Indian Patents
Title of Invention

PHARMACEUTICAL COMPOSITION CONTAINING NV!FGF FOR TREATING MYCORDIAL OR SKELETAL ANGIOGENIC DISORDERS OR DEFECTS ASSOCIATED WITH HYPERCHOLESTEROLEMIA OR DIABETES
Abstract The instant invention discloses a pharmaceutical composition containing NV1FGF for treating myocardial or skeletal angiogenic disorders or defects associated with hypercholesterolemia or diabetes in a patient suffering therefrom, wherein the administration of said composition does not induce VEGF-A factor expression in the myocardial or skeletal muscles.
Full Text PHARMACEUTICAL COMPOSITION CONTAINING NV1FGF FOR
TREATING MYOCARDIAL OR SKELETAL ANGIOGENIC DISORDERS OR
DEFECTS ASSOCIATED WITH HYPERCHOLESTEROLEMIA OR DIABETES
Field of the Invention and Introduction
The present invention relates to the use of a plasmid encoding a fibroblast growth
factor as therapeutic agent for the prevention and treatment of hypercholesterolemia or
diabetes associated myocardial or skeletal angiogenic defects. The present invention also
relates to a method for enhancing formation of both collateral blood vessels and artcrioles
in myocardial or skeletal ischemic tissues in a mammalian subject suffering from
hypercholesterolemia or diabetes. The present invention further relates to a method of
promoting collateral blood vessels in ischemic myocardial or skeletal tissues without
inducing VEGF-A factor (Vascular Endothelinl Growth Fnctor A) expression and
causing edema in the treated muscles.
Background of the invention
The blood vessels form a closed blood delivery system that begins and ends at the
heart, which comprises three major types of blood vessels, ie., arteries, capillaries, and
veins. As the heart beats, blood is forced into the large arteries from the ventricles. The
large-arteries branch into medium-sized arleries. which branch into smallers arteries that
deliver blood to various parts of the body. The arteries divide again and again until they
reach their smallest branches, the artcrioles. As arterioles enter tissue, they branch into
the microscopic vessels called capillaries, which lie ciose to tissue cells. The capillaries
have very thin walls. Oxygen and nutrients leave the blood in the capillaries and enter the
tissue cells, and carbon dioxide and other wastes leave the cells and enter the blood
within the capillaries. Before the capillaries leave the tissue, they merge to form small
veins called ver.ules. The venules merge to form progressively larger veins that
ultimately empty into the great veins that return blood to the heart.
Tnc walls of all blood vessels, except capillaries, arc composed of 3 distinct
layers, surrounding the lumen. The innermost layer that lines the vessel lumen is called
the tunica interma, and consists primarily of endothelium cells. The middle layer, the
tunica media, consists mostly of circularly arranged smooth muscle cells. The outermost
layer of the blood vessel wall, the tunica exterma is composed mostly of elastic fibers and
collagen fibers that protect the blood vessel and anchor it to surrounding structures. The
tunica extema is infiltrated with nerve fibers and, in the larger arteries and veins, a
system of tiny blood vessels.
Arterioles are the smallest arteries and have a lumen diameter smaller than 5Dum.
Wall of the arteriole consists of the tunica intema surrounded by scattered smooth muscle
fibers in the tunica media. Axterioles regulate blood flow from arteries into capillaries.
During vasoconstriction of arteriole walls, blood flow into capillaries is restricted and the
tissues served by the arteriole may be momentarily bypassed During vasodilatation of
arteriole walls, blood flow into the capillaries increases significantly.
In contrast, capillaries have extremely thin walls which only consist of one line of
endothelial cells, just the tunica intema. They form extensive networks that permeate
nearly all body tissues and almost near almost every cell of the body. The average lumen
diameter of a capillary is 0.01 mm (10µm), just large enough for red blood cells to slip
through b single file. The extremely thin walls make the capillaries perfectly suited for
their purpose, which is the exchange of nutrients, oxygen and waste products with the
cells of the body.
Collateral blood vessels play a significant role in supplying oxygen to an organ,
particularly when oxygen delivery is limited by disease in the normal vasculature.
Collateral vessels can be pre-existing vessels that normally have little or no blood flow.
Acute occlusion of normal vessels (e.g., thrombosis of a large artery) can cause a
redistribution of pressures within the vascular bed thereby causing blood Dow to occur in
collateral vessels. Collateral blood vessels are particularly important in the coronary and
skeletal muscle (e.g., human leg) circulations. In the heart, collateral vessels can help to
supply blood flow to ischemic regions due to stenosis or occlusion of epicardial arteries.
Collateral blood flow may be an important mechanism in limiting infarct size. Formation
of collateral blood vessels is triggered in the therapeutic angiogenesis.
Angiogenesis is a complex process which involves proliferation of endotheUal
cells, the degradation of the basement membrane, the migration through the surrounding
matrix, as well as the alignment, and differentiation into tube-like structures to form the
wails of blood vessels thus resulting in a newly formed capillary network.
Arteriogenesis, which refers to the outgrowth of collateral arterioles, is also
believed to be the most efficient process for restoration of blood perfusion because of the
high capacity of these vessels compared with the capillary network (Canneliet et al., Nat
Med, 2000; 6389-395; Van Royen et al., Cardiovasc. Res., 2001;49:543-553). In effect,
arterioles are considered as mature robust and functional vessels due to the presence of
both tunica intema and media, i.e., a layer of endothelial cells supported by a layer of
smooth muscle cells. Formation of arterioles is a preferred type for long term and
effective neovascularization.
Among the pathological conditions associated with a vascular endothelial
dysfunction is hypercholesterolemia, a disease characterized by abnormal vessel
formation, an impaired regulation of tissue perfusion, abnormal spatial distribution of
blood flow, as well as abnormal microvascular function. Also, kinetics of vessel growth
as well as the nature of resultant vessels are different from healthy tissues. These changes
can be the result of impaired vascular cndothelium which shows a reduced signal
transduction, a reduced availability of L arginine, a reduced expression of eNOS, NO
inactivation increased by superoxyde anion derived from macrophages other
inflammatory cells, release of several vasoconstricting factors, such as endothelin, and
smooth muscle vascular response. In subjects with hvpercbolesterolemia, capillary
density and distribution in the arterial wall change dramatically. Ultimately, they show
dense plexi of adventitial microvessels with marked disorieatation. It is believed that this
may result from different stimuli that may cause stronger or weaker angiogenic response.
Hypercholesterolemia in humans causes a vascular endothelial dysfunction and
ultimately a progressive narrowing of the main arteries. The unbalance in the coronary
blood flow at rest and during stress creates a furtive malfunction of the muscle that leads
to pain and hypocontractility. Usually the supplies are appropriate at rest, but when stress
occurs, the needs increase while the supplies cannot due to the arterial obstructive
lesions. This is the reason why the purpose of mimicking this human pathology, leads to
both the setup of a stenosis for example, on a major coronary artery and the use of a
stress test to reveal the unbalance created at stress by this stenosis. It is also known that
vascular function in patients with types I and II diabetes mellhus is characterized by
impaired endothelium-dependent relaxation. Diabetes mellitus is characterized by
premature development of microvascular and macrovascular disease (Kannel et al.,
Diabetes Care, 1979, 241:2035-2038). It is thus questionable whether such severe
endothelium impairment and abnormalities due to hypercholesterolemia, diabetes,
hypertension and hyperlipidemia in patients suffering from peripheral arterial disease
(PAD), peripheral arterial occlusive disease (PAOD), or cardiac artery disease (CAD)
might be rescued by using therapeutic angiogenesis.
Administration of several angiogenic factors such as for example VEGF, aFGF
and bFGF, HIF-la/VP16 and TGF-b to promote collateral blood vessels, also known as
therapeutic angiogenesis, has been proposed for the treatment of ischemic cardiovascular
and peripheral ischemia. However, the potent pleiotropic effects on various cell types
may limit the therapeutic applicability of some of these compounds. For example, VEGF-
A was one of the most potent candidate angiogenic factors. However, VEGF-A was
shown to generate edema, as well as disorganized, tortuous and leaky vessels, resembling
to those found in tumors (Lee et al., Circulation 2000,102:898-901; Springer et al., Mol
Cell, 1998,2:549-559).
Two distinct strategies for the therapeutic angiogenesis exist. Protein therapy
which involves delivery of the growth factor directly into the isehemic tissue is a possible
option. Angiogenic gene therapy is an alternative possible option aimed at improving
collateral development and overcoming perfusion defects and related ischemia through
the transfer of nucleic acids to somatic cells (8-10).
Animal studies have demonstrated the local angiogenic potential of many growth
factors including, for example, recombinant human VEGF, recombinant PDGF, ox
recombmant bFGF. However, delivery of recombinant proteins, and the systemic
administration of high doses of recombinant proteins was shown to lead to a multitude of
other negative side-effects. Furthermore, the quantity of the recombinant protein required
is important. If too little protein is delivered, angiogenesis will not be achieved. If too
much protein is delivered, the formation of disorganized vasculature beds and
promiscuous angiogenesis can result
Therapeutic angiogenesis via the administration of nucleic acids capable of
expressing an angiogenic protein either in a naked form or via liposomes or viral vectors
have been investigated. Viral vector delivery of angiogenic coding sequence allows for
high efficiency of delivery, but suffers from numerous disadvantages related to viral
vectors uses, such as the occurrence of an immune reaction as well as the possibility of
integration and dissemination. For example, adenovirus gene therapy methods have been
questioned following the death of Jesse Gelsinger in September 1999 at the University of
Pennsylvania after receiving, through intrahepatic artery infusion, El- and E4- deleted
recombinant adenovirus which expressed a correct form of the human ornithine
transcarbamylase. Pathological analyses have indicated that the official cause of death
was a multi-organ failure secondary to adult respiratory distress syndrome induced by a
systemic inflammatory response to recombinant adenovirus administered systematically.
More recently, two cases of leukemia have been reported in a trial aimed at treating
children with severe combined immunodeficiency disease (SCID) which were attributed
to the use of retro virus as a vector.
Both liposomes and naked DNA comprising a DNA encoding an angiogenic
peptide also suffer from a major disadvantage which is a lesser efficiency of delivery
when compared to virus and the level of the protein needed to achieve a therapeutic effect
may be difficult to reach.
Relating to naked DNA strategy, it was surprisingly demonstrated that
intramuscular injection of the NV1FGF a plasmid encoding an acidic Fibroblast Growth
Factor or Fibroblast Growth Factor type 1 (FGF-1), for patients with end-stage peripheral
arterial occlusive disease (PAOD) or with peripheral arterial disease (PAD), meets safety
requirements. Camerota et al. (J Vasc. Surg., 2002, 35, 5:930-936) describes that 51
patients with unreconstructible end-stage PAD, with pain at rest or tissue necrosis, have
been intramuscularly injected with increasing single or repeated doses of NV1FGF into
ischemic thigh and calf. Various parameters such as transcutaneous oxygen pressure,
ankle and toe brachial indexes, pains assessment, and ulcer healing have been
subsequently assessed. A significant increase of brachial indexes, reduction of pain,
resolution of ulcer size, and an improved perfusion after NV1FGF administration were
observed.
Induction of angiogenesis by VEGF gene transfer in patients with hindlimb
ischemia has also been demonstrated Naked plasmid DNA encoding VEGF-165 isoform
was administered in ischemic muscles of patients with non-healing ischemic ulcers
and/or pain at rest due to peripheral arterial disease. Newly developed collateral blood
vessels and improved perfusion could be seen angtographically at 8 weeks post-treatment
as well as capillaries. However, a significant elevation of the serum levels of VEGF at 5
to 6 weeks after treatment was also observed (Isner et al., Lancet, 1996; 348:370-4). Such
elevation of the serum VEGF level may cause promiscuous unwanted angiogenesis, and
serious negative side effects such as edema (26).
Vincent et al., (Circulation, 2000; 102(18) :2225-61) report that administration of
naked DNA plasmid encoding for the Hypoxia Induced Factor la (HIF-1a) transcription
factor was associated with significant improvements in calf blood pressure ratio,
angiogenic score, regional blood flow and capillary density. However, it is also reported
that HIF-1a activates expression of endogenous VEFG gene suggesting the enhancement
of VEGF-Dathway dependant angiogenesis as well as several targets in vivo.
Taniyama et ah, (Gene Therapy, 2001, 8: 181-189) have further reported
therapeutic angiogenesis using intramuscular injection of naked DNA plasmid coding for
a human Hepatocyte Growth Factor (HGF) in rat and rabbit ischemic hindlimb models.
An increase of the collateral blood vessels was identified by angiography and capillary
density as demonstrated by alkaline phosphatase as a marker of endothelial cells, HGF
which was first identified as a mitogen for hepatocytes, has also been shown to be a
mitogen for certain cell types including melanocytes, renal tubular cells, keratinocytes,
and certain endothelial cells and cells of epithelial origin (Matsumoto et al., BBRC, 1991,
176:45-51). HGF was also shown to stimulate growth of endothelial cells without
replication of vascular smooth muscle cells (Nakamura et al., Hypertension, 1996;
28:409-413; Hayashi et al., BBRC, 1996; 220:539-545). Finally, it was shown that HGF
can also act as a "scatter factor", an activity that promotes the dissociation of epithelial
and vascular endothelial cells (Giordano et al., PNAS, 1993, 90:649-653). Therefore,
HGF has been postulated to be involved in tumor formation.
In contrast, administration of a plasmid encoding acidic fibroblast growth factor
(aFGF or FGF-1) was proved not to increase the FGF-1 serum level thereby showing mat
the use of such human FGF-1 expression plasmid is particularly advantageous in terras of
safety as promiscuous angiogenesis or negative side effects are absent The absence of
circulating FGF-l provides a significant safety advantage over other angiogenic factors
such as for example, VEGF or FGF-2 which have been described to leak into the
circulation and lead to distant edema (Baumgartner et al., Circulation, 1998,97:1114-23),
The fibroblast growth factor (FGF) family is comprised of at least 23 structurally
related proteins (FGF 1-23) whose best known members are FGF-1, FGF-2, FGF-4, FGF-
7 and FGF-9. Members of this family stimulates late mitogenesis in most cells derived
from mesoderm and neuroectoderm and influence other biological processes, including
angiogenesis, neurite extension, osteoblast growth, neuronal survival, and myoblast
differentiation. In general, FGFs have a high affinity for heparin. Prior to resolution of
their nomenclature, some FGFs were referred to as heparin-binding growth factors -1,-2,
etc.), and many, but not all, are mitogens for fibroblasts. The members of the FGF family
possess roughly 25-55% iamino acid sequence identity within a core sequence and some
FGFs possess significant extensions, either C-terminal, N-terminal, or both, outside of
this core sequence. This structural homology suggests that the different genes encoding
known FGFs may be derived from a common, ancestral gene.
In addition to the 23 known members of the FGF family, additional complexity
results from the generation of several molecular forms of FGF from a single gene. For
example, the primary translation product of aFGF (FGF-1) consists of 155 residues.
However, the longest form of FGF-1 found in a natural source (e.g., bovine brain)
consists of 154 residues. This 154 residue form of FGF-1 lacks the NH2-terminal
methionine of the 155 residue form and has an acetylated amino terminus. Proteolytic
processing in vivo or during purification generates smaller active forms of FGF-1 in
which either the amino-terminal 15 (des 1-15) or 21 (des 1-21) amino acids are deleted.
As defined herein, FGF-1 refers to the 154 residue form of FGF-1 and shorter,
biologically active forms thereof such as the above described forms deleted of the
amino-terminal 15 (des 1-15) or 21 (des 1-21) amino acids. Historically, the 154 residue
form of FGF-1 was termed ß-endothelial cell growth factor (ß-ECGF), the des 1-15 form
was termed aFGF or FGF-1, and the des 1-21 form was termed alpha.-ECGF. Prior to
standardization of the terminology for this group of growth factors, several additional
terms were also applied to the same protein, including eye derived growth factor and
heparin binding growth factor 1. Similar forms of bFGF (FGF-2) have also been
described. In addition to cleaved forms, extended forms of bFGF have also been
described, resulting from initiation of translation at several different GTO codons located
upstream of the ATG translation initiation codon which generates the 155 residue form of
bFGF. All of these alternative forms of the FGFs contain the core region of structural
homology which defines the FGF family. Many of the various FGF molecules have been
isolated and administered to various animal models of myocardial ischemia with varying
and often times opposite results.
An angiogenic role for FGF-1 was suggested based on in vivo studies (Comerota
et al., J.Vasc.Surg., 2002, 35, 5:930-936). Intramuscular injections of FGF-1 expression
plasrnid demonstrated an unproved perfusion based on an increased in ankle brachial
index, reduction in pain, and an increased transcutanoous oxygen.
The Applicant has now been surprisingly discovered that intramuscular injection
of a FGF-1 expression plasrnid does not cause induction of VEGF in vascular eadothelial
cells, and thus constitues a very safe angiogenesis therapy in contrast with other of the
angiogenic factors, including other FGF factors, VEGF, HIF-1a VP16 and HGF.
Most therapeutic angiogenesis studies have been validated in animal models of
limb ischemia and performed in normal healthy animals, but few have been tested for
their capacity to reverse angiogenesis defects in a hypercholesterolemia or diabetes
setting, wherein endothelium function is greatly unpaired.
In this regard, known therapeutic angiogeoesis have not proved to be convincing
when tested in hypercholesterolemic rabbits model subjected to femoral artery excision,
as an impaired collateral vessel formation and capillary density that could be only
partially reversed by administration of VEGF was observed (26). In addition, therapeutic
angiogenesis were not proved to be convincing when tested in diabetes models, as
Roguin A. et al. (Cardiovascular Diabetology 2003, 2:18) showed that use of VEGF
expression plasmid failed to improve blood flow and to promote collateralization in a
diabetic iscbemic mouse.
In contrast, the Applicant has surprisingly found that a plasmid expressing the
human FGF-1 when administered intramuscularly in ischemk myocardial or skeletal
muscles was capable of efficiently reversing the hypercholesterolemla or diabetes
associated defects in collateral vessels and promoting the formation of mature vessels
such arterioles in a mammalian subject suffering from hypercholesterolemia or
diabetes.
In addition, the applicant has discovered that in contrast with similar angiogenic
factors, the FOF-1 expression plasmid intramuscular injection did not cause edema in the
treated skeletal or cardiac muscle and thus couid be used in an amount sufficient to
rescue angiogenesis defects of ischemic muscles in aggravated conditions such as
hypercholesterolemia or diabetes setting.
Summary of the Invention
The present invention concerns a method for treating myocardial or skeletal
angiogeaic defects associated with bypercholesterolemia or diabetes comprising the
administration to the subject of pharmaceutical compositions comprising a plasmid
carrying a gene encoding certain fibroblast growth factors in an amount which promote
reversal of eodothelium dysfunction and angiogenic defects.
The present invention also relates to method of treating myocardial or skeletal
angiogenic disorders or defects associated with hypercho/esterolemia or diabetes
comprising administering an effective amount of a plasmid encoding a fibroblast growth
factor, wherein VEGF-A factor expression is not induced in the myocardial or skeletal
muscle.
The present invention further concerns a method of treating vascular endothelium
dysfunction associated with hypercholesteroiemia or diabetes in a patient via the
administration in skeletal or myocardial muscles of an amount of a plasmid encoding a
fibroblast growth factor sufficient to reverse myocardial or skeletal angiogenic defects,
wherein VEGF-A factor expression is not induced and an edema is not generated.
It is also an object of the present invention to provide a method of stimulating
and/or promoting the formation of mature collateral vessels in ischemic cardiac or
skeletal muscle tissues in ischemic muscles in a hypercholesteroiemia or diabetes setting,
comprising injecting said tissues of said subject with an effective amount of a plasmid
encoding a fibroblast growth factor, wherein VEGF-A factor expression is not induced
and/or an edema is not generated.
Another object of the present invention is to provide a method for treating
iscbemic conditions such as PAD, PAOD or CAD in a mammalian subject suffering from
bypcrcholesterolemia or diabetes, without inducing expression of the VEGF factor, and
without causing formation of edema.
Still another object of the present invention is to provide a method for promoting
both collateral blood vessels and arterioles in ischemic tissues, wherein endothelial
function is impaired.
A further object is a method for reversing angiogenesis defects elicited by
hypercholesterolemia or diabetes, without inducing expression of the VEGF factor in a
mammalian subject in need for such treatment suffering from hypercholesterolemia or
diabetes.
Still a further object of the present invention is to provide a method of promoting
angiogenesis VEGF-independent pathway.
A further object of the present invention is to provide a method of promoting
angiogenesis with the provisio that VEGF is not upregulated in the treated cells.
Intramyocardial or intramuscular injection of the FGF expression plasmid is
preferably for the reversal of myocardial or skeletal angiogenic defects associated with
hypercholesterolemia or diabetes. The fibroblast growth factors preferred in the practice
of the present invention is FGF-1, and most preferably the human full-length FGF-1.
Other and further objects, features and advantages will be apparent from the
following description of the preferred embodiments of the invention given for the
purpose of the disclosure when taken in conjunction with the following drawings.
Brief Description of the Accompanying Figures
Figure 1: is a schematic of the design of the experiments.
Figures 2(A) (C): represent cross-sections (magnification X100) of hamster muscles
(Gracillis and Adductores) after HES staining; Fig 2(A) is a cross-section of non-
ischemic (contralateral) muscles; Fig 2(B) and (C) are cross-sections of ischemic
muscles; dashed lines in Fig. 2(B) illustrates the presence of mild necrosis; the arrow in
Fig. 2(C) points to centronucleation.
Figures 3 (A)-(C): set forth representative angiograms recorded in both non-ischemic
(left) and ischemic (right) hindltmbs from hamsters of LC (A); HC/21 (B); and
HC/28(C).
Figure 3 (D): illustrates the corresponding angiographic score obtained by quantification
of collateral formation after hindlimb ischemia.
Figures 4 (A)-(D): show representative cross-sections (magnification XI00) of mature
vessels labeled by smooth muscle a-actin (SMA) immunohistocbemistry from non-
ischemic (A and C) and ischemic (B and D) muscles (Adductores and Gracilis)
harvested at day 21 (A and B) or at day 28 (C and D) after induction of ischemia.
Figures 4 (E) and (F): represent a graph illustrating quantification of muscle area and
ischemic hindlimb. NS: not significant; **: p Figures 5 (A) and (B): set forth representative angiograms of both non-ischemic and
ischemic hindlimbs from hamsters treated with saline (A) or NV1FGF (B).
Figure 5 (C): illustrates quantification of collateral formation through angiographic
score seen after hindlimb ischemia.
Figures 6 (A) and (B): display representative cross-sections (magnification X100)
depicting mature vessels labeled by smooth muscle a-actin (SMA) immunochemistry
from ischemic muscles {Adduciores and Gracilis) of hamsters treated with saline (A) or
NV1FGF (B).
Figures 6 (C) and (D): represent graphs showing quantification of muscle area and
ischemic hindlimb. NS: Not significant; *: p0.01 saline vs. NV1FGF
Figure 7: are representative pictures (magnification X100) of immunochemical staining
with an anti-FGF-1 polyclonal antibody in muscles from the back part of the thigh
(Gracilis and Adductores) from non-ischemic (controlaterai) on injected limbs (A) and
ischemic limbs injected with saline (B) or NV1FGF (C). Arrows show immunoreactive
fibers identified by the brown staining of the immune complexes.
Figures 8 (A)-(C): are histological sections from Tibialis Cranialis muscles stained by
antibody to murine VEGF. (A): NaCl injected muscle section with a mosaic aspect of
myofiber positivity. (B): pCOR-CMV-empty injected muscle section with a similar
aspect (C) NaCl injected muscle section after adsorption of antibody to mVEGF-A with
mVEGF-A peptide.
Figures 8 (D) and (E): are histological serial sections of pCOR-CMV.rat-spFGF-I
injected Tibialis Cranialis muscle stained by antibody to mVEGF-A (D) or FGF-1 (E).
Figure 9A: displays a template showing the location of the 13 injections in the heart of
the rabbit with hypercholesterolemia.
Figure 9B: is a microscopic photo of HES stained sections of the left circumflex
coronary artery from hypercholesteromic Watanabe rabbit at a magnification of x100. A
corresponds to Adventicia; M corresponds to Media; Arrow heads correspond to Intima;
Arrow corresponds to atherosclerotic plaque;
Figure 10: displays an ECG at rest in humans according to the nomenclature of the ST
segment modifications during a stress test in humans (from Braunwald et al. Heart
Disease, 5* ed. pl59). On the left of Figure 10 is shown the ECG at rest in humans, and
on the right, from top to bottom, a progressively more serious modification of the ST
segment, depending on the slope of this segment: upsloping, horizontal, and
downsloping. The elevation shown at the bottom is the most serious.
Figure 11A.: displays an ECG in rabbits during a dobutamin stress test.
Figure 11B: displays an enlargement of the lead I. The ST depression, horizontal is
clearly seen just after the QRS complex by a large vertical peak.
Figure 12: displays an ECG scoring at the highest dobutamin dose
Figure 13A: displays a typical 12 lead ECO in a rabbit at rest
Figure 13B: displays a strong ischemia at maximun stress in the same rabbit.
Figure 14: is a schematic of the nomenclature of the 2D echocardiography.
Figures 15: show myocardial microscopic lesions and associated FGF-1 expression in
healthy rabbit heart 3 days after the injection of NV1FGF. HES staining demonstrating
myocardial degeneration and necrosis with active chronic inflammatory response (A) and
associated FGF-1 expression (arrow heads, B). Background (*) is relative to secondary
antibody conjugation (anti-rabbit) with endogenous IgG. Magnification: x100.
Figure 16: displays the evolution of the maximum ECG score during the stress test on
rabbits treated with empty plasmid (grey column) or NV1-FGF plasmids (hatched
column).
Figure 17: displays the quantification of 16 normal segments (grey) and 14 abnormal
segments (black). The qualification normal / abnormal was a visual evaluation.
Figure 18: displays a plot of the ECG maximum score versus the Echo maximum score.
The regression curve is shown in black. The two main zones (abnormal ECG and
abnormal echo, normal ECG and normal echo) are shaded in grey.
Figure 19: displays the evolution of the echocardiographic score with the time after
treatment. The NV1-FGF treated animals are shown in hatched, the empty plasmid-
treated animals are shown in grey. A • indicates a significant difference (p between groups.
Figure 20: presents a standardized procedure used for the preparation of heart sections
samples for histologic analysis with various sectors of the heart according to the 3 short
axis slices, e.g, apical, mid and basal segments.
Figure 21: displays a quantitative analysis of vascular density in the scar in viable
myocardium distant from the scar.
Detailed Description of the invention
The present invention provides a method for treating or repairing myocardial or
skeletal angiogenic defects associated with hypercholesterolemia or diabetes setting in
which endothelial functions are unpaired or inadequate.
The present method and composition are particularly useful in reversing
endothelium dysfunction associated with hypercholesterolemia or diabetes, following
direct intramuscular administration to promote a net increase of blood vessel formation in
the myocardial or skeletal muscle. The invention encompasses the use of a plasmid
encoding a biologically active fibroblast growth factor and pharmaceuticaUy acceptable
salts and derivatives thereof.
The present invention also provides a method of promoting the formation of
mature collateral vessels in ischemic cardiac or skeletal muscle tissues in a mammalian
subject in need of such treatment comprising injecting said tissues of said subject with an
effective amount of a plasmid encoding a fibroblast growth factor, wherein VEGF-A
factor expression is not induced in said subject Particularly, administration of PGF
expressing plasmid induces the formation of both collateral blood vessels and arterioles
in ischemic myocardial or skeletal muscle tissues, without inducing expression of the
VEGF-A factor. The FGF expression plasmid according to the present invention may not
cause side effect such as edema.
The present invention further provides a method of reversing defects in
angiogenesis elicited by hypercholesterolemia or diabetes, without inducing VEGF-A
factor expression, and/or causing the formation of edema, comprising injecting
myocardial or skeletal tissues of said patient with an effective amount of a plasmid
expressing a fibroblast growth factor to promote the formation of both collateral blood
vessels and arterioles.
The present invention further provides a method for enhancing revascularization
by promoting both collateral blood vessels and arterioles in ischemic tissues of a
mammalian subject in a hypercholesterolemic or diabetes setting, which comprises
injecting said tissues of said subject with an effective amount of a FGF expression
plasmid to reverse angiogeoesis defects. The delivery and expression of said plasmid
unexpectedly results in a significant improvement of the blood perfusion throughout the
ischemic muscles.
The term "subject" includes, but is not limited to, mammals, such as dogs, cats,
horses, cows, pigs, rats, mice, simians, and humans.
The term biologically active sequence means a nucleotide sequence encoding a
naturally occurring peptide or any biologically active analogues or fragments thereof.
Different forms exist in nature with variations in the sequence of the structural gene
coding for peptides of identical biological function. These biologically active sequence
analogues include naturally and non-naturally occurring analogues having single or
multiple amino acid substitutions, deletions, additions, or replacements. All such allelic
variations modifications and analogues resulting in derivatives which retain one or more
of the native biologically active properties are included in the scope of this invention.
The FGF encoding plasmid thus comprises a nucleotide sequence that encodes the
desired FGF protein. These molecules may be cDNA, genomic DNA, synthesized DNA
or a hybrid thereof or an RNA molecule such as mRNA. Preferably, the plasmid
comprises a nucleotide sequence encoding the FGF-1 and thus encompasses a nucleotide
sequence encoding the 154 residue form of FGF-1 acidic growth factor as described in
US patent 4,686,113.
The regulatory elements necessary for gene expression of a DNA molecule may
comprise a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In
addition, enhancers are often required for gene expression. It is necessary that these
elements be operable linked to the sequence that encodes the desired proteins and that the
regulatory elements are operable in the myocardium of the subject to whom they are
administered-
Initiation and stop codons are generally considered to be part of a nucleotide
sequence that encodes the desired protein. However, it is necessary that these elements
are functional in the subject to whom the gene construct is administered. The initiation
and termination codons must be in frame with the coding sequence.
Promoters and polyadenylation signals used must be functional within the
myocardial cells of the subject
Examples of promoters useful to practice the present invention include but are not
limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus
(MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long
Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such
as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus
(RSV) as well as promoters from human genes such as human alpha actin, human
Myosin, human Hemoglobin, human muscle creatine and human metalothionein.
In another preferred embodiment, the expression of tbe FGF genes is driven by
muscle specific promoter, such as the murine or human upstream sequence of the CARP
gene which is described in the US publication 2003/0039984, or the cardiac alpha actin
promoter sequence as described in the international publication WO01/11064.
Examples of polyadenylation signals useful to practice the present invention,
especially in the production of a genetic vaccine for humans, include but arc not limited
to SV40 polyadenylation signals, bovine or human Growth hormone polyadenyiaticn
signals, and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal
which is in pCEP4 plasmid (Invitrogen, San Diego Calif.), referred to as the SV40
polyadenylation signal is used.
In addition to the regulatory elements required for DNA expression, other
elements may also be included in the DNA molecule. Such additional elements include
enhancers. The enhancer may be selected from the group including but not limited to:
human Actin, human Myosin, human Hemoglobin, human muscle creatine and viral
enhancers such as those from CMV, RSV and EBV.
Genetic constructs can be provided with mammalian origin of replication in order
to maintain the construct extrachromosomally and produce multiple copies of the
construct in the cell. Plasmids pCEP4 and pREP4 from Invitrogen (San Diego, Calif.)
contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-1 coding
region which produces high copy episomal replication without integration. Other suitable
plasmids are well known to those skilled in the art, for example, plasmid pBR322, with
replicator pMBl, or plasmid pMKl6, with replicator ColEl (Ausubel, Current Protocols
in Molecular Biology, John Wiley and Sons, New York (1988) §II:1.52.
In a preferred embodiment the FGF encoding plasmid present a conditional origin
of replication pCOR as described in the International application WO 97/10343 and
Soubrier et al. (Gene Ther. 1999;6:1482-1488). The pCOR plasmid harbors an optimized
expression cassette encoding a secreted form of human FGF-1 (sphFGF-l) inserted into
an original backbone. The resulting plasmid is advantageously of small size of 2.4 kb.
The sequence encoding sphFGF-1 is a fusion between the sequences encoding the
secretion signal peptide (sp) from human fibroblast interferon and the naturally occurring
truncated form of human FGF-1 from amino acids 21 to 154 (US 4,686,113; US
5,849,538). Expression of sphFGF-1 was driven by the human cytomegalovirus (CMV)
immediate early enhancer/promoter (from nucleolide -522 to +72). The late
polyadenylation signal from simian virus 40 (nucleotides 2538 to 2759 from SV40
genome, GenBank locus SV4CG; US 5,168,062) was inserted downstream of the
sphFGF-l fusion to ensure proper and efficient transcription termination and subsequent
polyadeaylation of the sphFGF-1 transcript This prefemed plasmid is designated
NV1FGF and is devoid of any antibiotic resistancegene. plasmid selectiion relies on a
suppressor transfer RNA gene in the autotrophic recipient strain. Maintenance of high
copy number and strictly limited host range of the plasmid were obtained with the R6K ?
origin of plication. The sequence coding for this protein is not usually found in bactena
but was artificially inserted into the genome of the selected host strain. Thus, plasmid
potential dissemination was greatly limited.
plasmids according to the present invention can be administered to the vertebrate
by any method that delivers injectable materials to cells of the myocardium. Preferably,
the plasmid are administered as naked DNA plasmid in the sense that they are free from
any delivery vehicle that can act to facilitate entry into the cell, for example, the
polynuclcotide sequences are free of viral sequences, particularly any viral particles
which may carry genetic information. They are similarly free from, or naked with respect
to, any material which promotes transfection, such as liposomal formulations, charged
lipids such as Lipofectin™, or precipitating agents such as CaPO4. Plasmid may
otherwise be delivered to the animal with a pharmaceuticaily acceptable liquid carrier. In
preferred applications, the liquid carrier is aqueous or partly aqueous, comprising sterile,
pyrogen-free water. The pH of the preparation is suitably adjusted and buffered
Alternatively, the plasmid may be injected with the use of liposomes, such as cationic or
positively charged liposomes.
The following Examples clearly demonstrate that the plasmid NV1FGF allows a
slow release of the encoded FGF-1 protein at a concentration sufficient to promote a
sustained angiogenic response via the formation of capillary vessels as well as mature
vessels such as arterioles. In addition NV1FGF was shown to be particularly potent, as it
was demonstrated to efficiently promote angiogenesis at a non-detectable concentration
in treated muscles. NV1FGF may thus be used at concentrations which are within a
therapeutic window, thereby avoiding negative side effects due to dissemination to
surrounding tissues or organs or promiscuous angiogenesis.
In addition, it was demonstrated that due to such superior characteristics in terms
of safety and potency, NV1FGF was particularly useful for therapeutic angiogenesis in
aggravated conditions caused by hypercholesterolemia or diabetes.
The reversal of angiogenesis defects caused by attenuated blood supply regardless
of its origin which is aggravated in conditions such as hypercholesterolemia or diabetes
is thus contemplated by the present invention.
Within the context of the present invention, the target tissue thus comprises
muscle tissues suffering from or being at risk of suffering from ischemic damage which
results when the tissue is deprived of an adequate supply of oxygenated blood, further
aggravated in a hypercholesterolemia or diabetes setting. As demonstrated in the
Examples, the intramuscular injection of a plasmid NV1FGF may be efficiently used in a
therapeutic window which is compatible with required standard of safety in gene therapy
and is capable of inducing angiogenesis in an ischemic tissue further presenting an
impaired endothelial function.
According to one embodiment of the present invention, the NV1FGF plasmid is
administered in a localized manner to the target muscle tissue. While, any suitable means
of administering the NV1FGF plasmid to the target tissue can be used within the context
of the present invention, preferably, such a localized injection to the target muscle tissue
is accomplished by directly injecting the NV1FQF to the muscle using a needle.
By the term "injecting" it is meant that the NV1FGF is forcefully introduced into
the target tissue. Any suitable injection device can be used according to the present
invention.
While administration of a dose of the NV1FGF plasmid can be accomplished
through a single injection to the target tissue, preferably administration of the dose is via
multiple injection of NV1FGF. The multiple injections can be 2,3,4, 5, or more repeated
injections, and preferably 5 or more injections into the ischemic muscle of a mammalian
subject suffering from hypercholesterolemia or diabetes. Multiple injections present an
advantage over single injections in that they can be manipulated by such parameters as a
specific geometry defined by the location on the target tissue where each injection is
made. The injection of a single dose of the NV1FGF via multiple injections can be better
controlled, and the effectiveness with which any given dose is administered may be
maximized.
The specific geometry of the multiple injections may be defined either in two-
dimensional space, where the each application of the NV1FGF is administered The
multiple injections may be performed in or around the ischemic tissue, preferably are
spaced such that the points of injection are separated by 2 or 3 cm.
According to another embodiment of the present invention, each of the multiple
injections is performed within about 5 to 10 minutes of each other.
When administering the NV1FGF to the target tissue which is affected by
angiogenesis defects and wherein the endothelium function is severely impaired, it is
desirable that the administration is such that the NV1FGF is able to contact a region
reasonably adjacent to the source and the terminus for the collateral blood vessel
formation, as well as the area therebetween.
Administration of the composition according to the present invention to effect the
therapeutic objectives may be by local, intramuscular, parenteral, intravenous,
mtramyocardial, pericardial, epicardial or via intracoronary administration to the target
cardiac muscle tissue. Preferably, intramyocardial, epicardial, pericardial or
intracoronary administration is conducted using a needle or a catheter.
Preferably, intramuscular injection of NY 1FGF may~be performed into the distal
thigh and distal leg muscles, and in the region close and surrounding the ischemic she.
Also, when administration is performed by direct intramyocardial injection, those
may be performed during an open chest surgery or with the help of a catheter. Catheters
for heart delivery are well known in the art and include for example needle catheter as
described in US patents 5*045,565 or 4,661,133, with position sensor system as described
in US patents 6,254,573 and 6,309,370. Alternative catheters having a helix needle are
described in US patents 6,346,099 and 6,358,247.
In one advantageous aspect of the present invention, a therapeutiLcally effective
dose of NV1FGF is administered to reverse the defects in angiogenesis in a
hypercholesterolemic or diabetes setting. While the effective dose will vary depending on
the weight and condition of a given subject suffering from angiogenesis defects in
addition to hypercholesterolemia or diabetic subject, it is considered within the skill in
the art to determine the appropriate dosage for a given subject and conditions
According to a preferred embodiment of this aspect, treatment is performed with
dose of about 8000ug to about 16000ug of plasmid that is administered by multiple
injections of preferably 2 to 4 repeated intramuscular injections of KV1FGF with an
interval of time of around 1 to 2 weeks or more, in severe conditions of angiogenesis
defects, in order to promote a sustained formation of both collateral vessels and
arterioles, thereby allowing to reverse angiogenesis defects due to ischemia in a
mammalian subject suffering from hypercholesterolemia or diabetes.
The NV1FGF desirably is administered to the target ischemic muscle in a
pharmaceutical composition, which comprises a pharmaceutically acceptable earner and
the NVlFGFplasmid.
Any suitable pharmaceutically acceptable carrier can be used within the context
of the present invention, and such carriers are well known in the art. The choice of carrier
will be determined, in part, by the particular site to which the composition is to be
administered and the particular method used to administer the composition. Formulations
suitable for injection include aqueous and non-aqueous solutions, isotonic sterile
injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes
that render the formulation isotonic with the blood of the intended recipient, and aqueous
and non-aqueous sterile suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. The formulations can be presented in
unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored
in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid
carrier, for example, water, immediately prior to use. Extemporaneous injection solutions
and suspensions can be prepared from sterile powders, granules, and tablets of the kind
previously described. Preferably, the pharmaceutically acceptable carrier is a buffered
saline solution. Most preferably, the pharmaceutical composition comprises a solution of
sodium chloride (0.9%).
In a preferred embodiment the composition of the present invention is
administered in association with a low molecular weight heparin (LMWH). LMWH
molecules and method of preparation are well-known in the art and are described inter
alia in US 5,389,618; US 4,692,435, and US 4,303,651, European patent EP 0040 144,
and by Nenci GG (Vasc.Med, 2000; 5:251-258), which are herein incorporated by
reference.
In a second embodiment, the FGF expression plasmid is injected in the skeletal
muscle of a hypercholesterolemic or diabetic patient prior or after the administration of
an electrical stimulation to the treated skeletal muscle. The electrical stimulation used
according to this embodiment is as described in US2002/0031827, and is applied at a
voltage and a frequency that do not cause contraction of the skeletal muscle as well as no
pain to the patient, as it is below the threshold for muscle contraction. For example, the
frequency applied is around 50 Hfc, and the voltage is around O.lVolt "When used in
combination with the FGF expression plasmid, the electrical stimulation results in a
synergistic superior effect in terms of increase of blood flow.
In a third embodiment, the FGF-1 expression plasmid is BB is delivered in
combination with one or more angiogenesis-promoting factors. Without any limitation,
angiogenic fector may include PDGF-AA, M-CSF, GMCSF, VEGF-A, VEGFr-B, VEGF-
C. VEGF-D,. VEGF-E,. neuropilin, FGF2(bFGF), FGF-3, FGF-4, FGF-5, FGF-6,
Angiopoietin 1, Angiopoietin 2,1 5 erytbropoietin, BMP-2, BMP-4, BMP-7, TGF-beta,
IGF-I, Osteopontin, Pleiotropin, Activin, Endothelin-1 and combinations thereof.
Preferably, the NV1FGF is injected in skeletal or cardiac muscle with a PDGFBB
expression plasmid and results in a superior formation of collateral blood vessels and
arterioles in hypercholesterolemia or diabetes setting. This embodiment thus relates to a
method of promoting collateral blood vessels and arterioles comprising delivering
NV1FGF and a plasmid expressing PDGF-BB to a localized area of tissue in an amount
effective to induce angiogenesis within the area of tissue. The angiogenesfe-promoting
fector(s) is delivered by expression from isolated DNA encoding the factor following
delivery of the DNA to the localized area of tissue
The present invention also relates to a method of treating PAD and PAOD, CAD
or CHF pathologies in patients further suffering from hypercholesterolemia and diabetes.
Impaired perfusion in the hindlimb due to single or multiple large vessel
occlusions is the cause of PAD. At an early stage this results in discomfort in the muscles
of the leg with ambulation, leading at later stages to ulceration and gangrene (1). Chronic
cardiovascular disorders are aggravating factors is patients who are already suffering of
ischemic conditions, such as PAD, through mechanisms involving endothelium
dysfunction (2, 3). Pathologies such as hypercholestierolemia, hypertension and diabetes
have been investigated as possible targets for developing experimental models of PAD
(4-7). Nevertheless, in such models of hindlimb ischemia, a critical point is to negate a
spontaneous angiogenic response to allow efficacy of any revascularization treatment in
an attempt to mimic the clinical situation of hindlimb ischemia.
Genetic models of hypercholesterolemia have also been used to assess angiogenic
properties in hypercholesterolemia or diabetes conditions, however in such genetic
models a single alteration of a single receptor (deficit in the LDL receptor in Watanabe
heritable hyperlipidemic rabbits) or protein (deficit in the glycoprotein ApoE resulting in
inceased levels of VLDLs and IDLs in ApoE mice) is generated and do not actually
reproduce the conditions of the pathology.
In contrast, NV1FGF plasmid was demonstrated to be particularly potent in
reversing hypercholesterolemia-elicited defect in animal models which are very
comparable to the pathological conditions found in patients. Indeed, the pathology results
from a global lipid overload due to cholesterol-rich thet mimicking the situation
encountered in PAD patients suffering from hypercholesterolemia.
According to the present invention, the NV1FGF has been demonstrated to be
particularly potent for rescuing cholesterol-induced impairment of angiogenesis in
patients suffering from PAD, by promoting the growth of both collateral vessels and
arterioles.
Still another object of the present invention is to provide a method for promoting
both collateral blood vessels and arterioles in ischemic tissues, wherein endothelial
function is impaired.
As shown in the following examples, the KV1FGF is capable to effectively
induce the formation of mature large conductance vessels (>150µm collateral vessels)
and small resistance arteries ( posterior part of the thigh, which are required to convey and to deliver blood to tissues.
Induction of such mature vessels represent a particularly efficient treatment in most
severe cases where adverse angiogenesis defects are elicited by hypercholesterolemia or
diabetes.
Outgrowth of both collaterals and arterioles is of particular interest in therapeutic
angiogenesis. Indeed, collaterals are vessels forming bridges between arterial networks
while arterioles are mature vessels formed of a layer of endothelial cells stabilized by
mural cells (pericytes or smooth muscle cells) providing bulk flow to the tissue. Capillary
networks are therefore dependent on their presence for ensuring distribution of the flow.
(Carmeliet et si., Nat. Med., 2000; 6:389-395; Van Royen et aL, Cardiovasc. Res.,
2001;49;543-553).
A further object of the present invention is to provide a method of promoting
angiogenesis with the provisio that VEGF is not upregulated in the treated cells.
The following examples demonstrate that intramuscular administration of
injection of NV1FGF does not lead to local murine VEGF-A induction in injected
muscles and does not lead to murine VEGF-A secretion in the circulating blood of the
injected mice. This is an important aspect of the present invention, which is related to a
new method for promoting collateral blood vessels and arterioles, without inducing the
VEGF-A fector, in a VEGF-independent pathway, In effect, it is well known that VEGF-
A cause serious negative side effects such as promiscuous unwanted angiogenesis,
edema, and potential of tumorigenicity.
Throughout this application, various publications, patents and patent applications
have been referred to. The teaching and disclosures of these publications, patents and
patient applications in their entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which the present application
pertains.
It is also understood and expected that variations in the principles of invention
herein disclosed in an exemplary embodiment may be made by one skilled in the art and
it is intended that such modifications, changes and substitutions are to be included within
the scope of the present application.
Examples
Example 1: Animals and thets
Syrian Golden hamsters (n=S0) of 11-12 weeks (CERJ, Le Genest St Isle, France) were
used in the experiments. Animals were allowed to equilibrate in standard conditions at
least 7 days before initiation of the study protocol. All animals had free access to water
for the entire duration of the experiments. All animal procedures were approved by the
Animal Use Committee of Aventis Pharma and conducted in accordance with guidelines
published by the National Institute of Health (NIH publication No. 85-23, revised 1985).
In experiment 1, hamsters (n=37) were randomly divided into three groups
(Figure 1). Hamsters in the low cholesterol (LC) group (n=13) were fed ad libitum with
standard chow (ref. A04-C, UAR, Epinay-sur-Orge, France). Hamsters in both high
cholesterol (HC) groups during 21 days (HC/21) and 28 days (HC/28) post-treatment
(HC/21: n=12; HC/28: n=12) were given 20g per animal of cholesterol-enriched thet
daily, made of standard chow supplemented with 3% cholesterol and 15% cocoa butter
(ref. 1414C, UAR, Epinay-sur-Orge, France). In experiment 2, hamsters (n=13) were
randomly allocated to two groups (Figure 1). Hamsters in the saline group (n=5) and in
the NV1FGF group (n=8) were fed with cholesterol-enriched diet, as described in
experiment 1.
Example 2: Induction of hindlimb ischemia
After 35 days of LC or HC diet, animals Were subjected to hindlimb ischemia,
according to the following surgical procedure. Hindlimb ischemia was induced under gas
anesthesia with N2O (0.8 Limn-1), O2 (0.4 Lmin-1) and isofluorane (2%) according to a
procedure described in other animal species (19,20). Under sterile surgical conditions, a
longitudinal incision was performed on the medial thigh of the right hindlimb from the
inguinal ligament to a point proximal to the patella. Through this incision, using surgical
loops, the femoral artery was dissected free and its major branches were coagulated. The
femoral artery was completely excised from its proximal origin as a branch of the
external iliac artery to the point distal where it bifurcates into the saphenous and popliteal
branches (20). The incision was closed in one layer with a 4.0 silk wire.
Example 3; Gene transfer in hindlimb skeletal muscles
In experiment 2, saline (n=5) or plasmid encoding NV1FGF (180µg DNA, n=8)
was given blind 14 days after induction of ischemia, through three 50-µL injections each
in Tibialis cramalis, Adductores and Quadriceps muscles of the ischemic limb.
Example 4: Measurements of total cholesterol and triglyceride levels in sernm
On days -35, -7, and +21 or +28 related to the day of surgery, blood was obtained
from hamsters of HC/21, HC/28, saline and NV1FGF groups by retro-orbital puncture
under gas anesthesia with N20 (0.8 Lmin-1), O2 (0.4 Link-1) and isofluorane (2%). Total
cholesterol and triglyceride levels in serum were determined enzymatieally with
commercially available kits (Olympus Diagnostica GmbH, Hamburg, Germany).
Example 5: Quantification of collateral vessel formation by angiography
On day 21 (HC/21 group) or day 28 (LC, HC/28, saline and NV1FGF groups)
after induction of ischemia, an angiographic procedure was performed as follows.
Immediately after injection of ~300 µl of contrast medium (0.5 g.ml-1 sulfate
barium solution in water) through a catheter inserted into abdominal aorta, hamsters were
sacrificed with an overdose of sodium pentobarbital. Hamsters were placed in dorsal
decubitus into a radiography apparatus (model MX-20, Faxitron X-ray Corp., Wheeling,
IL, USA) and. post-mortem pictures of the vasculature from both limbs were collected and
digitalized (software Specimen, DALSA MedOptics, Tucson, AZ, USA). This
radiographic system allows visualization of vessels with diameters higher than 150 µm.
Pictures were analysed off-line by an investigator blinded to me treatment, with dedicated
software as previously described (22),
Briefly, for both ischemic and non-ischemic limbs, the extent of collateral vessels
in the posterior side of the thigh was determined as a percentage of the area analysed.
Angiographic score was calculated as the ratio ischemic/non-ischemic percentages. In
order to check the validity of the method, angiographic score was assessed in six separate
age-matched hamsters not subjected to hindlimb ischemia. As expected, angiographic
score calculated as the ratio right limb/left limb percentages was 1.04 ± 0.18, reflecting
similar vascularization in both limbs.
Example 6: Quantification of arteriolar formation by immunohistochemistry and
typical muscle lesions induced by hindlimb ischemia through excision of the femoral
artery of hypercholesterolemic hamsters
On day 21 (HC/21 group) or day 28 (LC, HC/28, saline and NV1FGF groups)
after induction of ischemia, skeletal muscles from the ischemic hindlimb were harvested
and fixed in a solution of PBS-3.7 % formaline, Muscles from the non-Ischemic hindlimb
were sampled similarly and served as control muscles. Two transverse slices composed
of different muscles (Gracilis, Semimembranosus, Adductores, Semitendinosus, Biceps
femoris), were processed from the back part of each thigh. Slices were dehydrated,
embedded in paraffin and 5-um thick sections were prepared for immunohistochemistry.
A mouse monoclonal antibody directed against smooth muscle a-actin (SMA; clone 1A4,
dilution 1:200, Dako, Carpinteria, CA, USA) was used as a marker for vascular smooth
muscle cells (VSMCs) since it is constitutively expressed in mature vessels. The SMA
antibody was detected with a commercially available kit (EnvisionTM+ System/Horse
Radish Peroxidase, Dako, Carpinteria, CA, USA) through an avidin-biotin-peroxidase
method. SMA-positive (SMA+) vessels were ranked by size (outer diameter) and
arterioles with diameter These muscles have been chosen for their susceptibility to histopathological
lesions in our hindlimb ischemia hamster model, conversely to other muscles from the
posterior side of the thigh (Semitendinosus, Semimembranosus and Biceps femoris).
Typical lesions induced by excision of femoral artery are shown in Figure 2,
Muscles from the back part of the thigh, i.e., Gracilis and Adductores were harvested 28
days after induction of ischemia, and 5µm thick sections of the muscles after HES
staining were observed. Figures 2B and 2C show the presence of mild necrosis (dashed
line) and centronucleation (arrows) in ischemic muscles, respectively. Figure 2A shows a
cross-section at magnification X100 of the non-ischemic controlateral muscles having no
lesions, as a control. Total area of Adductores and Gracilis muscles was determined to
investigate the impact of ischemia on muscle volume. Number of SMA+ arterioles was
determined for the total muscle area. For both parameters, the ratio ischemic/non-
ischemic values was then calculated. All procedures were performed by an investigator
blinded to the treatment
Example 7: Expression of FGF-1 after NV1FGF gene transfer in ischemic muscles
In experiment 2, 14 days after saline injection or NV1FGF gene transfer (ie., 28
days after induction of ischemia), muscles from the back part of the thigh (Gracilis and
Adductores) from non-ischemic and ischemic limbs were processed as follows. FGF-1
immunohistochemistry was performed using a classical streptavidin-biotin assay used to
detect FGF1 expression. The incubation with a primary polyclonal anti-FGF-1 rabbit
antibody (reference AB-32-NA, 1:30 dilution, R&D Systems, Abingdon, UK) was
followed by incubation with a biotinilated donkey anti-rabbit immunoglobulin (1:200
dilution, Amcrsham, Buckinghamshire, UK). The immune complexes were localized
using a chromogenic diaminobenzidine substrate, after adding peroxidase coupled to
streptavidine. The 5-um thick sections were counterstained with hematoxylin, dehydrated
and mounted with permanent mounting media, lmmunoreactive fibers were identified
(brown staining) under a microscope (Axioplan 2, Zeiss, Hallbergmoos, Germany).
Statistical analysis
Results are expressed as mean SD. Statistical significance was assumed at
p In experiment 1, serum lipid levels in groups HC/21 and HC/28 were compared at
the various timepoints by ANOVA followed by Tukey-Kramer post-test for comparison
between any 2 values. Angiograpbic scores calculated in groups LC, HC/21 and HC/28
were compared by ANOVA followed by Tukey-Kramer post-test Non-ischemic and
ischemic values of muscle area and number of SMA positive anterioles, as well as
corresponding ratios, were compared by unpaired t-test in groups HC/21 and HC/28.
In experiment 2, serum lipid levels in saline and NV1FGF groups were compared
at the various timepoints by ANOVA followed by Tukey-Kramer post-test for
comparison between any 2 values. Angiographic scores, non-ischemic and ischemic
values of muscle area and number of SMA positive arterioles, as well as corresponding
ratios, were compared by unpaired t-test in saline and NV1FGF groups.
Example 8: Measure of serum lipids in low cholesterol and cholesterol-rich thet
Tables 1 and 2 summarize serum lipid levels in experiment J and experiment 2,
respectively, before cholesterol-rich thet was given (day -35) and at the various
timepoints following thet modification (days -7 and +21 or +28). Cholesterol-rich diet
led to a time-dependent increase both in total cholesterol and triglyceride serum levels.
Example 9: Effect of cholesterol-rich thet on collateral development and arteriolar
density after hindlimb ischemia (Experiment 1)
As illustrated in Figures 3, collateral formation 28 days after hindlimb ischemia
was high in LC group (Fig. 3 A), leading to angiographic score of 0.93 ± 0.45 (Fig. 3D).
Conversely, in both HC groups formation of collaterals was dramatically impaired
21 or 28 days after hindlimb ischemia with angiographic scores decreased to 0.35 ± 0.38
and 0.37 ±021, respectively (see Figures 3B and 3C>. Decrease in angiographic score
was significant in both HC groups compared to LC group (PO.01). Nevertheless,
angiographic scores were not different between HC/21 and HC/28 groups (P>0.05) as
illustrated in Figure 3D.
Figures 4A-4D display representative cross-section at magnification X100,
depicting mature vessels labeled by smooth muscle a-actin (SMA)
immunohistochemistry from non-ischemic and ischemic muscles (Adductores and
Graeilis ) harvested at day 21 or day 28 after induction of ischemia and quantification of
muscle area and As illustrated in Figures 4E-F, area of Adductores and Gracilis muscles decreased
in the ischemic limb compared to the non-ischeraic limb, 21 days after ischemia
(P observed 28 days after hindlimb ischemia. In addition, the number of significantly decreased in the ischemic limb compared to the non-ischemic limb
(P significance in the intermediate steps of calculation of arteriolar density, density itself
was not different 21 and 28 days after initiation of thet modification. Muscle area, SMA+
arterioles and arteriolar density expressed as ratios ischemic/non-ischemic limb were not
different on days 21 and 28.
These results clearly demonstrate that hypercholesterolemia induces an inhibition
of the development of collateral vessels, and a chronic angiogenic disorder both at the
macro- and microvascular levels, as quantified by angiography, and of quantified by histological methods to the same extend, 21 and 28 days after hindlimb
ischemia (Figures 2B and 2C),
In addition, hypercholesterolemic hamsters used in this Study provide a
particularly severe model, as the lipid overload applied to our model is elevated and
clearly results in endothelial dysfunction and defect in angiogenesis response after
hindlimb ischemia. In addition histopathological analysis of arteries harvested from
hamsters 4 weeks after initiation of the cholesterol-rich thet revealed me presence of
foam cells. Furthermore, there is a continuous increase in the total cholesterol and
triglycerides levels lasting during the period of recovery from hindlimb ischemia,
thereby placing the model is the worst case scenario in terms of severity of the
endothelial impairment and angiogenesis defects.
Example 10: Effect of NV1FGF gene transfer on collateral development and
arteriolar density 14 days after intramuscular administration (i.e., 28 days after
hindlimb ischemia) in hypercholesterolemic hamsters (Experiment 2)
As indicated in Figure 5B, intramuscular NVlFGF gene transfer 14 days after
induction of hindlimb ischemia greatly improved collateral formation in the ischemic
limb, when compared with saline-treated hamsters (Figure 5A), Also, angiographic score
after NV1FGF geae transfer (0.75 ± 0.47) was indeed significantly higher (P that of saline-treated hamsters (022 ± 0.05).
Figures 6A and 6B which arc representative cross-sections (magnification X100)
depicting mature vessels labeled by smooth muscle a-actin (SMA)
immunohistochemistry from ischcmic muscles of hamsters treated with saline and
NV1FGF and quantification of muscle area and A decreased area of Adductores and Gracilis muscles in the ischemic limb was
observed in both saline and NV1FGP groups (P=0.2404 and P=0.0846, respectively). As
indicated in Figures 6C and 6D, muscle area expressed as ratio ischemic/non-ischemic
limb was similar in saline and NV1FGF groups (p=.4584). The number of arterioles decreased significantly in the ischemic limb compared to the non-ischemic limb
(P=0.0333) in saline-treated animals, whereas the difference was not significant in
NV1FGF group (P=0.1347). Calculation of the ratio ischemic/non-ischemic limb
revealed a statistically higher value in NVlFGF-treatcd muscles compared to saline
(P=0.0187). Nevertheless, arteriolar density was not different in muscles from non-
ischemic and ischemic limbs of either treatment group (P=0.9320 and P=0.1586 for
saline and NV1FGF, respectively). Axteriolar density expressed as the ratio
ischemic/non-ischemic limb was not different between saline and NV1FGF groups (P=
02724).
Example 11: Expression of FGF-1 in ischemic muscles after NV1FGF gene transfer
In contrast with gene therapy involving other angiogenic fectors, such as VEGF
or FGF-2, the inventors have evidenced that the expression of FGF-1 was advantageously
restricted to the ischemic muscles of animals treated with NV1FGF.
Also, as shown by representative pictures (magnification X100) of
immunohistochemical staining with an anti-FGF-1 polydonal antibody in muscles from
the back part of the thigh (Gracilis and Adductores) from non-ischemic (controlateral)
non injected limbs (Figure 7A) and ischemic limbs injected with saline (Figure 7B) or
with NV1FGF (in Figure 7C), the expression of FGF-1 could surprisingly be detected
neither in the ischemic muscles of saline-treated animals, nor in non-ischemic
(controlateral) muscles of saline and NV1FGF-treated animals. This clearly shows the
superior properties of the plasmid NV1FGF which allows a slow release of the encoded
FGF-1 protein within a therapeutic window sufficient to effect a sustained angiogenic
response via the formation of mature blood vessels, but at a concentration which does not
permit dissemination and promiscuous angiogenesis or negative side effects. NV1FGF
was thus proved to be particularly potent, as being capable of efficiently promoting
angiogenesis at a non-detectable concentration in treated muscles, thus allowing use of
concentrations of NV1FGF comprised within a therapeutic window and in conditions
characterized by aggravated endothelial dysfunctions. Due to such superior
characteristics in terms of safety and potency, the NV1FGF may advantageously be used
as angiogeaesis therapy in aggravated conditions caused by hypercholesterolemia or
diabetes.
These results clearly demonstrate that NV1FGF gene therapy is capable of
rescuing impaired by an increase of collateral vessels and arterioles. In effect, the growth
of >150µm collateral vessels has been evidenced angiographically and the growth of
thigh, which comprises Biceps femoris, Adductores, Gradlis, Semimembranosus, and
Semtiendtnosus muscles. Unexpectedly, the Applicant has demonstrated that formation of
collateral vessels was significantly stimulated into this region, 14 days after NV1FGF
gene transfer, as emphasized by angiographic score (Fig. 5C).
Though no in situ measurements of tissue oxygenation have been performed to
assert that ischemia occurred in that region after femoral artery excision, the presence of
bistological lesions such as centronucleation, dystrophy, necrosis and inflammation was
revealed (Fig. 2). More precisely, these lesions were restricted to Adductores and Gradlis
muscles whereas Biceps femoris, Semimembranosus, and Semitendinosus muscles were
not prone to lesions. Quantification of exclusively in Adductores and Gradlis muscles, just above the adductor canal. As
demonstrated in Fig. 6D, NV1FGF gene transfer increased the absolute number of
arterioles into these muscles of the ischemic limb.
These data thus unambiguously demonstrate the ability of NV1FGF to induce the
formation of mature large conductance vessels (>150µm collateral vessels) and small
resistance arteries ( the thigh, which are required to convey and to deliver blood to tissues.
Example 12: Absence of VEGF-A induction in NV1FGF treated muscles
The purpose of this experiment was to assess in vivo VEGF-A induction
following intramuscular (IM) administration of naked DNA encoding rat FGF-1 in mice.
Both murine VEGF-A (mVEGF-A) circulating levels and local gene expression were
investigated. The circulating levels were measured by ELIS A 3 and 7 days following IM
dosing, and local mVEGF-A gene expression was investigated by day 7 using two
assays: immunohistochemistry (IHC) against the protein and detection of mKNA by
Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR).
Fourty female mice were used in this study. The groups 1 to 3 (n = 8 per group)
received IM administrations of pCOR-CMV.rat-spFGF-1, pC0R-CMVJ3mpry (without
FGF-1 gene) or NaCl 0.9% respectively, in both right and left Tibialis Cranialis muscles.
The pCOR-CMVl.rat-spFGF-1 corresponds to the NV1FGF plasmid -wherein the human
FGF-1 was replaced by the corresponding rat-originated coding sequence. The injected
muscles were harvested on day 7 following dosing and were processed for mVEGF-A
and FGF-1 immunohistochemistry (right muscles) or mVEGF-A Real-Time RT-PCR
(left muscles). In groups 1 to 3, blood was collected on day 3 (D3) and day 7 (D7) post-
dosing for ELISA detection of mVEGF-A in freshly prepared serum samples.
As shown in Figure 8, no FGF-1 positive myofibers were detected in pCOR-
CMVEmpty I(Figure 8B) nor NaCl injected muscles (Figure 8C). A mean of 25 FGF-1
expressing myofibers/section was established for pCOR-CMV.sp-ratFGF-1 injected
muscles (Fig 8E).
Using immunohistochemistry, a similar and faint labeling of mVEGF-A was
observed in both groups of control muscles (NaCl and pCOR-CMV.Empty), radicating
endogenous expression of mVEGF-A. The immunostaining, localized within the
myofibers, displayed a mosaic pattern, as some fibers were more intensely labeled than
others. In muscles injected with pCOR-CMV.rat-spFGF-1, no increase of mVEGF-A
immunolabeling was observed by D7 post-dosing (Fig 8D) when compared to pCOR-
CMV2mpty or NaCl injected muscles. The same mosaic pattern of immunostaining was
observed.
Using Real-Time RT-PCR technology, similar levels of mVEGF-A mRNA
expression were evidenced in pCOR-CMV.Empty and NaCl treated muscles (6.84x103
and 4.31X103 cDNA copies per 2 ng of total RNA), indicating endogenous mVEGF-A
expression. In pCOR-CMV,sp-ratFGF-l injected muscles, no increase in mVEGF-A
mRNA level (6.51 103 cDNA copies per 2 ng of total RNA) was observed by D7 post-
dosing, when compared to pCOR-CMV. Empty or NaCl injected muscles.
' These results clearly indicate that intramuscular administration of pCOR-
CMV.tat-spFGF-1 did not lead to local mVEGF-A induction in injected muscles and did
not lead to mVEGF-A secretion in the circulating blood of the injected mice.
Example 13: Hypercholesterolemic Watanabe rabbit coronary artery disease
animal model
The validation of a gene therapy product as a potential treatment partly stands on
its biological activity. In order to assess it, the animal model of choice should be as close
as possible to the human disease it mimics as far as anatomy and function are concerned.
The anatomical relevance was biased on the species used, which should be as close
as possible to the human heart, as far as the coronary network is concerned. Moreover,
the bigger species the better, as it eased the various technical steps. On the other hand,
factors like the cost, handling facility, animal status contingency, and animal facility
compliance was addressed. The best compromise found in the present study is the rabbit,
small enough to be easily handled and stabulated, and big enough to allow a good
spotting of a particular artery, a precise coronarography, and a human-like comparison of
various anatomical and functional issues.
Indeed, me functional relevance was here based on the ability of the animal to
develop pathology as close as possible to its human counterpart, ie. angiogenic defects
associated with hypercholesterolemia.
Hypercholesterolemia in humans causes a vascular endothelial dysfunction and
ultimately a progressive narrowing of the main coronary arteries. The unbalance in the
coronary blood flow at rest and during stress creates a furtive malfunction of the
myocardium that leads to pain and hypocontractility. Usually the supplies are appropriate
at rest, but when stress occurs; the needs increase while the supplies cannot, due to the
coronary obstructive lesions. This is the reason why the purpose of mimicking this
human pathology, leads to both the setup of a stenosis ona major coronary artery and the
use of a stress test to reveal the unbalance created at stress by this stenosis.
Watanabe Heritable Hyperlipidemic Rabbits (WHHR) which lack of LDL
receptor, therefore developing a spontaneous atherosclerosis that leads to coronary
atheroma were thus used to assess ischemia and evaluate the effect on their ischemic
status of intramyocardial injections of NV1FGF during open chest surgery.
All experiments were conducted in accordance with a protocol approved by the
Animal Care and Use Committee and conform to the NIH Guidelines for the Use and
Care of Laboratory Animals.
WHH one-year-old rabbits weighing 3000 to 3500g were obtained from Covance
(PO Box 7200, Denver, PA, 17517, USA).
All the animals were kept in our animal quarters according to good animal care
practices for at least eight days preceding their utilization. Throughout this period, they
were housed one per cage, had free access to food (112C type from UAR) and
appropriately filtered drinking water. The animal house was maintained on a 12-h
light/dark cycle (lights on at 6 a.m.) with an ambient temperature of 20-24 °C and
humidity set at 35-75%.
Watanabe rabbits had cholesterol levels 7 to 10 times higher than the wild type
animals, while their triglycerides levels were 4 to 5 times higher than normal.
After sacrifice, four of them underwent an Oil Red O staining which evidences the
presence of lipid plaques in the aorta, the coronary ostia and the mitral valves.
Furthermore, one of those was submitted to histological analysis. Microscopic
examination of HES stained sections of the circumflex coronary artery identified an
atherosclerotic plaque covering about of 15 % of the lumen in one of the two stuthed
rabbit (Rabbit #CAD98R2, see Figure 9B).
The Watanabe Heritable Hyperlipidemic Rabbit possessed two interesting
properties: the size of its heart was well adapted to multiple intramyocardial injections,
and its coronary network was studded with atherosclerotic plaques. The latest explained
why the coronary blood flow is normal at rest (as assessed by a normal ECG and a
normal contractility), while a dobutamin stress test under anesthesia lead to a marked
pattern of ischemia.
In fact, both analysis, e.g., electrocardiography and echocardiography, evidences
at stress signs that can be compared to their human counterparts, for example ST
depression or hypokinesis.
Exanple 14: Surgery
Surgery was used to deliver the gene therapy product into the myocardium by
direct injection. The surgical procedure was performed under sterile conditions. The
animals were anesthetized with an intramuscular injection (1 ml/Kg) of a mixture of
ketamine (70mg/Kg) + Xylazine (7mg/Kg). After the animals were shaved, they were
vaporized with Lidocaine 5% spray in the throat to make easier the endotracheal
intubation, and the anesthesia was maintained throughout the experiment with a
mechanical ventilator (Siemens, Servo Ventilator 900D) with the following conditions:
> Insufflation volume: l,4L/min
> Breathing frequency: 40/min
> Halothane:0.4 to 0.6%
> Oxygen: 30-35%
A cannulation of the marginal car vein was done so that the animal is
continuously perfused with 5% of glucose. A monitoring by ECG was used to ensure a
stable rhythm throughout the surgical act. A left thoracotomy was performed on the
fourth intercostal space. After the opening of the pericardium, the heart was exposed for
plasmid injection.
The injections were made on 13 different locations on the free wall of the left
ventricle with a homemade needle: 250ul Hamilton syringe connected to a Steriflex G19
catheter (ref: 167.10) fixed with a short bevel needle BD (26G3/8). We injected 25ul per
location of a 1mg/ml solution of plasmid. Figure 1A show the location of each injection.
After the injections, a drainage tube was placed in the thoracic cavity and the ribs
were put side by side with two Mersurtures® (1.0) threads. The Halothane was stopped
and oxygen was maintained until the wakening of the animal. The drainage was set
around 200-400 mbar throughout the closing of the thorax. Two layers of muscles were
closed with a Vicryl® (4.0) thread. The skin was then stitched with a Suturamide (2/0)
thread. When the last stitch was done and before removal of the draining tube, the end
expiration positive pressure (PEEP) was increased in the lungs with the help of the
mechanical ventilator so that the lungs were well inflated throughout the thorax just
before the draining tube was pulled out,
A betadine gel was applied and a bandage was dressed on the wound. When the
animal woke up the mechanical ventilator was stopped.
Example 15: Post-surgery treatment
As soon as the animal was awake, the following compounds have been injected;
- an anti-aggregate and an anti-coagulate to avoid potential thrombosis of the
coronary artery during and after the surgery:
0.375 ml of Vetalgine (one day before surgery and then during 5 days):
intramuscular
0.1 ml heparin (for 4 days from next day of surgery): subcutaneous
an antibiotic with a large spectrum to prevent any infections:
0.2 ml of Baytril 2% (for 5 days): subcutaneous
- a strong analgesic so that the animal withstands the heavy surgical act:
0.5 ml of morphine (for 2 days): subcutaneous
Example 16: Electrocardiography
Electrocardiography was set up as close as possible to its human counterpart. Four
electrodes were set on the four limbs, and six (V1 to V6) were set on the precordium. A
classical 12 lead ECG was recorded on a HP Pagewriter It 4565A. The use of the ECG
follows enabled to monitor (1) the cardiac rhythm during the surgery, (2) the cardiac
rhythm during dobutamin stress test, (3) the detection of myocardial infarction, and (4)
the detection of the signs of ischemia.
4.1 Monitoring of cardiac rhythm during the surgery
An open chest surgery with total anesthesia could potentially lead to various per-
operatory problems that can be detected by ECG monitoring. Bradycardia was treated by
atropin injection, arythmia by Lidocaine 0,5% injection (0.5 to 1ml), and cardiac arrest
by energic cardiopulmonary resuscitation including Isoprenaline.
4.2 Monitoring of the cardiac rhythm during the dobutamin stress test
Even if the main pharmacological effect of dobutamin was its action on inotropy,
this product also seemed chronotropic, and the increase in cardiac rhythm thus appeared
the easiest way to monitor the stress test
4.3 Detection of myocardial infarction
Cardiac electric activity was recorded as a series of beats, each of them being a
succession of waves: p, q, r,s and t, for the most important part The p wave was used as a
witness of the atrial activity, the qrs complex the ventricular activity, and the t wave was
used as a marker of repolarization.
The most classical ECG sign of myocardial infarction in humans is a deep and
large q wave. The parallel could be done with rabbits, as the analysis of an exploratory
series of rabbits showed the presence of such a q wave in most of them when they
underwent a mechanical closure of a coronary artery.
4.4 Detection of signs of ischemia
This furtive phenomenon was usually not seen at rest During a stress test various
modifications were described. We took the assumption that a rabbit heart showed the
same electrical pattern when it undergoes the same mechanical / chemical stress. In fact,
a real ischemia in humans leads to an ST-segment elevation or depression, or / and
inversion of the T wave (Figure 10). The two main signs are the ST-segment depression
and the negative deep T wave, as shown in Figure 10 during a human stress test An
example of its rabbit counterpart was showed in Figures 11.
The method of scoring signs of ischemia was shown in Figure 12. A score from 0
(normal ECG) to 3 points (significant ischemia) for each lead as shown in Figure 12 was
given and recorded. The presence of a significant deviation in any lead was as important
as the number of leads where the deviation was found. Therefore, at first the sum of all
scores was recorded, then only the highest score found on a particular ECG was kept and
used for the inclusion of ischemic animals. The only exclusion criteria was the presence
of a q wave (larger than 1 square and deeper than 3 squares) indicating a transmural
necrosis.
A typical normal 12 lead ECG at rest was displayed in Figure 13A, while the
same animal at maximum stress (see Figure 13B) showed a significant downsloping
depression in lead I, II, aVF, V1, V2, V3, V4, and a non significant depression in lead HI,
V5 and V6. This particular ECG showed a very strong ischemia.
In conclusion, the ECG was the best first line method to detect either an exclusion
criteria (i.e. necrosis), or an inclusion criteria (ischemic response to dobutamin).
Example 17: Echocardiography
An Acuson Sequoia 256 and a linear 8L5 8MHz probe was used to assess the
myocardium contractility, as the small size of the rabbit thoracic area allowed using this
superficial probe in this particular analysis.
For each animal, once before the surgery, then every week before and during the
stress test, an echocardiography was performed. In each case, the overall kinetic behavior
of the heart was examined, as many segments as possible one after the other, in both long
axis and short axis. Then in long axis the largest diameter was spotted, and the apparatus
was switched in M mode to ensure a normal behavior of the left ventricle.
A similar 2D analysis was performed at each step of the stress test. Any detected
abnormality was recorded and further evaluated: the location was noted by using the
nomenclature below (Figure 14), and the type of defect was rated: 1 for normal, 2 for
hypokinesy, 3 for akinesy, 4 for dyskinesy. When there was a doubt on the presence of a
defect, the thickening fraction was rated using M mode. A sequence was thereafter scored
as 1 if all the segments have a normal thickening fraction (above 30%), 2 if hypokinetic
(one or more segments under 30%), 3 if akinetic (at least one segment with no
thickening), or 4 if dyskmetic (one segment with negative thickening fraction, ie. the
myocardium expends during the systole).
Only the highest defect score was kept for the final analysis.
Example 18: Dobutamin stress test
This test used dobutamin for its inotropic and chronotropic properties in order to
mimic the cardiac response to stress conditions. As ischemia Is a furtive phenomenon that
usually occurs during a stress, its unveiling was detected by an electric signature on the
ECG and its consequences on myocardial contractility, as evidenced by
echocardiogcaphy. The technical steps of the test are as follows.
- Anesthesia : Isoflurane was discarded as it leads to high heart rate at rest
Halothane was discarded as the addition of atropin lead to the appearance of ventricular
ectopies. Xylazinc-Ketamine was therefore chosen, as the kinetics of the heart rate
increase was good.
- Atropin: 0.5 mg/Kg of atropin methyl nitrate were injected in the ear catheter as
a preliminary step. Atropin sulfate was discarded at it lead to some degree of heart rate
increase.
- Scale up of the doses: The first dose was 2 µg/Kg/min. Every three minutes the
dose was increased to 5, 10, 20, 30, and 40 ug/kg/min. If the maximal heart was not
reached, a final dose of 80 µg/Kg/min was used.
ECO and echocardiography were recorded at every step. The heart rate of an
anesthetized rabbit at rest was 203 ± 22 (n=61 tests), 280 ± 31 (n=59) at 40 ug/kg/min,
and 299 ± 19 (n=29) at 80 µg/kg/min. The decision to go from 40 to 80 was taken only if
the heart rate was below 300 bpm at 40 µg/kg/min.
In conclusion, this test was used to enlighten the behavior of the myocardium
during a chemical stress, therefore unveiling an ischemic section of the heart
Example 19: Histological analysis - Detection of the atherosclerotic plaques,
tolerance and FGF-1 expression
The circonflex coronary artery of two rabbits was examined for the presence of
atherosclerotic plaques at the end of the experiment The hearts were removed and a
sample of the left ventricle containing the upper part of the circumflex coronary artery
was dissected and dipped in PBS buffered 3.7% formalin for further analysis. Each
sample was embedded in paraffin. 5 µm sections were performed each 300 µm and
stained with Hematoxilin-Eosin-Saffron (HES) for microscopic examination.
Two others rabbits were used to assess the efficiency and safety of a FGF-1
coding plasmid injection in the left ventricular wall. The latest were euthanized at day 3,
the heart was removed and washed, and the anterior wall was observed and kept in
formalin for one hour. Then it was divided in five samples, each of them fixed overnight
in 3.7% PBS-buffered formalin before to be embedded in paraffin.
For each heart, 5 samples presenting macroscopic lesions or supposed to contain
injection sites were collected and fixed overnight in 3.7% PBS-buffered formalin before
to be embedded in paraffin.
A standardized procedure was used for slide preparation. Two 5-µm serial
sections were performed from each block. One section was stained with Hematoxylin-
Eosin-Saffron (HES) for histopathological examination; the other section was processed
for FGF-1 immunohistochemistry (IHC). Injection sites were identified on HES stained
sections by the presence of histological changes related to needle and vector injection.
The IHC procedure was done using a classical streptavidin-biotin assay. The incubation
with a primary polyclonal anti-FGF-1 rabbit antibody (R&D Systems; # AB-32-NA, 1:30
dilution) was followed by incubation with a biotinilated donkey anti-rabbit
immunoglobulin (Amersham, 1:200 dilution). The immune complexes were localized
using a chromogenic diaminobenzidine substrate, after adding peroxydase coupled to
streptavidine. The sections were counterstained with hematoxylin, dehydrated and
mounted with permanent mounting media. With this method, immunoreactive fibers
appeared brown and the nuclei blue. Previous validation of FGF-1 IHC assay in rabbit
muscle demonstrated the ability to detect FGF-1 transgene in myofibers, even when using
anti-rabbit secondary antibothes on rabbit tissue samples. Negative control (omitting the
primary antibody, but using the secondary antibody) was used to discriminate non-
specific staining (mainly extracellular) to specific immunoreactrvity. Moreover, the
performances of FGF-1 IHC were controlled throughout the assay by using a positive
section from a rat muscle having previously demonstrated a high level of FGF-1
expression (slide P1056GNR2, study PAD31.2001). The immunoreactive fibers were
identified and counted under a microscope (Zeiss, Axioplan 2). The number of
immunoreactive cells given was the number of immunoreactive cardiomyofibers
observed around each injection site.
Example 20: Demonstration of therapeutic angiogenesis in hypercholesterolemic
settings
A total of 16 Watanabe one-year-old male rabbits were used to assess the efficacy
of NV1FGF on reversing myocardial ischemia associated with hypercholesterolemia. In
addition, two New Zealand rabbits underwent the same surgical procedure and they were
sacrificed at day 3 for the expression analysis.
20.1/ FGF-1 expression assessment and histopathological pattern
Two healthy New Zealand rabbits were sacrificed three days after the surgical
procedure and the injection of NV1FGF. The evaluation of histopathological changes
relative to the vector injection was successfully achieved, as 5 and 7 injection sites were
localized within the 5 samples analyzed from each heart Some samples evidenced up to
3 distinct injection sites, separated by 6 to 10 mm, corresponding to the distance between
2 injections. The myocardial lesions were more or less linear and consisted in
degeneration and necrosis with active chronic inflammatory response (see Figure 15A).
Some samples displayed diffuse infiltration of lymphocytes below the pericardium,
suggesting a limited pericarditis. Individual results are shown in table 3.
Three days after an intramyocardial injection of NV1FGF, FGF-1
hnmunoreactive myofibers were detected in all the samples displaying injection sites.
The expression, restricted to the periphery of the histological lesions, varied from 3 to 36
positive cardiomyofibers per injection site (see Figure 15B). Individual results were
indicated in the table 3.
Table 3: Histological observations and FGF-1 expression in healthy rabbit myocardium,
3 days after intramyocardial injections of NV1FGF
In conclusion, three days after direct injections of NV1FGF within healthy rabbit
myocardium, myofibers degeneration and subsequent inflammatory reaction were
observed. At such carry time points, histological damages were considered as non
5 specific of NV1FGF, as the severity and the type of the lesions were similar to the one
observed in pig myocardium following injection of naked DNA plasmid not encoding for
any transgene.
The efficiency of transgene expression in rabbit heart was established for
intramyocardial injections of NV1FGF as FGF-1 expression was found in all the samples
10 displaying an injection site. Taking into account the number of immunoreactive
myofibers per injection site, the level of expression was similar to the one observed after
intramyocardial injection of a same amount of NVlFGF in rat heart, assuming that one
injection site in rabbit equals a single injection in rat heart
15 20.2/ Electrocardiographic pattern of the injected Watanabe rabbits
16 rabbits were included in this study. Every animal underwent the stress test
before the surgery and then every week for four weeks. The highest score observed was
plotted for both groups (blue for empty plasmid injected animals, pink for NVlFGF
injected animals). As soon as day seven, most of the animals of the NV1FGF group got a
20 lower score, indicating that the ischemia tends to disappear under treatment. On the other
hand, most of the animals in the empty treated group remained deeply ischemic
(maximum ischemic score of 3).
The results as presented in Figure 16 showed the evolution of the maximum ECG
score during the stress test on rabbits treated with empty plasmid (blue dots for
25 individuals, blue column for the mean) or NV1 -FGF plasmids (red dots for individuals,
pink column for the mean). Treatment of NVlFGF plasmid clearly showed a significant
efficacious decrease of the ischemic size.
Starting with day 7, a statistical analysis (unpaired t-test) showed a significant
difference between both groups (p 30 presence of FGF-1 in the regression of the ischemic electrocardiographic pattern at stress.
203/Echocardiographic pattern of the injected Watanabe rabbits
A first step was to validate the accuracy of the correspondence between a
qualitative evaluation (classification normal, hypokinesis, akinesis) and the quantitative
analysis (fractional wall thickening). 30 segments were analysed, 16 as seen as normal
and 14 as abnormal. Their fractional wall thickening was thereafter calculated, and the
correspondence was shown in Figure 17.
As can be seen, the overlap between the two series is very narrow, indicating that
the visual evaluation was quite adequate. The abnormal segments included one dyskinetic
segment (good thickening fraction but abnormal move) and three akinetic segments, of
which one even got thinner during the systole. Subsequently our qualification were
considered valid as normal and hypokinetic / akinetic, thus enabling a second and wider
evaluation of the myocardial kinetic.
A second step was to correlate the ECG result with the echocardiographic
evaluation in each stress test The result was plotted on Figure 18. The regression curve
was shown in red, indicating that an abnormal ECG was roughly correlated with an
abnormal echo. The two shaded areas represented the two main sets of data: the first is
the normokinesis echo and normal / slightly ischemic ECG, and the second was the
abnormal echo (hypokinesis and akinesis) and the very ischemic ECG (score 3).
The six points where an ischemic ECG corresponded to a normal echo were due
to the technical difficulty of getting a good clip for every test, indicating probably that
defects on echo were missed. On the other hand, only three tests showed a slightly
ischemic ECG with an echo defect No normal ECG was correlated with an abnormal
echo, indicating a good sensitivity for the ECG.
In a third step, evolution of echocardiography in two groups of four treated
animals was followed. The score was rated as 1 (normal), 2 (hypokinetic) or 3 (akinetic).
No dyskinetic segment was observed in this set of animals. The raw data were plotted in
Table 4 As can be seen in this annexe, the baseline values were different (mean of 2.25
versus 1.5). Therefore, the whole set was normalized by using each baseline value as
100%. Then, the following points for each animal were expressed as a percentage of this
baseline. The final figure was displayed in Figure 19.
5 The NV1FGF treated animals score was significantly lower than the empty
plasmid treated animals score (analysis performed by unpaired t-test), indicating an effect
of the presence of FGF-1 on the myocardium contractility, while the empty plasmid
treated animals score increases.
Of note, as the final sacrifice step included other technical analysis was not
10 detailed here, no echo recording was available for these annuals at day 28.
The results as presented in Figure 19 clearly showed that treated animals with
multiple NV1FGF injections in the myocardium lead to a regression of the ischemia
pattern at stress both in ECG and echocardiography, while the empty plasmid injection
did not These results also clearly showed that NV1FGF effects helped the heart to adapt
15 to stress conditions in hypercholesteromic settings.
Example 21: Demonstration of the induction of arterioles in NV1FGF treated
cardiac muscle
20 21.1/ Animals
Mult male mini-swine (30kg) were used for the study. A total of 34 animals (n=8
final expected in each treated and control groups, 4 group total) were used. Animals were
housed under standard conditions and fed a regular thet. The Animal Care and Use
Committee of Duke University approved all procedures and protocols. Animals received
humane care in compliance with the "Principles of Laboratory Animal Care" formulated
by the National Society for Medical Research and the "Guide for the Care and Use of
Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and
published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Animals underwent anesthesia and orotracheal intubation. Continuous
dectrocardiographic and pulse-oximetric monitoring were used throughout the procedure
to ensure a stable cardiac rhythm and oxygenation. Under sterile conditions, a left
anterolateral thoracotomy was performed through the fourth intercostal space. The
pericardium was isolated longitudinally, and the left atrial appendage retracted to allow
exposure of the left circumflex (LCx) artery. The proximal LCx was dissected free to
allow placement of a hydraulic occluder and 2 mm ultrasonic flow probe (Transonic
Systems, Inc., Ithaca, NY) around the vessel. The flow probe was placed distal to the
occluder to record downstream flow through the LCx. The occluder and flow probe was
then exteriorized through a separate stab incision. A 20 French chest tube was placed and
the wound was closed in layers. The chest tube was removed at the conclusion of the
procedure. Three days postoperantively, the occluder was inflated to reduce resting blood
flow in the LCx to approximately 10% of baseline as assessed using the implanted flow
probe. The animals were kept in this low-flow state for two weeks with blood flow
recordings being performed three times per week to assure to same degree of vascular
occlusion prior to physiologic assessment
21.2/ Positron emission tomography, Dobutamine stress echocardiography
and Colored Microspheres
After 32 ± 11 days in the low-flow state, the animals underwent positron emission
tomography (PET) and dobutamine stress echocardiograpby (DSE) to characterize the
blood flow, metabolic, and functional status of the heart, and document the presence of
iscbemic, viable myocardium in the LCx distribution. PET scans were interpreted as
showing hibernating myocardium if a flow deficit is noted in the lateral and
posteroinferior walls of the left ventricle supplied by the LCx accompanied by normal or
increased glucose utilization in these same regions (both as compared to the non-ischcmic
septum). Using DSE, viability in the lateral and posteroinferior walls of the left ventricle
was defined as an improvement in systolic wall thickening with low dose dobutamine in
myocardial regions with severe hypocontractility at rest Viable segments were
considered ischemic if systolic wall motion was deteriorated with stress (biphasic
response).
Dosing was performed after PET and DSE confirm the presence of ischemic
myocardium, by direct intramyocardial injection of the FGF expression plasmid such as
NV1FGF, with an open chest approach (52 ± 16 days post LCx occlusion). The vectors
were administered in 10 sites (100 µg/ 100 µl/ injection site for plasmidic vectors)
distributed into the free left ventricular wall. 10 injections of 100 ul of saline were
performed for the control group. The treatments were performed by operators and
investigators which were blinded to the treatment All efficacy parameters were assigned
in a bunded manner, and the code was opened at the end of the study.
109 ± 13 days after the treatment, the hearts were excised for bistologic analysis
and perfusion assay. A standardized procedure was used for samples preparation as
shown in Figure 20. More precisely, the hearts were sectioned in 3 short axis slices
(apical, mid and basal segments). Each of these segments was subdivided in 6 (mid and
basal segments) or 4 orientated sectors (apical segment) giving a total of 16 sectors per
heart Each sector was then divided in 3 transmural samples; one sample was stored for
microsphere perfusion analysis, another was snap frozen in liquid nitrogen-cooled
isopentane while the latter sample was fixed in 3.7% formalin. A sample from the right
ventricle was collected as control.
21.4/ Histological analysis
A standardized procedure was used for slide preparation. Each formalin-fixed
sample was embedded in paraffin wax and three 5-µm serial sections were performed
from each block. One section was stained with Hematorylin-Eosin-Saffron (HES) for
evaluation of post-necrotic fibrosis by histopathological examination; the serial section
was stained with anti-a Smooth Muscle Actin (SMA) antibody to identify arteries and
arterioles. a-SMA is indeed expressed in both pericytes and smooth muscle cells
associated with endothelial cells in mature blood vessels (Benjamin et al., Development,
125, 1591-1598. 1998). Of note, some large veins can be stained with this antibody, but
are easily identified on morphological criteria.
21.4.1/a-SMA staining
All sectors from all the pigs were processed with a-SMA imrnunohistochemistry.
The procedure was done using the Dako EnVisionTM+HRP (Horse Radish Peroxidase)
detection system (Dako, Sabattini et al., J.Clin Patbol, 51:506-511, 1998). The sections
were incubated with an anti a-SMA monoclonal antibody (Dako, clone 1A4, 1:100
dilution). The second step consisted in incubation with goat anti-rabbit antibody
conjugated to an HRP labeled polymer. The immune complexes were localized using the
chromogenic diaminobenzidine substrate. The sections were counterstained with
hematoxylin, dehydrated and mounted with permanent mounting media. With this
method immunoreactive cells appeared brown and the nuclei blue.
21.4.2/ Morphometric analysis
The measurements were performed by a single observer blinded to the treatment
regimen. For each sector, the HES-stained section was first analyzed in order to
determine the scar area (post necrotic fibrosis). The amount of fibrosis in the sample was
scored at low magnification (x25) using the following scale:
+ : minimal (lees man 5% of the surface of the sample affected by fibrosis)
++: mild (~5-15% of the surface of the sample)
Evaluation of vascular density was performed on the serial a-SMA-stained
section. The number of a-SMA stained vessels was counted in 9 high-power microscopic
fields (037 mm2 each) located in l) the center of the scar (3 fields), ii) the border of the
scar (3 fields) and iii) distant from the scar, i.e.in viable myocardium (3 fields). For each
field, 3 categories of vessels were recorded: small unilayered vessels, multilayered
vessels with a diameter 100 µm (see
figure 2). Large veins with a a-SMA staining were excluded from the analysis, based on
their morphological features. Of note, the numerous myofibroblasts containing a-SMA
filaments were excluded from the morphometric analysis.
The vascular density (i.e. the number of each category of vessels per mm3) was
then calculated for each zone (scar, border zone, viable myocardium) by pooling data
from the 5 sectors supposed to be injected (BA, BAL, MA, MAL and AA sectors). To
serve as an additional control, vascular density in the non-injected zone (BIL, BI, BIS,
BAS, MIL, Ml, MIS, MAS, AL, AI, AS sectors) was also calculated.
21.4.3/ Transgene expression
BA, BAL, MA, MAL and AA sectors from the NVlFGF-treated pigs were processed
for FGF-1 immunohistochemistry. FGF-1 expression was assessed using a classical
streptavidin-biotin assay. The incubation with the primary polyclonal anti-FGF-1 rabbit
antibody (R&D Systems; # AB-32-NA, 1:30 dilution) was followed by incubation with a
biotinilated donkey anti-rabbit immunoglobulin (Amersham, 1:200 dilution). The
immune complexes were localized using a chromogenic diaminobenzidine substrate, after
adding peroxydase coupled to streptsvidine. The sections were counterstained with
hematoxylm, dehydrated and mounted with permanent mounting media. With this
method, immunoreactive fibers appeared brown and the nuclei blue. The performances of
the FGF-1 IHC were controlled throughout the assay by using a positive section from a
rat muscle having demonstrated a high level of pCOR-CMV.ratFGF-1 plasmid gene
transfer.
21.4.4/ Statistical analysis
Quantitative variations in vasculature were evidenced in the different zones (scar,
border, viable myocardium) between saline and plasmid-treated groups, arithmetical
means and standard deviation (Sd) were calculated for each category of vessels.
Intergroup data were compared using a one-way analysis of variance (ANOVA,
SigmaStat®, Jandel Scientific). When a significant difference was found, multiple
comparisons versus control group were performed using the Dunnett's method. All
differences were considered to be significant at a level of p=0.05.
21.5/ Evaluation of the vascular density
Comparisons of the means were made between the injected zone in the treated
animals with NV1FGF and NaCl injected zone in the controls animals (Figure 21).
Analysis was performed separately for each class of vessels (small, median, large)
in the viable myocardium. Whatever the treatment group, numerous arterial structures
with a diameter criteria only, these vessels were indistinguishable from native myocardial vessels. Most
of them were small, demonstrating a single layer of smooth muscle cells.
When compared to saline injected areas, the administration of NV1FGF to the
heart induced, in the viable part of myocardium located within the injected zone, a 22%
and 20% increase in the density of small unilayered vessels (p=0.017). This effect was
not demonstrated for larger vessels.
These results clearly showed that direct intramyocardial injection of a pCOR
plasmid encoding either the spFGF-1 transgene induced, in ischemic pig hearts, a
significant increase in the density of unilayered arteries, i.e. arterioles with a single layer
of smooth muscle cells. Also, these results demonstrated a long-lasting FGF-1 expression
following direct intramyocardial injection a NV1FGF. Indeed, some cardiomyofibers,
always located around the injection site, expressed detectable amount of FGF-1 104 to
132 days after the dosing. This long lasting expression is likely in fevor of the
recruitment and division of precursor cells needed for arteriogenesis and for the
maintenance of the induced new vessels. At the time of harvesting, these vessels are
believed to be at an early stage of arteriogenesis.


References
1. Ouriel K. Peripheral arterial disease. Lancet 2001;358:1257-1264.
2. Dormandy JA, Rutherford RB. TransAtlantic inter-consensus (TASC): management of
peripheral arterial disease (PAD). J Vase Surg. 2000;31 (suppl. 1, pt 2):S1-S278.
3. Yu HI, Sheu HH, Lai CJ, Lee WJ, Chen YT. Endothelial dysfunction in type 2
diabetes mellitus subjects with peripheral artery disease. Int J Cardiol. 2001 ;78:19-25.
4. Emanueli C, Salis MB, Stacca T, Gaspa L, Chao L, Piana A, Madeddu P. Rescue of
unpaired angiogenesis in spontaneously hypertensive rats by intramuscular human tissue
kallikrein gene transfer. Hypertension. 2001;38:136-141.
5. Duan J, Murohara T, Bceda H, Katoh A, Shintani S, Sasaki KI, Kawata H., Yamamoto
N, Imaizumi T. Hypercholesterolemia inhibits angiogenesis in response to hindlimb
ischemia. Nitric oxide-dependent mechanism. Circulation. 2000;102 (suppl IIT):III-370-
III-376.
6. Jang JJ, Ho HKV, Kwan HH, Fiajardo LF, Cooke JP. Angiogenesis is impaired by
hypercholesterolemia. Role of asymmetric dimethylarginine. Circulation.
2000;102:1414-1419,
7. Hirata K, Li TS, Nishida M, Ito H, Matsuzaki M, Kasaoka S, Hamano K. Autologous
bone marrow cell implantation as therapeutic angiogenesis for ischemic hindlimb in
diabetic rat model. Am. J. Physiol. Heart Circ. Physiol. 2003;284:H66-H70.
8. Yla-Herttuala S, Kari Alitalo. Nat Med 2003;6:694-701.
9. Ferrara N, Alitalo K. Clinical applications of angiogenic growth fectors and their
inhibitors. Nat. Med. 1999;5:1359-1364.
10. Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for
postnatal neovascularization. J. Clin, Invest. 1999;103:1231-1236.
11. Isner JM. Tissue responses to ischemia; local and remote responses for preserving
perfusion of ischemic muscle. J, Clin. Invest. 20G0;106:615-619.
12. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat, Med. 2000;6:389-
395.
13. Sordello S, Fons P, Malavaud B, Plouët J. Vascular endomelial growth fector femily
(VEGF). In Comprehensive Vascular Biology and Pathology. Bikfelvi A., editor. 2000.
Springer. Paris, France. 322-331.
14. Boilly B, Yercoutter-Edouart AS, Hondermarck H, Nurcombe V, Le Bourhis X. FGF
signals for cell proliferation and migration through different pathways. Cytokine Growth
Factor Rev. 2000; 11:295-302.
15. Tabata H, Silver M, Isner JM. Arterial gene transfer of acidic fibroblast growth fector
for therapeutic angiogenesis in vivo: critical role of secretion signal in use of naked
DNA. Cardiovasc. Res. 1997;35:470-479.
16. Nabel EG, Yang ZY, Plautz G, Forough R, Zhan X, Haudenschild CC, Maciag T,
Nabel GJ. Recombinant fibroblast growth fector-1 promotes intimal hyperplasia and
angiogenesis in arteries in vivo. Nature. 1993;362:844-846,
17. Rosengart TK, Budenbender KT, Duenas M, Mack,CA, Zhang QX, Isom OW.
Therapeutic angiogenesis: a comparative study of the angiogenic potential of acidic
fibroblast growth fector and heparin J.Vasc.Surg. 1997;26;302-312.
18. Comerota AJ, Throm RC, Miller KA, Henry T, Chronos N, Laird J, Sequiera R, Kent
CK, Bacchetta M, Goldman C, Salenius J-P, Schmieder FA, Pilsudski R. Naked plasmid
DNA encoding fibroblast growth factor type 1 for the treatment of end-stage
unreconstructible lower extremity ischemia: Preliminary results of a phase I trial.J. Vasc.
Surg. 2002;5:930-936.
19. Pu LQ; Jackson S, Lachapelle KJ, Arekat Z, Graham AM, Lisbona,R, Brassard R. A
persistent hindlimb ischemia model in rabbit. J. Invest Surg. 1992;7:49-60.
20. Takeshita S, Isshiki T., Sato T. Increased expression of direct gene transfer into
skeletal muscles observed after acute ischemic injury in rats. Lab. Invest. 1996;74;1061-
1065.
2L Soubrier F, Cameron B, Manse B, Somarriba S, Dubertret C, Jaslin G, Jung G, Le
Caer C, Dang D, Mouvault JM, Schennan D, Mayaux JF, Crouzet J. pCOR: a new design
of plasmid vectors for nonviral gene therapy. Gene Ther. 1999;6:1482-1488.
22. Silvestre IS, Mallat Z, Duriez M, Tamarat R, Bureau MF, Scherman D, Duverger N,
Branellec D, Tedgui A, Levy BI. Antiangiogenic effect of interleukin-10 in ischemia-,
induced angiogenesis in mice hindhmb. Circ. Res. 2000;87:448-452.
23. Lusis AJ. Atherosclerosis. Nature. 2000;407:233-241.
24. Dart AM, Chin-Dusting JPF. Lipids and the endothelium. Cardiovasc. Res.
1999;43:308.-322.
25. Chen CH, Cartwright J, Jr., Li Z, Lou S, Nguyen HH, Gotto AM, Jr., Henry PD.
Inhibitory effects of hypercholesterolemia and ox-LDL on angiogenesis-like endomelial
growth in rabbit aortic explants. Essential role of basic fibroblast growth factor,
Arterioscler. Thromb. Vase Biol 1997;17:1303-1312.
26. Van Belle E, Rivard A, Chen D, Silver M, Bunting S, Ferrara N, Symes JF, Bauters
C, Isner JM. Hypercholesterolemia attenuates angiogenesis but does not preclude
augmentation by angiogenic cytokines. Circulation. 1997;96;667-2674.
27. Morishita R, Sakaki M, Yamamoto K, Iguchi S, Aoki M, Yamasaki K, Matsumoto K,
Nakamura T, Lawn R, Ogihara T, Kaneda Y. Impairment of collateral formation in
lipoprotein(a) transgenje mice. Therapeutic angiogenesis induced by human hepatocyte
growth factor gene. Circulation. 2002; 105:1491-1496.
28. Couffinhal T, Silver M, Kearney M, Sullivan A, Witzenbichler B, Magner M, Annex
B, Peters K, Isner JM. Impaired collateral vessel development associated with reduced
expression of vascular endothelial growth (actor in ApoE mice. Circulation.
1999;99:3188-3198.
29. van Royen N, Hoefer I, Bottingen M, Hua J, Grundmann S, Voskuil M, Bodo C,
Schaper W, Buschmann I, Piek JJ. Local monocyte chemoattractant protein-1 therapy
increases collateral artery formation in apolipoprotein E-deficient mice but induces
systemic monocytic CD11b expression, neointimal formation, and plaque progression.
Circ Res 2003;92:218-225.
30. Yamanouchi J, Nishida E, Itagaki SI, Kawamura S, Doi K, Yoshikawa Y. Aortic
ameromatous lesions developed in APA hamsters with streptozotocin induced diabetes: a
new animal model for diabetic atherosclerosis. 1. Histopathological stuthes. Exp. Anton.
2000;49:259-266.
31. Yamanouchi J, Takatori A, Itagaki SI, Kawamura S, Yoshikawa Y. APA hamster
model for diabetic atherosclerosis. 2. Analysis of lipids and lipoproteins. Exp. Anim.
2000;49:267-274.
32. Simionescu M, Popov D, Sima A, Hasu M, Costache G, Faitar S, Vulpanovici A,
Stancu C, Stem D, Simionescu N. Pathobtochemistry of combined diabetes and
atherosclerosis stuthed on a novel animal model. The hyperlidemic-hyperglycemic
hamster. Am. J. Pathol. 1996;148:997-1014.
33. Dimmeler S, Aicher A, Vasa M, Mildner-Rihm C, Adler K, Tiemann M, Rotten H,
Fichtlscherer S, Martin H, Zeiher AM. HMG-CoA reductase inhibitors (statins) increase
endothelial progenitor cells via the PL3-kinase/Akt pathway. J. Clin. Invest.
2001;108:391-397.
34. Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, Walsh K.
The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and
promotes angiogencsis in normocholesterolemic animals. Nat Med. 2000;6:1004-1009.
35. Wecksell MB, Winchester PA, Bush HL, Jr., Kent KC, Prince MR, Wang YL Cross-
sectional pattern of collateral vessels in patients with superficial femoral artery occlusion.
Invest. Radio. 200l;36:422-429.
WECLAIM.1
1. Pharmaceutical composition containing NV1FGF for treating myocardial or skeletal
angiogenic disorders or defects associated with hypercholesterolemia or diabetes in a patient
suffering therefrom, wherein the administration of said composition does not induce VEGF-A
factor expression In the myocardial or skeletal muscles.
2. Pharmaceutical composition containing NV1FGF for treating vascular endothelium
dysfunction associated with hypercholesterolemia or diabetes in a patient suffering therefrom,
to reverse myocardial or skeletal angiogenic defects wherein the administration of said
composition does not induce VEGF-A factor expression in the myocardial or skeletal muscles.
3. Pharmaceutical composition containing NV1FGF for treating myocardial or skeletal
angiogenic disorders associated with hypercholesterolemia or diabetes in a patient suffering
therefrom, to promote blood vessels formation in myocardium or skeletal muscle of said patient,
wherein the administration of said composition does not induce VEGF-A tactor expression in
said muscle.
4. Pharmaceutical composition containing NV1FGF for treating myocardial or skeletal
angiogenic disorders associated with hypercholesterolemia or diabetes in a patient suffering
therefrom, to promote mature collateral blood vessels and arterioles formation in myocardium
or skeletal muscle of said patient.
5. Pharmaceutical composition containing NV1FGF for promoting the formation of mature
collateral vessels in ischemic cardiac or skeletal muscle tissues in a mammalian subject in need
thereof, wherein the administration of said composition does not induce VEGF-A factor
expression in said subject.
6. Pharmaceutical composition containing NV1FGF for promoting the
formation of mature collateral blood vessels in ischemic myocardial or skeletal muscle tissues
in a mammalian subject in need thereof.
7. Pharmaceutical composition as claimed in claim 6, wherein the the administration of said
composition does not induce VEGF-A factor expression in the myocardial or skeletal muscle of
said subject.
8. Pharmaceutical composition containing NV1FGF for reversing defects in angiogenesis
elicited by hypercholesterolemia or diabetes in a patient suffering therefrom without inducing
VEGF-A factor expression in said patient to promote the formation of both collateral blood
vessels and arterioles.
9. Pharmaceutical composition containing NV1FGF for promoting formation of mature
large conductance vessels ( arterioles ) in myocardial or skeletal muscle of hypercholesterolemic or diabetic patients.
10. Pharmaceutical composition as claimed in claim 9, wherein the VEGF-A factor
expression is not induced.
11. Pharmaceutical composition as claimed in any of claims 1 to 10, wherein the NV1FGF is
administrable by injection in skeletal muscles located in the posterior and/or front parts of the
thigh and the calf.
12. Pharmaceutical composition as claimed in claim 11, wherein the NV1FGF is
administrable by multiple injections around the ischemic site of said muscle.
13. Pharmaceutical composition as claimed in claims 1 to 10, wherein the NV1FGF is
administrable to the myocardium of said patient by intracoronary, intramyocardial, transthoracic,
pericardial, or epicardial injections.
14. Pharmaceutical composition as claimed in claim 13, wherein the NV1FGF is
administrable by multiple injections around the ischemic site of said cardiac muscle or by one
single injection.

15. Pharmaceutical composition as claimed in claim 13 or 14. wherein the injection is
administrable with a catheter.
16. Pharmaceutical composition as claimed in claim 15. wherein the catheter is a needle
catheter.
The instant invention discloses a pharmaceutical composition containing NV1FGF for
treating myocardial or skeletal angiogenic disorders or defects associated with
hypercholesterolemia or diabetes in a patient suffering therefrom, wherein the administration of
said composition does not induce VEGF-A factor expression in the myocardial or skeletal
muscles.

Documents:


« Previous Patent
Next Patent »
Patent Number 224741
Indian Patent Application Number 02451/KOLNP/2005
PG Journal Number 43/2008
Publication Date 24-Oct-2008
Grant Date 22-Oct-2008
Date of Filing 01-Dec-2005
Name of Patentee CENTELION
Applicant Address 72-82, RUE LEON GEFFROY, F-94400 VITRY-SUR-SEINE
Inventors:
# Inventor's Name Inventor's Address
1 FINIELS FRANCOISE 11, RUE JACQUES DORE, F-94430, CHENNEVIERES SUR MARNE
2 MICHELET SANDRINE 37 RUE DES TOURTERELLES, F-77340 PONTAULT-COMBAULT
3 ROUY DIDIER 31, RUE DES ROMAINS, F-57570 BOUST
4 BRANELLEC DIDIER 82, QUAI JOSEPH GILLET, F-69004 LYON
5 CARON ALEXIS 19 RUE DU 11 NOVEMBRE 1918, F-94240 LA HAY LES ROSES
6 EMMANUEL FLORENCE 43 AVENUE JEAN JAURES, F-93450 L'ILE SAINT DENIS
7 CARON ANNE 1, SQUARE BUFFALO, F-92120, MONTROUGE
8 SCHWARTZ BERTRAND 14, RUE GEORGES CLEMENCEAU, F-78350 JOUY EN JOSES
PCT International Classification Number A61K 48/00
PCT International Application Number PCT/EP2004/006903
PCT International Filing date 2004-06-04
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/560,915 2004-04-09 U.S.A.
2 60/475,959 2003-06-05 U.S.A.
3 60/566,193 2004-04-28 U.S.A.