Note: Descriptions are shown in the official language in which they were submitted.
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
METHOD OF PROMOTING MYOCYTE PROLIFERATION AND MYOCARDIAL
TISSUE REPAIR
Cross Reference
This application claims priority to U.S. Provisional Patent Applications
Serial Nos.
60/123,678 (filed March 9, 1999) and 60/151,874 filed August 31, 1999, both
incorporated
by reference herein in their entirety.
to Field of the Invention
This present invention relates to myocyte proliferation and differentiation
and to
myocardial tissue repair.
Background of the Invention
Techniques for harvesting and culturing myocardial cells from a range of
species,
including adult human atrial myocardiocytes, have been established. (Smith et
al., In Vitro
Cell. Dev. Biol. 27A:914-920 (1991)). However, cardiac myocytes proliferate
slowly if at all
in culture (Kardami, Mol. and Cell. Biochem. 92:129-135 (1990)). The ability
to manipulate
these cells in vitro is extremely important for identifying growth factors
pertinent to
2o regeneration of heart cells after myocardial infarction or other ischemic
injury.
Ventricular myocytes of the adult mammalian myocardium have traditionally been
considered to be terminally differentiated cells, incapable of proliferation.
(Kardami, Mol.
and Cell. Biochem. 92:129-135 (1990)). Soon after birth these cells stop
dividing and
subsequent muscle growth is brought about by increases in cell size
(hypertrophy) rather than
cell number. Id. However, evidence indicates that ventricular myocytes have
not lost their
proliferative potential irreversibly, since they can be induced to synthesize
DNA in culture.
(Claycomb and Bradshaw, Dev. Biol. 90:331-337 (1983)). Atrial myocytes of the
adult
heart retain mitotic potential to a significant extent (Rumyanchev, Int. Rev.
Cytol. 51:187-273
(1977)).
3o Techniques for the proliferation of human myocardial cells have been
utilized,
including the use of a platelet freeze-thaw extract (U.S. Patent Application
5,580,779, hereby
incorporated by reference in its entirety). However, these methods are
laborious, do not
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
utilize defined compounds for increasing myocyte proliferation, and provide
limited increases
in myocyte proliferation. Therefore, improved methods using defined compounds
for
inducing proliferation and differentiation of myocytes are needed.
Approximately 1.25 million Americans suffer from acute myocardial infarction
(MI)
each year, leading to more than 475,000 deaths per year. (Talbert, Am. J.
Health Syst.
Pharm. 54:59-S16 (1997)). The cost of MI is estimated at more than $50 billion
annually.
Recent advances in the management of acute cardiac diseases, including acute
MI, are
resulting in an expanding patient population that will eventually develop
chronic heart
failure.
1o Early recognition and treatment of MI are important if outcomes are to be
improved.
Infarct size and location are key prognostic factors for outcomes after acute
MI. (Talbert,
1997) Following MI, adverse ventricular remodeling by fibrous tissue occurs at
the site of
MI and remote to it. (Sun et al., Mol. Cell. Cardiol. 30:1559-1569 (1998)). It
has been
suggested that cardiac fibrosis plays a role in the development of congestive
heart failure in
post-MI heart. (Ju et al., Cardiovasc. Res. 35:223-232 (1997)). In the failing
human heart of
ischemic origin, fibrosis remote to the MI is considered the major feature of
adverse tissue
structure. Increased myocardial collagen concentration and abnormal matrix
structure
adversely alters myocardial stiffness, leading to ventricular diastolic
dysfunction. (Sun et al.,
1998). Damaged cardiac muscle is eventually replaced by scar tissue formed by
non-muscle
2o cells converging at the site of injury. This compromises cardiac
performance further and
shortens cardiac lifespan.
Reperfusion therapy is an accepted therapy for MI patients, and its
application early in
MI has been shown to reduce infarct size and increase survival. (Granger, Am.
J. Cardiol.
79(12A):44-48 (1997); Talbert, 1997) A number of drug classes administered in
this manner
have been shown to result in smaller infarct size, including free radical
scavengers, calcium
antagonists, (3 blockers, magnesium, inhibitors of white blood cell function,
inhibitors of
cellular adhesion selectin molecules, adenosine (Granger, 1997), and
fibroblast growth factor
(U.S. Patent No. 4,296,100). Despite the benefits conferred by reperfusion
therapy, there is
evidence that such treatment can lead to "reperfusion injury", including
microvascular
3o damage and dysfunction. (Granger, 1997)
These methods have proven to be of limited efficacy in promoting repair of
myocardial tissue after MI or for treating heart failure. Thus, there remains
a need for the
2
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WO 00/53211 PCT/US00/06198
development of methods to promote myocardial tissue repair following
myocardial infarction
that minimize fibrosis.
Recent studies have implicated angiotensin II (AII) and/or activation of the
All AT1
receptor in promoting fibrous tissue formation at MI and remote repair sites,
while All
receptor antagonism has been shown in animal models to improve wound healing
at MI and
remote repair sites. (Sun et al., 1998; Ju et al., 1997; Sun, Adv. Exp. Med.
Biol. 432:55-61
(1997); De Carvalho Frimm et al., Labor. and Clin. Med. 129:439-446 (1997);
Thai et al.,
Am. J. Physiol. 276:H873-880 (1999); Tomita et al., Hypertension 32:273-279
(1998))
Current therapy for heart failure is primarily directed to using angiotensin-
converting
to enzyme (ACE) inhibitors and diuretics (U.S. Patent No. 5,679,545). While
prolonging
survival in the setting of heart failure, ACE inhibitors appear to slow the
progression towards
end-stage heart failure, and substantial numbers of patients on ACE inhibitors
have functional
class III heart failure. Moreover, ACE inhibitors consistently appear unable
to relieve
symptoms in more than 60% of heart failure patients, and they reduce the
mortality of heart
failure only by approximately 1 S-20%. Heart transplantation is limited by the
availability of
donor hearts Id. Further, with the exception of digoxin, the chronic
administration of positive
inotropic agents has not resulted in a useful drug without accompanying
adverse side effects,
such as increased arrhythmogenesis, sudden death, or other deleterious side
effects related to
survival. These deficiencies in current therapy suggest the need for
additional therapeutic
2o approaches to heart failure.
The use of myocytes cultured in vitro as a therapeutic approach offers promise
for the
treatment of various cardiac disorders (U.S. Patent No. 5,679,545). As one
specific example,
myocytes may be implanted into a patient who has suffered a myocardial
infarction prior to
the onset of fibrosis, therefore potentially avoiding a weakening in the
myocardium that may
result in aneurysm formation. Alternatively, such myocytes may be used in
aneurysm repair.
In further embodiments, myocytes generated in culture may be used in
conjunction with
artificial materials to produce substrates for reconstructive cardiac surgery.
Summary of the Invention
3o In one aspect, the present invention provides methods, kits, and
pharmaceutical
compositions for increasing myocyte proliferation and differentiation by
contacting the cells
with angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and
analogues thereof,
3
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WO 00/53211 PCT/US00/06198
angiotensin II (AII), All analogues, All fragments or analogues thereof or All
ATZ type 2
receptor agonists, either alone or in combination with other growth factors
and cytokines.
The methods of this aspect of the invention may be used to treat heart
failure, to
provide myocardial cells that may be transplanted or implanted into a patient
that suffers
from a cardiac disorder, to study the physiology of cardiac muscle, or to
identify
pharmaceutical agents that may be useful in the treatment of heart disease.
In a further aspect, the present invention provides methods and kits to
promote
myocardial tissue repair following myocardial injury, comprising the
administration of
angiotensinogen, angiotensin I (AI), AI analogues, AI fragments and analogues
thereof,
to angiotensin II (AII), All analogues, All fragments or analogues thereof or
All AT2 type 2
receptor agonists to a patient in need thereof.
Detailed Description of the Preferred Embodiments
All references, patents and patent applications are hereby incorporated by
reference in
their entirety.
As defined herein, the term "myocyte" includes any myocardial cell, either
fetal or
adult in origin. Examples of myocytes include, but are not limited to, those
described in U.S.
Patent Application 5,580, 779; Smith et al., 1991 supra; and Kardami 1990,
supra, all
references hereby incorporated in their entirety. As defined herein,
"proliferation"
2o encompasses both cell self renewal and cellular proliferation with
accompanying
differentiation.
As defined herein, the term "myocardial injury" encompasses injuries
including, but
not limited to myocardial ischemia, myocardial infarction, cardiomyopathies,
coronary artery
disease, heart valve disease, myocaxditis, and heart failure.
As used herein, the term "myocardial infarction" refers to adverse changes in
the
myocardium or heart muscle that results from obstruction of a coronary artery.
As used herein, the term "repair following myocardial infarction" refers to a
decrease
in the fibrosis and scarnng that typically follows MI, and to promoting the
production of
healthy muscle and tissue at necrotic sites.
3o As used herein, the term "heart failure" refers to the failure of the heart
to pump blood
with normal efficiency and thus to provide adequate blood flow to other body
organs. Heart
failure may be due to failure of the right or left or both ventricles. The
signs and symptoms of
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CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
heart failure depend upon which side of the heart is failing. They can include
dyspnea,
cardiac asthma, pooling of blood in the systemic circulation or in the liver's
portal circulation,
edema, cyanosis, and hypertrophy of the heart. There are many causes of
congestive heart
failure including but not limited to coronary artery disease leading to heart
attacks and heart
muscle weakness, primary heart muscle weakness from viral infections or toxins
such as
prolonged alcohol exposure, heart valve disease causing heart muscle weakness
due to too
much leaking of blood or heart muscle stiffness from a blocked valve,
hypertension,
hyperthyroidism, vitamin deficiencies, and drug use. The aim of therapy for
heart failure is to
improve the pumping function of the heart.
to Unless otherwise indicated, the term "active agents" as used herein refers
to the group
of compounds comprising angiotensinogen, angiotensin I (AI), AI analogues, AI
fragments
and analogues thereof, angiotensin II (AII) analogues, All fragments or
analogues thereof or
All ATZ type 2 receptor agonists, either alone, combined, or in further
combination with
other compounds, for treating or preventing restenosis, such as
anticoagulants, platelet
aggregation inhibitors, smooth muscle cell proliferation inhibitors, calcium
channel blockers,
angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists,
and
antilipidemics.
Unless otherwise indicated, the term "angiotensin converting enzyme
inhibitors" or
"ACE inhibitors" includes any compound that inhibits the conversion of the
decapeptide
angiotensin I to angiotensin II, and include but are not limited to alacepril,
alatriopril, altiopril
calcium, ancovenin, benazepril, benazepril hydrochloride, benazeprilat,
benzazepril,
benzoylcaptopril, captopril, captopril-cysteine, captopril-glutathione,
ceranapril, ceranopril,
ceronapril, cilazapril, cilazaprilat, converstatin, delapril, delapril-diacid,
enalapril, enalaprilat,
enalkiren, enapril, epicaptopril, foroxymithine, fosfenopril, fosenopril,
fosenopril sodium,
fosinopril, fosinopril sodium, fosinoprilat, fosinoprilic acid, glycopril,
hemorphin-4, idapril,
imidapril, indolapril, indolaprilat, libenzapril, lisinopril, lyciumin A,
lyciumin B, mixanpril,
moexipril, moexiprilat, moveltipril, muracein A, muracein B, muracein C,
pentopril,
perindopril, perindoprilat, pivalopril, pivopril, quinapril, quinapril
hydrochloride, quinaprilat,
ramipril, ramiprilat, spirapril, spirapril hydrochloride, spiraprilat,
spiropril, spiropril
3o hydrochloride, temocapril, temocapril hydrochloride, teprotide,
trandolapril, trandolaprilat,
utibapril, zabicipril, zabiciprilat, zofenopril and zofenoprilat. (See for
example Jackson, et
al., Renin and Angiotensin in Goodman & Gilman's The Pharmacological Basis of
5
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
Therapeutics, 9th ed., eds. Hardman, et al. (McGraw Hill, 1996); and U.S.
Patent No.
5,977,159.)
Within this application, unless otherwise stated, the techniques utilized may
be found
in any of several well-known references such as: Molecular Cloning: A
Laboratory Manual
(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression
Technology
(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press,
San
Diego, CA), "Guide to Protein Purification" in Methods in Enzymology (M.P.
Deutshcer, ed.,
(1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and
Applications (Innis,
et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual
of Basic
Technique, 2n'~ Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene
Transfer and
Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc.,
Clifton, N.J.),
and the Ambion 1998 Catalog (Ambion, Austin, TX).
U.S. Patent No. 5,015,629 to DiZerega (the entire disclosure of which is
hereby
incorporated by reference) describes a method for increasing the rate of
healing of wound
tissue, comprising the application to such tissue of angiotensin II (AII) in
an amount which is
sufficient for said increase. The application of All to wound tissue
significantly increases the
rate of wound healing, leading to a more rapid re-epithelialization and tissue
repair. The term
All refers to an octapeptide present in humans and other species having the
sequence Asp-
Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID NO:l]. The biological formation of
angiotensin is
2o initiated by the action of renin on the plasma substrate angiotensinogen
(Circulation
Research 60:786-790 (1987); Clouston et al., Genomics 2:240-248 (1988);
Kageyama et al.,
Biochemistry 23:3603-3609; Ohkubo et al., Proc. Natl. Acad. Sci. 80:2196-2200
(1983)); all
references hereby incorporated in their entirety). The substance so formed is
a decapeptide
called angiotensin I (AI) which is converted to All by the converting enzyme
angiotensinase
which removes the C-terminal His-Leu residues from AI, Asp-Arg-Val-Tyr-Ile-His-
Pro-Phe-
His-Leu [SEQ ID N0:37]. All is a known pressor agent and is commercially
available.
Studies have shown that All increases mitogenesis and chemotaxis in cultured
cells
that are involved in wound repair, and also increases their release of growth
factors and
extracellular matrices (diZerega, U.S. Patent No. 5,015,629; Dzau et. al., J.
Mol. Cell.
3o Cardiol. 21:57 (Supp III) 1989; Berk et. al., Hypertension 13:305-14
(1989); Kawahara, et
al., BBRC 150:52-9 (1988); Naftilan, et al., J. Clin. Invest. 83:1419-23
(1989); Taubman et
al., J. Biol. Chem. 264:526-530 (1989); Nakahara, et al., BBRC 184:811-8
(1992); Stouffer
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WO 00/53211 PCT/US00/06198
and Owens, Circ. Res. 70:820 (1992); Wolf, et al., Am. J. Pathol. 140:95-107
(1992); Bell
and Madri, Am. J. Pathol. 137:7-12 (1990)). In addition, All was shown to be
angiogenic in
rabbit corneal eye and chick chorioallantoic membrane models (Fernandez, et
al., J. Lab.
Clin. Med. 105:141 (1985); LeNoble, et al., Eur. J. Pharmacol. 195:305-6
(1991)).
We have previously demonstrated that angiotensinogen, angiotensin I (AI), AI
analogues, AI fragments and analogues thereof, angiotensin II (AII), All
analogues, All
fragments or analogues thereof; All AT2 type 2 receptor agonists (hereinafter
referred to as
the "active agents") are effective in accelerating wound healing and the
proliferation of
certain cell types. See, for example, co-pending U.S. Patent Application
Serial Nos.
l0 09/012,400 (January 23, 1998); 09/198,806 (November 24, 1998); 09/264,563
(Filed March
8, 1999); 09/287,674 (Filed April 7, 1999); 09/307,940(Filed May 10, 1999);
09/246,162
(Filed February 8, 1999); 09/255,136 (Filed February 19, 1999); 09/245,680
(Filed February
8, 1999); 09/250,703 (Filed February 15, 1999); 09/246,525 (Filed February 8,
1999);
09/266,293 (Filed March 11, 1999); 09/332,582 (Filed June 14, 1999);
09/373,962 (Filed
August 13, 1999); and 09/352,191 (Filed March 11, 1999); as well as U.S.
Patent Serial Nos.
5,015,629; 5,629,292; 5,716,935; 5,834,432; and 5,955,430; all references
incorporated
herein by reference in their entirety.
The effect of All on a given cell type has been hypothesized to be dependent,
in part,
upon the All receptor subtypes) the cell expresses (Shanugam et al., Am. J.
Physiol.
268:F922-F930 (1995); Helin et al., Annals of Medicine 29:23-29 (1997); Bedecs
et al.,
Biochem J. 325:449-454 (1997)). These studies have shown that All receptor
subtype
expression is a dynamic process that changes during development, at least in
some cell types.
All activity is typically modulated by either or both the AT1 and AT2 All
receptors.
However, All has recently been shown to stimulate proliferation of primary
human
keratinocytes via a non-AT1, non-AT2 receptor. (Steckelings et al., Biochem.
Biophys. Res.
Commun. 229:329-333 (1996)). These results underscore the cell-type (ie: based
on receptor
expression) specific nature of All activity.
The effects of All receptor and All receptor antagonists have been examined in
two
experimental models of vascular injury and repair which suggest that both All
receptor
3o subtypes (AT1 and AT2) play a role in wound healing (Janiak et al.,
Hypertension 20:737-45
(1992); Prescott, et al., Am. J. Pathol. 139:1291-1296 (1991); Kauffman, et
al., Life Sci.
7
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WO 00/53211 PCT/US00/06198
49:223-228 (1991); Viswanathan, et al., Peptides 13:783-786 (1992); Kimura, et
al., BBRC
187:1083-1090 (1992).
Many studies have focused upon AII(1-7) (AII residues 1-7) or other fragments
of All
to evaluate their activity. AII(1-7) elicits some, but not the full range of
effects elicited by
AII. (Pfeilschifter, et al., Eur. J. Pharmacol. 225:57-62 (1992); Jaiswal, et
al., Hypertension
19(Supp. II):II-49-II-55 (1992); Edwards and Stack, J. Pharmacol. Exper. Ther.
266:506-510
(1993); Jaiswal, et al., J. Pharmacol. Exper. Ther. 265:664-673 (1991);
Jaiswal, et al.,
Hypertension 17:1115-1120 (1991); Portsi, et a., Br. J. Pharmacol. 111:652-654
(1994)).
Other data suggests that the All fragment AII(1-7) acts through a receptors)
that is
l0 distinct from the AT1 and AT2 receptors which modulate All activity.
(Ferrario et al., J. Am.
Soc. Nephrol. 9:1716-1722 (1998); Iyer et al., Hypertension 31:699-705 (1998);
Freeman et
al., Hypertension 28:104 (1996); Ambuhl et al., Brain Res. Bull. 35:289
(1994)). Thus,
AII(1-7) activity on a particular cell type cannot be predicted based solely
on the effect of All
on the same cell type. In fact, there is some evidence that AII(1-7) often
opposes the actions
is of AII. (See, for example, Ferrario et al., Hypertension 30:535-541 (1997))
Many studies have been conducted to assess the effect of All on the
cardiovascular
system. Some studies have suggested that All has a toxic effect on myocytes,
by inducing
cellular hypertrophy (in the absence of cell proliferation) in non-infarcted
myocardium
(Riegger, Cardiovasc. Drugs and Therapy 10:613-615 ( 1996); Kudoh et al.,
Circ. Res.
20 80:139-146 (1997); Hein et al., Proc. Natl. Acad. Sci. 94:6391-6396
(1997)). Other studies
suggest that All effects myocyte growth indirectly, via a fibroblast-derived
factor that is
increased by All (Sil and Sen, Hypertension 30:209-216 (1997). Other studies
suggest that
the AT2 receptor can serve to mediate the antigrowth effects of All (Booz and
Baker,
Hypertension 28:635-640, 1996). Thus, the effect of All on myocyte
proliferation is at best
2s controversial, and the effects of angiotensinogen, AI, and AI and All
fragments and
analogues is unknown.
Angiotensin II is also known as a potent stimulator of angiogenesis (Fernandez
et al.,
J. Lab. Clin. Med. 105:141-145 (1985)), and has been shown to activate
collateral circulation
via preformed blood vessels in rat kidneys (Fernandez et al., Am. J. Physiol.
243:H869-H875
30 (1982)). However, recent studies have implicated angiotensin II and/or All
AT1 receptor
activation in promoting fibrous tissue formation at MI and remote repair
sites, and
antagonism of the All receptor has been shown in animal models to influence
wound healing
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WO 00/53211 PCT/US00/06198
at MI and remote repair sites. (Sun et al., 1998; Ju et al., 1997; Sun, Adv.
Exp. Med. Biol.
432:55-61 (1997); De Carvalho Frimm et al., Labor. and Clin. Med. 129:439-446
(1997);
Thai et al., Am. J. Physiol. 276:H873-880 (1999); Tomita et al., Hypertension
32:273-279
(1998)) Thus, the use of the active agents of the invention to promote
myocardial tissue
repair following myocardial infarction would be unexpected.
A peptide agonist selective for the AT2 receptor (AII has 100 times higher
affinity for
AT2 than AT1) is p-aminophenylalanine6-All ["(p-NHZ-Phe)6-AII)"], Asp-Arg-Val-
Tyr-Ile-
Xaa-Pro-Phe [SEQ ID N0.36] wherein Xaa is p-NH2-Phe (Speth and Kim, BBRC
169:997-
1006 (1990). This peptide gave binding characteristics comparable to AT2
antagonists in the
to experimental models tested (Catalioto, et al., Eur. J. Pharmacol. 256:93-97
(1994); Bryson,
et al., Eur. J. Pharmacol. 225:119-127 (1992).
As hereinafter defined, a preferred class of AT2 agonists for use in
accordance with
the present invention comprises All analogues or active fragments thereof
having p-NH-Phe
in a position corresponding to a position 6 of AII. In addition to peptide
agents, various
nonpeptidic agents (e.g., peptidomimetics) having the requisite AT2 agonist
activity are
further contemplated for use in accordance with the present invention.
The active All analogues, fragments of All and analogues thereof of particular
interest in accordance with the present invention comprise a sequence
consisting of at least
three contiguous amino acids of groups R'-Rg in the sequence of general
formula I
2o R~-R2-R3-R4-RS-R6-R7 R8
in which Rl and R2 together form a group of formula
X-R''-RB-,
wherein X is absent, H or a one to three peptide group,
RA is suitably selected from H, Asp, Glu, Asn, Acpc (1-aminocyclopentane
carboxylic acid), Ala, Me2Gly, Pro, Bet, Glu(NHZ), Gly, Asp(NH2) and Suc,
RB is suitably selected from Arg, Lys, Ala, Citron, Orn, Ser(Ac), Sar, D-Arg
and D-Lys,
R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly,
Lys, Pro, Aib, Acpc and Tyr;
3o R4 is selected from the group consisting of Tyr, Tyr(P03)Z, Thr, Ser,
homoSer,
azaTyr, and Ala;
RS is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and
Gly;
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CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
R~ is His; Arg or 6-NHz-Phe;
R' is Pro or Ala; and
Rg is selected from the group consisting of Phe, Phe(Br), Ile and Tyr,
excluding sequences including R4 as a terminal Tyr group.
Compounds falling within the category of AT2 agonists useful in the practice
of the
invention include the All analogues set forth above subject to the restriction
that R6 is p-NHz-
Phe.
Particularly preferred combinations for RA and RB are Asp-Arg, Asp-Lys, Glu-
Arg
and Glu-Lys. Particularly preferred embodiments of this class include the
following: AIII or
to AII(2-8), Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID N0:2]; AII(3-8), also known
as desl-AIII or
AIV, Val-Tyr-Ile-His-Pro-Phe [SEQ ID N0:3]; AII(1-7), Asp-Arg-Val-Tyr-Ile-His-
Pro [SEQ
ID N0:4]; AII(2-7). Arg-Val-Tyr-Ile-His-Pro [SEQ ID NO:S]; AII(3-7), Val-Tyr-
Ile-His-Pro
[SEQ ID N0:6]; AII(5-8), Ile-His-Pro-Phe [SEQ ID N0:7]; AII(1-6), Asp-Arg-Val-
Tyr-Ile-
His [SEQ ID N0:8]; AII(1-5), Asp-Arg-Val-Tyr-Ile [SEQ ID N0:9]; AII(1-4), Asp-
Arg-Val-
Tyr [SEQ ID NO:10]; and AII(1-3), Asp-Arg-Val [SEQ ID NO:11]. Other preferred
embodiments include: Arg-norLeu-Tyr-Ile-His-Pro-Phe [SEQ ID N0:12] and Arg-Val-
Tyr-
norLeu-His-Pro-Phe [SEQ ID N0:13]. Still another preferred embodiment
encompassed
within the scope of the invention is a peptide having the sequence Asp-Arg-Pro-
Tyr-Ile-His-
Pro-Phe [SEQ ID N0:31]. AII(6-8), His-Pro-Phe [SEQ ID N0:14] and AII(4-8), Tyr-
Ile-
2o His-Pro-Phe [SEQ ID NO:15] were also tested and found not to be effective.
Another class of compounds of particular interest in accordance with the
present
invention are those of the general formula II
Rz_R3_R4_Rs_R6_R7_Rg
in which R2 is selected from the group consisting of H, Arg, Lys, Ala, Orn,
Citron, Ser(Ac), Sar, D-Arg and D-Lys;
R3 is selected from the group consisting of Val, Ala, Leu, norLeu, Ile, Gly,
Pro, Aib, Acpc and Tyr;
R4 is selected from the group consisting of Tyr, Tyr(P03)z, Thr, Ser, homoSer,
azaTyr, and Ala;
3o Rs is selected from the group consisting of Ile, Ala, Leu, norLeu, Val and
Gly;
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
R~ is His; Arg or 6-NHZ-Phe;
R' is Pro or Ala; and
Rg is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.
A particularly preferred subclass of the compounds of general formula II has
the
formula
R2-R3-Tyr-RS-His-Pro-Phe [SEQ ID N0:16]
wherein R2, R3 and RS are as previously defined. Particularly preferred is
angiotensin
III of the formula Arg-Val-Tyr-Ile-His-Pro-Phe [SEQ ID N0:2]. Other preferred
compounds
include peptides having the structures Arg-Val-Tyr-Gly-His-Pro-Phe [SEQ ID
N0:17] and
to Arg-Val-Tyr-Ala-His-Pro-Phe [SEQ ID N0:18]. The fragment AII(4-8) was
ineffective in
repeated tests; this is believed to be due to the exposed tyrosine on the N-
terminus.
In a preferred embodiment, the active agents comprise a sequence according to
the
general formula:
Asp-Arg-R 1-Tyr-Ile-His-Pro-R2
wherein R1 is selected from the group consisting of Val, Pro, Ile, norLeu,
Ile, Lys,
Ala, Gly, Aib, Acpc and Tyr; and
wherein R2 is selected from the group consisting of Phe, Phe(Br), Ile and Tyr.
Other particularly preferred embodiments include:
1GD Ala4-AII(1-7) DRVAIHP SEQ ID N0:38
2GD Pro3-AII(1-7) DRPYIHP SEQ ID N0:39
SGD Lys3-AII(1-7) DRKYIHP SEQ ID N0:40
9GD NorLeu-AII(1-7) DR(nor)YIHP SEQ ID N0:41
GSD 28 Ile$-All DRVYIHPI SEQ ID N0:42
Ala3aminoPhe6 DRAYIF*PF SEQ ID N0:43
AII:
Ala3-AIII RVAIHPF SEQ ID N0:44
Glyl-All GRVYIHPF SEQ ID N0:45
NorLeu4-AIII --RVYnLHPF SEQ ID N0:46
Acpc3-All DR(Acpc)YIHPF SEQ ID N0:47
GSD 37B Ornz-All D(Orn)VYIbIPF SEQ ID N0:48
3o GSD38B Citron2-All D(Citron)VYIHPFSEQ ID N0:49
3GD Pro3Ala4-AII(1-7)DRPAIHP SEQ ID NO:50
11
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In the above formulas, the standard three-letter abbreviations for amino acid
residues
are employed. In the absence of an indication to the contrary, the L-form of
the amino acid is
intended. Other residues are abbreviated as follows:
TABLE 1
Abbreviation for Amino Acids
Me2G1 N,N-dimeth 1 1 c 1
Bet 1-carboxy-N,N,N-trimethylmethanaminium hydroxide
inner
salt betaine
Suc Succin 1
Phe Br -bromo-L- hen lalan 1
azaTyr aza-a'-homo-L-t os 1
Ac c 1-aminoc clo entane carbox lic acid
Aib 2-aminoisobu ~c acid
Sar N-meth 1 1 c 1 sarcosine
Cit Citron
Orn Ornithine
It has been suggested that All and its analogues adopt either a gamma or a
beta turn
(Regoli, et al., Pharmacological Reviews 26:69 (1974). In general, it is
believed that neutral
side chains in position R3, R5 and R' may be involved in maintaining the
appropriate distance
between active groups in positions R4, R6 and R8 primarily responsible for
binding to
1o receptors and/or intrinsic activity. Hydrophobic side chains in positions
R3, R5 and R8 may
also play an important role in the whole conformation of the peptide and/or
contribute to the
formation of a hypothetical hydrophobic pocket.
Appropriate side chains on the amino acid in position RZ may contribute to
affinity of
the compounds for target receptors and/or play an important role in the
conformation of the
peptide. For this reason, Arg and Lys are particularly preferred as R2.
Alternatively, RZ may
be H, Ala, Orn, Citron, Ser(Ac), Sar, D-Arg, or D-Lys.
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For purposes of the present invention, it is believed that R3 may be involved
in the
formation of linear or nonlinear hydrogen bonds with RS (in the gamma turn
model) or R6 (in
the beta turn model). R3 would also participate in the first turn in a beta
antiparallel structure
(which has also been proposed as a possible structure). In contrast to other
positions in
general formula I, it appears that beta and gamma branching are equally
effective in this
position. Moreover, a single hydrogen bond may be sufficient to maintain a
relatively stable
conformation. Accordingly, R3 may suitably be selected from Lys, Val, Ala,
Leu, norLeu,
Ile, Gly, Pro, Aib, Acpc and Tyr.
With respect to R4, conformational analyses have suggested that the side chain
in this
1o position (as well as in R3 and RS) contribute to a hydrophobic cluster
believed to be essential
for occupation and stimulation of receptors. Thus, R4 is preferably selected
from Tyr, Thr,
Tyr (P03)Z, homoSer, Ser and azaTyr. In this position, Tyr is particularly
preferred as it may
form a hydrogen bond with the receptor site capable of accepting a hydrogen
from the
phenolic hydroxyl (Regoli, et al. (1974), supra). It has also been found that
R4 can be Ala.
In position R5, an amino acid with a (3 aliphatic or alicyclic chain is
particularly
desirable. Therefore, while Gly is suitable in position R5, it is preferred
that the amino acid in
this position be selected from Ile, Ala, Leu, norLeu, and Val.
In the active agents of particular interest in accordance with the present
invention, R6
is His, Arg or 6-NH2-Phe. The unique properties of the imidazole ring of
histidine (e.g.,
2o ionization at physiological pH, ability to act as proton donor or acceptor,
aromatic character)
are believed to contribute to its particular utility as R6. For example,
conformational models
suggest that His may participate in hydrogen bond formation (in the beta
model) or in the
second turn of the antiparallel structure by influencing the orientation of
R7. Similarly, it is
presently considered that R' should be Pro or Ala in order to provide the most
desirable
orientation of R8. In position R8, both a hydrophobic ring and an anionic
carboxyl terminal
appear to be particularly useful in binding of the analogues of interest to
receptors; therefore,
Tyr, Ile, Phe(Br), and especially Phe are preferred for purposes of the
present invention.
Analogues of particular interest include the following:
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WO 00/53211 PCT/US00/06198
TABLE 2
Angiotensin II Analogues
All Amino Sequence
Analogue Acid Identifier
Name Sequence
Analo ue As -Ar -Val-T-Val-His-Pro-Phe SE ID NO: 19
1
Analo ue Asn-Ar -Val-His-Pro-Phe SE ID NO: 20
2 -Val-T
Analo ue Ala-Pro-Gl -Ar -Ile-T r-Val-His-Pro-PheSE ID NO: 21
3 -As
Analo ue Glu-Ar -Ile-His-Pro-Phe SE ID NO: 22
4 -Val-T
Analo ue As -L s-Val-T-Ile-His-Pxo-Phe SE ID NO: 23
Analo ue As -Ar -Ala-T-Ile-His-Pro-Phe SE ID NO: 24
6
Analo ue As -Ar -Val-Thr-Ile-His-Pro-Phe SE ID NO: 25
7
Analo ue As -Ar -Val-T-Leu-His-Pro-Phe SE ID NO: 26
8
Analo ue As -Ar -Val-Tr-Ile-Ar -Pro-Phe SE ID NO: 27
9
Analo ue As -Ar -Val-T-Ile-His-Ala-Phe SE ID NO: 28
Analo ue As -Ar -Val-T-Ile-His-Pro-T SE ID NO: 29
11
Analo ue Pro-Ar -Ile-His-Pro-Phe SE ID NO: 30
12 -Val-T
Analo ue As -Ar -Pro-T-Ile-His-Pro-Phe SE ID NO: 31
13
Analo ue As -Ar -Val-TP03 2-Ile-His-Pro-Phe SE ID NO: 32
14
Analo ue As -Ar -norLeu-T SE ID NO: 33
r-Ile-His-Pro-Phe
Analo ue As -Ar -Val-T-norLeu-His-Pro-Phe SE ID NO: 34
16
Analo ue As -Ar -Val-homoSer-T SE ID NO: 35
17 -Ile-His-Pro-Phe
The polypeptides of the instant invention may be synthesized by any
conventional
5 method, including, but not limited to, those set forth in J. M. Stewart and
J. D. Young, Solid
Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford, Ill. (1984)
and J.
Meienhofer, Hormonal Proteins and Peptides, Vol. 2, Academic Press, New York,
(1973) for
solid phase synthesis and E. Schroder and K. Lubke, The Peptides, Vol. 1,
Academic Press,
New York, (1965) for solution synthesis. The disclosures of the foregoing
treatises are
1o incorporated by reference herein.
In general, these methods involve the sequential addition of protected amino
acids to a
growing peptide chain (U.S. Patent No. 5,693,616, herein incorporated by
reference in its
entirety). Normally, either the amino or carboxyl group of the first amino
acid and any
reactive side chain group are protected. This protected amino acid is then
either attached to an
15 inert solid support, or utilized in solution, and the next amino acid in
the sequence, also
suitably protected, is added under conditions amenable to formation of the
amide linkage.
After all the desired amino acids have been linked in the proper sequence,
protecting groups
14
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WO 00/53211 PCT/US00/06198
and any solid support are removed to afford the crude polypeptide. The
polypeptide is
desalted and purified, preferably chromatographically, to yield the final
product.
Preferably, peptides are synthesized according to standard solid-phase
methodologies,
such as may be performed on an Applied Biosystems Model 430A peptide
synthesizer
(Applied Biosystems, Foster City, Calif.), according to manufacturer's
instructions. Other
methods of synthesizing peptides or peptidomimetics, either by solid phase
methodologies or
in liquid phase, are well known to those skilled in the art.
Alternatively, the peptides can be produced by standard molecular biological
techniques.
to In one aspect of the present invention, methods, kits, and pharmaceutical
compositions for increasing in vivo, in vitro and ex vivo myocyte
proliferation by exposure to
angiotensinogen, AI, AI analogues, AI fragments and analogues thereof, All
analogues, All
fragments or analogues thereof or All ATZ type 2 receptor agonists
(hereinafter referred to as
the "active agents") is disclosed. Experimental conditions for the isolation,
purification, in
vitrolex vivo growth and in vivo mobilization of myocytes have been reported
(Smith et al.,
1991 supra; Kardami 1990, supra; U.S. Patent No. 5,580,779; Sil and Sen,
Hypertension
30:209-216, 1997; Jacobsen et al., Basic Res. Cardiol. 1:79-82 (1985); Sen et
al. J. Biol.
Chem. 263:19132-19136 (1988)).
Proliferation can be quantitated using any one of a variety of techniques well
known
2o in the art, including, but not limited to, bromodeoxyuridine incorporation
(Vicario-Abejon et
al., 1995); Lazarous et al. Biotechnol. and Histochem. 67:253-255 (1992)), 3H-
thymidine
incorporation (Fredericksen et al., 1988), or antibody labeling of a protein
present in higher
concentration in proliferating cells than in non-proliferating cells. In a
preferred
embodiment, proliferation of myocytes is assessed by reactivity to an antibody
directed
against a protein known to be present in higher concentrations in
proliferating cells than in
non-proliferating cells, including but not limited to proliferating cell
nuclear antigen (PCNA,
or cyclin; Zymed Laboratories, South San Francisco, California).
Differentiation of myocytes is detected by immunohistochemistry using
antibodies
specific for muscle myosin, myoglobin, and atrial natriuretic peptide (ANP),
as described in
3o U.S. Patent No. 5,580,779.
In one embodiment, myocytes are isolated from atrial tissue according to
standard
methods (Smith et al., 1991, supra), suspended in culture medium, and
incubated in the
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
presence of, preferably, between about 0.1 ng/ml and about 10 mg/ml of the
active agents of
the invention. The cells are expanded for a period of between 8 and 21 days
and cellular
proliferation is assessed as described above.
In a preferred embodiment, myocytes are isolated from atrial tissue obtained
from
cardiovascular surgery patients undergoing procedures requiring "heart-lung
bypass." Smith
et al., In Vitro Cell Dev. Biol. 27A:914-920 (1991). The samples are placed in
an ice-saline
slush immediately after removal, rinsed in saline, and the epicardial covering
is removed with
a scalpel to reduce the amount of connective tissue included in the cell
harvest. The
remaining "pure" atrial muscle is minced into 0.5 to 1.0 mm3 pieces and placed
in cold
to Hanks' balanced salt solution (HBSS) without calcium or magnesium
(Whitaker; Walkerville,
MD). The minced atrial tissue is digested in 0.14% collagenase solution
(Worthington,
Freehold, NJ) at a concentration of 1.43 mg/ml. The pieces are placed in 35 ml
of this
solution and digested in a shaker at 37°C at 125 rpm for one hour. The
supernatant is
removed from the atrial tissue and centrifuged at 3500 rpm for 10 minutes at
37°C. Another
35 ml of collagenase solution is placed on the minced tissue and the digestion
is continued for
another hour. Cell pellets are resuspended in 2 ml of Eagle's minimal
essential medium
(EMEM) with Earle's salts (Whitaker) containing 30% newborn bovine serum
(Whitaker) and
0.1% antibiotic solution. Several digestions are conducted in this manner, and
final cell
concentrations are checked using a hemocytometer and adjusted to 1 x 105 with
EMEM, and
2o then incubated with the active agents.
Myocytes exposed to the active agents as described above can be used, for
example,
for implantation or transplantation into a patient in need of such treatment.
In this manner,
autologous or heterologous cells may be implanted or transplanted into a
patient who suffers
from a caxdiac disorder. As one specific example, cells may be implanted into
a patient who
has suffered a myocardial infarction prior to the onset of fibrosis, therefore
potentially
avoiding a weakening in the myocardium that may result in aneurysm formation
(U.S. Patent
No. 5,580,779). Alternatively, such cells or artificially produced myocardial
tissue, may be
used in aneurysm repair. In further embodiments, cells generated in culture
may be used in
conjunction with artificial materials to produce substrates for reconstructive
cardiac surgery.
3o In further specific embodiments, atrial myocardial cells caused to
proliferate by the methods
of the invention may be used in vivo or in vitro as a source of atrial
natriuretic peptide. A
cellular implant comprising such cells may be introduced into a patient as a
source of atrial
16
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WO 00/53211 PCT/US00/06198
natriuretic peptide that is subject to biofeedback mechanism. The cells are
cultured in vitro
or ex vivo as described above. The cells are rinsed to remove all traces of
culture fluid,
resuspended in an appropriate medium and then pelleted and rinsed several
times. After the
final rinse, the cells are resuspended at between 0.7 x 10G and 50 x 106 cells
per ml in an
appropriate medium and used as described.
In another aspect of the present invention the active agents are used to
increase in vivo
myocyte proliferation. In a preferred embodiment of this aspect, the active
agent is
administered by systemic infusion directly into the heart using an osmotic
pump (Alza Palo
Alto, CA) attached to a 30 gauge cannulae implanted at the injection
coordinate, as described
to in Craig et al., J. of Neuroscience 16:2649-2658 (1996). In another
preferred embodiment,
the peptides are administered by infusion locally to the myocardium via an
indwelling
catheter.
A suitable injected dose of active agent is preferably between about 0.1 ng/kg
and
about 10 mg/kg administered twice daily. The active ingredient may comprise
from 0.001
to 10% w/w, e.g., from 1% to 2% by weight of the formulation, although it may
comprise as
much as 10% w/w, but preferably not more than 5% w/w, and more preferably from
0.1% to
1 % of the formulation.
In a further aspect, the present invention provides methods, pharmaceutical
compositions, and kits to promote myocardial tissue repair after myocardial
infarction,
2o comprising administration of the active agents of the invention to a
patient in need thereof to
promote myocardial tissue repair after MI.
According to this aspect of the invention, an area of myocardial tissue is
treated in
vivo following, or at the time of, myocardial infarction (MI) to promote
repair and lessened
fibrosis. An effective dose of the active agents is applied to the myocardial
tissue, preferably
immediately following MI, although it can also be applied when there is an
indication of
impending MI.
In a preferred embodiment, a catheter is placed into the coronary artery of a
subject
between about at the time of to about 24 hours after myocardial infarction and
injecting an
effective amount of the active agent into the heart of the subject. The
concentration of active
3o agent injected is between about 100 ng/kg body weight and about 10.0 mg/kg
body weight, as
described above. The injection can be repeated as needed to promote myocardial
tissue
repair following MI. Injections can also be by other routes, including but not
limited to by
17
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WO 00/53211 PCT/US00/06198
catheter via arterial angiography, intracoronary injection, or in a
cardioplegic solution by the
aortic route.
For in vivo delivery, the active agents may be administered by any suitable
route,
including parentally, topically, or by cardiovascular devices in dosage unit
formulations
containing conventional pharmaceutically acceptable carriers, adjuvants, and
vehicles. The
term parenteral as used herein includes, subcutaneous, intravenous,
intraarterial,
intramuscular, intrasternal, intracardiac, intratendinous, intraspinal,
intracranial, intrathoracic,
infusion techniques or intraperitoneally. Furthermore, the active agents can
be administered
by gene therapy techniques.
l0 The active agents of the invention may be made up in a solid form
(including
granules, powders or suppositories) or in a liquid form (e.g., solutions,
suspensions, or
emulsions). The compounds of the invention may be applied in a variety of
solutions.
Suitable solutions for use in accordance with the invention are sterile,
dissolve sufficient
amounts of the peptide, and are not harmful for the proposed application. In
this regard, the
compounds of the present invention are very stable but are hydrolyzed by
strong acids and
bases. The compounds of the present invention are soluble in organic solvents
and in
aqueous solutions at pH 5-8.
The active agents may be subjected to conventional pharmaceutical operations
such as
sterilization and/or may contain conventional adjuvants, such as Garners,
preservatives,
2o stabilizers, wetting agents, emulsifiers, buffers etc.
The active agents of the invention can be used alone or in a combination of
active
agents, or may be used in combination with other agents that promote
myocardial tissue
repair, including, but not limited to free radical scavengers, calcium
antagonists, (3-blockers,
magnesium, inhibitors of white blood cell function, inhibitors of cellular
adhesion selectin
molecules, adenosine, fibroblast growth factor, digoxin, and ACE inhibitors.
Similarly, the
active agents can be used in combination with other compounds that promote
myocyte
proliferation, differentiation, such as growth factors and cytokines including
but not limited
to epidermal growth factor, insulin-like growth factor, fibroblast growth
factor, platelet
derived growth factor, nerve growth factor, tumor necrosis factor, and
interleukin I. When
3o administered as a combination, the therapeutic agents can be formulated as
separate
compositions that are given at the same time or different times, or the
therapeutic agents can
be given as a single composition.
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WO 00/53211 PCT/US00/06198
For administration, the active agents are ordinarily combined with one or more
adjuvants appropriate for the indicated route of administration. The compounds
may be
admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic
acids, stearic acid,
talc, magnesium stearate, magnesium oxide, sodium and calcium salts of
phosphoric and
sulphuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidine,
and/or polyvinyl
alcohol, and tableted or encapsulated for conventional administration.
Alternatively, the
compounds of this invention may be dissolved in saline, water, polyethylene
glycol,
propylene glycol, carboxymethyl cellulose colloidal solutions, ethanol, corn
oil, peanut oil,
cottonseed oil, sesame oil, tragacanth gum; and/or various buffers. Other
adjuvants and
1o modes of administration are well known in the pharmaceutical art. The
Garner or diluent may
include time delay material, such as glyceryl monostearate or glyceryl
distearate alone or
with a wax, or other materials well known in the art.
The dosage regimen for the therapeutic methods of the invention is based on a
variety
of factors, including the age, weight, sex, medical condition of the
individual, the severity of
the condition, the route of administration, and the particular compound
employed. Thus, the
dosage regimen may vary widely, but can be determined routinely by a physician
using
standard methods. Dosage levels of the order of between 0.1 ng/kg and 10 mg/kg
body
weight active agent per body weight are useful for all methods of use
disclosed herein.
The treatment regime will also vary depending on the condition of the subject,
based
on a variety of factors, including the age, weight, sex, medical condition of
the individual, the
severity of the condition, the route of administration, and the particular
compound employed.
For example, an active agents is administered to a patient as soon as possible
after, or at the
time of, myocardial infarction and continuing for up to 30 days. The therapy
is administered
for 1 to 6 times per day at dosages as described above.
In a preferred embodiment, the active agent is administered via local delivery
using
cardiovascular devices. Local delivery of the active agents of the invention
can be by a
variety of techniques that administer the agent at or near the traumatized
vascular site.
Examples of site-specific or targeted local delivery techniques are not
intended to be limiting
but to be illustrative of the techniques available. Examples include local
delivery catheters,
3o such as an infusion catheter, an indwelling catheter, or a needle catheter,
synthetic grafts,
adventitial wraps, shunts and stems or other implantable devices, site
specific carriers, direct
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WO 00/53211 PCT/US00/06198
injection, or direct applications. (U.S. Patent 5,981,568, incorporated by
reference herein in
its entirety.)
Local delivery by an implant describes the surgical placement of a matrix that
contains the active agent into the lesion or traumatized area. The implanted
matrix can release
the active agent by diffusion, chemical reaction, or solvent activators. See,
for example,
Lange, Science, 249, 1527 (1990).
An example of targeted local delivery by an implant is the use of a stmt,
which is
designed to mechanically prevent the collapse and re-occlusion of the coronary
arteries or
other vessels. Incorporation of an active agent into the stmt permits delivery
of the active
1o agent directly to the lesion. Local delivery of agents by this technique is
described in Koh,
Pharmaceutical Technology (October, 1990). For example, a metallic, plastic or
biodegradable intravascular stmt is employed which comprises the active agent.
The stmt
may comprise a biodegradable coating, a porous or a permeable non-
biodegradable coating,
or a biodegradable or non-biodegradable membrane or synthetic graft sheath-
like coating,
e.g., PTFE, comprising the active agent. Alternatively, a biodegradable stmt
may also have
the active agent impregnated therein, i.e., in the stmt matrix.
A biodegradable stmt with the active agent impregnated therein can be further
coated
with a biodegradable coating or with a porous non-biodegradable coating having
a sustained
release-dosage form of the active agent dispersed therein. This stmt can
provide a differential
2o release rate of the active agent, i.e., there can be an initial faster
release rate of the active
agent from the coating, followed by delayed release of the active agent
impregnated in the
stmt matrix, upon degradation of the stmt matrix. The intravascular stmt also
provides a
mechanical means of providing an increase in luminal area of a vessel.
Another example of targeted local delivery by an implant is the use of an
adventitial
wrap. The wrap comprises a pharmaceutically acceptable Garner matrix,
including but not
limited to a Pluronic gel which is free, or contained by a collagen mesh,
which gel has
dispersed therein the active agent.
Another embodiment of the invention is the incorporation of the active agent
into the
expanded nodal spaces of a PTFE (Impra, Inc., Tempe, Ariz.) vascular graft-
like membrane
3o which can surround, or be placed on the interior or on the exterior surface
of, an interlumenal
vascular stmt, which comprises metal or a biodegradable or nonbiodegradable
polymer. The
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
active agent, or a sustained release dosage form of the active agent, fills
the nodal spaces of
the PTFE membrane wall and/or coats the inner and/or outer surfaces of the
membrane.
A suitable local delivery dose of active ingredient of active agent is
preferably
between about 0.1 ng/kg and about 10 mg/kg administered twice daily for a time
sufficient to
promote myocardial tissue repair following MI. In a more preferred embodiment,
the
concentration of active agent is between about 100 ng/kg body weight and about
10.0 mg/kg
body weight. In a most preferred embodiment, the concentration of active agent
is between
about 10 ~g/kg body weight and about 10.0 mg/kg body weight. This dosage
regimen
maximizes the therapeutic benefits of the subject invention while minimizing
the amount of
1o active agent needed. Such an application minimizes costs as well as
possible deleterious side
effects. when used in combination with existing therapies, the invention
further minimizes the
amount of other costly therapeutics, such as growth factors and cytokines.
In another preferred embodiment of the present invention, the active agent is
administered parentally. Suitable topical doses and active ingredient
concentration in the
formulation are as described for local delivery via cardiovascular devices.
In a further preferred embodiment of all of the aspects of the invention, the
active
agent is selected from the group consisting of angiotensinogen, SEQ ID NO:1,
SEQ ID N0:2,
SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8,
SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID
2o N0:16, SEQ ID N0:17, SEQ ID N0:18, SEQ ID N0:19, SEQ ID N0:20, SEQ ID
N0:21,
SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ ID N0:26, SEQ ID
N0:27, SEQ ID N0:28, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID NO: 32,
SEQ ID N0:33, SEQ ID NO: 34; SEQ ID N0:35, SEQ ID N0:36, SEQ ID N0:37, SEQ ID
N0:38, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:41, SEQ ID N0:42, SEQ ID N0:43,
SEQ ID N0:44, SEQ ID N0:45, SEQ ID N0:46, SEQ ID N0:47, SEQ ID N0:48, SEQ ID
N0:49, and SEQ ID N0:50.
In a further aspect, the present invention provides kits for promoting in vivo
myocyte
proliferation and differentiation, or myocardial tissue repair following MI,
wherein the kits
comprise an effective amount of active agent for promoting in vivo myocyte
proliferation or
3o myocardial tissue repair following MI, and instructions for using the
amount effective of
active agent as a therapeutic. In a preferred embodiment, the kit further
comprises a
pharmaceutically acceptable carrier, such as those adjuvants described above.
In another
21
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WO 00/53211 PCT/US00/06198
preferred embodiment, the kit further comprises a means for delivery of the
active agent to a
patient. Such devices include, but are not limited to infusion catheters,
indwelling catheters,
needle catheters, synthetic grafts, adventitial wraps, shunts, stems or other
implantable
devices, syringes, matrical or micellar solutions, bandages, wound dressings,
aerosol sprays,
lipid foams, transdermal patches, topical administrative agents, polyethylene
glycol
polymers, carboxymethyl cellulose preparations, crystalloid preparations
(e.g., saline,
Ringer's lactate solution, phosphate-buffered saline, etc.), viscoelastics,
polyethylene glycols,
and polypropylene glycols. The means for delivery may either contain the
effective amount
of active agent, or may be separate from the compounds, which are then applied
to the means
to for delivery at the time of use.
In a further preferred embodiment, the kits further comprise ~an amount
effective to
promote in vivo myocyte proliferation and differentiation, or repair of
myocardial tissue of at
least one compound selected from the group consisting of free radical
scavengers, calcium
antagonists, [3-blockers, magnesium, inhibitors of white blood cell function,
inhibitors of
cellular adhesion selectin molecules, adenosine, fibroblast growth factor,
other growth
factors, cytokines, digoxin, and ACE inhibitors.
In another aspect of the present invention, an improved cell culture medium is
provided for the proliferation and differentiation of myocytes, wherein the
improvement
comprises addition to the cell culture medium of an effective amount of the
active agents, as
2o described above. Any cell culture media that can support the growth of
myocytes can be
used with the present invention. Such cell culture media include, but are not
limited to Basal
Media Eagle, Dulbecco's Modified Eagle Medium, Iscove's Modified Dulbecco's
Medium,
McCoy's Medium, Minimum Essential Medium, F-10 Nutrient Mixtures, Opti-MEM~
Reduced-Serum Medium, RPMI Medium, and Macrophage-SFM Medium or combinations
thereof.
The improved cell culture medium can be supplied in either a concentrated (ie:
10X)
or non-concentrated form, and may be supplied as either a liquid, a powder, or
a lyophilizate.
The cell culture may be either chemically defined, or may contain a serum
supplement.
Culture media is commercially available from many sources, such as GIBCO BRL
(Gaithersburg, MD) and Sigma (St. Louis, MO)
In a further aspect, the present invention provides kits for the propagation
of
myocytes, wherein the kits comprise an effective amount of the active agents,
as described
22
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
above, and instructions for using the active agents to promote myocyte
proliferation and
differentiation.
In a preferred embodiment, the kit further comprises cell culture growth
medium. Any
cell culture media that can support the growth and differentiation of myocytes
can be used
with the present invention. Examples of such cell culture media are described
above.
The improved cell culture medium can be supplied in either a concentrated (ie:
lOX)
or non-concentrated form, and may be supplied as either a liquid, a powder, or
a lyophilizate.
The cell culture may be either chemically defined, or may contain a serum
supplement.
In another preferred embodiment, the kit further comprises a sterile
container. The
1o sterile container can comprise either a sealed container, such as a cell
culture flask, a roller
bottle, or a centrifuge tube, or a non-sealed container, such as a cell
culture plate or microtiter
plate (Nunc; Naperville, IL).
In a further preferred embodiment, the kit further comprises an antibiotic
supplement
for inclusion in the reconstituted cell growth medium. Examples of appropriate
antibiotic
supplements include, but are not limited to actimonycin D, Fungizone~,
kanamycin,
neomycin, nystatin, penicillin, streptomycin, or combinations thereof (GIBCO).
The present invention, by providing a method for enhanced proliferation of
myocytes,
will greatly increase the clinical benefits of myocyte cell therapy after
various ischemic
events, including but not limited to myocardial infarction. This is true both
for increased
2o "self renewal" of myocytes, which will provide a larger supply of myocytes
at the
appropriate site. Similarly, methods that increase in vivo proliferation of
myocytes are
beneficial in treating various ischemic events, including but not limited to
myocardial
infarction, aneurysm repair, and reconstructive cardiac surgery.
The method of the present invention also increases the potential utility of
myocytes as
vehicles for gene therapy in various ischemic events, including but not
limited to myocardial
infarction by more efficiently providing a large number of such cells for
transfection, and
also by providing a more efficient means to rapidly expand transfected
myocytes.
Administration of the active agents to accelerate in vivo myocyte
proliferation and/or to treat
myocardial injuries can be used to treat heart failure, cardiomyopathies,
inflammation,
3o infection, sepsis, ischemia, heart valve disease, myocarditis,
inflammation; or myocardial
ischemia and infarction; and for improvement of cardiac output by increasing
stroke volume.
23
CA 02364484 2001-09-07
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Examples
Example 1. Myocyte Proliferation
Monolayer cultures of 1-2 day old neonatal Sprague-Dawley rat myocytes were
prepared. Minced ventricular myocardium was placed into a Ca2+- and Mg2+-free
Hanks' salt
solution buffered with 30 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-
ethanesulfonic
acid), pH 7.4. The cells were dissociated and incubated at 37°C with a
mixture of partially
purified trypsin (2.4 ILT/ml, Worthington Biochemcial), a-chymotrypsin (2.7
IU/ml) and
elastase (0.94 IU/ml, Sigma Chemical, St. Louis, MO). After each of 5
successive 20 minute
incubations, the dissociated cells were mixed with Eagle's minimal essential
medium (MEM;
1o Gibco, MD) containing 10% newborn calf serum, and were centrifuged and
pooled. The
dissociated cells were enriched for cardiomyocytes by the technique of
differential adhesion
to tissue culture plastics for 90 minutes at 37°C in a humidified 5%
COz and air atmosphere,
and plated onto laminin-coated (20 ~g/ml) silicone dishes at a concentration
of approximately
4 x 105 cells/dish. Cultures were incubated in a humidified 5% C02, 95% air
atmosphere at
37°C. After an overnight incubation in MEM containing 10% newborn calf
serum and 0.1
mM 5-bromo 2 deoxyuridine (Sigma), the attached cells were rinsed in serum-
free medium.
Briefly, standard MEM was supplemented with MEM amino acids, vitamins,
penicillin-
streptomycin (GIBCO), and 2 mM glutamine. In addition, the medium contained 30
nM
NaSe04, 2.5 ~g/ml human insulin, 10 pg/ml human transferrin (Sigma), 0.25 mM
ascorbic
2o acid (Sigma) and 0.1 mM 5-bromo-2'-deoxyuridine to minimize the
proliferation of non-
myogenic cells. The medium was replaced every 2 days with fresh medium over
the course
of the experiments.
The results of these experiments (Table I) demonstrate that the active agents
of the
invention increased myocyte proliferation over that of culture medium alone.
% Increase in Cell Number
24
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
Example 2. Myocardial Repair
Rabbits underwent a surgical procedure under intramuscular anesthesia
(Ketamine
Rompum) after shaving with animal clippers and preparation with betadine and
isopropyl
alcohol. After induction of anesthesia, the rabbits were intubated and placed
on a ventilator
to assist respiration. Vital signs were monitored with a pulse oximeter. A
midline
sternotomy was then performed. After exposure of the pericardial sac, a 3 cm
incision was
made into the pericardium. After visualization of the epicardial surface, two
coronary
arteries, the left circumflex, and the left anterior descending artery were
exposed and ligated
by 4-0 Vicryl suture. Vehicle (10% Hydron; 60% ethanol, and 1% polyethylene
glycol), with
1o and without peptide (1 mg/ml, 0.05 ml) was injected in the cardiac muscle
distal to the site of
coronary occlusion. The sternum was then closed with 2-0 silk. The muscle and
skin were
then closed with 3-0 Dexon II suture. Twenty-eight days after surgery, the
animals were
euthanized and a necropsy was performed. The number of microscopic fields with
fibrosis
(scar) and the number of blood vessels/field present in the infarct site were
assessed by
microscopic evaluation. The presence of a blood vessel was defined as a
channel lined with
endothelial cells that contained red blood cells (indicating that the vessels
had a blood
source).
All (SEQ ID NO:1), AII(1-7) (SEQ ID N0:4), 2 GD (SEQ ID N0:39), and 9GD
(SEQ ID N0:41) all decreased the size of the infarct (as measured by the
number of
2o microscopic fields with scar) (Table 2), although the decrease associated
with administration
of 9GD was not statistically significant, possibly due to the smaller group
size. The
decreases with the other peptides ranged from 30% to 85% of the infarct size
found at
placebo-treated sites. In contrast with that observed on day 7, the
vascularization at the
infarct site was not changed at this latter time point (Table 3).
Table 2. Effect of Angiotensin Peptides on the Size of Myocardial Fibrosis
after Infarct
Repair
Treatment # of animals Mean +/- SEM P Value
H dron 12 17.83 +/- 2.66
2GD 4 6.00 +/- 2.94 0.032
9GD 2 11.50 +/- 6.5 >0.05
~I 3 5.67 +/- 0.88 0.045
All 107 4 3.20 +/- 1.28 0.004
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
Table 3. Effect of Angiotensin Peptides on the Formation of Collateral
Circulation in
Ischemic Myocardial Tissue
Treatment # of animals Mean +/- SEM P Value
H dron 12 52.6 +/- 14.6
2GD 4 8.8 +/- 3.53 0.110
9GD 2 15.95 +/- 0.95 0.337
All 3 41.9 +/- 13.3 0.729
All 107 4 82.0 +/- 22.2 0.287
The present invention is not limited by the aforementioned particular
preferred
embodiments. It will occur to those ordinarily skilled in the art that various
modifications
may be made to the disclosed preferred embodiments without diverting from the
concept of
to the invention. All such modifications are intended to be within the scope
of the present
invention.
26
CA 02364484 2001-09-07
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SEQUENCE LISTING
<110> Rodgers, Kathleen
diZerega, Gere
<120> Methods for Promoting Myocyte Proliferation and
Myocardial Tissue Repair
<130> 98068B
<140> To be assigned
<141> 2000-03-09
<160> 50
<170> PatentIn Ver. 2.0
<210> 1
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII
<400> 1
Asp Arg Val Tyr Ile His Pro Phe
1 5
<210> 2
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (2-8)
<400> 2
Arg Val Tyr Ile His Pro Phe
1 5
<210> 3
<211> 6
<212> PRT
<213> Artificial Sequence
1
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<220>
<223> Description of Artificial Sequence:AII (3-8)
<400> 3
Val Tyr Ile His Pro Phe
1 5
<210> 4
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-7)
<400> 4
Asp Arg Val Tyr Ile His Pro
1 5
<210> 5
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (2-7)
<400> 5
Arg Val Tyr Ile His Pro
1 5
<210> 6
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (3-7)
<400> 6
Val Tyr Ile His Pro
1 5
<210> 7
2
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (5-8)
<400> 7
Ile His Pro Phe
1
<210> 8
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-6)
<400> 8
Asp Arg Val Tyr Ile His
1 5
<210> 9
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-5)
<400> 9
Asp Arg Val Tyr Ile
1 5
<210> 10
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-4)
<400> 10
Asp Arg Val Tyr
3
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
1
<210> 11
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (1-3)
<400> 11
Asp Arg Val
1
<210> 12
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<220>
<221> MOD_RES
<222> (2)
<223> Nle
<400> 12
Arg Xaa Tyr Ile His Pro Phe
1 5
<210> 13
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<220>
<221> MOD_RES
<222> (4)
<223> Nle
<400> 13
4
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
Arg Val Tyr Xaa His Pro Phe
1 5
<210> 14
<211> 3
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (6-8)
<400> 14
His Pro Phe
1
<210> 15
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII (4-8)
<400> 15
Tyr Ile His Pro Phe
1 5
<210> 16
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
class
<220>
<221> UNSURE
<222> (1)
<223> Xaa at poistion 1 can be Arg, Lys, Ala, Orn, Ser,
MeGly, D-Arg, or D-Lys
<220>
<221> UNSURE
<222> (2)
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<223> Xaa at position 2 can be Val, Ala, Leu, Nle, Ile,
Gly, Pro, Aib, Acp, or Tyr
<220>
<221> UNSURE
<222> (4)
<223> Xaa at position 4 can be Ile, Ala, Leu, Nle, Val,
or Gly
<400> 16
Xaa Xaa Tyr Xaa His Pro Phe
1 5
<210> 17
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<400> 17
Arg Val Tyr Gly His Pro Phe
1 5
<210> 18
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue
<400> 18
Arg Val Tyr Ala His Pro Phe
1 5
<210> 19
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 1
6
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<400> 19
Asp Arg Val Tyr Val His Pro Phe
1 5
<210> 20
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 2
<400> 20
Asn Arg Val Tyr Val His Pro Phe
1 5
<210> 21
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 3
<400> 21
Ala Pro Gly Asp Arg Ile Tyr Val His Pro Phe
1 5 10
<210> 22
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 4
<400> 22
Glu Arg Val Tyr Ile His Pro Phe
1 5
<210> 23
<211> 8
<212> PRT
<213> Artificial Sequence
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<220>
<223> Description of Artificial Sequence:AII analogue 5
<400> 23
Asp Lys Val Tyr Ile His Pro Phe
1 5
<210> 24
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 6
<400> 24
Asp Arg Ala Tyr Ile His Pro Phe
1 5
<210> 25
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 7
<400> 25
Asp Arg Val Thr Ile His Pro Phe
1 5
<210> 26
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 8
<400> 26
Asp Arg Val Tyr Leu His Pro Phe
1 5
g
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<210> 27
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 9
<400> 27
Asp Arg Val Tyr Ile Arg Pro Phe
1 5
<210> 28
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 10
<400> 28
Asp Arg Val Tyr Ile His Ala Phe
1 5
<210> 29
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 11
<400> 29
Asp Arg Val Tyr Ile His Pro Tyr
1 5
<210> 30
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 12
<400> 30
9
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
Pro Arg Val Tyr Ile His Pro Phe
1 5
<210> 31
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 13
<400> 31
Asp Arg Pro Tyr Ile His Pro Phe
1 5
<210> 32
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 14
<220>
<221> MOD_RES
<222> (4)
<223> PHOSPHORYLATION
<400> 32
Asp Arg Val Tyr Ile His Pro Phe
1 5
<210> 33
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 15
<220>
<221> MOD_RES
<222> (3)
<223> Nle
1~
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<400> 33
Asp Arg Xaa Tyr Ile His Pro Phe
1 5
<210> 34
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 16
<220>
<221> MOD_RES
<222> (5)
<223> Nle
<400> 34
Asp Arg Val Tyr Xaa His Pro Phe
1 5
<210> 35
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:AII analogue 17
<220>
<221> MOD_RES
<222> (4)
<223> homo Ser
<400> 35
Asp Arg Val Ser Tyr Ile His Pro Phe
1 5
<210> 36
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial
11
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
Sequence:p-aminophenylalanine 6 All
<220>
<221> MOD_RES
<222> (6)
<223> p-aminophenylalanine
<400> 36
Asp Arg Val Tyr Ile Xaa Pro Phe
1 5
<210> 37
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:angiotensin I
<400> 37
Asp Arg Val Tyr Ile His Pro Phe His Leu
1 5 10
<210> 38
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
1GD:Ala4-AII(1-7)
<400> 38
Asp Arg Val Ala Ile His Pro
1 5
<210> 39
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: 2GD
Pro3-AII(1-7)
12
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<400> 39
Asp Arg Pro Tyr Ile His Pro
1 5
<210> 40
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: 5GD Lys
3-AII(1-7)
<400> 40
Asp Arg Lys Tyr Ile His Pro
1 5
<210> 41
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: 9GD
Norleu-AII(1-7)
<220>
<221> MOD_RES
<222> (3)
<223> Nle
<400> 41
Asp Arg Xaa Tyr Ile His Pro
1 5
<210> 42
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence GSD28 Ile8-All
<400> 42
Asp Arg Val Tyr Ile His Pro Ile
13
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
1 5
<210> 43
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Ala3aminoPhe6-All
<220>
<221> MOD_RES
<222> (6)
<223> aminophenyalanine
<400> 43
Asp Arg Ala Tyr Ile Xaa Pro Phe
1 5
<210> 44
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Ala3-AIII
<400> 44
Arg Val Ala Ile His Pro Phe
1 5
<210> 45
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:Glyl-All
<400> 45
Gly Arg Val Tyr Ile His Pro Phe
1 5
14
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<210> 46
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<221> MOD_RES
<222> (4)
<223> Nle
<220>
<223> Description of Artificial Sequence: Norleu4-AIII
<400> 46
Arg Val Tyr Xaa Leu His Pro Phe
1 5
<210> 47
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Acpc3-All
<220>
<221> MOD_RES
<222> (3)
<223> 1-aminocyclopentane carboxylic acid
<400> 47
Asp Arg Xaa Tyr Ile His Pro Phe
1 5
<210> 48
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Orn2-All
<220>
<221> MOD_RES
<222> (2)
<223> Orn
IS
CA 02364484 2001-09-07
WO 00/53211 PCT/US00/06198
<400> 48
Asp Xaa Val Tyr Ile His Pro Phe
1 5
<210> 49
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Citron2-All
<220>
<221> MOD_RES
<222> (2)
<223> Citron
<400> 49
Asp Xaa Val Tyr Ile His Pro Phe
1 5
<210> 50
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:
Pro3Ala4-AII(1-7)
<400> 50
Asp Arg Pro Ala Ile His Pro
1 5
16
