MXPA01010244A - 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

ISOLATED ANTIFUNGAL AGENTS OF PSEUDOMONAS SYRINGAE FIELD OF THE INVENTION The present invention relates to the depsidecapeptides, of P syringae, the method for making such a peptide, and the methods employing the antifungal activity of these peptides.

BACKGROUND OF THE INVENTION Fungal infections are a significant cause of disease, degradation of quality of life, and mortality among humans, particularly for immunocompromised patients. The incidence of fungal infections in humans has increased greatly in the last 20 years. This is partly due to the 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 or fungal pathogens that are prevalent throughout the population, but are kept stable by a functional immune system. These pathogens are difficult to control because some REF: 132849 existing antifungal or antifungal agents are either hy toxic or only inhibit fungal activity. For example, polyenes are fungicides but toxic; while azoles are-much less toxic but only fungistatic. More importantly, there have been recent reports of strains of Candida resistant to azole and polyene, which severely limits the therapeutic options against such strains. Pseudomonas syringae produce several classes of antifungal agents and antibiotics, such as pseudo iciñas, syringomycins, syringotoxins, and syringostatins, which are lipodepsinonapeptides. Natural strains and mutants generated by transposons of P. syringae produce these lipodepsinonapeptides. Several of the pseudomycins, syringomycins and other antifungal lipodepsipeptide agents have been isolated, chemically characterized and shown to possess broad spectrum antifungal activity, including activity against important fungal or fungal pathogens in humans and plants. Pseudomycins, syringomycins, syringotoxins, and syringostatins represent structurally distinct families of antifungal compounds. None of the lipodepsinonapeptides of P. syringae have been brought to the market for fungal therapy. The discovery of the undesirable side effects, the elaboration of the formulations, the large-scale production, and other problems of development have thus prevented the exploitation of the lipodepsinonapeptides of P. syringae against a wide range of fungal infections that They affect animals, humans and plants. There is a need for an antifungal or antifungal agent that can be used against infections not treated by existing antifungal or antifungal agents and for application against infections in animals, humans or plants. BRIEF DESCRIPTION OF THE INVENTION The present invention provides a depside-capeptide produced by P. syringae which contains the unusual amino acids homoserin (Hse), dehydroaminobutyric acid (Dhb) and dehydroalanine (Dha) as part of a depside-peptide ring. depside peptide of P. syringae includes a depside-peptide ring that has the amino acids arginine, threonine, homoserine, dehydroaminobutyric acid, and dehydroalanine, and a lactone formed from a carboxylic group of arginine and a hydroxyl group of threonine. of P. syringae, the depsidecapeptide is a lipodepsidecapeptide: a cyclic peptide coupled to a lipophilic moiety.The lipophilic moiety is typically a fatty acid moiety coupled to the amino group of threonine by an amide bond.Preferably, the fatty acid moiety is a portion of n-dodecanoic acid.The lipodepsidecapeptide is represented by the FORMULA I: where R is a lipophilic moiety. The lipophilic moiety includes alkyl of 9 to 15 carbon atoms, hydroxyalkyl of 9 to 15 carbon atoms, dihydroxyalkyl of 9 to 15 carbon atoms, alkenyl of 9 to 15 carbon atoms, hydroxyalkenyl of 9 to 15 carbon atoms, or dihydroxyalkenyl of 9 to 15 carbon atoms. Preferably, the lipophilic moiety is alkyl of 11 carbon atoms. The alkyl, hydroxyalkyl, dihydroxyalkyl, alkenyl, hydroxyalkenyl, or dihydroxyalkenyl groups can be branched or unbranched. Preferably, the amino acid sequence of the depsidecapeptide ring is threonine-alanine-threonine-glutamine-homoserine-acid-dehydroanebutyric-alanine-dehydroalanine-threonine-arginine, herein referred to as "decapeptide 25-B1" or * Thr-Ala- Thr-Gln-Xaa-Xaa-Ala-Xaa-Thr-Arg (SEQ ID No. 1) "As used herein, the term" antifungal agent A of decapeptide 25-B1"refers to the specific depsidecapeptide having the Preferred amino acid sequence of SEQ ID No. 1 and R = C 11 unbranched alkyl (for example, R = - (CH 2) 10 CH 3) The invention also relates to methods employing a P depside-peptide. syringae to inhibit the fungal activity or to reduce the symptoms of a fungal infection in a patient in need of it.These methods can kill the fungus, decrease the burden of a fungal infection, reduce fever and increase the general well-being of a patient, as a consequence a, the depsidecapeptides of P. syringae can be used in the manufacture of a medicament for the treatment of a patient as described herein. The methods and medicaments of the invention are effective against fungi such as Candida parapsilosis, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum. The invention provides the use of microorganisms in a method for producing an antifungal agent, such as the depsidecapeptides of P. syringae described above and including a 25-B1 decapeptide. The method involves the cultivation of Pseudomonas syringae in media containing three or fewer amino acids and recovering one or more depsidecapeptides of P. syringae from the culture. In one embodiment, the culture of P. syringae is a medium that includes glycine and a lipid, a potato product, or a combination thereof at a pH of about 4 to 6.5 until one or more depsidecapeptides of P. are produced. syringae at a concentration of at least about 10 μg / ml. In addition, the invention provides the depsidecapeptides of P. syringae prepared by the method described above. The invention also provides a method for treating or preventing the development of fungi in a plant, whereby a fungus is contacted with one or more of the depsidecapeptides of P. syringae described above.

DETAILED DESCRIPTION Antifungal Agents of Lipodepsidecapeptide As used herein "lipodepsidecapeptide antifungal agent" refers to an antifungal agent having a cyclic decapeptide ring closed by a lactone group and having an attached hydrophobic group, such as a fatty acid moiety. The antifungal agents of lipodepsidecapeptide are produced by Pseudomonas syringae. A representative of this class of compounds, the antifungal agent A of decapeptide 25-Bl, has been purified and its structure determined. As used herein the term "lipodepsidecapeptide of P. syringae" refers to an antifungal agent of lipodepsidecapeptide produced by P. syringae, and includes the antifungal agent A of decapeptide 25-B1 and related analogs. The lipodepsidecapeptides of P. syringae share several structural characteristics. For example, each of these antifungal agents includes the unusual amino acids such as serine (Hse), dehydroaminobutyric acid (Dhb) and dehydroalanine (Dha) as part of a depsidecapeptide ring. In each of the lipodepsidecapeptides of P. syringae, a carboxyl group of an arginine residue bound to the hydroxyl group of the N-terminal threonine forms a lactone which closes the depsidecapeptidic ring. The depsidecapeptidic ring sequence of the lipodepsidecapeptide of P. syringae 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 amino acids of natural origin. Conversely, the natural products of pseudomycin, the lipodepsidecapeptides of the present invention do not contain chlorotreonin which is suspected to be the cause for irritation at the injection site of pharmaceutical formulations containing pseudomycin compound. The depsidecapeptidic ring is linked to a lipophilic portion, 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 carbon atoms, typically carrying zero, one or two hydroxyl groups. The fatty acid may be branched or unbranched and may also contain at least one unsaturation. Preferred amino acid portions include a n-decanoic acid portion, a n-decanoic acid portion substituted with one or two hydroxyl groups, a n-dodecanoic acid moiety, a n-dodecanoic acid moiety substituted with one or two groups hydroxyl, a portion of n-tetradecanoic acid, and a portion of n-tetradecanoic acid substituted with one or two hydroxyl groups.

Antifungal Agents of the Decapeptide 25-B1 As used herein "de-peptide antifungal agent 25-Bl" refers to one or more members of a family of antifungal agents that have been isolated from the bacterium Pseudomonas syringae. -B1 is a lipodepsidecapeptide of Pseudomonas syringae.

Specifically, an antifungal agent of decapeptide 25-Bl is a lipodepside-peptide from Pseudomonas syringae having a depsidecapeptidic ring with the sequence: Thr-Ala-Thr-Gln-Hse-Dhb-Ala-Dha-Thr-Arg (SEQ ID No. 1 ) Each of the antifungal agents of decapeptide 25-B1 have the same cyclic peptide core, but these differ in the hydrophobic side chain coupled to this nucleus. Anti-fungal agents of decapeptide 25-B1 include antifungal agent A of decapeptide 25-Bl. The anti-fungal agents of decapeptide 25-B1 include a fatty acid linked through an amide bond with the amino group of the N-terminal threonine. The fatty acid portion of antifungal agent A of decapeptide 25-B1 is a portion of n-dodecanoic acid.

Biological Activities of the Lipodepsidecapeptides of P. syringae A lipodepside peptide of P. syringae, such as the antifungal agent A of decapeptide 25-B1, has various biological activities including the killing and inhibition activity of various fungi, such as pathogenic fungi for plants and animals. In particular, an antifungal agent of decapeptide 25-B1 is an antifungal agent active against fungi that cause opportunistic infections in immunocompromised individuals. These fungi include Cryptococcus neoformans, Histoplasma capsulatum and various species of Candida including C. parapsilosis and C. albicans.

Pseudomonas syringae Pseudomonas syringae include a wide range of bacteria that are generally associated with plants. Some of the Pseudomonas syringae are pathogenic for plants, while others are only weakly pathogenic or are saprophytes. Many different isolates of Pseudomonas syringae produce one or more cytotoxic agents that can help this bacterium survive in wildlife where it must compete with fungi and other bacteria. Cytotoxic agents produced by Pseudomonas syringae include antifungal agents such as Pseudomonas syringae lipodepsidecapeptides, including the antipuncture agent A of the decapeptide 25-B1, the pseudomycins, the syringomycins, the syringotoxins, and the syringostatins. Isolated strains of Pseudomonas syringae that produce one or more pseudomycins, syringomycins, syringotoxins, syringostins, are well known to those skilled in the art. The wild-type MSU174 strain and a mutant of this strain generated by transposon mutagenesis, MSU 16H (ATCC 67028) have been described in U.S. Patent No. 5,576,298, issued November 19, 1996 to G. Strobel and collaborators: Harrison et al., "Pseudomycins, a family of new peptides _ of Pseudomonas syringae that possess broad spectrum antifungal activity." J. Gen Microbiology 137.2857-2865 (1991); and Lamb et al. "Mutagenesis by Transposon and fluorescent pseudomonas labeling: Antimycotic production is necessary for the control of Dutch elm disease," Proc. Nati Acad. Sci. USA 84.6447-6451 (1987). Methods for the development of various strains of Pseudomonas syringae and their use in the production of antifungal agents such as pseudomycins, are also described in the application of United States Patent Serial No. PCT / US00 / 08728 by Matthew D. Hilton, and collaborators the headline "Production of Pseudomycin by Pseudomonas Syringae" sent on the same date with the present and described below. The descriptions of the references cited in this paragraph are incorporated herein by reference. Strains of P. syringae that are suitable for the production of one or more lipodepsidecapeptides of Pseudomonas syringae such as antifungal agent A of decapeptide 25-B1, can be isolated from environmental sources including plants such as barley plants, citrus plants and lilac plants, and peat or forest fertilizer, soil, water, air, and dust. The present invention includes a strain, an isolate and a biologically purified culture of Pseudomonas syringae that produces one or more lipodepsidecapeptides of Pseudomonas syringae such as antifungal agent A of decapeptide 25-B1, in amounts greater than about 10 μg / ml, preferably of at least about 10 μg / ml to about 50 μg / ml. Preferably, the biologically purified culture of a microorganism is from strains MSU 16H, 25-B1, 67H1, 7H9-1, from Pseudomonas syringae, or a pseudomycin-producing mutant, a variant, an isolate, or a recombinant of these strains. The MSU 16H crops are in storage at the Montana State University (Bozeman, Montana, United States) and available from the North American Collection of Species Crops (American Type Culture Collection; (Parklawn Drive, Rockville, MD, USA) Accession No. ATCC67028. A strain of Pseudomonas syringae that is suitable for the production of one or more lipodepsidecapeptides of Pseudomonas syringae, such as the antifungal agent A of decapeptide 25-B1, can be isolated from environmental sources including plants, such as barley plants, citrus plants , and lilaseas plants, and also from sources such as soil, water, air, and dust. A preferred strain is isolated from plants. These environmental isolates of Pseudomonas syringae can be referred to as wild type. As used herein, "wild type" refers to a dominant genotype that occurs naturally in the normal population of Pseudomonas syringae (e.g. strains or isolates of Pseudomonas syringae that are found in the wild and not produced by manipulation in the wild). laboratories). As is the case with other organisms, the characteristics of the lipodepsidecapeptide-producing cultures employed in this invention, strains of Pseudomonas syringae, such as MSU 174, MSU 16H, MSU 206, 25-B1 and 7H9 are subject to variation. Thus, the progeny of these strains, for example, the recombinants, mutants, and variants, can be obtained by methods well known to those skilled in the art. Mutant strains of P. Siringae are also suitable for the production of one or more lipodepsidecapeptides of P. Siringae, such as antifungal agent A of decapeptide 25-B1. As used herein, "mutant" refers to a sudden inheritable change in the phenotype of a strain, which may be spontaneous or induced by known mutagenic agents, including radiation and various chemicals. P. mutant Siringae 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 mutagenesis mediated by transposon. Examples of chemical mutagens are ethylmethyl sulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-N-nitrosoguanine (NTG), and nitrous acid. P. siringae suitable for the production of one or more lipodepsidecapeptides of P. Siringae, such as antifungal agent A of decapeptide 25-B1, according to the present invention, can be generated by treating the bacteria with an amount of effective utagénico agent to produce mutants that overproduce one or more lipodepsidecapeptides of P. Siringae, such as the antifungal agent A of decapeptide 25-B1, or that produce one or more lipodepsidecapeptides of P. syringae, such as antifungal agent A of decapeptide 25 -B1 under advantageous development conditions. While the type and amount of mutagenic agent to be used may vary, a preferred method is to dilute in series NTG to levels in the range of about 1 to about 100 μg / ml. Preferred mutants of the invention are those that overproduce the antifungal agent A of decapeptide 25-Bl, and develop in minimal medium. The mutants over-produce a lipodepside-peptide from P. Siringae, such as the antifungal agent A from depside-peptide 25-B1, preferably from at least about 10 μg / ml to about 50 μg / ml. Environmental isolates, mutant strains and other desirable strains of P. syringae may be subject to selection for the desirable traits of the developmental habit, the growth medium, the nutrient source, the carbon source, the growth conditions, and the amino acid requirements.

Preferably, a strain of P. syringae that produces the lipodepsidecapeptides of P. syringae such as the antifungal agent A of the depsidecapeptide 25-B1, is selected for development on the minimum defined medium. Preferred strains show the production characteristics of one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of depsidecapeptide 25-B1. When they are grown on a medium that includes more glycine, optionally a lipid, a potato product or both. Recombinant strains can be developed by transforming strains of P. Siringae using procedures well known to those skilled in the art. Through the use of recombinant technology, strains of P. Siringae can be transformed to express a variety of gene products in addition to the antibiotics that these strains produce. For example, strains can be transformed with a recombinant vector that confers resistance to an antibiotic to which the strains are normally sensitive. The transformants obtained in this way will not produce only a lipodepsidecapeptide of P. Siringae, such as the antifungal agent A of depsidecapeptide 25-B1, but also the enzyme that confers resistance, which allows the selection of the transformed cells from the wild-type ones. In addition, using similar techniques, the present strains can be modified to introduce multiple copies of the endogenous lipodepsidecapeptide biosynthesis genes to achieve greater performance of the lipodepsidecapeptide. Progeny, for example, natural and induced variants, mutants and recombinants of P. siringae strains 25-B1, 671, and 7H9-1 that retain the production characteristic of lipodepsidecapeptide, are part of this invention Development of Pseudomonas syringae As described herein, the "aqueous nutrient medium" refers to a water-based composition that includes minerals and organic compounds and their salts, necessary for the development of the bacteria 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 mixed food product of commercial mashed potatoes. Preferred minerals in the nutrient medium include mixtures of salts typically used in cell culture and fermentation, such as the Czapek mineral salts, which include potassium chloride, magnesium sulfate, and ferrous sulfate. The organic compounds in the nutrient medium preferably include glucose and may also optionally include soluble starch; Other similar 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 typically not critical for the development of the bacteria or for the production of a lipodepside-peptide from P. Siringae, such as the antifungal agent A of depside-peptide 25-B1, certain levels of nutrients are advantageous . A preferred amount of glycine is about 0.1 g / 1 to about 10 g / 1, more preferably about 0.3 g / 1 to about 3 g / 1, most preferably 1 g / 1. A preferred amount of lipid is about 1 g / 1 to about 10 g / 1 of an oily product such as soybean oil, more preferably about 0.5 g / 1 to about 2 g / 1 of soybean oil. A preferred amount of a fatty acid or fatty acid ester is from about 0.5 g / 1 to about 5 g / 1. Preferred amounts of the potato products include about 12 g / 1 to about 36 g / 1, more preferably about 24 g / 1 of potato dextrose broth; about 5 g / 1 to about 50 g / 1, preferably about 30 g / 1 of a commercial mixture of mashed potatoes; about 1 g / 1 to about 30 g / 1, preferably about 20 g / 1 of potato dextrin; and / or about 1 g / 1 to about 10 g / 1, preferably about 4 g / 1 of potato protein. A preferred nutrient medium includes minerals, preferably, potassium chloride at about 0.02 to about 2 g / 1, more preferably about 0.2 g / 1; magnesium sulfate, preferably magnesium sulfate heptahydrate (MgSO4 # 7H20) at about 0.02 to about 2 g / 1, more preferably about 0.2 g / 1; and ferrous sulfate, preferably ferrous sulfate heptahydrate (FeS04β7H20), at about 0.4 to about 40 mg / l, more preferably about 4 mg / l. When present, the soluble starch is preferably at about 0.5 to about 50 g / 1, more preferably about 5 g / 1. Glucose is preferably present at about 2 to about 80 g / 1, more preferably about 20 g / 1. P. Siringae are typically developed in the media described under the conditions of controlled or regulated pH, and controlled or regulated temperature. P. Siringae develop and produce one or more cytotoxic agents at temperatures between about 15 ° C and about 35 ° C, preferably about 20 ° C to about 30 ° C, more preferably about 25 ° C. P. syringae develop and produce one or more cytotoxic agents at pH between about 4 and about 9, more preferably between about 4 and about. 6, most preferably from about 4.5 to about 5.5. Typically, the development of P. syringae 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 the Production of Lipodepsidecapeptides of P. syringae To produce one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of depside-peptide 25-B1, from a wild-type or mutant strain of P. syringae, the organism is cultured with agitation in an aqueous nutrient medium which it includes an effective amount of three or fewer amino acids. The three or less amino acids are preferably glutamic acid, glycine, histidine, or a combination thereof. In a preferred embodiment, the amino acids include glycine and, optionally, one or more of a potato product and a lipid. The culture is conducted under conditions effective for the development of P. syringae, and the production of a desired lipodepsidecapeptide of P. syringae, such as the antifungal agent A of decapeptide 25-B1. 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 cultured on media such as those described herein, P. syringae can develop at cell densities up to about 10-15 g / 1 dry weight, and produce a lipodepside-peptide from P. syringae, such as the antifungal agent A depside peptide 25-B1, in a total amount of at least about 10 μg / ml, preferably at least about 50 μg / 1. The control of the concentration of oxygen in the medium during the culture of P. syringae is advantageous for the production of a lipodepsidecapeptide of P. syringae, such as the antifungal agent A of depsidecapeptide 25-Bl. Preferably, the oxygen levels are maintained at about 5% to about 50% saturation, more preferably about 30% saturation. Bleeding with air, with pure oxygen, or with gas mixtures that include oxygen can regulate the concentration of oxygen in the medium. In addition, the adjustment of the stirring speed can be used to adjust the oxygen transfer rate. Control of the pH of the medium during the cultivation of P. syringae is advantageous for the production of a lipodepsidecapeptide of P. syringae, such as the antifungal agent A of depsidecapeptide 25-B1. The pH of the culture medium can be maintained at less than about 6 and above about 4. P. syringae can produce a lipodepside-peptide from P. syringae, such as antifungal agent A from decapeptide 25-B1, when the culture is developed. by lots. However, batch feeding or semi-continuous feeding of the glucose and, optionally, an acid or base, such as ammonium hydroxide to control the pH, increases the production of the lipodepsidecapeptide of P. syringae, such as the antifungal agent. A of the depsidecapeptide 25-B1. A production of a lipodepside-peptide from P. syringae, such as the antifungal agent A of depside-peptide 25-B1, by P. syringae, can be further enhanced by the use of continuous culture methods in which glucose and, optionally, an acid base, such as ammonium hydroxide, to control the 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. The choice of strain of P. syringae may affect the amount and distribution of a lipodepsidecapeptide of P. syringae, such as the antifungal agent A of depsidecapeptide 25-B1, produced by culture under the conditions described in. the present. For example, strain Bl can predominantly produce the antifungal agent A of depsidecapeptide 25-B1. The cyclic decapeptide core of the lipodepsidecapeptides of P. syringae can be prepared by cleavage of the lipophilic portion, such as by deacylation. The cleavage and deacylation methods are well known to those skilled in the art, such as the use of the enzyme deacylase.

The Formulation and Antifungal Action of the Lipodepsidecapeptides of P. syringae A lipodepsidecapeptide of P. syringae, such as the antifungal agent A of depsidecapeptide 25-B1, shows both in vi tro and in vi ve activity and is useful in combating either systemic fungal activities or fungal skin infections. Accordingly, the present invention provides a method for inhibiting fungal activity, including contacting a lipodepsidecapeptide from P. syringae, such as an antifungal agent A from depsidecapeptide 25-B1, or a pharmaceutically acceptable salt thereof, with a fungus. A preferred method includes inhibiting the development or activity of various fungi such as Cryptococcus neoformans, Histoplasma capsulatum, and Candida species including C. parapsilosis and C. albicans. As used herein, "contacting" a compound of the invention with a parasite or fungus refers to a binding or addition, or apparent touch or mutual tangency of a compound of the invention with a parasite or fungus. However, the term of contact does not imply any mechanism of inhibition. The present invention further provides a method for the treatment of a fungal infection which includes the administration of an effective amount of a lipodepsidecapeptide of P. syringae, such as the antifungal agent A of depsidecapeptide 25-B1, or a pharmaceutically acceptable salt thereof. , to a guest in need of such treatment. A preferred method includes the treatment of an infection by various fungi, such as Cryptococcus neoformans, Histoplasma capsulatum, and Candida strains including C. parapsilosis and C. albicans. When administered in an effective antifungal amount, a formulation of one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of depsidecapeptide 25-B1, reduces the burden of a fungal infection, reduces symptoms associated with fungal infection and can result in the elimination of the fungal infection. Some patients in need of antifungal therapy with a formulation of one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of depsidecapeptide 25-Bl, have severe symptoms of infection, such as high fever, and are likely to be in care intensive or critical. Various fungi can cause such serious infections. Candida spp, for example, causes mucosal and serious systemic infections and may exist as the strains resistant to azole or polyene. Aspergillus causes systemic infections that threaten life. Cryptococccus is responsible for meningitis. Such serious fungal infections can occur in immunocompromised 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 the intravenous administration of a formulation of one or more lipodepsidecapeptides of P. syringae, (eg, the antiphungal agents of decapeptide 25-B1) over several days to stop or delay infection. With respect to antifungal activity, the term "effective amount" means an amount of a compound of the present invention that is capable of inhibiting fungal growth or activity thereof, or reducing the symptoms of fungal infection. For most fungal infections, the reduction of infection symptoms includes the reduction of fever, the return to consciousness, and the increased well-being of the patient. Preferably, the 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 "inhibition" refers to the inhibition of fungal activity, including the arrest, delay or prophylactically preventing or preventing the development of any present characteristics and results of the existence of a fungus. Typically, the compositions will be administered to a patient (human or other animal, including mammals such as cats, horses and livestock, and bird species) in need thereof, in an amount effective to inhibit fungal infection. The dose administered will vary depending on factors such as the nature and severity of the infection, age and general health for the host and the tolerance of the host to the antifungal agent. The particular dosage regimen can likewise vary according to such factors and can be administered in a single daily dose or in multiple doses during the day. The regimen can vary from approximately 2-3 days to approximately 2-3 weeks or more. A typical daily dose (administered in single or divided doses) will contain a dose level of about 0.01 mg / kg to about 100 mg / kg of the body weight of an active body of this invention. Preferred daily doses in general will be from about 0.1 mg to about 60 mg / kg and ideally from about 2.5 mg / kg to about 40 mg / kg. For serious infections, the compound can be administered by intravenous infusion using, for example, 0.01 to 10 mg / gk / hour of the active ingredient. The present invention also provides pharmaceutical formulations useful for the administration of the antifungal compounds of the invention. Accordingly, the present invention also provides a pharmaceutical formulation that includes one or more carriers, diluents, carriers, excipients, or other pharmaceutically acceptable additives, and one or more lipodepsidecapeptides of P. syringae, such as antifungal agent A of decapeptide 25- B1 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 carrier, diluent or excipient is compatible with the other ingredients of the formulation and not harmful to the container thereof. The formulation may also include additives such as various oils, including those of petroleum, animal, vegetable or synthetic origin oils, 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, skim milk powder, glycerol, propylene glycol, water and ethanol. The compositions may also be subject to conventional pharmaceutical procedures such as sterilization, and may contain conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting agents or emulsifiers, salts for adjusting the osmotic pressure, and buffers. Suitable pharmaceutical carriers and their formulations are described in Martin, "Remington's Pharmaceutical Sciences," 15a. Ed.; Mack Publishing Co. , Easton (1975): see, for example, pages 1405-1412 and pages 1461-1487. The term "pharmaceutically acceptable salt", as used herein, refers to the 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 the reaction of the compounds of the present invention with a mineral or organic acid or an inorganic base. Such salts are known as salts by addition of acid or addition of base. The acids commonly employed to form the 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 acid, methanesulfonic acid, oxalic acid, p-acid bromophenylsulphonic, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid. Examples of such pharmaceutically acceptable salts are the salts of sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monoacid phosphate, diacid phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate , isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butin-1,4-dioate, hexin-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate , phthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propansulfonate, naphthalene-1-sulfonate, naphthalene-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 maleic acid and methanesulfonic acid. The base addition salts include those derived from inorganic bases, such as carbonates, bicarbonates and ammonium hydroxides, or alkali metal or alkaline earth metal hydroxides. Such bases useful in the preparation of the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, and carbonate. of calcium. The potassium and sodium salts are particularly preferred. It should be recognized that the particular counter ion that forms a part of any salt of this invention is not of a critical nature, as long as the salt as a whole is pharmaceutically acceptable and as long as the counter ion or the counter ion does not. contribute to unwanted qualities of salt as a whole. A lipodepside peptide of P. syringae, such as antifungal agent A of decapeptide 25-B1, can be administered parenterally, for example using intramuscular, subcutaneous, or intraperitoneal injection, nasal or oral means. In addition to these methods of administration, the lipodepside peptide of P. syringae, such as the antifungal agent A of decapeptide 25-B1, can be applied topically for superficial infections of the skin or to inhibit the development of fungi in the mucus. For parenteral administration the formulation includes one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of the decapeptide 25-B1 and a physiologically acceptable diluent such as deionized water, physiological saline, 5% dextrose and other diluents commonly used. The formulation may contain a cyclodextrin and / or a solubilizing agent such as propylene glycol or polyethylene glycol or another known solubilizing agent. Such formulations can be constituted in sterile flasks containing the antifungal and excipient in a dry powder or lyophilized powder form. Before use, a physiologically acceptable diluent is added and the solution is removed by means of a 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 mixed with a carrier, or diluted by a carrier, or enclosed within a carrier, which may be in the form of a capsule, sachet, paper or another container. When the carrier serves as a diluent, it must 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, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or a liquid medium), ointments containing for example up to 10% by weight of the active ingredient, 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 suitable binder, dispersant or other excipients, suitable for the preparation of a tablet of suitable size for the dosing of a lipodepside-peptide of P. syringae, such as the antifungal agent A of the decapeptide 25-B1. For pediatric or geriatric use the antifungal compound can be formulated in a liquid solution, emulsion or liquid suspension flavored. A preferred oral formulation is linoleic acid, cremophor RH-60 and water, and preferably in the amount (by volume) of 8% linoeic acid, 5% cremophor RH-60, 87% sterile water and a lipodepside-peptide of P syringae, such as antifungal agent A of decapeptide 25-B1, in an amount of about 2.5 to about 40 mg / ml. For topical use the antifungal compound can be formulated with a dry powder for application to the surface of the skin or it can be formulated in a liquid formulation that includes an aqueous solubilizing liquid or a non-aqueous liquid, for example, an alcohol or glycol.

Uses of the Formulations of a Lipodepsidecapeptide of P. syringae The present invention also encompasses equipment that includes the present pharmaceutical compositions and for use with the methods of the present invention. The kit may contain a vial containing a formulation of the present invention and suitable carriers, either dry or in liquid form. The equipment also includes the instructions in the form of a label on the bottle and / or in the form of an insert included in a box in which the bottle is packaged, for the use and administration of the compounds. The instructions can also be printed on the box in which the bottle is packaged. The instructions contain information such as administration information and sufficient dosage, to allow a worker in the field to administer the drug. It is anticipated that a worker in the field includes any doctor, nurse or technician who can administer the drug. The present invention also relates to a pharmaceutical composition that includes a formulation of one or more lipodepsidecapeptides of P. syringae, such as antifungal agent A of decapeptide 25-B1, and which is suitable for administration by injection. According to the invention, a formulation of one or more lipodepsidecapeptides of P. syringae, such as antifungal agent A of decapeptide 25-B1, can be used for the manufacture of a composition or medicament suitable for administration by injection. The invention also relates to methods for the manufacture of compositions that include a formulation of one or more lipodepsidecapeptides of P. syringae, such as antifungal agent A of decapeptide 25-B1, in a form that is suitable for oral or oral administration. Topical For example, a liquid or solid formulation can be manufactured in various ways, using conventional techniques. A liquid formulation can be manufactured by dissolving one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of the decapeptide 25-B1 in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients.

AGRICULTURAL USES Antibiotics produced by P. syringae NRRLB-12050 have been shown to effectively treat Dutch elm disease (see, for example, U.S. Patent Nos. 4,342,746 and 4,277,462). In particular, P. syringae MSU 16H has been shown to confer greater protection than a wild-type strain in elms infected with Cera tocystis uli, the causative agent of Dutch elm disease (see, for example, Lam et al. Proc. Nati. Sci. USA, 84, 6447-6451 (1987)). More extensive tests on elms developed in the field confirmed the phenomenon of biocontrol at a prophylactic level. Therefore, the lipodepsidecapeptides of the present invention may be useful as a preventive treatment for Dutch Elm disease. Pseudomycins have been shown to be toxic to a wide range of plant pathogenic fungi including Rynchospori um secalis, Wax tocystis ulmi, Rizoctonia solani, Sclerotina sclerotiorum, Verticilli um alboculmorum (see Harrison, L., et al., "Pseudomycins, a family of new Pseudomonas syringae peptides that possess broad spectrum antifungal activity. "J. General Microbiology, 7, 2857-2865 (1991)). Accordingly, one or more lipodepsidecapeptides of P. syringae, such as antifungal agent A of decapeptide 25-B1 (including hydrates, solvates and esters thereof) may be useful in the treatment of fungi in plants (in particular, V. Albo-atrum Rhizoctonia solani and F. oxysporum) either as a direct treatment or preventive treatment. In general, infected plants are treated by injecting or spraying an aqueous suspension of the lipodepsidecapeptide compounds into or onto the plant. The means for the injection are well known to those skilled in the art (for example, piston with groove). Any spray medium of the suspension can be used which distributes an effective amount of the active material on the surface of the plant. The suspension may also include other additives generally used by those skilled in the art, such as solubilizers, stabilizers, wetting agents and combinations thereof.

The treatment of the plant can also be accomplished by using a dry composition containing one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of the decapeptide 25-B1. The anhydrous formulation can be applied to the surface of the plant by any means well known to those skilled in the art, such as spraying or stirring from a container. The present invention may also be understood with reference to the following examples. It is intended that these examples be representative of the specific embodiments of the invention, and are not intended to limit the scope of the invention.

EXAMPLES Biological Materials in Deposit P. syringae MSU 16H is publicly available from the American Type Culture Collection, Parklawn Drive, MD, USA as Accession No. ATCC 67028. P. syringae strains 25-Bl, 7H9, and 67 Hl were deposited with the American Type Culture Collection on March 23, 2000, and were assigned the following Access Nos: 25-B1 Accession No. PTA-1622 7H9-1 Accession No. PTA-1623 67 Hl Accession No. PTA-1621 Example 1 - Production of Antifungal Agent A of 25-B1 The fermentation methods were developed for the production of a lipodepsidecapeptide antifungal agent, the antifungal agent A of decapeptide 25-B1, in the fermentation broth of a strain of P. syringae.

Materials and methods Inoculation preparation: an aliquot of P. syringae strain 25-B1 stored in vapor phase of the liquid nitrogen was thawed and used to inoculate two 900 ml portions of the CSM broth. The CSM broth was composed of (g / 1): dextrose (5), maltose (4), Difco Tryptic soy broth (30), Difco yeast extract (3), and MgS0 »7H20 (2). Approximately 0.5 ml of cells were used to inoculate each 900 ml portion of the medium contained in a two-liter flask. The 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 the sterile fermentation broth.

Fermentation stage: the fermentation broth was composed of (g / 1): dextrose (20), soluble starch (5), Potato Pearls Basic American Foods Country Style in • time of mashed potatoes (30), glycine (1), MgS04 * 7H20 (0.2), potassium chloride (0.2), and FeS04 * 7H20 (0.004) in tap water. The pH was adjusted to 5.2 before sterilization. The fermentation was carried out at 25 ° C for 68 hours. The dissolved oxygen was maintained at or above 30% saturation with air by continuous adjustment of the air flow and the speed of agitation of the propellant. The pH was maintained between 4.0 and 5.4 through the addition of either sulfuric acid or sodium hydroxide. Various variations of simple batch processes were found to also produce the new cyclic peptide product. The dextrose can be fed to the fermenters beginning 24 hours after the initial inoculation at a rate of 60 ml per hour. The feeding can be continued throughout the course of the fermentation. Alternatively, a process has been used where dissolved oxygen is maintained at 5% saturation with air starting 24 hours after inoculation and continuing until the end of the fermentation period. The maintenance of dissolved oxygen at 5% was achieved through the addition of inert nitrogen gas (N2) to the air supply leading to the fermenter. In all cases, gas was supplied through a simple submerged purge tube, with an opening placed just below the bottom agitator turbine, in the fermenter.

Results and conclusions Several fermentation methods produce the antifungal agent A of the decapeptide 25-B1 from P. syringae.

Example 2 - Isolation and Purification of Antifungal Agent A of 25-B1 Methods for the isolation and purification of a lipodepsidecapeptide antifungal agent, antifungal agent A from decapeptide 25-B1, from the fermentation broth of a strain of Pseudomonas syringae are developed.

Materials and methods The complete fermentation broth produced according to Example 1, typically 100 1 after harvest, was filtered through a Membralox1 ^ (0.45 μm) ceramic filter. The resulting solid suspension was extracted with an equal volume of acetone containing 0.1% TFA for 90 minutes. 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 complete broth, and loaded onto an Amberchrom CG 300sd (4 1) resin column packed in water. The column was initially washed with 0.2% acetic acid (pH 4-8) until the effluent showed pH 4.5 followed by 10 1 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 1), and 35% acetonitrile containing 0.2% acetic acid (8 1) with a flow rate of 400 ml / minute. Fractions 11-16 were combined (4.8 1), concentrated in vacuo to 100 ml and centrifuged. The supernatant was separated and subjected to chromatography on a column of Amberchrom CG 300sd (11) using a linear gradient of 25-35% acetonitrile containing 0.2% acetic acid (pH 4.8) with a flow rate of 50 ml / minute. The fractions 20-25 (1.2 1) were combined and subjected again to chromatography on a reversed phase column (NovaPak Cis, 6 μm, 40x300 mm, flow rate 40 ml / minute, linear gradient of 30-60% acetonitrile which contained 0.2% TFA) to produce 21 mg of a compound (89% purity by UV). The ESIMS data showed a possible peak [M + H] "am / z 1165.7 which is different from the known antifungal agents that have been found so far from P. syringae." The 1 H-NMR spectrum showed signals reminiscent of lipopeptide similar to pseudomycin but indicated the presence of more than one compound In order to obtain antifungal agent A from decapeptide 25-B1 with high purity for structural determination and antifungal activity, an additional broth from the 4x100 1 fermentation was processed as described above and in addition, The final purification was carried out on a reverse phase column [Rainin C? 8, 6 μm, 24x250 mm, 0.1% TFA-acetonitrile-methanol (8: 1: 1 to 4: 3.3) gradient elution for 60 minutes: (4: 3.3 to 10.45: 45) gradient elution by 30 minutes] to provide 42 mg of the antifungal agent A of the decapeptide 25-B1 (purity of 93% by UV).

Results and Conclusion HPLC methods similar to those used to provide other lipodepsidecapeptic antifungal agents resulted in the purification of antifungal agent A from decapeptide 25-B1 from the fermentation broth.

Example 3 - Determination of the Structure of Antifungal Agent A of 25-B1 Mass spectroscopy and NMR data determined the structure of an antifungal agent lipodepsidecapeptide, the antifungal agent A of the decapeptide 25-B1.

Methods and Results The molecular formula of the antifungal agent A of the decapeptide 25-B1 was determined by high resolution FABMS as CsaHasN ^ Oie [m / z 1165. 6581 for C52H89N14O16 (M + H) + D + 0. 9 ppm].

According to this formula the spectra of 13C and DEPT-NMR showed 50 different resonances, which included twelve carbonyl carbons, five olefinic carbons, four oxygenated sp3 carbons, eight carbons of typical amino acids, eighteen methylene carbons and six carbons. of methyl. Among these, one of the methyl carbon signals at d 16.7 and one of the methylene carbon signals at d 28.9 each constituted a group of degenerate carbons, thus explaining the total number of 52 carbons observed in the molecular formula. The data of the detailed analysis of aH-13C and 2D-NMR (DQCOSY.TOCSY, HMQC, HMBC and ROESY) made it possible to determine the structure of antifungal agent A of decapeptide 25-B1 (IA) and unambiguously assigned all the protons and carbons (Table 1) ÍA Table 1. Chemical Displacements of ^ "H? 3C-NMR of Antifungal Agent A of Decapeptide 25-Bl in DMSO-d6 The results of the 1H, DQCOSY and TOCSY spectra measured in DMSO-de at 35 ° C revealed the presence of spin systems for seven amino acid residues of common origin-two alanines, one arginine, one glutamine and three threonines , and three amino acids of less common origin - a dehydroalanine (Dha), a dehydroaminobutyric acid (Dhb) and a homoserin. Amino acid residues of less common origin, namely Dha, Dhb and homoserin, were identified by the crossed peaks observed in the TOCSY spectrum from amide protons that resonate ad 9.01 (s broad), 9.07 (s broad ) and 8.22 to protons that resonate ad 5.86, 5.61 (dc 106.4), 6.40 (dc 128.5), 1.61 (dc 12.9) and 4.22 (dc 51.4), 1.82, 1.76 (dc 34.0), 3.47 (dc 57.3), respectively . Consistent with this, the 13 C-NMR spectrum showed eight carbon signals bound to the proton for the saturated amino acid residues and two quaternary carbons for the unsaturated amino acids of Dha and Dhb. Of the twelve amide or ester carbonyls, nine were assigned to the eight saturated amino acid residues (two to glutamine), two (d 164.0 and 163.7 ppm) for Dha and Dhb. The remaining carbonyl group was assigned to the dodecanoyl side chain, the presence of which was discerned from the terminal methyl signal (dH 0.83 and 13.9) and 10 methylene signals in the 13 C-NMR spectrum (Table 1) . The molecular formula requires sixteen degrees of unsaturation. The ten amino acids and the dodecanoyl side chain explained fifteen sites of unsaturation indicating that the antifungal agent A of the decapeptide 25-B1 is a monocyclic decapeptide. The comparison of the chemical shift of the protons ß of the threonines (dH 4.94, 4.11 and 4.00) indicated that the proton that resonates at d 4.94 was linked to a carbinol which is modified to an ester or a lactone. That this was so and that the antifungal agent A of decapeptide 25-B1 is a depsipeptide, was evidenced by NMBC and ROESY data. These data also established the amino acid sequence and the location of the dodecanoyl side chain in the antifungal agent A of the decapeptide 25-B1. With the amino acid Arg as a starting point, the wide-range 1H-13C correlations observed in the HMBC spectrum between the amide proton and the carbonyl of the adjacent amino acid and / or carbon (see Reaction Scheme I below) established unambiguously 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-aC, Glu-NH / Thr-CO, Thr-NH / Ala-CO and Ala-NH / Thr-CO. Reaction Scheme I NBC (H? C) Correlations The Thr NH adjacent to Ala did not show a broad-range 1H-13C correlation to the carbonyl of the Arg residue, rather it showed a correlation to a carbonyl assigned to the dodecanoyl side chain. The absence of the Thr-NH / Arg-CO correlation and the presence of a correlation between Thr-β-H (dH 4.94, dc 70.5) / Arg-CO clearly established an ester bond between Thr-β-OH and Arg-COOH . Consistent with these assignments are the ROESY correlations that were observed between the protons of the amide and the protons a of the adjacent amino acid (see Reaction Scheme II below).

Reaction Scheme II Selected ROESY correlations Conclusions Compound antifungal agent A of decapeptide 25-B1 represents a novel class of lipodepsidecapeptide possessing several amino acid residues that are not present in any of the pseudomycins, syringomycins, syringotoxin and syringostatins produced by the different isolates of P. syringae. The new depsipeptide is composed of ten amino acids which is also a deviation from the pseudomycins and singomycins that possess only nine amino acid residues. Conversely, to the natural products of pseudomycin, the new depsipeptide does not include the chlorotreonin that is suspected to be the cause for irritation at the injection site of pharmaceutical formulations containing pseudomycin compounds.

Example 4 - Antifungal Activity of Antifungal Agent A of Decapeptide 25-B1 Antifungal studies were conducted using a microtiter broth dilution assay according to the guidelines of the National Committee for Clinical Laboratory Standards in 96-well microtiter plates. The Sabouraud and dextrose broth was adjusted to contain 2.5 x 104 conidia / ml. The test compound was dissolved in water and tested in fifty percent dilutions starting at the highest concentration of 20 μg / ml. The plates were incubated at 35 ° C for 48 hours. The results in Table 2 show the minimum inhibitory concentration (MIC) of the compound that completely inhibited development compared to untreated growth controls.

Table 2. Antifungal activity of 1 Organism MIC (μg / ml) Candida albicans 10 C. parapsilosis 10 Cryptococcus neoformans 1.25 Aspergillus fumigatus > 20 Histoplasma capsulatum 20 The presence or amount of one or more lipodepsidecapeptides of P. syringae, such as antifungal agent A of decapeptide 25-B1, can be determined by measurement of the antifungal activity of a preparation. The antifungal activity can be determined in vi tro by obtaining the minimum inhibitory concentration (MIC) of the preparation using a standard agar dilution test or a disk diffusion test. A preparation of one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of the decapeptide 25-B1 may be an extract from a cell culture, or a more purified mixture. A typical fungus used in the test of antifungal activity is C. albicans. The antifungal activity is considered significant when the test preparation causes areas of inhibition of diameter of 10-12 mm on agar plates seeded with Candida albicnas x 657.

Example 5 - Isolation, Characterization and Mutagenesis of Pseudomonas syringae Environmental isolates and mutants of P. syringae were produced and used in the production of antifungal agents.

Materials and methods Strains MSU 174 and MSU 16-H were isolated and characterized as 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 Pseudomonas syringae peptides possessing broad spectrum antifungal activity" J. Gen. Microbiology 137. 2857-2865 (1991); and Lamb et al., "Mutagenesis by transposon and labeling of fluorescent pseudomonas: antifungal production is necessary for the control of Dutch elm disease", Proc. Nati Acad. Sci. USA 84, 6447-6451 (1987). The descriptions of the references cited in this paragraph are incorporated herein by reference. Additional strains were derived from wild type mutants and mutants generated by transposon, by chemical mutagenesis. The strain subject to mutagenesis include MSU 174, MSU 16H, and 25-B1. The strain to be mutagenized was developed in a medium containing a potato product, then divided into the medium that includes 0, 1, 2, 4, 16 or 32 μm of the chemical mutagen 1-methyl-3-nitro-l- nitrosoguanidine (NTG or 'MNNG). These cells were then frozen for future classification and selection. The mutagenized cells were selected for the desirable development conditions and / or the production of one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of the decapeptide 25-B1. The chemically mutagenized cells of P. syringae, such as the mutagenized strain 25-B1, were thawed and diluted to 6 cells / ml in the N21SM medium (Table 3). This medium sometimes contained one or more components for selection, such as varying concentrations of phosphate. A volume of 50 μl of mutagenized cells was filled into a well of a 96-well round bottom microtiter plate for an average distribution of 0.3 cells / well. Typically, silicone oil was added to each well to minimize evaporation. The plates were incubated with shaking for 6 to 12 days at 25 ° C.

Table 3 - The Composition of the N21SM Medium After this incubation, an aliquot, typically 5 μl, was taken from each well and serially diluted (eg 1:56, 1:96, 1: 320, 1: 686, and / or 1: 1715) and evaluated for the activity against Candida albi cans in a liquid microtitre plate bioassay. The plates were incubated at 37 ° C overnight and the wells were qualified for the inhibition of C. albicans development. Suitable strains were collected, inoculated in CSM medium (Table 4), and developed for 1 to 3 days at 25 ° C.

Table 4. Complete Streptomies Medium (CSM) Component Concentration (g / 1) Glucose 5 Maltose 4 Soy Broth Difco Trifold 30 Difco Yeast Extract 30 MgS04 »7H20 2 No pH adjustment The selected strains were preserved and inoculated into fermentation bottles containing 13 ml of NN21SM medium and developed for approximately 66 hours at 25 ° C. An aliquot of this fermentation was removed, extracted for one hour by a volume of acetonitrile equal to the volume of the aliquot, centrifuged and decanted for HPLC analysis of one or more of the lipodepsidecapeptides of P. syringae, such as the antifungal agent A of decapeptide 25-B1, as described in Examples 1-3. Strains that produce one or more lipodepsidecapeptides from P. syringae, such as antifungal agent A from decapeptide 25-B1, were re-isolated, referenced and prepared for development on a larger scale.

Results Strains that show production of one or more of P. syringae of lipodepsidecapeptides, such as antifungal agent A of decapeptide 25-B1, were produced using the methods described above. conclusion The methods and selection criteria described herein are effective for producing strains of P. syringae that grow on minimal medium and produce one or more lipodepsidecapeptides of P. syringae, such as the antifungal agent A of decapeptide 25-B1.

Example 6 - Formulations including the lipodepsidecapeptide of P. syringae.

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 lipodepside-peptide of P. syringae, or a pharmaceutically acceptable salt thereof.

Formulation I Hard gelatin capsules are prepared using the following ingredients: Ingredient Quantity (mg / capsule) Active ingredient 250 Dried starch 200 Magnesium stearate 10 Total 460 mg Formulation 2 A tablet was prepared using the following ingredients. The components are mixed and compressed to form tablets each weighing 665 mg.

Ingredient Quantity (mg / capsule) Active ingredient 250 Cellulose, microcrystalline 400 Silicon dioxide, smoked 10 Stearic acid 5 Total 665 mg Formulation 3 An aerosol solution containing the following components is prepared. The active compound is mixed with ethanol and the mixture is 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 vessel and diluted with the rest of the propellant. The valve units are then adjusted 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 the active ingredient are prepared as follows: Active ingredient 60 mg Microcrystalline cellulose 45 mg Polyvinylpyrrolidone (as 4 mg a 10% solution in water) Carboxymethyl sodium starch 4.5 mg Magnesium stearate 0.5 mg Talc 1 mg Total 150 mg The active ingredient, starch and cellulose are passed through a No. 45 mesh American screen and mixed thoroughly. The aqueous solution containing polyvinyl pyrrolidone is mixed with the resulting powder, and the mixture is then passed through a No. 14 mesh North American sieve. The granules thus produced are dried at 50 ° C, and passed through a No. 18 North American mesh screen. The carboxymethyl starch of sodium, magnesium stearate and talc, previously passed through the North American mesh screen No. 60 are then added to the granules which, after mixing, are compressed on a tabletting machine to produce tablets weighing each 150 mg.

Formulation 5 The following are capsules each containing 80 mg of the active ingredient: Active ingredient 80 mg Starch 59 mg Microcrystalline cellulose 59 mg Magnesium stearate 2 mg Total 200 mg The active ingredient, cellulose, starch and magnesium stearate are mixed, passed through a No. 45 mesh North American sieve and filled between the hard gelatin capsules in amounts of 200 mg.

Formulation 6 The following are prepared as suppositories each containing 225 mg of the active ingredient: Active Ingredient 225 mg Fatty Acid Glyceride 2,000 mg Saturated Total 2,225 mg The active ingredient is passed through a No. 60 mesh American sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat required. The mixture is then emptied into a suppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7 Suspensions containing each 50 mg of the active ingredient per 5 ml dose are prepared as follows: Active ingredient 50 mg Sodium carboxymethyl cellulose 50 mg Syrup 1.25 ml Benzoic acid solution 0.10 ml Taste q.s. Color c.s. Purified Water up to Total 5 ml The active ingredient is passed through a No. 45 mesh North American sieve and mixed with the sodium carboxymethyl cellulose and the syrup to form a smooth paste. The benzoic acid solution, flavor and color is diluted with a portion of water and added, with stirring. Sufficient water is then added to produce the required volume.

Formulation 8 An intravenous formulation can be prepared as follows. The solution of these ingredients is generally administered intravenously to a subject at a rate of 1 ml per minute.

Active ingredient 100 mg Isotonic saline 1,000 mg The invention has been described with reference to various specific and preferred modalities and techniques.

However, it should be understood that many modifications and variations may be made while remaining within the spirit and scope of the invention.

LIST OF SEQUENCES < HQ > Eli Lilly and Company < 120 > Antifungal agents isolated from Syringae Pseudomonas < 130 > X-11015 Sequence Listing < 140 > < 141 > < 150 > 60 / 129,446 < 151 > 1999-04-15 < 160 > 1 < 170 > PatentIn Ver. 2.1 < 210 > 1 < 211 > 10 < 212 > PRT < 213 > Pseudomonas syringae < 220 > < 223 > Xaa in position 5 is homoserin. < 220 > < 223 > Xaa in position 6 is dehydroaminobutyric acid < 220 > < 223 > Xaa in position 8 is dehydroalanine < 440 > 1 Thr Ala Thr Gln Xaa Xaa Ala Xaa Thr Arg 1 5 10 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (36)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A peptide decapeptide of isolated P. syringae, or a pharmaceutically acceptable salt, ester or hydrate thereof, characterized as comprising a depsidecapeptide ring wherein the ring comprises arginine, threonine, homoserine, desidroaminobutyric acid, and dehydroalanine, and a lactone is formed at from a carboxyl group of arginine and a hydroxyl group of threonine. 2. The depsidecapeptide of P. syringae according to claim 1, characterized in that the depsidecapeptide ring has the sequence: Thr-Ala-Thr-Gln-Hse-Dhb-Ala-Dha-Thr-Arg (SEQ ID No. 1) . 3. The depsidecapeptide of P. syringae according to claim 1, characterized in that the depsidecapeptide of P. syringae is a lipodepsidecapeptide of P. syringae. Four . The depside-peptide of P. syringae according to claim 3, characterized in that the lipodepside-peptide of P. syringae comprises a fatty acid portion coupled to an amino group of the threonine by an amide bond. 5. The depside peptide of P. syringae according to claim 4, characterized in that the fatty acid portion is a decanoic acid portion, a decanoic acid portion substituted with one or two hydroxyl groups, a portion of dodecanoic acid, a portion of dodecanoic acid substituted with one or two hydroxyl groups, a portion of tetradecanoic acid or a portion of tetradecanoic acid substituted with one or two hydroxyl groups. 6. The depsidepeptide of P. syringae according to claim 5, characterized in that the fatty acid portion is a portion of n-dodecanoic acid. 7. The depsidecapeptide of P. syringae according to claim 3, characterized in that the lipodepsidecapeptide of P. syringae is represented by the formula: where R is a lipophilic moiety, or a pharmaceutically acceptable salt, ester or hydrate thereof. 8. The depsidecapeptide of P. syringae according to claim 7, characterized in that the lipophilic portion is selected from the group consisting of alkyl of 9 to 15 carbon atoms, hydroxyalkyl of 9 to 15 carbon atoms, dihydroxyalkyl of 9 to 15 carbon atoms, alkenyl of 9 to 15 carbon atoms, hydroxyalkenyl of 9 to 15 carbon atoms, and dihydroxyalkenyl of 9 to 15 carbon atoms. 9. The depsidecapeptide of P. syringae according to claim 8, characterized in that the lipophilic portion is alkyl of 9 to 15 carbon atoms. 10. A depside peptide of isolated P. syringae, characterized in that it has the formula: or a pharmaceutically acceptable salt, ester or hydrate thereof. 11. A method for inhibiting fungal activity, characterized in that it comprises contacting a fungus with a depside-peptide of P. syringae isolated in accordance with claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The method according to claim 11, characterized in that the fungus comprises Candida parapsilosis, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum. 13. A method for reducing the symptoms of a fungal infection in a patient in need thereof, characterized in that the method comprises: administering to the patient an effective amount of a composition comprising a depside-peptide of P. syringae isolated in accordance with claim 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. The method according to claim 13, characterized in that the reduction of symptoms comprises the reduction of a burden of a fungal infection. The method according to claim 13, characterized in that the infection by fungi comprises the infection by Candida parapsilosis, Candida albicans, Cryptococcus neoformans, or Histoplasma capsulatum. The method according to claim 13, characterized in that it also comprises the steps of: determining the need to administer the depsidecapeptide of P. syringae isolated; and periodically check the patient for relief of the symptoms of fungal infection. The method according to claim 13, characterized in that the administration comprises the parenteral administration of about 1 to about 3 times per day from about 0.1 to about 5 mg / kg of the depsidecapeptide of P. syringae isolated. 18. The method according to claim 13, characterized in that reducing the symptoms of a fungal infection comprises reducing fever and increasing the general well-being of the patient. 19. The use of a depside-peptide of P. syringae in accordance with 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 according to 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 depsidecapeptides of P. syringae, characterized in that it comprises the steps of: cultivating 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 depsidecapeptides of P. syringae are produced at a concentration of at least about 10 μg / ml; and the recovery of one or more depsidecapeptides from P. syringae, or a pharmaceutically acceptable salt, ester or hydrate thereof, from the culture. 22. The method according to claim 21, characterized in that the amino acid comprises glutamic acid, glycine, histidine or a combination thereof. 23. The method according to claim 22, characterized in that the amino acid comprises glycine. 24. The method according to claim 22, characterized in that the nutrient medium further comprises soluble starch, yeast extract, or a combination thereof and a lipid. 25. The method according to claim 24, characterized in that the nutrient medium comprises a product of potato and a lipid selected from the group consisting of soybean oil, fatty acids and fatty acid esters. 26. The method according to claim 25, characterized in that the potato product comprises potato dextrose broth, crushed potato mixture, potato dextrin, potato protein or a combination thereof. 27. The method according to claim 21, characterized in that during the culture step the concentration of dissolved oxygen is contained at about 5% to 30%. 28. The method according to claim 21, characterized in that the culture step further comprises feeding glucose, ammonium hydroxide or a combination thereof. 29. The method according to claim 21, characterized in that the depside-peptide of P. syringae produced is a depside-peptide of P. syringae according to claims 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The method according to claim 21, characterized in that the Pseudomonas syringae comprises a strain derived from the strain MSU 16H, MSU 174, or MSU 206. 31. The method according to claim 21, characterized in that the Pseudomonas syringae comprise strain MSU 16H, strain 25-B1, strain 67H1, or strain 7H9-1. 32. The method according to claim 31, characterized in that the Pseudomonas syringae comprises strain 25-B1. 33. A peptide decapeptide of P. syringae according to claim 7, characterized in that it is prepared by the methods according to claims 21, 22, 23, 24, 25, 26, 27, 28, 30, 31 or 32. 34. A method for the treatment or prevention of the development of fungi in a plant, characterized in that it comprises contacting a fungus with a depside-peptide of P. syringae in accordance with claims 1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10. 35. The method according to claim 33, characterized in that 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 according to claim 34, characterized in that the fungus is V. albo-atrum, Rhizoctonia solani or F. oxysporum.