U.S. patent application number 09/764323 was filed with the patent office on 2001-08-16 for treatment of allograft rejection with anti-angiogenic agents.
Invention is credited to Briscoe, David M., Moulton, Karen, Sayegh, Mohamed H..
Application Number | 20010014663 09/764323 |
Document ID | / |
Family ID | 26890934 |
Filed Date | 2001-08-16 |
United States Patent
Application |
20010014663 |
Kind Code |
A1 |
Briscoe, David M. ; et
al. |
August 16, 2001 |
Treatment of allograft rejection with anti-angiogenic agents
Abstract
The invention relates a method of inhibiting allograft rejection
in a subject with an organ transplant. The method comprises
administering an effective amount of one or more anti-angiogenic
agents to a subject alone or in combination with one or more
immunosuppressive agents. The invention provides new and improved
methods of inhibiting allograft rejection.
Inventors: |
Briscoe, David M.; (Sharon,
MA) ; Moulton, Karen; (Weston, MA) ; Sayegh,
Mohamed H.; (Westwood, MA) |
Correspondence
Address: |
NIXON PEABODY LLP
101 Federal Street
Boston
MA
02115
US
|
Family ID: |
26890934 |
Appl. No.: |
09/764323 |
Filed: |
January 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09764323 |
Jan 17, 2001 |
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09200503 |
Nov 25, 1998 |
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6218361 |
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09200503 |
Nov 25, 1998 |
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09195375 |
Nov 18, 1998 |
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Current U.S.
Class: |
424/278.1 ;
514/13.3; 514/20.5 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 38/13 20130101; A61K 31/336 20130101;
A61K 31/335 20130101; A61K 38/13 20130101; A61K 31/336
20130101 |
Class at
Publication: |
514/2 ;
514/9 |
International
Class: |
A61K 038/16; A61K
038/13 |
Claims
What is claimed is:
1. A method of inhibiting allograft rejection in a subject with an
organ transplant comprising the step of administering an effective
amount of one or more anti-angiogenic agents, with the proviso that
the anti-angiogenic agent is not thalidomide.
2. The method of claim 1 wherein the subject is being treated for
acute allograft rejection.
3. The method of claim 2 wherein the anti-angiogenic agent is
endostatin.
4. The method of claim 2 wherein the anti-angiogenic agent is
angiostatin.
5. The method of claim 2 wherein the anti-angiogenic agent is AGM
1470 or an analogue.
6. The method of claim 5 wherein the anti-angiogenic agent is
TNP-470.
7. The method of claim 2 wherein the transplanted organ is a kidney
or heart.
8. The method of claim 2 wherein the anti-angiogenic agent is
co-administered with an effective amount of one or more
immunosuppressive agents.
9. The method of claim 8 wherein the immunosuppressive agent is
cyclosporine.
10. The method of claim 1 wherein the anti-angiogenic agent is a
polypeptide.
11. A method of inhibiting chronic allograft rejection in a subject
with an organ transplant comprising the step of administering an
effective amount of one or more anti-angiogenic agents.
12. The method of claim 11 wherein the anti-angiogenic agent is
endostatin.
13. The method of claim 11 wherein the anti-angiogenic agent is
angiostatin.
14. The method of claim 11 wherein the anti-angiogenic agent is AGM
1470 or an analogue.
15. The method of claim 14 wherein the anti-angiogenic agent is
TNP-470.
16. The method of claim 11 wherein the transplanted organ is a
kidney or heart.
17. The method of claim 11 wherein the anti-angiogenic agent is
co-administered with an effective amount of one or more
immunosuppressive agents.
18. The method of claim 17 wherein the immunosuppressive agent is
cyclosporine.
19. The method of claim 11 wherein the anti-angiogenic agent is a
polypeptide.
Description
BACKGROUND OF THE INVENTION
[0001] Acute and chronic allograft rejection are the most common
causes of allograft failure. A further understanding and improved
treatment of acute and chronic rejection are of critical importance
and will optimize transplantation as a successful treatment
modality in certain disease states such as kidney and heart
failure. The development of chronic rejection is reported to be
associated with acute rejection. Indeed, acute allograft rejection
episodes are most predictive for the development of chronic
rejection. Chronic allograft rejection is a complex multicellular
process which generally occurs months to years after organ grafting
and is characterized by pathological features such as graft
arteriosclerosis. Therefore, additional methods of suppressing
acute allograft rejection can also increase the longevity of
individuals with organ transplants.
[0002] Thus, there is a continued need to develop new and improved
methods of inhibiting allograft rejection to promote the quality of
life of subjects, such as human transplant patients.
SUMMARY OF THE INVENTION
[0003] This invention is based upon the discovery that
anti-angiogenic agents inhibit allograft rejection in a subject,
such as a mammal. As described herein acute and chronic allograft
rejection in subjects with organ transplants are inhibited with
anti-angiogenic agents. Thus, the present invention relates to a
method of inhibiting allograft rejection in a subject comprising
administering an effective amount of one or more anti-angiogenic
agents. When the method of the present invention is used to treat
acute allograft rejection the anti-angiogenic agent is not
thalidomide.
[0004] As described herein the anti-angiogenic agent is a
substance, such as a polypeptide, peptidomimetic, small organic
molecule, sugar or lipid, that interferes, either directly or
indirectly, with the development of new blood vessels. These
anti-angiogenic agents inhibit allograft rejection. Specifically
encompassed by the present invention is a method of treating a
subject such as a mammal, including humans, wherein the subject is
being treated for chronic allograft rejection. In another aspect of
the invention a subject is being treated for acute allograft
rejection. In one embodiment the anti-angiogenic agent is
angiostatin. In another embodiment the anti-angiogenic agent is
endostatin. In preferred embodiments, the anti-angiogenic agent is
O-chloroacetylcarbamoyl)fumagil- lol (referred to herein as
AGM-1470), or an analogue of AGM-1470, such as TNP-470.
Specifically encompassed by the present invention is a method of
inhibiting allograft rejection wherein the subject is being treated
for an organ transplant. In preferred embodiment the transplanted
organ is a kidney(s) or a heart.
[0005] A further embodiment of the present invention is a method of
inhibiting allograft rejection wherein the anti-angiogenic agent is
co-administered with an effective amount of one or more
immunosuppressive agents. Specifically encompassed is a method of
treating a subject wherein the subject is being treated for acute
allograft rejection by co-administering one or more anti-angiogenic
agents and one or more immunosuppressive agents.
[0006] The inventions described herein provide alternative and
improved methods for the successfully inhibiting, interrupting the
development of, or treating established allograft rejection in a
subject. The invention thereby offers new and effective methods
which are extremely useful in clinical settings for treating
mammals, such as humans, following the transplantation of organs
such as the heart and kidney.
BRIEF DESCRIPTION OF THE FIGURES
[0007] FIG. 1 is the chemical structure of AGM-1470.
[0008] FIG. 2 is a graph depicting the fraction or percent of
allografts which survive (surviving) over time in days (Days) from
Fisher strain rats ("F344 rats") transplanted with hearts from
Lewis strain rats ("LEW strain") in an acute allograft rejection
model. Transplant recipients were treated with TNP-470 or received
no treatment (control).
[0009] FIG. 3 is a graph depicting the fraction or percent of
allografts which survived (Graft Survival) over time in days (Days
Post Transplantation) from F344 rats transplanted with hearts from
LEW rats in a chronic allograft rejection model. Following
treatment with the immunosuppressive agent cyclosporine, the F344
rats were treated with the anti-angiogenic agent TNP-470 or vehicle
as a control.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present invention relates to the discovery that acute
and chronic allograft rejection can be inhibited by the
administration of an effective amount of one or more
anti-angiogenic agents, such as angiostatin, endostatin, AGM-1470,
or TNP-470, alone or in combination with one or more
immunosuppressive agents, such as cyclosporine, FK506, steroids, or
antiproliferative agents (e.g., azathioprine, mycophenolate
moefitil). Other suitable immunosuppressive agents can be those
currently under evaluation in Phase III human clinical trials
(e.g., Rapamycin). The methods of the present invention lead to
increased graft survival and an accompanying decrease in the mean
arteriosclerosis score of blood vessels in transplanted organs.
[0011] The term "allograft rejection" refers to a reaction within a
transplanted organ or tissue involving both immunologic and
non-immunologic responses that ultimately lead to damage or
necrosis of some or all of the transplanted organ or tissue. An
"organ" refers to a part of the body of a subject exercising a
specific function (such as a heart, kidney, liver, or lung). A
"tissue" refers to a collection of similar cell types (such as
epithelium, connective, muscle and nerve tissue). A "transplanted
tissue or organ" is meant to refer to a tissue or organ taken from
one subject and implanted into a subject other than the subject
from which the organ or tissue was taken.
[0012] The phrase "acute allograft rejection" as used herein is
intended to refer to a rejection reaction(s) that typically occurs
rapidly, within days to weeks after transplantation.
[0013] The phrase "chronic allograft rejection" as used herein
refers to a rejection reaction(s) that occurs over months to years.
Acute and chronic allograft rejection also have unique pathological
features. For example, in heart and kidney transplants, chronic
allograft rejection is characterized by the proliferation of the
smooth muscle cells in the intima of arteries in the graft leading
to a graft vascular arteriosclerotic lesion which resembles an
accelerated form of arteriosclerosis (Salomon, R. N., et al., Am.
J. Pathol. 138:791 (1991); Libby, L., et al., Clinical
Transplantation 8:327 (1994); and Ross, R., Nature Medicine 2:527
(1996)). Graft vascular arteriosclerotic lesions in chronic
allograft rejection can be determined using well-established
criteria as described in Example 2.
[0014] The term "anti-angiogenic agent" as used herein is intended
to refer to a substance (such as a polypeptide, peptide, small
organic molecule, peptidomimetic, sugar, lipid), that interferes,
either directly or indirectly, with angiogenesis (e.g., the
development of new blood vessels). As used herein "angiogenesis",
"endothelial cell proliferation" and the "development of new blood
vessels" are equivalent. An anti-angiogenic agent can, for example,
directly interact with an endothelial cell by binding, entering or
otherwise altering the endothelial cell thereby directly
interfering with the formation of blood vessels. Alternatively or
additionally, the anti-angiogenic agent can act indirectly by, for
example, binding, entering or otherwise altering a cell other than
an endothelial cell (e.g., a T-cell) thereby indirectly interfering
with angiogenesis by, for example, altering the production,
secretion or processing of other factors (e.g., vascular growth
factors such as VEGF, .beta.-FGF, TNF-.alpha., TNF-.beta.) involved
in angiogenesis. Graft-infiltrating T-cells and macrophages, for
example, result in smooth muscle cell proliferation (e.g., the
smooth muscle of the intimal wall of arteries in transplant organs)
and graft arteriosclerosis (Tullius, S. G., et al., Transplantation
59:313 (1995)). Thus, the anti-angiogenic agents of the present
invention can decrease the number of blood vessels through which
the infiltrating T-cells and macrophages migrate to the graft.
Alternatively and additionally, the anti-angiogenic agents
described herein can alter the ability of infiltrating T-cells and
macrophages to mediate the process of arteriosclerosis. In either
case allograft rejection is inhibited. Methods to assess
anti-angiogenic activity of a substance are routine and well-known
to one of skill in the art and include, for example, inhibition of
angiogenesis in a chicken chorioallantoic membrane assay or a
rabbit cornea micropocket assay (See, for example, Ingber, D., et
al., Nature 348:555-557 (1990); O'Reilly, M. S., et al., Cell
79:315 (1994); Folkman, J., New Eng J. Med 333:1757 (1995);
Holmgren, L., et al., Nature Medicine 1:149 (1995); O'Reilly, M.
S., et al., Cell 88:277 (1997); and O'Reilly, M. S., et al., U. S.
Pat. No. 5,639,725)
[0015] Where the anti-angiogenic agent is a polypeptide, the
polypeptide can be post-translationally modified (e.g., amidated,
demethylated, glycosylated or phosphorylated) Anti-angiogenic
agents that are polypeptides can be, for example, endostatin, or
angiostatin, or both.
[0016] In a preferred embodiment the anti-angiogenic agent, alone
or when co-administered with an immunosuppressive agent, is used to
inhibit the allograft rejection of a subject wherein the subject
has undergone an organ transplant. Specifically encompassed by the
present invention is the administration of one or more
anti-angiogenic agents, alone or when co-administered with
immunosuppressive agents, to inhibit the rejection of heart or
kidney transplants.
[0017] The phrase to "inhibit allograft rejection" as used herein
is meant to refer to suppressing, diminishing, limiting,
decreasing, reducing, preventing, retarding, or slowing down
allograft rejection. Indices to determine inhibition of allograft
rejection are well known to one of skill in the art and include
improvement in pathologic, clinical and biochemical criteria of
rejection as well as overall function of the allograft (Suki, N.,
"Primer on Transplantation", American Society of Transplantaion
Physicians, New York, N.Y. (1998)). For example, indices of
improving chronic allograft rejection can be increased graft
survival rates and decreased arteriosclerosis score of blood
vessels to the allograft organ (See Examples 1 and 2).
[0018] The invention also pertains to inhibiting or interrupting
the development of an allograft rejection by administering one or
more anti-angiogenic agents, alone or in combination with one or
more immunosuppressive agents. The treatment of established
allograft rejection with the methods described herein are also
within the scope of the invention.
[0019] In one embodiment of the present invention the
anti-angiogenic agents are the polypeptides angiostatin and
endostatin. The anti-angiogenic activity of endostatin and
angiostatin are well documented and have been used to successfully
inhibit the development of new blood vessels to tumors. See, for
example, O'Reilly, M. S., et al., Cell 79:315 (1994); Folkman, J.,
New Eng J. Med 333:1757 (1995); Holmgren, L., et al., Nature
Medicine 1:149 (1995); O'Reilly, M. S., et al., Cell 88:277 (1997);
and O'Reilly, M. S., et al., U.S. Pat. No. 5,639,725, the teachings
of which are incorporated herein in their entirety.
[0020] In another embodiment, the anti-angiogenic agent is AGM-1470
(FIG. 1), or an analogue of AGM-1470 such as TNP-470. The
anti-angiogenic activity of AGM-1470 and TNP-470 is known. See, for
example, Ingber, D., et al., Nature 348:555 (1990); Antoine, N., et
al., Am. J. Pathol. 148:393 (1996); and Griffith, E., et al., Chem.
Biol. 4:461 (1997); Folkman, J., et al., U.S. Pat. No. 5,135,919
(1992), the teachings of which are incorporated herein in their
entirety. TNP-470 inhibits DNA synthesis in endothelial cells by
inhibiting growth factor activation of cyclin dependent kinases
(Koyama, H., et al., Circulation Res. 79:757 (1996)) as well as
other enzymatic pathways (Antoine, N., et al., Am. J. Pathol.
148:393 (1996); and Griffith, E., et al., Chem. Biol. 4:461
(1997)).
[0021] TNP-470 inhibits acute and chronic allograft rejection
(Examples 1 and 2). The 30 day graft survival rate of rat heart
transplant recipients increased from 0% to greater than 50% when
allograft recipients were treated with the anti-angiogenic agent
TNP-470 (Example 1; FIG. 2). Thus, TNP-470 suppresses acute
allograft.
[0022] TNP-470 can also suppress chronic allograft rejection
(Example 2; FIG. 3). A 120 day increased graft survival rate
occurred when transplant recipients were treated with TNP-470 for
days 30-120 after allografting, following an initial 30 days of
treatment with cyclosporine, than recipients who received only
cyclosporine for 30 days (approximately 75% survival rate in
cyclosporine/TNP-470 treated recipients compared with approximately
45% in cyclosporine alone treated recipients; Example 2; FIG. 3).
In addition, TNP-470 treatment ameliorated the characteristic graft
arteriosclerotic lesions manifested in chronic allograft rejection
(Example 2). Decreased occlusion of cardiac vessels after 120 days
was observed in cyclosporine/TNP-470 treated animals (Example 2).
Thus, TNP-470 suppresses chronic allograft rejection.
[0023] In another aspect of the invention analogues of AGM-1470
(Griffith, E., et al., Chem. Biol. 4:461 (1997)), such as TNP-470
(Ingber, D., et al., Nature 348:555 (1990)), can be used to inhibit
allograft rejection. "Analogue" refers to a compound that resembles
AGM-1470 in structure (e.g., chemical structure), yet inhibits
allograft rejection as described herein for anti-angiogenic
agents.
[0024] Experimental methods to produce and purify the
anti-angiogenic agents of the present invention (e.g., angiostatin
or endostatin) are known to one of skill in the art. These
techniques include the production of recombinant angiostatin and
endostatin which have previously been shown to be anti-angiogenic
when produced in host cells such as yeast (such as Pichia pastorius
or Saccharomyces cerevisa), bacteria (such as Escherichia or
Bacillus) expression systems; animal cells or tissue, including
insect (such as baculoviruses) or mammalian cells (such as somatic
or embryonic cells, Chinese hamster ovary cells, HeLa cells, human
293 cells and monkey COS-7 cells) Appropriate vectors, transfection
and transformation protocols to generate recombinant
anti-angiogenic agents are well-known to the skilled artisan.
Exemplary discussions of recombinant DNA technologies can be found
in, for example, Ausubel, et al., "Current Protocols in Molecular
Biology", John Wiley & Sons, Inc., (1998). Nucleic acid
molecules coding for suitable anti-angiogenic agents (e.g.,
angiostatin and endostatin), in several mammalian species (e.g.,
mouse, human, rhesus monkey, pigs, cows), are known in the art and
can be obtained from, for example, the EMBL/GenBank data bases. For
example, the Accession numbers for angiostatin are 148501, 148503,
148504, 148505. Alternatively, other sequences can be employed,
such as related genes which encode structurally or functionally
equivalent polypeptides to anti-angiogenic agents described herein.
The related genes and the polypeptides encoded by the related genes
can share structural (e.g., nucleotide, or amino acid sequence, or
three dimensional structure) or activity (e.g., inhibit allograft
rejection) similarities to polypeptide anti-angiogenic agents
described herein (e.g., angiostatin, endostatin).
[0025] Related genes can encode functionally equivalent
polypeptides which inhibit allograft rejection as described for the
anti-angiogenic agents of the present invention. For example,
functionally equivalent polypeptides can increase graft survival
rates (Examples 1 and 2; FIGS. 2 and 3), or decrease mean
arteriosclerosis score of blood vessels in the allograft (Example
2), or both.
[0026] Thus, polypeptides or peptides which are structurally or
functionally equivalent to known polypeptide or peptide
anti-angiogenic agents (e.g., angiostatin, endostatin) can be used
in the methods of the present invention. Such polypeptides can show
nucleic acid or amino acid sequence identity with the
anti-angiogenic agents described herein (e.g., angiostatin,
endostatin). In one embodiment structurally equivalent
anti-angiogenic agents share at least about 40% sequence identity
(nucleic acid or amino acid identity) with the corresponding native
sequence, preferably, at least about 50-60% sequence. In a more
preferred embodiment, the percent sequence identity is at least
about 65-75%, and still more preferably, at least about 80%.
[0027] Methods to determine the identity of structurally equivalent
polypeptides by sequence identity are routine and familiar to one
of skill in the art. Nucleic acid or amino acid sequence identity
can be determined using database search strategies well known in
the art including, for example, Basic Local Alignment Search Tool
(BLAST) (Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1990))
and FASTA (Pearson, W. R., et al., Proc. Natl. Acad. Sci. U.S.A.
85:2444-2448 (1988)) algorithms. Structurally equivalent nucleic
acids, including DNA or RNA, can also be detected and/or isolated
by specific hybridization (e.g., under high stringency conditions).
"Stringency conditions" for hybridization is a term of art which
refers to the conditions of temperature, salt and buffer
concentrations which permit hybridization of a particular nucleic
acid to a second nucleic acid. "High stringency conditions",
"moderate stringency conditions", and "low stringency conditions"
for nucleic acid hybridizations are explained on pages
2.10.1-2.10.16 and pages 6.3.1-6.4.10 in Current Protocols in
Molecular Biology (Ausubel, F. M. et al., "Current Protocols in
Molecular Biology", John Wiley & Sons, (1998)) the teachings of
which are hereby incorporated by reference.
[0028] It is also envisioned that biologically active fragments of
the polypeptide ant-angiogenic agents described herein can be used
to inhibit allograft rejection. The term "fragment" is intended to
encompass a portion of the anti-angiogenic agent; or, in the case
of a polypeptide anti-angiogenic agent, a nucleic acid molecule or
sequence encoding an anti-angiogenic polypeptide which is at least
approximately 25 contiguous nucleotides to at least approximately
50 contiguous nucleotides or longer in length. "Nucleic acid
molecule" is meant to refer to chains of nucleotides joined
together by phosphodiester bonds to form nucleic acid sequences.
The nucleic acid molecules can be double stranded or single
stranded and can be deoxyribonucleotide (DNA) molecules, such as
cDNA or genomic DNA, or ribonucleotide (RNA) molecules. The
anti-angiogenic activity of such fragments can be determined using
techniques well-known to the skilled artisan (O'Reilly, M. S., et
al., Cell 79:315 (1994); Folkman, J., New Eng J. Med 333:1757
(1995); Holmgren, L., et al., Nature Medicine 1:149 (1995);
O'Reilly, M. S., et al., Cell 88:277 (1997); and O'Reilly, M. S.,
et al., U.S. Pat. No. 5,639,725). The ability of fragments of
anti-angiogenic agents to inhibit allograft rejection can also be
determined using routine, art-recognized criteria and methods
described herein including, for example, graft survival rates and
arteriosclerosis scores of blood vessels of the allografts. (See
Examples 1 and 2; and Russell, M. E., et al., J. Clin. Invest.
97:833 (1996))
[0029] In another aspect of the invention, peptidomimetics
(molecules which are not polypeptides, but which mimic aspects of
their structures to bind to the same site), that are based upon the
polypeptide anti-angiogenic agents of the present invention (e.g.,
angiostatin, endostatin) which can inhibit allograft rejection in a
similar manner (e.g., increased survival rate or decreased mean
arteriosclerosis score as discussed in Examples 1 and 2), can also
be used as anti-angiogenic agents.
[0030] The proteins, polypeptides and peptides of the present
invention can comprise naturally-occurring amino acids (e.g.,
L-amino acids), non-naturally-occurring amino acids (e.g., D-amino
acids), and small molecules that biologically mimic the
anti-angiogenic polypeptides or peptides, and are referred to
herein as peptide analogs, derivatives or mimetics (Saragovi, H.
U., et al., BioTechnology, 10:773-778 (1992)). The protein,
polypeptide, peptidomimetic of the present invention can be in a
linear or cyclic conformation.
[0031] The anti-angiogenic agents of the present invention can
comprise either the 20 naturally occurring amino acids or other
synthetic amino acids. Synthetic amino acids encompassed by the
present invention include, for example, naphthylalanine,
L-hydroxypropylglycine, L-3,4-dihydroxyphenylalanyl, .alpha.-amino
acids such as L-.alpha.-hydroxylysyl and D-.alpha.-methylalanyl,
L-.alpha.-methyl-alanyl, .beta. amino-acids such as .beta.-analine,
and isoquinolyl.
[0032] D-amino acids and other non-naturally occurring synthetic
amino acids can also be incorporated into the anti-angiogenic
agents of the present invention. Such other non-naturally occurring
synthetic amino acids include those where the naturally occurring
side chains of the 20 genetically encoded amino acids (or any L or
D amino acid) are replaced with other side chains, for instance
with groups such as alkyl, lower alkyl, cyclic alkyl, amide,
hydroxy, carboxy, lower alkyl carboxylic acid ester, sulfonic acid,
a lower alkyl sulfonic acid ester or a phosphorous acid or ester
thereof.
[0033] These peptide mimetics possess biological activity (e.g.,
anti-angiogenic activity) similar to the biological activity of the
corresponding polypeptide or peptide compound (e.g., angiostatin,
endostatin), but can possess a "biological advantage" over the
corresponding polypeptide or peptide anti-angiogenic agent with
respect to one, or more, of the following properties: solubility,
stability, and susceptibility to hydrolysis or proteolysis.
[0034] Methods for preparing peptide mimetics include modifying the
N-terminal amino group, the C-terminal carboxyl group, and/or
changing one or more of the amino linkages in the peptide to a
non-amino linkage. Modifications of peptides to produce peptide
mimetics are described in U.S. Pat. Nos. 5,643,873 and 5,654,276,
the teachings of which are incorporated herein by reference. The
anti-angiogenic agents can also be cyclic peptide mimetics. Such
cyclic test substances can be produced using known laboratory
techniques (e.g., as described in U.S. Pat. No. 5,654,276, the
teachings of which are herein incorporated in their entirety by
reference).
[0035] These peptidomimetic compounds can be manufactured by
art-known and art-recognized methods. Determining an appropriate
chemical synthesis route for the peptide mimetics can generally be
readily identified using no more than routine skill.
[0036] The anti-angiogenic agents of the present invention can be
synthesized using standard laboratory methods that are well-known
to those of skill in the art, including standard solid phase
techniques. Anti-angiogenic agents comprising polypeptides of
naturally occurring amino acids (e.g., angiostatin, endostatin) can
also be produced by recombinant DNA techniques known to those of
skill.
[0037] Anti-angiogenic agents can also be small organic molecules,
for example, molecules with a molecular weight less than about 1000
atomic mass units (amu), with the proviso that the anti-angiogenic
agent is not thalidomide when acute allograft rejection is being
treated. In one aspect, the small organic molecule has a molecular
weight between about 275 and about 1000 amu.
[0038] Thus, it is to be understood that the method of inhibiting
allograft rejection described herein can be performed with
anti-angiogenic agents other than those specifically described
herein. As such, anti-angiogenic agents not yet discovered can also
be used in this method of treatment, once it has been determined
that the agent is anti-angiogenic (e.g., inhibits or suppresses the
formation of new blood vessels) and inhibits allograft rejection.
These anti-angiogenic agents are also within the scope of the
present invention.
[0039] The method of the present invention can be used to inhibit
allograft rejection in a subject with transplanted solid organs or
tissues. Examples of solid organs include heart, kidney, lungs,
stomach, pancreas, liver, intestines, esophagus, and skin. Examples
of tissues include bone marrow, corneas. The method of the present
invention is especially useful for suppressing allograft rejection
of transplanted heart and kidneys. It is also envisioned that the
methods of the invention can be used to inhibit rejection of
transplanted limbs in a subject.
[0040] An "effective amount" is the quantity of an anti-angiogenic
agent which results in a longer life expectancy for subjects with
an organ or tissue transplant compared with subjects who did not
undergo the treatment. Alternatively or additionally, "an effective
amount" is the quantity of an anti-angiogenic agent which results
in a decreased manifestation or inhibition, or reversal of
pathological and/or the symptoms of allograft rejection, such as
those which prolong allograft survival, compared with subjects who
did not undergo the treatment. In the case of chronic allograft
rejection, improvements in graft arteriosclerosis scores (Example
2) can indicate an effective amount of an anti-angiogenic
agent.
[0041] It will be appreciated that the actual effective amount of
an anti-angiogenic agent in a specific case to inhibit allograft
rejection can vary according to the specific anti-angiogenic agent
being utilized, the particular composition formulated, the mode of
administration, the type of organ or tissue transplant and on the
characteristics of the subject, such as general health, age, sex,
body weight and tolerance to drugs. Dosages for a particular
subject can be determined by one of skill in the art using
conventional considerations (e.g., by means of appropriate,
conventional pharmacological protocol). Typically, an effective
amount of the anti-angiogenic agents can range from about 1 mg per
kilogram body weight per day to about 1000 mg per kilogram body
weight per day for an adult. Preferably, the dosage ranges from
about 1 mg per kilogram body weight per day to about 100 mg per
kilogram body weight per day.
[0042] A "subject" is preferably a mammal. The term "mammal", as
defined herein, refers to any vertebrate animal, including
monotremes, marsupials and placental, that suckle their young and
either give birth to living young (eutherian or placental mammals)
or are egg-laying (metatherian or nonplacental mammals). Examples
of mammalian species include primates (e.g., humans, monkeys,
chimpanzees, baboons), rodents (e.g., rats, mice, guinea pigs,
hamsters), companion animals (e.g., dogs, cats), ruminants (e.g.,
cows, horses) and monogastric farm animals (e.g., pigs).
[0043] An anti-angiogenic agent can be co-administered to a subject
with other pharmacologically active agents; for example, with other
immunosuppressive agents used to suppress allograft rejection. The
term "immunosuppressive agent" as used herein is meant to refer to
any substance (e.g., polypeptide, peptide, peptidomimetic, small
organic molecule, sugar, lipid) which inhibits or prevents the
development of an immunological response. "Co-administration" as
used herein is intended to refer to the administration of both an
anti-angiogenic agent and an immunosuppressive agent either
simultaneously or in sequence. Co-administration can result in
synergistic or additive effects of either the anti-angiogenic agent
alone or the immunosuppressive agent alone in preventing allograft
rejection. For example, the anti-angiogenic agent can be
administered to the subject at the same time as the
immunosuppressive agent or prior to or after the administration of
the immunosuppressive agents. The immunosuppressive agent can be,
for example, cyclosporin, or FK506, or steroids, or
antiproliferative agents (such as azathioprine, mycophenolate
moefitil), or any combination thereof. Other suitable
immunosuppressive agents can also be those currently under
evaluation in Phase III human clinical trials (e.g.,
Rapamycin).
[0044] In the methods of the present invention, anti-angiogenic
agents can be formulated into compositions with an effective amount
of the anti-angiogenic agent as the active ingredient. Such
compositions can also comprise a pharmaceutically acceptable
carrier, and are referred to herein as pharmaceutical compositions.
The anti-angiogenic agent compositions of the present invention can
be administered intramuscularly, subcutaneously, intraperitoneally,
intravenously, parenterally, orally, nasally, by inhalation, by
implant, by injection, or by suppository. Anti-angiogenic agents
which resist proteolysis can be administered orally, for example,
in capsules, suppositories, suspensions or tablets. For example,
the anti-angiogenic agents angiostatin, endostatin, AGM-1470 and
TNP-470, are preferably administered subcutaneously. The
anti-angiogenic agent composition can be administered in a single
dose or in more than one dose over a period of time to achieve a
level of inhibition which is sufficient to confer the desired
effect.
[0045] Anti-angiogenic agents in pharmaceutical compositions are
administered to the individual in conjunction with an acceptable
pharmaceutical carrier. Suitable pharmaceutical carriers may
contain inert ingredients which do not interact with the
anti-angiogenic agent. Standard pharmaceutical formulation
techniques may be employed such as those described in Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
Suitable pharmaceutical carriers include, but are not limited to
sterile water, physiological saline, bacteriostatic saline (saline
containing about 0.9% mg/ml benzyl alcohol), other saline solutions
(such as Ringer's-lactate solution, Hank's solution,
phosphate-buffered saline), alcohols, polyethylene glycols,
gelatin, carbohydrates such as lactose, amylose or starch,
magnesium stearate, talc, silicic acid, viscous paraffin, fatty
acid esters, hydroxymethylcellulose, or polyvinyl pyrolidone.
Methods for encapsulating compositions (such as in a coating of
hard gelatin or cyclodextran) are known in the art (Baker, et al.,
Controlled Release of Biological Active Agents, John Wiley and
Sons, 1986). The pharmaceutical preparations can be sterilized and
desired, mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, and/or aromatic
substances and the like which do not deleteriously react with the
anti-angiogenic agents. The anti-angiogenic agents can also be
combined where desired with other active substances, e.g., enzyme
inhibitors, to reduce metabolic or proteolytic degradation.
[0046] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, or implants,
including suppositories. Ampules are convenient unit dosages.
[0047] The following Examples are offered for the purpose of
illustrating the present invention and are not to be construed to
limit the scope of this invention. The teachings of all references
cited herein are hereby incorporated by reference.
EXAMPLE 1
[0048] Suppression of Acute Allograft Rejection in Rat Models
[0049] The Lewis strain (hereinafter "LEW") (Harlan Sprague
Laboratories) and Fisher strain (hereinafter "F344") (Harlan
sprague Laboratories) of rats were used to study acute and chronic
rejection. Procedures for transplantation of organs (e.g., heart,
kidney) and techniques to evaluate organ and animal survival as
well as criteria to score indices of graft rejection such as
arteriosclerosis are routine, well established and art-recognized
described by, for example, Adams, D. H., et al., Immunol. Rev.
134:5 (1993); Tilney, N. L., et al., Transplant Proc. 25:861
(1993); Nadeau, K. C., et al., Proc. Natl. Acad. Sci. USA 92:8729
(1995); Russell, M. E., et al., J. Clin. Invest. 97:833 (1996); and
Azuma, H., et al. , Proc. Natl. Acad. Sci. USA 93:12439 (1997), the
teachings of which are incorporated herein in their entirety.
[0050] F344 recipient rats underwent heterotopic abdominal
transplantation of hearts from LEW rats. Following surgery, one
group of allograft recipients was treated with a subcutaneous
injection of 20 mg/kg TNP-470, dissolved in sterile saline, every
other day for 30 days. A control group received no treatment.
[0051] Transplant recipients were monitored for graft survival by
external palpation of the transplant heart. A palpable heartbeat
was indicative of graft survival. The result of TNP-470 treatment
on survival of transplant recipients is depicted in FIG. 2. Greater
than 50% of grafts from TNP-470 treated animals survived for at
least approximately 22 days and as long as approximately 37 days,
whereas acute graft rejection developed in all of the controls
animals. None of the cardiac grafts from control animals survived
25 days. In this animal model, typically grafts fail due to acute
allograft rejection within the first 3 weeks (Adams, D. H., et al.,
Immunol. Rev. 134:5 (1993); and Russell, M. E., et al., J. Clin.
Invest. 97:833 (1996)). These results show that TNP-470 suppresses
acute allograft rejection.
EXAMPLE 2
[0052] Suppression of Chronic Allograft Rejection in Rat Models
[0053] Cyclosporine treatment can prevent the initial acute
allograft rejection and facilitate long term engraftment (Nadeau,
K. C., et al., Proc. Natl. Acad. Sci. USA 92:8729 (1995); Azuma,
H., et al., Proc. Natl. Acad. Sci. USA 93:12439 (1997)). However,
once cyclosporine treatment is discontinued, animals typically
develop progressive chronic allograft rejection (Russell, M. E., et
al., J. Clin. Invest. 97:833 (1996)). Rats undergoing chronic
allograft rejection develop moderate lymphocyte as well as monocyte
and macrophage infiltrates in the interstitium and perivascular
space in association with arteriovascular lesions in the allograft
(Adams, D. H., et al., Immunol. Rev. 134:5 (1993); Tilney, N. L.,
et al., Transplant Proc. 25:861 (1993); Russell, M. E., et al., J.
Clin. Invest. 97:833 (1996); and Azuma, H., et al., Proc. Natl.
Acad. Sci. USA 93:12439 (1997)). This characteristic
arteriovascular lesion begins as early as day 7-14. In the early
phase of chronic allograft rejection (between 28 and 75 days after
organ transplantation), the intima of blood vessels in the graft
thickens primarily as a result of macrophage accumulation and
smooth muscle cell proliferation. In the later phase of chronic
allograft rejection (between 90 and 120 days after organ
transplantation), smooth muscle cell proliferation in vessels of
the graft is very pronounced and the arteriovascular lesion becomes
characteristic of chronic allograft rejection. Accumulation of
macrophages and the activation of local macrophages is critical in
the development of this phase of chronic allograft rejection in
this animal model.
[0054] F344 recipient rats which had undergone heterotopic
abdominal transplantation of hearts from LEW rats were used as a
model for studying the effect of the co-administration of TNP-470
(an anti-angiogenic agent) and cyclosporine (an immunosuppressive
agent) in inhibiting chronic allograft rejection.
[0055] F344 transplant recipient rats were divided into three
groups (Prevention, Therapeutic and Interruption Groups)
Cyclosporine (5 mg/kg/day) and TNP-470 (20 mg/kg/every other day)
were administered subcutaneously. The "Prevention Group" (or
"Control Group") was treated for 30 days with cyclosporine alone
and then received no additional treatment for days 30-120 following
grafting. Animals in the "Therapeutic Group" were treated with
cyclosporine for 30 days after surgical transplantation to prevent
acute allograft rejection followed by treatment with TNP-470 until
day 120 after grafting. Animals in the "Interruption Group" did not
receive any treatment for 30 days (to develop acute allograft
rejection); after which they were treated with TNP-470 from days
30-120 following graft transplantation. The Therapeutic Group is
particularly applicable for the treatment of allograft rejection in
humans.
[0056] Grafts were monitored for survival by palpation to detect a
heartbeat and analyzed histologically using well-established
methods (Russell, M. E., et al., J. Clin. Invest. 97:833 (1996);
and Azuma, H., et al., Proc. Natl. Acad. Sci. USA 93:12439 (1997)).
The results for this study are depicted in FIG. 3. In addition,
cardiac grafts were harvested from recipients, fixed in 10%
buffered formalin, embedded in paraffin and analyzed
morphologically to assess the graft and the degree of occlusion of
the arterial lumen (e.g., an arteriosclerosis score). Methods for
tissue harvesting and analysis are well-known, routine and familiar
to one of ordinary skill in the art.
[0057] In the Control Group, animals were treated with a short
course of cyclosporine for 30 days. Cyclosporine was then
discontinued for 90 additional days. Analysis of the grafts from
animals in the Control Group revealed chronic allograft rejection
and occlusion of blood vessels in the cardiac graft (FIG. 3). In
the Interruption Group, administration of TNP-470 for the last 90
days inhibited the development of chronic rejection and increased
graft survival (FIG. 3--approximately 75% survival at day 120 for
the Interruption Group versus approximately 45% survival for the
Control Group) and improved arteriosclerosis scores of allografts
(2.06.+-.0.58 (n=5) for the Control Group versus 0.96.+-.0.14
(n=10) for the Interruption Group). An arteriosclerosis score of 0
indicates 0% occlusion of the cardiac vessels; 1 indicates 20%
occlusion; 2 indicates 40% occlusion; 3 indicates 60% occlusion; 4
indicates 80% occlusion; and 5 indicates 100% occlusion.
EQUIVALENTS
[0058] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
* * * * *