JP2002542258A - Antifungal substance isolated from Pseudomonas syringae - Google Patents

Abstract

(57) Abstract The present invention relates to P. syringae depsideca peptides, methods for producing such peptides, and methods using the antifungal activity of these peptides. P. syringae depsidecapeptide includes a compound of formula (a), wherein R is a lipophilic moiety, or a pharmaceutically acceptable salt, ester or hydrate thereof. Embedded image

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to P. syringae depsideca peptides, methods for producing such peptides, and methods utilizing the antifungal activity of these peptides.

BACKGROUND Fungal infections are a significant cause of disease, disrupted quality of life and death in humans, especially immunocompromised patients. The incidence of fungal infections in humans has increased significantly over the past two decades. This is partly due to organ transplantation, cancer chemotherapy, AIDS,
It is due to an increasing number of humans whose immune system is weakened or destroyed by age and other similar disorders or symptoms. Such patients are prevalent in people, but are susceptible to fungal pathogens that have been eliminated by checks by the functional immune system. These pathogens are difficult to control because some existing antifungal agents are highly toxic or only inhibit fungal activity. For example, polyenes are bactericidal but toxic; whereas azoles are much less toxic but only fungistatic. More importantly, azole and polyene resistant strains of Candida have recently been reported that greatly limit the choice of treatment for such strains.

[0003] Pseudomonas syringae produces several classes of antifungal or antibiotics, such as the lipodepsinona peptides pseudomycin, syringomycin, syringotoxin and syringostatin. Mutants produced by natural strains of P. syringae and transposons produce these lipodepsinona peptides. Some pseudomycins,
Syringomycin and other lipodepsipeptide antifungal agents have been isolated, chemically characterized and shown to have a wide range of antifungal activities, including activity against important fungal pathogens in both humans and plants . Pseudomycin, syringomycin, syringotoxin and syringostatin represent a family of structurally distinct antifungal compounds.

[0004] None of the P. syringae lipodepsinnonapeptides have been introduced to the market for antifungal therapy. The discovery of undesired side effects, formulation, expansion of production, and other development issues has led to the discovery of a full spectrum of fungal infections affecting animals, humans and plants.
. Syringae has greatly hindered the use of lipodepsinnonapeptide. Antifungal agents and animals that can be used against infections that cannot be treated by existing antifungal agents
There is still a need for applications for infections in humans or plants.

SUMMARY OF THE INVENTION The present invention relates to an unusual amino acid, homoserine (H), produced by P. syringae.
se), dehydroaminobutyric acid (Dhb) and dehydroalanine (Dha)
Depsidecapeptides are provided that are part of the depsidecapeptide ring. More specifically, the P. syringae depsideca peptide has an amino acid, arginine, threonine, homoserine, dehydroaminobutyric acid, and dehydroalanine, and has a lactone bond formed from a carboxyl group of arginine and a hydroxyl group of threonine. And a depsidecapeptide ring. When isolated from P. syringae, the depsidecapeptide is a lipodepsidecapeptide and the cyclic peptide is coupled to a lipophilic moiety. Typically, the lipophilic moiety is a fatty acid moiety coupled to the threonine amino group by an amide bond. Preferably, the fatty acid moiety is an n-dodecanoic acid moiety
It is. The lipodepsidecapeptide is that represented by Formula I.

Embedded imageWherein R is a lipophilic moiety. The lipophilic moiety contains C₉-C_FifteenAlkyl, C₉-C_FifteenHydroxyal
Kill, C₉-C_FifteenDihydroxyalkyl, C₉-C_FifteenAlkenyl, C₉-C _Fifteen Hydroxyalkenyl or C₉-C_FifteenContains dihydroxyalkenyl
You. Preferably, the lipophilic moiety is C₁₁Alkyl. Alkyl, hide
Loxyalkyl, dihydroxyalkyl, alkenyl, hydroxyalkenyl
Or a dihydroxyalkenyl group may be branched or unbranched
. Preferably, the amino acid sequence of the depsidecapeptide ring is threonine-alanine
-Threonine-glutamine-homoserine-dehydroaminobutyric acid-alanine-dehi
Doloalanine-threonine-arginine (herein referred to as "25-B1 decapeptide
Or "Thr-Ala-Thr-Gln-Xaa-Xaa-Ala-Xaa-Thr
-Arg (SEQ ID NO: 1) "). As used herein, the term "25-B
1-Decapeptide antifungal substance (antifungal agent) A "is a preferred amino acid sequence and amino acid sequence.
C having column number 1 and R is not branched₁₁Alkyl (ie, R =-(
CH₂)₁₀CH₃) Represents a particular depsidecapeptide.

The present invention also provides for inhibiting fungal activity in a patient in need thereof,
Alternatively, the present invention relates to a method using P. syringae depsidecapeptide for reducing symptoms of fungal infection. Such methods can kill the fungus, reduce the suffering of the fungal infection, lower the fever, and increase the patient's overall well-being. Consequently, the P. syringae depsidecapeptide can be used in the manufacture of a medicament for treating a patient described herein. The method and drug of the present invention may be a fungus, e.g.,
Candida parapsilosis, Candida albicans, Cryptococcus neoformans or
Effective against Histoplasma capsulatum.

[0007] The present invention relates to the aforementioned P. syri comprising an antifungal substance, for example a 25-B1 decapeptide.
ngae Depsidecapeptide is provided using a microorganism in a method of producing the peptide. This method involves using Pseudomon in a medium containing three or fewer amino acids.
culturing as syringae and recovering one or more P. syringae depsidecapeptides from the culture. In one embodiment, the P. syringae culture has a pH of about 4
~ 6.5 in a medium comprising glycine and lipids, potato products, or combinations thereof, wherein one or more P. syringae depsidecapeptides is produced at a concentration of at least about 10 μg / mL. . In addition, the present invention provides a P. syringae depsidecapeptide produced by the above method.

The present invention also provides a method of treating or preventing fungal growth in a plant,
A method is provided by contacting a fungus with one or more of the above P. syringae depsidecapeptides.

DETAILED DESCRIPTION Lipodepsidecapeptide antifungal substance As used herein, "lipodepsidecapeptide antifungal substance" means a cyclic decapeptide ring closed by a lactone group, Represents an antifungal substance having a modified hydrophobic group, for example, a fatty acid moiety. Lipodepsidecapeptide antifungal substance
Produced by Pseudomonas syringae. A representative compound of this class, 25-B1 decapeptide antifungal A, was purified and its structure determined.
As used herein, the term `` P. syringae lipodepsidecapeptide '' refers to P. syringae.
lipodepsidecapeptide antifungal produced by syringae, including 25-B1 decapeptide antifungal A and related analogs.

[0010] The P. syringae lipodepsidecapeptide shares several structural features. For example, each of these antifungal agents contains an unusual amino acid, homoserine (Hse), dehydroaminobutyric acid (Dhb) and dehydroalanine (Dha) as part of the depsidecapeptide ring. In each P. syringae lipodepsidecapeptide, the carboxyl group of the arginine residue is linked to the hydroxyl group of the N-terminal threonine,
A lactone is formed, which closes the depsidecapeptide ring. The sequence of the depsidecapeptide ring of P. syringae lipodepsidecapeptide may be as follows: Thr-Xaa-Xbb-Xcc-Hse-Dhb-Xdd-Dha-Xee-
Arg [where Xaa, Xbb, Xcc, Xdd and Xee are each independently a naturally occurring amino acid]. Unlike pseudomycin natural products, the lipodepsidecapeptides of the present invention do not contain chlorothreonine, which is suspected of causing inflammation at the site of injection of pharmaceutical formulations containing pseudomycin compounds.

[0011] The depsidecapeptide ring is linked to a lipophilic moiety, eg, a fatty acid, via an amide bond to the amino group of the N-terminal threonine. Fatty acids generally contain 10, 12, 14 or 16 carbons with typically 0, 1 or 2 hydroxyl groups. Fatty acids may be branched or unbranched, and at least one
May be included. Preferred fatty acid moieties include an n-decanoic acid moiety,
N-decanoic acid moieties substituted with one or two hydroxyl groups, n-dodecanoic acid moieties, n-dodecanoic acid moieties substituted with one or two hydroxyl groups, n
N substituted with a tetradecanoic acid moiety or one or two hydroxyl groups
-A tetradecanoic acid moiety is included.

25-B1 Decapeptide Antifungal Substance As used herein, “25-B1 decapeptide antifungal substance” refers to bacteria, Ps
Represents one or more members of the family of antifungal substances isolated from eudomonas syringae. The 25-B1 decapeptide antifungal is a P. syringae
It is a lipodepsidecapeptide. In particular, the 25-B1 decapeptide antifungal is a P. syringae lipodepsidecapeptide having a depsidecapeptide ring having the following sequence: Thr-Ala-Thr-Gln-Hse-Dhb- Ala-Dha-Thr-
Arg (SEQ ID NO: 1). Each 25-B1 decapeptide antifungal has the same cyclic peptide nucleus, but different hydrophobic side chains attached to this nucleus. 25-B1 decapeptide antifungal is 25
-Contains B1 decapeptide antifungal substance A.

[0013] 25-B1 decapeptide antifungal substances include fatty acids linked via an amide bond to the amino group of the N-terminal threonine. The fatty acid portion of the 25-B1 decapeptide antifungal A is an n-dodecanoic acid portion.

Biological activity of P. syringae lipodepsidecapeptide P. syringae lipodepsidecapeptide, such as the 25-B1 decapeptide antifungal substance A, is useful for a variety of fungi, such as plant and animal fungi. It has several biological activities, including biocidal and inhibitory activities of pathogens. In particular, 25-B1 decapeptide antifungal agents are antifungal agents active against fungi that cause opportunistic infections in immunocompromised individuals. These fungi include Cryptococcus neoformans
, Histoplasma capsulatum and various Candida, including C. parapsilosis and C. albicans.

Pseudomonas syringae Pseudomonas syringae generally comprises a wide range of plant-related bacteria. P. syri
Some ngae are phytopathogenic, while others are only weakly pathogenic or germs. Many different isolates of P. syringae produce one or more cytotoxic agents that aid the survival of fungi and other bacteria in the wild, which must compete with the bacteria. Cytotoxic substances produced by P. syringae include antifungal substances, such as the 25-B1 decapeptide antifungal substance A.
syringae lipodepsidecapeptide, pseudomycin, syringomycin, syringotoxin and syringostatin.

[0016] Isolates of P. syringae that produce one or more pseudomycins, syringomycins, syringotoxins, syringostatins are well known to those of skill in the art. This mutant, MSU 16H (ATCC 67028), created by wild-type strain MSU174 and transposon mutagenesis, was
G. Strobel et al, from U.S. Pat.
ison et al., "Pseudomycins, a family of novel peptides from Pseudomonas
syringae possessing broad-spectrum antifungal activity, "J. Gen. Microbi
137, 2857-2865 (1991); and Lamb et al., "Transposon mutagenesis
and tagging of fluorescent pseudomonas: Antimycotic production is neces
sary for control of Dutch elm disease, "Proc. Natl. Acad. Sci. USA 84, 6
447-6451 (1987). The method of culturing various P. syringae strains and their use in the production of antifungal substances, such as pseudomycins, is also described in Matthew D. Hilton, et al US Patent Application No. PCT / US0 filed on the same day as the present application.
0/08728 (title "Pseudomycin Production By Pseudomonas Syringae"
) And described below. The disclosure content of the references cited in this paragraph is incorporated herein by reference.

From the sources of the environment, including plants such as barley, citrus and hasti plants, and one or more P. sy from forest cover, soil, water, air and dust
P. syringae strains suitable for the production of the ringae lipodepsidecapeptide, for example the 25-B1 decapeptide antifungal A, can be isolated. The present invention provides about 10 μg / mL
A larger amount, preferably in an amount of at least about 10 μg / mL to about 50 μg / mL,
One or more P. syringae lipodepsidecapeptides, such as 25-B
Includes strains, isolates and biologically purified cultures of P. syringae that produce 1-decapeptide antifungal A. Preferably, the cultivation of the biologically purified microorganism is carried out by Pseudomonas syringae strain MSU 16H, 25-B1, 67H1, 7H9.
-1 or pseudomycin-producing mutants, variants, isolates or recombinants of these strains. Culture of MSU 16H is from Montana State University (
Bozeman, Montana, USA) and the American Type Culture Collection (Par
klawn Drive, Rockville, MD, USA) available from accession number ATCC 67028.

[0018] One or more P. syringae lipodepsidecapeptides, such as 25-
Suitable strains of P. syringae for the production of B1 decapeptide antifungal A include plants,
It can be isolated from surrounding sources including, for example, barley plants, citrus plants, and hasidium plants, and from sources such as soil, water, air, and dust. Preferred strains are those isolated from plants. These environmental isolates of P. syringae can be referred to as wild-type. As used herein, “wild-type” refers to P. syrin
Means the dominant genotype that occurs naturally in the normal population of gae (ie, a strain or isolate of P. syringae that has been found in nature and has not been produced by laboratory procedures). I do. As with other organisms, the characteristics of the cultures producing P. syringae, P. syringae strains used in the present invention, producing P. syringae, such as MSU 174, MSU 16H, MSU 206, 25-B1 and 7H9 are altered Receive. Thus, progeny of these strains, for example, recombinants, mutants and variants, can be obtained by methods well known to those skilled in the art.

The mutant strain of P. syringae is also suitable for the production of one or more P. syringae lipodepsidecapeptides, such as the 25-B1 decapeptide antifungal substance A. As used herein, "mutant" means a sudden heritable change in the phenotype of a strain, which is either spontaneous or a known mutation involving radiation and various chemicals. Can be induced by a substance that causes
The mutant P. syringae of the present invention employs various mutagenizing agents, including radiation, such as ultraviolet and x-rays; chemical mutagens, site-directed mutagenesis, and transposon-mediated mutagenesis. Can be created. Examples of chemical mutagens are ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-nitro-N'-nitrosoguanine (NTG) and nitrous acid.

In accordance with the present invention, one or more P. syringae lipodepsidecapeptides, such as P- suitable for producing 25-B1 decapeptide antifungal substance A
syringae overproduces the bacterium with one or more P. syringae lipodepsidecapeptides, such as the 25-B1 decapeptide antifungal substance A,
Alternatively, an amount of mutation effective to create a mutant that produces one or more P. syringae lipodepsidecapeptides, eg, 25-B1 decapeptide antifungal A, under favorable growth conditions. Can be created by treating with a substance that causes Although the type and amount of mutagen used may vary, the preferred method is to serially dilute NTG to a level in the range of about 1 to about 100 μg / mL. Preferred mutants of the invention are those that overproduce the 25-B1 decapeptide antifungal A and grow on minimal medium. The mutant preferably has at least about 10 μg / mL to about 50 μg / mL of P. syri
ngae lipodepsidecapeptide, eg 25-B1 decapeptide antifungal substance A
Overproduction.

[0021] Environmental isolates, mutants and other desirable strains of P. syringae are grown
A choice can be made regarding the desired characteristics of the culture medium, nutrient sources, carbon sources, culture conditions and amino acid requirements. Preferably, a strain of P. syringae that produces a P. syringae lipodepsidecapeptide, eg, a 25-B1 decapeptide antifungal substance A, is selected for growth on a minimally defined medium. Preferred strains are glycine and
Optionally, when cultured in a medium containing lipids, potato products or both, they exhibit a characteristic production of one or more P. syringae lipodepsidecapeptides, such as the 25-B1 decapeptide antifungal substance A. .

Recombinant strains can be developed by transforming P. syringae strains using techniques well known to those skilled in the art. Through the use of recombinant technology, P. syringae strains can be transformed to express various gene products in addition to the antibiotics produced by these strains. For example, the strain can be transformed with a recombinant vector that confers resistance to antibiotics against which the strain is normally sensitive. The transformant thus obtained is not only a P. syringae lipodepsidecapeptide, for example, a 25-B1 decapeptide antifungal substance A, but also a resistance-imparting agent that allows selection of transformants from wild-type cells. Will produce enzymes. In addition, similar techniques can be used to modify the current strain to introduce multiple copies of the endogenous lipodepsidecapeptide biosynthesis gene to achieve higher lipodepsidecapeptide yields. P. syringae strains 25-B1, 67H1 and 7H that achieve characteristic lipodepsidecapeptide production
The progeny of 9-1, ie, natural and derivatized mutants, mutants and recombinants, are part of the present invention.

Culture of Pseudomonas syringae The aqueous nutrient medium described herein refers to a water-based composition containing the inorganic and organic compounds and salts thereof required for the growth of the bacteria used in the present invention. Preferred nutrient media contain an effective amount of three or less amino acids, preferably glutamic acid, glycine, histidine or a combination thereof. 1
In one aspect, the medium comprises an effective amount of glycine and, optionally, one or more potato products and lipids. Glycine can be provided as a single amino acid or as part of a mixture of amino acids such as hydrolyzed proteins. Suitable lipids include soybean oil, fatty acids or fatty acid esters. Suitable potato products include potato dextrose broth, potato dextrin, potato protein or commercially available mashed potato mix. Preferred inorganic nutrient medium, typically a mixture of salt used in cell culture and fermentation, such as Czapek mineral salts solution (containing KCl, over MgSO ₄ and FeSO ₄₎ are included. The organic compounds in the nutrient medium preferably include glucose, and may optionally include soluble starch, and may also include other similar organic compounds. The pH of the medium is preferably in the range of about 4-6.5, more preferably in the range of about 4.5-5.7, and most preferably about 5.2.

The amount of each component in the nutrient broth is typically not critical for bacterial growth or production of the P. syringae lipodepsidecapeptide, eg, the 25-B1 decapeptide antifungal A, Certain levels of nutrients are advantageous. Preferred amounts of glycine are from about 0.1 g / L to about 10 g / L, more preferably from about 0.3 g / L to about 3 g.
/ L, most preferably about 1 g / L. Preferred amounts of lipids are from about 1 g / L to about 1
0 g / L oily product, such as soybean oil, more preferably about 0.5 g / L
~ 2 g / L soybean oil. Preferred amounts of fatty acids or fatty acid esters are from about 0.5 g / L to about 5 g / L. Preferred amounts of potato products are from about 12 g / L to
About 36 g / L, preferably about 24 g / L potato dextrose broth; about 5 g
/ L to about 50 g / L, preferably about 30 g / L, commercially available mashed potato mix; about 1 g / L to about 30 g / L, preferably about 20 g / L potato dextrin;
And / or from about 1 g / L to about 10 g / L, preferably about 4 g / L. Preferred nutrient media include minerals, preferably about 0.02 to about 2 g.
/ L, more preferably about 0.2 g / L KCl; about 0.02 to about 2 g / L, more preferably about 0.2 g / L MgSO _4, preferably MgSO ₄ · _7H 2 O; and from about 0 .4～ about 40 mg / L, more preferably from about 4mg / L FeSO _4, preferably includes a FeSO ₄ · 7H ₂ O. Soluble starch, if present, is about 0
. 5 to about 50 g / L, more preferably about 5 g / L. Glucose is preferably present at about 2 to about 80 g / L, more preferably at about 20 g / L.

[0025] P. syringae is typically cultured in the described media under controlled or regulated pH and temperature conditions. Range from about 15 ° C to about 35 ° C, preferably from about 20 ° C to about 30 ° C
At a temperature of about 25 ° C., more preferably, P. syringae grows and produces one or more cytotoxic agents. P. syringae grows at a pH in the range of about 4 to about 9, more preferably in the range of about 4 to about 6, and most preferably in the range of about 4.5 to about 5.5, and has one or more cytotoxicities. Produce substances. Typically, growth of P. syringae does not occur at temperatures above 37 ° C. or below 10 ° C., or at a pH above about 9 or below about 4.

Method for producing P. syringae lipodepsidecapeptide One or more P. syring from wild-type or mutant strains of P. syringae
The organism is cultured with stirring in an aqueous nutrient medium containing an effective amount of three or less amino acids to produce ae lipodepsidecapeptide, eg, 25-B1 decapeptide antifungal A. The three or less amino acids are preferably glutamic acid, glycine, histidine or a combination thereof.
In one preferred embodiment, the amino acids comprise glycine and, optionally, one or more potato products and lipids. The culture is performed under conditions effective for the growth of P. syringae and the production of the desired P. syringae lipodepsidecapeptide, for example, 25-B1 decapeptide antifungal substance A. Effective conditions include a temperature of about 22C to about 27C and a period of about 36 hours to about 96 hours. When cultured in a medium as described herein, P. syringae can grow at a cell density of about 10-15 g / L dry weight, with a total amount of at least about 10 μg / mL, preferably at least about 50 μg / mL. P. syringae lipodepsidecapeptide, eg 2
A 5-B1 decapeptide antifungal substance A can be produced.

Controlling the oxygen concentration in the medium during the culture of P. syringae is advantageous for the production of P. syringae lipodepsidecapeptide, for example, the 25-B1 decapeptide antifungal substance A. Preferably, the oxygen level is maintained at about 5% to about 50% saturation, more preferably at about 30% saturation. Sparging with air, pure oxygen, or a gas mixture containing oxygen can regulate the oxygen concentration in the medium. Further, the oxygen transport rate can be adjusted by adjusting the stirring speed.

Controlling the pH of the medium during the culture of P. syringae is advantageous for the production of P. syringae lipodepsidecapeptide, for example, the 25-B1 decapeptide antifungal substance A. The pH of the culture medium can be maintained below about 6 and above about 4.

P. syringae can produce a P. syringae lipodepsidecapeptide, for example, a 25-B1 decapeptide antifungal substance A, when cultured by a batch culture method. However, a fed-batch or semi-continuous feed of glucose and, optionally, an acid or base to control pH, such as ammonium hydroxide, is a P. syringae lipodepsidecapeptide, such as a 25-B1 decapeptide antifungal A Increase production.
P. syringae lipodepsidecapeptide by P. syringae, eg 25-B1
Production of the decapeptide antifungal substance A can be further enhanced by using a continuous culture method in which glucose and, optionally, an acid or base to control pH, such as ammonium hydroxide, are automatically fed. . pH is about 5
Preferably, it is maintained at about 5.4, more preferably at about 5.0 to about 5.2.

The selection of the P. syringae strain is based on the amount of P. syringae lipodepsidecapeptide, eg, 25-B1 decapeptide antifungal substance A, produced by culturing under the conditions described herein. And distribution. For example, strain 25 B1 can produce the predominant 25-B1 decapeptide antifungal A.

The cyclic decapeptide nucleus of P. syringae lipodepsidecapeptide can be prepared by cleaving a lipophilic moiety, for example, by deacylation. Methods for cleavage and deacylation, such as the use of deacylase enzymes, are well known to those skilled in the art.

Formulation and antifungal activity of P. syringae lipodepsidecapeptide A P. syringae lipodepsidecapeptide, such as the 25-B1 decapeptide antifungal substance A, shows activity in vitro and in vivo, Useful to fight infections or fungal skin infections. Accordingly, the present invention provides a method of inhibiting fungal activity comprising contacting a fungus with a P. syringae lipodepsidecapeptide, for example, a 25-B1 decapeptide antifungal substance A or a pharmaceutically acceptable salt thereof. I do. Preferred methods include various fungi such as Cryptococcus neoformans, Histopla
Inhibiting the growth or activity of sma capsulatum and Candida species, including C. parapsilosis and C. albicans. As used herein, "contacting" a compound of the present invention with a parasite or fungus means binding or linking, or making explicit contact or contact with, a parasite or fungus. . However, the term contact does not include any inhibitory mechanisms.

The present invention further provides an effective amount of a P. syringae lipodepsidecapeptide such as 2
Provided is a method for treating a fungal infection comprising administering a 5-B1 decapeptide antifungal substance or a pharmaceutically acceptable salt thereof to a host in need of such treatment.
Preferred methods include various fungi such as Cryptococcus neoformans, Histoplasma
a Treatment of infections with a capsulatum and strains of Candida, including C. parapsilosis and C. albicans. When administered an effective antifungal amount, the formulation of one or more P. syringae lipodepsidecapeptides, such as the 25-B1 decapeptide antifungal substance A, reduces the suffering of fungal infection and reduces Can be reduced, resulting in elimination of fungal infections.

[0034] One or more P. syringae lipodepsidecapeptides, such as 25-
Some patients in need of antifungal treatment with a formulation of the B1 decapeptide antifungal A have severe infections, such as high fever, and can be intensive or emergency treatment. Various fungi cause serious infections. For example, Candida spp. Causes mucosal and severe systemic infections and can exist as azole or polyene resistant strains. Aspergillus causes life-threatening systemic infections. Cryptococcus is the cause of meningitis. Such severe fungal infections occur in immunocompromised patients, for example, who have received organ or bone marrow transplants, have received chemotherapy for cancer, have recovered from major surgery, or have HIV infection. obtain. For such patients, antifungal treatments typically involve one or more P. syringae lipodepsidecapeptides to stop or retard infection over a period of days. For example, intravenous administration of a formulation of 25-B1 decapeptide (antifungal substance).

With respect to antifungal activity, the term “effective amount” refers to an amount of a compound of the present invention that can inhibit fungal growth or activity, or reduce the symptoms of a fungal infection. For most fungal infections, reducing symptoms of the infection includes lowering fever, restoring consciousness, and increasing patient well-being. Preferably, the symptoms are reduced by killing the fungus to eliminate the infection, or bringing the infection to a level that is tolerated by the patient or controlled by the patient's immune system. As used herein, “inhibition” means inhibition of fungal activity, including cessation, delay or prophylactic interference or prevention of growth or any attendant characteristics and consequences resulting from the presence of the fungus.

Typically, an effective amount of a composition that inhibits a fungal infection is administered to a patient in need thereof (human or other animals, including mammals and birds such as, for example, cats, horses and cows). I do. The dosage will vary depending on factors such as the nature and severity of the infection, the age and overall health of the host and the host's tolerance to antifungal agents. The specific dosing regimen may also vary with such factors,
Single or multiple doses can be given throughout the day. This treatment is about 2
It can take from 3 days to about 2-3 weeks or more. A typical daily dose (by single or divided doses) is from about 0.01 mg / kg to about 100 mg / k of body weight.
A g dose will contain the active compound of the invention. Preferably, the daily dose will generally be from about 0.1 mg / kg to about 60 mg / kg, ideally about 2.5 m / kg.
g / kg to about 40 mg / kg. For serious infections, e.g.
The compounds can be administered by intravenous infusion with the active ingredient from 01 to 10 mg / kg / hour.

The present invention also provides pharmaceutical formulations useful for administering the antifungal compounds of the invention.
Accordingly, the present invention also provides one or more pharmaceutically acceptable carriers, diluents, vehicles, excipients, or other additives with one or more P. syringae.
Provided is a pharmaceutical formulation comprising a lipodepsidecapeptide, for example, a 25-B1 decapeptide antifungal substance A. Such formulations contain from 0.1% to 99.9%, more usually from about 10% to about 30%, by weight of the formulation, of the active ingredient. "Pharmaceutically acceptable" means that the carrier, diluent or excipient, when mixed with the other ingredients of the formulation, does not cause a chemical reaction and is not harmful to the recipient.

The formulation can include additives such as various oils, including those of petroleum, animal, plant or synthetic origin, for example, peanut oil, soybean oil, mineral oil and sesame oil. Suitable pharmaceutical excipients include starch, cellulose, glucose, lactose,
Includes sucrose, gelatin, malt, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water and ethanol. The composition can be subjected to conventional pharmaceutical methods, such as sterilization, and includes conventional pharmaceutical additives such as preservatives, stabilizers, wetting or emulsifying agents, salts for adjusting osmotic pressure and buffers. Can be made. Suitable pharmaceutical carriers and their formulation are described in Martin,
"Remington's Pharmaceutical Sciences," 15th Ed .; Mack Publishing Co., E
aston (1975); see, for example, pp. 1405-1412 and 1461-1487.

As used herein, the term “pharmaceutically acceptable salt” means a salt of a compound of the above formula, which is substantially non-toxic to life. Typical pharmaceutically acceptable salts include those prepared by reacting a compound of the present invention with an inorganic or organic acid or base. Such salts are known as acid addition and base addition salts.

The acids commonly used to form acid addition salts are inorganic 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-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid. Examples of such pharmaceutically acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,
Monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride,
Bromide, iodide, acetate, propionate, decanoate, caprylate,
Acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate , Butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, Phthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate,
Citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate,
Methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,
There are naphthalene-2-sulfonate and mandelate. Preferred pharmaceutically acceptable acid addition salts include those formed with inorganic acids, such as hydrochloric acid and hydrobromic acid, and those formed with organic acids, such as maleic acid and methanesulfonic acid.

Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, and bicarbonates. Accordingly, bases useful in preparing such salts of the present invention include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide and calcium carbonate. Is included. The potassium and sodium salt forms are particularly preferred.

As long as the salt as a whole is pharmacologically acceptable and the counterion does not impart undesired properties to the salt as a whole, the particular counterion that forms part of any of the salts of the present invention may be of an important property. It should be understood that it is not.

The P. syringae lipodepsidecapeptide, eg, 25-B1 decapeptide antifungal substance A, is administered parenterally, eg, using intramuscular, subcutaneous, or intraperitoneal injections, by nasal or oral means. can do. In addition to these administration methods, P.
The syringae lipodepsidecapeptide, such as the 25-B1 decapeptide antifungal substance A, can be applied topically for epidermal infection or can inhibit fungal growth in mucus.

For parenteral administration, the formulation may contain one or more P. syringae lipodepsidecapeptides, such as 25-B1 decapeptide antifungal substance A and a physiologically acceptable diluent, such as deionized Include water, saline, 5% dextrose and other commonly used diluents. The formulation may include cyclodextrin and / or a solubilizing agent, such as polyethylene glycol or polypropylene glycol or other known solubilizing agents. Such formulations can be made in dry or lyophilized powder form in sterile vials containing the antifungal agent and excipients. Prior to use, a physiologically acceptable diluent is added and the solution is aspirated with 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 will generally be mixed with a carrier, or diluted with a carrier, or included in a carrier, which may be in the form of a capsule, sashimi, paper or other container. If the carrier acts as a diluent,
It can be a solid, semi-solid or liquid material that acts as a vehicle, excipient or medium for the active ingredient. Thus, the composition can be tablets, pills, powders,
Lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of active compound , Gelatin soft and hard capsules,
It may be in the form of suppositories, sterile injectable solutions and sterile packaged powders.

For oral administration, the antifungal compound is filled into gelatin capsules or formed into tablets. Such tablets may contain a binder, dispersant or other suitable excipient suitable to produce tablets of the proper size for the application dose and P. syringae lipodepsidecapeptide, e.g. B1 decapeptide antifungal substance A can be included. For pediatric or elderly use, the antifungal compound can be formulated in a flavored liquid suspension, solution or emulsion. Preferred oral formulations include linoleic acid, cremophor RH-60 and water, and preferably 8% linoleic acid, 5% cremophor RH-60, 87% sterile water, and about 2.5 to P. syringae lipodepsidecapeptide in an amount of about 40 mg / mL, for example 25-B1 decapeptide antifungal substance A.

For topical use, the antifungal compound may be formulated with a dry powder for application to the skin surface, and may be a solubilized aqueous or non-aqueous solution, such as a liquid formulation containing an alcohol or glycol It may be formulated as

Use of the P. syringae Lipodepsidecapeptide Formulation The present invention also includes a kit for use with the methods of the present invention, including the pharmaceutical compositions. The kit may include a vial containing the formulation of the invention in dry or liquid form and a suitable carrier. The kit may further comprise a vial label and / or an insert contained in the box in which the vial is packaged.
Includes instructions for use and administration of the compound. The instructions may be printed on the box where the vial is packaged. The instructions include information such as sufficient dosage and dosing information so that workers in the field can administer the drug. It is expected that workers in the art include any doctor, nurse or technician who may administer the drug.

The present invention also provides a pharmaceutical composition comprising a formulation of one or more P. syringae lipodepsidecapeptide, eg, 25-B1 decapeptide antifungal substance A, suitable for administration by injection. About things. In the present invention, one or more
Formulations of P. syringae lipodepsidecapeptide, eg, 25-B1 decapeptide antifungal substance A, can be used to produce compositions or medicaments suitable for administration by injection. The present invention also provides a composition comprising a formulation of one or more P. syringae lipodepsidecapeptides, eg, a 25-B1 decapeptide antifungal substance A, in a form suitable for oral or topical administration. About the method. For example,
Liquid or solid formulations can be manufactured by several routes using conventional techniques. Some liquid formulations contain one or more P. syringae lipodepsidecapeptides, such as 2
The 5-B1 decapeptide antifungal substance A can be prepared by dissolving in a suitable solvent containing a buffer or other excipients at a suitable pH, such as water.

Agricultural Use The antibiotic NRRL B-12050, produced from P. syringae, has been shown to effectively treat Dutch elm disease (eg, US Pat. Nos. 4,342,746 and 4). , 277,462). In particular, P. syringae MSU 16H has been shown to provide greater protection than wild-type strains in elm infected with Ceratocystis ulmi, the causative agent of Dutch elm disease (eg, Lam et al, Proc. Natl. Sci.
USA, 84, 6447-6451 (1987)). Larger tests on field grown elm confirmed the phenomenon of biocontrol at a preventive level. Therefore, the lipodepsidecapeptide of the present invention may be useful as a preventive treatment for Dutch elm disease. This pseudomycin is used for Rynchosporium secalis, Ceratocysti
s ulmi, Rizoctonia solani, Sclerotinia sclerotiorum, Verticillium albo-a
It has been shown to be toxic to a wide range of phytopathogenic fungi, including trum, Verticillium dahliae, Thielaviopis basicola, Fusarium oxysporum and Fusarium culmorum (Harrison, L., et al., "Pseudomycins, a family of novel p
eptides from Pseudomonas syringae possessing broad-spectrum antifungal a
ctivity, "J. General Microbiology, 7, 2857-2865 (1991). In conclusion, one or more P. syringae lipodepsidecapeptides, e.g.
The 5-B1 decapeptide antifungal A (including its hydrates, solvates and esters) can be used as a direct or prophylactic treatment for fungi in plants (particularly V. albo-
atrum, Rhizoctonia solani and F. oxysporum).
Generally, infected plants are treated by injecting or spraying an aqueous suspension of the lipodepsidecapeptide compound into or onto the plant. Means of injection are well known to those skilled in the art (eg, gouge pistol). Any suspension spraying technique that distributes an effective amount of the active substance to the plant surface can be used. The suspension may also contain other additives commonly used by those skilled in the art, such as solubilizers,
Stabilizers, wetting agents and combinations thereof may be included.

Treatment of plants can also be performed with a dry composition comprising one or more P. syringae lipodepsidecapeptides, eg, 25-B1 decapeptide antifungal A. The dry formulation can be applied to the plant surface by any means known to those skilled in the art, for example, by spraying or shaking from a container.

The present invention can be better understood with reference to the following examples. These examples are intended to illustrate specific embodiments of the present invention and are not intended to limit the scope of the present invention.

EXAMPLES Deposited Biological Material P. syringae MSU 16H was purchased from the American Type Culture Collection, Parklawn.
It is publicly available from Drive, Rockville, MD, USA under accession number ATCC 67028. P. syringae strains 25-B1, 7H9-1 and 67H1 were 200
Deposited with the American Type Culture Collection on March 23, 2004 and assigned the following accession number: 25-B1 accession number PTA-1622 7H9-1 accession number PTA-1623 67 H1 accession number PTA-1621

Example 1 Production of 25-B1 Antifungal Substance A In a fermentation broth of Pseudomonas syringae strain, fermentation method for production of lipodepsidecapeptide antifungal substance and 25-B1 decapeptide antifungal substance A Was developed.

Materials and Methods Preparation of Inoculum: An aliquot of P. syringae strain 25-B1 cells stored in the vapor phase of liquid nitrogen was thawed and used to inoculate two 900 mL volumes of two CSM broths. CSM broth consisted of the following (units g / L): dextrose (5), maltose (4), Difco Tryptic Soy Broth (30), Difco yeast extract (3).
And MgSO ₄ 7H ₂ O (2). Approximately 0.5 mL of cells were used to inoculate 900 mL each of media contained in two liter flasks. The flask was incubated at 25 ° C. with shaking for 24 hours. Combine the contents of the two flasks,
A 150 liter fermenter containing 115 liters of sterile fermentation broth was inoculated.

Fermentation stage: The fermentation broth consisted of the following (unit g / L): dextrose
(20), soluble starch (5), Basic American Foods Country Style Pot
ato Pearls Instant Mashed Potato (30), Glycine (1), MgSO
₄ 7H₂O (0.2), KCl (0.2) and FeSO₄ 7H₂O (0.0
04) (raw water). After adjusting the pH to 5.2, it was sterilized. Fermentation at 25 ° C 68
Time went. By continuously adjusting the air flow and impeller agitation speed,
Dissolved oxygen was maintained at 30% or more of air saturation. H₂SO₄Or Na
The pH was maintained between 4.0 and 5.4 by adding OH.

It has been found that several variations of simple batch processing can produce novel cyclic peptide products. Departure 24 hours after initial inoculation, 60m per hour
Dextrose can be transferred to the fermentor at the rate of L. Feed can be continued throughout the fermentation process. Alternatively, a method was used in which the dissolved oxygen was maintained at 5% of air saturation, starting 24 hours after inoculation and continuing until the end of the fermentation period. Dissolved oxygen could be maintained at 5% by adding inert nitrogen gas (N ₂ ) to the air supply connected to the fermenter. In all cases, gas was fed through a single submerged sparger tube whose opening was located directly below the bottom stirring turbine in the fermentor.

Results and Conclusions Several fermentation methods produce 25-B1 decapeptide antifungal A from P. syringae.

Example 2 Isolation and Purification of 25-B1 Antifungal Substance A A single lipodepsidecapeptide antifungal substance, 25-B1 decapeptide antifungal substance A from fermentation broth of Pseudomonas syringae strain A method was developed for separation and purification.

Materials and Methods The total fermentation broth, typically 100 L, produced according to Example 1 was recovered and filtered through a Membralox® ceramic filter (0.45 μm). The resulting solid slurry was extracted with an equal volume of acetone containing 0.1% TFA for 90 minutes. The acetone extract was separated by filtration and evaporated under reduced pressure to an aqueous solution.

This solution was mixed with the filtrate obtained from the ceramic filtration of the whole broth and charged to an Amberchrom® CG300sd resin column (4 L) packed in water. The column was first treated with 0.2% acetic acid (pH 4-8) until the effluent showed a pH of 4.5.
) And then 22% acetonitrile 1 containing 0.2% acetic acid (pH 4.8)
Washed with 0L. The column was then eluted with a 22-35% linear gradient of acetonitrile (32 L) containing 0.2% acetic acid and 35% acetonitrile (8 L) containing 0.2% acetic acid at a flow rate of 400 mL / min. Fractions 11-1
6 were combined (4.8 L), concentrated under reduced pressure to 100 mL, and centrifuged.

The supernatant was separated and chromatographed on an Amberchrom CG 300sd column (1 L) using a 25-35% linear gradient of acetonitrile containing 0.2% acetic acid (pH 4.8) at a flow rate of 50 mL / min. Graphed. Fractions 20 to 25 (1.
The 2 L) were combined, re-chromatographed on a reverse phase column (NovaPak _C18 , 6 μm, 40 × 300 mm, flow rate 40 mL / min, 30-60% linear gradient of acetonitrile with 0.2% TFA) and re-chromatographed 21 mg of compound (21 mg). (89% purity by UV).

The ESIMS data showed a possible [M + H] ⁻ peak at m / z 1165.7. This is different from known antifungal substances found outside of P. syringae. ^{The 1} H NMR spectrum showed a signal reminiscent of the pseudomycin-like lipopeptide, but indicated the presence of one or more compounds. To obtain a highly pure 25-B1 decapeptide antifungal substance A for structure determination and antifungal activity, additional broth from the 4 × 100 L fermentation was treated as described above and further subjected to a reverse phase column [Rainin C ₁₈ , 6 μm , 24 × 250 mm, 0.1% TFA-acetonitrile-MeOH (8: 1: 1 to 4: 3: 3) gradient elution (60 min)
: (4: 3: 3 to 10:45:45) gradient elution (30 minutes)] to obtain 42 mg of 25-B1 decapeptide antifungal substance A (93% purity by UV).

Results and Conclusions A 25-B1 decapeptide antifungal substance A could be purified from the fermentation broth by an HPLC method similar to that used to purify other lipodepsipeptide antifungal substances.

Example 3 Measurement of Structure of 25-B1 Antifungal Substance A The structures of the lipodepsipeptide antifungal substance and 25-B1 decapeptide antifungal substance A were determined by mass spectrometry and NMR.

[0066] Materials and methods molecular formula of 25-B1 decapeptide antifungal agent _{A, (with} respect to _{C 52 H 89 N 14 O 16} ) C 52 H 88 N 14 O 1 6 [m / z 1165.6581 (M + H
) ⁺ , Δ + 0.9 ppm] as determined by high resolution FABMS.

According to this formula, the ¹³ C and DEPT NMR spectra show 50 distinct resonances, including 12 carbonyl carbons, 5 olefinic carbons,
Number of oxygenated sp ³ carbon, eight typical amino acid α- carbon contained fifteen methylene carbons and six methyl carbons. Of these, one of the methyl carbon signals at δ 16.7 and one of the methylene carbon signals at δ 28.9 were each 1
It constitutes a set of modified carbons and thus accounts for the total number of 52 carbons observed in the molecular formula.

¹ H, ¹³ C, and 2D NMR (DQCOSY, TOCSY, HMQC,
Detailed analysis of the (HMBC and ROESY) data allowed the structure of the 25-B1 decapeptide antifungal A (IA) to be determined and its total protons and carbons to be clearly assigned (Table 1). .

Embedded imageTable 1. DMSO-d₆Of 25-B1 decapeptide antifungal substance A in¹H and ¹³ CNMR chemical shift

[Table 1]

[Table 2]

[Table 3]

DMSO-d₆Medium, measured at 35 ° C¹H, DQCOSY and TOCSY
The results obtained from the spectra show that seven commonly occurring amino acid residues-two amino acids.
Lanine, one arginine, one glutamine and three threonines and
Three commonly occurring amino acids—one dehydroalanine (Dha
), One dehydroaminobutyric acid (Dhb) and one homoserine
This indicates the existence of a system. Amino acid residues that are generally rarely present, ie Dh
a, Dhb and homoserine were found to be δ 9.01 (brs), 9.07 (brs) and
From the amide protons that resonate at 8.22 and 8.22, respectively. _C 106.4), 6.40 (δ_C128.5), 1.61 (δ_C12.9) and
4.22 (δ_C51.4), 1.82, 1.76 (δ_C34.0), 3.47
(Δ_C57.3) observed in the TOCSY spectrum to the proton resonating at
Identified by cross-peaks. Consistent with this,¹³C NMR spectrum
Have eight proton-linked α-carbon signals for saturated amino acid residues and
The two quaternary α-carbons for the sum amino acids Dha and Dhb are shown.
. Of the 12 amide or ester carbonyls, 9 are 8 saturated amino acids
Acid residues (two relative to glutamine), two (δ164.0 and 163.7p
pm) were assigned to Dha and Dhb. 1 remaining carbonyl
The group is assigned to the dodecanoyl side chain and its presence is¹³C NMR spectrum
The terminal methyl signal (δ in Table 1)_H0.83 and δ_C13.9) and
Discriminated from the 10 methylene signal.

This molecular formula requires 16 degrees of unsaturation. The 10 amino acids and dodecanoyl side chain account for 15 sites of unsaturation, indicating that the 25-B1 decapeptide antifungal A is a monocyclic decapeptide. Comparing the chemical shifts of the threonine β- protons (δ _H 4.94,4.11 and 4.00), δ4.9
The proton resonance at 4 has been shown to be associated with carbinols that are modified to esters or lactones. That this was the case and that the 25-B1 decapeptide antifungal A was a depsipeptide was proven by HMBC and ROESY data. These data also established the amino acid sequence and position of the dodecanoyl side chain in the 25-B1 decapeptide antifungal A.

Starting from amino acid Arg, the long-range ¹ H- ¹³ C correlation (see Scheme I below) observed in the HMBC spectrum between the amide proton and the adjacent amino acid carbonyl and / or α-carbon (see Scheme I below) The sequence was clearly established: Arg-NH / Thr-CO, Thr-NH / Dha-CO, Dha-NH / A
la-CO, Ala-NH / Dhb-CO, Dhb-NH / Hse-CO, Hs
e-NH / Glu-α-C, Glu-NH / Thr-CO, Thr-NH / Al
a CO and Ala-NH / Thr-CO.

Embedded image

The Thr NH adjacent to Ala did not show a long-range ¹ H- ¹³ C correlation to the carbonyl of the arginine residue, but instead showed a correlation with the carbonyl assigned to the dodecanoyl side chain. There is no Thr-NH / Arg-CO correlation,
The existence of a correlation between Thr-β-H (δ _H 4.94, δ _C 70.5) / Arg-CO clearly established the ester linkage between Thr-β-OH and Arg-COOH. The ROESY correlation observed between the amide proton and the adjacent amino acid α-proton is consistent with these assignments (see Scheme II below).

Embedded image

Conclusions Compound 25-B1 decapeptide antifungal A is not present in any of pseudomycin, syringomycin, syringotoxin and syringostatin produced by various isolates of P. syringae A new class of lipodepsipeptides having several amino acid residues. This novel depsipeptide is composed of another 10 amino acids apart from pseudomycin and syringomycin, which have only 9 amino acid residues. Unlike pseudomycin natural products, the novel depsipeptide does not contain chlorothreonine, which is suspected of causing inflammation at the site of injection of pharmaceutical preparations containing pseudomycin compounds

Example 4-Antifungal activity of 25-B1 decapeptide antifungal substance A Antifungal experiments were performed in 96 well microtiter plates in National Committee f
or Clinical Laboratory Standards guidelines, using a microtiter broth dilution assay. Sabourauds and dextrose broth were adjusted to contain 2.5 × 10 ⁴ conida / mL. Test compounds were dissolved in water and tested in 2-fold dilutions starting at the highest concentration of 20 μg / mL. Plates were incubated at 35 ° C. for 48 hours. The results in Table 2 show the minimum inhibitory concentration (MIC) of the compound, which completely inhibits growth compared to untreated culture controls. Table 21 Antifungal activity of 1

[Table 4]

[0075] One or more P. syringae lipodepsipeptides, such as 25-B1
The presence or amount of the decapeptide antifungal substance A can be determined by measuring the antifungal activity of the preparation. Antifungal activity can be measured in vitro by obtaining a minimum inhibitory concentration (MIC) of the preparation using a standard agar dilution test or a disk diffusion test. One or more P. syringae lipodepsidecapeptides, such as 2
The preparation of the 5-B1 decapeptide antifungal substance A may be an extract of a cell culture, or a more purified mixture. A typical fungus used for testing antifungal activity is C. albicans. Antifungal activity was considered significant if the test preparation caused inhibition of a 10-12 mm diameter area on Candida albicans x657 seeded agar plates.

Example 5-Isolation, characterization and mutagenesis of Pseudomonas syringae Environmental isolates and mutants of P. syringae were produced and used for the production of antifungal agents.

Materials and Methods US Pat. No. 5,576,298 issued Nov. 19, 1996 to G. Stro
bel et al .; Harrison et al., "Pseudomycins, a family of novel peptides f
rom Pseudomonas syringae possessing broad-spectrun antifungal activity. "
J. Gen. Microbiology 137, 2857-2865 (1991); and Lamb et al., "Transp.
oson mutagenesis and tagging of fluorescent pseudomonas; Antimycotic pro
duction is necessary for control of Dutch elm disease. "Proc. Natl. Acad
Sci. USA 84, 6447-6451 (1987). Strains MSU 174 and MSU 16-H were isolated and characterized. The disclosure content of the references cited in this paragraph is incorporated herein by reference.

Additional strains were derived from such wild type and transposon-generating mutants by chemical mutagenesis. Mutants were subjected to MSU 174
, MSU 16H and 25-B1. The strain to be mutated is grown in a medium containing the potato product and then 0, 1, 2, 4, 16 or 32 μl.
The cells were divided into media containing 1-methyl-3-nitro-1-nitrosoguanidine (NTG or MNNG), which is a chemical mutagen of M. These cells were then frozen for later screening and selection.

The mutated cells were selected for desired growth conditions and / or production of one or more P. syringae lipodepsidecapeptides, eg, 25-B1 decapeptide antifungal A. . P. s chemically mutated
yringae cells, such as the mutated strain 25-B1, are thawed and N21S
Diluted to 6 cells / mL in M medium (Table 3). The medium may contain one or more components for selection, such as various concentrations of phosphate.
A volume of 50 μL of mutated cells was distributed into wells of a 96-well round bottom microtiter plate with an average delivery of 0.3 cells / well. Typically,
Silicone oil was added to each well to minimize evaporation. 6-1 this plate
Incubate with shaking at 25 ° C. for 2 days. Table 3 Composition of N21SM medium

[Table 5]

After this incubation, an aliquot, typically 5 μL, from each well is serially diluted (eg, 1:56, 1: 196, 1: 320, 1: 686 and / or 1
: 1715) and assessed for activity against Candida albicans in a liquid microtiter plate bioassay. The plates were incubated overnight at 37 ° C. and the wells were scored for inhibition of C. albicans growth. Appropriate strains were selected and inoculated into CSM medium (Table 4) and grown at 25 ° C for 1-3 days. Table 4 Complete Streptomyces medium (CSM)

[Table 6]

The selected strain is stored and inoculated into a fermentation bottle containing 13 mL of N21SM medium,
Grow at 25 ° C. for about 66 hours. An aliquot is removed from the fermentation, extracted with an equal volume of acetonitrile for 1 hour, centrifuged, and one or more P. syringae lipodepsidecapeptides as described in Examples 1-3. , For example, 25-B
1 Decapeptide was decanted for HPLC analysis of antifungal substance A. Strains producing one or more P. syringae lipodepsidecapeptides, such as 25-B1 decapeptide antifungal A, were again isolated, refermented, and prepared for large-scale culture.

Results Using the methods described above, strains were produced that showed the production of one or more P. syringae lipodepsidecapeptides, eg, 25-B1 decapeptide antifungal substance A.

Conclusions The selection methods and criteria disclosed herein are based on the growth of one or more P. syringae lipodepsidecapeptides, such as the 25-B1 decapeptide antifungal, grown on minimal medium. It is effective in producing a strain of P. syringae that produces A.

Example 6-Formulations Containing P. syringae Lipodepsidecapeptide The following formulation examples are merely illustrative and are not intended to limit the scope of the invention in any way. The term "active ingredient" means P. syringae lipodepsidecapeptide or a pharmaceutically acceptable salt thereof.

Formulation Example 1 Hard gelatin capsules are prepared using the following ingredients:

[Table 7]

Formulation Example 2 A tablet is produced using the following ingredients. The ingredients are mixed and compressed into tablets, each 665 mg in weight.

[Table 8]

Formulation Example 3 An aerosol solution containing the following components is produced. 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 transferred to a stainless steel container and diluted with the remaining propellant. Next, the valve unit is attached to the container.

[Table 9]

Formulation Example 4 Tablets each containing 60 mg of the active ingredient are prepared as follows:

[Table 10] The active ingredients, starch and cellulose were no. Pass through a 45 mesh US sieve and mix well. An aqueous solution containing polyvinylpyrrolidone was mixed with the obtained powder, and the mixture was Pass through a 14 mesh US sieve. The granules thus produced were dried at 50 ° C. Pass through an 18 mesh US sieve. Then, in advance, The sodium carboxymethyl starch, magnesium stearate and talc that have been passed through a 60 mesh US sieve are added to the granules, which are mixed and then compressed on a tablet machine to give tablets each weighing 150 mg.

Formulation Example 5 Capsules each containing 80 mg of active ingredient are prepared as follows:

[Table 11] Mix active ingredients, cellulose, starch and magnesium stearate,
No. Fill a hard gelatin capsule with 200 mg amount through a 45 mesh US sieve.

Formulation Example 6 Suppositories each containing 225 mg of the active ingredient are prepared as follows:

[Table 12] The active ingredient was no. Pass through a 60 mesh US sieve and suspend in pre-melted saturated fatty acid glyceride with minimal heating required. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.

Formulation Example 7 Suspensions each containing 50 mg of active ingredient per 5 mL dose are prepared as follows:

[Table 13] The active ingredient was no. Pass through a 45 mesh US sieve and mix with sodium carboxymethylcellulose and syrup to form a smooth paste. The benzoic acid solution, flavor and color are diluted with some of the water and added, with stirring. Then add enough water to get the required amount.

Formulation Example 8 An intravenous formulation can be manufactured as follows. Solutions of these components are administered intravenously to the patient at a rate of 1 mL per minute.

[Table 14]

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications can be made without departing from the spirit and scope of the invention.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:38) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI) , FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN , TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, Z, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Matthew David Belvo United States 46140 Greenfield, Indiana Greenfield Co., No. 24 (72) Inventor James William Martin United States 46121 Mills Springs, Courtsville, Indiana No. 177 (72) Inventor Douglas Joseph Zechner United States 46143 Gris, Indiana Baywood, Bayside Drive No. 980 F term (reference) 4B064 AG37 CA02 CC03 CC07 CC09 CD01 CD07 CD09 CD12 CD19 CD21 DA03 4C084 AA02 AA06 AA07 BA01 BA17 BA25 BA27 BA28 CA04 NA14 ZB35 4H045 AA10 BA15 BA33 CA11 EA29 FA73

Claims (36)

[Claims] 1. An isolated P-protein containing an arginine, threonine, homoserine, dehydroaminobutyric acid and dehydroalanine, wherein the lactone comprises a depsidecapeptide ring formed from the carboxyl group of arginine and the hydroxyl group of threonine.
syringae depsidecapeptide or a pharmaceutically acceptable salt, ester or hydrate thereof. 2. The P. syringae depsi according to claim 1, wherein the depsidecapeptide has the sequence: Thr-Ala-Thr-Gln-Hse-Dhb-Ala-Dha-Thr-Arg (SEQ ID NO: 1). Decapeptide. 3. The P. syringae depsidecapeptide of claim 1, wherein the P. syringae depsidecapeptide is a P. syringae lipodepsidecapeptide. 4. The P. syringae depsidecapeptide of claim 3, wherein the P. syringae lipodepsidecapeptide comprises a fatty acid moiety coupled to an amino group of threonine by an amide bond. 5. A decanoic acid moiety in which the fatty acid moiety is substituted with one or two hydroxyl groups, a dodecanoic acid moiety, a dodecanoic acid moiety substituted with one or two hydroxyl groups, and tetradecanoic acid A P. moiety or a tetradecanoic acid moiety substituted with one or two hydroxyl groups.
syringae depsidecapeptide. 6. The P. syringae depsidecapeptide of claim 5, wherein the fatty acid moiety is an n-dodecanoic acid moiety. 7. The lipodepsidecapeptide of P. syringae has the formula: 4. The P. syringae depsidecapeptide of claim 3, wherein R is a lipophilic moiety, or a pharmaceutically acceptable salt, ester or hydrate thereof. 8. The lipophilic moiety is C ₉ -C ₁₅ alkyl, C ₉ -C ₁₅ hydroxyalkyl, C ₉ -C ₁₅ dihydroxyalkyl, C ₉ -C ₁₅ alkenyl, C ₉ -C ₁₅ hydroxyalkenyl and C _9. -C ₁₅ is selected from the group consisting of dihydroxy alkenyl, claim 7
The P. syringae depsidecapeptide described in 1 above. 9. The P. syringae depsidecapeptide of claim 8, wherein the lipophilic moiety is a C ₉ -C ₁₅ alkyl. 10. The formula: embedded image An isolated P. syringae depsidecapeptide represented by the formula: or a pharmaceutically acceptable salt, ester or hydrate thereof. 11. A fungus comprising contacting the fungus with the isolated P. syringae depsidecapeptide of claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. How to inhibit activity. 12. The method according to claim 12, wherein the fungus is Candida parapsilosis, Candida albicans, Crypt.
12. The method of claim 11, comprising ococcus neoformans or Histoplasma capsulatum. 13. A method for reducing the symptoms of a fungal infection in a patient in need of reducing the symptoms of a fungal infection, the method comprising the steps of: 1, 2, 3, 4, 5, 6, 7, 8
A method comprising administering to the patient an effective amount of a composition comprising the isolated P. syringae depsidecapeptide of claim 9, 9 or 10. 14. The alleviation of symptoms includes reducing the suffering of a fungal infection.
The method according to claim 13. 15. The fungal infection is Candida parapsilosis, Candida albicans,
Including infection by Cryptococcus neoformans or Histoplasma capsulatum,
The method according to claim 13. 16. The method of claim 13, further comprising determining the need for isolated P. syringae depsidecapeptide administration; and monitoring the patient for reduction of symptoms of a fungal infection. 17. The method according to claim 1, wherein the dose is about 1 to about 3 times per day, about 0.1 to about 5 mg.
14. The method of claim 13, comprising parenteral administration of P. syringae depsidecapeptide / kg of isolated. 18. The method of claim 13, wherein reducing the symptoms of a fungal infection comprises lowering fever and increasing the patient's overall health. 19. The method of claim 1 in the manufacture of a medicament for treating a fungal infection.
Use of an isolated P. syringae depsidecapeptide according to 2, 3, 4, 5, 6, 7, 8, 9, or 10. 20. The fungal infection is Candida parapsilosis, Candida albicans,
Including infection by Cryptococcus neoformans or Histoplasma capsulatum,
Use according to claim 19. 21. A method for producing one or more P. syringae depsidecapeptides, comprising the steps of: producing a biologically pure culture of Pseudomonas syringae having a pH of from about 4 to about 3 containing less than 3 amino acids. Culturing in the nutrient medium at 6.5 to produce one or more P. syringae depsidecapeptides at a concentration of at least about 10 μg / mL; and one or more P. syringae de. Recovering the psidecapeptide or a pharmaceutically acceptable salt, ester or hydrate thereof from the culture. 22. The method of claim 21, wherein the amino acid comprises glutamic acid, glycine, histidine, or a combination thereof. 23. The method of claim 22, wherein the amino acid comprises glycine. 24. The method of claim 22, wherein the nutrient medium further comprises soluble starch, yeast extract or a combination thereof, and a lipid. 25. The method of claim 24, wherein the nutrient medium comprises potato products and a lipid selected from the group consisting of soybean oil, fatty acids and fatty acid esters. 26. The method of claim 25, wherein the potato product comprises potato dextrose broth, mashed potato mix, potato dextrin, potato protein or a combination thereof. 27. The method of claim 21, wherein the dissolved oxygen concentration is maintained between about 5% and 30% during the culturing step. 28. The method of claim 21, wherein the culturing step further comprises feeding glucose, ammonium hydroxide, or a combination thereof. 29. The P. syringae depsidecapeptide that is produced.
22. The method of claim 21, which is a P. syringae depsidecapeptide according to 2, 3, 4, 5, 6, 7, 8, 9, or 10. 30. Pseudomonas syringae is strain MSU 16H, MSU 1
22. The method of claim 21 comprising a strain derived from 74 or MSU 206. 31. Pseudomonas syringae is strain MSU 16H, strain 25-B
22. The method of claim 21, comprising 1, strain 67H1 or strain 7H9-1. 32. The method of claim 3, wherein the Pseudomonas syringae comprises strain 25-B1.
2. The method according to 1. 33. The method of claim 21, 22, 23, 24, 25, 26, 27, 28.
The method according to claim 7, wherein the P is produced by a method according to claim 7, 30, 31 or 32.
syringae Depsidecapeptide. 34. A fungal control of a plant comprising contacting a fungus with a P. syringae depsidecapeptide according to claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. A method of treating or preventing growth. 35. The fungus is Rynchosporium secalis, Ceratocystis ulmi, Riz
octonia solani, Sclerotinia sclerotiorum, Verticillium albo-atrum, Verti
cillium dahliae, Thielaviopis basicola, Fusarium oxysporum or Fusariu
34. The method of claim 33, which is m culmorum. 36. The method according to claim 36, wherein the fungus is V. albo-atrum, Rhizoctonia solani or F. o.
35. The method of claim 34, wherein the method is xysporum.