Note: Descriptions are shown in the official language in which they were submitted.
CA 02198875 2001-08-O1
QUINAZOLINONE-CONTAINING PHARMACEUTICAL COMPOSITIONS
AND METHODS FOR THE USE THEREOF
The present. invention relates to compositions containing
quinazolinorAes. More particularly, the present invention
relates to a composition for the inhibition of restenosis,
comprising a quinazolinone derivative as herein defined as
active ingredient therein.
In U.S. Patent 3,320,124, issued in 1967, there is
described and claime<~ a method for treating coccidiosis with
quinazolinone deriva~:i.ves.
Halofuginone, otherwise know as 7 - bromo - 6 - chloro - 3
- [ 3 - ( 3 -hydroxy - 2 - piperidinyl ) - 2 - oxopropyl ] - 4
( 3H ) - quinazolinone, was first described and claimed in said
patent by ,zlmerican Cyanamid Company, and was the preferred
compound taught by said patent and the one commercialized from
among the derivatives described and claimed therein.
Subsequently, U.S" Reissue Patent 26,833 and U.S. Patents
4,824,847; X1,855,299; 9,861,758 and 5,215,993 all relate to the
coccidiocidal properties of halof:uginone, while U.S. Patent
4,340,596 teaches t.hai: it can also be used for combatting
theileriosis.
In U.S. Patent No. 5,449,678 there is described and
claimed an <~nti-fibroti.c: composition, comprising an amount of a
compound of formula I:
N
I Rl In
N. %
~ fH~C:CxH~ N
O R1
I
CA 02198875 2003-03-11
- 2 -
wherein: n=1 or 2
R1 is a member of the group consisting of hydrogen,
halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;
RZ is a member of the group consisting of hydroxy,
acetoxy, and lower alkoxy, and
R3 is a member of the group consisting of hydrogen and
lower alkenoxy-carbonyl;
effective to inhibit collagen type I synthesis, as active
ingredient therein.
After further research and development, it has now
been discovered that the above-identified compounds of
formula I are effective in the inhibition of restenosis,
which formally is not a fibrotic condition.
The pathogenesis of atherosclerosis involves abnormal
migration and proliferation of smooth muscle cells (SMCs)
infiltrated with macrophages and embedded in extracellular
matrix (ECM) of adhesive glycoproteins, proteoglycans and
collagens [V. Fuster, et al., "The Pathogenesis of Coronary
Artery Disease and the Acute Coronary Syndromes,". New Eng.
J. Med., Vol. 326, pp. 242-250 (2992); R. Ross, "The
Pathogenesis of Atherosclerosis: A perspective for the
1990's," Nature, Vol. 362, pp. 801-809 (1993)]. Under
physiological conditions, the majority of arterial SMCs
remains in the Go phase and cell growth is controlled by a
balance between endogenous proliferation-stimulating and
proliferation-inhibiting factors. Following endothelial cell
perturbation due to atherogenic risk factors (i.e.,
hypertension, hyperlipoproteinemia, diabetes mellitus),
platelets and non-platelet-derived growth factors and
cytokines are released and stimulate monocyte and SMC
migration as well as SMC proliferation (V. Fuster, et al.,
ibid.; R. Ross, ibid.). Among these growth factors are
a~~~~7~
WO 96/06616 PCT/US95111186
- 3 -
platelet-derived growth factor (PDGF) [G.A.A. Ferns, et
al., "Inhibition of Neoinitmal Smooth Muscle Accumulation
after Angioplasty by an Antibody to PDGF," Science,
Vol. 253, pp. 1129-1132 (1991)], basic fibroblast growth
factor (bFGF) [V. Lindner, et al., "Role of Basic Fibroblast
Growth Factor in Vascular Lesion Formations" Circ. Res.,
Vol. 68, pp. 106-lI3 (I99I) ] , and interleukin-1 (IL-I) [I~.
Loppnow and P. Libby, "Proliferating or Interleukin-1
Activated Human Vascular Smooth Muscle Cells Secrete Copious
Interleukin 6," J. Clin. Invest., Vol. 85, pp. 73I-738
(1990)]. Macrophages and platelets also release enzymes,
i.e., elastase, callagenase, heparanase) that digest various
constituents of the EG~I and release bFGF and possibly other
growth factors (TGFB) that are stored in basement membranes
and ECM [I. Vlodavsky, et al., "Extracellylar Matrix-bound
Growth Factors, Enzymes and Plasma Proteins," in: Molecular
and Cellular Asr~ects of Basement Membranes Monoctraohs i_n
Cel? Bioloav, D.H. Rohrbach and R. Timpl, Eds., Academic
Press, New York, New York, U.S.A., pp. 327-346 (1993)]. A
potent growth-promoting activity i:owards SMCs is also
exerted by thrombin, which, under certain conditions, may be
present within the vessel wall [R. Bar-Shavit, et al.,
"Thrombin Immobilized to Extracellular Matrix Is a Mitogen
for Vascular Smooth Muscle Cells: Non-Enzymatic Mode of
Action," Cell Rea., Vol. 1, pp. 453-463 (1990); S.M.
Schwartz, "Serum-Derived Growth Factor is~~Thrombin?"
Slin. Invest., Vol 91, p. 4 (1993)]. Molecules that
interfere with the growth-promoting activity of these growth
factors may attenuate the progression of the atherogenic
process.
H
Proliferation of arterial smooth muscle cells (SMC) in
response to endothelial injury is a basic event in the
process of restenosis of coronary arteries after
WO 9G/06616 PCT/US95/11186
.19~~7 ~
Q2.
- 4 -
percutaneous transluminal coronary angioplasty (PTCA) [V.
Fuster, et al., ibid.]. Coronary bypass surgery or
angioplasty are applied to reopen coronary arteries that
have been narrowed by heart disease. A major problem with
both procedures is that arteries rapidly reclog in about 3o%
of patients undergoing angioplasty and about 10% of bypass
surgery patients. Vascular SMC are ordinarily protected by
the smooth inner lining of the arteries, composed of
vascular endothelial cells. However, following bypass
surgery or angioplasty, SMC are often left exposed. In a
futile effort to repair the wound, the cells proliferate and
clog the artery.
According to the present invention, there is now
provided a pharmaceutical composition comprising a compound
of formula I as hereinbefore defined, in a pharmaceutically
effective amount for preventing restenosis by the inhibition
of vascular smooth cell proliferation and in combination
with a pharmaceutically acceptable carrier.
In preferred compositions of the present invention,
paid compound is halofuginone.
The invention further encompasses a pharmaceutical
composition as hereinabove described, wherein the carrier is
a liquid and the composition is a solution.
In the practice of the invention, the amount of
halofuginone incorporated in the pharmaceutical composition ,
may vary widely. Factors considered when determining the
precise amount are well-known to those skilled in the art.
Examples of such factors include, but are not limited to,
CA 02198875 2001-08-O1
-5-
the subject being treated, the specific pharmaceutical carrier,
route of adrninistration being employed, and the frequency with
which the composition is to be administered.
As stated above, the compounds of the present invention
are administered in a pharmaceutical composition which
comprises t:he compound and a pharmaceutically acceptable
carrier. As used herein, the term ~~pharmaceutically acceptable
carrier" encompasses any of the standard pharmaceutically
accepted carriers, such as a phosphate-buffered saline
solution, water, emuls=ions such as an oil/water emulsion or a
triglyceride emulsion ~iIld various types of wetting agents. An
example of an acceptable triglyceride emulsion useful in the
intravenous and intrape~ritoneal administration of the compounds
is the 1=righlycericle emulsion commercially known as
Intralipid.*
In the practice o.f the invention, the administration of
the pharmaceutical composition may be effected by any of the
well-know methods, inc:Luding, but not limited to, intravenous,
intraperitineal, intram.uscular, or subcutaneous administration.
In another aspect, the present invention provides use of a
compound of formula I:
N
(R1)~
/ n\
/ C'N,:COCH~ ~ N
G RJ
I
wherein: n= 1 or a'
Rl is a member of the group consisting of hydrogen,
halogen nitro, benzo, lower alkyl, phenyl and lower alkoxy;
*Trade-mark
CA 02198875 2003-03-11
-5a-
R, is a member of the group consisting of hydroxy,
acetoxy, and lower alkoxy; and
R, is a member of the group consisting of hydrogen and
lower alkenoxy-carbonyl
for preventing restenosis by the inhibition of vascular
smooth cell proliferation.
In another aspect, the present invention relates to use of
a compound of formula I:
'H
II/// ~ R= I
II
R::~ j' /
2 Z
O R~
I
wherein: n= 1 or 2
R_ is a member of the group consisting of hydroge_~.,
halogen, nitro, benzo, lower alkyl, phenyl and lower alkoxy;
R, is a member of the group consisting of hydroxy,
acetoxy, and lower alkoxy, and
R, is a member of the group consisting of hydrogen and
lower alkenoxy-carbonyl
for the manufacture of a pharmaceutical composition fcr
preventing restenosis by the inhibition of vascular smooth cel'_
proliferation, the composition comprising a pharmaceutically
acceptable carrier and the compound being an active ingredient
of the composition.
Preferably, the compound is halofuginone.
In another aspect, the present invention provides a
pharmaceutical composition for preventing restenosis by the
CA 02198875 2003-03-11
-5b-
inhibition of vascular smooth cell proliferation, comprising a
compound of formula I:
;R,:~ ,
N
\ CH~CL~H~ ~~ N
R7
I
wherein: n=1 or 2
R1 is a member of the group consisting of hydrogen,
halogen, nitro, benzo, lower alkyl having 1 to 6 carbon atoms,
phenyl and Lower alkoxy having 1 to 6 carbon atoms;
R2 is a member of the group consisting of hydroxy,
acetoxy, and lower alkoxy having Z to 6 carbon atoms; and
R3 is a member of the group consisting of hydrogen and
lower alkenoxy-carbonyl having 1 to 6 carbon atoms
as active ingredient therein, in combination with a
pharmaceutically acceptable carrier.
While the invention will now be described in connection
with certain preferred embodiments in the following examples
and with reference to the appended figures, so that aspects
thereof may be more fully understood and appreciated, it is not
intended to limit the invention to these particular
embodiments. On the contrary, it is intended to cover all
alternatives, modifications and equivalents as may be included
within the scope of the invention as defined by the appended
claims. Thus, the following examples which include preferred
embodiments will serve to illustrate the practice of this
invention, it being understood that the particulars
described are by way of example and for purposes of
6 PCT/US95111186
WO 96/0661
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illustrative discussion of preferred embodiments of the
present invention only, and are presented in the cause of
providing what is believed to be the most useful and readily ,
understood description of procedures as well as of the
principles and conceptual aspects of the invention.
In the drawings:
Fig. 1 is a characteristic curve showing the inhibitory
effect of halofuginone on SMC proliferation;
Fig. ~ is a characteristic curve showing reversion of the
antiproliferative effect of halofuginone on SMC;
Figs. 3a and 3b respectively are a bar graph and a
characteristic curve, showing the effect of
halofuginone on ~H-thymidine incorporation into
vascular SMCs;
Fig. 4 is a characteristic curve showing the effect of
halofuginone on vascular endothelial cell
proliferation;
Figs. 5a and 5b respectively are a bar graph and a
characteristic curve, showing the effect of
halofuginone on 3H-thymidine incorporation into
vascular endothelial cells;
Fig. 6 is a bar graph showing antiproliferative effect of
halofuginone on 3T3 fibroblasts;
Fic~. 7 is a bar graph showing the inhibitory effect of
halofuginone on the mitogenic activity of bFGF;
Figs. 8a and 8b are color light micrographs of the central
artery of a rabbit ear after being subj ected to crush
injury, respectively showing an untreated artery and
an artery treated according to the present invention;
and
Fic~. 9 is a graph showing the effect of halofuginone on
injury-induced artery stenosis.
WO 96/06616 PCT/US95/11186
.~~9~~75
_,_
~zEs
1) Exverimental Procedures
el s
SMC were isolated from the bovine aortic media as
previously described [see, e.g., J.J. Castellot, et al.,
"Structural Determinants of the Capacity of Heparin to
Inhibit the Proliferation of Vascular Smooth Muscle Cal7.so
Evidence for a Pentasaccharide Sequence that Contains a
3-0-Sulfates Group," J. Cell Biol., Vol. 102, pp. 1.879-1984
(1986); and A. Schmidt, et al., "The Antiproliferative
Activity of Arterial Heparan Sulfate Resides in Domains
Enriched with 2-0-Sulfated Uronic Acid Residues," J. Bio~=
C a ., Vol. 267, pp. 19242-19247 (1992)].
Briefly, the abdominal segment of the aorta was removed
and the fascia cleaned away under a dissecting microscope.
The aorta was cut longitudinally, and small pieces of the
media were carefully stripped from the vessel wall. Two or
three such strips, with average dimensions of 2-3 mm, were
placed in 100 mm tissue culture dishes containing DM~I
(4.5 g glucose/liter), supplemented with 10~ FCS, 100 U/ml
penicillin and 100 ~g/ml streptomycin. Within 7-14 days,
large patches of multilayered calls migrated from the
explants. Approximately 1 week later, the cells were
subcultured into 100-mm tissue culture plates (4-6x105
cells/plate). The cultures (passage 3-8) exhibited typical
~ morphological characteristics of vascular SMC and the cells
were specifically stained with monoclonal antibodies that
selectively recognize the muscle form of actin (HF-35).
This antibody does not recognize endothelial cells or
fibroblasts.
6 PCT/US95/11186
WO 96/0661
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Cultures of vascular endothelial cells were established '
from bovine aorta, as previously described by D.
Gospodarowicz, et al. ["Clonal Growth of Bovine Endothelial
Cells: Fibroblast Growth Factor as a Survival Agent,°' Proc.
u~~-i Acad Sci U.S.A., Vol. 73, p. 4120 (1979)]. Stock
cultures were maintained in DMEri (1 g glucose/liter)
supplemented with 10% calf serum, 50 U/ml penicillin, and
50 ~g/ml streptomycin at 37°C in 10% C02 humidified
incubators. Partially purified brain-derived bFGF
(100 ng/ml) was added every other day during the phase of
active cell growth [D. Gospodarowicz, et al., ibid., and I.
V~odavsky, et al., "Vascular Endothelial Cells Maintained in
the Absence of Fibroblast~Grawth Factor Undergo Structural
az~,d Functional Alterations That Are Incompatible with Their
Ire Vivo Differentiated Properties," J. Cell Biol., Vol 83,
pp. 468-486 (1979)].
Prol~.ferationt 3R- ~t~idine Ircflruoration
SMCs were plated (4x104 cells/16 mm well) in DMEri
supplemented with 10% FCS. 24 hours after seeding, the
medium was replaced wit.' medium containing 0.2% FCS, and 48
hours later, the cells were exposed to growth stimulants and
3~i-thymidine (1 ~cCi/well) for an additional 24-48 hours.
DNA synthesis was assayed by measuring the radioactivity
incorporated into trichloroacetic acid insoluble material
[~i. Benezra, et al., "Reversal of bFGF Autocrine Cell
Transformation by Aromatic Anionic Compounds," Cancer Res.,
Vol. 52, pp. 5656-5662 (1992)].
WO 96/06616 ~ PCT/US95/11186
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Growth Rate
SMCs (1.5x104 cells/well) were seeded into 24 well
culture plates and exposed to growth stimulants as described
above. 1 to 6 days after seeding, the calls were fixed with
2.5% formaldehyde in PBS. The plates were immersed in a
bath of 0.1 M borate buffer (pH 8.5), stained (1 h, 24°C)
with methylene blue (1% in 0.1 M borate buffer, pH 8.5) and
washed four times in water. This procedure removed
practically all non-cell-bound dye. Specific cell
incorporated methylene blue was dissolved with 0.5 ml of
0.1 N HC1 (1 h,. 25°C) and determined by measuring the
absorbency at 62o nm (Bar-Shavit, et al., ibid.). The
initial cell plating density was chosen to ensure a linear
relationship between cell number and absorbance at the end
of the experiment. In each experiment, 3 wells were fixed
before adding the test compound to determine the initial
average absorbance. This value was used to calculate
doubling times (DT) of control and drug-treated cells, using
the following equation:
DT = In 2/IN [(ODt/ODc)/h]
wherein:
DT = doubling time in hours;
ODt = optical density of a test well at the end of the
experiment;
ODc = optical density of a control well at the beginning of
the experiment;
h = duration of incubation in hours
The growth rate Was calculated by dividing the doubling
time of drug-treated calls by that of control cells [A.
Horowitz, et al., "In Vitro Cytotoxicity of Liposome-
O 6/06616 PCTlUS95/11186
W 9
- 10 -
Encapsulated Doxorubicin: Dependence on Liposome
Camposition and Drug Release," B~ochim. Biophvs. Acta, Vol.
1109, pp. 203-209 (1992)]. "
C~11 Dumber
SMCs were seeded (2.5x103 cells/well) into 24-well
plates in DMEM (4.5 g glucose/liter), supplemented with 10%
FCS and allowed to attach for 6 hours [A. Schmidt, et al. ,
'w'.Che Antiproliferative Activity of Arterial Heparan Sulfate
Resides in Domains Enriched with 2-0-Sulfated Uronic Acid
Residues," J. Biol. Chem., Vol. 267, pp. 19242-19247
(1992)]. ~ The medium was removed and experimental medium
(with or without halofuginone) containing 10% FCS was added
to quadruplicate wells. After 4 days of incubation, the
oell number was determined, using a Coulter counter
(Schmidt, et al., ibid.). The degree of inhibition was
calculated from the following formula:
% inhibition ~ 1-net growth in presence of halofuginone/net
growth in control x 100
The net growth was determined by subtracting the
initial cell number from the final cell number.
w
WO 96/06616
PCT/US95/11186
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' 2) Experimental Results
' i) Antiproliferative Effect of Halot~nciaone toward yascular
Growth Rate
Sparsely seeded vascular SMC were exposed to 10% FCS in
the absence and presence of increasing concentrations of
halofuginone. The cells were dissociated with STV and
counted daily. As shown in Fig. 1, 80-90% inhibition of SMC
proliferation was obtained in the presence of 75 ng/ml
halofuginone, with an almost complete inhibition at
125 ng/ml.
In another experiment, the SMCs were exposed to
halofuginone 48 hours, followed by removal of the d_~-ug
for
and subseguent growth in regular medium. As
growth
demonstrated Fig. Z, removal of the drug esulted in
in r a
gain of an accelerated similar that of the
growth rate to
untreated SMCs.
~H-Thvmidine Incorporation
Subcanfluent vascular SMCs maintained in a medium
containing 10% FCS were exposed (48 hours, 37°C) to
3H-thymidine in the absence and presence of increasinc
concentrations of halofuginone. As demonstrated in Fig. 3a,
complete inhibition of DNA synthesis was observed at
0.15 ~g/ml halofuginone, while 65% inhibition was obtained
at a concentration as low as 0.05 ~g/ml (Fig. 3b).
PCT/LTS95/11186
WO 9G/06616
_
12
ii) A~tiproliferative Effect too~ard Vascular Endothelial
Dells and 3T3 Fibroblasts
'~~,scular Endothelial Cells
Sparsely seeded bovine aortic endothelial cells were
cultured in medium containing 10% CS in the absence and
presence of increasing concentrations of halofuginone. The
cells were dissociated with 0.05% trypsin and 0.02% EDTA and
counted daily.
inhibition of endothelial cell proliferation was observed
primarily during the first 4 days, in cells treated with
relatively high concentrations (0.1-0.125 ~Cg/ml) of the drug
(Fig. 4). Unlike the results with SMCs, the endothelial
cells regained an almost normal growth rate (doubling time),
starting on day 5 (Fig. 4), indicating that vascular EC are
less susceptible than vascular SMCs to the inhibitory effect
of halofuginone. Thymidine incorporation studies revealed a
50% inhibition of DNA synthesis at 0.05 ~g/ml halofuginone
(Fig. 5) .
~T3 Fibroblasts
Fig. 6 demonstrates that 3H-thymidine incorporation by
actively growing 3T3 fibroblasts maintained in medium
containing 10% FCS was almost completely inhibited in the
presence of 0.025 ~ag/ml halofuginone, suggesting that
fibroblasts are even more sensitive to the drug as compared
to SMCS.
WO 96/06616 . ~ PCT/US95111186
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Effect on bFGF-Induced Cell Proliferation
Quiescent, growth arrested 3T3 fibroblasts maintained
n
(48 hours) in medium containing 0.5°s FCS are readily
stimulated to proliferate by low concentrations at basic
fibroblast growth factor (bFGF). Exposure to halofuginone
(0.025 ug/ml) resulted in an almost complete inhibition of
bFGF-stimulated thymidine incorporation in growth-arrested
3T3 fibroblasts (Fig. 7). This result suggests that
halofuginone efficiently antagonizes the gorwth-promoting
activity of bFGF.
iii) Arterial Stenosis Caused by Physical Injury
Adult New Zealand rabbits were anesthetized by
intramuscular injection of ketamine (50 mg/kg). Physical
injury was applied for 30 min. externally to the central
artery of each ear [Banai, et al., Circulation Res.,
Vol. 69, pp. 748-756 (1992)]. After the operation, the
rabbits were housed in accordance with Animal Welfare Act
specifications. Halofuginone (0.2 ml of 0.09 mg/ml) was
introduced subcutaneously around the physical crush area 1
hour after the crush and once every 24 hours during the
first 4 days. On day 14, the animals were sacrificed and
the ears fixed in 10°s buffered formaldehyde for 72 hours.
The crush sites were further trimmed at 1 mm intervals,
dehydrated in ethanol and xylene, and embedded in paraffin.
Serial (5~un) sections were stained by Movat pentachrome
' method. Computerized planimetry was performed at the site
of the lesion and at an adjacent normal arterial segment
displaced 2 mm from the location of the injury. Selection
of the normal site was random; approximately one-half were
proximal and one-half distal to the injury site. The lumen,
WO 9G/06616 PCTlUS95/11156
- 14
the area cricumscribed by the internal elastic lamina
("or~.ginal lumen") and the area circumscribed by the
external border of the media (total vessel area) were
h
traced, and the ratio between neointima and media was
calculated. In all cases, the single section demonstrating
the greatest extent of neointimal proliferation was selected
for planimetry.
Referring now to Figs. 8a and 8b, there are seen light
micrographs of the central artery of a rabbit ear 14 days
after external crush injury (Movat staining of
representative cross-sections).
In Fig. 8a, the SMCs are migrating from the media into
the neointima through the disrupted internal elastic lamina
a,nd the artery lumen is narrowed by the protruding neointima
in the untreated artery. As can be seen, there is striking
neointimal formation and an almost complete obliteration of
the arterial lumen.
In contradistinction, in Fig. 8b there is seen a rabbit
ear artery subjected to crush injury and treatment with
halofuginone. An almost complete inhibition of neo-intimal
formation is observed.
Fig. 9 shows a quantitative analysis of the ratio
between enointima to media performed in control rabbits and
rabbits treated with halofuginone (H) or a synthetic
heparin-mimicking compound (M). Each point represents one
rabbit.
WO 96106616 ~ ~ PCTlUS95111186
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Conclusions
Current approaches to inhibit the proliferation of
vascular SMC utilize heparin, suramin, antibodies to various
growth-promoting factors, anti-thrombin agents, and, mast
recently, antisense DNA technique. Heparin is a potent
anticoagulant and its anti-proliferative activity is
relatively small and subjected to major variations depending
on the source and manufacturing company. Suramin is highly
toxic at the effective dose, while antibodies are expensive,
have a short half life and may elicit an immune response.
Information on the antisense approach is new, and at present
very limited.
The present invention, in its most preferred
embodiment, utilizes a highly potent, inexpensive and
non-toxic compound which inhibits the activity of various
growth factors, including bFGF, and inhibits autocrine
growth of vascular SMC and fibroblasts. Moreover,
halofuginone is a low molecular weight compound which can be
administered orally. The compound has been approved by the
F.D.A. for use in farm animals. These characteristics make
halofuginone a most promising clinically useful drug to
inhibit restenosis.
Thus, the present invention provides for the use of
halofuginone as a non-toxic compound that efficiently
inhibits SMC proliferation, to provide an effective strategy
for inhibiting the pathophysiology of arteriosclerosis,
restenosis after coronary angioplasty, and neointimal
proliferation in saphenous vein grafts.
It will be evident to those skilled in the art that the
invention is not limited to the details of the foregoing
WO 96/06616 PCT/US95/11186
16
illustrative examples and that the present invention may be
embodied in other specific forms without departing from the
essential attributes thereof, and it is therefore desired
that the present embodiments and examples be considered in
all respects as illustrative and not restrictive, reference
being made to the appended claims, rather than to the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore
intended to be embraced therein.
s
