U.S. patent application number 10/023545 was filed with the patent office on 2002-08-22 for treatment of post-angioplasty restenosis and atherosclerosis with ras antagonists.
Invention is credited to George, Jakob, Keren, Gad, Kloog, Yoel.
Application Number | 20020115696 10/023545 |
Document ID | / |
Family ID | 26837949 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115696 |
Kind Code |
A1 |
Kloog, Yoel ; et
al. |
August 22, 2002 |
Treatment of post-angioplasty restenosis and atherosclerosis with
ras antagonists
Abstract
Disclosed are methods for inhibiting Ras activity such as cell
proliferation associated with vascular injury such as
post-angioplasty restenosis and atherosclerosis. Preferred Ras
antagonists are S-trans-trans farnesylthiosalicylic acid (FTS) and
structurally related compounds (or analogs) thereof.
Inventors: |
Kloog, Yoel; (Herzliya,
IL) ; Keren, Gad; (Kiryat Ono, IL) ; George,
Jakob; (Petach Tikva, IL) |
Correspondence
Address: |
LERNER, DAVID, LITTENBERG,
KRUMHOLZ & MENTLIK
600 SOUTH AVENUE WEST
WESTFIELD
NJ
07090
US
|
Family ID: |
26837949 |
Appl. No.: |
10/023545 |
Filed: |
December 18, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10023545 |
Dec 18, 2001 |
|
|
|
09597332 |
Jun 19, 2000 |
|
|
|
60140192 |
Jun 18, 1999 |
|
|
|
Current U.S.
Class: |
514/345 ;
514/352; 514/567 |
Current CPC
Class: |
A61K 31/185 20130101;
A61K 31/44 20130101; A61K 31/00 20130101; A61K 31/606 20130101;
A61K 31/196 20130101; A61K 31/465 20130101; A61K 31/18 20130101;
A61K 31/60 20130101; A61K 31/21 20130101 |
Class at
Publication: |
514/345 ;
514/567; 514/352 |
International
Class: |
A61K 031/44; A61K
031/195 |
Claims
1. A method of inhibiting Ras activity in a mammal suffering from
or at risk of vascular injury, comprising: administering to a
patient a Ras antagonist in an amount effective to inhibit the
activity.
2. The method of claim 1 wherein the Ras antagonist is represented
by the formula 6wherein R.sup.1 represents farnesyl, geranyl or
geranyl-geranyl; Z represents C--R.sup.6 or N; R.sup.2 represents
H, CN, the groups COOR.sup.7, SO.sub.3R.sup.7, CONR.sup.7R.sup.8,
COOM, SO.sub.3M and SO.sub.2NR.sup.7R.sup.8, wherein R.sup.7 and
R.sup.8 are each independently hydrogen, alkyl or alkenyl, and
wherein M is a cation; R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are
each independently hydrogen, carboxyl, alkyl, alkenyl, aminoalkyl,
nitroalkyl, nitro, halo, amino, mono- or di-alkylamino, mercapto,
mercaptoalkyl, axido, or thiocyanato; X represents O, S, SO,
SO.sub.2, NH or Se; and the quaternary ammonium salts and N-oxides
of the compounds of said formula when Z is N.
3. The method of claim 1 wherein the Ras antagonist is
farnesyl-thiosalicyclic acid (FTS).
4. The method of claim 1 wherein the Ras antagonist is
2-chloro-5-farnesylaminobenzoic acid (NFCB).
5. The method of claim 1 wherein the Ras antagonist is farnesyl
thionicoatinic acid (FTN).
6. The method of claim 1 wherein the Ras antagonist is
5-fluoro-FTS.
7. The method of claim 1 wherein the Ras antagonist is
5-chloro-FTS.
8. The method of claim 1 wherein the Ras antagonist is
4-chloro-FTS.
9. The method of claim 1 wherein the Ras antagonist is
S-farnesyl-methylthiosalicylic acid.
10. The method of claim 1 wherein the Ras antagonist is
administered parenterally.
11. The method of claim 1 wherein the Ras antagonist is
administered orally.
12. The method of claim 1 wherein the Ras antagonist is
administered prophylactically.
13. The method of claim 1 wherein the Ras antagonist is
administered in a formulation containing a cyclodextrin.
14. The method of claim 14 wherein the Ras is activated Ras.
15. The method of claim 1 wherein the vascular injury is
post-angioplasty restenosis.
16. The method of claim 15 wherein the Ras antagonist is
administered prophylactically.
17. The method of claim 1 wherein the vascular injury is
atherosclerosis.
18. A method of displacing Ras from its cell membrane anchor in a
mammal suffering from or at risk of vascular injury, comprising
administering to the mammal a Ras antagonist in an amount effective
to effect said displacing, wherein the Ras antagonist is
represented by the formula 7wherein R.sup.1 represents farnesyl,
geranyl or geranyl-geranyl; Z represents C-R.sup.6 or N; R.sup.2
represents H, CN, the groups COOR.sup.7, SO.sub.3R.sup.7,
CONR.sup.7R.sup.8, COOM, SO.sub.3M and SO.sub.2NR.sup.7R.sup.8,
wherein R.sup.7 and R.sup.8 are each independently hydrogen, alkyl
or alkenyl, and wherein M is a cation; R.sup.3, R.sup.4, R.sup.5
and R.sup.6 are each independently hydrogen, carboxyl, alkyl,
alkenyl, aminoalkyl, nitroalkyl, nitro, halo, amino, mono- or
di-alkylamino, mercapto, mercaptoalkyl, axido, or thiocyanato; X
represents O, S, SO, SO.sub.2, NH or Se; and the quaternary
ammonium salts and N-oxides of the compounds of said formula when Z
is N.
19. The method of claim 18 wherein said Ras antagonist is
farnesyl-thiosalicyclic acid (FTS).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/597,332, filed Jun. 19, 2000, which claims
priority under 35 U.S.C. .sctn.119(e) from U.S. application Ser.
No. 60/140,192, filed Jun. 18, 1999. The contents of these two
applications are hereby incorporated herein by reference in their
entireties.
FIELD OF THE INVENTION
[0002] The present invention relates to the inhibition of the onset
of or the treatment of non-malignant diseases, and particularly
diseases having pathologies involving Ras-induced proliferation of
cells.
BACKGROUND OF THE INVENTION
[0003] Autoimmune diseases include disorders involving dysfunction
of the immune system, which mediates tissue damage. Any organ may
be affected by such processes through precipitation of immune
complexes, cellular immunity, or inappropriate generation or action
of immuno-hormones such as cytokines. Epidemiologically, autoimmune
diseases are significant because of the numbers of patients that
they affect and the serious morbidity and mortality that they
cause. Common chronic systemic diseases in this group include
diabetes mellitus, thyroid disease, rheumatoid arthritis, systemic
lupus erythmatosus (SLE), primary antiphospholipid syndrome (APS),
and a variety of diseases that affect the central nervous system.
Neurological autoimmune diseases include disorders specific to the
nervous system such as myasthenia gravis, Lambert Eaton myasthenic
syndrome, Guillain-Barre syndrome, polymyositis, and multiple
sclerosis. In addition, there are neurological complications of the
systemic autoimmune diseases. Factors predisposing to autoimmune
diseases include genetic predisposition and environmental agents
such as certain infections and pharmaceutical products. Such
factors result in pathological activation of the immune response in
susceptible individuals, which is generally controlled by T
lymphocytes (T cells). The activation of T cells and B subtypes,
involves a complex interaction of cell surface receptors resulting
in equally complex signal transduction pathways which eventually
affect gene regulation. Full activation of lymphocytes requires
parallel stimulation of several signal transduction pathways. See
Ohtsuka et al., Biochim. Biophys. Acta. 1310:223-232 (1996).
[0004] Although there is growing understanding about the function
of T cells in the immune response, this knowledge has not explained
the basis of most autoimmune diseases. There are still questions to
be resolved such as how tolerance to self in normal individuals is
maintained; how tolerance is broken in autoimmunity; and which
autoantigens trigger the immune system to produce specific
diseases. A recent review by V. Taneja and C. S. David (J. Clin.
Invest. 101:921-926 (1998)) provides an overview of important
issues in this field and emphasizes how the generation of
transgenic mice expressing functional HLA molecules is important
for understanding the function of certain molecules in the
induction of autoimmune disease, as well as circumvention of the
xenogenic barrier. Regardless of the mechanisms involved in
induction of autoimmune disease or the rejection of grafts, the
common pathway for these events includes activation of a relatively
small number of T lymphocytes.
[0005] Several immunosuppressive and immunomodulating treatments
have been tested and subsequently applied in the treatment of
autoimmune diseases. Gana-Weisz, M., HaMai, R., Marciano, D.,
Egozi, Y., Ben-Baruch, G., and Kloog, Y. The Ras antagonist
S-farnesylthiosalicylic acid induces inhibition of MAPK activation.
Biochem. Biophys. Res. Commun. 1997; 239: 900-904; Marciano, D.,
Aharonson*, Varsano, T., Haklai, R., and K O, Y. Novel inhibitors
of the prenylated protein methyltransferase reveal distinctive
structural requirements. Bioerg. Med. Chem. Lett. 1997; 7,
1709-1714; Paterson P. Y. (1978) The demyelinating diseases:
clinical and experimental studies in animals and man. In:
Immunological Diseases, 3rd Edition, (ed. by M. Smater, N.
Alexander, B. Rose, W. B. Sherman, D. W. Talmage and J. H. Vaughn)
p. 1400. Little, Brown and Company, Boston.
[0006] The main drawback of immunosuppressive modalities is that
the induction of generalized suppression of all T-cells and immune
functions is associated with long-term and cumulative side effects.
In addition, it is now believed that broad suppression of immune
cells may also cancel or neutralize the potential beneficial
effects of down-regulatory cells such as suppressors and suppresor
inducers or cytokines such as IL-10, on the autoimmune lymphocytes.
Karussis, et al., supra; Gana-Weisz, et al., supra, Lieder, O., T.
Reshef, E. Berauud, A. Ben-Nun, and I. R. Cohen. 1988,
Anti-idiotypic network induced by T cell vaccination against
experimental autoimmune encephalomyelitis, Science 239:181; Varela,
F. J., and A. Coutinho, 1991, Second generation immune networks,
Immunol. Today 12:159; Cohen, I. R., and D. B. Young. 1991,
Autoimmunity, microbial immunity and the immunological homunculus,
Immunol. Today 12:105; Lehmann, D., D. Karussis, R. Mizrachi-Koll,
A. S. Linde, and O. Abramsky, 1997, Inhibition of the progression
of multiple sclerosis by linomide is associated with upregulation
of CD4+/CD45RA+ cells and downregulation of CD4+/CD45RO.vertline.
cells, Clin Immunol Immunopathol 85:202.
[0007] Therefore, current approaches for the treatment of
autoimmune diseases advocate the use of immunomodulators or
specific immunosuppressing medications. The goal of such research
is specific suppression of only the lymphocytes with the autoimmune
potential. The search for such specific suppressors is a formidable
challenge, particularly considering the complex networks of signal
transduction pathways associated with lymphocyte growth and
differentiation, where many such pathways are common to all
lymphoid lineages and to other cells.
[0008] In addition to autoimmune disease, there are several other
diseases in which proliferation of normal cells other than T-cells
constitutes part of the pathology.
SUMMARY OF THE INVENTION
[0009] An aspect of the present invention is directed to a method
of inhibiting Ras activity associated with vascular injury which
entails administration of a Ras antagonist in an amount effective
to inhibit the Ras activity. In a preferred embodiment, the present
invention is directed to a method of inhibiting Ras activity
associated with post-angioplasty restenosis, by administering to a
patient suffering from or at risk of restenosis, a Ras antagonist
in an amount effective to inhibit the Ras activity. In this
embodiment, a primary targeted activity of Ras in connection with
restenosis is the proliferation of vascular smooth muscle cells. In
another embodiment, the present invention is directed to a method
for inhibiting Ras activity in a patient with atherosclerosis or a
patient at risk for atherosclerosis. The patient is administered a
Ras antagonist in an amount effective to inhibit the Ras
activity.
[0010] Common to vascular injury is Ras. This protein becomes
activated by a series of biochemical events after it binds or docks
to a particular site on the inner surface of the cell membrane. The
activation of Ras then leads to another series of inter-related
biochemical reactions or signal transduction cascades that
ultimately produce cell growth and division. The Ras antagonists of
the present invention are believed to inhibit Ras activity by
inhibiting or reducing the binding of Ras to the cell membrane,
which in turn reduces or inhibits the unwanted cell
proliferation.
[0011] Preferred Ras antagonists include farnesyl thiosalicylic
acid (FTS) and structurally related compounds or analogs thereof,
which are believed to function by displacing or dislodging Ras from
its membrane anchor. Thus, in these respects, the invention may
also be described in terms of a method of displacing or dislodging
Ras from its cell membrane anchor in a mammal suffering from
vascular injury, by administering a Ras antagonist in an amount
effective to displace or dislodge Ras from its cell membrane
anchor. Ras antagonists may be administered parenterally or orally.
In a particularly preferred embodiment, the Ras antagonist is
formulated for oral or parenteral administration by complexation
with cyclodextrin or as nanocrystals.
[0012] FTS has been shown to affect the growth of cancers in
animals mediated by oncogenic forms of Ras, including melanomas and
lung, colon, pancreatic, uterine and Merkel cell cancers. The
results of these experiments showed that FTS achieved greater than
90% reduction in cancer cell growth in some cases without
significant toxic effects associated with standard cancer
chemotherapy. The results also showed that similar dosages of FTS
used in cancer treatment had very little, if any, effect on normal
cells. See Aharonson, et al., Biochim. Biophys. Acta 1406:40-50
(1998). It was known that the proliferation of normal cells
associated with various non-malignant diseases (e.g., T-cells
associated with various autoimmune diseases, stellate cells
associated with cirrhosis and smooth muscle cells associated with
post angioplasty restenosis) was mediated at least in part by
normal or non-oncogenic Ras. Still, it was not expected that FTS
and similarly active compounds could be used to achieve a
therapeutic benefit in patients afflicted with diseases
characterized by proliferations of normal cells.
[0013] The methods of the present invention offer several
advantages over current immunosuppressive and immunomodulatory
treatment modalities. They are generally non-cytotoxic to all
dividing cells, non-toxic at therapeutically effective doses, and
do not result in general immunosuppression.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIGS. 1A and B are bar graphs showing that FTS suppresses
early and advanced lesions in apoE deficient chow fed (A) and high
fat diet fed (B) mice.
[0015] FIG. 2A is a photograph of an immunoblott with Pan-Ras
Antibody showing amounts of total Ras (upper panel) and Ras-GTP
(lower panel) in aortas from FTS-treated and control mice.
[0016] FIG. 2B is a bar graph showing data from a densitometric
analysis of aorta samples obtained from 8 control and 6 FTS-treated
mice.
[0017] FIGS. 3A, 3B and 3C are bar graphs showing that FTS reduces
cellular and humoral immune responses to oxLDL in mice, wherein
FIG. 3A expresses the result in CPM, FIG. 3B expresses the results
in optical density (OD 405 nm(.times.1000), and FIG. 3C shows
anti-oxLDL isotype class distribution as a fraction of total
IgG-oxLDL binding as evaluated by ELISA.
[0018] FIGS. 4A and 4B are bar graphs showing that treatment with
FTS does not alter atherosclerotic plaque composition based upon
staining with monoclonal antibodies to CD3, Mac-1 and IL-2-R, using
plaque-positive cells from fatty streaks (FIG. 4A) and advanced
lesions (FIG. 4B).
[0019] FIG. 5A is a bar graph showing that FTS reduces VCAM-1
expression in atherosclerotic plaques in aortic sinus early and
advanced lesions compared to control mice.
[0020] FIG. 5B is a bar graph showing that FTS reduces NF-KB
expression in atherosclerotic plaques in aortic lesions obtained
from FTS compared to control mice.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The methods of the present invention are directed to the
treatment of non-malignant diseases, pathological states or other
disorders involving vascular injury that feature or otherwise
include Ras activity such as Ras-induced proliferation of cells.
One example of vascular injury is post-angioplasty restenosis.
Here, the insertion of an intra-arterial stent causes damage,
release of growth factors and proliferation of normal smooth muscle
cells.
[0022] Atherosclerosis is another example of a non-malignant
disease state characterized by abberant Ras activity. It involves
uninhibited accumulation of lipids in the vessel wall resulting in
considerable morbidity and mortality. Although conclusive data is
not yet available, it is becoming apparent that no single mechanism
is solely responsible for the development of the atherosclerotic
plaque, but rather an interplay of factors. See, Ross, N. Eng. J.
Med. 340:115-126 (1999). In the recent years, a growing body of
evidence has been presented supporting the participation of the
immune system in the initiation and progression of atherosclerosis,
Ross, supra; Libby, et al. Lab. Invest. 64: 5-11 (1991). This
notion is based on the idea that atherosclerosis is a form of a
chronic inflammatory state that involves interaction among
endothelial cells, macrophages, T lymphocytes and smooth muscle
cells, Ross, supra. According to the currently acceptable views and
without intending to be bound by any particular theory of
operation, turbulent flow within the arterial system imposes stress
on the vessel walls. Consequently, expression of endothelial
adhesion molecules ensues, resulting in recruitment of monocytes
that transform to macrophages. These macrophages which express
scavenger receptors initiate an unregulated intake of modified
lipoproteins (oxidized LDL) that culminate in plaque formation,
Ross, supra. Many of the diverse signals triggered by receptors
that are involved in the atherosclerotic process would require
intact Ras pathways, which play a key role in the control of cell
growth and differentiation, cell migration and adhesion and cell
survival, Boguski et al., Nature (London) 366:643-654 (1993);
Rebollo et al., Blood 94:2971-2980 (1999); Lange-Carter et al.,
Science 265:1458-1461 (1994). Thus, aberrant Ras functions
contribute to the atherosclerotic process by analogy to the cases
of human tumors that harbor activated ras genes or over-express
receptors that activate Ras. In many human tumors, active Ras
provides not only growth signals but also strong survival signals
and migration potential. A number of experiments also support the
direct involvement of active Ras in atherosclerosis. Shear forces
resulting from turbulent flow and oxidative stress are
contributory, possibly essential, to atherogenesis and have been
shown to activate Ras, Li et al., Mol. Cell. Biol. 6:5947-54
(1996). Moreover, oxidized LDL (oxLDL), considered crucial in
atherogenesis, has been shown to induce Ras activation in smooth
muscle cells, Chatterjee et al., Glycobiology 7:703-710 (1997).
[0023] For Ras to be activated (i.e., turned on) to stimulate the
regulatory cascades, it must first be attached to the inside of the
cell membrane. Ras antagonist drug development aimed at blocking
the action of Ras on the regulatory cascades has focused on
interrupting the association of Ras with the cell membrane,
blocking activation of Ras or inhibiting activated Ras. The details
of the approaches to development of Ras antagonists are reviewed in
Kloog et al., Exp. Opin. Invest. Drugs 8(12):2121-2140 (1999).
Thus, by the term "Ras antagonist," it is meant any compound or
agent that targets one or more of these phenomena so as to result
in inhibition of Ras activity e.g., that results in cell
proliferation.
[0024] Preferred Ras antagonist is represented by formula I: 1
[0025] wherein
[0026] R.sup.1 represents farnesyl, geranyl or geranyl-geranyl; Z
represents C-R.sup.6 or N;
[0027] R.sup.2 represents H, CN, the groups COOR.sup.7,
SO.sub.3R.sup.7, CONR.sup.7R.sup.8, COOM, SO.sub.3M and
SO.sub.2NR.sup.7R.sup.8, wherein R.sup.7 and R.sup.8 are each
independently hydrogen, alkyl or alkenyl, and wherein M is a
cation;
[0028] R.sup.3, R.sup.4, R.sup.5 and R.sup.6 are each independently
hydrogen, carboxyl, alkyl, alkenyl, aminoalkyl, nitroalkyl, nitro,
halo, amino, mono- or di-alkylamino, mercapto, mercaptoalkyl,
axido, or thiocyanato; X represents O, S, SO, SO.sub.2, NH or Se;
and
[0029] the quaternary ammonium salts and N-oxides of the compounds
of formula (I) wherein Z is N.
[0030] These compounds represent farnesyl-thiosalicylic acid (FTS)
(e.g. S-trans, trans-FTS) and its analogs. In embodiments wherein
R.sup.2 represents H, R.sup.3 is preferably a carboxyl group. The
structures of FTS and two preferred analogs are as follows: 2
[0031] (ii) 2-chloro-5-farnesylaminobenzoic acid (NFCB): 3
[0032] (iii) farnesyl thionicoatinic acid (FTN): 4
[0033] These compounds are the subject of U.S. Pat. No. 5,705,528.
Methods of synthesizing the compounds are also disclosed
therein.
[0034] Yet other FTS analogs embraced by formula I include
5-fluoro-FTS, 5-chloro-FTS, 4-chloro-FTS and
S-farnesyl-methylthiosalicylic acid (FMTS). Structures of these
compounds are set forth below. 5
[0035] A particularly preferred agent is FTS. This compound
destabilizes the proper attachment of Ras to the cell membrane
which is promoted by the Ras carboxy terminal S-farnesyl cysteine
required for Ras signaling. FTS and other compounds of the present
invention mimic Ras anchorage moieties and disrupt the interactions
of Ras with the cell membrane in living cells without being
cytotoxic. Without intending to be bound by any particular theory
of operation, it is believed that its mechanism of action involves
a dual effect on membrane Ras where initially (within 30 min) FTS
releases Ras from constraints on its lateral mobility which is
followed by release of Ras into the cytoplasm from its isoprenoid
dependent anchorage proteins, and then by Ras degradation. The
reduced amount of Ras and the altered membrane mobility of Ras in
FTS-treated fibroblasts and human tumor cells are then manifested
in the inhibition of Ras-mediated signaling to the mitogen
activated protein kinase (MAPK) Erk. This is also believed to
explain why FTS inhibits proliferation of Ras-transformed cells and
inhibits the mitogenic stimuli of T-cell antigens and of growth
factors such as thrombin and EGF, PDGF and FGF.
[0036] Compounds useful in the present invention are further
disclosed in Marciano, et al., 1995, J. Med. Chem. 38, 1267;
Haklai, et al., 1998, Biochemistry 37, 1306; Casey, et al., Proc.
Natl. Acad. Sci. USA 86, 8323; Hancock et al., 1989, Cell 57, 1167
and Aharonson, et al., 1998, Biochim. Biophys. Acta. 1406, 40.
[0037] Other Ras antagonists useful in the present invention may be
identified by using the cell free membrane assays and cellular
assays described in WO 98/38509. This patent publication describes
several assay systems designed to determine the ability of a
candidate agent to dislodge activated Ras from cell membranes. In
general, the assay material that contains specific membranes having
a known and detectable quantity of Ras anchored thereto is exposed
to the candidate agent. The assay material is then separated into a
membrane fraction containing the membranes and a cytosolic fraction
of a balance of the material remaining after the specific membranes
are removed. A fraction of the known quantity of the labeled Ras
contained in the membrane and cytosolic fraction is determined as a
measure of the ability of the candidate agent to disrupt membrane
association of Ras. A particularly convenient source of activated
Ras-anchored membranes is membranes isolated from Ras transformed
cancer cells such as Panc-1 cells. The Ras remaining in the
membranes after exposure to a candidate agent can be measured by
standard immuno-assays using anti-Ras antibodies.
[0038] Yet other Ras antagonists useful in the present invention
may be identified in accordance with the procedures described in
commonly owned application no. PCT/IL01/00918, filed Oct. 1, 2001,
entitled Isoprenoid-dependent Ras anchorage (IDRA) proteins, which
claims priority from commonly owned provisional Patent Application
No. 60/237,858. This patent application describes several assays,
designed to identify canidate agents that will disassociate
activated Ras from one or more anchor proteins with which Ras
proteins associate in the cell membrane. In certain assays, cells
in which either or both Ras or anchor protein are labelled with GFP
or another protein tag are visualized. The ability of a candidate
drug to disrupt Ras/anchor membrane protein interactions is
detected by a change in the location of Ras and/or the anchor
protein in the cell. Examples of the change and location include
movement from the cytoplasm to the cell membrane, from the cell
membrane to cytoplasm, and from membrane to a cellular compartment
other than the cell membrane. In the cell-free variant of these
assays, the candidate agent disrupts the interaction of Ras with
its anchor(s), the dimerization of Ras, the dimerization of the
anchor or the interaction of Ras with Raf protein.
[0039] The Ras antagonists or agents of the present invention tend
to be substantially insoluble in water and saline solutions such as
PBS. Thus, in one embodiment, the agents are salified [e.g., an
Na.sup.+, K.sup.+ or NH.sup.+ form] and formulated with an organic
solvent such as alkyl gallates and butylated hydroxyanisole
containing lecithin and/or citric acid or phosphoric acid. In these
formulations, the alkyl gallate, etc., is present in an amount of
from 0.02% to about 0.05%, and the citric or phosphoric acid is
present in an amount of about 0.01%. These formulations are
suitable for parenteral administration.
[0040] In addition to being insoluble in water, various Ras
antagonists such as FTS and its analogs are not active when
administered orally. It is believed that the standard crystals of
the drug do not dissolve in the gastrointestinal tract. In one
embodiment of the present invention, both of these shortcomings are
overcome by formulating the agent in cyclodextrin. This technology
is the subject of U.S. Pat. Nos. 5,681,828 and 5,935,941.
Cyclodextrins are a group of compounds consisting of, or derived
from, the three parent cyclodextrins--alpha-, beta- and
gamma-cyclodextrins. Alpha-, beta- and gamma-cyclodextrins are
simple oligosaccharides consisting of six, seven or eight
anhydroglucose residues, respectively, connected to macrocyles by
alpha (1 to 4) glycosidic bonds. Each of the glucose residues of a
cyclodextrin contains one primary (O6) and two secondary hydroxyls
(O2 and O3) which can be substituted, for example, methylated. Many
cyclodextrin preparations in practical use are mixtures of
chemically individual derivatives in which only a part of hydroxy
groups were substituted and which differ in number and position of
these substituents.
[0041] Cyclodextrins solubilize insoluble compounds into polar
media by forming what is known as an inclusion complex between the
cyclodextrin and the insoluble compound; cyclodextrin
solubilization power is directly proportional to the stability of
the complex. Inclusion complexes are non-covalent associations of
molecules in which a molecule of one compound, called the host, has
a cavity in which a molecule of another compound, called a guest is
included. Derivatives of cyclodextrins are used as the hosts, and
the insoluble compound is the guest.
[0042] In this invention, many different cyclodextrin derivatives
may be used. These include several types of mixtures of partially
methylated cyclodextrins. One type is a commercial preparation
(Wacker Chemie, Beta W7M1.8) in which the methyl groups are about
equally distributed between the primary and secondary hydroxyls of
glucopyranose residues; it is abbreviated as RAMEB. A second type
has methyls predominantly on the secondary hydroxyls. These
derivatives are described in U.S. Pat. No. 5,681,828. A third type
of methylated cyclodextrins is formed by those cyclodextrin
derivatives or their mixtures that have more than half of their
secondary hydroxy groups (i.e., O2 and O3) methylated. Other
mixtures of cyclodextrin derivatives are partial 2-hydroxypropyl
ethers, abbreviated as HPACD, HPBCD or HPGCD for derivatives of
alpha-, beta- and gamma-cyclodextrins, respectively.
[0043] To potentiate the formation of inclusion complexes between
the cyclodextrins and the Ras antagonists, highly methylated
cyclodextrins may be covalently or non-covalently complexed with
less substituted cyclodextrins.
[0044] Briefly, the Ras antagonist is salified and dissolved in an
appropriate solvent, and then added to a solution of methylated
cyclodextrin in PBS. The result of the solution is sterilized and
then the solvent is removed. To prepare a formulation suitable for
oral administration, the resultant cyclodextrin/FTS complex is
mixed with a suitable binder and then pressed into buccal tablets,
oral tablets or capsules. The buccal tablets dissolve when held in
the mouth against the mucus membrane. It is believed that as the
tablet dissolves, the cyclodextrin particles touch the membrane and
the drug is released and is passed across the membrane of the mouth
into the bloodstream. In other embodiments, the cyclodextrin/Ras
antagonist complex is reconsituted into solutions suitable for oral
or parenteral (e.g., intravenous or subcutaneous)
administration.
[0045] An alternative method of formulating the Ras antagonist of
the present invention such as FTS that are insoluble in water, is
to convert the crystals of drug, which are several .mu.m in
diameter, into crystals with a diameter in nanometers. The
production of nanocrystals may be accomplished by commercially
known procedures such as ones developed by Elan Pharmaceutical
Technologies (see e.g., U.S. Pat, Nos. 4,610,875 and 5,641,515).
See also U.S. Pat. No. 5,145,684. Such nanocrystals are suitable
for oral, parenteral and inhalation routes of administration.
Nanocrystal formulations enchance bioavailability, net onset of
drug action, improved drug proportionalities, reduce fed-fasted
effects, and increase the drug loading without increasing
toxicity.
[0046] Clinical effects may be achieved with dosages of the Ras
antagonist of about 1 mg/kg per day. In general, however, amounts
of the Ras antagonist effective for the present purposes, which are
treatment of the patients and inhibition of Ras activity, e.g.,
Ras-mediated cell proliferation, range from about 5 mg/kg every
other day to about 5 mg/kg per day. The response may be magnified
by increasing the dose up to about 20 mg/kg per day in a single
treatment as well as by increasing the frequency of treatment.
Alternatively, from about 20 to about 80 mg/kg can be administered
once weekly.
[0047] Timing of the administration of the Ras antagonist is
important to the extent that it is in circulation so as to be in
contact with the cells before or during proliferation. In the case
of restenosis, for example, the antagonist is preferably
administered prophylactically such as by way of i.v. infusion at
about the time of angioplasty. Administration is continued for
about 14 days. In addition to i.v. administration, the agent may be
formulated into a transdermal preparation such as a cream, gel or
patch, or in the form of a prodrug, optionally complexed with
cyclodextrin. In the case of atherosclerosis, it is preferable to
administer the Ras antagonist before or during accumulation of
lipid in the artery. Atherosclerotic patients, or those at risk can
be administered the Ras antagonists by way of an oral preparation
on a daily basis. Administration is preferably continued for a
year. The present invention will now be described by way of the
following examples. These examples demonstrate the efficacy of a
Ras antagonist of the present invention to inhibit or reduce the
proliferation of normal cells associated with various disease
states including animal models of atherosclerosis and restenosis.
They are presented solely for purposes of illustration, and are not
intended to limit the invention in any way. For ease of reading,
citations of the referenced scientific publications are listed at
the end of each example.
EXAMPLE 1
Functional Inhibition of Ras by FTS Attenuates
Atherosclerosis in Apolipoprotein E Knockout Mice
[0048] The following example demonstrates the efficacy of Ras
antagonist of the present invention, FTS, to inhibit or reduce
early and advanced atherosclerotic lesions. The experiments were
conducted on atherosclerosis-prone mice deficient in apolipoprotein
E and which were fed normal and high-fat diets.
[0049] Methods
[0050] Animals
[0051] Apo-E deficient mice on a C57BL/6 background (24-25) were
purchased from the Jackson Laboratories and grown at the local
animal house. Mice were either fed normal chow-diet containing 4.5%
fat by weight (0.02% cholesterol) or a Western-type diet containing
42% of calories from fat, 43% from carbohydrates, 15% from protein
(TD 96125, Harlan Teklad).
[0052] Experimental Design
[0053] In the first experiment, the effect of FTS was studied on
fatty streak formation (early atherosclerosis) in apoE mice. For
this purpose, 4 week-old male apoE mice were injected I.P. with FTS
(5 mg/Kg weight) 3 times a week for 6 weeks and fed a normal chow
diet. Upon sacrifice, plasma was obtained for anti-oxLDL antibody
level measurement and hearts were removed for estimation of lesion
size and plaque composition.
[0054] In the second experiment, the effect of FTS was studied on
advanced atherosclerotic plaque development. For this purpose, 4
week-old apoE mice were injected I.P. with FTS, or a control
vehicle employing the same protocol, yet they were fed a high fat
diet for 10 weeks. Upon sacrifice similar measurements were
performed as in the first experiment and spleen cells were obtained
for assessment of proliferation in the presence of oxLDL.
[0055] Lipid Profile.
[0056] Total plasma cholesterol and triglyceride levels were
determined using an automated enzymatic technique (Boehringer
Mannheim, Germany).
[0057] Splenocyte Proliferation Assays.
[0058] Splenocytes (1.times.10.sup.6 cells per ml) were incubated
in triplicates for 72 h in 0.2 ml of culture medium in microtiter
wells in the presence or in the absence of 10 .mu.g/ml mouse oxLDL
for 72 hrs. Proliferation was measured by the incorporation of
[.sup.-3H] thymidine into DNA during the final 12 h of incubation
as described previously.sup.26.
[0059] Detection of Anti-oxidized-LDL Antibodies and Isotypes by
ELISA.
[0060] Ninety-six well polystyrene plates (Nunc Maxisorp, Denmark)
were coated with either ox-LDL (at concentration of 10 .mu.g/ml in
PBS) or native LDL (both from humans) overnight at 4.degree. C.
Next steps were carried out as described previously (27). IgG
isotypes were determined employing an ELISA kit
(Southern-Biotechnology).
[0061] Measurement of Ras Expression
[0062] Levels of Ras protein content were determined in homogenates
of aortas obtained from FTS treated mice or controls, employing
anti-pan Ras antibodies (PanRas Ab03, Santa Cruz) as previously
described (11).
[0063] Aortas were homogenized (10% w/v) in cold homogenization
buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 7.6, 1 mM
dithiothriethol, 5 .mu.g/ml leupeptine, 5 .mu.g/ml pepstatine, 1 mM
benzamidine, 1 mM phenylmethylsulfonyl fluoride, 5 units/ml
aprotinine and 20 mM MgCl.sub.2. The nuclei and the cell debris
were then removed by a 10-min 1000.times.g spin. The resulting
supernatant was used for assays. Samples containing 25 .mu.g
proteins were used for the determination of total Ras protein (11).
Proteins were separated by 12.5% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) (mini-gel) and
blotted onto nitrocellulose paper. The paper was blocked with 1.5%
skim milk in Tris-buffered saline pH 7.6 (containing 0.05% Tween-20
and 150 mM NaCl) overnight, then incubated for 1.5 h with 1:2000
dilution of Pan-Ras Ab in the same buffer. Immunoblots were then
incubated for 1 h with 1:7500 dilution of anti mouse IgG
horseradish peroxidase conjugate and subjected to enhanced
chemiluminescence (ECL) assays. Bands were quantified by
densitometry on a BioImaging System 202D System (Dynco-Renium,
Jerusalem) using Tina 2.0 software (Ray Tests).
[0064] Determination of Active Ras in Aortic Samples from FTS or
Control Treated Mice
[0065] The above noted preparations of the aortas (500 .mu.g
protein) were adjusted to 0.5% NP-40 and used for the determination
of the levels of active GTP bound Ras (reference 28) as follows.
The Ras binding domain of human c-Raf-1 (RBD) fused to
glutathione-S-transferase (GST) in the expression vector pGEX-2T
was prepared in DH-10 B E.coli cells. The bacterial lysate was
rotated 30 min at 4.degree. C. with glutathione-agarose beads
(Sigma) in the homogenization buffer containing NP-40. The beads
were then washed with the same buffer and mixed with the above
aorta preparation. Samples were then rotated 30 min at 4.degree. C.
The active Ras was then precipitated and washed. SDS sample buffer
was then added to the precipitated Ras-GTP and the apparent amount
of the Ras was determined by immunoblotting with the Pan-Ras Ab as
detailed above.
[0066] Assessment of Atherosclerosis.
[0067] Quantification of atherosclerotic fatty streak lesions was
done by calculating the lesion size in the aortic sinus (29).
Briefly, the heart and upper section of the aorta were removed from
the animals and the peripheral fat cleaned carefully. The upper
section was embedded in OCT medium and frozen. Every other section
(10 .mu.m thick) throughout the aortic sinus (400 .mu.m) was taken
for analysis. Sections were evaluated for fatty streak lesions
after staining with oil-red O. Lesion areas per sections were
counted using a grid by an observer unfamiliar with the tested
specimen.
[0068] Immunohistochemistry.
[0069] Immunohistochemical staining for T-cells (CD3), T cell
activation marker (IL-2 receptor), macrophages (Mac-1), NFkB and
VCAM-1 were performed on aortic sinus 5 82 m thick frozen sections
(26). The sections were fixed for 4 min in methanol at -20.degree.
C. followed by 10 min incubation with ethanol at -20.degree. C. The
sections were then blocked with non-immune goat serum for 15 min.
at room temperature followed by incubation with CAS blocking
reagent for 30 min. at room temperature. Subsequently, the
rat-monoclonal anti-mouse antibodies were added for 1 hr at room
temperature. After washings, affinity purified biotinylated rabbit
anti-rat IgG antibodies (Jackson) were added for 30 min at room
temperature. After washings, the slides were incubated with 0.3%
H.sub.2O.sub.2, followed by additional rinses and incubation with
streptavidin-peroxidase conjugate for 30 min at room temperature.
After washings, the slides were developed with 3
amino-9-ethylcarbonasole (AEC) substrate (Dako) for 15 min.
Sections were counterstained with hematoxylin. Spleen sections were
used as a positive control. Staining in the absence of 1 st or 2nd
antibody were used as a negative control. Positive cells were
counted by 2 pathologists, blinded to the study protocol, and
averaged. VCAM-1 was evaluated by morphometry as previously
described (26).
[0070] Statistical Analysis
[0071] All parameters between the groups were evaluated by the
student's t-test. P<0.05 was considered statistically
significant. Results are expressed as mean+SEM unless otherwise
specified in the text.
[0072] Results
[0073] FTS does not Influence Lipid Profile
[0074] Treatment of chow fed mice with FTS did not appear to
influence total cholesterol levels (mean levels of 258.+-.61 mg/dl)
in comparison with control treatment (212.+-.60 mg/dl; p=n.s).
Similarly, total cholesterol levels were not significantly
different in the FTS and control treated mice fed a Western diet
(870.+-.252 mg/dl vs. 870.+-.185 mg/dl, respectively; p=n.s). FTS
administration in both experiments did not affect triglyceride
levels (data not shown).
[0075] FTS Attenuates Early and Advanced Atherosclerotic
Lesions
[0076] In the first experiment early atherosclerotic lesions were
significantly attenuated (a 52% reduction) by treatment with FTS
(mean aortic lesion size of 37000.+-.4300 .mu.m.sup.2) in
comparison with controls (77000.+-.17000 .mu.m.sup.2;
p<0.1)(FIG. 1A; photo not shown). Although less pronounced, FTS
reduced the more advanced plaques induced by high fat diet feeding
(mean lesion size of 285000.+-.15300 .mu.m.sup.2) as compared with
control group (348000.+-.25000 .mu.m.sup.2 in the control group;
p<0.05)(FIG. 1A; photo not shown). This was equivalent to a 28%
reduction.
[0077] The Effect of FTS on Ras Expression and Activation,
in-vivo.
[0078] To determine whether Ras expression was influenced by FTS
treatment, we evaluated Ras protein content in aortas from FTS
treated mice as compared with controls. Total Ras protein content
did not differ in atheromatous aortas from FTS treated mice in
comparison with non-treated (FIG. 2A). To evaluate whether active
Ras was diminished due to treatment, we evaluated Ras-GTP content
and found it reduced by 40%, in aortas of mice treated with FTS as
compared with controls (FIG. 2B).
[0079] FTS Reduces Cellular and Humoral Immune Responses to
Oxidized LDL
[0080] Next, we explored the effect of anti-Ras treatment on the
cellular and humoral immune response to oxLDL, known to prevail in
atherosclerotic apoE-deficient mice. Basal proliferative response
of splenocytes from FTS treated mice did not differ from control
treated animals (FIG. 3A). However, when primed with oxLDL, no
significant reactivity was evident in FTS treatment, whereas a 24%
(p<0.05) increase in thymidine uptake was obtained in control
treated mice (FIG. 3A). Splenocytes from FTS or control treated
mice did not differ with regard to their concavaline A induced
proliferation (data not shown).
[0081] Similar to the effects on cellular immunity, IgG anti-oxLDL
antibody levels were reduced in mice treated with FTS as compared
with levels in the controls (mean OD of 0.22.+-.0,08 Vs
0.76.+-.0.3; p<0.05)(FIG. 3b).
[0082] FTS does not Modify Plaque Composition.
[0083] To investigate whether reduction in atherosclerotic lesion
size induced by FTS, altered plaque composition, we evaluated the
relative density of macrophages, total lymphocytes and activated
(IL-2R+) lymphocytes. FTS treatment in both experiments did not
appear to influence the relative numbers of macrophages and
lymphocytes (FIGS. 4A and B). We then evaluated the relative
expression of a key adhesion molecule (VCAM-1) in plaques following
FTS treatment by morphometry. Administration of FTS considerably
reduced VCAM-1 expression in fatty streaks of mice from the 6-week
treatment schedule as compared with control (a 53% reduction; FIG.
5A). A similar, though less pronounced effect of FTS was evident on
VCAM-1 expression in more advanced plaques (a 43% reduction by FTS;
FIG. 5A; photo not shown).
[0084] As NF-KB is a transcription factor known to trigger VCAM-1
expression. We sought to evaluate its relative abundance in the
plaques and found that NF-KB positive cells were reduced in FTS
treated lesions in comparison with controls (FIG. 5B). The numbers
of NF-kB expressing cells were too low to obtain a meaningful count
in the first experiment.
Discussion
[0085] The results demonstrate the efficacy of functional Ras
inhibition by FTS of various Ras activity associated with
atherosclerotic plaque initiation and progression in murine
atherosclerosis. The effect was obtained in early fatty streaks,
and in more advanced plaques induced by high fat diet
supplementation.
[0086] It has been suggested that ras mutations are present in
samples from human atherosclerotic plaques (8). Ras has been shown
to activate NF-kB (15), a transcription factor that is known to
trigger production and expression of adhesion molecules that are
essential to the initiation of atherosclerotic plaques. NF-kB has
recently been shown to be expressed within atherosclerotic plaques
(16). We have shown here, that VCAM-1 expression within the plaques
is reduced following anti-Ras treatment. We have also shown that
NF-kB positive cells are reduced in lesions from FTS treatment
supporting our belief that interference with Ras activation and
signaling results in attenuation of NF-kB mediated induction of
adhesion molecules. Our observation that VCAM-1 plaque coverage was
reduced to a larger extent by FTS in early plaques in comparison
with more advanced plaques is consistent with the relatively more
important role of VCAM-1 in early atherosclerotic lesions.
[0087] We have also found the FTS treatment reduced Ras-GTP content
by approximately 40%, as compared with control aortas. As
homogenates from the aortas contain a heterogenous population of
cells, only part of which are actively proliferating, the effect of
FTS is apparently not complete. Yet, it is likely that Ras
inhibition in active Ras-expressing cells within the atheroma,
would be significantly more pronounced. Total Ras protein did not
differ between FTS and control treated aortas. This finding can
also be explained by the mixed population of cells within the
plaques, not all of which express Ras in a similar level. Thus, FTS
treatment was sufficient to inhibit active Ras in the aortas
without affecting total protein content. This lack of global effect
on Ras protein may well explain the lack of side effects that would
be expected to result from inhibition of such a key signaling
protein.
[0088] Atherogenesis is a process in which the immune system
appears to take an active part (1,2). Accordingly, activated
lymphocytes have been detected in human (17) and murine (18)
plaques, sometimes even preceding the infiltrating lipid-laden
macrophages. Ras protein expression has been shown to be involved
in regulating lymphocyte activation (19). Thus, interference with
lymphocyte activation by anti-Ras treatment could be a potential
beneficial anti-atherogenic mechanism. Herein, we failed to notice
differences in lymphocyte density within atherosclerotic plaques
between FTS and control treated mice, nor did we find evidence that
activation markers were reduced as a result anti-Ras therapy.
However, systemic immune responses towards oxLDL, which have been
associated with development of atherogenesis, were indeed altered.
Accordingly, proliferative response of splenocytes from FTS treated
mice to oxLDL were reduced as compared with responses recorded from
control lymphoyctes. Moreover, antibodies to oxLDL were also
diminished by FTS treatment. We believe that these findings
demonstrate that attenuation of immune reactions resulting from
oxidative stress is mediated by interference with Ras activation,
which contributes to the anti-atherogenic effect.
[0089] The atherosclerotic process entails a proliferative
phenotype, involving, apart from lymphocytes and macrophages, also
smooth muscle cells, which occupy lesions that are relatively more
advanced (1). Approaches designed to block Ras- mediated smooth
muscle cell proliferation and migration have been successful in
several in-vitro studies (20). Moreover, studies employing gene
delivery of dominant negative forms of ras have been shown
effective in suppressing neointimal formation after experimental
carotid injury (21-22). We believe that FTS induced suppression of
atherosclerotic lesions by altering the proliferative phenotype of
atherosclerotic lesions. Additionally, FTS inhibits cell migration
(14), a property that may be relevant to atherosclerosis
suppression that involves recruitment of monocyte and lymphocytes
followed by smooth muscle cells and fibroblasts.
[0090] FTS and analogs thereof (as represented by formula 1) bear
some advantages over other inhibitors that were designed to inhibit
Ras (9,23). FTS is the only compound that affects all isoforms of
activated Ras, namely activated H-, K-, and N-Ras (9). Other
compounds such as farnesyltransferase inhibitors, for example, are
good inhibitors of H-Ras but not of other Ras isoforms. This
property coupled with the lack of FTS toxicity in animal models
make FTS an attractive potential drug, particularly for the
treatment of multifactorial disorders such as atherosclerosis. It
is more probable that not that more than one Ras isoform (in
diverse cell types) is activated during the atherosclerotic
process. Our experiments lend support for a mechanism-based action
of FTS in atherosclerosis as expected from its in vitro inhibition
of Ras and its functions (9). First, they demonstrate that the in
vivo effects of FTS are manifested in the reduction in the contents
of active, GTP-bound Ras, in the atherosclerotic aortas. Second,
they demonstrate the reduced proliferative response of splenocytes
from FTS-treated mice to oxLDL. Third, they show the reduced levels
of antibodies to oxLDL in the FTS treated apoE mice, with no effect
of antibody class distribution. These observations support the
contention that FTS, at the doses employed in the current study,
would induce functional Ras blockade in vivo.
[0091] In conclusion, FTS, a functional Ras inhibitor, has been
shown to suppress early and more advanced atherosclerosis. The
effects brought about by FTS are probably multifactorial. In
certain embodiments, it is advantageous to employ the Ras
antagonist in combination with lipid lowering agents.
REFERENCES
[0092] 1. Ross, R., N. Eng. J. Med. 340:115-126 (1999).
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EXAMPLE 2
The Effect of FTS on Intimal Hyperplasia in a Model of Carotid
Balloon Injury in the Rat
[0121] The aim of this experiment was to investigate the effect of
FTS on intimal hyperplasia in a rat carotid balloon injury, as an
indication of whether FTS can ameliorate restenosis by reducing
smooth muscle cell proliferation and migration. The results
indicate that FTS appears to be a potent inhibitor of intimal
hyperplasia.
[0122] Atherosclerosis and restenosis are two processes that
involve cellular proliferation that eventually lead to functional
narrowing of blood vessels causing considerable morbidity and
mortality (1-4). The formation of neointimal hyperplasia following
balloon denudation is thought to involve proliferation and
migration of medial smooth muscle cells or modified adventitial
fibroblasts (4, 5, 6, 7). In recent years, evidence has also
accumulated pointing towards the involvement of the immune system
in atherosclerosis and restenosis, as manifested by the local
presence of activated T lymphocytes (3,4) and elevation of
inflammatory markers such as CRP, IL-6 and other markers
(8-10).
[0123] The long-term effectiveness of percutaneous balloon coronary
angioplasty (PTCA) and stent implantation is still largely limited
due to the occurrence of late lumen loss following intimal
thickening. Although several experimental strategies have provided
some success in reducing intimal thickening in animals, clinical
trials in humans performed so far failed to achieve significant
improvement (10-17). Effective clinical utility in reducing the
rate of restenosis was recently shown only for intracoronary
radiation therapy (18-19).
[0124] Methods
[0125] Animals
[0126] Male Wistar rats 6 weeks old (weighing 250-280 gr). The
animals were purchased from the Tel Aviv University and maintained
at the local animal house.
[0127] Study Design
[0128] Group A: Four rats received daily intraperitoneal injections
of FTS (5 mg/Kg) starting from the day of injury induction until
sacrifice, 14 days later.
[0129] Group B: Four rats received daily intraperitoneal injections
of control vehicle starting from the day of injury induction until
sacrifice, 14 days later.
[0130] Rat Carotid Injury Method
[0131] Animals were anesthetized by intraperitoneal injection of
Ketamin (80 mg/Kg) and Xylazine (5 mg/Kg). Endothelial denudation
and vascular injury was achieved in the left common carotid artery,
as described (6). Briefly, a balloon catheter (2F Fogarty) was
passed through the external carotid into the aorta; the balloon was
inflated with sufficient water to distend the common carotid artery
and then pulled back to the external carotid. This procedure was
repeated three times, and then the catheter was removed. After 14
days, the animals were sacrificed and the right and left carotid
arteries were taken out and fixed in 4% paraformaldehyde until
embedding in Paraffin. The arteries were cut in 10 .mu.m sections
and stained with H&E and computer-assisted morphometric
analyses were performed. The tested parameters were: intimal area,
medial area, intimal/medial ratio and lumen area. Additionally, the
%CSAN-N (% cross sectional area neointimal-neointamal) was
calculated [IEL area-Lumen area].times.100/IEL (a measure of the
degree to which the IEL area is reduced by neointimal hyperplasia
with greater normalization of the effect of changes in vessel wall
size. Vasular remodeling process were further evaluated by
computing the amount of plaque relative to the EEL (external
elastic lamina) area.
[0132] Immunohistochemistry
[0133] Paraffin fixed sections (10 .mu.m) were stained with a Pan
Ras antibody.
[0134] Statistical Analysis
[0135] Results of all parameters were computed employing 2 tailed
students t-test. Results are presented as means.+-.SEM. p<0.0.5
was considered significant.
[0136] Results
[0137] Intimal area was significantly reduced (76%) in rats treated
with FTS (0.38 mm.sup.2) in comparison with control treated animals
(1.61 mm.sup.2; p=0.02). FTS did not significantly influence medial
area (0.91 mm.sup.2) in the treated group as compared with the
control group (1.2.+-.0.14 mm.sup.2). Intimal to medial ratio was
significantly reduced in FTS treated rats (0.49.+-.0.19 mm.sup.2)
as compared with controls (1.29.+-.0.20 mm.sup.2; p=0.02). The
luminal area was significantly increased in FTS-treated rats
(1.45.+-.0.34 mm.sup.2) in comparison with control animals
(2.30.+-.0.32; p=0.015). %CSAN-N in the FTS rats was significantly
reduced (14.5.+-.4.3%) in comparison with control treated animals
(52.4.+-.7.4%; p=0.004). Increased amount of neointimal
proliferation was not associated with larger EEL area.
Immunostainable Ras was abundantly present in the neointimal cells
and only low expression was evident in the media and
adventitia.
[0138] Conclusions
[0139] FTS appears as a potent inhibitor of intimal hyperplasia
induced by carotid balloon injury in the rat. Increased patency of
the vessel lumen by FTS was mainly due to prevention of neointimal
proliferation and not due to the vessel wall remodeling processes.
Thus, the onset of restenosis may be inhibited, or restenosis may
be treated by coating or otherwise contacting the stent with the
Ras antagonist prior to deployment of the stent, systemic treatment
with the Ras antagonist following PTCA or administration of the Ras
antagonist following heart transplantation or coronary arterial
bypass graft to inhibit accelerated atherosclerosis.
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[0159] All patent and non-patent publications cited in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All these publications
are herein incorporated by reference to the same extent as if each
individual publication or patent application were specificaly and
individually indicated to be incorporated by reference.
[0160] Various modifications of the invention described herein will
become apparent to those skilled in the art. Such modifications are
intended to fall within the scope of the appending claims.
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