U.S. patent application number 11/359102 was filed with the patent office on 2006-08-31 for method of promoting natural bypass.
This patent application is currently assigned to Zimmer Orthobiologics, Inc.. Invention is credited to Rama Akella, James J. Benedict, John P. Ranieri, Marsha L. Whitney.
Application Number | 20060194729 11/359102 |
Document ID | / |
Family ID | 46277202 |
Filed Date | 2006-08-31 |
United States Patent
Application |
20060194729 |
Kind Code |
A1 |
Benedict; James J. ; et
al. |
August 31, 2006 |
Method of promoting natural bypass
Abstract
An angiogenic factor comprising a mixture of proteins derived
from bone. The angiogenic protein mixture is produced by a series
of steps that allow the proteins to be kept in solution. The
angiogenic mixture of bone proteins is produced by a multi-step
process that includes at least one ultrafiltration step, an anion
exchange chromatography step, a cation exchange chromatography step
and a high performance liquid chromatography (HPLC) purification
step.
Inventors: |
Benedict; James J.; (Arvada,
CO) ; Ranieri; John P.; (Austin, TX) ;
Whitney; Marsha L.; (Austin, TX) ; Akella; Rama;
(Austin, TX) |
Correspondence
Address: |
WILLIAMS, MORGAN & AMERSON
10333 RICHMOND, SUITE 1100
HOUSTON
TX
77042
US
|
Assignee: |
Zimmer Orthobiologics, Inc.
|
Family ID: |
46277202 |
Appl. No.: |
11/359102 |
Filed: |
February 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09748038 |
Dec 22, 2000 |
|
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11359102 |
Feb 22, 2006 |
|
|
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09173989 |
Oct 16, 1998 |
6211157 |
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09748038 |
Dec 22, 2000 |
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Current U.S.
Class: |
514/8.2 ;
514/17.2; 514/8.8; 514/8.9 |
Current CPC
Class: |
A61K 38/30 20130101;
A61K 38/1841 20130101; A61K 38/1841 20130101; A61K 38/1825
20130101; A61K 38/1858 20130101; A61K 9/0019 20130101; A61K 38/1875
20130101; A61K 38/30 20130101; A61K 38/1833 20130101; A61K 38/1875
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 38/1825 20130101;
A61K 38/1808 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 38/1808 20130101; A61P 9/10
20180101; A61K 38/1858 20130101; A61K 47/32 20130101; A61K 38/1833
20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 38/18 20060101
A61K038/18; A61K 38/17 20060101 A61K038/17; A61K 38/30 20060101
A61K038/30 |
Claims
1.-30. (canceled)
31. A composition, comprising: a mixture of proteins derived from
ground bone comprising an amount of at least two growth factors
selected from the group consisting of bone morphogenic protein-2
(BMP-2), bone morphogenic protein-3 (BMP-3), bone morphogenic
protein-4 (BMP-4), bone morphogenic protein-5 (BMP-5), bone
morphogenic protein-6 (BMP-6), bone morphogenic protein-7 (BMP-7),
transforming growth factor 1 (TGF-.beta.1), transforming growth
factor .beta.2 (TGF-.beta.2), transforming growth factor .beta.3
(TGF-.beta.3) and fibroblast growth factor 1 (FGF-1) effective to
promote angiogenesis in soft tissue in a mammal.
32. The composition of claim 1, wherein the mixture of proteins
further comprises a growth factor selected from the group
consisting of insulin-like growth factor-1 (IGF-1), epidermal
growth factor (EGF), hepatocyte growth factor (HGF), transforming
growth factor .alpha. (TGF-.alpha.), and platelet-derived growth
factor (PDGF).
33. The composition of claim 1, further comprising a preservative
or an adjuvant.
34. The composition of claim 1, wherein the mixture or proteins
comprises BMP-2, BMP-3, BMP-7, TGF-.beta., and FGF-1.
35. The composition of claim 1, further comprising a carrier
selected from the group consisting of polylactic acid, polyglycolic
acid, copolymers of lactic acid and glycolic acid, collagen,
polyoxyalkylene ether copolymer surfactant, and
polyvinylpyrrolidone.
36. The composition of claim 5, wherein the carrier is
polyvinylpyrrolidone.
37. The composition of claim 5, wherein the composition contains 10
.mu.g of the mixture of proteins per 0.1 cc of polyvinylpyrrolidone
or 100 .mu.g of the mixture of proteins per 0.1 cc of
polyvinylpyrrolidone.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/173,989, filed Oct. 16, 1998 and entitled "Protein Mixtures to
Induce Therapeutic Angiogenesis," which is incorporated herein by
reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The present invention relates to a method for inducing the
growth of blood vessels in instances where it is desirable to
increase the supply of blood to a portion of a living body. More
particularly, the present invention comprises a novel angiogenic
factor. Still more particularly, the present invention relates to
the use of mixtures of protein extracted from bone to cause a
natural vascular bypass effect.
BACKGROUND OF THE INVENTION
[0004] There are many medical circumstances in which an increase in
the supply of blood to living tissue is indicated. These include:
bums and wound healing, in which the incorporation of angiogenic
factors into artificial skin may facilitate the formation of blood
vessels in the healing wound and reduce the risk of infection;
cardiovascular disease, in which repair of anginal or ischemic
cardiac tissue can be effected by causing the ingrowth of new blood
vessels; stroke, where increased blood supply to the brain can
reduce the risk of transient ischemic attack and/or cerebral
arterial deficiency; and peripheral vascular disease, in which
blood flow in the extremities is diminished. In each case, it is
believed that the growth of new blood vessels will increase the
volume of blood circulating through the tissue in question, and
correspondingly increase the amount of oxygen and nutrients
available to that tissue.
[0005] One common cause of decreased blood flow is atherosclerosis.
Atherosclerosis affects the blood vessels, including those of the
heart, and is a major cause of cardiovascular disease, stroke and
peripheral vascular disease. This disease may have its beginnings
early in life and is first noted as a thickening of the arterial
walls. This thickening is an accumulation of fat, fibrin, cellular
debris and calcium. The resultant narrowing of the lumen of the
afflicted vessel is called stenosis. Stenosis impedes and reduces
blood flow. Hypertension and dysfunction of the organ or area of
the body that suffers the impaired blood flow can result. As the
buildup on the inner wall of a vessel thickens, the vessel wall
loses the ability to expand and contract. Also, the vessel loses
its viability and becomes weakened and susceptible to bulging, also
known as aneurysm. In the presence of hypertension or elevated
blood pressure, aneurysms will frequently dissect and ultimately
rupture.
[0006] Small vessels, such as the arteries that supply blood to the
heart, legs, intestines and other areas of the body, are
particularly susceptible to atherosclerotic narrowing. When an
artery in the leg or intestine is affected, the resultant loss of
blood supply to the leg or segment of the intestine may result in
gangrene. Atherosclerotic narrowing of one or more of the coronary
arteries limits and in some instances prevents blood flow to
portions of the heart muscle. Depending upon the severity of the
occlusion and its location within the coronary circulation system,
pain, cardiac dysfunction or death may result. Because the
consequences of blocked arteries are so serious, reliable
treatments are highly desirable.
[0007] In many instances, it is possible to correct aneurysms and
stenosis of major arteries using plastic reconstruction that does
not require any synthetic graft or patch materials. In other
instances, such as where the disease is extensive and the vessel is
no longer reliable, the blocked or weakened portion of the vessel
is usually replaced with a graft. In such case, the affected vessel
section is transected and removed and a synthetic patch, conduit or
graft is sewn into its place. These types of procedures, including
coronary artery bypass grafting (CABG) and percutaneous
transluminal coronary angioplasty (PTCA), are routinely performed
for the purpose of alleviating ischemia.
[0008] Nevertheless, coronary artery disease alone is responsible
for approximately 550,000 deaths each year in the United States.
Peripheral vascular disease results in lower limb amputation in
about 150,000 patients each year, with a subsequent mortality rate
of 40% within two years of amputation. Some of the difficulty in
treating arterial occlusion may lie in the fact that each of these
surgical procedures is associated with a certain incidence of
restenosis and may not be appropriate in certain instances. This is
particularly true when the patient is elderly or has undergone a
previous CABG or PTCA procedure. Furthermore, in such cases, a less
invasive technique would be preferred. In particular, it would be
advantageous to be able to stimulate the surrounding tissue to
produce for itself new vessels that would compensate for the
occluded vessels.
[0009] While angiogenic, or "vessel-growing," factors in general
have been the subject of much research, no angiogenic factor has
yet been found to be effective for promoting the desired natural
bypass effect. Examples of such growth factors are transforming
growth factor beta (TGF-.beta.), osteonectin or SPARC,
platelet-derived growth factor (PDGF), basic fibroblast growth
factor (bFGF) and vascular endothelial growth factor (VEGF). All of
these growth factors are either synthetic, meaning they are
manufactured chemically from non-living sources, or are produced by
recombinant manufacturing processes. Each of these angiogenic
factors comprises only a single protein and possesses only a single
functionality. In addition, many of the known angiogenic compounds
are exceedingly difficult and/or expensive to manufacture.
[0010] Hence, it is desired to provide an effective angiogenic
factor that is easy to manufacture from readily available
materials, easily administered by the surgeon and effective at
stimulating the growth of new blood vessels into the treated
tissue.
BRIEF SUMMARY OF THE INVENTION
[0011] The present invention comprises an angiogenic factor that is
easily manufactured from readily available materials, easily
administered by the surgeon and effective at stimulating the growth
of new blood vessels into the treated tissue. The angiogenic factor
of the present invention comprises a group of proteins extracted
from bone. It has been found that the mixtures of proteins produced
by certain processes are particularly effective angiogenic agents.
These angiogenic agents can be administered as part of the
treatment of an existing vascular disorder, or can play a role in
early intervention and prevention if administered in certain cases.
In particular, the present angiogenic agents can be introduced into
tissue in the vicinity of an occluded vessel so as to cause the
formation of new vessels that bypass the occluded vessel. In this
manner, a natural bypass mechanism is provided.
[0012] The angiogenic mixtures of bone proteins used according to
the present invention are produced by a multi-step process that
includes at least one ultrafiltration step, an anion exchange
chromatography step, a cation exchange chromatography step and a
high performance liquid chromatography (HPLC) purification
step.
[0013] In particularly preferred embodiments, the invention
provides a method for promoting natural bypass in a mammal so
provide increased blood flow to tissue served by an occluded or
partly occluded vessel, a method for promoting vessel growth to
heal a heart artery that has been blocked, or a method for
promoting angiogenesis to assist in recovery from tissue
damage.
[0014] In each instance, the method preferably comprises
administering to the mammal a mixture of proteins derived from
ground bone. The mixture of proteins preferably comprises at least
two growth factors selected from the group consisting of bone
morphogenic protein-2 (BMP-2), bone morphogenic protein-3 (BMP-3),
bone morphogenic protein-4 (BMP-4), bone morphogenic protein-5
(BMP-5), bone morphogenic protein-6 (BMP-6), bone morphogenic
protein-7 (BMP-7), transforming growth factor .beta.1
(TGF-.beta.1), transforming growth factor .beta.2 (TGF-.beta.2),
transforming growth factor .beta.3 (TGF-p3), and fibroblast growth
factor 1 (FGF-1).
[0015] The mammal to which the present method is applied can be a
human, and the mixture can be administered subcutaneously,
intramuscularly, or intravenously. The bone-derived protein mixture
may be derived from bovine bone. The mixture can be administered
discretely or continuously.
[0016] In a preferred embodiment, the mixture further comprises a
growth factor selected from insulin-like growth factor-1 (IGF-1),
epidermal growth factor (EGF), hepatocyte growth factor (HGF),
transforming growth factor .alpha. (TGF-.alpha.), or
platelet-derived growth factor (PDGF), and optionally includes a
preservative or an adjuvant. Particularly preferred mixtures
comprises BMP-2, BMP-3, BMP-7, TGF-.beta., and FGF, or the mixture
derived by (i) grinding mammalian bone, to produce ground bone;
(ii) cleaning the ground bone, to produce cleaned ground bone;
(iii) demineralizing the cleaned ground bone, to produce
demineralized cleaned ground bone; (iv) extracting protein from the
demineralized cleaned ground bone using a protein denaturant; to
yield extracted protein; (v) ultrafiltering the extracted protein
to separate out high molecular weight proteins; (vi) ultrafiltering
the extracted protein to separate out low molecular weight
proteins; (vii) transferring the extracted protein to a non-ionic
denaturant; (viii) subjecting the extracted protein to an anion
exchange process; (ix) subjecting the extracted protein to a cation
exchange process; and (x) subjecting the extracted protein to a
reverse phase HPLC process.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] For a more detailed description of the present invention,
reference will now be made to the accompanying Figures,
wherein:
[0018] FIG. 1 illustrates an SDS-PAGE of one embodiment of the
present angiogenic protein mixture, both in reduced and non-reduced
forms;
[0019] FIG. 2 is an SDS-PAGE gel of HPLC fractions 27-36 of a
protein mixture according to an embodiment of the present
invention.
[0020] FIG. 3 is an SDS-PAGE gel with identified bands indicated
according to the legend of FIG. 4;
[0021] FIG. 4 is an SDS-PAGE gel of a protein mixture according to
an embodiment of the present invention with identified bands
indicated, as provided in the legend;
[0022] FIG. 5 is a two dimensional (2-D) SDS-PAGE gel of a protein
mixture according to an embodiment of the present invention with
internal standards indicated by arrows;
[0023] FIG. 6 is a 2-D SDS-PAGE gel of a protein mixture according
to an embodiment of the present invention with circled proteins
identified as in the legend;
[0024] FIGS. 7A-O are mass spectrometer results for tryptic
fragments from one dimensional (1-D) gels of a protein mixture
according to an embodiment of the present invention;
[0025] FIG. 8 is a 2-D gel Western blot of a protein mixture
according to an embodiment of the present invention labeled with
anti-phosphotyrosine antibody;
[0026] FIGS. 9A-D are 2-D gel Western blots of a protein mixture
according to an embodiment of the present invention, labeled with
indicated antibodies. FIG. 9A indicates the presence of BMP-3 and
BMP-2. FIG. 9B indicates the presence of BMP-3 and BMP-7. FIG. 9C
indicates the presence of BMP-7 and BMP-2, and FIG. 12D indicates
the presence of BMP-3 and TGF-.beta.1;
[0027] FIG. 10 is a PAS (periodic acid schiff) stained SDS-PAGE gel
of HPLC fractions of a protein mixture according to an embodiment
of the present invention;
[0028] FIG. 11 is an anti-BMP-7 stained SDS-PAGE gel of a PNGase F
treated protein mixture according to an embodiment of the present
invention;
[0029] FIG. 12 is an anti-BMP-2 stained SDS-PAGE gel of a PNGase F
treated protein mixture according to an embodiment of the present
invention;
[0030] FIGS. 13A-B are bar charts showing explant mass of
glycosylated components in a protein mixture according to an
embodiment of the present invention (FIG. 13A) and ALP score (FIG.
13B) of the same components;
[0031] FIG. 14 is a chart showing antibody listing and
reactivity;
[0032] FIGS. 15A-B together comprise a chart showing tryptic
fragment sequencing data for components of a protein mixture
according to an embodiment of the present invention;
[0033] FIGS. 16A-F together comprise a chart showing tryptic
fragment mass spectrometry data for components of a protein mixture
according to an embodiment of the present invention;
[0034] FIGS. 17A-B are an SDS-gel (FIG. 17B) and a scanning
densitometer scan (FIG. 17A) of the same gel for a protein mixture
according to an embodiment of the present invention;
[0035] FIG. 18 is a chart illustrating the relative mass, from
scanning densitometer quantification, of protein components in a
protein mixture according to an embodiment of the present
invention;
[0036] FIGS. 19A-D together comprise a chart showing mass
spectrometry data of various protein fragments from 2D gels of a
protein mixture according to an embodiment of the present
invention;
[0037] FIGS. 20A-C show the results of a quail chorioallantoid
membrane (CAM) angiogenesis assay of a protein mix according to the
present invention;
[0038] FIG. 21 shows the vascular growth in the CAM of FIGS.
20A-C;
[0039] FIGS. 22A-E are histological sections of blood vessels
formed in the canine myocardium following treatment with a protein
mix in accordance with the present invention;
[0040] FIGS. 23 and 24 are in vivo angiograms showing blood flood
to a representative LAD after placement of an ameroid constrictor
on the LAD and occlusion the LAD but before treatment according to
the present invention; and
[0041] FIGS. 25 and 26 are in vivo angiograms showing blood flood
to a representative LAD after placement of an ameroid constrictor
on the LAD and occlusion the LAD and six weeks after treatment
according to the present invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0042] Angiogenesis is a complex process involving several
different cell types and molecular signaling events. Endothelial
cells must secrete proteases to dissolve cell-cell and cell-matrix
attachments, migrate and proliferate to form new vascular branches.
Although single factors such as bFGF and VEGF have shown promise as
angiogenic agents, it has been discovered that a more robust
angiogenic response may be obtained through the use of an agent
that comprises a mixture of proteins. This may be due in part to a
synergistic effect of the combined proteins on the subject tissue.
Thus, according to a preferred embodiment of the present invention,
a natural bypass effect is achieved by injecting an angiogenic
mixture of bone proteins into tissue in need of increased blood
flow.
[0043] In one embodiment, a natural bypass effect is promoted by
administering a mammal a mixture of growth factors derived from
bone. In another embodiment, vessel growth is promoted so as to
heal a heart artery that has been blocked. In still another
embodiment, angiogenesis is promoted in ischemic tissue so as to
assist in recovery.
[0044] The bone-derived angiogenic protein (BDAP) mixture preferred
for use in the present invention is preferably administered
directly to ischemic tissue in a suitable carrier. For example, in
some instances, it may be desired to apply the angiogenic factor in
a carrier that allows it to be absorbed quickly, while in other
instances it may be desired to apply the angiogenic factor in a
controlled, time-release manner. In other instances, a single dose
or other variation may be preferred. In general, the preferred
carrier material will vary depending on the desired clinical
application or site of administration. Polylactic acid,
polyglycolic acid and their copolymers, collagen, PLURONIC
(polyoxyalkylene ether co-polymer surfactant), and povidone
(polyvinylpyrrolidone) are all examples of biocompatible materials
that can be combined with BDAP mixtures to stimulate
angiogenesis.
Characterization of Preferred Growth Factors
[0045] A preferred angiogenic mixture of bone proteins is produced
by a multi-step process that includes an ultrafiltration step, an
anion exchange chromatography step, a cation exchange
chromatography step and a high performance liquid chromatography
(HPLC) purification step as described in detail below. Preferred
processes for producing the angiogenic protein mixtures of the
present invention are described in full detail in U.S. Pat. Nos.
5,290,763 and 5,371,191, which are incorporated herein in their
entireties. The processes can be summarized as follows. In a first
step, demineralized bone particles from a suitable source (such as
crushed bovine bone) are subjected to protein extraction using
guanidine hydrochloride. The extract solution is filtered, and
subjected to a two step ultrafiltration process. In the first
ultrafiltration step, an ultrafiltration membrane having a nominal
molecular weight cut off (MWCO) of 100 kD is preferably employed.
The retentate is discarded and the filtrate is subjected to a
second ultrafiltration step using an ultrafiltration membrane
preferably having a nominal MWCO of about 10 kD. The retentate is
then subjected to diafiltration to substitute urea for guanidine.
The protein-containing urea solution is then subjected to
sequential ion exchange chromatography, first anion exchange
chromatography followed by cation exchange chromatography. For the
anion exchange process, a strongly cationic resin is used,
preferably having quaternary amine functional groups. Typically,
the eluant for the anion exchange process has a conductivity from
about 10,260 micromhos (.mu.mhos) (1.026.times.10<-2> siemens
(S)) to about 11,200 .mu.mhos (1.120.times.10<31 2>S). For
the cation exchange process, a strongly anionic resin is used,
preferably having sulfonic acid functional groups. The eluant for
the cation exchange process typically has a conductivity from about
39,100 .mu.mhos (3.91.times.10<-2>S) to about 82,700 .mu.mhos
(8.27.times.10<-2>S) or more.
[0046] In the process described above, the proteins are
advantageously kept in solution. According to the present
invention, the proteins produced by the above process are then
subjected to HPLC. The HPLC process preferably utilizes a column
containing hydrocarbon-modified silica packing material. The
proteins can be loaded onto the HPLC column in a solution of
aqueous trifluoracetic acid or other suitable solvent, such as
heptafluorobutyric acid, hydrochloric or phosphoric acid.
Preferably, a trifluoracetic acid solution having a concentration
of from about 0.05 percent by volume to about 0.15 percent by
volume, and more preferably about 0.1 percent by volume
trifluoracetic acid is used.
[0047] Proteins are eluted from the HPLC column with an organic
solvent/water mixture suitable for obtaining the desired proteins.
A preferred eluant in the HPLC process is an acetonitrile solution.
The preferred eluant typically has an acetonitrile concentration
which varies, during elution, from about 30 percent by volume to
about 45 percent by volume. In preferred embodiments, the
acetonitrile concentration in the eluant is increased in increments
of between about 0.30 percent by volume and about 0.40 percent by
volume per minute until the desired highest concentration of
acetonitrile is achieved. Proteins can be recovered from the HPLC
process eluant by means generally known in the art. A preferred
angiogenic fraction of the eluted proteins occurs when the
acetonitrile concentration in the eluant is between about 33
percent by volume and about 37 percent by volume.
[0048] The purification processes described above yield novel
angiogenic protein mixtures. Because they comprise mixtures of
proteins, these angiogenic factors are most easily described in
terms of their properties. Hence, in one embodiment of the present
angiogenic factor, the factor is a mixture of a number of proteins
having the sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) profile shown in FIG. 1.
[0049] Another characterization of the present invention is a
mixture of proteins having a preferred amino acid composition of
about 20-25 mole percent of acidic amino acids [ASP(+ASN) and
GLU(+GLN)]; about 10-15 mole percent of hydroxy amino acids (SER
and THR); about 35-45 mole percent aliphatic amino acids (ALA, GLY,
PRO, MET, VAL, ILE, and LEU); about 4-10 mole percent aromatic
amino acids (TYR and PHE); and about 10-20 mole percent basic amino
acids (HIS, ARG and LYS). More particularly, this embodiment of the
angiogenic protein mixture amino preferably has an amino acid
composition of about 23.4 mole percent of acidic amino acids
[ASP(+ASN) and GLU(+GLN)]; about 13.5 mole percent of hydroxy amino
acids (SER and THR); about 40.0 mole percent aliphatic amino acids
(ALA, GLY, PRO, MET, VAL, ILE, and LEU); about 6.8 mole percent
aromatic amino acids (TYR and PHE); and about 16.6 mole percent
basic amino acids (HIS, ARG and LYS). (TRP, CYS and 1/2 CYS were
not measured and are not included in the calculation of mole
percent.)
[0050] An alternative embodiment of the present angiogenic factor
can be defined as a different fraction of the total protein stream
exiting the HPLC process. More particularly, the proteins eluted
when the eluant has an acetonitrile concentration of from about 37
to about 39.5 percent by volume have been found to have surprising
angiogenic activity. The mixture defined in this manner contains
hundreds of natural proteins. It is believed that the angiogenic
activity of proteins obtained in this manner may be further
enhanced by selecting smaller fractions of the eluant and
quantitatively comparing the angiogenic activity of each
fraction.
[0051] In addition to the foregoing, BP has been partially
characterized as follows: high performance liquid chromatography
(HPLC) fractions have been denatured, reduced the DTT, and
separated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). One minute HPLC fractions from 27 to 36
minutes are shown in FIG. 2. Size standards (ST) of 14, 21, 31, 45,
68 and 97 kDa were obtained as Low range size standards from
BIORAD.TM. and are shown at either end of the coomassie blue
stained gel. In the usual protocol, HPLC fractions 29 through 34
are pooled to produce BP (see boxes, FIGS. 2 and 3), as shown in a
similarly prepared SDS-PAGE gel in FIG. 17B.
[0052] The various components of BP were characterized by mass
spectrometry and amino acid sequencing of tryptic fragments where
there were sufficient levels of protein for analysis. The major
bands in the ID gel (as numerically identified in FIG. 3) were
excised, eluted, subjected to tryptic digestion and the fragments
were HPLC purified and sequenced. The sequence data was compared
against known sequences, and the best matches are shown in FIGS.
12A-B. These identifications are somewhat tentative, in that only
portions of the entire proteins have been sequenced and, in some
cases, there is variation between the human and bovine analogs for
a given protein.
[0053] The same tryptic protein fragments were analyzed by mass
spectrometry and the mass spectrograms are shown in FIGS. 7A-O. The
tabulated results and homologies are shown in FIGS. 16A-F, which
provide identification information for the bands identified in
FIGS. 3-4. As above, assignment of spot identity may be tentative
based on species differences and post translational modifications.
A summary of all protein identifications for 1d gels is shown in
FIG. 4.
[0054] The identified protein components of BP, as described in
FIGS. 15A-B, 16A-F and 19A-D, were quantified as shown in FIGS. 17A
and 17B. FIG. 17B is a stained SDS-PAGE gel of BP and FIG. 17A
represents a scanning densitometer trace of the same gel. The
identified proteins were labeled and quantified by measuring the
area under the curve. These results are presented in FIG. 18 as a
percentage of the total peak area.
[0055] Thus, there are 11 major bands in the BP SDS-PAGE gel,
representing about 60% of the protein in BP. The identified
proteins fall roughly into three categories: the ribosomal
proteins, the histones, and growth factors, including bone
morphogenic factors (BMPs). It is expected that he ribosomal
proteins may be removed from the BP without loss of activity, since
these proteins are known to have no growth factor activity. Upon
this separation, the specific activity is expected to increase
correspondingly.
[0056] It is expected that the histone and ribosomal proteins may
be removed from the BP with no resulting loss, or even with an
increase, in specific activity. It is expected that histones can
removed from the BP cocktail by immunoaffinity chromatography,
using either specific histone protein antibodies or a pan-histone
antibody. The histone depleted BP (BP-H) produced in this manner
may be suitable for wound healing. Similarly, the mixture produced
when the known ribosomal proteins are stripped from the BP cocktail
(BP-R) may be suitable for wound healing.
[0057] An SDS-PAGE gel of BP was also analyzed by Western
immunoblot with a series of antibodies, as listed in FIG. 14.
Visualization of antibody reactivity was by horseradish peroxidase
conjugated to a second antibody and using a chemiluminescent
substrate. Further, TGF-.beta.1 was quantified using commercially
pure TGF-.beta.1 as a standard and was determined to represent less
than 1% of the BP protein The antibody analysis indicated that each
of the proteins listed in FIG. 14 is present in BP.
[0058] The BP was further characterized by 2-D gel electrophoresis,
as shown in FIGS. 5-6. The proteins are separated in horizontal
direction according to charge (pI) and in the vertical direction by
size as described in two-dimensional electrophoresis adapted for
resolution of basic proteins was performed according to the method
of O'Farrell et al. (O'Farrell, P. Z., Goodman, H. M. and
O'Farrell, P. H., Cell, 12: 1133-1142, 1977) by the Kendrick
Laboratory (Madison, Wis.). Two-dimensional gel electrophoresis
techniques are known to those of skill in the art. Non-equilibrium
pH gradient electrophoresis ("NEPHGE") using 1.5% pH 3.5-10, and
0.25% pH 9-11 ampholines (Amersham Pharmacia Biotech, Piscataway,
N.J.) was carried out at 200 V for 12 hrs. Purified tropomyosin
(lower spot, 33,000 KDa, pI 5.2), and purified lysozyme (14,000
KDa, pI 10.5-11) (Merck Index) were added to the samples as
internal pI markers and are marked with arrows.
[0059] After equilibration for 10 min in buffer "0" (10% glycerol,
50 mM dithiothreitol, 2.3% SDS and 0.0625 M tris, pH 6.8) the tube
gel was sealed to the top of a stacking gel which is on top of a
12.5% acrylamide slab gel (0.75 mm thick). SDS slab gel
electrophoresis was carried out for about 4 hrs at 12.5 mA/gel.
[0060] After slab gel electrophoresis two of the gels were
coomassie blue stained and the other two were transferred to
transfer buffer (12.5 mM Tris, pH 8.8, 86 mM Glycine, 10% MeoH)
transblotted onto PVDF paper overnight at 200 mA and approximately
100 volts/two gels. The following proteins (Sigma Chemical Co., St.
Louis, Mo.) were added as molecular weight standards to the agarose
which sealed the tube gel to the slab gel: myosin (220,000 KDa),
phosphorylase A (94,000 KDa), catalase (60,000 KDa), actin (43,000
KDa), carbonic anhydrase (29,000 KDa) and lysozyme (14,000 KDa).
FIG. 5 shows the stained 2-D gel with size standards indicated on
the left. Tropomyosin (left arrow) and lysozyme (right arrow) are
also indicated.
[0061] The same gel is shown in FIG. 6 with several identified
proteins indicated by numbered circles. The proteins were
identified by mass spectrometry and amino acid sequencing of
tryptic peptides, as described above. The identity of each of the
labeled circles is provided in the legend of FIG. 6 and the data
identifying the various protein spots is presented in FIGS.
19A-D.
[0062] Because several of the proteins migrated at more than one
size (e.g., BMP-3 migrating as 6 bands) investigations were
undertaken to investigate the extent of post-translation
modification of the BP components. Phosphorylation was measured by
anti-phosphotyrosine immunoblot and by phosphatase studies. FIG. 8
shows a 2-D gel, electroblotted onto filter paper and probed with a
phosphotyrosine mouse monoclonal antibody by SIGMA (# A-5964).
Several proteins were thus shown to be phosphorylated at one or
more tyrosine residues.
[0063] Similar 2-D electroblots were probed with BP component
specific antibodies, as shown in FIGS. 9A-D. The filters were
probed with BMP-2, BMP-3 (FIG. 9A), BMP-3, BMP-7 (FIG. 9B), BMP-7,
BMP-2 (FIG. 9C), and BMP-3 and TGF-.beta.1 (FIG. 9D). Each shows
the characteristic, single-size band migrating at varying pI, as is
typical of a protein existing in various phosphorylation
states.
[0064] For the phosphatase studies, BP in 10 mM HCl was incubated
overnight at 37.degree. C. with 0.4 units of acid phosphatase
(AcP). Treated and untreated samples were added to lyophilized
discs of type I collagen and evaluated side by side in the
subcutaneous implant rat bioassay, as previously described in U.S.
Pat. Nos. 5,290,763, 5,563,124 and 5,371,191. Briefly, 10 (g of BP
in solution was added to lyophilized collagen discs and the discs
implanted subcutaneously in the chest of a rat. The discs were then
recovered from the rat at 2 weeks for the alkaline phosphotase
("ALP"--a marker for bone and cartilage producing cells) assay or
at 3 weeks for histological analysis. For ALP analysis of the
samples, the explants were homogenized and levels of ALP activity
measured using a commercial kit. For histology, thin sections of
the explant were cut with a microtome, and the sections stained and
analyzed for bone and cartilage formation.
[0065] Both native- and phosphatase-treated BP samples were assayed
for morphogenic activity by mass of the subcutaneous implant
(explant mass) and ALP score. The results showed that AcP treatment
reduced the explant mass and ALP score from 100% to about 60%.
Thus, phosphorylation is important for BP activity.
[0066] The BP was also analyzed for glycosylation. FIG. 10 shows an
SDS-PAGE gel stained with periodic acid schiff (PAS)--a
non-specific carbohydrate stain, indicating that several of the BP
components are glycosylated (starred protein identified as BMP-3).
FIGS. 11-12 show immunodetection of two specific proteins (BMP-7,
FIG. 14 and BMP-2, FIG. 15) treated with increasing levels of
PNGase F (Peptide-N-Glycosidase F). Both BMP-2 and BMP-7 show some
degree of glycoslyation in BP, but appear to have some level of
protein resistant to PNGase F as well (plus signs indicate
increasing levels of enzyme). Functional activity of PNGase F and
sialadase treated samples were assayed by explant mass and by ALP
score, as shown in FIGS. 13A and 13B, which shows that
glycosylation is required for full activity.
[0067] In summary, BMPs 2, 3 and 7 are modified by phosphorylation
and glycosylation. These post-translation modifications affect
protein morphogenic activity, 33% and 50% respectively, and care
must be taken in preparing BP not to degrade these functional
derivatives.
[0068] The methods disclosed and claimed herein can be made and
executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the method and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
[0069] The following examples are intended to be merely
illustrative, and do not limit the scope of the claimed
invention.
EXAMPLE 1
[0070] Quail chorioallantoic membrane (CAM) was in the manner
described in "A Novel Assay of Angiogenesis in the Quail
Chorioallantoic Membrane: Stimulation by bFGF and Inhibition by
Angiostatin According to Fractal Dimension and Grid Intersection,"
Parsons-Wingerter P., Dwai B., Yang M C., Elliot K E., Milaninia
A., Redlitz A., Clark J. and Sage E. H. Fertilized Japanese quail
eggs (Coturnix coturnix japonica) were opened onto Petri dishes on
day 3 post-incubation (FIG. 20A). After 4 days of culture, a BDAP
mixture, diluted in PBS/ovalubumin prewarmed to 37.degree. C., was
distributed evenly onto the surface of the CAM. After 24 hours of
incubation, the CAM's were fixed, dissected and photographed (FIG.
20B) at 10.times. magnification to visualize the arterial vascular
tree, including endstage vessels. Digital images of triplicate CAM
specimens were acquired at lOx magnification in grayscale,
binarized to black-and-white, and skeletonized (FIG. 20C). The
vessel branching pattern was analyzed and quantified by the fractal
dimension.
[0071] The photographs in FIG. 21 are representative digital
binarized images of CAMs exposed to 10 .mu.g/ml dose of growth
factor for 24 hours. Quantitative data corresponding to these
images were acquired by analyzing the skeletonized images and
determining the fractal dimension of the branched vascular pattern.
Data were pooled from two separate experiments consisting of three
CAMs per experiment. Exposure to BDAP resulted in 124% greater mean
angiogenic stimulation over the basal rate (defined as the change
in fractal dimension in untreated controls) versus a 43% increase
over basal rate for bFGF-treated CAMs. (p<0.006).
[0072] It is hypothesized that this combination of factors acts
synergistically to facilitate the proliferation, migration and
differentiation processes essential to angiogenesis more
effectively than a single factor.
[0073] Preliminary data suggest that other fractions of proteins
eluted from bone are also angiogenic. An assay of a second protein
mixture, BDAP-2, defined as the fraction eluting at an acetonitrile
concentration of from about 37 to about 39.5 percent, membrane was
performed on quail chorioallantoic membrane (CAM) using the same
protocol as that described above with respect to the BDAP assay.
The angiogenic response in the quail CAM assay was 86 percent
greater than the basal angiogenic rate after treatment with this
alternative protein mix.
EXAMPLE 2
Canine Myocardial Angiogenesis Pilot Study
[0074] Four adult mongrel dogs of either sex, weighing 21-26 kg,
were anesthetized and a left thoracotomy performed through the
fifth intercostal space. All visible epicardial collaterals
connecting LAD diagonals to circumflex or right coronary arteries
were ligated to minimize collateral flow to the LAD territory and
an ameroid constrictor was placed on the proximal to the first
diagonal branch. After completing the procedure, 0, 10 or 100 .mu.g
BDAP was injected in a 0.1 cc volume of povidone
(polyvinylpyrrolidone), as polymer microspheres suspended in
povidone, or in collagen gel for a total of nine injections. Each
series of injections was administered in the ischemic LAD region of
the left ventricle, as well as in a non-ischemic LCX region. The
chest was closed and the animal was allowed to recover.
[0075] In order to provide an index of cellular proliferation at
multiple time points after the initial surgery, bromodeoxyuridine
(BrdU, 25 mg/kg, Sigma, St. Louis, Mo.) was administered
subcutaneously on post-operative days 2, 4, 6, 8, 10, 12, 14 and
21. After two or six weeks, the dogs were euthanized and the hearts
explanted and cut into samples. Samples were fixed and serial
sections, 4-5 microns thick, were cut and stained with Masson's
trichrome stain to evaluate the general morphology of the
myocardium. Sister sections were stained using standard
immunohistochemical techniques with antibodies against
bromodeoxyuridine (BrdU), PC10 proliferating cell nuclear antigen
(PCNA), alpha smooth muscle actin (SMA) and Factor VIII using
standard techniques.
[0076] Initial histological data (FIGS. 22A-E) indicate that 10 or
100 micrograms of BDAP suspended in 0.1 cc povidone stimulated
blood vessel formation within two weeks post injection. Whereas
control sections showed no significant vessel formation and the
needle tract was visible, BDAP-treated sections had several newly
formed blood vessels, as evidenced by Masson's trichrome staining
(FIG. 22A). Immunohistochemical staining demonstrated that these
vessels are lined with endothelial cells (dark stain FIG. 22B) and
surrounded by a layer of smooth muscle cells (brown stain FIG.
22C). PCNA- and BrdU-stained sections (FIGS. 22D-E) indicated that
these vascular endothelial and smooth muscle cells are actively
proliferating. Thus, based on the qualitative results of the canine
study, it was concluded that BDAP stimulates formation of new
differentiated blood vessels approximately 50 -100 .mu.m in
diameter in canine myocardium. (Note, in FIG. 22A-C, 1
cm.apprxeq.200 .mu.m; in FIGS. 22D-E, 1 cm.apprxeq.40 .mu.m).
EXAMPLE 3
Large Scale Canine Myocardial Ischemia Study
[0077] The purpose of this study was to determine the effects of
intramyocardial injections of Sulzer's Growth Factor mixture (GFm,
also called ProVascTM). in a canine model of chronic myocardial
ischemia. 38 dogs underwent ameroid constrictor placement on the
proximal LAD and ligation of visible epicardial vessels
collateralizing the LAD territory. Three weeks later, during a
second surgery, animals had intramyocardial injections of either
placebo, GFm at a concentration of 1 mg/ml or GFm at a
concentration of 10 mg/ml. Each injection consisted of 0.15 ml,
injections were at a spatial density of .about.1/cm2 over the LAD
region. Group assignments were random and investigators were
blinded to group assignment until after the analysis of all test
results. Animal survived for an additional 6 weeks. Assessments of
regional blood flow (by color microspheres), angiography and
echocardiography (rest and stress) were performed prior to and
after treatment. Histology and necropsy were performed after
sacrifice. Results of this prospective, blinded, multifaceted
assessment of the effects of GFm showed that the agent has a
significant effect on vascular growth assessed histologically and
by angiographic criteria. There was no significant effect on blood
flow during maximal vasodilatory stress, though technical
limitations resulted in inclusion of only a small number of studies
for the analysis of maximal blood flow. There was a slight
reduction in regional wall motion score during maximum dobutamine
stress in the high concentration group, though global resting
function was not influenced by treatment.
Colored Microsphere Study
[0078] Dye-Trak.RTM. Colored Microspheres (15.+-.0.1 .mu.m
diameter, suspension in saline solution, 0.5% Tween 80 and 0.1%
Thimerosal as a bacteriostat; Triton Technology Inc., San Diego,
Calif.) provide a non-radioactive method of measuring regional
blood flow. These precision, highly uniform spheres are quantified
by spectrophotometry and were used to determine coronary blood flow
at rest and during maximum adenosine stress. After randomization of
colors, resting blood flow was assessed using rapid infusion of a
set of microspheres (COLOR 1, 2 ml, 6.times.10.sup.6 spheres)
through the previously placed left atrial line. Just prior to the
infusion, withdrawal of arterial blood from the descending aortic
line was instituted at a rate of 7 ml/min using a constant flow
pump (infusion and withdraw pump, Harvard Apparatus Inc., Millis,
Mass. that was calibrated prior to this set of studies); this
withdrawal was continued for a total of 2 minutes (14 ml). To
induce vasodilatory stress, adenosine (A-9251, Sigma Chemical Co.,
St. Louis, Mo.) was infused at a concentration and rate titrated to
cause an approximately 20% decrease in mean arterial pressure.
After achieving this blood pressure reduction, blood flow was
assessed through infusion of a second color microsphere (COLOR
2).
Dobutamine Stress Echocardiography:
[0079] Within the same 20-22 day window, if possible on a different
day, resting and stress echocardiography were performed in the
conscious state. These were done using protocols to standardize
echocardiographic windows and views. Animals were lying in a left
lateral position and a peripheral venous line with stop cock and
extension line was placed. A constant flow pump (Harvard Apparatus
Inc., model 22, South Natick, Mass.) was loaded with a 60 ml
syringe containing dobutamine in normal saline. The dobutamine
infusion solution was prepared immediately prior to the experiment.
The body weight multiplied by 0.048 provided the number of
milliliters of a 250 mg/ml stock solution that was diluted with
normal saline to a total volume of 60 ml. The syringe was connected
to the infusion line. Each echocardiogram was performed by one of 3
experienced echocardiographers completely blinded to group
assignment. Animal names, ID numbers, time and dobutamine infusion
rates were annotated on the video recording of each study. Baseline
recordings under resting conditions at 4 different levels (basal,
mid-papillary, low-papillary and apical; see FIG. 7) at each
dobutamine infusion rate were acquired (HP Sonos 5500.RTM., S4
transducer 2-2.5 MHz, Hewlett Packard, Andover, Mass.) from a
parastemal short axis window.
[0080] During dobutamine infusion, images of all four levels were
recorded on standard VHS tape for off-line analysis. The experiment
was terminated one dosing level after reaching a target heart rate
greater than 200/min, or when new onset of wall motion abnormality
was noted and persisted for longer than 3 minutes combined with a
decrease in mean aortic pressure.
Randomization and Blinding
[0081] After completing these assessments of flow and function
.about.3 weeks after the first surgery, animals were randomly
assigned to one of the 3 treatment groups: placebo, low
concentration GFm or high concentration GFm. By design, the study
intended to have a total of 21 animals reach the end of the
protocol, 7 in each treatment group, with males and females
approximately evenly distributed within each group. 21 envelopes
each containing a single treatment group assignment (7 for each
group, approximately equally distributed between male and female)
were prepared and randomly ordered; each envelope was labeled
"male" or "female" so as to guide animal recruitment through the
course of the study. On the day prior to the "second surgery" (see
next section), an envelope from the stack corresponding to the
gender of the animal was randomly chosen from those remaining and
was opened by an investigator independent of all other aspects of
the investigation. On the day of the second surgery, the assignment
group was reviewed by the independent investigator who prepared the
treatment solution. All solutions were prepared in a secluded
section of the laboratory. All solutions were identical in
appearances and were provided to the primary investigators during
the surgery approximately 30 minutes before injection. In the event
that an animal died at any time before the end of the study or
could not complete the study for any reason, the animal was
replaced by another animal of the same sex into the same treatment
group. As will be detailed, a total of 38 animals were enrolled in
order to obtain the 21 survivors who completed the entire study.
All animals are accounted for as detailed in Results. All
investigators involved in caring for the animals, performing tests,
analyzing data or making any interpretations of the test results
were completely blinded to group assignment.
Baseline Coronary Angiography and Second Surgery for
Intramyocardial Injections Of Gf.sub.m Or Placebo (Second
Operation, Op2)
[0082] Approximately 21 days after the first surgery and after
completing the baseline microsphere and stress echocardiographic
studies (study CEI), each dog was anesthetized using the same
anesthetic protocol as for the first surgery. Urine samples were
obtained using either a urinary catheter or a suprapubic cannula
and these were submitted for routine analysis. If possible, the
samples were taken prior to angiography. If the bladder was empty,
urine had to be withdrawn after surgery. The right femoral artery
was surgically exposed and a standard left coronary artery
catheter.sup.1 was introduced under fluoroscopic guidance through
the artery into the left main coronary artery. Angiography was
performed.sup.2 using standard views to visualize the left anterior
descending artery (LAD) and diagonal vessels. These sequences were
recorded on VHS videotape. After completing the angiography, the
catheter was withdrawn, the femoral artery ligated, and the skin
incision closed. .sup.1Cordis Super Torque Plus.TM. angiographic
catheter, JL3.5 .6F 100 cm, ref cat. no. 533-618, Cordis
Corporation, Miami Fla. or Schneider Guider.TM. Softip.RTM. Guiding
Catheter, Judkins Left 2.5 "Classic" JL3.5 6F 100 cm, model number
S6-JL3.5FC, Schneider (USA) Inc, Pfizer Medical Technology Group,
Minneapolis, Minn. or ACS Viking.TM. Guiding Catheter, Amplatz Left
AL I 6F 100 cm, Guidant Advanced Cardiovascular Systems, Inc.,
Temecula, Calif. .sup.2Visipaque.RTM. (iodixanol), nonionic,
iodinated x-ray contrast agent, Nycomed Inc., Princeton, N.J.
[0083] The chest was then prepared and draped in the usual sterile
fashion and the chest opened in the 5.sup.th intercostal space. At
this time the animal was then treated in a blinded manner according
to the randomization assignment described above. Animals in Group 1
received intramyocardial GF.sub.m injections at a GF.sub.m
concentration of 1 mg/ml, 0.15 ml/injection, 1 injection/cm.sup.2
to the LAD region, total of 15-20 injections per heart. Group 2
animals received intramyocardial GF.sub.m injections, at a GF.sub.m
concentration of 10 mg/ml, 0.15 ml/injection, 1 injection/cm.sup.2
to the LAD region, total of 15-20 injections per heart. Group 3
animals received intramyocardial injections of vehicle (1% LMW
Povidone) without GF.sub.m, 0.15 ml/injection, 1 injection/cm.sup.2
to the LAD region, total of 15-20 injections per heart. Injection
solutions were prepared by two individuals who were independent of
the group of investigators performing the surgeries and follow-up
tests. A shallow stitch (4-0 Prolene.RTM. taper RB-1 monofilament
polypropylene suture, Ethicon, Inc., Somerville, N.J.) was placed
over each injection site so that each site could be identified when
the heart was removed 6 weeks later. After completing the
injections, the heart surface was photographed in order to document
the injection site placement. After infiltration of the intercostal
musculature with 5 ml of Marcaine.RTM. (bupivacaine) the chest was
closed in layers (umbilical tape, Ethicon Inc. and 2-0 Vicryl.TM.
taper CT-1 polyglactin sutures, Ethicon Inc., Somerville, N.J.),
the pneumothorax was reduced and the animal was allowed to
recover.
[0084] Each dog received subcutaneous injections of
5-Bromo-2'-Deoxyuridine (BrdU, B-5002, Sigma-Aldrich, St. Louis,
Mo.; diluted in 0.9% saline solution, adjusted with KOH to pH 9.0)
starting the day before surgery (25 mg/kg), on the day of surgery
(15 mg/kg) and days 1, 3, 5, 7, 9, 13 and 20 after surgery (15
mg/kg) as a means of "marking" dividing cells [Boccadoro, 1986
#106] which can be detected using standard immunohistologic
techniques.
Physiologic Assessment of Blood Flow and Myocardial Function 3
Weeks After Treatment (Second Conscious Experiment, Ce2)
[0085] Between 20 and 22 days (approximately 3 weeks) after the
second surgery, blood flow was assessed in the conscious state only
during adenosine stress using the third colored microsphere (COLOR
3). Resting blood flow was not measured because there are only 5
different colored microspheres; 2 colors have been used at baseline
and 2 are required at the final time point (CE3, see next section).
Within the same 20-22 day time window, the resting echocardiogram
was repeated. The same protocols for performing microsphere
infusions, reference blood sample withdrawals and echocardiography
used during the initial evaluations were employed. Blood samples
were also obtained for routine analysis in a conventional
manner.
Physiologic Assessment of Blood Flow and Myocardial Function 6
Weeks After the Second Surgery (Third Conscious Experiment,
Ce3)
[0086] Between 40 and 44 days (approximately 6 weeks) after the
second surgery, blood flow was assessed at rest and during
adenosine stress using the fourth and fifth colored microsphere
(COLOR 4 and COLOR 5). Resting and dobutamine stress
echocardiographic tests were also performed. Blood flow and
echocardiographic tests were performed a minimum of 4 hours apart
from each other, preferably on different days within the 40-44 day
time window. The same protocols detailed above for performing
microsphere infusions, reference blood sample withdrawals,
echocardiograms and dobutamine infusion used during the initial
evaluations were employed. Blood samples were also obtained to
measure a host of chemical and hematologic parameters.
Coronary Angiography Followed by Sacrifice of the Animal and
Procurement of Tissue Samples (Terminal Experiment and Sacrifice,
Te/Sac)
[0087] After completing blood flow and myocardial functional
assessments with echocardiography, animals were anesthetized as
described above, urine samples were collected in the same manner as
during the second surgery and angiography was repeated using the
left femoral artery to introduce the coronary catheter. Images were
recorded on VHS tape for off-line analysis. After completing the
angiography, the animal was sacrificed with an overdose of
phenobarbital and the heart was removed. Three transmural tissue
blocks, each containing 1 or 2 injection sites (identified by the
previously placed epicardial stitches) were isolated in individual
transmural tissue blocks. These were cut into three approximately
equal thickness sections (epicardial, midwall and endocardial) and
placed in 10% neutral buffered formalin (buffered
Formalde-Fresh.phi., low odor 10% Formalin, cat. SF 93-20, Fisher,
Fair Lawn, N.J.) for fixation. These sections were taken from the
central region of the ischemic territory. The remainder of the
heart was cut into approximately 1 gram tissue blocks, with a map
of where individual samples were derived (including epicardial and
endocardial location; see FIG. 8) and these were submitted together
with samples from both kidneys for microsphere analysis. In
addition, other organs (lungs, liver, spleen, kidneys, brain, and
small intestines) were harvested, weighed and examined grossly and
histologically by a certified veterinary pathologist for signs of
remote tissue effects of GF.sub.m.
[0088] After completing the analysis of the study of all results, a
table was constructed which summarized the findings of angiography
(Angio), histology (Histo), descriptive echocardiographic findings
(Echo D), change in echocardiographic wall motion score (Echo WM),
change in fractional area shortening from echocardiography
(.DELTA.FAC, difference between baseline and final FAC in
percentage points) and percent change in blood flow from colored
microsphere analysis (CMS, .DELTA.%) were determined. The analysis
techniques were as follows:
[0089] Angiography was graded on a 3 point semiquantitative scale:
0, no improvement; 1+ mild improvement in distal LAD visualization;
2+ significant improvement in distal LAD visualization. As
summarized in the tables, there was a statistically significant
improvement in the angiographic score at both high and low
concentrations compared to the placebo group. In addition, there
was a nearly statistically significant difference between low and
high concentration treatments, suggesting a concentration-dependent
improvement in blood flow to the distal LAD.
[0090] In addition to the graded angiography results given in Table
2, FIGS. 23-26 illustrate the marked improvement in blood flow that
resulted from treatment with the inventive composition. In FIGS. 23
and 25, the angiograms are taken at the beginning of the marking
process, while the angiograms of FIGS. 24 and 26 are taken after a
significant of the radio-opaque marker has been injected. In each
of angiograms comprising FIGS. 23, 24, and 25, the visible portion
of the LAD is relatively short, indicating that the radio-opaque
marker has not entered the LAD. In marked contrast, the LAD is much
more visible in FIG. 26, indicating that blood is flowing in the
region. Comparing FIGS. 24 and 26, it can be seen that that blood
flow past the ameriod constrictor is small in FIG. 24, even well
into the injection, whereas blood flow past the ameroid constrictor
(via natural bypass mechanisms, i.e. new vessel growth) is greatly
improved followed treatment with the inventive composition, as seen
in FIG. 26. Hence, it is clear that the administration of an
angiogenic factor according to the present invention greatly
increases the natural bypass of the LAD or other occluded vessel
and allows significant blood flow into a previously ischemic
region.
[0091] The histologic findings from each animal were reviewed
globally and were graded semiquantitatively on a 3 point scale: 0,
no significant vascular growth detected; 1+ mild-to-moderate degree
of vascular growth detected; 2+, significant amount of vascular
growth detected. As summarized in the tables, there was a
statistically significant, concentration dependent increase the
semi-quantitative grading of vascular growth.
[0092] The echocardiograms were analyzed in 3 ways (as summarized
in Methods above). EchoD was a semiquantitative descriptive
parameter obtained by having an experienced echocardiographer
examining changes in the individual wall motion scores between
baseline and the final study (CE 3). EchoD was a 3 point scale: -1,
worsened wall motion during stress; 0, no change in wall motion
during stress; 1+ improved wall motion during stress. This
parameter tended to decrease in the high concentration group but
this was not statistically significant. EchoWM was the change in
the sum of wall motion scores; a lower number for this parameter
indicates better function. Similar to the EchoD parameter, there
was a slight decrease in function detected in the high
concentration group. The third parameter was the change in the
percent fractional shortening showed no significant difference
between groups.
[0093] Microsphere-derived blood flow measurements from all hearts
at all conditions measured are shown in Table 1. Prior to any
treatment (CE 1), blood flow at rest was similar in all groups and
only mildly decreased from the control area, reaching statistical
significance only in the Placebo group. During stress, by
definition, blood flow in the ischemic and border zones were
decreased compared to the control region. During the second
conscious experiments (3 weeks after treatment, CE 2), blood flow
during stress had not changed significantly in any group; blood
flow in the ischemic area was approximately half that in the
control region in all groups.
[0094] Similarly, 6 weeks after treatment blood flow at rest and
during stress was similar in all groups and had not changed
significantly over time in any group. TABLE-US-00001 TABLE 1
Myocardial Perfusion (ml/min/g tissue)-Results from All animals.
(all values are mean .+-. SEE) Number of animals Placebo 7 Low
Conc. GFm 7 High Conc. GFm 7 CE 1: Rest Ischemia 0.76 .+-. 0.06*
0.91 .+-. 0.17 0.90 .+-. 0.10 Borderzone 0.84 .+-. 0.11 0.92 .+-.
0.16 1.04 .+-. 0.13 Control area 0.94 .+-. 0.07 0.95 .+-. 0.15 1.18
.+-. 0.12 CE 1: Stress Ischemia 1.66 .+-. 0.18* 1.83 .+-. 0.19*
1.79 .+-. 0.15* Border zone 2.55 .+-. 0.26*# 2.39 .+-. 0.29*# 2.49
.+-. 0.17*# Control area 4.07 .+-. 0.53 3.70 .+-. 0.37 4.29 .+-.
0.36 CE 2: Stress Ischemia 1.73 .+-. 0.26* 2.17 .+-. 0.19* 1.79
.+-. 0.14* Border zone 2.98 .+-. 0.46*# 3.00 .+-. 0.28*# 2.46 .+-.
0.38*.sub.#=.056 Control 4.41 .+-. 0.69 4.32 .+-. 0.35 3.73 .+-.
0.53 CE 3: Rest Ischemia 0.70 .+-. 0.05* 1.07 .+-. 0.25* 0.90 .+-.
0.12* Border zone 0.85 .+-. 0.08 1.16 .+-. 0.25 1.04 .+-. 0.17
Control area 0.97 .+-. 0.07 1.30 .+-. 0.26 1.22 .+-. 0.16 CE 3:
Stress Ischemia 1.90 .+-. 0.18* 2.21 .+-. 0.12* 1.93 .+-. 0.19*
Border zone 2.81 .+-. 0.16*# 3.13 .+-. 0.16*# 2.73 .+-. 0.29*#
Control area 3.98 .+-. 0.37 4.51 .+-. 0.22 4.37 .+-. 0.43 *p <
0.05 vs. control area, #p < 0.05 vs. ischemic area. no
significant differences between groups or within groups CE1 vs.
CE3. Statistical comparisons done with one way ANOVA with Scheffe
post hoc test
[0095] Lack of uniform maximal vasodilation induced by intravenous
adenosine prohibited assessment of blood flow during maximal
vasodilatory stress in a large number of animals. As a result,
there was a small number values for comparison in each group and
there were no statistically significant differences between groups
with regard to how blood flow during stress changed in response to
treatment.
[0096] After unblinding, the results of these measurements were
sorted by group and are tabulated in Table 2. TABLE-US-00002 TABLE
2 Overview of results in each animal sorted by group. Group Name
Nr. ID # Angio Histo EchoD EchoWM .DELTA.FAC CMS GFm 1 Leoncavallo
8 3581 1+ 2+ 1+ -2 -15 n/i mg/ml Monteverdi 10 3879 1+ 1+ 1+ -1 17
1 Mozart 11 3605 1+ 1+ 1- 0 -5 -7 Puccini 16 3970 1+ 0/1+.sup.2 1+
-5 -4 -1 Rossini 18 11767 1+ 0/1+.sup.2 1-.sup.4 4 11 n/i Schumann
20 10568 1+ n/a.sup.2,3 1+ -3 1 -4 Lautrec 36 12545 2+ 1+ 1+ 4 -10
-1 Mean 1.1 1.0 0.4 -0.4 -4* -2.4 GFm 10 Vivaldi 25 4018 1+
1+.sup.3 1+ -3 5 -2 mg/ml Boticelli 27 11190 2+ 1+ 1- 4 2 1 Dali 29
11187 2+ 2+ 0 2 -10 -7 Degas 30 4070 2+ 2+ 0 -1 -6 n/i Gauguin 32
12311 2+ 2+ 1- 4 -4 n/i Matisse 38 4209 0 2+ 1- 5 3 n/i
Michelangelo 39 11745 2+ 2+ 1- 4 -5 6 Mean 1.6 1.7 -0.4 2.1 -4*
-0.5 Placebo Dvorak 5 10213 0 0 1+ -4 -3 n/i Gershwin 6 3711 1+
0/1+.sup.1 1+ -4 1 6 Mussorgsky 12 11334 1+ 1+ 1- -1 15 n/i
Offenbach 13 3889 1+ 0 1+ -4 0 5 Tchaikovsky 23 4130 0 0 1+ -4 7
n/i Klimt 35 4143 0 0 0 0 54 -14 Lichtenstein 37 4023 0 0 1+ 0 -2
18 Mean 0.4 0.2 0.6 -2.4 1* 3.75 *Median used instead of mean
because of large non-normal distribution. N/i, not included. See
.sup.1histology suggestive of presence of myocardial infarction
.sup.2technical problem with histologic staining .sup.3histology
suggestive of myocardial infarction alone .sup.4The score for this
animal was initially inadvertently entered onto the table sent to
the Sponsor as "0"; this should have been entered as "1-", as it
now appears
[0097] In summary, the general results of this prospective,
blinded, multifaceted assessment of the effects of GF.sub.m showed
that the agent has a concentration-dependent significant effect on
vascular growth assessed histologically and by angiographic
criteria. There was no significant effect on blood flow during
maximal vasodilatory stress, though technical limitations resulted
in inclusion of only a small number of studies for the analysis of
maximal blood flow rendering the results inconclusive. There was a
trend (not statistically significant) towards a slight reduction in
regional wall motion score during maximum dobutimine stress, though
global resting function was not influenced by treatment.
Nonethless, there is histologic and angiographic evidence of
significant vascular growth, though LV function during stress and
blood flow by color microsphere analysis did not improve.
[0098] Administration of angiogenic factors in accordance with the
present invention has several advantages over the alternative
methods for inducing angiogenesis, such as inflammation resulting
from laser injury. The growth factors of the present invention can
be delivered in a minimally invasive manner to ischemic tissues
either through a thoracotomy or percutaneous catheterization
without the use of expensive equipment. In addition, the process
for manufacturing the present angiogenic factors can be readily
scaled up to a commercial production scale. A further advantage is
that the proteins are kept in solution during the purification
steps and exhibit little deterioration during the production
process. Another advantage is that the resultant mixture of
proteins can be used directly, without the mixing that may be
required with proteins produced by other processes.
[0099] While the present angiogenic factor and methods for
producing and administering it have been described according to a
preferred embodiment, it will be understood that departures can be
made from some aspects of the foregoing description without
departing from the scope of the invention.
* * * * *