U.S. patent application number 09/988792 was filed with the patent office on 2003-02-13 for novel antimicrobial compounds.
Invention is credited to Carr, Daniel B., Lipkowski, Andrzej W..
Application Number | 20030032599 09/988792 |
Document ID | / |
Family ID | 22955723 |
Filed Date | 2003-02-13 |
United States Patent
Application |
20030032599 |
Kind Code |
A1 |
Lipkowski, Andrzej W. ; et
al. |
February 13, 2003 |
Novel antimicrobial compounds
Abstract
The invention features an antimicrobial composition comprising a
substance P peptide and methods of inhibiting growth of a
microorganism by contacting the microorganism with a substance P
peptide. Bacterial and fungal pathogens are inhibited by the
substance P compositions.
Inventors: |
Lipkowski, Andrzej W.;
(Warsaw, PL) ; Carr, Daniel B.; (Chestnut Hill,
MA) |
Correspondence
Address: |
Ingrid A. Bcattie
Mintz, Levin, Cohn, Ferris,
Glovsky and Popeo, P.C.
One Financial Center
Boston
MA
02111
US
|
Family ID: |
22955723 |
Appl. No.: |
09/988792 |
Filed: |
November 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60252369 |
Nov 21, 2000 |
|
|
|
Current U.S.
Class: |
514/2.4 ;
514/2.3; 514/3.3 |
Current CPC
Class: |
Y02A 50/475 20180101;
C07K 7/22 20130101; A61K 38/00 20130101; A61P 31/00 20180101; Y02A
50/473 20180101 |
Class at
Publication: |
514/15 |
International
Class: |
A61K 038/08 |
Claims
What is claimed is:
1. An antimicrobial composition comprising a substance P
peptide.
2. The antimicrobial composition of claim 1, wherein the amino acid
sequence of the peptide comprises residues 1-8 of SEQ ID No: 1.
3. The antimicrobial composition of claim 2, wherein the amino acid
sequence of the peptide comprises residues 1-8 of SEQ ID No: 2.
4. The antimicrobial composition of claim 2, wherein the sequence
is at least 50% identical to the amino acid sequence of SEQ ID Nos:
1 or 2.
5. The antimicrobial composition of claim 1, wherein the peptide
comprises the amino acid sequence
Xaa.sub.1-Pro-Xaa.sub.2-Pro-Xaa.sub.3-Xaa.sub.4-X-
aa.sub.5-Xaa.sub.6 (SEQ ID NO: 12), wherein Xaa.sub.1 and Xaa.sub.2
are positively charged amino acids, Xaa.sub.3 and Xaa.sub.4 are any
amino acids other than Pro, and Xaa.sub.5 and Xaa.sub.6 are
hydrophobic amino acids.
6. The antimicrobial composition of claim 5, wherein Xaa.sub.5 and
Xaa.sub.6 are aromatic amino acids.
7. The antimicrobial composition of claim 1, wherein the amino acid
sequence of the peptide comprises amino acids 1-10 of SEQ ID Nos: 1
or 2.
8. The antimicrobial composition of claim 1, wherein the amino acid
sequence of the peptide comprises
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu- -Xaa (SEQ ID NO: 13),
wherein Xaa is not a methionine residue.
9. The antimicrobial composition of claim 6, wherein Xaa.sub.5 and
Xaa.sub.6 are selected from the group consisting of Phe and
Trp.
10. The antimicrobial composition of claim 1, having at least one
dextrorotatory amino acid.
11. The antimicrobial composition of claim 1, wherein the peptide
inhibits growth of a bacteriim, fungus, or virus.
12. The antimicrobial composition of claim 11, wherein the peptide
inhibits growth of a cell selected from the genera consisting of
Staphylococcus, Streptococcus, Bacillus, Clostridium, Escherichia,
Shigella, Campylobacter, Hemophilus, Proteus, Yersinia, Klebsiella,
Pseudomonas, and Serratia.
13. The antimicrobial composition of claim 11, wherein the peptide
inhibits growth of a cell selected from the genera consisting of
Aspergillus, Candida, Cryptococcus, Epidermophyton, Histoplasma,
Microsporum, and Trichophyton.
14. The antimicrobial composition of claim 1, further comprising a
second antimicrobial agent.
15. A method for inhibiting growth or survival of a microorganism,
comprising directly contacting the microorganism with a substance P
peptide or a peptide mimetic thereof.
16. The method of claim 15, wherein the peptide comprises amino
acids 1-8 of SEQ ID Nos: 1 or 2.
17. The method of claim 16, wherein the peptide comprises amino
acids 1 -10 of SEQ ID Nos: 1 or 2.
18. The method of claim 15, wherein the microorganism is a bacteria
or a fungus.
19. The method of claim 18, wherein the bacteria is selected from
the group of cutaneous, mucosal, or enteric bacteria.
20. A method of inhibiting a microbial infection, comprising
identifying a mammal suffering from or at risk of developing the
infection and administering to the mammal a substance P peptide or
peptide mimetic thereof.
21. The method of claim 20, wherein the peptide or peptide mimetic
is administered topically.
22. A method of inhibiting a microbial infection, comprising
introducing into an articulating joint of an animal a substance P
peptide or peptide mimetic thereof.
23. A method of inhibiting a microbial infection, comprising
introducing directly into an abscess a substance P peptide or
peptide mimetic thereof.
24. A kit comprising at least one unit dose of an antimicrobial
substance P peptide having at least 50% identity to positions 1-8
of SEQ ID Nos: 1 or 2.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. patent
application Ser. No. 60/252,369, filed Nov. 21, 2000, which is
hereby incorporated herein by reference.
TECHNICAL FIELD
[0002] This invention relates to novel antimicrobial compounds
derived from peptides.
BACKGROUND
[0003] Widespread use of antibiotics in recent decades has led to
proliferation of pathogens having multiple drug resistance, often
encoded by transmissible plasmids, and therefore capable of
spreading rapidly between species. Many previously useful
antibiotics are no longer effective against infectious organisms
isolated from human and animal subjects. The specter of epidemic
forms of bacterial diseases such as tuberculosis and fungal
diseases, which are refractory to known antibiotic agents, may be
realized in the near future. Development of novel antimicrobial
compounds is a continuing urgent public health need.
SUMMARY OF THE INVENTION
[0004] The invention features an antimicrobial composition
comprising a substance P (SP) peptide or peptide mimetic thereof.
The amino acid sequence of the peptide contains at least residues
1-8 of Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met (SEQ ID No: 1),
or the amino acid sequence of the peptide contains at least
residues 1-8 of Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-Leu-Met
(SEQ ID No: 2). A SP peptide is a peptide with antimicrobial
activity, which contains an amino acid sequence that is at least
50% identical to the amino acid sequence of SEQ ID NO: 1.
[0005] The peptide contains levorotatory (L) and/or dextrorotatory
(D) forms of an amino acid. For example, the peptide has at least
one D amino acid. The antimicrobial composition further contains
one or more additional antimicrobial agents such as tetracycline,
penicillin, doxycycline, ampicillin, or CIPRO.TM..
[0006] Antimicrobial peptides contain the amino acid sequence
Xaa.sub.1-Pro-Xaa.sub.2-Pro-Xaa.sub.3-Xaa4-Xaa.sub.5-Xaa.sub.6 (SEQ
ID NO: 12). Referring to SEQ ID NO: 12, Xaa.sub.1 and Xaa.sub.2 are
positively charged amino acids, Xaa.sub.3 and Xaa.sub.4 are any
amino acids other than Pro, and Xaa.sub.5 and Xaa.sub.6 are
hydrophobic amino acids. Xaa.sub.5 and Xaa.sub.6 are preferably
aromatic amino acids. For example, Xaa.sub.5 and Xaa.sub.6 are Phe
or Trp. Preferably, the composition does not contain Saporin (U.S.
Pat. No. 6,063,758), neither in a free form, nor conjugated to to
the antimicrobial peptide.
[0007] The amino terminal portion of a SP peptide associates with a
membrane component of a microbe. Preferably, the SP peptide
associates with a microbial membrane component but does not
associate with an SP receptor, e.g., a tachykinin receptor.
Accordingly, the peptide contains amino acids 1-5, 1-6, 1-7, 1-8,
1-9, or 1-10 of SEQ ID NO: 1 or 2, and lacks 1, 2, 3, 4, or 5 amino
acids from the carboxy-terminal end of SEQ ID NO: 1 or 2. The amino
acid sequence of the peptide contains residues 1-10 of SEQ ID Nos:
1 or 2. For example, the amino acid sequence of the peptide
comprises Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Xaa (SEQ ID NO:
13), wherein Xaa is not a methionine residue.
[0008] The antimicrobial composition inhibits growth of a bacteria
(e.g., cutaneous, mucosal, or enteric bacteria), fungus, or virus.
For example with respect to bacteria, the peptide inhibits growth
of a cell selected from the genera consisting of Staphylococcus,
Streptococcus, Bacillus, Clostridium, Escherichia, Shigella,
Campylobacter, Hemophilus, Proteus, Yersinia, Klebsiella,
Pseudomonas, and Serratia. For example with respect to fungi, the
peptide inhibits growth of a cell selected from the genera
consisting of Aspergillus, Candida, Cryptococcus, Epidermophyton,
Histoplasma, Microsporum, and Trichophyton.
[0009] The invention also includes a method for inhibiting growth
or survival of a microorganism, by directly contacting the
microorganism (e.g., a membrane component of the microbe) with a SP
peptide or a peptide mimetic thereof. A peptide mimetic is an SP
analog in which one or more peptide bonds have been replaced with
an alternative type of covalent bond, and which is not susceptible
to cleavage by peptidases elaborated by the subject or by the
target microorganism. The peptide contains at least 8 consecutive
residues, e.g., residues 1-8, of the amino acid sequence of SEQ ID
Nos: 1 or 2. For example, the peptide contains at least positions
1-10 of the amino acid sequence of SEQ ID Nos: 1 or 2.
[0010] The invention also provides a method of inhibiting a
microbial infection by carrying out the following steps:
identifying a mammal suffering from or at risk of developing a
microbial infection and administering to the mammal a SP peptide or
peptide mimetic thereof. The peptide or peptide mimetic is
administered topically. In an alternative embodiment, the invention
provides a method of inhibiting a microbial infection, by
introducing directly into an articulating joint of an animal a SP
peptide or peptide mimetic thereof. The method is also carried out
by introducing the SP peptide or mimetic directly into an
abscess.
[0011] Also within the invention is a kit containing at least one
unit dose of an antimicrobial SP peptide or mimetic packaged
together with a label, instructions for use, or means of
administering the compound
[0012] "Antimicrobial" activity of an agent or composition shall
mean ability to inhibit growth of one or more microorganism. For
example, the antimicrobial compositions described herein inhibit
the growth of or kill bacterial, algal, fungal, protozoan, and
viral genera and species thereof.
[0013] It is well known to one of skill in the art of antibiotics
development that an agent that causes inhibition of growth can also
be lethal to the microorganism (bacteriocidal, for example in the
case of a microorganism that is a bacterium). The SP peptide or
mimetic is lethal, growth inhibitory, or both.
[0014] "Broad spectrum" antimicrobial activity means to ability to
inhibit growth of organisms that are relatively unrelated. For
example, ability of an agent to inhibit growth of both a Gram
positive and a Gram negative bacterial species is considered a
broad spectrum activity.
[0015] Various compositions and methods herein are useful for
preventing and treating Gram positive and Gram negative bacterial
infections in human and animal subjects. Gram positive bacterial
species are exemplified by, but not limited to, genera including:
Staphylococcus, such as S. epidermis and S. aureus; Micrococcus;
Streptococcus, such as S. pyogenes, S. equis, S. zooepidemicus, S.
equisimilis, S. pneumoniae and S. agalactiae; Corynebacterium, such
as C. pyogenes and C. pseudotuberculosis; Erysipelothrix such as E.
rhusiopathiae; Listeria, such as L. monocytogenes; Bacillus, such
as B. anthracis; Clostridium, such as C. perfringens; and
Mycobacterium, such as M. tuberculosis and M leprae.
[0016] Gram negative bacterial species are exemplified by, but not
limited to genera including: Escherichia, such as E. coli O157:H7;
Salmonella, such as S. typhi and S. gallinarum; Shigella, such as
S. dysenteriae; Vibrio, such as V cholerae; Yersinia, such as Y
pestis and Y enterocolitica; Proteus, such as P. mirabilis;
Bordetella, such as B. bronchiseptica; Pseudomonas, such as P.
aeruginosa; Klebsiella, such as K. pneumoniae; Pasteurella, such as
P. multocida; Moraxella, such as M. bovis; Serratia, such as S.
marcescens; Hemophilus, such as H. influenza; and Campylobacter
species. Further examples of bacterial pathogenic species that are
inhibited according to the invention are obtained by reference to
standard taxonomic and descriptive works such as Bergey's Manual of
Determinative Bacteriology, 9.sup.th Ed., 1994, Williams and
Wilkins, Baltimore, Md.
[0017] Prevention and treatment of fungal infections are embodied
by various compositions and methods provided herein. Suitable
fungal genera are exempflied, but not limited to: Candida, such as
C. albicans; Cryptococcus, such as C. neoformans; Malassezia
(Pityrosporum); Histoplasma, such as H. capsulatum; Coccidioides,
such as C. immitis; Hyphomyces, such as H. destruens; Blastomyces,
such as B. dermatiditis; Aspergillus, such as A. fumigatus;
Penicillium, such as P. marneffei; and Pneumocystis, such as P.
carinii. Subcutaneous fungi, such as species of Rhinosporidium and
Sporothrix, and dermatophytes, such as Microsporum and Trichophyton
species, are amenable to prevention and treatment by embodiments of
the invention herein.
[0018] Prevention and treatment of viral infections that are
topically manifested are embodied by various compositions and
methods provided herein. Suitable viral infections include but are
not limited to: viral warts (papilloma virus), Herpes simplex type
I and type II, varicella zoster (chicken pox), Molluscum
contagiosum (a pox virus), rubeola (measles), and rubella (German
measles).
[0019] A subject to be treated is an animal, preferably a
warm-blooded animal, including any bird or mammal species. Methods
and compositions embodied herein are envisioned for human and
veterinary use. Veterinary use includes application to cows,
horses, sheep, goats, pigs, dogs, cats, rabbits, and all rodents.
The methods of the invention are also useful to agricultural
workers and pet owners to combat infections contracted by exposure
to livestock or pet animals.
[0020] A subject is diagnosed as having a microbial infection by
inspection of a bodily tissue, e.g., epidermal and mucosal tissue,
including such tissue present in surfaces of oral, buccal, anal,
and vaginal cavities and glans penis. Diagnosis of infection is
made according to criteria known to one of skill in the medical
arts, including but not limited to, areas of inflammation or
unusual patches with respect to color, dryness, exfoliation,
exudation, prurulence, streaks, or damage to integrity of surface.
Conditions exemplary of those treated by the compositions and
methods herein, such as abscess, meningitis, cutaneous anthrax,
septic arthritis, emphysema, impetigo, cellulitis, pneumonia, sinus
infection and tubercular disease are accompanied by elevated
temperature. Diagnosis can be confirmed using standard ELISA-based
kits, and by culture, and by traditional stains and microscopic
examination of direct samples, or of organisms cultured from an
inoculum from the subject.
[0021] Other features and advantages of the invention will be
apparent from the following description of embodiments thereof, and
from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a diagram of the chemical structure of SP.
DETAILED DESCRIPTION
[0023] Naturally-occurring SP, a member of the tachykinin family,
is an undecapeptide (SEQ ID NO: 1, FIG. 1). SP plays a key role in
pain signal transmission. As it is widely distributed not only in
the central and peripheral nervous systems, but in other tissues as
well, it also mediates multiple homeostatic functions. Some classes
of immune cells, such as T and B cells, endothelial cells, and
macrophages, have the ability to express SP receptors; and some
classes of immune cells such as macrophages, eosinophils, and
endothelial cells, have the ability to produce SP.
[0024] The concentration of SP increases in inflamed tissues,
consistent with a role in mammalian host defense system.
Naturally-occurring SP is involved in direct stimulation of
lymphocytes and regulation of tissue repair via enhancement of
proliferation of fibroblasts and endothelial cells. In addition,
immune cells can be induced by SP to produce cytokines that
modulate hematopoiesis. SP is involved in activation of multiple
endogenous defense systems.
[0025] Surprisingly, the data herein show that compositions such as
SP peptides, SP-related molecules, SP fragments, and SP peptide
derivative compositions having a particular consensus amino acid
sequence, possess broad spectrum antimicrobial activities.
[0026] Determination of SP-mediated antimicrobial activity
[0027] Inhibition of growth (bacteriostasis, for example in the
case of a microorganism that is a bacterium) is determined by
turbidometric analysis of a liquid culture. For example, such that
a culture having a particular titer of cells per unit volume in the
presence of one or more concentrations of the agent fails to
increase in density as measured by light scattering in a
spectrophotometer or calorimeter, in comparison to the culture in
the absence of the agent or at a lower concentration of the agent.
Inhibition of growth is also determined using solid medium
containing a concentration of the agent, such that a reduced
ability to form a number of visible colonies (reduced titer of
bacterial cells) is observed in the presence of that concentration
of agent, in comparison to the absence of the agent or a lower
concentration. The assays are standardized by using a predictable
predetermined number of cells, and the lowest observed
concentration of the agent that inhibits growth is defined as the
minimum inhibitory concentration (MIC). With this parameter, an
agent having a lower MIC than another has a greater antimicrobial
activity for the organism used in the assay.
[0028] Anti-fungal activity is similarly assayed, using log phase
vegetative fungal cells. Vegetative cells of a model non-pathogenic
species such as Saccharomyces cerevisiae, a Kluyveromyces or a
Pichia species, or a pathogenic species such as Candida albicans
are grown in a nutrient broth such as a standard yeast medium, to
log phase, and then assayed by liquid or solid medium methods, as
described above for bacteria.
[0029] Methods of assay for antiviral activity are also known in
the art. A first method is direct measurement of lethality for
virions, such as mixing various concentrations of the antiviral
agent, e.g., for a fixed time, or as a function of time, with a
known number of "plaque forming units" (PFU) of the virus strain.
The treated virus PFUs are then diluted and mixed with an
appropriate number of sensitive cells, and the number of plaques
obtained in comparison to the same number of PFUs that have been
identically treated, in the absence of the antimicrobial compound.
This assay detects activity that directly disrupts virion structure
or initial function of interaction with a sensitive host cell.
[0030] A second method detects inhibition by the agent of ability
of the virus to successfully replicate in a sensitive cell. In this
method, a sample from each of a range of concentrations of the
antiviral agent is added to infected cells at a particular time
following the infection, e.g., simultaneous to infection, or within
10 min or one hour of infection. Progeny viruses are collected at
the end of the replication cycle, e.g., at 20 h after infection and
incubation of the infected cells. The reduction yield of progeny
viruses from infected cells in the presence of the agent, and as a
function of concentration of the agent, compared to the yield in
the absence of the agent, indicates that the agent, e.g., a SP
peptide, possesses antiviral activity.
[0031] SP peptides
[0032] SP peptides contain an amino acid sequence related to that
of positions 1-8 of the amino terminal amino acids in the sequence
of SP or SP antagonist (as shown in SEQ ID No: 1 or 2). The peptide
of SEQ ID NO: 2 is referred to as an SP antagonist because it
interferes with SP receptor-mediated SP activity. The antimicrobial
activity of an SP peptide is unrelated to SP receptor binding. SP
receptor binding involves the carboxy-terminal end of SP. The
carboxy-terminal 1, 2, or 3 amino acids of SP are not required for
antimicrobial activity.
[0033] SP peptides are at least 50% identical to the sequences of
SEQ ID NO: 1 or 2. Further, they are at least 75% identical, 85%,
95%, and 99% identical to the sequences of SEQ ID NO: 1 or 2.
Nucleotide and amino acid comparisons described herein are carried
out using the Lasergene software package (DNASTAR, Inc., Madison,
Wis.). The MegAlign module used is the Clustal V method (Higgins et
al., 1989, CABIOS 5(2):151-153). The parameter used is gap penalty
10, gap length penalty 10.
[0034] The one letter and three letter codes for the 20 naturally
occurring amino acids are well known to one of skill in the art,
and are indicated herein as appropriate. Amino acids which are
positively charged are lysine (lys, K) and arginine (arg, R).
Aromatic amino acids are tyrosine (tyr, Y), phenylalanine (phe, F)
and tryptophan (trp, W). The aromatic amino acids are classified
further as hydrophobic amino acids, a group which further includes
valine (val, V), leucine (leu, L), and isoleucine (ile, I). A
conservative substitution of one amino acid for another is a
replacement by an amino acid having a similar chemical functional
side group, e.g., replacement of a positively charged amino acid by
another positively charged amino acid, or replacement of a
hydrophobic amino acid by another hydrophobic amino acid.
[0035] The antimicrobial activity of the SP peptides and peptide
mimetics thereof are associated with the residues located at the
amino terminus. The amino-terminal four amino acids of SP peptides
are alternately basic residues, i.e., the positively charged amino
acids lysine (lys, K) and arginine (arg, R), and proline residues.
Reference to Table 1 shows that a consensus amino acid sequence
among the SP peptides in these first four positions is an
alternation of two basic amino acids and two proline residues. The
alternation of basic positively charged residues and proline
residues confers a particular structure to the SP peptide.
[0036] Peptide modification techniques are used in the manufacture
of drug analogs of biological compounds and are known to one of
ordinary skill in the art. Synthetic peptides having an
antimicrobial activity that is at least 50% of that of SP are
produced by either of two general approaches. In a first approach,
the peptides are produced by the well-known Merrifield solid-phase
chemical synthesis method wherein amino acids are sequentially
added to a growing chain. See, Merrifield (1963) J.Am. Chem. Soc.
85:2149-2156. Systems for manually synthesizing peptides on
polyethylene pegs are available from Cambridge Research
Biochemicals, Cambridge, Mass. Automatic peptide synthesis
equipment is available from several commercial suppliers, including
Applied Biosystems, Inc., Foster City, Calif.; Beckman Instruments,
Inc., Waldwick, N.J., and Biosearch, Inc., San Raphael, Calif.
Using such automatic synthesizers according to manufacturer's
instructions, peptides are produced in gram quantities for use in
the present invention. This method is preferred as yielding an SP
peptide in a substantially purified condition, free of
contaminating cell components, and substantially free of
contaminating chemicals used in the synthetic procedure.
[0037] A polypeptide is substantially pure when it constitutes at
least about 60%, by weight, of the protein in the preparation.
Preferably, the peptide in the preparation is at least about 75%,
more preferably at least about 90%, and most preferably at least
about 99%, by weight, of SP peptide or mimetic. Purity is measured
by any appropriate method, e.g., column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis. Accordingly,
substantially pure peptides include peptides purified from natural
sources, recombinant peptides derived from a eucaryote but produced
in E. coli or another procaryote, or in a eucaryote other than that
from which the peptide was originally derived as well as
chemically-synthesized peptides.
[0038] In a second approach, the synthetic SP peptides of the
present invention are prepared by recombinant techniques involving
the expression in cultured cells of recombinant DNA molecules
encoding a gene for a desired portion of a natural or analog SP
molecule. The gene encoding the peptide is natural or synthetic.
Polynucleotides are synthesized by well known techniques based on
the desired amino acid sequence. For example, short single-stranded
DNA fragments are prepared by the phosphoramidite method described
by Beaucage et al. (1981) Tetra. Lett. 22:1859-1862. A
double-stranded fragment is obtained either by synthesizing the
complementary strand using DNA polymerase under appropriate
conditions or by adding the complementary strand using DNA
polymerase with an appropriate primer sequence. The natural or
synthetic DNA fragments coding for the desired SP peptide is
incorporated into a suitable DNA construct capable of introduction
to and expression in an in vitro cell culture. A particular
technique for the recombinant DNA production of SP is described in
Yokota et al. (1989) J. Biol. Chem. 264:17649, the disclosure of
which is incorporated herein by reference. According to this
method, the peptide must be further purified away from
contaminating cellular materials. A peptide is considered purified
if it is at least about 90% free of material having a different
chemical composition, e.g., at least about 95% free, and at least
about 98-99% free of contaminating material, by weight.
[0039] The methods and the SP compositions in certain embodiments
of the present invention employ synthetic SP peptide derivative
compounds, which can comprise amino acid analogs such as D-amino
acids, or which can be non-peptide compositions or peptide mimetics
as described herein. The SP peptide derivative compounds and
peptide mimetics have functional antimicrobial activity comparable
to that of a known SP peptide. The antimicrobial activity is for
example, from about one-half of activity of SP peptide, to about
two-fold, about four-fold, or about ten-fold greater than that of
SP Peptide.
[0040] The derivative compounds have a shape, size, flexibility,
and electronic configuration such that the antimicrobial activity
of the molecule is similar to a natural antimicrobial peptide. Such
non-peptide molecules will typically be small molecules having a
molecular weight in the range from about 100 to about 1000 daltons.
The use of such small molecules is frequently advantageous both in
preparation of pharmacological compositions, and in pharmacological
properties such as bioavailability, permeability into the microbial
target, rate of metabolism by the subject and the target
microorganism, and stability.
[0041] The invention includes analogs in which one or more peptide
bonds have been replaced with an alternative type of covalent bond
(a "peptide mimetic") which is not susceptible to cleavage by
peptidases elaborated by the subject or by the target
microorganism. Where proteolytic degradation of a peptide
composition is encountered following administration to the subject,
replacement of a particularly sensitive peptide bond with a
noncleavable peptide mimetic renders the resulting peptide
derivative compound more stable and thus more useful as a
therapeutic. Such mimetics, and methods of incorporating them into
peptides, are well known in the art.
[0042] Similarly, the replacement of an L-amino acid residue by a
D-amino acid residue is a standard method for rendering the
compound less sensitive to enzymatic destruction. Other amino acid
analogs are known in the art, such as norleucine, norvaline,
homocysteine, homoserine, ethionine, and the like. Also useful is
derivatizing the compound with an amino-terminal blocking group
such as a t-butyloxycarbonyl, acetyl, methyl, succinyl,
methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,
benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyaselayl,
methoxyadipyl, methoxysuberyl, and a 2,3-dinitrophenyl group.
Blocking the charged amino- and carboxy-termini of the peptide
derived compound would have the additional benefit of enhancing
solubility of the compound in the hydrophobic environment of the
cell membrane of the target microorganism.
[0043] The design of such peptide mimetic SP compounds is performed
using techniques known in the art of drug design. Such techniques
include, but are not limited to, self-consistent field (SCF)
analysis, configuration interaction (CI) analysis, and normal mode
dynamics computer analysis, all of which are well described in the
scientific literature. See, e.g., Rein et al., Computer-Assisted
Modeling of Receptor-Ligand Interactions, Alan Liss, N.Y. (1989).
Preparation of the identified compounds will depend on the desired
characteristics of the compounds and will involve standard chemical
synthetic techniques. See, Cary et al. Advanced Organic Chemistry,
part B, Plenum Press, N.Y. (1983).
[0044] Antimicrobial Formulations and Therapeutic Administration
Thereof
[0045] The SP peptides, peptide analogs, and peptide mimetics are
incorporated in a physiologically acceptable carrier or salt,
suitable for topical application to the affected area, or for
direct injection into the affected areas such as a soft tissue
abscess or an infected joint, or for diffusion from a surgically
implanted device. Topical applications include lavage of body
cavities or lumens, e.g., pre- or post-surgical peritoneal lavage
or pulmonary lavage. Topical applications include use of gels,
creams, lotions, supposities, and use of devices and dressings such
as dissolving patches and bandages impregnated prior to use with
the antimicrobial peptide. Additional routes of delivery include
oral, and injection or infusion that is intramuscular, intravenous,
subcutaneous, intraperitoneal, intraspinal, and epidural.
Meningitis for example is treated by administration of an SP
peptide by several routes, including direct intraspinal
injection.
[0046] The compositions contain from about 0.1 nM to about 10 mM
peptide, usually containing from about 0.01 .mu.M to about 1 mM
compound, and more usually containing from about 0.1 .mu.M to about
100 .mu.M of SP peptide or peptide derived compound. The nature of
the carrier depends on the intended area of application. For
application to the skin, a cream lotion, or ointment base is
usually preferred, with suitable bases including lanolin,
Silvadene.TM., particularly for the treatment of burns;
Aquaphor.TM. (Duke Laboratories, South Norwalk, Conn.), and the
like. The peptides or derivative compounds are incorporated into or
onto natural and synthetic bandages and other wound dressings to
provide for continuous exposure of a wound to the peptide. Aerosol
applicators and inhaler devices are used, for delivery to sinuses
and deeper portions of the respiratory system. Peptides and
derivative compounds are also incorporated in or coated on
implantable devices, such as heart pacemakers, intralumenal stents,
and the like where the antimicrobial activity would be of benefit.
Coating is achieved by non-specific adsorption or covalent
attachment. Optionally, an anti-pruritic agent such as an opioid is
added to a antimicrobial composition to relieve pain at an infected
site. Additional antimicrobial agents can be combined with the SP
peptides, including but not limited to one or more of beta-lactam
antibiotics such as penicillin, macrolides such as erythromycin,
aminoglycosides such as lincomycin, tetracyclines such as
doxycycline, semi-synthetic antibiotics such as Ceclor, and
bacterially-derived peptide antibiotics such as gramicidin and
tyrocidin.
[0047] The contents of all cited patents and papers are hereby
incorporated by reference herein.
[0048] Example 1. Structure of SP and derivatives thereof
[0049] Peptides were synthesized by respective Boc- or Fmoc-
chemistry in solid phase by methods known in the art, e.g.,
Misicka, et al., Biochemical & Biophysical Research
Communications 1991:180(3):1290-7. Crude peptides were purified by
gel filtration on Sephadex LH-20 (in methanol), followed by
preparative HPLC. All peptides were confirmed to have correct amino
acid analyses and molecular weights by FAB-MS. For microbiological
study, peptides in acetate form were used. The sequences of the
peptides are: SP, Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2
(FIG. 1, SEQ ID NO: 1); SP antagonist,
Arg-D-Pro-Lys-Pro-Gln-Gln-D-Trp-Ph- e-D-Trp-Leu-MetNH2 (SEQ ID NO:
2); bradykinin, Arg-Pro-Pro-Gly-Phe-Ser-Pro- -Phe-Arg (SEQ ID NO:
3); neurotensin, Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg--
Pro-Tyr-Ile-Leu (SEQ ID NO: 4) or
Xaa-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-- Tyr-Ile-Leu (SEQ ID NO:
14; where Xaa is Pyr or Tyr); and indolicidin,
Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2 (SEQ ID NO:
5).
[0050] SP is expressed in a variety of different animals (see
Warren, L., http://www.wdv.com/Notebook/Biochemistrv/Substance P/).
Analysis of the sequences of these homologues, in comparison to
that of humans (SEQ ID NO: 1) yields insight into design of SP
peptides embodied herein. The sequence of SP native to the
following organisms has been reported:
1TABLE 1 SEQ ID NO:6 spotted dogfish
Lys-Pro-Arg-Pro-Gly-Gln-Phe-Phe-Gly-Leu-Met SEQ ID NO:7 guinea pig,
horse, cow Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met SEQ ID NO:8
alligator, chicken Arg-Pro-Arg-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met SEQ
ID NO:9 Atlantic cod Lys-Pro-Arg-Pro-Gln-Gln-Phe-Ile-Gly-Leu-Met
SEQ ID NO:10 rainbow trout
Lys-Pro-Arg-Pro-His-Gln-Phe-Phe-Gly-Leu-Met SEQ ID NO:11 sea
lamprey Ala-Lys-His-Asp-Lys-Phe-Tyr-Gly-Leu-Met SEQ ID NO:1 human
Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met
[0051] These data indicate a fully conserved consensus from
prevertebrate chordate animals to humans, located in the three
C-terminal residues. These three residues confer on the SP the
ability to interact with a specific SP receptor on immune cells.
Further, a consensus in the 8 N-terminal positions is found to be:
Xaa.sub.1-Pro-Xaa.sub.2-Pro-Xaa.sub.-
3-Xaa.sub.4-Xaa.sub.5-Xaa.sub.6 (SEQ ID NO: 12) where Xaa.sub.1 and
Xaa.sub.2 are positively charged amino acids, Xaa.sub.3 and
Xaa.sub.4 are Gln or Gly, and Xaa.sub.5 and Xaa.sub.6 are aromatic
amino acids, particularly Phe. This 8-residue fragment of SP has
antimicrobial activity but cannot bind to an SP receptor on a cell
of a subject, since the fragment lacks the portion of the peptide
that confers affinity to that receptor.
[0052] Without being bound by any particular mechanism,
antimicrobial activity of SP peptides requires the above 8-residue
consensus sequence. The aromatic residues confer solubility on the
peptide for the hydrophobic lipids of the membrane of target
microbe cells, and also confer the ability for self-aggregation and
multimerization of the SP peptide. The alternating positive
residues and prolines at positions 1-4 of the 8-mer peptide confer
a particular three dimensional configuration, for example, a
helical configuration, to the monomer, so that the monomer
associates with a lipid moiety of a cell membrane. SP peptides
interact with one another and with a component of a microbial
membrane to form a supramolecular structure, e.g., a pore.
[0053] Peptides having the above consensus offer advantages for use
as a novel antimicrobial agent. As SP is an endogenous peptide,
found in humans and other chordate and vertebrate animals, it is
not antigenic. Therefore continued administration of this agent
over time does not provoke an immune response. Further, deletion or
substitution one or more of the three carboxy-terminal residues
(Gly-Leu-Met) associated with affinity of the SP peptides to a
specific SP receptor on cells of the immune system assures that
possible undesired side affects of systemic SP administration
(e.g., SP-receptor mediated activities such as pain, inflammation,
and swelling) are reduced or eliminated. In addition, the
antimicrobial activity of SP peptides has a broad antimicrobial
spectrum as shown herein, including Gram positive and Gram negative
bacteria, and fungi. These data indicate that traditional targets
for antimicrobial agents, such as the prokaryotic ribosome or the
murein cross-bridges of a bacterial cell wall are not involved as
macromolecular targets. Therefore, the compounds described herein
cannot be evaded by enzymes associated with multiple drug
resistance factors. Topical administration of an SP peptide to an
epithelium of a subject offers the advantage that the peptide
remains external and does not become systemic.
[0054] Example 2. Antimicrobial activity of SP and derivatives
using bacterial test species
[0055] Antimicrobial activity, specifically antibacterial activity,
was assayed using cells of each of Staphylococcus aureus NCTC 4163,
Escherichia coli NCTC 8196, Pseudomonas aeruginosa NCTC 6749,
Proteus vulgaris NCTC 4635, and Enterococcus faecalis ATCC 19212.
To determine the minimum inhibitory concentration (MIC), the
microdilution broth method, well-known to one of ordinary skill in
the art of microbiology, was used. Cells of each bacterial strain
were collected in the logarithmic phase of growth, and resuspended
in nutrient broth. The concentration of colony-forming units (CFU)
per milliliter was quantified by measuring absorption of light at
600 nm (A.sub.600).
[0056] Peptide samples were dissolved in nutrient broth (pH 7.0)
and diluted serially. The sample solution (100 .mu.l) was mixed
with the diluted bacterial suspension (100 .mu.l). Mixtures
containing 10.sup.5 bacterial CFU, and from 1% to 0.003% of test
peptides, were incubated for 24 h at 37.degree. C. Antimicrobial
activities were expressed as the minimal inhibitory concentration
(MIC), which is defined as the concentration at which 100%
inhibition of growth of this number of cells was observed (Table
2). The indolicidin antibacterial property with cells of S. aureus
was used as a positive control reference.
2TABLE 2 Antimicrobial activities of SP peptides, neurotensin and
bradykinin Minimal Inhibitory Concentration MIC (%) S. aureus E.
coli E. faecalis P. vulgaris P. aeruginosa C. albicans SEQ ID NO: 1
0.007 0.06 0.13 0.13 0.13 0.25 SEQ ID NO: 2 0.13 0.13 0.13 0.13
0.13 0.03 Neurotensin 0.25 1.0 1.0 0.5 1.0 >1 Bradykinin 0.5 0.5
>1 0.5 1.0 0.25 Indolicidin 0.003 0.007 n.t* n.t. n.t. 0.015
*n.t. - not tested
[0057] Naturally-occurring SP binds to a specific SP receptor on
certain cells and plays an active role in the host defense system.
This peptide, and other regulatory peptides such as neurotensin and
bradykinin, exhibit coordinated actions in protecting mammals from
microbial infection. Previously, all activities of substance P have
been related to its effects on endogenous mechanisms activated by
NK receptors, such as antibody stimulation (Maszczynska, et al.,
Analgesia 2000;3:259-68; Hartung, et al., Journal of Immunology
1986;136(10):3856-63; Jeon, et al., Immunopharmacology 1999;41
(3):219-26; Pascual and Bost, Immunology 1990;71(1)52-5; Linnik and
Moskowitz, Peptides 1989; 10(5):957-62; Payan, Annual Review of
Medicine 1989;40:341-52); histamine release; (Shibata, et al.,
Biochimica et Biophysica Acta 1985;846(l):1-7); induction of NO
synthesis (Hartung, et al., Journal of Immunology
1986;41(3):219-26); vasodilation, and so on (Joos and Pauwels,
Trends in Pharmacological Sciences 2000;21(4):131-3).
[0058] In addition to SP, other peptides (e.g., neurotensin,
bradykinin), which are expressed also at sites and injured tissues
that form the frontiers of the host defense system, were tested.
Surprisingly, a variety of pathogenic microorganisms were found to
be inhibited by SP peptides with the amino acid sequence of SEQ ID
NO: 1 and 2. Bradykinin and neurotensin had significantly lower
antibacterial potency than either SP or SP antagonist against
nearly all microorganisms tested.
[0059] Data obtained using cells of S. aureus demonstrate that SP
and SP derivatives thereof have substantial antimicrobial activity.
The level of activity was comparable to that of indolicidin. The
antimicrobial property of SP was found to be very strong (Table 2),
i.e., equally potent to indolicidin for cells of the Gram positive
bacterial species S. aureus. Further, SP antagonist activity is
comparable to that of indolicidin for cells of the fungal pathogen
C. albicans.
[0060] The antimicrobial potencies of SP antagonist on the other
bacteria tested were weaker than SP (MIC was 10- to 20-fold
higher), but still significant. Observed differences in activity
correlate with endogenous recognition of pathogenic (S. aureus) and
symbiotic bacteria (E. coli). S. aureus is widely and normally
found on skin and so is "symbiotic" species, whereas infection by
some strains of E. coli, such as the enterotoxic strain O157:H7,
can be fatal.
[0061] Example 3. Antifungal activity of SP and SP antagonist
[0062] Antifungal activity was assayed using Candida albicans NCTC
10231 as a target fungus. Candida cells in the logarithmic phase of
growth were suspended in dextrose broth medium at a density of
10.sup.5 CFU/ml. A mixture of the sample solution (100 .mu.l ) and
the fungal suspension (100 .mu.l) was incubated for 24 h at
37.degree. C. Antifungal activity was assessed using turbidity
measurement as described above (Table 2).
[0063] SP was found to have antifungal properties against C.
albicans. Further, the SP antagonist was found to have
approximately 10 times higher potency against C. albicans than
either SP or bradykinin.
[0064] Example 4. SP antagonist does not affect the antimicrobial
activity of SP
[0065] Using mixing experiments, the antimicrobial effect of SP was
found not to be blocked by the presence of a SP antagonist. This
finding confirms the data in Table 2, and indicates that the
antimicrobial effect in vivo is not mediated by the SP receptor,
but is rather a direct effect on the microorganism. Data herein
indicate that substance P possesses previously unreported, direct
antimicrobial potency.
[0066] Other embodiments are within the following claims.
Sequence CWU 1
1
14 1 11 PRT Homo sapiens 1 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu
Met 1 5 10 2 11 PRT Homo sapiens VARIANT (2) Wherein Pro is
D-proline 2 Arg Pro Lys Pro Gln Gln Trp Phe Trp Leu Met 1 5 10 3 9
PRT Homo sapiens 3 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 5 4 13 PRT
Homo sapiens 4 Glu Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu
1 5 10 5 13 PRT Homo sapiens 5 Ile Leu Pro Trp Lys Trp Pro Trp Trp
Pro Trp Arg Arg 1 5 10 6 11 PRT Scyliorhinus canicula 6 Lys Pro Arg
Pro Gly Gln Phe Phe Gly Leu Met 1 5 10 7 11 PRT Cavia porcellus 7
Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met 1 5 10 8 11 PRT Gallus
gallus 8 Arg Pro Arg Pro Gln Gln Phe Phe Gly Leu Met 1 5 10 9 11
PRT Gadus morhua 9 Lys Pro Arg Pro Gln Gln Phe Ile Gly Leu Met 1 5
10 10 11 PRT Oncorhynchus mykiss 10 Lys Pro Arg Pro His Gln Phe Phe
Gly Leu Met 1 5 10 11 10 PRT Petromyzon marinus 11 Ala Lys His Asp
Lys Phe Tyr Gly Leu Met 1 5 10 12 8 PRT Artificial Sequence VARIANT
(1) Wherein Xaa is His or Lys or Arg 12 Xaa Pro Xaa Pro Xaa Xaa Xaa
Xaa 1 5 13 11 PRT Artificial Sequence VARIANT (11) Wherein Xaa is
not Met 13 Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Xaa 1 5 10 14 13
PRT Homo sapiens VARIANT (1) Wherein Xaa is Pyr or Tyr 14 Xaa Leu
Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu 1 5 10
* * * * *
References