U.S. patent application number 10/007363 was filed with the patent office on 2002-11-14 for psiepsilonrack peptide composition and method for protection against tissue damage due to ischemia.
Invention is credited to Mochly-Rosen, Daria.
Application Number | 20020168354 10/007363 |
Document ID | / |
Family ID | 26676880 |
Filed Date | 2002-11-14 |
United States Patent
Application |
20020168354 |
Kind Code |
A1 |
Mochly-Rosen, Daria |
November 14, 2002 |
psiepsilonRack peptide composition and method for protection
against tissue damage due to ischemia
Abstract
A method of reducing damage to cells and tissue caused by an
ischemic or hypoxic event is disclosed. The method includes
administering to the cell or tissue, either in vivo or ex vivo,
.psi..epsilon.RACK peptide. The peptide can be administered before,
during or after the ischemic or hypoxic event.
Inventors: |
Mochly-Rosen, Daria; (Menlo
Park, CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Family ID: |
26676880 |
Appl. No.: |
10/007363 |
Filed: |
November 9, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60247830 |
Nov 10, 2000 |
|
|
|
Current U.S.
Class: |
424/94.5 ;
514/15.1; 514/16.4; 514/17.7 |
Current CPC
Class: |
C12N 9/1205 20130101;
A61P 9/10 20180101; A61K 38/08 20130101 |
Class at
Publication: |
424/94.5 ;
514/12 |
International
Class: |
A61K 038/17; A61K
038/52 |
Goverment Interests
[0002] This work was supported in part by The National Institutes
of Health Grant HL52141. Accordingly, the United States Government
has certain rights in this invention.
Claims
It is claimed:
1. A method of reducing injury to a cell exposed to an ischemic or
an hypoxic condition, comprising administering to the cell a
.psi..epsilon.RACK peptide.
2. The method of claim 1, wherein said administering occurs prior
to exposing the cell to said ischemic or hypoxic condition.
3. The method of claim 2, wherein said administering prior to said
ischemic or hypoxic condition is for a period of time of between
about 1-180 minutes prior to said exposing.
4. The method of claim 1, wherein said administering occurs after
exposing the cell to said ischemic or hypoxic condition.
5. The method of claim 4, wherein said administering after exposure
to said ischemic or hypoxic condition occurs for between about
1-180 minutes after said ischemic or hypoxic condition.
6. The method of claim 1, wherein said administering occurs during
exposure of the cell to said ischemic or hypoxic condition.
7. The method of claim 1 wherein said administering includes
administering a peptide having a sequence identified as SEQ ID
NO:2.
8. The method of claim 1, wherein said administering includes
administering a peptide having a sequence selected from the group
consisting of SEQ ID NOS:6-18.
9. The method of claim 1, wherein said administering includes
administering a .psi..epsilon.RACK peptide linked to a moiety
effective to facilitate transport across a cell membrane.
10. The method of claim 9, wherein the moiety is selected from the
group consisting of a Tat-derived peptide (SEQ ID NO:5), an
Antennapedia carrier peptide (SEQ ID NO:3), and a polyarginine
peptide.
11. The method of claim 1, wherein said administering includes
administering the peptide by a route selected from the group
consisting or intraveneous, parenteral, subcutaneous, inhalation,
intranasal, sublingual, mucosal, and transdermal.
12. A method of reducing injury to tissue exposed to an ischemic or
an hypoxic condition, comprising administering to the tissue a
.psi..epsilon.RACK peptide.
13. The method of claim 12, wherein said administering occurs prior
to exposing the tissue to said ischemic or hypoxic condition.
14. The method of claim 13, wherein said administering prior to
said ischemic or hypoxic condition is for between about 1-180
minutes.
15. The method of claim 12, wherein said administering occurs after
exposing the tissue to said ischemic or hypoxic condition.
16. The method of claim 15, wherein said administering after
exposure to said ischemic or hypoxic condition occurs for between
about 1-180 minutes after said ischemic or hypoxic condition.
17. The method of claim 12, wherein said administering occurs
during exposure of the tissue to said ischemic or hypoxic
condition.
18. The method of claim 12, wherein said administering includes
administering a peptide having a sequence identified as SEQ ID
NO:2.
19. The method of claim 12, wherein said administering includes
administering a peptide having a sequence selected from the group
consisting of SEQ ID NOS:6-18.
20. The method of claim 12, wherein said administering includes
administering a .psi..epsilon.RACK peptide linked to a moiety
effective to facilitate transport across a cell membrane.
21. The method of claim 12, wherein the moiety is selected from the
group consisting of a Tat-derived peptide (SEQ ID NO:5), an
Antennapedia carrier peptide (SEQ ID NO:3), and a polyarginine
peptide.
22. The method of claim 12, wherein said administering includes
administering the peptide by a route selected from the group
consisting or intraveneous, parenteral, subcutaneous, inhalation,
intranasal, sublingual, mucosal, and transdermal.
23. The method of claim 12 wherein said administering is to a
tissue that is a whole organ ex vivo.
24. The method of claim 12 wherein said administering is to a
tissue that is a whole organ in vivo.
25. The method of claim 23 or 24, wherein said organ is selected
from the group consisting of heart, lung, liver, brain, and
kidney.
26. The method of claim 24, wherein said administering is by
infusion through coronary arteries to an intact heart.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/247,830 filed Nov. 10, 2000, incorporated herein
by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to a method of protecting
cells and tissues from damage due to an ischemic event. The method
involves administering a peptide agonist of protein kinase C, and
more specifically, administering a pseudo-epsilon RACK
(.psi..epsilon.RACK) peptide.
REFERENCES
[0004] Brew, E. C., et al., Am. J. Physiol 269 (Heart Circ.
Physiol. 38):H1370-H1378 (1995).
[0005] Colbert, M. C. et al, J. Clin. Invest. 100:1958 (1997).
[0006] Csukai, M., et al., 9.sup.TH INTERNATIONAL CONFERENCE ON
SECOND MESSENGERS AND PHOSPHOPROTEINS 112 (1995).
[0007] Csukai, M., et al, J. Biol. Chem., 272:29200-29206
(1997).
[0008] Disatnik, M. H., et al., Exp. Cell Res. 210:287-297
(1994).
[0009] Dorn, G., Proc. Natl. Acad. Sci. USA 96(22):12798
(1999).
[0010] Fawell et al., Proc. Natl. Acad. Sci. USA 91:664-68
(1994).
[0011] Gray, M. O. et al., J. Biol. Chem., 272:30945-3095
(1997).
[0012] Hu, K. and Nattel, S., Circulation 92:2259-2265 (1995).
[0013] Johnson, J. A., et al., J. Biol. Chem 271:24962-24966
(1996).
[0014] Johnson, J. A. et al., Circ. Res., 79:1086 (1996a).
[0015] Lindgren et al., Trends. Pharmacol. Sci., 21:99-103
(2000).
[0016] Liu, Y., et al., J. Mol. Cell. Cardiol. 27:883-892
(1995).
[0017] Mitchell, M. B., et al., Circulation 88:1633 (1993).
[0018] Mitchell, M. B., et al., Circ. Res. 76:73-81 (1995).
[0019] Mitchell et al., J. Peptide Res., 56:318-325 (2000).
[0020] Mochly-Rosen, D., et al., Molec. Biol. Cell (formerly Cell
Reg.) 1:693-706 (1990).
[0021] Mochly-Rosen, D., et al., Proc. Natl. Acad. Sci. USA
88:3997-4000 (1991).
[0022] Mochly-Rosen, D., et al., J. Biol. Chem., 226:1466-1468
(1991a).
[0023] Mochly-Rosen, D., et al., Science 268:247-251 (1995).
[0024] Murry, C. E., et al., Circulation 74:1123-1136 (1986).
[0025] Papadopoulos, V. and Hall, P. F. J. Cell Biol. 108:553-567
(1989).
[0026] Pitcher, J., et al., Science 257:1264-1267 (1992).
[0027] Rolhbard et al., Nature Med., 6:1253-1257 (2000).
[0028] Ron, D., et al., Proc. Natl. Acad. Sci. USA 91:839-843
(1994).
[0029] Ron, D., eta l., Biol. Chem. 279:24180-24187 (1995).
[0030] Saito, N. et al., Proc. Natl. Acad. Sci. USA 86:3409-3413
(1989).
[0031] Schultz, J. E. J., et al., Circ. Res. 78:1100-1104
(1996).
[0032] Schwarlze et al., Trends. Pharmacol. Sci., 21:45-48
(2000).
[0033] Smith, B. L. and Mochly-Rosen, D., Biochem. Biophys. Res.
Commun. 188:1235-1240 (1992).
[0034] Speechly-Dick, M. E., et al., Circ. Res. 75:586-590
(1993).
[0035] Stebbins, E. G., et al., J. Biol. Chem., 276:29644-29650
(2001).
[0036] Thodore L., et al., J. Neurosci. 15:7158 (1995).
[0037] Vives et al. J. Biol. Chem., 272:16010-16017 (1997).
BACKGROUND OF THE INVENTION
[0038] Protein kinase C (PKC) is a key enzyme in signal
transduction involved in a variety of cellular functions, including
cell growth, regulation of gene expression and ion channel
activity. The PKC family of isozymes includes at least 11 different
protein kinases which can be divided into at least three
subfamilies based on their homology and sensitivity to activators.
Members of the classical or cPKC subfamily, .alpha., .beta..sub.I,
.beta..sub.II and .gamma.PKC, contain four homologous domains (C1,
C2,C3 and C4) inter-spaced with isozyme-unique (variable or V)
regions, and require calcium, phosphatidylserine (PS), and
diacylglycerol (DG) or phorbol esters for activation. Members of
the novel or nPKC subfamily, .delta., .epsilon., .eta., and
.theta.PKC, lack the C2 homologous domain and do not require
calcium for activation. Finally, members of the atypical or
.alpha.PKC subfamily, .zeta. and .lambda./.iota.PKC, lack both the
C2 and one half of the C1 homologous domains and are insensitive to
DG, phorbol esters and calcium.
[0039] Studies on the subcellular distribution of PKC isozymes
demonstrate that activation of PKC results in its redistribution in
the cells (also termed translocation), such that activated PKC
isozymes associate with the plasma membrane, cytoskeletal elements,
nuclei, and other subcellular compartments (Saito, et al., 1989;
Papadopoulos and Hall, 1989; Mochly-Rosen, et al., 1990).
[0040] It appears that the unique cellular functions of different
PKC isozymes are determined by their subcellular location. For
example, activated .beta..sub.IPKC is found inside the nucleus,
whereas activated .beta..sub.IIPKC is found at the perinucleus and
cell periphery of cardiac myocytes (Disatnik, et al., 1994).
Further, in the same cells, .epsilon.PKC binds to cross-striated
structures (possibly the contractile elements) and cell-cell
contacts following activation or after addition of exogenous
activated .epsilon.PKC to fixed cells (Mochly-Rosen, et al., 1990;
Disatnik, et al., 1994). The localization of different PKC isozymes
to different areas of the cell in turn appears due to binding of
the activated isozymes to specific anchoring molecules termed
Receptors for Activated C-Kinase (RACKs).
[0041] RACKs are thought to function by selectively anchoring
activated PKC isozymes to their respective subcellular sites. RACKs
bind only fully activated PKC, but RACKs are not necessarily
substrates of the enzyme nor is the binding to RACKs mediated via
the catalytic domain of the kinase (Mochly-Rosen, et al., 1991).
Translocation of PKC reflects binding of the activated enzyme to
RACKs anchored to the cell particulate fraction and the binding to
RACKs is required for PKC to produce its cellular responses
(Mochly-Rosen, 1995). Inhibition of PKC binding to RACKs in vivo
inhibits PKC translocation and PKC-mediated function (Johnson, et
al., 1996; Ron, et al., 1995; Smith and Mochly-Rosen, 1992).
[0042] cDNA clones encoding RACK1 and RACK2 have been identified
(U.S. Pat. No. 5,519,003; Ron, et al., 1994; Csukai, et al., 1995).
Both are homologs of the .beta. subunit of G proteins, a receptor
for another translocating protein kinase, the .beta.-adrenergic
receptor kinase, .beta.ARK (Pitcher, et al., 1992). Similar to
G.beta., RACK1, and RACK2 have seven WD40 repeats (Ron, et al.,
1994; Csukai, et al., 1995). Recent data suggest that RACK1 is a
.beta..sub.IIPKC-specific RACK (Stebbins et al., 2001) whereas
RACK2 is specific for activated .epsilon.PKC (Csukai et al.,
1997).
[0043] Translocation of PKC is required for proper function of PKC
isozymes. Peptides that mimic either the PKC-binding site on RACKs
(Mochly-Rosen, 1991 a; Mochly-Rosen, 1995) or the RACK-binding site
on PKC (Ron, et al., 1995; Johnson, et al., 1996) are
isozyme-specific translocation inhibitors of PKC that selectively
inhibit the function of the enzyme in vivo. For example, an eight
amino acid peptide derived from .epsilon.PKC (peptide
.epsilon.V1-2; SEQ ID NO:1, Glu Ala Val Ser Leu Lys Pro Thr) is
described in U.S. Pat. No. 6,165,977. The peptide contains a part
of the RACK-binding site on .epsilon.PKC and selectively inhibits
specific .epsilon.PKC-mediated functions in cardiac myocytes.
[0044] Recently, PKC and more specifically .epsilon.PCK have been
shown to be involved in cardiac preconditioning to provide
protection from ischemic injury. Prolonged ischemia causes
irreversible myocardium damage primarily due to death of cells at
the infarct site. Studies in animal models, isolated heart
preparations and isolated cardiac myocytes in culture have
demonstrated that short bouts of ischemia of cardiac muscle reduce
such tissue damage in subsequent prolonged ischemia (Liu, Y., et
al., 1995, 1996; Hu, et al., 1995; Brew, et al., 1995; Schultz, et
al., 1996). This protection, which occurs naturally following
angina and has been termed preconditioning, can be mimicked by a
variety of non-specific PKC agonists (Mitchell et al., 1993;
Mitchell et al., 1995; Murry et al., 1986; Speechly-Dick et al.,
1994). Both .delta.PKC and .epsilon.PKC activation occurs following
preconditioning (Gray et al., 1997), however, .epsilon.PKC
activation is required for protection of cardiac myocytes from
ischemia-induced cell death (U.S. Pat. No. 6,165,977).
[0045] In a recent study, an .epsilon.PKC-selective peptide agonist
was shown to provide cardio-protection from ischemia when
administered intracellulary to isolated neonatal and adult
cardiomyocytes and when produced intracellulary in vivo in
transgenic mice (Dorn G. et al., 1999). In this work, a
.epsilon.PKC peptide agonist was administered intracellulary to
isolated cells in vitro by laboratory techniques suitable at the
cellular level or by genetic transfection. Unfortunately, neither
of these techniques are suitable or likely to be successful for
human therapy. Moreover, it is unknown from this work whether or
not the .epsilon.PKC peptide could be delivered extracellulary to
whole tissue or intact organs in vivo to achieve a therapeutic
effect.
SUMMARY OF THE INVENTION
[0046] Accordingly, it is an object of the invention to provide a
method of protecting tissue from damage due to an ischemic
event.
[0047] It is a further object of the invention to provide a method
of administering an .epsilon.PKC peptide agonist for induction of
ischemic preconditioning.
[0048] It is yet another object of the invention to provide a
method of ameliorating damage to tissue caused by an ischemic
event.
[0049] Accordingly, in one aspect, the invention includes a method
of reducing injury to a cell exposed to an ischemic or an hypoxic
condition by administering to the cell a .psi..epsilon.RACK
peptide. In one embodiment, the peptide is administered prior to
exposing the cell to the ischemic or hypoxic condition. For
example, the peptide administered for a period of time of between
about 1-180 minutes prior to exposing the cell to ischemia or
hypoxia. In another embodiment, the peptide is administered after
the cell is exposed to an ischemic or hypoxic condition. For
example, the peptide is administered for between about 1-180
minutes after the cell is exposed to an ischemic or hypoxic
condition. In another embodiment, the peptide is administered
during to the cell during the period of ischemia or hypoxia.
[0050] In one embodiment, the .psi..epsilon.RACK peptide has a
sequence identified as SEQ ID NO:2. In other embodiments, the
peptide has a sequence selected from the group consisting of SEQ ID
NOS:6-18.
[0051] In yet another embodiment, the .psi..epsilon.RACK peptide is
linked to a moiety effective to facilitate transport across a cell
membrane, such as a Tat-derived peptide (SEQ ID NO:5), an
Antennapedia carrier peptide (SEQ ID NO:3), or a polyarginine
peptide.
[0052] The peptide can be administered by a route selected from the
group consisting or intraveneous, parenteral, subcutaneous,
inhalation, intranasal, sublingual, mucosal, and transdermal.
[0053] In another aspect, the invention includes a method of
reducing injury to tissue exposed to an ischemic or an hypoxic
condition by administering to the tissue a .psi..epsilon.RACK
peptide, as described above.
[0054] These and other objects and features of the invention will
be more fully appreciated when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0055] FIG. 1A shows creatine phosphokinase (CPK) release as a
function of time during reverse perfusion in ex vivo rat hearts
treated with .psi..epsilon.RACK (open circles) or with scrambled
.psi..epsilon.RACK (control, open squares) prior to an ischemic
event. The treated hearts were compared to hearts subjected to the
ischemic period but left untreated (closed triangles) and to hearts
maintained under normoxia conditions (no ischemia, no peptide
treatment; closed squares) as controls.
[0056] FIG. 1B shows the total amount of CPK released in the study
described in FIG. 1A during 30 minutes of reperfusion.
[0057] FIG. 2A shows the functional recovery of a working heart
perfused with .psi..epsilon.RACK prior to global ischemia, where
the left ventricle developed pressure (LVP, in mmHg), its first
derivative (dP/dt, in mmHg/sec), and the coronary perfusion
pressure (PP, in mmHg) are shown. The right panel shows an
expansion of the same trace before (baseline) and after
reperfusion.
[0058] FIG. 2B is a scan similar to FIG. 2A for a working heart
perfused with scrambled .psi..epsilon.RACK prior to ischemia.
[0059] FIG. 3A shows CPK release as a function of time following
ischemic insult in ex vivo rat hearts treated for the first 20
minutes of reperfusion with .psi..epsilon.RACK (open circles) and
in hearts left untreated after ischemia (solid triangles).
[0060] FIG. 3B is a bar graph showing the total CPK release during
a 60 minute reperfusion period following an ischemic insult to
whole rat hearts (i) treated ex vivo with .psi..epsilon.RACK for 20
minutes after the ischemic insult, or (ii) left untreated.
[0061] FIGS. 4A-4B are computer generated photos of pig heart
slices taken from pigs five days after an in vivo treatment with
.psi..epsilon.RACK (FIG. 4A) or with scrambled .psi..epsilon.RACK
peptide as a control (FIG. 4B) during the initial 10 minutes of a
30 minute ischemic insult.
[0062] FIG. 4C is a bar graph showing the total infarct area as a
percent of area at risk, measured in grams of cardiac tissue, for
pigs treated with .psi..epsilon.RACK peptide or with scrambled
.psi..epsilon.RACK peptide (control).
[0063] FIG. 5 is a graph showing the ejection fraction, as measured
by left ventricurogram in pigs at three time points: (1) before
occlusion of left anterior descending artery by balloon catheter
(before occlusion); (2) immediately after reperfusion with
.psi..epsilon.RACK (post .psi..epsilon.RACK); and (3) before
sacrifice five days later (5 days post), for animals treated with
.psi..epsilon.RACK (solid triangles) and for control animals
treated with a scrambled peptide (open circles).
BRIEF DESCRIPTION OF THE SEQUENCES
[0064] SEQ ID NO: 1 is a prior art (U.S. Pat. No. 6,165,977)
.epsilon.PKC octapeptide.
[0065] SEQ ID NO:2 is a .psi..epsilon.RACK octapeptide
[0066] SEQ ID NO:3 is the Drosophila Antennapedia
homeodomain-derived carrier peptide.
[0067] SEQ ID NO:4 is a scrambled .psi..epsilon.RACK
octapeptide.
[0068] SEQ ID NO:5 is a Tat-derived carrier peptide.
[0069] SEQ ID NO:6 is a modification of SEQ ID NO:2.
[0070] SEQ ID NO:7 is a modification of SEQ ID NO:2.
[0071] SEQ ID NO:8 is a modification of SEQ ID NO:2.
[0072] SEQ ID NO:9 is a modification of SEQ ID NO:2.
[0073] SEQ ID NO:10 is a modification of SEQ ID NO:2.
[0074] SEQ ID NO:11 is a modification of SEQ ID NO:2.
[0075] SEQ ID NO:12 is a modification of SEQ ID NO:2.
[0076] SEQ ID NO:13 is a modification of SEQ ID NO:2.
[0077] SEQ ID NO:14 is a modification of SEQ ID NO:2.
[0078] SEQ ID NO:15 is a modification of SEQ ID NO:2.
[0079] SEQ ID NO:16 is a modification of SEQ ID NO:2.
[0080] SEQ ID NO:17 is a modification of SEQ ID NO:2.
[0081] SEQ ID NO:18 is a fragment of SEQ ID NO:2.
DETAILED DESCRIPTION OF THE INVENTION
[0082] I. Definitions
[0083] "Tissue" as used herein refers to a group of similarly
specialized cells that perform a common function. Tissues compose
the organs and structural components of living organisms. As used
herein, tissue is intended to include an organ composed of a given
tissue and to the cells, individually or collectively, that compose
the tissue.
[0084] "Ischemia" or an "ischemic event" refers to an insufficient
supply of blood to a specific cell, tissue or organ. A consequence
of decreased blood supply is an inadequate supply of oxygen to the
organ or tissue (hypoxia). Prolonged hypoxia may result in injury
to the affected organ or tissue.
[0085] "Anoxia" refers to a virtually complete absence of oxygen in
the organ or tissue, which, if prolonged, may result in death of
the cell, organ or tissue.
[0086] "Hypoxia" or a "hypoxic condition" intends a condition under
which a cell, organ or tissue receives an inadequate supply of
oxygen.
[0087] "Ischemic injury" refers to cellular and/or molecular damage
to an organ or tissue or cell as a result of a period of ischemia.
"Hypoxic injury" refers to damage to a cell, organ, or tissue due
to a period of inadequate oxygen supply.
[0088] "Reperfusion" refers to return of fluid flow into a tissue
after a period of no-flow or reduced flow. For example, in
reperfusion of the heart, fluid or blood returns to the heart
through a supply line, such as the coronary arteries in vivo, after
removal of an occlusion to the fluid or blood supply.
[0089] "Treating a disease" refers to administering a therapeutic
substance effective to reduce the symptoms of the disease and/or
lessen the severity of the disease.
[0090] "Conservative amino acid substitutions" are substitutions
which do not result in a significant change in the activity (e.g.,
.epsilon.PKC-agonist activity or .psi..epsilon.RACK-agonist
activity) or tertiary structure of a selected polypeptide. Such
substitutions typically involve replacing a selected amino acid
residue with a different residue having similar physico-chemical
properties. For example, substitution of Glu for Asp is considered
a conservative substitution since both are similarly-sized
negatively-charged amino acids. Groupings of amino acids by
physico-chemical properties are known to those of skill in the
art.
[0091] With respect to a specific sequence, "conservative
substitutions thereof" refers to sequences that differ from the
specific sequence by having conservative amino acid substitutions
at one or more positions. "Peptide" and "polypeptide" are used
interchangeably herein and refer to a compound made up of a chain
of amino acid residues linked by peptide bonds. Unless otherwise
indicated, the sequence for peptides is given in the order from the
amino termiums to the carboxyl terminus.
[0092] When a first peptide or polypeptide is said to "correspond"
or to be "homologous" to a second peptide or polypeptide fragment,
it means that the peptide or fragments have a similarity in amino
acid residues if they have an alignment score of >5 (in standard
deviation units) using the program ALIGN with the mutation gap
matrix and a gap penalty of 6 or greater (Dayhoff, M. O., in ATLAS
OF PROTEIN SEQUENCE AND STRUCTURE (1972) Vol. 5, National
Biomedical Research Foundation, pp. 101-110 and Supplement 2 to
this volume, pp. 1-10.) The two sequences (or parts thereof) are
more preferably homologous if their amino acids are greater than or
equal to 50%, more preferably 70%, still more preferably 80%,
identical when optimally aligned using the ALIGN program mentioned
above.
[0093] A polypeptide sequence or fragment is "derived" from another
polypeptide sequence or fragment when it has an identical sequence
of amino acid residues as a region of the another sequence or
fragment.
[0094] An ".epsilon.PKC agonist peptide" or ".epsilon.PKC specific
agonist peptide" is understood to mean a peptide between about 4
and about 30, preferably between about 5 and about 15, amino acids
in length that is derived from .epsilon.PKC. In one embodiment, the
.epsilon.PKC agonist peptide is derived from the region of
.epsilon.PKC between about amino acids 70 and 120, preferably
between about amino acids 80 and 100, more preferably between about
amino acids 85 and 92.
[0095] Abbreviations: "PKC", protein kinase C; "RACK", receptor for
activated C-kinase.
[0096] Abbreviations for amino acid residues are the standard
3-letter and/or 1-letter codes used in the art to refer to one of
the 20 common L-amino acids.
[0097] II. Administration of .psi..epsilon.RACK Peptide Agonist
[0098] In one aspect, the invention provides a method of protecting
a cell, a tissue, or an organ from damage due to an ischemic event
or a hypoxic condition by administering a peptide capable of
activating signaling proteins, such as PKC, that are activated in
vivo by binding to a cognate polypeptide such as a receptor protein
(RACK). Regions of homology between the PKC signaling peptide and
its RACK are termed "pseudo-RACK" sequences (.psi.-RACK; Ron et
al., 1994, 1995) and typically have a sequence similar to the
PKC-binding region of the corresponding RACK. A .psi.-RACK sequence
that acts as an .epsilon.PKC specific agonist peptide is identified
herein as SEQ ID NO:2. This peptide, referred to herein as
.psi..epsilon.RACK, is an .epsilon.PKC specific agonist peptide and
induces translocation of .epsilon.PKC. Heretofore, studies have
focussed on identifying drugs and peptidomnimetics that mimic the
action of .psi..epsilon.RACK as it was unknown if the peptide
itself could be administered in vivo or ex vivo to a whole organ to
induce translocation of .epsilon.PCK to confer protection from
ischemia. In studies performed in support of the invention,
.psi..epsilon.RACK was administered ex vivo and in vivo to whole
hearts prior to and after exposure to an ischemic condition. The
peptide reduced the extent of ischemic injury, as will now be
described.
[0099] A. Administration of .psi..epsilon.RACK Peptide Agonist
Prior to Ischemia
[0100] A peptide having the sequence identified herein as SEQ ID
NO:2 was synthesized and purified as described in the Methods
section. A scrambled .psi..epsilon.RACK peptide (SEQ ID NO:4) was
also prepared to serve as a comparison or a negative control to the
.psi..epsilon.RACK peptide. In some studies, the peptides were
conjugated to a carrier peptide, such as Drosophila Antennapedia
homeodomain (SEQ ID NO:3). It will be appreciated that
administration of the native peptide, that is the peptide
unmodified by attachment to a carrier, is also contemplated.
Carrier peptides other than Drosophila Antennapedia are also
contemplated, and other exemplary carrier peptides include
Tat-derived peptide (SEQ ID NO:5, Fawell et al.,1994, Vives et al.,
1997) or a polyarginine peptide (Mitchell et al., 2000; Rolhbard et
al., 2000), or other like carries described in the art (Lindgren et
al., 2000; Schwarlze et al., 2000).
[0101] Animals were anesthetized as described in Example 1 and
their hearts were rapidly removed and cannulated for perfusion on a
Langendorff apparatus. Hemodynamic parameters were monitored until
stabilized, typically for 10-20 minutes. After equilibration,
.psi..epsilon.RACK peptide (SEQ ID NO:2) or a scrambled
.psi..epsilon.RACK peptide (SEQ ID NO:4) were delivered to the
hearts via the coronary arteries using 0.5 .mu.M of peptide for 20
minutes.
[0102] To induce global ischemia, flow to the hearts was
interrupted for 45 minutes. The hearts were then reperfused for 30
minutes. During reperfusion, ischemia-induced cell damage was
determined by measuring the activity of creatine phosphokinase
(absorbance at 520 nm) in the perfusate. Creatine phosphokinase is
a cytosolic enzyme in cardiac myocytes and its presence in the
perfusate is proportional to the number of cardiomyocytes damaged
by the ischemia. The results are shown in FIGS. 1A-1B.
[0103] FIG. 1A shows the creatine phosphokinase (CPK) release as a
function of time during the 30 minute reperfusion of ex vivo hearts
treated with 500 nM (0.5 .mu.M) .psi..epsilon.RACK (open circles)
or with 0.5 .mu.M scrambled .psi..epsilon.RACK (open squares) prior
to the ischemic event.
[0104] Also shown is the CPK release for hearts subjected to the
ischemic period but untreated with a peptide (closed triangles) and
to hearts maintained under normoxia conditions (no ischemia, no
peptide treatment; closed squares), as controls. The hearts treated
with .psi..epsilon.RACK prior to ischemia have a release of CPK
close to the control hearts maintained under normoxia conditions.
In contrast, hearts treated with scrambled .psi..epsilon.RACK have
significant release of CPK, indicating significant cell damage.
[0105] FIG. 1B is a bar graph showing the total CPK release during
the reperfusion period for the hearts treated with
.psi..epsilon.RACK and with scrambled .psi..epsilon.RACK. The total
CPK release from hearts exposed to the ischemic event but left
untreated are also shown.
[0106] FIGS. 1A-1B show that hearts treated with .psi..epsilon.RACK
prior to an ischemic event provides protection from damage
resulting from a subsequent ischemic event. Accordingly, in one
embodiment the invention contemplates a method of reducing or
preventing injury to a tissue exposed to an ischemic or hypoxic
event by administering to the tissue an amount of
.psi..epsilon.RACK agonist peptide. The peptide can be administered
from between 1-180 minutes prior to the ischemic event, more
preferably from about 1-120 minutes prior to the ischemic event,
more preferably from about 1-60 minutes prior to the ischemic
event. In another embodiment, a time period of no more than about
180 minutes, more preferably no more than 120 minutes, still more
preferably no more than about 60 minutes, lapses between cessation
of peptide delivery and the ischemic event.
[0107] In another study in support of the invention, the functional
recovery of ex vivo hearts after an ischemic event was evaluated by
monitoring the isovolumic left ventricle pressure (LVP) at a
constant pacing (3.3 Hz) and at a constant coronary flow (10
mL/min), as described in Example 1. Prior to the ischemic event,
the hearts were treated with 500 nM .psi..epsilon.RACK (SEQ ID
NO:2) or with scrambled .psi..epsilon.RACK peptide (SEQ ID NO:4).
After the 30 minute global ischemia, the hearts were monitored
during a 30 minute reperfusion period. The results are shown in
FIGS. 2A-2B.
[0108] FIG. 2A shows the results for the hearts treated with
.psi..epsilon.RACK peptide prior to global ischemia, and FIG. 2B
shows the results for hearts treated with scrambled
.psi..epsilon.RACK prior to ischemia. In comparing the baseline
levels and reperfusion levels of the treated and untreated hearts,
it is seen that administration of .psi..epsilon.RACK peptide before
ischemia significantly reduced the ischemic injury. This is
particularly evidenced by comparing the recovery of left ventricle
developed pressure (LVP) in the hearts pre-treated with
.psi..epsilon.RACK to those pre-treated with the scrambled peptide.
A four-fold improvement in both the LVP recovery and its first
derivative (dP/dt) were achieved by pre-treating with
.psi..epsilon.RACK. Furthermore, .psi..epsilon.RACK reduced the
elevated LVP end diastolic pressure and the coronary perfusion
pressure (PP).
[0109] Accordingly, these studies (FIGS. 1 and 2) show that
cellular damage to a tissue due to ischemic or hypoxia is reduced
by administering .psi..epsilon.RACK prior to the ischemic event or
the hypoxic event. In this study, the .psi..epsilon.RACK was
administered through the coronary arteries to the organ and for a
period prior to exposure to the ischemic and/or hypoxic condition.
The time period, as well as the dose of peptide administered, can
vary considerably, as will be discussed in more detail below.
[0110] B. Administration of .psi..epsilon.RACK Peptide Agonist
Subsequent to Ischemia
[0111] In another study performed in support of the invention,
.psi..epsilon.RACK was administered to hearts ex vivo after a
prolonged ischemic period and was effective to provide protection
from ischemic injury. As described in Example 2, whole rat hearts
were perfused on a Langendorff apparatus. After a 30 minute
equilibration period, global ischemia was induced by stopping fluid
flow for 45 minutes. The hearts were then reperfused with or
without .psi..epsilon.RACK peptide for 20 minutes, followed by 40
minutes perfusion without peptide. During the 60 minutes following
ischemia (20 minutes of peptide reperfusion plus 40 minutes
perfusion), the CPK activity in the perfusate was analyzed. The
results are shown in FIGS. 3A-3B.
[0112] FIG. 3A shows the CPK release as a function of time
following ischemia for hearts treated with .psi..epsilon.RACK (open
circles) and for hearts left untreated after ischemia (solid
triangles). FIG. 3B is a bar graph showing the total CPK release
during the 60 minute perfusion period for the peptide treated and
untreated ex vivo hearts. The data shows that subsequent
administration of .psi..epsilon.RACK peptide to tissue previously
exposed to an ischemic or hypoxic condition is effective to reduce
the cellular damage. FIG. 3B shows there was a nearly 2-fold lower
total CPK release for tissue treated with .psi..epsilon.RACK
peptide.
[0113] C. Adminstration of .psi..epsilon.RACK Peptide In Vivo
[0114] In another study in support of the invention, the ability of
.psi..epsilon.RACK peptide to protect tissue from damage due to an
ischemic or hypoxic event was evaluated by administering the
peptide in vivo. As detailed in Example 3, .psi..epsilon.RACK
peptide (SEQ ID NO:2) or scrambled .psi..epsilon.RACK peptide (SEQ
ID NO:4) was administered to adult pigs during the first 10 minutes
of a 30 minutes ischemic insult. Five days later, the hearts were
analyzed for tissue damage. The results are shown in FIGS.
4A-4C.
[0115] FIGS. 4A-4B are computer-generated photos of pig heart
slices taken from the pigs treated in vivo with .psi..epsilon.RACK
(FIG. 4A) or with scrambled .psi..epsilon.RACK peptide as a control
(FIG. 4B). The hearts were stained with a double-staining technique
(Example 3) that allowed determination of the area at risk for
ischemic injury (area within the arrows, mainly in the mid wall, in
FIG. 4B) and infarcted area (white area in FIG. 4B). As seen in
FIG. 4B, control hearts treated with scrambled .psi..epsilon.RACK
peptide have a large infarct area within the area at risk (borders
shown with arrows). In contrast, pigs that received the
.psi..epsilon.RACK peptide (FIG. 4A) have a significantly reduced
infarct area. By measuring surface areas and the weights of regions
and total tissue weight of areas at risk and infarcted regions, it
was determined that the control hearts had an average of
36.5.+-.0.3% infarct of area at risk, whereas hearts treated with
.psi..epsilon.RACK peptide had an average of 14.9.+-.0.6% infarct
of area at risk (p<0.005).
[0116] FIG. 4C is a bar graph showing the infarct area as a percent
of area at risk, measured in grams of cardiac tissue. As seen, the
percent infarct was reduced by more than 2-fold for the animals
treated with .psi.RACK peptide. Accordingly, delivery of a
.psi..epsilon.RACK peptide in vivo prior to or during an ischemic
event is effective to reduce the percentage of infarct by at least
2-fold.
[0117] Blood samples and tissue samples of lung, liver, brain, gut,
kidney, etc. were collected from the animals and analyzed at a
pathology lab. All samples were normal and no inflammation or
tissue abnormalities were observed.
[0118] In another study, left ventricurogram (LVG) was performed in
pigs (n=5) at three time points: (1) before occlusion of left
anterior descending artery by balloon catheter (before occlusion);
(2) immediately after reperfusion with 5 .mu.M/10 mL of
.psi..epsilon.RACK (post .psi..epsilon.RACK); and (3) before
sacrifice five days later (5 days post), using 6 Fr. of pig-tail
catheter. LVG was recorded by two views, right anterior oblique and
left anterior oblique. Ejection fraction (EF), the percent of blood
ejected in a beat, during maximum contraction, of the total maximum
present in the left ventricle, was analyzed by the software, Plus
Plus (Sanders Data Systems), and the averages of two views were
evaluated. Ejection fractions were calculated based on left
ventricle dimensions and the results are shown in FIG. 5. Ejection
fraction is a measure of how well the heart is functioning, with a
higher ejection fraction indicative of a better functioning heart.
An ejection fraction of less than 50% in a short period of time can
suggest progression into a state of heart failure. As seen in FIG.
5, animals treated with .psi..epsilon.RACK (closed triangles) had a
higher ejection fraction after occlusion compared to the control
animals treated with a scrambled peptide (open circles), suggesting
the peptide reduces or prevents damage to the cells and tissue due
to ischemia. This is also evident from the data point at 5 days
post ischemia and treatment, where animals treated with
.psi..epsilon.RACK had an ejection fraction on par with that
measured prior to ischemia and about 10% higher than the untreated
animals. Accordingly, delivery of a .psi..epsilon.RACK peptide in
vivo after ischemia is effective to reduce cell and tissue damage,
as evidenced by an ejection fraction at least 10% greater than that
of untreated cells or tissues.
[0119] III. .psi..epsilon.RACK Peptide
[0120] As used herein, a ".psi..epsilon.RACK peptide" refers to the
peptide represented by SEQ ID NO:2 and to derivatives and fragments
of this peptide. Exemplary derivatives are given in SEQ ID
NOS:6-18, and include the following sequences: HEADIGYD (SEQ ID
NO:6); HDAPIGYE (SEQ ID NO:7); HDAPVGYE (SEQ ID NO:8); HDAPLGYE
(SEQ ID NO:9); HDAPIGDY (SEQ ID NO:10); HDAPIGEY (SEQ ID NO:11);
ADAPIGYD (SEQ ID NO:12); HDGPIGYD (SEQ ID NO:13); HDAAIGYD (SEQ ID
NO: 14), and combinations of these modifications.
[0121] In one preferred embodiment, the sequence "DAPIG" (SEQ ID
NO: 18) in SEQ ID NO:2 is has no more than two modifications at any
residue. One, two, or all three of the residues outside the
sequence "DAPIG" can be modified. For example, AEAPVGEY (SEQ ID
NO:15) is a derivative of SEQ ID NO:2 where all three residues
outside the "DAPIG" (SEQ ID NO: 18) sequence and two residues
within the "DAPIG" sequence are modified. Other examples include
HEAPIGDN (SEQ ID NO:16) and HDGDIGYD (SEQ ID NO:17).
[0122] It will also be appreciated that fragments of SEQ ID NO:2
and of the modifications described above may be suitable. An
exemplary fragment of SEQ ID NO:2 is DAPIG, (SEQ ID NO:18).
[0123] All of these exemplary peptides may be (i) chemically
synthesized or (ii) recombinantly produced in a host cell using,
e.g., an expression vector containing a polynucleotide fragment
encoding said peptide, where the polynucleotide fragment is
operably linked to a promoter capable of expressing mRNA from the
fragment in the host cell.
[0124] The dose of peptide administered will vary depending on the
tissue to be treated and the condition of the patient. Dosages are
readily determined by those of skill in the art based on animal and
human studies. Typically, between 0.05-5 .mu.M, more preferably
between 0.1-2 .mu.M, most preferably between about 0.1-1 .mu.M
peptide is administered. However, the upper and lower limits of
these ranges are merely exemplary.
[0125] The peptide can be administered by any route suitable, as
determine by the primary care provider. For example, administration
by intraveneous, parenteral, subcutaneous, inhalation, intranasal,
sublingual, mucosal, and transdermal, and the like, is
contemplated. Naturally, the route of administration will influence
the dose and timing of administration, as appreciated by those of
skill.
[0126] The peptide can be administered in the form of a fusion
protein or a transport protein conjugate. Typically, to form a
fusion protein, the peptide is bound to another peptide by a bond
other than a Cys-Cys bond. An amide bond from the C-terminal of one
peptide to the N-terminal of the other is exemplary of a bond in a
fusion protein. The second peptide to which the .delta.PKC
agonist/antagonist peptide is bound can be virtually any peptide
selected for therapeutic purposes or for transport purposes. For
example, it maybe desirable to link the .psi..epsilon.RACK peptide
to a cytokine or other peptide that elicites a biological
response.
[0127] Where the peptide is part of a conjugate, the peptide is
typically conjugated to a carrier peptide, such as Tat-derived
transport polypeptide (Vives et al., 1997), polyarginine (Mitchell
et al., 2000; Rolhbard et al., 2000) or Antennapedia peptide by a
Cys-Cys bond. See U.S. Pat. No. 5,804,604. In another general
embodiment, the peptide can be introduced to a cell, tissue or
whole organ using a carrier or encapsulant, such as a liposome in
liposome-mediated delivery.
[0128] It will also be appreciated that .psi..epsilon.RACK as well
as any compound having similar activity can be used in the methods
of treatment described herein. Other compounds, such as peptide
mimetics, chemical compounds, or other peptides, can be identified
by, for example, a screening method set forth in U.S. Pat. No.
6,165,977, and this portion on Col. 14, line 45-Col 15, line 54 is
incorporated by reference herein. In brief, and by way of example
for identifying a compound effective to protect a cell or tissue
from ischemia, .delta.PKC is immobilized inside the wells of a
multiwell plate by introducing a solution containing .delta.PKC
into the plate and allowing the .delta.PKC to bind to the plastic.
The wells may be precoated with substances that enhance attachment
of .delta.PKC and/or that decrease the level of non-specific
binding.
[0129] The plate is then incubated with a blocking solution
(containing, for example bovine serum albumin) and then washed
several times. A solution containing reporter-labelled (e.g.,
radiolabelled of fluorescently-tagged) peptide .psi..epsilon.RACK
(SEQ ID NO:2) and, in the test wells, as opposed to the control
wells, a test compound is added. Different wells may contain
different test compounds or different concentrations of the same
test compound. Each test compound at each concentration is
typically run in duplicate and each assay is typically run with
negative (wells with no test compound) as well as positive (wells
where the "test compound" is unlabeled peptide) controls. The free
peptide is then washed out, and the degree of binding in the wells
is assessed.
[0130] A test compound is identified as active it if decreases the
binding of the peptide, i.e., if its effect on the extend of
binding is above a threshold level. More specifically, if the
decrease in binding is a several-fold different between the control
and experimental samples, the compound would be considered as
having binding activity. Typically, a 2-fold or 4-fold threshold
difference in binding between the test and control samples is
sought.
[0131] Detection methods useful in such assays include
antibody-based methods, direct detection of a reporter moiety
incorporated into the peptide, such as a fluorescent label, and the
like.
[0132] A variety of test compounds may be screened, including other
peptides, macromolecules, small molecules, chemical and/or
biological mixtures, fungal extracts, bacterial extracts or algal
extracts. The compounds can be biological or synthetic in
origin.
[0133] IV. Utility and Routes of Administration
[0134] The present invention has application, for example, in
treatment of surviving heart attack victims, as well in treatment
of persons who presently die from heart disease after admission to
the hospital. Delivery of the .epsilon.PKC selective peptide
agonist, .psi..epsilon.RACK, is valuable in the management of these
patients, both acutely and chronically.
[0135] Acutely, in patients brought to hospital with impending
infarction, medical care has traditionally been directed towards
removing the cause of coronary occlusion either by thrombolytics or
by catheter angioplasty. However, reperfusion of the damaged areas
can be one of the major mechanisms of myocardial cellular injury.
Administration of a .psi..epsilon.RACK peptide PKC agonist
delivered to the site of occlusion by catheter or injected
intravenously to induce cardioprotection immediately before or
concurrently with thrombolysis or angioplasty is contemplated by
the invention.
[0136] Chronically, in patients with angina, the current medical
approach is to stop the symptoms of angina without replacement of
angina's preconditioning protective effect. A.epsilon.PKC selective
agonist, such as .psi..epsilon.RACK, can replace the
preconditioning effect induced by angina in these patients and
offer a higher rate of myocardial salvage during future episodes of
more severe ischemia.
[0137] Additional uses of the invention include clinical situations
in which the timing of ischemia is physician-controlled. In such
instances, pharmacologic enhancement of the preconditioning
response would provide a significant advantage to the patients
undergoing treatment. Specifically, each year, in the United States
alone, 600,000 adults and 12,000 children undergo open heart
operations utilizing cardiopulmonary bypass, during which the heart
is subjected to periods of controlled ischemia ranging from several
minutes to well over one hour. Despite advances in cardiac
protection, myocardial dysfunction during the immediate
post-operative period remains a leading cause of morbidity and
mortality in these patients. The exact timing of the ischemic
insult is known ahead of time in these patients, allowing for
administration of a .psi..epsilon.RACK peptide prior to ischemia.
Administration of .psi..epsilon.RACK will reduce myocardial damage
by inducing a preconditioning response in the hours, or days, prior
to surgery.
[0138] Similar benefits could be realized in the area of cardiac
transplantation, of which there are approximately 2500 cases
annually in the U.S. Prolonged graft ischemia is one of the factors
limiting long-distance donor organ acquisition for such cardiac
transplantation. Administration of .psi..epsilon.RACK peptide at
the time of organ procurement could extend the time between organ
harvest and implantation and reduce the risk of post-operative
myocardial dysfunction.
[0139] It will, of course, be understood that a .psi..epsilon.RACK
peptide may employed in the treatment of a variety of ischemic and
hypoxic conditions, in addition to cardiac ischemia. For example,
.psi..epsilon.RACK may be administered prior to, during or after an
ischemic or hypoxic event to a wide variety of cells and tissues.
Without intending to be limiting, examples include the kidney, the
vascular endothelium, the liver, the eye, and in the central
nervous system, where tissue damage to the brain and other tissues
of the central nervous system may result due to stroke.
[0140] As demonstrated by the studies described herein, the peptide
can be administered before, during or after an ischemic or hypoxic
event. When delivered before an ischemic insult, the peptide
effectively reduces the extent of cellular damage. Preferably, the
peptide is perfused over or through the tissue for about 1-180
minutes, more preferably for about 1-120 minutes, most preferably
for about 1-60 minutes, prior to the ischemic insult. In one
embodiment, a period of time not greater than about three hours,
more preferably not greater than about 120 minutes, and most
preferably not greater than about 60 minutes, lapses between
cessation of peptide perfusion and the ischemic or hypoxic event.
The peptide can be delivered for a 1 minute, 2 minute, 5 minute, 10
minute, 20 minute, 30 minute, or longer, period of time prior to
the ischemic insult.
[0141] When the peptide is delivered subsequent to an ischemic
event, in a preferred embodiment a period of time not greater than
about two hours, more preferably not greater than one hour, and
even more preferably not greater than 30 minutes lapses between the
ischemic event and initiation of administration of the peptide. The
peptide can be delivered for a 1 minute, 2 minute, 5 minute, 10
minute, 20 minute, 30 minute, or longer, period of time following
the ischemic insult.
[0142] The peptide can also be administered during an ischemic
event. Particularly, during time of controlled ischemia, such as
during surgery, the care provider can initiate administration of
the .psi..epsilon.RACK peptide just prior to or concurrent with
initiation of the ischemic event.
IV. EXAMPLES
[0143] The following examples further illustrate the invention
described herein and are in no way intended to limit the scope of
the invention.
[0144] Methods
[0145] 1. Peptide Preparation
[0146] .psi..epsilon.RACK (HDAPIGYD, SEQ ID NO:2) was synthesized
and purified (>95%) at the Stanford Protein and Nucleic Acid
Facility. Scrambled .psi..epsilon.RACK peptide (PDYHDAGI, SEQ ID
NO:4) was prepared similarly. In some studies, the peptides were
modified with a carrier peptide by cross-linking via an N-terminal
Cys-Cys bond to the Drosophila Antennapedia homeodomain-derived
peptide (C-RQIKIWFQNRRMKWKK, SEQ ID NO:3; Thodore, L. et al., 1995;
Johnson, J. A. et al., 1996a) or via an N-terminal Cys-Cys bond to
Tat protein-derived peptide (C-YGRKKRRQRRR, SEQ ID NO:6).
Example 1
Ex Vivo Administration of .psi..epsilon.RACK Prior to Ischemia
[0147] Mice or rats were anesthetized with i.p. avertin, and their
hearts were rapidly removed and cannulated via the aorta for
reperfasion as described in the art (Colbert et al, 1997). Care was
taken to have the hearts perfused within 90 seconds of removal. The
left ventricular pressure and real-time derivative (dP/dt) were
monitored via a catheter placed in the ventricular apex.
Hemodynamic parameters were archived every 20 seconds throughout
the procedure. The hearts were perfused with oxygenated
Krebs-Henseleit solution comprised of, in nmol/L, NaCl 120; KCl
5.8; NaHCO.sub.3 25; NaH.sub.2O.sub.4 1.2; MgSO.sub.4 1.2;
CaCl.sub.2 1.0; and dextrose 10, pH 7.4 at 37 C.
[0148] After a 10-20 minute equilibration period, the hearts were
treated with .psi..epsilon.RACK peptide (SEQ ID NO:2) or with
scrambled .psi..epsilon.RACK peptide (SEQ ID NO:4) for 20 minutes.
Perfusion was maintained at a constant flow of 10 mL/min with
Krebs-Henseleit solution containing 0.5 .mu.M of the appropriate
peptide. The Langendorff method employed used retrograde flow from
the ventricle to the aorta and into the coronary arteries,
bypassing the pulmonary arteries.
[0149] To induce global ischemia, flow was interrupted for 45
minutes. After the ischemic event, the hearts were reperfused with
Krebs-Henseleit solution for 30-160 minutes. During reperfusion,
ischemia-induced cell damage was determined by measuring the
activity of creatine phosphokinase (CPK) (absorbance at 520 nm) in
the perfusate using a Sigma kit. The results are shown in FIGS.
1A-1B.
Example 2
Ex Vivo Administration of .psi..epsilon.PKC After to Ischemia
[0150] Rat hearts were prepared as described in Example 1. After a
30 minute equilibration period, global ischemia was induced by
interrupting fluid flow for 45 minutes. The hearts were then
reperfused with 0.5 .mu.M of .psi..epsilon.RACK peptide for 20
minutes, followed by 40 minutes of reperfusion without the peptide.
As a control, some hearts were left untreated after ischemia.
During the 60 minute period following ischemia, ischemia-induced
cell damage was determined by monitoring the creatine phosphokinase
(CPK) activity (absorbance at 520 nm) in the perfusate collected
during reperfusion. The results are shown in FIGS. 3A-3B.
Example 3
In Vivo Administration of .psi..epsilon.PKC Prior to Ischernia
[0151] Young adult female pigs, 35-40 kg in weight, were
anesthetized and a catheter was introduced through the carotid
artery into the heart. Using conventional intervention cardiology
techniques, a wire was placed through a catheter and into the left
anterior descending artery (LAD). A balloon was run over this wire
to a site of occlusion where it was then inflated to block blood
flow for 30 minutes. During the first 10 minutes of the ischemic
insult, either the control scrambled .psi..epsilon.RACK peptide
(SEQ ID NO:4, n=5) or the biologically active .psi..epsilon.RACK
peptide (SEQ ID NO:2, n=5) was delivered by diffusion through the
balloon directly downstream of the occlusion. Approximately 20
.mu.g peptide (.about.400 ng per kg body weight) was
administered.
[0152] After 30 minutes of occlusion, the balloon was removed and
pigs were left to recover from surgery. Five days later, the pigs
were euthanized and hearts were harvested. After heart removal, the
LAD was occluded. With the occlusion in place, Evans Blue dye,
which stains all areas not at risk of infarct in blue while leaving
all areas with no access to blood flow red, was infused. Hearts
were then cut into slices and stained with a tetrazolium red dye
which stains all live areas red and infarcted dead tissue in white.
Each heart had multiple tissue slices with distinctive areas
marking the area at risk for ischemia and the infarcted area. From
this the percent infarct per area at risk for each slice and for
the entire heart was determined. The results are shown in FIGS.
4A-4C.
[0153] Although the invention has been described with respect to
particular embodiments, it will be apparent to those skilled in the
art that various changes and modifications can be made without
departing from the invention.
Sequence CWU 1
1
18 1 8 PRT Artificial Sequence epsilon V1-2, residues 14-21 of
epsilon-PKC 1 Glu Ala Val Ser Leu Lys Pro Thr 1 5 2 8 PRT
Artificial Sequence pseudo-epsilon RACK octapeptide 2 His Asp Ala
Pro Ile Gly Tyr Asp 1 5 3 16 PRT Artificial Sequence Drosophila
antennapedia homeodomain-derived carrier peptide 3 Arg Gln Ile Lys
Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 4 8 PRT
Artificial Sequence scrambled pseudo-epsilon RACK octapeptide 4 Pro
Asp Tyr His Asp Ala Gly Ile 1 5 5 11 PRT Artificial Sequence
Tat-derived carrier peptide 5 Tyr Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg 1 5 10 6 8 PRT Artificial Sequence modified pseudo-epsilon
RACK peptide 6 His Glu Ala Asp Ile Gly Tyr Asp 1 5 7 8 PRT
Artificial Sequence modified pseudo-epsilon RACK peptide 7 His Asp
Ala Pro Ile Gly Tyr Glu 1 5 8 8 PRT Artificial Sequence modified
pseudo-epsilon RACK peptide 8 His Asp Ala Pro Val Gly Tyr Glu 1 5 9
8 PRT Artificial Sequence modified pseudo-epsilon RACK peptide 9
His Asp Ala Pro Leu Gly Tyr Glu 1 5 10 8 PRT Artificial Sequence
modified pseudo-epsilon RACK peptide 10 His Asp Ala Pro Ile Gly Asp
Tyr 1 5 11 8 PRT Artificial Sequence modified pseudo-epsilon RACK
peptide 11 His Asp Ala Pro Ile Gly Glu Tyr 1 5 12 8 PRT Artificial
Sequence modified pseudo-epsilon RACK peptide 12 Ala Asp Ala Pro
Ile Gly Tyr Asp 1 5 13 8 PRT Artificial Sequence modified
pseudo-epsilon RACK peptide 13 His Asp Gly Pro Ile Gly Tyr Asp 1 5
14 8 PRT Artificial Sequence modified pseudo-epsilon RACK peptide
14 His Asp Ala Ala Ile Gly Tyr Asp 1 5 15 8 PRT Artificial Sequence
modified pseudo-epsilon RACK peptide 15 Ala Glu Ala Pro Val Gly Glu
Tyr 1 5 16 8 PRT Artificial Sequence modified pseudo-epsilon RACK
peptide 16 His Glu Ala Pro Ile Gly Asp Asn 1 5 17 8 PRT Artificial
Sequence modified pseudo-epsilon RACK peptide 17 His Asp Gly Asp
Ile Gly Tyr Asp 1 5 18 5 PRT Artificial Sequence pseudo-epsilon
RACK peptide fragment 18 Asp Ala Pro Ile Gly 1 5
* * * * *