NZ795498A - Antimicrobial peptides and methods of using same - Google Patents
Antimicrobial peptides and methods of using sameInfo
- Publication number
- NZ795498A NZ795498A NZ795498A NZ79549818A NZ795498A NZ 795498 A NZ795498 A NZ 795498A NZ 795498 A NZ795498 A NZ 795498A NZ 79549818 A NZ79549818 A NZ 79549818A NZ 795498 A NZ795498 A NZ 795498A
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- New Zealand
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- seq
- arginine
- amino acid
- acid sequence
- glycine
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Abstract
Antimicrobial peptides of general formula X0X1X2C X3X4X5CX6X7X8X9CYX10X11CX12X13 are provided. Also provided are certain formulations containing these peptides and methods of using these peptides for treating skin infections in an animal in need thereof.
Description
Antimicrobial es of general formula X0X1X2C X3X4X5CX6X7X8X9CYX10X11CX12X13 are
provided. Also provided are certain formulations containing these peptides and methods of using
these peptides for treating skin infections in an animal in need thereof.
NZ 795498
ANTIMICROBIAL ES AND METHODS OF USING SAME
RELATED ATIONS
The present application is a divisional of New d patent application 764838, which
is the national phase entry of PCT international application (published as
FIELD OF THE INVENTION
[0001A] This invention is in the field of antimicrobial peptides and uses of such peptides for
treatment of infections.
BACKGROUND
otics are chemical substances having the capacity, in a dilute solution, to kill or
inhibit growth of microorganisms. otics that are sufficiently nontoxic to the host are used as
chemotherapeutic agents to treat ious diseases of humans, animals, and plants. The term
was originally cted to substances produced by microorganisms, but has been extended to
include synthetic and semi-synthetic compounds of similar chemical activity.
Extensive and widespread use of antimicrobial drugs led to the emergence of resistant
strains of microorganisms. These microorganisms are no longer susceptible to currently
available antimicrobial drugs. In order to lower or prevent lethal infectious diseases and
maintain public health, new antimicrobial agents are required.
Antimicrobial Peptides (AMPs) are an essential component of the host defense system
of organisms throughout nature and offer protection from invading pathogens. They show potent
antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, tes and
viruses. The smaller AMPs (usually about 15-40 amino acids) act largely by disrupting the
structure or function of microbial cell membranes, they do not target single defined molecular
structures. ore, as opposed to conventional antibiotics, they are effective regardless of
the metabolic activity of bacteria. Human AMPs such as defensins and cathelicidin (LL-37) are
present in leukocytes and secreted by various epithelia in skin and mucosal surfaces. In
on to their antimicrobial ty, AMPs are important effector les in inflammation,
immune activation, and wound healing. AMPs are quite diverse in sequence and ary
structure, but share some common properties. They are usually cationic, amphipathic and exert
their microbicidal effect by compromising the bacterial membrane integrity. Interaction of AMPs
with the anionic membrane surface of the target microbes leads to membrane permeabilization,
cell lysis and death.
SUMMARY OF INVENTION
In the first aspect, the invention provides an amino acid sequence of 17-22 amino acids
long and comprising, at its N-terminus, SEQ ID NO:12
(X0X1XZCX3X4X5CX6X7X8XQCYX10X11CX12X13) wherein X0 is absent or proline; X1 is lysine,
arginine, glycine, or proline; X2 is alanine, tryptophan, or arginine; X3 is phenylalanine,
valine or tryptophan; X4 is ne, ne or alanine; X5 is valine or alanine; X6 is
tyrosine or arginine or lysine or tryptophan; X7 is arginine, phenylalanine, or glycine; X8 is
arginine, phenylalanine or glycine; X9 is isoleucine, alanine, phenylalanine, tyrosine or valine;
X10 is arginine or histidine; X11 is arginine or lysine; X12 is arginine, , or asparagine; X13 is a
0-4 amino-acid-long polypeptide; with provisos that if X0 is proline, then X1 is not proline; if said
amino acid sequence comprises SEQ ID NO: 13 (KWCFRVCYRGICYRRCR), or SEQ ID NO:
28 (KWCFRVCYRGICYRKCR) then X13 is 1-4 amino acids long; if X13 is 1 amino acid long or
longer, then the N-terminal amino acid in X13 is aspartic acid or glutamic acid; if the amino acid
at position corresponding to position 1 of SEQ ID NO: 1 is glycine, then said glycine is not acyl-
or palmitic acid — modified; if amino acid is X11 lysine then X6X7X8X9 (SEQ ID NO: 14) is not
RRRF (SEQ ID NO: 15); and if the amino acid is GFCWYVCYRGICYRRCN (SEQ ID NO: 16)
then the C-terminal asparagine is ed.
In certain embodiments, X0 is absent and X6 is arginine or lysine; and/or X7 is arginine or
lysine; X6X7X8X9 (SEQ ID NO: 14) is selected from the group ting of YRGI (SEQ ID NO:
17), YRGV (SEQ ID NO: 18), YRGF (SEQ ID NO: 19); and/or X10 is arginine; and/or X3X4X5
(SEQ ID NO: 20) is FRV (SEQ ID NO: 21), WYV (SEQ ID NO: 22); and/or X13 is 1 amino acid
long or longer, and the N-terminal amino acid in X13 is aspartic acid.
In a ular set of embodiments, the amino acid sequence is 17-21 amino acids long
and comprises, at its N-terminus, SEQ ID NO:12 (X0X1X2C X3X4X5CX6X7X8X9CYX1OX11CX12X13)
wherein X0 is absent; X1 is lysine, arginine or e; X2 is phenylalanine, tryptophan, or
argninine; X3 is phenylalanine, valine or tryptophan; X4 is tyrosine or phenylalanine; X5 is valine
or alanine; X6 is tyrosine or arginine; X7 is arginine or glycine; X8 is arginine, phenylalanine or
e; X9 is e, phenylalanine, tyrosine or valine; X10 is arginine or histidine; X11 is
arginine or lysine X12 is arginine, lysine, or asparagine; X13 is a 0-4 amino-acid-long polypeptide.
In a another set of embodiments according to the first aspect, the amino acid sequence
is 18—21 amino acids long and comprises, at its N-terminus, SEQ ID NO: 1
(KWCFRVCYRGICYRRCRD) or a peptide that differs from SEQ ID NO: 1 by one, two, three, or
four amino acids, wherein the amino acids differing from the amino acids of SEQ ID NO: 1 are
ndently selected from the group ting of arginine or glycine at position
corresponding to position 1 of SEQ ID NO: 1; phenylalanine or arginine at position
corresponding to position 2 of SEQ ID NO: 1; valine or tryptophan at position corresponding to
position 4 of SEQ ID NO: 1; tyrosine at position corresponding to on 5 of SEQ ID NO: 1;
arginine at position corresponding to position 8 of SEQ ID NO: 1; glycine at position
corresponding to position 9 of SEQ ID NO: 1; arginine at position corresponding to position 10
of SEQ ID NO: 1; alanine, phenylalanine, or valine at position corresponding to position 11 of
SEQ ID NO: 1; histidine at position corresponding to on 14 of SEQ ID NO: 1; lysine at
position corresponding to position 15 of SEQ ID NO: 1; asparagine at position corresponding to
position 17 of SEQ ID NO: 1.
More specifically, the amino acid sequence comprises aspartic acid at on
corresponding to position 18 of SEQ ID NO: 1; and/or asparagine at position corresponding to
on 17 of SEQ ID NO: 1; and/or glycine at on corresponding to position 1 of SEQ ID
NO: 1; alanine at position corresponding to position 11 of SEQ ID NO: 1; and/or arginine at
on corresponding to position 14 of SEQ ID NO: 1, at position corresponding to position 15
of SEQ ID NO: 1, or both.
In a set of embodiments, the amino acid sequence is selected from the group consisting
of amino acid sequences comprising, at the respective N-termini, SEQ ID NO: 1, SEQ ID NO: 2
(RWCFRVCYRGICYRRCRD), SEQ ID NO: 3 (GWCFRVCYRGICYRRCRD), SEQ ID NO: 4
(KFCFRVCYRGICYRRCRD); SEQ ID NO: 5 (KWCFYVCYRGICYRRCRD), SEQ ID NO: 6
(KWCFRVCRRGICYRRCRD), SEQ ID NO: 7 (KWCFRVCYRGVCYRRCRD), SEQ ID NO: 8
(KWCFRVCYRGACYRRCRD), SEQ ID NO: 9 (KWCFRVCYRGFCYRRCRD), SQE ID NO: 10
VCYRGICYHRCRD), or SEQ ID NO: 11 (KWCFRVCYRGICYRRCND).
In additional embodiments, the amino acid ce is selected from the group
consisting of SEQ ID NO: 97 (KRCFRVCYRGICYRRCRD); SEQ ID NO: 98
(KWCVRVCYRGICYRRCRD), SEQ ID NO: 99 (KWCFFVCYRGICYRRCRD), SEQ ID NO: 100
(KWCFWVCYRGICYRRCRD), SEQ ID NO: 102 ACYRGICYRRCRD), SEQ ID NO:
104 (KWCFRVCYFGICYRRCRD), SEQ ID NO: 105 (KWCFRVCYRGICYRRCRN), SEQ ID NO:
106 VCYRGICYRRCRD), SEQ ID NO: 107 (KWCFRVCWRGICYRRCRD), SEQ ID
NO: 108 (KWCFRVCYGGICYRRCRD), SEQ ID NO: 109 (KWCFRVCYRRICYRRCRD), SEQ
ID NO: 110 (KWCFRVCYRGYCYRRCRD), SEQ ID NO: 112 (KWCFRVCYRGICYRRCKD),
SEQ ID NO: 113 (KWCFRVCYRGICYRRCAD), SEQ ID NO: 114 VCYRGICYRRCRR),
SEQ ID NO: 115 (GWCFRVCYRGICYRRCND), SEQ ID NO: 116
(KWCFYVCYRGICYRRCND), SEQ ID NO: 117 (GWCFYVCYRGICYRRCRD), SEQ ID NO: 118
(GWCFYVCYRGICYRRCND).
In yet additional embodiments, the amino acid sequence is selected from the group
consisting of SEQ ID NO: 28, 29, 30, 31.
In the second aspect, the invention provides a multimer comprising a plurality of repeats
of the amino acid sequence according to the previous aspect of the invention, wherein further,
the N-terminal amino acid of said sequence is proline, and the C-terminal amino acid of said
sequence is ic acid. Advantageously, the repeats of the amino acid sequence are joined
each other directly, thereby forming D-P bonds. In certain ments, the plurality is
between 2 and 20.
The invention also provides a method of making the amino acid sequence that is
le for making the multimer as described in the second aspect of the invention. The
method comprises synthesizing the multimer and contacting the multimer with a mild acid (e.g.,
formic acid) whereby D-P bonds are broken.
In a third aspect, the invention es a method of treating infections in an animal in
need thereof, comprising stering to the animal a formulation comprising the amino acid
sequence according to the first aspect of the invention. In certain embodiments, the infection is
a skin infection. In other embodiments, the infection is mastitis, a respiratory infection, an ear
infection, urinary tract infection, or reproductive tract infection.
In certain embodiments, the animal is a companion animal, e.g., a dog, a cat, or a horse.
In a particular embodiment, the animal is a dog. In certain embodiments, the formulation is
formulated for a l application. In some embodiments, the formulation is a gel, a cream, an
emulsion, or a spray.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates toxicity of SEQ ID NOs: 1 and 13 in human, beagle, and rat red blood
cells.
DETAILED DESCRIPTION
Definitions
For a better understanding of the invention, the following miting tions are
"About" or "approximately," when used in connection with a measurable numerical
variable, refers to the indicated value of the variable and to all values of the variable that are
within the experimental error of the indicated value (e.g., within the 95% confidence interval for
the mean) or within 10 t of the indicated value, whichever is greater, unless about is used
in nce to time intervals in weeks where "about 3 weeks," is 17 to 25 days, and about 2 to
about 4 weeks is 10 to 40 days.
"Emulsion" means a composition of two immiscible liquids in which small droplets of one
liquid are suspended in a continuous phase of the other liquid.
teral administration" refers to the introduction of a substance, such as a vaccine,
into a subject's body through or by way of a route that does not include the digestive tract.
Parenteral administration includes subcutaneous, intramuscular, utaneous, ermal,
eritoneal, intraocular, and intravenous administration.
ion [in a sequence of interest] corresponding to” a n position of a reference
sequence is determined by aligning the reference sequence and the sequence of interest in
such a way that the cysteine residues of the sequence of interest and the reference sequence
are matched to each other, and then determining the position in the sequence of st that
matcher the desired position in the reference sequence.
"Pharmaceutically acceptable" refers to substances, which are within the scope of sound
medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity,
irritation, allergic response, and the like, commensurate with a reasonable benefit-to-risk ratio,
and effective for their ed use.
"Therapeutically effective amount" refers to an amount of the amino acid sequence
and/or the formulation containing same that would induce a response in a subject receiving the
amino acid or formulation which is adequate to prevent or reduce signs or symptoms of
infection.
"Treating" refers to preventing a er, condition, or disease, including, without
limitations, infections, to which such term applies, or to preventing or reducing one or more
symptoms of such disorder, condition, or disease.
"Treatment" refers to the act of "treating" as defined above.
Pepfldes
Generally, the invention provides an amino acid sequence of 17-22 amino acids long
and comprising, at its N-terminus, SEQ ID NO:12 (XOX1X2C X3X4X5CX6X7X8X9CYX1OX11CX12X13)
wherein
X0 is absent or proline;
X1 is , arginine, glycine, or proline;
X2 is phenylalanine, tryptophan, or arginine;
X3 is phenylalanine, valine or tryptophan;
X4 is arginine, ne or phenylalanine;
X5 is valine or alanine;
X6 is tyrosine or arginine;
X7 is arginine, phenylalanine, or glycine;
X8 is arginine, phenylalanine or glycine;
X9 is cine, alanine, phenylalanine, tyrosine or ;
X10 is arginine or histidine;
X11 is arginine or lysine;
X12 is arginine, lysine, or asparagine;
X13 is a 0-4 amino-acid-Iong polypeptide;
with provisos that if X0 is proline, then X1 is not proline; if said amino acid sequence comprises
SEQ ID NO: 13 (KWCFRVCYRGICYRRCR), or SEQ ID NO: 28 (KWCFRVCYRGICYRKCR)
then X13 is 1-4 amino acids long; if X13 is 1 amino acid long or , then the N-terminal amino
acid in X13 is ic acid or glutamic acid; if the amino acid at position corresponding to
position 1 of SEQ ID NO: 1 is glycine, then said glycine is not acyl- or palmitic acid — modified;
if amino acid is X11 lysine then X6X7X8X9 (SEQ ID NO: 14) is not RRRF (SEQ ID NO: 15); and if
the amino acid is GFCWYVCYRGICYRRCN (SQE ID NO: 16) then the C-terminal asparagine is
In certain embodiments, X0 is absent and X6 is arginine or lysine; and/or X7 is arginine or
lysine; X6X7X8X9 (SEQ ID NO: 14) is selected from the group consisting of YRGI (SEQ ID NO:
17), YRGV (SEQ ID NO: 18), YRGF (SEQ ID NO: 19); and/or X10 is arginine; and/or X3X4X5
(SEQ ID NO: 20) is FRV (SEQ ID NO: 21), WYV (SEQ ID NO: 22); and/or X13 is 1 amino acid
long or longer (e.g., 1, 2, 3, or 4 amino acids long), and the N-terminal amino acid in X13 is
aspartic acid.
In a particular set of ments according to the first aspect, the amino acid sequence
is 18—21 amino acids long and comprises, at its N-terminus, SEQ ID NO: 1
(KWCFRVCYRGICYRRCRD) or a peptide that differs from SEQ ID NO: 1 by one, two, three, or
four amino acids, wherein the amino acids differing from the amino acids of SEQ ID NO: 1 are
independently ed from the group consisting of arginine or glycine at position
corresponding to on 1 of SEQ ID NO: 1; alanine or argninine at position
corresponding to position 2 of SEQ ID NO: 1; valine or tryptophan at position corresponding to
position 4 of SEQ ID NO: 1; tyrosine at position corresponding to position 5 of SEQ ID NO: 1;
arginine at position corresponding to position 8 of SEQ ID NO: 1; glycine at position
corresponding to position 9 of SEQ ID NO: 1; arginine at position ponding to position 10
of SEQ ID NO: 1; alanine, alanine, or valine at position corresponding to position 11 of
SEQ ID NO: 1; histidine at position ponding to position 14 of SEQ ID NO: 1; lysine at
position ponding to position 15 of SEQ ID NO: 1; asparagine at position corresponding to
position 17 of SEQ ID NO: 1.
In ent embodiments, the amino acid sequence differs from SEQ ID NO: 1 by one,
two, or three amino acids.
In certain embodiments, the amino acid sequence comprises aspartic acid at position
corresponding to position 18 of SEQ ID NO: 1; and/or asparagine at position corresponding to
position 17 of SEQ ID NO: 1; and/or glycine at on corresponding to position 1 of SEQ ID
NO: 1; alanine at on corresponding to position 11 of SEQ ID NO: 1; and/or arginine at
position corresponding to position 14 of SEQ ID NO: 1, at position corresponding to position 15
of SEQ ID NO: 1, or both.
In a set of ments, the amino acid sequence is selected from the group consisting
of amino acid sequences comprising, at the respective N-termini, SEQ ID NO: 1, SEQ ID NO: 2
(RWCFRVCYRGICYRRCRD), SEQ ID NO: 3 (GWCFRVCYRGICYRRCRD), SEQ ID NO: 4
VCYRGICYRRCRD); SEQ ID NO: 5 (KWCFYVCYRGICYRRCRD), SEQ ID NO: 6
(KWCFRVCRRGICYRRCRD), SEQ ID NO: 7 (KWCFRVCYRGVCYRRCRD), SEQ ID NO: 8
(KWCFRVCYRGACYRRCRD), SEQ ID NO: 9 (KWCFRVCYRGFCYRRCRD), SQE ID NO: 10
(KWCFRVCYRGICYHRCRD), or SEQ ID NO: 11 VCYRGICYRRCND).
Additional amino acid sequences may be found among SEQ ID NO: 97
(KRCFRVCYRGICYRRCRD); SEQ ID NO: 98 (KWCVRVCYRGICYRRCRD), SEQ ID NO: 99
(KWCFFVCYRGICYRRCRD), SEQ ID NO: 100 (KWCFWVCYRGICYRRCRD), SEQ ID NO:
101 (KWCFRVYCYRGICYRRCRD), SEQ ID NO: 102 (KWCFRACYRGICYRRCRD), SEQ ID
NO: 103 (KWCFRVCKRGICYRRCRD), SEQ ID NO: 104 (KWCFRVCYFGICYRRCRD), SEQ ID
NO: 105 (KWCFRVCYRGICYRRCRN), SEQ ID NO: 106 VCYRGICYRRCRD), SEQ
ID NO: 107 (KWCFRVCWRGICYRRCRD), SEQ ID NO: 108 (KWCFRVCYGGICYRRCRD),
SEQ ID NO: 109 (KWCFRVCYRRICYRRCRD), SEQ ID NO: 110
(KWCFRVCYRGYCYRRCRD), SEQ ID NO: 111 (KWCFRVCYRGICRYRRCRD), SEQ ID NO:
112 (KWCFRVCYRGICYRRCKD), SEQ ID NO: 113 (KWCFRVCYRGICYRRCAD), SEQ ID NO:
114 (KWCFRVCYRGICYRRCRR), SEQ ID NO: 115 (GWCFRVCYRGICYRRCND), SEQ ID
NO: 116 (KWCFYVCYRGICYRRCND), SEQ ID NO: 117 (GWCFYVCYRGICYRRCRD), SEQ ID
NO: 118 (GWCFYVCYRGICYRRCND).
Thus, the amino acid sequence may be selected from the group consisting of SEQ ID
NO: 97 (KRCFRVCYRGICYRRCRD); SEQ ID NO: 98 (KWCVRVCYRGICYRRCRD), SEQ ID
NO: 99 (KWCFFVCYRGICYRRCRD), SEQ ID NO: 100 (KWCFWVCYRGICYRRCRD), SEQ ID
NO: 102 ACYRGICYRRCRD), SEQ ID NO: 104 (KWCFRVCYFGICYRRCRD), SEQ ID
NO: 105 (KWCFRVCYRGICYRRCRN), SEQ ID NO: 106 (KWCWRVCYRGICYRRCRD), SEQ
ID NO: 107 (KWCFRVCWRGICYRRCRD), SEQ ID NO: 108 (KWCFRVCYGGICYRRCRD),
SEQ ID NO: 109 (KWCFRVCYRRICYRRCRD), SEQ ID NO: 110
(KWCFRVCYRGYCYRRCRD), SEQ ID NO: 112 VCYRGICYRRCKD), SEQ ID NO:
113 (KWCFRVCYRGICYRRCAD), SEQ ID NO: 114 (KWCFRVCYRGICYRRCRR), SEQ ID NO:
115 VCYRGICYRRCND), SEQ ID NO: 116 (KWCFYVCYRGICYRRCND), SEQ ID
NO: 117 (GWCFYVCYRGICYRRCRD), SEQ ID NO: 118 (GWCFYVCYRGICYRRCND).
The peptides according to the invention can be manufactured by methods that are well-
known in the art, including, without tions, solid-phase peptide synthesis. The peptides may
also be synthesized using bioengineering ques (e.g., fermentation) in fungal, bacterial or
eukaryotic systems.
In certain embodiments, where the N-terminal amino acid of the antimicrobial peptide is
proline, and the C-terminal amino acid is aspartic acid, the method of manufacturing the anti-
ial peptide may entail sizing a multimer of the antimicrobial peptide. In different
embodiments, the number of monomers in the multimer may be 1 to about 20, e.g., about 5,
about 10, or about 15. Conveniently, the monomers of the antimicrobial peptide would be linked
via a peptide bond between the C-terminal aspartic acid of an upstream monomer and the N-
terminal proline of the downstream monomer (D-P bond). This D-P bond can conveniently be
cleaved via mild acid (e.g., formic or citric acid) hydrolysis. Thus, a le encompassed by
such description as, for example, (SEQ ID NO: 29)n or (SEQ ID NO: 31).,, wherein n is an
integer between 1 and 20, may be used in the itions and methods of the invention.
Formulations
The peptides according to the embodiments above may be formulated for delivery to the
target site (i.e., the site that is infected or the site that is in danger of being infected due to a
wound, irritation, to the like). Without limitation, the sites include skin, eyes, ears, mammary
gland, reproductive tract, urinary bladder, nasal and oral cavities. The composition comprising
the peptides of the instant invention is ated depending on the site of st.
Also provided are compositions that can be ed by mixing one or more
antimicrobial peptides described herein, with pharmaceutically acceptable carriers, excipients,
binders, diluents or the like, to treat or ameliorate a variety of bacterial infections. A
therapeutically effective dose or amount refers to that amount of one or more compounds
described herein ient to result in amelioration of symptoms of the infection. The
pharmaceutical compositions of the instant invention can be manufactured by methods well
known in the art such as conventional granulating, mixing, dissolving, encapsulating,
lyophilizing, or emulsifying processes, among others. The compositions can be in the form of,
for example, granules, powders, tablets, capsule syrup, suppositories, injections, emulsions,
elixirs, suspensions or ons. The instant compositions can be formulated for various routes
of administration, for example, by oral administration, by topical administration, by transmucosal
administration, by rectal administration, or aneous stration as well as intrathecal,
intravenous, intramammary, intramuscular, intraperitoneal, intranasal, intraocular or
intraventricular injection. The compound or compounds of the instant invention can also be
administered in a local fashion, such as injection as a sustained release formulation. The
following dosage forms are given by way of example and should not be construed as limiting the
instant invention.
For oral, buccal, and sublingual administration, powders, suspensions, granules, tablets,
pills, capsules, gelcaps, and caplets are acceptable as solid dosage forms. These can be
prepared, for example, by mixing one or more compounds of the t invention, or
pharmaceutically acceptable salts or tautomers f, with at least one additive or excipient
such as a starch or other additive. Suitable additives or excipients are sucrose, lactose,
cellulose sugar, mannitol, ol, dextran, sorbitol, , agar, alginates, s, chitosans,
pectins, tragacanth gum, gum arabic, gelatins, collagens, , albumin, synthetic or semisynthetic
polymers or glycerides, methyl cellulose, ypropylmethyl-cellulose, and/or
polyvinylpyrrolidone. Optionally, oral dosage forms can contain other ingredients to aid in
administration, such as an ve diluent, or lubricants such as magnesium stearate, or
preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol
or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or
perfuming agents. Additionally, dyestuffs or pigments can be added for fication. Tablets
and pills can be further treated with suitable coating materials known in the art.
Liquid dosage forms for oral administration can be in the form of pharmaceutically
acceptable emulsions, syrups, elixirs, suspensions, slurries and solutions, which can contain an
inactive diluent, such as water. Pharmaceutical formulations can be prepared as liquid
suspensions or solutions using a sterile liquid, such as, but not d to, an oil, water, an
alcohol, and combinations of these. ceutically suitable tants, suspending agents,
emulsifying agents, can be added for oral or parenteral administration.
As noted above, sions can include oils. Such oils include peanut oil, sesame oil,
cottonseed oil, corn oil, olive oil and mixtures of oils. Suspension ation can also n
esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and
ated fatty acid glycerides. Suspension formulations can include ls, such as, but not
limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers,
such as but not limited to, poly (ethyleneglycol), petroleum hydrocarbons such as l oil
and petrolatum; and water can also be used in suspension ations.
For certain routes of administration, the pharmaceutical formulations can be a spray or
aerosol containing and appropriate solvents and optionally other compounds such as, but not
limited to, stabilizers, antimicrobial , antioxidants, pH modifiers, surfactants, bioavailability
modifiers and combinations of these. A propellant for an aerosol formulation can include
ssed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent. The
compound or compounds of the instant invention are iently red in the form of an
aerosol spray presentation from a zer or the like.
lnjectable dosage forms generally include aqueous suspensions or oil suspensions
which can be prepared using a suitable dispersant or wetting agent and a suspending agent.
lnjectable forms can be in solution phase or in the form of a sion, which is prepared with
a solvent or diluent. able solvents or vehicles include sterilized water, Ringer's solution,
or an isotonic aqueous saline solution. Alternatively, sterile oils can be employed as solvents or
suspending agents. Generally, the oil or fatty acid is latile, including natural or synthetic
oils, fatty acids, mono-, di- or tri-glycerides.
For injection, the pharmaceutical formulation can be a powder suitable for reconstitution
with an appropriate solution as bed above. Examples of these include freeze dried, rotary
dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For
ion, the ations can optionally contain stabilizers, pH modifiers, surfactants,
bioavailability modifiers and combinations of these. The compounds can be formulated for
parenteral administration by injection such as by bolus injection or continuous infusion. A unit
dosage form for injection can be in ampoules or in multi-dose containers.
For rectal administration, the pharmaceutical formulations can be in the form of a
suppository, an ointment, an enema, a tablet or a cream for release of compound in the
intestines, d flexure and/or rectum. Rectal suppositories are prepared by mixing one or
more compounds of the instant ion, or pharmaceutically acceptable salts or tautomers of
the compound, with acceptable vehicles, for example, cocoa butter or polyethylene glycol, which
is present in a solid phase at normal storing temperatures, and present in a liquid phase at
those temperatures suitable to release a drug inside the body, such as in the rectum. Oils can
also be employed in the preparation of formulations of the soft gelatin type and suppositories.
Water, saline, aqueous dextrose and related sugar solutions, and ols can be employed in
the preparation of suspension formulations which can also contain suspending agents such as
pectins, carbomers, methyl cellulose, hydroxypropyl cellulose or carboxymethyl ose, as
well as buffers and preservatives.
Besides those representative dosage forms described above, pharmaceutically
acceptable excipients and carries are lly known to those skilled in the art and are thus
included in the instant invention. Such excipients and carriers are described, for example, in
"Remington's ceutical Sciences", Mack Pub. Co., New Jersey .
The formulations of the invention can be designed to be short-acting, fast-releasing,
long-acting, and sustained-releasing. Thus, the pharmaceutical formulations can also be
formulated for controlled release or for slow e.
The t compositions can also comprise, for example, micelles or liposomes, or
some other encapsulated form, or can be administered in an ed release form to provide a
prolonged storage and/or delivery effect. Therefore, the pharmaceutical ations can be
compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot
injections or as implants such as stents. Such implants can employ known materials such as
nes and biodegradable polymers.
The composition may also contain anti-pruritic medications, including, without limitations,
nib and salts thereof (e.g., APOQUEL® and anti-lL-31 antibodies (e.g., CYTOPOINTTM).
The composition can also comprise a steroid or an anti-fungal medicine. Suitable
ds include, without limitations, Betamethasone, triamcinolone acetonide, hydrocortisone
aceponate, hydrocortisone, triamcinolone, methylprednisolone acetate, and the like. Suitable
anti-fungal medicines include, without limitations chlotrimazole, econazole, itraconazole,
ketoconazole, miconazole.
The compositions can contain, for example, from about 0.1% by weight, to about 90% or
more by weight, of the antimicrobial peptide, depending on the method of administration. Where
the compositions comprise dosage units, each unit can contain, for example, from about 0.5 mg
to about 10 mg per dose of the antimicrobial e. For example, one dose of the
composition may contain about 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg,
.5 mg, 6 mg, 6.5 mg, 7mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg. The composition may n
about 1 to about 5 mg of the antimicrobial peptide per dose, or about 1.5 to about 5 mg of the
crobial peptide per dose, or about 2.5 mg to about 7.5 mg per dose, or about 1.5 mg to
about 2.5 mg per dose, depending on the severity of the wound and the size of the animal.
Methods
In yet another aspect, the invention also provides methods of treating or preventing a
bacterial infection in a t, comprising administering an effective amount of one or more
compounds described herein to the subject. Suitable subjects that can be treated e dogs,
cats, , cattle, sheep, pigs, poultry, primates (e.g., rhesus monkeys and lgus (also
known as crab-eating or long-tailed) monkeys, marmosets, nds, chimpanzees,
macaques), rabbits, and rodents (rats, mice, guinea pigs and the like). In certain embodiment,
the subject is a dog, and the antimicrobial peptide of the invention is delivered topically,
asally, intraocularly, or intraaurally. The antimicrobial peptide may be delivered in a form
of drops, spray, cream, gel, ointment and the like.
Infections that can be treated with the described compounds include external ear
infections, infections of the middle ear, such as acute otitis media, infections of the cranial
sinuses, eye infections, infections of the oral cavity, such as infections of the teeth, gums and
mucosa, upper atory tract infections, lower respiratory tract infections, genitourinary
infections, gastrointestinal infections, gynecological infections, emia, bone and joint
infections, skin and skin structure infections, burns, antibacterial prophylaxis of surgery, and
antibacterial prophylaxis in immunosuppressed ts, such as patients receiving cancer
chemotherapy, or organ lant ts. These infections can be treated in hospital or
community settings via various routes of administration as described herein.
The compounds or compositions described herein can also be used prophylactically.
Accordingly, one or more of the present compounds or compositions can be administered to a
subject deemed to be at risk for developing a microbial infection. Subjects at risk for developing
a microbial ion include individuals who have been exposed to a particular microorganism,
such as a pathogenic bacterial species; individuals having a compromised immune system, or
subjects that are particularly vulnerable to the infections due to compromised natural defenses
(e.g., where the skin is compromised due to burns or cuts).
The crobial peptides described herein can be used for the treatment or prevention
of infectious disorders caused by a variety of bacterial organisms, ing infections by
pathogenic bacterial species. miting examples of bacterial infection include Gram positive
and Gram ve aerobic and anaerobic bacteria, such as Staphylococci, e.g., S. aureus;
Enterococci, e.g., E. faecalis; ococci, e.g., S. pyogenes and S. pneumoniae; Escherichia
species, e.g., E. coli, including toxigenic, enteropathogenic, enteroinvasive,
enterohemorrhagic and enteroaggregative E. coli strains; Propionibacterium strains, e.g., P.
acnes; Haemophilus, e.g., H. influenza; Moraxella, e.g., M. catarrha/is. Other examples include
cteria, e.g., M. tuberculosis, M. avian-intracellulare, M. kansasii, M. bovis, M. africanum,
M. genavense, M. leprae, M. xenopi, M. simiae, M. scrofulaceum, M. ma/moense, M. celatum,
M. abscessus, M. chelonae, M. szulgai, M. gordonae, M. haemophi/um, M. fortuni and M.
marinum; Corynebacteria, e.g., C. diphtheriae; Pseudomonas species, e.g., P. aeruginosa;
Borrelia species, e.g., B. burgdorferi; Listeria species, e.g., L. monocytogenes; Bacillus species,
e.g., B. cereus; Bordetella species, e.g., B. bronchiseptica; Klebsiella species, Clostridium
species, e.g., C. perfringens, C. tetani; Chlamydia species, e.g., C. psittaci,; Rickettsia species,
e.g., B. rickettsii and B. prowazekii; Salmonella s, e.g., S. typhimurium; Yersinia species,
e.g., Y. enterocolitica and Y. pseudotuberculosis; Klebsiella s, e.g., K. pneumoniae; and
Mycoplasma, e.g., M. nia, Actinobacillus species, H. parasuis; and Truepere/Ia
pyogenes.
In certain aspects the bacteria are selected from Staphylococci, e.g, S.
ntermedius, S. aureus, 8. sch/eiferi, S. chromogenes, S. simulans, S. xylosus. The
bacteria may also be selected from Streptococci, e.g., S. uberis, S. aga/actiae, S. dysgalactiae,
S. suis. Further, the bacteria of family Pasteurellaceae are suitable for treatment with the
itions bed herein. Suitable Pasteurellaceae bacteria include M. haemolytica, P.
multocida, H. somni, Escherichia species, e.g., E. coli, and Klebsiella species.
In certain ments, the bacteria are S. pseudintermedius and/or P. aeruginosa.
The compositions described herein may be administered in different frequency
regiments. For example, suitable regimens include 4 times daily to once every week, e.g., three
times daily, twice daily, once daily, every two days, every three days, twice per week, every five
days and so on. Similarly, the inventions described herein may be stered in different
duration regimens, e.g., in a single administration, for two days, for three days, for four days, for
a week, for two weeks, for a month, for six weeks, and so on. The duration, the frequency and
the amount of the antimicrobial peptide per dose, as well as the species and the state of the
wound and/or state of the ion, may be considered together in determining the proper dose-
requency regimen for stration of the antimicrobial compositions claimed herein.
The following examples are presented as illustrative embodiments, but should not be
taken as limiting the scope of the invention. Many changes, variations, cations, and other
uses and ations of this invention will be apparent to those skilled in the art.
EXAMPLES
Example 1. Antimicrobial activity and safety in Vitro
Peptides according to SEQ ID NOs as listed in Table 1 were prepared by a commercial
manufacturer (CS Bio, Menlo Park, California) using solid phase synthesis. Antimicrobial
activity was assessed by determining the l Inhibitory Concentration (MIC) against S.
aureus and E. coli. Briefly, Microbroth MICs were performed using CLSI methodology (VET01-
S2). For S. aureus and E. coli ATCC strains, TSA with 5% |ysed horse blood agar was used for
ght culturing at 37°C ambient air. A 0.5 mM stock for each peptide was made with cell
e water, 0.01% acetic acid and serially diluted and spotted (10 uL) in a 96-well plate for in
assay dose titration concentration of 50 uM to 0.05 uM. 0.5 McFarland Standard of each strain
was diluted 1:250 in Mueller-Hinton Broth (MHB). 90 uL of culture suspension was then added
upon drug in the 96-well plate for overnight incubation for 18-20 hours. The MIC was
determined visually at the first well of no visible growth at the corresponding concentration.
The results of these experiments are provided in Table 1.
TABLE 1
SEQ S.aureus
ATCC 25922
ATCC 29213
SEQ ID NO: 23 KFCVYVCYRGICYRRCK 1.6
SEQ ID NO: 24 CYRGVCYRRCR 1.6
SEQ ID NO: 1 KWCFRVCYRGICYRRCRD 1.6
SEQ ID NO: 13 KWCFRVCYRGICYRRCR 3.1
GFCWYVCYRGFCYRRCN 3.1
RGGRLCYCRRRFCVCVGR 3.1
RRWCFRVCYRGFCYRKCR 3.1
KWCFRVCYRGICYRKCR 3.1
GFCWYVCRRRFCYRRCN 3.1
CRRRFCYRRCR 3.1
CYRGICYRRCN-NH2 3.1
GFCWYVCYRGFCYRRCN-NH2 3.1
GFCWYVCRRRFCYRRCN 6.2
PGFCWYVCRRRFCYRRCN 6.2
PFCWYVCRRRFCYRRCN 52
GFCWYVCRRRFCHRRCN 6.2
GVCVYVCRRRFCYRRCN 6.2
GVCVYVCRRRFCYRRCN 6.2
SEQ ID No: 38 6.2
SEQ ID No: 39 6.2
SEQ ID No: 40 6.2
SEQ ID No: 41 6.2
SEQ ID No: 42 6.2
WO 13181
SEQ ID NO: 45 *GFCWYVCYRGFCYRRCN-NHZ __6_._g ______________________________ }__Q_.__8_ _____________________________
GFCWYVCYRGICYRRCN £ng____________________________ __Q_._4_ _____________________________
GFCWYVCYRGFCYRRCN _§._g5_04_____________________________
SEQ ID N0147__1__2;5_08
_____________________________
SEQ ID _1__2;5_04
______________________________
SEQ ID No: 49 __I_2_.S_ 02
______________________________
SEQ ID No:50__1__2;5_04
______________________________
SEQ ID NO: 51 25 6.2
SEQ ID No: 52 2508
______________________________
SEQ ID No:532550
_______________________________
SEQ ID No:542504
________________________
SEQ ID No:552508
______________________________
SEQ ID No:562508
______________________________
SEQ ID No:572504
_____________________________
SEQ ID No: 58 _88________________________________ _Q_-__2_ _____________________________
SEQ ID No: 58 _88________________________________ _Q_-__8_ _______________________
SEQ ID No:602504
_____________________________
SEQ ID No: SI 2562
_____________________________
SEQ ID NO: 62 ____________________________ i__S_0__ ________________________
SEQ ID NO: 63 5050_______________________________
SEQ ID NO: 64 503I
SEQ ID NO 65 50825
____________________________
SEQ ID NO: 66 503I
_______________________________
SEQ ID NO 67 50I6
______________________________
SEQ ID NO: 68 50I8
______________________________
SEQ ID NO 69 5050
_______________________________
SEQ ID NO 70 5025
______________________________
SEQ ID NO:71503I
______________________________
SEQ ID NO 72 _59________________________________ __8_._I______________________________
SEQ ID NO: 73 _59________________________________ __8_._I______________________________
SEQ ID NO 74 __59 I_2__._S
_________________________ ___________________________
SEQ ID NO: 75 _59_______________________________ ?__S_o__ ______________________________
SEQ ID NO: 76 _59_______________________________ }__S_0__ ______________________________
SEQ ID NO 77 _59_______________________________ _8_._I______________________________
SEQ ID NO: 78 _59_______________________________ }__8_._I______________________________
SEQ ID NO 79 _59_______________________________ :__8_.__2_ _____________________________
SEQ ID NO: 80 __59
_______________________________ j___1__-__6_ _____________________________
SEQ ID NO81—89
_______________________________ :__S_0__ ______________________________
SEQ ID NO 82 _59_______________________________ :__8_.__2_ _____________________________
SEQ ID N0183—_S_o_
_______________________________ ;__I_.__8_ _____________________________
SEQ ID NO 84 503I
_______________________________
_I. 01
SEQ ID NO: 85 KKVCVNVCKQGICHKRCK
SEQ ID NO 86
SEQ ID NO 87
SEQ ID NO: 88
SEQ ID NO 89
SEQ ID NO: 90
GHCHHVCRRRHCHRRCN
* refers to N-acetylation
** refers to itic acid modification
SEQ ID NO: 1 was selected for further research. Toxicity of SEQ ID NO: 1 to eukaryotic
blood cells was compared to that of tachyplesin (SEQ ID NO: 13). A standard, well referenced
red blood cell hemolysis assay was employed t multiple s to test the lysis potential
of the peptides. Red blood cells (RBCs) were prepared and isolated by l centrifuge and
wash steps to remove the plasma fraction. A dose titration (50 [M to 0.05 uM) of test peptides
and control peptide in were spotted from 50 mM stocks in a 384 well plate. Prepared RBCs
were ted with peptide for one hour at 37°C. Percent hemolysis was measured by optical
density at 405nm and utilizing 1% TritonX100 as hundred percent effect (HPE) and phosphate
buffer alone as zero percent effect (ZPE).
The inventors have surprisingly discovered that SEQ ID NO: 1 had not only improved
anti-microbial activity but also decreased toxicity to red blood cells. The results of the
experiments using human, beagle, and rat red blood cells are rated in Fig. 1. Briefly, SEQ
ID NO: 1 was 2-4 times less toxic to human, beagle or rat red blood cells than SEQ ID NO: 13.
In mouse or bovine red blood cells, the differences were negligible.
Additional derivatives of SEQ ID NO: 1 have been synthesized by solid-phase synthesis
as described above. Antimicrobial activity was assessed by determining MICs against S.
aureus and E. coli, as described above. The results of these experiments are provided in Table
Table 2
Sequence S.aureus
SEQ ID E138I<IDZ5922
ATCC 29213
M “M
GWCFRVCYRGICYRRCRD
KFCFRVCYRGICYRRCRD
KWCFYVCYRGICYRRCRD 0790.07 N—LN _L_Lm
RWCFRVCYRGICYRRCRD COO‘)
||KWCFRVCRRGICYRRCRD
CYGRICYRRCRD
KWCFRVCYRGVCYRRCRD _L_L 0707;
KWCFRVCYRGACYRRCRD .9793 N—L
KWCFRVCYRGFCYRRCRD 97m N—L (AD—L LL07
SEQ ID NO: 10 KWCFRVCYRGICYHRCRD
SEQ ID NO: 11 CYRGICYRRCND 07.07 MN O_|. bob)
SEQ ID NO: 1 KWCFRVCYRGICYRRCRD to —L .0 oo
Antimicrobial activity of the peptides listed in Table 2 against different strains of MSSP
(Mathiciilin~Susceptible Staphyiomccus pseudmtermed’ius) and MRSP (Methicillin-Resistant
Staphylococcus pseudim‘ermedius) was further assessed. The results are in Tables 3 and 4,
respectively.
Table 3. MIC against selected strains of MSSP
L0098
HHHWH 1.6
HOE 1.6
Table 4. MIC against selected strains of MRSP
%’ 3 § §
0 go go to
A 01 m
---—“In
“—---
Im—n-
mama:
onal peptides were synthesized as described above. Antimicrobial properties of
these peptides have been determined and are summarized below.
Table 5. Effect of selected antimicrobial sequences on strains of MSSP
_|. O \l _|. 07 _|. 07 _|. 07 _|. 07 _|. 07 _|. 07 _|. 07 _|. 07 _|. 07
_L_L 00 L003 (:0 _L (:0 _L (:0 ' _L (:0 _|. (:0 _|. (:0 ' _|. 00 _L 9° _L 00 _L
_|. 07 _|. 07 _|. 07 _|. 07 _|. 07 _|. 07 _L _L 07 _L 07
_L _L o _|. 07 (A) —'- (A) '_. _|. 07 _|. 07 (A) '_. 407
_|. _|. N _|. 07 _|. 07 _|. 07 _L_L_L_L_Lm_|. 0707'—L07 O CO _|. 07 _|. 07
_|. _|. (:0 _|. 07 _|. 07 _|. 07 _|. 07 _|. 07 _|. 07 _L_Lm 0707 _L_Lm 0707—'- _L_Lm 0707—'-
_L _L .1; _|. 07 (A) —'- (A) '_. _|. 07 (A) —'- (A) '_. m-L-Lm-Lm-L —'-0707—'-07—'-07 (:0 _|. (:0 _|.
115 _|. 07 _|. 07 _|. 07 _|. 07 O (3007CO _LOO_L_L_L_L_L 07COCOO707070707 _LOO_L_L_L_L_L 0700000707070707 _|. 07 _|. 07 _|. 07 _|._|. . 0707
_|. _|. 07 _|. 07 _LO_L_L_L_L_L 07(200707070707 _L_L_L_L 07070707 _|. . 07 O_|. _|. 07 _|. 07 _|. 07 _|. .07
_|. _|. \l _|. 07 _|. 07 _|. 07
_|. _|. m _|. 07 _|. .07 _|. 07 9° _|.
(DCDNN AOLOGJ _|. 07 _|. 07 Am 07—'- _|. 07
_L 07 .09 COCO _|. 07
—|-O 07CO _|. 07
_I. 07 _L 07 _L_L_L_L_L_L 070707070707 _L_L_L_L_L_L 070707070707
Table 6
Effect of crobial peptides on different bacteria. MR = MRSP, SA = S. aureus, EC =
E. coli
SP = S. pseudintermedius
(I) (I)
g 6 6 6 6 6 6 6 3
\l \I oo oo oo 00 N 4>
6 07 w c: —k 07 \l
Q (I) N 0 O 07 g 8
B 8 E1 8 B 8 3 9
a 16 .6 1.6
a 1.6 .6 3.1
. 16 .8 1.6
102 3.1 . 1.6 .6 1.6
103 3.1 . 62 3.1
104 3.1 6 31 1.6
105 1.6 6 31 1.6
106 1.6 6 31 1.6
31 3.1
1.6 31 1.6
31 6.2 3.1
1.6 3.1 1.6
1.6 1.6
3.1 3.1
1.6 1.6
1.6 1.6
1.6 1.6
1.6 6. 1.6
1.6 3. 1.6
1.6 3. 1.6
1.6 3. 1.6
Safety of the peptides listed in table 2 was determining by measuring cell viability.
Canine-derived epithelial keratinocyte (CPEK) cells were propagated to determine cell viability
in the presence of peptides. Cells were grown from a frozen stock in CnT5 (with
ments) pre-warmed media in a T75 flask and incubated overnight at 37°C, 5% C02. Cells
were washed with phosphate buffer and replenished with rmed -9 media and
repeated for several days until cells reached a density of 6.6 x 104 cells/mL. Cells were then
transferred to a 384 well plate, allowed to settle and dosed with peptides and melittin control
peptide (50 uM to 0.05 uM) and incubated overnight at 37°C, 5% C02. 0.1% TritonX100 as
(HPE) and phosphate buffer alone as (ZPE) were added to the plates to calculate percent effect
once the assay was terminated with 10 uL CELLTITER-GLO® assay reagents for a luminescent
readout. The results are provided in Table 7.
Safety of the peptides listed in table 6 was determining by measuring cell ity as
described above. The results are provided in Table 8.
Table 8
@OHM)
These data demonstrate that the antimicrobial peptides of the t invention are not
only effective against the tested strains of bacteria but also safe, particularly for non-systemic,
e.g., topical, administration.
All publications cited in the specification, both patent publications and tent
publications, are indicative of the level of skill of those skilled in the art to which this invention
pertains. All these publications are herein fully incorporated by reference to the same extent as
if each individual publication were specifically and dually indicated as being incorporated
by reference.
Although the invention herein has been described with reference to ular
embodiments, it is to be understood that these embodiments are merely illustrative of the
principles and applications of the present invention. It is therefore to be understood that
numerous cations may be made to the illustrative embodiments and that other
arrangements may be devised t departing from the spirit and scope of the present
invention as defined by the following claims.
Claims (1)
1. An amino acid sequence of 17-22 amino acids long and comprising, at its N-terminus, SEQ ID No.12 (XOX1XZC X3X4X5CX6X7X8XQCYX10X11CX12X13) Wherein X0 is absent or proline X1 is lysine, arginine, glycine, or proline; X2 is phenylalanine, tryptophan, or arginine; X3 is alanine, valine or tryptophan; X4 is arginine, tyrosine or phenylalanine; X5 is valine or alanine; X6 is tyrosine, arginine, lysine or tryptophan; X7 is arginine, phenylalanine, or glycine; X8 is arginine or glycine; X9 is isoleucine, alanine, alanine, tyrosine or valine; X10 is arginine or histidine; X11 is arginine or lysine X12 is arginine, lysine, alanine, or asparagine; X13 is a 0-4 amino-acid-Iong polypeptide; with provisos that if X0 is proline, then X1 is not e; if said amino acid sequence ses SEQ ID NO: 13 (KWCFRVCYRGICYRRCR), or SEQ ID NO: 28 VCYRGICYRKCR) then X13 is 1-4 amino acids long; if X13 is 1 amino acid long or longer, then the N-terminal amino acid in X13 is asparagine, ne, aspartic acid or glutamic acid; it the amino acid at position corresponding to position 1 of SEQ ID NO: 1 is glycine, then said glycine is not acyl- or palmitic acid — modified; it amino acid is X11 lysine then X6X7X8X9 (SEQ ID NO: 14) is not RRRF (SEQ ID NO: 15); and if the amino acid is GFCWYVCYRGICYRRCN (SEQ ID NO: 16) then the C- terminal gine is ed. The amino acid sequence according to claim 1, wherein X0 is proline. The amino acid sequence according to claim 1 or 2, wherein X6 is arginine or tyrosine. The amino acid sequence according any one of claims 1-3, wherein X7 is arginine or glycine. The amino acid sequence according to claim 4, wherein X7 is arginine. The amino acid sequence according any one of claims 1-5 wherein X9 is isoleucine, alanine, phenylalanine, or . The amino acid sequence of claim 1, wherein X9 is isoleucine. WO 13181
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62/595,725 | 2017-12-07 |
Publications (1)
Publication Number | Publication Date |
---|---|
NZ795498A true NZ795498A (en) | 2022-12-23 |
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