Note: Descriptions are shown in the official language in which they were submitted.
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NOVISPIRINS: ANTIMICROBIAL PEPTIDES
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
This invention was made with Government support under Grant no. A143934,
awarded
by the National Institutes of Health. The Government may have certain rights
in this invention.
INTRODUCTION
Background
The development of effective antimicrobial agents was once seen as a
definitive cure for
bacterial diseases. But even before the development of the first antibiotics,
bacteria had
demonstrated an ability to adapt to stress in the environment, resulting in
the development of
resistance. In recent years, the variety of antimicrobial agents has increased
substantially,
along with a parallel increase in resistant pathogenic microorganisms.
Resistance is now
recognized against all clinically available antimicrobial agents. The response
to antimicrobial
resistance in the medical community has been to use new or alternative
antibiotics not
previously used against the resistant bacteria. This approach has required the
continuous
development of new antibiotics, either as modifications of currently existing
compounds or as
combinations of compounds that may inhibit or bypass the bacterial resistance
mechanisms.
Natural polycationic antibiotic peptides have been found in many different
species of
animals and insects and shown to have broad antimicrobial activity. In
mammals, these
antimicrobial peptides are mainly represented by two families, the defensins
and the
cathelicidins. Nearly all of these peptides have membrane affinity, and can
permeate and
permeabilize bacterial membranes, resulting in injury, lysis, and/or death to
the microbes. For
example, the human peptides termed alpha-defensins are produced by neutrophils
and
intestinal Paneth cells. In three-dimensions, defensins manifest an
amphiphilic, largely beta-
sheet structure, with a polar face formed largely by its arginines and with N-
and C-terminal
residues playing an important role in defining antimicrobial potency and
spectrum. (See
Gudmundsson et al. (1999) J Immunol Methods 232(1-2):45-54.) Antimicrobial
peptides are
reviewed by Hancocfc and Lehrer (1998) Trends in BiotechnoloaV 16:82.
Cystic fibrosis (CF) is an inherited disorder that occurs in one of every
3,300 U.S.
newborns. It affects some 30,000 Americans today. The median survival age of
patients with CF
is only 31.3 years, making CF the most common life-shortening inherited
disease in the U.S.
Most CF patients die from pulmonary failure that results from chronic,
progressive infection by
Pseudomonas aeruginosa - a Gram-negative bacterium that is widely distributed
throughout the
environment. P. aeruginosa has limited ability to infect normal individuals,
but can be a
devastating secondary invader in immunocompromised, severely burned, or
antibiotic-treated
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persons. Because Pseudomonas aeruginosa strains frequently are or become
resistant to
conventional antibiotics, infections caused by them are often difficult to
eradicate.
The in vitro activity of antimicrobial peptides, including cecropin P1,
indolicidin, magainin
II, nisin and ranalexin has been tested against clinical isolates of P.
aeruginosa. The peptides
were found to have a varied range of inhibitory values, and showed some
synergy when
combined with conventional antibiotics (Giacometti et al. (1999) J Antimicrob
Chemother.
44(5):641-5)
There is a clinical need for novel antibiotic agents that are active against
drug resistant
Gram-negative bacteria, and which have low toxicity against mammalian cells.
The present
invention addresses this need.
Relevant Literature
Saiman et al. (1999) Pediatr Pulmonol, Suppl. 17:320 report that drug
resistant
organisms from CF patients are inhibited by cathelicidin peptides; and Brogden
et al. (1999)
Pediatr Pulmonol, Suppl. 17:320 report on the efficacy of SMAP29 in an ovine
model of
pulmonary infection and its potential for treating P. aeruginosa infection in
patients with
cystic fibrosis (CF).
SUMMARY OF THE INVENTION
Methods and compositions are provided for the use of novispirin peptides.
Novispirin
peptides are small antimicrobial agents with potent activity against Gram-
negative bacteria,
including Chlamydia trachomatis, Pseudomonas aeruginosa, Escherichia coli and
Stenotrophomonas maltophilia. The peptides are nonhemolytic, exhibit reduced
in vitro
cytotoxicity relative to other antimicrobial peptides, and are well-tolerated
in vivo after
intravenous injection. Novispirins are equally effective against growing and
stationary phase P.
aeruginosa, and they retain activity in the presence of high concentrations of
salt or human
serum. Novispirins also bind lipopolysaccharide (LPS), a property that may
mitigate symptoms
associated with Gram-negative bacterial infection.
A pharmaceutical composition comprising novispirin as an active agent is
administered
to a patient suffering from a microbial infection, particularly bacterial
infections. The protein is
also effective at killing a variety of microbial organisms in vitro.
Novispirin may be administered
alone, or in combination with other bacteriocidal agents, e.g. antibiotics, as
a cocktail of
effective peptides, etc. Novispirin mediated killing of microbes is also
useful for modeling and
screening novel antibiotics.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1 B show the relative activity ofi ovispirin and representative
novispirins
against two Gram-negative bacteria.
Figure 2 shows the kinetics of antimicrobial peptides against P. aeruginosa.
Figure 3 shows the hemolytic activity of antimicrobial peptides against human
red blood
cells.
Figures 4A, 4B and 4C show the relative cytotoxicity of antimicrobial peptides
against
human cells.
Figure 5 shows the binding of LPS by novispirins.
Figures 6A and 6B show the permeabilization of bacterial membranes by
novispirins.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Methods are provided fior the use of novispirins as antimicrobial agents. The
peptides
are effective at killing a variety of microbial organisms in vitro and in vivo
by direct microbicidal
activity. Novispirin(s) are administered alone or in combination with other
active agents to a
patient suffering from or predisposed to an infection, in a dose and for a
period of time sufficient
to reduce the patient population of microbial pathogens.
There is a continuing need for new antimicrobial agents, particularly those
that are
effective in killing pathogens resistant to conventional antibiotics, e.g.
Pseudomonas
aeruginosa. Specific treatments of interest include, without limitation:
aerosol administration to
the lungs of patients with cystic fibrosis to treat infections caused, e.g. by
P. aeruginosa, S.
maltophilia, etc., and to forestall the emergence of resistance to other
inhaled antibiotics;
instillation into the urinary bladder of patients with indwelling catheters to
prevent infection;
application to the skin of patients with serious burns; opthalmic
instillation, directly or in
ophthalmic solutions, to treat or prevent infection; intravaginal application
to treat bacterial
vaginosis and/or prevent sexually transmitted disease, e.g. by preventing
infection with
Chlamydia trachomatis. The novispirins also find use in the treatment of plant-
pathogenic
pseudomonads, in agricultural applications designed to prevent disease in and
spoilage of food
crops.
The peptide form of novispirins provides a basis for further therapeutic
development, by
modification of the polypeptide structure to yield modified forms having
altered biological and
chemical properties. The native or modified forms are formulated in a
physiologically
acceptable carrier for therapeutic uses, or are otherwise used as an
antimicrobial agent.
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Novispirin Compositions
For use in the subject methods, any of the provided novispirins, modifications
thereof, or a combination of one or more forms may be used. Novispirins
include peptides
of the formula as follows:
SEQ ID N0:1 KNLRRX~X2RKX3X4HIIKKYG
wherein X~, XZ, X3 and X4 are independently selected from the group consisting
of the D
or L forms of glycine, threonione, serine and isoleucine, preferably glycine,
threonine, serine,
glutamic acid, aspartic acid, isofeucine, D-alanine and D-isoleucine, with the
proviso that not
more than 3 of the X residues are isoleucine. Preferred amino acids for the
non-isoleucine
residues are glycine, serine and threonine. Preferred are peptides wherein
only one of X~, X2,
X3 and X4 is selected from glycine, serine and threonine.
Without being limited by the theory for making these substitutions, it is
believed that the
cytotoxicity of the antimicrobial peptide ovispirin (SEQ ID N0:2) is related
to its high degree of
rigidity and amphipathicity. The substitution of glycine, threonine, serine,
glutamic acid, aspartic
acid, isoleucine, D-alanine or D-isoleucine for one of the isoleucine resides
at positions 6, 7,10
or 11 in ovispirin adds flexibility (glycine), breaks the alpha-helix (D-
alanine (DA) or D-isoleucine
(DI)) or adds a polar residue to disrupt the overly hydrophobic region.
Table I shows the primary sequences of ovispirin and exemplary novispirin
peptides.
Bolded letters indicate the residues that distinguish these novispirins from
ovispirin.
SEQ 1D
NO:
1 2 3 4 5 6 7 8 9 11 131415
10 12 16
17
18
OVISplfln 2 K N L R R I I R K I I I K K YG
I H
G6-nOVISplrln3 K N L R R G I R K I I I K K YG
I H
T6-nOVISplrln4 K N L R R T I R K I I I K K YG
I H
S6-nOVISplrln5 K N L R R S I R K I I I K K YG
I H
E6-noviSpirin6 K N L R R E I R K I I I K.K YG
I H
D6-nOVISplrln7 K N L R R D I R K I I I K K YG
I H
DA6-noViSpirirl8 K N L R R dA R K I I I K K YG
I I H
D16-novispirin9 K N L R R dlI R K I I I K K YG
I H
G7-nOVISplrln10 K N L R R I G R K I I I K K YG
I H
T7-nOVISplrln11 K N L R R I T R K I I I K K YG
I H
S7-nOVISplrln12 K N L R R I S R K I I I K K YG
I H
E7-novispirin13 K N L R R I E R K I I I K K YG
I H
D7-nOVISplrln14 K N L R R I D R K I I I K K YG
I H
DA7-nOVISplrln15 K N L R R I dAR K I I I K K YG
I H
D17-noviSpirin16 K N L R R I dIR K I I I K K YG
I H
G10-nOVISplrln17 K N L R R I I R K I I I K K YG
G H
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T10-nOVISpirln18 K N L RR I I R K T I H II K K Y G
S10-nOVISplrln19 K N L RR I I R K S I H II K K Y G
E10-nOVISplrln20 K N L RR I I R K E I H II K K Y G
D10-nOVISplfln21 K N L RR I I R K D I H II K K Y G
DA10-novispirin22 K N L RR I I R K dA H II K K Y G
I
DI10-nOVISplrln23 K N L RR I I R K dII H II K K Y G
G11-novispirin24 K N L RR I I R K I G H II K K Y G
T11-nOVISplrln25 K N L RR I I R K I T H II K K Y G
S11-novispirin26 K N L RR I I R K I S H II K K Y G
E11-nOVISplrln27 K N L RR I I R K I E H I'I K K Y G
D11-noVispirin28 K N L RR I I R K I D H II K K Y G
DA11-nOVISplrln29 K N L RR I I R K I dA H II K K Y G
D111-novispirin3o K N L RR I I R K T dI H II K K Y G
G10-nOVISplrln31 K N L RR I I R K G I I IK K Y G-CONHz
amide H
G10, R12- 32 K N L RR I I R K G I I IK K Y G
novis irin R
G10, R12- 33 K N L RR I I R K G I I IK K Y G-CONHz
novis irin R
R2,G10- 34 K R L RR I I R K G I I IK K Y G
novis irin H
R2, G10- 35 K R L RR I I R K G I I IK K Y G-CONHZ
novis irin H
amide
R1, R2, G10- 36 R R L RR I I R K G I I IK K Y G
novis irin R
R1, R2, G10- 37 R R L RR I I R K G I I IK K Y G-CONHz
novis irin R
amide
The sequence of the novispirin polypeptides may also be altered in various
ways known
in the art to generate targeted changes in sequence. The polypeptide will
usually be
substantially similar to the sequences provided herein, i. e. will differ by
one amino acid, and may
differ by two amino acids. The sequence changes may be substitutions,
insertions or deletions.
The protein may be joined to a wide variety of other oligopeptides or proteins
for a
variety of purposes. By providing for expression of the subject peptides,
various post-
translational modifications may be achieved. For example, by employing the
appropriate coding
sequences, one may provide farnesylation or prenylation. In this situation,
the peptide will be
bound to a lipid group at a terminus, so as to be able to be bound to a lipid
membrane, such as
a liposome. In another example, the carboxy terminus of the peptide is
amidated, thereby
increasing the positive charge of the peptide.
The novispirins for use in the subject methods may be produced from eukaryotic
or
prokaryotic cells by recombinant methods, or may be synthesized in vitro as
known in the art.
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In one embodiment of the invention, the antimicrobial peptide consists
essentially of the
polypeptide sequence set forth in any one of SEQ ID N0:1, or SEQ ID N0:3 to
SEQ ID N0:30.
By "consisting essentially of in the context of a polypeptide described
herein, it is meant that
the polypeptide is composed of the sequence set forth in the seqlist, which
sequence may be
flanked by one or more amino acid or other residues that do not materially
affect the basic
characteristics) of the polypeptide.
Methods of Use
Formulations of novispirins are administered to a host suffering from or
predisposed to a
microbial infection. Administration may be topical, localized or systemic,
depending on the
specific microorganism, preferably it will be localized. Generally the dose of
novispirin will be
sufficient to decrease the microbial population by at least about 50%, usually
by at least 1 log,
and may be by 2 or more logs of killing. The compounds of the present
invention are
administered at a dosage that reduces the microbial population while
minimizing any
side-effects. It is contemplated that the composition will be obtained and
used under the
guidance of a physician for in vivo use. Novispirins are particularly useful
for killing gram
negative bacteria, including Pseudomonas aeruginosa, and Chlamydia
trachomatis.
Novispirins are also useful for in vitro formulations to kill microbes,
particularly where
one does not wish to introduce quantities of conventional antibiotics. For
example, novispirins
may be added to animal and/or human food preparations. Novispirins may be
included as an
additive for in vitro cultures of cells, to prevent the overgrowth of microbes
in tissue culture.
The susceptibility of a particular microbe to killing with novispirins may be
determined by
in vitro testing, as detailed in the experimental section. Typically a culture
of the microbe is
combined with novispirins at varying concentrations for a period of time
sufficient to allow the
protein to act, usually between about one hour and one day. The viable
microbes are then
counted, and the level of killing determined.
Microbes of interest, include, but are not limited to Gram-negative bacteria,
for example:
Citrobacter sp.; Enterobacter sp.; Escherichia sp., e.g. E. coli; Klebsiella
sp.; Morganella sp.;
Proteus sp.; Providencia sp.; Salmonella sp., e.g. S. typhi, S. typhimurium;
Serratia sp.;
Shigella sp.; Pseudomonas sp., e.g. P. aeruginosa; Yersinia sp., e.g. Y.
pestis,
Y, pseudotuberculosis, Y enterocolitica; Franciscella sp.; Pasturella sp.;
Vibrio sp., e.g.
V. cholerae, V. parahemolyticus; Campylobacter sp., e.g. C. jejuni;
Haemophilus sp., e.g. H.
influenzae, H. ducreyi; Bordetella sp., e.g. B. pertussis, 8. bronchiseptica,
B. parapertussis;
Brucella sp., Neisseria sp., e.g. N. gonorrhoeae, N. meningitidis, etc. Other
bacteria of interest
include Legionella sp., e.g. L., pneumophila; Listeria sp., e.g. L.
monocytogenes; Mycoplasma
sp., e.g. M. hominis, M. pneumoniae; Mycobacterium sp., e.g. M. tuberculosis,
M. leprae;
Treponema sp., e.g. T. pallidum; Borrelia sp., e.g. B. burgdorferi;
Leptospirae sp.; Rickeftsia sp.,
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e.g. R. rickettsii, R. typhi; Chlamydia sp., e.g. C. trachomatis, C.
pneumoniae, C. psittaci;
Helicobacter sp., e.g. H. pylori, etc.
Non bacterial pathogens of interest include fungal and protozoan pathogens,
e.g.
Plasmodia sp., e.g. P, falciparum, Trypanosoma sp., e.g. T. brucei;
shistosomes; Entaemoeba
sp., Cryptococcus sp., Candida sp, e.g. C. albicans; etc.
Various methods for administration may be employed. The polypeptide
formulation may
be given orally, or may be injected intravascularly, subcutaneously,
peritoneally, by aerosol,
opthalmically, intra-bladder, topically, etc. For example, methods of
administration by inhalation
are well-known in the art. The dosage of the therapeutic formulation will vary
widely, depending
on the specific novispirin to be administered, the nature of the disease, the
frequency of
administration, the manner of administration, the clearance of the agent from
the host, and the
like. The initial dose may be larger, followed by smaller maintenance doses.
The dose may be
administered as infrequently as weekly or biweekly, or fractionated into
smaller doses and
administered once or several times daily, semi-weekly, etc. to maintain an
effective dosage
level. In many cases, oral administration will require a higher dose than if
administered
intravenously. The amide bonds, as well as the amino and carboxy termini, may
be modified for
greater stability on oral administration. For example, the carboxy terminus
may be amidated.
Formulations
The compounds of this invention can be incorporated into a variety of
formulations for
therapeutic administration. More particularly, the compounds of the present
invention can be
formulated into pharmaceutical compositions by combination with appropriate,
pharmaceutically
acceptable carriers or diluents, and may be formulated into preparations in
solid, semi-solid,
liquid or gaseous forms, such as tablets, capsules, powders, granules,
ointments, creams,
foams, solutions, suppositories, injections, inhalants, gels, microspheres,
lotions, and aerosols.
As such, administration of the compounds can be achieved in various ways,
including oral,
buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal,
intracheal, etc.,
administration. The novispirins may be systemic after administration or may be
localized by the
use of an implant or other formulation that acts to retain the active dose at
the site of
implantafiion.
In one embodiment, a formulation for topical use comprises a chelating agent
that
decreases the effective concentration of divalent cations, particularly
calcium and magnesium.
For example, agents such as citrate, EGTA or EDTA may be included, where
citrate is
preferred. The concentration of citrate will usually be from about 1 to 10 mM.
The compounds of the present invention can be administered alone, in
combination with
each other, or they can be used in combination with other known compounds
(e.g., perforin,
anti-inflammatory agents, antibiotics, etc.) In pharmaceutical dosage forms,
the compounds
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may be administered in the form of their pharmaceutically acceptable salts.
The following
methods and excipients are merely exemplary and are in no way limiting.
For oral preparations, the compounds can be used alone or in combination with
appropriate additives to make tablets, powders, granules or capsules, for
example, with
conventional additives, such as lactose, mannitol, corn starch or potato
starch; with binders,
such as crystalline cellulose, cellulose derivatives, acacia, corn starch or
gelatins; with
disintegrators, such as corn starch, potato starch or sodium
carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired, with diluents,
buffering agents,
moistening agents, preservatives and flavoring agents.
The compounds can be formulated into preparations for injections by
dissolving,
suspending or emulsifying them in an aqueous or nonaqueous solvent, such as
vegetable or
other similar oils, synthetic aliphatic acid glycerides, esters of higher
aliphatic acids or propylene
glycol; and if desired, with conventional additives such as solubilizers,
isotonic agents,
suspending agents, emulsifying agents, stabilizers and preservatives.
The compounds can be utilized in aerosol formulation to be administered via
inhalation.
The compounds of the present invention can be formulated into pressurized
acceptable
propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
The compounds can be used as lotions, for example to prevent infection of
burns, by
formulation with conventional additives such as solubilizers, isotonic agents,
suspending
agents, emulsifying agents, stabilizers and preservatives.
Furthermore, the compounds can be made into suppositories by mixing with a
variety of
bases such as emulsifying bases or water-soluble bases. The compounds of the
present
invention can be administered rectally via a suppository. The suppository can
include vehicles
such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body
temperature,
yet are solidified at room temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs,
and
suspensions may be provided wherein each dosage unit, for example,
teaspoonful,
tablespoonful, tablet or suppository, contains a predetermined amount of the
composition
containing one or more compounds of the present invention. Similarly, unit
dosage forms for
injection or intravenous administration may comprise the compound of the
present invention in a
composition as a solution in sterile water, normal saline or another
pharmaceutically acceptable
carrier.
Implants for sustained release formulations are well-known in the art.
Implants are
formulated as microspheres, slabs, etc. with biodegradable or non-
biodegradable polymers.
For example, polymers of lactic acid and/or glycolic acid form an erodible
polymer that is well-
tolerated by the host. The implant containing novispirins is placed in
proximity to the site of
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infection, so that the local concentration of active agent is increased
relative to the rest of the
body.
The term "unit dosage form", as used herein, refers to physically discrete
units suitable
as unitary dosages for human and animal subjects, each unit containing a
predetermined
quantify of compounds of the present invention calculated in an amount
sufficient to produce the
desired effect in association with a pharmaceutically acceptable diluent,
carrier or vehicle. The
specifications for the unit dosage forms of the present invention depend on
the particular
compound employed and the effect to be achieved, and the pharmacodynamics
associated with
the compound in the host.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants,
carriers or
diluents, are readily available to the public. Moreover, pharmaceutically
acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity adjusting
agents, stabilizers,
wetting agents and the like, are readily available to the public.
Typical dosages for systemic administration range from 0.1 ~.g to 100
milligrams per kg
weight of subject per administration. A typical dosage may be one tablet taken
from two to six
times daily, or one time-release capsule or tablet taken once a day and
containing a
proportionally higher content of active ingredient. The time-release effect
may be obtained by
capsule materials that dissolve at different pH values, by capsules that
release slowly by
osmotic pressure, or by any other known means of controlled release.
Those of skill will readily appreciate that dose levels can vary as a function
of the
specific compound, the severity of the symptoms and the susceptibility of the
subject to side
effects. Some of the specific compounds are more potent than others. Preferred
dosages for a
given compound are readily determinable by those of skill in the art by a
variety of means. A
preferred means is to measure the physiological potency of a given compound.
The use of liposomes as a delivery vehicle is one method of interest. The
liposomes
fuse with the cells of the target site and deliver the contents of the lumen
intracellularly. The
liposomes are maintained in contact with the cells for sufficient time for
fusion, using various
means to maintain contact, such as isolation, binding agents, and the like. In
one aspect of the
invention, liposomes are designed to be aerosolized for pulmonary
administration. Liposomes
may be prepared with purified proteins or peptides that mediate fusion of
membranes, such as
Sendai virus or influenza virus, etc. The lipids may be any useful combination
of known
liposome forming lipids, including cationic or zwitterionic lipids, such as
phosphatidylcholine.
The remaining lipid will be normally be neutral or acidic lipids, such as
cholesterol, phosphatidyl
serine, phosphatidyl glycerol, and the like.
For preparing the liposomes, the procedure described by Kato et al. (1991 ) J.
Biol.
Chem. 266:3361 may be used. Briefly, the lipids and lumen composition
containing peptides
are combined in an appropriate aqueous medium, conveniently a saline medium
where the total
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solids will be in the range of about 1-10 weight percent. After intense
agitation for short periods
of time, from about 5-60 sec., the tube is placed in a warm water bath, from
about 25-40° C and
this cycle repeated from about 5-10 times. The composition is then sonicated
for a convenient
period of time, generally from about 1-10 sec. and may be further agitated by
vortexing. The
volume is then expanded by adding aqueous medium, generally increasing the
volume by about
from 1-2 fold, followed by shaking and cooling. This method allows forthe
incorporation into the
lumen of high molecular weight molecules.
Formulations with Other Active Agents
For use in the subject methods, novispirins may be formulated with other
pharmaceutically active agents, particularly other antimicrobial agents. Other
agents of interest
include a wide variety of antibiotics, as known in the art. Classes of
antibiotics include
penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin,
carbenicillin, nafcillin, ampicillin,
etc.; penicillins in combination with (3-lactamase inhibitors, cephalosporins,
e.g. cefaclor,
cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams;
aminoglycosides;
tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones;
cloramphenical;
metronidazole; spectinomycin; trimethoprim; vancomycin; etc.
Anti-mycotic agents are also useful, including polyenes, e.g. amphotericin B,
nystatin; 5-
flucosyn; and azoles, e.g. miconazol, ketoconazol, itraconazol and fluconazol.
Antituberculotic
drugs include isoniazid, ethambutol, streptomycin and rifampin. Cytokines may
also be included
in a novispirins formulation, e.g. interferon y, tumor necrosis factor a,
interleukin 12, etc.
Synthesis of Novispirin
The subject peptides may be prepared by in vitro synthesis, using conventional
methods
as known in the art. Various commercial synthetic apparatuses are available,
for example
automated synthesizers by Applied Biosystems Inc., Foster City, CA, Beckman,
etc. By using
synthesizers, naturally occurring amino acids may be substituted with
unnatural amino acids,
particularly D isomers, e.g. D-alanine and Disoleucine, diastereoisomers, side
chains having
different lengths or functionalities, and the like. The particular sequence
and the manner of
preparation will be determined by convenience, economics, purity required, and
the like.
Chemical linking may be provided to various peptides or proteins comprising
convenient
functionalities for bonding, such as amino groups for amide or substituted
amine formation, e.g.
reductive amination, thiol groups for thioether or disulfide formation,
carboxyl groups for amide
formation, and the like.
If desired, various groups may be introduced into the peptide during synthesis
or during
expression, which allow for linking to other molecules or to a surface. Thus
cysteines can be
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used to make thioethers, histidines for linking to a metal ion complex,
carboxyl groups for
forming amides or esters, amino groups for forming amides, and the like.
The polypeptides may also be isolated and purified in accordance with
conventional
methods of recombinant synthesis. A lysate may be prepared of the expression
host and the
lysate purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity
chromatography, or other purification technique. For the most part, the
compositions which are
used will comprise at least 20% by weight of the desired product, more usually
at least about
75% by weight, preferably at least about 95% by weight, and for therapeutic
purposes, usually
at least about 99.5% by weight, in relation to contaminants related to the
method of preparation
of the product and its purification. Usually, the percentages will be based
upon total protein.
EXPERIMENTAL
The following examples are put forth so as to provide those of ordinary skill
in the art
with a complete disclosure and description of how to make and use the subject
invention, and
are not intended to limit the scope of what is regarded as the invention.
Efforts have been
made to ensure accuracy with respect to the numbers used (e.g. amounts,
temperature,
concentrations, etc.) but some experimental errors and deviations should be
allowed for.
Unless otherwise indicated, parts are parts by weight, molecular weight is
average molecular
weight, temperature is in degrees centigrade; and pressure is at or near
atmospheric.
Example 1
METHODS
Antimicrobial activity. Several different methods were used to examine the
antimicrobial
properties of novispirins, including: a) two-stage radial diffusion assays; b)
colony counting
assays; and c) standard microbroth dilution assays.
Antimicrobial activity was tested in media with low, normal or high salinity.
In radial
diffusion assays, this was accomplished by using a low salt "basic medium"
containing 0.3mg of
trypticase soy broth powder/ml of 10 mM sodium phosphate buffer, pH 7.4. Media
with "normal"
or "high" salinity were prepared by supplementing the basic medium with 100 mM
NaCI to obtain
media with normal salinity, or with 175-200 mM NaCI to obtain high salt media.
Retention of
activity against P. aeruginosa in high salt media is important because airway
fluids from cystic
fibrosis patients are reported to show hypersalinity, and other sites -
including skin surfaces and
the urinary bladder -may have locally high salt concentrations.
Microorganisms used in this study included 13 different Pseudomonas aeruginosa
strains. The AML-654 and LZ-1 strains are recent clinical isolates from the
UCLA Clinical
Microbiology laboratory. Nine strains (5 mucoid, 4 nonmucoid) were recent CF
isolates
provided by Dr. Lisa Saiman. All of the P. aeruginosa strains except PAO-1
strain were
resistant to multiple conventional antibiotics, and all 9 CF isolates were
resistant to 250 pg/ml of
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tobramycin. We have also tested 5 strains each of Stenotrophomonas maltophilia
and
Burkholderia cepacia from CF patients (also provided by Dr. Lisa Saiman), two
strains of
Escherichia coli (ML-35p and DH-5a), and single strains of Staphylococcus
aureus [MRSA
(methicillin-resistant S. aureus)] and C. albicans.
Cytotoxicity: Atetrazolium reduction assay (Boehringer-Mannheim, Indianapolis
IN) was
used to study cytotoxicity. ME-180 human cervical epithelial cells (ATCC HTB-
33) and A549
human lung epithelial cells (ATCC CCL-185) were grown to confluency in RPMI
Medium (ME-
180) or Ham's F12K medium with 2 mM L-glutamine (A-549) containing 10% fetal
bovine
serum, 2 mM L-glutamine and 50pg/mL gentamicin ("medium"). The cells were
harvested with
trypsin-EDTA, washed with medium, and after their concentration and viability
(trypan blue
exclusion) was determined, they were diluted to 5 x 104 cells/mL. Aliquots
(100 p1) were
dispensed into 96 well tissue culture plates (Costar) and incubated for 5 hr.
at 37 °C in room air
with 5% CO~. Then, serially diluted peptides were added and after 20 hr. of
additional
incubation, 10 p1 of MTT solution was added. Four hours later, 100 p1 of
solubilization buffer
was added and left overnight. The following day, MTT reduction was determined
by optical
density measurements at 600 and 650 nm, using a Spectramax 250
Spectrophotometer
(Molecular Devices, Sunnyvale, CA).
Hemolytic Activity: This was tested by incubating various concentrations of
peptide with
a suspension (2.8% v/v) of washed human, murine, sheep or bovine red cells in
PBS. After 30
min at 37C, the tubes were centrifuged and the percentage of total hemoglobin
released to the
supernatant was measured.
RESULTS
Antimicrobial Properties. Ovispirin and novispirins manifested potent
antimicrobial
activity against Gram-negative bacteria, including 13/13 strains of
Pseudomonas aeruginosa.
Eleven of these strains, including 9 (5 mucoid, 4 nonmucoid) from patients
with cystic fibrosis,
were recent clinical isolates. All of the CF strains were resistant to 250
pg/ml of tobramycin, and
most were highly resistant to multiple additional antibiotics. Novispirins
were also active against
5/5 strains of Stenotrophomonas maltophilia, an emerging pathogen in cystic
fibrosis that is
often resistant to conventional antibiotics. Like many other antibiotics and
antimicrobial
peptides, novispirins were inactive against Burkholderia cepacia.
Novispirin-mediated bactericidal activity occurred within 5-15 minutes, most
likely from
permeabilization of the inner and outer membranes of the bacteria.
Antimicrobial activity was
maintained under high salt (175-200 mM NaCI) conditions and was minimally
impacted by the
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presence of 20% serum. Novispirins were equally effective against growing or
quiescent P.
aeruginosa.
The relative activities of ovispirin and the novispirins against two
organisms, E. coli and
a multi-resistant strain of P. aeruginosa, are shown in Figure 1. The MICs
were obtained by
radial diffusion assays performed under low, normal, and high salt conditions
(which are
denoted along the X-axis as 0, 100 and 200 mM NaCI, respectively). The bars
show mean MIC
values ~ SEM (n=3). It can be seen that novispirins were more active than
ovispirin when
tested under conditions of low and normal salinity, and about equally active
under conditions of
high salinity.
Table 1 compares the activity of novispirins against these Gram-negative
bacteria with
its activity against Listeria monocytogenes, Staphylococcus aureus and C.
albicans. The
tabulated data derive from radial diffusion assays and show (in pg/ml) the
mean MIC ~ SEM,
n=3. Note that novispirins show activity against these Gram-positive bacteria
when tested in
physiological (100 mM NaCI) salt concentrations, and also that Novispirin G10
is quite active
against C, albicans. Although neither ovispirin nor novispirins retained
activity against C.
albicans or the Gram positives under very high salt conditions (200 mM NaCI),
they remained
active against the Gram-negatives under these conditions. Consequently, even
if the airways'
NaCI concentrations are exceptionally high in CF patients, this factor will
not impair the ability of
ovispirin or novispirins to 4eill P. aeruginosa.
TABLE 1.
MIC( pg/mL)
against
5 different
organisms,
[Mean SEM,
n=3]
Peptide E. coli P. aerugin.L. mono. S. aureus C. albicans
ML 35P MR 3007 EGD 930918-3 820
Low Salt M NaCI)
(+ 0 m
Ovispirin 0.7 0.0 2.7 1.1 2.0 0.5 2.1 0.3 2.2 0.2
G~Novispirin0.20.0 0.60.2 0.40.1 0.60.2 0.50.1
Goo Novispirin0.1 0.0 0.30.0 0.20.0 0.30.0 0.20.1
T~Novispirin0.30.1 1.30.6 0.50.2 0.70.3 0.50.2
T~oNovispirin0.50.2 1.50.9 0.60.3 1.00.6 0.80.4
Normal salinity
(+ 100 mM
NaCI)
Ovispirin 0.70.1 1.70.6 1.50.3 1.00.1 29.915.4
G~ Novispirin0.2 0.0 0.4 0.1 2.3 0.5 5.1 1.1 9.3 4.9
Goo Novispirin0.1 0.0 0.2 0.0 1.4 0.4 4.6 1.7 4.6 1.9
T~Novispirin0.20.1 0.50.3 2.41.5 3.31.5 18.65.9
T~oNovispirin0.30.2 0.90.5 5.54.0 9.74.6 23.03.9
Very high
salinity
(+ 200 mM
NaCI)
Ovispirin 1.5 0.6 2.7 0.1 3.3 0.1 2.9 0.7 >250
G~ Novispirin1.5 0.4 4.5 1.9 28.2 0.7 39.6 9.2 >250
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Goo Novispirin1.8 0.8 2.5 0.5 9.3 1.3 75.0 51.5 205
22.6
T~ Novispirin1.4 0.5 2.7 1.3 21.5 2.0 14.9 2.9 >250
T~ Novispirin2.7 1.1 3.9 2.1 26.8 1.6 75.3 56.2 >250
TABLE 2. Microbroth dilution assay P. aeruginosa PA01 (pg/ml, mean values,
n=3)
Peptide MIC MBC
mM NaCI 0 100 175 0 100 175
Ovispirin 3.1 3.1 4.7 4.7 7.1 9.4
G~ Novispirin 1.6 6.8 4.4 2.6 14.1 8.3
Gyp Novispirin 2.6 16.1 11.3 6.8 16.7 29.2
T~ Novispirin 2.1 3.9 5.7 7.3 6.3 12.5
Tao Novispirin 2.8 5.7 2.8 6.8 9.4 9.4
Protegrin PG1 2.1 2.6 2.6 3.2 2.7 3.5
P. aeruginosa within the airways of patients with cystic fibrosis can differ
in many ways
from free-living planktonic members of the species. Among these differences
are: their
entrapment in an alginate-rich biofilm, selection for auxotrophy and
antibiotic resistance, various
modifications to their lipid and LPS structures, and metabolism characteristic
of high density,
nutrient-limited cultures. Many conventional antibiotics (e.g., inhibitors of
cell wall or protein
synthesis) and many antimicrobial peptides act preferentially against rapidly
growing organisms
and might therefor be relatively inactive against high mass/low turnover
populations of bacteria.
We therefor tested the activity of ovispirin and novispirins against 3 strains
of P. aeruginosa,
PAO-1 , Liz-1 and AML654 to see if the peptides could kill stationary phase or
nutritionally-
starved organisms as well as rapidly growing ones. All three strains gave
equivalent results.
Table 3 shows data for PAO-1 and Liz-1 strains. Note that neither the growth
phase nor the
presence or absence of nutrients affected the susceptibility of P, aeruginosa
to novispirins.
TABLE 3
Activity of peptides against growing and stationary phase P. aeruginosa (2
strains), tested with
and without the presence of nutrients (TSB) in the underlay gel
MIC (pg/mL)
P. aeruginosa PA01/ P aeruginosa Liz-1
Peptide Mid-log Mid-log Stationary Stationary
1 % TSB No TSB 1 % TSB no TSB
Low Salt
Ovispirin 0.6 / 1.8 1.3/ 1.1 1.8 / 1.9 0.9 / 2.0
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G7 novispirin0.4 / 0.4 0.3 / 0.6 0.4 /0.3 0.3 /
0.6
G10 novispirin0.3 / 0.3 0.1 / 0.4 0.3/ 0.1 0.2 /
0.4
T7 novispirin0.4 / 0.6 0.5 / 0.6 0.6 /0.4 0.5 /
0.7
T10 novispirin0.6 / 0.7 0.6 / 1.0 0.9 /0.9 0.6 /
0.9
100 mM NaCI
Ovispirin 0.9 / 1.6 0.9 / 1.4 1.5 / 1.5 0.9 /
2.1
G7 novispirin0.2 / 0.2 0.5 / 0.6 0.1 / 0.2 0.5 /
1.2
G10 novispirin0.1 / 0.2 0.8 / 1.3 0.1 / 0.1 0.5 /
2.0
T7novispirin 0.3/0.3 0.6/0.4 0.4/0.4 0.5/0.7
T10 novispirin0.3 / 0.5 0.9 / 0.6 0.5 l 0.5 0.8 /
1.4
175 mM NaCI
Ovispirin 1.2/0.9 0.9/0.7 1.1 /1.2 0.8/0.8
G7 novispirin1.2 l 2.1 0.2 / 0.1 1.0 / 2.9 0.1 l
0.1
G10 novispirin3.6 / 5.9 0.1 / 0.1 4.7 / 5.9 0.1 /
2.3
T7 novispirin0.8 / 2.0 0.3 / 0.1 0.8 / 2.0 0.2 /
0.6
T10novispirin2.3/3.4 0.3/0.2 1.9/3.0 0.4/0.8
Effect ofserum. Human defensins and LL-37 (an a-helical human cathelicidin
peptide)
bind extensively to serum molecules. Consequently, their antimicrobial
activity is greatly
reduced if serum is present. Because serum can be present in an inflammatory
focus or
exudate, the ability to function in the presence of serum would be a desirable
property. We
recently found that SMAP-29, an a-helical cathelicidin found in sheep, retains
its antimicrobial
activity in the presence of serum, proving that serum inhibition is not an
inevitability but a
question of design. Table 4 shows the effect of serum on the activity (M1C) of
novispirins, LL-
37, SMAP-29 and other peptides against P. aeruginosa MR3007 (a serum-resistant
strain) and
E. coli ML-35P (sensitive to >5% serum). Ovispirin and novispirins lost some
activity against P.
aeruginosa in the presence of serum, but were affected much less than LL-37.
Table 4
Effect of normal human serum on antimicrobial activity
MIC (pg/mL)
Peptide E. coli P. aeruginosa MR
ML 35P 3007
0% NHS 2.5% NHS 0% NHS 2.5% NHS 20% NHS
Ovispirin 1.7 1.7 5.6 20.1 17.6
G7 Novispirin0.5 0.4 7.5 21.5 17.7
G10 Novispirin0.9 0.7 7.6 22.1 21.7
T7 Novispirin1.0 0.7 7.5 25.2 22.0
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T10 Novispirin 1.0 0.7 8.6 28.6 17.6
PG-1 (protegrin) 0.5 0.3 1.6 0.91 1.6
PGG 1.8 0.9 5.8 7.41 8.6
SMAP-29 0.6 0.3 1.4 1.5 1.2
LL-37 5.8 21.0 12.7 23.7 141
In these experiments, the underlay gel contained 60% normal
RPMI-1640, 2.5 or 20% human
serum (NHS). The remainder of the solution was phosphate-buffered
saline
Kinetics of Microbicidal activity. We measured the survival of P. aeruginosa
MR3007 at
intervals after it was exposed to 5 pg/ml of ovispirin or novispirins in a PBS
medium that
contained 1 % v/v trypticase soy broth. Figure 2 shows a rapid 2-3 logo fall
and that no
surviving bacteria were recovered by 30 minutes or thereafter.
Figure 3 shows the effect of ovispirin or novispirins in lysing red blood
cells. Although
ovispirin was about as hemolytic for human red blood cells as protegrin PG-1,
the novispirins
were non hemolytic. Similar results were obtained with murine red blood cells.
In contrast, none
of these peptides were appreciably hemolytic towards sheep or bovine red blood
cells.
Cytotoxic properties. Novispirins , especially novispirin G10, were markedly
less toxic
towards human epithelial cells or fibroblasts than ovispirin or other
antimicrobial peptides
previously studied, e.g. protegrin PG-1, magainin MSI-78, or PGG.
Representative assays for
cytotoxicity against ME-180 human cervical epithelial cells and A-549
pulmonary epithelial cells
are shown in Figure 4.
Figure 5 compares the cytotoxicity of these peptides for MRC-5 human lung
fibroblasts.
The G10 and T10 novispirins were virtually devoid of cytotoxicity,
demonstrating that they would
be unlikely to retard tissue repair or wound healing.
In vivo toxicity. Acute toxicity studies were performed in mice. 3/3 mice
survived an
intravenous dose of 10 mg novispirin G10 / kg body weight, and 1/3 survived a
dose of 20
mg/kg. If the blood volume of a mouse constitutes 5% of its mass, then the 10
mg/kg dose
would yield a peak level of approximately 200 micrograms/ml - some 20 to 50
fold higher than
its MIC concentrations.
LPS Binding. We measured the ability of ovispirin and novispirins to bind LPS
in two
assays. One assay is spectrophotomentric, and uses a quantitative Limulus
chromogenic assay.
The other assay is physical, and measures surface plasmon resonance changes.
The apparent binding constant of ovispirin for LPS was approximately 6.7 x 10-
6M. When
the binding assays were done under low salt conditions (0 mM NaCI), the
affinity of novispirins
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WO 02/00839 PCT/USO1/19094
was lower by a factor of 4-5, i.e., between 2.5 and 3.3 x 10'5M. When we
tested binding in
physiological NaCI, the affinity of novispirins for LPS increased.
Effects of novispirins on bacterial membranes. Alf of the novispirins, when
tested at 5
pg/ml, rapidly permeabilized the outer and inner membranes of E. coli M-35p.
The onset of
membrane permeabilization is rapid, beginning within 2 minutes and reaching
maximal extent
within 5 minutes. This experiment was performed with stationary phase E. coli,
suspended in
PBS (100 mM NaCI). Outer membrane permeabilization was measured by monitoring
the
hydrolysis of PADAC, a cephalosporin, and inner membrane permeabilization was
measured by
monitoring hydrolysis of ONPG (this E. coli strain has no lac permease.
Table 5 shows the activity of novispirins against S. maltophila.
Table 5
Novisoirins vs S. maltonf~ilia
LOW SALT CONDITIONS 51 47CG 18CP 42CK 36CN
CI
Protegrin PG-1 0.42 0.09 0.52 0.32 0.27
PGG 1.31 0.73 0.86 1.51 1.10
Polymyxin B 0.02 0.16 0.04 0.05 0.06
Ovispirin 1.60 0.07 1.46 1.73 1.01
G7 novispirin 0.54 0.25 0.61 0.42 0.17
G10 novispirin 0.45 0.08 0.40 0.43 0.15
T7 novispirin 0.88 0.18 0.74 0.56 0.21
T10 novispirin 0.97 0.21 0.88 0.77 0.32
+ 100 mM NaCI 51 47CG 18CP 42CK 36CN
CI
Protegrin PG-1 0.92 0.53 0.58 0.50 0.46
PGG 2.20 1.08 1.63 2.67 1.05
Polymxin B 0.04 0.08 0.04 0.02 0.02
Ovispirin 2.20 0.54 0.88 1.24 0.93
G7 nov. 0.89 0.39 0.38 0.92 0.31
G10 nov. 3.57 0.68 0.70 0.67 0.29
T7 nov. 1.75 0.47 0.58 0.73 0.32
T10 nov. 5.02 0.75 0.88 1.27 0.48
+ 175 mM NaCI 51 47CG 18CP 42CK 36CN
CI
Protegrin PG-1 1.08 0.74 0.70 0.79 0.57
j PGG 2.85 1.64 2.34 7.10 1.11
Polymyxin B 0.05 0.05 0.002 0.01 0.02
Ovispirin 1.67 0.77 0.91 2.03 0.91
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G7 nov. 1.94 0.29 0.70 2.85 0.29
G10 nov. 8.18 0.63 1.30 10.0 0.36
T7 nov. 2.74 0.48 0.62 1.92 0.52
T10 nov. 15.5 0.70 2.08 1.25 0.53
Protegrin PG-1, polymyxin B and PGG were used as controls. PGG is an a-helical
peptide, whose
sequence is: GLLRRLRKKIGEIFKKYG. It was designed and reported by Tossi, A.,
Tarantino, C. and
Romeo, D. 1997. Design of synthetic antimicrobial peptides based on sequence
analogy and
amphipathicity. Eur. J. Biochem. 250: 549-58.
Example 2
Susceptibility of Chlamydia trachomatis Serovars L2, D, and E to G-10
Novispirin
The susceptibility of three Chlamydia trachomatis serovars (L2, D and E) to
novispirin
(G-10), a novel 18-residue a-helical peptide was assayed in this study. G-10
is nonhemolytic
and has minimal cytotoxicity when tested against epithelial cells in vitro. It
is relatively selective
for Gram-negative organisms.
C. trachomatis serovar L2 causes invasive LGV, whereas serovars D and E
represent
typical genital strains. The effect of G-10 on Chlamydia trachomatis serovars
(L2, D and E) was
determined using standard and modified McCoy cell shell vial assays and
varying
concentrations of peptide. In the standard assay, the Chlamydia/peptide
mixture was pre
incubated for 2 hr., then removed from the host cell monolayer before the 48-
hr. incubation. In
the modified assay, the pre-incubation mixture was left on the monolayer for
the 48 hr.
incubation. In a third assay, G-10 and Chlamydia were mixed and infected to
cell monolayers
without pre-incubation, and examined after 48 hr. Inclusion-forming units
(IFUs) were scored to
measure the peptide's inhibitory effect.
G-10 completely inhibited C. trachomatis serovar E at 100 wg/ml in the pre-
incubation
assays. At 100 mg/ml, serovar L2 IFUs were reduced by 92.7-99.1 % and serovar
D IFUs were
reduced 99.4-100%. In general, omission of the pre-incubation step reduced
susceptibility of
Chlamydia to novispirin. Lack of removal of Chlamydia/peptide mixes did not
increase or
decrease susceptibility to G-10 significantly. G-10 is even more effective
than protegrins,
antimicrobial peptides found in porcine leukocytes that have excellent
activity against Chlamydia
trachomatis. In addition, all 3 serovars tested were susceptible to G-10.
Novispirin is therefore
shown to have utility as a chemoprophylactic agent to prevent chlamydial
infections.
Example 3
Effect of Divalent Cations on Novispirin Activity
The activity of G10 novispirin against certain bacteria is inhibited by
approximately 1 mM
concentrations of calcium and magnesium. This inhibition can be relieved by
adding a citrate-
containing buffer. Based on this finding, formulations of novispirins intended
for topical use
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WO 02/00839 PCT/USO1/19094
should contain citrate or another divalent cation binder, such as EDTA
(ethylene-diamine
tetraacetic acid). The beneficial effect of a citrate-containing formulation
on the activity of G10
Novispirin are shown below in Table 6.
Experiments which led to this finding involved the use of radial diffusion
assays. All
underlay gels contained 100 mM NaCI and 5 mM HEPES buffer (pH 7.4). In
addition, some
underlay gels were supplemented with 5 mM sodium citrate buffer, pH 7.4, while
others
contained 1 mM CaCl2 or 1 mM MgCl2. The numbers represent MIC values in pg/ml.
Note that
the enhancing effect of citrate was more prominent with Gram negative
organisms than with S.
aureus. EDTA, which is an even more effective chelator of calcium and
magnesium, is
expected to have the same effect as citrate.
Table 6
Organism Citrate No Ca/Mg +1 mM Ca + 1 mM
Mg
K. pneumoniae None 0.12 116.0 8.37
K. pneumoniae 5 mM 0.23 3.1 0.56
P. aeruginosa None 0.18 22.7 1.35
P. aeruginosa 5 mM 0.13 0.14 0.14
E. coli None 0.16 9.3 2.56
E. coli 5 mM 0.11 0.12 0.14
S. aureus none 0.46 250.0 7.7
S. aureus 5 mM 8.04 27.0 21.4
Example 4
Three new variants of novispirin G10 were prepared and tested to determine if
increasing the net positive charge of the peptide renders it more effective in
the presence of
divalent cations. Amidation (-NH2) of the carboxy terminus in some of the
novispirin G10
variants was designed to increase overall positive charge. The sequences of
these peptides
and their net charge is shown below in Table 7. The fractional (and pH
dependent) charge on
histidine~~ was ignored in this calculation.
Table 7
Peptide Net SEQ 1D Sequence
char NO
a
Goo Novispirin +7 17 K N L R R I I R K G I H I I
K K Y G-COOH
G,o Novispirin +8 31 K N L R R I I R K G I H I I
K K Y G-coNH~
amide
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Goo, R~2 Novispirin+8 32 K N L R R I I R I I K K Y
I R K G G-cooH
G~oR~2 Novi'n +9 33 K N L R R I I R I I K K Y
amide I R K G G-coNH
Results: MIC, mean t SEM, n=3. Shaded box indicates p <0.01, compared to G10,
by t-test.
Table 8
Organism Peptide No Ca/Mg +1 mM Ca + 1 mM
Mg
S. aureus Ovispirin 0.49 t 6.15 t 0.125.93 t
0.05 0.19
Novispirin G10 22.7 t 99.0 t 5.7 >79
2.1
Novispirin G10 amide2.72 >79 29.9 0.35
0.14
Novispirin G10, R12 23.0 t >79 87.0 t
0.13 5.7
Novispirin G10,R12 2.48 25.4 +_ 27.8 +_
amide 0.03 0.6 0.43
P. aeruginosaOvispirin 2.31 t 22.1 t 1.787.18 t
0.21 0.51
Novispirin G10 0.17 t >250 >250
0.04
Novispirin G10 amide0.12 t 83.5 t 6.4 >250
0.01
Novispirin G10, R12 0.10 t 22.9 1.2 26.9 0.78
0.01
Novispirin G10,R12 0.15 t 24.3 0.15 27.4 0.21
amide 0.04
E. coli Ovispirin 0.8 t 6.97 t 0.426.99 f
0.14 0.11
Novispirin G10 0.09 t 21.2 t 0.3620.3 t
0.02 0.1
Novispirin G10 amide0.06 t 7.26 t 0.0217.2 t
0.00 3.9
Novispirin G10, R12 '0.05 1.71 0.02 2.54 0.13
t 0.0
Novispirin G10, R12 0.06 t 1.69 0.15 2.91 0.29
amide 0.01
K. pneumoniaeOvispirin 2.31 t 22.1 t 1.8 7.18 t
0.02 0.5
Novispirin G10 0.17 t >250 >250
0.03
Novispirin G10 amide0.12 t 83.5 t 6.4 > 250
0.01
Novispirin G10, R12 0.10 t 22.9 1.20 26.9 0.8
0.1
Novispirin G10, R12 0.15 t 24.3 0.15 2.73 0.29
amide 0.04
In Table 8, MIC signifies minimal inhibitory concentration, as determined by
two stage
radial diffusion assays. These were done under standard conditions, except
that 10 mM HEPES
buffer was used in place of 10 mM phosphate to avoid solubility problems when
working with
calcium. Bold boxes show results that represent a significant improvement of
activity (p <0.01
by t-test), relative to novispirin G10. Note that G10, R10 novispirin amide
showed improved
-20-
CA 02415236 2002-12-19
WO 02/00839 PCT/USO1/19094
activity against all four test organisms in the presence of calcium and
magnesium and that G10,
R10 novispirin was more active against the three Gram negative organisms (P.
aeruginosa, E.
coli and K. pneumoniae) but was not more effective against S. aureus. None of
the above G10
novispirin derivatives caused any hemolysis of human red blood cells, even
when tested at 80
pg/ml. It is likely that other cationic amino acids (e.g., lysine, ornithine,
etc) could also be used
in position 12 to achieve better activity in the presence of calcium and
magnesium ions.
Example 5
Four variants of G10 novispirin were synthesized, to see if increasing the
positive charge
of the N-terminus can also increase resistance to calcium and magnesium. Amino
acid
sequence of these G10 variants are shown below in Table 9 with their
respective net charge.
Peptide Net SEQ ID Sequence
NO:
char
a
R2, Goo Novis +8 34 K R L R R I I R K G I H I I K
irin K Y G-cooH
R2, Goo Novispirin+9 35 K R L R R ( I R K G I H ( I K
K Y G-coNH2
amide
R1, R2,G10 +8 36 RRLRRIIRKG IRIIKKYG-cooH
Novis irin
R1, R2,G10 +8 37 RRLRRIIRKG IRIIKKYG-coNH~
Novis 'n amide
All publications and patent applications cited in this specification are
herein incorporated
by reference as if each individual publication or patent application were
specifically and
individually indicated to be incorporated by reference. The citation of any
publication is for its
disclosure prior to the filing date and should not be construed as an
admission that the present
invention is not entitled to antedate such publication by virtue of prior
invention.
Although the foregoing invention has been described in some detail byway of
illustration
and example for purposes of clarity of understanding, it will be readily
apparent to those of
ordinary skill in the art in light of the teachings of this invention that
certain changes and
modifications may be made thereto without departing from the spirit or scope
of the appended
claims.
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SEQUENCE LISTING
<110> Lehrer, Robert I.
blaring, Alan J.
Tack, Brian F.
<120> NOVISPIRINS: ANTIMICROBIAL PEPTIDES
<130> 06510-195W0
<140> Unassigned
<141>
<150> US 09/606,858
<151> 2000-06-28
<160> 37
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<221> VARIANT
<222> (1)...(18)
<223> Xaa = glycine, threonine, serine, glutamic acid,
aspartic acid, D-alanine, D-isoleucine
<400> 1
Lys Asn Leu Arg Arg Xaa Xaa Arg Lys Xaa Xaa His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 2
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 2 '
Lys Asn Leu Arg Arg Ile I1e Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 3
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
_1_
CA 02415236 2002-12-19
WO 02/00839 PCT/USO1/19094
<400> 3
Lys Asn Leu Arg Arg Gly Ile Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 4
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 4
Lys Asn Leu Arg Arg Thr Ile Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 5
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 5
Lys Asn Leu Arg Arg Ser Ile Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 6
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 6
Lys Asn Leu Arg Arg Glu Ile Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 7
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 7
Lys Asn Leu Arg Arg Asp Ile Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 8
<211> 18
_2_
CA 02415236 2002-12-19
WO 02/00839 PCT/USO1/19094
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<223> D-alanine
<400> 8
Lys Asn Leu Arg Arg Ala Ile Arg Lys Ile Ile His Ile I1e Lys Lys
1 5 10 15
Tyr Gly
<210> 9
<211> 18
<212> PRT
<2l3> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<223> D-isoleucine
<400> 9
Lys Asn Leu Arg Arg Tle Ile Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 10
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 10
Lys Asn Leu Arg Arg Ile Gly Arg Lys Ile Ile His Tle Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 11
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 11
Lys Asn Leu Arg Arg Ile Thr Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 12
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
-3-
CA 02415236 2002-12-19
WO 02/00839 PCT/USO1/19094
<223> Synthetic antimicrobial peptide
<400> 12
Lys Asn Leu Arg Arg Ile Ser Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 13
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 13
Lys Asn Leu Arg Arg Ile Glu Arg Lys Ile Ile His Ile I1e Lys Lys
1 5 10 15
Tyr Gly
<210> 14
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 14
Lys Asn Leu Arg Arg Ile Asp Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 , 10 15
Tyr Gly
<210> 15
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<223> D-alanine
<400> 15
Lys Asn Leu Arg Arg Ile Ala Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 16
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<223> D-isoleucine
<400> 16
-4-
CA 02415236 2002-12-19
WO 02/00839 PCT/USO1/19094
Lys Asn Leu Arg Arg Ile Ile Arg Lys I1e Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 17
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 17
Lys Asn Leu Arg Arg Ile Ile Arg Lys Gly Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 18
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 18
Lys Asn Leu Arg Arg Ile Ile Arg Lys Thr Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 19
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 19
Lys Asn Leu Arg Arg Ile Ile Arg Lys Ser Ile His Ile Tle Lys Lys
1 5 10 15
Tyr Gly
<210> 20
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 20
Lys Asn Leu Arg Arg Ile Ile Arg Lys Glu Ile His Ile Ile Lys Lys
1 5 10 15
Tyr G1y
<210> 21
<211> 18
<212> PRT
-5-
CA 02415236 2002-12-19
WO 02/00839 PCT/USO1/19094
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 21
Zys Asn Zeu Arg Arg Ile I1e Arg Zys Asp Ile His Ile Ile Zys Zys
1 5 10 15
Tyr Gly
<210> 22
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<223> D-alanine
<400> 22
Zys Asn Zeu Arg Arg Ile Ile Arg Zys Ala Ile His Ile Ile Zys Zys
1 5 10 15
Tyr Gly
<210> 23
<211> 18
<212> PRT
<213>~Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<223> D-isoleucine
<400> 23
Zys As.n Zeu Arg Arg Tle Ile Arg Zys Ile Ile His Ile Ile Zys Zys
1 5 10 15
Tyr Gly
<210> 24
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 24
Zys Asn Zeu Arg Arg Ile Ile Arg Zys Ile Gly His Ile Ile Zys Zys
1 5 10 15
Tyr Gly
<210> 25
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
-6-
CA 02415236 2002-12-19
WO 02/00839 PCT/USO1/19094
<400> 25
Zys Asn Zeu Arg Arg Ile Ile Arg Zys Ile Thr His Ile Ile Zys Zys
1 5 10 15
Tyr Gly
<210> 26
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 26
Zys Asn Zeu Arg Arg Ile Ile Arg Zys Ile Ser His Ile Ile Zys Zys
1 5 10 15
Tyr Gly
<210> 27
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 27
Zys Asn Zeu Arg Arg Ile Tle Arg Zys Ile Glu His Ile Ile Zys Zys
1 5 10 15
Tyr Gly
<210> 28
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 28
Zys Asn Zeu Arg Arg Ile Ile Arg Zys I1e Asp His Ile I1e Zys Zys
1 5 10 15
Tyr G1y
<210> 29
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<223> D-alanine
<400> 29
Zys Asn Leu Arg Arg Ile Ile Arg hys Ile Ala His Ile Ile Zys Zys
1 5 10 15
Tyr Gly
CA 02415236 2002-12-19
WO 02/00839 PCT/USO1/19094
<210> 30
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<223> D-isoleucine
<400> 30
Lys Asn Leu Arg Arg Ile Ile Arg Lys Ile Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 31
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<221> AMIDATION
<222> (18)...(18)
<223> G-CONH2
<400> 31
Lys Asn Leu Arg Arg Ile Ile Arg Lys Gly Ile His Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 32
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<400> 32
Lys Asn Leu Arg Arg Ile Ile Arg Lys G1y Ile Arg Ile Ile Lys Lys
1 5 10 15
Tyr Gly
<210> 33
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic antimicrobial peptide
<221> AMTDATION
<222> (18)...(18)
<223> G-CONH2
<400> 33
Lys Asn Leu Arg Arg Ile Ile Arg Lys Gly Ile Arg Ile Ile Lys Lys
1 5 10 15
Tyr Gly
_g_
CA 02415236 2002-12-19
WO 02/00839 PCT/USO1/19094
<210> 34
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic variant of G10 novispirin peptide
<400> 34
Zys Arg Zeu Arg Arg Ile Ile Arg Zys Gly Ile His Ile Ile Lys Zys
1 5 10 15
Tyr Gly
<210> 35
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic variant of G10 novispirin
<221> AMIDATION
<222> (18)...(18)
<223> G-CONH2
<400> 35
Zys Arg Zeu Arg Arg Ile Ile Arg Zys Gly Ile His Ile Tle Zys Zys
1 5 10 15
Tyr Gly
<210> 36
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic variant of G10 novispirin
<400> 36
Arg Arg veu Arg Arg Ile Ile Arg Zys Gly Ile Arg Ile Ile Zys Zys
1 5 10 15
Tyr Gly
<210> 37
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic variant of G10 novispirin
<221> AMIDATION
<222> (18)...(18)
<223> G-CONH2
<400> 37
Arg Arg Zeu Arg Arg Ile Ile Arg hys Gly Ile Arg Ile Ile Zys Zys
1 5 10 15
Tyr Gly
_g_
