U.S. patent application number 13/031295 was filed with the patent office on 2011-06-09 for novel antimicrobial agents.
This patent application is currently assigned to Technion Research & Development Foundation Ltd.. Invention is credited to Amram MOR, Inna Radzishevsky.
Application Number | 20110136726 13/031295 |
Document ID | / |
Family ID | 46124127 |
Filed Date | 2011-06-09 |
United States Patent
Application |
20110136726 |
Kind Code |
A1 |
MOR; Amram ; et al. |
June 9, 2011 |
NOVEL ANTIMICROBIAL AGENTS
Abstract
A novel class of antimicrobial polymeric agents which are
designed to exert antimicrobial activity while being stable,
non-toxic and avoiding development of resistance thereto and a
process of preparing same are disclosed. Further disclosed are
pharmaceutical compositions containing same and a method of
treating medical conditions associated with pathological
microorganisms, a medical device, an imaging probe and a food
preservative utilizing same. Further disclosed are conjugates of an
amino acid residue and a hydrophobic moiety residue and a process
of preparing same.
Inventors: |
MOR; Amram; (Nesher, IL)
; Radzishevsky; Inna; (Beer-Sheva, IL) |
Assignee: |
Technion Research & Development
Foundation Ltd.
Haifa
IL
|
Family ID: |
46124127 |
Appl. No.: |
13/031295 |
Filed: |
February 21, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11500461 |
Aug 8, 2006 |
7915223 |
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13031295 |
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11234183 |
Sep 26, 2005 |
7504381 |
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11500461 |
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60612778 |
Sep 27, 2004 |
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Current U.S.
Class: |
514/2.3 ;
530/328; 530/329; 530/330; 530/331; 548/339.1; 554/107;
562/562 |
Current CPC
Class: |
C12Q 1/04 20130101; A23L
3/3526 20130101; A61K 47/542 20170801; C12Q 1/18 20130101; Y02A
50/56 20180101; A61P 31/00 20180101; C07K 7/08 20130101; A61K 38/00
20130101; A61K 31/785 20130101; C07K 7/06 20130101; C07K 14/00
20130101; A61K 49/0002 20130101; Y02A 50/59 20180101; A23L 3/3481
20130101; A61K 45/06 20130101; C07K 14/001 20130101; Y02A 50/57
20180101; Y02A 50/30 20180101; C07C 237/22 20130101; C07K 7/00
20130101; C07K 14/4723 20130101; A61M 15/00 20130101; A61K 31/785
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/2.3 ;
530/328; 530/329; 530/330; 530/331; 548/339.1; 562/562;
554/107 |
International
Class: |
A61K 38/04 20060101
A61K038/04; C07K 5/00 20060101 C07K005/00; A61P 31/00 20060101
A61P031/00; C07K 7/00 20060101 C07K007/00; C07D 233/64 20060101
C07D233/64; C07C 229/26 20060101 C07C229/26; C07C 229/00 20060101
C07C229/00 |
Claims
1. A polymer comprising a plurality of positively charged amino
acid residues and at least one hydrophobic moiety residue, wherein
at least one of said at least one hydrophobic moiety residue is
being covalently linked to at least two amino acid residues in said
plurality of amino acid residues via the N-alpha of one amino acid
residue and via the C-alpha of the other amino acid residue in said
at least two amino acid residues.
2. The polymer of claim 1, wherein said plurality of amino acid
residues comprises from 2 to 50 amino acid residues.
3. The polymer of claim 2, wherein said plurality of amino acid
residues substantially consists of positively charged amino acid
residues.
4. The polymer of claim 3, wherein said positively charged amino
acid residues are selected from the group consisting of lysine
residues, histidine residues, ornithine residues, arginine residues
and combinations thereof.
5. The polymer of claim 3, said positively charged amino acid
residues are lysine residues.
6. The polymer of claim 1, wherein said at least one hydrophobic
moiety has a carboxylic group at one end thereof and an amine group
at the other end thereof.
7. The polymer of claim 6, wherein said at least one hydrophobic
moiety residue is linked to each of said at least two amino acid
residues via a peptide bond.
8. The polymer of claim 1, comprising from 1 to 50 hydrophobic
moiety residues.
9. The polymer of claim 1, wherein said at least one hydrophobic
moiety residue comprises a fatty acid residue.
10. The polymer of claim 1, wherein each of said at least one
hydrophobic moiety is an .omega.-amino-fatty acid residue.
11. The polymer of claim 10, wherein said hydrophobic moiety is
selected from the group consisting of 4-amino-butyric acid,
8-amino-caprylic acid and 12-amino-lauric acid.
12. The polymer of claim 1, comprising at least two hydrophobic
moiety residues, wherein at least one of said at least two
hydrophobic moiety residues is being linked to the N-alpha of an
amino acid residue at the N-terminus of said plurality of amino
acid residues.
13. The polymer of claim 5, comprising 2 to 50 lysine residues and
1 to 50 .omega.-amino-fatty acid residues, wherein: at least one of
said .omega.-amino-fatty acid residues is covalently linked to at
least two of said lysine residues via the N-alpha of one lysine
residue and via the C-alpha of the other lysine residue in said at
least two lysine residues, said at least one .omega.-amino-fatty
acid is linked to each of said lysine residues via a peptide bond,
and wherein: each of said lysine residues is independently selected
from the group consisting of a lysine residue and a lysine residue
having a fatty acid residue linked to a side-chain thereof; an
N-terminus of the polymer is selected from the group consisting of
a lysine residue, an .omega.-amino-fatty acid residue linked via a
peptide bond to a lysine residue and a fatty acid residue linked
via a peptide bond to said lysine residue; and a C-terminus of the
polymer is selected from the group consisting of a lysine residue
and a lysine residue terminated with an amide group.
14. The polymer of claim 13, wherein said .omega.-amino-fatty acid
residue is selected from the group consisting of 4-amino-butyric
acid residue, 6-amino-caproic acid residue, 8-amino-caprylic acid
residue, 10-amino-capric acid residue, 12-amino-lauric acid
residue, 14-amino-myristic acid residue, 16-amino-palmitic acid
residue, 16-amino-palmitoleic acid residue, 18-amino-stearic acid
residue, 18-amino-oleic acid residue, 18-amino-linoleic acid
residue, 18-amino-linolenic acid residue and 20-amino-arachidonic
acid residue.
15. The polymer of claim 14, having an amino acid sequence selected
from the group consisting of the amino acid sequences as set forth
in SEQ ID NOs: 1-83 and 86-94 as set forth herein.
16. The polymer of claim 15, each of said w-amino-fatty acid
residues covalently linked to said at least two of said lysine
residues is 8-amino-caprylic acid residue.
17. The polymer of claim 1, further comprising at least one active
agent attached thereto.
18. The polymer of claim 17, being capable of delivering at least
one active agent to at least a portion of the cells of a pathogenic
microorganism.
19. The polymer of claim 1, being selected from the group of
compounds presented in Table 3.
20. A pharmaceutical composition comprising, as an active
ingredient, the polymer of claim 1 and a pharmaceutically
acceptable carrier.
21. The pharmaceutical composition of claim 20, further comprising
at least one additional therapeutically active agent.
22. The pharmaceutical composition of claim 21, wherein said at
least one additional therapeutically active agent comprises an
antibiotic agent.
23. A medical device comprising the polymer of claim 1 and a
delivery system configured for delivering said polymer to a bodily
site of a subject.
24. A food preservative comprising an effective amount of the
polymer of claim 1.
25. An imaging probe for detecting a pathogenic microorganism, the
imaging probe comprising the polymer of claim 1, said polymer
further comprising at least one labeling agent attached thereto.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/500,461, filed Aug. 8, 2006, which is
continuation in part of U.S. patent application Ser. No.
11/234,183, filed Sep. 26, 2005, now U.S. Pat. No. 7,504,381,
issued on Mar. 17, 2009, which claims the benefit of priority from
U.S. Provisional Patent Application No. 60/162,778, filed Sep. 27,
2004. The contents of the above applications are incorporated
herein by reference in their entirety.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to novel antimicrobial agents
and, more particularly, to a novel class of polymers which are
designed to exert antimicrobial activity while being stable,
non-toxic and avoiding development of resistance thereto. The
present invention further relates to pharmaceutical compositions,
medical devices and food preservatives containing such polymers and
to methods of treating medical conditions associated with
pathogenic microorganisms utilizing same.
[0003] Antibiotics, which are also referred to herein and in the
art as antibacterial or antimicrobial agents, are natural
substances of relatively small size in molecular terms, which are
typically released by bacteria or fungi. These natural substances,
as well as derivatives and/or modifications thereof, are used for
many years as medications for treating infections caused by
bacteria.
[0004] As early as 1928, Sir Alexander Fleming observed that
colonies of the bacterium Staphylococcus aureus could be destroyed
by the mold Penicillium notatum. His observations lead Fleming to
postulate the existence and principle of action of antibiotic
substances. It was established that the fungus releases the
substance as a mean of inhibiting other organisms in a chemical
warfare of microscopic scale. This principle was later utilized for
developing medicaments that kill certain types of disease-causing
bacteria inside the body. In 1940's Howard Florey and Ernst Chain
isolated the active ingredient penicillin and developed a powdery
form of the medicine.
[0005] These advancements had transformed medical care and
dramatically reduced illness and death from infectious diseases.
However, over the decades, almost all the prominent
infection-causing bacterial strains have developed resistance to
antibiotics.
[0006] Antibiotic resistance can result in severe adverse outcomes,
such as increased mortality, morbidity and medical care costs for
patients suffering from common infections, once easily treatable
with antibiotics (Am. J. Infect. Control 24 (1996), 380-388; Am. J.
Infect. Control 27 (1999), 520-532; Acar, J. F. (1997), Clin.
Infect. Dis. 24, Suppl 1, S17-S18; Cohen, M. L. (1992), Science
257, 1050-1055; Cosgrove, S. E. and Carmeli, Y. (2003), Clin.
Infect. Dis. 36, 1433-1437; Holmberg, S. D. et al. (1987), Rev.
Infect. Dis. 9, 1065-1078) and therefore became one of the most
recognized clinical problems of today's governmental, medicinal and
pharmaceutical research (U.S. Congress, Office of Technology
Assessment, Impacts of Antibiotic-Resistant Bacteria, OTA-H-629,
Washington, D.C., U.S. Government Printing Office (1995); House of
Lords, Science and Technology 7th Report: Resistance to Antibiotics
and Other Antimicrobial Agents, HL Paper 81-II, session (1997-98);
and Interagency Task Force on Antimicrobial Resistance, A Public
Health Action Plan to Combat Antimicrobial Resistance. Part 1:
Domestic issues).
[0007] Due to the limitations associated with the use of classical
antibiotics, extensive studies have been focused on finding novel,
efficient and non-resistance inducing antimicrobial/antibacterial
agents.
[0008] Within these studies, a novel class of short, naturally
occurring peptides, which exert outstanding
antimicrobial/antibacterial activity, was uncovered.
[0009] These peptides, which are known as antimicrobial peptides
(AMPs), are derived from animal sources and constitute a large and
diverse family of peptides, which may serve as effective
antimicrobial agents against antibiotic-resistant microorganisms
(for recent reviews see, for example, Levy, O. (2000) Blood 96,
2564-2572; Mor, A. (2000) Drug Development Research 50, 440-447;
Zasloff, M. (2002) New England Journal of Medicine 347, 1199-1200;
Zasloff, M. (2002) Nature 415, 389-395; Zasloff, M. (2002) Lancet
360, 1116-1117). In the past 20 years, over 700 AMPs derived from
various sources, from unicellular organisms to mammalians and
including humans, have been identified (for recent reviews see, for
example, Andreu, D. and Rivas, L. (1998) Biopolymers 47, 415-433;
Boman, H. G. (2003) J. Intern. Med. 254, 197-215; Devine, D. A. and
Hancock, R. E. (2002) Curr. Pharm. Des. 8, 703-714; Hancock, R. E.
and Lehrer, R. (1998) Trends Biotechnol. 16, 82-88; Hancock, R. E.
(2001) Lancet Infect. Dis. 1, 156-164; Hancock, R. E. and Rozek, A.
(2002) FEMS Microbiol. Lett. 206, 143-149; Hoffmann, J. A. and
Reichhart, J. M. (2002) Nat. Immunol. 3, 121-126; Lehrer, R. I. and
Ganz, T. (1999) Curr. Opin. Immunol. 11, 23-27; Nicolas, P. and
Mor, A. (1995) Annu. Rev. Microbiol. 49, 277-304; Nizet, V. and
Gallo, R. L. (2002) Trends Microbiol. 10, 358-359; Shai, Y. (2002)
Curr. Pharm. Des. 8, 715-725; Simmaco, M. et al. (1998) Biopolymers
47, 435-450; Tossi, A. et al. (2000) Biopolymers 55, 4-30; Tossi,
A. and Sandri, L. (2002) Curr. Pharm. Des. 8, 743-761; Vizioli, J.
and Salzet, M. (2002) Trends Pharmacol. Sci. 23, 494-496; Brogden,
K. et al. (2003) Int. J. Antimicrob. Agents 22, 465-478 and
Papagianni, M. (2003) Biotechnol. Adv. 21, 465-499).
[0010] AMPs are now recognized to have an important role in the
innate host defense. They display a large heterogeneity in primary
and secondary structures but share common features such as
amphiphatic character and net positive charge. These features
appear to form the basis for their cytolytic function. Ample data
indicate that AMPs cause cells death by destabilizing the ordered
structure of the cell membranes, although the detailed mechanism
has not been fully understood yet (for recent reviews see, for
example, Epand, R. M. et al. (1995), Biopolymers 37, 319-338;
Epand, R. M. and Vogel, H. J. (1999), Biochim. Biophys. Acta 1462,
11-28; Gallo, R. L. and Huttner, K. M. (1998), J. Invest Dermatol.
111, 739-743; Gennaro, R. et al. (2002), Curr. Pharm. Des. 8,
763-778; Hansen, J. N. (1994), Crit. Rev. Food Sci. Nutr. 34,
69-93; Huang, H. W. (1999), Novartis. Found. Symp. 225, 188-200;
Hwang, P. M. and Vogel, H. J. (1998), Biochem. Cell Biol. 76,
235-246; Lehrer, R. I. et al. (1993), Annu. Rev. Immunol. 11,
105-128; Matsuzaki, K. (1999), Biochim. Biophys. Acta 1462, 1-10;
Muller, F. M. et al. (1999), Mycoses 42 Suppl 2, 77-82;
Nissen-Meyer, J. and Nes, I. F. (1997), Arch. Microbiol. 167,
67-77; Peschel, A. (2002), Trends Microbiol. 10, 179-186; Sahl, H.
G. and Bierbaum, G. (1998), Annu. Rev. Microbiol. 52, 41-79; Shai,
Y. (1995), Trends Biochem. Sci. 20, 460-464; and Yeaman, M. R. and
Yount, N.Y. (2003), Pharmacol. Rev. 55, 27-55). It is assumed that
disturbance in membrane structure leads to leakage of small solutes
(for example K.sup.+, amino acids and ATP) rapidly depleting the
proton motive force, starving cells of energy and causing cessation
of certain biosynthetic processes (Sahl, H. G. and Bierbaum, G.
(1998), Annu. Rev. Microbiol. 52, 41-79). This mechanism is
consistent with the hypothesis that antimicrobial activity is not
mediated by interaction with a chiral center and may thus
significantly prevent antibiotic-resistance by circumventing many
of the mechanisms known to induce resistance.
[0011] In addition to their direct well-documented cytolytic
(membrane-disrupting) activity, AMPs also display a variety of
interesting biological activities in various antimicrobial fields.
Some AMPs were shown to activate microbicidal activity in cells of
the innate immunity including leukocytes and monocyte/macrophages
(Ammar, B. et al. (1998), Biochem. Biophys. Res. Commun. 247,
870-875; Salzet, M. (2002) Trends Immunol. 23, 283-284; Scott, M.
G. et al. (2000), J. Immunol. 165, 3358-3365; and Scott, M. G. et
al. (2002), J. Immunol. 169, 3883-3891). Many cationic peptides are
endowed with lipopolysaccharide binding activity, thus suppress the
production of inflammatory cytokines and protect from the cascade
of events that leads to endotoxic shock (Chapple, D. S. et al.
(1998), Infect. Immun. 66, 2434-2440; Elsbach, P. and Weiss, J.
(1998), Curr. Opin. Immunol. 10, 45-49; Lee, W. J. et al. (1998),
Infect. Immun. 66, 1421-1426; Giacometti, A. et al. (2003), J.
Chemother. 15, 129-133; Gough, M. et al. (1996), Infect. Immun. 64,
4922-4927; and Hancock, R. E. and Chapple, D. S. (1999),
Antimicrob. Agents Chemother. 43, 1317-1323). Antimicrobial genes
introduced into the genome of plants granted the plant the
resistance to pathogens by expressing the peptide (Alan, A. R. et
al. (2004), Plant Cell Rep. 22, 388-396; DeGray, G. et al. (2001),
Plant Physiol 127, 852-862; Fritig, B., Heitz, T. and Legrand, M.
(1998), Curr. Opin. Immunol. 10, 16-22; Osusky, M. et al. (2000),
Nat. Biotechnol. 18, 1162-1166; Osusky, M. et al. (2004),
Transgenic Res. 13, 181-190; and Powell, W. A. et al. (2000), Lett.
Appl. Microbiol. 31, 163-168).
[0012] On top of the ribosomally synthesized antimicrobial peptides
that have been identified and studied during the last 20 years,
thousands of de-novo designed AMPs, were developed (Tossi, A. et
al. (2000), Biopolymers 55, 4-30). These de-novo designed peptides
are comprised of artificially designed sequences and were produced
by genetic engineering or by chemical peptide syntheses. The
finding that various antimicrobial peptides, having variable
lengths and sequences, are all active at similar concentrations,
has suggested a general mechanism for the anti-bacterial activity
thereof rather than a specific mechanism that requires preferred
active structures (Shai, Y. (2002), Biopolymers 66, 236-248).
Naturally occurring peptides, and de-novo peptides having
artificially designed sequences, either synthesized by humans or
genetically engineered to be expressed in organisms, exhibit
various levels of antibacterial and antifungal activity as well as
lytic activity toward mammalian cells. As a result, AMPs are
attractive targets for bio-mimicry and peptidomimetic development,
as reproduction of critical peptide biophysical characteristics in
an unnatural, sequence-specific oligomer should presumably be
sufficient to endow antibacterial efficacy, while circumventing the
limitations associated with peptide pharmaceuticals (Latham, P. W.
(1999), Nat. Biotechnol. 17, 755-757).
[0013] One of the challenges in designing new antimicrobial
peptides relies on developing peptidomimetics that would have high
specificity toward bacterial or fungal cells, and consequently,
would allow better understanding of the mechanism underlying the
peptide lytic specificity, i.e., discrimination between cell
membranes. Structure-activity relationships (SAR) studies on AMPs
typically involve the systematic modification of naturally
occurring molecules or the de-novo design of model peptidomimetics
predicted to form amphiphatic alpha-helices or beta-sheets, and the
determination of structure and activity via various approaches
(Tossi, A. et al. (2000), Biopolymers 55, 4-30), as follows:
[0014] Minimalist methods for designing de-novo peptides are based
on the requirement for an amphiphatic, alpha-helical or beta-sheet
structure. The types of residues used are generally limited to the
basic, positively charged amino acids lysine or arginine, and one
to three of the hydrophobic residues alanine, leucine, isoleucine,
glycine, valine, phenylalanine, or tryptophan (Blazyk, J. et al.
(2001), J. Biol. Chem. 276, 27899-27906; Epand, R. F. et al.
(2003), Biopolymers 71, 2-16; Hong, J. et al. (1999), Biochemistry
38, 16963-16973; Jing, W. et al. (2003), J. Pept. Res. 61, 219-229;
Ono, S. et al. (1990), Biochim. Biophys. Acta 1022, 237-244; and
Stark, M. et al. (2002), Antimicrob. Agents Chemother. 46,
3585-3590). While these approaches may lead to the design of potent
antimicrobial agents, subtleties to the sequence of AMPs that may
have been selected for by evolution are not considered and their
absence may lead to a loss of specificity.
[0015] Sequence template methods for designing and synthesizing
amphiphatic AMPs typically consists of extracting sequence patterns
after comparison of a large series of natural counterparts. The
advantage of this method, as compared with conventional sequence
modification methods, is that it reduces the number of peptides
that need to be synthesized in order to obtain useful results,
while maintaining at least some of the sequence based information.
As discussed hereinabove, the latter is lost in minimalist
approaches (Tiozzo, E. et al. (1998), Biochem. Biophys. Res.
Commun. 249, 202-206).
[0016] Sequence modification method includes all of the known and
acceptable methods for modifying natural peptides, e.g., by
removing, adding, or replacing one or more residues, truncating
peptides at the N- or C-termini, or assembling chimeric peptides
from segments of different natural peptides. These modifications
have been extensively applied in the study of dermaseptins,
cecropins, magainins, and melittins in particular (Scott, M. G. et
al. (2000), J. Immunol. 165, 3358-3365; Balaban, N. et al. (2004),
Antimicrob. Agents Chemother. 48, 2544-2550; Coote, P. J. et al.
(1998), Antimicrob. Agents Chemother. 42, 2160-2170; Feder, R. et
al. (2000), J. Biol. Chem. 275, 4230-4238; Gaidukov, L. et al.
(2003), Biochemistry 42, 12866-12874; Kustanovich, I. et al.
(2002), J. Biol. Chem. 277, 16941-16951; Mor, A. and Nicolas, P.
(1994) J. Biol. Chem. 269, 1934-1939; Mor, A. et al. (1994), J.
Biol. Chem. 269, 31635-31641; Oh, D. et al. (2000), Biochemistry
39, 11855-11864; Patrzykat, A. et al. (2002), Antimicrob. Agents
Chemother. 46, 605-614; Piers, K. L. and Hancock, R. E. (1994) Mol.
Microbiol. 12, 951-958; and Shepherd, C. M. et al. (2003),
Biochemistry 370, 233-243).
[0017] The approaches described above have been applied in many
studies aiming at designing novel AMPs. In these studies, the use
of alpha-helix and/or beta-sheet inducing building blocks, the use
of the more flexible beta-amino acid building blocks, the use of
mixed D- and L-amino acid sequences and the use of facially
amphiphilic arylamide polymers, have all demonstrated the
importance of induced amphiphatic conformations on the biological
activity of AMPs.
[0018] Antimicrobial peptides can act in synergy with classical
antibiotics, probably by enabling access of antibiotics into the
bacterial cell (Darveau, R. P. et al. (1991), Antimicrob. Agents
Chemother. 35, 1153-1159; and Giacometti, A. et al. (2000), Diagn.
Microbiol. Infect. Dis. 38, 115-118). Other potential uses include
food preservation (Brul, S, and Coote, P. (1999), Int. J. Food
Microbiol. 50, 1-17; Yaron, S., Rydlo, T. et al. (2003), Peptides
24, 1815-1821; Appendini, P. and Hotchkiss, J. H. (2000), J. Food
Prot. 63, 889-893; and Johnsen, L. et al. (2000), Appl. Environ.
Microbiol. 66, 4798-4802), imaging probes for detection of
bacterial or fungal infection loci (Welling, M. M. et al. (2000),
Eur. J. Nucl. Med. 27, 292-301; Knight, L. C. (2003), Q. J. Nucl.
Med. 47, 279-291; and Lupetti, A. et al. (2003), Lancet Infect.
Dis. 3, 223-229), antitumor activity (Baker, M. A. et al. (1993),
Cancer Res. 53, 3052-3057; Jacob, L. and Zasloff, M. (1994), Ciba
Found. Symp. 186, 197-216; Johnstone, S. A. et al. (2000),
Anticancer Drug Des 15, 151-160; Moore, A. J. et al. (1994), Pept.
Res. 7, 265-269; and Papo, N. and Shai, Y. (2003), Biochemistry 42,
9346-9354), mitogenic activity (Aarbiou, J. et al. (2002), J.
Leukoc. Biol. 72, 167-174; Murphy, C. J. et al. (1993), J. Cell
Physiol 155, 408-413; and Gudmundsson, G. H. and Agerberth, B.
(1999), J. Immunol. Methods 232, 45-54) and lining of
medical/surgical devices (Haynie, S. L. et al. (1995), Antimicrob.
Agents Chemother. 39, 301-307).
[0019] However, while the potential of AMPs as new therapeutic
agents is well recognized, the use of the presently known AMPs is
limited by lack of adequate specificity, and optional systemic
toxicity (House of Lords, Science and Technology 7th Report:
Resistance to antibiotics and other antimicrobial agents. HL Paper
81-II, session, 1997-98; and Alan, A. R. et al. (2004), Plant Cell
Rep. 22, 388-396). Thus, there is a clear need for developing new
antimicrobial peptides with improved specificity and toxicity
profile.
[0020] Moreover, although peptides are recognized as promising
therapeutic and antimicrobial agents, their use is severely limited
by their in vivo and ex vivo instability and by poor
pharmacokinetics. Peptides and polypeptides are easily degraded in
oxidative and acidic environments and therefore typically require
intravenous administration (so as to avoid, e.g., degradation in
the gastrointestinal tract). Peptides are further broken down in
the blood system by proteolytic enzymes and are rapidly cleared
from the circulation. Moreover, peptides are typically
characterized by poor absorption after oral ingestion, in
particular due to their relatively high molecular mass and/or the
lack of specific transport systems. Furthermore, peptides are
characterized by high solubility and therefore fail to cross
biological barriers such as cell membranes and the blood brain
barrier, but exhibit rapid excretion through the liver and kidneys.
The therapeutic effect of peptides is further limited by the high
flexibility thereof, which counteracts their receptor-affinity due
to the steep entropy decrease upon binding and a considerable
thermodynamic energy cost. In addition, peptides are heat and
humidity sensitive and therefore their maintenance requires costly
care, complex and inconvenient modes of administration, and
high-cost of production and maintenance. The above disadvantages
impede the use of peptides and polypeptides as efficient drugs and
stimulate the quest for an alternative, which oftentimes involves
peptidomimetic compounds.
[0021] Peptidomimetic compounds are modified polypeptides which are
designed to have a superior stability, both in vivo and ex vivo,
and yet at least the same receptor affinity, as compared with their
parent peptides. In order to design efficacious peptidomimetics, an
utmost detailed three-dimensional understanding of the interaction
with the intended target is therefore required.
[0022] One method attempting at achieving the above goal utilizes
synthetic combinatorial libraries (SCLs), a known powerful tool for
rapidly obtaining optimized classes of active compounds. Thus, a
number of novel antimicrobial compounds ranging from short peptides
to small heterocyclic molecules have been identified from SCLs
(Blondelle, S. E. and Lohner, K. (2000), Biopolymers 55,
74-87).
[0023] Several families of naturally occurring modified peptides
which exhibit strong antimicrobial activity, have been uncovered in
many organisms. These compounds, and their effective chemical
alterations, have proposed a lead towards a general solution to the
challenge of creating an antimicrobial compound devoid of the
disadvantages associated with natural AMPs.
[0024] Thus, for example, naturally occurring short antimicrobial
peptides characterized by a lipophilic acyl chain at the N-terminus
were uncovered in various microorganisms (Bassarello, C. et al.
(2004), J. Nat. Prod. 67, 811-816; Peggion, C., et al. (2003), J.
Pept. Sci. 9, 679-689; and Toniolo, C. et al. (2001), Cell Mol.
Life. Sci. 58, 1179-1188). Acylation of AMPs was hence largely used
as a technique to endow AMPs with improved antimicrobial
characteristics (Avrahami, D. et al. (2001), Biochemistry 40,
12591-12603; Avrahami, D. and Shai, Y. (2002), Biochemistry 41,
2254-2263; Chicharro, C. et al. (2001), Antimicrob. Agents
Chemother. 45, 2441-2449; Chu-Kung, A. F. et al. (2004), Bioconjug.
Chem. 15, 530-535; Efron, L. et al. (2002), J. Biol. Chem. 277,
24067-24072; Lockwood, N. A. et al. (2004), Biochem. J. 378,
93-103; Mak, P. et al. (2003), Int. J. Antimicrob. Agents 21,
13-19; and Wakabayashi, H. et al. (1999), Antimicrob. Agents
Chemother. 43, 1267-1269). However, some studies indicate that
attaching a hydrocarbon chain to the peptide, results in only
marginal increase in the affinity of the lipopeptide to the
membrane (Epand, R. M. (1997), Biopolymers 43, 15-24).
[0025] One family of AMPs capable of alluding towards the main goal
is the family of dermaseptins. Dermaseptins are peptides isolated
from the skin of various tree frogs of the Phyllomedusa species
(Brand, G. D. et al. (2002), J. Biol. Chem. 277, 49332-49340;
Charpentier, S. et al. (1998), J. Biol. Chem. 273, 14690-14697;
Mor, A. et al. (1991), Biochemistry 30, 8824-8830; Mor, A. et al.
(1994), Biochemistry 33, 6642-6650; Mor, A. and Nicolas, P. (1994),
Eur. J. Biochem. 219, 145-154; and Wechselberger, C. (1998),
Biochim. Biophys. Acta 1388, 279-283). These are structurally and
functionally related cationic peptides, typically having 24-34
amino acid residues. Dermaseptins were found to exert rapid
cytolytic activity, from seconds to minutes, in vitro, against a
variety of microorganisms including viruses, bacteria, protozoa,
yeast and filamentous fungi (Coote, P. J. et al. (1998),
Antimicrob. Agents Chemother. 42, 2160-2170; Mor, A. and Nicolas,
P. (1994), J. Biol. Chem. 269, 1934-1939; Mor, A. et al. (1994), J.
Biol. Chem. 269, 31635-31641; Mor, A. and Nicolas, P. (1994), Eur.
J. Biochem. 219, 145-154; Belaid, A. et al. (2002), J. Med. Virol.
66, 229-234; De Lucca, A. J. et al. (1998), Med. Mycol. 36,
291-298; Hernandez, C. et al. (1992), Eur. J. Cell Biol. 59,
414-424; and Mor, A. et al. (1991), J. Mycol. Med. 1, 5-10) as well
as relatively inaccessible pathogens such as intracellular
parasites (Efron, L. et al. (2002), J. Biol. Chem. 277,
24067-24072; Dagan, A. et al. (2002), Antimicrob. Agents Chemother.
46, 1059-1066; Ghosh, J. K. et al. (1997), J. Biol. Chem. 272,
31609-31616; and Krugliak, M. et al. (2000), Antimicrob. Agents
Chemother. 44, 2442-2451).
[0026] Since dermaseptins portray the biodiversity existing in a
very large group of antimicrobial peptides in terms of structural
and biological properties, they serve as a general model system for
understanding the function(s) of cationic antimicrobial
peptides.
[0027] The 28-residue peptide dermaseptin S4 is known to bind
avidly to biological membranes and to exert rapid cytolytic
activity against a variety of pathogens as well as against
erythrocytes (Mor, A. et al. (1994), J. Biol. Chem. 269(50):
31635-41).
[0028] In a search for an active derivative (peptidomimetic) of S4,
a 28-residue derivative in which the amino acid residues at the
fourth and twentieth positions were replaced by lysine residues,
known as K.sub.4K.sub.20--S4, and two short derivatives of 16 and
13 residues in which the amino acid residue at the fourth position
was replaced by a lysine residue, known as K.sub.4--S4(1-16) and
K.sub.4--S4(1-13), respectively, were prepared and tested for the
inhibitory effect thereof (Feder, R. et al. (2000), J. Biol. Chem.
275, 4230-4238). The minimal inhibitory concentrations (MICS) of
these derivatives for 90% of the 66 clinical isolates tested (i.e.,
MIC.sub.90 for S. aureus, P. aeruginosa and E. coli), varied
between 2 and 8 .mu.g/ml for the various species, whereby the
13-mer derivative K.sub.4--S4(1-13) was found to be significantly
less hemolytic when incubated with human erythrocytes, as compared
with similarly active derivatives of magainin and protegrin, two
confirmed antimicrobial peptide families (Fahrner, R. L. et al.
(1996), Chem. Biol. 3(7): 543-50; Zasloff, M. et al. (1988), Proc.
Natl. Acad. Sci. U S A 85(3): 910-3; Yang L. et al. (2000),
Biophys. J., 79 2002-2009). Additional studies further confirmed
that short, lysine-enriched S4 derivatives, are promising
anti-microbial agents by being characterized by reduced toxicity
and by showing efficacy also after pre-exposure of the subjects
thereto.
[0029] N-terminal acylation of the C-terminally truncated 13-mer S4
derivative K.sub.4--S4(1-13) also resulted in reduced hemolytic
activity, whereby several derivatives, such as its aminoheptanoyl
derivative, displayed potent and selective activity against the
intracellular parasite, i.e., increased antiparasitic efficiency
and reduced hemolysis. These studies indicate that increasing the
hydrophobicity of anti-microbial peptides enhance their
specificity, presumably by allowing such AMPs to act specifically
on the membrane of intracellular parasites and thus support a
proposed mechanism according to which the lipopeptide crosses the
host cell plasma membrane and selectively disrupts the parasite
membrane(s).
[0030] Overall, the data collected from in-vitro and in-vivo
experiments indicated that some dermaseptin derivatives could be
useful in the treatment of a variety of microbial-associated
conditions including infections caused by multidrug-resistant
pathogens. These agents were found highly efficacious, and no
resistance was appeared to develop upon their administration.
Nevertheless, the therapeutic use of these agents is still limited
by the in vivo and ex vivo instability thereof, by poor
pharmacokinetics, and by other disadvantageous characteristics of
peptides, as discussed hereinabove.
[0031] In conclusion, most of the presently known antimicrobial
peptides and peptidomimetics are of limited utility as therapeutic
agents despite their promising antimicrobial activity. The need for
compounds which have AMP characteristics, and are devoid of the
limitations associated with AMPs is still present, and the concept
of providing chemically and metabolically-stable active compounds
in order to achieve enhanced specificity and hence enhanced
clinical selectivity has been widely recognized.
[0032] There is thus a widely recognized need for, and it would be
highly advantageous to have, novel, metabolically-stable, non-toxic
and cost-effective antimicrobial agents devoid of the above
limitations.
SUMMARY OF THE INVENTION
[0033] The present inventors have now designed and successfully
prepared a novel class of polymeric compounds, which are based on
positively charged amino acid residues and hydrophobic moieties.
These novel polymers were found highly efficient as selective
antimicrobial agents, while being devoid of toxicity and resistance
induction.
[0034] Thus, according to one aspect of the present invention there
is provided a polymer which includes two or more amino acid
residues and one or more hydrophobic moiety residues, wherein one
or more of the hydrophobic moiety residues is being covalently
linked to at least two amino acid residues via the N-alpha of one
amino acid residue and via the C-alpha of another amino acid
residue.
[0035] According to further features in preferred embodiments of
the invention described below, the polymer is having an
antimicrobial activity.
[0036] According to still further features in the described
preferred embodiments the polymer is capable of selectively
destructing at least a portion of the cells of a pathogenic
microorganism.
[0037] According to still further features in the described
preferred embodiments the pathogenic microorganism is selected from
the group consisting of a prokaryotic organism, an eubacterium, an
archaebacterium, a eukaryotic organism, a yeast, a fungus, an alga,
a protozon and a parasite.
[0038] According to still further features in the described
preferred embodiments the polymer includes at least two hydrophobic
moiety residues, wherein one or more of the hydrophobic moiety
residues are linked to the N-alpha of an amino acid residue at the
N-terminus of one of the amino acid residues and/or the C-alpha of
another amino acid residue at the C-terminus.
[0039] According to still further features in the described
preferred embodiments the polymer includes two or more hydrophobic
moiety residues, wherein one or more of the hydrophobic moiety
residues are linked to the side-chain of an amino acid residue in
the polymer.
[0040] According to still further features in the described
preferred embodiments one or more of the amino acid residues is a
positively charged amino acid residue.
[0041] According to still further features in the described
preferred embodiments the positively charged amino acid residue is
selected from the group consisting of a histidine residue, a lysine
residue, an ornithine residue and an arginine residue.
[0042] According to yet further features of the present invention,
one or more of the hydrophobic moiety residues is linked to one or
more of the amino acid residues via a peptide bond.
[0043] According to still further features in the described
preferred embodiments one or more of the hydrophobic moiety
residues is linked to two amino acid residues via a peptide
bond.
[0044] According to still further features in the described
preferred embodiments one or more of the hydrophobic moiety
residues is linked to each of the amino acid residues via a peptide
bond.
[0045] According to still further features in the described
preferred embodiments one or more of the hydrophobic moiety
residues is linked to the N-alpha of the amino acid residue via a
peptide bond.
[0046] According to still further features in the described
preferred embodiments one or more of the hydrophobic moiety
residues is linked to the C-alpha of the amino acid residue via a
peptide bond.
[0047] According to still further features in the described
preferred embodiments one or more of the hydrophobic moieties has a
carboxylic group at one end thereof and an amine group at the other
end thereof.
[0048] According to still further features in the described
preferred embodiments the polymer includes from 2 to 50 amino acid
residues, preferably from 2 to 12 amino acid residues and more
preferably from 2 to 8 amino acid residues.
[0049] According to still further features in the described
preferred embodiments the polymer includes from 1 to 50 hydrophobic
moiety residues, preferably from 1 to 12 hydrophobic moiety
residues and more preferably from 1 to 8 hydrophobic moiety
residues.
[0050] According to still further features in the described
preferred embodiments the hydrophobic moiety residue includes one
or more hydrocarbon chains which have from 4 to 30 carbon
atoms.
[0051] According to still further features in the described
preferred embodiments the hydrophobic moiety residue includes one
or more fatty acid residues which are selected from the group
consisting of an unbranched saturated fatty acid residue, a
branched saturated fatty acid residue, an unbranched unsaturated
fatty acid residue, a branched unsaturated fatty acid residue and
any combination thereof, and the fatty acid residue has from 4 to
30 carbon atoms.
[0052] According to still further features in the described
preferred embodiments the fatty acid residue is selected from the
group consisting of a butyric acid residue, a caprylic acid residue
and a lauric acid residue.
[0053] According to still further features in the described
preferred embodiments one or more of the hydrophobic moieties is an
.omega.-amino-fatty acid residue. The .omega.-amino-fatty acid
residue is selected from the group consisting of 4-amino-butyric
acid, 6-amino-caproic acid, 8-amino-caprylic acid, 10-amino-capric
acid, 12-amino-lauric acid, 14-amino-myristic acid,
16-amino-palmitic acid, 18-amino-stearic acid, 18-amino-oleic acid,
16-amino-palmitoleic acid, 18-amino-linoleic acid,
18-amino-linolenic acid and 20-amino-arachidonic acid. Preferably,
the .omega.-amino-fatty acid residue is selected from the group
consisting of 4-amino-butyric acid, 8-amino-caprylic acid and
12-amino-lauric acid.
[0054] According to still further features in the described
preferred embodiments all of the amino acid residues of the polymer
are positively charged amino acid residues, such as lysine
residues, histidine residues, ornithine residues, arginine residues
and any combinations thereof.
[0055] According to still further features in the described
preferred embodiments all the positively charged amino acid
residues are lysine residues.
[0056] According to still further features of the preferred
embodiments of the invention described below, the polymer further
includes one or more active agent attached thereto.
[0057] According to still further features in the described
preferred embodiments the active agent is attached to a side chain
of an amino acid residue, either via the N-alpha of the amino acid
residue at the N-terminus and/or the C-alpha of the amino acid
residue at the C-terminus, and/or to one or more of the hydrophobic
moiety residues of the polymer.
[0058] According to still further features in the described
preferred embodiments the active agent is a labeling agent, which
is selected from the group consisting of a fluorescent agent, a
radioactive agent, a magnetic agent, a chromophore, a
phosphorescent agent and a heavy metal cluster.
[0059] According to still further features in the described
preferred embodiments the active agent comprises at least one
therapeutically active agent, which is selected from the group
consisting of an agonist residue, an amino acid residue, an
analgesic residue, an antagonist residue, an antibiotic agent
residue, an antibody residue, an antidepressant agent, an antigen
residue, an anti-histamine residue, an anti-hypertensive agent, an
anti-inflammatory drug residue, an anti-metabolic agent residue, an
antimicrobial agent residue, an antioxidant residue, an
anti-proliferative drug residue, an antisense residue, a
chemotherapeutic drug residue, a co-factor residue, a cytokine
residue, a drug residue, an enzyme residue, a growth factor
residue, a heparin residue, a hormone residue, an immunoglobulin
residue, an inhibitor residue, a ligand residue, a nucleic acid
residue, an oligonucleotide residue, a peptide residue, a
phospholipid residue, a prostaglandin residue, a protein residue, a
toxin residue, a vitamin residue and any combination thereof.
[0060] According to still further features in the described
preferred embodiments the polymer is capable of delivering one or
more active agents, such as a labeling agent or a therapeutically
active agent, to at least a portion of the cells of a pathogenic
microorganism as described herein.
[0061] According to still further features in the described
preferred embodiments the polymers are selected from the compounds
presented in Table 3 hereinbelow.
[0062] According to still further features in the described
preferred embodiments the polymer described herein can be
represented by the general formula I:
X--W.sub.0-[A.sub.1-Z.sub.1-D.sub.1]-W.sub.1-[A.sub.2-Z.sub.2-D.sub.2]-W-
.sub.2-- . . . [An-Zn-Dn]-Wn-Y Formula I
[0063] wherein:
[0064] n is an integer from 2 to 50, preferably from 2 to 12 and
more preferably from 2 to 8;
[0065] A.sub.1, A.sub.2, . . . , An are each independently an amino
acid residue, preferably a positively charged amino acid residue,
and more preferably all of A.sub.1, A.sub.2, . . . , An are
positively charged amino acid residues as discussed hereinabove,
such as histidine residues, lysine residues, ornithine residues and
arginine residues;
[0066] D.sub.1, D.sub.2, . . . , Dn are each independently a
hydrophobic moiety residue, as described herein, or absent,
provided that at least one such hydrophobic moiety residue exists
in the polymer, and preferably at least one of the hydrophobic
moiety residues is a .omega.-amino-fatty acid residue;
[0067] Z.sub.1, Z.sub.2, . . . Zn and W.sub.0, W.sub.1, W.sub.2, .
. . , Wn are each independently a linking moiety linking an amino
acid residue and a hydrophobic moiety residue or absent, preferably
at least one of the linking moieties is a peptide bond and most
preferable all the linking moieties are peptide bonds;
[0068] X and Y may each independently be hydrogen, an amine, an
amino acid residue, a hydrophobic moiety residue, another polymer
having the general Formula I or absent.
[0069] According to still further features in the described
preferred embodiments the polymer further includes one or more
active agent, as described herein, attached to one or more of
either X, Y, W.sub.0, A.sub.1, An and/or Wn.
[0070] According to another aspect of the present invention there
is provided a conjugate which includes an amino acid residue and a
hydrophobic moiety residue attached to the N-alpha or the C-alpha
of the amino acid residue, the hydrophobic moiety residue being
designed capable of forming a bond with an N-alpha or a C-alpha of
an additional amino acid residue.
[0071] According to further features in the preferred embodiments
of the invention described below, the hydrophobic moiety residue is
attached to the N-alpha or the C-alpha of the amino acid residue
via a peptide bond.
[0072] According to still further features in the described
preferred embodiments the hydrophobic moiety has a carboxylic group
at one end thereof and an amine group at the other end thereof and
further includes a hydrocarbon chain as described herein.
[0073] According to still further features in the described
preferred embodiments the hydrophobic moiety includes a fatty acid
residue as described herein.
[0074] According to still further features in the described
preferred embodiments the hydrophobic moiety is an
.omega.-amino-fatty acid residue as described herein.
[0075] According to still another aspect of the present invention
there is provided a process of preparing the conjugate described
hereinabove, the process comprises providing an amino acid;
providing a hydrophobic moiety having a first functional group that
is capable of reacting with an N-alpha of an amino acid residue
and/or a second functional group capable of reacting with a C-alpha
of an amino acid; linking the first functional group in the
hydrophobic moiety to the amino acid via the N-alpha of said amino
acid; or linking the second functional group in the hydrophobic
moiety to the amino acid via the C-alpha of the amino acid.
Preferably the hydrophobic moiety is linked to the amino acid via a
peptide bond.
[0076] According to further features in the preferred embodiments
of the invention described below, the amino acid is a positively
charged amino acid such as, for example, histidine, lysine,
ornithine and arginine.
[0077] According to further features in the preferred embodiments
all the positively charged amino acids are lysines.
[0078] According to still further features in the described
preferred embodiments the hydrophobic moiety has a carboxylic group
at one end thereof, an amine group at the other end thereof and a
hydrocarbon chain, as described herein.
[0079] According to still further features in the described
preferred embodiments the hydrophobic moiety includes a fatty acid
residue as described herein.
[0080] According to still further features in the described
preferred embodiments the hydrophobic moiety is an
.omega.-amino-fatty acid residue as described herein.
[0081] According to still further features in the described
preferred embodiments the hydrophobic moiety is a 8-amino-caprylic
acid.
[0082] According to still further features in the described
preferred embodiments, n is an integer from 6 to 8.
[0083] According to still further features in the described
preferred embodiments, the polymer has the formula:
##STR00001##
[0084] According to still further features in the described
preferred embodiments, the polymer has the formula:
##STR00002##
[0085] According to yet another aspect of the present invention
there is provided a pharmaceutical composition which includes as an
active ingredient the polymer of the present invention, described
herein, and a pharmaceutically acceptable carrier.
[0086] According to further features in the preferred embodiments
of the invention described below, the pharmaceutical composition is
packaged in a packaging material and identified in print, in or on
said packaging material, for use in the treatment of a medical
condition associated with a pathogenic microorganism such as a
prokaryotic organism, an eubacterium, an archaebacterium, a
eukaryotic organism, a yeast, a fungus, an alga, a protozon and a
parasite.
[0087] According to still further features in the described
preferred embodiments the pharmaceutical composition further
includes one or more additional therapeutically active agent as
described herein, whereby preferably the therapeutically active
agent includes an antibiotic agent.
[0088] According to another aspect of the present invention there
is provided a method of treating a medical condition associated
with a pathogenic microorganism, as described herein, the method
includes administering to a subject in need thereof a
therapeutically effective amount of the polymer described
herein.
[0089] According to further features in the preferred embodiments
of the invention described below, the administration is effected
orally, rectally, intravenously, topically, intranasally,
intradermally, transdermally, subcutaneously, intramuscularly,
intraperitoneally or by intrathecal catheter.
[0090] According to still further features in the described
preferred embodiments the method further includes administering to
the subject one or more therapeutically active agent as described
herein, preferably, an antibiotic agent.
[0091] According to still further features in the described
preferred embodiments the polymer of the present invention is
administered either per se or as a part of a pharmaceutical
composition; the pharmaceutical composition further includes a
pharmaceutically acceptable carrier, as described herein.
[0092] According to an additional aspect of the present invention
there is provided a medical device which includes the polymer of
the present invention and a delivery system configured for
delivering the polymer to a bodily site of a subject.
[0093] According to further features in the preferred embodiments
of the invention described below, the polymer forms a part of a
pharmaceutical composition, and the pharmaceutical composition
further includes a pharmaceutically acceptable carrier.
[0094] According to still further features in the described
preferred embodiments the delivery is effected by inhalation, and
the delivery system is selected from the group consisting of a
metered dose inhaler, a respirator, a nebulizer inhaler, a dry
powder inhaler, an electric warmer, a vaporizer, an atomizer and an
aerosol generator.
[0095] According to still further features in the described
preferred embodiments the delivery is effected transdermally, and
the delivery system is selected from the group consisting of an
adhesive plaster and a skin patch.
[0096] According to still further features in the described
preferred embodiments the delivery is effected topically and the
delivery system is selected from the group consisting of an
adhesive strip, a bandage, an adhesive plaster, a wound dressing
and a skin patch.
[0097] According to still further features in the described
preferred embodiments the delivery is effected by implanting the
medical device in a bodily organ. Preferably the delivery system
further includes a biocompatible matrix which in turn includes a
biodegradable polymer and further includes a slow release
carrier.
[0098] According to still an additional aspect of the present
invention there is provided a food preservative which includes an
effective amount of the polymer of the present invention, and
preferably further includes an edible carrier.
[0099] According to a further aspect of the present invention there
is provided an imaging probe for detecting a pathogenic
microorganism as described herein, which includes a polymer as
described herein, and one or more labeling agent, as described
herein, attached thereto.
[0100] According to further features in the preferred embodiments
of the invention described below, the labeling agent(s) is attached
to a side chain of an amino acid residue, a C-terminus and/or a
N-terminus of the polymer and/or one of the hydrophobic residues of
the polymer of the present invention.
[0101] The present invention successfully addresses the
shortcomings of the presently known configurations by providing a
novel class of antimicrobial polymers, which combine the merits of
therapeutically active antimicrobial peptides, e.g., high efficacy
and specificity, without exhibiting the disadvantages of
peptides.
[0102] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
case of conflict, the patent specification, including definitions,
will control. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0103] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0104] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0105] In the drawings:
[0106] FIG. 1 presents a cumulative bar graph demonstrating the
high correlation between the antimicrobial activity and the
hydrophobicity of exemplary polymers according to the present
invention, by marking the polymers which exhibited a significant
microbial activity (MIC value of less than 50 .mu.M) against E.
coli (in red bars), P. aeruginosa (in yellow bars),
methicilin-resistant S. aureus (in blue bars) and B. cereus (in
green bars), on the scale of the acetonitrile percentages in the
mobile phase at which the polymers were eluted on a reverse phase
HPLC column;
[0107] FIG. 2 presents a cumulative bar graph demonstrating the
lack of correlation between the antimicrobial activity and the net
positive charge of exemplary polymers according to the present
invention, by marking the polymers which exhibited a significant
microbial activity (MIC value of less than 50 .mu.M) against E.
coli (in red bars), P. aeruginosa (in yellow bars),
methicilin-resistant S. aureus (in blue bars) and B. cereus (in
green bars), over bins representing the net positive charge from +9
to +1;
[0108] FIGS. 3(a-c) presents a bar graph demonstrating the
non-resistance inducing effect of exemplary polymers according to
the present invention, by measuring MICs level evolution on E. coli
after 10 iterations of successive exposures of bacteria to
sub-lytic concentrations of K(NC.sub.12K).sub.3NH.sub.2 and
C.sub.12K(NC.sub.8K).sub.5NH.sub.2, as compared to exposures to
three classical antibiotic agents, tetracycline, gentamycin and
ciprofloxacin (FIG. 3a), and on methicilin-resistant S. aureus
after 15 iterations of successive exposures of bacteria to
sub-lytic concentrations of C.sub.12KKNC.sub.12KNH.sub.2, as
compared to exposures to two antibiotic agents, rifampicin and
tetracycline (FIG. 3b), and the development of resistance of E.
coli to C.sub.12K(NC.sub.8K).sub.7NH.sub.2, evaluated during 15
serial passages, as compared to exposures to three classical
antibiotic agents, ciprofloxacin, imipenem, and tetracycline (FIG.
3c) (the relative MIC is the normalized ratio of the MIC obtained
for a given subculture to the concomitantly determined MIC obtained
on bacteria harvested from control wells (wells cultured without
antimicrobial agent) from the previous generation;
[0109] FIG. 4 presents comparative plots demonstrating the kinetic
bactericidal effect of C.sub.12K(NC.sub.8K).sub.5NH.sub.2, an
exemplary polymer according to the present invention, on E. coli.
incubated in the presence of the polymer, with colony forming units
(CFU) counts performed after the specified incubation periods and
compared in a dose-dependent experiment at zero (control), 3 and 6
multiples of the minimal inhibitory concentration (MIC) value (3.1
.mu.M) in LB medium at 37.degree. C.;
[0110] FIG. 5 presents comparative plots demonstrating the kinetic
bactericidal effect of C.sub.12K(NC.sub.8K).sub.7NH.sub.2, an
exemplary polymer according to the present invention (black
triangles), on E. coli., compared with normal bacterial growth
control (black circles), and with kinetic bactericidal effect of
while Imipenem (white squares) and Ciprofloxacin (black squares),
as determined at a concentration corresponding to six multiples of
their respective MIC value (plotted values represent the
mean.+-.standard deviations obtained from at least two independent
experiments);
[0111] FIG. 6 presents comparative plots demonstrating the
hemolytic effect of C.sub.12K(NC.sub.8K).sub.7NH.sub.2, an
exemplary polymer according to the present invention, compared with
the hemolytic effect of bivalirudin, a synthetic peptide and FDA
approved thrombin inhibitor, and with the hemolytic effect of
MSI-78, a magainin derivative, determined against human RBC (10%
hematocrit) after 1 hour incubation at 37.degree. C. in the
presence of 31 .mu.M (striped bars), 94 .mu.M (gray bars) and 156
.mu.M (white bars) polymer/peptide concentration (plotted values
represent the mean.+-.standard deviations obtained from at least
four independent experiments);
[0112] FIG. 7 presents the circular dichroism spectra of two
exemplary polymers according to the present invention,
C.sub.12K(NC.sub.8K).sub.5NH.sub.2 and
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, taken in the designated media
at polymer concentration of 100 .mu.M (liposome concentration of 2
mM), expressed as mean residue molar ellipticity, and compared with
a 15-residue control peptide, an acylated dermaseptin S4 derivative
(data represent average values from three separate recordings);
[0113] FIG. 8 presents the circular dichroism spectra of
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, another exemplary polymer
according to the present invention (gray lines), and of a control
antimicrobial peptide K.sub.4S4(1-16) (black lines), taken in PBS
alone (dashed lines) or in the presence of 2 mM POPC:POPG (3:1)
liposomes concentration suspended in PBS (solid lines) (data
represent average values from three separate recordings);
[0114] FIG. 9 presents association and dissociation curves (binding
rates) obtained by surface plasmon resonance (SPR) measurements,
demonstrating the membrane binding properties of various doses
(0.21, 0.42, 0.84, 1.67, and 3.35 .mu.g) of
C.sub.12K(NC.sub.8K).sub.5NH.sub.2, an exemplary polymer according
to the present invention, to a model membrane (K.sub.app is the
resulting binding constants calculated assuming a 2-step
model);
[0115] FIG. 10 presents a bar graph demonstrating the binding of
exemplary polymers according to the present invention, denoted as
KNC.sub.8KNH.sub.2, K(NC.sub.8K).sub.2NH.sub.2,
K(NC.sub.8K).sub.3NH.sub.2, K(NC.sub.8K).sub.6NH.sub.2,
KNC.sub.12KNH.sub.2, K(NC.sub.12K).sub.2NH.sub.2 and
K(NC.sub.12K).sub.3NH.sub.2, to lipopolysaccharide, as measured by
SPR, wherein the weaker binding of the polymers to liposomes after
incubation with LPS substantiates that the polymers are bound to
the LPS;
[0116] FIG. 11 presents a photograph of a UV illuminated 1% agarose
gel electrophoresis, demonstrating the DNA binding characteristics
of C.sub.12KKNC.sub.12KNH.sub.2, K(NC.sub.4K).sub.7NH.sub.2 and
C.sub.12K(NC.sub.8K).sub.5NH.sub.2, exemplary polymers according to
the present invention, as measured by DNA retardation assay after
the polymers were incubated for 30 minutes at room temperature at
the specified DNA/polymer ratios (w:w) using 200 nanograms of
plasmid (normal migration in absence of the polymer of the plasmid
pUC19 is shown in leftmost lane);
[0117] FIG. 12 presents comparative plots demonstrating the
antimicrobial activity of C.sub.8K(NC.sub.8K).sub.7NH.sub.2, an
exemplary polymer according to the present invention (in black
circles), against the micro-flora found in human saliva, as
compared to 113-367, a peptide with known antimicrobial activity
(in white circles) and the vehicle buffer as control (white
triangle) in logarithmic units of CFU per ml versus incubation
time;
[0118] FIG. 13 presents a comparative plot demonstrating the
anti-malarial activity of C.sub.12K(NC.sub.12K).sub.3NH.sub.2, an
exemplary polymer according to the present invention, by showing
the effect of time of exposure of the malaria causing parasites to
the polymer on the stage-dependent effect on Plasmodium falciparum
parasite viability (chloroquine-resistant FCR3 strain versus
chloroquine-sensitive NF54 strain);
[0119] FIG. 14 presents a comparative plot demonstrating the
anti-malarial activity of C.sub.12KNC.sub.8KNH.sub.2, an exemplary
polymer according to the present invention, by showing the effect
of time of treatment at different parasite developmental stages
with the polymer, on parasite viability;
[0120] FIGS. 15a-b present the rate of survival, monitored over
time period of 7 days, of infected mice (n=10 per group) inoculated
intraperitoneally with 2.5.times.10.sup.6 CFUs of E. coli CI-3504
(FIG. 15a) and 5.times.10.sup.6 CFUs of E. coli (FIG. 15b), and
subsequently treated intraperitoneally with PBS (black circles),
with a single dose of 4 mg/kg C.sub.12K(NC.sub.8K).sub.7NH.sub.2
(gray squares) or with four doses of 2 mg/kg Imipenem (asterisk),
demonstrating the high in-vivo efficacy of the polymers of the
present invention; and
[0121] FIG. 16 presents the rate of survival, monitored over time
period of 6 days, of mice (n=12 per group) treated
intraperitoneally with a blank control (white bars), 4 mg/kg body
weight (sparsely striped bars), 10 mg/kg body weight (densely
striped bars) and 20 mg/kg body weight (black bars) of
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, demonstrating the low toxicity
of the polymers of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0122] The present invention is of a novel class of polymeric
antimicrobial agents, which are designed to exert antimicrobial
activity while being stable, non-toxic and avoiding development of
resistance thereto, and can therefore be beneficially utilized in
the treatment of various medical conditions associated with
pathogenic microorganisms. The present invention is further of
pharmaceutical compositions, medical devices and food preservatives
containing same. The antimicrobial polymers of the present
invention preferably include one or more positively charged amino
acid residues and one or hydrophobic moiety residues attached one
to another.
[0123] The principles and operation of the present invention may be
better understood with reference to the figures and accompanying
descriptions.
[0124] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or exemplified by the Examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0125] As discussed above, the use of classical modern antibiotic
agents such as tetracycline, gentamycin, ciprofloxacin and
methicillin has become during the years severely limited by the
development of resistance thereto. Extensive studies have therefore
been conducted in a search for novel antimicrobial agents that
would circumvent the resistance induction.
[0126] As further discussed above, naturally occurring
antimicrobial peptides (AMPs) are exceptionally potent
antimicrobial agents, but as pharmaceuticals they suffer from the
limitations associated with peptide production, maintenance and
modes of clinical administration for therapeutic use.
[0127] Based on the knowledge which accumulated over the years on
the nature of antimicrobial peptides and the limitations associated
with their use, the present inventors hypothesized that in order to
achieve a novel class of antimicrobial agents devoid of the
resistance-inducing drawbacks of classical antibiotic agents, and
those of AMPs, three key attributes of AMPs needs to be maintained:
a flexible structure, an amphiphatic character and a net positive
charge.
[0128] While conceiving the present invention, it was envisioned
that a flexible polymeric structure will serve the objective of
avoiding the development of resistance in the target microorganism.
It was further envisioned that use of amino acids, as defined
hereinbelow, can serve as a basis for both a polymer as well as a
source for net positive charge.
[0129] While further conceiving the present invention, it was
hypothesized that avoiding a pure amino acid polypeptide structure
would not only resolve the production and maintenance issues
limiting the use of polypeptides as drugs, but would also alleviate
the sever limitations restricting the administration of
polypeptides as drugs. Thus, it was envisioned that the desired
amphiphatic trait of the envisioned polymer may arise from
non-amino acid hydrophobic moieties, such as, but not limited to
fatty acids and the likes.
[0130] While reducing the present invention to practice, as is
demonstrated in the Examples section that follows, the present
inventors have developed and successfully produced a novel class of
polymers which were shown to exhibit high antimicrobial activity,
low resistance induction, non-hemolyticity, resistibility to plasma
proteases and high affinity to microbial membranes.
[0131] While further conceiving the present invention, it was
envisioned that conjugating an active agent to the polymeric
structure, such as a labeling agent and/or a therapeutically active
agent, will combine the affinity of the polymers of the present
invention to microbial cells, and the utility of the additional
active agent. In cases where the active agent is a labeling agent,
the combination will assist in locating and diagnosing
concentration of microbial growth in a host, and in cases where the
active agent is a therapeutically active agent, synergistic
therapeutic effects could be achieved, resulting from the dual
therapeutic effect of the therapeutically active agent and the
antimicrobial polymeric structure. In addition, targeted delivery
of the therapeutic agent could be achieved.
[0132] Thus, according to one aspect of the present invention,
there is provided a polymer, having an antimicrobial activity,
which comprises a plurality (e.g., two or more) amino acid residues
and one or more hydrophobic moiety residues, wherein at least one
of the hydrophobic moiety residues is covalently linked to at least
two amino acid residues via the N-alpha of one amino acid residue
and/or the C-alpha of the other amino acid residue. Therefore, the
polymer is a chain made of a sequence of amino acid residues,
interrupted by one or more hydrophobic moiety residues.
[0133] As used herein throughout the term "amino acid" or "amino
acids" is understood to include the 20 genetically coded amino
acids; those amino acids often modified post-translationally in
vivo, including, for example, hydroxyproline, phosphoserine and
phosphothreonine; and other unusual amino acids including, but not
limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine,
nor-valine, nor-leucine and ornithine. Furthermore, the term "amino
acid" includes both D- and L-amino acids and other non-naturally
occurring amino acids.
[0134] Tables 1 and 2 below list the genetically encoded amino
acids (Table 1) and non-limiting examples of
non-conventional/modified amino acids (Table 2) which can be used
with the present invention.
TABLE-US-00001 TABLE 1 Three-Letter One-letter Amino acid
Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N
Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid
Glu E Glycine Gly G Histidine His H Isoleucine Iie I Leucine Leu L
Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P
Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine
Val V
TABLE-US-00002 TABLE 2 Non-conventional amino acid Code
Non-conventional amino acid Code .alpha.-aminobutyric acid Abu
L-N-methylalanine Nmala .alpha.-amino-.alpha.-methylbutyrate Mgabu
L-N-methylarginine Nmarg aminocyclopropane-carboxylate Cpro
L-N-methylasparagine Nmasn aminoisobutyric acid Aib
L-N-methylaspartic acid Nmasp aminonorbornyl-carboxylate Norb
L-N-methylcysteine Nmcys Cyclohexylalanine Chexa
L-N-methylglutamine Nmgin Cyclopentylalanine Cpen
L-N-methylglutamic acid Nmglu D-alanine Dal L-N-methylhistidine
Nmhis D-arginine Darg L-N-methylisolleucine Nmile D-aspartic acid
Dasp L-N-methylleucine Nmleu D-cysteine Dcys L-N-methyllysine Nmlys
D-glutamine Dgln L-N-methylmethionine Nmmet D-glutamic acid Dglu
L-N-methylnorleucine Nmnle D-histidine Dhis L-N-methylnorvaline
Nmnva D-isoleucine Dile L-N-methylornithine Nmorn D-leucine Dleu
L-N-methylphenylalanine Nmphe D-lysine Dlys L-N-methylproline Nmpro
D-methionine Dmet L-N-methylserine Nmser D/L-ornithine D/Lorn
L-N-methylthreonine Nmthr D-phenylalanine Dphe L-N-methyltryptophan
Nmtrp D-proline Dpro L-N-methyltyrosine Nmtyr D-serine Dser
L-N-methylvaline Nmval D-threonine Dthr L-N-methylethylglycine
Nmetg D-tryptophan Dtrp L-N-methyl-t-butylglycine Nmtbug D-tyrosine
Dtyr L-norleucine Nle D-valine Dval L-norvaline Nva
D-.alpha.-methylalanine Dmala .alpha.-methyl-aminoisobutyrate Maib
D-.alpha.-methylarginine Dmarg .alpha.-methyl-.gamma.-aminobutyrate
Mgabu D-.alpha.-methylasparagine Dmasn
.alpha.-methylcyclohexylalanine Mchexa D-.alpha.-methylaspartate
Dmasp .alpha.-methylcyclopentylalanine Mcpen
D-.alpha.-methylcysteine Dmcys
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylglutamine Dmgln .alpha.-methylpenicillamine Mpen
D-.alpha.-methylhistidine Dmhis N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylisoleucine Dmile N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylleucine Dmleu N-(3-aminopropyl)glycine Norn
D-.alpha.-methyllysine Dmlys N-amino-a-methylbutyrate Nmaabu
D-.alpha.-methylmethionine Dmmet .alpha.-napthylalanine Anap
D-.alpha.-methylornithine Dmorn N-benzylglycine Nphe
D-.alpha.-methylphenylalanine Dmphe N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylproline Dmpro N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylserine Dmser N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylthreonine Dmthr N-(carboxymethyl)glycine Nasp
D-.alpha.-methyltryptophan Dmtrp N-cyclobutylglycine Ncbut
D-.alpha.-methyltyrosine Dmty N-cycloheptylglycine Nchep
D-.alpha.-methylvaline Dmval N-cyclohexylglycine Nchex
D-.alpha.-methylalnine Dnmala N-cyclodecylglycine Ncdec
D-.alpha.-methylarginine Dnmarg N-cyclododeclglycine Ncdod
D-.alpha.-methylasparagine Dnmasn N-cyclooctylglycine Ncoct
D-.alpha.-methylasparatate Dnmasp N-cyclopropylglycine Ncpro
D-.alpha.-methylcysteine Dnmcys N-cycloundecylglycine Ncund
D-N-methylleucine Dnmleu N-(2,2-diphenylethyl)glycine Nbhm
D-N-methyllysine Dnmlys N-(3,3-diphenylpropyl)glycine Nbhe
N-methylcyclohexylalanine Nmchexa N-(3-indolylyethyl) glycine Nhtrp
D-N-methylornithine Dnmorn N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylglycine Nala D-N-methylmethionine Dnmmet
N-methylaminoisobutyrate Nmaib N-methylcyclopentylalanine Nmcpen
N-(1-methylpropyl)glycine Nile D-N-methylphenylalanine Dnmphe
N-(2-methylpropyl)glycine Nile D-N-methylproline Dnmpro
N-(2-methylpropyl)glycine Nleu D-N-methylserine Dnmser
D-N-methyltryptophan Dnmtrp D-N-methylserine Dnmser
D-N-methyltyrosine Dnmtyr D-N-methylthreonine Dnmthr
D-N-methylvaline Dnmval N-(1-methylethyl)glycine Nva
.gamma.-aminobutyric acid Gabu N-methyla-napthylalanine Nmanap
L-t-butylglycine Tbug N-methylpenicillamine Nmpen L-ethylglycine
Etg N-(p-hydroxyphenyl)glycine Nhtyr L-homophenylalanine Hphe
N-(thiomethyl)glycine Ncys L-.alpha.-methylarginine Marg
penicillamine Pen L-.alpha.-methylaspartate Masp
L-.alpha.-methylalanine Mala L-.alpha.-methylcysteine Mcys
L-.alpha.-methylasparagine Masn L-.alpha.-methylglutamine Mgln
L-.alpha.-methyl-t-butylglycine Mtbug L-.alpha.-methylhistidine
Mhis L-methylethylglycine Metg L-.alpha.-methylisoleucine Mile
L-.alpha.-methylglutamate Mglu D-N-methylglutamine Dnmgln
L-.alpha.-methylhomo phenylalanine Mhphe D-N-methylglutamate Dnmglu
N-(2-methylthioethyl)glycine Nmet D-N-methylhistidine Dnmhis
N-(3-guanidinopropyl)glycine Narg D-N-methylisoleucine Dnmile
N-(1-hydroxyethyl)glycine Nthr D-N-methylleucine Dnmleu
N-(hydroxyethyl)glycine Nser D-N-methyllysine Dnmlys
N-(imidazolylethyl)glycine Nhis N-methylcyclohexylalanine Nmchexa
N-(3-indolylyethyl)glycine Nhtrp D-N-methylornithine Dnmorn
N-methyl-.gamma.-aminobutyrate Nmgabu N-methylglycine Nala
D-N-methylmethionine Dnmmet N-methylaminoisobutyrate Nmaib
N-methylcyclopentylalanine Nmcpen N-(1-methylpropyl)glycine Nile
D-N-methylphenylalanine Dnmphe N-(2-methylpropyl)glycine Nleu
D-N-methylproline Dnmpro D-N-methyltryptophan Dnmtrp
D-N-methylserine Dnmser D-N-methyltyrosine Dnmtyr
D-N-methylthreonine Dnmthr D-N-methylvaline Dnmval
N-(1-methylethyl)glycine Nval .gamma.-aminobutyric acid Gabu
N-methyla-napthylalanine Nmanap L-t-butylglycine Tbug
N-methylpenicillamine Nmpen L-ethylglycine Etg
N-(p-hydroxyphenyl)glycine Nhtyr L-homophenylalanine Hphe
N-(thiomethyl)glycine Ncys L-.alpha.-methylarginine Marg
penicillamine Pen L-.alpha.-methylaspartate Masp
L-.alpha.-methylalanine Mala L-.alpha.-methylcysteine Mcys
L-.alpha.-methylasparagine Masn L-.alpha.-methylglutamine Mgln
L-.alpha.-methyl-t-butylglycine Mtbug L-.alpha.-methylhistidine
Mhis L-methylethylglycine Metg L-.alpha.-methylisoleucine Mile
L-.alpha.-methylglutamate Mglu L-.alpha.-methylleucine Mleu
L-.alpha.-methylhomophenylalanine Mhphe L-.alpha.-methylmethionine
Mmet N-(2-methylthioethyl)glycine Nmet L-.alpha.-methylnorvaline
Mnva L-.alpha.-methyllysine Mlys L-.alpha.-methylphenylalanine Mphe
L-.alpha.-methylnorleucine Mnle L-.alpha.-methylserine mser
L-.alpha.-methylornithine Morn L-.alpha.-methylvaline Mtrp
L-.alpha.-methylproline Mpro L-.alpha.-methylleucine Mval Nnbhm
L-.alpha.-methylthreonine Mthr
N-(N-(2,2-diphenylethyl)carbamylmethyl-glycine Nnbhm
L-.alpha.-methyltyrosine Mtyr 1-carboxy-1-(2,2-diphenyl
ethylamino)cyclopropane Nmbc L-N-methylhomophenylalanine Nmhphe
N-(N-(3,3-diphenylpropyl)carbamylmethyl(1)glycine Nnbhe
D/L-citrulline D/Lctr
[0135] As used herein, the phrase "hydrophobic moiety" describes a
chemical moiety that has a minor or no affinity to water, that is,
which has a low or no dissolvability in water and often in other
polar solvents. Exemplary suitable hydrophobic moieties for use in
the context of the present invention, include, without limitation,
hydrophobic moieties that consist predominantly of one or more
hydrocarbon chains and/or aromatic rings, and one or more
functional groups which may be non-hydrophobic, but do not alter
the overall hydrophobicity of the hydrophobic moiety.
Representative examples include, without limitation, fatty acids,
hydrophobic amino acids (amino acids with hydrophobic side-chains),
alkanes, alkenes, aryls and the likes, as these terms are defined
herein, and any combination thereof.
[0136] As used herein, the phrase "chemical moiety" describes a
residue of a chemical compound, which typically has certain
functionality. As is well accepted in the art, the term "residue"
refers herein to a major portion of a molecule which is covalently
linked to another molecule.
[0137] As used herein, the phrase "functional group" describes a
chemical group that is capable of undergoing a chemical reaction
that typically leads to a bond formation. The bond, according to
the present invention, is preferably a covalent bond. Chemical
reactions that lead to a bond formation include, for example,
nucleophilic and electrophilic substitutions, nucleophilic and
electrophilic addition reactions, addition-elimination reactions,
cycloaddition reactions, rearrangement reactions and any other
known organic reactions that involve a functional group.
[0138] A polymer, according to the present invention, may have one
or more hydrophobic moiety residues, whereby at least one is linked
to one amino acid at one end and to another amino acid residue at
another end, and another may elongate the polymeric chain by being
linked to either one of the termini, i.e., the N-alpha of a
terminal amino acid residue and/or the C-alpha of a terminal amino
acid residue. Optionally, a second hydrophobic moiety may be linked
to the side-chain of an amino acid residue in the polymer.
[0139] The polymer, according to the present invention, preferably
includes from 2 to 50 amino acid residues. More preferably, the
polymer includes from 2 to 12 amino acid residues, more preferably
from 4 to 8 amino acid residues and more preferably from 5 to 7
amino acid residues.
[0140] The net positive charge of the polymer is maintained by
having one or more positively charged amino acid residues in the
polymer, optionally in addition to the positively charged
N-terminus amine, when present in its free form.
[0141] In one preferred embodiment of the present invention, all
the amino acid residues in the polymer are positively charged amino
acid residues. An exemplary polymer according to this embodiment
includes a plurality of lysine residues.
[0142] As used herein the phrase "positively charged amino acid"
describes a hydrophilic amino acid with a side chain pKa value of
greater than 7, namely a basic amino acid. Basic amino acids
typically have positively charged side chains at physiological pH
due to association with a hydronium ion. Naturally occurring
(genetically encoded) basic amino acids include lysine (Lys, K),
arginine (Arg, R) and histidine (His, H), while non-natural
(non-genetically encoded, or non-standard) basic amino acids
include, for example, ornithine, 2,3,-diaminopropionic acid,
2,4-diaminobutyric acid, 2,5,6-triaminohexanoic acid,
2-amino-4-guanidinobutanoic acid, and homoarginine.
[0143] In one embodiment of the present invention, each of the
components in the polymer according to the present embodiments is
preferably linked to the other by a peptide bond.
[0144] The term "peptide bond" as used herein refers to an amide
group, namely, a --(C.dbd.O)NH-- group, which is typically formed
by a condensation reaction between a carboxylic group and an amine
group, as these terms are defined herein.
[0145] However, the polymers of the present embodiments may have
other bonds linking the various components in the polymeric
structure. Such non-peptidic bonds may render the polymer more
stable while in a body or more capable of penetrating into cells.
Thus, peptide bonds (--(C.dbd.O)NH--) within the polymer may be
replaced, for example, by N-methylated amide bonds
(--(C.dbd.O)NCH.sub.3--), ester bonds
(--C(R)H--C(.dbd.O)--O--C(R)--N--), ketomethylen bonds
(--C(.dbd.O)CH.sub.2--), aza bonds (--NH--N(R)--C(.dbd.O)--),
wherein R is any alkyl, e.g., methyl, carba bonds
(--CH.sub.2--NH--), hydroxyethylene bonds (--CH(OH)--CH.sub.2--),
thioamide bonds (--CS--NH--), olefinic double bonds
(--CH.dbd.CH--), retro amide bonds (--NH--(C.dbd.O)--), peptide
derivatives (--N(R)--CH.sub.2--C(.dbd.O)--), wherein R is the
"normal" side chain, naturally presented on the carbon atom. These
modifications can occur at any of the bonds along the polymer chain
and even several (2-3) at the same time.
[0146] In a preferred embodiment, all of the bonds in the polymer,
linking the amino acid residues and hydrophobic moiety residues to
each other, are peptide bonds. For example, in one embodiment, the
polymer is made of an amino acid residue linked by a peptide bond
to a hydrophobic moiety residue which in turn is linked to a second
amino acid residue by another peptide bond. In another example, the
polymer of the previous example is elongated by a second
hydrophobic moiety residue which is linked to any one of the N- or
C-termini by a peptide bond, etcetera.
[0147] The polymer, according to the present invention, preferably
comprises from 1 to 50 hydrophobic moiety residues. More
preferably, the polymer comprises from 1 to 12 hydrophobic moiety
residues, more preferably from 4 to 10 hydrophobic moiety residues
and more preferably from 6 to 8 hydrophobic moiety residues.
[0148] The hydrophobic moieties that are used in the context of
this and other aspects of the present invention preferably have one
or more hydrocarbon chains, and are capable of linking to one or
two other components in the polymer (e.g., one or two of an amino
acid residue and another hydrophobic moiety) via two peptide bonds.
These moieties therefore preferably have a carboxylic group at one
end of the hydrocarbon chain (for linking a free amine group) and
an amine group at the other (for linking a carboxylic acid
group).
[0149] The hydrocarbon chain connecting the carboxylic and amine
groups in such a hydrophobic moiety preferably has from 4 to 30
carbon atoms.
[0150] In a preferred embodiment of the present invention, the
hydrophobic moiety residue is a fatty acid residue wherein the
hydrocarbon chain can be unbranched and saturated, branched and
saturated, unbranched and unsaturated or branched and unsaturated.
More preferably the hydrocarbon chain of the fatty acid residue is
an unbranched and saturated chain having from 4 to 30 carbon atoms,
preferably from 4 to 20 carbon atoms. Non-limiting example of such
fatty acid residues are butyric acid residue, caprylic acid residue
and lauric acid residue.
[0151] In a more preferred embodiment, the fatty acid residue has
an amine on the distal carbon of the hydrocarbon chain (with
respect to the carboxylic acid group). Such a fatty acid residue is
referred to herein as an .omega.-amino fatty acid residue. Again
here the hydrocarbon chain of the .omega.-amino fatty acid residue
may have from 4 to 30 carbon atoms.
[0152] Non-limiting example of such .omega.-amino fatty acids are
4-amino-butyric acid, 6-amino-caproic acid, 8-amino-caprylic acid,
10-amino-capric acid, 12-amino-lauric acid, 14-amino-myristic acid,
16-amino-palmitic acid, 18-amino-stearic acid, 18-amino-oleic acid,
16-amino-palmitoleic acid, 18-amino-linoleic acid,
18-amino-linolenic acid and 20-amino-arachidonic acid.
[0153] According to a preferred embodiment of the present
invention, the hydrophobic moiety is selected from the group
consisting of 4-amino-butyric acid, 8-amino-caprylic acid and
12-amino-lauric acid and more preferably is 8-amino-caprylic acid
and 12-amino-lauric acid.
[0154] The polymers described herein can be collectively
represented by the following general formula I:
X--W.sub.0-[A.sub.1-Z.sub.1-D.sub.1]-W.sub.1-[A.sub.2-Z.sub.2-D.sub.2]-W-
.sub.2-- . . . [An-Zn-Dn]-Wn-Y Formula I
[0155] wherein:
[0156] n is an integer from 2 to 50, preferably from 2 to 12 and
more preferably from 2 to 8;
[0157] A.sub.1, A.sub.2, . . . , An are each independently an amino
acid residue, preferably a positively charged amino acid residue,
more preferably all of A.sub.1, A.sub.2, . . . , An are positively
charged amino acid residues as discussed hereinabove, such as
histidine residues, lysine residues, ornithine residues and
arginine residues, and most preferably all the positively charged
amino acid residues are lysine residues;
[0158] D.sub.1, D.sub.2, . . . Dn are each independently a
hydrophobic moiety residue, as defined and discussed hereinabove,
or absent, provided that at least one such hydrophobic moiety
residue exists in the polymer, preferably at least one of the
hydrophobic moiety residues is a .omega.-amino-fatty acid
residue;
[0159] Connecting each monomer of the residue are linking moieties,
denoted Z.sub.1, Z.sub.2, . . . Zn and W.sub.0, W.sub.1, W.sub.2, .
. . Wn, each of which independently linking an amino acid residue
and a hydrophobic moiety residue or absent, preferably at least one
of the linking moieties is a peptide bond and most preferable all
the linking moieties are peptide bonds;
[0160] The fringes of the polymer, denoted X and Y, may each
independently be hydrogen, an amine, an amino acid residue, a
hydrophobic moiety residue, is another polymer having the general
Formula I or absent.
[0161] As discussed above, one or more of the hydrophobic moiety
residues may be attached to a side chain of one or more of the
amino acid residues of the polymer, i.e., act as a branch of the
main polymer.
[0162] The presently most preferred polymers are polymers in which
n is an integer from 5 to 7, the amino acid residues are all lysine
residues, the hydrophobic moiety residues are all 8-amino-caprylic
acid residues, X is a hydrophobic moiety such as, for example, a
fatty acid residue (a dodecanoic acid residue), and/or Y is amine
or absent.
[0163] Particularly preferred polymers according to the present
embodiments are those having the Formulae hereinbelow:
##STR00003##
which is also referred to herein as
C.sub.12K(NC.sub.8K).sub.7NH.sub.2; and
##STR00004##
which is also referred to herein as
C.sub.12K(NC.sub.8K).sub.5NH.sub.2.
[0164] The polymers according to the present embodiments can be
readily synthesized. For example, polymers in which the linking
moieties are peptide bonds, and hence resemble natural and
synthetic peptides in this respect, can be prepared by classical
methods known in the art for peptide syntheses. Such methods
include, for example, standard solid phase techniques. The standard
methods include exclusive solid phase synthesis, partial solid
phase synthesis methods, fragment condensation, classical solution
synthesis, and even by recombinant DNA technology. See, e.g.,
Merrifield, J. Am. Chem. Soc., 85:2149 (1963), incorporated herein
by reference. Solid phase peptide synthesis procedures are well
known in the art and further described by John Morrow Stewart and
Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce
Chemical Company, 1984).
[0165] The polymers of the present invention can be purified, for
example, by preparative high performance liquid chromatography
[Creighton T. (1983) Proteins, structures and molecular principles.
WH Freeman and Co. N.Y.].
[0166] Apart from having beneficial antimicrobial activity per se,
as detailed herein, the polymers of the present invention may
include an additional active agent such as a labeling agent and/or
a therapeutically active agent attached thereto. The conjugation of
the active agent to a polymer of the present invention can provide
a dual utility for the polymer. When the additional active agent is
a labeling agent, the conjugation thereof to an antimicrobial
polymer of the present invention, having a high affinity to
microbial cells, can assist in the location, diagnosis and
targeting of microbial growth loci in a host. When the additional
active agent is a therapeutically active agent, the conjugation
thereof to an antimicrobial polymer of the present invention will
exert a dual and possibly synergistic antimicrobial activity.
[0167] According to preferred embodiments of the present invention,
the one or more active agents may be attached to the polymer at any
substitutable position. Examples of such substitutable positions
include, without limitation, a side chain of any one or more of the
amino acid residues in the polymer, any one of the linking moieties
of the polymer, any one of the N- and C-termini of the polymer and
any one or more of the hydrophobic moiety residues in the
polymer.
[0168] Hence, as used herein, the phrase "a therapeutically active
agent" describes a chemical substance, which exhibit a therapeutic
activity when administered to a subject
[0169] As used herein, the phrase "labeling agent" refers to a
detectable moiety or a probe and includes, for example,
chromophores, fluorescent compounds, phosphorescent compounds,
heavy metal clusters, and radioactive labeling compounds, as well
as any other known detectable moieties.
[0170] Labeling of microbial growth loci in a host is critical for
the diagnosis and efficient targeting of the photogenic
microorganism and treatment thereof.
[0171] Adding a therapeutically active agent to the polymer can
provide a solution for many deficiencies of presently known
therapeutically active agent against photogenic microorganisms,
such as resistance of the photogenic microorganism to the
therapeutically active agent, specificity of the therapeutically
active agent to photogenic microorganism and general efficacy
weakness. The polymers of the present invention can exhibit not
only antimicrobial activity per se by virtue of their structure and
chemical properties, but can also provide targeting capacity as a
delivery vehicle to a presently know therapeutically active agents
and further provide membrane permeability to presently know
therapeutically active agents due to their capability to exert
disturbance in the membrane structure of photogenic
microorganisms.
[0172] Non-limiting examples of therapeutically active agents that
can be beneficially used in this and other contexts of the present
invention include, without limitation, one or more of an agonist
residue, an amino acid residue, an analgesic residue, an antagonist
residue, an antibiotic agent residue, an antibody residue, an
antidepressant agent, an antigen residue, an anti-histamine
residue, an anti-hypertensive agent, an anti-inflammatory drug
residue, an anti-metabolic agent residue, an antimicrobial agent
residue, an antioxidant residue, an anti-proliferative drug
residue, an antisense residue, a chemotherapeutic drug residue, a
co-factor residue, a cytokine residue, a drug residue, an enzyme
residue, a growth factor residue, a heparin residue, a hormone
residue, an immunoglobulin residue, an inhibitor residue, a ligand
residue, a nucleic acid residue, an oligonucleotide residue, a
peptide residue, a phospholipid residue, a prostaglandin residue, a
protein residue, a toxin residue, a vitamin residue and any
combination thereof.
[0173] The combined therapeutic effect is particularly advantageous
when the therapeutically active agent is an antimicrobial or an
antibiotic agent. The combined activity of the polymers of the
present invention and that of an additional
antimicrobial/antibiotic agent may provide the
antimicrobial/antibiotic agent the capacity to overcome the known
limitations of these drugs such as targeting, specificity,
efficacy, drug-resistance etcetera. Synergism may also be
achieved.
[0174] Non-limiting examples of antimicrobial and antibiotic agents
that are suitable for use in this context of the present invention
include, without limitation, mandelic acid,
2,4-dichlorobenzenemethanol,
4-[bis(ethylthio)methyl]-2-methoxyphenol, 4-epi-tetracycline,
4-hexylresorcinol, 5,12-dihydro-5,7,12,14-tetrazapentacen,
5-chlorocarvacrol, 8-hydroxyquinoline, acetarsol,
acetylkitasamycin, acriflavin, alatrofloxacin, ambazon, amfomycin,
amikacin, amikacin sulfate, aminoacridine, aminosalicylate calcium,
aminosalicylate sodium, aminosalicylic acid,
ammoniumsulfobituminat, amorolfin, amoxicillin, amoxicillin sodium,
amoxicillin trihydrate, amoxicillin-potassium clavulanate
combination, amphotericin B, ampicillin, ampicillin sodium,
ampicillin trihydrate, ampicillin-sulbactam, apalcillin, arbekacin,
aspoxicillin, astromicin, astromicin sulfate, azanidazole,
azidamfenicol, azidocillin, azithromycin, azlocillin, aztreonam,
bacampicillin, bacitracin, bacitracin zinc, bekanamycin,
benzalkonium, benzethonium chloride, benzoxonium chloride,
berberine hydrochloride, biapenem, bibrocathol, biclotymol,
bifonazole, bismuth subsalicylate, bleomycin antibiotic complex,
bleomycin hydrochloride, bleomycin sulfate, brodimoprim,
bromochlorosalicylanilide, bronopol, broxyquinolin, butenafine,
butenafine hydrochloride, butoconazol, calcium undecylenate,
candicidin antibiotic complex, capreomycin, carbenicillin,
carbenicillin disodium, carfecillin, carindacillin, carumonam,
carzinophilin, caspofungin acetate, cefacetril, cefaclor,
cefadroxil, cefalexin, cefalexin hydrochloride, cefalexin sodium,
cefaloglycin, cefaloridine, cefalotin, cefalotin sodium,
cefamandole, cefamandole nafate, cefamandole sodium, cefapirin,
cefapirin sodium, cefatrizine, cefatrizine propylene glycol,
cefazedone, cefazedone sodium salt, cefazolin, cefazolin sodium,
cefbuperazone, cefbuperazone sodium, cefcapene, cefcapene pivoxil
hydrochloride, cefdinir, cefditoren, cefditoren pivoxil, cefepime,
cefepime hydrochloride, cefetamet, cefetamet pivoxil, cefixime,
cefmenoxime, cefmetazole, cefmetazole sodium, cefminox, cefminox
sodium, cefmolexin, cefodizime, cefodizime sodium, cefonicid,
cefonicid sodium, cefoperazone, cefoperazone sodium, ceforanide,
cefoselis sulfate, cefotaxime, cefotaxime sodium, cefotetan,
cefotetan disodium, cefotiam, cefotiam hexetil hydrochloride,
cefotiam hydrochloride, cefoxitin, cefoxitin sodium, cefozopran
hydrochloride, cefpiramide, cefpiramide sodium, cefpirome,
cefpirome sulfate, cefpodoxime, cefpodoxime proxetil, cefprozil,
cefquinome, cefradine, cefroxadine, cefsulodin, ceftazidime,
cefteram, cefteram pivoxil, ceftezole, ceftibuten, ceftizoxime,
ceftizoxime sodium, ceftriaxone, ceftriaxone sodium, cefuroxime,
cefuroxime axetil, cefuroxime sodium, cetalkonium chloride,
cetrimide, cetrimonium, cetylpyridinium, chloramine T,
chloramphenicol, chloramphenicol palmitate, chloramphenicol
succinate sodium, chlorhexidine, chlormidazole, chlormidazole
hydrochloride, chloroxylenol, chlorphenesin, chlorquinaldol,
chlortetracycline, chlortetracycline hydrochloride, ciclacillin,
ciclopirox, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride,
citric acid, clarithromycin, clavulanate potassium, clavulanate
sodium, clavulanic acid, clindamycin, clindamycin hydrochloride,
clindamycin palmitate hydrochloride, clindamycin phosphate,
clioquinol, cloconazole, cloconazole monohydrochloride,
clofazimine, clofoctol, clometocillin, clomocycline, clotrimazol,
cloxacillin, cloxacillin sodium, colistin, colistin sodium
methanesulfonate, colistin sulfate, cycloserine, dactinomycin,
danofloxacin, dapsone, daptomycin, daunorubicin, DDT,
demeclocycline, demeclocycline hydrochloride, dequalinium,
dibekacin, dibekacin sulfate, dibrompropamidine, dichlorophene,
dicloxacillin, dicloxacillin sodium, didecyldimethylammonium
chloride, dihydrostreptomycin, dihydrostreptomycin sulfate,
diiodohydroxyquinolin, dimetridazole, dipyrithione, dirithromycin,
DL-menthol, D-menthol, dodecyltriphenylphosphonium bromide,
doxorubicin, doxorubicin hydrochloride, doxycycline, doxycycline
hydrochloride, econazole, econazole nitrate, enilconazole,
enoxacin, enrofloxacin, eosine, epicillin, ertapenem sodium,
erythromycin, erythromycin estolate, erythromycin ethyl succinate,
erythromycin lactobionate, erythromycin stearate, ethacridine,
ethacridine lactate, ethambutol, ethanoic acid, ethionamide, ethyl
alcohol, eugenol, exalamide, faropenem, fenticonazole,
fenticonazole nitrate, fezatione, fleroxacin, flomoxef, flomoxef
sodium, florfenicol, flucloxacillin, flucloxacillin magnesium,
flucloxacillin sodium, fluconazole, flucytosine, flumequine,
flurithromycin, flutrimazole, fosfomycin, fosfomycin calcium,
fosfomycin sodium, framycetin, framycetin sulphate, furagin,
furazolidone, fusafungin, fusidic acid, fusidic acid sodium salt,
gatifloxacin, gemifloxacin, gentamicin antibiotic complex,
gentamicin c 1a, gentamycin sulfate, glutaraldehyde, gramicidin,
grepafloxacin, griseofulvin, halazon, haloprogine, hetacillin,
hetacillin potassium, hexachlorophene, hexamidine, hexetidine,
hydrargaphene, hydroquinone, hygromycin, imipenem, isepamicin,
isepamicin sulfate, isoconazole, isoconazole nitrate, isoniazid,
isopropanol, itraconazole, josamycin, josamycin propionate,
kanamycin, kanamycin sulphate, ketoconazole, kitasamycin, lactic
acid, lanoconazole, lenampicillin, leucomycin A1, leucomycin A13,
leucomycin A4, leucomycin A5, leucomycin A6, leucomycin A7,
leucomycin A8, leucomycin A9, levofloxacin, lincomycin, lincomycin
hydrochloride, linezolid, liranaftate, l-menthol, lomefloxacin,
lomefloxacin hydrochloride, loracarbef, lymecyclin, lysozyme,
mafenide acetate, magnesium monoperoxophthalate hexahydrate,
mecetronium ethylsulfate, mecillinam, meclocycline, meclocycline
sulfosalicylate, mepartricin, merbromin, meropenem, metalkonium
chloride, metampicillin, methacycline, methenamin, methyl
salicylate, methylbenzethonium chloride, methylrosanilinium
chloride, meticillin, meticillin sodium, metronidazole,
metronidazole benzoate, mezlocillin, mezlocillin sodium,
miconazole, miconazole nitrate, micronomicin, micronomicin sulfate,
midecamycin, minocycline, minocycline hydrochloride, miocamycin,
miristalkonium chloride, mitomycin c, monensin, monensin sodium,
morinamide, moxalactam, moxalactam disodium, moxifloxacin,
mupirocin, mupirocin calcium, nadifloxacin, nafcillin, nafcillin
sodium, naftifine, nalidixic acid, natamycin, neomycin a, neomycin
antibiotic complex, neomycin C, neomycin sulfate, neticonazole,
netilmicin, netilmicin sulfate, nifuratel, nifuroxazide,
nifurtoinol, nifurzide, nimorazole, niridazole, nitrofurantoin,
nitrofurazone, nitroxolin, norfloxacin, novobiocin, nystatin
antibiotic complex, octenidine, ofloxacin, oleandomycin,
omoconazol, orbifloxacin, ornidazole, ortho-phenylphenol,
oxacillin, oxacillin sodium, oxiconazole, oxiconazole nitrate,
oxoferin, oxolinic acid, oxychlorosene, oxytetracycline,
oxytetracycline calcium, oxytetracycline hydrochloride, panipenem,
paromomycin, paromomycin sulfate, pazufloxacine, pefloxacin,
pefloxacin mesylate, penamecillin, penicillin G, penicillin G
potassium, penicillin G sodium, penicillin V, penicillin V calcium,
penicillin V potassium, pentamidine, pentamidine diisetionate,
pentamidine mesilas, pentamycin, phenethicillin, phenol,
phenoxyethanol, phenylmercuriborat, PHMB, phthalylsulfathiazole,
picloxydin, pipemidic acid, piperacillin, piperacillin sodium,
pipercillin sodium-tazobactam sodium, piromidic acid,
pivampicillin, pivcefalexin, pivmecillinam, pivmecillinam
hydrochloride, policresulen, polymyxin antibiotic complex,
polymyxin B, polymyxin B sulfate, polymyxin B1, polynoxylin,
povidone-iodine, propamidin, propenidazole, propicillin,
propicillin potassium, propionic acid, prothionamide, protiofate,
pyrazinamide, pyrimethamine, pyrithion, pyrroInitrin, quinoline,
quinupristin-dalfopristin, resorcinol, ribostamycin, ribostamycin
sulfate, rifabutin, rifampicin, rifamycin, rifapentine, rifaximin,
ritiometan, rokitamycin, rolitetracycline, rosoxacin,
roxithromycin, rufloxacin, salicylic acid, secnidazol, selenium
disulphide, sertaconazole, sertaconazole nitrate, siccanin,
sisomicin, sisomicin sulfate, sodium thiosulfate, sparfloxacin,
spectinomycin, spectinomycin hydrochloride, spiramycin antibiotic
complex, spiramycin b, streptomycin, streptomycin sulphate,
succinylsulfathiazole, sulbactam, sulbactam sodium, sulbenicillin
disodium, sulbentin, sulconazole, sulconazole nitrate,
sulfabenzamide, sulfacarbamide, sulfacetamide, sulfacetamide
sodium, sulfachlorpyridazine, sulfadiazine, sulfadiazine silver,
sulfadiazine sodium, sulfadicramide, sulfadimethoxine, sulfadoxine,
sulfaguanidine, sulfalene, sulfamazone, sulfamerazine,
sulfamethazine, sulfamethazine sodium, sulfamethizole,
sulfamethoxazole, sulfamethoxazol-trimethoprim,
sulfamethoxypyridazine, sulfamonomethoxine, sulfamoxol,
sulfanilamide, sulfaperine, sulfaphenazol, sulfapyridine,
sulfaquinoxaline, sulfasuccinamide, sulfathiazole, sulfathiourea,
sulfatolamide, sulfatriazin, sulfisomidine, sulfisoxazole,
sulfisoxazole acetyl, sulfonamides, sultamicillin, sultamicillin
tosilate, tacrolimus, talampicillin hydrochloride, teicoplanin A2
complex, teicoplanin A2-1, teicoplanin A2-2, teicoplanin A2-3,
teicoplanin A2-4, teicoplanin A2-5, teicoplanin A3, teicoplanin
antibiotic complex, telithromycin, temafloxacin, temocillin, tenoic
acid, terbinafine, terconazole, terizidone, tetracycline,
tetracycline hydrochloride, tetracycline metaphosphate,
tetramethylthiuram monosulfide, tetroxoprim, thiabendazole,
thiamphenicol, thiaphenicol glycinate hydrochloride, thiomersal,
thiram, thymol, tibezonium iodide, ticarcillin,
ticarcillin-clavulanic acid mixture, ticarcillin disodium,
ticarcillin monosodium, tilbroquinol, tilmicosin, tinidazole,
tioconazole, tobramycin, tobramycin sulfate, tolciclate, tolindate,
tolnaftate, toloconium metilsulfat, toltrazuril, tosufloxacin,
triclocarban, triclosan, trimethoprim, trimethoprim sulfate,
triphenylstibinsulfide, troleandomycin, trovafloxacin, tylosin,
tyrothricin, undecoylium chloride, undecylenic acid, vancomycin,
vancomycin hydrochloride, viomycin, virginiamycin antibiotic
complex, voriconazol, xantocillin, xibornol and zinc
undecylenate.
[0175] Major parts of the polymers of the present embodiments are
based on a repetitive element consisting of a conjugate between an
amino acid and a bi-functional hydrophobic moiety. The conjugate
may repeat several times in the sequence of the polymer and/or be
interrupted and/or flanked by a difference types of conjugates or
by single or repeats of amino acid residues and single or repeats
of hydrophobic moiety residues.
[0176] Hence, according to another aspect of the present invention,
there is provided a conjugate which includes an amino acid residue
and a hydrophobic moiety residue, as defined and described
hereinabove, attached to the N-alpha or the C-alpha of the amino
acid residue. The hydrophobic moiety residue in the conjugate of
the present invention is designed such that is it capable of
forming a bond with an N-alpha or a C-alpha of an additional amino
acid residue. Preferably, the hydrophobic moiety residue is
conjugated to the amino acid residue via a peptide bond.
[0177] The hydrophobic moiety of the conjugate of the present
invention is having a bi-functional design which allows the
conjugate to serve as a polymerizable conjugate that can form a
part of the polymers described and presented herein. Preferably,
the hydrophobic moiety which forms a part of the conjugate is
having a bi-functionality in the form of a carboxylic group at one
end thereof and an amine group at the other end thereof.
[0178] Hence, according to another aspect of the present invention,
there is provided a process of preparing the conjugate described
hereinabove, the general process is based on providing an amino
acid, preferably the amino acid is a positively charged amino acid,
such as histidine, lysine, ornithine and arginine; providing a
hydrophobic moiety as defined and discussed hereinabove having a
first functional group that is capable of reacting with an N-alpha
of an amino acid residue and a second functional group capable of
reacting with a C-alpha of an amino acid; linking the first
functional group in the hydrophobic moiety to the amino acid via
the N-alpha of the amino acid; or linking the second functional
group in the hydrophobic moiety to the amino acid via the C-alpha
of the amino acid.
[0179] Preferably, the link between the N-alpha or the C-alpha of
the amino acid and the hydrophobic moiety is via a peptide
bond.
[0180] In order to form a peptide bond linking the amino acid to
the hydrophobic moiety, the hydrophobic moiety preferably has a
carboxylic group at one end thereof and an amine group at the other
end thereof.
[0181] The antimicrobial polymers as described herein can be
beneficially utilized in the treatment of pathogenic microorganism
infections, as these are defined hereinbelow. As demonstrated in
the Example section that follows, such polymers are by themselves
capable of exerting antimicrobial activity. The option to include
an additional therapeutically active agent may thus act
synergistically as toxic agents against various bacteria, fungi and
other microorganisms.
[0182] Herein throughout, the phrase "pathogenic microorganism" is
used to describe any microorganism which can cause a disease or
disorder in a higher organism, such as mammals in general and a
human in particular. The pathogenic microorganism may belong to any
family of organisms such as, but not limited to prokaryotic
organisms, eubacterium, archaebacterium, eukaryotic organisms,
yeast, fungi, algae, protozoan, and other parasites. Non-limiting
examples of pathogenic microorganism are Plasmodium falciparum and
related malaria-causing protozoan parasites, Acanthamoeba and other
free-living amoebae, Aeromonas hydrophila, Anisakis and related
worms, Acinetobacter baumanii, Ascaris lumbricoides, Bacillus
cereus, Brevundimonas diminuta, Campylobacter jejuni, Clostridium
botulinum, Clostridium perfringens, Cryptosporidium parvum,
Cyclospora cayetanensis, Diphyllobothrium, Entamoeba histolytica,
certain strains of Escherichia coli, Eustrongylides, Giardia
lamblia, Klebsiella pneumoniae, Listeria monocytogenes,
Nanophyetus, Plesiomonas shigelloides, Proteus mirabilis,
Pseudomonas aeruginosa, Salmonella, Serratia odorifera, Shigella,
Staphylococcus aureus, Stenotrophomonas maltophilia, Streptococcus,
Trichuris trichiura, Vibrio cholerae, Vibrio parahaemolyticus,
Vibrio vulnificus and other vibrios, Yersinia enterocolitica,
Yersinia pseudotuberculosis and Yersinia kristensenii.
[0183] Hence, according to another aspect of the present invention,
there is provided a method of treating a medical condition
associated with a pathogenic microorganism, the method includes
administering to a subject in need thereof a therapeutically
effective amount of one or more of the polymers, as described
hereinabove
[0184] As used herein, the terms "treating" and "treatment"
includes abrogating, substantially inhibiting, slowing or reversing
the progression of a condition, substantially ameliorating clinical
or aesthetical symptoms of a condition or substantially preventing
the appearance of clinical or aesthetical symptoms of a
condition.
[0185] As used herein, the phrase "therapeutically effective
amount" describes an amount of the composite being administered
which will relieve to some extent one or more of the symptoms of
the condition being treated.
[0186] The method of treatment, according to an embodiment of the
present invention, may include the administration of an additional
therapeutically active agent, as this is defined and discussed
hereinabove.
[0187] As mentioned above and demonstrated in the Example section
that follows, the antimicrobial polymers of the present invention,
alone or in combination with any other therapeutically active
agents, can be designed and utilized to destroy pathological
microorganisms. The destruction of a pathogenic microorganism is
effected by selectively destructing a portion of the cells of a
pathogenic microorganism. While most known antibiotics act by
interfering selectively with the biosynthesis of one or more of the
molecular constituents of the cell-membrane, proteins or nucleic
acids, the polymers of the present invention also act by binding
and disrupting the outer membrane of the pathogenic microorganism
cells. Disrupting the outer membrane of a cell causes its death due
to membrane depolarization, leakage of metabolites and/or total
loss of cell integrity; therefore the polymers of the present
invention also act directly as effective antimicrobial agents by
disrupting the metabolism and/or the multiplication processes of
the pathogenic microorganism.
[0188] As mentioned above and demonstrated in the Example section
that follows, the polymers presented herein may act as
antimicrobial agents which do not evoke the appearance of
resistance thereto. The possible development of resistance to the
polymers of the present invention was tested by measuring the
minimal inhibitory concentration (MIC) levels following multiple
exposures of the bacteria to exemplary polymers according to the
present invention. The results obtained in the
antimicrobial-resistance studies in bacteria presented hereinbelow,
showed that exposing bacteria, and even strains that already
developed resistance to classical antibiotics, to the antimicrobial
polymers presented herein did not result in development of
resistance.
[0189] As is further mentioned above and demonstrated in the
Example section that follows, the polymers presented herein are
non-toxic to mammals.
[0190] As is further demonstrated in the Examples section that
follows, the polymers of the present invention can act
synergistically with another antibiotic or other therapeutically
active agent by permeabilizing the cells of the pathogenic
microorganism; hence exhibit additionally an indirect antimicrobial
activity. The results presented hereinbelow permit the conclusion
that the polymers of the present invention are potent
outer-membrane disintegrating agents. The permeabilizing action of
the polymers can increase the uptake of other therapeutically
active agents and therefore should be able to potentiate the
apparent antimicrobial activity of other drugs and antibiotics.
[0191] Medical conditions associated with a pathogenic
microorganism include infections, infestation, contaminations and
transmissions by or of pathogenic microorganism. In general, a
disease causing infection is the invasion into the tissues of a
plant or an animal by pathogenic microorganisms. The invasion of
body tissues by parasitic worms and other higher pathogenic
organisms is commonly referred to as infestation.
[0192] Invading organisms such as bacteria produce toxins that
damage host tissues and interfere with normal metabolism; some
toxins are actually enzymes that break down host tissues. Other
bacterial substances may inflict their damage by destroying the
host's phagocytes, rendering the body more susceptible to
infections by other pathogenic microorganisms. Substances produced
by many invading organisms cause allergic sensitivity in the host.
Infections may be spread via respiratory droplets, direct contact,
contaminated food, or vectors, such as insects. They can also be
transmitted sexually and from mother to fetus.
[0193] Diseases caused by bacterial infections typically include,
for example, actinomycosis, anthrax, aspergillosis, bacteremia,
bacterial skin diseases, bartonella infections, botulism,
brucellosis, burkholderia infections, campylobacter infections,
candidiasis, cat-scratch disease, chlamydia infections, cholera,
clostridium infections, coccidioidomycosis, cryptococcosis,
dermatomycoses, diphtheria, ehrlichiosis, epidemic louse borne
typhus, Escherichia coli infections, fusobacterium infections,
gangrene, general infections, general mycoses, gonorrhea,
gram-negative bacterial infections, gram-positive bacterial
infections, histoplasmosis, impetigo, klebsiella infections,
legionellosis, leprosy, leptospirosis, listeria infections, lyme
disease, malaria, maduromycosis, melioidosis, mycobacterium
infections, mycoplasma infections, necrotizing fasciitis, nocardia
infections, onychomycosis, ornithosis, pneumococcal infections,
pneumonia, pseudomonas infections, Q fever, rat-bite fever,
relapsing fever, rheumatic fever, rickettsia infections,
Rocky-mountain spotted fever, salmonella infections, scarlet fever,
scrub typhus, sepsis, sexually transmitted bacterial diseases,
staphylococcal infections, streptococcal infections, surgical site
infection, tetanus, tick-borne diseases, tuberculosis, tularemia,
typhoid fever, urinary tract infection, vibrio infections, yaws,
Yersinia infections, Yersinia pestis plague, zoonoses and
zygomycosis.
[0194] The polymers of the present embodiments can therefore be
used to treat medical conditions caused by pathogenic
microorganisms by virtue of their anti-microbial effects inflicted
upon the pathogenic microorganisms by one of the abovementioned
mechanism which mostly stem from their specific and selective
affinity to the membrane of the pathogenic microorganism, and
relative undamaging effect they have on mammalian cell, as
demonstrated for red blood cells and presented in the Examples
section that follows. This affinity can be used to weaken, disrupt,
puncture, melt, fuse and/or mark the membrane of a pathogenic
microorganism.
[0195] The pathogenic microorganism may be destroyed directly by
the disruption of its membrane as demonstrated and presented for a
series of bacterial strains in the Examples section that follows,
or be weakened so as to allow the innate immune system to destroy
it or slow down its metabolism and therefore its reproduction so as
to allow the innate immune system to overcome the infection.
[0196] The pathogenic microorganism may be destroyed by the
disruption of its membrane so as to allow a therapeutically active
agent, such as an antibiotic agent, to more easily penetrate the
cell of the microorganism and afflict its activity thereon.
[0197] The latter capacity of the antimicrobial polymer of the
present invention to assist the penetration of another
therapeutically active agent into the cells of the pathogenic
microorganism can be utilized to treat many infectious diseases,
such as, for example, malaria.
[0198] The experimental results presented in the Examples section
that follows suggests that secondary structure might not be an
absolute prerequisite for antimicrobial properties. On another
hand, evident from these results stems that the only property which
is shared by all typical AMPs, and also shared by the polymers of
the present invention, is the relative abundance of both
hydrophobic and positively charged amino acid residues. Thus,
according to the present invention, the antimicrobial polymers
presented are endowed with varied positive charge and
hydrophobicity and substantially lack secondary structure.
[0199] Malaria, also called jungle fever, paludism and swamp fever,
is an infectious disease characterized by cycles of chills, fever,
and sweating, caused by the parasitic infection of red blood cells
by the protozoan parasite, Plasmodium (one of the Apicomplexa),
which is transmitted by the bite of an infected vector for human
malarial parasite, a female Anopheles mosquito. Of the four types
of malaria, the most life-threatening type is falciparum malaria.
The other three types of malaria, vivax, malariae, and ovale, are
generally less serious and are not life-threatening. Malaria, the
deadliest infectious disease yet to be beaten, causes about half a
billion infections and between one and two millions deaths
annually, mainly in the tropics and sub-Saharan Africa. The
Plasmodium falciparum variety of the parasite accounts for 80% of
cases and 90% of deaths. The stickiness of the red blood cells is
particularly pronounced in P. falciparum malaria and this is the
main factor giving rise to hemorrhagic complications of
malaria.
[0200] To date there is no absolute cure for malaria. If diagnosed
early, malaria can be alleviated, but prevention still more
effective than treatment, thus substances that inhibit the parasite
are widely used by visitors to the tropics. Since the 17.sup.th
century quinine has been the prophylactic of choice for malaria.
The development of quinacrine, chloroquine, and primaquine in the
20.sup.th century reduced the reliance on quinine. These
anti-malarial medications can be taken preventively, which is
recommended for travelers to affected regions.
[0201] Unfortunately as early as the 1960s several strains of the
malarial parasite developed resistance to chloroquine. This
development of resistance, plus the growing immunity of mosquitoes
to insecticides, has caused malaria to become one the of world's
leading re-emerging infectious diseases. Mefloquine may be used in
areas where the disease has become highly resistant to chloroquine,
but some strains are now resistant to it and other drugs.
Artemisinin (derived from sweet wormwood) in combination with other
drugs is now in many cases the preferred treat for resistant
strains. Malarone (atovaquone and proguanil) is also used for
resistant strains. Vaccines against malaria are still
experimental.
[0202] While reducing the present invention to practice, the
present inventors have prepared and successfully used these
anti-microbial polymers as anti-malarial agents with reduced
hemolysis effect as demonstrated in the Examples section that
follows. It is shown that the polymers of the present invention
were able to kill the parasite in a manner that is clearly
dissociated from lysis of the host cell. These polymers were able
to enter the infected cell but to selectively permeabilize the
parasite cell membrane. These results are best explained by the
differential interaction of the peptides-like polymer with the
distinct properties of the structure and composition of the
membranes of intra-erythrocytic malaria parasite Plasmodium
falciparum as compared to those of the host and normal red blood
cells. These findings also established that the membrane active
polymers of the present invention could be engineered to act
specifically on the membrane of the intracellular parasite to
perturb its functions. The polymers of the present invention can
therefore overcome the problem of parasitic resistance to various
anti-malarial agents by, for example, weakening the parasite's
membrane and enabling the anti-malarial agents to penetrate the
parasite's membrane more rapidly.
[0203] Therefore, a preferred embodiment of the present invention
is the use of the antimicrobial polymers as an anti-malarial agent,
either per-se or in combination with a presently used anti-malarial
agent or any other anti-parasitic agent, as exemplified in the
Examples section that follows.
[0204] In any of the aspects of the present invention, the
antimicrobial polymers of the present invention can be utilized
either per se, or as an active ingredient that forms a part of a
pharmaceutical composition, with or without an additional
therapeutically active agent, and a pharmaceutically acceptable
carrier.
[0205] Hence, according to still another aspect of the present
invention, there are provided pharmaceutical compositions, which
comprise one or more of the polymers of the present invention as
described above having an antimicrobial activity and a
pharmaceutically acceptable carrier.
[0206] As used herein a "pharmaceutical composition" refers to a
preparation of the antimicrobial polymer described herein, with
other chemical components such as pharmaceutically acceptable and
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0207] Hereinafter, the term "pharmaceutically acceptable carrier"
refers to a carrier or a diluent that does not cause significant
irritation to an organism and does not abrogate the biological
activity and properties of the administered compound. Examples,
without limitations, of carriers are: propylene glycol, saline,
emulsions and mixtures of organic solvents with water, as well as
solid (e.g., powdered) and gaseous carriers.
[0208] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of a compound. Examples, without limitation, of
excipients include calcium carbonate, calcium phosphate, various
sugars and types of starch, cellulose derivatives, gelatin,
vegetable oils and polyethylene glycols.
[0209] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences" Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0210] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more pharmaceutically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
silver-coated enzymes into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen. Toxicity and therapeutic efficacy of the
silver-coated enzymes described herein can be determined by
standard pharmaceutical procedures in experimental animals, e.g.,
by determining the EC.sub.50, the IC.sub.50 and the LD.sub.50
(lethal dose causing death in 50% of the tested animals) for a
subject silver-coated enzyme. The data obtained from these activity
assays and animal studies can be used in formulating a range of
dosage for use in human.
[0211] The dosage may vary depending upon the dosage form employed
and the route of administration utilized. The exact formulation,
route of administration and dosage can be chosen by the individual
physician in view of the patient's condition. (See e.g., Fingl et
al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.
1).
[0212] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0213] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA (the U.S.
Food and Drug Administration) approved kit, which may contain one
or more unit dosage forms containing the active ingredient. The
pack may, for example, comprise metal or plastic foil, such as, but
not limited to a blister pack or a pressurized container (for
inhalation). The pack or dispenser device may be accompanied by
instructions for administration. The pack or dispenser may also be
accompanied by a notice associated with the container in a form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals, which notice is reflective of approval
by the agency of the form of the compositions for human or
veterinary administration. Such notice, for example, may be of
labeling approved by the U.S. Food and Drug Administration for
prescription drugs or of an approved product insert. Compositions
comprising a silver-coated enzyme of the invention formulated in a
compatible pharmaceutical carrier may also be prepared, placed in
an appropriate container, and labeled for treatment of an indicated
condition or diagnosis, as is detailed hereinabove.
[0214] Thus, according to an embodiment of the present invention,
depending on the selected polymers and the presence of additional
active ingredients, the pharmaceutical compositions of the present
invention are packaged in a packaging material and identified in
print, in or on the packaging material, for use in the treatment of
a medical condition associated with a pathogenic microorganism, as
is defined hereinabove and a parasite.
[0215] The pharmaceutical composition comprising a polymer of the
present invention may further comprise at least one additional
therapeutically active agent, as this is defined and presented
hereinabove.
[0216] The polymers of the present invention can be further
beneficially utilized as active substances in various medical
devices.
[0217] Hence, according to an additional aspect of the present
invention there is provided a medical device which includes one or
more of the polymers of the present invention, described
hereinabove, and a delivery system configured for delivering the
polymer(s) to a bodily site of a subject.
[0218] The medical devices according to the present invention are
therefore used for delivering to or applying on a desired bodily
site the polymers of the present invention. The polymers can be
incorporated in the medical devices either per se or as a part of a
pharmaceutical composition, as described hereinabove.
[0219] As used herein, the phrase "bodily site" includes any organ,
tissue, membrane, cavity, blood vessel, tract, biological surface
or muscle, which delivering thereto or applying thereon the
polymers of the present invention is beneficial.
[0220] Exemplary bodily sites include, but are not limited to, the
skin, a dermal layer, the scalp, an eye, an ear, a mouth, a throat,
a stomach, a small intestines tissue, a large intestines tissue, a
kidney, a pancreas, a liver, the digestive system, the respiratory
tract, a bone marrow tissue, a mucosal membrane, a nasal membrane,
the blood system, a blood vessel, a muscle, a pulmonary cavity, an
artery, a vein, a capillary, a heart, a heart cavity, a male or
female reproductive organ and any visceral organ or cavity.
[0221] The medical devices according to this aspect of the present
invention can be any medical device known in the art, including
those defined and classified, for example, by the FDA and specified
in http://www.fda.gov/cdrh/devadvice/313.html (e.g., Class I, II
and III), depending e.g., on the condition and bodily site being
treated.
[0222] Thus, for example, in one embodiment of this aspect of the
present invention, the medical device comprises a delivery system
that is configured to deliver the polymer(s) by inhalation. Such
inhalation devices are useful for delivering the polymers of the
present invention to, e.g., the respiratory tract.
[0223] The delivery system in such medical devices may be based on
any of various suitable types of respiratory delivery systems which
are suitable for administering a therapeutically effective dose of
the polymer(s) of the present invention to a subject.
[0224] The inhalation device may be configured to deliver to the
respiratory tract of the subject, preferably via the oral and/or
nasal route, the compound in the form of an aerosol/spray, a vapor
and/or a dry powder mist. Numerous respiratory systems and methods
of incorporating therapeutic agents therein, such as the polymers
of the present invention, suitable for assembly of a suitable
inhalation device are widely employed by the ordinarily skilled
artisan and are extensively described in the literature of the art
(see, for example to U.S. Pat. Nos. 6,566,324, 6,571,790,
6,637,430, and 6,652,323; U.S. Food & Drug Administration
(USFDA) Center For Drug Evaluation and Research (CDER);
http://www.mece.ualberta.ca/arla/tutorial.htm).
[0225] The respiratory delivery system may thus be, for example, an
atomizer or aerosol generator such as a nebulizer inhaler, a dry
powder inhaler (DPI) and a metered dose inhaler (MDI), an
evaporator such as an electric warmer and a vaporizer, and a
respirator such as a breathing machine, a body respirator (e.g.,
cuirass), a lung ventilator and a resuscitator.
[0226] In still another embodiment of this aspect of the present
invention, the medical device is such that delivering the
polymer(s) is effected transdermally. In this embodiment, the
medical device is applied on the skin of a subject, so as to
transdermally deliver the polymer(s) to the blood system.
[0227] Exemplary medical devices for transdermally delivering a
polymer according to the present invention include, without
limitation, an adhesive plaster and a skin patch. Medical devices
for transdermal or transcutaneous delivery of the polymer(s)
typically further include one or more penetration enhancers, for
facilitating their penetration through the epidermis and into the
system.
[0228] According to another embodiment of this aspect of the
present invention, the medical device is such that delivering the
polymer(s) is effected by topically applying the medical device on
a biological surface of a subject. The biological surface can be,
for example, a skin, scalp, an eye, an ear and a nail. Such medical
devices can be used in the treatment of various skin conditions and
injuries, eye and ear infections and the like.
[0229] Exemplary medical devices for topical application include,
without limitation, an adhesive strip, a bandage, an adhesive
plaster, a wound dressing and a skin patch.
[0230] In another embodiment of this aspect of the present
invention, the medical device is such that delivering the
polymer(s) is effected by implanting the medical device in a bodily
organ. As used herein, the term "organ" further encompasses a
bodily cavity.
[0231] The organ can be, for example, a pulmonary cavity, a heart
or heart cavity, a bodily cavity, an organ cavity, a blood vessel,
an artery, a vein, a muscle, a bone, a kidney, a capillary, the
space between dermal layers, an organ of the female or male
reproductive system, an organ of the digestive tract and any other
visceral organ.
[0232] The medical device according to this embodiment of the
present invention typically includes a device structure in which a
polymer according to the present invention is incorporated. The
polymer(s) can thus be, for example, applied on, entrapped in or
attached to (chemically, electrostatically or otherwise) the device
structure.
[0233] The device structure can be, for example, metallic structure
and thus may be comprised of a biocompatible metal or mixture of
metals (e.g., gold, platinum).
[0234] Alternatively, the device structure may be comprised of
other biocompatible matrices. These can include, for example,
plastics, silicon, polymers, resins, and may include at least one
component such as, for example, polyurethane, cellulose ester,
polyethylene glycol, polyvinyl acetate, dextran, gelatin, collagen,
elastin, laminin, fibronectin, vitronectin, heparin, segmented
polyurethane-urea/heparin, poly-L-lactic acid, fibrin, cellulose
and amorphous or structured carbon such as in fullerenes, and any
combination thereof.
[0235] In cases where a biodegradable implantable device is
desired, the device structure can be comprised of a biocompatible
matrix that is biodegradable. Biodegradable matrices can include,
for example, biodegradable polymers such as poly-L-lactic acid.
[0236] Optionally, the device structure may be comprised of
biocompatible metal(s) coated with other biocompatible matrix.
[0237] Further optionally, in cases where a device which releases
the polymer(s) of the present invention in a controlled manner is
desired, the device structure can be comprised of or coated with a
biocompatible matrix that functions as or comprises a slow release
carrier. The biocompatible matrix can therefore be a slow release
carrier which is dissolved, melted or liquefied upon implantation
in the desired site or organ. Alternatively, the biocompatible
matrix can be a pre-determined porous material which entraps the
polymer(s) in the pores. When implanted in a desired site, the
polymer(s) diffuse out of the pores, whereby the diffusion rate is
determined by the pores size and chemical nature. Further
alternatively, the biocompatible matrix can comprise a
biodegradable matrix, which upon degradation releases the
polymer(s) of the present invention.
[0238] The polymer(s) of the present invention can be incorporated
in the device structure by any methodology known in the art,
depending on the selected nature of the device structure. For
example, the polymer(s) can be entrapped within a porous matrix,
swelled or soaked within a matrix, or being adhered to a
matrix.
[0239] Much like their antimicrobial activity in the body, the
antimicrobial activity of the polymers of the present invention may
further be harnessed for the preservation of food ingredients and
products.
[0240] Hence, according to yet another aspect of the present
invention there is provided a food preservative comprising an
effective amount of the polymer of the present invention as
described herein.
[0241] The polymer(s) may be incorporated into the food product as
one of its ingredients either per se, or with an edible
carrier.
[0242] The polymers of the present invention have been shown to
have high and selecting affinity towards membranes of
microorganisms as demonstrated in the Examples section that
follows. This attribute is one of the main elements which
contribute to the effective and efficacious activity of the
polymers when utilized as an antimicrobial agent. When the polymer
is coupled with a labeling agent, this membrane binding attribute
can be further employed to label colonies and proliferation sites
of microorganisms, especially microbial growth loci in a host in
vivo.
[0243] Hence, according to another aspect of the present invention
there is provided an imaging probe for detecting a pathogenic
microorganism, the imaging probe comprising a polymer as defined
and described hereinabove, whereas the polymer further includes at
least one labeling agent, as defined hereinabove, attached thereto.
When released to the environment, these polymers, having a labeling
agent attached thereto will bind to the membrane of cell of
microorganisms and therefore attach the labeling agent to the cells
of the microorganism.
[0244] As used herein, the term "chromophore" refers to a chemical
moiety that, when attached to another molecule, renders the latter
colored and thus visible when various spectrophotometric
measurements are applied.
[0245] The phrase "fluorescent compound" refers to a compound that
emits light at a specific wavelength during exposure to radiation
from an external source.
[0246] The phrase "phosphorescent compound" refers to a compound
emitting light without appreciable heat or external excitation as
by slow oxidation of phosphorous.
[0247] A heavy metal cluster can be for example a cluster of gold
atoms used, for example, for labeling in electron microscopy
techniques.
[0248] According to preferred embodiments of the present invention,
one or more labeling agents may be attached to the polymer at any
substitutable position, as in the case of an active agent discussed
above. Examples of such substitutable positions are, without
limitation, a side chain of any one or more of the amino acid
residues in the polymer, any one of the linking moieties of the
polymer, any one of the N- and C-termini of the polymer and any one
or more of the hydrophobic moiety residues in the polymer.
[0249] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0250] Reference is now made to the following examples, which
together with the above descriptions; illustrate the invention in a
non limiting fashion.
Materials and Experimental Methods
Chemical Syntheses:
[0251] Materials:
[0252] Lysine having Fmoc ((9H-fluoren-9-yl)methyl carbonate)
protection on its main-chain amine group and Boc (tert-butyl
carbonate) protection on its side-chain amine group was purchased
from Applied Biosystems and from NovaBiochem.
[0253] .omega.-amino fatty acids such as 4-amino-butyric acid,
8-amino-caprylic acid and 12-animo-lauric acid having Fmoc
protection of the amine group were purchased from
Sigma-Aldrich/NovaBiochem.
[0254] All other solvents and reagents used were purchased from
Sigma-Aldrich/NovaBiochem/Applied Biosystems/J. T. Baker and were
used without further purification.
Preparation of Libraries of Antimicrobial Polymers--General
Procedure
[0255] The polymers according to the present invention were
prepared by a solid phase method and were purified to
chromatographic homogeneity according to methodologies described in
the art (Feder, R. et al. (2000) J. Biol. Chem. 275, 4230-4238).
Briefly, the polymers were synthesized by applying the Fmoc active
ester chemistry on a fully automated, programmable peptide
synthesizer (Applied Biosystems 433A). After cleavage from the
resin, the crude polymers were extracted with 30% acetonitrile in
water and purified to obtain a chromatographic homogeneity greater
than 95%, as determined by HPLC (Alliance Waters).
[0256] HPLC chromatograms were performed on C18 columns (Vydak, 250
mm.times.4.6 or 10 mm) using a linear gradient of acetonitrile in
water (1% per minute), both solvents contained 0.1% trifluoroacetic
acid. The purified polymers were subjected to mass spectrometry (ZQ
Waters) to confirm their composition and stored as a lyophilized
powder at -20.degree. C. Prior to being tested, fresh solutions
were prepared in water, mixed by vortex, solubilized by ultrasound,
centrifuged and then diluted in the appropriate medium.
[0257] In order to estimate the hydrophobicity of each polymer, the
polymer was eluted with a linear gradient of acetonitrile (1% per
minute) on an HPLC reversed-phase C18 column, and the percent of
acetonitrile at which the polymer was eluted was used for
hydrophobicity estimation (see, "ACN (%)" in Table 3 below).
[0258] Exemplary building units which were utilized in the
synthesis described above are presented in Scheme 1 below and
include: lysine and an .omega.-amino-fatty acid having m carbon
atoms (Compound I).
[0259] Synthesis of exemplary polymers according to the present
invention, which are comprised of lysine and Compound I, was
performed by adding an Fmoc/Boc-protected lysine and an
Fmoc-protected Compound I separately and sequentially to the resin
according to conventional peptide solid phase synthesis
protocols.
##STR00005##
In Vitro Studies:
[0260] Bacterial Strains and Sample Preparation:
[0261] Antibacterial activity was determined using the following
strains, cultured in LB medium (10 grams/liter trypton, 5
grams/liter yeast extract, 5 grams/liter NaCl, pH 7.4): Escherichia
coli (ATCC (American Type Culture Collection) 35218); methicilin
resistant Staphylococcus aureus (CI (clinical isolate) 15903);
Bacillus cereus (ATCC 11778); and Pseudomonas aeruginosa (ATCC
9027).
[0262] Minimal Inhibitory Concentration (MIC) Measurements:
[0263] Minimal inhibitory concentrations (MICs) were determined by
microdilution susceptibility testing in 96-well plates using
inocula of 10.sup.6 bacteria per ml.
[0264] Cell populations were evaluated by optical density
measurements at 600 nm and were calibrated against a set of
standards. Hundred (100) .mu.l of a bacterial suspension were added
to 100 .mu.l of culture medium (control) or to 100 .mu.l of culture
medium containing various polymer concentrations in 2-fold serial
dilutions. Inhibition of proliferation was determined by optical
density measurements after an incubation period of 24 hours at
37.degree. C.
[0265] Alternatively, MICs were determined using the microbroth
dilution assay recommended by the Clinical and Laboratory Standards
Institute (CLSI) using two-fold serial dilutions in cation-adjusted
Mueller-Hinton broth (CAMHB).
[0266] Clinical bacterial isolates were obtained from Tel Aviv
Sourasky Medical Center, Israel. Bactericidal kinetics was assessed
using the drop plate method [see, for example, Chen et al., Journal
of Microbiological Methods, November; 55(2):475-9, 2003; and
Skerman V. B. D., 1969, Abstracts of Microbiological Methods, p.
143-161, Wiley-Interscience, New York]. Statistical data for each
experiment were obtained from at least two independent assays
performed in duplicates.
[0267] The Effect of Physical Parameters (Charge and
Hydrophobicity) on Antimicrobial Activity:
[0268] A library of polymers was prepared to sample the effect of
increased charge and hydrophobicity on the antimicrobial activity.
The charge was serially sampled by increasing the number of the
.omega.-amino-fatty acid-lysine conjugates from 1 to 7. The
hydrophobicity was serially sampled by increasing the number of the
carbon atoms of the .omega.-amino fatty acid (4, 8 and 12). The
polymers in each series were tested for their antimicrobial
activity, as described hereinabove.
[0269] Development of Antimicrobial-Resistance in Bacteria:
[0270] The possible development of resistance to the antimicrobial
activity of the polymers of the present invention by bacteria, as
compared with known resistance-inducing classical antibiotic
agents, gentamycin, tetracycline and ciprofloxacin, which served as
controls for the development of antibiotic-resistant bacterial
strains, was studied.
[0271] Bacteria samples (E. coli strain ATCC 35218) at the
exponential phase of growth were exposed to an antimicrobial agent
for MIC determination as described above. Following incubation
overnight, bacteria were harvested from wells that displayed near
50% growth inhibition, washed and diluted in fresh medium, grown
overnight, and subjected again to MIC determination for up to 15
iterations (15 days). For each compound tested, the 0D.sub.620 of
one half the MIC well from the previous MIC assay was diluted to
yield 5.times.10.sup.5 cells/ml in LB (according to a calibration
curve) and was used again for MIC determination in subsequent
generations.
[0272] In parallel, MIC evolution in these subcultures was compared
concomitantly with each new generation, using bacteria harvested
from control wells (wells cultured without a polymer) from the
previous generation. The relative MIC was calculated for each
experiment from the ratio of MIC obtained for a given subculture to
that obtained for first-time exposure.
[0273] Kinetic Studies:
[0274] The kinetic assays were performed in test tubes, in a final
volume of 1 ml, as follows: 100 .mu.l of a suspension containing
bacteria at 2-4.times.10.sup.7 colony forming units (CFUs)/ml in
culture medium were added to 0.9 ml of culture medium or culture
medium containing various polymer concentrations (0, 3 and 6
multiples of the MIC value). After 0, 30, 60, 90, 120 and 360
minutes of exposure to the polymer at 37.degree. C. while shaking,
cultures were subjected to serial 10-fold dilutions (up to
10.sup.-6) by adding 50 .mu.l of sample to 450 .mu.l saline (0.9%
NaCl). Colony forming units (CFUs) were determined using the drop
plate method (3 drops, 20 .mu.l each, onto LB-agar plates, as
described in Yaron, S. et al. (2003), Peptides 24, 1815-1821). CFUs
were counted after plate incubation for 16-24 hours at 37.degree.
C. Statistical data for each of these experiments were obtained
from at least two independent assays performed in duplicates.
[0275] Antimicrobial Activity at Enhanced Outer-Membrane
Permeability Conditions:
[0276] The outer membrane permeability of gram-negative bacteria,
namely E. coli or P. aeruginosa, was enhanced by treating bacterial
cultures with EDTA (ethylenediaminetetraacetic acid) according to
the following procedure: 1 M EDTA solution in water (pH=8.3) was
diluted in LB medium to obtain a 4 mM concentration and the diluted
solution was used for polymer dissolution. Bacteria were grown
overnight in LB medium, and 100 .mu.l fractions containing 10.sup.6
bacteria per ml were added to 100 .mu.l of EDTA culture medium or
to EDTA culture medium containing various polymer concentrations
(2-fold serial dilutions) in 96-well plates. Growth inhibition was
determined against gram-negative bacteria as described above.
[0277] Susceptibility to Plasma Proteases:
[0278] The susceptibility of the polymers of the present invention
to proteolytic digestion was assessed by determining the
antibacterial activity after exposure to human plasma as follows:
250 .mu.l of polymer solutions in saline (0.9% NaCl) at a
concentration of 16 multiples of the MIC value were pre-incubated
with 50% (v/v) human plasma in culture medium at 37.degree. C.
After incubation periods of 3, 6, and 18 hours, the polymer
solutions were subjected to 2-fold serial dilutions in LB medium in
96-well plates. The susceptibility of the polymers of the present
invention to enzymatic cleavage was assessed by pre-incubating four
exemplary polymers according to the present invention,
C.sub.12K(NC.sub.8K).sub.5NH.sub.2, K(NC.sub.12K).sub.3NH.sub.2,
C.sub.12KNC.sub.12KNH.sub.2, and C.sub.12KKNC.sub.12KNH.sub.2, and
a 16-residues dermaseptin S4 derivative (S4.sub.16, an exemplary
AMP which served as the control), in human plasma (50%) for various
time periods. The antibacterial activity was thereafter determined
against E. coli and S. aureus, as described above. In parallel,
antibacterial activity was also determined in culture medium
conditions in the absence of plasma (referred to as 0 hours of
pre-incubation in the experimental results section below).
Statistical data was obtained from at least two independent
experiments performed in duplicates.
[0279] Hemolysis Assays:
[0280] The polymer's membranolytic potential was determined against
human red blood cells (RBC) in phosphate buffer solution (PBS).
Human blood samples were rinsed three times in PBS by
centrifugation for 2 minutes at 200.times.g, and re-suspended in
PBS at 5% hematocrit. A 50 .mu.l-fractions of a suspension
containing 2.5.times.10.sup.8 RBC were added to test tubes
containing 200 .mu.l of polymer solutions (2-fold serial dilutions
in PBS), PBS alone (for base-line values), or distilled water (for
100% hemolysis). After 3 hours incubation at 37.degree. C. under
agitation, samples were centrifuged, and hemolytic activity was
determined as a function of hemoglobin leakage by measuring
absorbance at 405 nm of 200 .mu.l aliquots of the supernatants.
[0281] Alternatively, a 10% hematocrit was used and hemolysis was
determined after one hour incubation. Hemolytic activity was
determined according to Antibacterial Peptides Protocols as
presented by Tossi, A. et al. in Methods Mol. Biol., 1997, 78, pp.
133-150.
[0282] Circular Dichroism (CD):
[0283] CD spectra in millidegrees were measured with an Aviv model
202 CD spectrometer (Aviv Associates, Lakewood, N.J.) using a 0.01
cm rectangular QS Hellma cuvette at 25.degree. C. (controlled by
thermoelectric Peltier elements with an accuracy of 0.1.degree.
C.). Polymer samples were dissolved in either PBS, 20% (v/v)
trifluoroethanol/water or titrated in PBS containing POPC
(2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine) and POPG
(1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol)) in a
3:1 ratio and concentration of up to 2 mM, to thereby obtain
liposomes. CD spectra of the polymers were scanned at a
concentration of 100 .mu.M as determined by UV using standard
curves of known concentrations for each polymer. The CD of the
N-terminus acylated S4 dermaseptin derivative
NC.sub.12K.sub.4S4(1-14) (Mor, A. et al. (1994), J. Biol. Chem.
269(50): 31635-41), an exemplary AMP, was measured under the same
conditions and was used as a reference compound in the CD studies.
The CD data presented herein represent an average of three separate
recordings values.
[0284] Surface Plasmon Resonance Assay:
[0285] Binding to model bilayer membranes was studied by surface
plasmon resonance (SPR) using a BIAcore 2000 biosensor system.
Liposomes composed of phospholipids mimicking bacterial plasma
membrane (POPC:POPG in a 3:1 ratio) were immobilized on the sensor
surface and polymer solutions were continuously flowed over the
membrane. The curve of resonance signal as a function of time
displays the progress of the interaction between the analyzed
polymer and the immobilized phospholipid membrane. The affinity of
the interaction was calculated from analysis of the resulting
curves as detailed in Gaidukov, L. et al. (2003), Biochemistry 42,
12866-12874. Briefly, the association and dissociation curves
(binding rates) were analyzed at five doses (0.21, 0.42, 0.84,
1.67, 3.35 .mu.g), and the K.sub.app (the resulting binding
constant) was calculated assuming a 2-step model).
[0286] Lipopolysaccharide Binding Assay:
[0287] In order to explore the mechanism by which the polymers of
the present invention exert the anti-bacterial activity, the
targeting of the polymers to the bacterial membrane was tested.
More specifically, the binding affinity of the positively charged
polymers to the negatively charged lipopolysaccharides (LPS)
present on the membrane of gram-negative bacteria was tested.
[0288] Thus, binding assays of the polymers of the present
invention to LPS were carried out with SPR technology using the
optical biosensor system BIAcore 2000 (BIAcore). A mixture of 50
.mu.M of the polymer samples in PBS and 100 .mu.g/ml LPS was
incubated for 30 minutes at room temperature. The binding assay was
performed by injecting 10 .mu.l of the mixture at a flow rate of 10
.mu.l per minute at 25.degree. C. over a POPC:POPG (3:1) bilayer
spread on an L1 sensor chip. 100 .mu.g/ml LPS without a polymer
sample were injected as a blank of LPS binding to the membrane and
50 .mu.M of a polymer sample was injected to determine the polymer
binding to membrane without LPS.
[0289] DNA Binding Assay:
[0290] Binding of the polymers of the present invention to nucleic
acids was studied by assessing their ability to retard migration of
DNA plasmids during gel electrophoresis in a 1% agarose gel.
DNA-retardation experiments were performed by mixing 200 nanograms
of the plasmid DNA (pUC19, 2683 base pairs) with increasing amounts
of various polymers in a final volume of 20 .mu.l doubly distilled
water (DDW). The reaction mixtures were incubated at room
temperature for 30 minutes. Subsequently, 2 .mu.l of loading dye
(20% Ficoll 400, 0.1 M EDTA, 0.25% bromophenol blue and 1% sodium
dodecyl sulfate) were added and an aliquot of 20 .mu.l was applied
to 1% agarose gel electrophoresis in TAE buffer (0.02 M Tris base,
0.01 M glacial acetic acid, 0.5 mM EDTA, pH=8.5) containing
ethidium bromide (0.25 .mu.g/ml). The plasmid used in this
experiment was isolated by the Wizard.RTM. Plus SV Minipreps DNA
Purification System (Promega).
[0291] Saliva Microbicidal Assays:
[0292] Antimicrobial activity of polymers of the present invention
against the melange of microorganisms in the saliva of healthy
human volunteers was studied by mixing fresh human saliva with the
polymers or IB-367 (both dissolved in 10 mM sodium acetate buffer
set at pH 5 to a final concentration of 100 .mu.M) at a 1:1 ratio.
A solution of the saliva with no anti-bacterial agent served as a
control. IB-367 is a positively charged protegrin peptide with
known in-vitro and in-vivo activities against the microflora
associated with human oral mucositis (Loury, D. et al., 1999, Oral
Surg Oral Med Oral Pathol Oral Radiol Endod 87(5): 544-51.). Each
of the solutions was spread over a LA plate, and the plated saliva
samples were incubated overnight at 37.degree. C. without aeration.
The colonies were enumerated and counted to determine the
microbicidal effect of the drug. The values of viable colony
forming units (CFU) were determined as described above.
[0293] Anti-Malarial Assays:
[0294] The investigation of the anti-malarial activity of the
polymers of the present invention was performed by screening part
of the library of the polymers presented hereinbelow in Table 3,
for anti-malarial and hemolytic activities as well as for their
toxic activities against mammalian cells in culture.
[0295] Parasite cultivation: Different strains of P. falciparum
were cultivated as described by Kutner and co workers [Kutner, S.,
Breuer, W. V., Ginsburg, H., Aley, S. B., and Cabantchik, Z. I.
(1985) J. Cell. Physiol. 125, 521-527] using human red blood cells
(RBC). The cultures were synchronized by the sorbitol method
[Lambros, C. J., and Vanderberg, J. P. (1979) J. Parasitol. 65,
418-420] and infected cells were enriched from culture by
Percoll-alanine gradient centrifugation [Kutner, S., Breuer, W. V.,
Ginsburg, H., Aley, S. B., and Cabantchik, Z. I. (1985) J. Cell.
Physiol. 125, 521-527].
[0296] Determination of IC.sub.50: Synchronized cultures at the
ring stage were cultured at 1% hematocrit and 2% parasitemia in the
presence of increasing concentrations of the tested polymers. After
18 hours of incubation parasite viability was determined by
[.sup.3H]hypoxanthine (Hx) uptake (final concentration was 2
.mu.Ci/ml) during 6 hours and compared to controls (without the
polymers). The 50% inhibitory concentration (IC.sub.50) was
determined by nonlinear regression fitting of the data using the
commercially available software suite Sigmaplot.TM..
[0297] Time- and stage-dependence action of the polymers:
Anti-malarial drugs are known to exert their action differentially
on different stages of parasite development. They also need a
minimal time of interaction with the parasite in order to inhibit
its growth. Therefore, cultures at the ring stage were seeded in
24-well plate at 1% hematocrit, 2% parasitemia in plate medium
(growth medium without hypoxanthine, 10 mM NaHCO3 and 7% heat
inactivated human plasma). Tested polymers were added at different
concentrations immediately and removed after 6, 24 and 48 hours.
Cultures without polymers were left to mature to the trophozoite
stage and dosed with compounds for 6 and 24 hours. Two .mu.Ci of Hx
per well were added to all cells after 30 hours from the onset of
the experiment and the cells were harvested after 24 hours.
[0298] Effect of the polymers on mammalian cells in culture: MDCK
(cell line from dog kidney) epithelial cells were grown to
confluence (about 3 days in culture). Parallel cultures were grown
with different concentrations of the tested polymers. Thereafter 10
.mu.l of Alamar blue was added and fluorescence was measured after
3.5 hours. For a positive control, 10 .mu.M of cycloheximide were
added to control samples at the beginning of cultivation.
In-Vivo Studies:
[0299] Activity Assay:
[0300] In-vivo prevention of E. coli-induced mortality using acute
peritonitis and sepsis model experiments were conducted using
female neutropenic ICR (Institute for Cancer Research) mice
(weighing 25 to 27 grams each).
[0301] A bacterial inoculum was prepared on brain heart infusion
(BHI) broth (Becton-Dickinson) with agitation (180 rpm) at
37.degree. C. for 5 to 6 hours. Cells were harvested when the
culture reached an optical density of 0.8 OD.sub.620 and
re-suspended in sterile BHI broth. The number of viable cells was
verified by plating serial dilutions of the injected inocula onto
BHI agar plates.
[0302] Infection was induced by intraperitoneal injection of
2.5.times.10.sup.6 or 5.times.10.sup.6 colony forming units (CFU)
of E. coli in the logarithmic growth phase (CI-3504, isolated from
a patient with peritonitis and bacteremia) in 0.5 ml of culture
media to groups of 10 mice each. One hour after infection, mice
were intraperitoneally injected with 0.5 ml of vehicle (PBS
control), one 4 mg/kg body weight dose of an exemplary polymer,
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, or 4 doses injection (after 1,
6, 20, and 28 hours) of 2 mg/kg body weight of imipenem (an
antibiotic drug which belongs to the carbapenems family and used to
treat severe or very resistant infections). Mice were monitored for
survival over 6 days period after infection.
[0303] Toxicity:
[0304] Acute toxicity was examined after intraperitoneal injection
of C.sub.12K(NC.sub.8K).sub.7NH.sub.2, an exemplary polymer
according to the present invention, to groups of 12 ICR mice. Each
mouse was injected with a 0.5 ml solution of freshly prepared
C.sub.12K(NC.sub.8K).sub.7NH.sub.2 in PBS. The doses of polymer
administered per mouse were 0 .mu.g (blank control), 100 .mu.g, 250
.mu.g, and 500 .mu.g (corresponding to 0 mg/kg, 4 mg/kg, 10 mg/kg,
and 20 mg/kg body weight). Animals were directly inspected for
adverse effects for 4 hour, and mortality was monitored for 7 days
thereafter.
Experimental Results
[0305] Preparation of Libraries of Polymers:
[0306] Several representative series of polymers according to the
present invention, which are substantially comprised of a plurality
of lysine residues and .omega.-amino-fatty acid residues and fatty
acid residues as hydrophobic moieties, were prepared according to
the general procedure described above, and are presented in Table 3
below.
[0307] These exemplary polymers are referred to in this section
according to the following formula:
T[NC.sub.iK].sub.jG
[0308] In this formula, NC.sub.i denotes an .omega.-amino-fatty
acid residue (an exemplary hydrophobic moiety according to the
present invention, represented by D.sub.1 . . . Dn in the general
formula I described herein), whereby i denotes the number of carbon
atoms in the fatty acid residue; K denotes a lysine residue (an
exemplary amino acid residue according to the present invention,
denoted as A.sub.1 . . . An in the general Formula I described
herein, such that [NC.sub.iK] denotes a residue of an
.omega.-amino-fatty acid-lysine conjugate (denoted as
[A.sub.1-Z.sub.1-D.sub.1] . . . [An-Zn-Dn] in the general Formula I
described herein); j denotes the number of the repeating units of a
specific conjugate in the polymer (corresponding to n in the
general Formula I described herein); and T and G each independently
denotes either a hydrogen (no denotation), a lysine residue
(denoted K), an .omega.-amino-fatty acid residue (denoted
NC.sub.i), a fatty acid residue (denoted C.sub.i), an
.omega.-amino-fatty acid-lysine conjugate residue (denoted
NC.sub.iK), a fluorenylmethyloxycarbonyl residue (denoted Fmoc), a
benzyl residue (denoted Bz), a cholate residue (denoted Chl), an
amine group (typically forming an amide at the C-terminus and
denoted NH.sub.2), and free acid residue (for the C-terminus no
denotation), an alcohol residue, and any combination thereof (all
corresponding to X and Y in the general formula I described
herein).
[0309] Thus, for example, a polymer according to the present
invention which is referred to herein as
NC.sub.12K(NC.sub.8K).sub.7NH.sub.2, corresponds to a polymer
having the general formula I described hereinabove, wherein: X is a
residue of a conjugate of an .omega.-amino-fatty acid having 12
carbon atoms (12-amino-lauric acid) and lysine; n is 6; A.sub.1 . .
. A.sub.6 are each a lysine residue; D.sub.1 . . . D.sub.7 are all
residues of an .omega.-amino-fatty acid having 8 carbon atoms
(8-amino-caprylic acid); Z.sub.1 . . . Z.sub.7 and W.sub.0--W.sub.7
are all peptide bonds; and Y is an amine. For clarity, the chemical
structure of NC.sub.12K(NC.sub.8K).sub.7NH.sub.2 is presented in
Scheme 2 below:
##STR00006##
[0310] Minimal Inhibitory Concentration Measurements:
[0311] The polymers in each series were tested for various
antimicrobial activities, as described hereinabove. The obtained
results are presented in Table 3 below, wherein:
[0312] "Q" represents the overall molecular charge at physiological
pH (column 3 in Table 3);
[0313] "ACN (%)" represents the percent of acetonitrile in the
HPLC-RP gradient mobile phase at which the polymer was eluted and
which corresponds to the estimated hydrophobicity of the polymer
(column 4 in Table 3);
[0314] "LC50" represents the lytic concentration of each tested
polymer in .mu.M obtained by the membranolytic potential
determination experiment of hemolysis of human red blood cells
measured as described hereinabove (column 5 in Table 3);
[0315] "MIC E.c." represents the minimal inhibitory concentration
of each tested polymer in .mu.M for E. coli, measured as described
hereinabove in the antibacterial activity assay (column 6 in Table
3);
[0316] "MIC EDTA E.c." represents the minimal inhibitory
concentration of each tested polymer in .mu.M for E. coli culture
in the presence of 2 mM EDTA, measured as described hereinabove for
the enhanced outer-membrane permeability assay (column 7 in Table
3);
[0317] "MIC P.a." represents the minimal inhibitory concentration
of each tested polymer in .mu.M for P. aeruginosa, measured as
described hereinabove for the antibacterial activity assay (column
8 in Table 3);
[0318] "MIC MR S.a." represents the minimal inhibitory
concentration of each tested polymer in .mu.M for
methicilin-resistant S. aureus, measured as described hereinabove
for the antibacterial activity assay of antibiotic-resistant
bacteria (column 9 in Table 3);
[0319] "MIC B.c." represents the minimal inhibitory concentration
of each tested polymer in .mu.M for Bacillus cereus, measured as
described hereinabove for the antibacterial activity assay (column
10 in Table 3); and
[0320] ND denotes "not determined".
[0321] Some values are presented with .+-.standard deviations from
the mean.
[0322] "Orn" and "Arg" in entries 84 and 85 denote ornithine and
arginine amino acid residues respectively.
[0323] Entries 96 to 99 present activity data of four control
antimicrobial peptides, namely MSI-78, a magainin derivative;
IB-367, a protegrin derivative; K.sub.4S.sub.4(1-16), a dermaseptin
derivative; and LL37, a cathelicidin derivative.
[0324] Entries 100 to 103 present activity data of four control
antibiotic agents, namely Ciprofloxacin, Imipenem, Tetracycline and
Rifampin.
TABLE-US-00003 TABLE 3 ACN MIC MIC MIC MIC MIC No. Polymer Q (%)
LC50 E. c. EDTA E. c. P. a. MR S. a. B. c. 1
C.sub.4KNC.sub.4KNH.sub.2 2 19.6 >100 >50 ND >50 >50
>50 2 C.sub.4K(NC.sub.4K).sub.2NH.sub.2 3 21.8 >100 >50 ND
>50 >50 >50 3 C.sub.4K(NC.sub.4K).sub.3NH.sub.2 4 21.5
>100 >50 ND >50 >50 >50 4
C.sub.4K(NC.sub.4K).sub.4NH.sub.2 5 22 >100 >50 ND >50
>50 >50 5 C.sub.4K(NC.sub.4K).sub.5NH.sub.2 6 22.9 >100
>50 ND >50 >50 >50 6 C.sub.4K(NC.sub.4K).sub.6NH.sub.2
7 23.5 >100 >50 ND >50 >50 >50 7
C.sub.4K(NC.sub.4K).sub.7NH.sub.2 8 24.3 >100 >50 ND >50
>50 >50 8 KNC.sub.4KNH.sub.2 3 0 ND >50 ND >50 >50
>50 9 K(NC.sub.4K).sub.2NH.sub.2 4 9 ND >50 ND >50 >50
>50 10 K(NC.sub.4K).sub.3NH.sub.2 5 18.8 ND >50 ND >50
>50 >50 11 K(NC.sub.4K).sub.4NH.sub.2 6 20.1 ND >50 ND
>50 >50 >50 12 K(NC.sub.4K).sub.5NH.sub.2 7 20.8 ND >50
ND >50 >50 >50 13 K(NC.sub.4K).sub.6NH.sub.2 8 21.7 ND
>50 ND >50 >50 >50 14 K(NC.sub.4K).sub.7NH.sub.2 9 22.2
ND >50 ND >50 >50 >50 15
C.sub.12K(NC.sub.4K).sub.1NH.sub.2 2 50.2 ND >50 ND >50
>50 >50 16 C.sub.12K(NC.sub.4K).sub.2NH.sub.2 3 47.6 ND
>50 ND >50 >50 >50 17
C.sub.12K(NC.sub.4K).sub.3NH.sub.2 4 46.4 ND >50 ND >50
>50 >50 18 C.sub.12K(NC.sub.4K).sub.4NH.sub.2 5 45.4 ND 50 ND
>50 >50 >50 19 C.sub.12K(NC.sub.4K).sub.5NH.sub.2 6 45.8
ND 12.5 6.3 >50 >50 >50 20
C.sub.12K(NC.sub.4K).sub.6NH.sub.2 7 45.1 ND 9.4 .+-. 3.1 4.7 .+-.
2.2 >50 >50 >50 21 C.sub.12K(NC.sub.4K).sub.7NH.sub.2 8
45.2 >100 9.4 .+-. 3.1 3.1 >50 >50 >50 22
NC.sub.12K(NC.sub.4K).sub.5NH.sub.2 7 29.3 ND >50 ND >50
>50 >50 23 NC.sub.12K(NC.sub.4K).sub.6NH.sub.2 8 29.8 ND
>50 ND >50 >50 >50 24
NC.sub.12K(NC.sub.4K).sub.7NH.sub.2 9 30.2 ND >50 ND >50
>50 >50 25 C.sub.8KNC.sub.8KNH.sub.2 2 38.9 >100 >50 ND
>50 >50 >50 26 C.sub.8K(NC.sub.8K).sub.2NH.sub.2 3 36
>100 >50 ND >50 >50 >50 27
C.sub.8K(NC.sub.8K).sub.3NH.sub.2 4 39.5 >100 >50 ND >50
>50 >50 28 C.sub.8K(NC.sub.8K).sub.4NH.sub.2 5 40.5 >100
>50 ND >50 >50 >50 29 C.sub.8K(NC.sub.8K).sub.5NH.sub.2
6 40.8 >100 25 3.1 >50 >50 >50 30
C.sub.8K(NC.sub.8K).sub.6NH.sub.2 7 40.3 >100 25 ND >50
>50 >50 31 C.sub.8K(NC.sub.8K).sub.7NH.sub.2 8 40.3 >100
12.5 ND >50 >50 >50 32 KNC.sub.8KNH.sub.2 3 21.6 ND >50
ND >50 >50 >50 33 K(NC.sub.8K).sub.2NH.sub.2 4 27.3 ND
>50 ND >50 >50 >50 34 K(NC.sub.8K).sub.3NH.sub.2 5 30
ND >50 ND >50 >50 >50 35 K(NC.sub.8K).sub.4NH.sub.2 6
31.7 ND >50 ND >50 >50 >50 36
K(NC.sub.8K).sub.5NH.sub.2 7 33.1 ND >50 >50 >50 >50
>50 37 K(NC.sub.8K).sub.6NH.sub.2 8 33.4 ND >50 >50 >50
>50 >50 38 K(NC.sub.8K).sub.7NH.sub.2 9 34.2 ND >50 37.5
>50 >50 >50 39 C.sub.12KNC.sub.8KNH.sub.2 2 50.9 ND >50
ND >50 >50 >50 40 C.sub.12K(NC.sub.8K).sub.2NH.sub.2 3 48
ND >50 ND >50 >50 >50 41
C.sub.12K(NC.sub.8K).sub.3NH.sub.2 4 46 ND >50 ND >50 >50
>50 42 C.sub.12K(NC.sub.8K).sub.4NH.sub.2 5 49 ND 25 4.7 .+-.
2.2 >50 37.5 .+-. 18 50 43 C.sub.12K(NC.sub.8K).sub.5NH.sub.2 6
49.7 >100 3.1 0.4 50 50 12.5 44
C.sub.12K(NC.sub.8K).sub.6NH.sub.2 7 50 >100 3.1 0.8 50 50 12.5
45 C.sub.12K(NC.sub.8K).sub.7NH.sub.2 8 47.5 >100 3.1 ND 6.2 50
12.5 46 NC.sub.12KNC.sub.8KNH.sub.2 3 29.6 ND >50 ND >50
>50 >50 47 NC.sub.12K(NC.sub.8K).sub.2NH.sub.2 4 35 ND >50
ND >50 >50 >50 48 NC.sub.12K(NC.sub.8K).sub.3NH.sub.2 5
33.7 ND >50 ND >50 >50 >50 49
NC.sub.12K(NC.sub.8K).sub.4NH.sub.2 6 36.2 ND >50 12.5 >50
>50 >50 50 NC.sub.12K(NC.sub.8K).sub.5NH.sub.2 7 36.6 ND 50
6.3 >50 >50 >50 51 NC.sub.12K(NC.sub.8K).sub.6NH.sub.2 8
37 ND 25 6.3 >50 >50 >50 52
NC.sub.12K(NC.sub.8K).sub.7NH.sub.2 9 36.9 ND 12.5 ND 50 >50
>50 53 C.sub.12KK(NC.sub.8K).sub.4NH.sub.2 6 47 >100 6.3 ND
50 37.5 .+-. 18 25 54 C.sub.12KNC.sub.12KNH.sub.2 2 59 45 .+-. 12
20.8 .+-. 7.2 ND 50 18.8 .+-. 7.2 >50 55
C.sub.12K(NC.sub.12K).sub.2NH.sub.2 3 52.9 ND >50 37.5 .+-. 18
>50 >50 >50 56 C.sub.12K(NC.sub.12K).sub.3NH.sub.2 4 53.5
ND >50 >50 >50 >50 >50 57
C.sub.12K(NC.sub.12K).sub.4NH.sub.2 5 53.4 ND >50 >50 >50
>50 >50 58 KNC.sub.12KNH.sub.2 3 32 >100 >50 >50
>50 >50 >50 59 K(NC.sub.12K).sub.2NH.sub.2 4 40 >100
>50 >50 >50 >50 >50 60 K(NC.sub.12K).sub.3NH.sub.2 5
44 >100 12.5 3.1 25 >50 >50 61 K(NC.sub.12K).sub.4NH.sub.2
6 46 6.5 .+-. 3.5 25 2.3 .+-. 1.1 >50 >50 >50 62
K(NC.sub.12K).sub.5NH.sub.2 7 47 ND >50 3.1 ND >50 >50 63
K(NC.sub.12K).sub.6NH.sub.2 8 48 ND >50 3.1 ND ND >50 64
K(NC.sub.12K).sub.7NH.sub.2 9 50 ND >50 6.3 ND ND >50 65
(NC.sub.12K).sub.2NH.sub.2 3 38.8 >100 >50 >50 >50
>50 >50 66 (NC.sub.12K).sub.3NH.sub.2 4 44.3 >100 25 6.3
50 >50 >50 67 (NC.sub.12K).sub.4NH.sub.2 5 46.8 4 .+-. 1.4
>50 6.3 >50 >50 >50 68 (NC.sub.12K).sub.5NH.sub.2 6
47.8 ND >50 12.5 >50 >50 >50 69
(NC.sub.12K).sub.6NH.sub.2 7 49 ND >50 3.1 ND >50 ND 70
(NC.sub.12K).sub.7NH.sub.2 8 50 ND >50 12.5 ND ND ND 71
(NC.sub.12K).sub.8NH.sub.2 9 51 ND >50 1.6 ND ND ND 72
KKNC.sub.12KNH.sub.2 4 30.9 >100 >50 ND >50 >50 >50
73 (KNC.sub.12K).sub.2NH.sub.2 5 38.1 >100 >50 ND 50 >50
>50 74 K(KNC.sub.12K).sub.2NH.sub.2 6 37.3 >100 >50 ND 25
>50 >50 75 C.sub.8KKNC.sub.12KNH.sub.2 3 40.3 >100 >50
ND >50 >50 >50 76 C.sub.8(KNC.sub.12K).sub.2NH.sub.2 4 45
>100 25 ND 12.5 >50 3.1 77
C.sub.8K(KNC.sub.12K).sub.2NH.sub.2 5 42.6 >100 37.5 .+-. 18 ND
12.5 >50 6.3 78 C.sub.12KKNC.sub.12KNH.sub.2 3 54 28.5 .+-. 9.2
18.8 .+-. 8.8 9.4 .+-. 4.4 25 3.1 3.1 79
C.sub.12(KNC.sub.12K).sub.2NH.sub.2 4 53.3 16.5 .+-. 6.4 3.1 ND 3.1
1.6 3.1 80 C.sub.12K(KNC.sub.12K).sub.2NH.sub.2 5 51 88 .+-. 3 3.1
ND 3.1 12.5 3.1 81 NC.sub.12KKNC.sub.12KNH.sub.2 4 38.9 >100
>50 ND >50 >50 >50 82
NC.sub.12(KNC.sub.12K).sub.2NH.sub.2 5 38.5 >100 50 ND 12.5
>50 3.1 83 NC.sub.12K(KNC.sub.12K).sub.2NH.sub.2 6 38.6 >100
25 ND 25 >50 6.3 84 C.sub.12OrnNC.sub.12OrnNH.sub.2 2 53.8 24
.+-. 6 10.4 .+-. 3.6 ND 25 12.5 16.7 .+-. 7.2 85
C.sub.12ArgNC.sub.12ArgNH.sub.2 2 57.1 9.5 .+-. 1 42 .+-. 14.4 ND
>50 12.5 42 .+-. 14.4 86 C.sub.12KNC.sub.12K 1 56.9 >100
>50 ND >50 >50 >50 87 C.sub.12K(NC.sub.12K).sub.2 2
56.4 ND >50 ND >50 31.3 .+-. 26.5 50 88
C.sub.12K(NC.sub.12K).sub.3 3 54.6 ND >50 ND >50 >50
>50 89 KNC.sub.12K 2 33.2 >100 >50 ND >50 >50 >50
90 K(NC.sub.12K).sub.2 3 36.4 >100 >50 ND >50 >50
>50 91 K(NC.sub.12K).sub.3 4 42.8 >100 25 ND 50 >50 >50
92 (NC.sub.12K).sub.2 2 38.7 >100 >50 ND >50 >50 >50
93 (NC.sub.12K).sub.3 3 43.8 >100 50 ND >50 50 >50 94
(NC.sub.12K).sub.4 4 45.8 ND 50 ND >50 >50 >50 95
FmocK(NC.sub.12K).sub.2 2 41 ND >50 ND >50 6.3 12.5 96 MSI-78
10 44 45 50 ND 3.1 >50 37.5 97 IB-367 5 45 7 3.1 ND 12.5 3.1 ND
98 K.sub.4S.sub.4(1-16) 6 47 10 3.1 ND 6.3 6.3 3.1 99 LL37 11 61 8
50 ND 12.5 ND 37.5 100 Ciprofloxacin ND ND ND 0.05 ND 0.3 >50
0.3 101 Imipenem ND ND ND 0.6 ND 16.4 >50 <0.03 102
Tetracycline ND ND ND 1.8 ND >50 0.4 0.07 103 Rifampin ND ND ND
7.7 ND 15.2 0.006 0.09
[0325] The Effect of Physical Parameters (Charge and
Hydrophobicity) on Antimicrobial Activity:
[0326] Charge and hydrophobicity may be viewed as two conflicting
physical characteristics of a molecule: charge facilitates
dissolution of a compound in aqueous media by interacting with the
polar water molecules, while hydrophobicity, which typically
corresponds to the number and length of non-polar hydrocarbon
moieties, hinders dissolution. Optimization of these physical
characteristics is crucial in the development of drugs in general
and antimicrobial agents in particular, as these characteristics
affect pharmaceutically important traits such as membrane
permeability and transport in and across biological systems.
[0327] Therefore, the library of polymers prepared to study the
effect of serial increases in charge and hydrophobicity properties
was measured for its antimicrobial activity against two
gram-negative bacteria: E. coli (results are presented in column 6
of Table 3 hereinabove) and P. aeruginosa (results are presented in
column 8 of Table 3), and two gram-positive bacteria:
methicilin-resistant S. aureus (results are presented in column 9
of Table 3) and Bacillus cereus (results are presented in column 10
of Table 3).
[0328] A serial increase in positive charge was achieved by
preparing polymers with serial elongation of the chain with respect
to the number of lysine residues. Serial increases in
hydrophobicity was achieved by preparing polymers with serial
rising of the number of fatty acid residues (as a representative
hydrophobic moiety) and/or with serial rising of the number of
carbon atoms in each fatty acid residue. Serial increases in both
positive charge and hydrophobicity were achieved by preparing
polymers with serial rising of the number of lysine-amino fatty
acid conjugates.
[0329] As can be seen in Table 3, increasing the hydrophobicity of
the polymers by increasing the number of the carbon atoms in the
fatty acid residue from 4 to 12, via 8 carbon atoms, was found to
affect the antimicrobial activity of the polymers. Series of
polymers in which the repeating hydrophobic moiety was a
4-amino-butyric acid (see, entries 1-24 in Table 3) was compared to
a series in which the repeating hydrophobic moiety was an
8-amino-caprylic acid (see, entries 25-53 in Table 3) and to a
series in which the repeating hydrophobic moiety was a
12-amino-lauric acid (see, entries 54-95 in Table 3). The results,
presented in Table 3, indicated that polymers in which the
repeating hydrophobic moiety was a 4-amino-butiric acid (see,
entries 1-14 in Table 3) and a 8-amino-caprylic acid (see, entries
25-38 in Table 3), generally did not show significant antimicrobial
activity up to the highest tested concentration of 50 .mu.M. The
only polymers which had no 12-animo-lauric acid residue in their
sequence and which showed significant antimicrobial activity at
lower concentrations were C.sub.8K(NC.sub.8K).sub.5NH.sub.2,
C.sub.8K(NC.sub.8K).sub.6NH.sub.2 and
C.sub.8K(NC.sub.8K).sub.7NH.sub.2 (see, entries 29-31 in column 6
of Table 3), whereas polymers containing one or more of the more
hydrophobic 12-amino-lauric acid residue, (see, entries 58-83 in
Table 3), showed significant activity at concentrations as low as
16 .mu.M.
[0330] Evaluation of the effect of the hydrophobicity of the
polymers in terms of the acetonitrile percentages of the HPLC
mobile phase in which the polymers were eluted further demonstrates
the correlation between this property and the antimicrobial
activity of the polymer. As can be seen in the data presented in
column 4 of Table 3, all the polymers which displayed a significant
level of antimicrobial activity against any one of the tested
bacteria were eluted in acetonitrile concentrations higher than
36%, whereby none of the polymers that were eluted in acetonitrile
concentrations lower than 36% exhibited such an activity.
[0331] FIG. 1 presents the distribution of polymers which exhibited
a significant microbial activity (MIC value of less than 50 .mu.M)
in any one of the four assays conducted. As is clearly seen in FIG.
1, antimicrobial activity against one or more of the tested
bacteria was exhibited only by polymers which were eluted at
acetonitrile concentrations of 36% and up and, furthermore,
polymers which were found active against all the tested bacteria
were eluted at acetonitrile concentrations of 51% and up.
[0332] As can further be seen in Table 3, increasing the positive
charge of the polymers by increasing the number of the lysine
residues in the polymer was found to affect the antimicrobial
activity of the polymers only marginally. Thus, polymers having net
charges raging from +1 to +9 in each series were tested for
antimicrobial activity. The results, presented in Table 3,
indicated, for example, that most of the polymers with the highest
net positive charge of +9, namely K(NC.sub.4K).sub.7NH.sub.2,
NC.sub.12K(NC.sub.4K).sub.7NH.sub.2, K(NC.sub.8K).sub.7NH.sub.2,
NC.sub.12K(NC.sub.8K).sub.7NH.sub.2, K(NC.sub.12K).sub.7NH.sub.2,
(NC.sub.12K).sub.8NH.sub.2, (see, respective entries 14, 24, 38,
52, 64 and 71 in Table 3), did not exhibit significant activity,
with only NC.sub.12K(NC.sub.8K).sub.7NH.sub.2 (see, entry 52 in
Table 3) exhibiting significant activity.
[0333] As can be concluded from the data presented in Table 3,
while none of the polymers series based on hydrophobic moieties
containing 4-carbon chains inhibited bacterial proliferation, three
polymers from the polymers series based on hydrophobic moieties
containing 8-carbon chains inhibited growth of E. coli, displaying
MIC values in the low micromolar (.mu.g/ml) range. Several
observations stem from the results shown in Table 3: the equivalent
4-carbon chain based polymers, whose hydrophobicity values,
represented by the percent of acetonitrile in the HPLC-RP gradient
mobile phase at which the polymer was eluted and which corresponds
to the estimated hydrophobicity of the polymer, varied between 20%
and 25% (indicating low hydrophobicity) had virtually no
antibacterial activity up to the highest concentration assayed
(MIC>50 .mu.M). The more hydrophobic 8-carbon chain based
polymers became active only when they reached hydrophobicity values
of >40% (see, C.sub.8K(NC.sub.8K).sub.5NH.sub.2, entry 29 in
Table 3). Elongating the polymer by one C.sub.8K subunit (see,
C.sub.8K(NC.sub.8K).sub.6NH.sub.2, entry 30 in Table 3) increased
the charge without significant change to hydrophobicity and did not
alter activity. Further addition of another C.sub.8K subunit (see,
C.sub.8K(NC.sub.8K).sub.7NH.sub.2, entry 31 in Table 3) had similar
charge and hydrophobicity effects but led to a two-fold enhanced
activity.
[0334] Overall, these results indicate that an optimal
antibacterial activity emerges when a polymer, as described herein,
attains an optimal window of charge and hydrophobicity, much as
observed with conventional AMPs.
[0335] Table 4 below presents a summary of the results obtained in
these experiments, in terms of the effect of the net positive
charge of the polymers and the antimicrobial activity thereof. Row
2 of Table 4 presents the number of polymers in 9 bins, wherein
each bin represents a net positive charge, starting from +9 to +1.
Row 3 of Table 4 presents the total number of activity assays which
were measured in the charge bin, namely, the number of polymers in
the bin multiplied by the four bacterial assays described above.
Row 4 of Table 4 presents the number of polymers in each of the
bins that were found active against any one of the four bacteria.
Row 5 of Table 4 presents the percentage of the active polymers
from the total number of assays measured in the charge bin. As can
be seen in Table 4, (row 4, for example), only a little if any
correlation between the net positive charge of the polymers and
their antimicrobial activity was found. It appears from these
results that the only feature that seems to affect the
antimicrobial activity of the tested polymers is a net positive
charge that is greater than +1.
TABLE-US-00004 TABLE 4 Charge +9 +8 +7 +6 +5 +4 +3 +2 +1 Number of
polymers 6 10 10 12 14 16 15 11 1 Number of assays 24 40 40 48 56
64 60 44 4 Number of actives 1 6 4 12 13 9 4 12 0 Percent actives
4.2% 15.0% 10.0% 25.0% 23.2% 14.1% 6.7% 27.3% 0.0%
[0336] FIG. 2 presents the distribution of polymers according to
the present invention which exhibited a significant microbial
activity (MIC value of less than 50 .mu.M) in any one of the four
assays mentioned above. As can be clearly seen in FIG. 2, polymers
which showed antimicrobial activity against any one of the four
bacteria are scattered across the entire range of charge values,
excluding the +1 charge, and thus demonstrating the lack of
correlation between the net positive charge of the polymers and the
antimicrobial activity thereof.
[0337] Overall, these results indicated that antibacterial activity
emerged when a polymer attained an optimal window of charge and
hydrophobicity, much as observed with conventional AMPs. These
results also suggested that a parallel increase in hydrophobicity
value might enhance potency.
[0338] Antimicrobial Activity Against Gram-Negative Bacteria
[0339] The activity of the novel polymers describes herein on
Gram-negative bacteria has also been tested. To date, new
antimicrobial agents that are effective against Gram-negative
bacteria are rarely found.
[0340] Table 5 below presents the results obtained in this study,
and clearly shows that C.sub.12K(NC.sub.8K).sub.7NH.sub.2, an
exemplary polymer as presented herein, displayed potent growth
inhibitory activity against 9 strains out of a panel of 10
Gram-negative bacterial strains.
[0341] "MIC" (column 3 in Table 5) represents the minimal
inhibitory concentration of the exemplary polymer
C.sub.12K(NC.sub.8K).sub.7NH.sub.2 in .mu.M for each of the tested
bacterial strains, which induced 100% inhibition of proliferation
after 24 hours incubation. Values represent the mean from two
independent experiments performed in duplicates.
[0342] As can be seen in Table 5, the MIC observed for
C.sub.12K(NC.sub.8K).sub.7NH.sub.2 ranged from 1.6 .mu.M to 12.5
.mu.M and in general the MIC value for most bacterial strains was
equal or inferior to 6.2 .mu.M (14 .mu.g/ml). These encouraging
results were also observed for clinically challenging species such
as Acinetobacter and Pseudomonas, for both of which a MIC value of
3.1 was observed (see, entries 5 and 6 in Table 5
respectively).
TABLE-US-00005 TABLE 5 Entry Gram-negative Bacteria Strain MIC 1
Enterobacter cloacae CI 730 1.6 2 Brevundimonas diminuta ATCC 19146
1.6 3 Yersinia kristensenii ATCC 33639 1.6 4a Escherichia coli CI
3504 1.6* 4b Escherichia coli ATCC 25922 3.1* 5 Acinetobacter
baumanii CI 1280 3.1 6 Klebsiella pneumoniae CI 1286 3.1 7 Proteus
mirabilis CI 1285 6.2 8 Pseudomonas aeruginosa CI 8732 6.2 9
Stenotrophomonas CI 746 12.5 maltophilia 10 Serratia odorifera ATCC
33077 >50 *MIC determined using the Clinical and Laboratory
Standards Institute (CLSI) recommended procedure as presented
hereinabove.
[0343] Development of Antimicrobial-Resistance in Bacteria:
[0344] The possible development of resistance to the polymers of
the present invention was tested by measuring the MIC levels
following multiple exposures of the bacteria to exemplary polymers
according to the present invention, as described hereinabove in the
Experimental Methods section. The tested polymers in these
experiments were K(NC.sub.12K).sub.3NH.sub.2,
C.sub.12K(NC.sub.8K).sub.5NH.sub.2, C.sub.12KKNC.sub.12KNH.sub.2
and C.sub.12K(NC.sub.8K).sub.7NH.sub.2, whereby the development of
resistance of E. coli to K(NC.sub.12K).sub.3NH.sub.2 and
C.sub.12K(NC.sub.8K).sub.5NH.sub.2 was compared with that of three
classical antibiotics: gentamycin, tetracycline and ciprofloxacin,
the development of resistance of methicilin-resistant S. aureus to
C.sub.12KKNC.sub.12KNH.sub.2 was compared with that of two
classical antibiotics: rifampicin and tetracycline, and the
development of resistance of E. coli to
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, evaluated during 15 serial
passages, was compared with that of three classical antibiotics,
ciprofloxacin, imipenem, and tetracycline.
[0345] The data obtained in these experiments is presented in FIGS.
3a, 3b and 3c. FIG. 3a presents the data obtained for
K(NC.sub.12K).sub.3NH.sub.2 and C.sub.12K(NC.sub.8K).sub.5NH.sub.2,
FIG. 3b presents the data obtained for C.sub.12KKNC.sub.12KNH.sub.2
and FIG. 3c presents the data obtained for
C.sub.12K(NC.sub.8K).sub.7NH.sub.2.
[0346] As is clearly seen in FIG. 3a, the relative MIC value of
K(NC.sub.12K).sub.3NH.sub.2 and C.sub.12K(NC.sub.8K).sub.5NH.sub.2
against E. coli remained stable for 10 successive subculture
generations following the initial exposure. In sharp contrast,
during the same period of time, the MIC values tested with the
reference antibiotic agents substantially increased, reflecting the
emergence of antibiotic-resistant bacteria. Thus, at the tenth
generation, the MIC values increased by 4-fold for tetracycline and
gentamycin, and by more than 16-fold for ciprofloxacin. These
results demonstrate that exposing bacteria to the antimicrobial
polymers of the present invention do not result in development of
resistance.
[0347] As is clearly seen in FIG. 3b, the relative MIC value of
C.sub.12KKNC.sub.12KNH.sub.2 against methicilin-resistant S. aureus
remained stable for 15 successive subculture generations following
the initial exposure. In sharp contrast, during the same period of
time, the MIC values tested with the reference antibiotic agents
substantially increased, reflecting the emergence of
antibiotic-resistant bacteria. Thus, at the fifteenth generation,
the MIC values increased by more than 230-fold for rifampicin, and
by 4-fold for tetracycline. These results demonstrate again that
exposing bacteria to the antimicrobial polymers of the present
invention do not result in development of resistance.
[0348] Similarly, it can be seen in FIG. 3c, the relative MIC value
of C.sub.12K(NC.sub.8K).sub.7NH.sub.2 against E. coli remained
stable for 15 successive subculture generations following the
initial exposure. In sharp contrast, during the same period of
time, the MIC values tested with the reference antibiotic agents
substantially increased, reflecting the emergence of
antibiotic-resistant bacteria. Thus, at the fifteenth generation,
the MIC values increased by more than 60-fold for ciprofloxacin,
and by 8-fold for imipenem and tetracycline. These results
demonstrate yet again that exposing bacteria to the antimicrobial
polymers of the present invention do not result in development of
resistance.
[0349] The development of antimicrobial resistance following
exposure to the polymers of the present invention was further
evaluated in a cross resistance experiment, in which a
methicilin-resistant strain of S. aureus was exposed to exemplary
polymers of the present invention. The results obtained in this
experiment are presented in Table 3 hereinabove, under column "MIC
MR S.a." and clearly demonstrate the persisting antimicrobial
activity of the polymers of the present invention against an
antibiotic-resistant bacteria, especially in the case of
C.sub.12(KNC.sub.12K).sub.2NH.sub.2, C.sub.12KKNC.sub.12KNH.sub.2,
FmocK(NC.sub.12K).sub.2, C.sub.12K(KNC.sub.12K).sub.2NH.sub.2,
C.sub.12OrnNC.sub.12OrnNH.sub.2, C.sub.12ArgNC.sub.12ArgNH.sub.2,
C.sub.12KNC.sub.12KNH.sub.2, C.sub.12K(NC.sub.12K).sub.2,
C.sub.12K(NC.sub.8K).sub.4NH.sub.2 and
C.sub.12KK(NC.sub.8K).sub.4NH.sub.2 (see, respective entries 79,
78, 95, 80, 84, 85, 54, 87, 42 and 53, in Table 3).
[0350] Kinetic Studies of Antimicrobial Activity at Time
Intervals:
[0351] The kinetic rates of bactericidal activity of a
representative polymer of the present invention,
C.sub.12K(NC.sub.8K).sub.5NH.sub.2, was tested as described in the
methods section above at concentrations corresponding to 3 and 6
times the MIC value. The results, presented in FIG. 4, clearly
reflect the antibacterial activity of the polymer. As is shown in
FIG. 4, the viable bacterial population was reduced by nearly seven
log units within 6 hours upon being exposed to the polymer at a
concentration of 3 multiples of the MIC, and within 2 hours upon
being exposed to the polymer at a concentration of 6 multiples of
the MIC.
[0352] FIG. 5 presents comparative plots demonstrating the kinetic
bactericidal effect of C.sub.12K(NC.sub.8K).sub.7NH.sub.2, an
exemplary polymer according to the present invention, on E. coli.
as compared with kinetic bactericidal effect of various classical
antibiotics as determined at a concentration corresponding to six
multiples of their respective MIC value.
[0353] As can be seen in FIG. 5,
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, (black triangles) was
responsible for rapid bacterial death namely,
C.sub.12K(NC.sub.8K).sub.7NH.sub.2 reduced bacterial population
from 106 to <50 CFU/ml within one hour compared to normal
bacterial growth control (black circles), while Imipenem (white
squares) and Ciprofloxacin (black squares) induced a weaker
bactericidal effect, and Tetracycline (white circles) was merely
bacteriostatic. The plotted values represent the mean.+-.standard
deviations obtained from at least two independent experiments. The
stared datum points indicate that bacteria were not detected at the
minimum level of sensitivity (<50 CFU/ml).
[0354] These remarkable results further demonstrate the efficacy of
the antimicrobial polymers of the present invention, in terms of an
efficient pharmacokinetic profile.
[0355] Antimicrobial Activity at Enhanced Outer-Membrane
Permeability Conditions:
[0356] The results obtained following addition of the
cation-chelator EDTA to the assay buffer, which was aimed at
enhancing the outer-membrane permeability of gram-negative bacteria
such as E. coli, are presented in Table 3 above under the column
headed "MIC EDTA E.c.". These results clearly show that the
activity profile of the polymers in the presence of EDTA is
different than that obtained without EDTA (presented in Table 3
above, column headed "MIC E.c."). Thus, polymers such as
K(NC.sub.8K).sub.7NH.sub.2, NC.sub.12K(NC.sub.8K).sub.4NH.sub.2,
NC.sub.12K(NC.sub.8K).sub.5NH.sub.2,
C.sub.12K(NC.sub.12K).sub.2NH.sub.2, K(NC.sub.2K).sub.5NH.sub.2,
K(NC.sub.12K).sub.6NH.sub.2, K(NC.sub.2K).sub.7NH.sub.2,
(NC.sub.12K).sub.4NH.sub.2, (NC.sub.12K).sub.5NH.sub.2,
(NC.sub.12K).sub.6NH.sub.2, (NC.sub.12K).sub.7NH.sub.2 and
(NC.sub.12K).sub.8NH.sub.2, which exhibited minor or no
antimicrobial activity in the absence of EDTA, became up to more
than 50 folds more active in its presence (see, respective entries
38, 49, 50, 55, 62, 63, 64, 67, 68, 69, 70 and 71, in Table 3).
Other polymers, such as K(NC.sub.12K).sub.4NH.sub.2,
C.sub.8K(NC.sub.8K).sub.5NH.sub.2,
C.sub.12K(NC.sub.8K).sub.4NH.sub.2,
NC.sub.12K(NC.sub.8K).sub.6NH.sub.2 and (NC.sub.12K).sub.3NH.sub.2,
which exhibited only marginal antimicrobial activity in the absence
of EDTA, became between 11-folds and 4-fold more active in its
presence, respectively (see, respective entries 61, 29, 42, 51 and
66 in Table 3).
[0357] These results illuminate the tight correlation between
membrane permeability of antimicrobial agents and their efficacy
and further demonstrate the complex relationship and delicate
balance between the positive charge and the hydrophobic
characteristics of the polymers of the present invention on the
antimicrobial activity thereof.
[0358] Susceptibility to Plasma Proteases Assays Results:
[0359] The susceptibility of the polymers of the present invention
to enzymatic cleavage was assessed by pre-incubating exemplary
polymers according to the present invention,
C.sub.12K(NC.sub.8K).sub.5NH.sub.2, K(NC.sub.12K).sub.3NH.sub.2,
C.sub.12KNC.sub.12KNH.sub.2, and C.sub.12KKNC.sub.12KNH.sub.2, and
an exemplary reference AMP, a 16-residues dermaseptin S4 derivative
(S4.sub.16), in human plasma (50%) for various time periods and
thereafter determining the antibacterial activity thereof against
E. coli and S. aureus. Statistical data were obtained from at least
two independent experiments performed in duplicates.
[0360] The results are presented in Table 6 hereinbelow, wherein
"MIC (E.c.) C.sub.12K(NC.sub.8K).sub.5NH.sub.2 (.mu.M)" is the
minimal inhibitory concentration in .mu.M of
C.sub.12K(NC.sub.8K).sub.5NH.sub.2, as measured for E. coli; "MIC
(E.c.) K(NC.sub.12K).sub.3NH.sub.2 (.mu.M)" is the minimal
inhibitory concentration in .mu.M of K(NC.sub.12K).sub.3NH.sub.2,
as measured for E. coli; "MIC (E.c.) S4.sub.16 (.mu.m)" is the
minimal inhibitory concentration in .mu.M of S4.sub.16, an
exemplary dermaseptin serving as a reference AMP, as measured for
E. coli; "MIC S.a. C.sub.12KNC.sub.12KNH.sub.2 (.mu.M)" is the
minimal inhibitory concentration in .mu.M of
C.sub.12KNC.sub.12KNH.sub.2, as measured for S. aureus; and "MIC
(S.a.) C.sub.12KKNC.sub.12KNH.sub.2 (.mu.M)" is the minimal
inhibitory concentration in .mu.M of C.sub.12KKNC.sub.12KNH.sub.2,
as measured for S. aureus.
TABLE-US-00006 TABLE 6 Incubation MIC (E.c.) MIC (E.c.) MIC MIC
(S.a.) MIC (S.a.) time C.sub.12K(NC.sub.8K).sub.5NH.sub.2
K(NC.sub.12K).sub.3NH.sub.2 (E.c.) S4.sub.16
C.sub.12KNC.sub.12KNH.sub.2 C.sub.12KKNC.sub.12KNH.sub.2 (hours)
(.mu.M) (.mu.M) (.mu.M) (.mu.M) (.mu.M) 0 3.1 12.5 3.1 12.5 3.1 3
3.1 12.5 >50 12.5 3.1 6 3.1 12.5 >50 25 6.3 18 3.1 12.5
>50 25 6.3
[0361] As is shown in Table 6, while the reference AMP, S4.sub.16,
was completely inactivated upon exposure to human plasma, the
polymers of the present invention maintained their activity, and
thus, the superior stability of the polymers according to the
present invention as compared with that of the highly active yet
unstable AMPs was clearly demonstrated. More specifically, as is
shown in Table 6, the dermaseptin S4.sub.16 did not display a
measurable MIC after 3 hours exposure to serum enzymes, even at a
concentration of more than 16-folds higher (greater than 50 .mu.M)
than the MIC value, indicating that the peptide was inactivated
probably due to enzymatic proteolysis.
[0362] In sharp contrast, the polymers of the present invention
exhibited prolonged resistance to enzymatic degradation. As is
further shown in Table 6, the activity of short polymers such as
C.sub.12KNC.sub.12KNH.sub.2 and C.sub.12KKNC.sub.12KNH.sub.2 was
reduced only by 2-folds after 6 hours exposure to plasma enzymes
while longer polymers such as K(NC.sub.12K).sub.3NH.sub.2 and
C.sub.12K(NC.sub.8K).sub.5NH.sub.2 did not display any degree of
inactivation even after 18 hours incubation.
[0363] Hemolysis Assays:
[0364] The toxic hemolytic effect of the polymers of the present
invention on human erythrocytes (red blood cells, RBC) was assayed
as described hereinabove. The results are presented in Table 3,
under the column headed "LC.sub.50", in terms of the lytic
concentrations that induced 50% (LC.sub.50) lysis of red blood
cells in phosphate buffer (PBS).
[0365] As shown in Table 3, polymers such as
C.sub.12K(NC.sub.8K).sub.7NH.sub.2,
C.sub.8(KNC.sub.12K).sub.2NH.sub.2,
C.sub.8K(KNC.sub.12K).sub.2NH.sub.2,
NC.sub.12K(KNC.sub.12K).sub.2NH.sub.2,
C.sub.12K(NC.sub.8K).sub.5NH.sub.2,
C.sub.12K(NC.sub.8K).sub.6NH.sub.2,
NC.sub.12(KNC.sub.12K).sub.2NH.sub.2,
C.sub.12KK(NC.sub.8K).sub.4NH.sub.2 and K(NC.sub.12K).sub.3NH.sub.2
(see, respective entries 45, 76, 77, 83, 43, 44, 82, 53 and 60 in
Table 3) which exhibited high antimicrobial activity, displayed low
hemolytic activity. As is further shown in Table 3, polymers
including various fatty acid moieties conjugated to the N-terminus
thereof and/or a relatively large number of lysine residues, were
particularly found to exhibit potent antibacterial activity along
with low hemolytic activity. These results clearly demonstrate the
low toxicity of the polymers of the present invention against human
red blood cells.
[0366] FIG. 6 presents comparative plots demonstrating the
hemolytic effect of C.sub.12K(NC.sub.8K).sub.7NH.sub.2, an
exemplary polymer as presented herein, as compared to the hemolytic
effect of bivalirudin, a synthetic 20 amino acid peptide, which is
clinically used as a specific and reversible direct thrombin
inhibitor approved by FDA for intravenous administration, and to
the hemolytic effect of MSI-78, a magainin derivative that was
recently assessed in human clinical trials for treatment of
diabetic foot ulcers, determined against human RBC (10% hematocrit)
after 1 hour incubation at 37.degree. C. in presence of three
polymer/peptide concentrations, namely 31 .mu.M (striped bars), 94
.mu.M (gray bars) and 156 .mu.M (white bars). Plotted values
represent the mean.+-.standard deviations obtained from at least
four independent experiments.
[0367] As can be seen in FIG. 6, C.sub.12K(NC.sub.8K).sub.7NH.sub.2
did not exhibited any hemolytic activity when tested against human
red blood cells at all three experimental concentrations, and did
not yield the characteristic dose-response profile observed with
conventional AMPs. Both bivalirudin, a non-antimicrobial peptide,
and C.sub.12K(NC.sub.8K).sub.7NH.sub.2 displayed merely a
"background level" activity at least up to 156 .mu.M, a
concentration that corresponds to about 100 folds the MIC value of
C.sub.12K(NC.sub.8K).sub.7NH.sub.2 against various bacteria.
Contrarily, MSI-78 exhibited a high degree of hemolysis at
concentrations as low as 31 .mu.M and 94 .mu.M. Circular dichroism
(CD):
[0368] The secondary structure of selected polymers according to
the present inventions was studied by circular dichroism (CD)
measurements in various media, as described hereinabove in the
Experimental Methods section. The CD profiles of
C.sub.12K(NC.sub.8K).sub.5NH.sub.2 and
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, exemplary antimicrobial
polymers according to the present invention, and
NC.sub.12K.sub.4S4.sub.(1-14), an exemplary dermaseptin derivative,
are presented in FIGS. 7 and 8. The CD data presented represent an
average of three separate recordings values.
[0369] FIG. 8 presents the circular dichroism spectra of
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, an exemplary polymer as
presented herein (gray lines), and control antimicrobial peptide
K.sub.4S.sub.4(1-16) (black lines), taken in PBS alone (dashed
lines) or in presence of 2 mM POPC:POPG (3:1) liposomes suspended
in PBS (solid lines) (data represent average values from three
separate recordings).
[0370] As is shown in FIG. 7 and FIG. 8, the CD spectra of the
polymers of the present invention displayed a minimum near 200 nm,
indicating a random structure. The same CD spectra were observed in
assays conducted in the presence and absence of liposomes. The CD
spectra of the control dermaseptin NC.sub.12K.sub.4S4.sub.(1-14)
and control antimicrobial peptide K.sub.4S.sub.4(1-16) showed a
typical spectrum characteristic of an alpha-helical secondary
structure. Similar results were observed in 20%
trifluoroethanol/water (data not shown). In general, secondary
structure imparts a distinct CD to their respective molecules.
Therefore, the alpha helix and beta sheet typically observed in
polypeptides and proteins have CD spectral signatures
representative of their structures. The lack of these
characteristic CD spectral signatures representative of a secondary
structure elements in the spectra obtain for the polymers presented
herein is indicative of their "random" secondary structure, or lack
thereof.
[0371] Surface Plasmon Resonance Assay:
[0372] The binding properties of exemplary polymers according to
the present invention to membranes were studied using surface
plasmon resonance (SPR) measurements, as described hereinabove in
the Methods section.
[0373] The obtained data indicated that the polymers according to
the present invention display high affinity binding to a model
membrane mimicking the bacterial plasma membrane, with K.sub.app
ranging from 10.sup.4 to 10.sup.7 M.sup.-1). FIG. 9, for example,
presents the data obtained with C.sub.12K(NC.sub.8K).sub.5NH.sub.2,
and demonstrates the high affinity binding of this exemplary
polymer according to the present invention (K.sub.app of
9.96.times.10.sup.4 M.sup.-1 to a model membrane.
[0374] An additional exemplary antimicrobial polymer according to
the present invention, K(NC.sub.12K).sub.3NH.sub.2, displayed an
even higher affinity binding (K.sub.app of 6.3.times.10.sup.5
M.sup.-1, data not shown).
[0375] These results substantiate the affinity of the polymers of
the present invention towards the membranes of a pathogenic
microorganism.
[0376] Lipopolysaccharide Binding Assay:
[0377] The binding affinity of the positively charged polymers
according to the present invention to the negatively charged
lipopolysaccharides (LPS) present on the membrane of gram-negative
bacteria was measured as described in the Methods section
hereinabove. The maximal binding levels of seven exemplary polymers
according to the present invention, KNC.sub.8KNH.sub.2,
K(NC.sub.8K).sub.2NH.sub.2, K(NC.sub.8K).sub.3NH.sub.2,
K(NC.sub.8K).sub.6NH.sub.2, KNC.sub.12KNH.sub.2,
K(NC.sub.12K).sub.2NH.sub.2 and K(NC.sub.12K).sub.3NH.sub.2, to
liposomal membranes before and after incubation with LPS, as
measured in these assays, are presented in FIG. 10.
[0378] As can be seen in FIG. 10, the binding affinity of a polymer
to the membrane is affected by the length of the polymer. Thus, for
example, the binding affinity of K(NC.sub.8K).sub.6NH.sub.2 is
higher than that of KNC.sub.8KNH.sub.2 and the binding affinity of
K(NC.sub.12K).sub.3NH.sub.2 was found higher than that of
KNC.sub.12KNH.sub.2.
[0379] As can be further seen in FIG. 10, the same correlation
between the polymer length and its binding affinity to LPS was
observed. Thus, for example, the polymers
K(NC.sub.8K).sub.6NH.sub.2 and K(NC.sub.12K).sub.3NH.sub.2 each
exhibits close to 2-fold reduction of affinity to liposomal
membrane following incubation with LPS, indicating binding of the
polymers to LPS during the incubation period, which interferes with
their binding to the membranal liposomes.
[0380] These results provide further support to a mechanism of
action of the polymers that involves strong interaction with LPS,
which promotes a destructive action against the bacterial membrane
and by which the risk of development of endotoxemia is reduced.
[0381] DNA Binding Assay:
[0382] The binding properties of exemplary polymers according to
the present invention to nucleic acids were studied by determining
their ability to retard migration of DNA plasmids during gel
electrophoresis in a 1% agarose gel. The obtained results show that
the polymers according to the present invention retard the
migration of various plasmids (e.g., pUC19, pGL3 Luciferase
Reporter Vector (Promega)) in a dose dependent manner.
Representative results, obtained with the plasmid pUC19 in the
absence and presence of three exemplary polymers of the present
invention, C.sub.12KKNC.sub.12KNH.sub.2, K(NC.sub.4K).sub.7NH.sub.2
and C.sub.12K(NC.sub.8K).sub.5NH.sub.2, are presented in FIG. 11
(Note: isolation of the plasmid from a bacterial culture results in
three major bands and several minor bands, as seen in the leftmost
slot of the gel's UV image). An apparent dose-dependent behavior
was evident in the presence of the shortest tested polymer
C.sub.12KKNC.sub.12KNH.sub.2. The dose-dependent behavior was
further accentuated with the longer tested polymers
K(NC.sub.4K).sub.7NH.sub.2 and C.sub.12K(NC.sub.8K).sub.5NH.sub.2.
Thus, at the lowest dose of C.sub.12KKNC.sub.12KNH.sub.2 (polymer
to DNA ratio of 1:1), the supercoiled plasmid DNA band disappeared
whereas the other bands displayed a smeared pattern. These results
suggest that the inhibitory effect of the polymers of the present
invention is higher with supercoiled DNA. Increasing the polymer
doses resulted in accentuated effect, such that the retardation
effect extended to all DNA species.
[0383] Furthermore, it was found that various polymer-DNA complexes
remained intact after exposure to either DNAse digestive enzymes or
peptidase digestive enzymes. These findings reveal a tight binding
between the polymers of the present invention and the DNA molecule,
exhibited by the mutual shielding exerted by the polymers to the
DNA molecules and vice versa.
[0384] Saliva Microbicidal Assays:
[0385] The antimicrobial activity of an exemplary polymer of the
present invention, C.sub.8K.sub.8, against microorganisms in human
saliva was studied as described above. FIG. 12 presents the results
obtained in this study in terms of the logarithmic units of CFU per
ml as a function of the incubation time of the samples with the
vehicle buffer (control), 113-367 (antimicrobial agent with known
activity control) and C.sub.8K.sub.8. The results show that while
in the control, untreated group the saliva microorganisms are
persistent and proliferate without any treatment, the growth of
saliva microorganisms treated is inhibited but proliferation is
resumed after 30 minutes; whereby the growth of the saliva
microorganisms treated with the polymer according to the present
invention is inhibited without recovery.
[0386] Anti-Malarial Assays:
[0387] A of a group of polymers, according to the present
invention, were tested for their anti-malarial effect on parasite
growth and on mammalian cells. The obtained results are presented
in Table 7 below, wherein:
[0388] "IC50 parasite (.mu.M)" represents the concentration of the
tested polymer in .mu.M that is required for 50% inhibition of the
growth of the malaria causing parasites, measured as described
hereinabove (column 3 in Table 7);
[0389] "IC50 MDCK (.mu.M)" represents the concentration of the
tested polymer in .mu.M that is required for 50% inhibition of
growth of MDCK cells, measured as described hereinabove (column 3
in Table 7); and
[0390] "IC50 Ratio" represents the ratio of IC50 MDCK over IC50
parasite, indicating the specificity of the polymer to parasitic
membranes over that of mammalian cells.
TABLE-US-00007 TABLE 7 Entry (entry in Table 3 IC.sub.50 parasite
IC.sub.50 MDCK above) Polymer (.mu.M) (.mu.M) IC.sub.50 Ratio A
(54) C.sub.12KNC.sub.12KNH.sub.2 3.54 156.8 44.29 B (65)
(NC.sub.12K).sub.2NH.sub.2 4.63 609.2 131.58 C (55)
C.sub.12K(NC.sub.12K).sub.2NH.sub.2 0.85 92.1 108.35 D (66)
(NC.sub.12K).sub.3NH.sub.2 0.14 48.3 352.55 E (56)
C.sub.12K(NC.sub.12K).sub.3NH.sub.2 0.08 37.3 449.40 F (67)
(NC.sub.12K).sub.4NH.sub.2 1.59 57.0 35.85 G (58)
KNC.sub.12KNH.sub.2 68.20 693.8 10.17 H (59)
K(NC.sub.12K).sub.2NH.sub.2 7.85 157.4 20.05 I (60)
K(NC.sub.12K).sub.3NH.sub.2 1.72 347.0 201.74
[0391] As shown in Table 7, some of the polymers have shown very
high activity against malarial parasites having an IC.sub.50 in the
sub-micromolar range, as presented in the column denoted IC.sub.50
parasite (.mu.M) (see entries D and E in Table 7 above). The
structure-activity relationship conclusion that emerges from this
series is that lengthening of the chain increases the anti-malarial
activity (reduces the IC.sub.50). The presence of the alkyl moiety
at the N-terminus of the lysine, invariably increases the
anti-malarial activity (see, entries G and A, entries H and C and
entries I and E in Table 7 above). For some polymers, the amino
alkyl adds further activity (see, entries C and D in Table 7 above)
but this performance is not always consistent (see, entries A and B
and entries E and F in Table 7 above).
[0392] There are similar consistencies for the effect of the
polymers of the present invention on the MDCK cells. Addition of an
alkyl at the N-terminus of the lysine results in a decrease in
activity (see, entries G and A, entries H and C, and entries I and
E in Table 7 above). The amino alkyl moiety usually results in
decreased activity (see, entries A and B, and entries C and D in
Table 7 above), but the opposite effect was observed for the
longest polymers (see, entries E and F in Table 7 above).
[0393] The ratio of IC.sub.50 is essentially equivalent to the
therapeutic ratio. Thus, entries D and E in Table 7 above show the
most therapeutically efficient polymers, according to the present
invention.
[0394] Similar results were obtained with the primary cultures of
cardio-fibroblasts (CF) and HepG2 transformed cells (results not
shown).
[0395] Another series of polymers was tested for anti-malarial
activity in order to further investigate the structure-activity
relationship with respect to polymer length and hydrophobic moiety
residue length.
[0396] The results, presented in Table 8 below, wherein "IC50
(.mu.M)" represents the concentration of the tested polymer in
.mu.M that is required for 50% inhibition of the growth of the
malaria causing parasites, measured as described hereinabove
(column 3 in Table 8), indicate that the addition of caprylic acid
(C.sub.8) to the N-terminus of the lysine residue increases the
anti-malarial potency considerably (up to 67 fold), but this
amplification diminishes as the chain length increases.
Substitution of C.sub.8 with lauric acid (C.sub.12) results in a
further increase the anti-malarial potency (up to 20-fold), whereas
further substitution at this terminus with .omega.-aminolauric acid
(NC.sub.12) reverts the potency considerably.
[0397] Among the most active polymers in the
C.sub.12K(NC.sub.8K).sub.nNH.sub.2 group, the anti-malarial potency
diminishes with increase polymer length (see, entries 15-21 in
Table 8 below). The opposite trend was observed for the
non-acylated (at the N-terminus) group K(NC.sub.8K).sub.nNH.sub.2
(see, entries 1-7 in Table 8 below) although they exhibit an
overall lower activity. No such consistent trends could be observed
for the other groups.
[0398] None of the polymers of this series caused lysis of infected
RBC at concentrations that are at least 2-fold higher than their
respective IC.sub.50 (data not shown).
TABLE-US-00008 TABLE 8 Entry (entry in Table 3 above) Polymer
IC.sub.50 (.mu.M) 1 (32) KNC.sub.8KNH.sub.2 260 2 (33)
K(NC.sub.8K).sub.2NH.sub.2 180 3 (34) K(NC.sub.8K).sub.3NH.sub.2
130 4 (35) K(NC.sub.8K).sub.4NH.sub.2 90 5 (36)
K(NC.sub.8K).sub.5NH.sub.2 84 6 (37) K(NC.sub.8K).sub.6NH.sub.2 71
7 (38) K(NC.sub.8K).sub.7NH.sub.2 47 8 (25)
C.sub.8KNC.sub.8KNH.sub.2 16 9 (26)
C.sub.8K(NC.sub.8K).sub.2NH.sub.2 2.7 10 (27)
C.sub.8K(NC.sub.8K).sub.3NH.sub.2 14.8 11 (28)
C.sub.8K(NC.sub.8K).sub.4NH.sub.2 48.9 12 (29)
C.sub.8K(NC.sub.8K).sub.5NH.sub.2 44.1 13 (30)
C.sub.8K(NC.sub.8K).sub.6NH.sub.2 30.5 14 (31)
C.sub.8K(NC.sub.8K).sub.7NH.sub.2 37.2 15 (39)
C.sub.12KNC.sub.8KNH.sub.2 0.38 16 (40)
C.sub.12K(NC.sub.8K).sub.2NH.sub.2 0.2 17 (41)
C.sub.12K(NC.sub.8K).sub.3NH.sub.2 1.16 18 (42)
C.sub.12K(NC.sub.8K).sub.4NH.sub.2 2.43 19 (43)
C.sub.12K(NC.sub.8K).sub.5NH.sub.2 5.57 20 (44)
C.sub.12K(NC.sub.8K).sub.6NH.sub.2 9.9 21 (45)
C.sub.12(NC.sub.8K).sub.7NH.sub.2 15.8 22 (46)
NC.sub.12KNC.sub.8KNH.sub.2 120.1 23 (47)
NC.sub.12K(NC.sub.8K).sub.2NH.sub.2 99.5 24 (48)
NC.sub.12K(NC.sub.8K).sub.3NH.sub.2 93.7 25 (49)
NC.sub.12K(NC.sub.8K).sub.4NH.sub.2 72.9 26 (50)
NC.sub.12K(NC.sub.8K).sub.5NH.sub.2 70.6 27 (51)
NC.sub.12K(NC.sub.8K).sub.6NH.sub.2 66.5 28 (52)
NC.sub.12K(NC.sub.8K).sub.7NH.sub.2 89.8
[0399] The anti-malarial effect of the polymer
C.sub.12K(NC.sub.12K).sub.3NH.sub.2 (see, entry E in Table 7 above)
has been tested by exposing parasite cultures at the ring and the
trophozoite stages for various lengths of time and different
polymer concentrations, the polymer has then been removed and after
48 hours all cultures that were subjected for the different
treatments were tested for parasite viability using the
hypoxanthine incorporation test.
[0400] The IC.sub.50 for each treatment has been calculated for the
chloroquine-resistant FCR3 strain versus chloroquine-sensitive NF54
strain, and the results are presented in FIG. 13. As seen in FIG.
13 the ring stage is more sensitive to the polymer than the
trophozoite stage where it also takes a longer time to exert the
inhibitory action. It also seems that the effect is cumulative in
that the IC.sub.50 values at 48 hours are lower than those observed
with shorter exposure times.
[0401] The effect of time of exposure of parasite cultures to
C.sub.12KNC.sub.8KNH.sub.2 (see, entry 15 in Table 8 above) at
different stages on parasite viability is shown in FIG. 14. As can
be seen in FIG. 14, the results indicate that ring and trophozoite
stages are almost equally sensitive to C.sub.12KNC.sub.8KNH.sub.2,
yet a period of 24 hours is required in order to exert the full
inhibitory activity on the rings and more so for the trophozoites
stage.
[0402] In-Vivo Therapeutic Efficacy:
[0403] The therapeutic efficacy of
C.sub.12K(NC.sub.8K).sub.7NH.sub.2 was assessed using a murine
peritonitis-sepsis model after intraperitoneal infection with E.
coli and intraperitoneal treatment with the tested and control
agents one hour post-infection.
[0404] FIGS. 15a-b present the rate of survival, monitored over a
time period of 7 days, of infected mice (n=10 per group) inoculated
intraperitoneally with 2.5.times.10.sup.6 CFUs of E. coli CI-3504
(FIG. 15a) and 5.times.10.sup.6 CFUs of E. coli CI-3504 (FIG. 15b),
and subsequently treated one hour after infection by
intraperitoneal administration of PBS (black circles), a single
dose of 4 mg/kg C.sub.12K(NC.sub.8K).sub.7NH.sub.2 (gray squares)
or four doses of 2 mg/kg imipenem at (asterisk).
[0405] As can be seen in FIGS. 15a-b, the polymer
C.sub.12K(NC.sub.8K).sub.7NH.sub.2 significantly prevented
mortality of mice infected with two different lethal inocula. In
these representative experiments, at the low dose inoculum
(2.5.times.10.sup.6 CFU/mouse), survival of infected mice treated
with polymer was 100% compared to 20% in the vehicle treated
control group (p<0.005). At the higher dose inoculum
(5.times.10.sup.6 CFU/mouse), survival was 80%, compared to 0%
survival in the vehicle-treated group (p<0.005). Treatment with
a classical antibiotic (imipenem, 4 doses during 28 hours, starting
one hour after infection) resulted in survival of 100% and 90% in
the two inocula studied, respectively. Overall, these results
clearly demonstrate the beneficial therapeutic potential use of the
polymers presented herein, and their efficacy in the treatment of
harsh bacterial infections.
[0406] The results obtained in this study, which reflect a
comprehensive efficacy profile, demonstrate that
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, a polymer according to the
present invention, although displaying less efficacious MIC levels
(MIC E. coli of 3.1) as compared to certain classical antibiotic
agents, such as imipenen (MIC E. coli of 0.6), the polymer still
proves a more efficacious antimicrobial agent.
[0407] In-Vivo Toxicity:
[0408] In-vivo acute toxicity of the polymers presented herein was
examined by intraperitoneal injection of 0 .mu.g (blank control),
100 .mu.g, 250 .mu.g, and 500 .mu.g of freshly prepared
C.sub.12K(NC.sub.8K).sub.7NH.sub.2, an exemplary polymer according
to the present invention, to groups of 12 mice; thus the dosage
corresponding to 0, 4, 10, and 20 mg/kg of body weight. Animals
were directly inspected for adverse effects for 4 hour, and
mortality was monitored for 7 days thereafter.
[0409] FIG. 16 presents the rate of survival, monitored over time
period of 6 days, of female ICR mice (n=12 per group) treated
intraperitoneally with 0 mg/kg of body weight (white bars) 4 mg/kg
of body weight (sparsely striped bars), 10 mg/kg of body weight
(densely striped bars) and 20 mg/kg of body weight (black bars) of
C.sub.12K(NC.sub.8K).sub.7NH.sub.2.
[0410] As can be seen in FIG. 16, only 25% of the mice treated with
the highest dose of 20 mg/kg of body weight died, while all the
mice treated with 4 and 10 mg/kg of body weight survived throughout
the duration of the experiment.
[0411] Based on the experimental results presented herein, the
polymers according to the present invention offer several
advantages over conventional AMPs, which are mostly of limited
utility as therapeutic agents due to their low bioavailability
and/or high toxicity. From pharmacologic, therapeutic and other
practical points of view, the polymers presented herein represent a
novel and promising family of antimicrobial agents that are devoid
of AMPs intrinsic disadvantages. They may therefore be beneficially
utilized in various antimicrobial fields including the treatment of
medical conditions associated with pathogenic microorganisms.
[0412] Moreover, the inherently simple and incremental nature in
designing polymer libraries provides a new alternative and a
systematic tool for the dissection of the relative importance of
charge and hydrophobicity, the parameters believed to be most
crucial to antimicrobial activity and their role in selective
activity.
[0413] The peptide-like backbone, the physico-chemical
characteristics, the broad antibacterial activity spectrum, the
rapid bactericidal kinetics and the bacterial challenge in
developing resistance, demonstrated herein for the polymers of the
present invention, are comparable and even superior to those of
conventional AMPs and thus reminiscent of their postulated membrane
disruptive properties and other eventual targets. In this respect,
the results presented herein may suggest that a defined secondary
structure does not necessarily play a determining role. Rather,
activity appears to depend substantially on a subtle interplay
between positive charge and hydrophobicity.
[0414] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0415] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0416] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
Sequence CWU 1
1
10212PRTArtificial sequenceSynthetic peptide 1Lys
Xaa123PRTArtificial sequenceSynthetic peptide 2Lys Xaa
Xaa134PRTArtificial sequenceSynthetic peptide 3Lys Xaa Xaa
Xaa145PRTArtificial sequenceSynthetic peptide 4Lys Xaa Xaa Xaa Xaa1
556PRTArtificial sequenceSynthetic peptide 5Lys Xaa Xaa Xaa Xaa
Xaa1 567PRTArtificial sequenceSynthetic peptide 6Lys Xaa Xaa Xaa
Xaa Xaa Xaa1 578PRTArtificial sequenceSynthetic peptide 7Lys Xaa
Xaa Xaa Xaa Xaa Xaa Xaa1 582PRTArtificial sequenceSynthetic peptide
8Lys Xaa193PRTArtificial sequenceSynthetic peptide 9Lys Xaa
Xaa1104PRTArtificial sequenceSynthetic peptide 10Lys Xaa Xaa
Xaa1115PRTArtificial sequenceSynthetic peptide 11Lys Xaa Xaa Xaa
Xaa1 5126PRTArtificial sequenceSynthetic peptide 12Lys Xaa Xaa Xaa
Xaa Xaa1 5137PRTArtificial sequenceSynthetic peptide 13Lys Xaa Xaa
Xaa Xaa Xaa Xaa1 5148PRTArtificial sequenceSynthetic peptide 14Lys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5152PRTArtificial sequenceSynthetic
peptide 15Lys Xaa1163PRTArtificial sequenceSynthetic peptide 16Lys
Xaa Xaa1174PRTArtificial sequenceSynthetic peptide 17Lys Xaa Xaa
Xaa1185PRTArtificial sequenceSynthetic peptide 18Lys Xaa Xaa Xaa
Xaa1 5196PRTArtificial sequenceSynthetic peptide 19Lys Xaa Xaa Xaa
Xaa Xaa1 5207PRTArtificial sequenceSynthetic peptide 20Lys Xaa Xaa
Xaa Xaa Xaa Xaa1 5218PRTArtificial sequenceSynthetic peptide 21Lys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5226PRTArtificial sequenceSynthetic
peptide 22Xaa Xaa Xaa Xaa Xaa Xaa1 5237PRTArtificial
sequenceSynthetic peptide 23Xaa Xaa Xaa Xaa Xaa Xaa Xaa1
5248PRTArtificial sequenceSynthetic peptide 24Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa1 5252PRTArtificial sequenceSynthetic peptide 25Lys
Xaa1263PRTArtificial sequenceSynthetic peptide 26Lys Xaa
Xaa1274PRTArtificial sequenceSynthetic peptide 27Lys Xaa Xaa
Xaa1285PRTArtificial sequenceSynthetic peptide 28Lys Xaa Xaa Xaa
Xaa1 5296PRTArtificial sequenceSynthetic peptide 29Lys Xaa Xaa Xaa
Xaa Xaa1 5307PRTArtificial sequenceSynthetic peptide 30Lys Xaa Xaa
Xaa Xaa Xaa Xaa1 5318PRTArtificial sequenceSynthetic peptide 31Lys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5322PRTArtificial sequenceSynthetic
peptide 32Lys Xaa1333PRTArtificial sequenceSynthetic peptide 33Lys
Xaa Xaa1344PRTArtificial sequenceSynthetic peptide 34Lys Xaa Xaa
Xaa1355PRTArtificial sequenceSynthetic peptide 35Lys Xaa Xaa Xaa
Xaa1 5366PRTArtificial sequenceSynthetic peptide 36Lys Xaa Xaa Xaa
Xaa Xaa1 5377PRTArtificial sequenceSynthetic peptide 37Lys Xaa Xaa
Xaa Xaa Xaa Xaa1 5388PRTArtificial sequenceSynthetic peptide 38Lys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5392PRTArtificial sequenceSynthetic
peptide 39Lys Xaa1403PRTArtificial sequenceSynthetic peptide 40Lys
Xaa Xaa1414PRTArtificial sequenceSynthetic peptide 41Lys Xaa Xaa
Xaa1425PRTArtificial sequenceSynthetic peptide 42Lys Xaa Xaa Xaa
Xaa1 5436PRTArtificial sequenceSynthetic peptide 43Lys Xaa Xaa Xaa
Xaa Xaa1 5447PRTArtificial sequenceSynthetic peptide 44Lys Xaa Xaa
Xaa Xaa Xaa Xaa1 5458PRTArtificial sequenceSynthetic peptide 45Lys
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5462PRTArtificial sequenceSynthetic
peptide 46Xaa Xaa1473PRTArtificial sequenceSynthetic peptide 47Xaa
Xaa Xaa1484PRTArtificial sequenceSynthetic peptide 48Xaa Xaa Xaa
Xaa1495PRTArtificial sequenceSynthetic peptide 49Xaa Xaa Xaa Xaa
Xaa1 5506PRTArtificial sequenceSynthetic peptide 50Xaa Xaa Xaa Xaa
Xaa Xaa1 5517PRTArtificial sequenceSynthetic peptide 51Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5528PRTArtificial sequenceSynthetic peptide 52Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5536PRTArtificial sequenceSynthetic
peptide 53Lys Lys Xaa Xaa Xaa Xaa1 5542PRTArtificial
sequenceSynthetic peptide 54Lys Xaa1553PRTArtificial
sequenceSynthetic peptide 55Lys Xaa Xaa1564PRTArtificial
sequenceSynthetic peptide 56Lys Xaa Xaa Xaa1575PRTArtificial
sequenceSynthetic peptide 57Lys Xaa Xaa Xaa Xaa1 5582PRTArtificial
sequenceSynthetic peptide 58Lys Xaa1593PRTArtificial
sequenceSynthetic peptide 59Lys Xaa Xaa1604PRTArtificial
sequenceSynthetic peptide 60Lys Xaa Xaa Xaa1615PRTArtificial
sequenceSynthetic peptide 61Lys Xaa Xaa Xaa Xaa1 5626PRTArtificial
sequenceSynthetic peptide 62Lys Xaa Xaa Xaa Xaa Xaa1
5637PRTArtificial sequenceSynthetic peptide 63Lys Xaa Xaa Xaa Xaa
Xaa Xaa1 5648PRTArtificial sequenceSynthetic peptide 64Lys Xaa Xaa
Xaa Xaa Xaa Xaa Xaa1 5652PRTArtificial sequenceSynthetic peptide
65Xaa Xaa1663PRTArtificial sequenceSynthetic peptide 66Xaa Xaa
Xaa1674PRTArtificial sequenceSynthetic peptide 67Xaa Xaa Xaa
Xaa1685PRTArtificial sequenceSynthetic peptide 68Xaa Xaa Xaa Xaa
Xaa1 5696PRTArtificial sequenceSynthetic peptide 69Xaa Xaa Xaa Xaa
Xaa Xaa1 5707PRTArtificial sequenceSynthetic peptide 70Xaa Xaa Xaa
Xaa Xaa Xaa Xaa1 5718PRTArtificial sequenceSynthetic peptide 71Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5723PRTArtificial sequenceSynthetic
peptide 72Lys Lys Xaa1734PRTArtificial sequenceSynthetic peptide
73Lys Xaa Lys Xaa1745PRTArtificial sequenceSynthetic peptide 74Lys
Lys Xaa Lys Xaa1 5753PRTArtificial sequenceSynthetic peptide 75Lys
Lys Xaa1764PRTArtificial sequenceSynthetic peptide 76Lys Xaa Lys
Xaa1775PRTArtificial sequenceSynthetic peptide 77Lys Lys Xaa Lys
Xaa1 5783PRTArtificial sequenceSynthetic peptide 78Lys Lys
Xaa1794PRTArtificial sequenceSynthetic peptide 79Lys Xaa Lys
Xaa1805PRTArtificial sequenceSynthetic peptide 80Lys Lys Xaa Lys
Xaa1 5813PRTArtificial sequenceSynthetic peptide 81Xaa Lys
Xaa1824PRTArtificial sequenceSynthetic peptide 82Xaa Xaa Lys
Xaa1835PRTArtificial sequenceSynthetic peptide 83Xaa Lys Xaa Lys
Xaa1 5842PRTArtificial sequenceSynthetic peptide 84Xaa
Xaa1852PRTArtificial sequenceSynthetic peptide 85Xaa
Xaa1862PRTArtificial sequenceSynthetic peptide 86Lys
Xaa1873PRTArtificial sequenceSynthetic peptide 87Lys Xaa
Xaa1884PRTArtificial sequenceSynthetic peptide 88Lys Xaa Xaa
Xaa1892PRTArtificial sequenceSynthetic peptide 89Lys
Xaa1903PRTArtificial sequenceSynthetic peptide 90Lys Xaa
Xaa1914PRTArtificial sequenceSynthetic peptide 91Lys Xaa Xaa
Xaa1922PRTArtificial sequenceSynthetic peptide 92Xaa
Xaa1933PRTArtificial sequenceSynthetic peptide 93Xaa Xaa
Xaa1944PRTArtificial sequenceSynthetic peptide 94Xaa Xaa Xaa
Xaa1953PRTArtificial sequenceSynthetic peptide 95Lys Xaa
Xaa19622PRTArtificial sequenceSynthetic peptide 96Gly Ile Gly Lys
Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe1 5 10 15Val Lys Ile
Leu Lys Lys 209717PRTArtificial sequenceSynthetic peptide 97Arg Gly
Gly Leu Cys Tyr Cys Arg Gly Arg Phe Cys Val Cys Val Gly1 5 10
15Arg9816PRTArtificial sequenceSynthetic peptide 98Ala Leu Trp Lys
Thr Leu Leu Lys Lys Val Leu Lys Ala Ala Ala Lys1 5 10
159937PRTArtificial sequenceSynthetic peptide 99Leu Leu Gly Asp Phe
Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu1 5 10 15Phe Lys Arg Ile
Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25 30Pro Arg Thr
Glu Ser 351008PRTArtificial sequenceSynthetic peptide 100Lys Lys
Lys Lys Lys Lys Lys Lys1 510114PRTArtificial sequenceSynthetic
peptide 101Ala Leu Trp Lys Thr Leu Leu Lys Lys Val Leu Lys Ala Ala1
5 1010216PRTArtificial sequenceSynthetic peptide 102Ala Leu Trp Met
Thr Leu Leu Lys Lys Val Leu Lys Ala Ala Ala Lys1 5 10 15
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References