HUP0200897A2 - Antifungal agents isolated from pseudomonas syringae - Google Patents

Description

¹ or 897 k

73.334/SZE ^SECRETARY

PELDaNY ^/antifungal agent isolated from pseudomonas syringae

TOOLS

SUBJECT OF THE INVENTION

The present invention relates to P. syringae depsidecapeptides, to the preparation of such peptides, and to a method of using these peptides antifungally.

BACKGROUND OF THE INVENTION

Fungal infections cause significant morbidity, reduced quality of life, and increased mortality in humans, especially in patients with immunocompromised conditions. The incidence of fungal infections in humans has increased dramatically over the past 20 years. This is partly due to the increasing number of patients with weakened immune systems or those with organ transplants, cancer chemotherapy, AIDS, age, and other disorders or conditions. Such patients are susceptible to attack by pathogenic fungi, which are widely distributed throughout the world. These pathogens are difficult to treat because some of the currently available antifungal agents are highly toxic or only inhibit fungal infections. For example, polyenes have fungicidal activity but are toxic, while azoles are less toxic but have only fungistatic activity. More importantly, recent research and publications indicate that Candida strains are resistant to azoles and polyenes, which seriously limits the therapeutic use of these compounds against Candida strains.

Pseudomonas syringae produces various classes of antifungal agents and antibiotics, such as pseudomycins, syringomycins, syringotoxins, and syringostatins, which are structurally lipodepsinone peptides. Natural strains and transposon-generated mutants of P. syringae produce these lipodepsinone peptides. Several pseudomycins, syringomycins, and other lipodepsipeptide antifungal agents have been isolated, their chemical structures identified, and their broad-spectrum antifungal activity, including activity against some fungi pathogenic to humans and plants, has been demonstrated. Pseudomycins, syringomycins, syringotoxins, and syringostatins constitute a distinct family of antifungal compounds based on their structure.

To date, no P. syringae lipodepsinonapeptide has been marketed as a drug. Undesirable side effects, formulation, scale-up, and other development problems have prevented the use of P. syringae lipodepsinonapeptides against the full range of fungal infections affecting animals, humans, and plants. There remains a need for an antifungal agent that can treat infections that are not treatable with currently available drugs in fungal infections of animals, humans, or plants.

SUMMARY OF THE INVENTION

The present invention relates to a depsidecapeptide produced by P. syringae, which contains the rare amino acids homoserine (Hse), dehydroaminobutyric acid (Dhb) and dehydroalanine (Dha), the latter being part of the depsidecapeptide ring. More specifically, the P. syringae depsidecapeptide contains a depsidecapeptide ring consisting of the amino acids arginine, threonine, homoserine, dehydroaminobutyric acid and dehydroalanine, and further contains a lactone ring formed from the carboxyl group of arginine and the hydroxyl group of threonine. The depsidecapeptide isolated from the P. syringae strain is a lipodepsidecapeptide, a cyclic peptide linked to a lipophilic moiety. Typically, the lipophilic moiety is a fatty acid moiety linked to the amino group of threonine via an amide bond.

Preferably, the fatty acid moiety is n-dodecanoic acid. The lipodepsidecapeptide is a compound of formula (I) wherein R is a lipophilic moiety. The lipophilic moiety comprises a C9-15 alkyl, C9-15 hydroxy-alkenyl, C9-15 dihydroxy-alkenyl, C9-15 alkenyl, C9-15 hydroxy-alkenyl, or C9-15 dihydroxy-alkenyl group. Preferably, the lipophilic moiety is a C11 alkyl group. The alkyl, hydroxy-alkenyl, dihydroxy-alkenyl, alkenyl, hydroxy-alkenyl, or dihydroxy-alkenyl groups may be straight or branched. Preferably, the amino acid sequence of the depsidecapeptide ring is: threonine-alanine-threonine-glutamine-homoserine-dehydroaminobutyric acid-alanine-dehydroalanine-threonine-arginine, which is called “25-B1 decapeptide” or “Thr-Ala-Thr-GIn-Xaa-Xaa-Ala-Xaa-Thr-Arg” (SEQ ID NO:1).

The term "25-B1 decapeptide A antifungal agent" as used herein refers to a specific depsidecapeptide having the above sequence [SEQ ID NO:1, R is a straight-chain alkyl group of 11 carbon atoms, (i.e. R = -(CH ₂ ) ioCH ₃ )].

The invention also provides a method for using P. syringae depsidecapeptide to inhibit fungal activity or to reduce the symptoms of fungal infection in patients as desired. Such methods are capable of killing the fungus, reducing the severity of the fungal infection, alleviating fever, and improving the general well-being of the patient. Consequently, P. syringae depsidecapeptides can be used in the manufacture of a pharmaceutical composition for treating the symptoms described above in patients. The methods and compositions of the invention are effective against fungi such as Candida parapsilosis, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum.

The invention also provides a method for producing a P. syringae depside decapeptide and 25-B1 decapeptide as described above. The method comprises culturing Pseudomonas syringae in a medium containing three or fewer amino acids, and recovering one or more P. syringae depside decapeptides from the culture. In one embodiment, the P. syringae culture is grown in a medium containing glycine, a lipid, and a potato product or combinations thereof at a pH of 4-6.5 until one or more P. syringae depside decapeptides are produced at a concentration of at least 10 ug/ml. The invention further provides P. syringae depside decapeptides as described above.

The invention also provides a method for treating and preventing fungal growth in plants, comprising treating the fungus with one or more of the P. syringae depsidecapeptides described above.

DETAILED DESCRIPTION OF THE INVENTION

Lipodepside decapeptide antitumor agents

The term "lipodepsidedecapeptide antifungal agent" as used herein means an antifungal agent comprising a cyclic decapeptide ring closed by a lactone group and a hydrophobic group, a fatty acid moiety attached thereto. Lipodepsidedecapeptide antifungal agents are produced by Pseudomonas syringae. A representative member of this family of compounds, the antifungal agent 25-B1 decapeptide A, has been purified and its structure determined. The term "P. syringae lipodepsidedecapeptide" as used herein means a lipodepsidedecapeptide antifungal agent produced by P. syringae, and the fermentation broth contains the antifungal agent 25-B1 decapeptide A and structurally related analogs.

P. syringae lipodepsidedecapeptides have distinctive structural features. For example, these antifungal agents contain the naturally occurring amino acids homoserine (Hse), dehydroaminobutyric acid (Dhb), and dehydroalanine (Dha) as parts of the depsidecapeptide ring. In each P. syringae lipodepsidedecapeptide, the carboxyl group of the arginine moiety combines with the hydroxyl group of the N-terminal threonine to form a lactone, which closes the lipodepsidedecapeptide ring. The sequence of the depsidecapeptide ring of P. syringae lipodepsidedecapeptide is as follows:

Thr-Xaa-Xbb-Xcc-Hse-Dhb-Xdd-Dha-Xee-Arg wherein each of Xaa, Xbb, Xcc, Xdd and Xee represents a naturally occurring amino acid. Unlike the natural pseudomycin materials, the lipodepside decapeptides of the present invention do not contain chlorothreonine, which is suspected to be the cause of irritation when pseudomycin-containing pharmaceutical compositions are administered by injection.

The depsidecapeptide ring is linked to a lipophilic moiety, which in this case is a fatty acid moiety linked to the amino group of the N-terminal threonine via an acid amide bond. The fatty acid moiety generally contains 10, 12, 14 or 16 carbon atoms and typically 0, 1 or 2 hydroxyl groups. The fatty acid chain may be straight or branched and may contain one or more double bonds. Preferably, the fatty acid moiety comprises n-decanoic acid, n-decanoic acid substituted with one or two hydroxyl groups, n-dodecanoic acid, n-dodecanoic acid substituted with one or two hydroxyl groups, n-tetradecanoic acid, n-tetradecanoic acid substituted with one or two hydroxyl groups.

25-B1 decapeptide antitumor agents

The term "25-B1 antifungal agent" as used herein refers to a member of the family of antifungal agents isolated from a culture of the bacterium Pseudomonas syringae. 25-

-B1 decapeptide antifungal agent is P. syringae lipodepside decapeptide. Specifically, 25-B1 decapeptide antifungal agent is a P. syringae lipodepside decapeptide containing a depside decapeptide ring and having the following amino acid sequence:

Thr-Ala-Thr-GIn-Hse-Dhb-Ala-Dha-Thr-Arg” (SEQ ID NO: 1)

Each 25-B1 decapeptide antifungal agent has the same cyclic peptide ring system, but differs in the structure of the hydrophobic side chain attached to the ring. The 25-B1 decapeptide antifungal agent contains a 25-B1 decapeptide A antifungal agent. The 25B1 decapeptide antifungal agent contains a fatty acid moiety linked to the amino group of the N-terminal threonine via an acid amide bond. The fatty acid moiety of the 25-B1 decapeptide A antifungal ^agent is an n-dodecanoic acid moiety. ¹ i

Biological activity of P. syringae lipodepsidekapeptides

P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A, has a multifaceted biological activity, including fungicidal activity, inhibition of the activity of various fungi, plant and animal pathogens. In particular, 25-B1 decapeptide A is an active antimycotic that is effective against fungi that cause infections in immunocompromised individuals. These fungi include Cryptococcus neoformans, Histoplasma capsulatum, and Candida, including various species of Candida parapsilosis and Candida albicans.

Pseudomonas syringae

Pseudomonas syringae includes a wide range of bacteria that are commonly associated with plants. Some Pseudomonas syringae are plant pathogens, while others are only weakly pathogenic or saprophytic. Different isolates of Pseudomonas syringae produce one or more cytotoxic substances that help the bacteria survive in a foreign environment where they coexist with fungi and other bacteria. The cytotoxic substances produced by Pseudomonas syringae include antifungal substances such as Pseudomonas syringae lipodepsidekapeptides, including 25-

-B1 decapeptide The antifungal substances pseudomycins, syringomycins, syringotoxins, and syringostatins.

Isolated strains of Pseudomonas syringae that produce one or more of pseudomycins, syringomycins, syringotoxins, and syringostatins are well known to those skilled in the art. The wild-type strain MSU 174 and a mutant of the strain generated by transposon mutation, MSU 16H (ATCC 67028), are described in U.S. Patent No. 5,576,298, issued November 19, 1996; G. Strobel 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. Sei. USA 84,64476451 (1987). A method for propagating various strains of P. Syringae and their use for the production of antifungal agents such as pseudomycins has also been described by Matthew D. Hilton et al. in United States Patent Application No. PCT/US00/08728, entitled "Production of Pseudomycin by Pseudomonas syringae." The entire disclosure and references therein are hereby incorporated by reference.

Strains of P. syringae capable of producing one or more P. syringae lipodepside decapeptides, such as the antifungal agent 25-B1 decapeptide A, can be isolated from plant environments such as barley, citrus and lilac, as well as from forest undergrowth, soil, water and air. The invention relates to a strain, an isolate and a biologically purified culture of P. syringae which produces one or more P. syringae lipodepside decapeptides, such as the antifungal agent 25-B1 decapeptide A, in an amount greater than 10 µg/ml, preferably in an amount of at least 10 µg/ml to 50 µg/ml. Preferably, the biologically purified culture of the microorganism is P. syringae, MSU 16H, 25-B1, 67H1, 7H9-1 strains, or a pseudomycin-producing mutant, variant, isolate, or recombinant of these strains. The MSU 16H culture is deposited at Montana State University (Bozeman, Montana, USA) and can be ordered from the American Type Culture Collection (Parklawn Drive, Rockville, MD, USA) under the number ATCC 67028.

Strains of P. syringae suitable for the production of one or more P. syringae lipodepside decapeptides, such as 25-B1 decapeptide A antifungal agent, can be isolated from plant environments such as barley, citrus, and lilac, as well as from forest undergrowth, soil, water, and air. A preferred strain is isolated from a plant. Isolates of P. syringae from these environments are referred to as wild-type isolates. The term "wild-type" as used herein refers to a dominant genotype that occurs naturally in a normal population of P. syringae (i.e., strains or isolates of P. syringae that occur in nature and are not the product of laboratory manipulation). Similar to other organisms, the characteristics of the production cultures used in this invention, P. syringae strains, such as MSU 174, MSU 16H, MSU 206, 25-B1 and 7H9, change over time. Thus, progeny, i.e. recombinants, mutants and variants of these strains can be obtained and produced by methods well known to those skilled in the art in the art.

Mutant strains of P. syringae are also suitable for producing one or more P. syringae lipodepside decapeptides, such as the antifungal agent 25-B1 decapeptide A. The term "mutant" as used herein refers to a rapidly heritable change in the phenotype of the strain, which may be spontaneous or induced by known mutagenic agents, including irradiation or induction by various chemical agents. The P. syringae mutants of the present invention can be produced using a wide variety of mutagenic agents, including irradiation such as ultraviolet radiation, X-rays; chemical mutagens, site-specific mutagens, and transposon-mediated mutagenesis. Examples of chemical mutagens include ethyl methyl sulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-N-nitrosoguanine (NTG), and nitric acid.

P. syringae is suitable for the production of one or more lipodepside decapeptides, such as 25-B1 decapeptide A antifungal agent. According to the present invention, production can be generated by subjecting the bacterium to an effective amount of a mutagenic agent sufficient to produce mutants that overproduce one or more lipodepside decapeptides, such as 25-B1 decapeptide A antifungal agent, or that produce one or more lipodepside decapeptides, such as 25-B1 decapeptide A antifungal agent under favorable growth conditions. While the type and amount of mutagenic agent to be used may vary, a preferred method is to use serially diluted NTG at a concentration range of about 1-100 ug/ml. Preferred mutants of the invention are those that overproduce the 25-B1 decapeptide A antifungal agent, preferably at least 10 ug/ml to 50 ug/ml.

Environmental isolates, mutant strains, and other desirable P. syringae strains can be selected for desired characteristics of growth habit, growth medium, nutrient source, carbon source, growth conditions, and amino acid requirements. Preferably, P. syringae lipodepsidedecapeptide, such as 25-B1 decapeptide A antifungal agent producing P. syringae strains are selected based on growth on minimal medium. Preferred strains exhibit the characteristics of one or more P. syringae lipodepsidedecapeptide, such as 25-B1 decapeptide A antifungal agent producing P. syringae strains when grown on a medium containing a glycine and optionally a lipid, a potato product, or both.

Recombinant strains can be developed by transforming P. syringae strains using methods well known to those skilled in the art from the literature. Using recombinant technologies, P. syringae strains can be transformed to express a large number of gene products, in addition to the fact that these strains produce antibiotics. For example, strains can be transformed with a recombinant vector that makes the strain resistant to an antibiotic to which it was previously susceptible. The transformants thus obtained not only produce P. syringae lipodepside decapeptide, such as the antifungal agent 25-B1 decapeptide A, but also a resistance-conferring enzyme that allows the selection of transformed cells from wild-type strains. Furthermore, using similar techniques, our current strains can be modified by introducing multiple copies of endogenous lipodepside decapeptide biosynthesis genes, thereby achieving higher lipodepside decapeptide production. The progeny, i.e., P. syringae strains 25-B1, 67H1 and 7H9-1, their natural and induced variants, mutants and recombinants, which retain the ability and characteristics of lipodepsidecapeptide production, are also part of this invention.

Reproduction of Pseudomonas syringae

The term "aqueous medium" as used herein refers to an aqueous medium composition comprising inorganic compounds and organic compounds and their salts, which are necessary for the growth of the bacteria used in the present invention. A preferred medium comprises an optimal amount of three or fewer amino acids, preferably glutamic acid, glycine, histidine, or combinations thereof. In one embodiment, the medium comprises a suitable amount of glycine, and optionally one or more potato starches and a lipid. Glycine may be provided alone or as one of a mixture of amino acids in the form of a hydrolyzed protein. Suitable lipids include soybean oil, fatty acids and fatty acid esters. Suitable potato products include potato dextrose medium, potato dextrin, potato protein, or a mixture of commercially available mashed potato food products. Preferred minerals for the medium include salt mixtures that are routinely used in cell cultures and fermentations, such as Czapek's mineral salt, which contains potassium chloride, magnesium sulfate, and iron sulfate. Among the organic compounds used in the medium, glucose is preferably used, and soluble starch and other similar organic substances may also be used. The pH of the medium is preferably 4-6.5, more preferably 4.5-5.7, particularly preferably 5.2.

Although the amount of each component is not critical for bacterial growth or the production of antifungal agents such as P. syringae lipodepside decapeptides such as 25-B1 decapeptide A, some level of the medium components is recommended.

The preferred amount of glycine is about 0.1 g/l to 10 g/l, more preferably about 0.3 g/l to 3 g/l, particularly preferably about 1 g/l. The preferred amount of lipids such as an oil, such as soybean oil, is about 1 g/l to 10 g/l, more preferably about 0.5 g/l to 2 g/l. The preferred amount of fatty acids or fatty acid esters is about 0.5 to 5 g/l. A preferred amount of potato products includes about 12 g/l to 36 g/l, more preferably about 24 g/l potato dextrose, about 5 g/l to 50 g/l, more preferably about 30 g/l mashed potato food product mixture, about 1 g/l to 30 g/l, more preferably about 20 g/l potato dextrin, and/or about 1 g/l to 10 g/l, more preferably about 4 g/l potato protein.

A preferred medium composition is for mineral salts, potassium chloride preferably 0.02-2 g/l, more preferably 0.2 g/l, magnesium sulfate, preferably magnesium sulfate x 7 H ₂ O about 0.02-2 g/l, more preferably 0.2 g/l, and iron sulfate, preferably iron sulfate x 7 H ₂ O about 0.4-40 g/l, more preferably about 4 g/l. If present among the medium components, the amount of soluble starch is preferably about 0.5-50 g/l, more preferably about 5 g/l. The amount of glucose is preferably about 2-80 g/l, more preferably about 20 g/l.

P. syringae growth occurs at controlled and regulated temperatures and pH. P. syringae grows and produces one or more cytostatic products at about 15-35 °C, more preferably at 20-30 °C, particularly preferably at about 25 °C. P. syringae grows and produces one or more cytostatic products at about 4-9 pH, preferably at 4-6 pH, particularly preferably at about 4.5-5.5 pH. P. syringae does not grow when the temperature is higher than 37 °C or lower than 10 °C, or when the pH is higher than 9 or lower than 4.

Process for producing P. syringae lipodepside decapeptide

One or more P. syringae lipodepside decapeptides, such as 25-B1 decapeptide A, are produced from a wild-type or mutant strain of P. syringae by growing the microorganism under agitation in an aqueous medium containing an effective amount of three or fewer amino acids. The three or fewer amino acids are preferably glutamic acid, glycine, histidine, or combinations thereof. In a preferred embodiment, the amino acids comprise a glycine, and optionally one or more potato products and a lipid. The fermentation is conducted under conditions effective for the growth of P. syringae and the production of the desired P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A. Effective conditions include a temperature of between 22°C and 27°C and a fermentation time of 36 to 96 hours. Under the conditions described herein, the P. syringae fermentation broth has a density of about 10-15 g/liter dry matter, and P. syringae produces lipodepside decapeptides, such as the antifungal agent 25-B1 decapeptide A, at a concentration of at least 10 µg/ml, preferably at a concentration of at least 50 µg/ml.

It is advantageous to control the oxygen concentration during P. syringae fermentation, during the production of P. syringae lipodepside decapeptides such as 25-B1 decapeptide A antifungal agent. Preferably, the oxygen level is maintained at 5-50% saturation, more preferably at 30% saturation. The oxygen concentration of the medium can be controlled by mixing air, pure oxygen or gas mixtures containing oxygen. Furthermore, the rate of oxygen transfer can be controlled by controlling the mixing rate.

It is advisable to control the pH of the medium during P. syringae fermentation, during the production of P. syringae lipodepside decapeptides such as 25-B1 decapeptide A. The pH of the medium is preferably maintained at pH = 4-6.

P. syringae is capable of producing the antifungal agent P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A, in batchwise, batchwise manner. However, continuous or semi-continuous addition of glucose, or optionally an acid or base, such as ammonium hydroxide, for pH control, increases the production of the antifungal agent P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A. The production of the antifungal agent P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A can be further increased by a continuous fermentation, during which glucose, and optionally an acid or base, such as ammonium hydroxide, are automatically added to adjust the pH. The pH is preferably maintained between 5 and 5.4, more preferably between 5.5.2.

The choice of P. syringae strain can affect the amount and distribution of the P. syringae lipodepside decapeptide produced as described herein, such as the antifungal agent 25-B1 decapeptide A. For example, strain 25-B1 is capable of producing almost exclusively the antifungal agent 25-B1 decapeptide A.

The cyclic decapeptide backbone of the P. syringae lipodepside decapeptide can be prepared by removing the lipophilic moiety, for example by deacylation. Cleavage and deacylation methods, as well as the use of deacylase enzymes, are well known to those skilled in the art.

P. syringae lipodepside decapeptide, its formulation and antifungal activity

P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A, exhibits antifungal activity in vitro and in vivo and is useful in combating both systemic fungal infections and fungal skin infections. Accordingly, the present invention provides a method for inhibiting fungal diseases, comprising treating a fungus with P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A, or a pharmaceutically acceptable salt thereof. In a preferred method, the growth or activity of various fungi, such as Cryptococcus neoformans, Histoplasma capsulatum, and Candida, including Candida parapsilosis and Candida albicans, is inhibited. As used herein, the term "treating" a parasite or fungus with a compound of the invention means an association, encounter, or apparent contact, or mutual contact between a compound of the invention and the parasite or fungus. However, the term treatment does not refer to any mechanism of inhibition.

The present invention further provides a method for treating a fungal infection comprising administering to a host an effective amount of a P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A antifungal agent, or a pharmaceutically acceptable salt thereof, for treating the host in need thereof. A preferred method comprises treating infections caused by various fungi, such as Cryptococcus neoformans, Histoplasma capsulatum, and Candida, including Candida parapsilosis and Candida albicans. Administration of an effective amount of one or more P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A antifungal agent, reduces the severity of the fungal infection, reduces the symptoms associated with the fungal infection, and may cause the fungal infection to resolve.

Some patients who require one or more P. syringae lipodepside decapeptides, such as 25-B1 decapeptide A, have severe symptoms of infection, high fever, and are likely to be in a critical condition. Several fungi can cause such serious infections. Candida spp., for example, can cause severe mucosal and systemic infections, and the fungus may be resistant to azoles and polyenes. Aspergillus causes life-threatening systemic infections. Cryptococcus is responsible for meningitis. Serious fungal infections can occur in patients with immune system disorders, in patients who have undergone organ or bone marrow transplants, in cancer patients receiving chemotherapy, in patients who have undergone major surgery, or who are HIV positive. In such patients, antifungal therapy typically involves the intravenous administration of one or more P. syringae lipodepside decapeptide preparations, such as 25-B1 decapeptide A. The antifungal agent can be used to stop or control the infection within a few days.

With respect to antifungal activity, the term "effective amount" as used herein means that amount of a compound of the present invention capable of inhibiting fungal growth or activity or reducing the symptoms of a fungal infection. Reducing the symptoms of most fungal infections includes reducing fever, returning to normal, and restoring the patient's continued health. Preferably, the symptoms are reduced by killing the fungus to eliminate the infection, or by reducing the infection to a level that is tolerable by the patient or the patient's immune system. The term "inhibition" as used herein means reducing the activity of the fungus, which includes stopping the infection, prolonging or prophylactically hiding or preventing growth, or any characteristic associated with the presence of the fungus, and results.

Typically, the compositions are administered to a subject (human or animal, including mammals such as cats, horses, cattle and poultry) in need thereof in an amount effective to inhibit the fungal infection. The dosage administered will vary depending on factors such as the nature and severity of the infection, the age and general condition of the subject, and the sensitivity of the subject to antifungal agents. The actual dosage range may likewise depend on these factors and may be administered in a single daily dose or in multiple daily doses. Administration may be for 2-3 days, or for up to about 23 weeks or longer. A typical daily dose (in single or multiple doses) will contain an amount of the active compound of the invention in the range of 0.01 mg/kg body weight to 100 mg/kg body weight. The preferred daily dose is generally 0.1 mg/kg body weight to 60 mg/kg body weight, ideally 2.5 mg/kg body weight to 40 mg/kg body weight. In severe infections, the compound is administered by intravenous infusion, e.g., the active ingredient at a rate of 0.01 to 10 mg/kg/hour.

The present invention also provides pharmaceutical compositions suitable for administering the antifungal compounds of the invention. Accordingly, the invention also provides a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, diluents, excipients, ingredients and further additives, and one or more P. syringae lipodepside decapeptides, such as the antifungal agent 25-B1 decapeptide A. The amount of active ingredient in such compositions is from 0.1 to 99.9% by weight, preferably from 10 to 30% by weight. The term "pharmaceutically acceptable" means that the excipients, carriers and ingredients are compatible with the other ingredients of the composition and are not deleterious to the patient.

The composition may contain further additives such as various oils, including mineral oils, animal, vegetable or synthetic materials, such as peanut oil, soybean oil, mineral oil, and sesame oil. Suitable pharmaceutical excipients include starch, cellulose, glucose, lactose, sugar, gelatin, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried milk powder, glycerin, propylene glycol, water and ethanol. The compositions may be prepared by conventional means and methods, such as sterilization, and the compositions may contain conventional pharmaceutical additives such as preservatives, stabilizers, wetting agents or emulsifiers, salts for achieving osmotic pressure, and buffers. Suitable pharmaceutical excipients and their preparation can be found in the following study: Martin: "Remington Pharmaceutical Sciences", ^15th Edition; Mack Publishing Co., Easton (1975): see for example pages 1405-1412 and pages 1461-1487.

The term "pharmaceutically acceptable salts" as used herein refers to salts of the compounds of the above formulae which are non-toxic to living organisms. Typical pharmaceutically acceptable salts are those salts which are prepared by reacting the compounds of the invention with an inorganic or organic acid or an inorganic base. Such salts are known as acid addition salts or base addition salts.

Inorganic acids commonly used to prepare acid addition salts include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, and organic acids include p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, gamma-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates. Preferred pharmaceutically acceptable acid addition salts with inorganic acids are chlorides and bromides, and with organic acids are maleates and methanesulfonates.

Base addition salts include salts formed with inorganic bases, such as ammonium hydroxide, alkali or alkaline earth metal hydroxides, carbonates and bicarbonates. Suitable bases for preparing the salts of the invention are sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide and calcium carbonate. The potassium and sodium salts are particularly preferred.

It is apparent that the cation or anion of any salt of the invention is not critical, as long as the salt is entirely pharmaceutically acceptable.

P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A antifungal agent, can be administered parenterally, for example, using intramuscular, subcutaneous or intraperitoneal injection, can be administered nasally or orally. In addition to these administration routes, P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A antifungal agent, can be administered topically to treat superficial skin infections or to inhibit fungal growth in mucus.

For parenteral administration, the composition comprises one or more P. syringae lipodepside decapeptides, such as the antifungal agent 25-B1 decapeptide A, and a physiologically acceptable solvent, such as deionized water, physiological saline, 5% dextrose solution, and other commonly used solvents. The composition may comprise a cyclodextrin and/or a solubilizing agent, such as polyethylene glycol, polypropylene glycol, or other known solubilizing agents. The compositions may be presented in sterile powder ampoules containing the antifungal agent and excipients in the form of a dry powder or lyophilized powder. Before use, a physiologically acceptable solvent is added to the powder ampoule, the agent is dissolved, and the solution is administered to the patients using a syringe.

The present pharmaceutical compositions are prepared by known methods and by known, readily available and readily available excipients. In preparing the compositions of the present invention, the active ingredient is generally mixed with excipients, or diluted with an excipient, or enclosed in a capsule, sachet, paper or other container. If an excipient serves as a diluent, it may be a solid, semi-solid, liquid, which serves as a carrier, excipient or medium for the active ingredient. Thus, the compositions may be tablets, pills, powders, lozenges, sachets, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solid forms, or in a liquid medium, ointments, for example, containing up to 10% by weight of active ingredient in soft and hard gelatin capsules, suppositories, sterile injectable solutions or sterile prepared powders.

For oral administration, the antifungal agent is filled into a gelatin capsule or prepared in tablet form. Such tablets may contain a binder, a dispersant or other suitable excipient to form tablets of the P. syringae lipodepside decapeptide, such as 25-B1 decapeptide A, of suitable size and dosage. For children and the elderly, the antifungal compound may be formulated as a flavored liquid suspension, solution or emulsion. A preferred oral formulation contains linolenic acid, cremophor RH-60, and water, preferably 8% by volume linolenic acid, 5% by volume cremophor RH-60, 87% by volume sterile water, and 2.5-40 mg/ml of the P. syringae lipodepside decapeptide, 25-B1 decapeptide A antifungal agent.

For topical application, the antifungal agent may be formulated as a dry powder on the skin surface, ^or it may be formulated as a liquid containing an aqueous or non-aqueous medium, such as an alcohol or glycol.

Use of P. syringae lipodepside decapeptide preparations

The present invention also provides a kit comprising a composition of the invention and a method for administering the same. The kit may comprise a vial containing the composition of the invention and a suitable carrier in the form of a powder or solution. The kit may further comprise instructions for use, either in the form of a label on the vial or in the form of separate instructions placed next to the vial, for the administration and administration of the compounds. The instructions may also be printed on the box containing the vial. The instructions for use may include information such as the appropriate dosage and dosage form, which will enable a healthcare professional to safely administer the agent. A healthcare professional is any medical professional, nurse or technician who is normally qualified to administer the drug.

The present invention also provides a pharmaceutical composition comprising one or more P. syringae lipodepsidedecapeptide 25-B1 decapeptide A antifungal agents suitable for administration by injection. According to the method of the invention, the one or more P. syringae lipodepsidedecapeptide 25-B1 decapeptide A antifungal agents can be used to prepare a composition or medicament suitable for administration by injection. The invention also provides a method for preparing compositions comprising one or more P. syringae lipodepsidedecapeptide 25-B1 decapeptide A antifungal agents, the composition being suitable for oral or topical administration. For example, a liquid or solid composition can be prepared in various ways using conventional methods. Liquid compositions can be prepared by dissolving one or more P. syringae lipodepsidedecapeptide 25-B1 decapeptide A antifungal agents in a suitable solvent, such as water, at a suitable pH, which also contains buffers and other additives.

Agricultural application

Antibiotics produced by P. syringae NRRL B-12050 have been reported to be effective in the treatment of Dutch elm blight (see, for example, U.S. Patents 4,342,746 and 4,277,462). In particular, P. syringae MSU 16H showed a higher degree of protection compared to the wild type when elms were infected with Ceratocystis ulmi, the causative agent of Dutch elm blight (see, for example, Lam et al., Proc. Natl. Sci. USA, 6447-6451 (1987)). Several intensive experiments with elms have demonstrated that this blight of trees can be prevented by appropriate treatment. Thus, the lipodepsidekapeptides of the present invention can be used for the preventive treatment of Dutch elm blight. It is known that pseudomycins are toxic to a number of plant pathogenic fungi, such as Rynchhosporium secalis, Ceratocystis ulmi, Rhizoctonia solani, Sclerotinia sclerotiorum, Verticillium alboatrum, Verticillium dahliae, Thielaviopis basicola, Fusarium oxysporum, and Fusarium culmorum (see, for example, Harrison L. et al., “Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broadspectrum antifungal activity,” J. General Microbiology, 7, 28572865 (1991). Consequently, one or more P. syringae lipodepside decapeptides, such as 25-B1 decapeptide A, may be effective against plant pathogenic fungi (especially V. albo-atrum, Rhizoctonia solani, and F. oxysporum) even in the presence of a bacterium. after infection, or for prevention. In general, infected plants are treated by spraying an aqueous suspension of the lipodepsidecapeptide onto the plant. The means and equipment for spraying are well known to those skilled in the art. Any form of spray may be used to deliver an effective amount of the active ingredient to the surface of the plant. The suspension may contain further additives known to those skilled in the art, such as solubilizers, stabilizers, wetting agents, and combinations thereof.

The plant can be treated with dry, solid formulations of one or more P. syringae lipodepside decapeptides, such as 25-B1 decapeptide A. The dry formulations can be applied to the surface of the plant by means and methods known to those skilled in the art, such as by spraying or by pouring from a container.

The invention may be better understood by reference to the following examples. These examples are intended to be representative of the invention and are not intended to limit the scope of the invention.

EXAMPLES

Deposit of biological materials

P. syringae MSU 16H is officially available and can be purchased from the American Type Culture Collection, Parklawn Drive, Rockville, MD, USA, accession number: No. ATCC 67028.

P. syringae strains 25-B1, 7H9-1, and 67H1 were officially deposited with the American Institute of Microbiology on March 23, 2000.

Type Culture Collection with the following identification numbers:

25-B1 identification number: PTA-1622

7H9-1 identification number: PTA-1623

67-H1 identification number: PTA-1621

Example 1 - Preparation of the antitumor agent 25-B1

The fermentation process was developed to produce the lipodepside decapeptide antifungal agent, 25-B1 decapeptide A, in the fermentation medium of Pseudomonas syringae strain.

Materials and methods

Preparation of the inoculum:

An aliquot of P. syringae strain 25-B1 cells stored in liquid nitrogen was thawed and inoculated into two 900 ml volumes of CMS medium. The composition of CMS medium in g/liter was: dextrose: 5, maltose: 4, Difco Trytic Soy Broth: 30, Difco yeast extract: 3, and magnesium sulfate x 7 H ₂ O: 2. Approximately 0.5 ml of cell suspension was used to inoculate each 900 ml portion, which was prepared in 2-liter flasks for inoculation. The flasks were incubated for 24 hours at 25 °C with shaking. The contents of the two flasks were combined and inoculated into a previously sterilized 115 liter volume of medium in a 150 liter apparatus.

Fermentation stage:

The composition of the fermentation broth was as follows, expressed in g/liter: dextrose: 20, soluble starch: 5, Basic American Foods Country Style Potato Pearls potato pulp: 30, glycine: 1, magnesium sulfate x 7 H ₂ O: 0.2, potassium chloride: 0.2, ferrous sulfate x 7 H ₂ 0:0.004 prepared in tap water. The pH was adjusted to 5.2 before sterilization. The fermentation was continued at 25 °C for 68 hours. The dissolved oxygen content was maintained at or above 30% by continuously adjusting the air flow and impeller speed. The pH was maintained between 4.0 and 5.4 by adding sulfuric acid or sodium hydroxide as needed.

Several variations of the simple fermentation process were developed to produce the new cyclic peptide product. Dextrose can be continuously added to the fermenter for 24 hours after inoculation at a rate of 60 ml/hour. Dextrose addition can be continued throughout the fermentation process. Alternatively, a procedure was developed in which the dissolved oxygen content was set to 5% 24 hours after inoculation and maintained at this level until the end of the fermentation. Maintaining the dissolved oxygen content at 5% was achieved by adding inert nitrogen gas. In each case, the gas was added through a simple dip tube, placed in the open position directly below the lower turbine mixer.

Results and conclusion

Several fermentation processes produce the antifungal substance 25-B1 decapeptide A from P. syringae.

Example 2 - Isolation and purification of the antitumor agent 25-B1

We have developed methods for the isolation and purification of the antifungal substance 25-B1 decapeptide A, a lipodepside decapeptide produced in the fermentation broth of Pseudomonas syringae strain.

Materials and methods

The volume of fermentation broth produced according to Example 1 was typically 100 liters after cutting, which was filtered through a Membralox™ 0.45 µm ceramic filter. The solids remaining in the filter were extracted with an equal volume of acetone containing 0.1% trifluoroacetic acid for 90 minutes. The acetone extract was filtered and evaporated in vacuo to give an aqueous solution.

This solution was combined with the original filtrate and continuously added to the top of a 4-liter chromatography column filled with water on Amberchrom™ CG300 sd resin. After adsorption, the column was washed with 0.2% acetic acid solution (pH 4-8) until the pH of the effluent reached 4.5, then washed with 10 liters of 0.2% acetic acid in 22% acetonitrile (pH 4.8). Then, a linear gradient elution was applied with 32 liters of 22-35% acetonitrile in 0.2% acetic acid and 8 liters of 0.2% acetic acid in 35% acetonitrile in 0.2% acetic acid at a flow rate of 400 ml/hour. Fractions 11-16 (4.8 liters) were combined, evaporated in vacuo to a volume of 100 ml, and then centrifuged.

The supernatant was separated and chromatographed on a 1 liter Amberchrom CG 300sd column with a linear gradient elution with 22-35% acetonitrile in water containing 0.2% acetic acid (pH 4.8) at a flow rate of 50 ml/hour. Fractions 20-25 were combined (1.2 liters) and rechromatographed on a reversed phase column (NovaPak Ci ₈ , 6 µm, 40x300 mm, flow rate 40 ml/minute, linear gradient elution 0.2%

with 30-60% acetonitrile aqueous solution containing TFA) thus 21 mg of product was obtained (89% purity, tested by UV method).

ESIMS data showed a possible [M + H]' peak at m/z 1165.7, which is different from the corresponding values of known antifungal substances isolated from P. syringae. The ¹ H NMR spectrum signals were similar to those of pseudomycin-like lycopeptides, but indicated the presence of more than one substance. In order to obtain the 25-B1 decapeptide A antifungal substance with sufficient purity for structure determination and antifungal activity measurement, another 4 x 100 liters of fermentation broth were purified as above, and in addition, a reverse phase column chromatography step was introduced for the final purification step [Rainin Ci ₈ 6 im, 24x250 mm, linear gradient elution 0.1 % TFA-t-acetonitrile methanol mixture (8:1:1 to 4:3:3) for 60 min, then (4:3:3 to 10:45:45) for 30 min]. Thus, 42 mg of 25-B1 decapeptide A antifungal substance was obtained with 93 % purity (based on UV).

Results and conclusion

Using a preparative HPLC method similar to that used for the purification of other lipodepsipeptide antifungals, the 25-B1 decapeptide A antifungal was obtained from the fermentation broth in sufficient purity.

Example 3 - Determination of the structure of the antifungal agent 25-B1

The structure of the lipodepside decapeptide antifungal agent, 25-B1 decapeptide A, was determined using mass spectroscopy and NMR spectroscopy.

Methods and results

The molecular formula of the antifungal agent 25-B1 decapeptide A was determined by high-resolution FABMS and found to be C52H88N14O16 [m/z 1165.6581 for C52H89N14O16 (M + H) ⁺ , λ+0.9 ppm].

Consistent with this empirical formula, the ¹³ C NMR spectrum and DEPT NMR spectrum showed 50 distinct resonance signals, which included 12 carbonyl carbons, 5 olefinic carbons, 4 oxygen-containing sp ³ carbons, 8 typical amino acid acene atoms, 15 methylene carbons, and 6 methyl carbons. Among these, one methyl carbon signal at 16.7 ppm and one methylene carbon signal at 28.9 ppm formed a degenerate set of carbon atoms, corresponding to the 52 carbons given by the empirical formula.

Based on ¹ H, ¹³ C, and 2D NMR (DQCOSY, TOCSY, HMQC, HMBC, and ROESY) data, we determined the structure of the antifungal agent 25-B1 decapeptide A (IA) and unambiguously identified all hydrogen and carbon atoms (Table 1).

Table 1 1 3

H and C NMR data of the antifungal agent 25-B1 decapeptide

In DMSO-d ₆

Amino acid Position of atoms OH father Arg NH 8.13 -

CO - 170.4 ά 4.23 51.7 lie 1.69 28.2 á2 1.63 ά 1.46 24.7 the 3.08 40.2 ΝΗ 7.59 - the - 156.7 Thr-1 ΝΗ 7.83 - CO - 169.8 ά 4.23 59.5 ά 4.00 66.4 ά 1.08 19.7 Dha ΝΗ 9.01 - CO - 163.7 ά - 135.3 lie 5.86 106.4 ά2 5.61 Ala-1 ΝΗ 7.96 - CO - 171.6 ά 4.22 49.9 ά 1.31 16.7 Amino acid Position of atoms _ΑΗ ÖC Dhb ΝΗ 9.07 - CO - 164.0 ά - 130.0 ά 6.40 128.5

ά 1.61 12.9 H-Ser NH 8.22 - CO - 171.1 ά 4.22 51.4 lie 1.82 34.0 á2 1.76 ά 3.47 57.3 Gin NH 7.59 - CO - 171.9 ά 4.31 52.0 lie 1.90 27.9 á2 1.73 a 2.11 31.1 CO - 173.9 nh ₂ 7.13 - 6.72 Thr-2 NH 7.80 - CO - 169.9 a 4.11 58.4 a 4.11 66.0 a 1.01 19.8 Amino acid Position of atoms the _H the _c Ala-2 NH 8.18 - CO - 172.4 a 4.32 48.5 a 1.16 17.6

Thr-3 NH 7.93 - CO - 168.6 ά 4.47 55.6 ά 4.94 70.5 ά 1.12 16.7 Website 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 1 1 1.21 22.0 12 0.83 13.9

^{The 1} H, DQCOSY and TOCSY spectra measured in DMSO- _d6 at 35 °C confirmed the presence of seven commonly occurring amino acid moieties, 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 spin systems. The less commonly occurring amino acid moieties, namely (Dha), (Dhb) and homoserine, were determined by cross-signals observed in the TOCSY spectrum, which shift from the amide protons at 9.01 (broad s), 9.07 (broad s) and 8.22 ppm, and the shifts of the additional protons and carbon atoms: a 5.86, 5.61 (a _c 106.4), 6.40 (a _c 128.5), 1.61 (a _c 12.9), 4.22 (a _c 51.4), 1.82, 1.76 (ö _c 34.0), 3.47 (a _c 57.3). According to this ¹³ C NMR spectrum, 8 protons are attached to the ά-position carbon atoms of the saturated amino acid moieties, and 2 quaternary ω-position carbon atoms appear in the case of the unsaturated amino acids Dha and Dhb. Nine of the 12 amide or ester-type carbonyl carbon atoms were identified as being attached to 8 saturated amino acid moieties (two to glutamine), and two (at 164.0 and 163.7 ppm) to Dha and Dhb. The remaining one carbonyl carbon atom is part of the dodecanoyl side chain, which is further confirmed by the terminal methyl signals (Öh 0.83 and öc 13.9) and the 10 methylene signals appearing in the ¹³ C NMR spectrum (Table 1).

Based on the molecular formula, the molecule contains 16 unsaturated bonds. The 10 amino acids and the dodecanoyl side chain show 15 unsaturated bonds, indicating that the antifungal agent 25-B1 decapeptide A is a monocyclic decapeptide. Comparison of the chemical shifts of the beta protons of threonine (ε _H 4.94, 4.11 and 4.00) indicates that the proton appearing at ε H 4.94 ppm is attached to a carbonyl group, which may be an ester or a lactone. Thus, the depsidecapeptide structure of the antifungal agent 25-B1 decapeptide A was confirmed by HMBC and ROESY data. These data also supported the amino acid sequence and the position of the dodecanoyl chain in the antifungal agent 25-B1 decapeptide A. In the case of the arginine amino acid as a starting point, a Jong range correlation was observed in the HMBC spectrum between the amide proton and the adjacent amino acid carbonyl and alpha-carbon atoms (see Figure I), which confirms beyond any doubt the amino acid sequence: ArgNH/Thr-CO, Thr-NH/Dha-CO, Dha-NH/Ala-CO, Ala-NH/Dhb-CO, Dhb-NH/Hse-CO, Hse-NH/Thr-CO, Thr-NH/Ala-CO and AlaNH/Thr-CO.

The threonine NH group adjacent to the alanine does not show a long-range correlation with the arginine carbonyl group, but instead shows a correlation with the carbonyl group of the dodecanoyl side chain. The lack of Thr-NH/Arg-CO correlation and the Thr-ά-Η (á _H 4.94, öc 70.5)/Arg-CO clearly indicate an ester group formed between the Thr-á-OH and Arg-COOH groups. These statements are supported by the ROESY correlation observed between the amide protons and the ά-protons of the neighboring amino acids (see Figure II).

Findings

The antifungal compound 25-B1 decapeptide A represents a new class of lipodepside decapeptides that contain several amino acid residues not found in the pseudomycins, syringomycins, syringotoxins, and syringostatins produced by various isolates of P. syringae. The new depsipeptide consists of 10 amino acids, a further difference from the pseudomycins and syringomycins, which contain only 9 amino acid residues. Unlike natural pseudomycin products, the new depsipeptide does not contain chlorothreonine, which is believed to be responsible for the irritating side effects of injectable preparations containing pseudomycin compounds.

Example 4 - The decapeptide 25-B1 is antifungal. The antifungal effect of the substance

Antifungal activity was measured using a microtiter dilution method in a 96-well microtiter plate according to the National Committee for Clinical Laboratory standards. Sabouraud and dextrose media were adjusted to contain 2.5 x 10 ⁴ conidia/ml. The test compound was dissolved in water and the test was started in two-fold dilution with the highest concentration of 20 ug/ml. The plates were incubated at 35 °C for 48 hours. The data in Table 2 show the minimum inhibitory concentration (MIC) of the compound that completely inhibits the growth of the microorganism compared to the untreated control.

Table 2. Antifungal activity of the compound of formula (I)

Microorganism MIC (pg/ml) Candida albicans 10 C. parapsilosis 10 Cryptococcus neoformans 1.25 Aspergillus fumigatus >20 Histoplasma capsulatum 20

One or more P. syringae lipodepsipeptides, such as 25-B1 decapeptide The presence or amount of the antifungal agent can be determined by measuring the antifungal activity of the composition. The antifungal activity can be determined in vitro by measuring the minimum inhibitory concentration (MIC) of the composition using a standard agar dilution method or a plate diffusion method. The preparation of one or more P. syringae lipodepsipeptides, such as 25-B1 decapeptide The antifungal agent can be an extract of a cell culture or a more purified mixture. A typical fungus used in testing antifungal activity is Candida albicans. Antifungal activity is considered significant if the composition produces a zone of inhibition of 10-12 mm in diameter on a Candida albicans x 657 agar plate.

Example 5 - Isolation, characterization and mutagenesis of Pseudomonas syringae

Environmental isolates and mutants of P. syringae were generated and used in the production of antifungal agents.

Materials and methods

The isolation and characterization of MSU 174 and MSU 16-H strains were described in U.S. Patent No. 5,576,298, issued November 19, 1996 to G. Strobel et al.; Harrison 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.Sei. USA 84, 6447-6451 (1987). The disclosures of these publications are hereby incorporated by reference.

Additional strains were derived from these wild-type and transposable mutants by chemical mutagenesis. The strains subjected to mutagenesis included strains MSU 174, MSU 16H, and 25-B1. The strain to be mutagenized was grown on a medium containing potato product and then distributed into a medium containing 0, 1, 2, 4, 16, or 32 µM of the chemical mutagen 1 -methyl-3-nitro-1 -nitrosoguanidine (NTG or MNNG). These cells were frozen for future measurements and selection.

Mutagenized cells were selected for desired growth conditions and/or production of one or more P. syringae lipodepsipeptides, such as 25-B1 decapeptide A. The chemically mutagenized P. syringae cells, the mutagenized 25-B1 strains, were thawed and diluted to 6 cells/ml with N21SM medium (Table 3). This medium sometimes contained one or more components for selection, such as phosphates at varying concentrations. A 50 µl volume of the mutagenized cell suspension was distributed into a 96-well circular microtiter plate at an average of 0.3 cells/well.

Typically, silicone oil was added to each well to minimize evaporation. The plates were incubated with shaking for 6-12 days at 25°C.

Table 3

Composition of N21SM medium

COMPONENTS GRAMS/LITRES glucose 20 ammonium sulfate 0.5 monosodium glutamate 2 L-histidine 2 glycine 0.5 soluble starch 5 KH ₂ PO ₄ 0.2 Czapek mineral salt solution 2 ml MES buffer 9.8 PH adjustment to 5.0

After incubation, an aliquot, usually 1:1, was serially diluted from each well (e.g., 1:56, 1:196, 1:320, 1:686, and/or 1:1715) and evaluated for activity against Candida albicans using a liquid microtiter plate bioassay. The plates were incubated overnight at 37°C and the wells were tested for C. albicans growth. Appropriate strains were picked, inoculated into CSM medium (Table 4) and incubated for 1-3 days at 25°C.

Table 4

Complete Streptomyces medium (CSM)

Ingredients Concentration (g/liter) glucose 5 maltose 4 Difco Tryptone Soy Medium 30 Difco yeast extract MgSO ₄ x 7H ₂ O 2 no pH adjustment The selected strains were preserved and inoculated into into fermentation flasks, which contain 13 ml of N21SM medium were contained and shaken about 66 hours at 25 °C. The during fermentation aliquot we took parts of the fermentation broth· extracted for 1 hour with the same volume as an aliquot with acetonitrile, centrifuged , and the supernatant was purified by HPLC we examined one or more, P. syringae described in examples 1-3 lipodepsi-decapeptide, as 25-B1 Decapeptide A Antifungal for the presence of a substance. The one or more P. syringae lipodepsi-

decapeptide 25--B1 decapeptide The strains producing the antifungal substance were re-isolated, re-fermented and prepared as inoculum for a larger-scale fermentation.

Results

Using the above methods, we generated strains that expressed one or more of the P. syringae lipodepside decapeptides 2539

-B1 decapeptide A antifungal substance is produced. ·;· « * » 'r-'l Λ · <Τί «

♦ Conclusion

The selection methods and criteria disclosed herein are effective for the production of P. syringae strains that grow in minimal media and produce one or more P. syringae lipodepside decapeptide 25-B1 decapeptide A antifungal agents.

Example 6 - Preparations containing P. syringae lipodepsidecapeptide

The following formulation examples illustrate the essence of the invention and do not limit the scope of the invention.

The term "active ingredient" means a P. syringae lipodepside decapeptide or a pharmaceutically acceptable salt thereof.

1. Preparation

We have prepared hard gelatin capsules containing the following ingredients:

Components Quantity (mg/capsule) active ingredient 250 dried starch 200 magnesium stearate 10 total 460 mg

2. Preparation A tablet was prepared with the ingredients listed below. The materials were mixed and tableted so that the average weight of the tablets was 665 mg.

Components Quantity (mg/capsule) active ingredient 250 microcrystalline cellulose 400 silicon dioxide 10 stearic acid 5 total 665 mg

3. Preparation

An aerosol solution was prepared containing the components listed below. The active ingredient was mixed with ethanol and the mixture was added to a portion of the propellant 22, cooled to -30 °C and poured into a measuring vessel. The required amount was then transferred to an acid-resistant steel container and diluted with the remaining propellant. The container was then closed with the valve.

Composition Weight (g) active ingredient 0.25 methanol 27.75 propellant 22 (chlorodifluoromethane) 74.00 total 100.00

4. Preparation

The tablets containing 60 mg of active ingredient were prepared in the manner and composition described below:

active ingredient 60 mg microcrystalline cellulose 45 mg Polyvinylpyrrolidone (10% aqueous solution) 4mg sodium carboxymethyl starch 4.5 mg magnesium stearate 0.5 mg talc 1mg total 150 mg

The active ingredient, starch and cellulose were sieved through a 45 mesh (US) sieve and mixed thoroughly. The powder thus prepared was mixed with an aqueous solution of polyvinylpyrrolidone and sieved through an 18 mesh (US) sieve. The granulate thus prepared was dried at 50 °C and sieved through an 18 mesh (US) sieve. Carboxymethylcellulose, magnesium stearate and talc were previously sieved through a 60 mesh (US) sieve, then added to the granulate, mixed and then formed into tablets with an average weight of 150 mg.

5. Preparation

Capsules containing 80 mg of active ingredient were prepared as follows:

active ingredient 80 mg starch 59 mg microcrystalline cellulose 59 mg magnesium stearate 2mg total 200 mg

The active ingredient, cellulose, starch, and magnesium stearate are mixed, sieved through a 45 mesh (US) screen, and 200 mg are filled into a hard gelatin capsule.

6. Preparation

Suppositories containing 225 mg of active ingredient were prepared as follows:

active ingredient 225 mg saturated fatty acid glycerides 2,000 mg total 2.225 mg

The active ingredient was sieved through a 60 mesh (US) screen and suspended in the saturated fatty acid glyceride previously melted with the minimum heat required. The mixture was then filled into a 2 g nominal capacity cone and allowed to cool.

7. Preparation

The suspension containing 50 mg of active ingredient in a 5 ml dose was prepared as follows:

active ingredient 50 mg sodium carboxymethyl cellulose 50 mg syrup 1.25ml benzoic acid solution 0.10 ml fragrance q.v. coloring agent q.v. purified water gives 5ml

The active ingredient is sieved through a 45 mesh (US) sieve and mixed with sodium carboxymethyl cellulose and syrup to a workable paste. While mixing, an aqueous solution of benzoic acid, fragrance and colorant are added. Finally, sufficient water is added to reach the desired volume.

8. Preparation

An intravenous formulation is prepared as follows: a solution of these ingredients is usually administered intravenously to the patient over 1 minute:

active ingredient 100mg isotonic saline solution 1,000 mg

Several specific and preferred embodiments and technical solutions of the invention have been described. However, it should be understood that many variations and modifications can be made without limiting the scope of the invention.

Claims (35)

PATENT CLAIMS 1. An isolated Pseudomonas syringae depsidecapeptide, or a pharmaceutically acceptable salt, ester or hydrate thereof, characterized in that the depsidecapeptide comprises a depsidecapeptide ring consisting of arginine, threonine, homoserine, dehydroaminobutyric acid and dehydroalanine, and a lactone ring formed from the carboxyl group of arginine and the hydroxyl group of threonine. 2. The depsidecapeptide according to claim 1, characterized in that the depsidecapeptide comprises the following sequence: Thr-Ala-Thr-GIn-Hse-Dhb-Ala-Dha-Thr-Arg” (SEQ ID NO: 1). 3. The Pseudomonas syringae depsidecapeptide according to claim 1, characterized in that the Pseudomonas syringae depsidecapeptide is Pseudomonas syringae lipodepsidecapeptide. 4. The Pseudomonas syringae depsidecapeptide according to claim 3, characterized in that the Pseudomonas syringae depsidecapeptide comprises a fatty acid moiety linked to the amino group of threonine by an amide bond. 5. The Pseudomonas syringae depsidecapeptide according to claim 4, wherein the fatty acid moiety is decanoic acid, decanoic acid substituted with one or two hydroxyl groups, dodecanoic acid, dodecanoic acid substituted with one or two hydroxyl groups, tetradecanoic acid, or tetradecanoic acid substituted with one or two hydroxyl groups. 6. The Pseudomonas syringae depsidecapeptide according to claim 5, characterized in that the fatty acid unit is a dodecanoic acid unit. 7. The Pseudomonas syringae depsidecapeptide according to claim 3, wherein the Pseudomonas syringae depsidecapeptide is the compound of formula (I), wherein R is a lipophilic moiety, or a pharmaceutically acceptable salt, ester or hydrate thereof. 8. The Pseudomonas syringae depsidecapeptide according to claim 7, wherein the lipophilic moiety is a C9-15 alkyl, C9-15 hydroxyalkyl, C9-15 dihydroxyalkyl, C9-15 alkenyl, C9-15 hydroxyalkenyl, or C9-15 dihydroxyalkenyl group. 9. The Pseudomonas syringae depsidecapeptide according to claim 8, characterized in that the lipophilic moiety is an alkyl group having 9-15 carbon atoms. 10. A Pseudomonas syringae depsidecapeptide of formula (II), or a pharmaceutically acceptable salt, ester or hydrate thereof. 11. A method for inhibiting fungal infections, comprising treating the fungus with an isolated Pseudomonas syringae depsidecapeptide, or a pharmaceutically acceptable salt, ester or hydrate thereof, wherein the depsidecapeptide comprises a depsidecapeptide ring consisting of arginine, threonine, homoserine, dehydroaminobutyric acid and dehydroalanine, and a lactone ring formed from the carboxyl group of arginine and the hydroxyl group of threonine. 12. The method of claim 11, wherein the fungus is Candida parapsilosis, Candida albicans, Cryptococcus neoformans or Histoplasma capsulatum. 13. A method for reducing fungal infections to the extent required by a patient, comprising treating the patient with a composition comprising an isolated Pseudomonas syringae depsidecapeptide, or a pharmaceutically acceptable salt, ester or hydrate thereof, wherein said depsidecapeptide comprises a depsidecapeptide ring consisting of arginine, threonine, homoserine, dehydroaminobutyric acid and dehydroalanine, and a lactone ring formed from the carboxyl group of arginine and the hydroxyl group of threonine. 14. The method of claim 13, wherein the severity of the fungal infection is reduced by alleviating symptoms. 15. The method of claim 13, wherein the fungal infection is caused by Candida parapsilosis, Candida albicans, Cryptococcus neoformans or Histoplasma capsulatum. 16. The method of claim 13, wherein the isolated Pseudomonas syringae depsidekapeptide is administered parenterally 1-3 times daily in an amount of about 0.1-5 mg/kg. 17. The method of claim 13, wherein the alleviation of said symptom results in a reduction in fever and an improvement in the general condition of the patient. 18. Use of an isolated Pseudomonas syringae depsidecapeptide, or a pharmaceutically acceptable salt, ester or hydrate thereof, in the manufacture of a composition for the treatment of fungal infection, wherein said depsidecapeptide comprises a depsidecapeptide ring consisting of arginine, threonine, homoserine, dehydroaminobutyric acid and dehydroalanine, and a lactone ring formed from the carboxyl group of arginine and the hydroxyl group of threonine. 19. The use according to claim 18, characterized in that the fungal infection is an infection caused by Candida parapsilosis, Candida albicans, Cryptococcus neoformans or Histoplasma capsulatum. 20. A method for producing one or more Pseudomonas syringae depsidecapeptides, comprising culturing Pseudomonas syringae on a medium containing three or fewer amino acids at a pH of 4 to 6.5 until one or more Pseudomonas syringae depsidecapeptides are produced at a concentration of at least 10 ug/ml; and isolating one or more Pseudomonas syringae depsidecapeptides, or a pharmaceutically acceptable salt, ester or hydrate thereof, from said culture. 21. The method according to claim 20, characterized in that the amino acid used is glutamic acid, glycine, histidine or a combination thereof. 22. The method according to claim 21, characterized in that the amino acid used is glycine. 23. The method according to claim 22, characterized in that the medium component comprises soluble starch, yeast extract, a combination thereof, and a lipid. 24. The method of claim 23, characterized in that a potato preparation and a lipid comprising soybean oil, fatty acids and fatty acid esters are used as the medium component. 25. The method of claim 24, wherein the potato preparation is potato dextrose medium, potato puree, potato dextrin, potato protein, or a combination thereof. 26. The method according to claim 20, characterized in that the dissolved oxygen concentration during fermentation is maintained between 5-30%. 27. The method according to claim 20, characterized in that glucose, ammonium hydroxide or a combination thereof is continuously added during the fermentation. 28. The method of claim 20, wherein the produced Pseudomonas syringae depsidecapeptide is an isolated Pseudomonas syringae depsidecapeptide, or a pharmaceutically acceptable salt, ester or hydrate thereof, wherein said depsidecapeptide comprises a depsidecapeptide ring consisting of arginine, threonine, homoserine, dehydroaminobutyric acid and dehydroalanine, and a lactone ring formed from the carboxyl group of arginine and the hydroxyl group of threonine. 29. The method of claim 20, wherein the Pseudomonas syringae comprises a strain derived from strains MSU 16H, MSU 174 or MSU 206. ARC 30. The method of claim 20, wherein Pseudomonas syringae MSU 16H, strain, 25-B1 strain, 67H1 strain or 7H9-1 strain is used. 31. The method according to claim 30, characterized in that Pseudomonas syringae strain 25-B1 is used. 32. Pseudomonas syringae depsidecapeptide or a pharmaceutically acceptable salt, ester, hydrate thereof, characterized in that the depsidecapeptide prepared by the process according to claim 20 comprises a depsidecapeptide ring consisting of arginine, threonine, homoserine, dehydroaminobutyric acid and dehydroalanine, and a lactone ring formed from the carboxyl group of arginine and the hydroxyl group of threonine. 33. A method for treating or preventing fungal growth in or on a plant with an isolated Pseudomonas syringae depsidecapeptide or a pharmaceutically acceptable acid, ester, or hydrate thereof, characterized in that the depsidecapeptide comprises a depsidecapeptide ring consisting of arginine, threonine, homoserine, dehydroaminobutyric acid, and dehydroalanine, and a lactone ring formed from the carboxyl group of arginine and the hydroxyl group of threonine. 34. The method of claim 33, wherein the fungus is Rynchosporium secalis, Ceratocycystis ulmi, Rhizoctonia solani, Sclerotinia sclerotiorum, Verticillum albo- * *· ** * • _Λ · ·· ** * ·2^· · atrum, Verticillum dahliae, Thielaviopis basicola, Fusarium · ?, * * oxysporum or Fusarium culmorum. « 35. The method of claim 34, wherein the fungus is Rhizoctonia solani, Sclerotinia sclerotiorum, Verticillum albo-atrum or Fusarium oxysporum. The authorized representative is Ádám Szentpéteri Jr., patent attorney at the SzabadalmiÁJgyvivfli of the S.B. way to Monday Phone: 461-1000 Fax: 4