U.S. patent application number 16/208731 was filed with the patent office on 2019-11-21 for ang (1-7) derviative oligopeptides for the treatment of pain and other indications.
The applicant listed for this patent is ARIZONA BOARD OF REGENTS ON BEHALF OF THE UNIVERSITY OF ARIZONA. Invention is credited to Brittany Forte, Meredith Hay, Evan Jones, John Konhilas, Tally Milnes, Robin L. Polt, Lajos Szabo, Todd Vanderah.
Application Number | 20190351009 16/208731 |
Document ID | / |
Family ID | 60089246 |
Filed Date | 2019-11-21 |
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United States Patent
Application |
20190351009 |
Kind Code |
A1 |
Hay; Meredith ; et
al. |
November 21, 2019 |
ANG (1-7) DERVIATIVE OLIGOPEPTIDES FOR THE TREATMENT OF PAIN AND
OTHER INDICATIONS
Abstract
The present invention provides oligopeptides, in particular,
Ang-(1-7) derivatives, and methods for using and producing the
same. In one particular embodiment, oligopeptides of the invention
have higher blood-brain barrier penetration and/or in vivo
half-life compared to the native Ang-(1-7), thereby allowing
oligopeptides of the invention to be used in a wide variety of
clinical applications including in treatment of cognitive
dysfunction and/or impairment, pain, and traumatic brain
injury.
Inventors: |
Hay; Meredith; (Tucson,
AZ) ; Konhilas; John; (Tucson, AZ) ; Polt;
Robin L.; (Tucson, AZ) ; Vanderah; Todd;
(Tucson, AZ) ; Forte; Brittany; (Tucson, AZ)
; Milnes; Tally; (Tucson, AZ) ; Jones; Evan;
(Tucson, AZ) ; Szabo; Lajos; (Tucson, AZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARIZONA BOARD OF REGENTS ON BEHALF OF THE UNIVERSITY OF
ARIZONA |
Tucson |
AZ |
US |
|
|
Family ID: |
60089246 |
Appl. No.: |
16/208731 |
Filed: |
December 4, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15401944 |
Jan 9, 2017 |
10183055 |
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16208731 |
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15134073 |
Apr 20, 2016 |
9670251 |
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15401944 |
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14801557 |
Jul 16, 2015 |
9796759 |
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15134073 |
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62027219 |
Jul 21, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 7/14 20130101; C07K
7/06 20130101; A61K 38/085 20130101; C07K 9/001 20130101 |
International
Class: |
A61K 38/08 20060101
A61K038/08; C07K 9/00 20060101 C07K009/00; C07K 7/06 20060101
C07K007/06; C07K 7/14 20060101 C07K007/14 |
Claims
1. A method for providing analgesia to a subject having a
neuropathy, the method comprising administering a therapeutically
effective amount of an oligopeptide derivative having the formula:
A.sup.1-A.sup.2-A.sup.3-A.sup.4-A.sup.5-A.sup.6-A.sup.7-A.sup.8
(SEQ ID NO:1) wherein A.sup.1 is selected from the group consisting
of aspartic acid and glycosylated forms thereof, glutamic acid and
glycosylated forms thereof, and alanine; A.sup.2 is selected from
the group consisting of arginine, histidine, and lysine; A.sup.3 is
selected from the group consisting of valine, alanine, isoleucine,
and leucine; A.sup.4 is selected from the group consisting of
tyrosine and glycosylated forms thereof, phenylalanine, and
tryptophan; A.sup.5 is selected from the group consisting of
isoleucine, valine, alanine, and leucine; A.sup.6 is selected from
the group consisting of histidine, arginine, and lysine; A.sup.7 is
serine or a glycosylated form thereof; and A.sup.8 is absent.
2. The method of claim 1, wherein the neuropathy is selected from
the group consisting of HIV-induced neuropathy, diabetic
neuropathy, and chemotherapeutic neuropathy.
3. The method of claim 1, wherein the neuropathy is diabetic
neuropathy.
4. The method of claim 1, wherein at least one amino acid is
glycosylated with a monosaccharide or disaccharide.
5. The method of claim 4, wherein at least one of the
monosacharides or disaccharides is selected from the group
consisting of glucose, galactose, xylose, fucose, rhamnose,
lactose, cellobiose, and melibiose.
6. The method of claim 1, wherein A.sup.7 is glycosylated.
7. The method of claim 6, wherein A.sup.7 is glycosylated with a
saccharide selected from the group consisting of glucose,
galactose, xylose, fucose, rhamnose, lactose, cellobiose, and
melibiose.
8. The method of claim 7, wherein the saccharide is glucose or
lactose.
9. The method of claim 1, wherein A.sup.7 is terminated with an
amino group.
10. The method of claim 7, wherein A.sup.7 is terminated with an
amino group.
11. The method of claim 1, wherein the oligopeptide is Ang
1-6-Ser(OGlc)-NH.sub.2 (SEQ ID NO: 10).
12. The method of claim 1, wherein the oligopeptide is Ang
1-6-Ser(OLac)-NH.sub.2.
13. The method of claim 1, wherein the oligopeptide comprises at
least one D-amino acid.
14. The method of claim 1, wherein each of A.sup.1-A.sup.8 is a
D-amino acid.
15. A method for providing analgesia to a subject having a
neuropathy, the method comprising administering to the subject a
therapeutically effective amount of an oligopeptide comprising an
amino acid sequence consisting of the formula:
A.sup.1-A.sup.2-A.sup.3-A.sup.4-A.sup.5-A.sup.6-A.sup.7-A.sup.8
(SEQ ID NO:1) wherein A.sup.1 is selected from the group consisting
of aspartic acid and glycosylated forms thereof, glutamic acid and
glycosylated forms thereof, and alanine; A.sup.2 is selected from
the group consisting of arginine, histidine, and lysine; A.sup.3 is
selected from the group consisting of valine, alanine, isoleucine,
and leucine; A.sup.4 is selected from the group consisting of
tyrosine and glycosylated forms thereof, phenylalanine, and
tryptophan; A.sup.5 is selected from the group consisting of
isoleucine, valine, alanine, and leucine; A.sup.6 is selected from
the group consisting of histidine, arginine, and lysine; A.sup.7 is
selected from the group consisting of proline, glycine, and serine
and glycosylated forms thereof; and A.sup.8 is serine or a
glycosylated form thereof.
16. The method of claim 15, wherein the neuropathy is selected from
the group consisting of HIV-induced neuropathy, diabetic
neuropathy, and chemotherapeutic neuropathy.
17. The method of claim 16, wherein the neuropathy is diabetic
neuropathy.
18. The method of claim 15, wherein A.sup.8 is glycosylated.
19. The method of claim 18, wherein A.sup.8 is glycosylated with a
saccharide selected from the group consisting of glucose,
galactose, xylose, fucose, rhamnose, lactose, cellobiose, and
melibiose.
20. The method of claim 15, wherein A.sup.8 is terminated with an
amino group.
21. The method of claim 19, wherein A.sup.8 is terminated with an
amino group.
22. The method of claim 15, wherein the oligopeptide is selected
from the group consisting of Ang 1-7-Ser-NH.sub.2 (SEQ ID NO: 7),
Ang 1-7-Ser(OGlc)-NH.sub.2 (SEQ ID NO: 8), and Ang
1-6-Ser(OLac)-NH.sub.2 (SEQ ID NO: 9).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/401,944, filed Jan. 9, 2017, which is a continuation-in-part
of U.S. application Ser. No. 15/134,073, filed Apr. 20, 2016, now
U.S. Pat. No. 9,670,251, which is a continuation of U.S.
application Ser. No. 14/801,557, filed Jul. 16, 2015, now U.S. Pat.
No. 9,796,758, which claims the priority benefit of U.S.
Provisional Application No. 62/027,219, filed Jul. 21, 2014, each
of which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 4, 2018, is named UAPN_002_X1C1_SL.txt and is 4,996 bytes
in size.
FIELD OF THE INVENTION
[0003] The present invention relates to oligopeptides, such as
Ang-(1-7) and related derivative oligopeptides and Mas
receptor-binding non-peptides, and methods for using the same for
the treatment of pain of various etiologies and other clinical
indications.
BACKGROUND OF THE INVENTION
[0004] Bone pain is experienced by 75-90% of late-stage metastatic
cancer patients. Metastatic cancer-induced bone pain (CIBP) is
frequently reported but poorly managed. The World Health
Organization implemented a three-tiered Pain-Relief-Ladder for
cancer pain that recommends mild-to-strong opioids as the cancer
progresses. Yet, moderate-to-severe cancer pain is not adequately
managed in many patients with current analgesic therapy. Opioid
therapy is associated with a host of challenging side effects
contributing to their failure, while diversion of prescribed
opioids have led to an addiction epidemic. Recent reports suggest
that opioids may exacerbate bone loss in humans and experimental
animal models, indicating that opioid therapy may be
counterproductive to anti-osteolytic co-therapies and CIBP
management. Furthermore, prolonged opioid therapy may increase the
proliferation/migration of certain cancers.
[0005] Preclinical modeling of CIBP has revealed mechanisms driving
this complex disease state and lead to the identification of
potential therapeutic targets. Although the bone is innervated by
both sympathetic and nociceptive nerve fibers, many human tumors of
the bone lack detectable nerve fibers in the tumor itself and
adjacent peripheral bone. Contributors to nociceptive signaling
associated with CIBP include an acidic tumor environment and the
secretion of growth factors, cytokines, and chemokines from the
tumor and tumor-associated cells, as well as enhanced nerve
sprouting in the local environment.
[0006] The bone is innervated by both sympathetic and nociceptive
nerve fibers. However, many human bone tumors lack detectable nerve
fibers within the tumor itself and the adjacent peripheral bone.
Contributors to nociceptive signaling associated with CIBP include
an acidic tumor environment and the secretion of growth factors,
cytokines, and chemokines from the tumor and tumor-associated
cells, as well as enhanced nerve sprouting in the local
environment. Thus, there is a need to develop non-opioid analgesics
for the treatment of pain including cancer-induced bone pain.
[0007] The present inventions are based on the discovery that
native Ang(1-7), related derivative polypeptides, and/or
non-peptide agonists that have affinity and agonistic efficacy for
the Mas receptor improve a variety of biologic, physiologic, and
pathologic parameters. Specifically, it is shown that Mas receptor
activation attenuates spatial memory and object recognition
impairment caused by congestive heart failure (CHF), pain of
various etiologies including cancer-induced bone pain and the
neurological sequelae of traumatic brain injury (TBI).
SUMMARY OF THE INVENTION
[0008] Some aspects of the invention provide an oligopeptide that
is angiotensin-(1-7), i.e., "Ang-(1-7)", derivative. Oligopeptides
of the invention have a longer in vivo half-life and/or increased
blood-brain barrier penetration than Ang-(1-7). In some
embodiments, the oligopeptides of the invention have seven or eight
amino acids.
[0009] One particular aspect of the invention provides an
oligopeptide derivative of the formula:
A.sup.1-A.sup.2-A.sup.3-A.sup.4-A.sup.5-A.sup.6-A.sup.7-A.sup.8
(SEQ ID NO:1), where A.sup.1 is selected from the group consisting
of aspartic acid, glutamic acid, alanine, and a derivative thereof;
A.sup.2 is selected from the group consisting of arginine,
histidine, lysine, and a derivative thereof; A.sup.3 is selected
from the group consisting of valine, alanine, isoleucine, leucine,
and a derivative thereof; A.sup.4 is selected from the group
consisting of tyrosine, phenylalanine, tryptophan, and a derivative
thereof; A.sup.5 is selected from the group consisting of
isoleucine, valine, alanine, leucine, and a derivative thereof;
A.sup.6 is selected from the group consisting of histidine,
arginine, lysine, and a derivative thereof; A.sup.7 is selected
from the group consisting of proline, glycine, serine, and a
derivative thereof; and A.sup.8 can be present or absent, wherein
when A.sup.8 is present, A.sup.8 is selected from the group
consisting of serine, threonine, hydroxyproline, and a derivative
thereof, provided (i) at least one of A.sup.1-A.sup.8 is optionally
substituted with a mono- or di-carbohydrate; or (ii) when A.sup.8
is absent: (a) at least one of A.sup.1-A.sup.7 is substituted with
a mono- or di-carbohydrate, (b) A.sup.7 is terminated with an amino
group, or (c) a combination thereof.
[0010] In some embodiments, carbohydrate comprises glucose,
galactose, xylose, fucose, rhamnose, lactose, cellobiose,
melibiose, or a combination thereof. In another embodiment, A.sup.8
is serine or a derivative thereof.
[0011] Still in other embodiments, (i) A.sup.8 is terminated with
an amino group; or (ii) when A.sup.8 is absent, A.sup.7 is
terminated with an amino group. Within these embodiments, in some
instances (i) A.sup.8 is serine that is glycosylated with glucose
or lactose; or (ii) when A.sup.8 is absent, A.sup.7 is serine that
is glycosylated with glucose or lactose. Still in other instances,
when A.sup.8 is absent and A.sup.7 serine that is glycosylated with
glucose. Within the latter instances, in some cases A.sup.7 is
terminated with an amino group.
[0012] Yet in other embodiments, A.sup.1 is aspartic acid; A.sup.2
is arginine; A.sup.3 is valine; A.sup.4 is tyrosine; A.sup.5 is
isoleucine; A.sup.6 is histidine; and (i) A.sup.8 is absent and
A.sup.7 is terminated with an amino group or A.sup.7 is a
glycosylated serine, or (ii) A.sup.8 is serine terminated with an
amino group. Within these embodiments, in some cases A.sup.8 is a
glycosylated serine. Still in other cases, A.sup.8 is absent and
A.sup.7 is a glycosylated serine that is terminated with an amino
group.
[0013] Another aspect of the invention provides a glycosylated
Ang-(1-7) derivative having eight amino acids or less, typically
seven or eight amino acids (e.g., amino acid residues). In some
embodiments, the glycosylated Ang-(1-7) derivative is glycosylated
with xylose, fucose, rhamnose, glucose, lactose, cellobiose,
melibiose, or a combination thereof. Still in other embodiments,
the carboxylic acid end of said glycosylated Ang-(1-7) derivative
is substituted with an amino group.
[0014] Other aspects of the invention provide methods for treating
cognitive dysfunction and/or impairment in a subject by
administering a therapeutically effective amount of an
oligonucleotide of the invention. In general, oligopeptides of the
invention can be used to treat any clinical condition that can be
treated with Ang-(1-7).
[0015] In some embodiments, the oligopeptides of the invention may
be used to treat (i.e., reduce or eliminate) pain of any etiology
(i.e., a painful condition). Specific pain syndromes and painful
conditions amenable to treatment include, for example,
cancer-induced bone pain from caused by either a primary or
metastatic tumor, post-surgical pain, post herpatic neuralgia,
fibromyalgia, inflammatory pain, stroke-induced pain, trauma-based
neuropathic pain, multiple sclerosis-induced pain, rheumatoid
arthritis, and complex regional pain syndrome (CRPS). The
oligopeptides of the invention also may be used to treat pain and
other symptoms and conditions associated with HIV-induced
neuropathy, diabetic neuropathy, and chemotherapeutic
neuropathy.
[0016] In other embodiments, the oligopeptides of the invention may
be used to reduce or eliminate one or more symptoms of cognitive
impairment including, for example, reduced attention, memory loss,
psychomotor slowing, and diminished executive function. Specific
conditions that are associated with cognitive impairment and that
are amenable to treatment using the inventive oligopeptides
include, for example, congestive heart failure, cardiovascular
disease, hypertension, stroke, post-operative cognitive defects
and/or delerium, dementia including age-related dementia,
Alzheimer's disease, and traumatic brain injury including
concussion and penetrating brain injury.
[0017] In some embodiments, the inventive oligopeptides are
administered at a dosage of about 0.1-50 mg/kg, including for
example at least about 0.25, 0.50, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0,
4.0, 5.0, 10, 15, 20, 25, 30, or 40 mg/kg. The oligopeptides may be
administered QD, bid, tid, qid, or more as necessary to obtain the
desired clinical outcome. The oligopeptides may be administered
orally or by injection (intravenous, subcutaneous, intramuscular,
intraperitoneal, intracerebroventricular, or intrathecal), or by
inhalation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a graph showing some of the oligopeptides of the
invention and native Ang-(1-7) to activate human umbilical vascular
endothelial cells (HUVEC) in culture.
[0019] FIG. 2 is a graph showing NO production assay results for
native Ang-(1-7) and oligopeptides PN-A3, PN-A4 and PN-A5 of the
invention.
[0020] FIG. 3A is a graph showing the select Mas receptor
antagonists A779 blocks NO production induced by oligopeptide PN-A5
of the invention.
[0021] FIG. 3B is a graph showing the averaged effect of the select
Mas receptor antagonists A779 on NO production induced by
oligopeptide PN-A5.
[0022] FIG. 4 is a graph showing the effects of oligopeptide PN-A5
on heart failure induced object recognition memory impairment.
[0023] FIG. 5 is a graph showing the effects of oligopeptide PN-A5
on heart failure induced spatial memory impairment.
[0024] FIG. 6A is a graph showing oligopeptide PN-A5 attenuates
spontaneous pain in CIBP acutely.
[0025] FIG. 6B is a graph showing the results of tactile allodynia
test using von Frey filaments.
[0026] FIGS. 7A-7F is a series of graphs showing the pain responses
(guarding and flinching) of mice having a bone intramedullary
transplantation of a breast cancer cell line under various
single-dose therapeutic treatment regimens and the blockade by the
Mas receptor antagonist A779.
[0027] FIGS. 8A-8D is a series of bar graphs showing the pain
responses (guarding and flinching) of mice having a bone
intramedullary transplantation of a breast cancer cell line under
various chronic therapeutic treatment regimens and the blockade by
the Mas receptor antagonist A779.
[0028] FIGS. 9A-D is a series of graphs showing the pain responses
of mice having an intramedullary transplantation of a breast cancer
cell line under various chronic treatment regimens using either
Ang-(1-7) or antagonists of the AT1 or AT2 receptor.
[0029] FIGS. 10A-10B is a series of graphs showing the nesting
behavior of experimental mice with or without bone cancer under
various treatment regimens.
[0030] FIG. 11A is a Western blot showing Mas receptor expression
in dorsal root ganglia of nociceptive fibers in experimental
animals.
[0031] FIG. 11B is a bar graph showing the relative expression of
the Mas receptor between the ipsilateral and contralateral dorsal
root ganglia in experimental animals.
[0032] FIG. 11C is a Western blot showing Mas receptor expression
in femoral exudate of experimental animals.
[0033] FIG. 11D is a bar graph showing the relative expression of
the Mas receptor between the ipsilateral and contralateral femoral
exudate in experimental animals.
[0034] FIG. 12A is a series of photomicrograph of experimental
animal femurs following various treatment regimens.
[0035] FIG. 12B is a bar graph showing the quantification of tumor
tissue in the experimental animal femurs.
[0036] FIG. 13A is a series of radiographs taken from experimental
animals.
[0037] FIG. 13B is a graph quantifying bone lesion scoring in
experimental animals.
[0038] FIG. 13C is a bar graph showing the amount of
carboxy-terminal collagen crosslinks as a measure of bone integrity
in experimental animals.
[0039] FIG. 14 is a line graph showing the discrimination ratio of
experimental animals in a novel object recognition test after an
acute traumatic brain injury (TBI).
[0040] FIG. 15A is model of the three-dimensional structure of
native Ang(1-7). FIG. 15B is a computational model of various
glycosylated Ang(1-7) derivatives.
[0041] FIG. 16 is a line graph showing the in vitro serum half-life
of native Ang(1-7) and various derivatives.
[0042] FIG. 17A is a line graph showing the serum concentration of
native Ang(1-7) and PN-A5. FIG. 17B is a line graph showing the CSF
concentration of native Ang(1-7) and PN-A5.
DETAILED DESCRIPTION OF THE INVENTION
[0043] Definitions
[0044] The term "native" refers to any sequence of L amino acids
used as a starting sequence or a reference for the preparation of
partial or complete retro, inverso or retro-inverso analogues.
Thus, the term "native Ang-(1-7)" refers to an oligopeptide having
the same amino acid sequence as that of endogenous Ang-(1-7). It
should be appreciated that the use of the term "native" does NOT
imply naturally occurring, although it can include naturally
occurring Ang-(1-7). The term "native" merely refers to having the
same amino acid sequence as that of Ang-(1-7) without any
modification of the amino acid residues. Accordingly, the term
"native Ang-(1-7)" includes both synthetic Ang-(1-7) and naturally
occurring Ang-(1-7) as long as the amino acid residues are the same
and are not modified.
[0045] The term "Ang-(1-7) derivative" refers to oligopeptide in
which one or more amino acid residue is either modified or
different than the amino acid residue of the corresponding native
Ang-(1-7). The term "Ang-(1-7) derivative" also includes
oligopeptide of eight amino acid residues as discussed in more
detail below.
[0046] The term "retro modified" refers to a peptide which is made
up of L-amino acids in which the amino acid residues are assembled
in opposite direction to the native peptide with respect the which
it is retro modified. The term "inverso modified" refers to a
peptide which is made up of D-amino acids in which the amino acid
residues are assembled in the same direction as the native peptide
with respect to which it is inverso modified. The term
"retro-inverso modified" refers to a peptide which is made up of
D-amino acids in which the amino acid residues are assembled in the
opposite direction to the native peptide with respect to which it
is retro-inverso modified. Thus, native Ang-(1-7) (L-amino acids,
N.fwdarw.C direction) is: Asp-Arg-Val-Tyr-Ile-His-Pro, i.e.,
DRVYIHP (SEQ ID NO:2). Retro-inverso Ang-(1-7) (D-amino acids,
C.fwdarw.N direction) is: DRVYIHP (SEQ ID NO:3). Retro Ang-(1-7)
(L-amino acids, C.fwdarw.N direction) is: DRVYIHP (SEQ ID NO:4).
And inverso Ang-(1-7) (D-amino acids, N.fwdarw.C direction) is:
DRVYIHP (SEQ ID NO:5). The use of D-amino acids in the context of
inverso modified and retro-inverso modified Ang-(1-7) derivatives
is not intended to be limiting on the use of D-amino amino acids in
the oligopeptides. As discussed in more detail below, fewer than
all of the amino acids in an Ang-(1-7) derivative may be D-amino
acids.
[0047] The term "carbohydrate" refers to pentose and hexose of
empirical formula (CH.sub.2O).sub.n, where n is 5 for pentose and 6
for hexose. A carbohydrate can be monosaccharide, disaccharide,
oligosaccharide (e.g., 3-20, typically 3-10, and often 3-5
monomeric saccharides are linked together), or polysaccharide
(e.g., greater than 20 monomeric saccharide units). More often, the
term carbohydrate refers to monosaccharide and/or disaccharide.
However, it should be appreciated that the scope of the invention
is not limited to mono- or di-saccharides. Often the terms
"carbohydrate" and "saccharide" are used interchangeably
herein.
[0048] The term "oligopeptide" as used throughout the specification
and claims is to be understood to include amino acid chain of any
length, but typically amino acid chain of about fifteen or less,
often ten or less, still more often eight or less, and most often
seven or eight.
[0049] It should be appreciated that one or more of the amino acids
of Ang-(1-7) can be replaced with an "equivalent amino acid", for
example, L (leucine) can be replaced with isoleucine or other
hydrophobic side-chain amino acid such as alanine, valine,
methionine, etc., and amino acids with polar uncharged side chain
can be replaced with other polar uncharged side chain amino acids.
While Ang-(1-7) comprises 7 amino acids, in some embodiments the
oligopeptide of the invention has eight or less amino acids.
[0050] The term "derivative" refers to any chemical modification of
the amino acid, such as alkylation (e.g., methylation or
ethylation) of the amino group or the functional group on the side
chain, removal of the side-chain functional group, addition of a
functional group (e.g., hydroxyl group on proline), attachment of
mono- or di-carbohydrate (e.g., via glycosylation) etc. Exemplary
glycosylated derivatives include hydroxyl group on serine that is
glycosylated with glucose, galactose, ribose, arabinose, xylose,
lyxose, allose, altrose, mannose, gulose, iodose, talose, fucose,
rhamnose, etc. as well as disaccharides and amino sugars such as
galactosamine, glucosamine, sialic acid, N-acetyl glucosamine, etc.
Amino acid derivatives also include modified or unmodified D-amino
acids.
[0051] The term "combinations thereof," which reference to any
modifications (e.g, carbohydrate modifications) of Ang-(1-7)
derivatives refers to oligopeptides in which two, three, four,
five, six, seven, or eight of the individual amino acids are
modified by the attachment of a carbohydrate. For Ang-(1-7)
derivatives having a plurality of carbohydrate modifications, the
modifying carbohydrates may be the same on every modified amino
acid, or the several modified amino acids may comprise a mixture of
different carbohydrates.
[0052] "A therapeutically effective amount" means the amount of a
compound that, when administered to a mammal, at an appropriate
interval and for a sufficient duration for treating a disease, is
sufficient to effect such treatment for the disease. The
"therapeutically effective amount" will vary depending on the
compound, the disease and its severity, physiological factors
unique to the individual including, but not limited to the age,
weight, and body mass index, the unitary dosage, cumulative dosage,
frequency, duration, and route of administration selected.
[0053] As used herein, the term "treating", "contacting" or
"reacting" when referring to chemical synthesis means to add or mix
two or more reagents under appropriate conditions to produce the
indicated and/or the desired product. It should be appreciated that
the reaction which produces the indicated and/or the desired
product may not necessarily result directly from the combination of
two reagents which were initially added, i.e., there may be one or
more intermediates which are produced in the mixture which
ultimately leads to the formation of the indicated and/or the
desired product.
[0054] As used herein, the terms "treating" and "treatment" refer
to effecting an improvement of any symptom or physiological,
cognitive, or biochemical indicium of the condition or disease
being treated. For example, treatment of a cognitive dysfunction
and/or impairment may refer to: (1) preventing cognitive
dysfunction and/or impairment from occurring, i.e., causing the
clinical symptoms of cognitive dysfunction and/or impairment not to
develop in a subject that may be or predisposed to developing
cognitive dysfunction and/or impairment but does not yet experience
or display symptoms of cognitive dysfunction and/or impairment; (2)
inhibiting cognitive dysfunction and/or impairment, i.e., arresting
or reducing the development of cognitive dysfunction and/or
impairment or its clinical symptoms; or (3) relieving cognitive
dysfunction and/or impairment, i.e., causing regression of
cognitive dysfunction and/or impairment or its clinical
symptoms.
[0055] The terms "approximately" or "about" in reference to a
number are generally taken to include numbers that fall within a
range of 5%, 10%, 15%, or 20% in either direction (greater than or
less than) of the number unless otherwise stated or otherwise
evident from the context (except where such number would be less
than 0% or exceed 100% of a possible value).
[0056] The term "subject" or "patient" refers to any organism to
which a composition of this invention may be administered, e.g.,
for experimental, diagnostic, and/or therapeutic purposes. Typical
subjects include animals (e.g., mammals such as mice, rats,
rabbits, dogs, cats, non-human primates, and humans).
[0057] Unless defined otherwise, all technical and scientific terms
used herein have the meaning commonly understood by a person
skilled in the art to which this invention belongs.
[0058] Oligopeptides of the Invention:
[0059] The renin-angiotensin system (RAS), well known for roles in
blood pressure regulation and fluid homeostasis, was recently
implicated in metastatic bone disease including inflammation,
angiogenesis, tumor cell proliferation, and migration. Angiotensin
II (Ang II) is the major end product of the RAS through cleavage by
Angiotensin Converting Enzyme (ACE). This nonapeptide binds to and
activates two G-protein coupled receptors (GPCRs): angiotensin II
receptor type 1 (AT1) and type 2 (AT2). Physiological effects such
as vasoconstriction, inflammation, fibrosis, cellular
growth/migration, and fluid retention are reported for AT1 and AT2.
Ang II is cleaved by ACE2 to yield Angiotensin-(1-7) (Ang-(1-7)), a
biologically active heptapeptide. In contrast to Ang II, Ang-(1-7)
binds to the GPCR, Mas receptor (MasR; Kd=0.83 nM) with 60-100 fold
greater selectivity over the AT1 and AT2 receptors. Activation of
the MasR elicits effects opposite to those of the Ang II/AT1/AT2
axis including having anti-inflammatory and antidepressant
activities.
[0060] Some aspects of the invention provide oligopeptides that are
derivatives of Ang-(1-7). As discussed above, the term "derivative"
of Ang-(1-7) refers to an oligopeptide whose amino acid sequence of
any one or more of Ang-(1-7) is modified (e.g., via methylation,
presence of a functional group, such as hydroxy group on proline),
attached to a carbohydrate, is replaced with corresponding D-amino
acid or an "equivalent amino acid" as defined above, and/or the
terminal amino group end or the carboxyl end of Ang-(1-7) is
modified, for example, the carboxylic acid end can be modified to
be an amide, an amine, a thiol, or an alcohol functional group, or
one in which an additional amino acid residue is present compared
to native Ang-(1-7). It should be appreciated that the term
"Ang-(1-7) derivative" excludes the native Ang-(1-7), i.e., amino
acid sequences of endogenous Ang-(1-7) without any
modification.
[0061] In some embodiments, oligopeptides of the invention have the
amino group on the carboxylic acid terminal end (i.e., the --OH
group of the carboxylic acid is replaced with --NR.sup.aR.sup.b,
where each of R.sup.a and R.sup.b is independently hydrogen or
C1-C6 alkyl) and/or have one or more amino acid residues that are
(i) replaced with a corresponding D-amino acid, (ii) glycosylated,
(iii) replaced with another amino acid, (iv) or a combination
thereof
[0062] Still in other embodiments, the oligopeptide of the
invention is retro-inverso Ang-(1-7). Yet in other embodiments, the
oligopeptide of the invention is retro Ang-(1-7). In other
embodiments, the oligopeptide of the invention is inverso
Ang-(1-7).
[0063] Other embodiments of the invention include Ang-(1-7)
derivatives in which at least one or more, typically one or two,
and often only one amino acid is attached to a carbohydrate.
Generally, the carbohydrate is attached to the amino acid via
glycosylation. Typically, the carbohydrate is a mono- or
di-carbohydrate. Exemplary mono- and di-carbohydrates that can be
used in the invention include, but are not limited to, xylose,
fucose, rhamnose, glucose, lactose, cellobiose, melibiose, and a
combination thereof.
[0064] In one particular embodiment, the oligopeptide of the
invention is Ang-(1-7) derivative of the formula:
A.sup.1-A.sup.2-A.sup.3-A.sup.4-A.sup.5-A.sup.6-A.sup.7-A.sup.8
(SEQ ID NO:1), where A.sup.1 is selected from the group consisting
of aspartic acid, glutamic acid, alanine, and a derivative thereof;
A.sup.2 is selected from the group consisting of arginine,
histidine, lysine, and a derivative thereof; A.sup.3 is selected
from the group consisting of valine, alanine, isoleucine, leucine,
and a derivative thereof; A.sup.4 is selected from the group
consisting of tyrosine, phenylalanine, tryptophan, and a derivative
thereof; A.sup.5 is selected from the group consisting of
isoleucine, valine, alanine, leucine, and a derivative thereof;
A.sup.6 is selected from the group consisting of histidine,
arginine, lysine, and a derivative thereof; A.sup.7 is selected
from the group consisting of proline, glycine, serine, and a
derivative thereof; and A.sup.8 can be present or absent, wherein
when A.sup.8 is present, A.sup.8 is selected from the group
consisting of serine, threonine, hydroxyproline, and a derivative
thereof, provided (i) at least one of A.sup.1-A.sup.8 is optionally
substituted with a mono- or di-carbohydrate; or (ii) when A.sup.8
is absent: (a) at least one of A.sup.1-A.sup.7 is substituted with
a mono- or di-carbohydrate, (b) A.sup.7 is terminated with an amino
group, or (c) a combination thereof.
[0065] In some embodiments, A.sup.1 is the amino terminal end of
the oligopeptide and A.sup.8 (or A.sup.7 when A.sup.8 is absent) is
the carboxyl terminal end. Still in other embodiments, A.sup.1 is
the carboxyl terminal end and A.sup.8 (or A.sup.7 when A.sup.8 is
absent) is the amino terminal end. Yet in other embodiments, the
carboxylic acid functional group of the carboxyl terminal end is
modified as an amide functional group, an amine functional group, a
hydroxyl functional group, or a thiol functional group. The amide
and the amine functional groups can be non-alkylate, mono-alkylated
or di-alkylated.
[0066] Yet in other embodiments, the carbohydrate comprises
glucose, galactose, xylose, fucose, rhamnose, or a combination
thereof. In some instances, the carbohydrate is a
mono-carbohydrate, whereas in other instances, the carbohydrate is
a di-carbohydrate.
[0067] In other embodiments, at least one of A.sup.1-A.sup.8 is
substituted with a mono-carbohydrate. Still in other embodiments,
at least one of A.sup.1-A.sup.8 is substituted with a
di-carbohydrate. It should be appreciated that the scope of the
invention also includes those oligopeptides having both mono- and
di-carbohydrates.
[0068] Exemplary di-carbohydrates that can be used in oligopeptides
of the invention include, but are not limited to, lactose,
cellobiose, melibiose, and a combination thereof. However, it
should be appreciated that the scope of the invention includes
oligopeptides that are substituted with any dicarbohydrates known
to one skilled in the art.
[0069] In one particular embodiment, A.sup.8 is serine or a
derivative thereof. In some instances, the carboxylic acid moiety
of the serine is modified as an amide or an amine. In one case,
serine is terminated as an amino group. Still in other embodiments,
the serine residue of A.sup.8 is glycosylated with glucose or
lactose.
[0070] Yet in other embodiments, at least one, typically at least
two, generally at least three, often at least four, still more
often at least five, yet still more often at least six, and most
often all of A.sup.1-A.sup.8 is D-amino acid.
[0071] In particular, in some specific embodiments, said
oligopeptide is retro modified, inverso modified, or retro-inverso
modified.
[0072] Another aspect of the invention provides oligopeptides, such
as Ang-(1-7) derivatives, having eight amino acids or less,
typically seven or eight amino acid residues. In some embodiments,
one or more amino acids have attached thereto a carbohydrate group.
Often the carbohydrate group is attached to the oligopeptide via
glycosylation. The carbohydrate can be attached to the oligopeptide
via any of the side chain functional group of the amino acid or the
amide group. Accordingly, the scope of the invention includes, but
is not limited to, O-glycosylate, N-glycosylate, S-glycosylated
oligopeptides. The term "X-glycosylated" refers to having a
carbohydrate attached to the oligopeptide via the heteroatom "X" of
the amino acid. For example, for serine whose side-chain functional
group is hydroxyl, "O-glycosylated" means the carbohydrate is
attached to the serine's side-chain functional group, i.e., the
hydroxyl group. Similarly, "N-glycosylation" of leucine refers to
having the carbohydrate attached to the amino side-chain functional
group of leucine. Typically, the glycosylation is on the side-chain
functional group of the amino acid.
[0073] In some embodiments, the Ang-(1-7) derivative is
glycosylated with xylose, fucose, rhamnose, glucose, lactose,
cellobiose, melibiose, or a combination thereof.
[0074] Yet in other embodiments, the carboxylic acid terminal end
of said glycosylated Ang-(1-7) derivative is substituted with an
amino group. When referring to the carboxyl acid terminal end being
substituted with an amino group, it means --OH group of the
carboxylic acid is replaced with --NH.sub.2 group. Thus, the actual
terminal end functional group is an amide, i.e., rather than having
the oligopeptide being terminated at the carboxylic acid terminal
end with a functional group --CO.sub.2H, the carboxylic acid
terminal end is terminated with an amide group (i.e.,
--CO.sub.2NR'.sub.2, where each R' is independently hydrogen or
C.sub.1-C.sub.12 alkyl). Still in other embodiments, the carboxylic
acid terminal group is terminated with a hydroxyl or a thiol
group.
[0075] In some embodiments, the modified carboxylic acid terminal
group is used to attach the carbohydrate, e.g., via
glycosylation.
[0076] One of the purposes of the invention was to produce
Ang-(1-7) derivatives to enhance efficacy of action, in vivo
stabilization, and/or penetration of the blood-brain barrier.
Improved penetration of the blood-brain barrier facilitates
cerebral entry of the Ang-(1-7) derivative of the invention, and,
consequently, Mas activation, or intrinsic-efficacy. To improve
(i.e., increase) penetration of the blood-brain barrier, in some
embodiments the Ang-(1-7) derivative is attached to at least one
mono- or di-carbohydrates.
[0077] Without being bound by any theory, it is believed that the
oligopeptide of the invention that are glycosylated exploits the
inherent amphipathicity of the folded Ang-(1-7) glycopeptides
(i.e., glycosylated oligopeptides of the invention) and the
"biousian approach" to deliver the glycosylated oligopeptides of
the invention across the blood-brain barrier. In some instances,
the amount of increase in crossing the blood-brain barrier by
oligopeptides of the invention is at least 6%, typically at least
10%, and often at least 15% compared to native Ang-(1-7). In some
instances, the amount of increase in the Cmax for oligopeptides of
the invention in cerebral-spinal fluid is 2-10 fold, 3-8 fold, or
5-8 fold compared to native Ang-(1-7). In some instances, the
amount of increase in the Cmax for oligopeptides of the invention
in cerebral-spinal fluid is 2, 3, 4, 5, 6, 7, 8, 9 or 10 fold
compared to native Ang-(1-7). In other instances, oligopeptides of
the invention have in vivo half-life of at least 20 min, at least
30 min, at least 40 min, at least 50 min, at least 60 min, or at
least 2, hours, at least 3 hours, at least 4 hours, at least 5
hours or at least 6 hours. In some instances, the amount of
increase in the in vivo half-life for oligopeptides of the
invention is 2-30 fold, 3-25 fold, 4-20 fold, 4-10 fold, 10-25
fold, 15-25 fold, or 20-25 fold compared to native Ang-(1-7).
Alternatively, compared to native Ang-(1-7), oligopeptides of the
invention exhibit at least 50 fold, typically at least 75 fold, and
often at least 100 fold increase in in vivo half-life.
[0078] In other embodiments, oligopeptides of the invention exhibit
enhanced vascular efficacy. Without being bound by any theory, it
is generally recognized that blood-brain barrier transport occurs
via an absorptive endocytosis process on the blood side of the
endothelium of the brain capillaries followed by exocytosis on the
brain side, leading to overall transcytosis. It is also believed
that for this process to be efficient, the oligopeptide must bind
to the membrane for some period of time, and must also be able to
exist in the aqueous state for some period of time (biousian
nature). Based on previous work from one of the present inventors,
it is believed that effective drug delivery and blood-brain barrier
transport requires a biousian glycopeptide that has at least two
states: (1) a state defined by one or more membrane-bound
conformations that permit or promote endocytosis; and (2) a state
defined by a water-soluble, or random coil state that permits
"membrane hopping" and, presumably, vascular efficacy.
[0079] In general, the degree of glycosylation does not have a
large effect on the structure of the individual microstates. Thus,
altering the degree of glycosylation allows for the modulation of
aqueous vs. membrane-bound state population densities without
significantly affecting the overall structure of the oligopeptide.
Moreover, it is believed that glycosylation also promotes stability
to peptidases, thereby increasing the half-life of the Ang-(1-7)
derivatives in vivo.
TABLE-US-00001 TABLE 1 Some of the representative oligopeptides of
the invention. carboxyl terminal end 1 2 3 4 5 6 7 8 functional
group Native AT.sub.1-7 Asp Arg Val Tyr Ile His Pro -- OH (SEQ ID
NO: 2) PN-A1 Asp Arg Val Tyr Ile His Pro -- NH.sub.2 (SEQ ID NO: 6)
PN-A2 Asp Arg Val Tyr Ile His Pro Ser.degree. NH.sub.2 (SEQ ID NO:
7) PN-A3 Asp Arg Val Tyr Ile His Pro Ser* NH.sub.2 (SEQ ID NO: 8)
PN-A4 Asp Arg Val Tyr Ile His Pro Ser** NH.sub.2 (SEQ ID NO: 9)
PN-A5 Asp Arg Val Tyr Ile His Ser* -- NH.sub.2 (SEQ ID NO: 10)
PN-A6-PN-A11 Ala .fwdarw. scan Tyr Ile etc. Pro
Ser.degree..sup.\*.sup./** NH.sub.2 (SEQ ID NO: 11) PN-A12 Asp Arg
Xxx Tyr Yyy His Pro Ser.degree..sup.\*.sup./** NH.sub.2 (SEQ ID NO:
12) PN-AXX Asp Arg Xxx Zzz Yyy His Pro Ser.degree..sup.\*.sup./**
NH.sub.2 (SEQ ID NO: 13)
[0080] Table 1 above shows some of the representative oligopeptides
of the invention. In particular, these oligopeptides can be
considered Ang-(1-7) derivatives. In Table 1, the "n-x", where x is
an integer, represents the oligonucleotide identifier. For example,
PN-A1 means oligopeptide number 1, PN-A2 means oligopeptide number
2, PN-A6-PN-A11 means oligopeptide numbers 6 through 11, and so
forth. Thus, the term "A-x" is used for identification purposes
only. As shown in Table 1, some of the oligopeptides have
carbohydrate attached to the native Ang-(1-7) peptide. These
peptides are sometimes referred to as glycopeptides.
[0081] Studies have shown that inherent binding of the glycopeptide
to the native receptor is minimally affected. Therefore, the
glycosylated Ang-(1-7) derivatives, at a minimum, maintain Mas
binding similar to that of the native Ang-(1-7) peptide. In
addition, promoting the aqueous nature of the glycopeptide can
further enhance vascular efficacy of Ang-(1-7) derivatives. The
degree of glycosylation (e.g., Table 1: unglycosylated Ser.degree.,
glucosylated Ser* or lactosylated Ser**) for optimal blood-brain
barrier transport is determined using the best binding compounds
from these using the in vivo mouse model. Besides the disaccharide
.beta.-lactose, the more robust disaccharide .beta.-cellobiose is
examined using these first few structures. Based on the amino acid
sequence of Ang-(1-7) and the potential modification strategies,
there are at least about 200 possible derivatives of Ang-(1-7) that
are rapidly generated using the well known oligopeptide synthesis,
including automated peptide synthesis as well as combinatorial
synthesis.
[0082] Cognitive Dysfunction
[0083] Cognitive dysfunction or impairment is a common neurological
complication of congestive heart failure ("CHF") and post cardiac
surgery affecting approximately 50-70% of patients at hospital
discharge and 20-40% of patients six months after surgery. The
occurrence of CHF and postoperative cognitive dysfunction is
associated with increased duration of hospitalization and impaired
long-term quality of life. Without being bound by any theory, it is
believed that in general any clinical condition associated with an
increase in inflammatory cytokines and/or increase in reactive
oxygen species in central nervous system, in particular in the
brain, can lead to cognitive dysfunction.
[0084] Other aspects of the invention provide methods for treating
cognitive dysfunction and/or impairment in a patient using an
oligopeptide of the invention. Typically, methods of the invention
include administering to a patient in need of such a treatment a
therapeutically effective amount of an oligopeptide of the
invention. It should be appreciated that the oligopeptides of the
invention can be used to treat any clinical conditions that are
known to be treatable or appears to be treatable using Ang-(1-7).
However, for the sake or clarity and brevity, the invention will
now be described in reference to treating cognitive dysfunction
and/or impairment in a patient.
[0085] The cognitive dysfunction that occurs in congestive heart
failure (CHF) patients includes decreased attention, memory loss,
psychomotor slowing, and diminished executive function, all of
which compromises patients' ability to comply with complex medical
regimens, adhere to dietary restrictions and make self-care
decisions. Mechanisms thought to contribute to cognitive impairment
in patients with CHF include changes in cerebral blood flow,
altered cerebrovascular autoregulation and microembolisms. In one
study, cerebral blood flow was measured with single-photon emission
computed tomography (SPECT) and found to be reduced by 30% in
patients with severe heart failure. The causes for decreased
cerebral perfusion in CHF have been attributed to low cardiac
output, low blood pressure and altered cerebrovascular reactivity.
In some cases, the cognitive impairment seen in CHF is improved
following either heart transplant or improvement in cerebral blood
flow via optimal management of CHF. However, for many patients with
CHF, management is rarely optimal and the cognitive impairment
persists. Interestingly, long-term follow up studies have revealed
that cognitively normal CHF patients have a significantly higher
risk of dementia or Alzheimer's disease compared to age-matched
non-CHF controls, suggesting that CHF and cardiovascular disease
predispose patients to further cognitive impairment and
dementia.
[0086] During CHF, the well characterized changes in the
circulating neurochemical milieu and increases in inflammatory
factors are also seen in the brain. Most of the studies on
CHF-induced changes in inflammatory cytokines and ROS have focused
on brain regions involved in sympathetic outflow regulation and not
on cognition. CHF elevates sympathetic tone and causes abnormal
cardiac and sympathetic reflex function. In the rat,
ischemia-induced CHF significantly increases pro-inflammatory
cytokines and Angiotensin II type 1 receptors (AT1) in the
paraventricular nucleus (PVN) of the hypothalamus. Further, in CHF
rabbits, the increase in sympathetic outflow is blocked by ICV
injection of the super oxide dimustase (SOD) mimetic tempol,
presumably by inhibition of ROS. CHF in this model is associated
with increased expression of NADPH oxidase subunits and ROS
production in the rostral ventral lateral medulla (RVLM) and
increases in NO.
[0087] The role of ROS in learning and memory has been extensively
studied. All of the NAD(P)H oxidase subunits, including NOX2 and
NOX4, have been localized within the cell bodies and dendrites of
neurons of the mouse hippocampus and perirhinal cortex and are
co-localized at synaptic sites. These are key regions of the brain
in learning and memory. In the brain, superoxide production via
actions of NAD(P)H oxidase are known to be involved in
neurotoxicity, age related dementia, stroke and neurodegenerative
diseases and have been identified throughout the brain including
the hippocampus, thalamus, cerebellum and amygdala. In younger,
healthy animals ROS and NAD(P)H oxidase is shown to be required for
normal learning and hippocampal long-term potentiation (LTP).
Recent studies in mice lacking Mas have shown that Ang-(1-7) and
Mas are essential for normal object recognition processing and
blockade of Mas in the hippocampus impairs object recognition. In
addition, Ang-(1-7) facilitates LTP in CA1 cells and this effect is
blocked by antagonism of Mas. In older animals or in CHF animals,
an increase in ROS is linked to LTP and memory impairments.
[0088] Over the last decade, it has become recognized that renin
angiotensin system (RAS) involves two separate enzymatic pathways
providing a physiological counterbalance of two related peptides
acting at distinct receptors. The well described ACE-AngII-AT1
receptor system is thought to be physiologically opposed and
balanced by the ACE2-Ang-(1-7)-Mas system. Functionally, these two
separate enzymatic pathways of RAS are thought to be involved in
balancing ROS production and nitric oxide (NO) in the brain,
microvasculature and peripheral tissues. Increases in AT1 receptor
activation are known to increase NAD(P)H oxidase and ROS generation
which are both known to contribute to abnormal increases of
sympathetic nerve activity observed in CHF and hypertension. This
increase in AT1 receptor-induced ROS formation is thought to be
opposed by ACE2-Ang-(1-7)-Mas inhibition of ROS formation.
Ang-(1-7), the majority of which is produced from ACE2 cleavage of
Ang II, decreases ROS production and increases NOS in the brain via
activation Mas and, possibly through AT2 receptor.
[0089] Within the brain, the Mas receptor is known to be expressed
on neurons, microglia and vascular endothelial cells. Further, all
three of these key components that make up the "neurovascular unit"
(neurons, microglia and endothelial cells) are central players in
neurogenic hypertension and CHF-induced increases in brain
inflammation and ROS production. Both CHF and hypertension increase
circulating cytokines promoting ROS production within the
"neurovascular unit". The end-result of this feed-forward cascade
is neuronal dysfunction and cognitive impairment. The ideal
therapeutic candidate to treat cognitive impairment would be
designed to interrupt this cascade by working at both sides of the
blood-brain barrier, the brain vascular endothelium and neuronal
cells. Ang-(1-7), acting at the Mas receptor, is known to have
effects at both endothelial cells and neurons. However, using a
native Ang-(1-7) for treating cognitive dysfunction and/or
impairment is not suitable because native Ang-(1-7) is susceptible
to enzymatic degradation. Moreover, native Ang-(1-7) does not
readily cross the blood-brain barrier to be suitable as a
therapeutic agent.
[0090] Without being bound by any theory, it is believed that one
of the advantages of using oligopeptides of the invention in
treating cognitive dysfunction and/or impairment is that
oligopeptides of the invention have enhanced endothelial
"interaction" and brain penetration. It is believed that
oligopeptides of the invention act at both endothelial cells and
neurons thus inhibiting inter alia neurovascular ROS production and
mitigating the brain inflammatory cascade.
[0091] Accordingly, oligopeptides the invention can be used to
treat cognitive impairment and/or dysfunction (1) associated with
pre- and/or post-surgery dementia, or (2) observed in patients with
congestive heart failure, cardiovascular disease, or hypertension.
More generally, oligopeptides of the invention are useful in
treating cognitive dysfunction and/or impairment in a subject whose
cognitive dysfunction and/or impairment is clinically associated
with an increase in inflammatory cytokines and/or increase in
reactive oxygen species ("ROS") in the central nervous system, in
particular the brain. As used herein, the term "clinically
associated" refers to the root cause or underlying cause of
cognitive dysfunction and/or impairment (such as, but not limited
to, memory loss) that when ameliorated results in reduction,
prevention, treatment or reversal of cognitive dysfunction and/or
impairment. Exemplary clinical conditions associated with an
increase in inflammatory cytokines and/or increase in reactive
oxygen species that can cause cognitive dysfunction and/or
impairment include, but are not limited to, circulatory compromise,
cardiovascular disease, hypertension, hypotension, congestive heart
failure, stroke, embolism, surgery (e.g., postoperative recovery
condition), dementia, Alzheimer's disease, disease related
cognitive impairment, trauma related cognitive impairment,
age-related dementia, postoperative related delirium and/or
increase in inflammatory cytokine and/or increase in reactive
oxygen species within the central nervous system of said subject or
a combination thereof.
[0092] Anti-Nociception and Analgesia
[0093] The inventions described herein are based, in part, on the
discovery that Mas receptor agonists, including the prototypical
native Ang(1-7) polypeptide, induce analgesia. As described herein,
the analgesic properties of Ang-(1-7) was evaluated in a CIBP as a
representative model of nociceptive conditions. It is demonstrated
that acute and chronic systemic administration of Ang-(1-7)
significantly reduced the spontaneous pain behaviors associated
with CIBP. Importantly, repeated administration attenuated CIBP
without loss of efficacy after 7 days. However, no significant
change in nesting behaviors with or without treatments was
observed, suggesting that the nesting is not representative of
possible anxiety or depression in mice with CIBP.
[0094] To confirm that the effects of Ang(1-7) are mediated by the
Mas receptor, control experiments using the Mas receptor
antagonist, A-779, were performed. The inhibition of guarding and
flinching by Ang(1-7) were significantly prevented by
administration of A-779.
[0095] Repeated Ang-(1-7) administration did not significantly
alter the expression of MasR in the DRGs or femur extrudate
demonstrating that repeated Ang-(1-7) dosing does not significantly
alter MasR expression in the DRGs containing soma of fibers
innervating the bone-tumor microenvironment. Consistent with these
findings, analgesic tolerance was not observed over the treatment
paradigm.
[0096] It also was discovered that pre-administration of an AT1
receptor antagonist, Losartan potassium, further alleviates
cancer-induced bone pain, yet by itself had no significant effect.
It is hypothesized that Losartan augments the effect to Ang-(1-7)
in CIBP because AT1 antagonism inhibits Ang-(1-7) from acting
similarly to Ang II at AT1, thereby allowing Ang-(1-7) to bind
primarily to MasR to induce analgesia. The AT2 antagonist, PD
123319, did not attenuate the effects of Ang-(1-7) nor result in
enhanced pain relief, indicating that the AT2 receptor does not
play a role in CIBP. In addition, the Ang-(1-7) did not demonstrate
any changes in motor acitivty by measuring the amount of time
aniamls that received Ang-(1-7) remained walking on a slow rotating
rod.
[0097] In sum, these data demonstrate that Ang-(1-7) at the Mas
receptor is for inhibiting pain in the tumor-nociceptor
microenvironment. Ang-(1-7) did not significantly change the
tumor-induced degradation of the bone nor did it significantly
alter tumor proliferation, further suggesting the analgesic effect
is directly towards inhibiting nociceptive activation and not due
to changes in tumor burden. Mas receptor agonists such as Ang(1-7)
therefore induce primary analgesia through pharmacologic mechansims
rather than secondarily through effects on the bone tissue or
anti-neoplastic activity. Thus, Mas receptor agonists may attenuate
pain and the behavioral signs of pain in conditions other than
CIBP.
[0098] Methods of Administration
[0099] Oligopeptides of the present invention can be administered
to a patient to achieve a desired physiological effect. Preferably
the patient is an animal, more preferably a mammal, and most
preferably a human. The oligopeptide can be administered in a
variety of forms adapted to the chosen route of administration,
i.e., orally or parenterally. Parenteral administration in this
respect includes administration by the following routes:
intravenous; intramuscular; subcutaneous; intraocular;
intrasynovial; transepithelially including transdermal, ophthalmic,
sublingual and buccal; topically including ophthalmic, dermal,
ocular, rectal and nasal inhalation via insufflation and aerosol;
intraperitoneal; and rectal systemic.
[0100] The active oligopeptide can be orally administered, for
example, with an inert diluent or with an assimilable edible
carrier, or it can be enclosed in hard or soft shell gelatin
capsules, or it can be compressed into tablets. For oral
therapeutic administration, the active oligopeptide may be
incorporated with excipient and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparation can
contain at least 0.1% of active oligopeptide. The percentage of the
compositions and preparation can, of course, be varied and can
conveniently be between about 1 to about 10% of the weight of the
unit. The amount of active oligopeptide in such therapeutically
useful compositions is such that a suitable dosage will be
obtained. Preferred compositions or preparations according to the
present invention are prepared such that an oral dosage unit form
contains from about 1 to about 1000 mg of active oligopeptide.
[0101] The tablets, troches, pills, capsules and the like can also
contain the following: a binder such as gum tragacanth, acacia,
corn starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, lactose or saccharin can be added
or a flavoring agent such as peppermint, oil of wintergreen, or
cherry flavoring. When the dosage unit form is a capsule, it can
contain, in addition to materials of the above type, a liquid
carrier. Various other materials can be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules can be coated with shellac,
sugar or both. A syrup or elixir can contain the active
oligopeptide, sucrose as a sweetening agent, methyl and
propylparabens a preservatives, a dye and flavoring such as cherry
or orange flavor. Of course, any material used in preparing any
dosage unit form should be pharmaceutically pure and substantially
non-toxic in the amounts employed. In addition, the active
oligopeptide can be incorporated into sustained-release
preparations and formulation.
[0102] The active oligopeptide can also be administered
parenterally. Solutions of the active oligopeptide can be prepared
in water suitably mixed with a surfactant such as
hydroxypropylcellulose. Dispersion can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0103] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It can be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacterial and fungi. The carrier can be a solvent of dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, e.g.,
sugars or sodium chloride. Prolonged absorption of the injectable
compositions of agents delaying absorption, e.g., aluminum
monostearate and gelatin.
[0104] Sterile injectable solutions are prepared by incorporating
the active oligopeptide in the required amount in the appropriate
solvent with various other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredient into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and the freeze drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from previously sterile-filtered solution
thereof.
[0105] The therapeutic oligopeptides of the present invention can
be administered to a mammal alone or in combination with
pharmaceutically acceptable carriers, as noted above, the
proportion of which is determined by the solubility and chemical
nature of the oligopeptide, chosen route of administration and
standard pharmaceutical practice.
[0106] The physician will determine the dosage of the present
therapeutic agents which will be most suitable for prophylaxis or
treatment and it will vary with the form of administration and the
particular oligopeptide chosen, and also, it will vary with the
particular patient under treatment. The physician will generally
wish to initiate treatment with small dosages by small increments
until the optimum effect under the circumstances is reached. The
therapeutic dosage can generally be from about 0.1 to about 1000
mg/day, and preferably from about 10 to about 100 mg/day, or from
about 0.1 to about 50 mg/Kg of body weight per day and preferably
from about 0.1 to about 20 mg/Kg of body weight per day and can be
administered in several different dosage units. Higher dosages, on
the order of about 2.times. to about 4.times., may be required for
oral administration.
[0107] Additional objects, advantages, and novel features of this
invention will become apparent to those skilled in the art upon
examination of the following examples thereof, which are not
intended to be limiting. In the Examples, procedures that are
constructively reduced to practice are described in the present
tense, and procedures that have been carried out in the laboratory
are set forth in the past tense.
EXAMPLES
Example 1
Ang-(1-7) Derivative High-Throughput Screening (HTS)
[0108] For HTS, a sensitive and direct measure of nitric oxide (NO)
production in 2 separate cell lines is utilized, primary CA1
hippocampal neurons and human umbilical vein endothelial cells
(HUVEC). The use of primary CA1 cells is self-evident for the study
of central effects. In addition, the contribution of endothelial
dysfunction to the progression of CHF and to the induction of
cognitive impairment is clinically appreciated. The emerging
picture that the Ang-(1-7) singling axis holds promise as a
therapeutic target for endothelial dysfunction strongly indicates
that reversal of CHF-induced endothelial dysfunction as mechanism
cannot be ruled out. HUVEC are isolated from the human umbilical
vein and cryo-preserved after primary culture. HUVEC is included as
a second line for the primary screen because these cells are the
model in vitro system for the study of endothelial cell function
and can be used to directly measure Mas-dependent NO
production.
[0109] Cell culture. To isolate primary hippocampal CA1 neuronal
cells, whole brain was removed from neonatal rat pups (1-2 day old)
and the cortices dissected away. The hippocampus was isolated and
the CA1 field was excised and placed in buffer. After gentle
disruption in digestion buffer, the cells were counted, placed in
culture media, and plated in a 96-well format coated with
poly-d-lysine. At the time of plating, cells were approximately at
50% density and were allowed to culture to 70-80% density before
starting the assay. Commercially available HUVEC (Life
Technologies/Thermo Fisher) was thawed and plated (5000-10,000
cells/well) in a 96-well format coated with gelatin. HUVEC cells
were allowed to culture overnight before starting the assay.
[0110] Cell Activation: The xCELLigence system Real-Time Cell
Analyzer (RTCA), developed by Roche Applied Science, uses
microelectronic biosensor technology to do dynamic, real-time,
label-free, and non-invasive analysis of cellular events including
G-protein receptor activation of cells. The RTCA analysis was
utilized to measure the potency and relative ability of
oligopeptides of the invention and native Ang-(1-7) to activate
human umbilical vascular endothelial cells (HUVEC) in culture.
Following uniform cellular adherence based on a linear increase in
cell impedance (CI), HUVECs were treated with Ang-(1-7) and
oligopeptides of the invention. Each trace of the CI over time in
FIG. 1 represents the average of 4 wells normalized to CI at the
time of compound addition. FIG. 1 shows the results from data
acquired using the xCELLigence RTCA to measure the relative potency
of PN-A3, PN-A4, PN-A5 and native Ang-(1-7). A 100 nM
administration of PN-A3, PN-A4 and PN-A5 and 10 nM of PN-A3 and
PN-A5 resulted in a significant (-2-fold) increase in CI over the
native Ang-(1-7) demonstrating that the oligopeptides of the
invention have greater potency for cell activation than native
Ang-(1-7).
[0111] NO production assay. As a screen for mechanisms of action of
oligopeptides of the invention, the ability to increase NO
production of three oligopeptides of the invention (PN-A3, PN-A4
and PN-A5) were characterized and compared to native Ang-(1-7).
Human umbilical vascular endothelial cells (HUVEC) culture plates
received fluorescence reaction buffer (0.2 M phosphate buffer, pH
7, 1 mM EDTA, 0.1% glucose) containing diaminofluorescein-FM
diacetate (DAF-FM, 1 .mu.M) to measure real-time NO production.
Time-resolved (10 minutes) fluorescent intensity was detected using
a BioTek Synergy 2 microplate reader with excitation at 485 nm and
emission at 535 nm. DAF-FM is a sensitive flourometric derivative
for the selective detection of NO in live cells.
[0112] FIG. 2 shows relative peak fluorescence intensity following
5 minutes exposure to native Ang-(1-7) and three oligopeptides of
the invention. Values were normalized to control fluorescence. As
expected, native Ang-(1-7) induced a significant elevation of NO
over control levels. More importantly, as shown in FIG. 2,
oligopeptides of the invention (namely PN-A3, PN-A4 and PN-A5) also
elicited a significant elevation of NO over control levels, with
PN-A5 significantly enhancing NO production over that seen with
native Ang-(1-7), *=p<0.05. These results demonstrate that
oligopeptides of the invention increase NO production similar to or
greater than that of native Ang-(1-7).
[0113] FIG. 3A illustrates the ability of the select Mas receptor
antagonists, A779, (C.sub.39H.sub.60N.sub.12O.sub.11) which is
known to block native Ang-(1-7) NO production, to also block NO
production induced by the oligopeptide of the invention, namely
PN-A5. In these studies, HUVEC cells were incubated with DAF-FM, 1
.mu.M to measure real-time NO production. Cells were treated with
either PN-A5 alone (1.0 mM, n=10), PN-A5+A779 (n=6). Measurements
were obtained using an Olympus 550 Confocal Microscope and analyzed
using Image J. Images were obtained every 10 sec. These results
indicate that the oligopeptide PN-A5 actions are due to activation
of the Mas receptor.
[0114] FIG. 3B shows the averaged effect of the select Mas receptor
antagonists, A779, which is known to block native Ang-(1-7) NO
production, to also block NO production induced by the oligopeptide
of the invention, PN-A5. In these studies, HUVEC cells were
incubated with DAF-FM, 1 .mu.M to measure real-time NO production.
Cells were treated with either PN-A5 alone (1.0 mM, n=10),
PN-A5+A779 (n=6), or the NO donor S-nitroso-N-acetylpenicillamine
(SNAP). Fluorescent measurements were obtained using an Olympus 550
Confocal Microscope and analyzed using Image J. Images were
obtained every 10 sec. The NO response produced by PN-A5 was
completely blocked by A779 demonstrating that PN-A5's ability to
increase NO is due to PN-A5 actions on the Mas receptor.
Example 2
Effects of Ang-(1-7) Derivative on Heart Failure (HF) Induced
Cognitive Impairment
[0115] A total of 33, male C57B1/6J adult mice (Harlan, 8-10 weeks
old) were used. Mice were randomly assigned to either the sham
(n=12) or congestive heart failure (CHF) group (n=21). Experimental
groups are described as follows: sham+saline, CHF+saline,
CHF+PN-A5. All mice prior to surgery were weighed and anesthetized.
For the CHF mice, MI was induced by ligation of the left coronary
artery (LCA). Under anesthesia (2.5% isoflurane in a mixture of air
and O.sub.2) a thoracotomy was performed at the fourth left
intercostal space and the LCA permanently ligated to induce a
myocardial infarction (MI). Occlusion of the LCA was confirmed by
observing blanching, a slight change in color of the anterior wall
of the left ventricle downstream of the ligature. Sham mice
underwent the same procedure with the exception of ligating the
LCA.
[0116] Following 8 weeks post MI surgery, CHF mice were treated
with either daily subcutaneous injections of the Ang-(1-7)
derivative PN-A5 (1 mg/kg/day) for 28 days or saline. After 21
days, animals were tested for object recognition using a standard
NOR test as described below. After approximately 25 days of
treatment, animals were tested for spatial memory using the
standard Morris water task as described below.
[0117] Novel Object Recognition (NOR): The apparatus consisted of
an evenly illuminated Plexiglas box (12 cm.times.12 cm.times.12 cm)
placed on a table inside an isolated observation room. All walls of
the apparatus were covered in black plastic, and the floor was grey
with a grid that was used to ensure that the location of objects
did not change between object familiarization and test phases. The
mouse behavior and exploration of objects was recorded with a
digital camera. The digital image from the camera was fed into a
computer in the adjacent room. Two digital stopwatches were used to
track the time the mouse spent interacting with the objects of the
test. All data was downloaded to Excel files for analysis.
Triplicate sets of distinctly different objects were used for the
test.
[0118] The novel object recognition task included 3 phases:
habituation phase, familiarization phase, and test phase. For the
habituation phase, on the first and second day, mice were brought
to the observation room habituated to the empty box for 10 min per
day. On the third day, each mouse had a "familiarization" trial
with two identical objects followed by a predetermined delay period
and then a "test" trial in which one object was identical to the
one in the familiarization phase, and the other was novel. All
stimuli were available in triplicate copies of each other so that
no object needed to be presented twice. Objects were made of glass,
plastic or wood that varied in shape, color, and size. Therefore,
different sets of objects were texturally and visually unique. Each
mouse was placed into the box the same way for each phase, facing
the center of the wall opposite to the objects. To preclude the
existence of olfactory cues, the entire box and objects were always
thoroughly cleaned with 70% ethanol after each trial and between
mice. During the familiarization phase, mice were allowed to
explore the two identical objects for 4 min and then returned to
their home cages. After a 2 hour delay, the "test phase" commenced.
The mice were placed back to the same box, where one of the two
identical objects presented in the familiarization phase was
switched to a novel one and the mouse was allowed to explore these
objects for another 4 min. Mouse "exploratory behavior" was defined
as the animal directing its nose toward the object at a distance of
.about.2 cm or less. Any other behavior, such as resting against
the object, or rearing on the object was not considered to be
exploration. Exploration was scored by an observer blind to the
mouse's surgical group (CHF vs. Sham). Finally, the positions of
the objects in the test phases, and the objects used as novel or
familiar, were counterbalanced between the 2 groups of mice.
[0119] Discrimination ratios were calculated from the time spent
exploring the novel object minus time spent exploring the familiar
object during the test phase divided by the total exploration time.
DRatio=(t novel-t familiar)/(t novel+t familiar). Data were
analyzed from first 2 minutes of `test phase`. A positive score
indicates more time spent with the novel object, a negative score
indicates more time spent with the familiar object, and a zero
score indicates a null preference. All NOR data was examined using
one-way analysis of variance, between subjects (ANOVA). Individual
group differences were tested using the post hoc Tukey HSD test. In
comparisons between groups of different sample sizes, equal
variance was tested using a modified Levene's test. All statistical
tests and p-values were calculated using MS Excel with Daniel's
XLtoolbox and alpha was set at the 0.05 level. Error bars represent
SEM.
[0120] Morris Water Task: Testing Spatial Learning and
Memory/Visual Test: The apparatus used was a large circular pool
approximately 1.5 meters in diameter, containing water at
25.degree. C. made opaque with addition of non-toxic white Crayola
paint. An escape platform was hidden just below the surface of the
water. Visual, high contrast cues were placed on the walls of the
test room. A digital camera connected to a computer in the adjacent
room is suspended over the tank to record task progress. For
spatial testing prior to MI at 4 and 8 weeks post-MI or sham
surgery, the platform was located at different sites in the
pool.
[0121] During the spatial version of the Morris water task, all
animals were given 6 training trials per day over 4 consecutive
days. During these trials, an escape platform was hidden below the
surface of water. Mice were released from seven different start
locations around the perimeter of the tank, and each animal
performed two successive trials before the next mouse was tested.
The order of the release locations was pseudo-randomized for each
mouse such that no mouse was released from the same location on two
consecutive trials. Performance on the swim task was analyzed with
a commercial software application (ANY-maze, Wood Dale, Ill.).
Because different release locations and differences in swimming
velocity produce variability in the latency to reach the escape
platform, a corrected integrated path length (CIPL) was calculated
to ensure comparability of mice performance across different
release locations. The CIPL value measures the cumulative distance
over time from the escape platform corrected by an animal's
swimming velocity, and is equivalent to the cumulative search
error. Therefore, regardless of the release location, if the mouse
mostly swims towards the escape platform the CIPL value will be
low. In contrast, the more time a mouse spends swimming in
directions away from the platform, the higher the CIPL value.
[0122] Following approximately 21 days of treatment with
oligopeptide PN-A5, CHF mice showed object recognition memory
improvement. FIG. 4 illustrates the effects of three weeks
treatment with oligopeptide PN-A5 on object recognition memory as
determined by the Novel Object Recognition Test (NOR). The mean
performance of CHF mice with oligopeptide PN-A5 treatment (n=11)
was similar to sham mice with saline (n=6), (CHF-Ang-(1-7)
derivative PN-A5 M=+0.38, SE 0.11 vs. Sham-saline M=+0.52, SE 0.06)
and significantly greater in comparison to CHF mice treated with
saline (n=10) (M=-0.05, SE 0.09, *=p=0.009. These results
demonstrate that oligopeptide PN-A5 acts to attenuate and even
rescue object recognition memory impairment in mice with CHF.
[0123] Following approximately 25 days of treatment with
oligopeptide PN-A5, CHF mice showed spatial memory improvement.
FIG. 5 shows the mean CIPL of CHF+oligopeptide PN-A5 mice (n=11),
CHF-saline treated mice (n=10) and Sham+saline mice (n=6). The
CHF+oligopeptide PN-A5 mice showed significant improvement in
spatial memory day 3 of the Morris swim task as compared to
CHF-saline mice. CHF mice treated with saline had a significantly
higher CIPL score as compared to CHF-oligopeptide PN-A5 treated
mice (CHF-saline M=32.5, SE=2.1 vs CHF-oligopeptide PN-A5 M=23.5,
SE 2.2, *=p=0.003. These results demonstrate that oligopeptide
PN-A5 improves spatial memory.
Example 3
Effect of Oligopeptide PN-A5 on Nitric Oxide Bone Pain
[0124] Female BALB/cfC3H mice (Harlan, Ind., USA) were 15 to 20 g
prior to initiation of study (n=5 animals per treatment group).
Clinical signs of morbidity were monitored and mice not meeting
inclusion parameters (e.g. paralysis, rapid weight loss of >20%
in 1 week) were removed from the study.
[0125] Mice were anesthetized with ketamine:xylazine (80 mg:12
mg/kg, 10 ml/kg injection volume; Sigma-Aldrich). An arthrotomy was
performed. The condyles of the right distal femoris were exposed
and a hole was drilled to create a space for injection of
4.times.10.sup.4 66.1 cells in 5 .mu.L Opti-MEM or 5 .mu.L Opti-MEM
without cells in control animals within the intramedullary space of
the mouse femoris. Injections were made with an injection cannula
affixed via plastic tubing to a 10-.mu.L Hamilton syringe (CI31,
Plastics One). Proper placement of the injector was confirmed
through use of Faxitron X-ray imaging. Holes were sealed with bone
cement.
[0126] Spontaneous pain (flinching and guarding), and tactile
allodynia were measured 0, 15, 30, 60, 90 and 120 minutes after a
single dose of drug was administrated in a blinded fashion on Day
7. Breast cancer-induced hypersensitivity returned to baseline
levels 2 hours after drug administration. Flinching and guarding
were observed for duration of 2 minutes during a resting state.
Flinching was characterized by the lifting and rapid flexing of the
right hind paw when not associated with walking or movement.
Flinches were recorded on a five-channel counter. Guarding was
characterized by the lifting the right hind limb into a fully
retracted position under the torso. Time spent guarding over the
duration of 2 minutes was recorded.
[0127] The assessment of tactile allodynia consisted of measuring
the withdrawal threshold of the paw ipsilateral to the site of
tumor inoculation in response to probing with a series of
calibrated von Frey filaments using the Chaplan up-down method with
the experimenter blinded to treatment groups. The 50% paw
withdrawal threshold was determined by the nonparametric method of
Dixon.
[0128] On day 7, mice received an intraperitoneal (i.p.) injection
of either saline or 0.8 .mu.g/.mu.L (200 .mu.L) for a total dose of
800 .mu.g/kg. The in-vivo efficacy of PN-A5 was measured for a
total of 2 hours.
[0129] Within group data were analyzed by non-parametric one-way
analysis of variance. Differences were considered to be significant
if P.ltoreq.0.05. All data were plotted in GraphPad Prism 6.
[0130] FIG. 6 shows the results on the effects of oligopeptide
PN-A5 on cancer induced bone pain (CIBP). Cancer implanted into the
distal femoralis of mice induced a significant increase in the
number of spontaneous flinches (FIG. 6A) and time spent guarding
(FIG. 6B) after 7 days. Administration of a bolus of PN-A5 (800
.mu.g/kg, i.p.) significantly reversed cancer induces spontaneous
pain for nearly one hour in duration (flinching: 60 min; guarding:
30 min; p<0.001). Similarly, cancer-induced tactile
hypersensitivity was significantly attenuated 30 minutes after
injection (p<0.01). For all measurements, the time of peak
effect was 15-30 min. Behaviors returned to post-surgery values 90
min post-injection. Media inoculated, sham control animals did were
not statistically different from pre-surgery baselines at any point
during time-course.
Example 4
Effect of Ang(1-7) on Cancer-Induced Bone Pain
[0131] The effect on cancer-induced bone pain of the archetypical
Mas receptor agonist, native Ang(1-7) was investigated. It was
discovered that the action of Ang-(1-7) at the Mas receptor
inhibits pain via the tumor-nociceptor microenvironment and not the
tumor-bone environment because Ang-(1-7) induced analgesia but did
not significantly change the tumor-induced degradation of the bone
or otherwise reduce bone loss. Chronic treatment with Ang-(1-7) did
not significantly alter tumor proliferation, further suggesting the
analgesic effect is directly towards inhibiting nociceptive
activation and not due to changes in tumor burden.
[0132] Cell Culture: A murine mammary adenocarcinoma cell line,
66.1, was cultured in Eagle's minimum essential medium with 10%
fetal bovine serum, 100 IU-1 penicillin, and 100 .mu.g mL-1
streptomycin (P/S). The 66.1 cells were plated in T-75 tissue
culture flasks, allowed to grow exponentially in an incubator at
37.degree. C. and 5% CO.sub.2. The viability of cells cultured with
treatments described below was measured using the XTT assay (ATCC,
Manassas, Va.).
[0133] Animals: Female BALB/cAnNHsd mice (Harlan, Ind., USA)
between 15 and 20 g were used in this study. Mice were housed in a
climate control room on a 12-hour light/dark cycle and allowed food
and water ad libitum. Animals were monitored on days 0, 7, 10, and
14 of the study for clinical signs of rapid weight loss and signs
of distress.
[0134] Drug Treatment: Animals received Angiotensin-(1-7) (Tocris,
Ellisville, Mo.), the MasR antagonist A-779 (Abcam, Cambridge,
Mass.), the AT1 antagonist Losartan potassium (Tocris Bioscience,
Minneapolis, Minn.), or the AT2 antagonist PD 123319
ditrifluoroacetate (Tocris Bioscience, Minneapolis, Minn.)
dissolved in 0.9% saline. All intraperitoneal (i.p.) injections
were made at a volume of 10 mL/kg. Systemic doses as follows:
Ang-(1-7) =0-100 .mu.g/kg, A-779=0.19 .mu.g/kg, Losartan
potassium=0.4 mg/kg, PD 123319 ditrifluoroacetate=0.4 mg/kg. In
antagonist studies, A-779, Losartan potassium, or PD 123319
ditrifluoroacetate was administered 30 minutes prior to
Ang-(1-7).
[0135] Tail Flick: A warm water (52.degree. C.) tail flick test was
used to determine the effects of Ang-(1-7) on acute nociception.
The distal third of the tails of naive mice were submerged into the
water bath. The withdraw latency, defined as the time for the tail
to be withdrawn from the water bath, was recorded. A cutoff time of
10 seconds was enforced to prevent tissue damage. Baseline
latencies were recorded prior to drug administration. Animals were
dosed (i.p.) with Ang-(1-7) (0-100 .mu.g/kg). Tail flick latencies
were reassessed 15, 30, 60, 90, 120, 150, and 180 minutes
post-treatment.
[0136] Rotarod: A rotarod performance test was used to determine
the motor and/or sedative effects of Ang-(1-7) (Rotamex 4/8,
Columbus Instruments, Columbus, Ohio, USA). Three days prior to
testing, naive mice were subjected to 5 trials in which they were
able to acclimate to the rotating rod (10 revolutions/min). On the
day of testing, animals were allowed one trial and then baselined.
The amount of time the animal remained on the rod was recorded,
with a cutoff time of 120 seconds to prevent exhaustion. Animals
were dosed (i.p.) as previously described and reevaluated 15, 30,
60, and 120 minutes post-administration.
[0137] Arthrotomy-Intramedullary implantation of 66.1 cells: To
induce CIBP, an arthrotomy was performed. Briefly, animals were
anesthetized with 80 mg/kg ketamine-12 mg/kg xylazine (in a 10
mL/kg volume). The surgical area was shaved and cleaned with 70%
ethanol and betadine. The condyles of the right femur were exposed
and a burr-hole (0.66 mm) was drilled to create a space for the
66.1 cell inoculation. A 5 .mu.l volume of 66.1 cells (8,000 cells
per 1 .mu.l) in MEM (or 5 .mu.l MEM without cells in sham animals)
was injected into the intramedullary space of the mouse femora.
Proper placement of the injector was confirmed by radiograph
(Faxitron X-ray imaging). Holes were sealed with bone cement and
the patella reset. Muscle and skin were closed in separate layers
with 5-0 vicryl suture and wound autoclips, respectively. Animals
were given 8 mg/kg (10 mL/kg volume) gentamicin to prevent
infection. Staples were removed 7 days post-surgery.
[0138] Acute Behavioral Testing: Fourteen days post-surgery,
baseline behaviors of spontaneous flinching/guarding were recorded.
Flinching was characterized by the lifting and rapid flexing of the
hind paw ipsilateral to femoral inoculation when not associated
with walking or other movement. Guarding was characterized by the
lifting the inoculated hind limb into a fully retracted position
under the torso. The total number of flinches and the time spent
guarding 2 min duration was recorded. Mice were then separated into
treatment groups and dosed systemically with Ang-(1-7) (0-10
.mu.g/kg), A-779 (0.19 .mu.g/kg), Losartan potassium (0.4 mg/kg),
PD 123319 (0.4 mg/kg), vehicle (0.9% saline), or a combination of
Ang(1-7) and each antagonist. Antagonists were administered 30
minutes prior to Ang-(1-7). Following administration, animals were
tested at over a three-hour time course until their pain behaviors
returned to baseline
[0139] Chronic Behavioral Testing: Seven days post-surgery,
baseline behaviors of spontaneous pain, as described above, were
recorded. Mice were treated (i.p.) with Ang-(1-7) (0.058 .mu.g/kg),
A-779 (0.19 .mu.g/kg), vehicle (0.9% saline), or a combination.
Antagonist was administered 30 minutes prior to Ang-(1-7). Animals
were dosed at the same time each day 7 to 14 days post-surgery. On
day 10, pain behaviors were assessed 15 minutes following
treatment, based on the time of peak effect determined by the acute
studies. Fourteen days post-surgery, behaviors were again recorded
pre- and post-treatment. Animals were sacrificed following
treatment and testing on the fourteenth day post-surgery, and the
following tissues were collected for biochemical analyses: serum,
femur extrudate, and lumbar dorsal root ganglia.
[0140] Nesting: Nesting behaviors of naive, media, and
cancer-inoculated mice were assessed using the protocol described
by Negus et al. Animals were acclimated to individual cages,
without an existing nest, for 30 minutes prior to drug
administration. Cotton fiber nestlets were cut into 6 equal pieces,
and each piece was placed in the cage in 6 zones in the manner
previously described following drug administration. Throughout the
duration of the 100-minute time course, the number of cleared zones
was recorded; upon completion the height (mm) of each fluffed
nestlet was measured.
[0141] Western Blot Analysis: Dorsal root ganglia (DRG) and femur
extrudates from mice used in behavioral studies were analyzed for
expression of MasR. DRGs were homogenized in modified
radioimmunoprecipitation assay (RIPA) buffer with protease
inhibitor cocktail and EDTA (Pierce, Rockford, Ill., USA) via
sonication. 10 .mu.g of each sample was resolved on a 10%
SDS-polyacrylamide gels (TGX Criterion XT; Bio-Rad, Hercules,
Calif.) and transferred to a polyvinylidene difluoride membrane
(PVDF, Bio-Rad, Hercules, Calif.). Ipsilateral and contralateral
femurs were removed from each animal. For each femur, the proximal
and distal ends were clipped and the intramedullary extrudate was
flushed six times with 700 .mu.L phosphate-buffered saline
containing protease inhibitor cocktail and EDTA (Pierce, Rockford,
Ill., USA). Femur marrow from five animals was pooled per sample
and 15 .mu.g of sample was resolved and transferred in the same
manner as DRGs. Protein transfer was verified by staining blots
with Ponceau S (Sigma, St. Louis, Mo.), and PVDF membranes were
blocked with 5% non-fat dry milk in Tris-buffered saline containing
0.05% (v/v) Tween-20 (TBST) for one hour at room temperature.
Membranes were then incubated with primary antibody: rabbit
polyclonal anti-Angiotensin-(1-7) Mas Receptor (Alomone Labs
AAR-013; 1:200 dilution for DRGs or 1:800 for femurs) or mouse
monoclonal anti-actin AC40 (Cell Signaling 7076S; 1:4,000 dilution)
in 1% milk in TBST overnight at 4.degree. C. The membranes were
washed in TBST and incubated with appropriate secondary antibodies
(Cell Signaling 7074 Anti-rabbit IgG HRP-Linked, 1:10,000 dilution;
Cell Signaling 7076 Anti-mouse IgG HRP-Linked, 1:5000 dilution) for
1 hour at room temperature. Membranes were again washed and
developed using enhanced chemiluminescence (Clarity ECL Substrate,
Bio-Rad, Hercules, Calif.), and bands were detected using GeneMate
Blue-Ultra Autorad films (BioExpress, Kaysville, Utah. Bands were
quantitated and corrected for background using ImageJ densitometric
software (Wayne Rasband, Research Services Branch, National
Institute of Mental Health, Bethesda, Md.). All data were
normalized to actin in each lane and reported as fold change over
untreated control.
[0142] Ang-(1-7) Administration in Established CIBP Attenuates
Spontaneous Pain in a MasR Dependent Manner.
[0143] The antinociceptive efficacy of Ang-(1-7) in a model of
established CIBP in which 66.1 tumor cells were injected into the
right femurs of syngeneic BALB/cAnNHsd mice was investigated. Prior
to surgery, mice did not display behavioral signs of pain (data not
shown). Animals showed a significant amount of flinching (FIG. 7A
at "post-Sx") and guarding (FIG. 7B at "post-Sx") compared to
media-treated controls (p<0.0001, n=8). A single systemic
injection of Ang-(1-7) (0.036, 0.360, 1, and 10 .mu.g/kg) or
vehicle was administered, and pain behaviors were assessed. Animals
given an acute i.p. administration of Ang-(1-7) showed a
significant (p<0.01, n=8) reduction in spontaneous pain
behaviors with an onset 15 min after injection of either 0.36 or 1
.mu.g/kg which persisted for nearly 2 hours (FIGS. 7A and 7C).
[0144] Dose response curves were constructed from data collected at
the time of peak effect, 15 min, for guarding and flinching
behavior (FIGS. 7B and 7D, respectively). At 15 min, the maximum
effect of Ang-(1-7) in reducing guarding behavior was 52.75%
(p<0.01, n=8) with a corresponding A90 dose of 0.058 .mu.g/kg
(FIG. 1B). Flinching displayed less of a dose-dependency and a more
significant inhibition at the lower dose (0.036 .mu.g/kg). Thus, a
single injection of Ang-(1-7) is effective in reducing spontaneous
pain behavior by more than 50% in animals with established
CIBP.
[0145] To confirm that the observed Ang-(1-7) effect is mediated by
the Mas receptor, A-779 (0.19 .mu.g/kg), a selective MasR
antagonist, or vehicle was administered 30 minutes prior to
Ang-(1-7) (0.058 .mu.g/kg) 14 days post-femur inoculation.
Inhibition of MasR with A-779 alone did not alter spontaneous or
evoked pain thresholds; however, pretreatment with A-779
significantly inhibited Ang-(1-7) attenuation of guarding (FIG. 7E,
p<0.01) and flinching (FIG. 7F, p<0.001). These data
demonstrate that Ang-(1-7) elicits antinociception in established
CIBP through actions at MasR.
[0146] Antinociceptive Effects of Ang-(1-7) through MasR are
Maintained After Repeated Administration.
[0147] The antinociceptive activity of repeated Ang-(1-7) was
investigated to determine whether, like other analgesics, chronic
administration results tolerance. Ang-(1-7) (0.058 .mu.g/kg, i.p.)
was administered daily, beginning 7 days post implantation of 66.1
cells into the femur. Mice were evaluated for CIBP spontaneous pain
behaviors on day 7 prior to drug administration, and on days 10 and
14 post-surgery 15 minutes post-treatment. Cancer inoculation
significantly increased the amount of time spent guarding and
number of flinches 7 days post-surgery (p<0.0001, n=12). Animals
experienced significant (p <0.0001, n=12) reduction in guarding
(FIG. 8A) and flinching (FIG. 8B) following Ang-(1-7) treatment on
days 10 and 14 post-surgery. Vehicle treatment had no significant
effect.
[0148] A-779 was again used to confirm that the antinociceptive
effects of Ang-(1-7) are mediated by the Mas receptor. A-779 (0.19
.mu.g/kg) was administered 30 minutes prior to Ang-(1-7) (0.058
.mu.g/kg) daily 7-14 days post-cancer inoculation (FIGS. 8C and
8D). Administration of A-779 alone had neither a pro- or
anti-nociceptive effect on the mice, and similar to earlier
observations following a single injection, the chronic
pre-treatment with A779 before Ang-(1-7) prevented attenuation of
CIBP by the latter.
[0149] Effects of AT1/AT2 Antagonists on Ang-(1-7)/MasR-Mediated
Antinociception in Established CIBP.
[0150] Ang II is the precursor molecule to Ang-(1-7). Accordingly,
the role of the AngII receptors in mediating Ang-(1-7)
antinociception was investigated using a pre-treatment of the AT1
or AT2 selective antagonists, Losartan potassium (Ki=10 nM) and PD
123319 (also known as EMA200) (IC50=34 nM), respectively. Animals
were inoculated with 66.1 cells or media, as previously described,
and pain behaviors were assessed 14 days post-femur inoculation.
Mice received either the AT1 or AT2 antagonist (0.4 mg/kg, i.p.) 30
minutes prior to Ang-(1-7) (0.058 .mu.g/kg, i.p.), or vehicle (0.9%
saline). Spontaneous pain behaviors of flinching and guarding were
recorded 15, 30, 60, 90, 120, and 150 minutes post-administration.
Confirming the previous results, Ang-(1-7) administration alone
reduced pain behaviors (p<0.01, n=7), while neither Losartan
potassium nor PD 123319 significantly altered pain behaviors when
administered alone. Interestingly, administration of the AT1
receptor antagonist, Losartan potassium, prior to Ang-(1-7) yielded
a 77.527% maximal possible efficacy (MPE) in reducing guarding
(p<0.0001, n=7) 30 minutes post-administration (FIG. 9A) and an
80.56% MPE in reducing flinching (p<0.0001, n=7) 15 minutes
post-administration (FIG. 9B). However, use of the AT2 antagonist,
PD 123319, prior to Ang-(1-7) did not further increase nor decrease
guarding or flinching of animals with established CIBP (FIGS. 9C
and 9D) as compared to the animal group treated with solely
Ang-(1-7). The additive effect of Losartan potassium to potentiate
Ang-(1-7)-induced antinociception was not further investigated but
may result from the ability of Losartan to prevent Ang-(1-7)
binding to the AT1 receptor, thereby increasing the free
concentration of Ang-(1-7) to bind to the Mas receptor.
[0151] Ang-(1-7) Administration in Established CIBP Does Not Change
Nesting Behaviors.
[0152] Nesting is an innate behavior in mice that is hindered by
various states of pain. The effect of both the arthrotomy and
Ang-(1-7) administration on nesting behavior was evaluated. Six
equally sized pieces of nestlet were placed into 6 zones of the
animals' individual cages. The number of zones that the animals
cleared of the nestlet pieces was recorded over the 100-minute time
course (FIG. 10A). During the first hour of the study on
post-surgical day 6, the 66.1-inoculated animals cleared
significantly fewer zones than the naive animals (p<0.05). After
the second hour of the study, the media animals cleared fewer zones
than both the naive and 66.1-inoculated animals (p<0.05). A
second study was conducted on post-surgical day 15 in which
66.1-inoculated animals were treated with Ang-(1-7) (0.058
.mu.g/kg, i.p.) or vehicle (0.9% saline) (FIG. 10B). The nesting
behaviors of both treated cancerous groups did not differ
significantly from the naive group. However, at both the 75 and
90-minute time points, the media animals cleared fewer zones than
the other groups (p<0.05). These data demonstrate that while the
nesting of animals with established CIBP alters the nesting
behaviors of mice, Ang-(1-7) administration does not further alter
these complex behaviors.
[0153] Ang-(1-7) Administration Results in Antinociception but not
Motor Impairment in Naive Mice
[0154] Ang-(1-7) was systemically administered (0.360, 1, 10
.mu.g/kg, i.p.) to naive mice. Small but significant increase in
thermal tail flick latencies were observed (data not shown).
Ang-(1-7) effects peaked between 15 and 30 min post administration
with 1 .mu.g/kg Ang-(1-7) (MPE=27.8%, p<0.001) and 10 .mu.g/kg
Ang-(1-7) (MPE=20.2%, p<0.01) that returned to baseline between
90 to 120 min.
[0155] To exclude the possibility that Ang-(1-7) administration
reduced mobility in order to increase tail withdraw latency,
rotarod testing was performed. Naive animals were trained to walk
on a rotating rod for 2 min. After training, animals were injected
with Ang-(1-7) by either spinal (0.3 pmol/5 .mu.L) or systemic
routes (0.058 and 10 .mu.g/kg). No significant differences in
rotarod latencies were observed between vehicle and Ang-(1-7)
treated mice (results not shown; p=0.99 i.t.; p=0.18 i.p.).
Together, these data suggest that systemic Ang-(1-7) is
antinociceptive after a single administration without noticeable
impact on motility.
[0156] MasR is Expressed in the Dorsal Root Ganglion and Femur
Extrudate.
[0157] Ipsilateral lumbar dorsal root ganglion (DRG) and femur
extrudate were collected from naive, sham, cancer (66.1), and 66.1
Ang-(1-7) treated mice. In naive mice, MasR is expressed in the
dorsal root ganglia (FIG. 11A); MasR bands were observed at
.about.50 kDa and .about.40 kDa. Sham surgery (i.e. media only) or
the introduction of the murine mammary adenocarcinoma line 66.1
into the femoral intramedullary space did not significantly alter
MasR expression levels in ipsilateral DRGs relative to the
contralateral control DRGs (FIG. 11B). MasR was also found to be
expressed in the femur extrudates of the same mice (FIG. 11C).
Inoculation with the 66.1 cells significantly increased the
expression of MasR in the femur extrudate at both .about.50 kDa and
.about.40 kDa (p<0.001 compared to sham group), while sham
surgery did not significantly alter the expression of MasR in the
femur extrudate as compared to the naive animal group (FIG.
11D).
[0158] Repeated Dosing of Ang-(1-7) Does Not Alter Tumor Burden of
Mice with Established CIBP or Alter Cell Viability In Vitro.
[0159] Tumor burden was evaluated to determine whether the
antinociceptive effect of repeated Ang-(1-7) administration results
from an antineoplastic activity. Following chronic administration
studies, femurs were harvested from animals, decalcified, and
embedded in paraffin blocks prior to sectioning (5 micron) and
hemotoxylin/eosin staining (FIG. 12A). The region of the bone
containing cancer cells was quantified as a measure of total
intramedullary content and represented as a percent of the entire
cells within the bone. Repeated Ang-(1-7) administration did not
significantly increase nor decrease the percent tumor of the bone
(p=0.3, n=3-5) as compared to the saline-treated group (FIG. 12B).
Thus, repeated Ang-(1-7) administration did not significantly
impact tumor burden within the femur making it unlikely that the
antinociceptive effect is secondary to an antineoplastic
activity.
[0160] To verify in vivo findings, the effect of Ang-(1-7) on 66.1
cell viability was assessed in vitro. 66.1 cells were treated with
vehicle, or increasing concentrations of Ang-(1-7) (1, 10, 100, or
1000 ng) for 24 hr and an XTT cell viability assay was performed.
As compared to vehicle treated cells (relative absorbance
(RA.+-.SD)=1.02.+-.0.09), each of the four Ang-(1-7) treatments did
not significantly change cell viability (1 ng: 0.93.+-.0.11; 10 ng:
0.93.+-.0.07; 100 ng: 0.78.+-.0.11, 1000 ng: 0.93.+-.0.13).
Together, these data indicate that Ang-(1-7) at the
doses/concentrations tested neither promotes tumor cell
proliferation nor causes tumor cell death both in vivo and in
vitro.
[0161] Repeated Dosing of Ang-(1-7) Does Not Affect Bone Remodeling
of Mice with Established CIBP.
[0162] Radiographic images of all chronically treated animals were
taken on day 0, 7, 10, and 14 post-surgery to determine whether
repeated Ang-(1-7) administration affected bone remodeling in mice
with established CIBP (FIG. 13A). Day 14 images were scored by
three blinded observers with the following scale: 0--healthy bone,
1--1-3 lesions; 2--4-6 lesions; 3--unicortical fracture;
4--bicortical fractures (FIG. 13B). A healthy bone was defined as
one without any visible lesions or fractures, and a lesion was
defined as a dark hole-like spot below the epiphyseal plate. While
no animals experienced bicortical fractures, both saline and
Ang-(1-7)-treated cancer-inoculated animals experienced unicortical
fractures. Sham-treated (media) animals (7 out of 16) received
scores of 0. As another marker of bone remodeling, levels of
carboxy-terminal collagen crosslinks (CTX) in the serum were
quantified (FIG. 13C). While cancer-inoculation significantly
increased (p<0.05, n=3-4) CTX levels in the bone compared to
both media controls, Ang-(1-7) repeated administration did not
significantly alter CTX levels compared to the 66.1 saline treated
group. Overall, daily Ang-(1-7) administration in mice with
established CIBP did not significantly influence bone remodeling of
the ipsilateral femur.
Example 5
Ang-(1-7)Mitigates Cognitive Deficits Caused by Traumatic Brain
Injury
[0163] Twenty-four C57/B16 mice (5.5 weeks, mass=18 to 20 g) were
used for the duration of the study. The animals were housed in a
humidity- and temperature-controlled environment and maintained on
a 12:12 light:dark cycle (7:00 am-7:00 pm). Standard food and water
were available ad libitum. The mice were divided into two main
treatment groups: 1.) intraperitoneal (i.p.) injections of a normal
saline (0.90%) vehicle (n=12) and (2.) i.p. injections of 0.1 mg/mL
Ang-(1-7) (1 mg/kg) (n=12). A traumatic brain injury (TBI) model of
closed head injury in mice using a pneumatic impactor capable of
delivering a blow of a predetermined velocity, depth, and dwell
time (duration of cortical depression) to a defined, 7.07 mm.sup.2
area of the skull (Xiong, Mahmood, & Chopp, 2013) was used.
Mice were first anesthetized using a 5% isoflurane vapor for
induction. Once a response to toe-pinch was no longer observed, the
mice were secured in the ear bars of a stereotaxic frame beneath
the head impactor (TBI-0310 Impactor, Precision Systems) during
which time 2.5% isoflurane was administered for maintenance of
anesthesia. The parameters of each administered impact were set to
the following: diameter of tip of cylindrical piston=3 mm; velocity
of piston (v.sub.p)=4.0 m/s; depth of impact (d.sub.i)=1 mm; dwell
time (t.sub.dwell)=0.5 s. The point of impact was universalized in
the medio-lateral plane to 1.5 mm left of the sagittal suture (as
estimated by the mid-sagittal line of the mouse's head), and in the
antero-posterior plane to an imaginary line intersecting the
anterior point of insertion of the mouse's ears (approximately 1-2
mm anterior to the lambdoid suture). This point was chosen so as to
avoid rupture of the superior sagittal sinus and the confluence of
sinuses.
[0164] Immediately after being subjected to impact, mice were
monitored for recovery of spontaneous respiration. Once noted to be
breathing normally, mice were placed on the bedding of their normal
enclosures and allowed to recover for 24 hours prior to their
first, post-TBI novel object recognition trial.
[0165] The novel object recognition (NOR) task, as it pertains to
the study of working memory and attention, is predicated on rodent
preference of novel stimuli, whether spatial or otherwise
(Ennaceur, Cavoy, Costa, & Delacour, 1989; Ennaceur &
Delacour, 1988; Goulart et al., 2010; Silvers, Harrod, Mactutus,
& Booze, 2007). When novel objects are paired simultaneously
with familiar ones in an environment to which the animal has been
habituated, it is possible to use the difference in exploration
times of each object to make determinations of the degree of
cognitive impairment relative to a measured baseline (Aggleton,
Albasser, Aggleton, Poirier, & Pearce, 2010; Antunes &
Biala, 2012; Olarte-Sanchez, Amin, Warburton, & Aggleton,
2015). The primary metric used to compare mice of different groups
is the discrimination ratio (DR)--a value calculated as the ratio
of time spent exploring the novel object (NO) to the total time
spent exploring the familiar objects (FO) in addition the NO, i.e.
DR=Time at NO/(Time at NO+Time at FO). In slight contrast to
definitions of exploration used by previous authors (Aggleton et
al., 2010; Aubele, Kaufman, Montalmant, & Kritzer, 2008;
Ennaceur & Delacour, 1988; Goulart et al., 2010; Silvers et
al., 2007), exploration in this investigation was defined as the
directing of the nose toward an object at a distance of <2 cm
from the object, touching an object with the nose or mouth,
touching the object with both front paws, or standing on the object
itself
[0166] The overall structure of the NOR task, including associated
familiarization trials, is as follows: mice from both groups
underwent a two-day, combined habituation/familiarization phase,
wherein they were allowed to roam freely in an evenly-lit, plastic,
rectangular enclosure with walls 19.05 cm in height, containing
three identical objects made of either glass or plastic, for five
minutes. On the third day, the same test was run, but with one of
the three "familiar" objects replaced with the NO, all spatial
characteristics of the enclosure and objects therein remaining the
same. Data collected on the third day constituted each mouse's
baseline DR. On the fourth day, mice in both groups were subject to
TBI as delineated in the previous section. The fifth day
constituted the 24-hour post-TBI time point, wherein mice were
administered an i.p. injection of either normal saline (vehicle
group) or Ang-(1-7) solution (drug group) 30 minutes prior to
undergoing the NOR task (NOs were rotated such that no animal saw
the same NO twice). This pattern of injection and subsequent NOR
trial was repeated to five days post-TBI. Both groups were run
through two additional NOR tasks on post-TBI days 8 and 16 without
prior drug or saline administration, again on post-TBI day 18 with
prior drug or saline administration, and again on post-TBI day 25
without prior drug or saline administration. All NOR trials were
filmed in high definition and manually reviewed using two
stopwatches to determine the time spent at either a novel or
familiar object.
[0167] Temporal data collected from each NOR trial was tabulated in
a Microsoft Excel (2016) spreadsheet and individual discrimination
ratio values calculated therein. Two-way ANOVA followed by a Tukey
range test was performed using GraphPad Prism version 7.00 for
Windows, GraphPad Software, La Jolla Calif. USA.
Example 6
Glycosylation of Ang(1-7) and its Derivatives Improves
Pharmacokinetic Properties
[0168] One known limitation of therapeutically administering native
Ang(1-7) is its relatively short half-life and relatively poor
blood-brain-barrier permeability. The following experiments used a
rational drug design approach to assess the effect of adding
various glycosides to Ang(1-7) and its derivatives on serum
half-life and BBB permeability. Stability in vivo is affected by a
number of factors, including susceptibility to peptidases and
glycosidases, as well as aggregation phenomena in solution, and a
wide array of binding events, including membrane absorption.
Interaction of the glycopeptide drug with biological membranes is
greatly influenced by both the geometry and degree of
glycosylation. Our previous experience with glycopeptide GPCR
agonists of a similar size indicates that the degree of
glycosylation (mono- vs disaccharide) will not greatly affect
interaction with the MAS receptor or its activation.
[0169] Membrane-bound conformations of the Ang(1-7)-based
glycopeptides were modeled in silico by .sup.1H-NMR NOESY
measurements in the presence of d.sub.25-SDS micelles. Using
derived H--H distance constraints, a highly amphipathic folded
structure was characterized. As illustrated in FIG. 15A, a Solvent
Accessible Surface Area was constructed using the MOE.RTM. software
package with the AMBER-99 force field to illustrate the resulting
amphipathicity of the U-shaped folded structure. The uncharged
lipophilic residues Val-Tyr-Ile are at the bottom of the "U" and
insert into the membrane while charged "ends" protrude into the
aqueous compartment. The "amphipathic moment" is suggested by the
arrow.
[0170] FIG. 15B illustrates the MOE.RTM. calculations indicating
that the linkage geometries of the saccharide and peptide chain can
modify interactions of the resulting amphipathic glycopeptide with
biological membranes prior to "docking" with the Mas receptor. D-
or L-Serine, D- or L-Threonine, and D- or L-allo-Threonine, as well
as D- or L-Cysteine orient the glycoside at different angles
relative to the surface of the membrane.
[0171] Based on these calculations, native Ang(1-7), Ang(1-7)
having a C-terminal amino group (Ang 1-7-NH.sub.2; SEQ ID NO: 2;
"PN-A2"), PN-A5 (Ang 1-6-Ser(OGlc)-NH.sub.2; SEQ ID NO: 10), and
Ang 1-6-Ser(OLac)-NH.sub.2 (i.e., PN-A5 in which lactose is
substituted for the C-terminal glucose moiety) were produced and
the serum half-life tested. Serum half-life was assessed by
incubating 100 .mu.M of each peptide in mouse serum for eight
hours. Aliquots were withdrawn at the indicated time intervals and
the peptide concentration was determined using HPLC-MS and
expressed as a percentage of the initial concentration. As
illustrated in FIG. 16 and Table 2, glycosylation significantly
improved the serum half-life of the Ang(1-7) derivatives.
TABLE-US-00002 TABLE 2 In Vitro Serum Half-Life Assay Peptide
Half-life Native Ang(1-7) 14 min Ang 1-7-NH.sub.2 (PN-A2 21 min Ang
1-6-Ser(OGlc)-NH.sub.2 (PN-A5) 1 hour Ang 1-6-Ser(OLac)-NH.sub.2
5.8 hours
[0172] Based on these findings, the in vivo serum stability and BBB
penetration was assessed in vivo for Ang(1-7) and PN-A5. The
peptides (10 mg/kg) or vehicle control were individually
subcutaneously injected into naive mice. Serum concentrations were
determined every 10 minutes by HPLC-MS using a 20-30 .mu.l blood
sample. Ang(1-7) and PN-A5 were found to reach a maximum serum
concentrations of about 200 nM and about 3,500 nM, respectively
(FIG. 17A). CSF samples were simultaneously withdrawn from the same
animals via a microdialysis probe and assayed for the peptide
concentration and corrected for basal CSF levels. Ang(1-7) and
PN-A5 were found to reach a maximum CSF concentrations of about 50
nM and about 400 nM, respectively (FIG. 17B).
[0173] The foregoing discussion of the invention has been presented
for purposes of illustration and description. The foregoing is not
intended to limit the invention to the form or forms disclosed
herein. Although the description of the invention has included
description of one or more embodiments and certain variations and
modifications, other variations and modifications are within the
scope of the invention, e.g., as may be within the skill and
knowledge of those in the art, after understanding the present
disclosure. It is intended to obtain rights which include
alternative embodiments to the extent permitted, including
alternate, interchangeable and/or equivalent structures, functions,
ranges or steps to those claimed, whether or not such alternate,
interchangeable and/or equivalent structures, functions, ranges or
steps are disclosed herein, and without intending to publicly
dedicate any patentable subject matter. All references cited herein
are incorporated by reference in their entirety.
Sequence CWU 1
1
1318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(1)..(1)Asp, Glu, Ala or a derivative
thereofMOD_RES(2)..(2)Arg, His, Lys or a derivative
thereofMOD_RES(3)..(3)Val, Ala, Ile, Leu or a derivative
thereofMOD_RES(4)..(4)Tyr, Phe, Trp or a derivative
thereofMOD_RES(5)..(5)Ile, Val, Ala, Leu or a derivative
thereofMOD_RES(6)..(6)His, Arg, Lys or a derivative
thereofMOD_RES(7)..(7)Pro, Gly, Ser or a derivative
thereofMOD_RES(8)..(8)Ser, Thr, hydroxyproline, a derivative
thereof or absent 1Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 527PRTHomo
sapiens 2Asp Arg Val Tyr Ile His Pro1 537PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(7)D-amino acid 3Pro His Ile Tyr Val Arg Asp1
547PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Pro His Ile Tyr Val Arg Asp1 557PRTArtificial
SequenceDescription of Artificial Sequence Synthetic
peptideMOD_RES(1)..(7)D-amino acid 5Asp Arg Val Tyr Ile His Pro1
567PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideC-term -NH2 6Asp Arg Val Tyr Ile His Pro1
578PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideC-term -NH2 7Asp Arg Val Tyr Ile His Pro Ser1
588PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(8)..(8)Glucosylated-SerC-term -NH2 8Asp
Arg Val Tyr Ile His Pro Ser1 598PRTArtificial SequenceDescription
of Artificial Sequence Synthetic
peptideMOD_RES(8)..(8)Lactosylated-SerC-term -NH2 9Asp Arg Val Tyr
Ile His Pro Ser1 5107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic
peptideMOD_RES(7)..(7)Glucosylated-SerC-term -NH2 10Asp Arg Val Tyr
Ile His Ser1 5118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptideMOD_RES(2)..(3)Any amino
acidMOD_RES(6)..(6)Any amino acidMOD_RES(8)..(8)Ser,
Glucosylated-Ser, or Lactosylated-SerC-term -NH2 11Ala Xaa Xaa Tyr
Ile Xaa Pro Ser1 5128PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(3)..(3)Any amino
acidMOD_RES(5)..(5)Any amino acidMOD_RES(8)..(8)Ser,
Glucosylated-Ser, or Lactosylated-SerC-term -NH2 12Asp Arg Xaa Tyr
Xaa His Pro Ser1 5138PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptideMOD_RES(3)..(5)Any amino
acidMOD_RES(8)..(8)Ser, Glucosylated-Ser, or Lactosylated-SerC-term
-NH2 13Asp Arg Xaa Xaa Xaa His Pro Ser1 5
* * * * *