U.S. patent application number 10/442671 was filed with the patent office on 2004-04-22 for method and composition for inhibiting cardiovascular cell proliferation.
Invention is credited to Cooke, John P., Fathman, Garrison C., Kown, Murray H., Robbins, Robert C., Rothbard, Jonathan B., Uemura, Shiro.
Application Number | 20040074504 10/442671 |
Document ID | / |
Family ID | 22479193 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040074504 |
Kind Code |
A1 |
Cooke, John P. ; et
al. |
April 22, 2004 |
Method and composition for inhibiting cardiovascular cell
proliferation
Abstract
Cardiovascular cell proliferation in a blood vessel subjected to
trauma, such as angioplasty, vascular graft, anastomosis, or organ
transplant, can be inhibited by contacting the vessel with a
polymer consisting of from 6 to about 30 amino acid subunits, where
at least 50% of the subunits are arginine, and the polymer contains
at least six contiguous arginine subunits. Exemplary polymers for
this purpose include arginine homopolymers 7 to 15 subunits in
length.
Inventors: |
Cooke, John P.; (Palo Alto,
CA) ; Fathman, Garrison C.; (Portola Valley, CA)
; Rothbard, Jonathan B.; (Woodside, CA) ; Uemura,
Shiro; (Nara, JP) ; Robbins, Robert C.;
(Stanford, CA) ; Kown, Murray H.; (Menlo Park,
CA) |
Correspondence
Address: |
REED & EBERLE LLP
800 MENLO AVENUE, SUITE 210
MENLO PARK
CA
94025
US
|
Family ID: |
22479193 |
Appl. No.: |
10/442671 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10442671 |
May 20, 2003 |
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09587647 |
Jun 5, 2000 |
|
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6605115 |
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60137826 |
Jun 5, 1999 |
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Current U.S.
Class: |
128/898 ;
623/916 |
Current CPC
Class: |
A61K 38/03 20130101;
A61K 38/08 20130101; A61K 38/16 20130101; A61L 27/227 20130101;
A61L 33/12 20130101; A61P 9/14 20180101; A61P 9/00 20180101; A61P
7/04 20180101; A61L 27/34 20130101; A61P 43/00 20180101; A61K 38/10
20130101; A61L 33/128 20130101; A61L 27/34 20130101; C08L 77/04
20130101 |
Class at
Publication: |
128/898 ;
623/916 |
International
Class: |
A61F 002/06 |
Goverment Interests
[0002] This invention was made with the support of NIH grant number
CA 65237. Accordingly, the U.S. Government may have certain rights
in this invention.
Claims
It is claimed:
1. A method for inhibiting trauma-induced intimal hyperplasia in a
blood vessel, comprising: contacting with said vessel, in a
pharmaceutically acceptable vehicle, a polymer consisting of from 6
to about 30 amino acid subunits, wherein at least 50% of said
subunits are arginine, and said polymer contains at least six
contiguous arginine subunits.
2. The method of claim 1, wherein at least 70% of the subunits in
the polymer are arginine.
3. The method of claim 1, wherein at least 90% of the subunits in
the polymer are arginine.
4. The method of claim 1, wherein no arginine subunit is separated
from another such subunit by more than one non-arginine
subunit.
5. The method of claim 1, wherein the arginine subunits are
L-arginine.
6. The method of claim 1, wherein non-arginine subunits are natural
or unnatural amino acid subunits which do not significantly reduce
the rate of membrane transport of the polymer.
7. The method of claim 6, wherein said non-arginine subunits are
selected from the group consisting of glycine, alanine, cysteine,
valine, leucine, isoleucine, methionine, serine, threonine,
.alpha.-amino-.beta.-guanidino- propionic acid,
.alpha.-amino-.gamma.-guanidinobutyric acid, and
.alpha.-amino-.epsilon.-guanidinocaproic acid.
8. The method of claim 1, wherein said polymer is an arginine
homopolymer.
9. The method of claim 8, wherein said polymer is an L-arginine
homopolymer.
10. The method of claim 8, wherein said polymer contains 7 to 15
arginine residues.
11. The method of claim 1, wherein the trauma comprises an incision
to the vessel, excessive or prolonged pressure applied to the
vessel, transplant of an organ containing the vessel, or a
combination thereof.
12. The method of claim 1, wherein the vessel is a vascular conduit
or patch to be grafted into or onto an endogenous vessel.
13. The method of claim 11, wherein the vessel is an endogenous
vessel receiving a graft.
14. The method of claim 11, wherein the vessel is a vein undergoing
an arterial venous anastomosis procedure for the purpose of
dialysis.
15. The method of claim 11, wherein the vessel is subjected to
angioplasty.
16. The method of claim 11, wherein the vessel is contained within
a transplanted organ.
17. The method of claim 16, wherein said contacting comprises
immersion of the organ in a solution of the polymer.
18. A method of preparing a vascular conduit for a vascular graft
procedure, comprising: contacting an isolated vessel conduit with
an arginine polymer as recited in claim 1, in a pharmaceutically
acceptable vehicle, for a time sufficient for the polymer to be
transported into the wall of the vessel conduit, such that the
level of the polymer in the conduit wall is effective to reduce the
level of post-graft intimal hyperplasia in and/or adjacent to the
conduit, relative to the level of post-graft intimal hyperplasia
that would occur in the absence of such contacting with the
polymer.
19. The method of claim 18, wherein said contacting is limited to
contacting the polymer-containing liquid with the interior of the
vessel conduit.
20. The method of claim 18, wherein the polymer is an arginine
homopolymer.
21. The method of claim 20, wherein the polymer is an L-arginine
homopolymer.
22. The method of claim 20, wherein the polymer contains 7 to 15
arginine residues.
23. An isolated vessel conduit comprising, within the wall of the
conduit, a polymer as recited in claim 1, present at a level
effective to reduce the level of post-graft intimal hyperplasia in
and/or adjacent to the conduit, relative to the level of post-graft
intimal hyperplasia that would occur in the absence of the
polymer.
24. The vessel conduit of claim 23, wherein the polymer is an
arginine homopolymer.
25. The vessel conduit of claim 24, wherein the polymer is an
L-arginine homopolymer.
26. The vessel conduit of claim 24, wherein the polymer contains
from 7 to 15 arginine residues.
27. A method of increasing NO production in a vascular cell or
tissue, comprising contacting with said cell or tissue, a polymer
as recited in claim 1, in a pharmaceutically acceptable
vehicle.
28. The method of claim 27, wherein said polymer is an L-arginine
homopolymer containing 7 to 15 L-arginine residues.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 09/587,647, filed Jun. 5, 2000, which claims priority to U.S.
Provisional Application Serial No. 60/137,826, filed Jun. 5, 1999,
which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to methods and compositions
for inhibiting cardiovascular cell proliferation. The invention
provides methods for improving the longevity and quality of
arterial grafts, for enhancing vascular NO production, and for
reducing post-graft intimal hyperplasia, stenosis, and
restenosis.
BACKGROUND OF THE INVENTION
[0004] Myointimal hyperplasia is a vascular response to injury that
contributes to the development of vein graft disease, restenosis
after angioplasty, and atherosclerosis (Motwani et al. (1998)
Circulation 97:916-931). Myointimal hyperplasia involves the
migration and proliferation of vascular smooth muscle cells (VSMC)
as well as the elaboration of extracellular matrix in the intima
(Demeyer et al. (1997) Vasc. Med. 2:179-189; Kraiss et al.,
"Response of the Arterial Wall to Injury and Intimal Hyperplasia,"
in The Basic Science of Vascular Disease, Surnpio et al., Eds.,
Futura Publishing, NY, N.Y., Pp 317 (1997)). Vascular nitric oxide
(NO), an endogenous regulator of vascular function, opposes the
development of myointima formation by inhibiting VSMC proliferation
and by inducing VSMC apoptosis (Cooke et al. (1997) Ann. Rev. Med.
48:489-509; Best et al. (1999) Arterioscler. Thromb. Vasc. Biol.
19:14-22). Failure of endogenous biological processes to control
myointimal hyperplasia can lead to formation of vascular occlusions
which seriously compromise tissue function.
[0005] Autologous vein grafting constitutes a major tool in
coronary bypass procedures. About 400,000 to 500,000 first-time
coronary graft procedures are performed every year in the United
States alone. Although patient survival rates exceed 90% over the
first five years after treatment, about 20% to 40% of the grafts
fail during this time due to occlusive phenomena. Thus,
80,000-100,000 graft replacement procedures are needed in the U.S.
yearly to avoid premature mortality. Vascular occlusive phenomena
also lead to failures in other vascular grafts, such as
arterial-venous anastomosis used for kidney dialysis, and in organ
transplants.
[0006] In light of the significant costs to patients and insurers
engendered by repeated graft procedures, there is a need to improve
the longevity and quality of first-time vascular grafts. Ideally,
such a procedure should be simple to carry out, without requiring
extensive manipulation or lengthy processing. Furthermore, the
procedure preferably involves materials that are relatively easy to
prepare in therapeutically effective forms.
SUMMARY OF THE INVENTION
[0007] In one aspect, the invention provides a method for
inhibiting trauma-induced intimal hyperplasia in a blood vessel. In
accordance with the method, a polymer consisting of from 6 to about
30 amino acid subunits, wherein at least 50% of the subunits are
arginine, and containing at least six contiguous arginine subunits,
contained in a pharmaceutically acceptable vehicle, is contacted
with the vessel, typically with the interior of the vessel. Such
contacting is effective to reduce the level of intimal hyperplasia
in and/or adjacent to the vessel, relative to the level of intimal
hyperplasia that would occur in the absence of the contacting.
[0008] The hyperplasia-inducing trauma may comprise an incision to
the vessel, excessive or prolonged pressure applied to the vessel,
transplant of an organ containing the vessel, or a combination
thereof. The contacting may occur prior to the trauma (as in
preparation of a vessel segment for grafting), concurrent with, or
following the trauma (as in an angioplasty procedure). The vessel
may be a vessel conduit to be grafted into (as in a bypass
procedure) or onto (as in an anastomosis) an endogenous vessel, or
it can be an endogenous vessel receiving a graft. Also included are
vein "patches" used in arterial repair. In preferred embodiments,
the above noted procedures take place in a human subject.
[0009] The invention provides, for example, a method for repairing
an arterial vessel site in a human subject. Accordingly, an
isolated vessel conduit, such as a saphenous vein segment or an
internal mammary artery segment, is contacted with a polymer as
described above, in a pharmaceutically acceptable vehicle, and the
vessel conduit is then grafted into a selected arterial vessel site
in need of repair.
[0010] In one embodiment, the vessel is a vein which undergoes an
arterial venous anastomosis procedure for the purpose of dialysis.
In another embodiment, the vessel is subjected to angioplasty. In a
further embodiment, the vessel is contained within a transplanted
organ, such as, for example, a heart or kidney, where the
contacting is preferably carried out by immersion of the organ in a
solution of the polymer.
[0011] Preferably, at least 70%, and more preferably at least 90%,
of the subunits in the polymer are arginine. When non-arginine
subunits are present, preferably no arginine subunit is separated
from another arginine subunit by more than one non-arginine
subunit. The non-arginine subunits are preferably amino acid
subunits which do not significantly reduce the rate of membrane
transport of the polymer. In preferred embodiments, the arginine
subunits are L-arginine. In particularly preferred embodiments, the
polymer is an arginine homopolymer, preferably containing 7 to 15
arginine residues.
[0012] Also provided is an isolated vessel conduit, comprising,
within the wall of the conduit, a polymer as described above,
present at a level effective to reduce the level of post-graft
intimal hyperplasia in and/or adjacent to the conduit, relative to
the level of post-graft intimal hyperplasia that would occur in the
absence of the polymer. The vessel conduit may be a venous or
arterial segment, or it may be an artificial vessel segment made
from a physiologically compatible material.
[0013] The invention also provides a method of preparing a vascular
conduit for a vascular graft procedure, wherein an isolated vessel
conduit, preferably the interior of the conduit, is contacted with
an arginine polymer as described above, in a pharmaceutically
acceptable vehicle, for a time sufficient for the polymer to be
transported into the wall of the vessel conduit to a level
effective to reduce post-graft intimal hyperplasia in and/or
adjacent to the conduit, relative to the level of post-graft
intimal hyperplasia that would occur in the absence of such
contacting with the polymer. Preferred embodiments of the polymer
are as described above.
[0014] The invention also provides a method of increasing NO
production in a vascular cell or tissue, by contacting a polymer
consisting of from 6 to about 30 amino acid subunits, wherein at
least 50% of the subunits are L-arginine, and containing at least
six contiguous arginine subunits, in a pharmaceutically acceptable
vehicle, with the cell or tissue.
[0015] In vascular tissue, the polymers as described above are
shown to translocate through the vascular wall and into the
cytoplasm and nuclei of vascular cells. In addition to their
utility in inhibiting myointimal hyperplasia, the oligomers are
useful as transporters of vascular therapeutics.
[0016] These and other objects and features of the invention will
become more fully apparent when the following detailed description
of the invention is read in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1A-F show translocation of biotin labeled
heptaarginine (R7) into cultured rat vascular smooth muscle cells
(VSMC). Each figure is representative of three separate
experiments. The cells were treated for 30 minutes with
biotin-labeled heptalysine (bK7; 10 .mu.M; FIG. 1A) and
biotin-labeled heptaarginine (bR7) at 0.1 .mu.M (FIG. 1B) and 10
.mu.M (FIG. 1C). Note intense staining in the nuclei and cytoplasm
of all cells in FIGS 1B-C. FIG. 1D shows bR7 translocation at
4.degree. C.; there is no apparent reduction in efficacy of
transport at this temperature. FIG. 1E shows inhibition of bR7
translocation by sodium azide, indicating that the transport
process is energy-dependent. FIG. 1F shows lack of inhibition of
bR7 translocation by addition of 10 mM free L-arginine to the
medium, indicating that the y+ transporter is not involved in
polymer uptake.
[0018] FIGS. 2A-C show dose, incubation time, and temperature
dependence of R7 translocation in rabbit carotid artery segment.
The magnitude of R7 translocation is expressed as percent stained
nuclei (stained nuclei/total nuclei). (FIG. 2A) Dose dependence of
bR7 translocation. (FIGS. 2B-C) Time dependence of bR7
translocation at 4.degree. C. and 37.degree. C., in intimal cells
and medial cells, respectively. Data are from 4 independent
experiments. *, p<0.5.
[0019] FIGS. 3A-B show the time course of disappearance of
internalized biotin signal in bR7 treated artery segments; (FIG.
3A) intimal cells, (FIG. 3B) medial cells. Vascular segments
exposed to biotin-labeled L-R7 or D-R7 (10.0 .mu.M) were incubated
in serum containing medium for up to 5 days. The magnitude of R7
translocation is expressed as percent stained nuclei. Data are from
4 independent experiments. *, p<0.05.
[0020] FIGS. 4A-B show dependence of NO biosynthesis on cytokine
concentration and L-arginine availability. In FIGS. 4-7,
extracellular NO production was measured as its stable oxidative
metabolite, NO.sub.2. (FIG. 4A) Effect of IFN-.gamma. dose on NO
production by rat VSMC, incubated in medium containing 400 .mu.M
free L-arginine, stimulated with a mixture of IFN-.gamma. (100
U/ml) and LPS (100 .mu.g/ml). (FIG. 4B) Effect of extracellular
free L-arginine on NO production. NO.sub.2 accumulation in the
culture medium was quantified after 24 hours. NO.sub.2 production
was corrected as 10.sup.5 cells.
[0021] FIG. 5 shows the effect of arginine oligomers on NO
synthesis in cytokine-stimulated VSMC. VSMCs were pretreated with
arginine oligomers for 30 minutes, and then stimulated with a
mixture of IFN-.gamma. (100 U/ml) and LPS (100 .mu.g/ml). NO
production is expressed as a percentage of that observed in vehicle
treated cytokine-stimulated cells. R5, penta-L-arginine; R7,
hepta-L-arginine; R9, nona-L-arginine; D-r7, hepta-D-arginine; K7,
hepta-L-lysine. *; p<0.05 vs. vehicle treated cells.
[0022] FIG. 6 shows the effect of N-terminal acetylation of the
arginine oligomers on NO production. Rat VSMCs were pretreated with
each polymer for 30 minutes, then stimulated with a mixture of
IFN-.gamma. (100 U/ml) and LPS (100 .mu.g/ml). NO production is
expressed as a percentage of that observed in vehicle treated
cytokine-stimulated cells. *; p<0.05 vs. vehicle treated
cells.
[0023] FIG. 7 shows the effect of L-NMMA on NO production from
arginine oligomer treated VSMC. NO production is expressed as a
percentage of that observed in vehicle treated cytokine-stimulated
cells. *; p<0.05 vs. vehicle treated cells.
[0024] FIGS. 8A-C show representative photomicrographs of cross
sections of (FIG. 8A) vehicle treated, (FIG. 8B) L-R7 treated (10.0
.mu.M), and (FIG. 8C) D-R7 treated (10.0 .mu.M) vein grafts
harvested on the 28th day postsurgery. Arrowheads indicate the
internal elastic lamina. (Hernatoxylin Eosin staining, X100,
bars=100 .mu.m).
[0025] FIGS. 9A-D show planimetric measurements of vein graft
segments harvested on the 28th day after surgery. (FIG. 9A) luminal
area; (FIG. 9B) medial area; (FIG. 9C) intimal area; (FIG. 9D) I/M
ratio. I/M represents the ratio of intima to media area. Each
experimental group was composed of 6 animals. *, p<0.05.
[0026] FIG. 10A shows NOx (nitrate and nitrite) production measured
from vein graft segments harvested 3 days after surgery. Graft
segments were incubated in medium in either the absence (basal) or
presence (stimulated) of calcium ionophore. Each experimental group
is composed of 3 vessel segments. *, p<0.05. FIG. 10B shows
NO-independent effects of heptaarginines on cell proliferation. Rat
VSMC were incubated with either L-R7 or D-r7 (10.0 .mu.M, 30
minutes), then incubated with growth medium containing 0.5% FBS for
48 hours. Cell proliferation was measured using XTT assay; percent
OD of each treatment group to group treated with serum free medium
(DSF) was calculated. Data are from 6 experiments. *, p<0.05 vs.
growth medium (GM) control group.
DETAILED DESCRIPTION OF THE INVENTION
[0027] I. Definitions
[0028] The term "poly-arginine" or "poly-Arg" refers to a polymeric
sequence composed of contiguous arginine residues; poly-L-arginine
refers to all L-arginines; poly-D-arginine refers to all
D-arginines. Poly-L-arginine is also abbreviated by an upper case
"R" followed by the number of L-arginines in the peptide (e.g., R8
represents an 8-mer of contiguous L-arginine residues).
Poly-D-arginine is abbreviated by a lower case "r" followed by the
number of D-arginines in the peptide (r8 represents an 8-mer of
contiguous D-arginine residues). "Ac" indicates a sequence having
an acetylated N-terminal residue (e.g. AcR7), while "b" indicates a
sequence having a biotinylated N-terminal residue (e.g. bR7).
[0029] An "arginine polymer" (or "oligomer"), as used herein,
refers to an arginine homopolymer or a peptide copolymer in which
arginine is the major component (at least 50%, preferably at least
70%, and more preferably at least 90% arginine). An "arginine
homopolymer", wherein all residues are arginine, may contain a
mixture of L-arginine and D-arginine residues.
[0030] A "vessel" as used herein refers to a blood vessel or any
segment thereof, including a segment used as a vascular patch.
[0031] Amino acid residues are referred to herein by their full
names or by standard single-letter or three-letter notations: A,
Ala, alanine; C, Cys, cysteine; D, Asp, aspartic acid; E, Glu,
glutamic acid; F, Phe, phenylalanine; G, Gly, glycine; H, His,
histidine; 1, Ile, isoleucine; K, Lys, lysine; L, Leu, leucine; M,
Met, methionine; N, Asn, asparagine; P, Pro, proline; Q, Gln,
glutamine; R, Arg, arginine; S, Ser, serine; T, Thr, threonine; V,
Val, valine; W, Trp, tryptophan; X, Hyp, hydroxyproline; Y, Tyr,
tyrosine.
[0032] II. Arginine Polymers
[0033] The present invention utilizes arginine homopolymers, or
copolymers having arginine as their major component, that are
efficiently transported into vascular tissues, and that reduce
intimal hyperplasia when treated vessel segments, such as arterial
segments, venous segments, or artificial vessel conduits, are
grafted into vascular sites in need of repair. The polymers are
also useful in reducing post-transplant hyperplasia, such as GCAD
(graft coronary artery disease), and inhibiting intimal hyperplasia
in vein segments used as "patches", e.g. for arterial walls damaged
during edarterectomy for atherosclerotic plaques. The polymers are
also useful for reducing hyperplasia in vessels subjected to high
pressures, as in angioplasty or blood dialysis.
[0034] The arginine polymer or copolymer contains at least 6, more
preferably at least 7, and up to 30 amino acid residues, where at
least 50% of these residues are arginine, and at least 6 contiguous
residues are arginine. Preferably, at least 70%, and more
preferably at least 90%, of the residues are arginine. Non-arginine
residues, if present, are amino acid subunits (including unnatural
amino acids) which do not significantly reduce the rate of membrane
transport of the polypeptide. These are preferably selected from
the group consisting of glycine, alanine, cysteine, valine,
leucine, isoleucine, methionine, serine, and threonine, and more
preferably selected from the group consisting of glycine, alanine,
cysteine, and valine. Non-naturally occurring amino acids which are
homologs of arginine may also be used. These include
.alpha.-amino-.beta.-guanidinopropionic acid,
.alpha.-amino-.gamma.-guani- dinobutyric acid, or
.alpha.-amino-.epsilon.-guanidinocaproic acid (containing 2, 3 or 5
linker atoms, respectively, between the backbone chain and the
central guanidinium carbon).
[0035] In a preferred embodiment, all of the residues are arginine.
In further embodiments, the polymer is an arginine homopolymer
containing 6 to 25, more preferably 6 to 15, and most preferably 7
to 10 arginine residues. The arginine residues may be
L-stereoisomers, D-stereoisomers, or a combination thereof.
Preferably, all of the arginine residues have the
L-configuration.
[0036] The terminal ends of the arginine polymer can be capped or
uncapped. Preferably, the terminal ends are uncapped, meaning that
the N-terminus has a free amino group and the C-terminus has a free
carboxylic acid. However, the polymer can be capped at either or
both terminal ends with selected terminal moieties, if desired,
provided that the capping groups do not adversely affect the
therapeutic benefits of the polymer. For example, the N-terminus
can be capped with an N-acetyl, N-methyl, N-dimethyl, N-ethyl,
N-diethyl, N-Boc, N-benzyl group, or the like. Similarly, the
C-terminus can be capped with an amino group of the form NR.sub.2
(free amino, alkylamino, or dialkylamino) to form a terminal amide
moiety (CONR.sub.2), wherein each R group is separately H or a
linear, cyclic or branched C.sub.1-C.sub.10 alkyl group, preferably
C.sub.1-C.sub.5 alkyl, and more preferably C.sub.1-C.sub.2 alkyl);
or an alkyl alcohol of the form OR, to form a carboxylic acid ester
(CO.sub.2R), wherein R is a linear, cyclic or branched
C.sub.1-C.sub.10 alkyl group, preferably C.sub.1-C.sub.5 alkyl, and
more preferably C.sub.1-C.sub.2 alkyl, or the like. Preferably,
such N- and C-capping groups contain no more than 20 carbon atoms,
and preferably no more than 10 carbon atoms.
[0037] In one embodiment, the polymer has the formula
X-Arg.sub.n-Y, wherein X is NH.sub.2 or an N-terminal capping
group, Y is COO.sup.- or a C-terminal capping group, and n is an
integer from 6 to 30, such that the Arg residues are L-arginine
residues, D-arginine residues, or combinations thereof. In selected
embodiments, n is an integer from 6 to 30, 7 to 30, 6 to 25, 7 to
25, 6 to 15, 7 to 15, 6 to 10, or 7 to 10. Preferably, the arginine
residues all have an L-configuration.
[0038] The polymers formula also include all protonated variants
that may occur, with associated counterions. The arginine polymer
may be provided as a pharmaceutically acceptable salt with one or
more counterions, such as phosphate, sulfate, chloride, acetate,
propionate, fumarate, maleate, succinate, citrate, lactate,
palmitate, cholate, mucate, glutamate, camphorate, glutarate,
phthalate, tartrate, laurate, stearate, salicylate,
methanesulfonate, benzenesulfonate, sorbate, picrate, benzoate,
cinnamate, and the like.
[0039] The arginine polymers can be prepared by any method
available in the art. Conveniently, the polymers are produced
synthetically, e.g., using a peptide synthesizer (PE Applied
Biosystems Model 433) (See Example 1), or they can be synthesized
recombinantly using a biological expression system by methods well
known in the art.
[0040] For use in transport of biological agents, the polymers of
the invention may be conjugated to compounds to be transported, by
methods known in the art. Exemplary methods of synthesis, including
incorporation of cleavable linkers, exemplary classes of biological
agents to be transported, and methods and formulations for
administration, are described in copending and co-owned application
Ser. No. 09/083,259, entitled "Method and Composition for Enhancing
Transport Across Biological Membranes", which is incorporated
herein by reference in its entirety.
[0041] III. Transport of Arginine Polymers Across Vascular Cell
Membranes
[0042] In the studies described below, transmembrane transport and
cellular uptake of ofigomers was assessed by incubating cells or
tissue with biotin-oligomer conjugates, followed by treatment with
horseradish peroxidase (HRP)-conjugated streptavidin and subsequent
incubation with DAB (3,3'diaminobenzidine), a substrate of the
enzyme which produces a highly colored product.
[0043] A. Translocation of Arginine Polymers into Cultured VSMC and
Endothelial Cells
[0044] Cultured VSMC (vascular smooth muscle cells) were treated,
as described in Example 2, with biotinylated oligomers of arginine
or lysine. After incubation, the cells were treated with
HRP-streptavidin conjugate, then with DAB. Staining by the
oxidation product was observed using light microscopy.
[0045] Results are shown in FIG. 1. With polylysine (bK7; 10
.mu.M), no internalized biotin signal was observed (FIG. 1A). On
the other hand, even at the lowest concentration of biotinylated
heptaarginine (bR7; 0.1 .mu.M), internalized biotin was observed in
all VSMCs (FIG. 1B). Incubation with 10 .mu.M bR7 showed very
intense staining, not only in the cytoplasm, but also in the
nucleus of all VSMC (FIG. 1C).
[0046] When cells were exposed to 1% sodium azide for 30 minutes
prior to incubation with bR7, neither cytoplasmic nor nuclear
staining was observed (FIG. 1E), indicating that the cellular
uptake of arginine polymers is an energy dependent process.
However, when the experiments were performed at 4.degree. C., no
apparent reduction was observed in the efficacy of bR7
translocation (FIG. 1D), which is incompatible with known
endocytotic pathways. Nor was translocation of bR7 inhibited by the
addition of free L-arginine up to 10 mM (FIG. 1F). High
concentrations of extracellular L-arginine would be expected to
compete for binding to the y+transporter, which is known to
transport monomers of basic amino acids (Deves et al. (1998)
Physiol. Rev. 78:485-545). Because exogenous L-arginine did not
block the effect of the arginine polymers, the latter are not
utilizing the transport system y+ to gain intracellular access.
These results indicate that transport is not mediated by the basic
amino acid transport system y+ or by classical endocytotic
pathways.
[0047] Translocation of the polymers into endothelial cells (EC)
was similarly examined, as described in Example 3. No internalized
biotin signals were detected in control cultures treated with
vehicle or with biotin alone, or in cells treated with biotin
labeled heptalysine (bL-K7). However, even at the lowest
concentration of b-R7 (0.1 .mu.M), internalized biotin was observed
in the cytoplasm of all EC. After treatment with 10.0 .mu.M b-R7
for 30 minutes, internalized biotin was detected not only in the
cytoplasm but also in the nucleus of virtually all exposed
endothelial cells.
[0048] There were no observable differences in the distribution or
intensity of internalized biotin between the L or D forms of
heptaarginine (R7 and r7), indicating that polymer uptake is not
significantly affected by the chirality of the arginine residues in
the polymer under the conditions tested. These findings indicate
that both L-R7 and D-r7 are very efficient at translocating across
both cytoplasmic and nuclear membranes of VSMC and endothelial
cells in culture, and act as a carrier for a second molecule,
biotin.
[0049] B. Ex Vivo Translocation of Arginine Polymers
[0050] Studies using rabbit carotid artery and jugular vein, as
described in Example 4, were performed to evaluate the
translocation ex vivo of the polymers. Microscopic examination of
the treated carotid artery segments revealed a
concentration-dependent uptake of biotin in both the cytoplasm and
nucleus of all vascular cells. Following incubation for 30 minutes
at a dose of 10.0 .mu.M bL-R7, a distinct biotin signal was
observed in virtually all intimal cells, medial cells, and
adventitial cells. See FIG. 2A, where the magnitude of bL-R7
translocation is expressed as percent stained nuclei (stained
nuclei/total nuclei). Jugular vein segments incubated with bL-R7
exhibited a similar staining pattern. When the carotid artery and
jugular vein were incubated with biotinylated polylysine (bL-K7; 10
.mu.M), no stained cells were apparent.
[0051] The extent and the intensity of staining of the
polyarginine-treated tissues, as well as the depth of penetration
within the tissue segment, increased in a time-dependent manner, so
that within 30 minutes, virtually all vascular cells exhibited a
distinct biotin signal in both the cytoplasm and nucleus (FIGS.
2B-C). These results are consistent with first-order kinetics for
arginine polymer uptake.
[0052] When bR7 was instilled intraluminally, as described in
Example 4, biotin signals were detected throughout the vessel,
including the adventitial cells (outermost layer of the vessel),
staining them intensely after 30 minutes of intraluminal exposure.
There were no differences between D and L forms of heptaarginine in
their ability to penetrate the vascular wall and translocate into
all cells.
[0053] There was no apparent reduction in the speed or efficacy of
bL-R7 translocation into vascular tissue when the experiments were
performed at 4.degree. C. (FIGS. 2B-C), indicating that R7
translocation was not dependent on classical endocytosis. Note that
incubations of arginine polymer with vessel segments at lower
temperatures (e.g., just above the freezing point) may be desired
to preserve the vascular segments prior to grafting.
[0054] To estimate the relative stability of the D and L forms of
heptaarginine in vivo, the disappearance of the biotin signal over
time from vascular segments was observed by microscopic
examination. At days 1 and 2 after exposure, residual nuclear
biotin in both endothelial and medial cells was greater in vascular
segments treated with bD-r7 than those treated with bL-R7 (FIGS.
3A-B). No significant positive staining was observed with either
form by day 5, but it was not established whether this observation
was the result of cellular degradation of the biotin moiety.
[0055] IV. Effect of Arginine Polymers on NO Production and
Myointimal Hyperylasia
[0056] A. Enhancement of NO Production in Cytokine-Stimulated
VSMC
[0057] Vascular nitric oxide (NO) is synthesized from L-arginine by
endothelial cells, and contributes to vascular relaxation as well
as maintenance of normal vascular structure (Lloyd et al. (1996)
Ann. Rev. Med. 47:365-375; Cooke, supra). It is well established
that vascular NO inhibits nionocyte adherence and chemotaxis (Tsao
1997), platelet adherence and aggregation (Radomski et al. (1990)
Proc. Natl. Acad. Sci. USA 87:5193-5197; Wolf et al. (1997) J. Am.
Coll. Cardiol. 29:479-485), and vascular smooth muscle cell (VSMC
proliferation (Garg et al. (1989) J. Clin. Invest.
83(5):1774-7).
[0058] Vascular endothelium normally expresses endothelial NO
synthase (eNOS). In disease states, vascular cells also express
inducible NO synthase (iNOS). Derangement of NO synthesis
contributes to the development of vascular proliferative disorders,
including atherosclerosis, restenosis after balloon angioplasty or
other injury, and vein graft disease (Lloyd, supra; Cooke, supra).
Recent evidence suggests that preservation or enhancement of NO
synthesis can prevent or reverse some of the pathophysiological
processes that contribute to vascular proliferative diseases.
[0059] Because intracellular levels of L-arginine normally greatly
exceed the K.sub.m of the NOS enzyme, NO synthesis is ordinarily
not dependent on extracellular supplementation (Harrison (1997) J.
Clin. Invest. 100:2153-2157). However, under certain circumstances,
local L-arginine concentration can become rate-limiting. Such
circumstances include elevated plasma or tissue levels of the
endogenous NO synthase antagonist ADMA (asymmetric
dimethylarginine) (Boger et al. (1998) Circulation 98:1842-1847)
and inflammation-induced expression of the inducible NO synthase
(iNOS) (Guoyao et al. (1998) Biochem. J. 366:1-17). Both of these
abnormalities are operative in the setting of vascular injury
(Dattilo et al. (1997) Am. J. Surg. 174:177-180).
[0060] Inducible NOS is also stimulated, in a dose-dependent
manner, by IFN-.gamma. and LPS, as described in Example 5. Addition
of extracellular L-arginine confirmed that L-arginine is a limiting
factor for NO production in cytokine-stimulated VSMC. Both of these
effects are shown in FIG. 4.
[0061] In accordance with the invention, poly(L)arginine oligomers
enhance NO synthesis in vascular tissue. As described in Example 6,
cytokine-stimulated VSMC incubated in physiological levels (100
.mu.M) of extracellular (L)-arginine were treated with different
length (L)-arginine oligomers, then stimulated with IFN-.gamma. and
LPS. Pretreatment with L-R5 for 30 minutes gave no significant
enhancement of NO production. Pretreatment with L-R7 and L-R9,
however, resulted in dose-dependent increases in NO production at
doses as low as 10 .mu.M (FIG. 5). The degree of enhancement was
significantly greater in cells treated with R9 than those treated
with R7 (24.+-.3.8 vs.44.+-.5.2%, p<0.05). Treatment with D-r7
or K7 (polylysine) (10- .mu.M, 30 minutes) did not increase the NO
production (FIG. 5). N-terminal acetylation of the L-peptides (R5,
R7 and R9), which delays intracellular degradation, had no
significant effect on NO production after 24 hours (FIG. 6). When
R7 treated cells were subsequently treated with the NO synthase
inhibitor, L-NMMA, the enhancement of NO production was abolished
(FIG. 7).
[0062] The arginine oligomers were found to be significantly more
efficacious, on a mass basis, than equivalent amounts of free
arginine monomer, which is not significantly taken up by the walls
of arterial and venous segments. When VSMCs were exposed to R7 for
5 minutes, a sustained increase in NO biosynthesis was observed for
24 hours. By contrast, exposure of VSMCs to high levels (1 mM) of
free L-arginine for 5 minutes did not significantly increase NO
biosynthesis (data not shown).
[0063] It is proposed that the (L)-arginine oligomers enhance NO
production by supplementing intracellular (L)-arginine levels. In
order to clarify whether R7 translocation affects iNOS protein
expression in cytokine-stimulated VSMC, iNOS protein expression was
examined by western blotting (Example 7). Inert rat VSMC did not
express detectable iNOS protein (130 KDa). When the cells were
stimulated by IFN-.gamma. and LPS, demonstrable expression of iNOS
protein was observed, as expected. Treatment with R7 (10 .mu.M, 30
minutes exposure), however, had no effect on the expression levels
of iNOS. Therefore, the enhancement of NO production by the
L-arginine oligomers, R7 and R9, was not due to an increase in iNOS
expression.
[0064] As demonstrated above, arginine polymers of the invention
are extremely efficient at translocating into vascular cells. The
cellular translocation is energy dependent, but does not involve
classical endocytosis, nor the basic amino acid transporter.
Pretreatment of cells with R7 (10 .mu.M) caused a significant
elevation of NO production which was not observed when cells were
treated with high concentrations of free L-arginine (up to 1 mM).
These findings indicate that cellular uptake of the short polymers
of arginine is uniquely efficient, with different kinetics than the
y+ transport system.
[0065] B. Effects of D- and L-Arginine Polymers on NO Production
and on Myointima Formation in Vein Grafts
[0066] Myointimal hyperplasia involves the migration and
proliferation of VSMCs in the intima, accompanied by elaboration of
extracellular matrix (DeMeyer, supra). Within 24 hours of a
vascular injury (e.g., interposition of a vein graft or balloon
angioplasty), VSMC express iNOS (Morris et al. (1994) Am. J.
Physiol. 266:E829-E839; Hansson et al. (1994) J. Exp. Med.
180:733-738). Vascular NO, derived largely from iNOS in the setting
of vascular injury, may play an important role in suppressing VSMC
hyperplasia (Lloyd, supra; Cooke, supra), by inhibiting monocyte
adherence and infiltration and VSMC proliferation and by inducing
VSMC apoptosis (Tsao et al (1996) Circulation 94:1682-1689; Tsao et
al. (1997) Circulation 96:934-940; Garg, supra).
[0067] In view of the extremely efficient transport of arginine
oligomers into vascular cells, as demonstrated above, and the
enhancement of intracellular NO levels by poly(L)arginine, the
ability of these polymers to inhibit intimal hyperplasia was
investigated. Vein grafts were carried out on male New Zealand
white rabbits, as described in Example 8. After excision and prior
to grafting, the jugular vein was gently flushed and immersed in
PBS (control) or in PBS containing either L-R7 or D-r7 (10.0 .mu.M)
for 30 minutes. L-R5 and D-r5 (10.0 .mu.M), which have been
previously demonstrated not to translocate across the cell
membrane, were also used as controls.
[0068] All vein grafts treated with vehicle alone developed
significant myointimal hyperplasia 28 days after surgery (FIG. 8A,
arrowheads indicate internal elastic lamina). By contrast, vessel
segments treated with either L-R7 (FIG. 8B) or D-r7 (FIG. 8C) had
substantially less myointima formation (intimal area: control;
1.7.+-.0.8, L-R7; 0.5.+-.0.2, D-i-7; 1.1.+-.0.4 mm.sup.2,
p<0.05). Treatment with L-R7 was more effective than D-r7,
reducing intimal area by more than 70%, vs. about 35% for Dr7. The
intima/media ratio (I/M) of L-R7-treated vein grafts was also
significantly less than both control and D-r7-treated grafts (I/M:
Control; 1.5.+-.0.5, L-R7; 0.4.+-.0.2, D-R7; 0.8.+-.0.2,
p<0.05). Treatment using the smaller oligopeptide (R5), which
translocates poorly across membranes, was not effective in
inhibiting myointima formation, suggesting that the observed
inhibitory effects were due to translocation of the heptamers of
arginine and not simply the availability of polyarginine, e.g.,
complexed to the cell membrane.
[0069] The greater activity of the L-polymer is consistent with
proteolysis to form L-arginine monomers, which can promote
formation of NO. Thus, it will be appreciated how the composition
of the arginine polymer can be modified to achieve variations in
the rate of arginine release. The rate of arginine release may be
attenuated by including one or more D-arginine residues, which slow
the rate of proteolytic breakdown of the polymer. In addition,
D-Arg must be converted to LArg (see below) before it can serve as
a substrate for NO synthase. In one embodiment, all of the arginine
residues in the polymer have an L-configuration, for more rapid
biological activity.
[0070] Grafts were harvested after 3 days for assessment of NO
production, as described in Example 9. Basal NO.sub.x production
from L-R7 treated vein grafts was significantly higher than that of
both control and D-r7 treated vein grafts, as shown in FIG. 10A
(control; 35.+-.6, L-R7; 80.+-.14, D-r7; 48.+-.8 nM/mg tissue/hr,
p<0.05). There was no significant difference in basal NO.sub.x,
production between D treated and vehicle-treated vein grafts.
[0071] Calcium ionophore stimulation of eNOS did not significantly
affect the NO.sub.x production by the vein grafts (FIG. 10A). Graft
segments were incubated in medium in either the absence (basal) or
presence (stimulated) of calcium ionophore. This finding suggests
that the majority of NO was generated by the calcium-independent
inducible form of NO synthase (rather than the endothelial
isoform), and is compatible with previous reports of iNOS
expression in VSMC after vein grafting.
[0072] Because D-arginine is not a substrate for NO synthase, its
inhibitory effect on myointimal hyperplasia was surprising. The
effect of D-r7 could be due to NO production after epirnerization
of D- to L-arginine (Wang et al. (1999) J. Pharmacol. Exp. Ther.
288:270-273; D'Aniello et al. (1993) Comp. Biochem. Physiol.[B]
105:731-734). It is also known that D-arginine may be oxidized to
D-citrulline and NO by a non-enzymatic reaction involving hydrogen
peroxide (Nagase et al. (1997) Biochem. Biophys. Res. Commun.
233:150-153).
[0073] Alternatively, there may be NO-independent mechanism(s) of
polyarginine action. Both L-R7 and D-r7 treatment inhibited
proliferation of VSMC in vitro. Because non-stimulated VSMC express
neither constitutive nor inducible NO synthase, this inhibitory
effect seems to be NO-independent. One possible NO-independent
effect of the arginine polymers might be mediated by a cationic
interaction with nucleic acid; arginine rich sequences are often
found in RNA-binding proteins.
[0074] Direct cytostatic properties of arginine oligomers were
investigated, as described in Example 10. Rat VSMC were incubated
with either L-R7 or D-r7 (10.0 .mu.M, 30 minutes), then incubated
with growth medium containing 0.5% FBS for 48 hours. Cell
proliferation assays revealed that, in the absence of NOS enzyme,
VSMC proliferation was significantly inhibited by pretreatment with
either L-R7 or D-r7, as compared to vehicle incubation. There were
no significant differences between L-R7 and D-r7 treatment groups
in this NO-independent cytostatic effect (FIG. 10B).
[0075] C. Effects of L-Arginine Polymers on GCAD in Heart
Transplant Model
[0076] Graft coronary artery disease (GCAD) is characterized by
diffuse neointimal hyperplasia in the coronary arteries of the
transplanted heart. As discussed above, nitric oxide (NO) limits
the development of neointima formation by inhibiting vascular
smooth muscle cell proliferation.
[0077] In this study, PVG rat donor hearts (n=48) were transplanted
heterotopically into the abdomen of ACI recipients. Donor hearts
received either intracoronary PBS or intracoronary 50 uM
(L)-arginine polymer (L-R5 or L-R9) for 30 minutes. (D-arginine
polymers were not included in this study.) Each of these groups was
further divided into 60 and 90 day study animals (n=6 each).
Percent luminal narrowing, intima to media ratio (I/M.), and
percent affected vessels were determined as described in Example 11
below. Transfection efficiency was determined by infusing
biotinylated R5 and R9, as in the preceding studies, and
calculating the percentage of biotin positive nuclei divided by
total number of nuclei.
[0078] Results are shown in Table 1 below. As the data show, the R5
oligomer demonstrated minimal transfection of coronary vessels and
essentially no difference in GCAD compared to PBS controls at both
60 and 90 days. The R9 groups, however, demonstrated both marked
transfection of the intima and media of coronary vessels and a
significant reduction in GCAD (p values <0.05) at post op days
60 and 90.
1TABLE 1 Transplant Study Treatment % luminal narrowing I/M ratio %
affected vessels PBS-60 days 11.3 .+-. 4.2 0.12 .+-. 0.05 22.9 .+-.
9.5 PBS-90 days 18.5 .+-. 13.7 0.13 .+-. 0.08 22.9 .+-. 15.7 R5-60
days 12.6 .+-. 6.7 0.13 .+-. 0.07 18.9 .+-. 7.0 R5-90 days 14.2
.+-. 12.0 0.16 .+-. 0.14 18.5 .+-. 11.9 R9-60 days 3.2 .+-. 3.8
0.03 .+-. 0.04 6.5 .+-. 6.9 R9-90 days 1.6 .+-. 3.3 0.01 .+-. 0.02
4.9 .+-. 7.1
[0079] V. Isolated Vessel Conduit
[0080] In a further embodiment, the invention provides an isolated
vascular vessel conduit, such as an arterial segment, venous
segment, or an artificial vessel segment, which is prepared to
contain a polymer of arginine as described herein. Any suitable
conduit can be used. As used herein, "isolated" refers to a conduit
that, prior to grafting, exists outside of the subject's body.
Exemplary arterial conduits include segments of internal mammary
artery (IMA), internal thoracic artery, and gastroepiploic artery.
Venous segments can be prepared from various sources, preferably
from a cutaneous vein from a subject's arm or leg, such as a
saphenous vein. Preferably, the vessel segment is an autologous
saphenous vein or an internal mammary artery segment. However,
venous and arterial segments from other human donors (allografts)
can also be used, as well as vessel segments obtained from other
animals (xenografts), such as pigs. Conveniently, the segment is
obtained from the subject who is to receive the vessel conduit
graft. In addition, the vessel conduit can also be provided as an
artificial vessel segment made from a physiologically compatible
material, such as "DACRON".TM., PTFE, or other non-tissue graft
materials, and which preferably is prepared or derivatized, e.g.,
by carboxylation, sulforiation, or phosphorylation, to contain
negatively charged groups for adsorbing the positively charged
arginine polymer. The artificial vessel segment can also be
partially porous in its internal wall to provide a reservoir region
from which the arginine polymer can gradually diffuse after the
conduit has been grafted into the subject. Although intimal
hyperplasia would not occur within artificial segments, it can be
especially problematic at the anastomotic junctions where the
terminal ends of the artificial segment join to the subject's
vascular system. Thus, such intimal hyperplasia adjacent to the
grafted vessel conduit can be inhibited by the arginine
polymer.
[0081] For grafting, the vessel conduit may be of any suitable
length, e.g., 3 to 12 inches in length. Multiple vessel segments
from the same subject may be used, including both arterial and
venous segments.
[0082] The arginine polymer is preferably dissolved in a sterile,
physiologically suitable liquid that minimizes disruption of the
physical and biological function of the vessel conduit. Exemplary
liquids include serum-free culture media, such as serum-free
Dulbecco's minimal essential medium (DMEM), aqueous solutions such
as 0.9% (w/v) saline (NaCl), and any other sterile liquid medium or
solution that is used in vessel grafting procedures. The polymer is
provided at a concentration that achieves the desired effect.
Typically, the polymer concentration is from 0.01 .mu.M to 100
.mu.M, preferably 1 .mu.M to 50 .mu.M, or 1 .mu.M to 25 .mu.M,
although concentrations above or below these ranges may also be
used.
[0083] The vessel conduit is contacted with the arginine
polymer-containing liquid for a time sufficient for the arginine
polymer to be taken up into the wall of the vessel, so that a
reduction in intimal hyperplasia is obtained after graft. For
example, the vessel conduit can be immersed in the solution so that
the arginine polymer penetrates both the interior and exterior
walls of the vessel. Alternatively, the polymer solution can be
placed inside the vessel with both ends closed by ligation,
clamping, or the like, so that only the intraluminal wall is
exposed to the polymer. Usually, the polymer liquid is contacted
with the vessel segment for from 60 seconds to 120 minutes, more
typically between 5 and 45 minutes, and preferably for a time that
is less than 30 minutes. Generally, less contact time is necessary
with higher concentrations of arginine polymer. The contacting step
can be performed at any appropriate temperature, typically at a
temperature from 4.degree. C. to 37.degree. C., and conveniently at
ambient room temperature.
[0084] Before or after the vessel conduit is contacted with the
arginine polymer solution, the vessel segment may be stored in the
same types of liquids mentioned above, without the arginine
polymer. Preferably, the venous and arterial vessel segments are
maintained ex vivo for as brief a time as possible, to help avoid
degradation of their function.
[0085] The site where the vessel conduit is to be grafted can be
prepared by conventional methods, e.g., for a coronary bypass or an
above-knee or below-knee femoro-popliteal arterial bypass
procedure. Damaged or necrotic tissue is removed, and the site is
surgically prepared for attachment of the new vessel conduit,
preferably during the time that the vessel conduit is being
contacted with the arginine polymer. Following the graft procedure,
the subject may be monitored periodically to verify physiological
acceptance of the graft and to assess the level of blood flow
through the grafted vessel over time.
[0086] VI. Treatment Methods
[0087] As shown above, the polymers of the invention are useful in
reducing post-graft and post-transplant hyperplasia. Accordingly,
the invention provides a method in which one or both of the
vascular vessel regions adjacent to an incoming vessel conduit are
contacted with a solution of an arginine polymer as described
herein, to inhibit post-graft intimal hyperplasia in these regions.
For example, after a necrotic vessel segment (lesion) has been
removed (transected), the remaining vascular region downstream from
the excision site (distal to the heart) is clamped at a point
several centimeters (e.g., 4 cm) from the proximal end of the
downstream (distal) region, the clamped region is filled with
arginine polymer solution, and the proximal end is then clamped to
create a "sausage" containing the polymer solution. After the
arginine polymer solution has been incubated in the clamped region
for an appropriate time, the proximal clamp is removed, and the
arginine polymer solution is optionally removed, followed by
removal of the remaining clamp.
[0088] A similar procedure can be performed on the vascular region
upstream of the excision site where the vessel conduit is to be
grafted. By contacting the inner walls of the distal and proximal
vascular regions in the subject adjacent to the graft site, before
the vessel conduit is grafted into the subject, the zone of
protection against intimal hyperplasia can be extended around the
graft site, thereby increasing the probability of success. This
method is particularly useful for artificial vessel grafts, to
inhibit intimal hyperplasia at the anastomotic junctions.
[0089] Inhibition of intimal hyperplasia in a vessel extends to the
use of the polymers of the invention to treat "patches" of arterial
walls, e.g., vein patches used to repair arteries which have
undergone endarterectomy for atherosclerotic plaques. Such patches,
when untreated, often undergo intimal hyperplasia.
[0090] Another application in which the invention finds use is in
the vascular access model of kidney dialysis, where a surgically
formed arterial-venous anastomosis or shunt provides access to the
artery and vein used for dialysis. During dialysis, the rate of
blood flow, turbulence and stress at the venous junction is much
higher than in a normal vein. Repeated exposure to these pressures
frequently leads to hyperplasia and stenosis within the vein,
causing dialysis access failure (see, for example, reviews by
Himmelfarb (1999) J Curr. Opin. Nephrol. Hypertens. 8(5):569-72 and
Woods et al. (1997) Nephrol. Dial. Transplant. 12(4):657-9). Under
these circumstances, repeated surgeries must be performed on fresh
vessel segments. In accordance with the present method, in the
anastomosis procedure, the vein segment to be grafted is exposed to
an arginine polymer solution, typically by infusion into the
clamped segment, prior to attachment to the target artery. This
treatment significantly reduces hyperplasia and extends the useful
lifetime of the anastomosis, thus reducing the need for further
surgery.
[0091] The method and compositions of the invention may also be
used in prevention of vasculopathy, or chronic rejection of
transplanted organs. Prevention of GCAD in a heart transplant model
is demonstrated above. Preferably, the organ, such as a heart or
kidney, is retrieved from the donor into an arginine polymer
solution. Currently used preservation/transport media, such as
Cardioplegin.TM., could be supplemented with the polymer. The
polymer is provided at a concentration that achieves the desired
effect. Typically, the polymer concentration is from 0.01 .mu.M to
100 .mu.M, preferably 1 .mu.M to 50 .mu.M, or 1 .mu.M to 25 .mu.M,
although concentrations above or below these ranges may also be
used. The organ to be transplanted is contacted with the arginine
polymer-containing liquid for a time sufficient for the arginine
polymer to be taken up into the vascular tissues of the organ, so
that a reduction in intimal hyperplasia is obtained after
transplant.
[0092] The grafting method of the invention also contemplates use
in conjunction with any other ameliorative procedures which may
facilitate the success of the graft. For example, the subject can
be placed on an arginine-rich diet to increase vascular NO levels,
as taught in U.S. Pat. Nos. 5,428,070, 5,852,058, 5,861,168, and
5,891,459 by J. P. Cooke and coworkers, which are incorporated
herein by reference. In addition, anti-thrombotic drugs such as
heparin may be administered shortly before and after grafting to
help reduce the possibility of thrombus formation.
[0093] VII. Formulations
[0094] Compositions and methods of the present invention have
utility in the area of human and veterinary vascular therapeutics.
The intrinsic biological effects of the subject oligomers, in
enhancing NO synthesis and/or inhibiting intimal hyperplasia, are
useful in preventing or treating vascular disease or injury,
particularly in treating vascular proliferative disorders such as
post-operative restenosis. In addition, the polymers are useful in
the area of intracellular delivery of therapeutic agents which show
limited cell entry in unconjugated form.
[0095] Stability of the oligomers can be controlled by the
composition and stereochemistry of the backbone and sidechains. For
polypeptides, D-isomers are generally resistant to endogenous
proteases, and therefore have longer half-lives in serum and within
cells. For use of the subject oligomers in inhibition of intimal
hyperplasia, oligomers which generate L-arginine in vivo, e.g.,
L-arginine oligomers, are preferred. As shown above, however,
D-arginine oligomers, which are not readily degraded to arginine,
were also effective in this area.
[0096] The compositions typically include a conventional
pharmaceutical carrier or excipient and may additionally include
other medicinal agents, carriers, adjuvants, and the like.
Preferably, the composition will be about 5% to 75% by weight of a
compound or compounds of the invention. As noted above, media used
for heart transplants may include a cardioplegic agent such as
Cardioplegin.TM., a mixture of magnesium aspartate, procaine, and
sorbitol, or similar compositions (see, e.g., Isselhard et al.
(1980) Thoracic and Cardiovascular Surg. 28(5):329-36). Appropriate
excipients can be tailored to the particular composition and route
of administration by methods well known in the art.
[0097] Liquid compositions can be prepared by dissolving or
dispersing the polymer (about 0.5% to about 20%), and optional
pharmaceutical adjuvants in a carrier, such as, for example,
aqueous saline (e.g., 0.9% w/v sodium chloride), aqueous dextrose,
glycerol, ethanol and the like, to form a suspension or,
preferably, a solution. As discussed above, the polymer solution
can be used to immerse a vessel or organ to be grafted or
transplanted, prior to surgery.
[0098] The polymers may also be delivered to a vessel
post-surgically or following an angioplasty procedure. Devices for
delivery of a medicament to the lumen of a vessel are known in the
art and include, for example, perforated or porous catheter
balloons containing the medicament. Such a delivery device may also
incorporate a biocompatible polymeric carrier, such as a
Pluronic.TM. hydrogel, containing the medicament.
[0099] The compounds of the invention are administered in a
therapeutically effective amount, i.e., a dosage sufficient to
effect treatment, which will vary depending on the individual and
condition being treated. Typically, a therapeutically effective
daily dose is from 0.1 to 100 mg/kg of body weight per day of drug.
Most conditions respond to administration of a total dosage of
between about 1 and about 30 mg/kg of body weight per day, or
between about 70 mg and 2100 mg per day for a 70 kg person.
[0100] Administered dosages will generally be effective to deliver
picomolar to micromolar concentrations of the therapeutic
composition to the site. Appropriate dosages and concentrations
will depend on factors such as the therapeutic composition or drug,
the site of intended delivery, and the route of administration, all
of which can be derived empirically according to methods well known
in the art.
[0101] For certain applications, e.g., delivery to a site of
angioplasty, the surface area of tissue to be treated may also be
considered. For delivery to the site of vessel injury, in vivo
models such as described in Edelman et al. (1995) Circ. Res.
76(2):176-182, maybe used. Further guidance can be obtained from
studies using experimental animal models for evaluating dosage, as
are known in the art. Methods for preparing such dosage forms are
known or will be apparent to those skilled in the art; for example,
see Remington's Pharmaceutical Sciences (19th Ed., Williams &
Wilkins, 1995). The composition to be administered will, in any
event, contain a quantity of the active compound(s) in a
pharmaceutically effective amount for relief of the condition being
treated when administered in accordance with the teachings of this
invention. Compositions for use in the methods described herein may
also be enclosed in kits and/or packaged with instructions for
use.
EXAMPLES
[0102] The following examples are intended to illustrate but not
limit the present invention.
Materials and Methods
Cell Culture
[0103] Rat VSMCs were prepared from the media layer of thoracic
aorta of Sprague-Dawley rats by the explant method. The cells were
cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco BRL,
Gaithersburg, Md.) containing 10% fetal calf serum, 100 U/ml
penicillin and 100 .mu.g/ml streptomycin at 37.degree. C. under a
humidified atmosphere containing 5% CO.sub.2. After subconfluent
growth, cells were cultured with MEM Select Amine Kit (Gibco BRL,
Gaithersburg, Md.) to be treated by specific concentrations of
extracellular free L-arginine. Experiments were performed using
cultured cells at passage levels of 5-10.
Histological Detection of Internalized Biotin Labeled Oligomers
[0104] After incubation with biotin labeled oligomer, vascular
segments were washed, then frozen in OCT compound (Miles
Scientific). Frozen sections, 5 .mu.m thick, were fixed with
acetone for 10 minutes. Internalized biotin was detected using the
staining procedure described in Example 2. Methyl green was used
for nuclear counter staining.
[0105] To quantify the efficiency of nuclear translocation, the
numbers of both DAB positive nuclei and total nuclei were counted
in the intima and media separately at X400 magnification with a
video image analysis system (Automatrix). The frequency of nuclear
translocation was expressed as the percent staining of nuclei,
defined as the ratio of the number of DAB positive nuclei that of
all nuclei, in the intima and media. Each protocol was repeated 4
times.
NO.sub.2 Measurement
[0106] Extracellular NO production was measured as its stable
oxidative metabolite, nitrite (NO.sub.2). At the end of each
incubation, samples of the medium (80 .mu.l) were collected, and
NO.sub.2 measurement was performed using the Griess reaction
facilitated by a commercial colorimetric assay (Cayman Chemical,
Ann Arbor, Mich.). Values of NO.sub.2 production were corrected
with relative cell count assessed by a cell proliferation kit II
(XTT) (Boeheringer Mannheim, Germany).
Statistical Analysis
[0107] All values are expressed as mean .+-.SEM. Means were
compared using an analysis of variance, and p values less than 0.05
were accepted as statistically significant.
[0108] Recombinant rat recombinant interferon-.gamma., E. coli
lipopolysaccharide (0111:B4), and L-NMMA (N.sup.G-monomethyl
L-arginine) were purchased from Sigma Chemical (St. Louis, Mo.). A
monoclonal anti-iNOS antibody was purchased from Transduction
Laboratories (Lexington, Ky.), and gout anti-mouse IgG antibody
conjugated with horseradish peroxidase was obtained from Kirkegaard
and Perry Laboratories (Gaithersburg, Md.).
[0109] Experimental protocols were approved by the Administrative
Panel on Laboratory Animal Care of Stanford University, and were
performed in accordance with the "Guide for the Care and Use of
Laboratory Animals" issued by National Institute of Health (NIH
Publication No. 80-23, revised 1985).
EXAMPLE 1
Peptide Synthesis
[0110] Peptides were synthesized using solid-phase techniques and
commercially available Fmoc amino acids, resins, and reagents (PE
Biosystems, Foster City, Calif., and Bachem, Torrance, Calif.) on a
Applied Biosystems 433 peptide synthesizer as previously described
(Hill et al. (1994) J. Immunol. 152:2890-2895).
EXAMPLE 2
Translocation of Biotin-Labeled Peptides into VSMC
[0111] Rat VSMCs were grown on glass microscope slide chambers
(Nunc Inc., Naperville, Ill.). Subconfluent cells were washed and
placed in serum-free medium. After 2 hours, cells were treated with
bR7, or bK7 (0.1 .mu.M, 1.0 .mu.M, 10 .mu.M), at 37.degree. C. for
30 minutes. To assess the role of endocytosis in cellular uptake of
the peptides, experiments were performed at 4.degree. C., and also
in the presence of sodium azide (1.0%) for 30 minutes prior to
exposure to the peptides. To assess the involvement of the basic
amino acid transport system y+ in the translocation of peptides
into the cell, experiments were performed in the presence of excess
extracellular L-arginine (10 mM).
[0112] After 30 minutes of incubation with the peptides, cells were
washed 3 times with phosphate-buffered saline (PBS), fixed for 5
minutes at -20.degree. C. in ethanol/acetone, washed 3 times in
PBS, incubated for 30 minutes with a peroxidase suppressor
(ImmunoPure, Pierce, Rockford, Ill.) to block endogenous peroxidase
activity and nonspecific binding, washed, and then incubated with 5
.mu.g/ml of horseradish peroxidase (HRP) conjugated streptavidin
(Pierce, Rockford, Ill.) for 30 minutes. Cells were washed 3 times
with PBS, and a substrate of HRP, DAB (Sigma, St. Louis, Ill.), was
added to the cells. The reaction was terminated by washing in
distilled water after a 60-second incubation with DAB. Cell
preparations were observed by conventional light microscopy (see
FIG. 1). This experimental protocol was repeated 3 times.
EXAMPLE 3
In Vitro Translocation Study
[0113] Spontaneously transformed human umbilical vein endothelial
cells (ECV304, ATCC) were cultured in medium M199 (Irvine
Scientific) containing 10% fetal bovine serum (FBS), 100 IU/ml
penicillin and 100 .mu.g/ml of streptomycin (Gibco BRL). Confluent
cells were washed and placed in serum-free medium. After 2 hours,
the cells were incubated in presence of biotin labeled peptides, as
bL-R7, or bD-R7, or bL-K7 (0.1, 1.0, and 10.0 .mu.m). After 30
minutes of incubation, cells were washed 3 times with
phosphate-buffered saline (PBS), fixed in ethanol/acetone; washed
in PBS; incubated for 30 minutes with peroxidase suppressor
(ImmunoPure, Pierce) to block endogenous peroxidase activity; and
then incubated with 5 .mu.g/ml of horse-radish peroxidase (HRP)
conjugated streptavidin (Pierce) for 30 minutes. Substrate of HRP,
diaminobenzidine (DAB, Sigma), was added to the cells. The reaction
was terminated by washing in distilled water after a 60-second
incubation. This experiment was repeated 3 times.
EXAMPLE 4
Ex Vivo Translocation Study
[0114] Both carotid artery and jugular vein segments of male New
Zealand white rabbits were used. To test the dose dependence of R7
translocation, vascular segments were incubated for 30 minutes with
either bL-R7 or bD-R7 solution (0.1, 1.0, and 10.0 .mu.M) in
serum-free Dulbecco's minimal essential medium (DMEM) (Gibco BRL).
To test the incubation time dependence, vascular segments were
incubated with 10.0 .mu.M of biotin labeled R7 solution for 10
seconds, 60 seconds, 5 minutes, 10 minutes, and 30 minutes.
Furthermore, to determine the ability of R7 to penetrate through
the vessel wall, vascular segments were ligated one end, R7
containing medium was instilled, and the other end ligated so as to
expose only the luminal surface to R7. The luminal surface of the
vascular segment was exposed to bL-R7 or bD-R7 (10.0 .mu.M) for 30
minutes. To test the temperature dependence of translocation,
vascular segments were incubated with 10.0 .mu.M of biotin labeled
R7 solutions at 37.degree. C. or 4.degree. C. To determine the
disappearance time course of translocated R7, vascular segments
were incubated with biotin labeled R7 solutions (10.0 .mu.M) at
37.degree. C. for 30 minutes, and then reincubated in DMEM with 10%
FBS up to 5 days. Vascular segments were harvested at 1, 2 and 5
days after initial incubation.
EXAMPLE 5
Stimulation of Cells with Interferon-y and LPS
[0115] Cells were plated at a density of 5.times.10.sup.3 per well
into 96-well plates. For experiments assessing the effect of
extracellular L-arginine concentration on NO synthesis from
cytokine stimulated VSMC, subconfluent cells were washed twice with
arginine free medium, then incubated for 24 hours with the medium
containing the desired concentration of L-arginine (0, 10 .mu.M,
100 .mu.M, 1 mM, 10 mM). After 24 hours of incubation, the cells
were then treated with a mixture of IFN-.gamma. (100 U/ml) and LPS
(100 .mu.g/ml) in the medium containing the same dose of L-arginine
for another 24 hours, and nitrite (NO.sub.2) accumulation in the
culture medium was quantified.
[0116] No detectable NO.sub.2 was measured in the medium of
non-stimulated VSMC. When cells were stimulated with a mixture of
IFN-.gamma. (100 U/ml) and LPS (100 .mu.M), a significant amount of
NO.sub.2 was detected in the medium (14.7.+-.0.3 .mu.M/10.sup.5
cells/24 hours). The effect of IFN-.gamma. on NO biosynthesis was
dose-dependent (FIG. 4A).
[0117] Cells were stimulated with IFN-.gamma. (100 U/ml) and LPS
(100 .mu.g/ml) for 24 hours to assess the dose-dependence on
substrate availability. Increases in extracellular L-arginine led
to a progressive increase in NO.sub.2 synthesis by cytokine
stimulated VSMC over the range of 0 to 10 mM (FIG. 4B).
EXAMPLE 6
Effect of Arginine Oligomers on NO Production in VSMC
[0118] Subconfluent cells were pre-incubated with medium containing
100 .mu.M arginine for 24 hours. The cells were then transiently
pretreated with each arginine polymer for 30 minutes. After
translocation, the cells were incubated with a mixture of
IFN-.gamma. (100 U/ml) and LPS (100 .mu.g/ml) in medium containing
100 .mu.M L-arginine for another 24 hours. In some experiments,
L-NMMA (1 mM), a nitric oxide synthase inhibitor, was added to the
medium.
EXAMPLE 7
Assay for iNOS Protein Expression
[0119] In order to clarify the effects of arginine polymer
translocation on iNOS expression, iNOS protein concentrations of
rat VSMC were analyzed by western blotting. Samples were analyzed
from non-stimulated cells, cells stimulated with IFN-.gamma. (100
U/ml) and LPS (100 .mu.g/ml), or R7 (10 .mu.M) pretreated cells
which were stimulated with IFN-.gamma. and LPS. Treated cells were
washed twice with PBS, and total cell lysates were harvested in 150
.mu.l of lysis buffer containing 150 mM NaCl, 50 mM Tris/Cl, pH
8.0, 1% NP40, and 0.1% SDS. Samples were centrifuged for 5 minutes
at 14,000 g, 4.degree. C., to remove insoluble material, and the
supernatant was collected. Protein concentrations were measured
with the Lowry method. Cell lysates containing 50 .mu.g of protein
were boiled for 5 minutes and separated on an 8.0%
SDS-polyacrylamide minigel. Eluted proteins were electroblotted
onto nitrocellulose membrane (HyBond, Amersham, England). The blots
were incubated for 1 hour in 5% non-fat dry milk/0.05% Tween.RTM.
in Tris buffered saline (TBS) to block non-specific binding of the
antibody. Blots were incubated for 3 hours with primary monoclonal
antibodies against iNOS protein diluted 1:2,500 in TBS/Tween.RTM..
The blots were then incubated with peroxidase labeled gout
anti-mouse IgG in the same buffer for 1 hour. Peroxidase labeled
protein was visualized with an enhanced chemiluminescence detection
system (Amersham, England) on X-ray film.
EXAMPLE 8
Effect of Treatment with Arginine Oligomers on Vein Graft
Segments
[0120] A. Surgical Procedure
[0121] Male New Zealand white rabbits (3.0-3.5 kg) fed standard
diets were anesthetized with a mixture of ketamine (40 mg/kg) and
xylazine (5 mg/kg) intramuscularly. The left external jugular vein
was exposed through a longitudinal neck incision. The jugular vein
was excised, gently flushed and immersed in PBS (control) or PBS
containing either L-R7 (10.0 .mu.M) or D-r7 (10.0 .mu.M) for 30
minutes. L-R5 and D-r5 (10.0 .mu.M), which have been previously
demonstrated not to translocate across the cell membrane, were used
as controls.
[0122] The right common carotid artery was exposed and clamped at
the both proximal and distal ends. The treated vein segment was
washed with PBS and then anastomosed in a reverse end-to-side
fashion into the carotid artery, using continuous 8-0 polypropylene
sutures. The common carotid artery was ligated and dissected
between the two anastomoses, and the wound was closed with 3-0
nylon suture.
[0123] B. Vessel Morphometry
[0124] Vein graft segments were harvested on the 28th surgical day.
Graft segments were fixed in 10% buffered formalin with gentle
intraluminal pressure to maintain the physiological graft
configuration. The middle portion of the paraffin samples were
sectioned (5 .mu.m) and stained with hematoxylin/eosin for light
microscopic examination (FIGS. 8A-C). Three sections of each graft,
taken at 0.5 mm intervals, were analyzed by planimetry by a
observer blinded to the treatment group. The cross-sectional areas
of the lumen, intima and media was digitized with the use of the
Image Analyst program (Automatrix). The ratio of intima to media
(I/M) area was calculated.
EXAMPLE 9
Ex Vivo NOx Production from Vein Graft
[0125] Vein grafts were harvested 3 days after surgery, as
described in Example 7. Vein graft segments were incubated in 1 ml
of Hanks' buffered saline solution (HBSS, Irvine Scientific)
containing Ca.sup.2+ (1.0 mM) and L-arginine (100 .mu.M, Sigma) at
37.degree. C. for 2 hours. NOx (nitrate and nitrite) production was
measured either in the absence (basal) or presence (stimulated) of
calcium ionophore (A23187, 10.0 .mu.M, Sigma). Samples of the
medium (80 .mu.L) were collected, and NO.sub.x measurement was
performed using the Griess reaction facilitated by a commercial
colorimetric assay (Cayman Chemical).
EXAMPLE 10
VSMC Proliferation Assay
[0126] Rat aortic VSMC were grown to 50% confluence in 96-well cell
culture plates. VSMC were then washed and incubated with serum free
DMEM for 48 hours to obtain quiescent nondividing cells.
Thereafter, VSMC were treated with vehicle, L-R7 (10.0 .mu.M), or
D-R7 (10.0 .mu.M) for 30 minutes. After the treatment, cells were
washed, and further incubated with serum containing DMEM (0.5%,
FBS). Cells were harvested after 48 hours of incubation. Cell count
was performed with commercial cell proliferation assay kit using
spectrophotometer (XTT, Boeheringer Mannheim). As a negative
control, cells treated with vehicle and incubated with DSF were
used. As an index of cell proliferation, the OD ratio of each
treatment group to negative control group was calculated as an
index of cell proliferation.
EXAMPLE 11
Heart Transplant Study
[0127] PVG rat donor hearts (n=48) were transplanted
heterotopically into the abdomen of ACI recipients. Donor hearts
received either intracoronary PBS or intracoronary 50 uM
(L)-arginine polymer (L-R5 or L-R9) for 30 minutes. Each group was
further divided into 60 and 90 day study animals (n=6 each). Tissue
cross sections (5 A) were stained with EVG (Elastica-van Gieson)
preparation, and vessels were scored using computerized morphometry
for analysis of % luminal narrowing, intima to media ratio (I/M),
and % affected vessels. Transfection efficiency was determined by
infusing biotinylated R5 and R9, as in preceding examples, and
calculating the percentage of biotin positive nuclei divided by
total number of nuclei. Results are described in Section IV C,
above.
[0128] While the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications and changes may be made without departing
from the spirit of the invention.
* * * * *