Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1997-10-10
2002-03-19
Moezie, F. T. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S317000, C530S318000
Reexamination Certificate
active
06358921
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a class of cyclic peptides that exhibit gram-positive and gram-negative bactericidal activity, as well as fungicidal and anti-mycoplasma activity, and are useful as antimicrobial agents.
BACKGROUND OF THE INVENTION
The emergence of many medically relevant resistant strains of bacteria is a major issue in human health (Neu, 1992). It is becoming essential that new therapeutic agents be developed to combat microorganisms resistant to traditional antibiotics.
Gramicidin S (GS) is a naturally occurring cyclic peptide antibiotic (cyclo(Val-Orn-Leu-D-Phe-Pro)
2
) first isolated from
Bacillus brevis
. There may be a reduced incidence of resistance developed against GS analogs, allegedly because the target of these analogs is the cell membrane of sensitive microorganisms; nonetheless, their mechanism of action is still not well understood. Furthermore, only two proteases are known to degrade GS, GS and its analogs are predicted to be stable in vivo (Maeda, et al., 1993; Yukioka, et al., 1966).
X-ray and NMR studies of GS have confirmed that it forms a two-stranded antiparallel &bgr;-sheet structure with the strands fixed in place by two type II′ &bgr;-turns (Izumiya, et al., 1979; Rackovsky and Scheraga, 1980; Hull, et al., 1978). Based on studies of GS and its analogs, a number of structural requirements believed to be important for GS activity have been determined. These include: i) the requirement for an amphipathic structure containing basic residues on the hydrophilic face of the molecule (Izumiya, et al., 1979); ii) a &bgr;-sheet structure, or the ability to achieve a &bgr;-sheet structure in the presence of lipid bilayers (Izumiya, et al., 1979; Ando, et al., 1993, 1995), and iii) a high overall hydrophobicity (Kondejewski, et al., 1996; Katayama, et al., 1994; Tamaki and Ludescher, 1969).
A drawback to the use of GS as an antibiotic is its ability to lyse certain eukaryotic cells, which can result in a high level of hemolysis. Consequently GS is generally restricted to topical administration.
GS analogs containing more than ten residues have been shown to exhibit a change in activity profile (i.e., a change from gram positive specificity to gram negative specificity) when evaluated using agar-based assays, and to exhibit reduced hemolytic activity compared to GS (Ando, et al., 1993, 1995; Aoyagi, et al., 1988). Further, Aoyagi, et al. (1988) reported on a cyclic 14-mer having activity against gram positive and gram negative bacteria. Tamaki, et al. (1988) present CD data that shows that a 14-mer having two D-Phe residues as a non-L amino acid exists in a beta-sheet conformation, which can be disrupted by incorporation of a third D-amino acid in the peptide.
As mentioned above, despite its desirable properties as a broad spectrum antibiotic, the use of GS is limited by its side effects, specifically, its propensity for hemolysis. Therefore it would be useful to have a broad spectrum, cyclic peptide antibiotic that possess a high level of broad spectrum antimicrobial activity with much reduced hemolytic activity. Peptides having such properties form one aspect of the present invention.
More specifically, it is the discovery of the present invention that cyclic peptides that do not conform to the &bgr;-pleated sheet conformation that is characteristic of GS, exhibit superior properties, including broad spectrum antimicrobial activity and a high therapeutic index. The present invention provides guidance for the design and testing of cyclic peptides that exhibit such exceptional properties.
SUMMARY OF THE INVENTION
The present invention is generally directed to cyclized peptides that have antimicrobial activity. Such peptides are generally greater than 11 amino acids in length. The invention provides guidance for the design, production, testing and use of such peptides.
In one embodiment, the invention includes an analog of the 14-mer cyclic peptide having the amino acid sequence V1-K2-L3-K4-V5-
Y6
-P7-L8-K9-V10-K11-L12-
Y13
-P14 also identified by SEQ ID NO: 1, where Y represents D-tyrosine, numerals represent relative positions in the sequence of the peptide analog, and P14 is linked to V1. The analogs are further characterized by the presence of an amino acid substitution selected from the group consisting of
(i) a D-amino acid in at least one of positions 1-5, 7-12 and 14 or an L-amino acid in position 6 or 13;
(ii) same class substitution at any of positions 1, 3, 5, 8, 10 or 12 with a class I hydrophobic sidechain;
(iii) same class substitution at any of positions 2, 4, 9 or 11 with a class II basic sidechain;
(iv) same class substitution at any of positions 6 or 13 with a D-amino acid comprising a class I hydrophobic sidechain;
(v) transposition of adjacent positions with substitutions of a class I sidechain at any of positions 1, 3, 5, 8, 10 or 12 with a class II basic sidechain and substitution of an adjacent class II sidechain with a class I sidechain to form a non-amphipathic cyclic peptide analog with respect to a plane formed by the peptide backbone of the cyclic structure;
(vi) substitution of class I sidechains for class II side chains at one end of the cyclic structure and substitution of class II sidechains for class I sidechains at the opposite end of the cyclic structure to form a polarized non-amphipathic cyclic peptide having basic residues at one end of the cyclic peptide and hydrophobic residues at the opposite end of the structure; and
(vii) combinations of any of (i)-(vi).
In one embodiment the analog referred to above excludes the peptide known as GS14 (SEQ ID NO: 1). In another embodiment, the analog is further characterized by a disrupted &bgr;-sheet structure, as evidenced by a circular dichroism spectrum that is shifted relative to a spectrum measured from a cyclic peptide having the sequence SEQ ID NO: 1 (GS14). In another embodiment, the analog has a specified hydrophobicity, defined in terms of a hydrophobicity window, herein. In a preferred embodiment, the peptide analog is further characterized by a therapeutic index of greater than 1, where the therapeutic index value is determined as a ratio of the concentration of analog required to produce hemolysis of human red blood cells divided by the concentration of analog required to inhibit growth of a specified microbe.
According to further preferred embodiments, the peptide analog is effective against one or more microbes selected from the group consisting of Pseudomonas,
Escherischia coli
, Salmonella, Staphylococcus, Bacillus, Enterococcus, Corynebacterium, Candida, and mycoplasma.
In another preferred embodiment, amino acid substitution to form the analog comprises substitution of a D-valine at a position selected from the group consisting of position 1 and position 5. In a further preferred embodiment, the amino acid substitution comprises substitution of a D valine at position 10. In still a further embodiment, the amino acid substitution comprises substitution of a D-leucine at a position selected from the group consisting of position 8 and position 12. The amino acid substitution may also include substitution of a D-leucine at a position 3.
In a related embodiment, the peptide analog of the invention has an amino acid substitution of a D-lysine at a position selected from the group consisting of position 2, position 4, position 9 and position 11. A further embodiment includes substitution at any of positions 1, 3, 5, 8, 10 or 12 with an amino acid selected from the group consisting of alanine, valine, leucine, norvaline, isoleucine, norleucine, methionine, phenylalanine, tyrosine and tryptophan. Here, the amino acid substitution is preferably selected from the group consisting of a leucine at each of positions 1, 5 and 10 (V3/L3), an alanine at each of positions 3, 8 and 12 (L3/A3), an alanine at each of positions 1, 5 and 10 (V3/A3), and an alanine at each of positions 1, 3, 5, 8, 10, and 12. Within this group, the amino acid substitution will preferably consist of a leucine at each of positions 1, 5 and 10 and further comprising a D-p
Hancock Robert E. W.
Hodges Robert S.
Kondejewski Leslie H.
Lewis Ruthven N. A. H.
McElhaney Ronald N.
Moezie F. T.
Pence Inc.
SEED Intellectual Property Law Group PLLC
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