U.S. patent application number 17/314473 was filed with the patent office on 2021-08-26 for theta defensin analogs and methods of use.
The applicant listed for this patent is The University of Southern California. Invention is credited to Justin B. Schaal, Michael E. Selsted, Dat Q. Tran.
Application Number | 20210261623 17/314473 |
Document ID | / |
Family ID | 1000005572064 |
Filed Date | 2021-08-26 |
United States Patent
Application |
20210261623 |
Kind Code |
A1 |
Selsted; Michael E. ; et
al. |
August 26, 2021 |
THETA DEFENSIN ANALOGS AND METHODS OF USE
Abstract
Uses of novel peptide analogs of a .theta.-defensin that have
been developed which provide a biphasic effect in treating
infection and/or sepsis are described. These analogs are active at
concentrations below those needed to provide a bactericidal or
bacteriostatic effect, and function by initially recruiting
effector cells of the immune system to address the infective
organism followed by regulation of the immune system to down
regulate the inflammatory response characteristic of sepsis and
septic shock. These novel .theta.-defensin analogs are protective
at concentrations where naturally occurring .theta.-defensins have
no apparent effect, and include a core set of structural and
sequence features not found in native .theta.-defensin.
Inventors: |
Selsted; Michael E.;
(Pasadena, CA) ; Tran; Dat Q.; (Alhambra, CA)
; Schaal; Justin B.; (Orange, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Southern California |
Los Angeles |
CA |
US |
|
|
Family ID: |
1000005572064 |
Appl. No.: |
17/314473 |
Filed: |
May 7, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16914038 |
Jun 26, 2020 |
11021518 |
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17314473 |
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62867000 |
Jun 26, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/00 20180101;
C07K 7/64 20130101; A61K 38/00 20130101 |
International
Class: |
C07K 7/64 20060101
C07K007/64; A61P 31/00 20060101 A61P031/00 |
Goverment Interests
[0002] This invention was made with government support under Grant
Nos. R01 AI125141 and R44 AR068833, awarded by the National
Institutes of Health (NIH). The government has certain rights in
the invention.
Claims
1. A method of treating a condition resulting from infectious
disease or dysregulation of an immune or inflammatory response in
an individual, comprising: identifying that the individual is in
need of treatment for the infections disease or the condition
resulting from dysregulation of an immune or inflammatory response;
and administering a cyclic peptide to the individual wherein the
cyclic peptide has the following structure: ##STR00001## wherein
AA3 and AA12 are cysteines joined by a disulfide bond, AA5 and AA10
are cysteines joined by a disulfide bond, AA4 is a first
hydrophobic amino acid, AA11 is a second hydrophobic acid, AA6 is
arginine, AA7 is arginine, AA8 is arginine, wherein AA1, AA2, AA9,
AA13 and AA14 are amino acids, and wherein the cyclic peptide has
four arginine residues that provide a positively charged content of
about 28% at physiological pH.
2. The method of claim 1, wherein the first hydrophobic amino acid
and the second hydrophobic amino acid are leucine or
isoleucine.
3. The method of claim 1, wherein AA1 is glycine.
4. The method of claim 1, wherein AA2 is a third hydrophobic amino
acid.
5. The method of claim 1, wherein AA9 is a fourth hydrophobic amino
acid.
6. The method of claim 1, wherein AA13 is glycine.
7. The method of claim 1, wherein AA14 is arginine.
8. The method of claim 1, wherein at least one of AA4 and AA11 is
not alanine or serine.
9. The method of claim 1, wherein the cyclic peptide is MTD12813
(SEQ ID NO. 2).
10. The method of claim 1, wherein the method provides a biphasic
response on administration to an organism, wherein the biphasic
response comprises a phase of moderation of host inflammatory
response.
11. The method of claim 1, wherein the method inhibits TACE
activity.
12. The method of claim 1, wherein the method suppresses at least
one of expression, processing, and release of a proinflammatory
cytokine.
13. The method of claim 1, wherein the cyclic peptide retains
activity following exposure to environmental extremes of
temperature, low pH, freezing and/or thawing, and dissolution in a
biological matrix.
14. The method of claim 1, wherein the cyclic peptide is
non-immunogenic at doses effective to treat the condition resulting
the infectious disease or dysregulation of an immune or
inflammatory response.
15. The method of claim 1, wherein the condition results from
dysregulation of an immune or inflammatory response is selected
from the group consisting of rheumatoid arthritis, inflammatory
bowel disease, and sepsis.
16. The method of claim 1, wherein the condition results from the
infectious disease, wherein the infections disease results from one
of the group consisting of a viral pathogen and a bacterial
pathogen.
17. The method of claim 1, wherein the cyclic peptide is
administered in combination with pharmaceutical agent selected from
the group consisting of an anti-viral drug, an anti-bacterial drug,
an anti-fungal drug, an anti-inflammatory drug, a vasopressor, and
a biologic.
18. The method of claim 17, wherein the cyclic peptide and the
pharmaceutical agent in combination provide a synergistic effect
that exceeds the additive effects of the cyclic peptide and the
pharmaceutical agent when administered individually.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/914,038, filed Jun. 26, 2020, which claims
the benefit of U.S. Provisional Patent Application No. 62/867,000
filed on Jun. 26, 2019. This and all other referenced extrinsic
materials are incorporated herein by reference in their entirety.
Where a definition or use of a term in a reference that is
incorporated by reference is inconsistent or contrary to the
definition of that term provided herein, the definition of that
term provided herein is deemed to be controlling.
FIELD OF THE INVENTION
[0003] The field of the invention is biomedicine, specifically
peptide drugs and their applications.
BACKGROUND
[0004] The background description includes information that may be
useful in understanding the present invention. It is not an
admission that any of the information provided herein is prior art
or relevant to the presently claimed invention, or that any
publication specifically or implicitly referenced is prior art.
[0005] Under some circumstances the body's protective inflammatory
response can result in injury or even death. For example, sepsis or
septic shock is a result of an inflammatory immune response.
Treatment of septic shock is primarily by the administration of
antibiotics and provision of supportive care and vasopressive drugs
to stabilize blood pressure. Morbidity, however, remains
significant. Death rates from such inflammatory responses range
from about 30% for sepsis to about 80% for septic shock.
Accordingly, there is significant interest identifying therapeutic
compounds that are effective in treating various aspects of sepsis
and of septic shock.
[0006] Defensins are a diverse family of small antimicrobial
proteins that are part of the body's nonspecific defense against
infection. There are three different and structurally distinct
classes of defensin proteins: alpha, beta, and theta defensins. The
.alpha. and .beta. defensins are linear, tri-disulfide containing
peptides having molecular weights of about 2.6 kDa or 4.5 kDa,
respectively. In contrast, .theta.-defensins are cyclic peptides
(i.e. circular peptides wherein the backbone is formed by
sequential peptide bonds with neither a free amino or carboxyl
terminus) composed of 18 amino acids.
[0007] .theta.-defensins are expressed in tissues of rhesus
monkeys, baboons, and other Old World monkeys. They are not present
in humans and other hominids. Naturally occurring .theta.-defensins
are composed of 18 backbone cyclized (i.e. through the alpha-amine
groups rather than side chain moieties) peptides stabilized by
three disulfide bonds. These three disulfide bonds are conserved
among all known .theta.-defensins. .theta.-defensins were
originally discovered and classified as defensins based on the
antimicrobial properties of the peptides. More recently it has been
found that .theta.-defensins can have potent immunomodulatory
effects.
[0008] International Patent Application Publication No. WO
2007/044998 (to Leherer et al) describes relationships between
structure and biological activity for retrocyclin peptides and
analogs of such peptides that include varying degrees enantiomer
content in an attempt to derive structure/activity relationships.
These analogs, however, retain the length and structure of the
native retrocyclin. In addition, the reference is only instructive
for antibacterial activity.
[0009] Peptide analogs of various defensins have been investigated.
For example, European Patent Application EP2990415 (to Colavita et
al) describes circularized analogs of a .beta.-defensin that show
improved antibiotic effectiveness relative to the parent protein.
Such .beta.-defensins, however, have been shown to stimulate
release of pro-inflammatory cytokines, which raises safety concerns
and limits their utility.
[0010] United States Patent Application Publication No. US
2003/0022829 (to Maury et al) describes synthesis and biologic
activity of chimeric .theta.-defensins and speculates on the
possibility of making conservative amino acid substitutions,
however these appear to retain the length and structure of native
.theta.-defensins. U.S. Pat. No. 10,512,669 (to Selsted et al)
describes several tetradecapeptide .theta.-defensin analogs derived
from RTD-1, and their biological properties.
[0011] There remains, therefore, a need for safe and effective
compounds for the management and/or treatment of sepsis/septic
shock and the physiologically related disorders resulting from
dysregulated inflammatory reactions.
SUMMARY OF THE INVENTION
[0012] The inventive subject matter provides synthetic analogs of
.theta.-defensins that have improved activity in treating sepsis
and/or septic shock relative to native .theta.-defensins, at
concentrations that are below those at which the analogs have
direct bactericidal and/or bacteriostatic effect.
[0013] One embodiment of the inventive concept is a cyclic peptide
consisting of 14 amino acids and having a structure as shown in
FIG. 2A, which includes two disulfide bonds between two pairs of
cysteines. In such a peptide AA3 and AA12 are cysteines joined by a
disulfide bond, AA5 and AA10 are cysteines joined by a disulfide
bond, AA4 is a first hydrophobic amino acid, AA11 is a second
hydrophobic acid, AA6 is arginine, AA7 is arginine, AA8 is
arginine. The cyclic peptide has a total of four arginine residues
that provide a positive charge content of about 28% at
physiological pH. The first hydrophobic amino acid and the second
hydrophobic amino acid can be leucine or isoleucine. AA1 can be
glycine. AA2 can be a third hydrophobic amino acid, such as valine
or phenylalanine. AA9 can be a fourth hydrophobic amino acid, such
as valine or phenylalanine. AA13 can be glycine. AA14 can be
arginine. In some embodiments AA4 cannot be alanine or serine. In
some embodiments AA11 cannot be alanine or serine. In some
embodiments the cyclic peptide is MTD12813 (SEQ ID NO. 2).
[0014] Such a cyclic peptide can be an analog of a .theta.-defensin
that provides improved survival when applied systemically in a
murine sepsis model relative to the .theta.-defensin itself. In
some embodiments the cyclic peptide provides a biphasic response on
application to a murine model of sepsis. Such a biphasic response
includes a first phase of recruitment of host effector cells having
antimicrobial activity and a second phase of moderation of host
inflammatory response. In some embodiments the cyclic peptide has a
TACE inhibiting activity, and/or suppresses at least one of
expression, processing, and release of TNF.
[0015] Such a cyclic peptide retains activity following exposure to
environmental extremes of temperature, low pH, freezing and/or
thawing, and dissolution in a biological matrix (such as blood,
plasma, or serum. In some embodiments such a cyclic peptide is
non-immunogenic at doses effective to treat or prevent sepsis
and/or septic shock. Such cyclic peptides can activate a host
immune system to enhance host clearance of pathogens, and can also
have an activity that modulates inflammation to enhance disease
resolution and survival at doses effective to treat or prevent
septic shock.
[0016] Another embodiment of the inventive concept is a method of
treating or preventing septic shock by administering a cyclic
peptide as described above to an animal at risk of septic
shock.
[0017] Another embodiment of the inventive concept is the use of a
cyclic peptide as described above in treating or preventing sepsis
and/or septic shock, or the use of such a cyclic peptide in
preparing a medicament that is effective in treating or preventing
septic shock.
[0018] Various objects, features, aspects and advantages of the
inventive subject matter will become more apparent from the
following detailed description of preferred embodiments, along with
the accompanying drawing figures in which like numerals represent
like components.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1: FIG.1 shows a schematic depiction of exemplary
cyclic peptides referred to throughout. RTD-1 (SEQ ID NO. 1) is a
naturally occurring octadecapeptide .theta.-defensin. Remaining
peptides are .theta.-defensins analogs.
[0020] FIGS. 2A and 2B: FIG. 2A shows a schematic of a cyclic
defensin analog, showing numeric designations for amino acids by
position along the cyclic chain. FIG. 2B provides an example of the
application of these designations to amino acids of the MTD12813
(SEQ ID NO. 2) peptide.
[0021] FIG. 3: Shows the results of efficacy studies of macrocyclic
peptides in a murine carbapenem resistant Klebsiella pneumoniae
sepsis model.
[0022] FIG. 4: Shows the results of potency studies of MTD12813
(SEQ ID NO. 2) in a murine carbapenem resistant Klebsiella
pneumoniae sepsis model.
DETAILED DESCRIPTION
[0023] The inventive subject matter provides novel peptides that
induce a biphasic effect in treating infection and/or sepsis, by
initially recruiting effector cells of the immune system to address
the infective organism followed by regulation of the immune system
to prevent a systemic inflammatory response as found in sepsis and
septic shock. The novel peptides are analogs of naturally occurring
.theta.-defensins with sequences that have been modified to provide
an indirect antimicrobial effect via recruitment of effector cells
of the host immune system and to prevent and/or treat sepsis/septic
shock. These novel .theta.-defensin analogs are effective at
subantimicrobial plasma concentrations that do not provide a direct
anti-microbial effect (i.e. that do not generate a bactericidal or
a bacteriostatic effect) in the absence of host innate immune
effectors. Such .theta.-defensin analogs are protective at
concentrations where native .theta.-defensins have no apparent
effect, and include a core set of structural and sequence features
not found in native .theta.-defensins.
[0024] Within the context of this application, a "subantimicrobial"
concentration in regard to a should be understood to be a
concentration at which the compound so described has no
antimicrobial effect when applied to the a representative microbial
pathogen in vitro (e.g. in a liquid culture medium), e.g. in the
absence of host immune effectors For example, a subantimicrobial
concentration of a compound in regard to Klebsiella pneumoniae
would be a concentration that is less than that which demonstrates
an antimicrobial effect against the organism in an in vitro setting
(e.g. in the absence of host immune effectors). Such
subantimicrobial concentrations can be determined experimentally
(for example, by culture from a patient sample) or, preferably,
from historical data.
[0025] The following description includes information that may be
useful in understanding the present invention. It is not an
admission that any of the information provided herein is prior art
or relevant to the presently claimed invention, or that any
publication specifically or implicitly referenced is prior art.
[0026] In some embodiments, the numbers expressing quantities of
ingredients, properties such as concentration, reaction conditions,
and so forth, used to describe and claim certain embodiments of the
invention are to be understood as being modified in some instances
by the term "about." Accordingly, in some embodiments, the
numerical parameters set forth in the written description and
attached claims are approximations that can vary depending upon the
desired properties sought to be obtained by a particular
embodiment. In some embodiments, the numerical parameters should be
construed in light of the number of reported significant digits and
by applying ordinary rounding techniques. Notwithstanding that the
numerical ranges and parameters setting forth the broad scope of
some embodiments of the invention are approximations, the numerical
values set forth in the specific examples are reported as precisely
as practicable. The numerical values presented in some embodiments
of the invention may contain certain errors necessarily resulting
from the standard deviation found in their respective testing
measurements.
[0027] As used in the description herein and throughout the claims
that follow, the meaning of "a," "an," and "the" includes plural
reference unless the context clearly dictates otherwise. Also, as
used in the description herein, the meaning of "in" includes "in"
and "on" unless the context clearly dictates otherwise.
[0028] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member can be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. One or more members of a group can be included in, or
deleted from, a group for reasons of convenience and/or
patentability. When any such inclusion or deletion occurs, the
specification is herein deemed to contain the group as modified
thus fulfilling the written description of all Markush groups used
in the appended claims.
[0029] The recitation of ranges of values herein is merely intended
to serve as a shorthand method of referring individually to each
separate value falling within the range. Unless otherwise indicated
herein, each individual value is incorporated into the
specification as if it were individually recited herein. All
methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g. "such as") provided with respect to certain embodiments
herein is intended merely to better illuminate the invention and
does not pose a limitation on the scope of the invention otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element essential to the practice of the
invention.
[0030] The following discussion provides many example embodiments
of the inventive subject matter. Although each embodiment
represents a single combination of inventive elements, the
inventive subject matter is considered to include all possible
combinations of the disclosed elements. Thus if one embodiment
comprises elements A, B, and C, and a second embodiment comprises
elements B and D, then the inventive subject matter is also
considered to include other remaining combinations of A, B, C, or
D, even if not explicitly disclosed.
[0031] One should appreciate that the disclosed peptides provide
many advantageous technical effects, including provision of a
biphasic response that is effective in reducing mortality from
sepsis/septic shock when administered in low, subantimicrobial
amounts.
[0032] Inventors have described synthetic cyclic tetradecapeptide
analogs of the theta defensin RTD-1 that showed some of the
activities of the parent peptide, despite their smaller size and
reduced number of disulfide bonds. The structures of natural theta
defensin RTD-1 (SEQ ID NO. 1) and some exemplary synthetic cyclic
tetradecapeptide analogs are shown in FIG. 1. As shown, RTD-1
(which is expressed naturally in rhesus monkeys) is a cyclic
octadecapeptide that includes 3 pairs of cysteines coupled by
disulfide bonds that transit the circular primary structure of the
peptide. A number of examples of synthetic (i.e., non-naturally
occurring) analogs of RTD-1 are shown. Each of the exemplary
synthetic analogs is a tetradecapeptide that includes 2 pairs of
cysteines coupled by disulfide bonds. These disulfide bonds transit
the circular primary structure of the synthetic peptides to form a
"box" substructure that incorporates additional amino acids. It
should be appreciated that these exemplary analogs show varying
degrees of sequence identity with RTD-1, and in some instances show
conservative amino acid substitutions near and between the "box"
defined by cysteines of the synthetic peptide analogs.
[0033] Inventors have prepared and screened a series of
.theta.-defensin analogs based on the analog designated MTD1280
(see FIG. 1, SEQ ID NO. 3) a synthetic peptide that provides
substantially improved effects (relative to RTD-1) in long term
survival of mice in a model of sepsis, and that provides these
effects at surprisingly low concentrations. It should be
appreciated that long term survival of sepsis requires both
management of the infecting organism and of the shock induced by
the host response to the infection, either of which can lead to
death.
[0034] While examples of activity against sepsis and/or septic
shock are provided, Inventors believe that .theta.-defensin analogs
as described herein can be effective at treating a variety of
conditions resulting from dysregulation of the immune or
inflammatory response, including chronic conditions. Examples of
such chronic conditions include rheumatoid arthritis and
inflammatory bowel disease.
[0035] The Inventors note that .theta.-defensins have been found to
have antiviral activity, and believe that .theta.-defensin analogs
of the inventive concept can similarly provide anti-viral activity,
and can prove useful in treating viral disease and inflammatory
sequelae of viral infection. Such treatment includes prophylaxis
and/or active disease. In some embodiments active disease so
treated is symptomatic. In other embodiments active disease so
treated is asymptomatic.
[0036] Surprisingly, .theta.-defensin analogs were identified that
provide a biphasic response in modulating the immune system. The
initial effect is opsonic, recruiting effector cells to the sepsis
site. This serves to combat infection, and surprisingly was found
to occur at concentrations of the .theta.-defensin analog that
demonstrated neither a bactericidal nor a bacteriostatic effect
(i.e., subantimicrobial concentrations). Following this initial
opsonic effect these synthetic .theta.-defensin analogs exhibit a
longer term immunomodulatory effect (for example, reducing TNF,
IL-6 and other inflammatory cytokines) that contributes to long
term survival in preventing septic shock.
[0037] As noted above, examples of a naturally occurring
.theta.-defensin and exemplary .theta.-defensin analogs are shown
in FIG. 1. It should be appreciated that these are cyclic peptides
that lack conventional amino- and carboxyl- termini; as such amino
acid sequence information as provided in accompanying amino acid
sequence listings should not be construed as based on a discrete
N-terminus or C-terminus. The primary structure of the naturally
occurring .theta.-defensin RTD-1 (SEQ ID NO. 1) is shown at the top
of FIG. 1. The remaining peptides are exemplary non-natural analogs
of .theta.-defensins. In the 14-amino acid analog series, it should
be appreciated their three dimensional structures include a first
.beta.-turn formed by amino acids 6 to 9 and a second .beta.-turn
formed by amino acids 13, 14, 1, and 2 as designated using a
numbering system adapted for use with cyclic .theta.-defensins and
their analogs and as shown in FIGS. 2A and 2B.
[0038] Although these cyclic peptides do not have free amino- or
carboxyl-termini, amino acid positions within the cyclic structure
can be designated based on their positions relative to certain
structural features (such as disulfide bonds and/or a distinctive
`triplet` of arginines). Such a set of designations as utilized for
this purpose within this application is illustrated in FIG. 2A,
where amino acids are designated 1 to 14 (AA1, AA2, etc.) in a
cyclic teradecapeptide structure having a circular and continuous
chain of peptide bonds through the primary amines of the individual
amino acids (i.e. not through side chain groups), and in which two
intra-peptide covalent bonds occur between side chains of cysteine
amino acids designated AA3 and AA12 and between side chains of
cysteine amino acids designated amino acids AA5 and AA10. FIG. 2B
depicts application of amino acid positions as within the context
of this application, as applied to an exemplary synthetic cyclic
tetradecapeptide (MTD12813 peptide, SEQ ID NO. 2). Amino acid
identity is designated using single-letter amino acid code.
[0039] FIG. 3 shows the results of application of RTD-1 (SEQ ID NO.
1) and exemplary novel synthetic tetradecapeptides in a murine
model of sepsis utilizing an antibiotic-resistant bacteria that
results in 75% mortality (i.e., 25% survival) if untreated. BALB/c
mice were infected interperitoneally with 3-5.times.10.sup.8 CFU of
a carbapenem resistant strain of Klebsiella pneumoniae (KPC+-Kp
BAA-1705 (ATCC)) and treated with peptide one hour post infection.
The left panel of FIG. 3 shows exemplary results of comparative
studies between the synthetic peptide MTD12813 (SEQ ID NO. 2)and
naturally occurring RTD-1 administered at 5 mg/kg. The right panel
of FIG. 3 shows exemplary results of comparative studies between
the synthetic peptides MTD12813 and MTD1280 (SEQ ID NO. 3)
administered at 0.5 mg/kg. P-values were determined by Fisher's
exact test. The therapeutic peptides were provided
intraperitoneally 1 hour after induction of sepsis.
[0040] As shown in the left panel of FIG. 3, at 5 mg/kg RTD-1 (SEQ
ID NO. 1) provides only partial protection (70% survival vs 25% for
sham controls), whereas MTD12813 (SEQ ID NO. 2) provides complete
protection from sepsis and septic shock. As shown in the right
panel of FIG. 3, at 0.5 mg/kg the effects of MTD1280 (SEQ ID NO. 3)
on survival are essentially identical to that of the sham control,
whereas MTD12813 provides almost 90% survival. This difference is
highly significant (P=0.0031).
[0041] It should be appreciated that at these dosages a
.theta.-defensin and/or its analog does not produce a drug Cmax
sufficient to have an appreciable direct antimicrobial (e.g.
bactericidal, bacteriostatic) effect. Without wishing to be bound
by theory, Inventors believe that the antimicrobial effects of
MTD12813 (SEQ ID NO. 2) characteristic of the first phase of the
biphasic response are the result of a systemic immune activating
mechanisms that result in recruitment and stimulation of cells from
the host immune system, which in turn phagocytose, kill, and clear
the pathogen. In addition, MTD12813 moderates the inflammatory
response to mitigate deleterious effects of hyperactivated and/or
unresolved systemic inflammation, such as cytokine storm.
[0042] FIG. 4 shows typical results for survival studies in such a
murine survival study for various concentrations of MTD12813 (SEQ
ID NO. 2), utilizing the same murine sepsis model as used in
studies shown in FIG. 3. BALB/c mice were infected i.p. with
KPC+-Kp BAA-1705 (ATCC) and treated with a single dose of MTD12813,
at the levels indicated, 1 hour post infection. The significance
(P-values determined by Fisher's exact test) of the therapeutic
effect for each dose is shown. As shown, 1.25 mg/kg of this novel
.theta.-defensin analog is as effective as 5 mg/kg, and a dose of
MTD12813 as low as 0.5 mg/kg is also highly effective. It should be
appreciated that 0.5 mg/kg is the lowest dose tested, and that
Inventors believe that MTD12813 is effective at still lower doses,
for example down to 0.25 mg/kg, 100 mg/kg, 50 mg/kg, 25 mg/kg, or
10 .mu.g/kg.
[0043] As noted above, MTD12813 (SEQ ID NO. 2) was identified in
screening studies of a range of cyclic peptide analogs of the
.theta.-defensin RTD-1 (SEQ ID NO. 1). RTD-1 is a cationic,
arginine-rich cyclic peptide that includes 18 amino acids and 3
disulfide bonds between pairs of cysteines (FIG. 1). Other active
.theta.-defensin analogs are also shown in FIG. 1.
[0044] Inventors have identified a number of novel .theta.-defensin
analogs that show superior performance relative to MTD1280 (SEQ ID
NO. 3), despite having similar covalent structures (e.g., length,
cyclic configuration, two pairs of disulfide bonds, and cationic
character). Features evaluated included survival efficacy in
antibiotic resistant K. pneumoniae sepsis, biocompatibility (lack
of toxicity), in vitro suppression of TNF-.alpha. (TNF) release,
and inhibition of TACE. Amino acid sequences of exemplary cyclic
peptides are shown in Table 1. It should be appreciated that amino
acids identities are indicated using the numerical designation for
corresponding positions within the cyclic structures as established
in FIG. 2. Properties and activities associated with these peptides
are shown in Table 2
TABLE-US-00001 TABLE 1 Analog 1.sup.st .beta. turn 2.sup.nd .beta.
turn SEQ name 3 4 5 6 7 8 9 10 11 12 13 14 1 2 ID NO. MTD12813 C I
C R R R V C I C G R G V 2 MTD1280 C I C R R R F C L C R R G V 3
MTD1284 C L C R R G V C L C R R G V 4 MTD1281 C I C R R G V C I C R
R G V 5 MTD12815 C A C A R R F C A C R R G V 6 MTD12811 C S C R R R
F C I C R R G V 7 MTD12812 C I C R R R F C S C R R G V 8 MTD12810 C
S C R R R F C L C R R G V 9 MTD1286 C I C R R R F C L C R R G A 10
MTD1289 C A C R R R F C A C R R G V 11 Amino acid positions are
designated according to the convention shown in FIG. 2A.
TABLE-US-00002 TABLE 2 Properties and activities of cyclic peptide
analogs of the .theta.-defensin RTD-1 as listed in Table 1. Kp
sepsis TACE TNF % Analog Positive Molecular % survival IC.sub.50
suppression name charge Weight at 5 mg/kg .mu.g/mL at 5 mg/kg
MTD12813 4 1572 100 0.858 95.6 MTD1280 5 1720 80-100 2.202 98.7
MTD1284 4 1572 53.3 1.150 78.8 MTD1281 4 1572 40 0.546 83.0
MTD12815 4 1550 toxic 1.828 not tested MTD12811 5 1693 30 3.192
37.5 MTD12812 5 1693 20 1.869 14.9 MTD12810 5 1693 0 2.613 2.0
MTD1286 5 1691 toxic 0.515 not tested MTD1289 5 1635 toxic 2.737
not tested
[0045] A number of sequence features were identified that confer
superior activity to RTD-1- and MTD1280-derived analogs compared to
these reference peptides. All active .theta.-defensin analogs can
have at least: [0046] Two disulfide bonds, between Cys3 and Cys12
and between Cys5 and Cys10, respectively. [0047] A hydrophobic
amino acid positioned between Cys3 and Cys5 and a hydrophobic amino
acid positioned between Cys10 and Cys12 in the primary structure of
the .theta.-defensin analog (i.e. at positions 4 and 11),
preferably leucine or isoleucine. In combination with the disulfide
bonds noted above this defines a feature referred to as the "C-X-C
box" within the circular primary structure of the peptide, where
"C" is a cysteine and "X" is preferably either leucine or
isoleucine. [0048] In some embodiments, a C-X-C box that includes
arginine. [0049] A total of four arginine residues that provide the
peptide with a charge of +4 at physiological pH. [0050] A triplet
of adjacent arginines at positions 6, 7, and 8, i.e. within the
first .beta.-turn. In some embodiments active .theta.-defensin
analogs can also include one or more of the following features:
[0051] A glycine at position 1 and a glycine at position 13. [0052]
Hydrophobic amino acids at position 2 and position 9, preferably
valine or phenylalanine. [0053] An arginine within the second
.beta.-turn (e.g. at position 14).
[0054] Toxicity of candidate peptides suggests that active
.theta.-defensin analogs should not include one or more of: [0055]
An alanine at position 4. [0056] An alanine at position 11.
[0057] Accordingly, Inventors believe a .theta.-defensin analog
that include a "C-X-C box" structure as described above, a triplet
of adjacent arginine residues at positions 6, 7, and 8, a
hydrophobic amino acid (e.g., valine or phenylalanine) at position
9, and having a net positive charge of +4 (about 28% of total amino
acid content) due to arginine content will be effective in reducing
mortality and/or improving long term survival in sepsis, and can be
effective in treating other conditions characterized by
dysregulation of an inflammatory or immune response.
[0058] Analogs of .theta.-defensins as described herein can be
applied using any suitable method. For example, such analogs can be
provided by injection or infusion. The high degree of effectiveness
observed for some .theta.-defensin analogs indicates that these can
be provided to an individual in need of treatment in effective
amounts by simple subcutaneous, intradermal, subdermal,
intravenous, and/or intramuscular injection.
[0059] Alternatively, the low molecular weight and high degree of
stability conferred by circular structure and the presence of
disulfide bonds can allow for oral administration of
.theta.-defensin analogs of the inventive concept. Such oral
administration can include administration of a solution or
suspension of the .theta.-defensin analog in a liquid
pharmaceutical carrier suitable for oral administration. In some
embodiments a .theta.-defensin analog can be provided in a dry or
lyophilized form that is reconstitute in a liquid media prior to
oral administration. Such dry or lyophilized formulations can
include a stabilizer. Suitable stabilizers include carbohydrates
(e.g., mannitol, sucrose, trehalose) and/or proteins (e.g.,
albumin).
[0060] Alternatively, analogs of .theta.-defensin can be provided
in a tablet, capsule, pill, or other suitable solid and compact
form for oral administration. Such formulations can include
coatings, shells, or similar components that provide for delayed
release of the .theta.-defensin analog (for example, delaying
release until reaching the small intestine). Such formulations can
include the .theta.-defensin in liquid form within an enclosure or
coating. Alternatively, such formulations can include a
.theta.-defensin analog in a dry or lyophilized form. Suitable dry
or lyophilized forms include powders, granules, and compressed
solids. Such dry or lyophilized formulations can include a
stabilizer. Suitable stabilizers include carbohydrates (e.g.,
mannitol, sucrose, trehalose) and/or proteins (e.g., albumin).
[0061] As noted above, .theta.-defensin analogs of the inventive
concept can effectively treat sepsis and/or septic shock. In some
embodiments such treatment is in response to an ongoing, acute
condition. In other embodiments such treatment is prophylactic, for
example used to prevent the development of septic shock when the
individual is suspected of having sepsis or a high probability of
developing sepsis. Treatment can be provided by administration of a
.theta.-defensin analog of the inventive concept on any suitable
schedule. For example, a .theta.-defensin analog can be provided as
a single dose, periodic doses, or as a continuous infusion.
Periodic doses can be administered at any suitable intervals.
Suitable intervals can be hourly, every 2 hours, every 4 hours, 4
times a day, 3 times a day, twice a day, once daily, every 2 days,
every 3 days, twice a week, weekly, every 2 weeks, every 4 weeks,
every 2 months, every 3 months, every 4 months, 3 times a year,
twice a year, or annually.
[0062] In some embodiments the mode of administration for a
.theta.-defensin analog can be modified during the course of
treatment. For example, a .theta.-defensin analog of the inventive
concept can initially be administered by intravenous injection or
infusion (e.g., to rapidly provide effective concentrations in
acute sepsis or septic shock), followed by intradermal injection,
intramuscular injection, and/or oral administration in order to
maintain an effective concentration over a remaining period of
treatment.
[0063] For prophylactic use, a .theta.-defensin analog can be
administered prior to the onset of observable symptoms. For
treatment of an active disease or condition a .theta.-defensin
analog can be administered for a period of suitable to effectively
treat the disease or condition. Such a period can be over for a
controlled period of time, or can be long term (e.g., for treatment
of chronic conditions).
[0064] In some embodiments of the inventive concept a
.theta.-defensin analog can be used in combination with other
pharmaceutically active compounds. Suitable compounds include a
.theta.-defensin, a different .theta.-defensin analog, an
antibacterial antibiotics, an antiviral, an antifungal antibiotic,
an anti-inflammatory drug (e.g., steroids, non-steroidal
anti-inflammatory drugs), a vasopressor, and/or a biologic (e.g.,
antibodies or antibody fragments). Such additional pharmaceutical
compounds can be provided on the same schedule as the
.theta.-defensin analog, or on an independent schedule. In some
embodiments a .theta.-defensin analog-containing formulation can be
provided that incorporates one or more of such additional
pharmaceutically active compounds. Inventors believe that such
cotherapy can provide a synergistic effect in which the cumulative
effect of administration of the .theta.-defensin analog in
combination with the additional pharmaceutically active compound
exceeds the sum of the individual effects observed with treatment
using the .theta.-defensin analog and the additional
pharmaceutically active compound in amounts corresponding to those
used for cotherapy.
[0065] It should be apparent to those skilled in the art that many
more modifications besides those already described are possible
without departing from the inventive concepts herein. The inventive
subject matter, therefore, is not to be restricted except in the
spirit of the appended claims. Moreover, in interpreting both the
specification and the claims, all terms should be interpreted in
the broadest possible manner consistent with the context. In
particular, the terms "comprises" and "comprising" should be
interpreted as referring to elements, components, or steps in a
non-exclusive manner, indicating that the referenced elements,
components, or steps may be present, or utilized, or combined with
other elements, components, or steps that are not expressly
referenced. Where the specification claims refer to at least one of
something selected from the group consisting of A, B, C . . . and
N, the text should be interpreted as requiring only one element
from the group, not A plus N, or B plus N, etc.
Sequence CWU 1
1
11118PRTMacaca mulatta 1Gly Phe Cys Arg Cys Leu Cys Arg Arg Gly Val
Cys Arg Cys Ile Cys1 5 10 15Thr Arg214PRTArtificial
SequenceSynthetic cyclic peptide MTD12813 2Gly Val Cys Ile Cys Arg
Arg Arg Val Cys Ile Cys Gly Arg1 5 10314PRTArtificial
SequenceSynthetic cyclic peptide MTD1280 3Gly Val Cys Ile Cys Arg
Arg Arg Phe Cys Leu Cys Arg Arg1 5 10414PRTArtificial
SequenceSynthetic cyclic peptide MTD1284 4Gly Val Cys Leu Cys Arg
Arg Gly Val Cys Leu Cys Arg Arg1 5 10514PRTArtificial
SequenceSynthetic cyclic peptide MTD1281 5Gly Val Cys Ile Cys Arg
Arg Gly Val Cys Ile Cys Arg Arg1 5 10614PRTArtificial
SequenceSynthetic cyclic peptide MTD12815 6Gly Val Cys Ala Cys Ala
Arg Arg Phe Cys Ala Cys Arg Arg1 5 10714PRTArtificial
SequenceSynthetic cyclic peptide MTD12811 7Gly Val Cys Ser Cys Arg
Arg Arg Phe Cys Ile Cys Arg Arg1 5 10814PRTArtificial
SequenceSynthetic cyclic peptide MTD12812 8Gly Val Cys Ile Cys Arg
Arg Arg Phe Cys Ser Cys Arg Arg1 5 10914PRTArtificial
SequenceSynthetic cyclic peptide MTD12810 9Gly Val Cys Ser Cys Arg
Arg Arg Phe Cys Leu Cys Arg Arg1 5 101014PRTArtificial
SequenceSynthetic cyclic peptide MTD1286 10Gly Ala Cys Ile Cys Arg
Arg Arg Phe Cys Leu Cys Arg Arg1 5 101114PRTArtificial
SequenceSynthetic cyclic peptide MTD1289 11Gly Val Cys Ala Cys Arg
Arg Arg Phe Cys Ala Cys Arg Arg1 5 10
* * * * *