U.S. patent application number 14/311523 was filed with the patent office on 2014-11-27 for angiogenically effective unit dose of fgf-2 and method of use.
This patent application is currently assigned to NOVARTIS VACCINES AND DIAGNOSTICS, INC.. The applicant listed for this patent is Novartis Vaccines and Diagnostics, Inc.. Invention is credited to Martha Jo Whitehouse.
Application Number | 20140349932 14/311523 |
Document ID | / |
Family ID | 27805342 |
Filed Date | 2014-11-27 |
United States Patent
Application |
20140349932 |
Kind Code |
A1 |
Whitehouse; Martha Jo |
November 27, 2014 |
ANGIOGENICALLY EFFECTIVE UNIT DOSE OF FGF-2 AND METHOD OF USE
Abstract
The present invention provides a unit dose composition
comprising 0.2 .mu.g/kg to 48 .mu.g/kg of an FGF-2 of SEQ ID NO:2,
or an angiogenically active fragment or mutein thereof in a
pharmaceutically acceptable carrier. Also provided is a method for
treating a human patient for coronary artery disease, comprising
administering into one or more coronary vessels or a peripheral
vein of said patient a safe and angiogenically effective dose of a
recombinant FGF-2, or an angiogenically active fragment or mutein
thereof. Also provided is a pharmaceutical composition comprising a
therapeutically effective amount of FGF-2, alone or in combination
with heparin, in a therapeutically effective carrier.
Inventors: |
Whitehouse; Martha Jo; (San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis Vaccines and Diagnostics, Inc. |
Emeryville |
CA |
US |
|
|
Assignee: |
NOVARTIS VACCINES AND DIAGNOSTICS,
INC.
Emeryville
CA
|
Family ID: |
27805342 |
Appl. No.: |
14/311523 |
Filed: |
June 23, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13088132 |
Apr 15, 2011 |
8796211 |
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14311523 |
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|
10184708 |
Jun 28, 2002 |
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13088132 |
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09385114 |
Aug 27, 1999 |
6440934 |
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10184708 |
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60104102 |
Oct 13, 1998 |
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60104103 |
Oct 13, 1998 |
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Current U.S.
Class: |
514/9.1 |
Current CPC
Class: |
A61K 38/1825 20130101;
A61P 9/10 20180101; A61K 38/1825 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/9.1 |
International
Class: |
A61K 38/18 20060101
A61K038/18 |
Claims
1. A method for treating a human patient for a myocardial
infarction, comprising administering a single unit dose of a
recombinant fibroblast growth factor-2 (FGF-2) or an angiogenically
active fragment or an angiogenically active mutein thereof into one
or more coronary vessels or into a peripheral vein in a human
patient in need of treatment for coronary artery disease or a
myocardial infarction, said unit dose comprising from about 0.008
mg to 7.2 mg of said recombinant FGF-2 or said angiogenically
active fragment or said angiogenically active mutein thereof,
wherein said recombinant FGF-2 has the amino acid sequence of human
FGF-2, and wherein said angiogenically active mutein has at least
90% amino acid sequence identity with human FGF-2.
2. The method of claim 1, further comprising the step of
administering 10 U/kg to 80 U/kg of heparin to said patient within
30 minutes of administering said unit dose, wherein said heparin is
administered by intravenous or intracoronary administration.
3. The method of claim 1, wherein said unit dose is administered by
infusion.
4. A method for inducing angiogenesis in a heart of a human
patient, comprising administering a single unit dose of a
recombinant fibroblast growth factor-2 (FGF-2) or an angiogenically
active fragment or an angiogenically active mutein thereof into one
or more coronary vessels or into a peripheral vein in a human
patient in need of treatment for coronary artery disease, said unit
dose comprising from about 0.008 mg to 7.2 mg of said recombinant
fibroblast growth factor-2 (FGF-2) or said angiogenically active
fragment or said angiogenically active mutein thereof, wherein said
recombinant FGF-2 has the amino acid sequence of human FGF-2, and
wherein said angiogenically active mutein has at least 90% amino
acid sequence identity with human FGF-2.
5. The method of claim 4, wherein said single unit dose produces an
improvement in one or more clinical endpoints in said human patient
that lasts at least four months.
6. The method of claim 5, wherein said single unit dose produces an
improvement in one or more clinical endpoints in said human patient
that lasts 6 months.
7. The method of claim 4, wherein said unit dose is administered by
infusion.
8. A method for providing a human patient with relief from symptoms
of angina, comprising administering a single unit dose of a
recombinant fibroblast growth factor-2 (FGF-2) or an angiogenically
active fragment or an angiogenically active mutein thereof into one
or more coronary vessels or into a peripheral vein in a human
patient in need of relief from symptoms of angina, said unit dose
comprising from about 0.008 mg to 7.2 mg of said recombinant
fibroblast growth factor-2 (FGF-2) or said angiogenically active
fragment or said angiogenically active mutein thereof, wherein said
recombinant FGF-2 has the amino acid sequence of human FGF-2, and
wherein said angiogenically active mutein has at least 90% amino
acid sequence identity with human FGF-2.
9. The method of claim 8, wherein said unit dose is administered by
infusion.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 13/088,132, filed Apr. 15, 2011 (the content
of which is herein incorporated by reference in its entirety),
which is a continuation application of U.S. application Ser. No.
10/184,708, filed Jun. 28, 2002 (the content of which is herein
incorporated by reference in its entirety), now abandoned, which is
a continuation application of U.S. application Ser. No. 09/385,114,
filed Aug. 27, 1999 (the content of which is herein incorporated by
reference in its entirety), now U.S. Pat. No. 6,440,934, issued
Aug. 27, 2002, which claims the benefit of U.S. Provisional
Application Ser. No. 60/104,102, filed Oct. 13, 1998 and U.S.
Provisional Application Ser. No. 60/104,103, filed Oct. 13,
1998.
FIELD OF THE INVENTION
[0002] The present invention is directed to a unit dose composition
for inducing cardiac angiogenesis in a human comprising a
therapeutically effective amount FGF-2 or an angiogenically active
fragment or mutein thereof. The present invention is also directed
to a method for administering a single unit dose composition to a
human to induce cardiac angiogenesis while minimizing systemic risk
to the patient. The present invention is useful because the
disclosed unit dose composition, and method for its administration,
provide an alternative to angioplasty or surgical intervention for
the treatment of coronary artery disease (CAD) and further provide
an adjunct for reducing post myocardial infarct (MI) injury in
humans.
BACKGROUND OF THE INVENTION
[0003] The fibroblast growth factors (FGF) are a family of at least
eighteen structurally related polypeptides (named FGF-1 to FGF-18)
that are characterized by a high degree of affinity for
proteoglycans, such as heparin. The various FGF molecules range in
size from 15-23 kD, and exhibit a broad range of biological
activities in normal and malignant conditions including nerve cell
adhesion and differentiation [Schubert et al., J. Cell Biol.
104:635-643 (1987)]; wound healing [U.S. Pat. No. 5,439,818
(Fiddes)]; as mitogens toward many mesodermal and ectodermal cell
types, as trophic factors, as differentiation inducing or
inhibiting factors [Clements, et al., Oncogene 8:1311-1316 (1993)];
and as an angiogenic factor [Harada, J. Clin. Invest., 94:623-630
(1994)]. Thus, the FGF family is a family of pluripotent growth
factors that stimulate to varying extents fibroblasts, smooth
muscle cells, epithelial cells and neuronal cells.
[0004] When FGF is released by normal tissues, such as in fetal
development or wound healing, it is subject to temporal and spatial
controls. However, many of the members of the FGF family are also
oncogenes. Thus, in the absence of temporal and spatial controls,
they have the potential to stimulate tumor growth by providing
angiogenesis.
[0005] Coronary artery disease is a progressive condition in humans
wherein one or more coronary arteries gradually become occluded
through the buildup of plaque (atherosclerosis). The coronary
arteries of patients having this disease are often treated by
balloon angioplasty or the insertion of stents to prop open the
partially occluded arteries. Ultimately, many of these patients are
required to undergo coronary artery bypass surgery at great expense
and risk. It would be desirable to provide such patients with a
medicament that would enhance coronary blood flow so as to reduce
the need to undergo bypass surgery.
[0006] An even more critical situation arises in humans when a
patient suffers a myocardial infarction, wherein one or more
coronary arteries or arterioles becomes completely occluded, such
as by a clot. There is an immediate need to regain circulation to
the portion of the myocardium served by the occluded artery or
arteriole. If the lost coronary circulation is restored within
hours of the onset of the infarction, much of the damage to the
myocardium that is downstream from the occlusion can be prevented.
The clot-dissolving drugs, such as tissue plasminogen activator
(tPA), streptokinase, and urokinase, have been proven to be useful
in this instance. However, as an adjunct to the clot dissolving
drugs, it would also be desirable to also obtain collateral
circulation to the damaged or occluded myocardium by
angiogenesis.
[0007] Accordingly, it is an object of the present invention to
provide a medicament and a mode of administration that provides
human patients with cardiac angiogenesis during coronary artery
disease and/or post acute myocardial infarction. More particularly,
it is a further object of the present invention to provide a
therapeutic dose of an FGF and a mode of administration to humans
that provide the desired property of cardiac angiogenesis, while
minimizing adverse effects.
[0008] 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. According to Battler et
al., "the canine model of myocardial ischemia has been criticized
because of the abundance of naturally occurring collateral
circulation, as opposed to the porcine model, which `excels` in its
relative paucity of natural collateral circulation and its
resemblance to the human coronary circulation." Battler et al.,
"Intracoronary Injection of Basic Fibroblast Growth Factor Enhances
Angiogenesis in Infarcted Swine Myocardium," JACC, 22(7): 2001-6
(December 1993) at page 2002, col. 1. However, Battler et al., who
administered bovine bFGF (i.e., FGF-2) to pigs in a myocardial
infarct model, considered the varying results that are obtained
from one animal species to another, and expressly discloses that
the divergent results "thus emphasiz[e] the caution that must be
exercised in extrapolating results from different animal models."
Battler et al., at page 2005, col. 1. Further, Battler points out
that "the dosage and mode of administration of bFGF [i.e., bovine
FGF-2] may have profound implications for the biologic effect
achieved." Battler, et al., at page 2005, col. 1. Thus, it is a
further object of this invention to discover a dosage and a mode of
administration of a fibroblast growth factor that would provide for
the safe and efficacious treatment of CAD and/or post MI injury in
a human patient. More generally, it is an object of the present
invention to provide a pharmaceutical composition and method for
inducing angiogenesis in a human heart.
SUMMARY OF THE INVENTION
[0009] The Applicants have discovered that administering a single
unit dose of about 0.2 .mu.g/kg to about 48 .mu.g/kg of rFGF-2 or
an angiogenically active fragment or mutein thereof into one or
more coronary vessels (IC) or a peripheral vein (IV) of a human
patient in need of coronary angiogenesis, unexpectedly provided the
human patient with a rapid and therapeutic coronary angiogenesis
that resulted in an unexpectedly large increase (i.e., 96 and 100
seconds of increase in the mean change from baseline for all groups
at 2 and 6 months) in the treated patient's exercise tolerance time
(ETT) that persisted for an unexpectedly long duration (i.e., 6
months as of this writing). These changes should result in a
decreased need for standard revascularization procedures. By the
term "coronary angiogenesis," as used herein, is meant the
formation of new blood vessels, ranging in size from capillaries to
arterioles which act as collaterals in coronary circulation. By way
of comparison, angioplasty is considered a therapeutic success if
it provides an increase in a patient's ETT of greater than 30
seconds compared to the placebo.
[0010] Accordingly, in one aspect, the invention is directed to a
unit dose of rFGF-2 comprising a safe and therapeutically effective
amount of rFGF-2 or an angiogenically active fragment or mutein
thereof. Typically, the safe and therapeutically effective amount
comprises about 0.2 .mu.g/kg to about 48 .mu.g/kg of rFGF-2 or an
angiogenically active fragment or mutein thereof, based upon ideal
body weight. In other embodiments, the safe and therapeutically
effective amount of the unit dose comprises 0.2 .mu.g/kg to 2.0
.mu.g/kg, greater than 2.0 ug/kg to less than 24 .mu.g/kg, or 24
.mu.g/kg to 48 .mu.g/kg IC of rFGF-2 or an angiogenically active
fragment or mutein thereof. In another embodiment, the safe and
therapeutically effective amount of the unit dose comprises 18
.mu.g/kg to 36 .mu.g/kg IV of rFGF-2 or an angiogenically active
fragment or mutein thereof. Expressed in absolute terms, the unit
dose of the present invention comprises 0.008 mg to 7.2 mg, more
typically 0.3 mg to 3.5 mg, of FGF-2 or an angiogenically active
fragment or mutein thereof. A suitable FGF-2 is the rFGF-2 of SEQ
ID NO:2 or an angiogenically active fragment or mutein thereof.
[0011] In another aspect, the present invention is directed to a
method of treating a human patient for CAD or to induce coronary
angiogenesis therein. The method comprises administering into one
or more coronary vessels or a peripheral vein of a human patient in
need of treatment for coronary artery disease (or in need of
angiogenesis) a safe and therapeutically effective amount of a
recombinant FGF-2 (rFGF-2) or an angiogenically active fragment or
mutein thereof. Typically, a portion of the safe and
therapeutically effective amount is administered to each of two
coronary vessels. The safe and therapeutically effective amount
comprises about 0.2 .mu.g/kg to about 48 .mu.g/kg of rFGF-2 or an
angiogenically active fragment or mutein thereof in a
pharmaceutically acceptable carrier. In other embodiments, the safe
and therapeutically effective amount comprises 0.2 .mu.g/kg to 2
.mu.g/kg, >2 .mu.g/kg to <24 .mu.g/kg, or 24 .mu.g/kg to 48
.mu.g/kg of rFGF-2 an angiogenically active fragment or mutein
thereof in a pharmaceutically acceptable carrier. In absolute
terms, the amount of rFGF-2 or angiogenically active fragment or
mutein thereof that is used in the above method comprises 0.008 mg
to 7.2 mg, more typically 0.3 mg to 3.5 mg of rFGF-2 or an
angiogenically active fragment or mutein thereof.
[0012] Because FGF-2 is a glycosoaminoglycan (e.g., heparin)
binding protein and the presence of a glycosoaminoglycan optimizes
activity and AUC (see FIGS. 3 and 4), the IC dosages of RFGF-2 of
the present invention typically are administered from 0-30 minutes
prior to the administration of a glycosoaminoglycan, such as a
heparin. The heparin is administered IC or IV, typically IV.
Optionally, the heparin is combined with the unit dose
composition.
[0013] Because rFGF-2 releases nitric oxide, which is a potent
vasodilator, aggressive fluid management prior to (proactively) and
during the infusion is critical to patient's safety. Administration
of IV fluids (e.g., 500-1000 mL of normal saline) to establish a
wedge pressure of 12 mm Hg prior to infusion and administration of
boluses of IV fluids (e.g., 200 mL normal saline) for decreases of
systolic blood pressure (e.g., <90 mm Hg) associated with
infusion optimized the safety of administration of rFGF-2 by IC or
IV infusion to human patients.
[0014] Because EDTA is a potent chelator of calcium that is
required for normal myocardial contraction and cardiac conduction,
minimizing the concentration of EDTA is critical to patient's
safety. A concentration of EDTA less than 100 :g/ml in the unit
dose composition optimized the safety of administration of rFGF-2
by IC or IV infusion to human patients.
[0015] Because a sudden bolus of rFGF-2 is associated with profound
hypotension in animals, the rate of infusion is critical to
patient's safety. Administration at 0.5 to 2 mL per minute,
typically 1 mL per minute, optimized the safety of administration
of rFGF-2 by IC or IV infusion to human patients.
[0016] The unexpected magnitude and duration of the therapeutic
benefit that was provided to human patients in need of coronary
angiogenesis by the unit dose composition and method of
administration was seen as early as two weeks after the single unit
dose was administered, and persisted for 6 months after the single
unit dose was administered IC or IV, as determined by measuring
art-recognized clinical endpoints such as ETT, the "Seattle Angina
Questionnaire" (SAQ) and MRI of the target areas of the heart. In
particular, when the ETT of 58 human CAD patients was assessed by
treadmill at baseline, and at 1 month, 2 months, and 6 months after
administration of a single unit dose of rFGF-2 by IC or IV routes,
clinical benefit was observed in some patients in all dosage
groups. See Table 1. Increases in exercise capacity appear between
1 and 2 months. The mean ETT increased to greater than 60 seconds
at 2 and 6 months with greater benefit being seen in the higher
dose group (24-48 .mu.g/kg) than in the mid (6-12 .mu.g/kg) or low
(0.33-2.0 .mu.g/kg) dose groups. (See Table 1.) Particularly
unexpected and unpredicted by animal models, were the mean
increases in ETT in human patients of 93.4 and 87.5 seconds that
were observed at 2 and 6 months, respectively, post-dosing for
those patients administered a unit dose of rFGF-2 by IV. Even
assuming a placebo effect, the mean change from baseline for the
ETT seconds still allowed an unexpectedly favorable comparison of
results with angioplasty.
[0017] When the quality of life of 48 human CAD patients was
assessed by a validated, disease specific questionnaire, the
Seattle Angina Questionnaire (SAQ), at baseline (i.e., prior to
dosing), and at 2 and 6 months after a single receiving a single
unit dose of rFGF-2 of the present invention by IC or IV routes,
the mean change from baseline for the 5 scales measured by the SAQ
increased in a clinically significant manner for all dosage ranges
whether administered IC or IV (Tables 2-6). In particular, the five
scales assessed by the SAQ are exertional capacity, angina
stability, angina frequency, treatment satisfaction, and disease
perception. Relative to the baseline, the mean score for exertional
capacity increased by 10.9 to 20.2 at 2 months; and by 16.5 to 24.1
at 6 months. For angina stability, the mean score increased by 32.1
to 46.2 at 2 months; and by 16.7 to 23.2 at 6 months. For angina
frequency, the mean score increased by 20.0 to 32.9 at 2 months;
and by 11.4 to 36.7 at 6 months. For treatment satisfaction, the
mean score increased by 8.5 to 19.8 at 2 months; and by 6.3 to 19.8
at 6 months. For disease perception, the mean score increased by
20.2 to 27.8 at 2 months; and by 23.8 to 34.0 at 6 months.
Generally, a change of 8 points on any scale is considered
clinically significant. Thus, the observed changes of 8.5-46.2 are
clinically significant for each of the five scales that were
assessed. Even assuming a placebo effect whereby a mean change from
baseline of 14 points is considered clinically significant, the
results still provide for an unexpectedly superior effect at almost
all scales that were assessed.
[0018] As part of this study, MRI was also performed on 33 human
patients diagnosed with CAD to assess the effect of administering a
single unit dose of rFGF-2 on their cardiac ejection fraction,
regional myocardial function and perfusion (delayed arrival zone).
Specifically, the patients were administered a single unit dose of
0.33 .mu.g/kg to 48 .mu.g/kg IC or 18 .mu.g/kg to 36 .mu.g/kg IV of
rFGF-2 of SEQ ID NO:2. When the 33 human CAD patients were assessed
by resting cardiac magnetic resonance imaging (MRI) at baseline
(i.e., prior to treatment), and 1, 2 and 6 months after treatment
with a single unit dose of rFGF-2 of the invention by IC or IV
routes, the patients exhibited a highly statistically significant
response to the method of treatment as objectively measured by
increased target wall thickening, target wall motion, and target
area collateral extent, and by decreased target area delayed
arrival extent. By way of summary, at 1, 2 and 6 months, the target
wall thickening increased relative to baseline at 4.4%, 6.3% and
7.7%, respectively; the target wall motion increased relative to
baseline at 2.7%, 4.4% and 6.4%, respectively; the target area
collateral extent increased relative to baseline at 8.3%, 10.9% and
11.2%, respectively; and the target area delayed arrival extent
decreased relative to baseline at -10.0%, -8.3% and -10.0%,
respectively.
[0019] The above data demonstrates the clinical efficacy in humans
of the present unit dose composition of rFGF-2 or an angiogenically
active fragment thereof when administered IC or IV in accordance
with the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1A is a plot of the mean rFGF-2 plasma concentration
versus time profiles for eight different doses of rFGF-2 (SEQ ID
NO:2) administered by IC infusion in humans over a 20 minute
period. The eight doses of rFGF-2 presented in FIG. 1A are 0.33,
0.65, 2, 6, 12, 24, 36, and 48 .mu.g/kg of lean body mass
(LBM).
[0021] FIG. 1B is a plot of the mean FGF-2 plasma concentration
versus time profiles for 2 different doses of rFGF-2 (SEQ ID NO:2)
administered by IV infusion in humans over a 20 minute period. The
two IV doses of rFGF-2 in FIG. 1B are 18 and 36 .mu.g/kg. The mean
concentration-time profile following IC administration of 36
.mu.g/kg rFGF-2 is included for comparison.
[0022] FIG. 2 is a plot of mean FGF-2 area under the curve (AUC) in
pg*min/ml corresponding to FIGS. 1A and 1B. This plot shows the
dose linearity of systemic rFGF-2 exposure following IC or IV
infusion. The systemic exposure for the IC route is similar to that
observed following IV administration.
[0023] FIG. 3 is a plot of individual human patient FGF-2 plasma
clearance (CL) values as a function of the time of heparin
administration in "minutes prior to rFGF-2 infusion" and shows the
influence of timing of heparin administration on rFGF-2 plasma
clearance (CL).
[0024] FIG. 4 is a plot individual human patient FGF-2 dose
normalized area under curves (AUCs) as a function of the time of
heparin administration in "minutes prior to rFGF-2 infusion" and
shows the influence of timing of heparin administration on FGF-2
AUC.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The Applicants have discovered that a single dose of rFGF-2
or an angiogenically active fragment or mutein thereof, when
administered in a safe and therapeutically effective amount into
one or more coronary vessels or into a peripheral vein of a human
patient diagnosed with CAD provides the patient with a safe and
therapeutically efficacious treatment for the patient's coronary
artery disease that lasts at least 4 to 6 months, more typically at
least six months, before a further treatment is needed. This
duration of the effect and the magnitude of the improvements in
ETT, SAQ and MRI were unexpected for a single unit dose of
medicament.
[0026] By the phrase "therapeutically effective amount" or "safe
and therapeutically effective amount" as used herein in relation to
rFGF-2 is meant an amount of rFGF-2 or an angiogenically active
fragment or mutein thereof that when administered in accordance
with this invention, is free from major complications that cannot
be medically managed, and that provides for objective cardiac
improvement in patients having symptoms of CAD despite optimum
medical management. Thus, acute hypotension that can be managed by
administration of fluids, is considered "safe" for the purpose of
this invention. Typically, the safe and therapeutically effective
amount of rFGF-2 comprises about 0.2 .mu.g/kg to about 48 .mu.g/kg
of rFGF-2 or an angiogenically active fragment or mutein thereof. A
suitable FGF-2 for use in the present invention is the rFGF-2 of
SEQ ID NO:2 or an angiogenically active fragment or mutein
thereof.
[0027] Accordingly, the present invention has multiple aspects. In
its first aspect, the present invention is directed to a unit dose
composition for inducing angiogenesis in a human patient, the unit
dose comprising a therapeutically effective (i.e., an
angiogenically effective) amount of rFGF-2 or an angiogenically
active fragment or mutein thereof, the amount comprising about 0.2
.mu.g/kg to about 48 .mu.g/kg of rFGF-2 or an angiogenically active
fragment or mutein thereof.
[0028] By the term "unit dose composition" as used herein is meant
a composition that when administered to a human patient in
accordance with the method of the present invention provides a
typical human patient in need of angiogenesis with an angiogenic
effect of significant efficacy so as not to require retreatment for
at least 4-6 months, typically 6 months. The unit dose composition
of the present invention is typically provided in combination with
one or more pharmaceutically acceptable excipients or carriers. In
other embodiments of the unit dose composition, a safe and
therapeutically effective amount comprises about 0.2 .mu.g/kg to
about 2 .mu.g/kg, about 2 .mu.g/kg to about 24 .mu.g/kg, or about
24 .mu.g/kg to about 48 .mu.g/kg of rFGF-2 or an angiogenically
active fragment or mutein thereof.
[0029] It is convenient to define the unit dose composition of the
present invention in more absolute terms that are not dependent
upon the weight of the patient to be treated. When so defined, the
unit dose composition comprises from 0.008 mg to 7.2 mg of rFGF-2
or an angiogenically active fragment or mutein thereof. In this
embodiment, the unit dose composition contains a sufficient amount
of FGF-2 to accommodate dosing any one of the majority of human CAD
patients, ranging from the smallest patient (e.g., 40 kg) at the
lowest dosage (about 0.2 .mu.g/kg) through the larger patients
(e.g., 150 kg) at the highest dosage (about 48 .mu.g/kg). More
typically, the unit dose comprises 0.3 mg to 3.5 mg of rFGF-2 or an
angiogenically active fragment or mutein thereof. The unit dose
composition is typically provided in solution or lyophilized form
containing the above referenced amount of rFGF-2 and an effective
amount of one or more pharmaceutically acceptable buffers,
stabilizers and/or other excipients as later described herein.
[0030] The active agent in the Applicants' above described unit
dose composition is a recombinant FGF-2 or an angiogenically active
fragment or mutein thereof. Methods for making recombinant FGF-2
are well-known in the art. The recombinant FGF-2 of SEQ ID NO:2 is
made as described in U.S. Pat. No. 5,155,214, entitled "Basic
Fibroblast Growth Factor," which issued on Oct. 13, 1992, and which
is expressly incorporated herein by reference in its entirety.
Moreover, all other references cited herein, whether occurring
before or after this sentence, are expressly incorporated herein by
reference in their entirety. As disclosed in the '214 patent, a DNA
of SEQ ID NO:1, which encodes a bFGF (hereinafter "FGF-2") of SEQ
ID NO:2, is inserted into a cloning vector, such as pBR322, pMB9,
Col E1, pCR1, RP4 or X-phage, and the cloning vector is used to
transform either a eukaryotic or prokaryotic cell, wherein the
transformed cell expresses the FGF-2. In one embodiment, the host
cell is a yeast cell, such as Saccharomyces cerevisiae. The
resulting full length FGF-2 that is expressed has 146 amino acids
in accordance with SEQ ID NO:2. Although the FGF-2 of SEQ ID NO:2
has four cysteines, i.e., at residue positions 25, 69, 87 and 92,
there are no internal disulfide linkages. ['214 at col. 6, lines
59-61.] However, in the event that cross-linking occurred under
oxidative conditions, it would likely occur between the residues at
positions 25 and 69.
[0031] The FGF-2 of SEQ ID NO:2, which has 146 amino acid residues,
differs from naturally occurring human FGF-2 by only two amino acid
residue. In particular, the amino acids at residue positions 112
and 128 of the FGF-2 of SEQ ID NO:2 are Ser and Pro, respectively,
whereas in human FGF-2, they are Thr and Ser, respectively. In
nature, bovine FGF-2, like the corresponding human FGF-2 is
initially synthesized in vivo as a polypeptide having 155 amino
acid residues. Abraham et al. "Human Basic Fibroblast Growth
Factor: Nucleotide Sequence and Genomic Organization," EMBO J.,
5(10):2523-2528 (1986). When the FGF-2 of SEQ ID NO:2 is compared
to the full length 155 residue bovine FGF-2 of Abraham, the FGF-2
of SEQ ID NO:2 lacks the first nine amino acid residues, Met Ala
Ala Gly Ser Ile Thr Thr Leu (SEQ ID NO:3), at the N-terminus of the
corresponding full length molecule. The recombinant FGF-2 employed
in the present compositions and method was purified to
pharmaceutical quality (98% or greater purity) using the techniques
described in detail in U.S. Pat. No. 4,956,455, entitled "Bovine
Fibroblast Growth Factor" which issued on Sep. 11, 1990 and which
is incorporated herein by reference in its entirety. In particular,
the first two steps employed in the purification of the recombinant
FGF-2 of Applicants' unit dose composition are "conventional
ion-exchange and reverse phase HPLC purification steps as described
previously." [U.S. Pat. No. 4,956,455, citing to Bolen et al., PNAS
USA 81:5364-5368 (1984).] The third step, which the '455 patent
refers to as the "key purification step" ['455 at col. 7, lines
5-6], is heparin-SEPHAROSE.RTM. affinity chromatography, wherein
the strong heparin binding affinity of the FGF-2 is utilized to
achieve several thousand-fold purification when eluting at
approximately 1.4M and 1.95M NaCl ['455 at col. 9, lines 20-25].
Polypeptide homogeneity was confirmed by reverse-phase high
pressure liquid chromatography (RP-HPLC). Buffer exchange was
achieved by SEPHADEX.RTM. G-25(M) gel filtration
chromatography.
[0032] In addition to the 146 residue rFGF-2 of SEQ ID NO:2, the
active agent in the unit dose of the present invention also
comprises an "angiogenically active fragment" of FGF-2. By the term
"angiogenically active fragment" of FGF-2 is meant a fragment of
FGF-2 that has about 80% of the 146 residues of SEQ ID NO:2 and
that retains at least 50%, preferably at least 80%, of the
angiogenic activity of the FGF-2 of SEQ ID NO:2.
[0033] To be angiogenically active, the FGF-2 fragment should have
two cell binding sites and at least one of the two heparin binding
sites. The two putative cell binding sites of the analogous human
FGF-2 occur at residue positions 36-39 and 77-81 thereof. See
Yoshida, et al., "Genomic Sequence of hst, a Transforming Gene
Encoding a Protein Homologous to Fibroblast Growth Factors and the
int-2-Encoded Protein," PNAS USA, 84:7305-7309 (October 1987) at
FIG. 3. The two putative heparin binding sites of hFGF-2 occur at
residue positions 18-22 and 107-111 thereof. See Yoshida (1987) at
FIG. 3. Given the greater than 98% similarity between the amino
acid sequences for naturally occurring human FGF-2 (hFGF-2) and
rFGF-2 (SEQ ID NO:2), it is expected that the two cell binding
sites for rFGF-2 (SEQ ID NO:2) are also at residue positions 36-39
and 77-81 thereof, and that the two heparin binding sites are at
residue positions 18-22 and 107-111 thereof. Consistent with the
above, it is well known in the art that N-terminal truncations of
the FGF-2 of SEQ ID NO:2 do not eliminate its activity in cows. In
particular, the art discloses several naturally occurring and
biologically active fragments of the FGF-2 that have N-terminal
truncations relative to the FGF-2 of SEQ ID NO:2. An active and
truncated bFGF-2 having residues 12-146 of SEQ ID NO:2 was found in
bovine liver and another active and truncated bFGF-2, having
residues 16-146 of SEQ ID NO:2 was found in the bovine kidney,
adrenal glands and testes. [See U.S. Pat. No. 5,155,214 at col. 6,
lines 41-46, citing to Ueno, et al., Biochem and Biophys Res.
Comm., 138:580-588 (1986).] Likewise, other fragments of the bFGF-2
of SEQ ID NO:2 that are known to have FGF activity are FGF-2
(24-120)-OH and FGF-2 (30-110)-NH.sub.2. [U.S. Pat. No. 5,155,214
at col. 6, lines 48-52.] These latter fragments retain both of the
cell binding portions of FGF-2 (SEQ ID NO:2) and one of the heparin
binding segments (residues 107-111). Accordingly, the
angiogenically active fragments of FGF-2 typically encompass those
terminally truncated fragments of FGF-2 that have at least residues
that correspond to residues 30-110 of FGF-2 of SEQ ID NO:2; more
typically, at least residues that correspond to residues 18-146 of
FGF-2 of SEQ ID NO:2.
[0034] The unit dose of the present invention also comprises an
"angiogenically active . . . mutein" of the rFGF-2 of SEQ ID NO:2.
By the term "angiogenically active . . . mutein" as used herein, is
meant an isolated and purified recombinant protein or polypeptide
that has 65% sequence identity (homology) to any naturally
occurring FGF-2, as determined by the Smith-Waterman homology
search algorithm (Meth. Mol. Biol. 70:173-187 (1997)) as
implemented in MSPRCH program (Oxford Molecular) using an affine
gap search with the following search parameters: gap open penalty
of 12, and gap extension penalty of 1, and that retains at least
50%, preferably at least 80%, of the angiogenic activity of the
naturally occurring FGF-2 with which it has said at least 65%
sequence identity. Preferably, the angiogenically active mutein has
at least 75%, more preferably at least 85%, and most preferably, at
least 90% sequence identity to the naturally occurring FGF-2. Other
well-known and routinely used homology/identity scanning algorithm
programs include Pearson and Lipman, PNAS USA, 85:2444-2448 (1988);
Lipman and Pearson, Science, 222:1435 (1985); Devereaux et al.,
Nuc. Acids Res., 12:387-395 (1984); or the BLASTP, BLASTN or BLASTX
algorithms of Altschul, et al., Mol. Biol., 215:403-410 (1990).
Computerized programs using these algorithms are also available and
include, but are not limited to: GAP, BESTFIT, BLAST, FASTA and
TFASTA, which are commercially available from the Genetics
Computing Group (GCG) package, Version 8, Madison Wis., USA; and
CLUSTAL in the PC/Gene program by Intellegenetics, Mountain View
Calif. Preferably, the percentage of sequence identity is
determined by using the default parameters determined by the
program.
[0035] The phrase "sequence identity," as used herein, is intended
to refer to the percentage of the same amino acids that are found
similarly positioned within the mutein sequence when a specified,
contiguous segment of the amino acid sequence of the mutein is
aligned and compared to the amino acid sequence of the naturally
occurring FGF-2.
[0036] When considering the percentage of amino acid sequence
identity in the mutein, some amino acid residue positions may
differ from the reference protein as a result of conservative amino
acid substitutions, which do not affect the properties of the
protein or protein function. In these instances, the percentage of
sequence identity may be adjusted upwards to account for the
similarity in conservatively substituted amino acids. Such
adjustments are well-known in the art. See, e.g., Meyers and
Miller, "Computer Applic. Bio. Sci., 4:11-17 (1988).
[0037] To prepare an "angiogenically active mutein" of an
angiogenic agent of the present invention, one uses standard
techniques for site directed mutagenesis, as known in the art
and/or as taught in Gilman, et al., Gene, 8:81 (1979) or Roberts,
et al., Nature, 328:731 (1987). Using one of the site directed
mutagenesis techniques, one or more point mutations are introduced
into the cDNA sequence of SEQ ID NO: 1 to introduce one or more
amino acid substitutions or an internal deletion. Conservative
amino acid substitutions are those that preserve the general
charge, hydrophobicity/hydrophilicity, and/or steric bulk of the
amino acid being substituted. By way of example, substitutions
between the following groups are conservative: Gly/Ala,
Val/Ile/Leu, Lys/Arg, Asn/Gln, Glu/Asp, Ser/Cys/Thr, and
Phe/Trp/Tyr. Significant (up to 35%) variation from the sequence of
the naturally occurring angiogenic FGF-2 is permitted as long as
the resulting protein or polypeptide retains angiogenic activity
within the limits specified above.
[0038] Cysteine-depleted muteins are muteins within the scope of
the present invention. These muteins are constructed using site
directed mutagenesis as described above, or according to the method
described in U.S. Pat. No. 4,959,314 ("the '314 patent"), entitled
"Cysteine-Depleted Muteins of Biologically Active Proteins." The
'314 patent discloses how to determine biological activity and the
effect of the substitution. Cysteine substitution is particularly
useful in proteins having two or more cysteines that are not
involved in disulfide formation. Suitable substitutions include the
substitution of serine for one or both of the cysteines at residue
positions 87 and 92, which are not involved in disulfide formation.
Preferably, substitutions are introduced at the FGF-2 N-terminus,
which is not associated with angiogenic activity. However, as
discussed above, conservative substitutions are suitable for
introduction throughout the molecule.
[0039] The unit dose composition of the present invention comprises
a safe and an angiogenically effective dose of rFGF-2 or an
angiogenically active fragment or mutein thereof, and a
pharmaceutically acceptable carrier. Typically, the safe and
angiogenically effective dose of the pharmaceutical composition of
the present invention is in a form and a size suitable for
administration to a human patient and comprises (i) 0.2 .mu.g/kg to
48 .mu.g/kg of rFGF-2 or an angiogenically active fragment or
mutein thereof, (ii) and a pharmaceutically acceptable carrier. In
other embodiments, the safe and angiogenically effective dose
comprises 0.2 .mu.g/kg to 2 .mu.g/kg, >2 .mu.g/kg to <24
.mu.g/kg or 24 .mu.g/kg to 48 .mu.g/kg of FGF-2 or an
angiogenically active fragment or mutein thereof, and a
pharmaceutically acceptable carrier. Expressed in absolute terms
for the majority of human CAD patients, the unit dose of the
present invention comprises 0.008 mg to 7.2 mg, more typically 0.3
mg to 3.5 mg, of the FGF-2 or an angiogenically active fragment or
mutein thereof.
[0040] The second recited component of the unit dose composition of
the present invention is a "pharmaceutically acceptable carrier."
By the term "pharmaceutically acceptable carrier" as used herein is
meant any of the carriers or diluents that are well-known in the
art for the stabilization and/or administration of a proteinaceous
medicament that does not itself induce the production of antibodies
harmful to the patient receiving the composition, and which may be
administered without undue toxicity. The choice of the
pharmaceutically acceptable carrier and its subsequent processing
enables the unit dose composition of the present invention to be
provided in either liquid or solid form.
[0041] When the unit dose composition is in liquid form, the
pharmaceutically acceptable carrier comprises a stable carrier or
diluent suitable for intravenous ("IV") or intracoronary ("IC")
injection or infusion. Suitable carriers or diluents for injectable
or infusible solutions are nontoxic to a human recipient at the
dosages and concentrations employed, and include sterile water,
sugar solutions, saline solutions, protein solutions or
combinations thereof.
[0042] Typically, the pharmaceutically acceptable carrier includes
a buffer and one or more stabilizers, reducing agents,
anti-oxidants and/or anti-oxidant chelating agents. The use of
buffers, stabilizers, reducing agents, anti-oxidants and chelating
agents in the preparation of protein based compositions,
particularly pharmaceutical compositions, is well-known in the art.
See, Wang et al., "Review of Excipients and pHs for Parenteral
Products Used in the United States," J. Parent. Drug Assn.,
34(6):452-462 (1980); Wang et al., "Parenteral Formulations of
Proteins and Peptides: Stability and Stabilizers," J. Parent. Sci.
and Tech., 42:S4-S26 (Supplement 1988); Lachman, et al.,
"Antioxidants and Chelating Agents as Stabilizers in Liquid Dosage
Forms-Part 1," Drug and Cosmetic Industry, 102(1): 36-38, 40 and
146-148 (1968); Akers, M. J., "Antioxidants in Pharmaceutical
Products," J. Parent. Sci. and Tech., 36(5):222-228 (1988); and
Methods in Enzymology, Vol. XXV, Colowick and Kaplan Eds.,
"Reduction of Disulfide Bonds in Proteins with Dithiothreitol," by
Konigsberg, pages 185-188. Suitable buffers include acetate,
adipate, benzoate, citrate, lactate, maleate, phosphate, tartarate
and the salts of various amino acids. See Wang (1980) at page 455.
Suitable stabilizers include carbohydrates such as threlose or
glycerol. Suitable reducing agents, which maintain the reduction of
reduced cysteines, include dithiothreitol (DTT also known as
Cleland's reagent) or dithioerythritol at 0.01% to 0.1% wt/wt;
acetylcysteine or cysteine at 0.1% to 0.5% (pH 2-3); and
thioglycerol at 0.1% to 0.5% (pH 3.5 to 7.0) and glutathione. See
Akers (1988) at pages 225 to 226. Suitable antioxidants include
sodium bisulfite, sodium sulfite, sodium metabisulfite, sodium
thiosulfate, sodium formaldehyde sulfoxylate, and ascorbic acid.
See Akers (1988) at pages 225. Suitable chelating agents, which
chelate trace metals to prevent the trace metal catalyzed oxidation
of reduced cysteines, include citrate, tartarate,
ethylenediaminetetraacetic acid (EDTA) in its disodium,
tetrasodium, and calcium disodium salts, and diethylenetriamine
pentaacetic acid (DTPA). See e.g., Wang (1980) at pages 457-458 and
460-461, and Akers (1988) at pages 224-227. Suitable sugars include
glycerol, trehalose, glucose, galactose and mannitol, sorbitol. A
suitable protein is human serum albumin.
[0043] In liquid form, a typical unit dose composition of the
present invention comprises from about 0.001 mg to 8 mg, more
typically 0.03 to 5 mg rFGF-2 or an angiogenically active fragment
or mutein thereof, dissolved a pharmaceutically acceptable carrier.
A suitable pharmaceutically acceptable carrier comprises 10 mM
thioglycerol, 135 mM NaCl, 10 mM sodium citrate, and 1 mM EDTA, pH
5. A suitable diluent or flushing agent for the above-described
unit dose composition is any of the above-described carriers.
Typically, the diluent is the carrier solution. rFGF-2 or an
angiogenically active fragment or mutein thereof is unstable for
long periods of time in liquid form. To maximize stability and
shelf life of the liquid form, the unit dose composition should be
stored frozen at -60.degree. C. When thawed, the solution is stable
for 6 months at refrigerated conditions. A typical unit dose would
comprise about 1-40 ml, more typically 10-40 ml, of the
above-described composition having 0.008-7.2 mg of rFGF-2 or an
angiogenically active fragment or mutein dissolved therein. A
suitable rFGF-2 for use in the unit dose is the rFGF-2 of SEQ ID
NO:2 or an angiogenically active fragment or mutein thereof.
[0044] In another embodiment, the unit dose composition is provided
in lyophilized (freeze-dried) form. In this form, the unit dose of
rFGF-2 is capable of being stored at refrigerated temperatures for
substantially longer than 6 months without loss of therapeutic
effectiveness. Lyophilization is accomplished by the rapid freeze
drying (i.e., removing water) under reduced pressure of a plurality
of vials, each containing the above described liquid form of the
unit dose of the rFGF-2 of the present invention therein.
Lyophilizers, which perform the above described lyophilization, are
commercially available and readily operable by those skilled in the
art. The resulting lyophilized unit dose composition, in
lyophilized cake form, is formulated to contain within the
resulting lyophilized cake one or more of the buffers, stabilizers,
anti-oxidants, reducing agents, salts and/or sugars described above
for the corresponding liquid formulation. A lyophilized unit dose
composition containing all such other components need only be
reconstituted to a known volume or concentration with sterile
aqueous diluent such as sterile water, a sterile sugar solution, or
a sterile saline solution. Alternatively, it could be reconstituted
with a sterile buffer solution as described above, but lacking a
chelating agent, such as EDTA. As a lyophilized cake, the unit dose
composition is stable from 6 months to two years at refrigerated
temperatures. Thus, storage of the unit dose composition in
lyophilized form is readily accommodated using conventional
refrigeration equipment.
[0045] Because the unit dose composition of the present invention
is administered via a cardiac catheter or other injection device,
which has dead space, it is convenient to formulate the vial
containing the unit dose composition so that it contains about
10-50% more of the rFGF-2 or angiogenically active fragment or
mutein thereof than is to be administered to the patient. For
example, when the unit dose of the rFGF-2 to be administered is 7.2
mg, the vial is optionally formulated to contain up to 50% extra
(e.g., a total of about 10.8 mg) of rFGF-2 or angiogenically active
fragment or mutein thereof. The extra solution is suitable for
filling the dead space in the delivery equipment. In an alternative
embodiment that does not allow for dead space, the pharmaceutical
composition is loaded in the cardiac catheter in front of a
pharmaceutically acceptable buffer, diluent or carrier, which is
then used to deliver the appropriate amount of the one or more
dosages to the one or more sites in the myocardium that are in need
of angiogenesis.
[0046] As discussed above, the pharmaceutically acceptable carrier
for the above described unit dose composition comprises a buffer
and one or more stabilizers, reducing agents, anti-oxidants and/or
anti-oxidant chelating agents. It is also within the scope of the
present invention that the unit dose composition contain an amount
of a glycosoaminoglycan (also known as a "proteoglycan" or a
"mucopolysaccharide"), such as heparin, that is effective to bind
to the FGF-2 and to the endothelial cell receptors so as to enhance
the angiogenic effectiveness of the FGF-2 or angiogenically active
fragment or mutein thereof. The amount of heparin that is
administered is about 10-80 Upper kg of patient weight (U/kg),
typically about 40 U/kg. Expressed in absolute terms, the total
amount of heparin administered to any one patient does not exceed
5,000 U. Thus, upon reconstitution, the unit dose composition of
the present invention would not only contain an angiogenically
effective amount of rFGF-2 or an angiogenically active fragment or
mutein thereof, it would also contain from about 10-80 U/kg of
heparin, typically about 40 U/kg. The typical volume of diluent is
from about 1 to 40 ml. While larger volumes of diluent could be
used, such larger volumes would typically result in longer
administration times. Depending upon the weight of the patient in
kg, a single dose comprising from 0.2 .mu.g/kg to 48 .mu.g/kg of
the rFGF-2 or an angiogenically active fragment or mutein thereof
is withdrawn from the vial as reconstituted product for
administration to the patient. Thus, an average 70 kg man that is
being dosed at 24 .mu.g/kg would have a sufficient volume of the
reconstituted product withdrawn from the vial to receive an IC
infusion of (70 kg.times.30 .mu.g/kg) 2100 .mu.g (i.e., 2.1
mg).
[0047] In its second aspect, the present invention is directed to a
method for treating a human patient for CAD or MI, using the above
described unit dose composition. In particular, in one embodiment,
the present invention is directed to a method for treating a human
patient for coronary artery disease, comprising administering a
safe and therapeutically effective amount of a recombinant FGF-2 or
an angiogenically active fragment or mutein thereof to one or more,
typically two, patent coronary vessels or a peripheral vein of a
human patient in need of treatment for coronary artery disease. The
human patient in need of treatment for coronary artery disease is
typically a human patient with coronary artery disease who remains
symptomatic with angina despite optional medical management. A
preferred coronary vessel is a coronary artery, although grafted
saphenous veins and grafted internal mammary arteries, as provided
by coronary angioplasty, are also suitable. Suitable peripheral
veins for administering the unit dose composition include those
peripheral veins found throughout the human body that are routinely
used by treating physicians and nurses for administration of fluids
and medicaments. Examples of such veins include the cephalic, the
median cubital, and the basilic of the arm.
[0048] When administered as an intracoronary (IC) infusion, the
unit dose of rFGF-2 or angiogenic fragment or mutein thereof is
typically administered within an hour, more typically over a period
of about 20 minutes into one or more (typically, two) patent
coronary vessels. When administered over a twenty minute period,
the unit dose composition is typically administered at a rate of
0.5 to 2.0 ml/minute, more typically at about 1 ml/minute. The
coronary vessels can be native vessels or grafts, so long as they
are not occluded. The volume of the unit dose of rFGF-2 or
angiogenic fragment or mutein thereof is typically 10-40 ml; more
typically 20 ml. The length of time for infusion of the unit dose
is not critical and can be shortened or lengthened depending on the
rate and volume of infusion.
[0049] When administered as an intravenous (IV) infusion, the unit
dose of rFGF-2 or angiogenic fragment or mutein thereof is
administered typically within an hour, more typically over a 20
minute period, into a peripheral vein using a conventional IV
setup. When administered over a twenty minute period, the unit dose
composition is typically administered at a rate of 1 ml/minute.
[0050] In the phase I clinical trial of the above described method
for treating CAD, a single unit dose composition was administered
IC or IV to human patients having CAD who remained symptomatic with
angina despite optional medical management. Because the method of
the present invention induces angiogenesis, the method of the
present invention provides treatment of the underlying condition in
CAD or MI and not merely transitory relief from the symptoms, such
as provided by nitrates. Typically, the safe and therapeutically
effective amount of the method of the present invention comprises
0.2 .mu.g/kg to 48 .mu.g/kg of rFGF-2 or an angiogenically active
fragment or mutein thereof in a pharmaceutically acceptable
carrier. In other embodiments, the safe and therapeutically
effective amount comprises 0.2 .mu.g/kg to 2 .mu.g/kg, >2
.mu.g/kg to <24 .mu.g/kg, or 24 .mu.g/kg to 48 .mu.g/kg of
rFGF-2 or an angiogenically active fragment or mutein thereof in a
pharmaceutically acceptable carrier. In absolute terms, the safe
and therapeutically effective amount is about 0.008 mg to about 7.2
mg of rFGF-2 or an angiogenically active fragment or mutein
thereof; more typically, 0.3 mg to 3.5 mg of rFGF-2 or an
angiogenically active fragment or mutein thereof. A suitable rFGF-2
is the rFGF-2 of SEQ ID NO:2 or an angiogenically active fragment
or mutein thereof.
[0051] In another aspect, the present invention is also directed to
a method for inducing angiogenesis in a heart of a human patient
comprising, administering a single unit dose composition of a
recombinant FGF-2 or an angiogenically active fragment or mutein
thereof to one or more coronary vessels or to a peripheral vein in
a human patient in need of coronary angiogenesis, said unit dose
composition comprising from about 0.008 mg to 7.2 mg of recombinant
rFGF-2 or an angiogenically active fragment or mutein thereof in a
pharmaceutically acceptable carrier. More typically, the unit dose
composition comprises about 0.3-3.5 mg rFGF-2 or an angiogenically
active fragment or mutein thereof in a pharmaceutically acceptable
carrier. As described above, a single unit dose composition
containing a therapeutically effective amount of an rFGF-2 or an
angiogenically fragment or mutein thereof is administered to at
least one coronary vessel of a human patient in need of
angiogenesis, using standard cardiac catheterization techniques
already known and used in the art for the intracoronary
administration of medicaments, e.g., thrombolytics, streptokinase,
or radio-opaque dyes or magnetic particles used to visualize the
coronary arteries. By way of example, a coronary catheter is
inserted into an artery (e.g., femoral or subclavian) of the
patient in need of treatment and the catheter is pushed forward,
with visualization, until it is positioned in the appropriate
coronary vessel of the patient to be treated. Using standard
precautions for maintaining a clear line, the pharmaceutical
composition in solution form is administered by infusing the unit
dose substantially continuously over a period of 10 to 30 minutes.
Although the pharmaceutical composition of the invention could be
administered over a longer period of time, the Applicants perceive
no benefit and a potentially increased risk of thrombosis in doing
so. Typically, a portion (e.g., one half) of the unit dose is
administered in a first coronary vessel. Then, the catheter is
repositioned into a second secondary coronary vessel and the
remainder of the unit dose is administered with flushing of the
catheter. Using the above-described repositioning procedure,
portions of the unit dose may be administered to a plurality of
coronary vessels until the entire unit dose has been administered.
After administration, the catheter is withdrawn using conventional
art known protocols. In the phase I clinical trials described
herein, therapeutic benefit was reported by patients as early as
two weeks following the IC rFGF-2 administration of a single unit
dose. Clinically significant improvement was demonstrable by
objective criterion (ETT and/or SAQ) as early as 30 days following
IC or IV administration of a single unit dose of the present
invention, and was maintained for six months following dosing. In
certain patients with progressive CAD disease, it may be necessary
or appropriate to administer additional unit doses of rFGF-2 at six
or 12 month intervals after the initial unit dose, to overcome the
progression of the CAD during that interim period. In some patients
with very progressive CAD, unit doses of present invention would be
readministered at 4 month intervals. In any instance, the treating
physician would be able to determine the time, if any, for
readministration based upon routine assessment of the clinical
symptoms of the patient.
[0052] One of the benefits of the method of the present invention
is cardiac angiogenesis. Accordingly, in another aspect, the
present invention is directed to a method for inducing angiogenesis
in a heart of a human patient, comprising administering into one or
more coronary vessels (IC) or into a peripheral vein (IV) of a
human patient in need of coronary angiogenesis, a single unit dose
composition comprising an angiogenically effective amount of rFGF-2
or an angiogenically active fragment or mutein thereof in a
pharmaceutically acceptable carrier. In the above method, the
angiogenically effective amount comprises about 0.2 .mu.g/kg to
about 48 .mu.g/kg (or in absolute terms about 0.008 mg to about 7.2
mg) of a recombinant FGF-2 or an angiogenically active fragment or
mutein thereof. More typically, the angiogenically effective amount
comprises about 0.3 mg to 3.5 mg of a recombinant FGF-2 or an
angiogenically active fragment or mutein thereof. A suitable rFGF-2
for use in the above-identified method is the rFGF-2 of SEQ ID NO:2
or an angiogenically active fragment thereof. In one embodiment of
the above method, the unit dose composition is administered IC to
patent coronary vessels or IV to a peripheral vein. In another
embodiment, the unit dose composition is administered with heparin
as described herein.
[0053] The above described method for providing coronary
angiogenesis is also beneficial in human patients that have
undergone a myocardial infarction (MI) in one or more coronary
arteries. Accordingly, in another aspect, the present invention is
also directed to a method for treating a human patient for an MI
comprising, administering into one or more coronary vessels or into
a peripheral vein of said human patient, a single unit dose
composition comprising a therapeutically effective amount of rFGF-2
or an angiogenically active fragment or mutein thereof. In the
above method, the unit dose composition typically comprises about
0.2 .mu.g/kg to about 48 .mu.g/kg (or in absolute terms about 0.008
mg to about 7.2 mg) of a recombinant FGF-2 or an angiogenically
active fragment or mutein thereof in a pharmaceutically acceptable
carrier. A suitable rFGF-2 for use in the above-identified method
is the rFGF-2 of SEQ ID NO:2 or an angiogenically active fragment
thereof.
[0054] In the event of unstable angina or acute myocardial
infarction, requiring angioplasty, the same doses of rFGF-2 or
angiogenic fragment or mutein thereof that are disclosed herein
would also be useful as an adjunct therapy in treating those
conditions. Accordingly, in another aspect, the present invention
is directed to an improved method for treating a patient for
unstable angina or acute myocardial infarction, requiring
angioplasty, the method comprising providing angioplasty to the
patient in need of treatment; the improvement comprising
administering into one or more coronary vessels or into a
peripheral vein of said human patient, a single unit dose
composition comprising a therapeutically effective amount of rFGF-2
or an angiogenically active fragment or mutein thereof. In the
above method, the unit dose composition comprises about 0.2
.mu.g/kg to about 48 .mu.g/kg (or in absolute terms about 0.008 mg
to about 7.2 mg) of a recombinant FGF-2 or an angiogenically active
fragment or mutein thereof in a pharmaceutically acceptable
carrier. A suitable rFGF-2 for use in the above-identified method
is the rFGF-2 of SEQ ID NO:2 or an angiogenically active fragment
thereof.
[0055] In any of the above-described methods of the present
invention, the rFGF-2 or the angiogenically active fragment or
mutein thereof is associated with release of nitric oxide, a
recognized smooth muscle dilator, which upon administration to the
patient causes a sudden drop in the patient's blood pressure.
Accordingly, in the methods of the present invention, it is
preferable to hydrate the patient with IV fluids prior to
administering the unit dose of the present invention. Moreover, for
safety and tolerability of the unit dose, aggressive fluid
management during and after rFGF-2 administration is also
preferred. Finally, it is also within the scope of the above
described methods to include the step of administering an effective
amount of a glycosoaminoglycan (also known as a "proteoglycan" or a
"mucopolysaccharide"), such as heparin from 0-30 minutes prior to
administering the unit dose composition of the present invention.
Typically, the effective amount of glycosaminoglycan (such as
heparin) that is administered is about 10-80 U/kg, more typically,
about 40 U/kg. However, the total amount of heparin administered to
any one patient immediately prior to dosing generally does not
exceed 5,000 U.
[0056] Because EDTA is a potent chelator of calcium which is
required for normal myocardial contraction and cardiac conduction,
minimizing the concentration of EDTA is critical to patient's
safety. A concentration of EDTA less than 100 .mu.g/ml optimized
the safety of administration of rFGF-2 by IC or IV infusion to
human patients.
[0057] Because a sudden bolus of rFGF-2 is associated with profound
hypotension in animals, the rate of infusion is critical to
patient's safety. Administration at 0.5 to 2 mL per minute,
typically 1 mL per minute, optimized the safety of administration
of rFGF-2 by IC or IV infusion to human patients.
[0058] A Phase I clinical trial directed to treating human patients
for CAD by administering a single unit dose composition of the
present invention was conducted and is described in Examples 1-3
herein. In that trial, sixty-six (66) human patients diagnosed with
CAD, who satisfied the criteria of Example 2 herein, received a
single unit dose of rFGF-2 in accordance with the method of the
present invention. Specifically, fifty-two human patients were
administered a unit dose of 0.33 .mu.g/kg to 48 .mu.g/kg of rFGF-2
by IC infusion over about a 20 minute period. Fourteen human
patients were administered a unit dose of either 18 .mu.g/kg or 36
.mu.g/kg of rFGF-2 by IV infusion over about a 20 minute period.
The 66 treated patients were then assessed relative to baseline
(i.e., prior to treatment with the single unit dose), and again at
1 month, 2 months and 6 months after treatment with the single unit
dose, using three sets of art-recognized assessment criteria: 1)
changes in their exercise tolerance time (ETT); 2) the Seattle
Angina Questionnaire, which provides an assessment based upon a
mixed combination of objective and subjective criteria; and 3) the
measurement of physical changes in the heart as assessed by
MRI.
[0059] For ETT of the 66 patients of the Phase I clinical trial of
Examples 1-3 was measured at baseline, and at 1 month, 2 months and
6 months after dosing (with a single unit dose composition of the
invention) using a Bruce treadmill protocol. Subjects were excluded
from the analysis if the treadmill protocol was not the same as
used at baseline. Therefore, the number of subjects varied over
time. In addition, any patients who had emergency revascularization
were excluded from the analysis. A dose was considered effective if
the mean change in ETT from baseline increased by greater than 60
seconds. The results of the ETT assessment are provided in Table
1.
TABLE-US-00001 TABLE 1 Exercise Tolerance Time (ETT)-Change from
Baseline Change from Change from Change from FGF-2 Baseline
Baseline Baseline Dose Group at One Month at Two Months at Six
Months 0.33 to 2.0 .mu.g/kg IC N = 8 N = 6 N = 5 (N = 16) 45.1 sec
130.0 sec* 60.8 sec (low) (-105 to 180) (19 to 240) (-45 to 210)
6.0 and 12 .mu.g/kg IC N = 2 N = 4 N = 2 (N = 8) -24.0 sec -2.5 sec
6.5 sec (mid) (-48 to 0) (-90 to 120) (-0 to 13) 24.0 to 48.0
.mu.g/kg IC N = 18 N = 21 N = 16 (N = 28) 51.9 sec 107.9 sec* 133.1
sec* (high) (-188 to 399) (-30 to 385) (-195 to 386) 18.0 &
36.0 .mu.g/kg IV N = 12 N = 12 N = 12 (N = 14) 45.1 sec 93.4 sec*
87.5 sec* (-75 to 237) (0 to 285) (-60 to 285) ALL GROUPS N = 40 N
= 43 N = 35 (N = 66) 45.0 sec 96.0 sec 100.0 sec N = number of
subjects; mean; (range in seconds); *= p < 0.05
[0060] Referring to Table 1, the mean change from baseline at one
month was less than 60 seconds for all dose groups. However, the
percentage of patients stopping their treadmill test because of
angina decreased in all groups over time. At 2 months and 6 months
after dosing, the mean changes from baseline were greater in the
high dose IC and IV groups of patients than in the low and mid dose
IC groups. The persistence of increased ETT at 6 months (133.1 sec
and 87.5 sec) in the high dose IC (24-48 .mu.g/kg) and IV (18 &
38 .mu.g/kg) groups, respectively, was unexpected. The greatest
mean increases in ETT of 107.9 and 133.1 seconds at 2 and 6 months,
respectively, occurred in the high dose (24-48 .mu.g/kg) IC group.
The IV group exhibited significant mean increases in ETT of 93.4
seconds and 87.5 seconds, at 2 months and 6 months respectively,
which was not predicted by the rat and pig animal models used
herein. Overall, the persistence of the effect (increase in ETT) at
six months and its magnitude for both the IC and IV groups was
wholly unexpected.
[0061] The 66 human patients of the Phase I clinical trial
described in Examples 1-3 herein were also evaluated using the
Seattle Angina Questionnaire (SAQ). The SAQ is a validated,
disease-specific, quality of life instrument which assesses the
following five scales: 1) "exertional capacity"=limitation of
physical activity; 2) "disease perception"=worry about MI; 3)
"treatment satisfaction"; 4) "angina frequency"=number of episodes
and sublingual nitroglycerin usage; and 5) "angina
stability"=number of episodes with most strenuous physical
activity. The possible range of scores for each of the five scales
is 0 to 100 with the higher scores indicating a better quality of
life. Typically, a mean change of 8 points or more between the mean
baseline scores (i.e., before treatment) and the post-treatment
scores is recognized as being "clinically significant." However, in
the present analysis, a dose was considered "effective" if the mean
change in score from baseline increased by greater than 14 points.
The reason that 14 was chosen (instead of 8) was to allow for the
improvement that was seen in the placebo group at 2 months in a
clinical trial of another growth factor--VEGF.
[0062] In performing the SAQ evaluation, the patients were
categorized according to the same dosage groups that were evaluated
for ETT, i.e., 0.33-2.0 .mu.g/kg IC (low) 6.0-12.0 .mu.g/kg IC
(mid); 24-48 .mu.g/kg IC (high); and 18 and 36 .mu.g/kg W. The
questionnaire was administered to subjects in each dosage group at
baseline (prior to dosing), and at 2 months and 6 months after
being administered a single unit dose composition of rFGF-2 in
accordance with the method of the present invention.
[0063] The first SAQ scale is "exertional capacity." The data on
exertional capacity is summarized in Table 2 herein.
TABLE-US-00002 TABLE 2 Exertional Capacity (EC)-Change from
Baseline FGF-2 Change from Baseline Change from Baseline Dose Group
at Two Months at Six Months 0.33 to 2.0 .mu.g/kg IC N = 14 N = 7 (N
= 16) 15.0* (-25 to 53) 23.2* (0 to 53) 6.0 and 12 .mu.g/kg IC N =
7 N = 6 (N = 8) 20.2* (-14 to 44) 24.1 (-11 to 69) 24.0 to 48.0
.mu.g/kg IC N = 26 N = 23 (N = 28) 14.6* (-33 to 75) 22.9* (-14 to
75) 18.0 and 36.0 .mu.g/kg IV N = 12 N = 14 (N = 14) 10.9 (-8 to
67) 16.5* (-19 to 63) N = number of subjects; mean (range); *= p
< 0.05
[0064] As reflected in Table 2, the change from baseline in mean
score increased at 2 and 6 months for each of the three IC dosage
groups and at 6 months for all dosage groups (IC and IV). All
scores at all dosage levels increased with time in going from 2
months to 6 months with the best increases (23.2, 24.1, 22.9 and
16.5) relative to baseline being seen at 6 months post-dosing.
[0065] The second SAQ scale to be evaluated was "angina stability."
The data summarizing the angina stability is presented in Table 3
herein.
TABLE-US-00003 TABLE 3 Angina Stability (AS)-Change from Baseline
FGF-2 Change from Baseline Change from Baseline Dose Group at Two
Months at Six Months 0.33 to 2.0 .mu.g/kg IC N = 13 N = 7 (N = 16)
46.2* (0 to 100) 21.4* (0 to 50) 6.0 and 12 .mu.g/kg IC N = 7 N = 6
(N = 8) 32.1* (0 to 50) 16.7 (-25 to 50) 24.0 to 48.0 .mu.g/kg IC N
= 27 N = 24 (N = 28) 34.3* (-25 to 75) 17.7* (-25 to 75) 18.0 and
36.0 .mu.g/kg IV N = 12 N = 14 (N = 14) 39.6* (0 to 100) 23.2* (0
to 75) N = number of subjects; mean (range); *= p < 0.05
[0066] According to Table 3, the change in score for angina
stability increased relative to baseline at both 2 and 6 months for
each group. The improvements in angina stability seen at 2 months
after dosing (46.2, 32.1, 34.3 and 39.6) were significantly greater
than those scores seen at 6 months (21.4, 16.7, 17.7 and 23.2).
However, the scores found at both 2 months and 6 months after
dosing showed that all dosages were found to be effective (>14)
in increasing angina stability. Moreover, the magnitude of the
increases and their duration for 6 months were unexpected.
[0067] The third SAQ scale to be evaluated was "angina frequency."
The data summarizing the angina frequency is presented in Table 4
herein.
TABLE-US-00004 TABLE 4 Angina Frequency (AF)-Change from Baseline
FGF-2 Change from Baseline Change from Baseline Dose Group at Two
Months at Six Months 0.33 to 2.0 .mu.g/kg IC N = 14 N = 7 (N = 16)
27.9* (-10 to 80) 12.9 (-40 to 50) (low) 6.0 and 12 .mu.g/kg IC N =
7 N = 6 (N = 8) 32.9* (0 to 80) 36.7 (-10 to 90) (mid) 24.0 to 48.0
.mu.g/kg IC N = 27 N = 24 (N = 28) 28.9* (-40 to 80) 25.8* (-30 to
80) (high) 18.0 and 36.0 .mu.g/kg IV N = 12 N = 14 (N = 14) 20.0*
(0 to 90) 11.4 (-30 to 60) ALL GROUPS N = 60 N = 51 (N = 66) 27.3
21.4 N = number of subjects; mean (range); *= p < 0.05
[0068] According to Table 4, the mean patient scores (27.9, 32.9,
28.9 and 20.0) for angina frequency increased at 2 months (relative
to baseline) by an effective amount (>14) for all dosage groups
and for all modes of administration (IC or IV). The mean patient
scores continued to increase at 6 months only for the mid dose
(6.0-12.0 .mu.g/kg) group, suggesting a peak effect at 2 months
post-dosing. However, for the mid dose (6.0-12.0 .mu.g/kg) and high
dose (24.0-48.0 .mu.g/kg) groups, the changes at 2 months and 6
months were similar, suggesting a persistent effect at 6 months on
angina frequency. The third SAQ scale to be evaluated was "angina
frequency." The data summarizing the angina frequency is presented
in Table 4 herein.
[0069] The fourth SAQ scale to be evaluated was "angina frequency."
The data summarizing the angina frequency is presented in Table 5
herein.
TABLE-US-00005 TABLE 5 Treatment Satisfaction (TS)-Change from
Baseline FGF-2 Change from Baseline Change from Baseline Dose Group
at Two Months at Six Months 0.33 to 2.0 .mu.g/kg IC N = 14 N = 7 (N
= 16) 8.5* (-19 to 31) 6.3 (-25 to 25) (low) 6.0 and 12 .mu.g/kg IC
N = 7 N = 6 (N = 8) 17.9 (-13 to 44) 19.8 (0 to 63) (mid) 24.0 to
48.0 .mu.g/kg IC N = 27 N = 24 (N = 28) 18.8* (-38 to 69) 13.0 (-75
to 63) (high) 18.0 and 36.0 .mu.g/kg IV N = 12 N = 14 (N = 14)
19.8* (-13 to 63) 13.4* (-19 to 56) N = number of subjects; mean
(range); *= p < 0.05
[0070] According to Table 5, the score for treatment satisfaction
increased by an effective amount at 2 months for the mid and high
dose IC groups as well as the IV group. At six months post-dosing,
only the score for the mid dose group IC had a score that was
greater than 14, suggesting a peak effect for treatment
satisfaction at 2 months.
[0071] The fifth SAQ scale to be evaluated was "disease
perception." The data summarizing the disease perception is
presented in Table 6 herein. According to Table 6, the scores for
disease perception increased from baseline to scores of 20.2-29.2
at 2 months and 23.8-34.0 at 6 months. These scores showed that
administering a single unit dose composition in accordance with the
method of the present invention was considered to be as effective
(or more effective) at 6 months as at two months. These scores
suggest a persistence of the effectiveness of the method of the
present invention on disease perception out to six months following
administration of a single unit dose composition.
TABLE-US-00006 TABLE 6 Disease Perception (DP)-Change from Baseline
Change from Baseline Change from Baseline Dose Group at Two Months
at Six Months 0.33 to 2.0 .mu.g/kg IC N = 14 N = 7 (N = 16) 29.2*
(-8 to 58) 26.2* (0 to 42) (low) 6.0 and 12 .mu.g/kg IC N = 7 N = 6
(N = 8) 20.2* (-8 to 50) 30.6* (0 to 58) (mid) 24.0 to 48.0
.mu.g/kg IC N = 27 N = 24 (N = 28) 27.8* (-33 to 92) 34.0* (-33 to
100) (high) 18.0 and 36.0 .mu.g/kg IV N = 12 N = 14 (N = 14) 22.9*
(-8 to 92) 23.8* (-8 to 75) N = number of subjects; mean (range);
*= p < 0.05
[0072] Up to 60 of the human patients of the Phase I clinical trial
described in Examples 1-3 herein were also evaluated using resting
magnetic resonance imaging (MRI) scans of their heart. The resting
MRI scans were performed on the patients at baseline, and at 1
month, 2 months and 6 months after dosing with a single unit dose
composition of the present invention. The doses were considered
"effective" based upon statistical significance (p<0.05). The
objective criteria assessed by the resting MRI scans are the
following: (1) ejection fraction; (2) myocardial infarct extent
(%); (3) normal wall thickening (4) normal wall motion (%); (5)
target wall thickening (%); (6) target wall motion (%); (7) target
wall area collateral extent (%); and (8) target area delayed
arrival extent (%).
[0073] Based upon the resting MRI, no change in "ejection fraction"
was observed at one month for any one group. The mean change from
baseline for all groups (n=33) at 1 month was an increase of 2.0%
(p=0.042). At two months, the mean change from baseline for the low
dose IC group (n=13) was an increase of 8.1% (p=0.007); and for all
groups (n=54), the mean change from baseline was an increase of
3.8% (p=0.001). At six months, the mean change from baseline for
the high dose IC group (n=19) was 5.3% (p=0.023); for the IV group
(n=3) was 11.1% (p=0.087); and for all groups (n=33) was 5.7%
(p=0.001).
[0074] Based upon the resting MRI, there was no statistically
significant change in the "myocardial infarct extent" (%) for any
group, or for all groups in combination at 1 month, 2 months or 6
months post-dosing. When the normal wall motion (%) and normal wall
thickening were assessed, there was no statistically significant
change from baseline at 1 month, 2 months or 6 months for any one
group. However, there was a statistically significant change from
baseline in target wall motion for all groups at one (n=60), two
(n=54) and six (n=33) months, which was reflected as a mean
increase from baseline of 2.7% (p=0.015), 4.4% (p=<0.001) and
6.4% (p<0.001), respectively. However, there was also a
statistically significant change from baseline in target wall
thickening for all groups at one (n=60), two (n=54) and six (n=33)
months, which was reflected as a mean increase from baseline of
4.4% (p=0.015), 6.3% (p=<0.001) and 7.7% (p<0.001),
respectively.
[0075] The next criteria assessed by MRI was "target area
collateral extent" (%). The mean increase from baseline in target
area collateral extent for all groups was highly statistically
significant at one month (n=31), two months (n=27) and six months
(n=16), wherein the increases were 8.3% (p<0.001), 10.9%
(p<0.001) and 11.2% (p<0.001), respectively. The greatest
collateral extent increases were observed for the low and mid IC
doses, i.e., at one month (10.4% and 18.3%, respectively), two
months (14.7% and 18.0%, respectively) and six months (16.0% and no
value for mid dose, respectively), which was wholly unexpected. At
one month, two months and six months post-dosing, the corresponding
% increases in target area collateral extent that were observed for
the IC high dose group were 6.3%, 8.0% and 9.0%, respectively.
[0076] The final criteria assessed by MRI was "target area delayed
arrival extent" (%). The mean decrease from baseline in target area
delayed arrival extent for all groups was highly statistically
significant at 1 month (n=60), 2 months (n=54) and 6 months (n=34),
wherein the decreases were -5.8% (p<0.001), -8.3% (p<0.001)
and -10.0% (p<0.001), respectively. The greatest target area
delayed extent decreases were observed for the low dose IC group,
which was also highly unexpected.
[0077] Thus, providing CAD patients with a single IC or IV infusion
of rFGF-2 in accordance with the present invention provided the
patients with a statistically significant physical improvement as
objectively measured by MRI and other conventional criteria.
Pharmacokinetics and Metabolism
[0078] The molecular structure of FGF-2 contains a positively
charged tail that is known to bind to proteoglycan chains (heparin
and heparin-like structures) on cell surfaces and on the
endothelial wall of the vasculature. See Moscatelli, et al.,
"Interaction of Basic Fibroblast Growth Factor with Extracellular
Matrix and Receptors," Ann. NY Acad. Sci., 638:177-181 (1981).
[0079] The kidneys and liver are the major organs for the
elimination of rFGF-2. In particular, the kidneys have a protein
cutoff of about 60 kD and thus retain serum albumin (MW 60 kD).
However, FGF-2 (146 residues) has a molecular weight of about 16.5
kD. Accordingly, renal excretion is to be expected. In a
radiolabelled biodistribution study of commercially available
bovine FGF-2 (bFGF-2), both the liver and the kidney were shown to
contain high counts of the radiolabelled bFGF-2 at 1 hour after IV
or IC injection. In a published study, wherein another recombinant
iodinated form of bFGF-2 was given to rats, the liver was
identified as the major organ of elimination. Whalen et al., "The
Fate of Intravenously Administered bFGF and the Effect of Heparin,"
Growth Factors, 1:157-164 (1989). It is also known that FGF-2 binds
in the general circulation to .alpha..sub.2-macroglobulin and that
this complex is internalized by receptors on the Kupffer cells.
Whalen et al. (1989) and LaMarre et al., "Cytokine Binding and
Clearance Properties of Proteinase-Activated
Alpha-2-Macroglobulins," Lab. Invest., 65:3-14 (1991). Labelled
FGF-2 fragments were not found in the plasma, but they were found
in the urine and corresponded in size to intracellular breakdown
products.
[0080] In preclinical testing, we determined the pharmacokinetics
of rFGF-2 (SEQ ID NO:2) after intravenous (IV) and intracoronary
(IC) administration in domestic Yorkshire pigs, and after IV
administration dosing in Sprague Dawley ("SD") rats. The pig models
demonstrated linear pharmacokinetics (0.65 .mu.g/kg-20 .mu.g/kg) IC
and IV. The terminal half-life of the FGF-2 in the pig model was
3-4 hours. The rat models demonstrated linear pharmacokinetics over
the range of 30-300 .mu.g/kg IV. The terminal half-life of the
FGF-2 in the rat model was 1 hour. Both species showed plasma
concentration suggesting a two-compartment model.
[0081] Likewise, in humans, the FGF-2 plasma concentrations after
IV and/or IC infusion followed a biexponential curve with an
initial steep slope and considerable decrease over several log
scales (the distribution phase) during the first hour, followed by
a more moderate decline (the elimination phase). FIG. 1A provides a
plasma concentration versus time curve showing these phases in
humans after IC administration of rFGF-2 of SEQ ID NO:2 as a
function of each of the following eight doses: 0.33 .mu.g/kg, 0.65
.mu.g/kg, 2 .mu.g/kg, 6 .mu.g/kg, 12 .mu.g/kg, 24 .mu.g/kg, 36
.mu.g/kg, and 48 .mu.g/kg of lean body mass (LBM). FIG. 1A shows
the plasma dose linearity for the eight doses of rFGF-2 that were
administered by IC infusion over a twenty minute period. FIG. 1A
also shows a biphasic plasma level decline, i.e., a fast
distribution phase during the first hour, followed by an
elimination phase with an estimated T.sub.1/2 of 5-7 hours. The
plasma concentrations of FGF-2 of SEQ ID NO:2 were determined by a
commercially available ELISA (R&D Systems, Minneapolis Minn.)
that was marketed for analysis of human FGF-2. The ELISA assay
showed 100% cross-reactivity with the rFGF-2 of SEQ ID NO:2. Other
members of the FGF family, as well as many other cytokines, were
not detected by this assay. Further, heparin does not interfere
with the assay.
[0082] FIG. 1B is a plot of the mean FGF-2 plasma concentration as
a function of time for 18 .mu.g/kg and 36 .mu.g/kg rFGF-2
administered IV, as compared to 36 .mu.g/kg rFGF-2 administered IC.
The plasma concentration versus time profiles in FIG. 1B for the 36
.mu.g/kg doses by the IV and IC routes are superimposible. However,
a first-pass effect with the IC route is not eliminated.
[0083] FIG. 2 is a plot of mean FGF-2 area under the curve (AUC) in
pg*min/ml corresponding to FIGS. 1A and 1B. FIG. 2 shows the dose
linearity of systemic rFGF-2 exposure (AUC) following IC or IV
infusion. In particular, FIG. 2 shows that the systemic exposure to
rFGF-2 following administration by the IC and IV routes is
substantially similar.
[0084] FIG. 3 is a plot of individual human patient plasma
clearance (CL) values (ml/min/kg) as a function of the time of
heparin administration in "minutes prior to rFGF-2 infusion." FIG.
3 shows the influence of timing of heparin administration on FGF-2
plasma clearance (CL). Although FIG. 3 shows that administering
heparin up to 100 minutes prior to rFGF-2 decreases FGF-2
clearance, the preferred time for administering heparin is from
0-30 minutes prior the rFGF-2 administration, wherein the effect of
the heparin on decreasing FGF-2 clearance is greatest.
[0085] FIG. 4 is a plot OF individual human patient rFGF-2 dose
normalized area under curves (AUCs) as a function of the time of
heparin administration in "minutes prior to rFGF-2 infusion" and
shows the influence of timing of heparin administration on rFGF-2
AUC. FIG. 4 shows that the greatest AUC/dose was achieved when an
effective amount of a glycosoaminoglycan, such as heparin, was
preadministered within 30 minutes or less of IC rFGF-2 infusion,
more preferably within 20 minutes or less of IC or IV rFGF-2
infusion. Typically, an effective amount of a glycosoaminoglycan is
10-80 U/kg heparin.
[0086] The mean pharmacokinetic parameters for rFGF-2 in humans as
a function of dosage and mode of administration are summarized in
Table 8 herein. Referring to Table 8, the T.sub.1/2 for FGF-2 in
humans was determined to range from 2.2.+-.3.7 hours at low dose
(0.33-2.0 .mu.g/kg) IC to 7.0.+-.3.5 hours at a dose of 18-36
.mu.g/kg IV; given the limitations of the assay, the terminal
half-life is estimated at 5-7 hours for all groups. The clearances
of FGF-2 ranged from 13.2 to 18.2 L/hour/70 kg man. Finally, the
steady state volume (V.sub.ss) was determined to range from
11.3.+-.10.4 L/70 kg man to 16.8.+-.10.7 L/70 kg man.
TABLE-US-00007 TABLE 8 Mean rFGF-2 PK Parameters in Humans FGF-2 CL
V.sub.ss Dose .mu.g/kg N Route (L/hr/70 kg) t.sub.1/2 (h) (L/70 kg)
0.3-2 16 IC 18.2 .+-. 13.4 2.2 .+-. 3.7 11.3 .+-. 10.4 6-12 8 IC
13.2 .+-. 7.3 3.1 .+-. 2.5 12.1 .+-. 4.9 24-48 28 IC 14.7 .+-. 8.3
6.3 .+-. 1.8 16.8 .+-. 10.7 18-36 14 IV 13.9 .+-. 7.9 7.0 .+-. 3.5
16.4 .+-. 8.6
[0087] Although the binding of FGF-2 to heparin-like structures is
strong (dissociation constant .about.2.times.10.sup.-9 M), the
binding of FGF-2 to a specific tyrosine kinase receptor is
approximately two orders of magnitude higher (dissociation constant
.about.2.times.10.sup.-11 M). Moscatelli et al., (1991). Thus,
without being bound to any theory, the complexation of rFGF-2 with
a glycosoaminoglycan, such as a heparin, might increase signal
transduction and mitogenesis, and/or protect the rFGF-2 from
enzymatic degradation.
[0088] The examples, which follow, provide more details on the
selection criterion and the Phase I clinical trial that gave rise
to the data discussed above.
Example 1
Unit Dose of rFGF-2 Employed in the Phase I Clinical Trial
[0089] The rFGF-2 of SEQ ID NO:2 was formulated as a unit dose and
pharmaceutical composition and administered to rats, pigs and
ultimately to humans in the Phase I clinical trial referenced
herein. The various formulations are described below.
[0090] The rFGF-2 unit dose was provided as a liquid in 3 cc type I
glass vials with a laminated gray butyl rubber stopper and red
flip-off overseal. The rFGF-2 unit dose contained 1.2 ml of 0.3
mg/ml rFGF-2 of SEQ ID NO:2 in 10 mM sodium citrate, 10 mM
monothioglycerol, 1 mM disodium dihydrate EDTA (molecular weight
372.2), 135 mM sodium chloride, pH 5.0. Thus, in absolute terms,
each vial (and unit dose) contained 0.36 mg rFGF-2. The vials
containing the unit dose in liquid form were stored at 2.degree. to
8.degree. C.
[0091] The rFGF diluent was supplied in 5 cc type I glass vials
with a laminated gray butyl rubber stopper and red flip-off
overseal. The rFGF-2 diluent contains 10 mM sodium citrate, 10 mM
monothioglycerol, 135 mM sodium chloride, pH 5.0. Each vial
contained 5.2 ml of rFGF-2 diluent solution that was stored at
2.degree. to 8.degree. C.
[0092] The rFGF-2 pharmaceutical composition that was infused was
prepared by diluting the rFGF-2 unit dose with the rFGF diluent
such that the infusion volume is 10-40 ml. In order to keep the
EDTA concentration below a preset limit of 100 .mu.g/ml, the total
infusion volume was increased to a maximum of 40 ml when
proportionately higher absolute amounts of FGF-2 were administered
to patients with higher body weights.
Example 2
Selection Criteria for Patients with Coronary Artery Disease for
Treatment with rFGF-2
[0093] The following selection criteria were applied to Phase I
patients with coronary artery disease, whose activities were
limited by coronary ischemia despite optimal medical management,
and who were not candidates for approved revascularization
therapies:
[0094] Inclusion Criteria:
[0095] Subject is eligible if: [0096] Male or female, greater than
or equal to 18 years of age [0097] Diagnosis of coronary artery
disease (CAD) [0098] Suboptimal candidates for approved
revascularization procedures, e.g., angioplasty, stents, coronary
artery bypass graft (CABG) (or refuses those interventions) [0099]
Able to exercise at least three minutes using a modified Bruce
protocol and limited by coronary ischemia [0100] Inducible and
reversible defect of at least 20% myocardium on pharmacologically
stressed thallium sestamibi scan [0101] CBC, platelets, serum
chemistry within clinically acceptable range for required cardiac
catheterization [0102] Normal INR, or if anticoagulated with
Coumadin, INR <2.0 [0103] Willing and able to give written
informed consent to participate in this study, including all
required study procedures and follow-up visits
[0104] Exclusion Criteria:
[0105] Subject is not eligible if: [0106] Malignancy: any history
of malignancy within past ten years, with the exception of
curatively treated basal cell carcinoma [0107] Ocular conditions:
proliferative retinopathy, severe non-proliferative retinopathy,
retinal vein occlusion, Eales' disease, or macular edema or
funduscopy by ophthalmologist: history of intraocular surgery
within six months [0108] Renal function: creatinine clearance below
normal range adjusted for age; protein >250 mg or microalbumin
>30 mg/24 h urine [0109] Class IV heart failure (New York Heart
Association) [0110] Ejection fraction <20% by echocardiogram,
thallium scan, MRI or gated pooled blood scan (MUGA) [0111]
Hemodynamically relevant arrhythmias (e.g., ventricular
fibrillation, sustained ventricular tachycardia) [0112] Severe
valvular stenosis (aortic area <1.0 cm.sup.2, mitral area
<1.2 cm.sup.2), or severe valvular insufficiency [0113] Marked
increase in angina or unstable angina within three weeks [0114]
History of myocardial infarction (MI) within three months [0115]
History of transient ischemic attack (TIA) or stroke within six
months [0116] History of CABG, angioplasty or stent within six
months [0117] History of treatment with transmyocardial laser
revascularization, rFGF-2, or vascular enodothelial growth factor
(VEGF) within six months [0118] Females of child-bearing potential
or nursing mothers [0119] Any pathological fibrosis, e.g.,
pulmonary fibrosis, scleroderma [0120] Known vascular malformation,
e.g., AV malformation, hemangiomas [0121] Coexistence of any
disease which might interfere with assessment of symptoms of CAD,
e.g., pericarditis, costochondritis, esophagitis, systemic
vasculitis, sickle cell disease [0122] Coexistence of any disease
which limits performance of modified Bruce protocol exercise stress
test, e.g., paralysis or amputation of a lower extremity, severe
arthritis or lower extremities, severe chronic obstructive
pulmonary disease (COPD) [0123] Participation in clinical trials of
investigational agents, devices or procedures within thirty days
(or scheduled within sixty days of study drug) [0124] Known
hypersensitivity to rFGF-2 or related compounds [0125] Any
condition which makes the subject unsuitable for participation in
this study in the opinion of the investigator, e.g., psychosis,
severe mental retardation, inability to communicate with study
personnel, drug or alcohol abuse.
Example 3
Phase I Clinical Study on Recombinant FGF-2 (SEQ ID NO:2)
Administered to Humans
[0126] The Phase I CAD trial of this example is an open label, dose
escalation study of recombinant fibroblast growth factor-2 (rFGF-2)
for safety, tolerability and pharmacokinetics. The study was
conducted at two sites: Beth Israel Deaconess Hospital (Harvard) in
Boston, Mass. and Emory University Hospital in Atlanta, Ga.
Enrollment is complete. Subjects were treated with a single
infusion of rFGF-2 on Day 1 and followed for 360 days; follow-up is
not yet complete in some subjects.
[0127] The subject population consists of patients with advanced
CAD who are exercise limited by coronary ischemia and are
considered suboptimal candidates for (or do not want to undergo)
one of the established revascularization procedures (e.g., CABG,
angioplasty--with or without stent). The major exclusion criteria
were history or suspicion of malignancy, uncompensated heart
failure or left ventricular ejection fraction <20%, renal
insufficiency or proteinuria, and various ocular conditions (e.g.,
proliferative diabetic retinopathy, severe non-proliferative
retinopathy).
[0128] Sixty-six subjects have received rFGF-2 of SEQ ID NO:2 in
this trial: fifty-two received the rFGF-2 as an IC infusion and
fourteen received it as an IV infusion. Each subject was observed
in the hospital for at least twenty-four hours, and then followed
as an outpatient for 360 days with follow-up visits (Days 15, 29,
57, 180 and 360). At least four subjects were studied at each dose;
if no subject experienced dose-limiting toxicity as defined by the
protocol within six days, the dose was escalated. The drug was
administered as a single 20 minute infusion divided between two
major sources of coronary blood supply (IC), using standard
techniques for positioning a catheter into the patient's coronary
artery (such as already employed in angioplasty) or in a peripheral
vein (IV). The doses in .mu.g/kg of rFGF-2 administered IC (and the
number of patients) were 0.33 (n=4), 0.65 (n=4), 2.0 (n=8), 6.0
(n=4), 12.0 (n=4), 24 (n=8), 36 (n=10) and 48 (n=10) of rFGF-2 of
SEQ ID NO:2. The doses in .mu.g/kg of rFGF-2 administered IV (and
the number of patients) were 18.0 (n=4) and 36.0 (n=10) or rFGF-2
of SEQ ID NO: 2.
[0129] Angina frequency and quality of life was assessed by the
Seattle Angina Questionnaire (SAQ) at a baseline (before rFGF-2
administration) and at 2 months and 6 months after rFGF-2
administration. Exercise tolerance time (ETT) was assessed by the
treadmill test at 1, 2, and 6 months. Rest/exercise nuclear
perfusion and gated sestamibi-determined rest ejection fraction
(EF), and resting magnetic resonance imaging (MRI) were assessed at
baseline, and at 1 month, 2 months and 6 months post rFGF-2
administration. MRI measurements which were thought to objectively
measure changes in % in cardiac function and perfusion included:
(1) ejection fraction; (2) myocardial infarct extent (%); (3)
normal wall thickening (4) normal motion (%); (5) target wall
thickening (%); (6) target wall motion (%); (7) target wall area
collateral extent (%); and (8) target area delayed arrival extent
(%).
[0130] If one of four subjects experienced dose-limiting toxicity
(as defined by the protocol), four additional subjects were studied
at that dose; if none experienced toxicity, the dose was escalated
and another group was studied. The maximally tolerated dose (MTD)
was defined as the IC dose which was tolerated by 9/10 subjects,
i.e., 36 .mu.g/kg IC.
[0131] Careful fluid management pre-infusion was prescribed using a
Swan-Ganz catheter and vital signs were monitored frequently during
dosing. Heparin was administered IV prior to the infusion of rFGF-2
in all groups. The EDTA concentration was less than 100 .mu.g/ml in
the unit dose composition. Volume of study drug administered varied
with dose and subject's weight, and ranged from 10 ml at lower
doses to 40 ml at higher doses.
Preliminary Results
[0132] The results presented here are unaudited and are based on a
third interim analysis for sixty-six subjects with six months
follow-up for all groups (1-10) and the serious adverse events
(SAE) report of 29 Jul. 1999 from Chiron Drug Safety. Data
collection for the last visit (Day 360) and final analysis is in
progress.
[0133] The starting dose of 0.33 .mu.g/kg IC was escalated over
eight sequential groups to 48 .mu.g/kg IC, at which dose 2 of ten
subjects experienced dose-limiting toxicity (hypotension) as
defined by the protocol. Hypotension was manageable with fluids
alone in all subjects (no vasopressors or assistive devices). At 36
.mu.g/kg IC, only 1 of 10 subjects had dose-limiting toxicity which
defined this dose as the maximally tolerated dose (MTD). Two
additional groups were studied by IV infusion; four subjects of
half the MTD (18 .mu.g/kg) and ten subjects at the MTD (36
.mu.g/kg).
[0134] Hypotension was dose-limiting in humans, as predicted by the
animal model in Yorkshire pigs. However, 36.0 .mu.g/kg rFGF-2 IC
was tolerated in humans; whereas in pigs, 20.0 .mu.g/kg rFGF-2 IC
caused profound hypotension in one of two animals. Better
tolerability in humans was attributed to aggressive fluid
management and absence of general anesthesia.
[0135] As of 29 Jul. 1999, thirty-three serious adverse events
(SAEs) have occurred in 24/66 subjects, but were not dose-related.
Fifteen (15) SAEs were considered at least possibly related to
rFGF-2; whenever there was a difference between relatedness
assigned by the investigator and the medical monitor, the more
conservative relationship was assigned. SAE's were multiple in five
subjects: 01103 (0.33 .mu.g/kg IC), 01106 (0.65 .mu.g/kg IC), 01113
(2.0 .mu.g/kg IC), 01137 (36.0 .mu.g/kg IV), 02101 (0.65 .mu.g/kg
IC).
[0136] The most frequent treatment-emergent adverse events (AEs) on
Day 1 were transient systolic hypotension and transient
bradycardia. The hypotension was dose-dependent and occurred more
frequently at doses greater than or equal to (.gtoreq.) 24 .mu.g/kg
IC; bradycardia was not dose-dependent. Other adverse events (AEs)
which were deemed at least possibly related and appeared
dose-related occurred within the first several days or week post
infusion and included chest pain, shortness of breath, insomnia,
anxiety, and nausea. These events were mild to moderate in
severity, and most did not require specific intervention.
[0137] When administered IC, the drug was administered over
approximately 20 minutes as a single infusion divided between two
major sources of coronary blood supply (IC), using standard
techniques for positioning a catheter into the patient's coronary
artery (such as already employed in angioplasty). When administered
IV, the drug was administered as an infusion over 20 minutes into a
peripheral vein.
[0138] The preliminary safety results indicate that serious events
were not dose related. Thus far, of the eight IC dosage groups,
there were three deaths in the lower dosage groups, i.e., at 0.65
.mu.g/kg (Day 23), at 2.0 .mu.g/kg (Day 57) and at 6.0 .mu.g/kg
(Day 63), and one death in the highest dose group, i.e., at 48.0
.mu.g/kg (approximately 4 months post-dosing). Three of the deaths
were cardiac; one death was due to a large B cell lymphoma that was
diagnosed three weeks after dosing in the patient in group 4 (6.0
.mu.g/kg) who patient died at two months post-dosing.
[0139] Acute myocardial infarction (MI) occurred in four patients,
i.e., one patient from each of groups 1 (0.33 .mu.g/kg), 3 (2.0
.mu.g/kg), 4 (6.0 .mu.g/kg) and 7 (36.0 .mu.g/kg). Multiple MIs
occurred in two patients, i.e., one from group 1 (0.33 .mu.g/kg)
and one from group 3 (2.0 .mu.g/kg). Emergency revascularization
procedures (CABG or angioplasty with or without stent) were
performed during follow-up in 4 patients: one each from groups 1
(0.33 .mu.g/kg), 3 (2.0 .mu.g/kg), 4 (6.0 .mu.g/kg), and 7 (36.0
.mu.g/kg).
[0140] Acute hypotension, seen at higher doses during or just
subsequent to infusion, was managed by administration of IV fluids
without need for a vasopressor. The maximally tolerated dose (MTD)
in humans was defined as 36 .mu.g/kg IC. (In contrast, in pigs, the
MTD was 6.5 .mu.g/kg IC.) Doses of rFGF-2 up to 48 .mu.g/kg IC were
administered in human patients with aggressive fluid management,
but were defined by the protocol as "not tolerated" due to acute
and/or orthostatic hypotension in two out of ten patients. The
terminal half-life of the infused rFGF-2 was estimated at 5 to 7
hours.
[0141] The human patients in this study that were treated with a
single IC or IV infusion of rFGF-2 of SEQ ID NO:2 exhibited a mean
increase in ETT of 1.5 to 2 minutes. See Table 1. This is
especially significant because an increase in ETT of greater than
(>) 30 seconds is considered significant and a benchmark for
evaluating alternative therapies, such as angioplasty. The angina
frequency and quality of life, as measured by SAQ, showed a
significant improvement at 2 months in all five subscales for the
66 patients (n=66) tested. See Tables 26. In Tables 2-6, a mean
change of 14 or more was considered "clinically significant."
[0142] When 33 human CAD patients were assessed by resting cardiac
magnetic resonance imaging (MRI) at baseline, and at 1, 2, and 6
months after receiving a single unit dose composition of the
present invention by IC or IV routes, a highly statistically
significant increase was observed in target wall thickening, target
wall motion and target area collateral extent; a highly
statistically significant decrease was observed in target area
delayed arrival extent; and no statistically significant changes
were observed in normal wall motion, normal wall thickening or
myocardial infarct extent.
[0143] In addition to the above criterion (i.e., ETT, SAQ, MRI), a
treatment is considered very successful if the angiogenic effects
last at least six months. In the present Phase I study, the
unexpectedly superior angiogenic effects were observed to last up
to 6 months in some patients in all dosage groups. Based upon the
results already obtained, it is expected that the angiogenic
effects may last twelve months or more but do last at least six
months in the patients, at which time the procedure could be
repeated, if necessary.
Example 4
Proposed Phase II Clinical Study on rFGF-2 (SEQ ID NO:2)
Administered to Humans to Treat Coronary Artery Disease
[0144] The Phase II clinical trial of rFGF-2 for treating human
patients for coronary artery disease is performed as a double
blind/placebo controlled study having four arms: placebo, 0.3
.mu.g/kg, 3.0 .mu.g/kg, and 30 .mu.g/kg administered once IC. This
study is ongoing and results are not yet available.
Example 5
Unit Dose and Pharmaceutical Composition of rFGF-2 for the Phase II
Human Clinical Trial
[0145] The rFGF-2 of SEQ ID NO:2 was formulated as a unit dose
pharmaceutical composition for administration to humans in the
Phase II clinical trial referenced herein. The various formulations
are described below.
[0146] The rFGF-2 unit dose was prepared as a liquid in 5 cc type I
glass vials with a laminated gray butyl rubber stopper and red
flip-off overseal. The rFGF-2 formulation contains 0.3 mg/ml rFGF-2
of SEQ ID NO:2 in 10 mM sodium citrate, 10 mM monothioglycerol, 0.3
mM disodium dihydrate EDTA (molecular weight 372.2), 135 mM sodium
chloride, pH 5.0. Each vial contained 3.7 ml of rFGF-2 drug product
solution (1.11 mg rFGF-2 per vial). The resulting unit dose in
liquid form is stored at less than -60.degree. C. The above
described unit dose is diluted with the "rFGF-2 placebo." Depending
on the size of the patient, the contents of several of the vials
may be combined to produce a unit dose of 36 .mu.g/kg for the Phase
II study.
[0147] The rFGF-2 placebo is supplied as a clear colorless liquid
in 5 cc type I glass vials with a laminated gray butyl rubber
stopper and red flip-off overseal. The rFGF-2 placebo is
indistinguishable in appearance from the drug product and has the
following formulation: 10 mM sodium citrate, 10 mM
monothioglycerol, 0.3 mM disodium dihydrate EDTA (molecular weight
372.2), 135 mM sodium chloride, pH 5.0. Each vial contains 5.2 ml
of rFGF-2 placebo solution. Unlike the unit dose, the rFGF-2
placebo is stored at 2.degree. to 8.degree. C.
[0148] The rFGF-2 pharmaceutical composition that is infused is
prepared by diluting the rFGF-2 unit dose with the rFGF diluent
such that the infusion volume is 20 ml for Phase II.
[0149] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
[0150] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
present invention described herein.
Sequence CWU 1
1
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tcc ggg gcc ttc cca cca ggg cac 48Pro Ala Leu Pro Glu Asp Gly Gly
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Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu 20 25 30cga atc cac cca gat
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Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp 35 40 45cca cac atc aaa
cta caa cta caa gcc gaa gaa cga ggg gta gta tcc 192Pro His Ile Lys
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Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly 65 70 75 80cga
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95cga cta gaa tcc aac aac tat aac acc tat cga tcc cga aaa tat tcc
336Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Ser
100 105 110tcc tgg tat gta gcc cta aaa cga acc ggg caa tat aaa cta
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cca atg tcc gcc 432Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu
Pro Met Ser Ala 130 135 140aaa tcc taag 442Lys Ser1452146PRTBovis
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Gly Phe Phe Leu 20 25 30Arg Ile His Pro Asp Gly Arg Val Asp Gly Val
Arg Glu Lys Ser Asp 35 40 45Pro His Ile Lys Leu Gln Leu Gln Ala Glu
Glu Arg Gly Val Val Ser 50 55 60Ile Lys Gly Val Cys Ala Asn Arg Tyr
Leu Ala Met Lys Glu Asp Gly 65 70 75 80Arg Leu Leu Ala Ser Lys Cys
Val Thr Asp Glu Cys Phe Phe Phe Glu 85 90 95Arg Leu Glu Ser Asn Asn
Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Ser 100 105 110Ser Trp Tyr Val
Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Pro 115 120 125Lys Thr
Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala 130 135
140Lys Ser14539PRTBovis bovinus 3Met Ala Ala Gly Ser Ile Thr Thr
Leu 1 5
* * * * *