Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
2001-10-15
2003-10-07
Navarro, Mark (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S234100, C424S260100, C514S011400, C514S015800, C530S300000, C530S317000, C530S328000
Reexamination Certificate
active
06630147
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to pseudomycin natural products including pseudomycins A′ and B′, methods for making such pseudomycins, and methods employing antifungal activity of these pseudomycins.
BACKGROUND
Fungal infections are a significant cause of disease, degradation of quality of life, and mortality among humans, particularly for immune compromised patients. The incidence in fungal infections in humans has increased greatly in the past 20 years. This is in part due to increased numbers of people with immune systems weakened or devastated by organ transplants, cancer chemotherapy, AIDS, age, and other similar disorders or conditions. Such patients are prone to attack by fungal pathogens that are prevalent throughout the population but are kept in check by a functioning immune system. These pathogens are difficult to control because some existing antifungal agents are either highly toxic or only inhibit fungal activity. For example, the polyenes are fungicidal but toxic; whereas, the azoles are much less toxic but only fungistatic. More importantly, there have been recent reports of azole and polyene resistant strains of Candida which severely limits therapy options against such strains.
Pseudomonas syringae
produce several classes of antifungal or antibiotic agents, such as the pseudomycins, syringomycins, syringotoxins, and syringostatins, which are lipodepsinonapeptides. Natural strains and transposon generated mutants of
P. syringae
produce these lipodepsinonapeptides. Several of the pseudomycins, syringomycins and other lipodepsipeptide antifungal agents have been isolated, chemically characterized, and shown to possess wide spectrum antifungal activity, including activity against important fungal pathogens in both humans and plants. For example, pseudomycins A, B, C and C′ have each been isolated and purified and their structures have been characterized by methods including amino acid sequencing, NMR, and mass spectrometry. See. e.g. Ballio et al., “Novel bioactive lipodepsipeptides from
Pseudomonas syringae:
the pseudomycins,”
FEBS Lett.
355. 96-100 (1994) and U.S. Pat. No. 5,576,298. The pseudomycins, the syringomycins, the syringotoxins, and the syringostatins represent structurally distinct families of antifungal compounds.
None of the pseudomycins, syringomycins, syringotoxins, or syringostatins has been brought to market for antifungal therapy. Discovery of undesirable side effects, making formulations, scaling up production, and other development problems have thus far prevented exploitation of the pseudomycins, syringomycins, syringotoxins, or syringostatins against the full range of fungal infections that affect animals, humans and plants. There remains a need for an antifungal agent that can be used against infections not treated by existing antifungal agents and for application against infections in animals, humans, or plants.
SUMMARY OF THE INVENTION
The present invention provides a pseudomycin natural product produced by
P. syringae.
The pseudomycin natural product includes a depsinonapeptide ring with the sequence Ser-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr, more specifically, L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-Dhb-L-Asp(3-OH)-L-Thr(4-Cl), with the carboxyl group of the ClThr and the hydroxyl group of the serine closing the ring with a lactone bond. Pseudomycin A′ (IA) includes a 3,4-dihydroxypentadecanoic acid moiety, the carboxyl group of which forms an amide bond with the amine group of the N-terminal serine.
Pseudomycin B′ (IB) includes a 3-hydroxydodecanoic acid moiety, the carboxyl group of which forms an amide bond with the amine group of the N-terminal serine.
The invention also relates to methods employing a pseudomycin natural product, such as pseudomycin A′, pseudomycin B′ or a mixture thereof, for inhibiting fungal activity or for reducing the symptoms of a fungal infection in a patient in need thereof. Such methods can kill the fungus, decrease the burden of a fungal infection, reduce fever and/or increase the general well being of a patient. The methods of the invention are effective against fungi such as
Candida parapsilosis, Candida albicans, Cryptococcus neoformans,
and/or
Histoplasma capsulatum.
DETAILED DESCRIPTION
Pseudomycins
As used herein, pseudomycin or pseudomycin natural product refers to one or more members of a family of antifungal agents that has been isolated from the bacterium
Pseudomonas syringae.
A pseudomycin is a lipodepsipeptide, a cyclic peptide including one or more unusual amino acids and having one or more appended hydrophobic or fatty acid side chains. Specifically, the pseudomycins are lipodepsinonapeptides, with a cyclic peptide portion closed by a lactone bond and including the unusual amino acids 4-chlorothreonine, 3-hydroxyaspartic acid, dehydro-2-aminobutyric acid, and 2,4-diaminobutyric acid. It is believed that these unusual amino acids are involved in biological characteristics of the pseudomycins, such as stability in serum and their killing action.
Each pseudomycin has the same cyclic peptide nucleus, but they differ in the hydrophobic side chain attached to this nucleus. Each pseudomycin has a cyclic nonapeptide ring having the sequence Ser-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr (i.e., Serine; 2,4-Diaminobutyric acid; Aspartic acid; Lysine; 2,4-Diaminobutyric acid; alloThreonine; Dehydro-2-aminobutyric acid; 3-hydroxyAspartic acid; 4-chloroThreonine), with the carboxyl group of the ClThr and the hydroxyl group of the serine closing the ring with a lactone bond. The lipophilic moiety is attached to the amine group of the N-terminal serine. The amine group of the serine forms an amide bond with the carboxyl of a 3,4-dihydroxytetradecanoyl moiety in pseudomycin A, a 3-monohydroxytetradecanoyl moiety in pseudomycin B, a 3,4-dihydroxyhexadecanoyl moiety in pseudomycin C and a 3-monohydroxyhexadecanoyl moiety in pseudomycin C′. The carboxyl group of the serine forms an amide bond with the Dab of the ring.
Pseudomycins A′ and B′
As used herein the terms pseudomycin A′ and pseudomycin B′ refer to antifungal agents that have been isolated from the bacterium
Pseudomonas syringae.
Pseudomycins A′ and B′ are pseudomycins having the characteristic depsinonapeptide ring with the sequence Ser-Dab-Asp-Lys-Dab-aThr-Dhb-HOAsp-ClThr, with the carboxyl group of the ClThr and the hydroxyl group of the serine closing the ring with a lactone bond. Pseudomycin A′ includes a 3,4-dihydroxypentadecanoic acid moiety, the carboxyl group of which forms an amide bond with the amine group of the N-terminal serine. Pseudomycin B′ includes a 3-hydroxydodecanoic acid moiety, the carboxyl group of which forms an amide bond with the amine group of the N-terminal serine.
Biological Activities of Pseudomycins
A pseudomycin has several biological activities including killing various fungi, such as fungal pathogens of plants and animals. In particular, a pseudomycin is an active antimycotic agent against fungi that cause opportunistic infections in immune compromised individuals. These fungi include various species of Candida including
C. parapsilosis, C. albicans, C. glabrata, C. tropicalis,
and
C. krusei.
They also incldue other genera such as
Cryptococcus neoformans, Aspergillus fumigatus,
and
Histoplasma capsulatum.
Killing, rather than inhibiting the growth of fungi, particularly of fungal pathogens, is a desirable and preferred biological activity of an antifungal, such as pseudomycin A′ and/or B′.
The pseudomycins have been shown to be toxic to a broad range of plant-pathogenic fungi including
Rynchosporium secalis, Ceratocystis ulmi, Rizoctonic solani, Sclerotinia sclerotiorum, Verticillium albo
-
atrum, Verticillium dahliae, Thielaviopis basicola, Fusarium oxysporum
and
Fusarium culmorum.
(see Harrison, L., et al., “Pseudomycins, a family of novel peptides from
Pseudomonas syringae
possessing broad-spectrum antifungal activity,”
J. of General Microbiology,
7, 2857-28
Belvo Matthew David
Kulanthaivel Palaniappan
Martin James William
Perun Thomas John
Zeckner Douglas Joseph
Eli Lilly and Company
Navarro Mark
Tucker Tina M.
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