CA2371363A1 - Antifungal agents isolated from pseudomonas syringae - Google Patents

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
J
FIELD OF THE INVENTION
The present invention relates to P. svringae 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 polvenes 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 svringae produce several classes of antifungal or antibiotic agents, such as the pseudomycins, syringomycins, syringotoxins, and syringostatins.
which are lipodepsinonapeptides. Natural strains and transposon generated mutants of P.
svrin,~ae produce these lipodepsinonapeptides. Several of the pseudomycins, syringomvcins and other lipodepsipeptide antifungal agents have been isolated, chemically characterized. and shown to possess wide spectrum antifungal activity. including activity against important fungal pathogens in both humans and plants. The pseudomycins, the syringomycins. the svringotoxins, and the svringostatins represent structurally distinct families of antifungal compounds.
None of the P. svringae lipodepsinonapeptides has been brought to market for antifungal therapy. Discovery of undesirable side effects, making formulations, scaling i up production, and other development problems have thus far prevented exploitation of the P. svringae lipodepsinonapeptides 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 in animals, humans, or plants.

SUMMARY OF THE INVENTION
The present invention provides a depsidecapeptide produced by P. svringae which contains the unusual amino acids homoserine (Hse), dehydroaminobutyric acid (Dhb) and dehydroalanine (Dha) as part of a depsidecapeptide ring. More specifically, the P.
15 svringae depsidecapeptide includes a depsidecapeptide ring having the amino acids, arginine, threonine, homoserine, dehydroaminobutyric acid, and dehydroalanine, and a lactone formed from a carboxyl group of the arginine and a hydroxyl group of the threonine. As isolated from P. svringae, the depsidecapeptide is a lipodepsidecapeptide: a cyclic peptide coupled to a lipophilic moiety. Typically the lipophilic moiety is a fatty 20 acid moiety coupled to the amino group of the threonine by an amide bond.
Preferably.
the fatty acid moiety is an n-dodecanoic acid moiety. The lipodepsidecapeptide is represented by formula I:
-, O
O O
H N

H ~ H
NH ~ O
HO OH O NH
O O
U wN NH2 HN H
O HN
N N O
'"

HO
where R is a lipophilic moiety. The lipophilic moiety includes C9-C,~ alkyl, C9-CI;
hydroxyalkyl, C9-C~~ dihydroxyalkyl, C9-Ci; alkenyl, C9-C,; hydroxyalkenyl, or C9-C,5 dihydroxvalkenyl. Preferably, the lipophilic moiety is C~, alkyl. The alkyl, hydroxyalkyl, dihydroxyalkyl, alkenyl, hydroxyalkenyl, or dihydroxyalkenyl groups may be branched or unbranched. Preferably, the amino acid sequence of the depsidecapeptide ring is threonine-alanine-threonine-glutamine-homoserine-dehydroaminobutyric acid-alanine-dehydroalanine-threonine-arginine, referred to herein as "25-B 1 decapeptide"
or "Thr-Ala-Thr-Gln-Xaa-Xaa-Ala-Xaa-Thr-Arg (SEQ 1D NO: 1)".
As used herein, the term "25-B 1 decapeptide antifungal agent A" refers to the specific depsidecapeptide having the preferred amino acid sequence SEQ 1Z7 NO: 1 and R
=
unbranched C,1 alkyl (i.e., R = -(CH~),oCH:).
1 ~ The invention also relates to methods employing a P. svrifzgae 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.
.svringae depsidecapeptides may be used in the manufacture of a medicament for treatment of a patient as described herein. The methods and medicaments of the invention are effective against fungi such as CafZdida parapsilosis, Candida albica~zs, Cn~ptococcus neoformans, or Histoplasma capsulatum.
The invention provides using microorganisms in a method for producing an anti-fungal agent, such as the P. svringae depsidecapeptides described above and including a 25-B 1 decapeptide. The method involves culturing Pseudomonas svringae in media including three or fewer amino acids and recovering one or more P. syringae depsidecapeptides from the culture. In one embodiment, P. svringae culture is in medium including alycine and a lipid, a potato product, or a combination thereof at a pH of about 4 to 6.~ until one or more P. svringae depsidecapeptides is produced at a concentration of at least about 10 ~,g/mL. In addition, the invention provides P. svringae depsidecapeptides prepared by the method described 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 P. svringae depsidecapeptides described above.
DETAILED DESCRIPTION
Linode~sidecane~tide AntifunQal Agents As used herein "lipodepsidecapeptide antifungal agent" refers to an antifunaal agent having a cyclic decapeptide ring closed by a lactone group and having an appended hydrophobic group, such as a fatty acid moiety. Lipodepsidecapeptide antifungal agents are produced by Pseudomonas svringae. A representative of this class of compounds, 25-B 1 decapeptide antifungal agent A, has been purified and its structure determined. As used herein the term "P. svringae lipodepsidecapeptide" refers to a lipodepsidecapeptide antifungal agent produced by P. svringae, and includes 25-B1 decapeptide antifungal anent A and related analogs.
P. svringae lipodepsidecapeptides share several structural features. For example.
each of these antifungal agents includes the unusual amino acids homoserine (Hse), dehvdroaminobutyric acid (Dhb) and dehvdroalanine (Dha) as part of a depsidecapeptide ring. In each of the P. svringae lipodepsidecapeptides, a carboxyl group of an arainine residue linked to the hydroxyl group of the N-terminal threonine forms a lactone that closes the depsidecapeptide ring. The sequence of the depsidecapeptide ring of the P.
svringae lipodepsidecapeptide 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 occurring amino acids. Unlike the pseudomycin natural products, the lipodepsidecapeptides of the present invention do not contain chlorothreonine which is suspected to be the cause for irritation at the injection site of pharmaceutical formulations containing pseudomycin compounds.
The depsidecapeptide ring 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 bearing 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 ra-tetradecanoic acid moiety, or an n-tetradecanoic acid moiety substituted with one or two hydroxyl groups.
25-B 1 Decapeptide Antifungal Agents As used herein, "25-B 1 decapeptide antifungal agent" refers to one or more members of a family of antifungal agents that has been isolated from the bacterium Pseudomonas syringae. A 25-B 1 decapeptide antifungal agent is a P. svringae lipodepsidecapeptide. Specifically, a 25-B 1 decapeptide antifungal agent is a P. syringae lipodepsidecapeptide having a depsidecapeptide ring with the sequence:
Thr-Ala-Thr-Gln-Hse-Dhb-Ala-Dha-Thr-Arg (SEQ >D NO: 1 i Each of the 2~-B 1 decapeptide antifungal agents has the same cyclic peptide nucleus, but thev differ in the hydrophobic side chain attached to this nucleus. The 25-B 1 decapeptide antifungal agents include 2~-B 1 decapeplide antifungal agent A .
J

The 25-Bl decapeptide antifungal agents include a fatty acid linked through an amide bond with the amino group of the N-terminal threonine. The fatty acid moiety of 25-B 1 decapeptide antifungal agent A is an n-dodecanoic acid moiety.
Biological Activities of P. svrineae Lipodepsidecapeptides A P. svringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, has several biological activities including killing and inhibiting activity of various 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 various species of Candida including C.
parapsilosis and C. albicans.
Pseudomonas syrin ,tae Pseudomonas svringae include a wide range of bacteria that are generally associated with plants. Some of the P. syringae are plant pathogens, while others are only weakly pathogenic or are saprophytes. Many different isolates of P. syringae produce one or more cytotoxic agents that can help this bacterium survive in the wild where it must compete with fungi and other bacteria. The cytotoxic agents produced by P.
svringae include anti-fungal agents such as the P. svringae lipodepsidecapeptides, including 25-B 1 decapeptide antifungal agent A, the pseudomycins, the syringomycins, the syringotoxins.
and the syringostatins.
Isolated strains of P. syringae that produce one or more pseudomycins, syringomycins, syringotoxins, syringostatins are well-known to those skilled in the art.
Wild type strain MSU 174 and a mutant of this strain generated by transposon mutaaenesis, MSU 16H (ATCC 67028) have been described in U.S. Patent No.
5,576.298, issued November 19, 1996 to G. Strobel et al.: Harnson et al., "Pseudomycins, a family of novel peptides from Pseudomonas svringae possessing broad-spectrum antifun~al 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 various strains of P.
syringae and their use in production of antifungal agents such as pseudomycins are also disclosed in U.S. Patent Application Serial No. PCT/US00/08728 by Matthew D. Hilton, et al.
entitled "Pseudomycin Production By Pseudomonas Syringae" submitted evendate herewith and described below. The disclosures of the references cited in this paragraph are incorporated herein by reference.
Strains of P. syringae that are suitable for production of one or more P.
syringae lipodepsidecapeptides, such as 25-B1 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-purified culture of P.
syringae that produce one or more P. syringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, in amounts greater than about 10 ug/mL, preferably from at least about 10 p.g/mL to about 50 ~Cg/mL. Preferably, thebiologically-purified culture of a microorganism is of Pseudomonas syringae strains MSU 16H, 25-B1, 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 lipodepsidecapeptides, such as 25-B1 decapeptide antifungal agent A, can be isolated from environmental sources including plants, such as barley plants, citrus plants, and Iilac 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._ As used herein, "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). As is the case with other organisms, the characteristics of the lipodepsidecapeptide-producing cultures employed in this invention, P. syringae strains such as MSU 174, MSU 16H, MSU
206, RECTIFIED SHEET (RULE 91) ISAlEP

~~-B1. and 7H9 are subject to variation. Thus: progeny of these strains, e.g..
recombinants, mutants and variants, may be obtained by methods well-known to those skilled in the art.
Mutant strains of P. svringae are also suitable for production of one or more P.
svringae lipodepsidecapeptides, such as 2~-B 1 decapeptide antifungal agent A.
As used herein. "mutant" refers to a sudden heritable change in the phenotype of a strain, which can be spontaneous or induced by known mutagenic agents, including radiation and various chemicals. Mutant P. svringae of the present invention can be produced using a variety 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-vitro-N'-nitrosoguanine (NTG), and nitrous acid.
P. svringae suitable for producing one or more P. syringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, according to the present invention can be generated by treating the bacteria with an amount of a mutagenic agent effective to produce mutants that overproduce one or more P. syringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, or that produce one or more P. syringae lipodepsidecapeptides, such as 25-B 1 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 serially dilute NTG to levels ranging from about 1 to about 100 q,g/mL. Preferred mutants of the invention are those that overproduce 2~-B 1 decapeptide antifungal agent A, and grow in minimal medium. The mutants overproduce a P.
svringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, preferably from at least about 10 ~, g/mL to about 50 ~C g lmL.
?5 Environmental isolates, mutant strains. and other desirable strains of P.
syringae can be subjected to selection for desirable traits of growth habit, growth medium, nutrient source. carbon source, growth conditions, and amino acid requirements.
Preferably, a strain of P. svringae producing P. svringae lipodepsidecapeptide, such as 2~-B

decapeptide antifungal agent A. is selected for growth on minimal defined medium.
Preferred strains exhibit the characteristlos of producing one or more P.
syringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A. when grown on a medium including glycine plus, optionally, a lipid. a potato product, or both.
Recombinant strains can be developed by transforming the P. svringae strains.
using procedures well-known to those skilled in the art. Through the use of recombinant technology, the P. svriragae strains can be transformed to express a variety 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. syringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, but also the resistance-conferring 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 lipodepsidecapeptide-biosynthesis genes to achieve greater lipodepsidecapeptide yield. Progeny, i.e. natural and induced variants, mutants and recombinants, of the P. svringae strains 25-Bl, 67H1, and 7H9-1 which retain the characteristic of lipodepsidecapeptide production are part of this invention.
Growth of Pseudomonas svrin~
As described herein, "aqueous nutrient media" refers to a water-base composition including minerals and organic compounds and their salts necessary for growth of the bacterium used in the present invention. Preferred nutrient media contain an effective amount of three or fewer amino acids, preferably, glutamic acid, glycine, histidine, or a combination thereof. In one embodiment, the medium contains an effective amount of glycine and, optionally, one or more of a potato product and a lipid. Glycine can be provided as a single amino acid or as part of a mixture of amino acids, such as hydrolyzed protein. Suitable lipids include soybean oil, fatty acids, or fatty acid esters. Suitable potato products include potato dextrose broth, potato dextrin, potato protein, or a commercial mashed potato mix food product. Preferred minerals in the nutrient medium include salt mixtures typically used in cell culture and fermentation, such as Czapek mineral salts. which includes KC1, MgS04, and FeSOs. Organic compounds in the nutrient 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 nutrient broth is not typically critical to growth of the bacteria or to production of a P. svringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, certain levels of nutrients 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 lipid 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/I. of potato dextrin; and/or about 1 g/L to about 10 glL, preferably about 4 g/L of potato protein. A
preferred nutrient medium includes minerals, preferably, KCI at about 0.02 to about 2 g/LL., more preferably about 0.2 g/L; MgS04, preferably MgSO~~7H~0, at about 0.02 to about 2 g/L, more preferably about 0.2 g/L; and FeS04, preferably FeSOa~7H~0, at about 0.4 to about 40 mglL, 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 described under conditions of controlled or regulated pH, and temperature. P. syringae 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.
svringae grow and produce one or more cvtotoxic 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 P.
svringae 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. svrin~ae Lipodepsidecapeptide To produce one or more P. svringae lipodepsidecapeptides, such as 2~-B 1 decapeptide antifungal agent A, from a wild type or mutant strain of P.
svringae, 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. Culturing is conducted under conditions effective for growth of P.
syringae and production of a desired P. syringae lipodepsidecapeptide, such as 25-B 1 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 described herein, P. svringae can grow at cell densities up to about 10-15 g/L dry weight and produce a P. syringae 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 during culturing of P.
svringae is advantageous for production of a P. svringae lipodepsidecapeptide, 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 in the medium. Further, adjustment of the agitation rate can be used to adjust the oxvaen transfer rate.
Controlling the pH of the medium during culturing of P. syringae is advantageous for production of a P. syringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A. The pH of the culture medium can be maintained at less than about 6 and above about 4.
P. svringae can produce a P. svringae lipodepsidecapeptide, such as 25-B 1 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 P. svringae lipodepsidecapeptide, such as 2~-B 1 decapeptide antifungal agent A. Production of a P. svringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, by P.
svringae 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. svringae strain can affect the amount and distribution of a P.
svringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, produced by culturing under the conditions described herein. For example, strain 25 B 1 can produce predominantly 2~-B1 decapeptide antifungal agent A.
The cyclic decapeptide nucleus of the P. syringae lipodepsidecapeptides can be prepared by cleaving off the lipophilic moiety, such as by deacylation.
Cleavage and deacvlation methods are well-known to those skilled in the art, such as the use of deacylase enzymes.
Formulation and Antifun~al Action of P. svringae Lipodepsidecapeptides A P. syringae lipodepsidecapeptide, 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 skin infections. Accordingly, the present invention provides a method of inhibiting fungal activity including contacting a P. syringae lipodepsidecapeptide, such as a 25-B 1 decapeptide antifungal agent, or a pharmaceutically acceptable salt thereof, with a fungus. A preferred method includes inhibiting growth or activity of various 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 administering an effective amount of a P. svringae 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 various 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 P. syringae lipodepsidecapeptides, such as 25-B 1 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 P.
svringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, have severe symptoms of infection, such as high fever, and are likely to be in intensive or critical care. Various fungi can cause such serious infections. Carzdida spp., for example, causes mucosal and serious systemic infections and may exist as azole- or polvene-resistant strains. Aspergillus causes life-threatening systemic infections.
Cryptococcus is responsible for meningitis. Such serious fungal infections may occur in immune compromised patients, such as those receiving organ or bone marrow transplants, undergoing chemotherapy for cancer, recovering from major surgery, or suffering from HIV infection. For such patients, antifungal therapy typically includes intravenous administration, of a formulation of one or more P. svringae lipodepsidecapeptides (e.g., the 2~-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 bring 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 prophylactically hindering or preventing the growth or anv attending characteristics and results from the existence of a fungus.

Typically, the compositions will be adrriinistered 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 severity 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 during 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 mglkg to about 40 mg/kg. For serious infections, the compound can be administered by intravenous infusion using, for example, 0.01 to 10 mglkg/hr of the active ingredient.
The present invention also provides pharmaceutical formulations useful for administering the antifungal compounds of the invention. Accordingly, the present invention also provides a pharmaceutical formulation including one or more pharmaceutically acceptable Garners, diluents, vehicles, excipients, or other additives and one or more P. svringae lipodepsidecapeptides, 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. By "pharmaceutically acceptable" it is meant that the Garner, diluent or excipient is compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The formulation can include additives such as various oils, including those of petroleum, animal, vegetable or synthetic origin, 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 chloride, dried skim milk, glycerol, propylene Glycol, water, and ethanol. The compositions can be subjected to conventional pharmaceutical expedients, such as sterilization, and can contain conventional pharmaceutical additives, such as preservatives, stabilizing agents, wetting, or erriulsifying agents. salts for adjusting osmotic pressure, and buffers. Suitable pharmaceutical carriers and their formulations are described in Martin. "Remington's Pharmaceutical Sciences," 15th Ed.; Mack Publishing Co., Easton f 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 hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. Examples of such pharmaceutically acceptable salts are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, 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. Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as malefic acid and methanesulfonic acid.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides. carbonates, and bicarbonates.
Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium ~0 hydroxide, ammonium hydroxide. potassium carbonate, sodium carbonate, sodium l bicarbonate, potassium bicarbonate, calcium Hydroxide. and calcium carbonate.
The potassium and sodium salt forms are particularly preferred.
It should be recognized that the particular counterion forming a part of any salt of this invention is not of a critical nature. so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
A P. syringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A, may be administered parenterally, for example using intramuscular, subcutaneous, or infra-peritoneal injection, nasal, or oral means. In addition to these methods of administration, a P. syringae lipodepsidecapeptide, such as 25-B1 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. svringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, and a physiologically acceptable diluent such as deionized water, physiological saline, 5°Io dextrose and other commonly used diluents. The formulation may contain a cyclodextrin and/or a solubilizing agent such as a polyethylene glycol or polypropylene glycol or other known solubilizing agent. Such formulations may be made up in sterile vials containing the antifungal and excipient in a dry powder or lyophilized powder form. Prior to use, a physiologically acceptable diluent is added and the solution withdrawn via syringe 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 carrier, or diluted by a carrier, or enclosed within a Garner which may be in the form of a capsule, sachet, paper or other container. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material 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 mediumi. ointments containing, for example, up to 10~/o by weight of the active compound, soft and hard gelatin capsules, suppositories. sterile injectable solutions, or sterile 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 preparing a proper size tablet for the dosage and a P.
syringae lipodepsidecapeptide, such as 25-B 1 decapeptide antifungal agent A.
For pediatric or geriatric use the antifungal compound may be formulated into a flavored liquid suspension, solution or emulsion. A preferred oral formulation is linoleic acid.
cremophor RH-60 and water and preferably in the amount (by volume) of 8%
linoleic acid. 5°Io cremophor RH-60, 87% sterile water and a P. syringae lipodepsidecapeptide, such as 25-Bl decapeptide antifungal agent A, in an amount of from about 2.~
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 solubilizing aqueous liquid or non-aqueous liquid, e.g., an alcohol or glycol.
Uses of Formulations of a P. svrinQae 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 carriers, either dried 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 in which the vial is packaged. for the use and administration of the compounds. The instructions can also be printed 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. svringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, and that is suitable for administration by injection.
According to the invention, a formulation of one or more P. svi-ingae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A. can be used for manufacturing a composition or medicament suitable for administration by injection. The invention also relates to methods for manufacturing compositions including a formulation of one or more P.
svrir2gae lipodepsidecapeptides, such as 25-B 1 decapeptide antifunaal anent 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 P.
syringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients.
Agricultural Uses Antibiotics produced from P. svringae 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. 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. Hence, the lipodepsidecapeptides of the present invention may be useful as a preventative treatment for Dutch Elm disease. The pseudomvcins have been shown to be toxic to a broad range of plant-pathogenic fungi including Rvnchosporium secalis, Ceratocystis ulmi, Rizoctonia solani, Sclerotinia sclerotiorum, Verticillium albo-atrum, Verticillium dahliae, Thielaviopis basicola, Fusarium oxysporum and Fusarium culmorum. (see Hatnson, L., et al., "Pseudomycins, a family of novel peptides from Pseudomonas svringae possessing broad-spectrum antifungal activity, " J.
General Microbiology, 7, 2857-2865 (1991).) Consequently, one or more P. svringae lipodepsidecapeptides, such as 25-B1 decapeptide antifungal agent A (including hydrates.
solvates, and esters thereof) may be useful in the treatment of fungi in plants (in particular. V. albo-atruna, RhizoctorZia solani and F. orvsporum) either as a direct treatment or preventative treatment. Generally, the infected plants are treated by 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.Q., Qouge pistol). Any means of spraying the suspension may be used that distributes an effective amount of the active material onto the plant surface. The suspension may also include other additives generally used by those skilled in the art, such as solubilizers, stabilizers. wetting agents. and combinations thereof.
Treatment of the plant may also be accomplished using a dry composition containing one or more P. svringae lipodepsidecapeptides, such as 25-B 1 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 P. syringae MSU 16H is publicly available from the American Type Culture Collection, Parklawn Drive, Rockville, MD, USA as Accession No. ATCC 67028. P.
svringae strains 25-B1, 7H9-1, and 67 Hl were deposited with the American 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 H1 Accession No. PTA-1621 Example 1 - Production of 25-B1 Antifungal Agent A
Fermentation methods were developed for producing a lipodepsidecapeptide antifungal agent, 25-B 1 decapeptide antifungal agent A, in the fermentation broth of a Pseudomonas svringae strain.

Materials and Methods Preparation of inoculum: An aliquot of P. svrin~ae strain 25-B 1 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 MgS04 7H~0 (2). Approximately 0.~
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 sterile fermentation broth.
Fermentation Stage: Fermentation broth was composed of (g/L): dextrose (20), soluble starch (5), Basic American Foods Country Style Potato Pearls instant mashed potatoes (30), glycine (1), MgS04 7H~0 (0.2), KCl (0.2), and FeS04 7H~0 (0.004) in tap water. The pH was adjusted to ~.2 before sterilization. Fermentation was carried 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~S04 or NaOH.
Several variations 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 period. 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.
Results and Conclusion Several fermentation methods produce 25-Bl decapeptide antifungal agent A from P. svrin,Qae Example 2 - Isolation and Purification of 25-B1 Antifun~al Agent A
;Methods were developed for isolation and purification of a lipodepsidecapeptide antifungai agent. 2~-B 1 decapeptide antifungal agent A. from the fermentation broth of a Pseudomonas svrinQae strain.
J
Materials and Methods The whole fermentation broth produced according to Example l, typically 100 L
after harvest, was filtered through a MembraloxTM ceramic filter (0.45 ~.m).
The resulting solid slurry was extracted with an equal volume of acetone containing 0.1 %
TFA for 90 min. 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 AmberchromTM CG 300sd resin column (4 L) packed in water. The column was initially washed with 0.2% acetic acid (pH 4-8) until 1~ the effluent showed pH 4.5 followed by 10 L of 22% acetonitrile containing 0.2% acetic acid (pH 4.8). Then the column was eluted with a linear gradient of 22-35%
acetonitrile containing 0.2% acetic acid (32 L) and 35% acetonitrile containing 0.2% acetic acid (8 L) with 400 ml/min flow rate. Fractions 11-16 were combined (4.8 L), concentrated in vacuo to 100 ml and centrifuged.
The supernatant was separated and chromatographed over an Amberchrom CG
300sd column (1 L) using a linear gradient of 25-35% acetonitrile containing 0.2% acetic acid (pH 4.8) with 50 ml/min flow rate. Fractions 20-25 (1.2 L) were combined and rechromatographed over a reversed-phase column (NovaPak C,B, 6 ~.m. 40X300 mm, flow rate 40 ml/min, linear gradient 30-60% acetonitrile containing 0.2% TFA) to yield 21 ma of a compound (89% purity by LTV).
The ESIMS 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.
Syringae. The 'H
NMR spectrum showed signals reminiscent of pseudomycin-like lipopeptide but indicated the presence of more than one compound. In order to obtain 2~-B 1 decapeptide antifungal agent A in high purity for structure determination and antifungal activity. an additional broth from 4X100 L fermentatiomvas processed as described above and in addition. the final purification was carried out on a reversed-phase column [Rainin C,B, 6 ~,m, 24X250 mm. 0. l~'o TFA-acetonitrile-MeOH (8:1:1 to 4:3:3) gradient elution for 60 min: (4:3:3 to 10:45:40 Gradient elution for 30 min] to afford 42 mg of 25-B 1 decapeptide antifungal agent A (93% purity by UV).
Results and Conclusion HPLC methods similar to those used to purify other lipodepsipeptide antifungal agents resulted in purification of 25-B 1 decapeptide antifungal agent A from fermentation broth.
Example 3 - Determination of the Structure of 25-B1 Antifungal Agent A
Mass spectrometry and NMR determined the structure of a lipodepsidecapeptide antifungal agent, 25-B 1 decapeptide antifungal agent A.
Methods and Results The molecular formula of 25-B 1 decapeptide antifungal agent A was determined by high resolution FABMS as C;~HggN,4O,6[m/Z 1165.6581 for CszHs9N~a0~6(M+H)+,4+0.9 ppm].
In accordance with this formula the 1'C and DEPT NMR spectra showed 50 distinct resonances, which included twelve carbonyl carbons, five olefinic carbons, four oxygenated sp' carbons, eight typical amino acid a-carbons, fifteen methylene carbons and six methyl carbons. Among these, one of the methyl carbon signals at 8 16.7 and one of the methylene carbon si finals at 8 28.9 each constituted a set of degenerate carbons, 2~ thus accounting for the total number of 52 carbons observed in the molecular formula.
Detailed analysis of 'H. ''C. and 2D NMR (DQCOSY, TOCSY, HMQC, HMBC
and ROESY) data enabled to determine the structure of 25-B 1 decapeptide antifungal agent A (IA) and unambiguously assign all the protons and carbons (Table 1).

(CHZ)~oCH~
NH
' 'NH2 IA
Table 1. 1H and''C NMR Chemical Shifts of 25-Bl decapeptide antifungal agent A in DMSO-d6 Amino acid Position ~ $c Art NH 8.13 -CO - 170.4 a 4.23 51.7 (31 1.69 28.2 (32 1.63 1.46 24.7 b 3.08 40.2 NH 7.59 -- 16.7 Thr-1 NH 7.83 -CO - 169.8 4.23 59.5 4.00 66.4 1.08 19.7 Dha NH 9.01 -CO ~ - 163.7 - 13.3 (31 J.S6 106.4 (32 5.61 Amino acid Position ~ $H $~

I

Aia-1 NH 7.96 CO - 171.6 4.22 49.9 1.31 16.7 Dhb ~ NH 9.07 -CO - 164.0 130.0 6.40 128.5 1.61 12.9 HSer NH 8.22 -CO - 171.1 a, 4.22 51.4 (31 1.82 34.0 (37 1.76

3.47 X7.3 Gln NH 7.~9 -CO - 171.9 a, 4.31 52.0 1.90 27.9 (j2 1.73 2.11 31.1 CO - 173.9 NH~ 7.13 -6.72 Thr-2 NH 7.80 -CO - 169.9 a, 4.11 58.4

4.11 66.0 1.01 19.8 Ala-2 NH 8.18 -CO - 172.4 4.32 48.5 1.16 17.6 Amino acid Position $H

Thr-3 NH 7.93 CO - 168.6 4.47 55.6 4.94 70.5 v 1.12 16.7 Side chain CO 172.2 2 2.08 35.1 3 1.44 25.1 4 1.21 28.5

5-10 1.21 31.1. 28.9X2, 28.8, 28.7, 28.6 11 1.21 22.0 12 0.83 -- 13.9 I

The results from ~H, DQCOSY and TOCSY spectra measured in DMSO-d6 at 35°C revealed the presence of spin systems for seven commonly occurring amino acid residues - two alanines, one arginine, one glutamine and three threonines, and three less commonly occurring amino acids - one dehydroalanine (Dha), one dehydroaminobutyric acid (Dhb) and one homoserine. The less commonly occurring amino acid residues, viz.
Dha. Dhb and homoserine, were identified by the cross peaks observed in the TOCSY
spectrum from the amide protons resonating at 8 9.01 (brs), 9.07 (brs) and 8.22 to protons resonating at 8 5.86, 5.61 (8c 106.4), 6.40 (8c 128.5), 1.61 (8c 12.9) and 4.22 (8c 51.4).
1.82. 1.76 (8~ 34.0), 3.47 (8~ 57.3), respectively. Consistent with this the spectrum displayed eight proton bound a-carbon signals for the saturated amino acid residues and two quaternary a-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 (8 164.0 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 (8H 0.83 and cSc 13.9) and methvlene signals in the ~-C NMR spectrum (Table 1).
~J

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 2~-B 1 decapeptide antifungal agent A is a monocyclic decapeptide.
Comparison of the chemical shift of the (3-protons of threonines (8H 4.94, 4.11 and 4.00) indicated that the proton resonating at b 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-B 1 decapeptide antifungal agent A.
With amino acid Arg as a starting point, the long range 'H -'3C correlations observed in the HMBC spectrum between the amide proton and the adjacent amino acid carbonyl and or a-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-a-C, Glu-NH/Thr-CO. Thr-NH/Ala CO and Ala-NHIThr-CO.

O
O
O CH3 O v v v O N~ ~ NH~ H3C
'H~ ~ 'H~
NH~ O H C
H3~\OH
HO ~~O H NH
O
HN ~ 1 NH2 H3~ O CH2 HN~~
H o N ~' O ~ H

HO ~H3 IiMBC (H -~ C) Correlations ?6 Scheme I
The NH of Thr adjacent to Ala did not show a long range 1H - ~'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 cotTelation and presence of a correlation between the Thr-(3-H (8H 4.94, b~ 70.5)/Arg-CO clearly established an ester linkage between the Thr-~3-OH and Arg-COOH. Consistent with these assignments are the ROESY correlations that were observed between the amide protons and the adjacent amino acid a-protons (see Scheme II below).
HZN
O
O
O ~CH3 O
O N NH H,3C
J ~" ~' ~ "'~'~
NH O
,~ c °" O
O H NH
o N
N~
HN
O ~~2 O 1~
'H
Selected ROESY Correlations Scheme II
Conclusions Compound 25-B 1 decapeptide antifungal agent A represents a novel class of lipodepsipeptide which possesses several amino acid residues that are not present in any of the pseudomycins, svringomycins. svringotoxin and syringostatins produced by different isolates of P. syringae. The new depsipeptide is composed of ten amino acids ?7 which is also a departure from the pseudomycins and syringomycins which possess only nine amino acid residues. Unlike the pseudomycin natural products, the new depsipeptide does not include chlorothreonine which is suspected to be the cause for irntation at the injection site of pharmaceutical formulations containing pseudomycin compounds.
Example 4 -- Antifun~al activity of 25-B1 Decapentide Antifun~al 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
10'~
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.
Table 2. Antifungal activity of 1 Organism MIC (~Cg/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 P. svringae lipodepsipeptides, such as B 1 decapeptide antifungal agent A, can be determined by measuring 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 lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, can be an extract of a cell culture, or a more purified 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.
'' 8 Example S -- Isolation, Characterization and Muta~enesis of Pseudomonas syrin~ae Environmental isolates and mutants of P. svringae were produced and employed in production of antifungal agents.
Materials and Methods Strains MSU 174 and MSU 16-H were isolated and characterized as described in L'.S. Patent No. 5,576,298, issued November 19, 1996 to G. Strobel et al.;
Harnson et al., "Pseudomycins, 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 $4, 6447-6451 (1987). The disclosures of the references cited in this paragraph are incorporated herein by reference.
Additional strains were derived from such wild type and transposon generated mutants by chemical mutagenesis. Strains subjected to mutagenesis include MSU
174, MSU 16H, and 25-B 1. 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 1-methyl-3-vitro-1-nitrosoguanidine (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. svringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A. Chemically mutagenized cells of P. syringae, such as mutagenized strain 25-B l, 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 ~0 ~,L 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, silicone oil was added to each well to minimize evaporation. The 0 plates were incubated with shaking for 6 to 12 davs at '?~ °C.
?9 Table 3 -- The Composition of N21SM Medium GRAMS
INGREDIENT PER LITER

Glucose 20 Ammonium Sulfate 0.~

Monosodium Glutamate 2 L-Histidine 2 Glvcine 0.5 Soluble Starch 5 KH~P04 Czapek Mineral Salts 2 mL
Solution MES Buffer 9.8 Adjust pH to 5.0 After this incubation, an aliquot, typically 5 ~L, from each well was serially 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.

Table 4. Complete Streptbmyces Medium (CSM) Component Concentration (g/L) Glucose Maltose 4 Difco Tryptic Sov Broth 30 Difco Yeast Extract MgS047H,0 2 No pH adjustment The selected strains were preserved and inoculated into fermentation bottles containing l3mL 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 acetonitrile equal to the volume of the aliquot, centrifuged, and decanted for HPLC
analysis of one or more P. syringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, as described in Examples 1-3. Strains producing one or more P.
syringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, were reisolated, refermented, and prepared for growth on a larger scale.
Results Strains exhibiting production of one or more P. syringae lipodepsidecapeptides, such as 25-B 1 decapeptide antifungal agent A, were produced using the methods described above.
Conclusion The selection methods and criteria disclosed herein are effective for producing strains of P. syringae that grow on minimal medium and produce one or more P.
syringae lipodepsidecapeptides, such as 2~-B1 decapeptide antifungal agent A.

Example 6 - Formulations Including P. syrin~ae Lipodepsidecapeptide The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way. The term "active ingredient"
means a P.
svringae lipodepsidecapeptide or a pharmaceutically acceptable salt thereof.
J
Formulation 1 Hard gelatin capsules are prepared using the following ingredients:
Quantity Ingredient (mg/capsule) Active ingredient 250 Starch, dried 200 Ma nesium stearate 10 Total 460 m~

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, microcrystalline400 Silicon dioxide, fumed 10 Stearic acid Total 665 m~

1 ~ 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:
Active ingredient 60 m Microcrvstalline cellulose 45 ma Polyvinylpyrrolidone (as 10% 4 mg solution in water) Sodium carbox methyl starch 4.5 m~

Magnesium stearate 0.5 m Talc 1 m Total _ _- 150 mg The active ingredient, starch and cellulose are passed through a No. 45 mesh U.S.
sieve and mixed thoroughly. The aqueous solution containing polyvinyl-pyrrolidone 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 dried at SOo 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 m~.
Formulation 5 Capsules. each containing 80 mg of active ingredient, are made as follows:
., -, JJ

Active ingredient 80 mg i, Starch ~9_ma ~

Microcrvstalline cellulose ~9 ma Magnesium stearate 2 ma I, Total 200 ma 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 Suppositories, each containing 225 mg of active ingredient, are made as follows:
Active in redient 225 m~
Saturated fatt acid 1 cerides 2,000 m Total 2 225 ma The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides 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 in ~edient _ ~0 mg Sodium carboxymethyl ~0 mg cellulose S ru 1.25 ml Benzoic acid solution 0.10 ml Flavor q.v.

Color .v.

Purified water to total ~ 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 stirring.
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 rrunute.
Active in redient 100 m Isotonic saline 1,000 mg The invention has been described with reference to various specific and preferred 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 (36)

WE CLAIM:

1. An isolated P. syringae depsidecapeptide, or a pharmaceutically acceptable salt, ester, or hydrate thereof, comprising a depsidecapeptide ring wherein the depsidecapeptide ring comprises arginine, threonine, homoserine, dehydroaminobutyric acid, and dehydroalanine, and a lactone is formed from a carboxyl group of the arginine and a hydroxyl group of the threonine.

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

3. The P. syringae depsidecapeptide of claim 1, wherein the P. syringae depsidecapeptide is a P. syringae lipodepsidecapeptide.

4. The P. syringae depsidecapeptide of claim 3, wherein the P. syringae lipodepsidecapeptide comprises a fatty acid moiety coupled to an amino group of the threonine by an amide bond.

5. The P. 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 P. syringae depsidecapeptide of claim 5, wherein the fatty acid moiety is an n-dodecanoic acid moiety.

7. The P. syringae depsidecapeptide of claim 3, wherein the P. syringae lipodepsidecapeptide is represented by the formula:

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

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

9. The P.syringae depsidecapeptide of claims 8, wherein the lipophilic moiety is C9-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 P. 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 P. 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 comprises infection by Candida parapsilosis, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum.

16. The method of claim 13, further comprising the steps of:
determining the need for administering isolated P. syringae depsidecapeptide;
and monitoring the patient for relief of symptoms of the fungal infection.

17. The method of claim 13, wherein administering comprises parenteral administration about 1 to about 3 times per day of about 0.1 to about 5 mg/kg of isolated P. svringae depsidecapeptide.

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

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

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

21. A method for producing one or more P. syringae depsidecapeptides comprising the steps of:
culturing a biologically pure culture of Pseudomonas syringae in nutrient medium comprising three or fewer amino acids at a pH of about 4 to about 6.5 until one or more P.
syringae depsidecapeptide is produced at a concentration of at least about 10 µg/mL; and recovering 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 comprises glutamic acid, glycine, histidine, or a combination thereof.

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

24. The method of claim 22, wherein the nutrient medium further comprises soluble starch. yeast extract, or a combination thereof and a lipid.

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

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

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

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

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

30. The method of claim 21, wherein the Pseudomonas syringae comprises a strain derived from strain MSU 16H, MSU 174, or MSU 206.

31. The method of claim 21, wherein the Pseudomonas syringae comprises strain MSU 16H, strain 25-B1, strain 67H1, or strain 7H9-1.

32. The method of claim 31, wherein the Pseudomonas syringae comprises strain 25-B1.

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 comprising contacting a fungus with a P. syringae 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, Verticillium albo-atrum, Verticillium dahliae, Thielaviopis basicola, Fusarium oxysporum or Fusarium culmorum.

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