides, such as 25-Bl decapeptide antitungal 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 R svnngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A. in a form that is suitable for oral or topical administration. For example, 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 R. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, in a suitable solvent, such as water, at an appropnate pH, including buffers or other excipients.

Agricultural Uses Antibiotics produced from R. 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) In particular, P synngae MSU 16H has been shown to confer a greater protection than the wild-type strain in elms infected with Ceratocystis 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. Hence, 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, Ceratocystis ulmi, Rizoctoma solani, Sclerotinia sclerotwrum, Venicillium albo- atrum, Venicillium dahliae, Thielavwpis basicola, Fusanum oxysporum and Fusanum culmorum. (see Harπson, L., et al., "Pseudomycms, a family of novel peptides from Pseudomonas synngae possessing broad-spectrum antifungal activity, " J. General Microbiology, 7, 2857-2865 (1991) ) Consequently, one or more P. svnngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A (including hydrates, solvates. and esters thereof) may be useful in the treatment of fungi in plants (in particular. V albo-atrum, Rhizoctonia solani and F oxvsporum) either as a direct treatment or preventative treatment Generally, the infected plants are treated by injecting or spraying an aqueous suspension of the hpodepsidecapeptide 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 distπbutes 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 R. synngae 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.

EXAMPLES Biological Materials on Deposit

R. syringae MSU 16H is publicly available from the Ameπcan Type Culture Collection, Parklawn Dnve, 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. PTA- 1622

7H9- 1 Accession No. PTA- 1623

67 HI Accession No. PTA-1621

Example 1 - Production of 25-Bl Antifungal Agent A Fermentation methods were developed for producing a hpodepsidecapeptide antifungal agent, 25-B 1 decapeptide antifungal agent A, in the fermentation broth of a Pseudomonas svnngae strain. Mateπals and Methods

Preparation of inoculum: An aliquot of P. svnngae strain 25-Bl cells stored in the vapor phase of liquid nitrogen was thawed and used to inoculate two 900 mL portions of CSM broth. CSM broth was composed of (g/L): dextrose (5), maltose (4), Difco Tryptic Soy Broth (30), Difco yeast extract (3), and MgSO₄ 7H₂0 (2). Approximately 0.5 mL of cells was used to inoculate each 900 mL portion of medium contained in a two liter flask. Flasks were incubated with shaking for 24 hours at 25°C. The contents of two flasks were combined to inoculate a 150 liter fermentor containing 115 liters of stenle fermentation broth. Fermentation Stage: Fermentation broth was composed of (g/L): dextrose (20), soluble starch (5), Basic Ameπcan Foods Country Style Potato Pearls instant mashed potatoes (30), glycine (1), MgSO₄ 7H₂0 (0.2), KC1 (0.2), and FeSO₄ 7H₂0 (0.004) in tap water. The pH was adjusted to 5.2 before steπ zation. 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 H₂SO₄ or NaOH.

Several vanations of the simple batch process were also found to produce the novel cyclic peptide product. 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. Alternatively, 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 peπod. Maintenance of dissolved oxygen at 5% was achieved through addition of inert nitrogen gas (N₂) 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.

Results and Conclusion

Several fermentation methods produce 25-B 1 decapeptide antifungal agent A from P svnngae Example 2 - Isolation and Purification of 25-Bl Antifungal Agent A

Methods were developed for isolation and puπfication of a hpodepsidecapeptide antifungal agent. 25-B 1 decapeptide antifungal agent A. from the fermentation broth of a Pseudomonas svnngae strain.

Matenals and Methods

The whole fermentation broth produced according to Example 1, typically 100 L after harvest, was filtered through a Membralox™ ceramic filter (0.45 μm). The resulting solid slurry was extracted with an equal volume of acetone containing 0.1% TFA for 90 mm. The acetone extract was separated by filtration and evaporated in vacuo to an aqueous solution.

This solution was combined with the filtrate obtained from the ceramic filtration of the whole broth and charged on to an Amberchrom™ CG 300sd resm column (4 L) packed in water. The column was initially washed with 0.2% acetic acid (pH 4-8) until the effluent showed pH 4.5 followed by 10 L of 22% acetonitnle containing 0.2% acetic acid (pH 4.8). Then the column was eluted with a linear gradient of 22-35% acetonitnle containing 0.2% acetic acid (32 L) and 35% acetonitnle containing 0.2% acetic acid (8 L) with 400 ml/mm flow rate. Fractions 11-16 were combined (4.8 L), concentrated in vacuo to 100 ml and centnfuged. The supematant was separated and chromatographed over an Amberchrom CG

300sd column (1 L) using a linear gradient of 25-35% acetonitnle containing 0.2% acetic acid (pH 4.8) with 50 ml/mm flow rate. Fractions 20-25 (1.2 L) were combined and rechromatographed over a reversed-phase column (NovaPak Cι₈, 6 μm, 40X300 mm, flow rate 40 ml/min, linear gradient 30-60% acetonitnle containing 0.2% TFA) to yield 21 mg of a compound (89% punty by UN).

The ESHVIS 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 R. Syringae The Η ΝMR spectrum showed signals reminiscent of pseudomycm-like lipopeptide but indicated the presence of more than one compound. In order to obtain 25-Bl decapeptide antifungal agent A in high punty for structure determination and antifungal activity, an additional broth from 4X100 L fermentation was processed as descπbed above and in addition, the final punfication was earned out on a reversed-phase column [Rainin C_{) 8}, 6 μm. 24X250 mm, 0. 1% TFA-acetomtnle-MeOH (8:1:1 to 4:3:3) gradient elution for 60 mm; (4:3:3 to 10:45:45) gradient elution for 30 mm] to afford 42 mg of 25-Bl decapeptide antifungal agent A (93% punty by UN).

Results and Conclusion

HPLC methods similar to those used to punfy other hpodepsipeptide antifungal agents resulted m puπfication of 25-B 1 decapeptide antifungal agent A from fermentation broth.

Example 3 - Determination of the Structure of 25-Bl Antifungal Agent A

Mass spectrometry and ΝMR determined the structure of a hpodepsidecapeptide antifungal agent. 25-Bl decapeptide antifungal agent A.

Methods and Results

The molecular formula of 25-B l decapeptide antifungal agent A was determined by high resolution FABMS as C₅₂H₈₈Νι₄Oι₆[m/z 1165.6581 for C₅₂H₈₉N_I4O_!6(M+H)⁺.Δ+0.9 ppm]. In accordance with this formula the ¹³C and DEPT NMR spectra showed 50 distinct resonances, which included twelve carbonyl carbons, five olefinic carbons, four oxygenated sp³ carbons, eight typical ammo acid -carbons, fifteen methylene carbons and six methyl carbons Among these, one of the methyl carbon signals at δ 16 7 and one of the methylene carbon signals at δ 28.9 each constituted a set of degenerate carbons. thus accounting for the total number of 52 carbons observed in the molecular formula.

Detailed analysis of Η. ^{I 3}C. and 2D NMR (DQCOSY, TOCSY, HMQC. HMBC and ROESY) data enabled to determine the structure of 25-Bl decapeptide antifungal agent A (IA) and unambiguously assign all the protons and carbons (Table 1).

T)

IA

Table 1. H and 13, C NMR Chemical Shifts of 25-Bl decapeptide antifungal agent A in DMSO-de

The results from Η, DQCOSY and TOCSY spectra measured in DMSO-c at 35°C revealed the presence of spin systems for seven commonly occurring amino acid residues - two alanmes, one arginine, one glutamine and three threomnes, and three less commonly occurnng amino acids - one dehydroalanme (Dha), one dehydroammobutync acid (Dhb) and one homosenne. The less commonly occurnng amino acid residues, viz. Dha. Dhb and homosenne, were identified by the cross peaks observed m the TOCSY spectrum from the amide protons resonating at δ 9.01 (brs). 9.07 (brs) and 8.22 to protons resonating at δ 5.86. 5.61 (δ_c 106.4), 6.40 (δ_c 128.5), 1.61 (δc 12.9) and 4.22 (δ_c 51.4), 1.82. 1.76 (δ_c 34.0), 3.47 (δc 57.3), respectively. Consistent with this the ¹³C NMR spectrum displayed eight proton bound α-carbon signals for the saturated amino acid residues and two quaternary α-carbons for the unsaturated amino acids Dha and Dhb. Of the twelve amide or ester type carbonyls, nine were assigned to the eight saturated amino acid residues (two to glutamine), two (δ 164.0 and 163.7 ppm) to Dha and Dhb. The remaining one carbonyl group was assigned to the dodecanoyl side chain, the presence of which is discerned from the terminal methyl signal (O_H 0.83 and δc 13.9) and 10 methylene signals in the ^{l j}C NMR spectrum (Table 1) The molecular formula requires sixteen degrees of unsaturation. The ten amino acids and the dodecanoyl side chain accounted for fifteen sites of unsaturation indicating that 25-Bl decapeptide antifungal agent A is a monocychc decapeptide. Companson of the chemical shift of the β-protons of threonines (S_H 4.94. 4.11 and 4.00) indicated that the proton resonating at δ 4.94 was attached to a carbinol which is modified to an ester or a lactone. That this was so and 25-B 1 decapeptide antifungal agent A is a depsipeptide was evidenced by the HMBC and ROESY data. These data also established the amino acid sequence and the location of the dodecanoyl side chain in 25-Bl decapeptide antifungal agent A.

With amino acid Arg as a starting point, the long range Η - ¹³C conelations observed the HMBC spectrum between the amide proton and the adjacent ammo acid carbonyl and or α-carbon (see Scheme I below) unambiguously established the amino acid sequence Arg-NH/Thr-CO, Thr-NH/Dha-CO. Dha-NH/Ala-CO, Ala-NH/Dhb-CO, Dhb-NH/Hse-CO, Hse-NH/Glu-α-C, Glu-NH/Thr-CO, Thr-NH/Ala CO and Ala-NH/Thr-CO.

HMBC (H → C) Conelations Scheme I

The NH of Thr adjacent to Ala did not show a long range 1H - ' ^JC correlation to the carbonyl of Arg residue, instead it showed a conelation to a carbonyl assigned to the dodecanoyl side chain. The absence of Thr-NH/Arg-CO correlation and presence of a conelation between the Thr-β-H (δ_H 4.94, δ_c 70.5)/Arg-CO clearly established an ester linkage between the Thr-β-OH and Arg-COOH. Consistent with these assignments are the ROESY correlations that were observed between the amide protons and the adjacent amino acid α-protons (see Scheme π below).

Selected ROESY Correlations Scheme II

Conclusions Compound 25-Bl decapeptide antifungal agent A represents a novel class of hpodepsipeptide which possesses several ammo acid residues that are not present in any of the pseudomycms, syπngomycins. syπngotoxm and synngostatms 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 am o acid residues. Unlike the pseudomycin natural products, the new depsipepude does not include chlorothreonme which is suspected to be the cause for lrntation at the injection site of pharmaceutical formulations containing pseudomycin 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 in 96 well microtiter plates. Sabourauds and dextrose broth was adjusted to contain 2.5 X 10⁴ conida/ml. Test compound was dissolved in water and tested in two-fold dilutions starting with the highest concentration of 20 μg/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.

Table 2. Antifungal activity of 1

Organism MIC (μg/ml)

Candida albicans 10

C. parapsilosis 10

Cryptococcus neoformans 1.25

Aspergillus fumigatus >20

Histoplasma capsulatum 20

The presence or amount of one or more R. svnngae podepsipeptides, such as 25- B 1 decapeptide antifungal agent A, can be determined by measunng 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 R. synngae 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 syringae

Environmental isolates and mutants of R. syringae were produced and employed in production of antifungal agents

Mateπals and Methods

Strains MSU 174 and MSU 16-H were isolated and characteπzed as descπbed m U.S. Patent No. 5,576,298, issued November 19, 1996 to G. Strobel et al.; Harπson et al., "Pseudomycms, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity," J Gen. Microbiology 137, 2857-2865 (1991); and Lamb et al , "Transposon mutagenesis and tagging of fluorescent pseudomonas: Antimycotic production is necessary for control of Dutch elm disease," Proc. Natl. Acad. Sci. USA 84, 6447-6451 (1987) The disclosures of the references cited m this paragraph are incorporated herein by reference.

Additional strains were deπved from such wild type and transposon generated mutants by chemical mutagenesis. 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 μM of the chemical mutagen l-methyl-3-nιtro-l-nιtrosoguanιdιne (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. syringae hpodepsidecapeptides. such as 25-Bl decapeptide antifungal agent A Chemically mutagenized cells of R. 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. A 50 μL volume of mutagenized cells was dispensed into a well of a 96-well round bottom microtiter plate for a delivery of an average of 0.3 cells/well Typically, sihcone oil was added to each well to minimize evaporation. The plates were incubated with shaking for 6 to 12 days at 25 °C Table 3 — The Composition of N21SM Medium

After this incubation, an aliquot, typically 5 μL, from each well was seπally diluted (e.g. 1:56, 1:196, 1:320, 1:686, and/or 1:1715) and evaluated for activity against Candida albicans m 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.

Table 4. Complete Streptomyces Medium (CSM) Component Concentration (g/L)

Glucose 5

Maltose 4

Difco Tryptic Soy Broth 30

Difco Yeast Extract

MgSO₄7H₂O 2

No pH adjustment

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 ahquots was removed from this fermentation, extracted for 1 hour with a volume of acetonitnle equal to the volume of the aliquot, centπfuged, and decanted for HPLC analysis of one or more P. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, as descπbed m Examples 1-3. Strains producing one or more R. syringae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, were reisolated, refermented, and prepared for growth on a larger scale.

Results

Strains exhibiting production of one or more P. synngae hpodepsidecapeptides, such as 25-Bl decapeptide antifungal agent A, were produced using the methods descπbed above.

Conclusion

The selection methods and cπtena 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 Example 6 - Formulations Including P. syringae Lipodepsidecapeptide

The following formulation examples are illustrative only and are not intended to limit the scope of the invention m any way. The term "active ingredient" means a R. syringae hpodepsidecapeptide or a pharmaceutically acceptable salt thereof.

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

Quantity

Ingredient (mg/capsule)

Active ingredient 250

Starch, dπed 200

Magnesium stearate 10

Total 460 mg

Formulation 2

A tablet is prepared using the ingredients below. The components are blended and compressed to form tablets each weighing 665 mg.

Quantity

Ingredient (mg/capsule)

Active ingredient 250

Cellulose, microcrystalhne 400

Silicon dioxide, fumed 10

Steaπc acid 5

Total 665 mg

Formulation 3

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 Component Weight (g)

Active ingredient 0.25

Methanol 27.75

Propellant 22 (Chlorodifluoromethane) 74.00

Total 100.00

Formulation 4

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-pyrrohdone 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 dned 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.

Formulation 5

Capsules, each containing 80 mg of active ingredient, are made as follows: Active ingredient 80 mg

Starch 59 mg

Microcrvstalhne cellulose 59 mg

Magnesium stearate 2 mg

Total 200 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.

Formulation 6

Suppositones, each containing 225 mg of active ingredient, are made as follows:

Active ingredient 225 mg

Saturated fatty acid glycendes 2,000 mg

Total 2,225 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended m 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

Formulation 7

Suspensions, each containing 50 mg of active ingredient per 5 ml dose, are made as follows-

Active ingredient 50 mg

Sodium carboxymethyl 50 mg cellulose

Syrup 1.25 ml

Benzoic acid solution 0.10 ml

Flavor q v

Color q v

Puπfied water to total 5 ml The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor and color are diluted with a portion of the water and added, with stirπng. Sufficient water is then added to produce the required volume.

Formulation 8

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.

Active ingredient 100 mg

Isotonic saline 1 ,000 mg

The invention has been described with reference to various specific and prefened embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

WE CLAIM:

1. An isolated R. synngae depsidecapeptide. or a pharmaceutically acceptable salt, ester, or hydrate thereof, compnsmg a depsidecapeptide nng wherein the depsidecapeptide πng compπses arginine, threonine, homoseπne, dehydroammobutync acid, and dehydroalanme. and a lactone is formed from a carboxyl group of the argin e and a hydroxyl group of the threonine.

2. The R. svnngae depsidecapeptide of claim 1, wherein the depsidecapeptide nng has a sequence: Thr -Ala-Thr-Gln-Hse-Dhb-Ala-Dha-Thr-Arg (SEQ ID NO: 1).

3. The R. synngae depsidecapeptide of claim 1, wherein the R. syringae depsidecapeptide is a P. synngae podepsidecapeptide.

4. The R. synngae depsidecapeptide of claim 3, wherein the R. synngae hpodepsidecapeptide compπses a fatty acid moiety coupled to an ammo group of the threonine by an amide bond.

5 The R syringae depsidecapeptide of claim 4, wherein the fatty acid moiety is a decanoic acid moiety, a decanoic acid moiety substituted with one or two hydroxyl groups, a dodecanoic acid moiety, a dodecanoic acid moiety substituted with one or two hydroxyl groups, a tetradecanoic acid moiety, or a tetradecanoic acid moiety substituted with one or two hydroxyl groups.

6 The R synngae depsidecapeptide of claim 5, wherein the fatty acid moiety is an tt-dodecanoic acid moiety

7 The R svnngae depsidecapeptide of claim 3, wherein the R. synngae podepsidecapeptide is represented by the formula

where R is a lipophilic moiety, or a pharmaceutically acceptable salt, ester, or hydrate thereof.

8. The P. syringae depsidecapeptide of claim 7, wherein the lipophilic moiety is selected from the group consisting of C -C₁₅ alkyl, C₉-Ci5 hydroxyalkyl, C₉-C₎ dihydroxyalkyl, C -Cι₅ alkenyl, C -Cι₅ hydroxyalkenyl, and C -Cι₅ dihydroxyalkenyl.

9. The P. syringae depsidecapeptide of claims 8, wherein the lipophilic moiety is C -C₁₅ alkyl.

10. An isolated P. syringae depsidecapeptide having the formula:

or a pharmaceutically acceptable salt, ester, or hydrate thereof.

11. A method of inhibiting fungal activity comprising contacting a fungus with an isolated R. syringae depsidecapeptide of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

12. The method of claim 11, wherein the fungus comprises Candida parapsilosis, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum.

13. A method of reducing the symptoms of a fungal infection in a patient in need thereof comprising: administering to the patient an effective amount of a composition comprising an isolated R. syringae depsidecapeptide of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

14. The method of claim 13, wherein reducing the symptoms comprises decreasing a burden of a fungal infection.

15. The method of claim 13, wherein the fungal infection compnses infection by Candida parapsilosis, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum.

16. The method of claim 13, further compπsmg the steps of: determining the need for administeπng isolated R. synngae depsidecapeptide; and momtonng the patient for relief of symptoms of the fungal infection.

17. The method of claim 13, wherein adrmnistenng compπses parenteral administration about 1 to about 3 times per day of about 0.1 to about 5 mg/kg of isolated R. synngae depsidecapeptide.

18. The method of claim 13, wherein reducing the symptoms of a fungal infection compnses reducing fever and increasing general well being of the patient.

19. Use of an isolated R. syringae depsidecapeptide of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 m the manufacture of a medicament for the treatment of a fungal infection.

20. The use of Claim 19 wherein the fungal infection compπses infection by Candida parapsilosis, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum.

21. A method for producing one or more R. svnngae depsidecapeptides compnsing the steps of: cultunng a biologically pure culture of Pseudomonas svnngae in nutnent medium compnsing three or fewer amino acids at a pH of about 4 to about 6.5 until one or more R synngae depsidecapeptide is produced at a concentration of at least about 10 μg/mL; and recovenng one or more P. syringae depsidecapeptides. or a pharmaceutically acceptable salt, ester, or hydrate thereof, from the culture.

22. The method of claim 21, wherein the amino acid compnses glutamic acid, glycine, histidine, or a combination thereof.

23 The method of claim 22, wherein the amino acid compnses glycine

24. The method of claim 22, wherein the nutnent medium further compπses soluble starch, yeast extract, or a combination thereof and a hpid.

25. The method of claim 24, wherein the nutπent medium compπses a potato product and a hpid selected from the group consisting of soybean oil, fatty acids, and fatty acid esters.

26. The method of claim 25, wherein the potato product compnses potato dextrose broth, mashed potato mix, potato dextnn, potato protein, or a combination thereof.

27. The method of claim 21, wherein dunng the cultuπng step the dissolved oxygen concentration is maintained at about 5% to 30%.

28 The method of claim 21, wherein the cultunng step further compnses feeding of glucose, ammonium hydroxide, or a combination thereof.

29. The method of claim 21, wherein the R. synngae depsidecapeptide produced is a R. syringae depsidecapeptide of Claims 1. 2, 3, 4, 5, 6, 7, 8, 9 or 10.

30. The method of claim 21, wherein the Pseudomonas synngae compnses a strain denved from strain MSU 16H, MSU 174, or MSU 206

31 The method of claim 21. wherein the Pseudomonas svnngae compnses strain MSU 16H, strain 25-Bl, strain 67H1. or strain 7H9-1

32. The method of claim 31. wherein the Pseudomonas syringae compπses strain 25-Bl.

33. A P. syringae depsidecapeptide of Claim 7 prepared by the methods of

Claims 21, 22, 23, 24, 25, 26, 27, 28, 30, 31 or 32.

34. A method for treating or preventing fungal growth in a plant compnsing contacting a fungus with a R. synngae depsidecapeptide of Claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

35. The method of Claim 33 wherein the fungus is Rynchosporium secalis, Ceratocystis ulmi, Rizoctonia solani, Sclerotinia sclerotiorum, Venicillium albo-atrum, Venicillium dahliae, Thielaviopis basicola, Fusarium oxysporum or Fusarium culmorum.

36. The method of Claim 34 wherein the fungus is V. albo-atrum, Rhizoctonia solani or E. oxysporum.